PB83-263665
Toxicity and Metabolism Studies with EPA
(Environmental Protection Agency)
Priority Pollutants and Related
Chemicals in Freshwater Organisms
Wisconsin Univ.-Superior
Prepared for
Environmental Research Lab.-Duluth, MN
Sep 83
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NTIS
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EPA-600/3-85-09S
September 1983
TOXICITY AND METABOLISM STUDIES WITH EPA PRIORITY
POLLUTANTS AND RELATED CHEMICALS IN FRESHWATER ORGANISMS
by
Daniel J. Call, Larry T. Brooke, Nasim Ahmad, and
Joseph E. Richter
Center for Lake Superior Environmental Studies
University of Wisconsin-Superior, Superior, WI 54880
; U.S. EPA Grant No. R 880020010
U.S. EPA Cooperative Agreement Nos. CR 806864020 & CR 806864030
Project Officer
John I. Teasley
Environmental Research Laboratory - Duluth
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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TECHNICAL REPORT DATA
(Please read Instructions on the rtvene brfore completing)
REPORT NO.
EPA-600/3-83-095
3. RECIPIENT'S ACCESSION NO.
263665
TITLE AND SUBTITLE
Toxicity and Metabolism Studies with EPA Priority
Pollutants and Related Chemicals in Freshwater Organisms
5 REPORT DATE
September 1983
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
D.J. Call, L.T. Brooke, N. Ahmad, and J.E. Richter
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Lake Superior Environmental Studies
University of Wisconsin-Superior
Superior, Wisconsin 54880
10. PROGRAM ELEMENT NO.
II. CONTRACT/GRANT NO.
806196, 80020010, 806864
2. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/03
19. SUPPLEMENTARY NQTES
J-
Towlcologlcal rtxidle* were conducted I" two areas: (1) the to*lclty, bloconcentratlon
toxlcl'ty and/or metabolism of priority
16. ABSTRACT
potential tnd metabol I 9> of five herbicide* In fish; »nd (2)
pollutants and related chemicals In various aquatic organisms,
The test herbicides Included »l »chl or |2-chl»rc-2' ,6'-dlethy l-f»-(metnoxymethy I ) actt»ni I Icel , bromacll
(S-bromo-S-sec-butyl-^—nethyluracI I) , dlnoseb 1 2-(5»c-butyl 1-4,6-dlnl trapn»nal I , dluran ( 3-(3,4-dlch loro-
phenyl)-t ,l-dln»thylur»al , end propanll ( 3,4-dl chlaroproplonjn 1 1 ld») , Acut* ^»•lclty (through C72 hr) ,
••rly llf»-»taq» to'lclty (58-64 d*y), and bloconcantr»rion studies «ar» conducted »ltrt f»rn*«d -nfnnavs
(Plm»ph»l»» promelis) In L»K» Suptrlar »«ter. M»rblcld» fl»t»bollsm »as Invastlgited In rjlnbow tr^ut (Salm
g»lrjn»rl) both I n vlv» «nd I n vl tra,
T»»nty-two chemicals from th» EPA priority pollirt»nt list «er» studied for tn»lr acift* »nd/or chronic
toKlctty to selected freshwater »rg»nlsms. These Included 1 ,2-d Ichloro«th»ne, 1 , 1 ,2-tr Ichloroethane,
1 ,t ,2,2-tetr»chloroeth*ne, t»tr»chl»ro«thylen», 1 ,2-dlchlorobenc*ne, 1 ,3-dlchlerobenzene, 1 ,4-dlchloro-
benzene, hevachlor^banzene, hexachlor9but»dlen», dl-njbutylphttialate, pentachloraphenol , haptachlor,
chlardtne, to*«ph»n», arsenic , chromium , lead* , mercury*2, nlcKel* , silver* ,
selenlun , and cy»nld», Fresh»et»r species tested Included the fathead minnov, r*|nbov trout, blueglll
sunftsh (Lapomls macrochlrus), ftagflsh (Jordanella flarld»e), Dtphnla magn», scud (Ga^marus
Piaudol Imnaeus) , wldge (T«nyt»rsus dlsslmllls) «nd green ilga (Selan»strum capr Icornurum) . Todclfy tests
•ere *lso conducted vlftt pentachloroetriane, hevichloroethine, 1 ,2,4-tr Ichlvrabenttne, pent»chlor»b«nc»ne,
methan»l and dimethyl formamlde. The uptake by fish of dl-n-butylphth*! »ta from «ater. Its metabolism ind
elimination »«re 1 nve»tlgated. Cooperative metabolise et 1 , 1 ,2-tr lchloro«tti»n», chlor»b«nzene,
— l,t,2-trlc*loro»t1«yleA«, chloroform, end carbon tetrachlorld* -m» studied In rel«bo» trout end 0 aphn I e,
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
*.j^^-v*..-»|VV'>.*y V'
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATi Field/Croup
• «. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (Till* Reportf
UNCLASSIFIED
20. SECURITY CLASS
UNCLASSIFIED
CP* P*rm 2220-1 (••••. 4-77) ••«v>ou» CBITI»M n
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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FOREWORD
The Environmental Research Laboratory-Duluth is concerned with effects of
chemical pollutants upon aquatic life. Many chemicals are presently in use
without adequate knowledge of their effects on aquatic life, and new chemicals
are continuously being developed and marketed.
This report contains information on thirty-two chemicals and their effects
on freshwater life. Included are values for acute toxicity, chronic toxicity,
and metabolism of these chemicals with various species of organisms. These
values can be used to provide guidance for the protection of aquatic life.
Norbert Jaworski, Ph. D.
Di rector
Environmental Research Laboratory
Duluth, MN
111
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ABSTRACT
Twenty-two chemicals from the EPA priority pollutant list were studied for
their acute and/or chronic toxicity to selected freshwater organisms. These
included 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane,
tetrachloroethylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichloro-
benzene, hexachlorobenzene, hexachlorobutadiene, di-n_-butylphthalate, penta-
chlorophenol, heptachlor, chlordane, toxaphene, arsenic^ , chromium , lead ,
+2 +2 +1 +4
mercury , nickel , silver , selenium , and cyanide. Freshwater species
tested included the fathead minnow CPimephales promelas), rainbow trout CSalmo
gairdneri), bluegill sunfish (.Lepomis macrochirus), flagfish (Jordanella
floridae), water flea CDaphm'a magna), scud CGammarus pseudol imnaeus), midge
(Tanytarsus dissimilis), and green alga (Selenastrum capricornutum).
Toxicity tests were also conducted with pentachloroethane, hexachloroethane,
1,2,4-trichlorobenzene, pentachlorobenzene, dimethylformamide and methanol.
Di-rv-butylphthalate uptake from water, elimination and metabolism by fish was
studied. A comparison was made of the metabolism and binding of carbon tetra-
chloride, chloroform, 1,1,2-trichloroethane, 1,1,2-trichloroethylene and
monochlorobenzene by microsomal fractions of rainbow trout livers and of daphnid
whole bodies.
IV
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CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables 1X
Acknowledgments xii
1. Introduction 1
2. Conclusions 3
3. Recommendations 7
4. Materials and Methods 3
Water Supply and Environmental Control 8
Test Organisms 8
Acute Toxicity Tests 10
Chronic and Subchronic Toxicity Tests 21
Chemical Analysis of Toxicants 2^
Statistical Analysis of Test Results . 26
Di-n_-butylphthalate Uptake, Elimination and Metabolism . . . 27
Di-rv-butylphthalate Protein Binding 31
Microsomal Metabolism and Binding of Chlorinated
Hydrocarbons by Trout and Daphnia 32
Mixed Function Oxidase Enzyme Assays 35
5. Results 37
Acute Toxicity Tests 37
v
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5. Results Cont.
Chronic and Subchronic Toxicity Tests ............ 45
Di-n_-butylph thai ate Uptake, Elimination and
Metabolism by Fish ........ ........... . "
Di-n_-butylphthalate Binding ................ . 53
Microsomal Metabolism and Binding of Chlorinated
Hydrocarbons by Trout and Daphni a ........... . 6 '
Mixed Function Oxidase Levels ................ 68
6. Discussion ......................... . 70
Chlorinated Ethanes .................... . 70
Tetrachloroethylene .................... . 71
Chlorinated Benzenes ................... . 73
Hexachlorobutadiene ..................... 75
Di-n_-butylphthalate .................... . 75
Pentachlorophenol ...................... 77
Keptachlor ........................ •'. 73
Chlordane .............. . ......... . 78
Toxaphene ........................ . 79
Arsenic ........................ . 80
+fi
Chromium ........................ . 81
+2
Lead * .......................... : . 82
+2
Mercury ......................... . 82
Nickel"1"2 .................... ..... . 83
Silver"1"1 ......................... . 84
+4
Selenium ......................... 85
Cyanide .......................... 85
VI
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6. Discussion Cont.
Microsomal Metabolism and Binding of
Chlorinated Hydrocarbons 85
Mixed Function Oxidase Activity 86
References 88
Appendices 95
A. Summaries of Conditions and Water Characteristics 95
for Toxicity Tests
B. Toxicity Test Chemical Concentrations 104
C. Purity Levels, Analytical Parameters and Procedures,
and Analytical Quality Control Data for Toxicity
Test Chemicals 115
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FIGURES
Number Page
14
1 Log mean exposure water concentrations of C-
labeled di-n-butylphthalate Cug-mL~') and log
mean (± S-07) whole fish total 14-c residues
during uptake (days 1-11) and depuration (days
12-32) phases 57
VTIl
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TABLES
Number Page
1 LCcg Values 195% Confidence Intervals) for Pooled
Replicates of Acute Tests in Which Fathead Minnows
(Pimephales promelas) were Exposed to Arsenic 3,
Mercury "^, Si 1 ver"1"!, Dimethyl formamide and Methanol 38
2 LCgQ Values (95% Confidence Intervals) for Pooled
Replicates of Acute Tests in Which Rainbow Trout
were Exposed to Selected Organic Compounds 39
3 Results from Flow-Through Measured Acute Toxicity Tests
in Which Bluegill Sunfish (Lepomis macrochirus) were
Exposed to Hexachlorobutadiene, Hexachlorobenzene/DMF,
DimethyIformamide, and Methanol (Replicates Pooled) 40
4 LCcQ Values (95% Confidence Intervals) for Pooled
Replicates of Acute Tests in Which Flagfish were
Exposed to Arsenic+3 and Silver"*"' 42
5 48 Hr LC5Q and EC5Q Values (95% Confidence Intervals)
for Pooled Replicates of Daphnia magna Exposed to
Selected Test Chemicals 43
6 LCcQ Values (95% Confidence Intervals) for Pooled
Replicates of Acute Tests in Which Scuds (Gammarus
pseudplimnaeus) were Exposed to Pentachlorophenol,
Arsenic"1"-5, Silver"1"1, Lead*2, and Chromium"1"6 44
7 48 Hr LCsq Values (95% Confidence Intervals) for Pooled
Replicates of Acute Tests in Which (Tanytarsus
dissimilis) were Exposed to Selected Inorganic and
Organic Chemicals 46
8 Percent Inhibition of Selenastrum capricornutum Growth
when Exposed to Several Concentrations of Toxaphene
for 96 Hr 47
9 Percent Inhibition of Selenastrum capricornutum Growth
at 96 Hr Following Exposure to Several Concentrations
of Heptachlor and its Breakdown Product, 1-Hydroxy-
chlordene (Test 1) 48
ix
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Number Page
10 Percent Inhibition of Selenatrum capricornutum Growth
at 96 Hr Following Exposure to Several Concentrations
of Heptachlor and its Breakdown Product, 1-Hydroxy-
chlordene (Test 2) 49
11 Mean Exposure Concentrations of Selected Test Chemicals
and Effects Upon Reproductive Success and Growth in
Daphnia magna During 28 Day Chronic Tests .. . 50
12 Hatchability, Development, Survival and Growth of Fathead
Minnows (Pimephales promelas) Exposed to Arsenic"1"3
(NaAs02) for 30 Days Post-Fertilization 53
13 Hatchability, Development, Survival and Growth of Fathead
Minnows (.Pimephales promelas) Exposed to Inorganic
Mercury (HgCl2) for 35 Days Post-Fertilization . 54
14 Hatchability, Development, Survival and Growth of Flagfish
(Jordanella floridae) Exposed to Arsenic 3 (NaAs02) for
30 Days Post-Fertilization . . 56
15 Distribution of Radioactivity in Fathead Minnows (Pimephales
promelas) Exposed to '4C-Di-rv-butylphthai ate 59
16 Distribution (% ± S.D.) of 14C after Incubation of 14C-
Di-n_-butylphthalate for Various Time Intervals with
Microsomal Fractions of Rainbow Trout (Salmo gairdneri)
Liver and Post-Mitochondria! Supernatant of Daphnia
magna 60
17 Distribution (% ± S.D.) of 14C after Incubation with 14C-
Carbon Tetrachloride for Various Time Intervals with
Microsomal Fractions of Rainbow Trout (Salmo gairdneri)
Liver and Post-Mitochondrial Supernatant of Daphnia
magna 62
18 Distribution (% t S.D.) of 14C after Incubation with 14C-
Chloroform for Various Time Intervals with Microsomal
Fractions of Rainbow Trout (.Salmo gairdneri) Liver and
Post-Mitochondrial Supernatant of Daphnia magna 64
19 Distribution (% ± S.D.) of 14C after Incubation with 14C-
Chlorpbenzene for Various Time Intervals with Microsomal
Fractions of Rainbow Trout (Salmo gairdneri) Liver
and Post-Mitochondrial Supernatant of Daphnia magna. 65
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Number Page
20 Distribution (% ± S.D.) of 14C after Incubation with 14C-
1,1,2-Trichloroethylene for Various Time Intervals
with Microsomal Fractions of Rainbow Trout (Salmo
gairdneri) Liver and Post-Mitochondrial Supernatant
of Daphnia magna 66
21 Distribution (% ± S.D.) of 14C after Incubation with 14C-
1,1,2-Trichloroethane for Various Time Intervals with
Microsomal Fractions of Rainbow Trout CSalmo gairdneri)
Liver and Post-Mitochondria! Supernatant of Daphnia
magna 67
22 Mixed Function Oxidase Systems of Rainbow Trout (Salmo
gairdneri) Liver and Daphnia magna 69
23 Comparison of Mixed Function Oxidase Measurements Between
Mammals and Several Non-Mammalian Aquatic Organisms 87
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ACKNOWLEDGEMENTS :
We would like to thank our Project Officer, John Teasley, from the
Environmental Research Laboratory-Duluth, MN (ERL-D), U.S. Environmental
Protection Agency for his cooperation in this study. We are appreciative of
assistance and advice from the following ERL-D staff members: William Brungs,
Steven Broderius, Charles Stephan, John Poldoski, Roll Syrett, Larry Herman,
Gary Phipps, Gary Hoi combe, Anthony Carlson, James Fiandt, and Carolanne
Curtis. Glenn Endicott and the facilities staff of ERL-D were very helpful.
We gratefully recognize the assistance of the following University of Wisconsin-
Superior technical staff members: Michael Knuth, Steven Poirier, Catherine
Moriarity, Cheryl Anderson, Pamela Shubat, James Huot, Ann Lima, Marilynn
Hoglund, Dean Hammermeister, Tom Markee, Taryl Felhaber and Debra Svejskovsky.
We thank representatives from Monsanto Corporation for supplying technical
grade and radiolabeled di-rv-buty1phthalate for our studies. We gratefully
acknowledge the work of our secretary, Joyce Barnes, in the preparation of
this report.
XI1
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SECTION I
INTRODUCTION
A 1978 court settlement referred to as the "EPA Consent Decree" between EPA
and several environmentally concerned organizations as plaintiffs resulted in
the publication of a list of toxic pollutants for which effluent limitations and
guidelines were to be developed (.Keith and Telliard, 1979). This list of
"priority pollutants" initially consisted of 65 chemicals (.or groups of
chemicals), and was later expanded to 129 entries. EPA was charged with the
responsibility of determining the hazard potentials of these "priority pollu-
tants" to aquatic life and human health.
In a formal Cooperative Agreement with EPA, the University of Wisconsin-
Superior contracted to perform toxicity tests with selected "priority pollutants"
utilizing various species of freshwater organisms in an effort to provide some
of the data necessary for the development of water quality criteria statements
for the protection of freshwater aquatic life. Toxicity tests were also con-
ducted with several haloalkanes and halobenzenes closely related to "priority
pollutants" and with methanol and dimethylformamide which are sometimes used as
carrier solvents in toxicity tests.
Studies with mammalian systems have suggested that carbon tetrachloride,
chloroform and other chlorinated alkanes are converted to toxic metabolites
by the microsomal mixed function oxidase system of the liver CDocks and Krishna,
1976; Watanabe et, al_., 1978). However, information is limited concerning the
1
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metabolic disposition and protein binding of such compounds In fish and aquatic
food chain organisms. Therefore, one aspect of this study was to investigate
the comparative metabolism and protein binding potential of carbon tetrachloride,
chloroform, 1,1,2-trichloroethylene, 1,1,2-trichloroethane and monochlorobenzene
by microsomal fractions of rainbow trout CSalmo gairdneri) liver and the water
f 1 ea (Daphnla magna).
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SECTION II
CONCLUSIONS
Acute toxicity tests were conducted with fathead minnows (Pimephales
promelas), rainbow trout (Salmo gairdneri)> bluegill sunfish CLepomis
macrochirus), flagfish (Jordanella floridae), water fleas (Oaphnia magna),
scuds (Gammarus pseudolimnaeus), midge larvae (Tanytarsus dissimilis
Johannsen 1937), and green algae (Selenastrum capricornutum). Fathead minnows
were exposed to arsenic , mercury , silver , dimethylformamide (DMF), and
methanol with resulting estimated 96 hr LC5Q values of 14.2, 0,150, 0.0107,
10,700, and 28,100 mg-L , respectively. Rainbow trout were exposed to hexa-
chloroethane, tetrachloroethylene, tetrachloroethylene with DMF as a carrier
solvent, DMF, 1,2-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene,
pentachlorobenzene with DMF, hexachlorobenzene with DMF, hexachlorobutadiene,
and methanol with resultant 96 hr LC5Q estimates of 0.94, 4.99, 5.84, 10,000,
1.58, 1.12, 1.53, >0.71, >0.0809, 0.320, and 20,000 mg-L"1, respectively,
Bluegill sunfish were exposed to hexachlorobutadiene, hexachlorobenzene
with DMF, DMF, and methanol with resultant 96 hr LC5Q estimates of 0.324,
>0.0784, 7,100, and 15,500 mg-L , respectively. Flagfish were exposed to
arsenic and silver with resultant 96 hr LC5Q estimates of 14.4 and 0-0092
mg-L , respectively.
Water fleas were exposed to hexachloroethane, pentachloroethane, 1,1,2,2-
tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, 1,3-dichloro-
3
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benzene, 1,2,4-trichlorobenzene, tetrachloroethylene, di-n-butylphthalate,
+2
DMF, chlordane and nickel with resultant unfed 48 hr LC5Q estimates of 2.90,
7.32, 62-1, 186, 268, 7.43, 2.09, 18.1, 3.70, 14,530, 0.035, and 0.915 mg-L"1,
respectively. LC™ estimates were also made for most of the same compounds in
exposures where the organisms were fed. ECrQ estimates were made for fed and
unfed exposures with the chlorinated compounds and with arsenic .
Scuds were exposed to pentachlorophenol, arsenic , silver , lead"1"2, and
chromium with resultant 96 hr LC5Q estimates of 280, 875, 4.49, 140, 67.1
and 94.1 yg-L" , respectively. Midge larvae were exposed to hexachloroethane,
tetrachloroethylene, 1,2-dichlorobenzene, 1,4-dichlorobenzene, hexachlorobenzene
with DMF, pentachlorophenol, DMF, chromium , lead , silver , selenium ' and
cyanide with resultant 48 hr LCgQ estimates of 5.85, 30.8, 12.0, 13.0, >0.0581,
46.0, 36,000, 57.3, 224, 3.17, 42.5, and 2.36 as HCN or 2.49 as CN" mg-L"1,
respectively.
Green algae were exposed to toxaphene and heptachlor for 96 hr with resul-
tant EC5Q estimates C50% reduction of growth) of 0.33 mg-L for toxaphene and
38.1 and 28.2 ug-L"' for two tests with heptachlor.
Chronic and subchronic toxicity tests were conducted using water fleas,
fathead minnows, and flagfish. Water fleas were exposed to 1,1,2,2-tetra-
chloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, 1,2,4-trichlorobenzene,
1,3-dichlorobenzene, 1,2,4-trichlorobenzene, 1,3-dichlorobenzene, tetrachloro-
+3
ethylene, and arsenic for 28 days with significant (p<0.05 or p<0.01) reduc-
tions in production of young at concentrations at or above 14.4, 41.8, 20.7,
0.694, 1.45, 1.11, and 1.32 mg-L , respectively.
Fathead minnows were exposed to arsenic for 30 days post-fertilization
+2
and mercury for 35 days post-fertilization. The "no-effect" concentration
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+3 -1
for arsenic was between 2.1 and 4.3 mg-L based upon significant (p^O-Ol)
+2
reductions in wet weight and body length. Mercury exposures resulted in
significant (p<0.01) reductions in wet weight and length at all exposure con-
+2
centrations. A "no-effect" concentration for mercury was less than the lowest
exposure concentration of 0.23 ug-L" . Flagfish were exposed to arsenic* for
30 days post-fertilization with a resultant "no-effect" concentration between
2.13 and 4.12 mg-L based upon a reduction in body length.
Uptake, elimination, and metabolism of di-n_-butylphthalate was studied with
14
fathead minnows. A steady-state level of C equivalents of di-n_-butylphthalate
was attained within 4 hr in the whole-body. Bioconcentration factors in C
equivalents of di-n_-butylphthalate were 2,068 and 2,125 for the two measured
exposure concentrations. Estimated bioconcentration factors for parent di-rv-
butylphthalate were 570 and 590 for the two tests based upon a mean value of
27-6% unmetabolized compound over an 11 day exposure.
Seven metabolites of di-n_-buty1phthalate were separated by thin-layer
chromatography after three days of exposure. The only metabolite identified
was phthalic acid.
Binding of di-n_-butylphthalate to proteins was studied using rainbow trout
liver microsomes and water flea post-mitochrondrial supernatant (PMS).
Irreversible binding to proteins occurred with 9% of the compound bound to the
rainbow trout liver microsomes in 2hr and <1% irreversibly bound to water flea
PMS in 1 hr.
Microsomal metabolism and binding of carbon tetrachloride, chloroform,
chlorobenzene, 1,1,2-trichloroethylene, and 1,1,2-trichloroethane were studied
with rainbow trout liver microsomes and water flea PMS. The compounds were
metabolized by both species with rainbow trout appearing to have a greater
5
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capacity for metabolizing them. The compounds were metabolized by rainbow trout
in the following order: chloroform > 1,1,2-trichloroethane, > 1,1,2-trichloro-
ethylene > chlorobenzene > carbon tetrachloride. The compounds were metabolized
by water fleas in the following order: chloroform > chlorobenzene > 1,1,2-tri-
chloroethylene > 1,1,2-trichloroethane % carbon tetrachloride.
Mixed function oxidase assays were performed on the microsomal fraction
from rainbow trout liver and the water flea PMS fraction. Rainbow trout liver
microsomes had 0.28 and 0.19 nM-mg of cytochrome P-450 and cytochrome b,-,
respectively. The level of NADPH cytochrome c reductase activity was 16 nM of
cytochrome c reduced-min" -mg" protein. Water flea PMS had 42 nM of cytochrome
c reductase activitymin -rag protein.
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SECTION III
RECOMMENDATIONS
Adequate assessment of a particular chemical's toxicity would be enhanced
by exposing the chemical to many species of aquatic organisms representing the
taxa likely to be impacted in the environment. Particular effort should be
expended with species shown to be generally sensitive to the class of compound
of immediate interest. Organisms such as Tanytarsus dissimilis (midge) with
consistently high tolerances to a broad range of chemical classes should receive
minimal effort.
Physical properties (.i-e. hardness, pH, temperature) of the laboratory
test water need to be carefully monitored, and several waters with different
natural chemical characteristics used for exposures to assess the impacts of
these parameters. The effects that water hardness and organic ligands have
upon toxicity of some metals are known but not completely understood. Studies
should be conducted to elucidate the relationships between chemical characteris-
tics of natural waters and pollutant toxicity.
A better understanding is needed of compound metabolism and enzyme induc-
tion in aquatic organisms. Additional research on the capabilities of animals
from various taxonomic groups to metabolize foreign chemicals would be valuable.
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SECTION IV
METHODS
Water Supply and Environmental Control
Water for the toxicity tests was either directly from Lake Superior or was
dechlorinated city water from Superior, WI. CSuperior, WI derives its water
from shallow wells beneath. Lake Superior.) Several chemical parameters (.dis-
solved oxygen, pH, hardness, acidity, and alkalinity) were monitored during the
fish and scud toxicity tests by standard analytical methods (.American Public
Health Association, 1975), A portion of the water was heated before being
distributed to the test systems. Lighting for the toxicity tests was artificial,
supplied by fluorescent bulbs centered above the exposure chambers.
Test Organisms
Fathead minnow CPimephales promelas) brood fish were received from stock
maintained by the Environmental Research Laboratory-Duluth, MN, U.S. EPA. Brood
fish were maintained at 25 C, and were fed twice daily a diet of frozen adult
brine shrimp.
Asbestos pipe 02.5 cm O.D.) cut in half, longitudinally, was used as the
spawning substrate. The spawning substrates (.tiles) were checked daily for egg
deposition. Eggs were removed from the tiles the same day Cl 24 hr) that
spawning occurred when used in early life-stage tests. Eggs were allowed to
remain on the tiles and were cared for by brood stock males until 50% or more
were "eyed up" for later use in acute toxicity tests. Tiles with "eyed up"
8
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eggs were placed in temperature controlled (25 C) hatching chambers to complete
incubation. Once they had hatched (approximately 96 hrs after spawning at 25 C),
fry were transferred to rearing chambers where they were fed fresh, newly hatched
brine shrimp nauplii (Artemia sp.) three times daily.
Rainbow trout (Salmo gairdneri) fingerlings used in the study were received
from Lake Mills, WI National Fish Hatchery and from the Fattig Hatchery of Brady,
NE. The fish were acclimated and maintained in Lake Superior water at 12 C until
used in testing. They were maintained on a diet of Glencoe Mills trout chow.
Bluegill sunfish (Leponris macrochirus) were obtained from the Newtown, OH
Laboratory of the U.S. EPA. The fish were acclimated and maintained in Lake
Superior water at 25 C. They were maintained on a diet of Glencoe Mills trout
chow until used in acute tests.
Flagfish (Jordanella floridae) were obtained through brood stock maintained
at the Environmental Research Laboratory-Duluth, MM,U.S. EPA. Brood stock were
fed frozen adult brine shrimp. Young flagfish were fed freshly hatched brine
shrimp and were raised in continuously flowing water at 25 C.
Stainless steel mesh grids covered with yarn were used as spawning substrates
for flagfish brood parents. Eggs were removed from the yarn the same day
(f. 24 hr) that spawning occurred when used in early life-stage tests. If the
hatched fry were to be used for acute tests, the eggs and fry were handled as
described for the fathead minnow.
Water flea (Daphnia magnal brood stock was obtained from the Environmental
Research Laboratory-Duluth, MN, U.S. EPA. The brood stock was maintained at a
temperature of approximately 20 C on a diet consisting of a mixture of finely -
ground trout chow and baker's yeast.
Scuds (Gammarus pseudolimnaeus) were collected from the Eau Claire River
a
-------
in Douglas County, WI. They were acclimated and reared in 56 L glass chambers
with continuously flowing Lake Superior water at 20 C. The organisms were fed
leaves of various deciduous species of trees native to St. Louis County, MN,
that had been soaked in lake water for at least one month. Reproduction of the
scuds occurred in the rearing chambers. At the start of a toxicity test
organisms were selected based upon size uniformity with no attempt to determine
age.
A midge (Tanytarsus dissimilis Johannson 1937) culture was maintained from
stock organisms received from the Environmental Research Laboratory-Duluth, MN,
U.S. EPA. The colony was maintained at a water temperature of approximately
20 C on a diet of CerophylV* and trout pellets as described by Anderson et al.
0980).
A stock culture of green algae (Selenastrum capricornutum) was received
from the Environmental Research Laboratory-Corvallis, OR,U.S. EPA. It was
maintained in an environmental chamber according to the procedure of Miller e_t
al. (1978) with some modifications. Modifications that were employed included a
doubling of the nutrient solution concentration to allow for greater algal
biomass production and stock culture transfers every 4-5 days.
Acute Toxicity Tests
Fathead Minnows - Fathead minnows were used as test organisms in acute
tests with arsenic , mercury , and silver . The test with arsenic was
+2 +1
conducted with dechlorinated city water while the mercury and silver tests
were conducted with Lake Superior water. These were flow-through tests using
a proportional diluter system CMount and Brungs, 1967), in which there were
five toxicant concentrations plus a control all in duplicate. Individual
exposure chambers measured either 26 x 17 x 15 on, or 20 x 35 x 15 cm and
10
-------
contained 3.1 L and 6.3 L of water. The diluter system delivered 0.5 L of fresh
water alone to the controls or 0.5 L of fresh water plus toxicant in the case of
the exposure groups every 15-19 min. Water temperature was maintained at 25 C
and a 16 hr light photoperiod was used- Ten to 20 fish of age 30-32 days were
placed into each chamber. Fish standard lengths and weights for the individual
tests were: arsenic - 21.0 t 2.5 mm, 0.139 t 0.140 g Cn=20); mercury*2 - 20.0 ±
1.8 mm, 0.098 ± 0.027 g (n=20) and silver*1 - 19.2 ±3.0 mm, 0.079 ± 0-031 g
(n=40). Fish were not fed during the acute tests. Observations were made at
regular intervals through 96 hrs for mortalities and other gross behavioral
effects. Death was defined as cessation of opercular movement in all acute tests
with fish.
Several water quality parameters were monitored throughout the tests. These
included temperature, dissolved oxygen, hardness, alkalinity, acidity, and pH.
These values are presented in Appendix A, Table A-l.
Toxicant concentrations were measured daily in acute tests. All twelve
chambers were analyzed at 0 and 96 hr, with alternating replicates analyzed at
24, 48 and 72 hr. Measured concentrations of toxicants are presented in Appendix
B, Table B-l.
Rainbow Trout - Rainbow trout were used as test organisms in acute tests
with the following priority pollutants: tetrachloroethylene, 1,2-dichlorobenzene,
1,4-dichlorobenzene, hexachlorobenzene, and hexachlorobutadiene- Acute tests
with rainbow trout and hexachloroethane, 1,2,4-trichlorobenzene, and pentachloro-
benzene were also conducted.
All rainbow trout tests with the exception of penta- and hexachlorobenzene
were flow-through tests conducted in the same type of diluter system as
described for fathead minnows. The diluter cycled every 10-16 min, providing
11
-------
from 6.8 to 10.6 volume additions per day. Water temperature was maintained at
approximately 12 C. Ten fish were tested per chamber. Fish were of the follow-
ing sizes for the individual tests: tetrachloroethylene - 6.1 ± 1.0 cm, 3-2 ±
1.5 g (n=19); tetrachloroethylene with dimethylformamide (.DMF) carrier-solvent
7.3 + 1.0 cm, 5.86 ± 2.45 g (n=19); 1,2-dichlorobenzene - 5.6 ± 0-8 cm, 2.69 ±
1.24 g Cn=10)i 1,4-dichlorobenzene - 5.3 ± 0.6 cm, 2.1 ± 1.0 g (.n=20); hexa-
chlorobutadiene - 5.6 ± 0.6 cm, 2.6 ± 0.9 g Cn=19); hexachloroethane - 6.6 i 1.0
cm, 4.3 ± 1.8 g (n=20); 1,2,4-trichlorobenzene - 4.7 ± 0.4 cm, 1.6 ± 0.4 g
Cn=20).
Water quality parameters were routinely measured CAppendix A, Table A-l).
Toxicant concentrations were measured daily as described with fathead minnows
CAppendix B, Table B-l).
Penta- and hexachlorobenzene acute tests were conducted in a different type
of flow-through system due to their limited water solubilities. Pentachloro-
benzene was tested with dimethyl formamide (.DMF) as a carrier solvent. Penta-
chlorobenzene/DMF stock stolutions of known concentrations were pumped into 5
test chambers with fluid metering pumps. The control chambers received DMF only.
DMF concentrations were nominally equal and averaged 395 mg-L" between exposure
chambers. Test chambers were 30 x 60 x 30 cm, and contained 27 L of water Cdepth
of 15 cm}. Pumps were set to deliver every time the system cycled and delivered
1 L of Lake Superior water to each chamber. The cycle time of the system
averaged 16.5 min, providing 3.2 volume additions of water per day. The mean
water temperature was 12.7 C.
The test was run with 10 fish [mean standard length, 6.9 ± 1.2 cm; mean
weight, 5.2 ± 2.5 g (n=20)] per chamber. Replicates were separated in time by
11 days. Since there were insufficient deaths at 96 hr to calculate an LCgQ
12
-------
concentration, the exposures were continued through 144 hr.
Hexachlorobenzene was tested in the system as described for pentachloro-
benzene, also with DMF as a carrier-solvent. Two toxicant concentrations plus
a control in duplicate were used. The lower concentration was at or near the
solubility limit and the higher concentration exceeded water solubility. DMF
concentrations were nominally equal in all chambers, and averaged 932 mg-L .
Test duration was 96 hr at a mean water temperature of 11.2 C. The cycle time
of the system averaged 8.25 min, providing 6.5 volume additions of water per day.
Ten fish per tank were tested. Mean fish length and weight values were 3.3 ± 0.3
cm and 0.5+0.1 g (n=20), respectively.
Toxicant concentrations were measured daily CAppendix B, Table B-l). Water
quality parameters for penta- and hexachlorobenzene tests are presented in
Appendix A, Table A-l.
Bluegill Sunfish - Bluegill sunfish were used as test organisms in acute
tests with hexachlorobutadiene and hexachlorobenzene. In the test with hexa-
chlorobutadiene, a proportional diluter system was used. Chamber dimensions
were 51 x 15 x 15.5 cm, with a 10 cm water depth for a volume of 7.7 L- The
diluter cycle time was 9.1 min, providing 10.3 volume additions per day. Ten
fish were exposed per chamber. Mean standard length of the fish was 3.9 ± 0-5
cm, and mean weight was 1.5 ± 0.6 g (n=20). Water temperature was maintained
at 25.2 C-
i
Hexachlorobenzene was tested with bluegill sunfish in the system as
described for hexachlorobenzene and rainbow trout, with two toxicant concen-
trations and a control in duplicate. DMF was used as a carrier-solvent, and .
averaged 884 mg-L in all chambers. The delivery system cycled every 9.7 min,
providing 5.5 volume additions of water daily. The test was conducted at a
13
-------
mean water temperature of 23.3 C. Ten fish were tested per chamber at a mean
standard length of 2.9 ± 0.4 cm and a mean weight of 0.4 ± 0.1 g (n=10). Water
quality parameters and toxicant concentrations are presented in Appendices A
and B, Tables A-l and B-l, respectively.
Flagfish. Flagfish were used in acute tests with arsenic and silver .
Flagfish 34 days of age Cstandard length, 13 ± 2.0 mm; weight, 0.058 t 0.027 g,
+3
n=20) were exposed to arsenic in a proportional diluter system using dechlori-
nated city water. They were tested simultaneously with fathead minnows in glass
exposure chambers C20-5 x 30 x 25 cm) divided with screen into two sections.
Flagfish sections contained an average of 2.1 L of water. Twenty fish per
chamber were tested. The diluter cycle time of 19 min provided 6.9 volume
additions of water per day. The test was conducted at a mean water temperature
of 25.8 C.
Flagfish 30 days of age Cstandard length, 12.7 t 1-6 mm; weight, 0.044 t
0.021 g, n-30) were exposed to silver in a proportional diluter system using
Lake Superior water. They were tested simultaneously with fathead minnows in
glass chambers 05 x 30 x 25 cm) divided by screen into two sections. Fifteen
fish per chamber were tested. The diluter cycle time of 16 min provided about
8 volume additions of water per day. Mean test water temperature was 24.7 C.
Water quality parameters and toxicant concentrations were measured routinely
throughout the tests CAppendices A and B, Tables A-l and B-l, respectively).
Daphnia magna - Daphnia magna was used as the test species in static acute
tests conducted with 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetra-
chloroethane, pentachloroethane, hexachloroethane, tetrachloroethylene, 1,3-
dichlorobenzene, 1,2,4-trichlorobenzene, di-n_-butylphthalate, chlordane,
+2 +3
nickel , and arsenic , Acute tests with chlorinated ethanes, ethylene, and
14
-------
benzene were conducted using first instar C± 24 hr old) daphnids. Adult daphnids
were originally obtained from the laboratory stock reared at the Environmental
Research. Laboratory-Duluth, MN. Both stock and test animals were maintained in
a constant temperature water bath C20 ± 1 C).
A combination of Gro-Lux and Duro-Test (.Optima FS) fluorescent bulbs pro-
vided 32 ft-candles of light at the air-water interface, and were set for a
16L:8D photoperiod coupled with a 15 min transition period between light and dark
phases. All culturing and testing was done with Lake Superior water which was
filtered (5 pm) and aerated. All chemical stock solutions were prepared by
saturating lake water with the test chemical on a stirring plate.
Acute tests were conducted according to the "Proposed Standard Practice of
Conducting Basic Acute Tests with Fishes, Macroinvertebrates, and Amphibians -
Draft No. 8". Test containers were 200 ml erlenmeyer flasks filled to 200 or 160
mL for tests in which the daphnids were unfed or fed, respectively. The flasks
were tightly stoppered with foil wrapped neoprene stoppers. Food concentration
was 20 rng-L . The measure of acute toxicity was the 48 hr median effective con-
centration C48 hr EC,-Q) based upon complete immobilization and the 48 hr median
lethal concentration C48 hr LC5Q) based upon death as determined by cessation
of heart beat and gut movement. Both were determined using a SOX dissection
scope.
Toxicant concentrations were measured during each test (Appendix B,
Table B-l). Water quality parameters are presented in Appendia A, Table A-2.
The Daphnia acute test with di-n_-butylphthalate was a renewed static
test using Lake Superior water, in which toxicant solutions were renewed daily.,
and test organisms were transferred daily by wide-mouthed pipettes into the
new solutions. Exposures were conducted in 200 mL erlenmeyer flasks containing
15
-------
150 ml of solution. Five first instar (<24 hr old) daphnids per duplicate flask
were tested. The test organisms were obtained from the Environmental Research
Laboratory-Duluth, MN (U-S. EPA) stock culture. Culture and test organisms were
maintained at 20 ± 2 C. A 16L:8D photoperiod was used with a light intensity of
45 ft-candles. Flasks were observed daily for mortalities. Water quality para-
meters and toxicant concentrations are presented in Appendices A and B, Tables
A-2 and B-l, respectively.
The Daphnia acute static test with chlordane was conducted with Lake
Superior water Cfirst instar, <24 hr old from the Environmental Research Labora-
tory-Duluth, MN stock cultures). Ten organisms were placed into solutions in
200 mL erlenmeyer flasks. Five concentrations and a control were tested, in
duplicate. The flasks were kept in a 21 C water bath, and a 16L:8D photoperiod
was used. Flasks were observed daily for mortalities. Water quality parameters
and toxicant concentrations were measured CAppendices A and B, Tables A-2 and
B-l, respectively).
+2
The Daphnia acute test with nickel was conducted in Lake Superior water,
using 10 organisms Cfirst instar <24 hr old, from the Environmental Research
Laboratory-Duluth, MN stock culture) per flask. Five concentrations plus a
control in duplicate, were tested in a 20 C water bath. The flasks were 300 mL
erlenmeyers containing approximately 250 mL of water. A 16L:8D photoperiod was
used, with a light intensity of 60 ft-candles. Water quality parameters and
toxicant concentrations are presented in Appendices A and B, Tables A-2 and
B-l, respectively. Flasks were observed daily for mortalities.
The Daphnia acute test with arsenic was conducted in Lake Superior water
using 9-11 organisms Cfirst instar, 24 + 12 hr old, from the University of
Wisconsin-Superior, WI stock culture) per flask. Six concentrations plus a
16
-------
control in duplicate were tested. The flasks were maintained at a temperature
of 14.8 ± 0.8 C, with a photoperiod of 16L:8D. Acute tests were run with
organisms both fed throughout exposure and not fed. Flasks were observed daily
for mortalities. Water quality parameters and toxicant concentrations were
measured CAppendices A and B, Tables A-2 and B-l, respectively).
Scuds - Gammarus pseudolimnaeus was tested in acute tests conducted with
+3 +1 +2 +fi
pentachlorophenol, arsenic , silver , lead , and chromium , All were flow-
through tests conducted in proportional diluter systems CMount and Brungs, 1967)
using Lake Superior water.
In the pentachlorophenol test, 15 organisms C* = 0.050 ± 0.016 g) per
chamber were tested. Chamber dimensions were 25.5 x 17 x 15 cm, with a water
depth of 9 cm, for a volume of 3.9 L. The diluter cycled every 16 min, pro-
viding 11.5 volume additions per day. A 16L:8D photoperiod was used, with a
light intensity of 20-31 ft-candles. The test was conducted at a water tempera-
ture of 17.1 ± 0.5 C. Five exposures plus a control, in duplicate, were used.
Organisms were considered dead when movement ceased and they would not respond
to prodding. Water quality parameters and toxicant concentrations were monitored
throughout the test (Appendices A and B, Tables A-l and B-l, respectively).
+3
In the test with, arsenic , 10 organisms (0.3 - 1.1 cm, total length) per
chamber were tested. Chamber dimensions were 6.3 x 6.3 x 9,3 cm. These chambers
were placed inside larger glass chambers (.26 x 17 x 15 cm) containing exposure
water 8 cm deep. The inner chambers were constructed of glass on two sides and
the bottom. Two sides were made of 202 Nitex^mesh to allow exchange of
exposure water from the larger chambers. Exposure water passively entered each
chamber containing the test organisms, and toxicant concentrations were identical
both inside and outside of the inner chambers. The diluter cycle time averaged
17
-------
16 min, providing 12.9 volume additions per day. The photoperiod and light
intensity were the same as above. Water temperature was 18.4 ± 0.9 C. Five
exposures, plus a control, in duplicate were tested. Water quality parameters
and toxicant concentrations are presented in Appendices A and B, Tables A-l
and B-l, respectively.
An acute test with Gammarus and silver was conducted under the same
conditions as described for the arsenic test. Ten organisms (.0-3 - 1.1 cm,
total length! per chamber were tested. The diluter cycle time was 15.8 min,
providing 13.1 volume additions of water per day. The test water temperature
was 19.9 t 0.5 C. Water quality parameters and toxicant concentrations were
monitored throughout the test CAppendices A and B., Tables A-l and B-l,
respectively).
+2
An acute test with Gammarus and lead was conducted under identical condi-
tions as described for the arsenic test. Fifteen organisms CO.053 + 0.021 g)
per chamber were tested at a water temperature of 17,6 ± 0.4 C. Water quality
parameters and toxicant concentrations are presented in Appendices A and B,
Tables A-l and B-l, respectively.
Midges - Tanytarsus dissimilis was the test species for acute tests con-
ducted with tetrachloroethylene, 1,2-dichlorobenzene, 1,4-dichlorobenzene,
+6 +2
hexachlorobenzene, pentachlorophenol, hexachloroethane, chromium , lead ,
+1 +4
silver , selenium , and cyanide. All tests were conducted using Lake Superior
water. Midge exposures were conducted in 8,5 cm diameter glass crystallizing
dishes filled to a depth of 2.6 cm (>200 ml volume) with test solutions. Each
chamber with a 3 mm glass overflow tube contained lake water plus a small amount
of food (ratio of 11:0.0025 L), along with a fine layer of sand on the bottom.
Food was a mixture of Cerophyl r*and trout pellets blended with water.
18
-------
Ten to 20 midge larvae in their 3rd or 4th instar stage of development
(2,0 - 3.5 mm total length) were placed into each dish containing Lake Superior
water, plus food, and were allowed to acclimate overnight Cor for 48 hr in the
silver and selenium tests) in a 20 C water bath. Hexachlorobenzene and
pentachlorophenol tests were acclimated and run at room temperature C22.5 -'
25.6 C). A photoperiod of 16L:8D at an intensity of 1.9 ft-candles was main-
tained throughout the test- In the case of the silver test, the light was
turned off at 24 hr due to an observable color change (graying) at the higher
+4
toxicant concentrations. In the selenium test, the midges were exposed to room
light only during working hours.
Each dish was examined after 24-48 hrs of acclimation for normal movement
and case building. Midges were replaced if they were immobile or were building
pupation cases. The water was siphoned off to a depth of 2-3 mm, and toxicant
slowly dripped in from a separatory funnel at a rate of approximately 2 mL-min .
Five toxicant concentrations plus a control, in duplicate, were tested, with
the exception of hexachlorobenzene.
Hexachlorobenzene was tested at two concentrations plus a control, in
duplicate. The crystallizing dishes contained 150 mL of solution and were
covered. Nominal toxicant concentrations were 5.2 and 94.1 yg-L" . DMF was
used as a solvent carrier at a mean concentration of 1086 mg-L" . The
crystallizing dishes were twice siphoned and replaced with fresh hexachloro-
benzene/DMF solutions at the beginning of the test in an attempt to maintain
nominal concentrations. This test was run at a temperature of 23-9 ± 1-2 C.
Midges were observed on a light table at various time intervals through -
48 hr. Effects and deaths were recorded. Death was defined as complete lack
of movement when prodded.
19
-------
The water was analyzed daily for toxicant concentrations (Appendix B,
Table B-l). Water quality parameters were also monitored (Appendix A, Table
A-l).
Green Algae - Selena'strum capricornutum was tested in 96 hr toxicity tests
using toxaphene and heptachlor. Algal tests were conducted in 125 ml erlenmeyer
flasks stoppered with foam plugs on a shaker platform. The flasks were placed
in an environmental chamber and incubated under controlled conditions of light
and temperature. Light intensity was 400 ft-candles and the test temperature
was 24 C.
The nutrient solution concentration was twice the concentration used in
the 1978 Selenastrum Bottle Test Procedure (.Miller et al., 1978), providing for
greater biomass production and more reliable dry weight measurements. Ethanol
was used as a carrier-solvent in the toxaphene test, and all test flasks con-
tained a concentration of 0.4% ethanol. No carrier-solvent was used in the
heptachlor test.
Nutrient solutions containing toxaphene or heptachlor were inoculated with
a 4-5 day-old culture of Selenastrum to yield an initial density of 20,000
cells-mL in each flask. Triplicate flasks were inoculated at each exposure
level (control plus 5 toxicant concentrations) for biomass determinations after
96 hr of exposure. After 96 hr, individual control and exposure flask solutions
containing algae were filtered through pre-weighed 0-45 pm filters, dried, and
weighed to determine algal biomass. Mean algal weights at various exposure
levels were compared to the mean algal weight of the control group, and expressed
as percentage inhibition of growth. The initial toxicant concentration that
inhibited growth by 50% UC5Q) was determined from interpolation by the trimmed
Spearman-Karber method.
20
-------
Toxicant concentrations were measured during each test (Appendix B, Table
B-l). A portion of each initial test concentration was analyzed to determine
the starting concentration. One additional flask, which was not inoculated with
algae, was carried through the 96 hr period for each exposure concentration.
These flasks were used to determine final concentrations. Six additional flasks
Ccontaining no algae) were used at a middle toxicant concentration in each test.
Three of these flasks were analyzed after 24 hr and the other three after 96 hr.
Aliquots from the three flasks containing algae at the middle toxicant concen-
tration for each test were filtered through glass fiber filters at the end of
the test period to remove the algae to determine the amount of toxicant remaining
in solution.
Chronic and Subchronic Toxicity Tests
Daphnia magna - Daphnia magna was used in chronic toxicity tests with
1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, tetra-
chloroethylene, 1,3-dichlorobenzene, 1,2,4-trichlorobenzene, and arsenic .
Chronic tests were conducted according to the ASTM "Proposed Standard Practice
for Conducting Renewal Life Cycle Toxicity Tests with the Oaphnid, Daphnia
magna "(.Draft No. 4, 19781, with minor modifications to control the losses of
volatile chemicals. Test containers were 200 mL erlenmeyer flasks filled to
150-160 ml, with the exception of tetrachlorethylene which was filled to 175 ml.
The flasks were tightly stoppered with foil wrapped neoprene stoppers to reduce
chemical losses. Tests were conducted at a temperature of approximately 20 C
with a 16L.-8D photoperiod.
Tests with 1,1,2,2-tetrachloroethane, 1,3-dichlorobenzene, 1,2,4-tri-
chlorobenzene and arsenic had 7 replicates at each of 6 test chemical con-
centrations, whereas tests with 1,2-dichloroethane, 1,1,2-trichloroethane,
21
-------
and tetrachloroethylene had 10 replicates at each of 6 concentrations. Each
flask initially contained 1 daphnid (<24 hr old). The food concentration was
20 mg-L" .
Young daphnids were filtered from each flask after the transfer of adults
and washed onto a watch glass to be counted alive. If less than 20 animals were
present they were counted manually. If more than 20 animals were present, they
TM
were counted with an Artek counter. Chronic toxicity was determined by
reproductive success and length of animals surviving the 28 day test. Length
was determined using a 30X dissection scope and measuring from the top of the
head to the base of the spine with an ocular microscope.
Toxicant concentrations were measured both before and after new solutions
were added, and the mean of the "before" and "after" measurements represented
the concentration for a particular time interval. Water quality parameters
and toxicant concentrations are presented in Appendices A and B, Tables A-2
and B-l, respectively.
Fathead Minnows - Early life-stage toxicity tests were conducted using
+3 +2
the fathead minnow as test species for tests with arsenic and mercury
The arsenic test was performed using dechlorinated city water and the
+2
mercury test with Lake Superior water. Both tests were performed in
proportional diluter systems with five toxicant concentrations plus a control,
all in duplicate. Test chambers for the arsenic test contained an average
of 1.9L of water, and had an average of 7.4 volume additions per day. Test
+O
chambers for the mercury test contained 3.0 L of water, and had an average
of 12.6 volume additions per day. The arsenic test was conducted at a mean.
+2
water temperature of 23.0 t 2.7 C, and the mercury test at a temperature of
25.0 ± 0.6 C.
22
-------
Fathead minnow eggs <24 hr old were placed into incubation jars containing
200 urn mesh nylon, screen bottoms. Fifty eggs per jar were added to each of 2
jars per treatment replicate. The eggs were incubated in the test chambers by
slowly oscillating (6 revolutions per minute) the jars in the test solutions to
enhance water movement past the eggs. Dead and fungused eggs were removed
daily. Upon completion of hatch C5-6 days), dead, live and abnormal fry were
counted, and healthy fry C20 total for the arsenic test and 30 total for the
+2
mercury test) were released into the test chambers. Feeding was begun the
day after hatching and continued to the end of the test. Finely granualted
dry fish food (retraining and newly hatched brine shrimp were fed for the entire
test. Equal volumes of food were provided to each chamber.
Throughout the tests, observations were regularly made for mortalities and
abnormal development. At the end of the test, all survivors were measured for
wet weight and standard length to determine effects upon growth. Toxicant
concentrations were measured in all tanks twice weekly (Appendix B, Table B-l).
Water quality parameters were routinely measured throughout the tests (Appendix
A, Table A-l).
Flagfish - Two early life-stage toxicity tests were conducted with
arsenic and flagfish. One was conducted using Lake Superior water and one
using dechlorinated city water. Both tests were conducted in proportional
diluter systems. The test with Lake Superior water was conducted in chambers
containing 2.7 L of water, and had a mean of 16.7 volume additions of water
daily. The test with dechlorinated city water was conducted in test chambers
containing an average of 2.1 L with 7.4 volume additions daily. Mean water
temperatures were 24.8 ± 1.3 C and 24.4 t 2.8 C for tests in Lake Superior
water and dechlorinated city water, respectively.
23
-------
Flagfish eggs (<24 hr old) were placed into incubation jars as described
for fathead minnows. In the test with Lake Superior water, 50 eggs per jar were
added, while in the test with dechlorinated city water, 34 eggs per jar were
added. In the test using city water, the eggs were treated for 10 min with a
0.4 mg-L" solution of malachite green on the first and second days of incuba-
tion to reduce fungal growth.
Upon hatching (6-7 days), the numbers of dead, live, and abnormal fry were
determined, and 20 healthy fry were released into each chamber. Fish were fed
d?)
Tetramin-'once daily and newly hatched live brine shrimp 3 times daily. Equal
volumes of food were provided to each chamber.
Throughout the test, observations were regularly made for mortalities and
abnormal development. Weight (wet) measurements were taken at the conclusion
of the test. Toxicant concentrations were measured in all tanks twice weekly
(Appendix B, Table B-l). Water quality parameters are presented in Appendix A,
Table A-l.
Toxicity Test Chemicals
The sources of test chemicals and chemical purity levels are presented in
Appendix C, Table C-l. All chemicals had purity levels >95% except for
chlordane and toxaphene, which were of technical grade.
Chemical Analysis of Toxicants
Organic Chemicals. Exposure chamber concentrations of most of the organic
chemicals tested were determined by extraction from water with an organic sol-
vent (hexane, isooctane, or petroleum ether) and analysis by gas-liquid
chromatography (GLC). GLC parameters used are presented in Appendix C,
Table C-2. The extraction procedures and percentage recoveries are given in
24
-------
Appendix C, Table C-3. Electron capture CNi) detectors were used for most
analyses on the different instruments.
Methanol concentrations in the exposure water for the rainbow trout and
bluegill sunfish acute tests and dimethyIformamide concentrations in the rain-
bow trout acute test were determined by GLC using direct aqueous injection on
a Tenax Gc^packed column and flame-ionization detection. Methanol concentra-
tions in exposure chambers for the fathead minnow acute test were determined by
using Rhodamine B dye as a tracer. The stock solution contained 25 mg-L
Rhodamine B which was delivered to the exposure tanks. The concentration of
alcohol in the exposure tanks was calculated from the analysis of Rhodamine B.
Rhodamine B was measured using a Baird-Atomic Model SFR100 spectro-
fluorometer. The excitation and emission wavelengths were 554 nm and 578 nm,
respectively. Standards were prepared in Lake Superior water and lake water
was used as the reference blank. The exposure tanks contained from 0.1 to 1.3
ug-L Rhodamine B.
DimethyIformamide concentrations in exposure chambers for acute tests with
bluegill sunfish, fathead minnows, midges, and Daphnia magna were determined by
direct measurement on a spectrophotometer at a wavelength of 200 nm.
Inorganic Chemicals. Exposure chamber concentrations of arsenic,
chromium, lead, mercury, nickel, silver and selenium were determined by atomic
absorption analytical techniques. Samples were collected, preserved, and
analyzed as stated in "Methods for Chemical Analysis of Water and Wastes"
(U.S. EPA, 1979) and in "Analytical Methods for Atomic Absorption Spectro-
photometry" (Perkin-Elr-er Corp., 1976).
Arsenic, chromium, lead, nickel, selenium, and silver were analyzed with
a Perkin-Elmer Model 306 atomic absorption spectrophotometer, employing a
25
-------
deuterium arc background corrector, an HGA-2100 graphite furnace atomizer or
flame atomization burner head and a Model 56 recorder. Hollow cathode lamps
were used for chromium, lead, nickel and silver. Electrodeless discharge lamps
were used for arsenic and selenium. Flame or furnace atomization was used
depending on sample concentration.
Mercury was analyzed on a Perkin-Elmer Model 403 atomic absorption
spectrophotometer by a cold vapor technique (U.S. EPA, 1979), using a hollow
cathode lamp. Methods of analyses and quality control parameters for the
previous tests are summarized in Appendix C, Table C-4.
Cyanide was analyzed in the midge toxicity test exposure chambers by the
colorimetric method described in "Standard Methods for the Examination of Water
and Wastewater" (American Public Health Association, 1975). Absorbance readings
were taken on a spectrophotometer at 578 nm.
Statistical Analysis of Test Results
LC5Q values for all of the acute exposures with fish, scuds, and midges,
and for some of the acute exposures with Daphnia were determined by the trimmed
Spearman-Karber method (Hamilton et al_., 1977). EC™ values for the algal
toxicity tests with heptachlor and toxaphene were also determined by this
method, with the EC5Q values being the interpolated concentrations at which
algal biomass was 50% of the biomass of controls. LC5Q and EC™ values for the
remainder of the Daphnia acute exposures were calculated by probit, moving
average or binomial formulas depending upon the characteristics of the data.
Fish early life-stage test results and Daphnia chronic test results were
analyzed by one-way analysis of variance and Dunnett's procedure (Steel and
Torrie, 1960). "No effect" concentration ranges were determined for the
parameters measured. The ranges encompass the highest mean exposure concen-
26
-------
,tration at which no parameter was significantly affected (p>0.05) and the lowest
mean exposure concentration at which one or more of the parameters was signifi-
cantly affected (p<0.05).
Di-n-butylphthalate Uptake, Elimination and Metabolism
Fathead minnows were exposed in glass test chambers with inside dimensions
of 29.5 x 59.0 x 29.5 cm, and a water depth of 15-0 cm. Each chamber contained
26.1 L of water. The chambers were glass covered and the outer sides were
covered with fiber board to minimize visual disturbance to the fish.
The water delivery system was the water metering cell portion (w cells)
of a proportional diluter (Mount & Brungs, 1967) adjusted to deliver 1 L of
dilution water per cycle to mixing chambers before entering the exposure
chambers. Each tank had a mixing chamber which was a 1 L beaker with a self-
starting siphon which began to empty when nearly 1 L of dilution water entered.
Simultaneously with the delivery of the dilution water to the mixing chamber,
di-n_-butylphthalate dissolved in methyl alcohol was delivered to the mixing
chambers by metering pumps (Fluid Metering, Inc., Oyster Bay, NY, Model RFC,
0-1,6 mL-min" ). Methyl alcohol alone was delivered to the mixing chambers in
the control tanks. The compound was mixed and then administered through a
self-starting siphon to exposure chambers just beneath the water surface in a
strong downward jet. This caused a mixing action within the test chambers.
Concentrations of di-n_-butylphthalate averaged 34.8 and 4.83 ug-L for the
higher and lower exposure concentrations, respectively. The delivery cycle
time was 17 min.
Test water temperature was 25.4 t 0.5 C (n=34). Hardness, alkalinity and
acidity had mean concentrations of 44.3 t 0.89 (n=2), 38.2 ± 0.35 (.n=2), and
2.25 ± 0.071 (n=2) mg-L as CaC03> respectively. Percent saturation of
27
-------
dissolved oxygen tWinkler Method) averaged 54.2 (n=2). The test water pH
averaged 7.22 (n=2).
14
C labeled di-n_-butyl phthalate was supplied by California Bionuclear
Corporation, 7654 San Fernando Road, Sun Valley, CA 91352, and had radio-
chemical purity of 98%. The compound was uniformly ring-labeled. Unlabeled
phthalate was supplied by Monsanto and was 99.76% pure. Labeled and unlabeled
stock compounds were mixed proportionally to yield a minimal activity of
approximately 5000 counts-min in 100 ml of test water.
Di-n_-butyl phthalate (.labeled and unlabeled) was dissolved in reagent grade
methyl alcohol to give nominal low and high toxicant test concentrations of 5
and 50 ug-L , respectively. Approximately 0.1 ml (± 5%) of stock solution
containing the test compound was mixed with 1.0 L of water before entering each
chamber containing fish. The control chamber received 0.1 mL (± 5%) of methyl
alcohol mixed with 1.0 L of water. This gave an approximate concentration of
100 mg'L of methyl alcohol to all chambers, and 5 or 50 ug-L of test com-
pound to the low and high chambers, respectively.
Fathead minnows used for the study were from the EPA Environmental Research
Laboratory-Duluth, MN stock culture. The fish used were 28-29 days old with an
average weight of 0.091 ± 0.035 g (n=49). Fish were fed freshly hatched brine
shrimp CArtemia sp.) in equal volumes per tank during the study. Artificial
lighting was supplied by florescent lamps with a 16L:8D photoperiod.
Before the exposure, fish were randomly withdrawn from a common pool of
fish and placed into each test and control chamber. The chambers contained
130, 200, and 300 fish in the control, low, and high concentrations, respec-
tively. Fish were exposed for 11 days, during which 80 fish from the high
exposure concentration were saved for metabolite characterization, and the re-
28
-------
maining fish transferred to clean water for depuration studies. Depuration
lasted 21 days. Fish were randomly sampled from control and test chambers on
day 0 at 0, 4, 8, and 12 hrs and on days 1, 2, 3, 5, 8 and 11 during uptake;
and at 0, 4, 8, and 12 hrs on day 0 and on days 1, 2, 3, 4, 7, 14 and 21 during
depuration.
Water samples were collected in duplicate with a glass siphon from near
the center of the test chambers and approximately mid-depth in the water column.
Water sampling dates coincided with fish sampling dates. Samples of test water
C50 ml} were added to 100 ml volumetric flasks containing 50 ml of hexane. The
samples were stirred vigorously for 45 min on a magnetic stirring plate, and
allowed to stand for 15 rain. A 5 mL aliquot of the solvent layer was added to
15 ml of scintillation cocktail (Permafluor*'III, Tritorr^X-100 and scintillized
toluene, 10:33:57 v/v) and counted in a Packard Model 3375 liquid scintillation
spectrometer for 5.0 min.
Extraction efficiencies were measured several times during the testing
period. Water was spiked with known amounts of radiolabeled stock solution and
extracted. Aliquots of 5.0 ml from the solvent extract were directly added to
15.0 ml of scintillation cocktail and counted for 5.0 min. The counts were
then compared to counts from equal amounts of radiolabeled stock solution added
directly into the scintillation cocktail which contained 5.0 ml of hexane.
Recovery from water was 97.6 t 1-3% (n=19).
Fish were blotted dry on paper toweling and weighed to the nearest 0.1 mg.
14
Whole fish were analyzed for C content by oxidation in a Packard Sample
Oxidizer CModel B306}. Labeled carbon dioxide was trapped in Carbosoro^II
and diluted with scintillation fluid. Recovery efficiencies were determined
by adding radioactive stock compound to a combustion cup containing an un-
29
-------
exposed fish. The recovery averaged 83.6 t 10.1% (n=34).
Water and fish were counted for 5 min along with known concentrations of
spiked samples. Concentration-count relationships were determined for each
compound using 5 duplicated standards for both water and fish. Background counts
and instrument quench curve readings were made with each run. A computer program
corrected sample counts for differences- in instrument quench readings, and
14.
calculated yg quantities of C parent compound equivalents in water and fish
based on a regression line fit (/least squares) from parent compound standard
curves. Sample concentrations were expressed in ug-L of water or yg-g of
fish tissue.
Fish measured with unusually high and low concentrations of test compound
(differing from the mean by more than one standard deviation) were statistically
analyzed for outlying results (PlO.05) by the method of Grubbs and Beck (1972).
This resulted in the elimination of data on 4 fish out of the 285 analyzed.
Fish sampled from days 1, 9, 16, and 21, and also fish from 2 days after
the test was completed were analyzed for lipid content. These samples were
frozen until analysis. The fish were weighed and collectively homogenized with
Na2SO.. The homogenate was extracted for 5 hr in a Soxhlet apparatus with a
1:1 mixture of methylene chloride and hexane. The extract was concentrated to
20 ml, and an aliquot placed in a rinsed, dried and pre-weighed weighing pan.
The solvent was evaporated at 100 C, the lipid residue weighed, and percent
lipids calculated.
Analytical Methods
Metabolism - Fathead minnows were sampled on days 0, 1, 2, 3, and 11 of
uptake from the higher exposure concentration (.34.8 ug-L" ). A "pooled" weight
of the fish from each, day was taken and the fish were frozen until analysis.
30
-------
Fish were thawed and homogenized in 5.0 ml of distilled water using a Potter-
El vehjem tissue homogenizer. The homogenate was centrifuged at 2,150 x g for
20 min. The supernatant was decanted and the pellet saved, A 50 yL aliquot
of supernatant was analyzed for radioactivity using a Packard Tricarb Liquid
Scintillation Counter CModel 3375). The remaining supernatant was frozen in
liquid nitrogen and freeze-dried. The freeze-dried sample was dissolved in
0.1 ml of acetone, and chromatographed on glass thin-layer chromatography (TLC)
plates. The TLC plates were coated with silica gel. Standards of di-n_-
butylphthalate and phthalic acid were spotted alongside the fish extract. The
solvent system consisted of ethanol: H^O: 25£ acetic acid (.100 mL:12 mL:16 mL).
TLC plates were air dried and examined under short-wave ultraviolet light.
Radioactive spots and bands were also examined by autoradiography. The
TLC plates were exposed to Kodak "No-Screen" X-ray plates for 6 weeks. The
plates were processed for 5 min in Kodak X-ray developer, 10 min in Kodak
fixer, and rinsed in distilled water for one-half hour. Radioactive areas
appeared as dark bands or spots on the X-ray plates.
TLC plates from extracts of fish exposed for 0, 1, 3, and 11 days were
used to quantify radioactivity for each day. Bands were numbered, then the
silica gel was scraped off and added to scintillation cocktail. The samples
were counted in a scintillation counter for 5 min.
Pellets remaining from the initial homogenates were analyzed for radio-
activity by combustion with a Packard Sample Oxidizer. The samples were
counted for 5 min by liquid scintillation technique.
Protein Binding - Radiolabeled (_ C) di-n_-butylphthai ate was incubated
with a protein substrate and NADPH generating system to determine metabolism
and irreversible protein binding of di-n-butylphthalate. Rainbow trout liver
31
-------
microsomes (4 mg protein, Lowry's Method) and Daphnia post-mitochondrial
supernatant (PMS), (4 mg protein, Lowry's Method) were used as the protein sub-
strates. The NADPH generating system consisted of 3 pM NADP, 30 pM glucose-6-
phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 1 yM MgCl2- The
reaction mixture was brought to a total volume of 5.0 ml with 0.07 M sodium
phosphate buffer, pH 7.45. The reaction mixture was incubated at 20 t 2 C.
Aliquots of reaction mixture were taken at 0, 15, 30, 45 and 60 min for Daphnia
PMS, and 0, 15, 30, 45, 60 and 120 min for rainbow trout liver. Small portions
of the reaction mixture were counted for total radioactivity. Reaction mixture
aliquots were extracted twice with 15.0 ml of hexane. Protein in the reaction
mixture was precipitated with 1.0 ml of 6M trichloroacetic acid, and the
reaction mixture was then centrifuged at 2,150 x g for 20 min, forming a
floating protein pellet. The floating protein pellet was added directly to
scintillation cocktail. Aliquots of the hexane unextractable aqueous phase
and hexane extract were added to scintillation cocktail. All samples were
counted in a Packard Tricarb Liquid Scintillation Counter for 5 min. Data was
analyzed with a Hewlett-Packard Automation Data System.
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons by Rainbow
Trout and Daphnia
Chemicals - Uniformly C labeled 1,1,2-trichloroethylene and 1,1,2-tri-
chloroethane were purchased from California Bionuclear Corporation, 7654 San
14
Fernando Road, Sun Valley, CA 91352. Uniformly C labeled 1-chlorobenzene,
chloroform and carbon tetrachloride were purchased from New England Nuclear
Corporation, 549 Albany Street, Boston, MA 02118. Purity of these compounds
ranged between 98-99 percent as determined by gas-liquid chromatography.
NADPH, NADP, glucose-6-phosphate monosodium salt, glucose-6-phosphate dehydro-
32
-------
genase from torula yeast, and cytochrome C were purchased from Sigma Chemical
Company, St. Louis, MO.
Tissue Preparations - Livers were dissected from 3-5 rainbow trout (350-
400 g}, weighed, and cut into thin slices in cold (4 C) 0-15 M KC1 solution.
Liver slices were washed several times with KC1 (0-15 M) to remove hemoglobin
and red blood cells, transferred to 0.1 M pH 7.5 sodium phosphate buffer and
homogenized by 6-8 passes of a teflon pestle in a Potter-Elvehjem glass
homogenizer. Homogenates of 30-40% liver by weight in phosphate buffer were
centrifuged twice at 10,000 x g for 15 min in a Beckman L5-50 ultra-centrifuge
to remove nuclear and mitochondrial fractions, which were discarded. The 10,000
x g supernatant was centrifuged at 105,000 x g for 60 min using a T150 rotor.
The supernatant was discarded and the pellet was stored at -20 C until used.
Adult Daphnia (approximately 21 days old) were reared in the laboratory
form U.S. EPA Environmental Research Laboratory-Duluth, MN brood stock, and
from 0-5 - 2.5 g (total wet weight) homogenized with a teflon pestle homogenizer.
The hornogenate was filtered through loose glass wool to remove chitinous
materials, and centrifuged twice at 10,000 x g to remove nuclear and mito-
chondrial fractions. The PMS was then frozen at -20 C until used for in vitro
metabolic studies.
Protein Determination - Protein determinations were made for Daphnia
PMS and rainbow trout liver microsomes according to the method described by
Lowry et. al_. (19511. This enabled known concentrations of protein to be used
in the reaction mixture for metabolic studies.
In Vitro Metabolism Studies - Due to the highly volatile nature of carbon
tetrachloride, chloroform, chlorobenzene, 1,1,2-trichloroethane and 1,1,2-
trichloroethane, an incubation system was designed to study their binding to
33
-------
microsomal protein and their metabolism. This enclosed system consisted of
an erlenmeyer flask 025 ml) which was fitted with a glass column (5 mm i.d.)
containing two glass wool plugs with approximately 5 cm of silica gel between
them to trap the parent compounds being volatilized from the reaction mixture.
Another glass column connected the erlenmeyer flask to a CCL absorbing system
£R^
containing a solution of Carbosorb Ii^X The reaction mixture in the erlenmeyer
glask contained an NADPH-generating system (consisting of 3 uM glucose-6-phos-
phate, 1 unit -' glucose-6-phosphate dehydrogenase, and 1 uM MgClp), 8 mg
microsomal protein from rainbow trout liver or 4 mg PMS protein from Daphnia,
in 0.07 M sodium phosphate buffer (pH 7.5) and 0.1 ml of test compound with
known amount of radioactivity made to a final volume of 5 ml. The reaction
mixture was incubated in a shaking water bath at a temperature of 24 ± 2 C.
The reaction was initiated by addition of radioactive compound (0-1 mL) and
was continued for 0, 15, 30, 45, 60 and 120 min with rainbow trout liver
microsomes or 0, 15 and 30 min with PMS from Daphnia. The reaction was
terminated at various time intervals by addition of 1 ml of 3 M trichloro-
acetic acid (TCAJ solution. The reaction mixture was then extracted thrice
with 10 ml of hexane and the extracts pooled. Total percent recovery was
determined by summation of the radioactivity in the various fractions as
compared to the known amount of radioactivity added initially. Recoveries
ranged from 91.4 to 29.2% with recovery efficiency decreasing with time. The
14
loss likely occurred by escape of C through the silica gel column and the
air space within the reaction vessel becoming saturated with parent compound
or metabolites.
One unit will oxidize 1.0 pM of D-glucose 6-phosphate to 6-phospho-D
gluconate per min in the presence of NADP at pH 7.4 at 25 C.
34
-------
Aliquots of the hexane extract (representing parent compound) were trans-
(&\
ferred to scintillation cocktail UO ml Permaflour IIP; 33 ml Triton X-100,
57 ml scintillized toluene) and C radioactivity was counted with a Packard
Model 3375 liquid scintillation spectrometer for 5 min. Background and quench
corrections were made for all counts. The aqueous phase (representing water
soluble metabolites) was then centrifuged at 2200 x g with an International
Model PR-2 centrifuge for 20 min and the radioactivity determined in the super-
natant and the floating protein pellet. This method distinguished between
protein-bound and free radioactivity present in the aqueous phase which was
unextractable in hexane.
The silica gel trap was extracted with 30 ml of hexane to determine the
amount of radioactivity volatilized from the reaction mixture. The carbon
dioxide absorbing solution was counted to determine radioactivity evolved as
C02 during metabolic reactions or volatilized as parent compound. The
analysis was performed three times with three batches of tissue preparations.
Enzyme Activity - Cytochrome P-450 and cytochrome bg, were determined by
difference spectroscopy with a Beckman DB-G spectrophotometer according to the
methods of Omura and Sato (1964). NADPH-cytochrome c reductase activity was
determined by the method described by Williams and Kamin (.1962). Aniline
hydroxylase activity was determined by measuring the amount of £-aminophenol
produced during a 30 min incubation of the liver microsomes on the PMS with
aniline hydrochloride at 24 C. The reaction mixture contained an NADPH-
generating system as described previously, 1 uM of aniline hydrochloride and
5 mg microsomal protein. The reaction was stopped by addition of 0.5 ml of -
3 M TCA. After centrifugation of the reaction mixture at 2200 x g for 20 min,
a 1 mL aliquot of the reaction mixture was made basic with 0.5 ml of 10% Na-CO^
35
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and a blue phenol-Indophenol complex was formed by addition of 1 ml of 2%
phenol in 0.2 N NaOH. Absorbance was measured using a Beckman DB-G spectro-
photometer at 630 nm.
36
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SECTION V
RESULTS
Acute Toxlclty Tests
Fathead Minnows - 96 hr LC5Q values with 955 confidence intervals for
pooled replicate data in acute tests with arsenic , mercury , silver ,
dimethylformamide, and methanol were 14.2 (.12.5-16-0), 0.150 (0.128-0.176),
0.0107 CO-0106-0.0108), 10,700 00,500-10,900), and 28,100 (27,200-29,000)
mg-L , respectively (Table 1).
Rainbow Trout - LC5Q values at selected time intervals for rainbow trout
were determined for the following chemicals: hexachloroethane, tetrachloro-
ethylene, tetrachloroethylene with dimethyl formamide (.DMF) as carrier solvent,
DMF, 1,2-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, hexa-
chlorobutadiene, and methanol (Jable 2). Values for 96 hr LC5Qs ranged from
0.320 mg-L for hexachlorobutadiene to 20,100 mg-L for methanol. LC5Q
values at 96 hr were not calculable for pentachloro- and hexachlorobenzene due
to insufficient mortalities at the highest exposure concentrations tested of
0.71 and 0.0809 mg-L , respectively.
Bluegill Sunfish - The pooled 96 hr LC5Q with 95% confidence interval for
bluegill sunfish exposed to hexachlorobutadiene was 0.324 (0-312-0.337) mg-L
(Table 3). Mortalities were insufficient to yield an LC5Q value at 96 hr with
hexachlorobenzyne/DMF. Only 1 of 20 fish died at the highest mean exposure
concentration of 78.4 yg-L" . Dimethylformamide and methanol produced 96 hr
37
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TABLE 1. LC50 VALUES (95% CONFIDENCE INTERVALS) FOR POOLED
REPLICATES OF ACUTE TESTS IN WHICH FATHEAD MINNOWS
(Pimephales promelas) WERE EXPOSED TO ARSENIC*3,
MERCURY"1"2, SILVER"1"1, DIMETHYLFORMAMIDE AND METHANOL.
LC5Q (mg-L'1)
Chemical 24 hr 48 hr 72 hr 96 hr
Arsenic*3 19.0 (17.4-20.7) 15.9 (14.3-17.8) 14.7 (13.1-16.5) 14.2 (12.5-16.0)
Mercury*2 0.240 (0.181-0.317) 0.196 (0.170-0.226) 0.155 (0.132-0.181) 0.150 (0.128-0.176)
Silver*1 0.0152 (0.0137-0.0168) 0.0116 (0.0109-0.0124) 0.0107 (0.0107-0.0107) 0.0107 (0.0106-0.0108)
01methy1-
forniamlde 11,500(10,900-12,000) 10,800(10,500-11,100) 10,700 (10,500-10,900) 10,700 (10,500-10,900)
Methanol 28,400 (27,600-29,200) 28,400 (27,600-29,200) 28,400 (27,600-29,200) 28,100 (27,200-29,000)
-------
TABLE 2. LC5Q VALUES (95% CONFIDENCE INTERVALS) FOR POOLED
REPLICATES OF ACUTE TESTS IN WHICH RAINBOW TROUT
(SalIPO yalrdnerl) WERE EXPOSED TO SELECTED ORGANIC COMPOUNDS.
LC5Q (mg-L'1)
Chemical 24 hr 48 hr 72 hr 96 Hr 192 hr
Hexachloroethane 1.80 (1.69-1.92) 1.17 (1.09-1.26) 1-05 (0.96-1.15) 0.94 (0.85-1.04) 0.77(0.72-0.83)
Tetrachloroethylene 4.99 (4.73-5.27) 4.99 14-73-5.27) 4.99 (4.73-5.27) 4.99 (4.73-5.27) n.d.37
Tetrachloroethylene/DMF 6.31 (5.54-7.18) 5.95 (5.23-6.78) 5.81 (5.06-6.67) 5.84 (5.05-6.76) n.d.
Dimethylfonnamlde (DMF) 11,000 10,000 10.000 10,000 n.d.
U 1,000-11,000) (9,100-11,000) (9,100-11,000) (9,100-11,000)
1,2-Dichlorobenzene 1.65 (1.49-1.84)^1.58 (1.44-1.73) 1.58 (1.44-1.73) 1.58 (1.44-1.73) 1.54 11.42-1.68)^
1,4-Dlchlorobenzene 1.37 (1.25-1.49) 1.24 (1.13-1.35) 1.24 (1.13-1.35) 1-12 (1.05-1.20) n.d.
1,2,4-Trlchlorobenzene 2.30 (2.17-2.43) 2.00 (1.84-2.17) 1.73 (1.54-1.94) 1.53 (1.35-1.73) 1.28 (1.11-1.47)
Pentachlorobenzene/DMF n.c.^ n.c. n.c. n.c. 0.28 (0.21-0.37)^
Hexachlorobenzene/DMF n.c. n.c. n.c. n.c. n.d.
'Hexachlorobutadlene n.c. n.c. 0.429 (0.372-0.495) 0.320 (0.268-0.381) 0.121 (0.098-0.149)
Hethano1 20,300 20,100 20,100 20,100 n.d.
(19,800-20,700) (19,500-20,700) (19,500-20,700) (19,500-20,700)
ay Not determined.
b/ 22 hr LC5Q value.
c/ 144 hr LC5Q value.
d/ LC5Q value not calculable due to insufficient mortalities at exposure concentrations tested.
-------
TABLE 3. RESULTS FROM FLOW-THROUGH MEASURED ACUTE TOXICITY TESTS IN WHICH
BLUEGILL SUNFISH (Leponns macrochirus) WERE EXPOSED TO
HEXACHLOROBUTADIENE, HEXACHLOROBENZENE/DMF,
DIMETHYLFORMAMIDE, AND METHANOL (REPLICATES POOLED).
Chemical
Hexachlorobutadiene
Hexachlorobenzene/DMF
Dimethyl formami de
24 hr
n.c.^
n.c.
7,500
48 hr
n.c.
n.c.
7,500
LC5Q (mg-L'1)
72 hr
0.307 (0.322-0.466)
n.c.
7,400
96 hr
0.324 (0.312-0.337)
n.c.
7,100
192 hr
0.318 (0.318-0.318)
n.d>
n.d.
(7,200-7,800) (7,200-7,800) (7,000-7,700) (6,700-7,500)
^ Methanol 19,230 19,230 17,720 15,500 n.d.
0 (17,310-21,360) (17,310-21,360) (15,510-20,240) (13,540-17,740)
a/ LC5U value not calculable due to Insufficient mortalities at exposure concentrations tested.
b/ Not determined.
-------
LC5Q values of 7,100 16,700-7,500) mg-L"1 and 15,500 (13,540-17,740) mg-L"1,
respectively.
Flagflsh - Pooled 96 hr LC5Q values (95% confidence interval) for acute
tests with arsenic"1"3 and silver"1"1 were 14.4 (.12.7-16.3) and 0.0092 (0.0080-
0.0107) mg-L , respectively (Table 4).
Daphnia magna - LC5Q values C48 hr, unfed) for Daphnla magna exposed to
thirteen selected test chemicals ranged from 0.035 (0.032-0.038) mg-L"1 for
chlordane to 14,530 (13,260-15,920) mg-L"1 for dimethylformamide (Table 5).
EC50 values in addition to LC5Q values were determined for eight chlorinated
ethanes, benzenes and ethylene. EC5Q values (48 hr, unfed) ranged from 2.10
(1.82-2.45) mg-L"1 for hexachloroethane to 155 (137-188) mg-L"1 for 1,2-
dichloroethane.
Generally, little difference was noted between endpoints U-C50 or EC5Q)
whether test animals were fed or not fed during the exposures. Two exceptions
were tetrachloroethylene, in which the LC5Q value in the unfed test was twice
that for the fed test, and arsenic , in which the EC5Q value for the fed test
was about 3 times the value for the unfed test.
Gammarus pseudolimnaeus - LC5Q values (96 hrs) for scuds exposed to
pentachlorophenol, arsenic , silver , lead"*"2, and chromium ranged from
4.49 (3.67-5.49) ug-L'1 for silver to 875 (846-904) ug-L"1 for arsenic"1"3
(Table 6). The static chromium test that was run with nominal exposure con-
centrations resulted in a 96 hr LC5Q value (94.1 ug-L ) that was approximately
-1 +6
40% higher than the value of 67.1 ug-L for the chromium test that was run
in a flow-through system with measured exposure concentrations.
Tanytarsus dissimilis - LC5Q values (48 hr) for midges exposed to hexa-
chloroethane, tetrachloroethylene, 1,2-dichlorobenzene, 1,4-dichlorobenzene,
41
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TABLE 4. LC5Q VALUES (95% CONFIDENCE INTERVALS) FOR POOLED
REPLICATES OF ACUTE TESTS IN WHICH FLAGFISH .,
(Jordanella floHdae) WERE EXPOSED TO ARSENIC*3 AND SILVER '.
LC5Q Img-L'1)
Chemical 24 hr 48 hr 72 hr 96 hr
Arsenic*3 18.3 (17.0-19.7) 16.3 (14.7-18.0) 15.9 (14.3-17.8) 14.4 (12.7-16.3)
Silver"*"1 0.0441 (0.0418-0.0465) 0.0259 (0.0222-0.0301) 0.0138 (0.0119-0.0160) 0.0092 (0.0080-0.0107)
4*
IM
-------
TABLE 5. 48 HR LC50 AND EC50 VALUES (95% CONFIDENCE
INTERVALS) FOR POOLED REPLICATES OF DAPHNIA
MAGNA
EXPOSED TO SELECTED TEST CHEMICALS.
Chemical
Hexachloroe thane
Pentachloroethane
1 ,1 ,2,2-Tetrachloroethane
1 ,1 ,2-Trichloroethane
1 ,2-Dichloroethane
1 ,3-Dichlorobenzene
1 ,2,4-Trichlorobenzene
Tetrachl oroethyl ene
Di -n_-butyl phthal ate
Di methyl f ormami de
Chlordane
Nickel +2
A +3
Arsenic
LC
Unfed
(nig
2.90s7
(2.50-3.33)
7.32s/
(5.98-8.99)
62.1^-
(55.9-70.7)
186s/
1164-214)
268^/
(246-293)
7.43s/
(6.29-8.77)
2.09s/
(1. 80-2. 63)
18.1^
(15.5-21.8)
3.70d-/
(3.70-3.70)
50
Fed
•L'1)
2.35^
(1.99-2.86)
8.02s/
(6.89-9.39)
56.9s/
(49.9-66.3)
174s/
(154-201)
315s/
(265-414)
7.23s/
(6.14-8.50)
1.63&/
(1.52-1.35)
D /
Q OQ T
(7.70-11.0)
n.d.
14,530 (13,260-
15,920)S/ n.d.
0.035^/
(0.032-0.038)
0.915^
(0.782-1.070)
n.d.
n.d.
n.d.
n.d.
EC
Unfed
(mg
2.10s/
(1.82-2.45)
4.69s/
(3.99-5.50)
23- O^/
(16.3-34.5)
30.6-X
(57.5-113)
155^
(137-188)
4.23^
(3.28-5.89)
n.d.^
8.50^/
(7.00-11.5)
n.d.
n.d.
n.d.
n.d.
1.54*
(1.20-1.97)
50
Fed
-L"1)
i.siby
(1.61-2.07)
6.88s/
(6.07-7.85)
25.2s/
(22.2-28-2)
77.8^
(56.6-107)
183^
(154-225)
5.98^
(4.85-9.53)
n.d.
7.49^/
(6.08-9.03)
n.d.
n.d.
n.d.
n.d.
4.33^
(3.71-6,29)
a/ LC50 or EC5Q calculated by binomial method.
b_/ LC5Q or EC5Q calculated by moving average method.
c/ LC5Q or EC50 calculated by probit method.
d/ LCgQ or ECcQ calculated by trimmed Spearman-Karber method.
e/ No determinations were i.iade.
43
-------
TABLE 6. LC5n VALUES (95% CONFIDENCE INTERVALS) FOR POOLED
REPLICATES OF ACUTE TESTS IN UHICH SCUDS (Ganiniarus ,
pseudolimnaeus) WERE EXPOSED TO PENTACHLOROPIIENOL, ARSENIC ,
SILVER*1, LEAD*2, AND CHROMIUM*5.
Chemical
24 hr
48 hr
72 hr
96 hr
Pentachlorophenol
Arsenic
Silver
Lead*2
-F» +6
*» Chromium
Chromium static^
600 (540-650)^ 400 (350-460)^ 290 (250-340)
n.c.5/ 1990 (1780-2220)^ 875 (846-904)
4.71 (3.82-5.79)^ 4.49 (3.67-5.49) 4.49 (3.67-5.49)
n.c. 275 (234-322) n.d.^
n.c. 288 (252-329)9/ 164 (139-192)^
n.c. 609 (680-962) 415 (313-551)
280 (240-330)
875 (846-904)
4.49 (3.67-5.49)
140 (140-140)
67.1 (55.0-81.S)
94.1 (65.1-135.8)
ay 26 hr LC&Q value.
b/ 50 hr LC5Q value.
c/ Not calculable due to insufficient mortalities at highest exposure.
d/43.5 hr LC5Q value.
§/ 22.0 hr LC5Q value.
£/ Not determined.
gy 51.5 hr LC50 value.
h/ 73.5 hr LC5() value.
i/ Test run with nominal exposure concentrations.
-------
hexachlorobenzene/DMF, pentachlorophenol, dimethylformamide, chromium"1"6,
lead , silver"*" , selenium"1"4, and cyanide ranged from 2.36 (2.10-2.66) mg-L'1
for cyanide to 36,000 C33,000-40,000) mg-L'1 for dimethylformamide (Table 7).
Insufficient mortalities occurred with hexachlorobenzene to yield an LCj-n value
at 48 hr.
Selenastrum capricornutum - Exposure of Selenastrum to initial toxaphene
concentrations of from 0.25 to 1.93 mg-L'1 resulted in growth inhibition
ranging from 30.4 to 88.8% (Table 8). The 96 hr EC5Q value for green algae
(5Q% reduction in dry weight as compared to controls) exposed to toxaphene was
0.38 mg-L" , based on initial toxaphene concentrations.
Exposure of Selenastrum to initial concentrations of heptachlor from 8.6
to 107 pg-L"1 reduced growth from 2-4 to 85.3% (Tables 9 and 10). The 96 hr
ECgQ values for algae exposed to heptachlor were 38.1 and 28.2 ug-L based
on initial heptachlor concentrations for tests 1 and 2, respectively. The
heptachlor conversion product 1-hydroxychlordene was readily formed and had
initial concentrations as high as 70.9% of the initial heptachlor concentra-
tions. 1-hydroxychlordene concentrations did not vary much over the 96 hr
exposure. Heptachlor concentrations decreased to less than 0.8 pg-L"1 at all
exposure levels.
Chronic and Subchronic Toxicity Tests
Daphnia Magna - Production of young Daphnia magna was significantly
(p<0-05 or p<0.01) reduced by the test chemicals at or above the following
mean concentrations CTable 11): 1,1,2,2-tetrachloroethane (14.4 mg-L ), 1,1,2-
trichloroethane (.41.8 mg-L), 1,2-dichloroethane (20-7 mg-L"1), 1,2,4-tri-
chlorobenzene (0-694 mg-L"1), 1,3-dichlorobenzene (1.45 mg-L), tetrachloro-
45
-------
TABLE 7. 48 HR LC50 VALUES (95% CONFIDENCE INTERVALS) FOR
POOLED REPLICATES OF ACUTE TESTS IN WHICH MIDGES
(Tanytarsus dissimilis) WERE EXPOSED TO
SELECTED INORGANIC AND ORGANIC CHEMICALS
Chemical
Hexach To roe thane
Tetrachl oroethy 1 ene
1 ,2-Dichlorobenzene
1 ,4-Dichlorobenzene
Hexachl orobenzene/DMF
Pentachlorophenol
Dimethylformamide
Chromium
Lead+2
Silver"1"1
+4
Selenium
Cyanide
(free cyanide as HCN)
Cyanide
(free cyanide as CN")
24 hr
n.c.^
54.6 (47.4-62.8)
19.9 (16.7-23-7)
22.1 (19.2-25-6)
n.c.
84.8 (71.6-100)
47,000(43,000-51,000)
206 (167-254)
n.c.
5.03 (4.47-5.65)
56.7 (56.7-56.7)
6-02 (5.75-6.30)
8-99 (8.46-9.55)
LC5Q (mg-L-1)
48 hr
5.85 (3.77-9.09) 1.68
30.8 (28.7-33.0)
12.0 (10.0-14.5)
13.0 (10.9-15.6)
n.c.2'
46.0 (39.0-54.3)
36,000(33,000-40,000)
57.3 (46.4-70.8)
224 (108-468)
3.16 (2.49-4.01)
42.5 (36.7-49.2)
2.36 (2.10-2-66)
2.49 (2.20-2.82)
72 hr
(1.31-2-14)
n.d>
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a/ Not calculable due to insufficient mortalities at highest exposure.
b_/ Not determined.
c/ The highest concentration tested was 0.0581 mg-L .
46
-------
TABLE 8. PERCENT INHIBITION OF SELENASTRUM CAPRICORNUTUM GROWTH WHEN
EXPOSED TO SEVERAL CONCENTRATIONS OF TOXAPHENE FOR 96 HR.
Nominal _,
Cone. (mg-L )
0.0
0.3
0.5
1.0
1.5
2.0
Initial
Cone. (mg-L )
0.0
0.25
0.48
0.85
0.87
1.93
Final
Cone. (mg-L" )
0.0
0.14
0.23
0.68
0.64
1.56
Mean ,
Cone. (mg-L )
0.0
0.195
0.355
0.765
0.755
1.745
Mean Algal
Weight ± S.D. (g)
0.00240 t 0.0005
0.00167 t 0.0002
0.00085^
0.00057 ± 0.0002
0.00050 t 0.0001
0.00027 ± 0.0602
Mean %
Inhibition
o-.o
30.4
64.6
76.3
79.2
88.8
Only two samples were included for consideration at this concentration.
-------
TABLE 9. PERCENT INHIBITION OF SELENASTRUM CAPRICORNUTUM GROWTH AT
00
96 HR FOLLOWING EXPOSURE TO SEVERAL CONCENTRATIONS OF HEPTACHLOR
AND ITS BREAKDOWN PRODUCT, 1 -HYDROXYCHLORDENE (Test 1)
Nominal Heptachloi
Cone. (% of Stock
Solution)
0 (control)
20%
40%
60%^
80%
100%
r Heptachlor Cone. (yg-L )
Initial
<0,8
8.6
17.6
28.4
38.5
44.4
24 hr Final
<0.8
cO. 8
<0.8
<0.8 <0.8
<0,8
<0.8
1 -Hydro xychlordene Cone. (yg'L )
Initial 24 hr
<1.0
6.1
11.5
14.8 21.4
17.8
30.5
Final
<1.0
6.1
14.9
19.9
20.1
28.7
Mean Algal Wt.
± S.D. (g)
0.0042 ± 0.0006^
0.0038 ± 0.0004^
0.0041 ± 0.0003
0.0040 ± 0.0007
0.0020 ± 0.0008
0.0012 i 0.0006
Mean %
Inhibition
0.0
9.5
2.4
4.8
52.4
71.4
a/ Only two samples were Included for consideration at this concentration.
by Heptachlor and 1-hydroxychlordene concentrations are means of triplicate determinations
at this exposure. Other exposure concentrations are from single determinations.
-------
TABLE 10. PERCENT INHIBITION OF SELENASTRUM CAPRICORNUTUM GROWTH AT
96 HR FOLLOWING EXPOSURE
AND ITS BREAKDOWN
TO SEVERAL CONCENTRATION OF HEPTACHLOR
PRODUCT, 1-HYDROXYCHLORDENE (Test 2)
Nominal Heptachlor
Cone. (% of Stock
Solution)
0 (control)
20%
40%
60%^
80%
100%^
Heptachlor Cone. <
Initial 24 hr
<0.8
13.6
22.8
44.4 <0.8
57.0
107
[yg-L-1)
Final
<0.8
<0.8
<0.8
<0.8
<0.8
<0.8
1-Hydroxychlordene Cone. (yg'L )
Initial 24 hr Final
<1.0 <1.0
4.5 6.7
11.4 13.2
17.0 18.4 20.5
25.3 28.6
27.8 23.7
Mean Algal Wt.
t S.D. eg)
0.0034 + 0.0010
0.0032 + 0.0007
0.0017 + 0.0004
0.0013 + 0.0003
0.0009 + 0.0003
0.0005 + 0.0004
Mean %
Inhibition
0.0
5.9
50.0
61.8
73.5
85.3
ay Heptachlor and 1-hydroxychlordene concentrations are means of triplicate determinations
at this exposure. Other exposure concentrations are from single determinations.
b/ This exposure level was not included in ejaculation of the EC5Q value due to t
unusually high analytical value for the Initial concentration of heptachlor.
the
-------
TABLE II. MEAN EXPOSURE CONCENTRATIONS OF SELECTED
TEST CHEMICALS AND EFFECTS UPON REPRODUCTIVE
SUCCESS AND GROWTH IN DAPHNIA MAGNA
DURING 28 DAY CHRONIC TESTS
Compound
1 ,1 ,2,2-Tetrachloroethane
1 ,1 ,2-Trichloroethane
1 ,2-Dichloroethane
1 ,2,4-Trichlorobenzene
1 ,3-Dichlorobenzene
Chemical
Concentration
mg-L (x ± s-d.)
0.0 (Controls)
0.419 t 0.036
0-859 t 0.085
1.71 t 0.17
3.43 t 0.39
6.85 t 0-90
14.4 t 1.4
0.0 (Controls)
1.72 ± 0.16
3.40 ± 0.29
6.35 t 0.52
13.2 t 1.7
26.0 ± 2.2
41.8 ± 3.0
0.0 (Controls)
10.6 ± 0.8
20.7 ± 1.7
41.6 t 2.4
71.7 t 4.8
94-4 t 5.5
137.0 ± 9.0
0.0 (Controls)
0.018 ± 0.003
0.039 t 0.005
0.079 t 0.011
0.162 ± 0.028
0.363 t 0.056
0.694 t 0.140
0.0 (.Controls)
0-044 t 0.012
0.102 * 0.023
0.182 t 0.039
0.373 t 0.053
0.689 t 0.156
1.45 t 0.28
50
Number of
Young Produced
Per Adult
(x ± s.d.)
162 t 49
84 t 50
69 ± 39
71 ± 40
78 t 37
78 ± 18
23 ± 5**
150 + 42
95 ± 53
132 ± 57
146 i 55
163 ± 59
114 ± 31
11 t 4**
164 t 45
128 t 37
88 t 51*
54 ± 24**
43 ± 22**
19 t 21**
-
166 t 51
151 ± 60
159 ± 38
157 t 25
125 ± 27
107 ± 30
32 t 20**
165 * 23
167 ± 34
178 ± 30
212 ± 37
137 ± 46
190 t 39
93 t 30**
Length (mm)
of Adults
(x + s.d.)
No data
No data
No data
No data
No data
No data
No data
4.1 ± 0.2
3.9 ± 0.2
3.8 ± 0.2
4.1 t 0.2
4.0 t 0-2
3.9 ± 0-2*
3.9 t 0.2*
3.9 ± 0.3
3.9 t 0.2
3.8 t 0.2
3.6 ± 0.2
3.4 t 0.2**
3.1 t 0.4**
2.3 t 0.1**
3.9 ± 0.2
4.2 ± 0.2
3.9 t 0.1
3.7 ± 0.1
3.6 ± 0.5
3.6 ± 0.2
3.0 t 0.2**
4.2 t 0.1
4.4 t 0-1
4.3 t 0.1
4.5 t 0.2
4.1 t 0.2
4.3 t 0.2
3.5 ± 0.2**
-------
TABLE 11 Cont. MEAN EXPOSURE CONCENTRATIONS OF SELECTED
TEST CHEMICALS AND EFFECTS UPON REPRODUCTIVE
SUCCESS AND GROWTH IN DAPHNIA MAGNA
DURING 28 DAY CHRONIC TESTS
Chemical
Concentration
Compound mg-L" (x ± s.d.)
Tetrachloroethylene 0.0 (Controls)
0.75 ± 0.036
0.159 ± 0.085
0.254 ± 0.094
0.505 + 0.250
1.11 ± 0.48
1.75 t 1-10
Arsenic 0.0 (Controls)
0.073 ±0.006
0.132 ± 0.004
0.270 ±0.014
0.633 ± 0.034
1.32 ± 0.03
2.68 ± 0.06
Number of
Young Produced
Per Adult
tx ± s.d.)
154 ± 47
165 ± 45
111 ± 76
169 ± 46
169 ± 43
58 ± 26**
0
83 ± 28
126 ± 27
81 ± 31
115 ± 19
132 ± 10
0**
0**
Length (mm)
of Adults
Cx ± s.d.)
3.9 ± 0.2
4.1 ± 0.2
3.9 ± 0.4
4.0 ± 0.2
4.1 ± 0.1
3.6 ± 0.1**
0
3.6 ± 0.2
3.7 ± 0.2
3-6 ± 0.2
3.7 ± 0.2
3.7 ± 0.3
3.2 ± 0.3**
3.2 ^
* Significantly different from controls (p<0.05).
** Significantly different from controls (p<0.01).
a/ Value for one animal only.
51
-------
-1 +3 1
ethylene (1.11 mg'L ), and arsenic 0-32 mg-L). Length of adults was
significantly (p^Q.05 or p<0.01) less than that of controls at or above the
following mean concentrations (Table 11): 1,1,2,2-tetrachloroethane (no data),
1,1,2-trichloroethane (.26.0 mg-L"1), 1,2-dichloroethane (71.7 mg-L"1), 1,2,4-
trichlorobenzene (0-694 mg-L" ), 1,3-dichlorobenzene (1.45 mg-L" ), tetrachloro-
1 +3 1
ethylene (1.11 mg-L ), and arsenic 0-32 mg-L ). "No effect" concentration
ranges for these chemicals were: 1,1,2,2-tetrachloroethane (6-85-14.4 mg-L ),
1,1,2-trichloroethane 03.2-26.0 mg-L"1), 1,2-dichloroethane 00.6-20.7 mg-L"1),
1,2,4-trichlorobenzene CO-363-0.694 mg-L" ), 1,3-dichlorobenzene (0.689-1.45
mg-L ), tetrachloroethylene 0-11-1-75 mg-L"1), and arsenic (0.63-1.32 mg-L" ).'
Fathead Minnows - Arsenic did not adversely affect fathead minnow hatch-
ing success, nor the percentage of newly hatched fry that died immediately after
hatch at the exposure concentrations tested (Table 12). Arsenic exposures
did not result in any abnormal morphological characteristics of the fry. Sur-
vival of the fish through 24 days post-hatch was significantly (p<0.01) reduced
at the highest exposure concentration (16.5 mg-L" ). Wet weight and length of
the fish at 24 days post-hatch were both significantly (pi.0.01) reduced at con-
centrations of 4.30 mg-L" and above. The "no-effect" concentration was between
2.13 and 4.30 mg-L" .
+2
Mercury did not affect hatching success nor the percentage of fry that
died immediately after hatch at the exposure concentrations tested (Table 13).
The two highest exposures (1.85 and 3.70 ug-L" ) resulted in significantly
greater (jxO.Ol) percentages of fry with gross deformities, mainly in the form
of spinal curvatures; and in reduced survival of juvenile fish through 30 days
post-hatch. All exposures resulted in significant (p<).01) reductions in
weight and length at test termination. Therefore, the "no effect" concentra-
52
-------
TABLE 12, HATCHABILITY, DEVELOPMENT. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Plmephales
en
CJ
promelas)
EXPOSED TO ARSENIC
+J (NaAs02)
FOR 30 DAYS
POST-FERTILIZATION
Mean As Concentration (mg-L"^)
Mean % hatch
Mean % dead fry at transfer
from egg cups
Mean % normal fry^at transfer
from egg cups
Mean % survival of fish at 24
days post-hatch
Mean fry wet wt (g) at 24
days post-hatch
Mean fry length (mm) at 24
days post-hatch
Control
0.00 t 0.00 1
(n=13)
86.0
0.0
97.5
95.0
0.058
17.0
.06 ± 0.28
(n=18)
92.5
0.1
94.7
70.0
0.01)6
17.0
2.13 ± 0.39
(n=18)
91.5
1.1
94.9
90.0
0.050
16.3
4.30 ± 0.50 7
(n=18)
93.1
2.6
95.2
77.5
0.041**
**
15,6
.37 ± 0.49
(n=18)
91.1
3.6
92.3
97.5
**
0.026
**
13.3
16.5 ± 1.03
(n=18)
86.4
2.9
94.9
22.5
**
0.012
**
11.2
a/ Fry were considered abnormal when they exhibit!ed gross morphological anomalies
such as spinal curvatures, or were dead after hatching.
** Significantly different from controls (piO.Ol).
-------
TABLE 13. HATCHABILITY, DEVELOPMENT, SURVIVAL AND GROWTH OF FATHEAD MINNOWS
(Piniephales promelas) EXPOSED TO INORGANIC MERCURY (HgCl,.)
FOR 35 DAYS POST-FERTILIZATION
Control
0.01 t 0.01
(n-22)
Mean % hatch37 67.3
Mean % dead fry at transfer^
from egg cups 4.3
Mean % normal fry-^at transfer^
from egg cups 100.0
Mean % survival of fish at 30
days post-hatch3' 100.0
Mean fish wet wt (g) at 30
days post-hatch 0.215
Mean fish length (mm) at 30
days post-hatch 22.4
+2 1
Mean Hg Concentration (yg-L )
0.23 ± 0.03 0.48 ± 0.07 0.87 ± 0.08 1.85 ± 0.2 3.70 ± 0.6
(n=22) (n=22) (n-22) (n=22) (n=21)
73.2 69.6 75.8 77.1 63.1
4.7 4.1 3.8 2.8 5.4
** **
100-0 100.0 100.0 25.0 0.0
100.0 100.0 100. 027 56.7**^ 11.6**^
** ** r/ (•/ **
0.193 0.193 -V -^ 0.013
** ** **r/ **r / **
21.5 21.2 19.9 ^ 17.2 v 9.4
a/ Percentage values were subjected to arcsin transformation for statistical analysis.
b/ Abnormal fry had gross morphological anomalies such as spinal curvatures.
c/ All fish at the 0.87 and 1.85 vig-L exposures died on day 29 post-hatch due to a diluter malfunction,
and weight data were not available. Survival and length data were from 29 days post-hatch.
**
Significantly different from controls (p<0-01).
-------
tion was less than 0.23 ug-L" , the lowest exposure concentration.
Flagfish - The early life-stage tests were conducted with arsenic and
flagfish. None of the parameters were significantly affected in the first test
at mean As concentrations of 0.30, 0.60, 1.20, 2.34, and 5.04 mg-L . How-
ever, the weight of juvenile fish at the end of the exposure period at the
exposure concentration of 5.04 mg-L was considerably less (x = 0.118 g) than
the weight of control fish (x = 0.135 g). Arsenic did not adversely affect
hatching success at any of the concentrations tested (Table 14). A significant
(p<0.05) increase in hatching success was observed at a concentration of 4.12
mg-L , but higher exposures did not result in significant changes. There were
no significant (p>0.05) differences from controls in the percentages of dead
fry or normal fry at the time of transfer from the egg cups, nor in the survival
of fish through 25 days post-hatch. Wet weight was significantly CPlP-01)
reduced at concentrations of 7.57 mg-L and above. Length of the fish was
significantly (p<).05) reduced at concentrations of 4.12 mg-L" and above.
The "no-effect" concentration was between 2-13 and 4.12 mg-L .
Di-n-butylphthalate Uptake. Elimination and Metabolism by Fish
14
C-labeled di-n_-butylphthalate was rapidly taken up from the water and
accumulated in the tissues of fathead minnows (Fig. 1). A steady-state level
14
of total C was attained within 4 hrs of exposure. This steady-state level
was maintained, with some fluctuations, throughout the 264 hr (11 day)
exposure. The mean bioconcentration factor (BCF) in C equivalents of
di-n_-butylphthalate for the exposure period from 4 hr to 11 days with the lower
exposure was 2,068. The mean BCF for the higher exposure between 4 and 120 hr
was 2,125. The time interval for determining the BCF at steady-state in the
higher exposure was not considered beyond 120 hr due to a decrease in exposure
55
-------
TAULE 14. IIATCHAIHLITY, DEVELOPMENT, SURVIVAL AND GROWTH OF FLAGFISH
Mnrrlanolla fln*-irlaa\ FYDMQFn TH flDCPMTT"1"J /MaficD ^ FflD
30 DAYS POST-FERTILIZATION
+3 -1
Mean As Concentration (mg-L )
Control
0.00 + 0.00 1.2410.35 2.1310.38 4.1210-29 7.5710.61 16. 3 10. 85
(n=17) (n=20) (n=20) (n=20) (n=20) (n=20)
Mean % hatch
Mean % dead fry at transfer
from egg cups
Mean % normal^ fry at transfer
from egg cups
Mean % survival of fish at 25
days post-hatch
Mean fry wet wt (g) at 25
days post-hatch
Mean fry length (mm) at 25 days
post-hatch
56.4 66.0 64.4 07.4* 78.3 76.1
2.2 2.2 1.4 0.0 0.9 1.0
95.6 96.5 97.2 98.3 99.1 99.0
77.5 85.0 80.0 95.0 82-5 75.0
** **
0.056 0.043 0.055 0.046 0.031 0.031
13.2 12.6 12.7 12.1* 10.8** 7.9**
ay Fry were considered abnormal when they exhibited gross morphological anomalies such
as spinal curvatures, or were dead after hatching.
* Significantly different from controls (p^O.05).
** Significantly different from controls (p<0.01).
-------
4.0
en
0 4 812 24 48 144 284268272276 286312 408 528 648
Figure
Log mean exposure water concentrations (ua-mL" ) and log mean,
concentrations (± S.D.) of whole body '^C equivalents (yg-g )
of di-n-butylphthai ate 1n fathead minnows (Pimephales promelas)
exposed for 11 days (264 hrs) and allowed to depurate 21 days
(500 hrs).
-------
concentrations beyond 120 hr and the slow depuration rate.
14C residues were eliminated slowly in the fish tissues upon transfer to
clean flowing water. The half-lives (t^) for total C residues in fish tissue
were 216 hr (9 days) and 243 hr 00 days) for lower and higher exposure concen-
trations, respectively.
After 1 and 3 days of exposure, dt-n_-butylphthalate contributed 21.6 and
53.0%, respectively, of the total radioactivity measured in the fish. These
percentages were determined from the percentages of supernatant radioactivity
that were due to di-n_-butylphthalate CTable 15). After 11 days of exposure,
di-n_-butylphthalate contributed only 8-]% of the total radioactivity. Applica-
tion of the mean of these three percentages (27.6%} gave di-n_-butylphthalate
BCF values of 570 and 590 for lower and higher exposures, respectively.
After 1 day of exposure, 7 radioactive spots were present on the TLC
plate. No phthalic acid was detected. After 3 days of exposure, 8 radioactive
spots were observed on the TLC plate, with 1.9% of the radioactivity contributed
by the metabolite phthalic acid- Only 4.4% of the radioactivity remained at the
TLC plate origin, while 79.3% had the same Rf as di-n_-butylphthalate. After 11
days of exposure, 8 radioactive spots were present, with 26.0% of the radio-
activity remaining at the origin, 2.2% as phthalic acid, and only 14.6% as
di-n-butylphthai ate. On day 11, the percentage of total radioactivity
associated with the pellet increased to 44.4%.
Di-n-butylphthalate Binding
Di-n_-butylphthalate was rapidly converted by rainbow trout liver micro-
somes to unextractable compound present in the aqueous phase (Table 16).
Approximately 40% of the radio!abeled compound was converted within 15 min,
and 60% within 1 hr. Irreversible protein binding occurred throughout the
58
-------
01
U3
TABLE 15. DISTRIBUTION OF RADIOACTIVITY IN FATHEAD MINNOWS
(Pimephales proinelas) EXPOSED TO 14C-DI-n_-BUTYLPHTHALATE
Exposure Duration
(uays)
1
2
3
11
Total 14C
66.9
61.2
66.9
55.6
Supernatant
% of C as ,
Di-n.-butylphthalate^'
32.3
n.d.^/
79.3
14.6
% of 14C as .
Phthallc Acid57
0
n.d.
1.9
2.2
Pellet^
%of
Total . C
33.1
38.8
33.1
44.4
a/ Percentages of supernatant C contributed by di-r^-butylphthalate and phthalic acid
were determined by thin-layer chromatography, autoradiography, and scintillation
counting techniques.
b/ Pellet produced by centrifugation of fish tissue homogenate at 2,150 x g for 20 min.
c/ Hot determined.
-------
TABLE 16. DISTRIBUTION (% ± S-D-) OF 14C AFTER INCUBATION
OF 14C-DI-n_-BUTYLPHTHALATE FOR VARIOUS TIME INTERVALS
WITH MICRQSOMAL FRACTIONS OF RAINBOW TROUT (Salmo
gairdneri) LIVER AND POST-MITOCHONDRIA!. SUPERNATANT OF
DAPHNIA I'lAGNA. (Values are the Means of Three Separately
Prepared Tissue Fractions)
Time
(Min)
0
Ib
30
45
60
120
0
15
30
45
60
Hexane37
Extracted
90.3 t 1.2
54.3 t 4.3
44.3 t 6.7
34.2 t 2.5
32.7 ± 0.6
27.5 ± 1.3
97.9 t 0.7
94.7 ± 0.5
92.4 ± 1.7
93.2 ± 1.8
88.2 t 6.7
Rainbow Trout
Aqueous or^
Unextracted
in Hexane
7.7 t 1.1
38.1 t 3.7
46.3 ± 3.0
57.3 ± 2.8
57.7 t 1.5
63.1 t 2.8
Daphnia
1.9 t 0.7
4.7 ± 0.7
7.0 ± 1.7
6.2 ± 1.8
11.3 ± 6.1
Protein -'
Bound
2.0 t 0.3
7.0 ± 1.0
3.7 ± 3.1
8.5 ± 1.7
9.6 ± 1.9
9.4 ± 1.8
0.22 ± 0.07
0.54 ± 0.39
0.57 ± 0.32
0.55 t 0.09
0.48 t 0.47
Total &
Recovery
90.8 ± 4.9
91.5 ± 5.9
95.4 t 2.3
95-1 ± 4.7
94.0 t 5.5
91.2 ± 5.5
36.3 ± 12.5
37.1 ± 3.6
37.0 t 14.4
89.7 ± 3.0
78.6 ± 13.9
14
a/ Percent of total added C in 0.1 mL solution which was recovered in hexane
after extraction of reaction mixture.
by Percent dpm in aqueous fraction Csoluble) and protein pellet relative to
dpm extractable in hexane.
c/ Total percent recovery is based on dpm recoverable in all fractions divided
by totalJ4C in the reaction mixture.
60
-------
2 hr incubation with C labeled di-n_-buty1phthalate. Approximately 9% of the
radiolabeled compound was bound to the trout liver microsomes during a 2 hr
incubation with 7% of the total bound in the first 15 min of incubation.
Radiolabeled di-n_-butylphthalate was converted more slowly by Daphnia PMS
during a 1 hr incubation than by trout liver microsomes. Only about 10% of the
radiolabeled compound was converted (Table 16) to water soluble hexane un-
extractable compounds. Less than 1% of the di-n_-butylphthalate was irreversibly
bound to protein.
*
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons
by Rainbow Trout and Daphnia
Measurements were made of the distribution of radioactivity after C-
labeled carbon tetrachloride was incubated with rainbow trout liver microsomes
and Daphnia PMS tissue fractions. Most of these compounds were readily
volatilized from the reaction mixture in spite of a silica gel trap. This
resulted in low recoveries of the compound at the termination of chemical
reaction. Most of the radioactivity was present in the hexane extract at each
incubation time interval (Table 17). This indicated that most of the radio-
activity remained as parent carbon tetrachloride. However, the amount re-
coverable with hexane decreased with incubation time. Radioactivity in the
aqueous phase and the CO^ trap increased with concomitant decrease of the
hexane-extracted radiocarbon. Carbon tetrachloride bound slowly with the
microsomal protein fractions of rainbow trout liver and Daphnia PMS. Forma-
tion of the aqueous metabolites and the protein binding of carbon tetrachloride
did not appear to be linear with time of incubation. Both species were capable
of slowly metabolizing this hepatoxin in vitro via microsomal mixed function
oxidases.
61
-------
TABLE 17. DISTRIBUTION (% + S.D.) OF 14C AFTER INCUBATION
OF UC-CARBON TETRACHLORIDE FOR VARIOUS TIME INTERVALS
WITH MICROSOMAL FRACTIONS OF RAINBOW TROUT (Salrco
gai rdneri) LIVER AND POST-MITOCHONDRIAL SUPERNATANT OF
DAPHNIA MAGNA. (Values are the Means of Three Separately
Prepared Tissue Fractions)
Rainbow Trout
Time
(Min)
0
15
30
45
60
120
0
15
30
45
60
Hexane^
Extracted
91.0 t 5.8
92.6 ± 4.2
88.1 ± 3.0
90.1 ± 0.8
87. 1 ± 1.8
84.7 t 5.9
95.9 i 1.8
91.4 ± 1.3
93.5 t 2.3
89.1 t 1-2
87.5 t 0.9
Aqueous or-'
Unextracted
in Hexane
0.56 ± 0.15
0.69 ± 0.13
0.73 ± 0.15
0.89 ± 0.05
1.20 ± 0.28
1.10 + 0-16
0.72 ± 0.43
0.99 t 0.38
0.91 ± 0.24
1.30 t 0.21
1-50 ± 0.56
C02^
Trap
0.012 t 0.016
O.lb ± 0.13
0.097 t 0.095
0.15 ± 0.15
0-25 t 0.21
0-13 t 0.20
Daphni a
0.09 ± 0.02
0-15 ± 0.14
0.06 ± 0.08
0.38 t 0.13
0.13 ± 0.18
Protein^
Bound
0.26 t 0.10
0.35 ± 0.10
0.37 ± 0.15
0.53 ± 0.13
0-61 ± 0.30
0.60 i 0.15
0.061 ±0.03
0-074 ± 0.03
0-12 ± 0.10
0.10 t 0.06
0.09 t 0.10
Total %&
Recovery
61.8 ± 11.9
52.7 ± 8.9
47.9 ± 16.8
45.6 i 7.4
37.5 ± 8.3
40.0 ± 4.7
55.8 ± 16.2
45.9 ± 9.3
47.1 ± 4.7
38.3 ± 3.6
37.5 ± 2.9
a/ Percent of total added dpm in 0.1 mL solution which could be recovered
in hexane after the extraction of reaction mixture.
by Percent dpm in aqueous fraction (soluble and protein pellet) relative
to dpm extractable in hexane.
cy Percent radioactivity trapped in Carbosorb Irrelative to dpm extractable
in hexane.
dy Total percent recovery is based on the dpms recoverable in all fractions
including the silica gel trap divided by the total added dpm in the
reaction mixture.
62
-------
Chloroform was rapidly converted to hexane-unextractable water soluble
metabolites in rainbow trout liver microsomes and Daphnia PMS (Jable 18).
Approximately 40% of the radioactivity was found in the aqueous phase and only
53% was extracted in hexane within 1 min of incubation with rainbow trout liver
microsomes. Similarly, the aqueous phase from Daphnia contained more than 50%
of the radioactivity as compared to 25-40% in the hexane extract. Radioactivity
in the aqueous phase increased with incubation time to 70% in the case of
Daphnia and to about 45% in rainbow trout. Measureable radioactivity was also
found in the carbon dioxide traps of both animal species. Rainbow trout liver
microsomes showed increased protein binding with incubation time. However,
Daphnia showed little change in protein bound radioactivity with incubation time.
Most (87-94*) of the radioactivity spiked in the microsomal tissue
preparation with C chlorobenzene was extractable in hexane even after 120 min
of incubation time with rainbow trout liver microsomes and 60 min with Daphnia
PMS (Table 19). The aqueous phase of the reaction mixture, in both species,
showed small percentages (0.6-2.3) of water soluble products of metabolism.
Higher amounts of protein bound radioactivity were found with rainbow trout
liver than with Daphnia tissue preparations.
Rainbow trout appeared to show higher metabolic activity than Daphnia
for 1,1,2-trichloroethylene and 1,1,2-trichloroethane (Tables 20 and 21).
More polar metabolites of 1,1,2-trichloroethylene and 1,1,2-trichloroethane
were formed by rainbow trout liver microsomes than Daphnia PMS. Trichloro-
ethane was more readily converted to water soluble products than trichloro-
ethylene in the case of rainbow trout. On the other hand, Daphnia converted
both of the compounds to aqueous metabolites at similar rates, but at much
slower rates than rainbow trout. Both compounds showed protein binding with
63
-------
TABLE 18. DISTRIBUTION (% + S.D.) OF 14C AFTER INCUBATION
OF 14C-CHLOROFORM FOR VARIOUS TIME INTERVALS
WITH MICROSOMAL FRACTIONS OF RAINBOW TROUT (Salmo
gairdneri) LIVER AND POST-MITOCHONDRIAL SUPERNATANT OF
DAPHNIA MAGNA. (Values are the Means of Three Separately
Prepared Tissue Fractions)
Rainbow Trout
Time
(Min)
0
15
30
45
60
120
0
15
30
45
60
Hexane^
Extracted
53.2 t 25.3
45.9 t 27.2
44.3 ± 20.7
46.3 ± 25.9
42.1 ± 27.7
45.3 ± 16.7
39.7 t 23.4
40.3 ± 20.7
37.8 t 22.8
25.4 t 7.3
26.2 t 15.4
Aqueous or^
Unextracted
in Hexane
38.6 ± 25.8
43.4 ± 27.5
44.3 t 28.7
44.3 t 26.8
44.8 ± 26.3
42.4 t 17.5
54.0 ± 25.9
53.0 ± 22.8
56.1 ± 23.9
70.1 ± 6,5
69.3 ± 16.5
t
Trap
0.25 ± 0.32
0.09 ± 0-04
0.19 ± 0-23
0.18 ± 0.29
1.2 t 1.7
0.73 ± 1.0
Daphnia
2.4 ± 3.8
2.3 ± 3.5
1.7 ± 2.1
0.52 ± 0.54
0.11 ± 0.12
Protein^
Bound
1.1 ± 0.6
4.2 ± 2.6
3.5 t 3.2
4.6 ± 1-7
4.6 * 2.0
5.9 ± 4.2
1.6 ± 2.4
1.5 ± 2.5
1.3 ± 1.9
1.5 t 2.0
1.5 ± 1.5
Total %&
Recovery
87.9 t 25.3
86.8 t 14.0
91.4 ± 15.7
83.6 t 12.5
81.9 ± 14.9
83.7 t 11.6
83.0 ± 14.1
77.0 t 14.8
85.8 t 11.5
73.3 ± 28.8
74.9 ± 14.2
a/ Percent of total added dpm in 0.1 mL solution which could be recovered
in hexane after the extraction of reaction mixture.
b/ Percent dpm in aqueous fraction Csoluble and protein pellet) relative
to dpm extractable in hexane.
cy Percent radioactivity trapped in Carbosorb
extractable in hexane.
relative to dpm
dy Total percent recovery is based on the dpms recoverable in all fractions
including the silica gel trap divided by the total added dpm in the
reaction mixture.
64
-------
TABLE 19. DISTRIBUTION (% + S.D.) OF 14C AFTER INCUBATION
OF l^c-CHLOROBENZENE FOR VARIOUS TIME INTERVALS
WITH MICROSOHAL FRACTIONS OF RAINBOW TROUT (Salmo
gai rdneri) LIVER AND POST-MITOCHONDRIAL SUPERNATANT OF
DAPHNIA MAGNA. (Values are the Means of Three Separately
Prepared Tissue Fractions)
Rainbow Trout
Time
(Win)
0
15
30
45
60
120
0
15
30
45
60
Hexane^
Extracted
92.2 ± 2.3
94-4 ± 1.8
92.5 ± 1.2
93.4 ± 2.1
92.3 ± 2.6
86.9 ± 1.0
91.2 ± 4.0
94.4 ± 2.0
92.5 ± 2.7
90.6 ± 2.1
92.5 ± 2.7
Aqueous or-'
Unextracted
in Hexane
0.56 ±'0.06
0.95 ± 0.15
1.0 ± 0.3
1.3 ±0.2
1.5 ±0.2
1.9 ± 0.2
1.2 ± 0.17
1.7 ± 0-15
2.1 ± 0.06
2.1 ± 0.07
2.3 ± 0.36
Trap
0.006 ± 0.004
0.03 ± 0.01
0.015 ± 0.009
0.070 ± 0.014
0.014 ± 0.010
0.21 ±0.25
Daphnia
0-060 ± 0-004
0.024 ± 0.006
0.042 ± 0.030
0.070 ± 0.014
0.090 ± 0.010
Protein^/
Bound
0.4 ± 0.1
0.60 ± 0.27
0.79 ± 0.17
0.8 ± 0-0
0.56 ± 0.40
1.2 ± 0.70
0.06 ± 0.026
0.13 ± 0.08
0.11 ± 0.08
0.15 ± 0.02
0.053 ± 0-006
Total %4/
Recovery
75.2 ± 13.4
58.4 ± 8-3
50.0 ± 8.3
42.6 ± 2.5
39.1 ± 6.6
29.2 ± 3.5
66.4 ± 15.6
49.1 ± 8.4
41.3 ± 16-6
33.3 ± 11.2
35.7 ± 6.1
a/ Percent of total added dpm in 0.1 mL solution which could be recovered
in hexane after the extraction of reaction mixture.
b/ Percent dpm in aqueous fraction (.soluble and protein pellet) relative
to dpm extractable in hexane.
relative to dpm extractable
cy Percent radioactivity trapped in Carbosorb I
in hexane.
# Total percent recovery is based on the dpms recoverable in all fractions
including the silica gel trap divided by the total added dpm in the
reaction mixture.
65
-------
TABLE 20. DISTRIBUTION (% ± S.D.) OF 14C AFTER INCUBATION
OF 14C-1,1,2-TRICHLOROETHYLENE FOR VARIOUS TIME INTERVALS
WITH MICROSOMAL FRACTIONS OF RAINBOW TROUT (Salmo
galrdneri) LIVER AND POST-MITOCHONDRIAL SUPERNATANT OF
DAPHNIA MAGNA. CValues are the Means of Three Separately
Prepared Tissue Fractions)
Rainbow, Trout
Time
(Min)
0
15
30
45
60
120
Hexane^
Extracted
89.0 ± 3.4
92.2 ± 4.9
34.5 ± 5.8
88.3 ± 3.5
85.4 ± 3.6
82.8 ± 3.2
Aqueous or**
Unextracted
in Hexane
1.1 ± 0.11
1.6 ± 0.06
6.3 ± 7.5
2.2 t 0.15
1.8 ± 0.35
2.6 ± 0-80
C02^
Trap
0.032 ± 0.007
0.096 ± 0.090
0.063 ± 0.046
0.21 ± 0.27
0-11 ± 0.11
0.19 ± 0.10
Protein-'
Bound
0.09 ± 0.01
0.08 ± 0.08
0.4 ± 0-5
0.16 ± 0.09
0.14 ± 0.05
0-31 ± 0.20
Total $
Recovery
63-3 ± 15.9
54.0 ± 9.2
49.2 ± 11.9
39.9 ± 4.4
46.5 ± 8.2
32.7 ± 5.9
0
15
30
45
60
88.8 ± 2.1
89.4 ± 1.7
90.5 ± 3.7
91.5 t 5.0
89.1 ± 1.3
1.03 ± 0.24
1.56 ± 0.16
1.8 ± 0.23
1.9 ± 0.42
1.95 ± 0.64
Daphnia
0.06 ± 0.01
0.10 ± 0.04
0.14 ± 0.09
0.03 ± 0.01
0.10 ± 0.01
0.024 ± 0.032
0.023 ± 0.017
0.020 ± 0.014
0.013 ± 0-011
0.012 ± 0-011
54.6 ± 0-6
42.4 * 4.1
42.7 ± 5.5
37.4 ± 0.6
34.5 ± 3.2
a/ Percent of total added dpm in 0.1 mL solution which could be recovered
in hexane after the extraction of reaction mixture.
B/ Percent dpm in aqueous fraction (soluble and protein pellet) relative to
dpm extractable in hexane.
rt?\
c/ Percent radioactivity trapped in Carbosorb Ir* relative to dpm extractable
in hexane.
d/ Total percent recovery is based on the dpms recoverable in all fractions
including the silica gel trap divided by the total added dpm in the
reaction mixture.
66
-------
TABLE 21. DISTRIBUTION (% t S.D.) OF 14C AFTER INCUBATION
OF 14C-1,1,2-TRICHLOROETHANE FOR VARIOUS TIME INTERVALS
WITH MICRQSOMAL FRACTIONS OF RAINBOW TROUT CSalmo
gairdneri) LIVER AND POST-MITOCHONDRIAL SUPERNATANT OF
DAPHNIA HAGNA. (Values are the Means of Three Separately
Prepared Tissue Fractions)
Rainbow Trout
Time
(Min)
0
15
30
45
60
120
0
15
30
45
60
Hexane3'
Extracted
81.5 ± 24.7
79.3 ± 31.3
76.3 ± 34.1
77.8 ± 28.7
77.9 ± 31.7
76.4 ± 30.9
96.3 ± 0.6
97.2 ± 1.0
96.8 + 0.8
97.0 ±0.8
97.0 ± 0.0
Aqueous or-/
Unextracted
in Hexane
12.4 t 20.3
16.7 ± 27.4
15.1 ± 24.5
15.8 ± 25.6
15.5 ± 24.9
16.8 ± 26.7
0.77 + 0.30
0.96 ± 0.30
1.1 ±0.4
1-1 ±0.14
0.98 ± 0.04
^ '
Trap
0.22 ± 0.35
0.049 t 0.07
0.67 t 0.25
0.18 ± 0.28
1.1 ± 1.8
0.70 ± 1.00
Daphnla
0.005 ± 0.004
0.007 ± 0.005
0.036 t 0.029
0.015 ± 0.007
0.02 ± 0.00
Protein**
Bound
0.65 ± 0.91
1.6 ± 2.5
1.3 ± 2.1
1-46 ± 2.30
1.47 ± 2.20
1-26 ± 1.50
0.004 ± 0.004
0.012 ± 0.008
0.009 t 0.001
0.010 ± 0.014
0.012 ± 0.011
Total 1?
Recovery
77.6 ± 21.7
75.6 ± 18.6
68.9 ± 16.3
69.4 t 13.3
66.7 ± 11.1
59.7 ± 9.6
71.0 ± 14.7
60.1 ± 19.9
49.6 ± 7.6
47.3 ± 13.4
50.0 ± 18.8
a/ Percent of total added dpm in 0.1 nt solution which could be recovered
in hexane after the extraction of reaction mixture.
by Percent dpm in aqueous fraction (soluble and protein pellet) relative
to dpm extractable in hexane.
^ relative to dpm
c/ Percent radioactivity trapped in Carbosorb 1
extractable in hexane.
d/ Total percent recovery is based on the dpms recoverable in all fractions
including the silica gel trap divided by the total added dpm in the
reaction mixture.
67
-------
rainbow trout or Daphnia mlcrosomal mixed function oxidase system in vitro.
Both rainbow trout and Daphnia metabolized chloroform most readily and
carbon tetrachloride least readily, based upon the percentages of total radio-
activity present in the aqueous phase and in the protein bound phase. For the
remaining three compounds, the orders for rate of metabolism were not the same
between species. The order for rainbow trout was chloroform > 1,1,2-trichloro-
ethane > 1,1,2-trichloroethylene > chlorobenzene > carbon tetrachloride. The
order for Daphnia was chloroform > chlorobenzene > 1,1,2-trichloroethylene >
1,1,2-trichloroethane <± carbon tetrachloride.
Mixed Function Oxidase Levels
Microsomal monooxygenase or mixed function oxidase assays of rainbow trout
liver and Daphnia PMS fractions were performed. Rainbow trout liver microsomes
had mean values of 0.28 and 0.19 nM-mg of cytochrome P-450 and cytochrome b-,
respectively (Table 22). The level of NADPH cytochrome c reductase activity
in rainbow trout liver microsomes was 16 nM of cytochrome c reduced -mi n -ing
protein. Rainbow trout liver microsomes metabolized aniline at a very slow
rate of 0.04-0.05 nM-mg protein-min (Table 22). PMS from adult Daphnia
showed a mean value of 42 ± 5.3 nM of cytochrome c reductase activity-min-
ing" protein, which was higher than rainbow trout.
68
-------
TABLE 22. MIXED FUNCTION OXIDASE SYSTEMS OF RAINBOW
TROUT CSalmo galrdneri) LIVER AND DAPhNIA MAGMA
Enzymes
Cytochrome^
P-450
Cytochrome bJ^
Rainbow
•0.
0.
28
19
t 0.
t 0.
Trout Daphnia
1
05
(4)^ N.D.
(4) N.D.
NADPH Cytochrome^
c-reductase 15.9 + 2-2 (8) 42 ± 5.3 (3)
Aniline hydroxylase5/ 0.05 + 0.01 (.3)
a/ Nanomoles-rag microsomal protein ± S.D.
b/ Nanomoles of cytochrome c reduced•min" -mg" protein ± S.D.
c/ Nanomoles of £-aminophenol formed-min -rug protein ± S.D.
d/ Numbers in parentheses are the number of tissue preparations from
separate animal batches.
69
-------
SECTION VI
DISCUSSION
EPA PRIORITY POLLUTANTS
Chlorinated Ethanes. In this study, the acute toxicities of a series of
chlorinated ethanes from 1,2-dichloroethane to hexachloroethane were determined
with Daphnia magna liable 5). Chronic toxicity to Daphnia was determined for
1,2-dichloroethane, 1,1,2-trichloroethane, and 1,1,2,2-tetrachloroethane
(Table 11). The acute toxicity of hexachloroethane was also determined for
rainbow trout and midges (Tables 2 and 7).
Toxicity to Daphnia increased with degree of chlorination in acute and
chronic tests. This direct relationship between toxicity and degree of chlori-
nation has also been noted with fathead minnows (.U-S. EPA, 1980b) and bluegill
sunfish (Buccafusco et.al,., 1981). However, this clear relationship was not
evident with Daphnia magna in a study by LeBlanc (1980).
In the present study, 48 hr LC5Q values for Daphnia ranged from 268 mg-L
for 1,2-dichloroethane to 2.90 mg-L for hexachloroethane. Comparison of our
results with those of LeBlanc (1980) show good agreement of LC5Q values for 1,2-
dichloroethane (268 vs. 218 mg-L ), but poor agreement for the remainder of
the series from tri-through hexachloroethane. In a similar study of Adema (1978),
LC5Q values based on nominal concentrations of 1,1,2-trichloroethane were 43
mg-L for fed and unfed Daphnia magna. which were about one-fourth the values
reported here. In agreement with our acute test results, feeding did not affect
70
-------
the toxicity of this compound. Feeding did not appear to greatly affect the
toxicity of any of the chlorinated ethanes tested here.
The 96 hr rainbow trout LC5Q for hexachloroethane of 0.982 mg-L was
almost identical to the value of 0.980 for bluegill sunfish (Buccafusco et al.,
of
-1
1981) and somewhat lower than the LC5Q of 1.53 mg-L for fathead minnows (U.S.
EPA, 1980b). A 48 hr LC5Q of 5.85 mg-L ' for the midge indicated that
Tanytarsus was less sensitive to hexachloroethane in acute exposures than Daphnia
magna, rainbow trout, or bluegill sunfish.
"No-effect" concentration ranges from the Daphnia magna chronic exposures
were: 1,2-dichloroethane - 10.6 - 20.7 mg-L"', 1,1,2-trichloroethane - 26,0 -
41-8 mg-L"1 and 1,1,2,2-tetrachloroethane - 6.85 - 14.4 mg-L"1 (Table 11). The
chronic "no-effect" level for reproduction of 18 mg-L"1 for 1,1,2-trichloroethane
by Adema 0978) was close to our value of 26.0 mg.L. Similar to our work,
Adema (.1978) used completely filled and closed flasks to minimize the loss of
volatile compounds. These same compounds have been studied in early life-stage
exposures with the fathead minnow (U.S. EPA, 1980b). "No-effect" concentration
ranges for fathead minnows were: 1,2-dichloroethane - 14.0 - 29.0 mg-L" , 1,1,2-
trichloroethane - 6.0 - 14.8 mg.L, and 1,1,2,2-tetrachloroethane - 1.4 - 4.0
mg-L .
Tetrachloroethylene. The acute toxicity of tetrachloroethylene was deter-
mined with rainbow trout (.Table 2), Daphnia magna (.Table 5), and midges
(Table 7). Tetrachloroethylene toxicity to rainbow trout was determined both
with and without the carrier solvent, dimethylformamide (DMF).
Rainbow trout were the most sensitive of the three species and midges the
least sensitive. The 96 hr LC5Q of 4.99 mg-L for tetrachloroethylene alone
with rainbow trout was similar to the value of 5.84 mg-L" for tetrachloro-
71
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ethylene with DMF, and the 95% confidence intervals overlapped. Fish exposed
to tetrachloroethylene with DMF were larger than those exposed to tetrachloro-
ethylene alone, which may explain the slightly greater tolerance in the former
test. Increased tolerance of rainbow trout to permethrin with an increase in
body size was observed by Kumaraguru and Beamish (.1981). The 96 hr LC5Q values
determined here for rainbow trout indicate that trout are more sensitive than
other fish species tested. LC5Q values of 13.5 and 18.4 mg-L were obtained
for fathead minnows in flow-through measured tests (U.S. EPA, 1980b; Alexander
et.al., 1978), and 12-9 mg-L for bluegill sunfish in a static unmeasured test
(Buccafusco et. al_., 1981).
A 48 hr LC5Q value of 18.1 mg-L" for Daphnia magna in an unfed test was
almost twice the value of 9.09 mg-L in a fed test. This difference between
results from fed and unfed tests was greatly reduced when the toxic responses
were expressed as EC5Q values. 48 Hr EC5Q values of 8.50 and 7.49 mg-L" were
obtained for unfed and fed tests, respectively. LeBlanc (1980) obtained a 48
hr LC50 of 17.7 mg-L in a static unmeasured test.
Midges had a 48 hr LC5Q of 30.8 mg-L . Of the five species of freshwater
animals used in acute tests, midges were the most tolerant to tetrachloroethylene
(U.S. EPA 1980c).
A 28 day chronic exposure of Daphnia magna to tetrachloroethylene resulted
in a "no-effect" concentration range of 0.505 - 1.11 mg-L"1 (Table 11). Pro-
duction of young and length of adult daphnids were both significantly reduced
(p<0.01) at a mean exposure concentration of 1.11 mg-L" . The "no-effect"
range for Daphnia was very similar to the range of 0.5 - 1.4 mg-L reported
for the fathead minnow (.U.S. EPA, 1980b).
72
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Chlorinated Benzenes. Acute toxicity tests were conducted with rainbow
trout for 1,2- and 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, pentachloro-
benzene with DMF as a carrier solvent, and hexachlorobenzene with DMF (Table 2).
One acute test was conducted with bluegill sunfish and hexachlorobenzene with
DMF (Table 3). Daphnia magna was tested in acute and chronic exposures to 1,3-
dichlorobenzene and 1,2,4-trichlorobenzene (Tables 5 and 11). Midges were used
in acute tests with 1,2-dichlorobenzene, 1,4-dichlorobenzene, and hexachloro-
benzene with DMF (Table 7).
The 96 hr LC5Q values for rainbow trout were 1.58 and 1.12 mg-L for 1,2-
and 1,4-dichlorobenzene, respectively. 1,4-Dichlorobenzene produced a 96 hr
LC^ of 4.0 mg-L" in a flow-through measured test with the fathead minnow (U.S.
*)U • •
EPA 1980b), Static unmeasured tests with bluegill sunfish and 1,2-dichloro-
benzene gave 96 hr LC5Qs of 27.0 mg-L (Dawson et_ al_., 1977) and 5.6 mg-L
(Buccafusco e_t al_., 1981). A static unmeasured test with 1,4-dichlorobenzene
and bluegills produced an LC5Q of 4.3 mg-L (Buccafusco e_t al_., 1981).
The 48 hr LC5Q values for 1,3-dichlorobenzene and Daphnia magna in the
present study were 7.43 and 7.23 mg-L in unfed and fed tests, respectively.
Feeding had virtually no effect upon the response. LeBlanc (1980) obtained an
LC5Q of 28 mg-L" in a static unmeasured test with this species.
The 48 hr LC5Qs for midges exposed to 1,2- and 1,4-dichlorobenzene were
12.0 and 13.0 mg-L, respectively. Daphnia magna was the only other inverte-
brate species that had been tested with these compounds (U.S. EPA, 1980d) where
LC5Qs of 2.4 and 11 mg-L were obtained in static unmeasured tests with 1,2-
and 1,4-dichlorobenzene, respectively (.LeBlanc, 1980).
1,2,4-Trichlorobenzene produced a 96 hr LCj-Q of 1.53 mg-L" in rainbow
trout and 48 hr LC5Qs of 2.09 and 1.68 in Daphnia magna for unfed and fed tests,
73
-------
respectively. A 96 hr LC5Q of 2.87 mg-L was reported with 1,2,4-trichloro-
benzene and fathead minnows in a flow-through measured test (II.S. EPA, 1980b),
and 3.4 mg-L" for bluegill sunfish in a static unmeasured test (Buccafusco et
al., 1981). In a static unmeasured test with Daphnia magna, a 48 hr LCCO of
— —• ••— ou
50 mg-L was obtained (LeBlanc, 1980), a value about 24 times greater than ours.
Pentachlorobenzene (with DMF) did not yield sufficient mortalities in rain-
bow trout at 96 hr to allow for an estimation of the LC5Q. However, by 144 hr
sufficient mortalities had occurred to produce an LC5Q estimate of 0.28 mg-L.
Buccafusco e_t al_ (.1981) obtained a 96 hr LC-Q of 0.25 mg-L with bluegill sun-
fish in a static unmeasured test.
Hexachlorobenzene was insufficiently soluble in water to yield concentra-
tions high enough to result in acute test mortalities and LC5Q estimates. It
was tested using the carrier solvent DMF with rainbow trout, bluegill sunfish
and midges. The same observation was made in tests with fathead minnows (U.S.
EPA, 1980b).
Chronic (28 day) exposure of Daphnia magna to 1,3-dichlorobenzene resulted
in a "no-effect" concentration range of 0.689 to 1.45 mg-L" . Production of
young and length of adults were both significantly reduced (p<0.01) at the
highest exposure of 1.45 mg-L" . The "no-effect" range for fathead minnows
exposed to 1,3-dichlorobenzene in an early life-stage test was 1.0 to 2.3
mg-L"1 (U.S. EPA, 1980b).
Chronic exposure of Daphnia magna to 1,2,4-trichlorobenzene resulted in a
"no-effect" concentration range of 0.363 to 0.694 mg-L . Significant re-
ductions (p<0-01) in both production of young and length of adults occurred at
the highest exposure of 0.694 mg-L. The "no-effect" range for fathead minnows
in an early life stage test was 0.499 to 0-995 mg-L"1 (U.S. EPA, 1980fa).
74
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A general trend of increased toxicity with increased degree of chlorination
in the chlorinated benzenes had been noted (U.S. EPA, 1980e). In this study,
1,2,4-trichlorobenzene was more toxic to Daphnia than 1,3-dichlorobenzene in
both acute and chronic tests. In the rainbow trout acute tests, pentachloro-
benzene (.144 hr LC^Q) was more toxic than the di- and trichlorobenzenes. However,
1,2-di-, 1,4-di-, and 1,2,4-trichlorobenzene had quite similar LC5Q values.
Hexachlorobutadiene. LC5Q values (96 hr) of 0.320 and 0.324 mg-L"1 were
determined for rainbow trout and bluegill sunfish, respectively (Tables 2 and
3). Only four other freshwater species have had acute values reported (U.S.
EPA, 1980f; Leeuwangh et_ al_., 1975). In a flow-through measured test, the
fathead minnow had a 96 hr LC5Q of 0.102 mg-L"1 (U.S. EPA, 1980b). LC5Q values
(96 hr) for the goldfish (Carassius auratus) and for the invertebrates Asellus
aquaticus (.Crustacea) and Lymnaea stagnalis (Mollusca) were 0.09, 0.13 and 0.21
mg-L" , respectively, in measured static tests where the solutions were renewed
daily (J-eeuwangh e_t al_., 1975).
Di-n-Butylphthalate. In this study a 48 hr LC-Q of 3.70 mg-L"1 was
obtained with Daphnia magna (Table 5), This value may be compared to 96 hr LCcn
— bU
values of 2.10 mg-L for the scud (Gammarus pseudolircnaeus) and >10.00 mg-L
for the crayfish (Orconectes nais) in static unmeasured tests (Mayer and
Sanders, 1973). The fathead minnow, bluegill, channel catfish CIctalurus
punctatus), and rainbow trout had 96 hr LC5Qs of 1.30, 0.73, 2.91, and 6.47
mg-L, respectively (Mayer and Sanders, 1973). Johnson and Finley (1980)
reported the same values for these species except for rainbow trout for which
they reported a 96 hr LC5Q concentration of 2-6 mg-L . Sensitivities to
di-rv-butylphthalate have generally been similar amongst the different fresh-
water species tested (U.S. EPA, 1980g).
75
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The mean bioconcentration factor (JJCF) for di-n_-butyl phthalate (measured
as C equivalents) in whole body fathead minnows was -v2,100. When expressed as
parent compound alone, the mean BCF was 580. An equilibrium between the water
and fish tissue was reached within 4 hr (Fig. 1). C residues were eliminated
very slowly (half-life = 9-10 days), indicating that much of the di-n_-butyl-
14
phthalate taken up was being metabolized and the C retained.
Mayer and Sanders (1973) exposed four invertebrate species to C di-n_-
butylphthalate. In Daphnia magna and Gammarus pseudolimnaeus, an equilibrium
in C residues between water and tissue was reached within 7 days. Tissue
residues 400 and 1,350 times greater than water concentrations were obtained
on day 7 for Daphnia and Gammarus, respectively. The midge, Chironomus
plumpsus, and the mayfly, hexagenia bilineata, had residue concentration
factors 720 and 430 tines the water concentration on day 7 (.last day of
measurements), but an equilibrium was not evident. Following 7 days of
14
exposure to C di-n_-butyl phthalate, Daphnia transferred to fresh flowing water
eliminated 50% of the total radioactivity after 3 days, but still retained 25%
after 7 days (Mayer and Sanders, 1973).
14
Somewhat different results for di-n_-butyl phthalate accumulation of C
equivalents in the whole body were reported by Sanders et al_. (1973). Tests
with midge larvae (.Chi ronomus plumps us), water flea CDaphnia magna), scud
(Gammarus pseudolimnaeus), mayfly (Hexagenia bilineata), grass shrimp
(Palaemonetes kadiakensis), and damselfly (Ischnura vertical is) showed bio-
magnification values at seven days of 6,600, 5,000, 6,500, 1,900, 5,000 (3
days) and 2,700, respectively. There did not seem to be further uptake be-
tween days 7 and 14 for the two species tested (water flea and scud) at this
time interval.
76
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Metabolism of di-n-butylphthalate has been measured In vitro using
microsomal preparations from channel catfish Uctalurus punctatus) liver.
Stalling e_t al_. (1973) found four metabolites after 2 hr incubation with liver
microsomes. Three unidentified metabolites required NADPH for their production.
These unidentified metabolites comprised 42% of the total metabolites present.
The monoester of di-n_-butylphthalate was the dominant metabolite accounting for
55% of the total metabolites and required no NADPH or oxygen for its production.
Only 3% of the radioactivity in the microsomal preparation remained as parent
compound.
In our metabolism study with fathead minnows exposed to 34.3 ug-L of
di-n_-butylphthalate, 7 radioactive spots were separated by TLC from the
homogenized whole fish supernatant after 1 day of exposure. After 72 hr of
exposure, 8 spots were distinguishable by TLC. Only standards of parent com-
pound and phthalic acid were co-chromatographed for possible identification of
unknowns. Phthalic acid became detectable (Table 15) by the third day of
exposure (.1-9%) and remained nearly constant to the eleventh aay (2-2%). The
presence of parent di-n-butylphthalate on days 1 and 3 of exposure and a
greatly reduced percentage on day 11 indicates that induced metabolism of
di-n_-butylphthai ate may have occurred.
Protein binding of di-n_-butylphthalate occurred in rainbow trout liver
microsomes (Table 16) and to a more limited extent in Daphnia PMS. It was
assumed that a portion of the radioactivity in the pellet (Table 15) from the
fathead minnow metabolism study was protein bound di-n_-butylphthalate.
Pentachlorophenol. LC5Q values of 0.280 mg-L at 96 hr and 46.0 mg-L
at 48 hr were obtained for Gammarus pseudolimnaeus and Tanytarsus dissimilis,
respectively (Tables 6 and 7). Daphnia magna was the only other freshwater
77
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invertebrate species for which acute toxicity information was available where
exposure concentrations were measured (U.S. EPA, 1980h). A 48 hr LC5Q value of
0.6 mg-L" for Daphnia magna was reported (Adema, 1978). Static unmeasured
48 hr LCgQ values for Daphnia magna have ranged from 0.24 to 0.80 mg-L , while
Daphnia pulex and Daphnia cucullata had 48 hr LC5Qs of 2.0 and 1.5 mg • L ,
respectively (Canton and Adema, 1978). Acute toxicities to 9 species of fresh-
water fish ranged from 0.034 to 0.600 mg-L"1 (11-S. EPA, 1930h).
Heptachlor. Exposure of Selenastrum capricornutum to heptachlor in two
separate tests resulted in 96 hr EC™ values of 0.0381 and 0.0282 mg-L , based
upon initial measured concentrations. Heptachlor concentrations declined rapidly,
and the conversion product 1-hydroxychlordene was readily formed in considerable
quantity (Tables 9 and 10). Values of 0.0394 and 0-0267 mg-L were reported
for our results in the heptachlor criteria document (U-S. EPA, 1980i), where
ECg0 values had been calculated by a different method. No other plant toxicity
data have been published.
Eighteen freshwater species of animals, including fish and various
invertebrates, had been used in static unmeasured acute tests with heptachlor
(U.S. EPA, 1980i). No measured tests were reported. Toxicity values ranged
from 0.0009 mg-L for the stonefly (Pteronarcella badia) to 0-320 mg-L for
the goldfish. Selenastrum sensitivity to heptachlor on an acute basis appears
to be similar to that for the bluegill sunfish, the scud (Gammarus 1acustris),
and Daphnia pulex.
Chlordane. A 48 hr LC5Q estimate of 0.035 mg-L" to Daphnia magna was
found in this study with technical chlordane. In a static unmeasured test,
'50
technical chlordane produced a 48 hr LC,-n value of 0.097 mg-L" (Randall e_t
al., 1979). A 96 hr EC5Q value of 0.0284 mg-L" with Daphnia magna was
78
-------
observed in a measured test (Cardwell e_t a]_., 1977). Of 14 freshwater species
of animals ranked for their sensitivities to chlordane, Daphnia magna was ranked
number 11 (U.S. EPA, 1980J). However, almost all of the tests were static tests
with unmeasured chlordane concentrations. Three fish tests had measured concen-
trations of chlordane, and in those tests the sensitivities of the fish species
were quite similar to the sensitivity,of Daphnia magna in this study. Flow-
through measured tests with brook trout (Salvelinus fontinalis), fathead minnows,
and bluegills gave 96 hr LC5Q values of 0.047, 0.037, and 0.059 mg-L of
technical chlordane, respectively (Cardwell et. al_., 1977).
Toxaphene. The 96 hr ECj-g value for Selenastrum capricornutum (50*
reduction in dry weight as compared to controls) exposed to toxaphene was 0.38
mg-L , based upon initial measured toxaphene concentrations. Our results were
incorrectly reported as 0.38 ug-L" in the toxaphene criteria document (U.S.
EPA, 1980k). Toxaphene did not dissipate from the test solutions as rapidly as
heptachlor (Tables 3, 9 and 10). No other toxicity tests with freshwater plants
have been reported in the literature (U.S. EPA, 1980k). Studies with marine
algae have shown that the productivity of natural phytoplankton communities
is inhibited 90.8% with 1.0 mg-L" toxaphene present (Butler, 1963). In
another study, death or complete inhibition of growth of marine algae occurred
at much lower toxaphene concentrations ranging from 0.00015 to 0.150 mg-L
(Ukeles, 1962).
The acute toxicity of toxaphene to freshwater animals ranged from 0.0013
mg-L for the stonefly, Claassenia sabulosa, to 0-180 mg-L" for the midge,
Chironomus plumosus (U-S. EPA, 1980k). Comparison of our results using
Selenastrum with animal species sensitivities indicates that this species of
green algae is less sensitive than freshwater animals.
79
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Arsenic . In this study, 96 hr LCgQ values of 14-2 and 14.4 mg-L were
obtained for the fathead minnow and flagfish, respectively (Tables 1 and 4).
The 48 hr EC™ value for Daphnia magna was 1.54 mg-L (Table 5), and the 96 hr
LC5Q value for Gammarus pseudolimnaeus was 0.875 mg-L" (Table 6).
From a review of the acute toxicity of trivalent inorganic arsenic to
seven species of freshwater fish, LC5Qs,ranged from 13.3 to 41.8 mg-L (U.S.
EPA, 1980£). These values were obtained from static unmeasured tests as well
as flow-through measured tests (Clemens and Sneed, 1959; Cardwell et al.,
1976; Inglis and Davis, 1972; Fish Pesticide Research Laboratory, 1980).
Other Cladocean species have been tested for their acute sensitivities to
+3 -1
arsenic . Daphnia pulex had 48 hr ECrQ values of 1.0 and 1.7 mg-L (Sanders
and Cope, 1966; Fish Pesticide Research Laboratory, 1980), and Simocephalus
'50
serrulatus had a 48 hr ECcn of 0.812 mg-L (Sanders and Cope, 1966).
Our acute value for Gammarus pseudolimnaeus is reported as 0.879 mg-L
in the arsenic criteria document (U.S. EPA, 1980£). This value was a mean of
two replicates, whereas 0.875 mg-L is a single value for the two replicates
pooled. Exposure of Gammarus pseudolimnaeus to an arsenic concentration of
0.961 mg-L for 7 days resulted in SOS mortality (Spehar e_t al_., 1980). Of
12 species of fish and invertebrates combined, Gammarus pseudolimnaeus was the
second most sensitive species in acute tests (U.S. EPA, 1980£).
In the chronic study with Daphnia magna, significant reductions (p<0.01)
in the number of young produced and length of adults were observed at mean
arsenic concentrations of 1.32 mg-L and above (Table 11). The "no-effect"
range was 0.633 to 1.32 mg-L . Thus, the concentration that caused signifi-
cant adverse chronic effects was not much different from the 43 hr EC-Q of
1.54 mg-L" . No other chronic studies with invertebrates have been reported
80
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(U.S. EPA, 1980£).
In the early life-stage test with fathead minnows and arsenic , wet weight
and length of the juvenile fish at 24 days post-hatch were significantly reduced
(p<0.01) at concentrations of 4.30 mg-L" and above (Table 12). The "no-effect"
range was from 2.13 to 4.30 mg-L" , or from 15 to 30 percent of the 96 hr LC5Q
concentration of 14.2 mg-L .
In the first early life-stage test using flagfish, the exposure concen-
trations were too low to cause any effects (Table 14). When this test was
repeated (Table 15). a reduction (p<0.05) in length of juvenile fish at 25 days
post-hatch was observed at 4.12 mg-L" . At the two higher exposures length and
weight were both reduced at the 99% probability level. The "no-effect" range
was 2.13 to 4.12 mg-L , or from 15 to 29 percent of the 96 hr LC5Q concentra-
tion of 14.4 mg-L . No chronic toxicity tests with fish have been reported
for either freshwater or marine species (U.S. EPA, 1980£).
Chromium . LC5Q values (96 hr) of 0.0671 and 0.0941 mg-L were deter-
mined for Gammarus pseudolimnaeus with flow-through measured and static unmeas-
ured tests, respectively (Table 6). A 48 hr LC5Q value of 57.3 mg-L was
determined for the midge, Tanytarsus dissimilis (Table 7).
From a review on hexavalent chromium (U.S. EPA, 1980m), the acute
toxicity to freshwater animals is highly variable with species and extends over
four orders of magnitude. The lowest LC^Q value reported was 0.031 mg-L" for
Daphnia magna at 72 hr (Debelak, 1975). The highest LC5Q value reported was
249 mg-L for goldfish at 96 hr (Dowden and Bennett, 1965). Invertebrate
species were generally more sensitive to chromium than fish species (U.S. EPA,
'50
1980m). However, Tanytarsus with a 48 hr LC™ value of 57.3 mg'L" (rather
than 59.9 mg-L" as listed in the criteria document), was the most tolerant
81
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freshwater invertebrate with acute sensitivity similar to that for brook and
rainbow trout (Benoit, 1976).
+2 -1
Lead . A 96 hr LC5Q value of 0.140 mg-L was determined for Gammarus
pseudolimnaeus (Table 6) and a 48 hr LC_Q value of 224 mg-L was obtained for
Tanytarsus dissimilis (Table 7). A flow-through measured test with the same
scud species was conducted by Spehar e_t al_. 0978). They obtained a 96 hr
LC50 value of 0.124 mg-L , and a 28 day LC5Q value of 0.028 mg-L"1. Of the
freshwater animals tested, Gammarus was the most sensitive species to lead in
acute tests (U.S. EPA, 1980n).
The toxicity of lead decreases with increased water hardness (U.S. EPA,
1980n). For test water with hardness below 150 mg-L as CaCO-, Tanytarsus
dissimilis was the most tolerant of freshwater animals (U.S. EPA, 1980n).
+2 -1
Mercury . A 96 hr LC5Q value of 150 pg-L was obtained with the fathead
minnow (Table 1). The "no-effect" concentration from an early life-stage study
with this si
(Table 13).
A 96 hr LC50 value of 168 yg-L was found for fathead minnows of approx-
imately 3 months in age (.Snarski and Olson, 1982). The fish in our study were
approximately 1 month old. Mercuric chloride 96 hr LC^n values for rainbow
trout fingerlings were 400, 280, and 200 ug-L at temperatures of 5, 10, and
with this species was less than the lowest exposure concentration of 0-23 ug-L
20 C (Macleod and Pessah, 1973). In other tests with HgCl, and rainbow trout,
'50
a 96 hr LC,-n value of 210 ug-L was obtained at 16.7 C (Matida e_t al_., 1971)
and a 24 hr LC5Q value of 903 ug-L at 10 C (Wobeser, 1975). LC5Q values for
various freshwater invertebrate species exposed to HgCl,, ranged from 5 yg-L"
for Daphnia magna (Biesinger and Christensen, 1972) to 2.0 mg-L for a
stonefly (Acroneuria lycorius), a mayfly CEphemerella subvaria), and a
82
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caddisfly (Hydropsyche betteni) CWarnick and Bell, 1969).
In a full life-cycle chronic exposure of fathead minnows to HgClg, no
spawning occurred at or above 1,02 ug-L" , and total egg production was only
46 and 54% of controls at mean exposure concentrations of 0.26 and 0.50 ug-L ,
respectively CSnarski and Olson, 1982). Progeny after 30 days of exposure were
shorter and lighter than controls at all exposure concentrations. The lowest
mean exposure level was 0.26 ug-L . Therefore, the "no-effect" range based
on growth parameters of offspring was below the lowest exposure of 0.26 ug-L" .
Rainbow trout exposed to HgCl2 demonstrated growth retardation at concentrations
c<
-1
of 21 ug-L or greater (Matida e_t al_., 1971). A permissible concentration
range based on growth was reported at between 2.1 and 21 ug-L
Embryonic or larval forms of aquatic animals have generally been more
sensitive to mercury than adults (Taylor. 1979). HgCU was toxic to embryos
and teratogenic in marine killifish (Fundulus heteroclitus) at 10 yg-L
(Weis and Weis, 1977) and in Japanese medaka (Qryzias latipes) at 15 ug-L"
(Heisinger and Green, 1975). Scoliosis Glateral spinal curvature) occurred
in 18 and 38% of the smaller undeveloped fathead minnows exposed to 2.01 and
3.69 ug-L HgCl2, respectively, for 41 weeks CSnarski and Olson, 1982).
Significant teratogenic effects were observed in the present study at concen-
trations of 1.8 and 3.7 ug'L" .
The "no-effect" ranges for Daphnia magna exposed to HgCl2 in flow-through
and renewed static tests were 0.7 to 1.3 ug-L" and 0.9 - 1.8 ug-L ,
respectively (Biesinger et al_., 1982). No other chronic studies with HgCU
and freshwater organisms have been published (U.S. EPA, 1980 o).
+2 -1
Nickel . A 48 hr LC-n value of 0.915 mg-L with Daphnia nagna was
-^——i^^——— jjy
determined (Table 5). This was reported as 0.365 mg-L in the criteria
83
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document when determined by a different method (U.S. EPA, 1980p). The acute
toxicity of nickel decreases with increased water hardness (U.S. EPA, 1980p).
Acute toxicity values for freshwater invertebrates from studies with hardness
similar to ours (40 - 50 mg-L ) have ranged from 0.510 mg«L in Daphnia magna
(Biesinger and Christensen, 1972} to 33.5 mg-L in a stonefly, Acroneuria
lycorias (Warnick and Bell, 1969).
Silver'1'1. LC^ values (96 hr) of 0.0107 mg-L"1, 0.0092 mg-L"1, and 0.00449
mg-L were determined for the fathead minnow, flagfish, and scud, respectively
(Tables 1, 4 and 6). The 48 hr LC5Q for Tanytarsus dissimilis was 3.17 mg-L
(Table 7). In the silver criteria document (U.S. EPA, 1980q), our LC5Q values
are reported as 0.011 mg-L" and 0.0096 mg-L" for fathead minnows and flagfish,
respectively. The 48 hr LCcQ for Tanytarsus dissimilis was listed as 3.2 mg-L .
Deviations in values reported here from those published in the criteria document
are due to rounding or the use of a different approach in calculating LC5Q
values. However, the scud LC5Q value is incorrectly listed as 4.5 mg-L . It
should be 4.5 yg-L .
The acute toxicity of silver decreases as water hardness increases (U.S.
EPA, 1980q). From a review of the acute toxicities of silver to other fresh-
water invertebrates (II.S. EPA, 1980q), LC5Q values ranged from 0.25 yg-L
for Daphnia magna in an unpublished study by Chapman and co-workers from the
EPA laboratory in Corvallis, Oregon, to 1.4 mg-L for the rotifer, Philodena
acuticornis (Buikema ejt al_., 1974). The midge LC5Q value of 3.17 mg-L
represents the least sensitive acute response.
Most of the acute toxicity studies with freshwater fish have been con-
ducted with rainbow trout and fathead minnows (U.S. EPA, 1980q). In water of
similar hardness as in our study, rainbow trout had LCcn values ranging from
84
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0.0069 to 0.110 mg-L in flow-through measured tests (.Lemke, 1981). In softer
water (20 - 31 mg-L as CaC03), rainbow trout LC5Q values ranged from 0.0053
50
to 0.0081 mg-L in three tests CDavies e_t al_., 1978). Fathead minnow LC
values ranged from 0.0039 to 0.030 mg-L in water with a hardness range of
46 - 54 mg-L"1 as CaC03 (J-emke, 1981).
+4 -1
Selenium . The 48 hr LC5Q value was 42-5 mg-L for Tanytarsus dissimilis
(Table 7). Our test result was reported as 42-4 mg-L" in the selenium criteria
document (U.S. EPA, 1980r). The midge was the most tolerant of freshwater in-
vertebrates from acute tests (.U.S. EPA, 1980r). LC5Q values for other inverte-
brate species range from 0.340 mg-L for the scud, Myall el a azteca CHalter et_
al., 1980) to 24.1 mg-L" in a snail, Physa sp. (Reading, 1979).
Cyanide. The 48 hr LCcn value for Tanytarsus dissimilis was 2.36 mg-L"
•"•"• -•"- • DU ~~-~—"~~~~'
for free cyanide expressed as HCN and 2.49 mg-L when expressed as CN" (Table 7)
The criteria document for cyanides CU.S. EPA, 1980s) has reported our test
result as 2.24 mg-L" . Upon recalculation, we have arrived at a final 48 hr
LC5Q of 2.49 mg-L"1.
Four other invertebrate species had been used in acute toxicity tests with
cyanide (.U.S. EPA 1980s). LC5Q values were 0.431 mg-L for the snail, Physa
heterostropha, 0.083 mg-L for Daphnia pulex, 0.167 mg-L" for the scud,
Gammarus pseudolimnaeus, and 2.326 mg-L" for the isopod, Asellus communis
(Cairns and Scheier, 1958; Patrick e_t al_., 1968; Lee, 1976; Oseid and Smith,
/
1979). Of 15 species of freshwater fauna tested for acute sensitivity to
cyanide, Tanytarsus dissimilis was the most tolerant (.U-S. EPA, 1980s).
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons
It is apparent that rainbow trout and Daphnia both possess an active
85
-------
mixed function oxidase system which may play an important role in detoxication
of chlorinated hydrocarbons. Perhaps the initial oxidation of these compounds
occurs via the nixed function oxidase system. Toxicity may be related to
irreversible protein binding, and lipid peroxidation causing disruption of
the endoplasmic membrane. Further metabolic studies of these chemicals should
be conducted to determine their interaction with cellular components, and to
identify specific metabolites.
Mixed Function Oxidase Activity
Our data indicate (Table 23) that aquatic organisms have measurable but
lower mixed function oxidase activity than mammals. However, with similar
metabolic systems, the mechanisms leading to toxicity and neoplasia are pre-
sumed to be qualitatively similar in all organisms. Therefore, studies with
aquatic organisms can be used for important functions. The first is for
\
laboratory screening. Because they are relatively easy to rear, they are
economically attractive test organisms. The second is for environmental
monitoring. Aquatic organisms are currently being used as sentinels to signal
environmental contamination (£1 ack et_ a_K , 1980}. In summary, both laboratory
and field studies using aquatic organisms are recommended for programs in
comparative pharmacological testing, short-term screening and environmental
monitoring.
86
-------
TABLE 23. COMPARISON OF MIXED FUNCTION OXIDASE MEASUREMENTS
BETWEEN MAMMALS AND SEVERAL NON-MAMMALIAN AQUATIC ORGANISMS
Enzymes^
Cytochrome P-450
Cytochrome br
HADPH Cytochrome c
reductase
Aniline hydroxylase
Human
0.60 t 0.10^
0.49 ± 0.06^
102.6 ± 14.6^
8.7 ± 6.Q&
Male Rat^
0.72 ± 0.08
0.30 ± 0.08
96 ± 20
22 ± 5
Rainbow Trout** Daphnia^
0.28 ±0.10 ND^
0.19 ± 0.05 ND
15.9 ± 2.2 42.0 t 5.3
0.55 ± 0.01
Blue Crab^
0-18 ± 0.08
-
5.2 ± 4.8
0.016 t 0.008
a/ Activities expressed as in Table 22.
^ b7 Ahmad and Black, 1977.
£/ Kato, 1979.
d/ This study.
e/ James e_t a]_. , 1979.
f/ Not detectable.
-------
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94
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APPEIIUIX A
SUMMARIES OF CUHUITIOUS Mti WATER CHARACTERISTICS FUR TOXICITV TtSTS
TAULE A-l. SUMMARY OF TEST CONDITIONS AND TEST WATER CHARACTERISTICS FOR MEASURED
TOXICITV TESTS WITH FISH, SELECTED INVERTEURATLS AND ALGAE
Test Compound
llexa tl> luruc thane
llexathloroe thane
us
cr.
Tetrachloroethylcne
Tetrachloroethylene/DMF
Tetrachloroethylene
1,2-Dichlorobcnzene
1 ,2-Dichlorubenzene
1 ,4-Dlchlorubenzene
1 ,4-Dichlorobenzene
1 ,2,4-Trichlorobenzens
Test Test
Organism Duration
Tanytarsus dlsslnllis 48 hr
SalniQ galrdnerl 192 hr
Salnio gairdnerl 96 hr
Saliiio galrdnerl 96 hr
Tanytarsus disslmilis 48 hr
Salkn gatrdiieri 144 hr
Tanytarsus disslmllls 48 hr
Saliiio yalrdneri 96 hr
Tanytarsus dlsslmllls 48 hr
Salino ualrdneri 192 hr
Water
Supply
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
Superior
Superior
Superior
Superior
Superior
Superior
Superior
Superior
Superior
Superior
% Dissolved
Temperature C Oxygen
x t s.d. x i s.d.
(range) (range)
11
(10
11
(11
12
12
(11
11
(11
- -
12
(11
. 20
.6 t 0.4
.4 - 13.4)
n-96
93.4 t 3.2
(88.7 - 98.7)
n=36
69.2 t 6.5
(59.0 - 77.1)
n=13
81.6 t 5.2
(73.9 - 92.4)
80.7 t 5.4
(71.2 - 86.2)
n-10
97.0 t 1.2
(95.3 - 99.8)
n--22
82.4 t 7.4
(73.8 - 99.4)
87.2 i 3.3
(80.9 - 90.9)
84.0 t 7.6
(73.7 - 100.3)
n=10
97.5 t 2.1
(95.3 - 99.8)
n=22
86.4 t 7.6
(66.5 - 95.3)
n=16
Hardness*/
J i s.d.
46.7 t 0.9
n-5
42.5 t 1.0
44.1 t 0.5
46.2 t 0.8
46.4 t 1.0
n=5
47.3 t 0.1
n=7
47.0 t 2.0
45.3 i 0.8
45.9 ± 1.6
47.7 t 1.3
Alkalinity*/
x t s.d.
44.0 t 1-9
n=6
46.6 i 0.5
n=4
46.9 t 0.6
n=4
46.5 t 0.5
n-3
43.0 t 1.0
n»5
47.7 t 1.1
n>7
46.0 t 3.0
n--6
44.6 1 1.7
44.8 t 2.4
53.1 t 10.1
o-9
Acidity*/
x t s.d.
2.9 t 0.7
2.4 t 0.1
2.1 t 0.1
n=4
2.0 t 0.1
2.8 t 0.3
n=5
2.0 t 0.1
2.3 t 0.3
n-6
4.1 t 1.0
2.7 i 0.5
2.6 t D.7
n=8
PH.
x t s.d.
7.6 i 0.
n=6
7.2 t 0.
n=4
7.1 t 0.
n-4
7.2 t 0.
n*3
7.5 i 0.
n=5
7.5 t 0.
n=7
7.6 t 0.
n-6
6.8 t 0.
n-7
7.6 t 0.
7.4 t 0.
0
0
0
0
0
1
0
I
0
1
-------
TAiiLE A-l Cont. SUMMARY OF TtST CONDITIONS AMU TEST WATER CHARACTERISTICS FUR MEASURED
TOXICITY TESTS WITH FISH. SELECTED INVERTEBRATES AND ALGAE
X Dissolved
Test Compound
Pentaclilorobunzene
HcxacMorobenzene
llexachlorobenzene
llexaclilorobenzene
llbxachlorobutadiene
llexacblorobutadfene
Ptntathlorophenol
Fentachloruplicnol
lluptacblor
Toxapbene
Test Test
Organism Duration
Saliuo gatrdnerl
Salnio gairdnerl
Lepomis inacrochtrus
Tanytarsus dlssinlHs
Salnio gairdnerl
lepoiiils niacraclitrus
Gamnarus pseudolltnnaeus
Tanytarsus disslniUs
Selenastruin
caprlcornutuni
Selenastrum
capricormitum
144 hr
96 hr
96 hr
48 hr
168 hr
192 hr
96 hr
48 hr
L
L
L
L
L
L
L
L
1
Water
Supply
. Superior
. Superior
. Superior
. Superior
. Superior
. Superior
. Superior
. Superior
1 CIHpCI 0 bUP C I,
x t s.d.
(range)
12.7 t 0.8
(12.2 - 13.5)
n-79
11.2 t 0.8
(10.5 - 12.8)
n=30
23.3 t 0.5
(21.1 - 23.8)
n-36
23.9 t 1.2
(22.5 - 25.6)
n-8
11.9 i 0.4
(10.9 - 13.0)
n-87
25.2 t 0.2
(24.7 - 25.8)
n=102
17-1 i 0.5
(16.3 - 18.0)
n*56
23.8 t 0.3
(23.5 - 24.0)
n=3
UAJTlJk.PI
x t s.d.
(range)
03.4 t 7.8
(67.7 - 96.0)
n=l4
n.d.
89.9 t 11.3
(72.8 - 100)
n-15
85.9 t 0.0
(85.9 - 85.9)
n=4
81.2 t 0.7
(80.4 - 82.1)
n=7
93.0 t 3.1
(89.2 - 99.9)
n=17
95.2 i 3.7
(84.9 - 98.9)
n=ll
79.0 i 4.6
(68.7 - 84.3)
n-12
Hardness*7'
x t s.d.
46.2 t 1.8
n=4
44.3 t 2.1
n-3
45.4 t 1.5
n=3
40.7 i 2.8
n'4
45.9 i 0.6
n=7
41.7 t 2.7
n=12
47.5 i 2.2
nz4
44.5k/
n-1
Alkalinity*/
x t s.d.
45.4 t 1.9
nM
49.0 t 1.0
n=3
42.5 t 0.7
n-3
43.2 ! 1.3
n=4
45.7 i 0.8
n-7
46.5 t 6.0
n=12
62.8 i 8.1
n=4
55.0^
n=l
Acidity*/
x i s.d.
3.6 i 0.4
n-4
0.3 i 0.1
n-3
1.9 t 1.1
n=3
1.5 1 0.6
n=4
2.0 t 0.1
n=7
2.2 t 0.5
n-12
4.8 t 2.0
n-4
0.5b/
n=l
. P"
x t s.d
7.4 t 0
n-4
7.5 i 0
n-3
7.7 t 0
n=3
7.8 J 0
n=4
7.5 i 0
n=7
7.6 t 0
n-12
7.2 t 0
n=4
7.9 t 0
n-12
.0
.4
.1
.0
.0
.2
.2
.2^
reconstituted
96 hr
deionized
^ 24
n.d.
n.d.
n.d.
n.d.
n.d.
reconstituted
96 hr
deionized
-v. 24
n.d.
n.d.
n.d.
n.d.
n.d.
-------
Test Compound
Arsenic
Arsenic
Arsenic
Arsenic'3 (test 1)
Arsunic<3 (test 2)
Arsenic
Chromium'6
Chromium
Lead*2
Lead'2
TABLE A-l tont. SUMHARY
TOXICITV
Test Test
Organise Duration
Plmephales pronielas 96 hr
Piniepliales promelas 31 days
Jordanella flortdae 96 hr
Jordanella florldae 38 days
Jordanella florldae 31 days
Ganinariis pseudo- 96 hr
lininaeus
Ga arus pseudo- 96 hr
H milieus
Tanytarsus dlsslmlUs 48 hr
Ganmarus pseudo- 96 hr
liinaeus
Tanytarsus dlsslmllls 48 hr
UF TEST CONDITIONS ANU TEST WATER CHARACTERISTICS FOR MEASURED
TESTS MITII FISH. SELECTED INVERTEBRATES ANU ALGAE
I Dissolved
- Temperature C Oxygen
Mater J l s.d. x 1 s.d. Hardness3' Alkalinity3'
Supply (range) (range) x t s.d. x t s.d.
City of
Superior
City of
Superior
City of
Superior
L. Superior
City of
Superior
L, Superior
L. Superior
L. Superior
L. Superior
L. Superior
24.2 1 0.6 83.8 t 2.5
(23.0 - 25.3) (77.8 - 88.8)
n-49 n=9
23.0 t 2.7 79.6 i 7.4
(13.5 - 26.7) (63.7 - 93.3)
n*360 n-108
25.8 t 0.7 86.9 1 2.5
(24.1 - 27.2) (82.0 - 89.2)
n=50 ns9
24.8 t 1.3 83.6 1 5.9
(21.8 - 27.5) (70.2 - 92.5)
n*168 n=96
24.4 1 2.8 85.8 1 4.7
(13.4 - 28.6) (73.1 - 95.8)
n=360 n'108
18.4 t 0.9 99.3 1 1.3
(17.3 - 19.5) (97.3 - 100.2)
n-54 n=6
17.4 1 0.5 96.7 1 0.7
(16.5 - 18.0) (95.1 - 97.4)
n=54 r>--\0
20.4 1 0.1 86.6 1 1.4
(20.3 - 20.5) (84.3 - 89.8)
n*3 n*10
17.6 1 0.4 94.7 1 2.1
(17.0 - 18.5) (91.7 - 96.8)
n-52 n-11
18.6 t 0.6 78.9 1 4.2
(17.6 - 19.5) (70.0 - 83.9)
n~36 n»10
49.9 1 0.7 37.2 1 0.8
n*5 nc5
49.2 i 1.4 38.0 t 2.2
n=8 n*8
49.9 1 0.7 37.2 1 0.8
n=5 n=5
47.0 l 0.0 n.d.
n'4
49.1 1 1.4 38.1 1 2.1
n=8 n=8
46.3 1 0.5 43.4 1 1.5
n=4 n=4
47.8 1 0.6 53.8 t 1.0
n-4 n=4
47.0^ 187. 5^
n=l n-1
48.3 1 0.9 40.8 1 4.3
n=4 n«4
n.d. 12.1^
n=l
Acidity47 pH
i 1 s.d. x 1 s.d.
3.4 1 O.I 7.2 1 0.1
n-5 n=8
3.3 1 0.3 7.2 t 0.1
n=8 n--2Q
3.4 t 0.1 7.2 1 0.1
n=5 n=8
4.2 1 0.3 7.4 1 0.0
r
n-4 n=4
3.3 t 0.2 7.2 1 0.1
n-8 n-20
3.0 1 0.0 7.7 1 0.2
n=4 n=4
6.9 1 1.2 7.6 1 0.1
n=4 n-4
2.5^ 7.5 1 0.2
n=l n-4
1.4 1 0.2 6.5 1 0.2
nM n-4
65. 0^ 4.9 - 6.9^
n-1 n=12
-------
TABLE A-l Cunt. SUMMARY OF TEST CONDITIONS AND TtST UATER CHARACTERISTICS FOR MEASURED
TOX1CITY TESTS WITH FISH. SELECTED INVERTEUKATES AND ALGAE
or « Dissolved
00
Test Compound
Mercury
Mercury
Silver'1
Silver*1
Silver'1
Silver'1
Selenium
Cyanide
Test Test
Organism Duration
Pliiepliales
Piniepliales
Piinephales
Jordanella
pronielas 96 hr
pronielas 35 days
promelas 96 hr
florldae 96 hr
Gamnarus pseudo- 96 hr
Hinnaeus
Tanytarsus
Tanytarsus
Tanytarsus
dlsslmilis 48 hr
dissiiiillls 48 hr
dlssinillls 48 hr
Hater
Supply
L. Superior
L. Superior
City of
Superior
L. Superior
L. Superior
L. Superior
L. Superior
L. Superior
1 WMIf *> * V Vt»l •* *
x t s.d.
(range)
25.
(24.
25.
(23.
23.
(18.
24.
(23.
20.
(19.
19.
(19-
19.
(18.
7 t 0.6
7 - 27.3)
n=51
0 t 0.6
7 - 26.3)
n=>132
4 t 2.6
5 - 26.1)
n»36
7 t 0.5
9 - 25.8)
n=51
0 t 0.5
1 - 20.7)
n-44
8 t 0.3
5 - 20.0)
n=3
0 t 0.8
1 - 19.8)
n«4
"• 20
-Oxygen
(range)
93.
(88.
81.
(65.
85.
(79.
92.
(82.
95.
(91.
94.
(88.
97.
(93.
99.
(97.
0 t 3.6
5 - 97.9)
r,= 10
0 + 9.6
1 - 91.2)
n»17
5 t 3.5
4 - 89.4)
n-8
4 t 5.9
7 - 98.8)
n-10
0 t 2.6
2 • 98.5)
n=JO
5 t 4.0
7 - 97.6)
n-4
4 t 2.3
9 - 100.3)
n-10
0 t 1.2
0 • 99.8)
n-5
Hardness^
x t s.d.
42.8 t 0.0
n-3
45.6 i 3.2
n=2
46.0 t 1.4
ns2
44.5 t 4.0
n=4
48.1 t 1.4
Alkalinity^
x t s.d.
41.2 t 0.2
n-3
41.3 t 0.2
n=2
36.2 t 3.9
n=2
43.5 t 1.7
n=4
45.2 t 1.3
Acidity^ pit
i i s.d. x i
2.4 t 0.0 7.6 t
n=3 n-
2.6 t 0.3 7.5 t
n=2 n=
3.5 i 0.1 7.2 t
n-2 n-
2.5 t 0.0 7.8 t
n=4 n-
1.1 i 0.2 7.5 i
s.d.
0.0
3
0.1
2
0.1
7
0.0
4
0.2
n-4 n-4 n-4 n-4
47.9*
n=l
48. 0^
n=l
46.8 t 1.6
n-6
42.0^
n-1
n.d.
46 - 82^
n=6
1.5^ 7.6 t
n-1 n-
44. <$ 3.2 -
n^l n=
0 - 2.0^ 7.6 -
n=6 n-
0.1
12
7.6*
10
9.2^
6
a/ Values expressed as ntg-L of
I)/ Analysis performed on single pooled sample from selected exposure concentrations.
cy Values increased with Increased toxicant concentration.
d/ Values decreased with increased toxicant concentration.
-------
TABLE A-2, SUMMARY OF CONDITIONS AND WATER CHARACTERISTICS FOR MEASURED
TOXICITY TESTS WITH DAPHNIA MAGNA IN LAKE SUPERIOR WATER
VO
Compound
1,2-Dichloroethane
1 ,2-D1chloroethane
1 ,1 ,2-Trichloroethane
1 ,1 ,2-Tr1chloroethane
1 ,1 ,2,2-Tetrachloroethane
1 ,1 ,2,2-Tetrachloroethane
(test 1)
1 ,1 ,2,2-Tetrachloroethane
(test 2)
Pentachloroethane
Hexachloroe thane
Test
Duration
48 hr
28 day
48 hr
28 day
48 hr
28 day
28 day
48 hr
48 hr
Temperature C
x t s,d.
(Range)
20 ± 1
20 ± 1
20 t 1
20 ± 1
20 ± 1
20 ± 1
20 ± 1
20 ± 1
20 ± 1
% Dissolved
Oxygen In
New Solutions
x ± s.d.
(Range)
Fed or Unfed
n.d.
97.3 ± 3.4
(89,8 - 102.2)
n=32
90.1 ± 1.4
(88.9 - 91.8)
n=3
94.1 ± 6.0
(79.5 - 102.5)
n=30
91.2 ± 0.9
(90.2 - 92.2)
n=4
n.d.
92.4 ± 4.4
(87.7 - 97.2)
n=4
93.4 ± 1.7
(92.0 - 95.4)
n=3
87.1 ± 0.8
(86.1 - 87.9)
% Dissolved Oxygen
1n Old
Solutions
x ± s.d.
(Range)
Fed
n.d.
73.8 t 9.6
(55.5 - 96.3)
n=57
74.2 ± 4.9
(67.2 - 78.5)
n=4 .
69.1 ± 10.2
(39.8 - 86.2)
n=63
74.7 ± 4.3
(69.4 - 77.4)
n=3
33.5 ± 18.8
( 5.5 - 72.1)
n=136
64.4 ± 16.8
(22.6 - 79.2)
n=29
82.2 t 4.9
(75.0 - 86.1)
n=4
69.0 ± 7,0
(59.0 - 74.5)
Unfed
n.d.
_
109,1 ± 4.3
(104.1 - 114.2)
n=4
-
92.6 ± 1.3
(91.8 - 94.1)
n=3
_
94.8 t 1.4
(93.0 - 96,3)
n=4
87.5 t 1.6
(80.2 - 89-4)
n=4
n=4
n=4
-------
TABLE A-2 Cont. SUMMARY OF CONDITIONS AND WATER CHARACTERISTICS FOR MEASURED
TOXICITY TESTS WITH DAPHNIA MAGNA IN LAKE SUPERIOR WATER
o
o
Compound
Tetrachloroethylene
Tetrachloroethylene
1 ,3-Dlchlorobenzene
1 ,3-D1chlorobenzene
i
i
1
1 ,2,4-Trichlorobenzene
1 ,2,4-Trlchlorobenzene
Dl-r^-butylphthalate
Chlordane
Arsenic
Test
Duration
48 hr
28 day
48 hr
28 day
48 hr
28 day
48 hr
48 hr
28 day
Temperature C
x t s.d.
(Range)
20 ± 1
20 ± 1
20 ± 1
20 ± 1
20 ± 1
20 ± 1
20-9 ± 0.1
(20.8 - 21.0)
n=3
-v-21
21.5 ± 3.0
% Dissolved
Oxygen In
New Solutions
x ± s.d,
(Range)
Fed or Unfed
n.d.
98.1 ± 8.6
(80.4 - 114.7)
n=39
n.d.
92.6 ± 4.4
(84.4 - 99.3)
n=27
n.d.
90-6 ± 3.4
(81.5 - 94.7)
n=19
n.d.
n.d.
n.d.
% Dissolved Oxygen
in Old Solutions
x ± s.d.
(Range)
Fed
45.2 ± 7.1
(37.5 - 53.3)
n=4
59.6 ± 13.7
(26.2 - 74.8)
n=50
83.7 ± 1.8
(82.5 - 85.7)
n=3
71-2 ± 9.2
(52.4 - 83.4)
n=25
n.d.
72.0 ± 9.5
(48.8 - 91.8)
-
_
86.9 t 9.9
Unfed
98.6 t 6.2
(89.4 - 102.5)
n=4
_
94.9 ± 0.6
(94.4 - 95.4)
n=3
_
n.d.
93.3 ± 1.5
(90.5 - 95.0)
n=9
88. 2b
n=l
(15 - 26)
(70.1 - 101.8)
n=71
-------
TABLE A-2 Cont. SUMMARY OF CONDITIONS AND WATER CHARACTERISTICS FOR MEASURED
TOXICITY TESTS WITH DAPHNIA MAGNA IN LAKE SUPERIOR WATER
Compound
Arsenic ^
Nickel*2
Test
Duration
48 hr^
48 hr
Temperature C
x ± s.d.
(Range)
14,8 ± 0.8
(13.4 - 16.0)
n=140
•x-20
% Dissolved
Oxygen in
New Solutions
x ± s.d.
(Range)
Fed or Unfed
91.6 ± 0.0
(91.6 - 91.6)
n=8
n.d.
%
Dissolved Oxygen
in Old Solutions
Fed
90.4 ±
(90.4 -
n=4
_
x t s.d.
(Range)
Unfed
0.0 91.0 ± 0.3
90.4) (90.7 - 91.4)
n=4
87.7 t 1.6
(85.8 - 89.0
n=4
-------
TABLE A-2 Cont. SUMMARY OF CONDITIONS AND WATER CHARACTERISTICS FOR MEASURED
TOXICITY TESTS WITH DAPHNIA MAGNA IN LAKE SUPERIOR WATER
Test
Compound Duration
1 ,2-Dichloroethane
1 ,2-Dichloroethane
1 ,1 ,2-Trlchloroethane
1 ,1 ,2-Trlchloroethane
1,1,2,2-Tetrachloro-
ethane
1,1,2,2-Tetrachloro-
ethane (test 1)
1 ,1 ,2,2-Tetrachloro-
e thane (test 2)
Pentachloroethane
Hexachloroe thane
Tetrachloroethylene
Tetrachloroethylene
1 ,3-Dichlorobenzene
1 ,3-Dichlorobenzene
1 ,2,4-Trichlorobenzene
48 hr
28 day
48 hr
28 day
48 hr
28 day
28 day
48 hr
48 hr
48 hr
28 day
48 hr
28 day
48 hr
Hardness^
44.5
44.0 t 0.0
n=2
43.0
44.4 t 0.9
n=4
45.0
44.1 ± 0.9
n=4
45.3 ± 0.3
n=3
44.5
45.0
44.0
44.5 t 0.7
n=2
43.5
45.5 ± 1.8
n=3
45.0
pH of New
Solution
Alkalinity37 Acidity*7 x ± s.d.
x + s.d. x t s.d. Fed or Unfed
37.0
41.5 ± 0.7
n=2
37.0
39.9 ± 3.5
n=4
42.0
41.6 t 1.2
n=4
43.3 ± 1.6
n=3
42.0
42.0
42.0
42.0 t 0.0
n=2
41.0
42.2 ± 0.8
n=3
44.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7.5 ± 0.1
n=33
n.d.
7.6 t 0.1
n=27
7.1 ± 0.2
n=4
n.d.
7.9
n=l
7.2 ± 0.1
n=3
7.4 ± 0.1
n=4
n.d.
7.4 ± 0.2'
n=32
n.d.
7.4 ± 0.2
n=7
n.d.
pH of Old
Solution
x t s.d.
Fed Unfed
n.d.
7.1 ± 0.1
n=32
7.4 ± 0.1
n=4
7.0 ± 0.1
n=59
7.0 ± 0.1
n=3
n.d.
7.6 ± 0.1
n=2
7.0 ± 0.0
n=4
7.0 ± 0.1
n=4
n.d.
7.0 ± 0.2
n=34
n.d.
6.9 t 0.1
n=8
n.d.
n.d.
-
7.7 ± 0.1
n=4
-
7.3 ± 0.3
n=3
-
-
7.2 ± 0.2
n=4
7.6 ± 0.1
n=4
n.d.
n.d.
n.d.
-
n.d.
-------
TABLE A-2 Cont. SUMMARY OF CONDITIONS AND WATER CHARACTERISTICS FOR MEASURED
TOXICITY TESTS WITH DAPHNIA MAGNA IN LAKE SUPERIOR WATER
Compound
1 ,2 ,4-Tr1chloroben-
zene
Di-n_-butylphthalate
Chlordane
Arsenic
-^
c*> +3 Cy
Arsenic
Nickel*2
Test
Duration
28 day
48 hr
48 hr
28 day
48 hr
48 hr
Hardness^
44.8 ± 0.6
n=4
45.4^
n=l
42.2^
n=l
47.2 ± 0.4
n=4
48,7 ±2.0
n=16
51.1 ± 1.0
n=2
pH of New
Solution
Alkalinity*7 Acidity*' x ± s.d.
x ± s.d. x ± s.d. Fed or Unfed
41.9 ± 0-9
n=4
.,. cb/
46-5
n=l
58. 0^
n3!
45.5 ± 1.3
n=4
46.8 ± 2.4
n=16
53.4 ± 2.8
n=4
n.d. 7.3 ± 0.1
n=7
5.1 n.d.
n=l
2,5^ n,df
n=l
n.d,
n.d. 8.1 ± 0.3
n=8
n.d. n.d.
pH of Old
Solution
x ± s.d.
Fed Unfed
6.6 ± 0.1
n=8
-
_
7.4 ± 0.2
n=73
8.1 ± 0.4
n=4
_
_
7.1^
7.5^
n=l
_
7.9 ± 0.2
n=4
8.8 ± 0.1
n=4
&/ Values expressed as mg-L of CaCO-.
b/ Analysis performed on single pooled sample from selected exposure concentrations
c/ Test conducted in dechlorinated city water from the City of Superior. Wisconsin.
-------
ArrtNinx b
TOXICITY TEST ClltMICAL CONCENTRATIONS
TABLE B-l. MEASURED CONCENTRATIONS OF TOXICANTS IN EXPOSURE WATER CHAMBERS (M»*n ± S.D.)
Chuiilcal Organ! tin
1 ,2-Ulcl>lori>elliunu l)t)|>l>nla niagnq
1,2-Ulclilorocthan* " "
l,2-Ulclili>rei;tli*ne " "
l,l,2-Trlcl>li>r»ctlmne " "
O
** 1,1,2-TrlcltlorvetliBne " "
1,1,2-TricliloroetliiMte " "
l.l,2,2-Tutr»cliloroi:tl>uiie " "
1,1,2,2-lttruL-lilorueLltinc " "
l,i,2,2-TL-tr«cl>lor»elli»ne " "
Pt:nl*cliloroetl>iuiii " "
PentuclilurueLliiiii* " "
lluxiiclilurvL-tluim " "
Ik'HULl.lffrOUtlliKL" " "
lluM«rl)l»roi;lli»nc Saluio cilnlnerl
llDucliloruulliune ' Tunyturvu*
dlkvlMllU
Duration
48 h (Unfed)
48 h (Fed)
28 d
48 h (Unfed)
48 b (Fed)
28 d
48 h (Unfed)
48 h (Fed)
28 d
48 h (Unfed)
48 h (Fed)
48 l> (Unfed)
48 1. (Fed)
192 li
72 h
Control
0.0 * 0.0
(n-2)
0.0 t 0.0
(n-2)
0.0 ± 0.0
(n-22)
0.0 t 0.0
(•-2)
0.0 t 0.0
(n-2)
0.0 t 0.0
(n-34)
0,0 t 0.0
(n-2)
0.0 t 0.0
(n-2)
0.0 t 0.0
(n-48)
0.0 * 0.0
(n-2)
0.0 * 0.0
(n-2)
0.0 i 0.0
0>-2)
0.0 t 0.0
(n-2)
0.00 * 0.00
(n-10)
0.004 t 0.005
(n-4)
Concentration* (»g-L )
A B
70.4 t 2.8
(n-2)
72.4
(n-1)
10.6 t 0.8
(n-16)
24.3 t 1.8
(n-2)
23 t 3.S
(n-2)
1.72 t 0.16
(n-21)
16.4 * 1.3
(n-2)
16.3 t 1.4
(n-2)
0.41'J ± 0.036
(n-2/,)
1.7 * 1.7
(-2)
2.0 t 1.3
(n-2)
0.805 t 0.14
(n-2)
0.631 t 0.39
(n-2)
0.35 * 0,06
(n-7)
0.248 * 0,038
(n-8)
99.2 t 5.3
(n-2)
94.8 t 11.6
(n-2)
20.7 t 1.7
(•)-15)
40.1 t 2.6
(n-2)
40.1 t 2.5
(n-2)
3.40 t 0.29
(n-20)
22.8
(n-1)
23.1
(n-1)
0.859 t 0.085
(n-24)
3.4 t 2.8
(n-2)
3.83 t 2.1
(n-2)
1.08 t 0.16
(n-2)
0.844 ± 0.49
(n-2)
0.64 1 0.10
(n-7)
0.619 t 0.080
(n-8)
C
137 + 8.5
(n-2)
143
(n-1)
41.6 i 2.4
(n-14)
57.5 t 2.9
(n-2)
56.6 t 4.2
(n-2)
6.35 t 0.52
(n-20)
33.3 t 3.7
(n-2)
34.5 t 2.1
(n-2)
1.71 t 0.17
(n-24)
5.2 t 3.7
(n-2)
5.91 t 2.7
(n-2)
1.54 t O.OB
(n-2)
1.12 t 0.67
(n-2)
0.92 t 0.05
(n-7)
1.26 t 0.14
(n-8)
-------
TAUl.t U-l Cool. MEASUKED CONCENTRATIONS OF TOXICANTS IH EXH>bUHE WATCH CHAMBERS (Heun ± S.U.)
LliL-u»Uul Oreuni» Duration
1,2-Ultliloroetliune Dubinin mo BOB 48 li (Unfvd)
1 ,2-Uitliloroelliim* " " 40 It (Fed)
l,2-Ulclilt>rui;i.|iui)u " " 28 d
1,1,2-lrUl.luroeil.Jni; " " 48 h (Unfed)
_, 1,1 ,2-Trlcl.li>r»eil.»»i: " " 48 h (Fed)
O
(Ji
1 ,1 ,2-lrU!.lL.r»L-ll.y.n» " " 28 d
l,l,2,2-Tcirucl>l»ruetlian>! " " 48 » (Unfed)
1 ,1,2,2-iLti Jcl.loroelliy.ie " " 48 It (Fed)
1,1,2,2-TBtrnchloroell.om: " " 28 d
Puntutlilurottliune " " 48 h (Unfed)
t'uiiliiclilvruclliune " " 40 li (Fed)
liuKaelili>riM.-ili>ni! " " 48 h (Unfed)
lluKuclili>ri>L-iliunc " " 48 li (Fed)
HLXUI liluiLiLil.uoi; Scliuo galrdnerl 192 li
IlL'KULliluruL-lhunu Tinytarvu* 72 lir
Jlbiululll*
U
1B8 t 9.2
(»-2)
17i * 27.6
(n-2)
71.7 ± 4.8
(n-16)
113 t 7.1
(n-2)
107 t 15.2
13.2 ± 1.7
(n-18)
46. !> i 4.2
(n-2)
46. B ± 4.5
(»-2)
3.43 t 0.39
(n-24)
9.2 t 5.9
(n-2)
9.53 ± 5.5
(n-2)
2.55 t 0.35
(n-2)
2.04 t I.Ob
(n-2)
1.58 ± 0.19
(,,-6)
2.75 i 0.48
(n-8)
Concent rut iuny (•£•!•
E
258 i 17,7
(n-2)
244 t 36.8
(••-2)
94.4 ± 5.5
(n-13)
159 ± 4.9
(n-2)
149 t 18/4
(n-2)
26.0 t 2.2
(n-19)
64.7 i 4.6
(n-2)
63.8 t 5.9
(n-2)
6.85 t 0.90
(n-23)
17 t 8.2
(n-2)
16.4 t 9.1
(n-2)
4.65 t 0.42
(n-2)
3.81 i 1.62
(n-2)
1.83 t 0.35
(n-3)
6.16 ± 0.54
(n-8)
F
405 t 43.8
(n-2)
390 t 65
(n-2)
137 ± 9
(n=17)
258 ± 9.1
(n-2)
241 ±32.5
(n-2)
41.8 t 3.0
(n-19)
94.9 t B.7
(n-2)
93.1 ± 11.1
(n-2)
14.4 t 1.4
(n-8)
30 t 10
(n-2)
28.5 t 12.4
(n-2)
5.44 t 0.63
(n=2)
3.87 ± 2.84
(n-2)
-------
TAUIt B-l Cont. MEASUKKO CONCENTRATIONS OF TOXICANTS IN EXl'ObUKt WAI EH CIIAHUEUS (Me«n t S.U.)
Clitmlcul UrganluB
Tutrucblorouiliylenu l)j)ilint« magn»
Tuir»clileroutbylene " "
I'uirtfcliloruetliylene " "
TL-I raclilaroutliylen* Tunyt*r>u«
divninlll*
Tetrtfdilaroetliylenc Sulmo e*lrdneri
O
O> Telrocl.luroulhyleiic/UHP " "
1,2-Ulclilorobcnzvnt; " "
1 ,2-Dlcblurobci>:cena Tvny Iffunt
dliulBlllw
1,3-Dlcblorobunzcne l)j|>lintp magna
1,3-Ulcblnrobenzene " "
1,-l-Ulthlurobcnitnc " "
l,2,4-Trlclilnri>bcnzt:>iii " "
l,2,4-Trlchlurobi:nzeni: " "
1,2,4-Tricl.lurDbenzeiu: " "
1,2,4-irlclilorabenzenc Salino galrdncrl
Duration
48 li (Unfed)
48 l> (Fed)
28 d
48 h
96 h
96 h
96 h
48 h
48 1. (Unfed)
48 h (Fed)
28 d
48 h (Fed)
48 b (Unfed)
28 d
192 li
Control
0.0 t 0.0
(n-2)
0.0 i 0.0
(n-2)
0.0009 t 0.0026
(n-20)
0.00 * 0.00
(n-3)
0.00 t 0.00
(n-5)
0.00 t O.OO/
0.0 ± 0.0
(n-3/5)
0.00 t 0.00
(n-10)
0.00 t 0.00
(n-3)
0.0 t 0.0
(n-2)
0.0 t 0.0
(n-2)
0.001 t 0.004
(»-37)
0.001 t 0.001
(n-2)
0.001 t 0.001
(n-2)
0.000 1 0.001
(n-31)
0.00 i 0.00
(n-9)
Concentration!!
A
2.S6 t 0.6U
(n-2)
3.04
(•'-!)
0.0759 t 0.036
(n-10)
4.38 t 0.63
(n-6)
2.41 t 0.22
(n-7)
2.23 t 0.46/
75.8 t 13.9
(•••7/6)
0.7S t 0.04
(n-7)
2.02 1 0.49
(n-6)
1.27 t O.Oi
(n-2)
1.30
(n-1)
0.044 t 0.012
(n-21)
0.293 i 0.031
(n-2)
0.276 t 0.055
(n-2)
0.018 t 0.003
(n"20)
0.44 t 0.03
(n-12)
(•g-L'1)
B
3.61 t 1-33
(n-2)
4.55
(n-1)
0.159 t 0.085
(n-12)
9.91 t 0.48
(n-6)
3.69 t 0.20
(n-7) ,
3.53 t O.B4/
122 1 12
(n-7/6)
1.29 i 0.07
(n-7)
5.53 t 1.55
(n-6)
2.14 t 0.04
(n-2)
2.11
(n=l)
0.102 t 0.023
(n-21)
0.45 * 0.017
(••-2)
0.428 t 0.049
(n-2)
0.039 t 0.005
(n-19)
0.57 t 0.05
(n-12)
C
7.00 t 1-61
(n-2)
5.88 * 3.20
(n-2)
0.254 t 0.094
(n-8)
21.5 ± 1.5
(n-6)
6.39 t 0.65
(n-6)
5.95 t 1.41/
220 ± 11
(n-7/6)
2.05 t 0.17
(n=7)
13.2 t 2.2
(n-6)
3-28 + 0.21
(n=2)
2.93 t 0.70
(n-2)
0.182 t 0.039
(n-20)
0.850 * 0.056
(n-2)
0.762 ± 0.18
(n-2)
0.079 t 0.011
(n-20)
1.09 * 0.05
(n-12)
-------
TAULK B-l Coi.t. HKASUKKU CONCtNTHATIONS OF TOXICANTS IN KXHUbUKE WATtK CIlAJIUtHS (Mc-.n t S.U.)
I'liciuicul OrganlvB
TL'triitliluroiitl.ylL-ne Uupl.nla magn»
TelruL-l.luructhylene " "
Tctr.cl.Ur.etl.ylcn.
TelruL'hloruetl.ylene Tunytaruuu
dlavlBlllu
TelrJtlilarautliylene Sulmo gwlrdnerl
T»lr.cl.lVr.etl.yUnv/»ir
1,2-UU-lilurobenzL'nii " "
1 ,2-OUIilarobupizunu Tuipyt >run*
dlvilBlll*
1 ,3-l'lf blurobenze.ie Uapl.nln nmena
l,J-lUcl,lon>bLnzene
1 , J-DiL'ltlurobtfHzeiiu " "
1 2 4~Tr icliitttrubtfiizciitf " "
1 r 2 ,A"'i'i" tc It] »ri>bttnii;piti " "
1,2,4-Trlcl.lo.uben.cn,
1 ,2 ,4-Ti iililurobtn/BUL- SH!UH> K»lrdnerl
Duration
48 h (Unfed)
48 li (Fed)
28 d
48 I)
96 li
96 h
96 li
48 h
48 h (Unfed)
48 li (Fed)
28 d
48 I. (Fed)
48 li (Unfed)
28 d
192 li
Concentration* (•£•!• )
D E
1J.5 t 2.30
(n-2)
'J.61 t 4.94
(n-2)
0.505 t 0.250
(n-10)
47.2 t 6.0
11.2 t 0.2
(n-2)
11.3 1 0.9
J26 t 16
(n-3/2)
3.07 i 0.03
(n-2)
21.9 t 7-4
(n-5)
5.89 i 0.89
(n-2)
4.85 t 2.36
(n-2)
0.373 i 0.053
(n-21)
1.32 t 0,057
(n-2)
1.21 ± 0.21
(n-2)
0.162 t 0.028
(n-19)
1.70 t 0.12
(n-12)
19.1 i 2,33
(n-2)
15.6 1 7.28
(n-2)
1.11 t 0.48
101.8 * 0.8
(n-2)
17.3 t 1.0
(n-2)
16.4 t 1.5/
513 i 17
(n-3/2)
3.81 t 0.43
("-3)
45.8 i 16.1
(n-3)
10.2 t 1.52
(n-2)
9.53 i 2.50
0.689 t 0.156
(n-20)
1,68 * 0.092
(n-2)
1.61 i 0.18
0.363 ± 0.056
(n-20)
2.82 t 0.17
(n-4)
F
31.0 t 3.04
(n-2)
25.6 t 10.6
(n-2)
1.75 i 1.10
(n-6)
-
-
17.2 t 2.05
(n-2)
16.0 t 3.68
(n-2)
1.45 t 0.28
2.64 i 0.16
(n-2)
2.63 t 0.17
(n-2)
0.694 t 0.140
(n-19)
-
-------
TADLt 11-1 Com. MCASUUKU COriCUNTUAlIONS OF TOXICANTS IN bXPOSUUK WATER CIlAMlitKS (Kean ± S.U.)
««....!
l'i.iil.iLlilorubi.'0/iJiic/UMF
llu*iobeiui!Di!/l>MI''
lluvuclilorobiitudiene
l»l-n- bul yl|>lil|>j la le
u/
J/
Sal 100 giiirdneil
»' M
iiwcrnclilrui
TJI1 lar»Ui,
dl»»l»lll>
Sal 100 gatrdncrl
uacruiJlilruv
Gi>nwii)ruy
I'veudol liuiftiM.tf
Tunyiarsuu
dl w •!•! 1 !•
t>i?lcntffltruiB
capricorniiiua
(Tent 1)
Sclcnai truii
i: jpr Icornutiiii
(Tt»t 2)
C3i>ricornut uni
Duration
144 li
96 h
96 h
48 h
96 li
96 h
48 li (Unfed)
96 li
48 l>
96 li
96 li
48 li (Unfed)
96 li
Control
0.00 ± O.OO/
437 t 136
(n-14/4)
0.00014 t 0.00016/
922 i 114
(n-9/3)
0.0000 ± O.OOOO/
943 t 106
(n-10/6)
0.0000 t O.OOOO/
0
(n-4/1)
0.00 t 0.00
(n-10)
0.0000 i 0.0000
(n-12)
< 0.32 + 0.13
(n-4)
0.000 i 0.000
(n-7)
< 0.05 i 0.00
(n-4)
< 0.0008
(n-1)
< 0.0008
0.0000 t 0.0000
(n-6)
0.0
(n-1)
Cancen tru t long
A
0.06 t 0.02/
365 ± 193
0.0038 i 0.0003/
947 t 40
(n-10/3)
0.0041 t 0.0003/
878 ± 144
(n-10/6)
0.0000 t O.OOOO/
1391
(n=4/l)
0.067 t 0.01
(n=7)
0.0462 t 0.0064
(n-12)
0.54 t 0.14
0.108 t 0.004
(•-7)
11.6 t 0.6
(n-4)
0.0086
0.0136
(n-1)
0.0118 i 0.0019
(n-5)
0.25
(n-1)
B
0.12 t 0.03/
408 t 138
(n-14/4)
0.096 t 0.01
0.0846 t 0.0137
(n-12)
1.16 t 0.31
0.184 t 0.008
(n-7)
18.0 t 6.5
(n-4)
0.0176
(n-1)
0.0228
(n-1)
0.0227 ± 0.0018
(n-6)
0.48
(n-1)
C
0.28 t 0.08/
380 t 172
(n-14/4)
0.0028 t 0.0009/
1018
(n-4/1)
0.229 t 0.02
(n-7)
0.126 t 0.016
(n-12)
2.51 ± 0.40
(n-12)
0.287 t 0.023
(n-7)
46.4 ± 6.4
(n-4)
0.0284
(n=l)
0.0444
(n-1)
0.0463 ± 0.0039
(n-6)
0.85
(n-1)
-------
TABLK 11-1 Cviit. M£ASUH1U> CONCKNTBAT10N OP TOXICANTS IN EXPOSURE UATEtt CHAMBERS (He»n ± S.U-)
Concentration* (ag-L )
1'liL'iulL Jl OrgunlvB Duration U E F
CLIII jcliltfrubi.'iwciiu/DHt'' Silaio galrdnerl 144 1) 0.44 i 0.09/
402 i 146
(n-14/4)
IlL'KiicbloirubL'nzL'nL'/I'MF " " 96 It .
lU-ijcliliirubtn^une/UMK tf|H»i»l» 96 li -
Ducrncltlru*
lltKULblurubi.'iixi.iiL'/UMI'' Tuitytarvni 48 I)
dlv»lBlll»
Ik'XuchlurobiitudJenc Sulmo giilrdnurl 96 It 0.468 ± 0.08
(n-7)
IlL-KuLhlurubutadlcni; LL-|.I>«I|I> 96 li 0.228 t 0.025
uuicruciiiruu (n-12)
lll-n-bulylpli(liulutc Uj(»lnilu magi)* 48 l> (Unfed) 5.45 t U.98
(n-9)
Fi;»[ jt|p|i>r»|/lii;i>»l i;juunaru» 96 b 0.462 t 0.036
|>UL>Ulll>l luUIUl'UV (l>'7)
FL'.)i jLbluro^bL-nul TunyturtfuH 48 li 83.4 i 13-2
illwilulll* (»-4)
Ik-i'tucl.lur-' Selunmtrum 96 1) 0.0385
ca|>rlc»ntu(uB (n-1)
(Te«t 1)
llu|.i jtlilor-' SL-lenuutruB 96 b 0.0570
cu|>rlcurnuiuu (n-1)
(Test 2)
i;iiU>rdjin. Uupbiiln BUgna 48 b (Unfed) 0.0871 i 0.0076
(,,-6)
u/
li>K.i|>bLin.— bfleiiaul rum 96 li O.B7
cuprici>rnutuui (n-1)
0.71 t 0.09/
372 t 133
(n-14/4)
0.0809 1 0.0078/
929 ± 53
(n-9/3)
0.0774 t 0.0057/
862 t 147
(n-10/6)
0.0581 t 0.0169/
850
0.670 i 0.10
(n-7)
0.445 t 0.036
(n-7)
11.2 ± 2.32
(n-8)
0.761 ± 0.141
(n-4)
197 ±9.3
(n-4)
0.0444
0.107
(n-1)
0.1733 ± 0.0118
(n-6)
1.93
(n-1)
-------
TAilLK U-l Coot. MEASURED CONCeUTKATIONS OF TOXICANTS IN EXPOSURE WATER CHAMBERS (Hcuit ± S.D.)
ClicniLul
DiiiwtliylforiuaBlde
UiuiL-l liyl f urmauildii
UluictliyKuruuuulde
UluiL tliylfonujmide
Uliui/lliylfuriaaiuldi;
Mcilunol
+3
Ari»Li>lL
-»j
OrgnnliM Duration
Suliua gulrdnerl 96 li
maiToclilru*
Pluteplivlev 96 li
Tunytcruuu 48 h
dlitflBlllv
|)»l>linli> Bagm 48 li (Unfed)
Sulno tjiilrdnerl 96 It
l.L|.o»i» 96 h
•ucrDcliiruK
Pl»li;pll*lCH 96 ll
pronicl**
Jord»nell« 96 h
florldae
JorJjnclla 30 d (Teit 1)
HorJdue
Joidjnell* 30 d (Teut 2)
florldae
Pluic'pluiJes 96 li
proraclis
30 d
Cjiimuruw 96 li
l>seud»Hiui)Jcu»
l)j|>linlu maKiia 48 li (Unfed)
Control
<80 ± 27
(n-6)
0.0 t 0.0
(n-8)
3.1 ± 1.4
(••-7)
0.0 t 0.0
0.0
(n-2)
0.0 ± 0.0
(n-8)
0.0 t 0.0
(n-8)
0.0 t 0-0
(••=10)
o.oo t o.oo
(•••7)
o.oo t o.oo
(n-24)
0.00 i 0.00
o.oo t o.oo
(n-7)
0.00 t 0.00
(n-13)
0.00 t 0.00
(n-5)
< 0.002 t 0,000
(••-6)
Cone en t r • t lonu
A
1900 t 100
(n-7)
3,500 + 790
(n-8)
5,100 t 360
(n-7)
25,000 t 3.800
(n-4)
2,200
(n»2)
3.720 t 560
(n-7)
3,500 t 140
3.900 t 500
(n-10)
5.06 + 1.19
0.30 t 0.12
(n-24)
1.24 t 0.35
(n-20)
5.06 t 1-19
(n-B)
1.06 t 0.28
(n-18)
0.30 ± 0.03
(n-6)
O.U32 t 0.039
(n-6)
B
2,800 t 170
5,200 t 500
(n-8)
6.400 t 470
(n-7)
33,000 t 2,600
(n-4)
6,000 <
(n-2)
6.400 t 550
(n-7)
6,180 t 140
7,500 t 1.800
(n-10)
13.13 ± 1.11
(n-7)
0.60 t 0.24
(n-24)
2.13 ± 0.38
(n-20)
13.13 t 1.11
(n-7)
2.13 i 0.39
(n-18)
0.58 i 0.07
(n»7)
1.11 t 0.05
(n-6)
C
4,900 t 240
(n-7)
6,600 t 260
(n-B)
7,800 t 150
(n-7)
47,000 ± 1,700
(n-4)
11,000
10,700 ± 520
(n-7)
10,850 t 540
(n-7)
13,400 t 2,000
(n'lO)
25.91 ± 3.49
(n-7)
1.20 t 0.43
(n-24)
4.12 t 0.29
(n-20)
25.91 ± 3.49
(n-7)
4.30 t 0.50
(n-18)
1.34 t 0.18
(n-6)
1.83 t 0.08
(n-6)
-------
TABLE B-l Cant. HKASURKO CONCENTUATION OF TOXICANTS 1U KXTOSURK WATER CUAMBEKS (Mean t S.D.)
Ulieu icul
OliiMzl liy 1 f ti mui'lilt?
DiiMulliylforinauilde
Dimethyl furuuiuldu
l>iinut|iylf»rnuiulde
Ulnu-'lliyltoruiauiidi:
Mt.-lli.jiml
He tli Jim 1
Hcllunul
Aruenlc
Aruenlt:
Arwenlc
ArwL'itlc
Arsenic
Arsenic
1 J
Or^jnluiB Duration
Sjluiu galrdncrl 96 1)
Lupuulu nacrechlruu 96 li
Plujei'lialta graoelav 96 I)
Tanytarau* dlailBlllu 48 h
l)j|>linla umt;n» 48 h (Unfed)
Suliuo jjalrdncri 96 h
lu|>L»l» macroclilruK 96 h
Piinciilialeu |>romela» 96 h
Jord»ntlla florldae 96 h
luidjnella flurldJu 30 d (Test 1)
30 d (Tot 2)
30 d
CdDBUjruu 96 It
pjL-udol ImnatuM
l)j|'lmla auifcna 48 li (Unfed)
U
7,800 i 370
8,800 t 470
(n-8)
9,500 t 440
(n-7)
66,000 t 2,500
22,000
(n-2)
16,300 t 420
(••-7)
16,300 t 450
23,200 t 2,000
(n-10)
52.05 t 1.26
(n-3)
2.34 t 0,81
(n-24)
7-57 t 0.61
(n-20)
52.05 + 1.26
(n-3)
7.37 i 0.49
(n-18)
2.40 ± 0.24
2.55 t 0.23
(n-5)
Concentration* (*g'L )
K F
15,000 t 60
(n-3)
9,400 t 320
(••-8)
12,000 t 340
(n-7)
93,000 1 2,200
(n-4)
41,000
(n-2)
25,800 i 140
(n-2)
28,590 t 1,320
(n-2)
36,200 t 2,700
(n-4)
99.69 t 10.18
(n-3)
5.04 t 1.82
(n-24)
16.32 ± 0.85
(n-20)
99.69 t 10.18
(n-3)
16.48 t 1.03
(n-18)
5.25 ± 1.63
(n-4)
4.19 t 0.16 6.46 t 0.92
-------
TABLE B-l Cunt. MEASURED CONCBNTUATlOriS Ot TOXICANTS IN BXFOSUKfc UAfEII CIIAMBKUS (Muun ± S.O.)
Clicmlcjl
Arkeiiic
Arvcnlc
+6
Clirowluiu
+6
Uiruu>lu»
U-d*2
i*au*2
Mercury
Mercury
rtlckel*2
Silver*1
Silver*1
Silver*1
Silver*1
tie lenlum
OrgaoluB
Uaplmla •»gn»
i» »»
'iJinlaaru*
pyeudullnuneuK
Taiiytamui)
dlvulBlllv
CuMDuruw
puendul lunaeuii
Tanytaruuw
dluvlBllU
Plv>e|>liulc>
uroi»L'l»s
it ii
Uaiilmla niaena
Plujujilialeti
proiiel»»
Jordanell*
flvrldae
Tmiytarnu*
dUtiulll*
Caiiiniaru*
l>»cudol IBUHJUU*
Tanylursui
dtM«I»lll»
Duration
48 Ii (Fed)
2U d
96 Ii
4tt 1.
96 It
48 I.
96 h
35 d
48 Ii (Unfed)
96 h
96 Ii
48 h
96 h
48 Ii
Cancrul
<0.002 t 0.000
(n-4)
< 0.002 t 0.001
(pi-12)
0.0008 t 0.0008
0-5)
1.00 t 0.00
(n-4)
0.0018 t 0.0010
(n-4)
0.0 t 0.0
(n-2)
o.oooo t o.oooo
(n-7)
0.00001 t 0.00001
(n-22)
0.030 t 0.041
(••-2)
0.00013 t 0.00017
(n-5)
0.00013 t 0.00017
(»-5)
0.000 i 0.000
(n-6)
0.00017 t o.oaoos
(n-6)
0.0 + 0.0
(n-2)
Concent rut Ion* (Bg-L"1)
A It
1.04 t 0.11
(n-6)
0.073 t 0.006
0>-12>
0.0290 i 0.0028
(•«-7)
26.0 t 0.4
(i,-4)
0.047 t 0.009
(n-6)
8.7 ± 5.9
(n-4)
0.0630 t 0.0086
(n-7)
0.00023 t 0.00003
(n-22)
0.249 ± 0.031
(n-4)
0.003J4 ± 0.00102
(n-7)
0.00334 ± 0.00102
(n-7)
0.371 t 0.054
(n-6)
0.00080 t 0.00017
(n-5)
19.8 ± 0.4
(n-4)
1.54 t 0.10
(n-4)
0.13 t 0.005
(n-12)
0.0635 t 0.0057
(n-7)
51.0 ± 1.6
(n-4)
0.097 t 0.010
(n-4)
40 t 9.2
(n-4)
0.134 t 0.0119
(n-7)
0.00048 t 0.00007
(n-22)
0.453 t 0.008
(n-4)
0.00838 t 0.00492
(»-7)
0.00838 t 0,00492
(n-7)
0.842 t 0.047
(n-6)
0.00215 t 0.00065
(,.-B)
39.7 t 0.4
(n-4)
C
2.21 t 0.03
(»-6)
0.27 t 0.014
(n-12)
0.137 t 0.0073
(n-7)
104 ± 2
(n-4)
0.202 t 0.019
(n-4)
210 ± 14
(n-4)
0.207 + 0.028
(n-7)
0.00087 t 0.00008
(n-22)
0.723 t 0.221
(n-4)
0.01374 t 0.00376
(n-7)
0.01374 i 0.00376
(n-7)
1.870 ± 0.1JO
(n-6)
0.00488 t 0.00056
(»-7)
80.9 t 0.4
(n-2)
-------
TABLE U-l Co.it. UKASURED CONCENTRATION OF TOXICANTS IN EXPOS UK t UATtK CHAMBERS (Mean t S.U.)
«M.,U,1
Arsenic*1
+3
Clu oiuluui
>6
Lejd42
Le^d*2
Hci tury
Mercury
Nickel42
SI 1 > ^'
Silver41
Silver41
Silver41
"*
Orgunlin Our (lien
D»|.lml» raatsn* 48 li (Fed)
28 d
Cjiumdiu» 96 li
l>»eud«lluntfei»
Tunyt«rtu» dln>lBlll» 48 li
(iucimurus 96 h
|j»eudi»U di»«l.Ul» 49 It
Cumuiaruy 96 It
P9eud»llmn>eu>
Tjnytarvu* di»»l»llls 48 h .
I)
4.22 t 0.53
(n-6)
0.63 t 0.034
(n-11)
0.280 t 0.014
(n-7)
205 ± 6
(n-4)
0.420 t 0.036
(n-4)
650 t 29
(n-4)
0.482 i 0.024
(n-4)
0.0018 t 0.0002
(••-22)
1.7? t 0.019
(n-4)
OnjQss •*• n nofcft?
r \J& :fJJ X U > UUOO i
(n-7)
0.02955 t 0.00662
(n-7)
3.350 t 0.064
(n-6)
0.01527 t 0.00385
(n-3)
164 t 2
(n-2)
-I.
Concent rdlvnu (ug-L )
E f
7.U5 t 0.21 13.3 t 0.52
(n-4) (n-4)
1.32 t 0.032 2.68 -f 0.059
(n-12) (n-11)
0.548 t 0.013
(n-4)
412 i 11
(n-4)
0.878 t 0.029
(n-4)
1800 i 95
(n-4)
. k/
0.0037 t 0.0006
(n-21)
3.77 ± 0.032
(n-4)
OAft7Ql -4- A ftl AQ&
• UD/7 A x " * wti yo
(n-3)
0.06791 t 0.01496
(n-3)
7.190 t 0.290
(n-6)
0.03561 t 0.00800
(n-3)
325 t 10
(n-2)
-------
: 11-1 Cant. HBASUKGU CONCtNlHATIONS OF TOXICANTS IN liXpOSUHU UATtU ClUMIiKllS (Mean t S.D.)
J .
Cyunldi;
(us ItCN)
Cyunidii
(o« CN")
Organ !«• Dumt Ion
iunyi jrtfuii 48 l>
dluulmllU
" " 48 I)
Conlial
0.00 t 0.00
0.00 i 0.00
(n-3)
Conctnlrui IODM
A
0.86 t 0-01
O.HB t O.O/
(-8-1"1)
B
1.66 1 0.01
(n-2)
1.72 t 0.11
(n-6)
C
3.27 + 0.06
(n-2)
3. SO t 0.18
(n-6)
-------
TABU B-l Gout. HKASURKD CONCENTRATION Of TOXICANTS IN EXPOSURE WA'lEtt CIIAM11ERS (Item ± S.U.)
Clieulcul
CN )
Orgjnlvw
Duff I ion
Concentration*
U E
tyjnlile Tunytjriuv dl»lBlll» 4tt li
(JK IICN)
Cyanide " " 48 li
5.31 t 0.25 7.14 ± 0.23
(n-2) (»'2)
6.56 ± 1.16 13.1 t 0.4 -*
(,,-6)
u/ litiilul cunce»tr»Liu>iB of lieptichlor or t»i»pliei>i:.
—> b/ Uue i» imulytlcul proLK-ms untl lilgli vcrivblllcy of cone tut rctlonw, this unpovure v»t> not uaud In
t" determination of I|IL LC,,. concenlrctIon.
£/ DHL: tu 111 nit |>ll »nd ruvultint effect upon I lit equilibrium between IICN Hnd CN , tlilw concent rat li>n
Wiiv not lined In del trwlnut lo» of I lit LC _ concentritlon.
-------
APPENDIX C
PURITY LEVELS, ANALYTICAL PARAMETERS AND PROCEDURES, AND ANALYTICAL
QUALITY CONTROL DATA FOR TOXICITY TEST CHEMICALS
TABLE C-l. SOURCES AND PURITY LEVELS OF CHEMICALS
USED IN TOXICITY TESTS
Chemical
Source
Lot No.
Puri ty
1,2-Dichloroethane
1,1,2-Trich.loroethane
1,1,2,2-Tetrach.loroethane
Pentachloroethane
Hexachloroethane
Tetrachloroethylene
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
1,2,4-Trichlorobenzene
Pentachlorobenzene
Hexachlorobenzene
Hexachlorobutadiene
Di -rv-butyl phthal ate
Pentachlorophenol
Heptachlor
Chlordane
Toxaphene
DimethyIformamide
Methanol
As"1"3 (NaAs02)
Cr+6
Pb+2 [PbCN03)23
Hg*2 (HgCl2)
Ni+2 [Ni(N03)23 - 6
Ag*1 (AgNO.)
4-A
Se 4 CSe02)
CN"1 (NaCN)
Aldrich. Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem. Co.
Aldrich Chem, Co.
Monsanto Co.
Aldrich Chem. Co.
EPA/RTP
EPA/FDA
Hercules
Burdick & Jackson
Burdick & Jackson
Fisher
Fisher
Fisher
Ventron CAlpha)
Mallinckrodt
Fisher
Fisher
Fisher
JB070177/HD061197
LB090677
CD072484
ED080787
JB072677
PCI10987
073034
JC010487
PB082017
P260-4
20(69-18)
X-16189-49
AD665
AB468/AE562
776308
787060
791928
021971
6376
732462
764220
702579
99%
95%
98%
96%
98%
99%
99%
98%
97%
99%
98%
97%
98%
99.8%
99+%
98%
Techni cal
grade
Technical
grade
98%
99+%
ACS Grade
99.96%
ACS Grade
ACS Grade
99-9%
99.994%
95-2%
98.8%
116
-------
TABLE C-2. SUMMARY OF GAS-LIQUID CIIROHATOGRAPII PARAMETERS
FOR ORGANIC TEST CHEMICALS
Compound
Column
Packing
Isothermal
Colunn
Temp (°C)
Retention
Time (Mini
1,2-Dichloroethant
1,1,2-Trtchloroethano
1,1,2,2-Tetrachloroethane
Pentachloroethane
llexachloroe thane
Tetrachloroethylene
1 ,2-D1chlorobenzene
1,3-Okhlorobenzene
1,4-Dlchlorobenzene
1,2,4-Trlchlorobenzene
1,2,4-Trlchlorobenzene
1,2,4-Trlchlorobenzene
Pentachlorobenzene
Hexachlorobenzene
llexachlorobutadlene
Dl-n-butylphthalate
Pentachlorophenol
lleptachlor
Chlordane
Toxaphene
Methanol
Otmetliylfonnamtde
Hewlett-Packard 5710A
Hewlett-Packard S710A
Itewlttt-Packard 5710A
Hewlett-Packard S710A
Tracor 550
Tracer 650
Tracor SSO
Hewlett-Packard 5710A
Tracor SSO
Tracor SSO
Hewlett-Packard S710A
Hewlett-Packard 5710A
Tracor HT 160
Tracor MT 160
Tracor SSO
Tracor HT-160
Hewlett-Packard 5730A
Tracor MT-220
Tracor 550
Tracor MT-220
Hewlett-Packard S730A
Hewlett-Packard 5730A
41 SE-30/61 OV-210 SO
4X SE-30/6X OV-210 50
1.5X OV-17/1.95X QF-1 75
l.SX OV-17/1.95X QF-1 90
31 OV-101 130
3X OV-101 65
31 OV-101 120
4X SE-30/6X OV-210 100
3X OV-101 120
3X OV-101 120
1.5X OV-17/1.95X QF-1 110
4X SE-30/6X OV-210 120
3X OV-101 205
3X OV-101 205
3X OV-101 120
3X OV-101 225
1.5X SP2250/1.95XSP2401 160
4X SE-30/6X OV-210 185
3X OV-101 205
4X SE-30/6% OV-210 185
Tenax G@ 80
Tenax GC® 160
3.03
2.68
1.68
1.80
1.30
1.60
1.44
2.36
1.35
2.48
2.29
2.94
1.18
2 41
2.35
2.7
5.2
0.90
7.57,
.£/
1.1
1.6
8.25"
*/ M electron-capture detectors used for analysis of compounds, except for iiethanol and dime thy Iformamlde
where flauie-lonlzatton detectors were used.
b/ Ritentlon times for trans- and cis- chlordane Isomers, respectively.
c/ Multiple confounds present In toxaphene mixture eluted over a range of retention times.
-------
TABLE C-3. SUMMARY OF EXTRACTION PROCEDURES AND PERCENTAGE RECOVERIES FOR
ORGANIC TEST CHEMICALS ANALYZED BY GAS-LIQUID CHROMATOGRAPHY
Compound
Volume of Mater
Sample (mL)
Extraction Solvent
(Volume Used)
Extraction
Apparatus
% Recovery of
Spiked Samples
x ± s.d.
00
1,2-Dichloroethane
1,1,2-Tr1chloroethane
75
75
1,1,2,2-Tetrachloroethane 75
Pentachloroethane 75
Hexachloroethane 5-50
Tetrachloroethylene 5-50
1,2-Dichlorobenzene 2-50
1,3-D1chlorobenzene 75
1,4-Dichlorobenzene 2-50
1,2,4-Tr1chlorobenzene 2-50
1,2,4-Trlchlorobenzene 75
Pentachlorobenzene 1-5
Hexachlorobenzene 1-5
Isooctane (25 mL)
Isooctane (25 mL)
Isooctane (50 nl)
Isooctane (25 mL)
Hexane (50 mL)
Hexane (50 mL)
Hexane (50 mL)
Isooctane (25 mL)
Hexane (50 mL)
Petroleum ether
(50 mL)
Isooctane (25 mL)
Hexane (5
Hexane (5
100 mL vol. flask
on magnetic stlrrer
100 mL vol. flask
on magnetic stlrrer
200 mL vol. flask
on magnetic stlrrer
100 mL vol. flask
on magnetic stlrrer
100 mL vol.
on magnetic
100 mL vol.
on magnetic
100 mL vol.
on magnetic
100 mL vol.
on magnetic
flask
stlrrer
flask
stlrrer
flask
stlrrer
flask
stlrrer
100 mL vol. flask
on magnetic stlrrer
100 mL vol. flask
on magnetic stlrrer
100 mL vol. flask
on magnetic stirrer
18 mL glass-stoppered
test tube
18 mL glass-stoppered
test tube
103.1 ± 5.6 (n=18)
96.6 ± 7.0 (n=15)
100.2 ± 8.2 (n=8)
98
(n=l)
96.7 ± 2.9 (n=16)
89.9 ± 6.2 (n=23)
103.7 ± 2.6 (n=15)
95.4 ± 4.7 (n=15)
100.1 ± 3.0 (n=19)
98.8 ± 2.3 (n=18)
98.5 ± 8.4 (n=15)
94.3 t 4.0 (n=21)
96.6 ± 3.1 (n=23)
-------
TABLE C-3 Cont. SUMMARY OF EXTRACTION PROCEDURES AND PERCENTAGE RECOVERIES FOR
ORGANIC TEST CHEMICALS ANALYZED BY GAS-LIQUID CHROMATOGRAPHY
Compound
Volume of Water
Sample (mL)
Extraction Solvent
(Volume Used)
Extraction
Apparatus
% Recovery of
Spiked Samples
x ± s.d.
Hexachlorobutadiene 75
D1-n_-butylphthalate 50
Pentachlorophenol 100
(Midge and Gammarus)
Heptachlor 10
Chlordane 5-50
Toxaphene 50
Methanol -^
Dimethylformamide -
Isooctane (25 mL)
Hexane (50 mL)
Hexane (100
Hexane (10 mL)
Hexane (50 mL)
Hexane (50 mL)
_C/
d/
100 mL vol. flask
on magnetic stirrer
100 mL vol. flask
on magnetic stirrer
200 mL vol. flask
on magnetic stirrer
50 mL vol. flask
on magnetic stirrer
100 mL vol. flask
on magnetic stirrer
100 mL vol. flask
on magnetic stirrer
.£/
d/
100.7 ± 5.5 (n=16)
96.9 t 9.2 (n=17)
99.3 ± 4.6 (n=4
97.2 ± 6.0 (n=6
100.7 ± 7.5 (n=3)
99.9 ± 7.6 (n=21)
93.3 ± 6.9 (n=4)
102.2 ± 3.1 (n=10)
100.2 ± 0.4 (n=6)
a/ Three drops saturated NaCl solution added.
b/ Concentrated H^SO. (2.0 mL) added prior to extraction, and extract derlvatized with diazomethane.
cy Analyzed by direct aqueous injection GLC technique for rainbow trout and bluegill sunfish acute tests.
d/ Analyzed by direct aqueous injection GLC technique for rainbow trout acute test.
-------
TABLE C-4. SUMMARY OF ANALYTICAL TECHNIQUES AND QUALITY CONTROL PARAMETERS FOR
TOXIC1TV TESTS WITH SELECTED ELEMENTS AND AQUATIC ORGANISMS
Element
Arsenic
Chromium
Chromium
Lead
Lead
Nickel
Selenium
Silver
Mercury
Salt
NaAsO,
2
K,Cr,07
£. t 1
K,Cr 0,
£ L. 1
Pb(H03)2
J £,
Pb(WK),
J t
N i ( NO -1)0* oH«(
SeO
£
AgllO,
J
HgCl
c
Organlsn
Fathead minnow
Flagftsh
Midge
Ganniarus
Midge
Gamnarus
) Oaphnja
Midge
Fathead Minnow
Flauflsh
Midge
Gamnarus
Fathead minnow
% LSW*' in
Method Atomizatlon Samples & Stds.
EPA^ Furnace < 2*c/
1206.2
EPA Flame 25
1218.1
EPA Furnace 25
I21B.1
EPA Flaiie 50
1239.1
EPA Furnace 25
1239.1
EPA Furnace 50
1249.2
Parkin- Flame 99.5
Elmer
(1976)fi/
EPA Furnace 10-99.5
1272.2
EPA Cold vapor -#
1245.1
Modified
X Spike
Recovery
100.4 i 6.1
(n-57)
106.0
(n-2)
100.5 t 2.0
(n-4)
99.7
(n-2)
n.d.d-/
100.8 t 2.9
(n-6)
96.0
t nc2 \
96.7 t 9.0
(n=24)
102.5 i 7.1
(Acute test)
(n-12)
95.7 t 5.0
(Early life-
stage test)
(n-12)
% Agreement
of Duplicates
96.2 t 3.7
(n-25)
99.6
(n=2)
97.3 t 3.5
(n-5)
98.4 (Filtered-
0.45 P)
91.7 (Unfiltered)
(n-2)
93.4 i 5.9
(n-5)
96.0 t 4.2
(n=3)
96.8
/na?\
/
95.9 t 2.8
(n-12)
,9/
tl Lake Superior water used in samples and standards to overcome any i.iatrtx Interference.
b' U.S. EPA. 1979. Methods for Chemical Analysis of Water and Waste. tPA 600-4-79-020.
c/ Controls contained itO'i and all saiiiples contained ±2X of dechlorinated city water.
d/ dot determined because spiked sai.iples and standards were prepared identically.
i/ Pcrkiii-EUier Corp. 1976. Analytical Methods for Ator.ilc Absorption Spectropliotouietry (Revised
Sept., 1976). Perkln-Lluier Corp. Publ., Horwslk, Conn.
U Ueionizcd water was used for standard preparation and sample dilution.
ij/ The mean coefficient of variation from triplicate analyses of the same sauples was 5.3S (n-8).
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