Document EqwRNMVbdLYVd5aE01K1VO87b
TECHNICAL REPORT DATA
(Please read Instructions on the reverse Before completing)
l. REPORT NO.
EPA-6Q0/3-83-095
.4 TITLE AND SUBTITLE
pm26366 53. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
Toxicity and Metabolism Studies with EPA Priority
September 1983
Pollutants and Related Chemicals in Freshwater Organisms 6. PERFORMING ORGANIZATION CODE
7. AUTHORISE
D,J. Call, L.T. Brooke, N, Ahmad, and J.E. Richter
8. PERFORMING ORGANIZATION HEPORT NQ.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Lake Superior Environmental Studies University of Wisconsin-Superior Superior, Wisconsin 54880
12. SPONSORING AGENCY NAME AND ADORESS
U.S. Environmental Protection Agency Environmental Research Laboratory-Du.1uth 6201 Congdon Boulevard Duluth, Minnesota 55804
IS. SUPPLEMENTARY NOTES
IQ. PROGRAM ELEMENT NO.
XU CONTRACT/GRANT n o .
806196, 80020010, 806864
13. t y p e OF REPORT AND PERIOD COVERED 14. SPONSORING AGENCY CODE
EPA/600/03
16. ABSTRACT
Tq-jflcploglcel studlee were conducted In two areas: (I) the (toxicity, bloconcentrat Ion
potential end metsbol! sm ot five harblcldas In fish; and (21 the toxicity and/or metaboi Ism of priority
pol 1 utants and related cha.ailc.als In various aquatic organisms.
The tost herbicides Included alachlor Jl-cblpro-Z'.fi'rdlathyl-tMmethoxyraethyf) acetanilide!, orcmac 11
(5-brqmo-3-sa.;-lbu'ty I -6-mathy I urac 11}, d I nosob 12-(sac-bufy I )-4,6-dlnl Troptianol I, dturon 13-(3,4-dIch loro-
phenyl )-l,t-d laathyl urea?., and propanl! (3,4-dtchlproprapIananII Ids). Acuta toxicity (through IT2 hr),
; early life-stags toxicity (58-64 day), and bloconcantrarlon studies ware conducted w Ifh fathead minnows
(Pi mepha I es p.roroe las) In Lake Superior water, Herbicide metabolism eats Investigated In ra Inbox trput (Salmq
galrdnerl) both In vlyQ and In vitro,
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-dIchloroefhane, 1 I,2-trIcnloronthana,
1,1 t2,2-tefrach.locoat:hafte, fetraehlor.oethylsna, 1 ,.2-dt chlorobenzene, 1 ,3-d (chlorobenzene, 1,4-dl chloro
benzene, hexaoMorobonzene, hexpchforabutadlene, dl-n-butytphthal ate, pentach I prophenol , heptaeh.tor.
chlcrdane, toxapKene, arsenic. '''3"', chromium*, Iead,+T2i, mercury.".+1*2, nlcke.l+T2<, silver
selenium**.. Bind cyanide, freshwater species tested Included The fathead minnow, rainbow trout, bluagl 11
sun fish (Leponls macroch lirusl, flagf Ish (Jordanel I a f lor I daa), Oaphnla magna. scud (Gammarus
ftSeudoUmnaeus), midge (Teoytarsus dlsslml I Is) and green alga (SelehastriKn caprlcormtum). Toxicity tests
were also conducted with pentachloroethana, hexachloroothane, l,2,<-trIchlorpbenzana, pentachlprobenzana,
methanol end dlmethylfomamlde. The uptake by fish of dl-n-butylphfhalate from water. Its metabolism and
elimination ware Investigated, Comparative metabolIsi of 1,t,2-trlchloroethane, chlorobenzene,
<--Vfl.2-trlcRlo(roerti!tyleAei, chloroform, and carbon tetrachloride mae studied In rainbow trout and Oaphnla.
7.
KEY WORDS AND OOCUMENT ANALYSIS
~
DESCRIPTORS
b.lOENTlFIERS/OPEN ENDED TERMS c. COSa t i Field/Croup
I
I'i
IiI!
118. DISTRIBUTION STATEMENT RELEASE TO PUBLIC EfA Fwrai 2220--1 (flu*. 4--77) p r e v io u s e o it io m i o b io u e t e
IB. SECURITY CLASS (This Report)
UNCLASSIFIED 20. SECURITY CLASS (This page)
UNCLASSIFIED
FOREWORD
The Environmental Research Laboratory-Duluth is concerned with effects,of r
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,
Nprbert Jaworski, Ph. D. Director Environmental Research Laboratory Duluth, MN
iii
DUP04001I2538
ABSTRACT Twenty-two chemicals from the ERA priority pollutant list were studied fpr their acute and/or chronic toxicity to selected freshwater organisms. These included 1,2~dichioroethane, 1,1 ,2-trichloroethane, 1,1,2,2-tetracbloroethane, tetrachloroethylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, hexachlorobenzene, hexachlorobutadiene, di-n-butylphthalate, pentachlorophenol,, heptachlor, chlprdane, toxaphene, arseni|:+3, chromium46, lead42, mercury42, nickel42, silver4^, selenium^, and cyanide. Freshwater species tested included the fathead minnow (.Pimephales promelas 1, rainbow trout (Salmo galrdneri\, bluegill sunfish (lepomls macrochirus), flagfish (Jordanella floridae), water flea (Daphnia magna), scud (Gammarus pseudolimnaeus), midge (Tanytarsus digsimi 1is), and green alga (Selenastrum capricornutum), Toxicity tests were also conducted with pentachloroethane, hexachloroethane, 1,2,4-trichiorobenzene, pentachlorobenzene, dimethylformamide and methanol . Di-n-butylphthalate uptake from water, elimination and metabolism by fish was studied. A comparison was made of the metabolism and binding pf carbon tetra chloride, chloroform, 1,1,2-trichloroethane, 1,1,2-trichloroethylene and nranochiorobenzene by microsomal fractions of rainbow trout livers and of daphnid whole bodies.
iv
DUP040012539
CONTENTS
I'orfiworrf . . . > <. .4 *
/tbS "tv'rflCt a m . - * -4 ; * . .
FigUrCS
> . ' :**.
* >. *
ill fv
viii
1 Sli?T@5 * 4 4 i * 4 4 t 4 Acknowledgments . , . . < . * *
*-* *
**
U xii
1, Introduction
* , . , . . . * 9 4 * :
2. Conclusions ...........
3, Recommendations ,. . ... . . . . ... . * . . . . .
4. Materials and Methods ........ .......... .
Water Supply and Environmental Control
Test Organisms ......................
Acute Toxicity Tests . . . . . ,
,
Chronic and Subchronic Toxicity Tests ........ . . .
Chemical Analysis of Toxicants ,. ........ . * .
Statistical Analysis of Test Results ...........
Oi-n-butylphthalate Uptake, Elimination and Metabolism . .
Di-iv-butylphthalate Protein Binding . . . .................... . . .
1 3 7 8 8 8 TO 21 24 26 27 31
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons by Trout and Oaphnia . . ............................... . 32
Mixed Function Oxidase Enzyme Assays
35
5, Results ........ . ......
37
Acute Toxicity.Tests .........
37
v
DUP040012540
$,, Results Cent.
Chronic and Subchronic Toxicity Tests , . . , . . . . . . . . 45
Di-nr-butyl phthalate Uptake, Elimination and Metabolism by Fish , .
__
Di -rr-butyl phthalate Binding......................................................... * 58
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons by Trout and Daphnia , . > , . . , , . ,. .
Mixed Function Oxidase Levels . . > ,, , . * . * , , . . . . 68
B-* Discussion . * . , . * . . p p * . . * p ' .% TO
Chlorinated Ethanes ..............
. . 7$
Tetrachlorpethylene
i-. .
is* . . 71
Chlorinated Benzenes
p . 73
Hexachlorobutadiene ... . , . . . . . -. .... . ... . . 75
Di*-n-butyl phthalate .......... , . . . . . , , * . 75
Pentachlorophenol . , ...... . ............. 77
Heptach1 or
78
Chlordane
, . .. . . .... . . .... 78
Toxaphene Arsenic+3
..**.
. . ..... . .... * .. 79
...................... . . . 80
Chromium
81
Lead*2 ............ ................... ......... 82
Mercury*2 . , . .. . ............. . ...... 82
Ni ckel *2
ft1!
Silver*^ ........... ....................... ........ Selenium+4 .* > :
84 85
Cyflmds *.*##*.*; ****.* .* 85
vt
DUP040012541
6. Discussion Coot.
Microsomal Metabolism and Binding of Chlorinated Hydrocarbons ........ . . .' , , . , . . 85
Mixed Function Oxidase Activity .... . . ........ 86
Deferences .. .. . . .1. ..
* . . * > . * 58
AppGndi
. ...
. , . 95
A, Summaries of Conditions and Water Characteristics for Toxicity Tests ...............
95
.
B. toxicity Test Chemical Concentrations . , . . . . .
. . 104
G. Purity Levels, Analytical Parameters and Procedures, and Analytical Quality Control Data for Toxicity Test Chemicals ....... . .... . . .... .
. . 116
vii
DUP040012542
FIGURES
Number 1
Log mean exposure water concentrations of 14C-
1 abeled dt-n-butylphthalate Cjig*mL-*) and log mean ( S.OT) whole fish total )4-c residues during uptake (days 1-11) and depuration (days 12-32) phases ........................... ............................................. . . .
Page 57
viii
DUP040012543
TABLES
Humber
1 LCcg Values (55% Confidence Intervals) for Pooled ..eplicates of Acute Tests In Whi eh Fathead Mirjqows (Pimephales promelas) were Exposed to Arsenic,
Mercury4*, Si Tver43, Dimethyl formami de and Methanol . .
LCc q Values (95% Confidence Intervals) for Pooled Replicates of Acute Tests in Which Rainbow trout were Exposed to Selected Organic Compounds
Results from Flow-Through Measured Acute Toxicity Tests in Which Bluegin Sunfish (lepomls macrocfelrus) were Exposed to Hexachlorobutadiene, Hexachlorobenzene/DMF, Dimethylfortnamide, and Methanol (Replicates pooled),* .
LC Values (55% Confidence Intervals) for Pooled _plicates of Acute Tests in Which Flagfish were
Exposed to Arsenic43 and Silver4-'. . . . . . .
5 48 Hr LC5o and ECgg Values (.95% Confidence Intervals) for Pooled Repucates of Daphnia magna Exposed to Selected Test Chemicals ....... , , , . , . ,
5 LC -q Values (95% Confidence Intervals) for Pooled . Implicates of Acute Tests in Which Scuds (Gammarus pseudo!imnaeus ) were Exposed to Pentach1orophenpl, Arsenic4'3, Silver4-', Lead+2, and Chromium46 . . . . .
7 48 Hr LC5Q Values (95% Confidence Intervals) for Pooled Replicates of Acute Tests in Which (Tanytarsus dissimjlis) Were Exposed to Selected Inorganic and Organic Chemicals . ...................................... ... . . . , .
8 Percent Inhibition of Se1enastrum capricornuturn Growth when Exposed to Several Concentrations of Toxaphene for 95 Hr. . .. . . .... . . . ... . ... . . .
9 Percent Inhibition Of Selenastrum capricornuturn Growth at 96 Hr Following Exposure to Several Concentrations of Heptachlor and its Breakdown Product* 1-Hydraxychlordene (Test T) ...........................
ix
Page y
, . 38 . . 39
. . 40 . . 42 . . 43
. . 44
. . 46 . . 47
, , 48
DUP040012544
Sim
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
Oaphnia magna During 28 Day Chronic Tests .......... 50
.. .
;
12 Hatchability, Development, Survival and Growth of Fathead
X
Minnows (Pimephales promelas) Exposed to Arsenic+.
(NaAsC^) for 30 Days Post-Fertilization . .... .... 53
13 Hatchability, Development, Survival and Growth of Fathead Minnows,(Pimephales promelas) Exposed to Inorganic Mercury (HgCT^) for 35 Days Post-Fertilization ........ 54
14 Hatchability, Development, Survival and Growth of Flagfish (Jordanel 1 a floridae) Exposed to Arseni c^jHaAsO^) for 30 Days Post-Fertilization . . . . ... . . . . . . , . . , . 56
15 Distribution of Radioactivity in Fathead Minnows (Pimephales promelas) Exposed to '4C-PI-n-butylphtbalate . . . . . . . . , 59
16 Distribution {% S.D.) of
after Incubation of^C-
Di-n_-butylphthalate for Various Time Intervals with
Microsomal Fractions of Rainbow Trout (Salmo gairdneri)
Liver and Post-Mitochondrial Supernatant of Oaphnia
magna
60
17 Distribution {% + S.D.) of 14C after Incubation with ^CCarbon Tetrachloride for Various Time Intervals with
Microsomal Fractions of Rainbow Trout (Salmo gairdneri) Liver and Post-Mitqchondrial Supernatant of Paphnia magna ................ .............. . . ....... 62
18 Distribution (% + S.D.) of ^C after incubation with ^CChloroform for Various Time intervals with Microsomal Fractions of Rainbow Trout (.Salmo gairdneri) Liver and Post-Mitochondrial Supernatant of Oaphnia magna .................... ... 64
19 Distribution (% + S.D.) of ^C after Incubation with ^4C-
Chlorobenzene for Various Time Intervals with Microsomal Fractions of Rainbow Trout (Salmo gairdneri) Liver and Post-Mitochondrial Supernatant of Djaphnla magna. .... , 65
i
x
DUP040012545
Humber
20 Distribution i$ S.D.} of C after Incubation With ^C1.1.2-Trichloroethylene for Various Time Intervals with Microsomal Fractions of Rainbow Trout (Salino gairdneri) liver and Post-Mitochondria] Supernatant of Daphnia magna................... , ..............................................
Page 66
21 Distribution (% + S.D.) of
after Incubation with
1.1.2-Trich!oroethane for Various Time Intervals with
Microsomal Fractions of Rainbow Trout (Salmo gairdneri)
Li ver and Postr-Mitochondri al 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
xi
DUP040012546
We would like to thank our Project Officer, John Teasley, from the ' Environmental Research Laboratory-Duluth, MN (ERL-D), O.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 Holcombe, Anthony Carl son, James F|andt, 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 WisconsinSuperior technical staff members: Michael Knuth, Steven Poirier, Catherine Mpriarity, Cheryl Anderson, Pamela Shubat, James Huot, Ann Lima, Marilynn HogTund, Dean Hammermeister, Tom Markee, Taryl Felhaber and Debra Svejskovsky. We thank representatives from Monsanto Corporation for supplying technical grade and radiolabeled di-n-butylphthalate for our studies. We gratefully acknowledge the work of our secretary, Joyce Barnes, in the preparation of this report.
xii
DUP04Q012547
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 TelTiard, 1979). This list of "priority pollutants" initially consisted of 65 chemicals (or groups of
i
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 WisconsinSuperior 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 hatoalkanes 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 (Docks and Krishna, 1976; Watanabe et al., 1978). However, Information is limited concerning the
1
DUP040012548
metabolic disposition and protein binding of such compounds ill 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 (.Salmo gairdneri 1 liver and the water flea (Daplinla magna).
2
DUP040012549
SECTION II CONCLUSIONS
Acute toxicity tests were conducted with fathead minnows (Pimephales promelas) rainbow trout (Salmo gairdnerl), bluegill sunfish (.Lepomis macrochlms), flagfish (Jordanella floridae), water fleas (Daphnia magna), scuds (Gammarus pseudo!Imnaeus), midge larvae (.Tanytarsus dissimllis Johannsen ]937), and green algae (Selenastnin) capricornutum). Fathead minnows
+3 +2 +i were exposed to arsenic , mercury , silver , dimethylformamide (OMF), and methanol with resulting estimated 96 hr LCgg values of 14.2, 0.150, 0.0107, 10,700, and 28,100 mg*L"V respectively. Rainbow trout were exposed to hexachloroethane, tetrachloroethy1ene, tetrachloroethylehe with DMF as a carrier solvent, OMF, 1,2-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, pentachlorobenzene with OMF, hexachlorobenzene with OMF, hexachlorobutadiene, and methanol with resultant 96 hr LC50 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-^, respectively.
Bluegill sunfish were exposed to hexachlorobutadiene, hexachlorobenzene with DMF, DMF, and methanol with resultant 96 hr IC5Q estimates of 0.324, >0,0784, 7,100, and 15,500 mg-L-1 , respectively. Flagfish were exposed to arsenic *1*3 and silver*^*1 with resultant 96 hr LCcn estimates of 14.4 and 0.0092 mg*L'^, respectively.
Water fleas were exposed to hexachloroethane, pentachloroethane, 1,1,2,2tetreichl oroethane, 1,1,2-trichloroethane, 1,2-di chi oroethane, 1,3-dichloro-
3
DUP040Q12550
benzehe# 1,2,4-trichlorobenzene, tetrachloroethylene, di-n-butylphthalate, DMF, chlordane and nickel +2 with resultant unfed 48 hr LCgg 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. LCgg estimates were also made for most of the same compounds in
exposures where the organisms were fed. Cgg estimates were made for fed and
unfed exposures with the chlorinated compounds and with arsenic+3 ,
/
Scuds were exposed to pentachlorophenol, arsenic , silver 1, lead , and
chromium* with resultant 96 hr LCgg estimates of 280, 875, 4.49, 140, 67.1
and 94.1 jig-L^, respectively. Midge larvae were exposed to hexachlOroethane,
tetraehloroethylene 1,2-dichlorobenzene, 1,4-dichlorobenzene, hexachlorobenzene with DMF, pentachlorophenol, DMF, chromium*6, lead+^ysilver*1, selenium**1, and
cyanide with resultant 48 hr LCgg 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 Cff mg-L-1,
respectively. Green algae were exposed to toxaphene and heptachlor for 96 hr with resul
tant ECgg estimates (,50% reduction of growth) of 0.38 mg-L"1 for toxaphene and 38,1 and 23.2 yg-L-1 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-tetrachloroethane, 1,1,2-trichlorpethane, 1,2-dichloroethane, 1,2,4-trichiorohenzene, 1,3-sili chlorobenzene, 1,2,4-trichlorobenzene, 1,3-dichlorobenzene, tetrachloroethylene, and arsenie+3 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*1, respectively.
, +3 Fathead minnows were exposed to arsenic for 30 days post-fertilization and mercury for 35 days post-fertilization. The "no-effect" concentration
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DUP040012551
'*for arsenic+3 -was between 2.1 and 4.3 mg*L-1 based upon significant (p<O,01) reductions in wet weight and body length. Mercury*f2 exposures resulted in
significant (p<Q.Q1) reductions in wet weight and length at all exposure concentrations. A "nn-effect" concentration for mercury+2 was less than the lowest exposure concentration of 0.23 n9*b-T FTagfish were exposed to arsenic+3 for
30 days post-fertilization with a resultant "no-effect" concentration between 2,13 and 4.12 mg*L7^ based upon a reduction in body length.
Uptakei elimination, and metabolism of di-ji-butylphthalate was studied with
fathead minnows,- A steady-state level of
equivalents of di-n.-butyiphthalate
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-n.butyiphthalate were 570 and 590 for the two tests based upon a mean value of 27.6# unmetaboiized 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-e-butylphthalate to proteins was studied using rainbow trout liver raierosomes 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
DUP040012552
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-trichioroethylane > 1,1,2-trichlorgethane % 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 micrpspmes had 0,28 and 0.19 nM-nig"^ of cytochrome P-450 and cytochrome bg,
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 activity*min^*mg"^ protein.
* 6
! DUP040012553
se*.
SECTION III r e c o mmen d a t io n s 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 Tanvtarsus dissimilis (midge) with consistently high tolerances to a broad range of chemical cl asses should receive minimal effort. Physical properties (j.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 animalsfrom various taxonomic groups to metabolize foreign chemicals would be valuable.
7
DU P040012554
#
s ec t io n 0 METHODS
Water Supply and Environmental Control Water for the toxicity tests was either directly from Lake Superior or was
dechlurinated city water from Superior* WI. (Superior, WI derives Its water from shallow wells heneath Lake Superior.) Several chemical parameters (dis solved oxygen, pH, hardness, acidity* and alkalinity) were monitored during the
'i
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. lest Organisms
Fathead minnow (Pimephales promelas) brood fish were received from stock maintained by the Environmental Research Laboratory-Duluth, MM, U.S. EPA, Brood fish were maintained at 25 C, and were fed twice daily a diet of frozen adult brine shrimp.
Asbestos pipe (12.5 cm 0.0.) 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 (<_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
DUP040012555
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 Take 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 dlssimi1is 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 9n C on a diet of CerophylP^and trout pellets as described by Anderson et al.
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: tetrachlorpethylene - 6.1 1.0 cm, 3,2 t 1.5 g tn^lS); tetrachloroethylene with dimethylformamide (.OMF) carrier-solvent 7.3 + 1.0 cm, 5.86 t 2.45 g (.n-19); T ,2-dichlorobenzene - 5.6 0.8 cm, 2.69 1.24 g (n=I0); 1,4-dlchlorobenzene - 5.3 0.6 cm, 2.1 1.0 g (n=20); hexachlorobutadiene - 5.6 0.6 cm, 2.6 0,9 g (n=V9}j hexachloroethane - 6.6 +1.0 cm, 4,,3 i: 1.-8 g (n=20) j 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-1). Toxicant concentrations were measured daily as described with fathead minnows . (Appendix 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* Pentachlorobenzene was tested with dimethyl formamide (0MF) as a carrier solvent* Penta-' chlorobenzene/DMF stock stolutions of known concentrations were pumped into PUP04001, test chambers with fluid metering pumps. The control chamho*.- ----- -
nuc-----
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 dimethylformarnide (DMF); carrier-solvent 7,3 i 1.0 cm, 5.86 + 2.45 g (0=19); 1,2-dichlorobenzene - 5,6 0.8 cm, 2.69 1.24 g (n=10); 1,4-dichTorobenzene - 5.3 0.6 cm, 2.1 1.0 g (n=2Q); hexachlorobutadiene - 5,6 0.6 cm, 2.6 0.9 g (n=19}; hexachloroethane - 6.6 t 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*2d).
Water quality parameters were routinely measured (Appendix A, Table A-T). Toxicant concentrations were measured daily as described with fathead minnows , (Appendix B, Table B-t).
Penta- and hexaehlorobenzene acute tests were conducted in a different type of flow-through system due to their limited water solubilities. Pentachlorobenzene was tested with dimethyl formarnide (DMF). as a carrier sol vent. Pentachlorobenzene/OMF 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 (depth Of 15 cm}.. Pumps were set to deliver every time the system cyclecj and delivered I 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 (0=20)3 per chamber. Replicates were separated in time by II days. Since there were insufficient deaths at 96 hr to calculate an LC^q
12 *
DUP040012557
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 fcn=20), respectively.
toxicant concentrations were measured daily (Appendix 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 hexachlorobutadiene, a proportional di1uter system was used. Chamber dimensions were 51 x 15 x 15.5 cm, with a JO 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=2Q). Water temperature was maintained at 25,2 C.
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
18
DUP040012558
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^lO), 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*3 and silver*^. Flagfish 34 days of age (.standard length, 13 2.0 ran; weight, 0,058 0.027 g, n=20) were exposed to arsenic+3 in a proportional dilater system using dech'lorinated city water. They were tested simultaneously with fathead minnows in glass exposure chambers (20.5 x 30 x 25 cm) divided with screen into two sections, Flagf ish sections contained an average of 2A ( of water. Twenty fish per chamber were tested. The diluter cycle time of 19 min provided 6.9 volume
V: additions of water per day. The test was conducted at a mean water temperature of 25,8 C>
Flagfish 30 days of age (standard length. 12.7 + 1,6 ran; weight, 0.044 0.021 g n-30). were exposed to silver+1 in a proportional diluter system using Lake Superior water. They were tested simultaneously with fathead minnows in glass chambers (15 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 (Appendices A and B, Tables A-l and B-l, respectively).
Daphnia magna - Daphm'a magna was used as the test species in static acute tests conducted with 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetrachloroisthane , pentachloroethane, hexachloroethane, tetrachloroethylene, 1,3- di chlorobenzene, 1,2,4-tri chlorobenzene, di-n.-butylphthaTate, chlordane, nickelH2, and arsenic*3. Acute tests with chlorinated ethanes, ethylene, and
14
DUP040012559
benzene were conducted using first instar (< 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 animal's were maintained in a constant temperature water bath (20 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 photoperpod coupled with a IS 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 '4-
Conducting Basic Acute Tests with Fishes, Macroinvertebrates, and Amphibians Draft Mo, 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 mg*L The measure of acute toxicity was the 48 hr median effective con centration (48 hr EC^q ) based upon complete immobilization and the 48 hr median lethal concentration (48 hr LCgQ) based upon death as determined by cessation of heart beat and gut movement. Both were determined using a 30X dissection scope.
Toxicant concentrations were measured during each test (Appendix B, Table B-l). Water quality parameters are presented in Appendix A, Table A-2.
The Daphitia 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
DUP040012560
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 MM (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-candies- Flasks were observed daily for mortalities. Water quality para meters and toxicant concentrations are presented in Appendices A and 0, Table* . A-2 and B-l, respectively.
The Daphnia acute static test with chlordane was conducted with Lake Superior water (first instar, <24 hr old from the Environmental Research Laboratory-Duluth, MM stock cultures}. Ten organisms were placed into solutions in 200 ml. erlenraeyer flasks. Five concentrations and a control were tested* in
' t-
duplicate. The flasks were kept in a 21 C water bath, and a 16L:BD photoperiod was used. Flasks were observed daily for mortalities. Water quality parameters and toxicant concentrations were measured (.Appendices A and B, Tables A-2 and B-l, respectively).
The Daphnia acute test with nickel was conducted in Lake Superior water, using 10 organisms (first instar <24 hr old, from the Environmental Research Laboratory-Duluth, MM 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 ni of water. A 16L:8D phptoperiod 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 arsenic4^ was conducted in Lake Superior water using 9-11 organisms (first instar, 24 12 hr old, from the University of Wisconsin-Superior, WI stock culture) per flask. Six concentrations plus a
16
DUP040012561
control in duplicate were tested. The flasks were maintained at a temperature of 14.8 0.8 C, with a photpperidd 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 {Appendices A and b , Tables A-2 and B-l, respectively).
Scuds - Gamfflarus pseudoliiiinaeus was tested in acute tests conducted with pentachJoropbenol, arsenic*^, silver^, lead+^, and chromium^. All were flow through tests conducted in proportional diluter systems (Mount and Brungs, 1967) using Lake Superior water.
In the pentachlorophenol test, IS organisms (jc = 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).
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 NiteP^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
DUP040012562
16 min, providing 12.9 volume additions per day. The photoperiod and light
Intensity were the same as above. Water temperature was IS.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+1 was conducted under the same
conditions as described for the arsenic+3 test. Ten organism's- CD-3 -1.1 tan,,y
total length1 per chamber were tested. The diluter cycle time was 15,8 rain,
providing 13.1 volume additions of water per day. The test- water temperature
was 13.9 + 0.5 C, Water quality parameters and toxicant concentrations were
monitored throughout the test {.Appendices A and B, Tables A-1 and B-1,
respectively).
'ir
An acute test with Gammarus and lead+2 was conducted under identical condi-
tions as described for the arsenic*f*3 test. Fifteen organisms {.0,053 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 fcetrachldrqethylene, 1,2-dichlorobenzene, 1,4-dichlorobenzene,
hexachlorobenzene, pentachloropheno}, hexachloroethane, chromium*6, lead*2, -Kl +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 {>208 mi, volume), with test solutions. Each
chamber with a 3 mm glass overflow tube contained lake water plus a small amount
of food {.ratio of 1L:0.OO25 L), along with a fine layer of sand on the bottom.
Food was a mixture of Cerophyl and trout pellets blended with water.
18
DUP040012563
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 silver1'1 and selenium44 tests) in a 20 C water hath. Hexaehlorobenzene and
pentactiTorophenoI tests were acclimated and run at room temperature (22.5 25.6 C). A photoperiod of 16L:8p at an Intensity Of 1,9 ft-candle$ s/as main- . tained throughout the test. In the case of the silver41 test, the Tight was
turned off at 24 hr due to an observable color change (graying) at the higher toxicant concentrations. In the selenium+4 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 ram, and toxicant slowly dripped in from a separatory funnel at a rate of approximately 2 mL-min"1.
Five toxicant concentrations plus a control, in duplicate, were tested, with
the exception of hexaehlorobenzene. '7
Hexaehlorobenzene 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-1. DMF was V
used as a solvent carrier at a mean concentration of 1086 mgL~1. The
crystallizing dishes were twice siphoned and replaced with fresh hexachlpro-
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 Tack
of movement when prodded.
19
DUP040012564
The water was analyzed daily for toxicant concentrations (Appendix B>
Table B-l). Water quality parameters were also monitored (Appendix A, Table
A-l),
Sreen Algae - Selenastrum 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, tight 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"1 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 pro-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% (EC^q ) was determined from interpolation by the trimmed
Spearman-Karber method.
'
20
DUP040012565
I
; t8l8i3-i26r38.6.5..
EPA-600/S-83^095 September 1983
' 4^
..*
"< \ v5
*1
\
TOXICITY AND METABOLISM STUDIES WITH ERA 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
1
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
REPRODUCED BY
. .,
u s. DEPARTMENT OF COMMERCE
NATIONAL TECHNICAL
INFORMATION SERVICE
SPRINGFIELD, VA 2216}
1 i
DUP040012566