EPA-600/3- -84-009
January 1984
AQUATIC TOXICITY TESTS TO CHARACTERIZE THE HAZARD OF VOLATILE OKCAK7C
•&- CHEMICALS IN WATER: A TOXICITY DATA SUMMARY -- PARTS I AND H
by
*K. Alinad, D. Bcnoit, L. Brooke, D. Call, A. Carlson, D. DeFoe, J. liuot,
A. Moriarity, J. Richter, P. Shubat, C. Veith, and C,
Co-Projsct Coordinators:
J. M. McKiia and R. A. Drunmond
Environmental Research Laboracr.ry
U.S. Environmental Protection Agency
Duluth, MN 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MN 55804
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PB34-14150S
Aquatic 7o:iicity Tests to Characterise the
Ea^ard of Volatile Organic Chemicals in Wat?::
A Toxicifcy Data Eurasiacy. Parts 1 and 2
(U.S.) Environmental Research Lab.-Duluth, MN
Jan 34
Ssparfcnent of
Tech?scw
on Service
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TECHNICAL REPORT DATA
(fiafa raid Infjvericnt an fa mmi bffryv cv*&itf*s}
». MC7OAT NO.
EPA-600/3-84-009
2. WSCIPiBNT'S ACC&S4ION NO.
4. TITLE ANO SUCTITUi
Aquatic Toxicity Tests to Characterize the Hazard of
Volatile Organic Chemicals in Water: A Toxicity Data
_Surn-nary — Parts I and II t
». KEPOHT BATO
January 1984
O. PSaPORS.IMC OMCAMlZATtCM CXIDfl
7. AUTHORS* H> Ahmad**, D. Benoit*, L. Brooke*, D. Call*,
A. Carlson'-, D. DeFoe*. J. Huot**, A. Morlarity**,
J^Rxchter*. P. Shubat*. G. Veith*. and C. Vtel|bridge*.
H8FCKT NO.
10. PfiOCNAM H.2M8MY WO.
*U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
G201 Congiion Boulevard
Duluuh, MN 55804
ii.
2. SfOHiONINO AOCNCV NAMC AMD ADOPltiS
Saae as above
IS. TVPC OP REPONT AND f eKttif) COVERED
14. SrONSOiMNO AOCNCV COUE
EPA/600/03
1. SUPPLEMENTARY NOT£»
**Center for Lake Superior Environmental Studies, University of Wisconsin-Superior
Superior, Wisconsin 54880
ia. ABSTRACT —^^———~——
This summary presents acute and chronic toxicity test data and bioconcentra-
tion factors cor ^.led over a 2-year p
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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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TABLE OF CONTENTS
Subiect Page
Introduction • 2
Expected publications 3
Methods 5
Acute toxicity with fish 5
Acute toxicitv to rainbow trout 7
Acute r.oxicity with Papbnia 12
Development of earlv life stage, mini-diluter apparatus 16
Chronic toxicity with fish 19
Chronic toxicity with Dapbnia 22
Results and Discussion. 24
Acute toxicity fish 24
Acute toxicity - invertebrates 30
Chronic toxicir.y fish 30
Bioconcentrat ion 36
Chronic toxicity - invertebrates 36
Summary and Conclusions 43
References Cited 53
Appendix A: Mini-Di luter Design Manual (end of te;:t)
ill
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LIST OF TABLES
Number Page
1 Summary of Analytical Conditions and Recoveries 8
2 Results of Flow-Through Acute Toxicity Tests (mg/1) with
Fathead Minnows 25
3 Mean and 95X Confidence Intervals for the 96-Hr 50% Effect
Concentration ('..C50) and 5C2 Lethal Concentrations (LC50) for
Rainbow Trout Exposed in Lake Superior Water to Various
Organic Compounds 27
4 Acute Toxicity Values for Daphnjq maena Exposed to Eight
Chlorinate" Aliphatic Compounds for AS Hrs 31
5 Effects of Chlorinated Ethylenes, Propanes, and Butadienes
as Survival and Growth of Fathead Minnows in 32 Day
Embryo-Larval Tests 32
6 Effects of Chlorinated Benzenes on Survival and Growth of
Fathead Minnows in 32 Day Embryo-Larval Tests 33
7 Effects of Chlorinated Ethanes on Survival and Growth of
Fathead Minnows in 32 Day Embryo-Larval Tests 34
8 Bioconcentration Factors Determined for Ten Chlorinated
Aliphatic Compounds in Fathead Minnows Exposed for 32 Days . . 37
9 Chronic Effect/No Observed Effect Concentration Ranges for
Daohnia magna Based on Recroductwe Success and Growth
During 28 day Test? 38
10 Chemicalc Teste-'l in Fathead Minnow Careinogenesis Study. ... 42
11 Summary of Acute Toxcity Data for Fathead Minnows, Rainbow
Trout and Daphnia 45
iv
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OF TABLES (Continued).
Number
.*•
12 Sumrrary of Fathead Minnow and Daohnia Chronic Toxicity Data. . . 48
13 A Comparison of BioconcentratLon Factors for Chemicals Tested
in Present Study in Fathead Minnows vs. Other Species of 'fish
in Other Studies 50
Aprsendix A: Tables in Text
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LIST OF FIGURES
Figure 1: Mini-diluter exposure system for conducting early life sta^e ... 17
toxicity tests.
Appendix A: Figures in text.
. vi
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INTRODUCTION
The objectives of this study were to develop hioassav methods, and
provide information on the relative toxicity and metabolic relationships
between selected aquatic organisms and higher animals. Investigations were
divided into three major areas. The first phase involved determining
similarities and differences in metabolism of selected xenobiotics between
aquatic and mammalian organisms. Results of this work have been reported
under separate cover (Ahmad et al., 1981). The second phase of the study was
to develop methods for testing volatile chemicals, and to evaluate the
sensitivity and similarity among daohnids, embryo-larval fish and mammals.
The third phase was directed toward evaluating the use of a fish carcino-
genesia modei, involving the fathead minnow, as a predictor of environmental
carcinogenesls.
Chemicals selected for testing were chosen from four classes of
compounds - halomethanes, chlorinated ethanes, chlorinated benzenes'and
chlorinated ethylenes. These classes were suggested by personnel at
HERL-Cincinnati who planned to study many of these same chemicals in
mammalian systems. These data should be of oarticular interest to these
people.
This report represents an overview of research results obtained by a
number of different investigators. All data has been, or is scheduled to be,
oiblished iii Deer-reviewed scientific iournals. A listing of scientific
reports expected as a direct, or indirect, result of monies allocated for
this unit of studv follows.
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PUBLICATIONS EXPECTED AS A RESULT OF THESE STUDIES
Ahmad, N., Catherine Horiarity and James Huot. 1981. Microsooal metabolism
and binding of carbon tetrachloride, chloroform, 1,1,2-trichloroethane ,
1,1,2-trichloroethylene and monochlorobenzene by microsoaal fractions of
rainbow trout and water flea. (Manuscript in preparation).
Kenoit, D. A., F. A. PuRlisi, and D. L. Olson. 1981. A compact continuous-
flow mini-diluter exposure system for testing early life stages of fish
and invertebrates in single chemicals and complex effluents. Water Res.
(In oress).
Renoil, D. , F. Put-lisi, and D. ("ilson. 1981. A fathead B-.ir.now early life
sta^e toxicity test method evaluation and exposure to four organic
. chemicals. J. Environ. Pollut. (in press).
Renoit, D., R. Syrett, and F. Freeman. 1981. Design manual for construction
of a continuous flow mini-diluter exposure system. (Submitted to USEPA
for approval, April 24, 1981).
Carlson, A., and P. Kosian. 1981. Toxicity and bioconcentration of several
chlorinated benzenes in fathead minnows. (Manuscript in preparation).
Carlson, A. 1981. Effects of low dissolved oxygen concentrations on the
toxicity and bioconcentration of 1,2,4-trichlorobenzene in early life
fathead minnows. (Manuscript in preparation).
OeFoe, I). 1981. Effects of four chlorinated ethanes and one chlorinated
ethylene on survival and growth of fathead minnows. ("anuscript in
preparat ion),
Richter, J. E. , S. F. Peterson, and C. F. Kleiner. 1981. Acute and chronic
toxicity of some chlorinated benzenes, ethanes, and tetrachloroethylene
to Paphnia magna. (Manuscript in preparation).
3
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Shubat, P., S. Poi^er, M. Knuth, D. Hammermeister, A. Lima, L. Brooks, D.
Call, and T. Felhaber. 1981. Acute toxicitiee of nine chlorinated
organic compounds to selected freshwater organisms. (Jlanuscript in
preparation).
Shubat, P., S. Poirer, M. Knuth, and I. Srooke. 1981. Acute toxicity of
.tetrachloroethylene and teirachloro^thylene with dimethylfonnamide to
rainbow trout. Bull. Environ. Contam. Toxicol. (In press).
Veith, G., D. Call, and L. Brooke. 1981. Structure-activity relationship
for estimating bioconcentraticn factors and acute toxicity with fish.
(Manuscript in preparation).
Walbrtdge, C., J. Fiandt, G. Phipps, and G. Holcombe. 1981. Acute toxicity
of ten chlorinated aliphatic hydrocarbons to the fathead minnow.
(Manuso-ipt in preparation).
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METHODS
Acute Toxicity with Fish
Exposure Systeo—Proportional -diluters (Mount and Brungs, 1967) were
used to carry out these tests. The. dilution factors were 0.6 (that is each
concentration, except the control, was 0.6 times the next higher
concentration). With the five test concentrations used this covered a range
of 2 orders of magnitude. All the chambers were duplicated. Flows were 3.2
to 10 tank-volumes per day. In general, all methods followed closely those
of the committee for toxicity tests with aquatic organisms (1975).
Physical an
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When start inr, a test 10-50 fish were randomly assigned to each of the 12
exposure tanks. Dead fish were counted and removed at least twice during the
first day, and twice daily after that. The nnparatus used was described in
Phipps et al. (1981).
Methods not discussed here followed those specified by the U.S.
Environmental Protection Agency (1975).
Chemical Methods—The chemical analyses for these compounds were
performed by gas chromatography; 1,1,2-trichloroethane,
1,1,2,2-tetrachloroethane, tetrach loroethvlene, pentachloroethane ,
hexachloroethane, and hexachlorcbutadier.e were all run on a Hewlett-Packard
5730A automatic gas chromatograph equipped with a Model 3552A data system and
a Ni electron capture detector. The column was packed with 100/120
raesh Supelcoport® coated with 1.5% SP2250/1.952 SP-2401. The carrier gas was
5% methane in argon and the column temperature was adjusted between 40 and
80*C depending on the compound. Retention times varied between 1.50 and 5.00
minutes. The 1,2-dichloroethane, 1,2-dichloropropane, 1,3-dichloropropane,
and 1,1,2-trichloroethy.lene were run on a Tracor MT-220 manual gas
chromatograph with a Ni electron capture detector. The column was
/
packed with 80/100 mesh '.Jas-Chrom Q® coated with 4% SE-30/5% OV-210. The
carrier gas, column temperatures, and retention times were the same as above.
Gas chromatographic analyses on the benzene compounds were performed on a
model 5730A Hewlett-Packard gas chromatograph equipped.with an auto sampler,
a Ni electron capture detector, and a Hewlett-Packard Model 3354B
laboratory automation data system. The column was 2.3 mm (l.D.) x 2 m packed
with 1.5/1.95 percent SP-2250/SP-2401 coated Supelcoport (100-120 mesh). The
carrier gas was 5 percent methane in argon. The injector and detector
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temperatures were 250 and 300 0, respectively, and the oven temperature for
each chemical is presented in Table 1.
Water samples were added directly to 100 ml volumetric flasks to which
50 ml of hexane had already heen added. The samples were then stirred
vigorously on magnetic stirring devices for 1.5 h and allowed to separate for
1 h. The samples were then diluted with hexnne if necessary and analyzed as
outlined above by comparison to known hexane standards. Known amounts of the
chemicals were added to water samples to determine the efficiency of this
extraction procedure and the recoveries exceeded 95%. Final results were not
corrected for recovery.
Statistical—The. LC50 concentrations were calculated bv using the
Trimmed Spearman-Karber method for estimating median lethal concentrations
(Hamilton et al., 1977).
Acute Toxicitv to Rainbow Trout
Exposure System—Lake Superior water was used for all tests. It was
modified only by heating or cooling portions and mixing them together in the
proportions necessary to yield the desired test temperatures. Temperature
was controlled within^ 1.0°C of nominal test temperatures.
Fish were exposed in a flow-through diluter with room air temperature
maintained at 12*C, and a controlled photoperiod of 16 hr light (28-29
ft.c.). Test chamber water exchange rates ranged from 3.2 to 9.3 times per
24 hr. Test chamber dimensions (l.D.) ware 21.0 x 35.0 x 24.5 era, and
contained water at a depth of 9.0 cm (pentachlorobenzene and hexachloro-
benzene were exceptions). Ten fish were exposed per chamber resulting in
chamber loadings ranging from 1.3 to 4.1 g/L. Five exposure concentrations
and a control were used for. all tests except hexachlorobenzene.
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TABLE 1. SUMMARY OF ANALYTICAL CONDITIONS AND RECOVERIES.
Chemical
Hex achloroe thane
Pent achloroe thane
1 ,1 , 2, 2 -Tetr achloroe thane
Tetrachloroethylene
1 , 1 ,2-Tr ichloroethane
^exachl or o-l,3 -butadiene
Hexachlorobenzene
1,2,3 ,4-Tetrachlorobenzene
1 , 2 ,4-Trichlorobenzene
1 ,3-Diuhlorobenzene
1 , 3-Dich lorobenzeno
Sample
Vol (ml)
200
200
200
200
200
200
100
100
100
100
100
Extract
Vol (ral)
150
150
150
150
150
150
50
50
50
50
50
GLC
Temp. (*C)
80
60
60
40
40
100
160
80
130
80
80
Spiked Recoveries (a)
Water (%)
109
N.D.
96
N.D.
96
95
98
100
99
102
107
Tissue (%}
89
86
82
74
N.D.
96
91
94
92
95
99
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Rainbow trout tests with pentachlorobentene and hexachlorobenzene were
run in a diluter at 12 C (nominal) water temperatures. Two toxicant
concentrations (saturation and 10 times saturation, nominally) and a control,
all in duplicate, were tested in 30.0 x 60.0 x 15.0 cm glass chambers
,r
containing 27.0 L of water. The water metering cells delivered l.C L of
water to each test chamber every 8.2 minutes and the toxicant was delivered
simultaneously by metering pumps into mixing chambers before entering the
exposure chamber. Stock solutions of dimethylformamide (DMF) containing the
appropriate amounts of hexachlorobenzene were prepared for each o'.imo enabling
each exposure chamber to receive the same amount of DMF and different amounts
of hexachlorobenzene. Roth hexachlorobenzene and DMP concentrations were
measured in the water. Ton fir-h were tested in each chamber for 96 hrs with
a photoperiod of 16 hr light. Tor «ach test, all exposures and controls were
duplicated. Mortalities were observed and recorded at 1, 2, 4, 8, 12 and 24
hr and daily thereafter.
Physical Chemical Conditions—Total hardness, acidity, total alkalinity
(all as mg/L as CaCOj), pH, and dissolved oxygen (mg/L) weve measured
several times at 3 or more exposure concentrations in test chambers during
each test. Exposure chamber water temperatures were measured daily.
The ranges for all water chemistries were: total hardness - 50.6 to
56,8 mg/L as CaC03; total alkalinity - 44.6 to 53.1 mg/L as CaC03;
acidity - 1.97 to 4.1 mg/L as CaCC^; oH - 6.8 to 7.5; dissolved oxygen -
8.0 to 9.6 mg/L; and temperature - 11.6 to 12.7*C.
Chemical Methods—Chamber water concentrations of the toxicants were
measured in all chambers twice during each test (i.e., at: the start and at 96
hrs). On the other days of exposure one chamber of each replicate was
measured for toxicant concentrations. 1,2 ,4-trich lorobenzc.ne was extracted
9
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into petroleum ether and 1,2-dichlorobenzen«, and 1,4-dichlorobenzene were
extracted into hexane and analyzed by GLC. Water samples of 2-50 ml are
placed into 100 mL volumetric flasks with 50 mL of organic solvent. The
total volume is brought up to 100 mL with distilled water, stirred for 20 min
on a magnetic stirrer, and diluted as appropriate for GLC analysis.
GLC analyses were performed on a Tracer 550 gas chromatograph with a
63Ni detector and column packing of 3% OV-101 on 100/120 mesh Gas Chrom Q
and *?2 carrier eas flow rate of 50 mL/min. At a column temperature of
120 r, i,2,4-trichlorobenzene had a retention time of 0.61 min.
Recovery of 1,2,4-trichlorobenzene from Lake Superior water spiked over
a range of concentrations from 0.1 to 10 mg/L was 96.8 _+_ 2.0% for 12
analyses. Recovery of 1 ,2-dichlorobenzene from Lake Superior war.er spiked
over a concentration range of 8.4 yg/L to 3.4 rag/L was 103.7 +_ 2.6% for 15
determinations, and recovery of 1,4-dichlorobenzene spiked into Lake Superior
water over a concentration range between 0.2 to 20 ng/L was 100.1 _+ 3.0% for
19 determinations.
Hexachloro- and pentachlorobenzene concentrations in vater were
determined by extraction into hexane and analysis by GLC. Water samples of
1-5 mL are placed into 18 mL glass-stoppered test tubes, 3 drops of saturated
NaCl solution was added, followed by the addition of 5.0 mL hexane. The
tubes were shaken for 3 minutes, and diluted as necessary for GLC analysis.
GLC analyses were performed on a Tracer MT 160 gas chromatograph
equipped with a Ni electron - capture detector and a column packing of
3% OV-101 on 100/120 mesh Gas Chrom 0. At a column temperature of 205 C and
a N2 Carrier eas flow rate of 50 mL/rain, retention times were 2.41 and 1.18
min for hexachloro- and pentachlorobenzene, respectively.
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Recovery of hexachlorobenzcne from Lake Superior water spiked with test
compound over a concentration range from 1.0 ug/L to 1.0 mg/L was 96.6 _+ 3.12
for 23 dctc-rminations. Recovery of pentachlorobenzene spiked into Lake
Superior water over a concentration range from 10.0 ug/L to 10.0 mg/L was
94.3 ^4.0% for 13 determinations.
For the hexachlorobenzene, peritachlorobenzene, and letrachloroathylene
acute tests ir. which DKF was the stock solvent, levels of DMF in exposure
ch&ftbers were determined. DMF was analyzed on a. UV-visible double-beam
spec trophotonieter at a wavelength of 200 nm.
Water samples containing hexchloroethane and tetrachloroethvlene ware
extracted by adding 5.0 to 50.0 mL of sample to a 100 mL volumetric flask
containing 50.0 mL of hexane. Samples less than 50.C mL were diluted to
volume with distilled water. The samples were stirred vigorously for 20
minutes on a magnetic st.irrer. Samples were allowed to stand 15 minutes,
then diluted as necessary for GLC analvsis.
GLC analvsis was performed on a Tracer 550 instrument equipped with a
"Hi electron-capture detector. The 180 cm x 4 mm column was packed with
3Z OV-101 on 100/120 mesh Chroraosorb® W. The carrier gas was argon-methane
(95:5) at a flow rate of 50 mL/min. All peak area calculations were
performed by a Hewlett-Packard Laboratory Automation flata System. Detector
and inlec temperatures were 300 C and 225 C, respectively. Column
temperatures for hexachlorof i har.e and tetrachloroethylene were 130 and 65 C,
respectively.
The retention time of hexachloroethane was 1.30 minutes and the
sensitivity was about 1 pg at an attenuation of 32X. The retention time of
tetrachloroethylene was 1.60 minutes and the sensitivity was about 1 pg at an
attenuation of 64X.
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Recovery of hexachloroethane from Lake Superior water spiked with the
test compound over a concentration range of 0.20 to 2.0 ug/mL was 96.7 * 2.92 ,
for 16 determinations.
Recovery of tetrachloroethylenfc from Lake Superior water spiked with the
test compound over a concentration renge of 0.086 to 43.2 ue/mL was 89.9 2.
6.2X for 23 determinations.
Water samples containing 1,3-hexachlorobutadiene were extracted by
adding 75.0 ml of samples to a 100 mL volumetric flask containing 25.0 raL of
isooctane, and stirring vigorously for 20 minutes on a magnetic stirrer. The.
samples were allowed to stand for 15 minutes, then diluted as necessary for
GLC analvsis.
GLC analysis was performed on a Tracor 550 instrument equipped with a
° Ni electron-capture detector. The 183 cm x 6 mm column was packed vith
3Z OV-101 on 80/100 mesh Chromosorb* W. The column oven was operated
isothermally at 220 C. Detector and inlet temperatures were 300 C and 215 C,
respectively. The carrier gas was argon-methane (95:5) at a flow rate of 50
tnl/min. A Hewlett Packard automatic samoler was modified to fit the Tracer
GLC and all calculations were performed by a Hewlett Packard Laboratory
Automation Data system. The senstivity of 1,3-hexachlorobutadiene was about
1 pg at an electrometer attenuation of 16X. The retention time with the
above conditions was 2.35 minutes.
Stat istical—The LC50 concentrations were calculated by using the
trimmed Spearman-Karber method for estimating median lethal concentrations
(Hamilton et al., 1977).
Acute Toxicitv with Tiaohnia
Exposure System—Adult daphnids (Daphnia magna) were originally obtained
from the laboratory stock reared at the U.S. Environmental Protection
12
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Agency, Duluth, MN. All culcuring and testing were done using Lake Superior
water which was filtered «5ym), heated to 20°C, and aerated with filtered
air. Means and ranges for total hardness and total alkalinity of test waters
were 44.7 (43.5-47.5) and 41.5 (37.0-45.5) ng/L as CaCo3» respectively.
x
Chemical measurements were made in accordance vlth procedures in American
Public Health Association (1975). Additional chemical characteristics of
Lake Superior water are summarized In Eieslnger and Christensen (1972).
Cultur'lng and testing were done in an enclosed constant temperature water
bath (20 + 1 C). A combination of Gro-Lux and Duro-Test (Optima KS)
fluorescent bulbs provided 344 lumens at the air water interface and were on
a 16L:8D photoperlod coupled with a 15 minute transition period between light
and dark phases. Brood cultures of 25 animals in 1 L beakers were maintained
by renewing, food (30 mg/L) and water three times each week. For acute and
chronic testing, first instar daphnlds (<24 hours old) were collected froa
brood animals of approximately 3 weeks in age.
Chemical stock solutions were prepared by saturating lake water with the
test chemical on a magnetic stirrer plate.
Acute bioassays were conducted according to the ASTtl "Standard Practice
of Conducting Basic Acute Toxlcity Tests with Fishes, Macroinvertebrates, and
Amphibians" (ASTM 1979). Test containers were 200 raL erlenmeyer flasks
filled to 200 or 160 raL for unfed and fed tests, respectively. The flasks
were tightly stoppered with foil wrapped neoprer.e stoppers. Food
concentration was 20 mg/L. Acute toxicity endpcints were the 48 hr median
effective concentiacion (48 hr EC50) determined by complete immobilization,
and the 48 hr lethal concentration (48 hr LC50) based on death, deterrcined by
cessation of heart beat and gut movement. Both endpoints were determined
using a 30x dissection scope.
13
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Physical-Chemical Conditions—Oxygen was measured with either a Beckman
Model 0260 Oxygen Analyzer or by Winkler titration. pH measurements were made
with a Corning Model 12 pH meter. These measurements were generally made at
low, medium, and high toxicant concentration of both new and old samples.
Total alkalinity und total hardness measurements were made according to API1A
(1975). All values of these measurements fell within the ranges given for
rainbow trout on page 9.
Chemical Method"—All chemicals used in preparing standards were taken
from the sane stock bottle as those used for the exposure test system. The
chemicals were purchased from the Aldrich Chemical Company and ranged in
purity from 95 to 99 percent. The solvents, hexane, iso-octane, and acetone
were purchased from Burdick and Jackson Laboratories, Inc. and were glasc
distilled gas-chromatography grade. Standards and spike solutions v/ere
weighed with a Sartorius analytical balance and prepared in 100 niL volumetric
flasks. Because of the volatility of the chemicals being tested, both the
standards and the spike solutions were refrigerated while not in use and
renewed after one month.
Water samples were taken three times a week. The samples included both
the initial and final concentrations of the exposure water in the renewal
static test system. Seventy-five mL of sample from selected test bottles were
transferred with the aid of a funnel to 100 mL volumetric flasks containing 25
mL of hexane. A Teflon-coated magnetic stirring bar was ^placed in each flask
and stirred rigorously for one hour with at least half of the solvent in
suspension. When necessary the samples were stored in a refrigerator for no
longer than three days.
The effectiveness of the extraction method was examiied by determining
the percent recovery of a known amount of chemical in water. The recoveries
14
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for several chemicals ranged from 91 to 103 percent. Duplicate samples of
the same test bottle and concentration were also used to determine the
accuracy of the overall analytical method. Accuracy of the analytical aethod
was within 92 percent.
A Hewlett-Packard 5710A gas' chromatograph equipped with an autosampler,
a Hewlett-Packard 3354C data systen, and a Ni pulsed electron capture
detector was used for the analysis. The computer systen was capable of auto-
matically Injecting the samples, integrating the detector response, calibrat-
ing standards, analyzing a set of samples, and storing the data. A 6 foot by
2 mra (ID) glass column packed with 80/100 mesh Gas Chrora 0® coated with 1.5%
OV-17 plus 1.95% QF-1 was used with the following compounds and their
respective isothennal over, temperatures: 1,1, 2, 2-tetrachloroethane (75 C) ,
hexachlorobenzene (150 C), 1,2,4-tricMorobenzene (110 C) , pentachloroethane
(90 C), and hexachloroef.hane (100 C). A 6 fcot by 2 ram (ID) glass column
packed with 80/10O mesh Gas Chrcm QS coated with 4% SE 30/6% OV-210 was used
for the additional compounds and their respective isothermal oven tempera-
tures: 1,3-dichlorobenzene (HOC), 1,1,2-trichloroethar.e (50 C),
1,2-dichloroethane (50 C), and tetrachloroethylene (50 C). For all compounds
the Injection port temperature was 200°C and the detector temperature was
300 C. The carrier gas was 5% methane In argon with a flow rate of 41.7
mL/min.
Stat1stleal--EC50 and LC50 values were derived using the measured mean
effective toxicant concentrations (averag~ initial and final test solution
concentrations) and we're calculated by probit, moving average, or biuonial
formulas depending on the characteristics of the data.
15
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Development of Early Life Stage, Mini-Diluter Apparatus
In order to successfully and safely test volatile chemicals it was
necessary to develop specialized exposure systems and methods for testing air
and water leaving these systems. Interest in developing the early life stage
(ELS) fish toxicity test led to the "esign of a compact continuous flow
nini-diluter exposure system which accurately delivers as little as 3 liters
of test water per hour to each of 5 concentrations plus a control. This
system can be used to tesr. the effects of either single chemicals or treated
complex effluents on young fish in the laboratory or in the field. The small
ELS test apparatus takes less space and requires smaller volumes of test
water which is a critical factor when shipping effluents to the laboratory or
conducting on-site toxicitv tests. Smaller volumes of test water ;»lso
reduces filtration costs when one i* reauired to remove hazardous test
chemicals before discharging waste water to the sewer.
The ELS test system has been tested and evaluated in th* laboratory and
on-site in a mobile trailer. This apparatus has been used to conduct fathead
minnow (Pimephales^ promelas) ELS exposures to various toxicants including
volatile organic compounds, metals, nesticides, and- treated complex effluents
from metal plating, oil refinery, and sewage treatment plants. The system
also has been successfully used for testing macroinvertebrates.
Figure 1 shows a photograph of the compact stationary vented exposure
system for conducting ELS tests. The vented plywood enclosure is sealed with
fiberglass or epoxy paint on the inside and measures 76 cm wide x 120 en long
with a height of 112 cm over the exposure chambers and 159 cm over the
dilter. Both apparatus ancl test fish can easily be observed through viewing
windows located on the sides and top. One 5 cm hole located near the bottom
of each side allows a continuous flow of air to be drawn through the
16
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FIGURE 1:. Mlni-diluter e/:pc?ure system for conducting early life stage
toxicity tests.
17
-------
enclosure and out a 10 cm exhaust vent located over rhe diluter. Exhaust air
can be purified through a charcoal filter if necessary. Ample space is
available in the bottom of the enclosure to install apparatus such as
chemical saturators, stock bottles, metering pumps, or special filters used
to remove the test chemical before discharging waste water to the sewer.
Tliis systera is ideally suited for use in testing hazardous volatile
chemicals and was designed to protect the investigator from possible harmful
exposures to toxic fumes. Negative pressure createo on the inside of the
enclosure enables one to safely service the system and take care of the test
fish through small sliding glass doors. During tests conducted at our
laboratory the enclosure was vented through the laboratory air exhaust system
which drew an average of 0.7 cubic meters per min through the enclosure
(aporoximate ly one air volume: every 2 min). Air samples taken vith one 30 x
30 cm sliding glass door open have shown measurable quantities of volatile
test chemical inside the enclosure but no detectable concentrations were
found outside.
Another feature of the enclosure is if the diluter leaks or overflows,
the soilled test water can be diverted directly to the system's drain lines
and will not flood the room. Due to the fiberglass or epoxy paint, the
enclosure bottom is also water tight and can hold up to 100 liters if a leak
should occur in some other part of the systera. An alarm can he installed in
the enclosure base to warn the investigator of major leaks. The accumulated
test water in the base can then be drained off through a discharge valve and
if necessary passed through a, filter.
Design details of the mini-diluter exposure system has been written
(Bo.noit, Syrett and Freeman, 1981) and submitted to U.S. EPA for approval as
a design manual (for Appendix A). This manual compliments a paper,
18
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undergoing peer-review by scientific journal editors, titled "A Compact
Continuous Flow Mini-Diluter Exposure System for Testing Early Life Stages of
Fish and Invertebrates in Single Chemicals and Complex Effluents" by Benoit,
Mattson, and Olson (1981).
Chronic Toxicity with Fish
Exposure system—All tests were conducted with the above described
raini-diluter exposure system installed in a vented enclosure. The small.
system incorporated four renlicatc glass exposure chambers (18.7 y. 7 x 9.2 cm
high) at each of five concentrations plus control. The mini-diluter
delivered 15 mL of test water per min to each replicate 500 mL chamber. Test
water delivery tubes were positioned by stratified random assignment.
Replacement time for 90% of the test water was calculated to be approximately
75 rain in exposure chambers (Sprague, 1969). Water depth in each chamber
measured 4.5 cm. All test chambers were carefully siphoned daily with a
large pipette and squeeze bulb, after larvae began feeding. Cleaning was
done just before the last feeding of the day. Cool white fluorescent lamps
were used as the main source of illumination and a constant daylight
photoperiod of 16 hr was maintained. Light intensity at the water surface
ranged from 30 to 60 lumens.
Physical-Chemical Conditions—Water obtained .lirectly from Lake Superior
was passed through a sand tilter and ultraviolet sterilizer; and then heated
to a test temperature of 25^1 C. Total hardness, alkalinity, acidity, and pH
were determined on water from the control chambers once a week; dissolved
oxygen was measured in each treatment twice a week. The means for total
hardness, alkalinity, did acidity determinations were 45, 42, and 3 me/L as
C03, respectively. The mean dissolved oxygen was measured at 7 tng/L and
19
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the mean pH equaled 7.4. Chemical measurements were made according to the
American Public Health Association et al. (1975).
Chemical Methods~-Saturated water solutions of hexachlorobutadiene,
1,2-dichloropropane, 1,3-dichloroprooane, 1,2--dichloroethane,
*r
hexachlorcethane, pentachloroethane, 1,1,2,2-tetrachloroethane ,
tetrachloroethvlene, 1,1,2-trichloroethane, hexachloro-1,3-hutadiene,
hexachlorobenzene, 1,2,3,4-tetrachlorobenzene, 1,2,4-trichlorobenzene,
1,3-dichlorobenzene, 1,4-dichlorobenzene (98-992 purity) ' were used
as the toxicant source to avoid the use of solvents. Test chemical stock
solutions during'each test were continuously made up with a chemical
saturator similar to one described by Gingerich et al. (1979). Stock
solutions were delivered from the. saturator to the diluter by an FMl
metering pump. Test concentrations were assigned to each exposure chamber by
stratified random assignment.
All test water treatments were measured twice a week in alternate
replicate exposure chambers. Chemical analyses of the test water samples
containing hexachlorobutad iene , 1, 2-dichloropropan'3 , 1,3-dichloropropane , and
1,2-dichloroethane were done by solvent extraction followed by gas
chromatography as described for acute tests with fish. Comparisons of
chemical concentrations within each group of four replicates, sampled
simultaneously, showed that concentrations were within 90% of each other
(range, 85-98Z). Samples from selected replicates were also split and
*• The U.S. Environmental Protection Aijency neither recommends nor endorses
any commercial product; trade names are usea only for identification.
2 Aldrich Chemiccl Co., Milwaukee, WI 53233
3 Fluid Metering, Inc., Oyster Bay, NY 11771
20
-------
analyzed separately to check the variability of the method used to measure
test water concentrations. Results from eight split samples fhowed that
reproducibility of the chemical analysis was within 98% (range 96-992).
Detection limits and percentage recovery of 8-11 spiked samples for
hexachlorobutadiene, 1,2-dichlorooropane, 1,3-dichloropropane, and
1,2-dichloroethane were 0.03 ug/L and 97% (range 82-1092), 0.09 rag/L and 992
(range 93-106%), 0.1 mg/L and 100X (range 97-107%), and 0.1 mg/L and 102%
(range 97-105%). respectively. Because of the similarity of the other eleven
chemicals tested the methods are described in general terms and specific
conditions for each chemical are presented in Table 1. The individual
measurements are presented in subsequent tables.
Water samples were siphoned directly from the tanks into volumetric
flasks to which hexane had previously been added. After filling to the
volumetric mark, a Teflon stirring bar was added and the sample was extracted
by vortex mixing with a magnetic stirrer for 1.5 hours. The chases were
allowed to separate for 0.5 hours, and an aliquot was removed, diluted if
necessary, and transferred to a GLC sample injection vial for analysis. The
calculation of water concentration was based on original volume of hexane
pipetted into the volumetric flask before filling with water.
Gas chromatographic analyses were performed on a model 5730A
Hewlett-Packard gas chromatograph equipped with an auto sampler, a Ni
electron capture detector, and a Hewlett-Packard Model 3354B labortory
automation data system. The column was 2.3 mm (l.D.) x 2m packed with
1.5/1.95 percent SP-2250/SP-2401 coated Supelcoport (100-120 mesh). The
carrier gas was 5 percent methane in argon. The injector and detector
temperatures were 250°C and 300°C, respectively, and the oven temperature for
each chemical is presented in Table 1.
21
-------
Statist ical—Effect, no effect endpoints were determined as described in
Benoit et al. (1981).
Rioconcentration—Bioconcentration factors were calculated for those
chemicals listed in Table 1 on fathead minnows exposed for 28-days. Because
of the large number of samples and the small size of the individual fish,
surviving fish from each test concentration were composited into single
samples for the determination of tissue residues. Whole fish samples were
homogenized with 70 gras of anhydrous sodium sulfate previously cooled to
about ~5°C. The horaoeenate was transferred to a 300 mL Shell column and
extracted by elnting the columri with 250 mL hexane collected in a 250 mL
volumetric flask. An aliquot was diluted to an approoriatf. volume for
analysis. Gas chromato^rarhic *r.alvses ware performed on these samples as
described earlier for water sa-noles.
Chronic Toxicitv with Daphnia
Exposure system—Chrcnir bioassays (28-day) were conducted according to
the ASTM "Proposed Standard Practice for Conducting Static Renewal Life Cycle
Toxicity Tests with the Daphni-J, Daphnia magna" (ASTM, 1979), with minor
modifications to control volatile chemical losses. Test containers were 200
mL Erlenmeyer flasks filled to 160 mL, with the exception of
tetrachloroethylene v.hich was filled to 175 mL. The flasks were tightly :
stoppered with foil wrapped neoprene stoppers. All of the flasks were held
in a constant temoerature bath under a specified photoperiod as descirbed
earlier under acute toxicity with Daohnia.
Physical-chemical conditons--Identifical to those described for acute
toxicitv with Daphnia.
Biological, methods—Each flask contained one daphnid. Food
concentration was 20 mg/L. The tests with 1,1,2,2-tetrachloroethane,
22
-------
1,3—dichlorobenzene, 1,2,4-trichlorobenzenp. had seven replicates at. each of
six test chemical concentrations, whereas 1,2-diochloroethane,
1,1,2-trichloroethane, and tttrachloroethylene had 10 replicates at each of
six chemical concentrations. Young daphnids were filtered from each flask
after the transfer of the adults and washed onto a watch glass to be counted
alive with an Artek Counter*. If less than 20 animals are present they were
counted visually. Chronic toxicity was determined by reproductive success
and length ot: animals surviving the 28 day test. Counting the animals alive
eliminated the additional steps of poisoning and stirring to redisperse them.
This technique also allowed the determination of live from dead animals.
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 micrometer.
Statist ical—Both reproductive success and length were treated
statistically by analysis of variance and Dunnett's test. A NOEC (no
observable effect concentration) was determined to be the highest
concentration tested which was not significantly different from the control
values at either P
-------
RESULTS AND DISCUSSION
Acute Toxicity Fish
Fathead minnows—The 96-hr LC50 values and 95% confidence intervals of
these chlorinated aliphatic compounds are given, in Table 2.
The most acutely toxic compounds tested were hexachlorobutadiene,
1,2 , 3,4-tetrachlorobenzene, hexachloroethane, and 1,2 ,4-trichlorobenzene with
96-hr LC50s of 0.10, 1.07, 1.53, and 2.76 mg/L, resoectively. All other
compounds in the group were considerably less toxic. Two of the compounds,
hexachlorobenzene and pentachlorobenzene, were found to be acutely non-toxic
near water saturation; therefore, no 96-hr LC50 could be determined (Table
2). Acute toxicity increased in direct relation to the number of chlorines
on the molecule for the ethanes, bsnsenes, and ethylenes. The oosition of
the chlorine on the molecule made a difference in acute toxicity with 1,3 and
1,4-dichlorobenzene, but seemed to have little effect on 'the 1,2 and
1,3-dichloropropanes.
Rainbow trout: 1,2-Dichlorobenzene—Rainbow trout from Lake Mills,
Wisconsin, National Fish Hatchery (mean standard length, 5.6 cm; mean weight
2.7 g) were exposed to five concentrations (0.72, 1.26, 2.01, 3.07, and 3.81
mg/L) in duplicate, plus controls. Only one fish died beyond 48 hrs of
exposure. The 96-hr LC50 value was 1.61 mg/L (Table 3). ' Fish that were
unable to swim and Laid motionless on the exposure chamber bottom were
considered affected. The 96 hr EC50 value was 1.55 mg/L.
1,4~Dichlorohenzene--Rainbow trout fingerlings from Lake Mills,
Wisconsin, National Vish Hatchery (mean length 52.7 +_ 6.4 cm, mean weight 2.1
•*• 1.0 g) were exposed to five concentrations of d ich lorobenzene (1.74, 1.36,
0.83, 0.52, and 0.37 mg/L) in duplicate. The 96-hr LC50 was 1.12 mg/L (Table
24
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TABLE 2. RESULTS OF FLOW-THROUGH ACUTE TOXICITY TESTS (MG/L) WITH FATHEAD MINNOWS
EXFOSED TO 16 CHLORINATED ALIPHATIC COMPOUNDS.
Compound
Chlorinated Ethanes
Hexachloroe thane
Pen tachloroe thane
1,1* ,2, 2'-tetrachloroeth.ar,e
1,1-', 2-trichloroethane
1 ,2-dichlo roe thane
Chlorinated Benzenes
Hexachlorobenzeue
Pentachlorobenzane
1,2,3,4-trichlorobenzene
1 , 2, 4-trichlorobenzene
1 ,3-dichlorobenzene
1 ,4-dichlorobenzene
Chlorinated Ethylenes
Tetrachloroethylene
1,1* ,2-trichloroethylene
Chlorinated Propanes
1 , 3-d ichloropropane
1 , 2-dichloropropane
24 h LC50
(ng/L)
1.80a '
(1.70-1.91)
7.72
(7.45-7.99)
22.3
(21.9-23.8)
81.6
141
(131-153)
17.9
(17.3-18.4)
58.8
(57.8-59.7)
133
(126-139)
194
(184-205)
48 h LC50
(mg/L)
1.55
(1.47-1.63)
7.43
(7.16-7.71)
22.2
(21.2-23.1)
81.6
118
(111-125)
15.9
(15.0-16.8)
57.9
(57.2-58.6)
131
(124-137)
154
(144-166)
72 h LC50
(mg/L)
1.55
(1.47-1.63)
7.34
(^.07-7.63)
20.4
(20.0-20.8)
81.6
116
(110-123)
14.9
(13.9-15.8)
55.4
(53.0-57.8)
131
(124-137)
141
(132-151)
96 h LC50
(ng/L)
1.51
(1.43-1.58)
7.34
(7.07-7.63)
20.4
(20.0-20.9)
81.6
116
(110-123)
_c
1.07
2.76
(2.62-2.91)
7.79
4.16
13.4
(12.4-14.4)
45.0
(41.9-48.4)
131
(124-137)
140
(131-150)
25
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TABLE 2. (Continued)
Compound
Chlorinated Hutadienes
Hexachlorobutadiene
24 h LC50 43 h LC50
(mg/L) W/L)
*r
0.23
(0.20-0.26)
72 h LC50
(mR/L)
0.13
(0.09-0.18)
96 h LC50
(raj>/L)
0.10
(0.09-0.11)
a 95% confidence limits.
^ Here it was not possible to calculate confidence limits. There were no partial
kills. Mortality was either 0 or 100%.
c Not toxic at the highest concentrations that could be maintained in the. chambers.
26
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TABLE 3. MEAN AND 9f.Z CONFIDENCE INTERVALS FOR THE 96 HR 50Z EFFECT
CONCENTRATIONS (EC50) AND 50% LETHAL CONCENTRATIONS (LC50) FOR
RAINBOW TROUT (SALMO GAIRDNERl) EXPOSED IN LAKE SUPERIOR WATER TO
VARIOUS ORGANIC COMPOUNDS.
Comoound
96-hr EC50J (tn?t/l)
Mean
95% Confidence
Interval
96 hr-LC50 (ms/l)
Mean
95? Confidence
Interval
Chlorinated Ethanes
Hexachloroethane
O.R4
0.75-0.94
0.84
0.75-0.94
Chlorinated Benzenes
Hexachlorobenzene
Pentachlorobenzene/DMFc
1,2 ,4 -Trichlorobenzene
1,4-Dichlorobenzene
1,2-Dichlorobenzene
b
0.10d
1.27
1.10
1.55
0.09-0.12
1.11-1. .46
1.05-1.16
1.44-1.65
b
0.27d
1.52
1.12
1.61
0.20-0.37
1.34-1.72
1.05-1.20
1.48-1.77
Chlorinated Ethylenes
Tetrachloroethylene 4.86 (?) 4.99 4.73-5.27
Tetrachloroethylene/DMFc 5.76 4.71-7.05 5.84 5.05-7.67
Chlorinated FuLadienes
Hexachlorobutadiene
0.14
0.13-0.15
0.32
0.13-0.15
a Abnormal swimming behavior, usually loss of equilibrium.
** No effects on lethality observed at water saturation.
c Compound was administered as a mixture with dimethylforaraide to facilitate
solubility.
d 144-hr LC50 due to insufficient d>jath at 96 hrs to coraute a LC50.
27
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3). Loss of equilibrium occurred, as much as 12 hrs before death and was
recorded as an effect. The 96-hr EC50 concentration was 1.10 mg/L.
1,2,4-Trichlorobenzene—Rainbow trout fingerlings from Lake Mills,
Wisconsin, National Fish Hatchery (mean length 47.0 + A.O era, mean weight
1.55 +_ 0.42 g) were exposed to five concentrations of trichlorobenzene (2.82,
1.68, 1.10, 0.58, and 0.43 mg/L) in duplicate. The 96-hr LC50 concentration
was 1.52 mg/L (Table 3). Loss of equilibrium in the fish occurred as much as
48 hrs before death and was recorded as an effect. The 96—hr EC50
concentration was 1.27 rag/L.
Pentachlorohenzfene/rWF—Rainbow trout from Fattig Hatchery, Brady,
Nebraska (mean standard length, 6.9 cm; mean weight, 5.2 g) were exposed to
five concentrations of pentachlorobenzene (59, 120, 277, 435, £.id 714 ug/L)
in duplicate, plus controls. Saturation of Lake Superior water vith
pentach lorobenzene at 16.3*0 was 325 Ug/L. DMF was used as a solvent in a).I.
concentrations. DMF concentrations were nominally equal and averaged 395
rag/L between exposure chambers. The first death occurred after 48 hrs of
exposure but there were insufficient deaths to calculate a 96-hr LC50
concentration. The test was run for 144 hrs and the LC50 concentration for
this time was 0.27 mg/L (Table 3). Fish that had lost equilibrium or were
motionless on the chamber bottom were considered affected. The 144-hr EC50
concentration was 0.10 rag/L respectively.
Hexachlorobenzene/DMF—Rainbow trout from Lake Mills, Wisconsin National
Fish Hatchery (mean standard length, 33 + 3 cm; mean weight 0.46 _+_ 0.11 g)
were exposed to two concentrations (3.8 ana 80.9 ue/L) of hexachlorobenzene
in duplicate plus controls. All exposure chambers including controls
contained similar concentrations of DMF (932 +_ 12.9 mg/L). Fish did not die
or show signs of distress in any test concentrations in a 96-hr exposure.
28
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Hexachloroethane—Rainbow trout from Fattig Hatchery, Brady, Nebraska
(mean standard length, 66.A +_ 9.9 cm; mean weight, 4.3 +_ 1.8 g) were exposed
to five concentrations of hexachloroethane (0.34, 0.67, 0.97, 1.58, and 1.83
mg/L) in duplicate, plus controls. The 96-hr LC50 concentration was 0.84
mg/L (Table 3). Fish that lost equilibrium or were motionless on the chanber
bottom were considered affected. The 96-hr EC50 concentration was also 0.84
me/L. Tetrachloroethylene—Rainbow trout from Fattig Hatchery, ^rady,
Nebraska (mean standard length, 6.1 cm; mean weight, 3.2 g) were exposed to
five concentrations of tetrachloroethylene (2.41, 3.69, 6.39, J.I.2, and 17.3
mg/L) in duplicate, plus controls. All mortal i'; Ltes occurred during the
first 28 hours of exposure. The 96-hr LC50 value «as 4.99 mg/L (Table 3).
Fish that swam abnormally or laid motionless on the chamber bottom were
considered affected. The 96-hr KC30 was 4.86 mg/L.
Tetract. loroethylene/DMF—Rainbow trout from Fattig Hatchery, Brady,
Nebraska (mean standard length, 7.3 cm; mean weight, 5.9 g) were exposed to
five concentrations of tetrachloroethylene (2.23, 3.53, 5.95, 11.29, and
16.43 tng/L) in duplicate dissolved in dimethylformamide (DMF), plus controls.
The measured concentrations of DMF in the respective exposure chambers
beginning with the lowest'exposure (2.23 mg/L) were 75.8, 121.7, 220.3,
326.3, and 513.0 mg/L. The 96-hr LC50 and EC50 values were 5.84 and 5.76
mg/L, respectively. Although the results of the two tetrachloroethylene
tests with and without DMF are in reasonable agreement, there was concern
that the test fish in this test were unhealthy and showed symptoms of
distress at the termination of the test.
Hexachlorobutadiene--Rainoow trout from Lake Mills, Wisconsin, National
Fish Hatchery (mean standard length, 56 cm; mean weight 3.2 g) were exposed
to five concentrations of hexachlorobutadiene (66, 96, 229, 468, and 670
29
-------
ug/L) in duplicate, plus controls. Fish deaths occurred throughout the. 168
hrs of exposure. The 96-hr LC50 value was 320 ug/L (Table 3). Affected fish
swam erratically, lost equilibriunr and laid on the chamber bottom. The 96-hr
EC50 value was 140 ug/L.
Acute Toxicity - Invertebrates
Daphnia magna—The 48-hr LC50s and ECSOs values, including 952
confidence intervals, for eight chlorinated aliphatic compounds are presented
in Table 4.
The chlorinated ethanes increased in i'cute toxic ity with an increase in.
chlorine substitution (Table 4). The LC50 values ranged frora 268 rag/L for
' 1 ,2-dichloroethan^ to 2.9 rag/L for hexachloroethane. This trend also htld
for the 48-hr LC50 values obtained for 1,3-dichlorobenzene and
1 ,2,4-trich lorobenzene (Table 4) of 7.43 and 2.09 mg/L, respectively.
In general, feeding of the animals during acute tests had no apparent
effect on toxicity, with the exception of the results with tetrachloro-
ethylene in which feeding appeared to reduce toxicity.
Chronic Toxicitv-Fish - .
Fathead minnow early lite stage (F.LS) test—Larval growth vias the most.
sensitive indicator of toxic stress during the 32-day ELS toxicity tests
(Tables 5-7). Retarded growth of larval fish is critical, ana could have a
very profound effect on their ability to obtain food and compete with other
organisms in the natural ecosystem. Mean replicate control weights of
fathead minnows varied somewhat between tests. These differences in growth
were probably due to differences in the quality and quantity of food offered
to the fish between tests. Because of the difficulties in standardizing
quantities of live food fed to fathead minnows, such differences in growth
can be expected between tests, investigators, and laboratories. Regardless
30
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TABLE 4. ACUTE TOXICITY VALUES FOR PAPHNIA HACNA, EXPOSED TO EIGHT
CHLORINATED ALIPHATIC COMPOUNDS FOR 48 HRS.
I.C50
Unfed Fed
(mg/l)
Chlorinated Ethanes
Hexachloroer.ha..e
Pent ach lor oe thane
1,1 ,2,2-Tetrachloro-
c thane
1,1, 2-Tr ichloroethane
1 , 2-Dichloroethane
Chlorinated Benzenes
1,3-Dichlorohenzene
1 ,?. ,4-Tr ichlorobenzene
Chlorinated Ethyleues
Tctr achloroethyleae
2.903
2.50-3.33
7.323
5.98-8.99
62. I2
55.9-70.7
1863
164-214
2682
246-29 34
7.433
6.29-C.77
2.093
1.80-2.63
18. 12
15.5-21.8
2.351
1.99-2.86
8.023
6.89-9.39
56. 93
49.9-66.3
1743
154-201
3153
265-414
7.233
6.14-8.50
1.682
1.52-1.85
9.092
7.70-11.0
EC50
Unfed Fed
W/1)
2.103
1.82-2.45
4.693
3.99-5.50
23. 01
16.3-34.5
80. 61
57.5-113
J551
137-188
4.231
3.28-5.89
Nd
8.501
7.0U-11.5
1.812
1.61-2.07
6.883
6.07-7.85
25. 23
22.2-28.2
77. 81
56.6-107
!«32
154-225
5.981
4.85-9.53
>Jd
7.492
6.08-9.03
Nd = No determination.
2 Moving average method
3 Probit method
95% Confidence intervals
31
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TABLE 5. EFFECTS OF CHLORINATED ETHYLF.NES , PROFANES, AMD BUTADIF.NKS ON
SURVIVAL AND GROWTH OF FATHEAD MINNOWS IN 32 UAY EMBRYO-LARVAL
TESTS.
Chemical
Tested
Tetrachloroethylene
1 ,2-Dichloropropane
1 ,3-Dichlorupropane
Hexachlorobutadiena
Mean Chemical
Concentrat ion
Cwg/l>
0.0 (Controls)
500
1,400
2,800
4,100
8,600
100 (Controls)
6,000
11,000
25,000
51,000
110,000
200 (Controls)
4, .000
8,000
16,000
32,000
65,000
0.08
1.7
3.2
6.5
13.0
27.0
Percent
Survival
95
55 (explair.
83
38**
0**
0**
95
92
95
58**
27**
0**
93
98
93
97
98
49**
100
98
97
85
53**
55**
Me«n Individual
Wet Weight:'.
(mg)
258
able) 255
185**
118**
i 0**
0**
145
140
126*
79*
18*
0*
125
115
111
98*
79**
23** .
130 .
127
125
125
104**
32**
* Significantly different from controls (P = .05).
** Significantly different from controls (P = .01).
32
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TABLE 6. EFFECTS OF CHLORINATED BENZENES ON SURVIVAL AND CROUTH OF FATHEAD
MINNOWS IN 32 DAY EMBRYO-LARVAL TESTS.
Chcraical
Tested
Hexachlorobenzene
Pen t a chloro benzene
1,2, 3,4-Tetrachlorohenzene
1 , 2 ,4-Trichlorobenzene
1 ,4-Dichlorobenzene
.
1,3-Dichlorobenzenc
Mean Chemical
Concencratlon Percent
(yg/1) Survival
.03 (Controls)
.31
.66
1.16
2.58
4.76a Saturatlon=lO
0.5 (Controls)
3.3
6.7
13.0
27.7
54. 9a Saturation=l20
0.35 (Controls)
19
39
110
245
412
15 (Controls)
75
134
304
499
1,001
19 .
565
1,040
2,000
4,090
8,720
31 (Controls)
304
555
1,000
2,267
3,913
93
100
97
87
97
97
88
89
85
82
76
73
92
83
90
93
82
60*
92
83
92
91.5
88
62*
95
93
78*
0*
0*
0*
97
98
97
95
93
7*
Mean Individual
Wet Weight
(nig)
170
159
172
164
150
165
104
103
111
108
107
99
112
114
114
102
98
57
95
96
89
85
86
67*
101
100
87*
0*
0*
0*
100
99
99
102
67*
10*
a Highest concentration that could be maintained in chambers.
* Significantly different from controls (P = .05).
33
-------
TABLE 7. EFFECTS OF CHLORINATED ETHANES ON SURVIVAL AND GROWTH OF FATHEAD
MINNOWS IN 32 DAY EMBRYO-LARVAL TESTS.
Gieralcal
Tested
Hexachloroe thane
Pentachlc roe thane
1, 1 ,2,2-Tetrachloroethane
1 , 1 ,2-Trichloroethane
1,2-Dichloroethane
Mean Chemical
Concentration
(yg/D
0.9 (Control)
28
69
207
608
1,604
10.0 (Control)
900
1,400
2.9CO
4,100
13,900
12.0 (Control)
1,400
4,000
6,800
13,700
28,400
50 (Control)
2,000
6,000
14,800
48,300
147,000
300 (Control)
4,000
7,000
14,000
29,000
59,000
Percent
Survival
87.5
67.5
75
82.5
90
0**
85.0
82.5
77.5
92.5
45.0**
0**
95
100
95
95
12.5**
0**
100
100
95
100
77.5**
0**
92
95
92
92
97
90
Mean Individual
Wet Weight
(nig)
172
188
163
121*
38**
0**
218
226
147**
95**
46**
0**
' 191
186
150*
144**
25**
0**
144
152
140
122*
43**
0**
134
126
126
134
120
51*
* Significantly different from -ontrols (P = .05).
** Significantly different fiom controls (P = .01).
34
-------
of the different feedings rates between tests, one of the most importint
considerations when conducting an ELS toxicity test is that all groups of
fish within a test are offered similar amounts of food at each feeding:. Food
volumes must also be adjusted accordinp,ly when survival in any - repl icate is
reduced by 25, 50, or 75%. "The,foregoing feeding method will ensure that
significant growth differences (or lack of differences) between the control
and test concentrations were not due simply to poor feeding technique.
Larval survival was either equal or slightly less sensitive than growth
(Tables 5-7). Daily counts of live fish during each test revealed that all
reductions in survival occurred within two weeks after hatch. Replicate
control survival, ranging from 80-'002, was excellent during each of the
exposures (Tables 5-7).
The estimated MATC for fathead minnows exposed to hexachlorobutadiene
lies between 6.5 and 13.0 ug/L, and is based on reduced larval survival and
weight (Table 5). The estimated MATCs for fathead minnows exposed to
1,2-dichloropropane, 1,3-dichloropropane, and tetrachloroethylene lie between
6,000 and 11,000 ug/L, 8,000 and 16,000 Ug/L, and 500 and 1,400 ug/L,
respectively; and are based on reduced larval weight (Table 5).
The effects of chlorinated- benzenes on ELS are presented in Table 6.
Hexachlorobenzene and pentachlorobenzene were not toxic near saturation, 4.76
Ug/L and 54.9 iig/L respectively, therefore no estimate of MATC could be made.
The estimated MATC for 1. ,2,3,4-tetrachlorobenzene lies between i45 and 412
ug/L based on survival. The MATC ranges for 1,2,4-trichlorobenzene (499 to
1,001 ug/L) and 1,4-dichlorobenzene (565 to 1,040 ug/L) are b
-------
All estimated MATCs for chlorinated ethanes were based on wet weight
data (Table 7). These MATCs are as follows: hexachloroethane (69-207 ug/L;
pentschloroethane (900-1.400 ug/L); 1,1,2,2-tetrach loroethane (1,400-4,000
Ug/L); 1,1,2-crichloroethane (6,000-14,800 ue/L>; and 1,2-dichloroethane
(29,000-59.000 ug/L).
Results obtained from the preceding ELS test method evaluations and the
estimated MATCs derived from these evaluations demonstrate the usefulness and
consistency of the ELS toxicity test procedures for fathead minnows currently
being adooted as standards by the U.S. EPA and ASTM. These ELS test methods
produced good replication; and when used to oredict long-term chronic
toxicity, will provide an economical, means to O) develop water quality
criteria and (2) screen large numbers of single chemicalc, complex effluents,
or aqueous mixtures containing potentially hazardous chemicals.
Sioconcentrat ion factors—The most, readily bioaccuTiu lated chemical in
Table 8 was hexachlorobenzene and the least bioaccumulated was
1,1,2,2-tetrachloroethane. The bioconcentration potential of both the ethane
and the benzene groups was directly related to the number of chlorine atoms
on the molecule as shown in Table 8 by the calculated POP values (Cp/Cy). '.•
Chronic Toxicity - Invertebrates
Paohnia 28-dav tests—The 28-day no observable effect concentrations
(NOEC) were determined for Daohnia magna with three different groups of
chemicals (Table 9). The chronic NOEC based on growth were identical to
those based on reproduction for 1,3-dichlorobenzene, 1,2,4-trichlorobenzene
and tetrachloroethylene, but varied somewhat for 1,2-dichloroethane and
1, 1,2-trichloroethane. The toxicity generally increased with incressins; •
36
-------
TABLE 8. BIOCON'CENTRATION FACTORS DETERMINED FOR TEN
CHLORINATED ALIPHATIC COMPOUNDS IN FATHEAD MINNOWS
EXPOSED FOR 32 DAYS.
Chemical BCF Log BCF
Hexachlorosthane 756 2.88
Pentachloroethanc 6? 1.79
1,1,2,2-Tetrachloroethane 7 0.37
Tetrachloroethylene 74 1.87
Hexachloro-l,3-butadiene 6988 3.84
Hexachlorobenzene • 23391 4.37
1,2,3,4-Tetrachlorobenzene 2567 3.41
1,2,4-Trichlorobenzene 393 2.GO
1,3-Dichlorobenzene 97 1.99
1,4-Dichlorobenzene 112 2.05
37
-------
TABLE 9. CHRONIC EFFECT/NO OBSERVED EFFECT CONCENTRATION RANGES1 FOR
DAPHNIA MAGMA BASED ON REPRODUCTIVE SUCCESS AND GROWTH DURING 23
DAY TESTS
Compound
1,1 ,2,2-Tetrachloroethane
1,1, 2-Tr ichloroethane
1, 2 -D ichloroethane
Chemical
Concentration
MR/ l' (X+S.D.)
0
0
0.0
.419
.859
1.71
3.43
6.85
14.4
0.0
1.72
3.40
6.35
13.2
26.0
41.8
0.0
10.6
20.7
41.6
7
1.7
94.4
(Controls)
+ .036
+ .035
+ 17
+ .39
+ .90
+ 1.4
(Controls)
+ .16
+ .29
+ .52
+ 1.7
+ 2.2
1 3-°
(Controls)
+ 0.8
+ 1.7
+ 2.4
+
•f
137.0^
1 , 2 ,4-Trichlorobenzene.
1 ,3-Dichlorobenzene
0
0
0
0
0
0
0
f
.
4
^
•
•
0
0.
0
0
0
0
.
,
.
1
.0
018
039
079
162
363
694
.0
044
102
182
373
689
.45
4.8
5.5
9.0
(Controls)
•f
~
+
+
~
.003
.005
.011
.028
.056
.140
(Controls)
+
T
~
T
T
T
.012
.023
.039
.053
.156
.23
Number of
Young Produced
(x+s.n.)
162
84
69
71
78
78
23
150
95
132
146
163
114
1.1
164
128
88
54
43
19
166
151
159
157
125
107
32
165
167
178
212
137
190
93
+ 49
+ 50
+ 39
+ 40
+ 37
± 18
•t- 5**
+ 42
+ 53
+ 57
+ 55
+ 59
± 31
•f 4**
+ 45
+ 37
+ 51*
+ 24**
T 22**
+_ 21**
-
+ 51
> 60
+ 38
+ 25
+ 27
+ 30
+_ 20**
+ 23
+ 34
+ 30
+ 37
+ 46
+ 39
+ 30**
Length (mm)
of Adults
Cx+s.o.)
No Data
No Data
No Data
No Data
No Data
No Data
No Data
4.1 + .2
3.9 + .2
3.8 + .2
4.1 - .2
4.0 * .2
3.9 + .2*
3.9 + .2*
3.9 + .3
3.9 + .2
3.3 + .2
3.6 + .2
3.
3.
2.
3.
4.
3.
3.
3.
3.
3.
4.
4.
4.
4.
4.
it.
3.
4
1
3
9
2
9
7
6
6
0
2
4
3
5
1
3
C
+
T
_
+
+
~
~
+
+
4-
+
•f
+
7
+
T
+
.2**
.4**
.1**
.2
.2
.1
.1
.5
.2
.2**
.1
.1
.1
.2
.2
.2
.2**
38
-------
TABLE 9. (Continued)
Comoounc!
Tetrachioroethylene
Chemical
Concentration
mg/1 (Ic+S.D.)
0.0 (Controls)
0.75 +.036
0.159 + .085
0.254 + .094
0.505 + .250
1.11 + .480
1.75 + 1. 10
Nuir.be r of
Youn^ Produced
(X+S.D.)
154 + 47
165 «• 45
111 + 76
169 + 46
169 + 43
58 + 26**
0
Length (mm)
of Adults
(XjfS.D.)
3.9 + .2
4.1 + .2
3.9 + .4
4.0 + .2
4.1 + .1
3.6 + .1**
0
1 Chronic ranges comparable to MATCs for fathead minnows.
* Significant difference (P = .05).
** Significant difference (P = .01).
39
-------
chlorin.ition. Chlorinated ethanes were less toxic chronically, than the
chlorinated benzenes and ethylenss tested (Table 9).
Evaluation of fathead minnow model to detect carcinogenesis—Recent
studies with fish have indicated that certain trout strains are thousands of
times mere sensitive than raanmials-to a selected carcinogen administered \,i
the diet (Sinnhuber et al., 1977). Further investigations have shown that
the exposure of trout embryos to low pptn concentrations of a known carcinogen
for one hour will produce tumors in the juvenile fish (Wales et al., 1978).
The purpose of this project was to examine the possibility of using the
fathead minnow to screen volatile organic chemicals found in drinking water
chemicals for potential careinogenicity.
Surviving fathead minnows from 30-day ELS exposures to organic chemicals
were weighed alive at the termination of each test. Control, fish ar.d fish
from the highest toxicant concentration which showed no effect v,-cre moved to
freshwater aquaria. These two groups of fish (12-54 fish per group) were
held until they reached sexual maturity (5-6 mos.). They were then weighed,
sacrificed and examined grossly with the dissecting microscope. Special
attention was given to the liver and kidney of each fish, noti'ig general
appearance and/or any abnormalities, e.g. color, texture, nodules.
Liver and kidney tissue will be prepared for histological examination by
fixation in neutral buffered formalin, dehydration in graded alcohol
dilutions, and embedding in JE-4 plastic. Sections 3-5u thick were cut,
placed on slides and stained with methylene blue-Azure II-basic fuchsia.
These sections will be examined microscopically for tumors and/or
abnormal cells. A significant increase, in timor incidence above that seen in
control animals could red flag the chemical being tested for further in-depth
exposure studies.
40
-------
Histological studies—All fish from the 14 exposures (Table 10) have
been examined grossly and fixed in neutral buffered formalin. Twenty fish
(10 controls and 10 exposed) were chosen at random from each of the
1,1,2,2-tetrachlcroethane and hexachiorobenzene exposures and the livers and
kidneys' were dissected out and embedded in JB-4 plastic. Tissues from the
1,1,2,2-tetrachloroethane exposure have been sectioned, stained and examined
microscopically.
Because of the carcinogenicity status of the ethane chemical group, fish
from those exoosurcs will be. chosen next for histological examination.
41
-------
TABLE 10. CHEMICALS TESTED IN FATHEAD MINNOH CAKCINOGENESIS STUDY.
Chemical
Number of Fish
Control Exposed
Carcinogen Status
1,2-flichloroathane 48
(ethylene dichloride)
1,1 ,.2-trichloroethane **
(vinyl trichloride)
1,1,2,2-tetrachloroethane 25
(acetylene tetr^chloride)
Pentachloroethane 21
Hexachloroethane 13
Hexachlorobutadiene 50
1,3-dichlorobenzene (isotner of 13
1,4-d ichlorobenzenc)
1,4-dichlorobenzene 24
(p-dichlorobenzene)
1,2,3,4-tetrachlorobenzene 16
Pentachlorobenzene 15
Hexachlorobenzene 41
Tetrachloroethyler.e 27
(PerchlorcethyIene)
1,2-dichloropropane 51
(Propylene dichloride)
1,3-dichloropropane 37
**
54
21
i6
40
17
24
24
14
44
12
50
53
NCI +rat, mouse
NCI *roouse
NCI -Hnouse
Currently being
tested by NCI
NCI -Hnouse; Being
re-tested
EPA TSCA Inventory
Currently being
tested by NCI
*
*
NCI -Hnouse; Being
re-tested
Currently being
tested by NCI
EPA TSCA Inventory
* Benzene is a human suspect carcinogen; animal studies are inadequate.
** 56 fish total; control and exposed fish were -.nixed in aquarium.
42
-------
SUMMARY AND CONCLUSIONS
These data have been used in a number of important ways from criteria
documents and the structure-activity data base on aquatic toxicology to an
evaluation of the use of aquatic organisms in a screening program to serve as
an early warning system for higher animals including man. Many or these
chemicals have been detected in surface and subsurface drinking water
supplies of major cities, but at concentrations well beiow those causing
obvious acute toxic effects on higher animals or man. There was, however,
concern over the long term chronic effects of these chemicals continually
available to a population in the drinking water supply. It has long been
known that aquatic animals are extremely sensitive to chemicals of all kinds
at very low iig/L to ng/L concentrations in the aqueous environment. This
knowledge led us to the position of evaluating both acute and chronic
toxicity tests with several sensitive aquatic species in an effort to
determine the range of sensitivities and the possible application of the data
to the red flagging of chemicals, which after short inexpensive tests with
selected aquatic species were shown to be extremely toxic, highly
bioaccumulatable, and/or cause an increased incidence of tumors in exposed
animals.
Phase I of these studies, submitted as a separate report, was designed
to give us some preliminary information on the metabolic capabilities of
several of the lower animals (rainbow trout, Salmo gairdneri and water flea,
Daphnia raagna). These studies coupled wir.h earlier studies on MFO activity
in mammals and lower animals indicate the metabolic s- 'terns to be similar
qualitatively, therefore, the mechanisms leading to toxicity and neoplasia,
for example, are presumed to be similar in all organisms. Hence, aquatic
43
-------
animals are being used in laboratory screening and in environmental
tnoni coring.
Phase II was involved with the acute and chronic toxicity of five
classes of chlorinated org.mic compounds to selected fish and invertebrate
animals. In addition, the bioconcentration potential of these chemicals was
important in the determination of possible food-chain problems involving
man.
Of the five chemical classes tested the most acutely toxic to fish was
the one representative of the butadiene class hexachlorobutadiene followed in
decreasing order of toxic ily bv the chlorinated benzenes, ethylene.s , ethanes,
and the propane.s (Table 11). The invertebrate Daphp.ia magna showed the same
order of sensitivity as the fish for those classes of chemicals tested.
A comparison of species sensitivities in Table 11 indicated that Daphnia
was slightly more resistant than the fathead minnow, although quite similar,
while the rainbow trou1: was considerably more sensitive than either the
fathead or Daphnia except for the hexachlorobutadiena exposures.
Ona of the more interesting findings of the acute studies was the
increased toxicity of the ethanes, benzenes and ethylenes as the nuraber of
chlorines on the molecule incre'ased. This was true for both fathead minnows
and Daphnia (Table 11).
These data indicate that either fathead minnows or Daphnia would provide
essentially tha same acute values for these particular chemicals. It is also
true that these chemicals .>re not considered to be verv toxic to aquatic
species, since their 96-hr LCSOs are one to two orders of magnitude above
those environmental chemicals considered as extreraaly toxic.
Fifteen chronic toxicity tests with fish were also conducted on
chemicals in the five chemical classes. As with the acute toxicity tests the
44
-------
TABLE 11. SUMMARY OF ACUTE TOXICITY DATA FOR FATHF.AD MINNOWS, RAINBOW TROUT,
AND DAPHNIA.
Compound
Chlorinated Ethanes
Hexach loroethane
Pent achl or oe thane
1 , 1 , 2, 2-Tetracti loroethane.
1 ,1 ,2-Trich loroethane
1 ,2-Dich loroethane
Chlorinated Benzenes
Hexach lor obenzene
Pentachlorobcnzene
1 ,2,3,4-Tetrachlorohenzene
1 , 2, 4 -Tricolor obenzene
1 , 3-Dichlorobenzene
1 ,4-Dichlorobenzene
Chlorinated Ethvlenes
Tetrachloroethylene
1 , 1 ,2-Tr ichloroethylene
Chlorinated Propanes
1 ,2-Dich lor op ro pane
1 ,3-Dichloropropane
Fathead Minnow Rainbow Trout
96-hr LC50 96-hr LC50
(ra«;/l) (ing/ 1)
1.53 0.84
7.30 *
20.30 a
81.70 a
117.80 a
b b
fa b
1.07 a
2.76 1.52
7.79 1.61
4.16 1.12
13.50 4.99
44.1'. a
139.30 a
131.10 a
Daphnia
48-hr LC50
(m^/1)
2.90
7.32
62.10
186.0
268.0
a
a
a
2.09
7.43
a
18.10
a
a
a
45
-------
TABLE 11. (Continued)
Compound
Fathead Minnow
96-hr LC50
(rag/1)
Rainbow Tvout
96-hr LC50
(m$>/l)
Daphn ia
48-hr LCf>0
(mg/ 1)
Chlorinated Butadienes
Hexachlorobutadiene
0.10
0.32
Not tested.
Not toxic at saturation.
-------
chlorinated butadiene - hexachlorobutadiene was the most toxic follwed by
benzenes, ethylenes, ethanes, and oropanes in order of decreasing to/.icity
(Table 12). Six chronic values were also determined for Daphnia and in most
cases the sensitivity was similiar except for the ethanes where there seemed
to be considerable variation between the fathead and Daphnia results. Again,
chronic toxicitv increased considerably for both species as the number of
chlorines on ths molecule increased (Table 12).
The bioconcentration potentials of these chemicals were determined by
establishing a bioconcentrat ion factor (BCF) (C - ./C ) for
" Fish Water
fathead minnows exposed for 32-days to each chemical during the early
life-stage toxicity test. Those PCFs were then compared to BCF values for
other species of fish found in the literature (Table 13). In this study vith
the fathead minnows Che benzenes hiocor.cent rated the nost followed by
hexachlorobutadiene, the ethanes, and the ethylenes. It is interesting to
note again that bioconcentration also increases as the number of chlorines on
the molecule increases just as toxicity increased. The literature values for
other fathead minnow studies as well as bluegill and guppys all agree very
closely with the BOFs generated during the 32-day early life-stage toxicity
tests. This is an important finding in that it indicates age, size or
species of fish has little effect on the BCF generated over a 30-day period
of water exposure. Based on BCF values hcxachlorobenzene, hexachlorobuta-
diene, and 1,2,3,4-tetrachlorobenzene are the chemicals in the group which
might pose the greatest bioconcentration problem in the environment.
Phase III
A careinogenesis model using fathead minnows 'was designed to establish
whether or not fish might be a sensitive indicator of care ino^enesis in the
environment. Previous studies of fish had indicated exposure in the low ppm
47
-------
TABLE 12. SUMMARY OF FMHF.AD MINNOW AND PAPKNIA CHRONIC TOXICITY 1WTA.
Fathead Minnow Daphnia
32-day (ELS) MA^C 28-daya Chronic
Compound ( PR/ 1) ( lig/ 1)
Chlorinated Ethanes
Hexachloroethane 69-207
Pentachloroethane 900-1,400
1,1,2,2-Tetrachloroethane 1,400-4,000 6,850-14,400
1,1,2-Trichloroethane 6,000-14,800 13,200-26,000
1,2-Dichloroethane 29,000-59,000 10,600-20,700
Chlorinated Benzenes
Hexachlorobenzene 4.76 -
PentachLorobenzene 54.9 -
1,2,3,4-Tetrachlorobenzeno 245-412
1,2,4-Trichlorobenaene 499-1,003 363-694
1,3-Dichlorobenzene 1,000-2,267 • '.,450
1,4-Dichlorobenzene 565-1,040
Chlorinated Ethvlenes
Tetrachloroethylene 500-1,400 505-1,110
1,1,2-Trichloroethylene - -
Chlorinated Propanes
1,2-Dichloroprop^ne 6,000-11,000
1,3-Dichloropropane 8,000-16,000
48
-------
TABLE 12. (Continued)
Fathead Minnow Daphnia
32-day (ELS) MATC 28-day01 Chronic
Compound (ug/1) (viR/l)
Chlorinated Butadienes
Hexachlorobutadiene 6.5-13.0
° Effect - no effect concentrations.
b Saturation - no effects noted.
-------
TABLE 13. A COMPARISON OF BIOC01JCENTRATION FACTORS FOR CHEMICALS TESTED IN'
PRESENT STUDY IN FATHEAD MINNOWS VS. OTHER SPECIES OF FISH IN OTHER
STUDIES.
Chemicals
Present Study
Fathead minnows3
BCF
Log BC?
Literature Values
"Fathead
Minnow0 Bluegillc Guppyc
Chlorinated Ethanes
Hexachloroethane . 757 2.85
Pentachloroethane 62 1.78
1,1,2,2-Tetrachloroethane 7 0.91
Chlorinated Ethylenes
Tetrachloroethylene 75 1.79
Chlorinated Butadienes
HexachLoro-1,3-butadiene
6,988
3.84
a 32-day exposure ELS toxicity test.
b G. Veith, D. Call, and L. Brooke, (In preparation),
c 30-day old fish exposed f^r 30-days.
<* Adult fish exposed for 30-days.
138
68
8
49
Chlorinated Benzenes
Hexachlorobenzene
1,2,3 ,4-Te trachlorobenzene
1 ,2,4-Trichlorobenzene
1 ,3-T)ichlorobenzene
1 ,4-Dichlorobenzenc
23,391
2,567
398
97
112
4.37 21,878
3.41
2.60 1,698
1.99
2.05
14,454
1,820 3,631
646
66
60 91
50
-------
ran^e to developing embrvos was sufficient to induce liver tumors (Wales et
al., 1978). The present studies on 14 chemicals representing 5 classes of
organic chemicals indicated that gross tumors in the liver or kidney were not
present 4 months after hatching, however, a microscopic work up on the ethane
group (many of which are known, carcinogens) is underway now and will provide
more information on the usefulness of this approach as an early warning
experiment for environmental carcinogenesis.
The acute toxicity tests run with both fish and invertebrates
established a relative order of toxicity of the individual chemicals that
was identical to the order seen in the more sensitive chronic exposures.
Therefore, the short 4-day 96-hr 1.C50 fish exposures or the 48-hr Daphnia
exposures could be used to establish a priority list of chemicals found to
occur in drinking water to initially concentrate the more expensive chronic
testing on the more toxic materials.
The Daphnia acute test would be better than a fish acute, since it is
only 48-hrs long and does not require the more difficult flow-through system
required for a fish 96-hour LC50, yet it gives the same relative order of
chemical sensitivity (Tables 11 and 12).
Early life-stage toxicity tests with fish or 28-day Daphnia chronics
would provide the most sensitive tests for drinking water; however, the fish
exposures would also allow the determination of a BCF and the possibility of
determining an increased incidence of tumors in exposed fish, both of which
would provide further information for red flagging (prioritizing) chemicals
for more in-depth testing on mammals.
Since the amounts of these chemicals in drinking waters are in low vg/L
amounts it would be necessary to concentrate samples for testing, since these
51
-------
chemicals or groups of these chemicals would noL be toxic to aquatic animals
at most ambient levels now reported for U.S. or inking water supplies.
The usefulness of aquatic tests for red flagging chemicals in these
particular classes in drinking water may be somewhat limited, because of
their low toxicity and low ambient water concentrations. Howevar, this
approach for other more toxic chemicals has considerable promise as an early
warning system for higher animals including man.
52
-------
REFERENCES
Ahmad, N., D. J. Call, L. T. Brooke, and C. A. Moriarity. (Unpublished
Manuscript). Microsomal metabolism and binding of carbon tetrachloride,
chloroform, 1,1,2-trichloroethane, 1,1,2-trlchloroethylene, and
monochlorobenzene by microsotaal fractions of rainbow trout (Salno
gairdne.ri) and water flea (Daphnia magna).
Anerican Public Health Association, American Water Works Association, and
Water Pollution Control Federation. 1975. Standard methods for the
examination of water and wastewater. 14th ed. American Public Health
Association, Washington, D.C. 20036.
American Society for Testing and Materials. 1980. Standard practice for
conducting acute toxicity tests with fishes, macroinvertebrates, and
amphibians. ASTM E729-80. American Society for Testing and Materials,
Philadelphia, Penn. 25 pp.
Benolt, D. A., V. R. Mattson, and D. L. Olson. I981a. A compact continuous
flow mini-diluter exposure system for testing early life stages of fish
and invertebrates in single chemicals and complex effluents. Water
Res., In press.
Benoit, D. A., R. F. Syrett, and F. B. Freeman. 1981b. Design manual for
construction of a continuous flow mini-diluter exposure system. USEPA,
ERL, Duluth, Minn. Unpublished report.
Biesinger, K. E., and G. M. Christensen. 1972. Effects of various metals on
survival, growth, reproduction, and metabolism of Daphnia magna. J.
Fish. Res. Board Can., Vol. 29, pp. 1691-1700.
53
-------
Gingerich, W. H., W. K. Seim, and R. D. Schonbrod. 1979. An apparatus for
the continuous generation of stock solutions of hydrophobia chemicals.
Bull. Environ. Contam. Toxicol. , Vol. 23, pp. 685-689.
Hamilton, M. A., R. C. Russo, R. V. Thurston. 1977. Trimmed Spearman-Karber
method for estimating median lethal concentrations in toxicity
bioassays. Environ. Sci. Technol. 11: 714-719.
Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res., Vol. 1, pp. 21-29.
Phipps, G. L., G. W. Kolcombe, and J. T. Fiandt. 1981. Acute toxicity of
phenol and substituted phenols to the fathead minnow. Bull. Environ.
Contam. Toxicol. 26: 585-593.
Sinnhuber, R. 0., J. D. Hendricks, J. H. Wales, and G. B. Putnam. 1977.
Neoplasms in rainbow trout, a sensitive animal model for environmental
carclnogenesis. Ann. ?J.Y. Acad. Sci. 295: 389-408.
Sprague, J. B. 1969. Measurement of pollutant toxicity to fish. I.
Bioassay methods for acute toxicity. Water Res. 3: 793-821.
U. S. Environmental Protection Agency, Committee on Methods for Toxicity
Tests with Aquatic Organisms. 1975. Standard practice for conducting
acute toxicity tests with fishes and macroinvertebrates, and amphibians.
EPA-660/3-75-009. Duluth, Minn. 67 p.
Wales, J. H., R. 0. Sinnhuber, J. D. Hendricks, J. E. Nixon, and T. A.
Eisele. 1978. Aflatoxin B., induction of hepatocellular carcinoma in
the embryos of rainbow trout (Salmo gairdneri). J. Natl. Cancer Inst.
60: 1133-1139.
54
-------
APPENDIX A:. Early Life Stage Hlni-Diluter Design Manual
This manual was designed Co be used as a supplemental guide for the
crnstruct of a continuous-flcw mini-diluter system for toxiclty testing which
has been described and evaluated by Benoit et al. (1982 - see references).
Additional studies conducted by Anderson (Manuscript), Carlson and Kosian
ff
(Manuscript), Spehar »t al. (1230), and Benoit et al. (1982 - see references)
have demonstrated the usefulness of this test system and have also
illustrated the type of data one cm expect to obtain with young fish and
macroinvertebrates•
The following text on glass cutting, assembly, and equipment was taken
from Lercke et al. (1978). This information was included to familiarize the
reader with techniques currently used at the U.S. EPA Environmental Research
Laboratory-Duluth.
Glass Construction Equipment
Recommended equipment for diluter construction includes :. sharp glass
cutters, a glass cutting table, a glass saw, a set of glass drills, designed
for use on a standard heavy duty drill press, and a power stopper borer.
Sharp glass cutters are needed to obtain straight, smooth cuts to
prevent leaks. An optional piece of equipment is the glass cutting board,
similar to those used by hardware stores to cut window panes. One style is
available from Fletcher Terry Co., Bristol, Conn. 06010. A large flat
surface and a good straight edge- may be substituted. The glass saw is used
for cutting glass tubing and is generally useful for a variety of cutting
purposes. It is used to make cut ends on tubing, both square and angled, as
required during diluter construction. A rolling table model, such as the
Model C manufactured by Pistorius Machine Co., Hicksville, N.Y. 11801, is
desirable, but if only diluter and other glass tubing is to be cut, their
Model CC12, which has a tilting table, is satisfactory. The glass drills are
1
-------
necessary to drill holes In the various glass cells and are listed as diamond
iwpregnated tube drills In the catalog of Sommer and Maca, Glass Machinery
Co., 5501 W. Ogden Ave., Chicago, 111. 60650. Theje drills are relatively
expensive, but enable the dlluter builder to also drill drain holos in
aquaria and test chambers The drill press can be of any type, but should be
sturdy and vibration frt.- Turpentine is an excellent cooling lubricant to
use when drilling glass, and is recommended over water.
The boring of stoppers for various parts of a diluter is tiros consuming,
and a powar stopper-borer, such as that manufactured by E. H. Sargent Co.
(Model Mo. S-232DT), is very useful. Some glass bending is necessary,
therefore, an air-blast-type burner, such as that manufactured by Fisher
Scientific Co., is very convenient. This burner enables the operator to
apply sufficient heat re the tubing to allow uniform bending.
Accurate rulers and steel tapes, a micrometer for inside and outside
measurement, felt marking pens, and a sufficiently large work area to prevent
moving of assembled parts during construction and assembly also save time and
increase efficiency.
Glass Cutting
The primary skill needed to be successful in building a diluter is the
ability to cut glass with straight edges and parallel sides. A commercial
glass cutting board, if well maintained, is particularly good for long cuts.
A second technique is to use a large flat sturdy table and a heavy ruler or
other straight edge to guide the cutter. This latter technique is faster
and more versatile once mastered. All pieces should be cut with minimum
tolerance. After cutting, all edges should be dulled with a stone or
fine-grit sand paper to prevent hand cuts. The pieces should be cleaned by
washing in a detergent solution and then rinsed thoroughly and dried.
2
-------
Removal of grime Is necessary to ensure good glue adhesion. Glass should be
double strength (3 nun thick), but the "B" or second grade is satisfactory.
Flint glass tubing is preferred to Pyrex because the lower melting point of
the former makes bending and cutting the glass easier.
Glass Assembly
The most important construction material is the silicone sealant or
glass glue. Dow Corning Class and Ceramic Cement and General Electric
Corporation RTV are both satisfactory. Glues that are listed as dish-water
safe are preferable so that cleaning the assembled diluter with hot water
will not cause the joints to fail. Disposable 10- or 15-ml plastic syringes
with enlarged bores in the tips for faster application are useful for
applying a thin bead of glue as needed and can be used with one hand (Figure
1). Application with the original collapsible tube requires two hands to
maintain a steady and constant flow of glue from tube to the edges of the
glass. If the bead of glue is too thin, any irregularities in glass cutting
will not be filled by glue and will leak.
Lines are drawn to show the location of the cell dividers during
assembly. A wax pencil or felt pen can be used. It is important to renember
during assembly, however, that these marked surfaces should be on the outside
of the cells so that glue adhesion is not affected by these lines. Waxed or
other paper is placed on the table top to catch any excess glue. The paper
can be removed easily after the glue has dried. Slight pressure at all glued
joints distributes the glue, helps prevent leaks, and places the glass
surfaces in closer contact. To ensure against leaks, a pencil eraser or
rounded wooden dowel may.be used to spread the freshly applied, excess glue
along each seam. Use care to avoid moving the glass. After all cells have
-------
dried overnightj they should be tested for leaks after plugging the drilled
holes.
-------
REFERENCES
Anderson, R. L. Effect of pydrin on non-target aquatic invertebrates. U.S.
Environ. ?rot. Agency, Environmental Research Laboratory, Duluth, Mn.
55804. (Manuscript).
Benoit, D. A., V. R. Mattson, and D. L. Olson. 1982. A continuous- flow
mini-diluter system for toxicity testing, Water Research (In press).
Eenoit, D. A., F. A. Puglisi, and D. L. Olson. 1982. A fathead minnow
(Ptmephales proaelas) early life stage toxicity test method evaluation
and exposure to four organic chemicals. J. Environ. Pollut. (In
Press).
Carlson, A. R., and P. A. Kosian. Toxicity and bioconcentration of several
chlorinated benzenes in fathead minnows. U.S. Environ. Prot. Agency,
Environmental Research Laboratory-Duluth, Duluth, Mn. 55804.
(Manuscript).
Leoke, A. E., W. A. Brungs, and B. J. Halligan. 1978. Manual for
construction and operation of toxicity-testing proportional diluters.
U.S. Environmental Protection Agency, Ecological Research Series
EPA-600/3-7S-072.
^pehar, R. L., D. K. Tanner, and J. H. Gibson. 1980. The effects of
kelthane and pydrin on early life stages of fathead minnows (Pimephales
promelas) and amphipods (Hyallela azteca). Presented at The American
Society for Testing and Materials, 5th Annual Proceedings of Aquatic
Toxicology.
-------
(SIDE VEW)
TABLE TOP
(TOP VIEW)
GLASS
ADHESIVE
TABLE TOP
Figure i. Glue-application system. Syringe tip to be bored out to
approximately 4 ntn to give sufficient bead size.
-------
AQCD ARCt, ABCD
Figuro 2. Scliema''ic dra/ing and flow pattern of continuous flow n;ii: i-d i InLur . Legend: (C), concentration
flow tube; (DC), dilution call; (EO), emergency out lot; (FIJC), flow booster cell; (FSC), : '• >u
split' -»r cell; (FV), float valve; (T), toxicaiTt flow tub .2; (TC), toxicant cell; (W) , water Clow
(WC), wate' cell; (WO), water outlet; (IX), one \;oljme; (2X), two volumes.
-------
Figure 3. Diluter cells attached to back board: (A), toxicant and water
cell; (B), dilution cell; (C), flow booster cells: (D), flow
splitter cells.
-------
•68.6cm-
33
cm
I
12.7.
cm
7.5
cm
18
cm
0
Q
28
cm
0
Q
B
1-D
i
5.1
cm
i
0 0
0 0
0
D
\XXVV\SX\\\\N\X\\\\\\\\\\V\\X\\V\\XVSX\\\\V\X\\S\\X\\S\V\\XV
Figure 4. Oiluter back board made of 1.9 cm exterior plywood: (A), plastic
scorm window clips; (B), metal shelf bracket (5.1 cn); (C), netal
shelf bracket (3.8 cm); (D), sheet plastic, plywood or metal shelf
(0.3 ca wide).
-------
Screen insert bracket
Toxicant oei
ilufiint water cell
5.1cm
TOP
VIEW
k9.4cmJ
IT
W, W2
•SB
W4 W5;
-133cm—
-14.9cm-
!•*-
cm
.$cm-
•353cm-
•45.4cm
Figure 5. Toxicant and water cell. A constant depth is maintained at 3 cm
in each cell to obtain the prescribed flow rates. Arrows showing
location of drilled holes denote distance from left edge of glass
to center of hole. (T, toxicant; W, water; EO, emergency outlet -
1.4 cm holes).
Capillary
flow tube
T-
1
ana
W-5
W-l through
W-4
Adjusted
flow rate
100 mL/rain
50 mL/min
Size (ID) Length Stopper
2 mm 3 cm -:-0 1
1
1.5 mm 2.5 cm #0 1
1
Drill
.4
.6
.4
.6
cm
cm
cm
cm
°d hole
cenc
from
cent
from
3 red
edge
ered
edge
-------
TOP
VIEW
C2
« 9.7 cm—*
« 16.7 cnv
•23.7cm
\vo
-30.5cm-
•32.6cm-
•34.8cm-
Figure 6. Dilution cell. Arrows showing location of drilled holes denote
distance from left edge of glass to center of hole. (C, concen-
tration; WO, water outlet - 1.4 cm hole).
Capillary
flow tube
C-l through
C-6
Adjusted
flow rate
50 mL/min
Size (ID)
1.5 tnm
Length
3.5 cm
Stoncer
#0
Drilled hole
1 .A cm centered
1.3 cm from edge
-------
Flow booster cells:
FB; FB2
s ize ,
2.5 x 4.5 x
5 .4 cm
hole , 1.3 cm
s copper, v?00
12345 6
AECD ASCD A6CD ABCD ABCD ABC'J
Delivery Tube and Exposure Designation
Flow splitter cells:
size, 2.5 x 5.7 x
4.8 cm
hole, 1 ca
stopper, ?'000
capillary flow tube:
size, 1.5 aaa (ID) ,
length, 2.5 cm
Flow booster siphon
siphon standpipe:
siphon sleeve:
6 mm (OD) glass tube cut 8.3
cm long with glass sav notches
(3 imn deep) on upper end and
lower end tapered.
11 mm (OD) glass tube cut
3.8 cm long with a #000
stopper.
and
i*
flow
7igure ~i. Flow booster cells with siphon.
splitter cells. The lower ends of all
capillary flow splitter tubes fit loosely into
1.3 cm (OD) Nalgene elbows which are attached
with Bev-A-Line® tubing to 6 ran (OD) glass
delivery tubes.
-------
0.6cm Noigene
Elbow
9.2
cm
I8.7cm •
Water depth, 4.5cm
Drain hole, 1.4cm
Stopper, # 0
Standpipe, 6mm (CO) glass tube
cut 7cm long
Stainless steel screen, 4Omesh,
.010 wire
A, 6mm (OD) glass tube
B, pinch clamp
C, flexible Teflon tube
Figure 8. Exposure chamber with 6 mm (OD) glass delivery tube ana water
sampling siphon.
-------
Table 1. Dimensions and Nuaber of Double Strength Glass Pieces Needed to
Construct One Mini-Diluter and 24 Exposure Chambers As Shovn i.n Figures 2-8.
. • *
Toxicant and Mater Cell Unit:
bottom - 7.6 x 54.6 cm (1)
sides - 5.1 x 54 ctn (2)
ends - 5.1 x 7.6 cm (2)
full divider -5.1x7 cm (1)
partial divider - 3.8 x 7 cm (2),
screen holder - 3.2 x 1.3 cm (4)
stainless steel screen (20 mesh, .016 wire) - 5.1 x 7 CQ (2)
DiluE ion Cell:
bottom - 3.8 x 37.5 cm (1)
sides - 5.1 x 37.5 cm (2)
ends - 3.8 x 5.4 cm (2)
upper dividers - 3.2 x 4.5 cai (10)
lower dividers - 2.5 x 3.2 cm (10)
Flow Booster Cells:
bottom - 2.5 x 4.5 cm (6)
sides - 3.8 x 5.1 cm (12)
ends - 2.5 x 5.1 cm (12)
Flow Splitter Cells;
•bottom - 2.5 x 5.7 cm (6)
sides - 4.5 x 5.1 cm (12)
ends - 2.5 x 4.5 cm (12)
Exposure Chambers;
bottom - 7 x 18.8 cm (24)
sides - 8.9 x 18.1 cm (48)
ends - 7 x 8.9 cm (48)
divider - 1.9 x 6.4 cm (24)
stainless st«.-el screen (40 mesh, .010 wire) - 6.4 x 7 cm (24)
If glass drilling is not convenient, the bottoms of each diluter cell and
exposure chamber may be made from £316 stainless steel (3 mm thick).
-------
Figure 9. Diluter float valve used to maintain a constant head pressure in
the toxicant and water cell.
(A), alucinuo Flexafraoe© fittings; (B), extension clamp, medium;
(C), 7 jam (OD) glass tube water inlet, 8 ca long: (D), 14 no (OD)
glass tube stationary sleeve, 10 cm long; (hole notched in each
side to let water out and to clean valve orifice); (E), neoprene
tapered micro-stopper; (F), 11 mm (OD) glass tube sliding sleeve,
4 cm long; (G), float made from 30 ml Nalgene® bottle with 1.4 cm
hole bored in center of one side.
-------
roe
(Reinovoble)
203
" cm
Oilutcr
Bo.
737
cm
...
76.2
cm
RIGHT SIDE
Dilutee Flood
nciffte QOroin
45cm
6 6t-in
-895cm
235cm
33
on
87.6cm
3I.S
cm
.r..
Shell
._..!"
iv.vr.
I13«n
467
an
3.1 cm hole
oir Intel
8.6cm
.,__ ^JSffi .
| \ Bose
191
3E
112.4
cm
«••• I2l.9c:n
'^
t_J
;•;••---•--
-'SI-
< .162 „
cm
of
31.1
C 11
LEFT SIDE
116.1cm -
., "3 2 ..
cm
j /'l
D
46
2 9cm dole
tcf droin tine
It
l
xm
Icm Robbel
9cm — »•
.6cm
1219cm-
Figurii 10. Vented enclosure used with the mini-il iluter system to test
hazardous volatile chemicals. (Dilulur box nncl exposure box)
-------
FRONT
DILUTERBOX FRAME
Mem
350cm
-SOOctr-
(.Sdilulcr
flood droin ^
_
Icm flabhtl
rm ------ »•
L ___ __1
.._,.
89im
7G tin
(Removable)
X - 2.3cm: OIUJTER WATER INLET
Y-K>2em: AIR EXHAUST
Z-G.4tm: TOH ELECTRICAL CORDS PASSING
92.6cm
LEG SUPPORT
Figure 10. (Continued)
-------
~
r-\ J.
102cm \..x
i---/ n
' / "i
I ' 15.2cm 1 1
1 A ''
'f~cin"vl) II
/ 1
/i.^\<.m rtroin hole !
w/lcm rtohtiel !
i
|
1 •
1
I
K :
\ 'i
... .V Jl
SHELF
(Removable)
Slcin, XI
51
3cm
, HA |fm - • . ^
— ir TI — *; —
II M v
n ii
n n \
n "
* ii
n
n
n jj
\\ \\
MSE jj
1 1 ' !
jj |;
,i M
I !!
1 1 1 ^
t • *
i M /
J Ii ./....
7?.-1cm
T
-HS.Zr.rn
M 9cm
19cm-
5cmi_
112-1cm
\—
^
~\
Figure 10. (Continued)
-------
Table 2. Dimensions and Number of 1.9 en Exterior Plywood Pieces and Other
Materials Needed co Conscrucc One Vented Enclosure Shown in Figura 10
Exposure Box*: Diluter Box*:
base - 72.4 x 118.1 cm (1) sides - 13.4 x 72.4 (2)
3.8 x 118.1 cm (2) , top - 18.4 x 76.2 CD (1)
3.8 x 68.6 cm (4) botcoia - 18.4 x 76.2 cm (1)
front - 76.2 x 167.6 cm (1) front frame - 7 x 76.2 cm (4)
side - 112.4 x 118.1 cm (2) flood baffle - 5 x 72.4 cm (1)
raar - 76.2 x 112.4 cm (1)
top - 76.2 x 120 cm (1)
shelf - 53.3 x 117.5 cm (1)
10.2 x 117.5 cm (2)
shelf bracket - 5 x 49 cm (2)
Sliding 6 mm Plate Glass Doors: (all edges rounded)
sides2 (upper) - 30.5 x 30.5 cm (6)
right side2 (lower) - 45.7 x 45.7 cm (2)
top - 25.4 x 91.4 cm (2)
diluter box2 - 31.8 x 61 cm (2)
6 nan Sheet Plastic for Lower Front and Left Side: 50.8 x 50.8 cm (2)
E-Z Glide^ Aluminum Track; Upper channel, 3.66 M; lower channel, 7.32 M
Miscellaneous Equipment: Snap holders for Plexiglass, (8): leg levelers (4);
weatherstrip for removable diluter frame and exposure box top, 1.9 en wide;
weatherstrip for exposure box top glass, 1 cm wide; FVC bulkhead for waste
water drain (1.3 cm) and base drain (2.5 cm).
1 Bottom must be water tight.
2 Sliding glass doors with finger notches cut in on one side.
-------
6.4cm
-47cmOD —
505cm DIA-
I5.2cm DIA—H
25cm
2.5 err.
A
S.lcm
6.4cm
I
T
-NEOPRENE SEAL STRIPS
AIR FLOW
MATERIAL:24 GA GALVANIZED METAL
UNIT: 47CM »47CM
FRONT ond REAR OPEN FOR PANEt INSERTION end REMOVAL
SIDES WITH PADDED SEALS FOR AIRTIGHT FIT
Figure 11. Carbon panel adsorber frame used with the vented enclosure for
purification of exhaust air.
-------
Adjustable
Standpipe.
2.5cm
PVC P!pe
Waste -
Wafer
Screen Baffle to
prevent channeling
Plexiglass Plaie
with holes and
5.S. Screen
Recirculatlng Pump
Figure 12. Waste valve carbon filter for low-flow exposure systems (0.5
l./min). Two or more units con be used in series to increase
filtration. Debris siphoned off exposure chnmbei bottoms should
net be dumped on top of the carbon, but cnn he filtered out of the
siphoned test water with glass wool.
-------
gj' ta^.^.V^
-f -i «• r-I '7 ' 'fssaaaj
Figure 13. Continuous flow mini-diiuter for use wich either siugle chemicals
or treated complex effluents.
Figure 14, Portable early life stage exposure system
-------
Figure 15. Stationary vented early life
stage exposure system.
Figure 16. Vented enclosure for
testing hasardous volacile
chemicals.
-------
Figure 17-19. Steel support frame (66 x 102 x 74
cm high) and hardware for portable
exposure system.
Figure 20, Carbon panel absorber
rrame attached to a 5.1 x
41 cm sheetinetal adapter
for vented enclosure.
-------
Figure 21. Vent pipes for enclosure
systems.
Figure 22. Headboxes for either diluent
water or created complex effluents.
Legend (A) headbox with float valve;
(B) insulated headbox with issuers ion
heater. Water flows by gravity from A
to B through an interconnecting pipe.
' '"«fc
Figure 23 and 24. Fifty five gallon
effluent holding drum.
-------
Figure 25. Rocker arm assembly and light
attached to removable top of
vented enclosure.
Figure 26. Delivery tubes showing
stratified random assignment.
Figure 27 and 28. Exposure chambers with egg
cups on rocker arm.
-------
Figure 29 and 30. Saturator for either solid or
liquid hydrophobic chemicals showing reservoir with
float valve for make-up water, recirculating pump, —»••••.*
chemical flask, and liquid chemical transfer flask.
Figure 31. Air tight soda carbonation
can saturators can safely be
be used outside of enclosure.
Figure 32. Chemical saturator in lower
section in vented enclosure.
Chemical flask is inverted for
those chemicals lighter than
water.
-------
Figure 33-36. Vented enclosure protects the
investigator from exposure to
toxic fumes.
$&?%g*m
-'i.,
•'>5gS£a****rj*j '•'• ''^i^----,-^^f^?^,,^
.
-------
Table 3. Source List For Major Equipment Used With The Hir.i-Dilucer Exposure /
System
I. Test Water Temperature Control:
A. Syrett-Dawson temperature controller:
(Maximum rating: 500 watt with 8 amp fuse)
R. F. Syrett, and W. F.-Dawson. 1975. An inexpensive solid-state
temperature controller. Prog. Fish-Cult. 37: 171-172.
B. Heavy duty mercury relay: Model 760
Quick-set therzio-regulator: Model 7501
(Optional temperature controller)
H-B Instrument Co., American 4 Bristol St., Philadelphia 40, Pa.
C. Stainless steel immersion heater with 1.3 cm pipe threads:
500 watt-RIS-505
750 watt-RIS-755
Voice Co., 831 S. 6th St., Minneapolis, Mn. 55415
II. Vented Exposure Air Exhaust:
A. Exhaust booster fan: Auto-draft inducer model DJ-2
Tjernlund Products Inc., 1620 Terrance Drive, St. Paul, Mn. 55113
B. Carbon panel adsorber: Model PRC, 45.7 x AS.7 x 2.5 era
Barnebey Cheney, N. Cassady at E. 8th Ave., Columbus, Oh. 43219
III. Light:
A. Light timer: Model 101-G
Tork Time Controls Inc., Mount-Vernon, N.Y.
B. Fluorescent light fixture 61 cm: Model 5-120-TS-120-ACLC
Lithonis.Lighting, Box A, Conyers, Ga. 30207
-------
IV. Chemical Saturator:
A. Metering pump drive: RP-G50 or 150
Metering pump head: SSY 1 or 2
Fluid Metering Inc., 29 Orchard St., Oyster Bay, N.Y. 11771
B. Combination Open Air - Submersable Pump: Model 1-1C
March Manufacturing Co., 18-19 Pickwick Ave. , Glenview III. 60025
V. Waste Water Filter:
A. Combination Open Air - Submersable Pump: Model 1-1C
March Manufacturing Co., 1819 Pickwick Ave., Glenview, 111 60025
B. Activated Carbon: 6x8 pellets, coconut base
Union Carbide Corp. (Linde Division), P. 0. Box 372, 51 Cragwood
Rd., South Plainfield, N.J. 07080
VI. Alarm System:
A. Float switch: Model LS-1950
Load-Pak Relay: Model ST-22160
Gems Division, Farmington, Conn. 06032
B. Alarm bell: Model 340
Edwards Co., Inc., Horwalk, Conn.
VII. Effluent Holding Drum:
A. Combination open air - submersible pump: Model 1-1C
March Manufacturing Co., 1819 Pickwick .Ave., Glenview, 111. 60025
B. 208 1 polyethylene storage tank with spigot and cover: Number
04032
U.S. Plastic Corp., 1390 Neubrecht Rd., Lima, Oh. 45801
VIII. Effluent or Diluent Water Headbox:
304 .stainless steel, 20 gauge with welded seams and 1.3 cm stain-
less steel couplings welded in place: headbox A - 30.5 en wide x
-------
30.5 long x 40.6 cm high; headbox B - 30.5 cm wide x 45.7 en long x
40.6 ca high. A. G. O'Brien or Chester Ziom Sheet Metal Works,
Duluth, Mn.
IX. Egg Cup Rocker Arm Assembly (as shown in Fig. 17-19 and 25):
A. 2 RPM induction geared motor (RMS Motor Corp.) •
Blan Electronics Corp., 52 Warren St., New York, N.Y. 10007
B. Aluminum Flexafranie rods and fictings
Fisher Scientific, 1600 W. Gler.Lake Ave., Itasca, 111. 60143
X. Magnetic Stirrer for Diluter:
Stir-mate: Model 214-957
Curtis Matheson Scientific Inc.
XI. Inert Flexible Tubing for Diluter System and Saturator:
Bev-A-Line: 5 mm ID, 1 mm wall thickness
: 8 mm ID, 2 mm wall thickness
Thermoplastic Scientifics Inc., 57 Stirling Rd., Warren, N.J.
07060
XII. Stainless Steel Screen: (20 mesh, .016 wire; 40 mesh, .010 wire)
W. S. Tyler Co. Inc., 8200 Tyler Blvd., Mentor, Oh. 44060
XIII. Diluter Float Valves:
A. Aluminum Flexaframe rods and fittings
Fisher Scientific, 1600 W. GlenLake Ave., Itasca, 111. 60143
B. Tapered micro stopper
Scientific Products, 1210 Leon Place, Evanston, 111. 60201
C. Nalgene® 30 mL bottle
Scientific Products, 1210 Leon Place, Evanston, 111. 60201
-------
Aquatic Toxicity Tests to Characterize the Hazard of Volatile Organic
Chemicals in Water: A Toxicity Data Summary -- Part II
Final Data Summary Report: Phase 1,
Microsomal Metabolism and Binding of Carbon Tetrachloride, Chloroform,
1,1,2-Trichloroethane, 1,1,2-Trichloroethylene and Monochlorobenzene
by Microsomal Fractions of Rainbow Trout (Salmo gairdneri) and
Water Flea (Daphnia magna)
-------
INTRODUCTION
Halogenated hydrocarbons are among the most widely utilized industrial
chemicals. They are used as solvents, degreasers and intermediates in
chemical synthesis. Because of their desirable chemical and physical properties
and reasonable cost, a large volume of chlorinated aliphatic and benzene com-
pounds are used for manufacturing a variety of products. Some of these halo-
alkanes are known central nervous system depressants, hepatotoxins, nephrotoxins
and proven carcinogens (Anderson and Scott, 1981). Many haloalkanes are listed
as priority pollutants by the Environmental Protection Agency.
The present study was designed to assess' the in vitro comparative metabolism
and protein binding of carbon tetrachloride, chloroform, 1,1,2-trichloroethylene,
1,1,2-trichloroethane and monochlorobenzene by microsomal fractions cf rainbow
trout (Salmo gairdneri) liver and by post-mitochrondrial supernatant (PMS)
fractions of the water flea (Daphnia magna). Hepetotoxic and nephrotoxic
effects of some of these compounds in mammals have been extensively studied
£,nd reviewed in recent years (Plaa, 1977; Ahmed et aj_., 1980; Tsyrlov and
Lyakhovich, 1975; Rechnagel, 1967) but very little information is available
concerning the metabolic disposition or protein binding in fish species and
aquatic food chain organisms, such as Daphnia sp. Previous studies have shown
that carbon tetrachloride and other chlorinated benzenes have hepatotoxic effects
on fish liver (Pfeifer end Weber, 1979; Gingerich and Weber, 1979; Gingerich
et^ aj!_., 1978; Statham e_t aJL , 1978). Recent evidence suggests that carbon
tetrachloride, chloroform and other chlorinated alkanes are converted to toxic
metabolites by the microsomal mixed function oxidase system (Docks and Krishna,
1976; Watanabe et al,, 1978) in mammalian liver. Therefore, in this investiga-
-------
tion rainbow trout liver microsomes iind Daphnia PHS v;ere used to determine
the formation of water soluble metabolites and protein bindings of these
haloalkanes.
METHODS AND MATERIALS
Chemicals: Uniformly C labelled 1,1,2-trichloroethylene and 1,1,2-
trichloroethane were purchased from California Bionuclear Corporation, 7654
San Fernando Road, Sun Valley, California 91352. Uniformly C labt'led
1-chlorobenzene, chloroform and carbon tetrachloride were purchased from New
England Nuclear Corporation, 549 Albany Street, Boston, Massachusetts 02118.
Purity of these compounds ranged between 98-99 percent as determined by gas-
liquid chrornatography. . NADPH, NADP, glucose-6-phosphate nionosodium salt,
glucose-6-phosphate dehydrogenase fron. torula yeast, and cytochrome C were
purchased from Sigma Chemical Company, St. Louis, Missouri.
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 we'ight in phosphate buffer
were centrifuged twice at 10,000 g for 15 min in a Beckman L5-50 ultra-
centrifuge with a 50 Tirotor to remove nuclear and mitochondrial fractions,
which were discarded. The 10,000 g supernatant was centrifuged at 105,000 g
for 60 min using a T150 rotor. The supernatant was discarded and the pellet
was stored at -20 C until used.
-------
3.
Adult Daphnia (approximately 21 days old) were reared in the laboratory
from U.S. EPA Environmental Research Laboratory-Duluth brood stock. They
were collected on Whatman #1 filter paper, dried, weighed, and 0.5 - 2.5 g
of Daphnia homogenized with a teflon pestle homogenizer. The homogenate was
filtered through loose glass wool to remove chitinous materials, and centrifuged
twice at 10,000 g to remove nuclear and mitochondria! 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 arid rainbow trout liver microsomes according to tl.e method described by
Lowry et^ aj_. (1951). 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 l,'i,2-
trichloroethane, an incubation systen was designed to study their binding to
microsor.ial protein and their metabolism. This enclosed system consisted of
en erlenmeyer flask (.125 ml) which was fitted with a glass column (5 mn 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 (XL absorbing sytem
containing a solution of Carbosorb IIM The reaction mixture in the erlenmeyer
flask contained an NADPH-generating system (consisting of 3 pM Glucose-5-
phosphate, 1 unit-- Glucose-6-phosphate dehydrogenase, aid 1 pM MgCl2), 8 mg
microsomal protein from rainbow trout liver or 4 mg PMS protein from Daphnia,
in 0.07 M souiurn phosphate buffer(pH 7.5)and 0.1 ml of test compound with
known cmoun^ of radioactivity made to a final volume of 5 ml. The reaction
One unit will oxidize 1.0 wM of D-glucose 6-phosphate to 6 phospho-D-
gluconate per minute in the presence of NADP at pH 7.A at 25 C.
-------
4.
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, f>0 and 120 min with rainbow trout liver
microsomes or 0, 15 and 30 ir.in with PMS from Daphnia. The reaction was
terminated at various time intervals by addition of 1 ml of 3 M trichloro-
acetic acid (TCA) solution. The reaction mixture was then extracted thrice
with 10 ral 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 29.25. with recovery efficiency decreasing with time. Ths
14
loss 'ikely 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.
Aliquots of the hexane extract (representing parent compound) were
transferred to scintillation cocktail (10 ml Permaflour IlPK 33ml 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 at220Dgwith International Model PR-2
centrifuge for 20 minutes and the radioactivity determined in the supernatant
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 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
-------
CO- during the metabolic reaction or volatilized as parent compound. The
analysis was performed three times with three batches of tissue creparation.
Enzyme Activity: Cytochrome P-450 and chtochrome br> 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 p_-aminophenol
produced during a 30-min incubation of the liver microscmes or the PMS with
aniline hydrochloride at 24 C. The reaction mixture contained an NADPH-generating
system as described previously 1 pM mole of aniline hydrochloride and 5 mg
microsomal protein. The reaction was stopped by addition of 0.5 ml 3 M TCA.
After centrifugation of the reaction mixture at 2200 g for 20 ruin., a 1 ml aliquot
of the reaction mixture was made basic with 0.5 ml of 10» Na?CO^ 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 spectrophotometer at 630 nm.
RESULTS AND DISCUSSION
Measurements were made showing the distribution of radioactivity after
C-labeled carbon tetrachloHde incubation with trout liver microsomes and
Daphnia PMS tissue fractions It was also found that most of these compounds
were "readily volatilized from the reaction mixture in spite of a silica gel
trap (Figure 1). This resulted in lower recoveries of the compound at the
termination of chemical reaction. The results (Table -jj indicate that parent
carbon tetrachloride could be extracted with hexane after incubation with trout
liver microsomes or Daphnia PMS for various time intervals. However, the radio-
activity in the aqueous phase and the CO- traps increased with concomitant
decrease of the hexane extracted radiocarbon. The data also indicate that
carbon tetrachlorioe binds slowly with the nrfcrosomal protein fractions of the
-------
6.
trout liver and Daphr.ia PMS. Formation of the aqueous metabolites and the
protein binding of carbon tetrachloride does not appear to be linear with
time of incubation. It is evident from our results that both species are
capable of metabolizing this hepatotoxin in vitro via microsomal mixed
function oxidases.
14
Metabolism of C-labeled chloroform in fish and Daphnia shows the
metabolism of chloroform by microsomal mixed function oxidase system of trout
liver and Daphn.ia. The data indicate (Table 2) a rapid conversion of
chloroform to hexane-unext^ctable water soluble metabolites in trout liver
and Daphnia PMS. Approximately 40* of 80 to 90% radioactivity was found in
the aqueous phase and 53% was extracted in hexane within 1 min of its incubation
with trout ln"ver microsomes. Similarly, the aqueous phase, from Daphnia had
more than 50% of the radioactivity in the aqueous phase as compared to about
40% in the hexane extract. The radioactivity in the aqueous phase increased
to 70% in the case of Daphnia, while about 45% was found in trout. Measur-
able radioactivity was also found in the carbon dioxide traps of both animal
species. Trout liver microsomes showed increased protein binding with incuba-
tion time. However, Daphnia showed little change in protein bound radio-
activity with incubation time.
Most (87-94%) of the radioactivity spiked in the microsomal tissue
14
preparation with C chlorobenzene was extractable in hexane even after 120
min of incubation time with trout liver microsomes and 60 min with Daphni a
PMS. The aqueous phase of the reaction mixture, in both species, showed
small percentages (0.6 - 2.3) water soluble products of metabolism. Higher
-------
7.
amounts of protein bound radioactivity were found with trout livsr than
with Daphnia tissue preparations.
Trout appear to show higher metabolic activity than Daphnia for 1,1,2-
trichloroethylene and 1,1,2-trichloroethane (Tables 4 and 5). More polar
metabolites of 1,1,2-trichloroethylene and 1,1,2-trichloroethane were formed
by trout liver microsomes than Daphnia PM$. Trichloroethane was more readily
converted to water soluble products than trichloroethylene In the case of
trout. On the other hand, Daphnia converted both of the compounds to aqueous
metabolites at similar but at much slower rates than rainbow trout. Both
compounds showed protein binding with rainbow trout or Daphnia microsonial
mixed function oxidase system in vitro.
Both rainbow trout and Daphnia metabolized chlorofrom 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 were not the same between species.
The order for rainbow trout was chloroform > 1,1,2-trichloroethane > 1,1,2-
trichloroethylene > chlorobenzene > carbon tetrachloride. The order for
Daphnia was chloroform > chlorobenzene > 1,1,2-trichloroethylene > 1,1,2-
trichloroethane Mrarbon tetrachloride.
Microsonial monooxygenase or mixed function oxidase assays of trout liver
and Daphnia PMS-fractions were performed. Trout liver microsomes had mean
values of 0.28 and 0.19 nanomoles-mg , of cytochrome P-450 and cytochrome br,
respectively (Table 6). The level of NADPH cytochrome c reductase activity in
trout liver microsomes was 16 nanomoles of cytochrome c reduced-min" -mg~
protein. This activity appears to be low in rainbow trout as compared to
mammalian liver tissue (Table 7). Trout liver microsomes metabolized aniline
-------
8.
at a very slow rate of 0.04 - 0.05 nanomoles'mg protein-min (Table 6).
PMS from adult Daphnia shov/ed a mean value of 42 ± 5.3 nanomoles of cytochrome
c reductase activitymin" -mg" protein, which was higher than rainbow trout.
Based on the present information, it is apparent that both species
possess an active 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 mixed function oxidase system in
rainbow trout and Daphnla. Toxicity may be related to irreversible protein
binding, and lip-Id 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.
Our data indicate (Table 7) that aquatic organisms have measurable but lo.ver
mixed function oxidase activity than mammals. However, with similar metabolic
systems, the mechanisms leading to toxicity arid neoplasia are presumed 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 easier, cheaper and faster to rear than mammals,
they are economically attractive test organisms. The second is for environ-
mental monitoring. Aquatic organisms are currently being used as sentinels
to signal environmental contamination (Black e_t aJL, 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.
-------
TABLE 1. Distribution (% + standard deviation) of C
after incubation with ^C carbon tetrachloride
for various time intervals with microsomal fractions
of rainbow trout (Salmo gairdneri) liver and
post-mitochrondrial supernatant of Daphnia^
magna. (Values are the means of three
separately prepared tissue fractions.)
Rainbov.- Trout
Time
(Min)
0
15
30
45
60
120
0
15
30
45
60
Hexane a
Extracted
91.0 t 5.8
92.6 t 4.2
88.1 ± 3.0
90.1 ± 0.75
87.1 t 1-8
84.7 + 5.9
95.9 ± 1.8
91.4 t 1.3
93.5 ± 2.3
89.1 ± 1.2
87.5 ± 0.92
Aqueous or ^
Unextracted
in Hexane
0.56 ± 0.15
0.69 ± 0.13
0.73 ± 0.15
0.89 ± 0.046
1.2 ± 0.28
1.1 i 0.16
•0.72 i 0.43
0.99 t 0.38
0.91 i 0.24
1.3 ± 0.21
1.5 t 0.56
C02C
Trap
0.012 ± 0.016
0.15 ± 0.132
0.097 ± 0.095
0.15 ± 0.15
0.25 ± 0.21
0.18 ± 0.20
Daphnia
0.09 ± 0.02
0.15 i 0.14
0.06 t 0.08
0.38 ± 0.13
0.13 ± 0.18
Proteinb/
Bound
0.26 t 0.096
0.35 ± 0.10
0.37 i 0.15
0.53 ± 0.13
0.61 t 0.30
0.60 ± 0.15
0.061 t 0.03
0.074 ± 0.03
0.12 i 0.10
0.1 ± 0.06
0.09 ± 0.10
Total %d
Recovery
61.8 ± 11.9
52.7 ± 8.9
47.9 ± 16.8
45.6 ± 7.4
37.5 ± 8.3
40.0 ± 4.7
55.8 + 16.2
45.9 ± 9.3
47.1 t 4.7
38.3 t 3.6
37.5 ± 2.9
Percent of total added dpm in 0-1 ml solution which could be recovered in hexane
after the extraction of reaction mixture.
Percent dpm in aqueous fraction (soluble and protein pellet) relative to dpm
extractable in hexane.
c R
Percent radioactivity trapped in Carbosorb II relative to dpm extractable in hexane.
Total percent recovery is based on the dpms recoverable in all fractions including
the silica ge'i trap divided by the total added dpm in the reaction mixture.
-------
TABLE 2. Distribution (% ± standard deviation) of 14C
after incubation with '^C chloroform
for various ti.me intervals with microsorcal fractions
of rainbow trout (Salmo gairdneri) liver and
post-mitochrondrial supernatant of Dajhnla
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 a
Extracted
53.2 ± 25.3
45.9 ± 27.2
44.3 ± 20.7
46.3 ± 25.9
42.1 ± 27.7
45.3 ± 16.7
39.7 + 23.4
40.3 ± 20.7
37.8 ± 22.8
25.4 ± 7.3
26.2 ± 15.4
Aqueous or b
Unextracted
in Hexane
38.6 ± 25.8
43.4 t 27.5
44.3 J 28.7
44.3 ± 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
C02 C
Trap
0.25 ± 0.32
0.09 i 0.04
0.19 ± 0.23
0.18 ± 0.29
1.2 ± 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
b
Protein
Bound
1.1 ± 0.61
4.2 ± 2.6
3.5 ± 3.2
4.6 + 1.7
4.6 ± 2.0
5.9 ± 4.2
1.6 ± 2.4
l.C ± 2.5
1.3 t 1.9
1.5 + 2.0
1.5 ± 1.5
Total % d
Recovery
87.9 ± 25.3
86.8 ± 14.0
91.4 i 15.7
83.6 ± 12.5
81.9 ± 14.9
83.7 ± 11.6
83.0 ± 14.1
77.0 ± 14.8
85.8 t 11.5
73.3 ± 28.8
74.9 ± 14.2
Percent of total added dpm in 0-1 ml solution which could be recovered in hexane
after the extraction of reaction mixture.
Percent dpm in aqueous fraction (soluble and protein pellet) relative to dpm
extractable in hexane.
R
Percent radioactivity trapped in Carbosorb II relative to dpm extractable 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.
-------
TABLE 3. Distribution (% ± standard deviation) of 1/!(
after incubation with l^C chlorobenzene
for various time intervals with microsomal fractions
of rainbow trout (Salmo gairdneri) liver and
post-mi tochrondrial supernatant of Daphnia
maana. (Values are the moans of three
separately prepared tisr.ue fractions.)
Time
(Min)
0
15
30
45
60
120
0
15
30
45
60
a
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 i 2.7
90.6 ± 2.1
92.5 ± 2.7
b
Aqueous or
Unextracted
in Hexane
0.56 ± 0.06
0.95 ± 0.15
1.0 ± 0.28
1.3 t 0.21
1.5 ± 0.17
1.9 ± 0.20
1.2 ± 0.17
1.7 ± 0.15
2.1 ± 0.06
2.1 ± 0.07
2.3 ± 0.36
Rainbow Trout
CO, °
Tr3p
0.006 ± 0.004
0.03 t 0.01
0.015 ± 0.009
0.07 ± 0 01
0.014 ± 0.010
0.21 ± 0.25
Daphnia
0.06 ± 0.004
0.024 ± 0.006
0.042 t 0.03
0.07 ± 0.014
0.09 ± 0.010
b
Protein
Bound
0.4 ± 0.12
0.60 ± 0.27
0.79 i 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 % d
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 t 15.6
49.1 ± 8.4
41.3 ± 16.6
33.3 ± 11.2
35.7 ± 6.1
Percent of total added dpm ir, 0.1 ml solution which could be recovered in hexane
after the extraction of reaction mixture.
Percent dpm in aqueous fraction (soluble and protein pellet) relative to dpm
extractable in hexane.
cPercent radioactivity trapped in Carbosorb II relative to dpm extractable 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.
-------
TABLE 4. Distribution (% ± standard deviation) of C
after incubation with 14C 1,1,2-trichloroethylene
for various time invervals with microsonial fractions
of rainbow trout (Salmo gairdneri) liver and
post-mitochrondrial 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
a
Hexane
Extracted
89.0 ± 3.4
92.2 i 4.9
84.5 ± 5.8
88.3 ± 3.5
85.4 ± 3.6
82.8 i 3.2
88.8 ± 2.1
89.4 ± 1.7
90.5 ±3.7
91.5 ± 5.0
89.1 ± 1.3
Aqueous or
Unextracted
in Hexane
1.1 + 0.11
1.6 ± 0.06
6.3 ±7.5
2.2 ± 0.15
1.8 ± 0.35
2.6 + 0.80
1.03 ± 0.24
1.56 ± 0.16
1.8 ± 0.23
1.9 ± 0.42
1.95 ± 0.64
c
co2
Trap
0.032 ± 0.007
0.096 ± 0.09
0.063 ± 0.046
0.21 ± 0.27
0.11 ± 0.11
0.19 ± 0.10
Daphnia
0.06 ± 0.01
0.10 + 0.04
0.14 ± 0.09
0.03 ± 0.014
0.095 ± 0.007
b
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
0.024 ± 0.032
0.023 ± 0.017
0.020 ± 0.014
0.013 ± 0.011
0.012 ± 0.011
d
Total %
Recovery
63.3 ± 15.9
54.0 ± 9.2
49.2 ± 11.9
39.9 i 4.4
46.5 t 8.2
32.7 ± 5.9
54.6 ± 0.6
42.4 ± 4.1
42.7 ± 5.5
37.4 ± 0.6
34.5 ± 3.2
Percent of total added dpm in 0.1 ml solution which could be recovered in hexane
after the extraction of reaction mixture.
Percent dpm in aqueous fraction (soluble and protein pellet) relative to dpm
extractable in hexane.
c R
Percent radioactivity trapped in Carbosorc II relative to dpm extractable in hexane.
Total percent recovery is based on the dpms recoverable in all fractions including
the silica gel trap divided by the total adJed dpm in the reaction mixture.
-------
TABLE 5. Distribution (% i standard deviation). of 14C
after incubation with 'flC 1 ,1 ,2-Trichloroethane
for various time intervals with nricrosonial fractions
of rainbow trout (Salmo gairdneri ) liver and
post-rcitochrondrial supernatant of Daphnia
magna. (Values are the means of three
separately prepared tissue fractions.)
Rainbow Trout
Time
(M1n)
0
15
30
45
60
120
0
15
30
45
60
a
Hexane
Extracted
81.5 ± 24.7
79.3 ± 31.3
76.3 ± 34.1
77.8 + 28.7
77.9 i 31.7
76.4 ± 30.9
96.3 + 0.56
97.2 ± 0.96
96.8 ± 0.75
97.0 ± 0.78
97.0 ± 0.0
Aqueous or
Unextracted
in Hexane
12.4 ± 20.3
16.7 ± 27.4
15.1 ± 24.5
15.8 ± 25.6
15.5 ± 24.9
16.8 ± 26.7
0.77 i 0.30
0.96 ± 0.30
1.1 ± 0.35
1,1 ± 0.14
0.98 ± 0.035
c
co2
Trap
0.22 t 0.35
O.C49 ± 0.07
0.67 t 0.25
0.18 t 0.28
1.1 ± l.T.
0.70 ± l.u
Daphnia
0.005 ± 0.004
0.007 ± 0.005
0.036 + 0.029
0.015 ± 0.007
0.02 ± 0.0
b
Protein
Bound
0.65 t 0.91
l.G ± 2.5
1.3 ± 2.1
1.46 + 2.3
1.47 ± 2.2
1.26 t 1.5
0.004 ± 0.004
0.012 ± 0.008
0.009 + 0.001
0.01 ± 0.014
0.012 + 0.011
d
Total %
Recovery
77.5 t 21.7
75.6 ± 18.6
68.9 ± 16.3
69.4 ± 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 i 18.8
Percent of total added dpm in 0.1 ml solution which could be recovered in hexane
after the extraction of reaction mixture.
Percent dpm in aqueous fraction (soluble and protein pellet) relative to dpm
extractable in hexane.
c • R
Percent radioactivity trapped in Ccrbosorb II relative to dpm extractable 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.
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TABLE 6. Mixed Function Oxidase System of Rainbow
Trout (Salmo gairdneri) liver and Daplmia
Enzymes
Cytochrome2
P-450
Cytochrome bKa
Rainbow Trout
6.28 ± 0.1 (4)d
0.19 i 0.05 (4)
Oaphnia
N.D.
N.D.
NADPH Cytochrome5
c-reductase 15.9 ± 2.2 (8) 42 ± 5.3 (3)
Aniline hydroxylasec 0.05 ± 0.01 (3)
nanomoles-mg" microsomal protein ± S.D.
nanomoles of cytochrome c reduced-min" -mg~ protein ± S.D.
c nanomoles of £-aminophenol formed-min" -mg' protein ± S.D.
numbers in parentheses are the number of tissue preparations frcm
separate animal batches
-------
TABLE 7. Comparison of mixed function oxidase measurements
between mammals and several non-mammalian aquatic organisms
Enzymes3
Cytochrome P-450
Cytochrome b,.
NADPH Cytochrome c
reductase
Aniline hydroxylase
Human
0.
0.
102.
8.
60 ±
49 i
6 ±
7 ±
0.10b
0.06b
14. 6b
6.8C
Male
0.72 ±
0.30 ±
96 ±
22 ±
Retc
0.08
0.08
20
5
Rainbow
0.
0.
15.
0.
28 ±
19 ±
9 ± 2
05 ±
Troutd
0.10
0.05
.2
C.01
Daphnia
NDf
NO
42.0 ± 5.3
-
Blue Crab
0.18 t 0.
-
5.2 ± 4.
0.016 ± 0
e
08
8
.008
a Activities expressed as in Table 6.
b.Ahmad and Black, 1977
c Katb, 1979; Tables 27 and 38.
d This study.
e James e_t ^1_., 1979; Tables 3 and 4.
f Not detectable.
-------
: 10
i X
; D
OJ
O
I x
c
O
u
(O
CD
T3
Ol
o Trichloroethane
Chloroform
Carbon tetrachloride
Trichloroethylene
Monochlorobenzene
10 20
30 40 50 60 70
Incubation Tiroe (Ilinutes)
90 100 110 120
Ficjure 1. Disappearance of the added radioactivity from the reaction
mixture at different time intervals.
-------
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