PRPO-101637
EPA-560/11-79-007
TOXICITY OF ORGANIC CHEMICALS
TO EMBRYO-LARVAL STAGES OF FISH
June 1979
Final Report
Contract No. 68-01-4321
Wesley J. Birge
Jeffrey A. Black
Donald H. Bruser
Project Officer
Arthur M. Stern
U.S. Environmental Protection Agency
Office of Toxic Substances
Washington, D.C. 20460
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TECHNICAL REPORT DATA
(fttau nod latauetiont an tfi* rcvent before committing)
1. REPORT NO. 2,
•PA-560/ 11-79-007
4. TITLE AND SUBTITLE
oxi city of Organic Chemicals to Embryo-Larval
Stages of F1sh
7. AUTHOfl(S)
Lesley J. Birge, Jeffrey A. Black, and Donald H. Bruser
9. PERFORMING ORGANIZATION NAMS AND ADDRESS
Thomas Hunt Morgan School of Biological Sciences
Jni varsity of Kentucky
Lexington, Kentucky 40506
12. SPONSORING AGENCY NAME AND ADDRESS
Jffice of Toxic Substances
KS. Environmental Protection Agency
Washington, D.C. 20460
3. RECIPIENT'S ACCESS1OWNO.
9. REPORT DATE '
June 1979 (Date of Issue)
9. PERFORMING ORGANIZATION
8. PERFORMING ORGANIZATION
10. PROGRAM ELEMENT NO.
CODE
REPORT NC
11. CONTRACT/GRANT NO.
68-01-4321
13. TYPE OF REPORT AND PERIOD COVERED
Final (Oct. 1976-Feb. 1979)
14. SPONSORING AGENCY COOE
19. SUPPLEMENTARY NOTES
is. ABSTRACT
continuous flow procedure was developed for evaluating effects of insoluble
and volatile organ 1cs on embryo,- larval stages of fish. Test compounds were selected
for different combinations of solubility and volatility and included aniline, atrazlne,
chlorobenzene, chloroform, 2,4-dichlorophenol , 2,4-dichl or ophenoxy acetic acid, dioctyl
phthalate, malathlon, tri sodium nltrilotri acetic acid, phenol, and poly chlorinated bi-
phenyl (Capacitor 21). A closed system devoid of standing air space greatly reduced
volatility as a test variable. Mechanical homogenization proved highly effective in
suspending hydrophobia compounds in influent water. Continuous agitation in the test
chamber 'and regulation of detention time further precluded the need for carrier sol-
vents. Test results Indicated good reproducibiHty of exposure concentrations. The
most toxic compounds included Capacitor 21, chlorobenzene, 2,4-dichlorophenol, and
phenol. Chlorobenzene at 90 yg/1 produced complete lethality of trout eggs. The three
other compounds gave log probit LCso's of 2 to 70 ug/1 when trout stages were exposed in
hard water, and LCj's were 0.3, 1.0, and 1.7 yg/1 for phenol, Capacitor 21, and 2,4-di-
chlorophenol. Chloroform also was highly toxic to trout stages and LCi's ranged from
4.9 to 6.2 pg/1 . When bass and goldfish stages were exposed to chlorobenzene, LC^'s
ranged from 8 to 33 ug/1. Compared to other species, trout developmental stages gener-
ally exhibited the greatest sensitivity. The LCj values determined in embryo-larval
tests compared closely with maximum acceptable toxicant concentrations developed In
1 1
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NOTICE
This report has been reviewed by the Office of
Toxic Substances, EPA, and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute Indorsement or
recommendation for use.
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ABSTRACT
A continuous flow procedure was developed for evaluating effects of
Insoluble and volatile organics on embryo-larval stages of fish. A closed system
devoid of standing air space was used to minimize volatility as a test variable.
Insoluble compounds were suspended in influent water by mechanical homogeniza-
t1on» without the use of carrier solvents. Tests were performed on aniline,
atrazine, chlorobenzene, chloroform, 2,4-d1chlorophenol, 2,4-dichlorophenoxy-
acetic acid (2,4-0), dioctyl phthalate (OOP), malathion, trisodium nitrilo-
triacitic acid (NTA), phenol, and polychlorinated biphenyl (Capacitor 21).
Maintaining water hardness at 50 and 200 mg/1 CaC03, exposure was continuous
from fertilization through 4 days posthatching for largemouth bass, blueglll
sunflsh, channel catfish, goldfish, rainbow trout, and redear sunfish.
Exposure levels which produced S0% (LCg*) and 1% (IC*) control-adjusted
impairment (lethality, teratogenesis) of test populations were calculated by
log probit analysis. The LC,'s were used is a basis for estimating threshold
concentrations for toxic effects. To determine reliability of LC| values, they
were compared with maximum acceptable toxicant concentrations (MATC) developed
in partial and complete life-cycle studies. Good correlations were obtained
when data were adequate to permit comparisons, and the findings indicated that
LC, values determined in embryo-larval tests carried through 4 days posthatching
were useful 1n estimating long-term effects of aquatic pollutants.
Test results indicated good repreducibility of exposure concentrations
for both volatile and insoluble toxicants. The most toxic compounds Included
Capacitor 21, chlorobenzene, 2,4-dichlorophenol, and phenol, Chlorobenzene at
90 yg/1 produced complete lethality of trout eggs, and LCj's ranged from
8 to 33 yg/1 in tests with the largemouth bass and goldfish. The tnree
other compounds gave log probit LCcn's of 2 to 70 pg/1 when trout stages were
exposed in hard water, and LC,'s were 0.3, 1.0, and 1.7 pg/1 for phenol,
Capacitor 21, and 2,4-d1chloropheiiol. Phenol was less toxic to developmental
stages of the goldfish and bluegill. When tests were conducted in hard water,
the LCgg's were 0.34 and 1.69 mg/1 and the LC,'s varied from 2.0 to 8.8 pg/1.
Depending on water hardness, LC,'s determined in yg/1 with the rainbow trout
iii
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ranged from 4.9 to 6.2 for chloroform, 21.9 to 32.5 for 2,4-D, and 29.0 to
77.2 for atrazlne. Though not tested on the trout, LC,'s determined with the
goldfish ranged from 143.2 to 215.0 yg/1 for aniline and 141.1 to 439.6 wg/1
for malathion. The organics least toxic to the trout included NTA ind OOP, and
the LCgg's varied from 90.5 to 114.0 and 139.5 to 149.2 rag/1, respectively.
Compared to the other species, trout developmental stages generally exhibited
the greatest overall sensitivity. Though water hardness did not substantially
alter toxiclty of the selected organic compounds, phenol was somewhat more
toxic 1n hard water. All compounds produced appreciable frequencies of teratic
larvae.
iv
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TABLE OF CONTENTS
Page
ABSTRACT Hi
LIST OF TABLES vf
LIST OF FIGURES vii
ACKNOWLEDGMENTS vltl
INTRODUCTION 1
CONCLUSIONS 3
RECOMMENDATIONS 5
DEVELOPMENT OF TEST SYSTEM AND PROCEDURES 7
Materials and Methods 7
Selection of animal species 7
Selection of organic toxicants 7
Test conditions and expression of data 9
Test water .......... ..... 10
Embryo-larval test system ... 12
Analytical procedures .............. 14
Initial Performance Evaluation 20.
APPLICATION OF TEST SYSTEM 24
Embryo-Larval Toxicity Tests ..... . 24
Aniline . 24
Atrazine 26
Capacitor 21 26
Chlorobenzene 27
Chloroform 28
2,4-Dichlorophenol 28
2,4-Dichlorophenoxyacetic acid ...... 29
Dioctyl phthertate 30
Malathion 30
Trisodium nltHlotriacetic acid 31
Phenol 32
Teratogenesfs in Fish Embryos 35
SUMMARY . 36
REFERENCES 56
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LIST OF TABLES
Table Page
1 Organic compounds used In toxlcity tests ..... 2
2 Text city tests performed on embryo-larval stages of fish .... 8
3 Water quality characteristics observed
during toxldty tests with organic compounds 11
4 Reconstituted test water 13
5 Regulation of Sudan IV-chlorobenzene
In continuous flow tests . 22
6 Regulation of organic compounds In continuous flow
toxiclty tests with fish embryo-larval stages 23
7 Log problt LCsg values for organic compounds .......... 38
8 Log problt LCj values determined at
4 days posthatching for organic compounds 41
9 Toxiclty of aniline to embryo-larval stages of fish ....... 43
10 Toxiclty of atrazine to embryo-larval stages of fish 44
11 Toxiclty of Capacitor 21 to embryo-larval stages of fish .... 45
12 Toxiclty of chlorobenzene to embryo-larval stages of fish . . . . 46
13 Toxiclty of chloroform to embryo-larval stages of rainbow trout . 47
14 Toxiclty of 2,4-d1chlorophenol (DCP)
to embryo-larval stages of fish . 48
15 Toxiclty of 2,4-d1chlorophenoxyacetic acid (2,4-0)
to embryo-larval stages of fish 49
16 Toxiclty of dloctyl phthalate (OOP)
to embryo-larval stages of fish 50
17 Toxiclty of malathion to embryo-larval stages of goldfish .... 51
18 Toxicity of tHsodium nitHlotrlacetic acid (NTA)
to embryo-larval stages of fish 52
19 Toxicity of phenol to embryo-larval stages of fish 54
20 Comparison of LCi's determined in embryo-larv":
tests with MATC's derived from life-cycle stiriies 55
v1
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LIST OF FIGURES
1 Embryo-larval test system 15
2 Multichannel assembly of toxlcity test units . . . 17
3 Exposure chamber 19
4 Toxlcity of aniline to fish eggs • 25
5 Effect of water hardness on phenol toxicity to trout eggs .... 33
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ACKNOWLEDGMENTS
The authors are grateful to A.G. Westsrman, W.E. McDonnell, H.C. Parekh,
and J.E. Hudson for technical support and to Barbara A. Ramey for preparation
of the manuscript and figures. We also are appreciative of Dr. Arthur Stern
for his assistance during this study. The research facilities used to conduct
these tests were provided 1n part by research funds from the U.S. Department of
the Interior, Office of Hater Research and Technology (grant no. A-074-KY) and
the National Science Foundation (grant no. AEN 74-08768-A01).
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INTRODUCTION
The toxicological characterization of organic pollutants 1s frequently
complicated by physical and chemical properties of the test compounds (Schoor,
1975; Veith and Comstock, 1975). Volatility or low water solubility may
preclude adequate regulation of exposure concentrations in aquatic test systems*
especially when open test chambers are used. Though emulsifiers or carrier
solvents may be of some aid in testing hydrophobic organics, they generally
introduce undesirable variables. The initial objective of this investigation
was to develop a continuous flow system designed for testing volatile and
insoluble organic compounds which are difficult to stabilize with conventional
techniques. Using fish embryo-larval stages as test organisms, a closed flow-
through test chamber devoid of an air-water interface was used to minimize
evaporative loss of volatile organics. Insoluble compounds were suspended in
influent water by mechanical homogenization, and maintained by continuous
agitation in the exposure chamber and regulation of detention time. Fish
embryos were selected as test organisms because of their simple culture require-
ments, suitability for use in a closed test system, and high sensitivity to
aquatic contaminants. Reconstituted water, with physicochemical characteristics
representative of natural freshwater, was formulated to provide stable,
reproducible test conditions and to minimize problems with background contami-
nants. In the process of developing the new procedures, tests were performed
with eleven organic compounds, selected for varying degrees of volatility and
water solubility (Table 1).
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Table 1. Organic compounds used in toxicity tests.
Compound
Aniline
Atrazine
Capacitor,^ 211
Chlorobenzene
Formula
CHCHCHCHCHCNH9
NCClNC(NHC2H5)NCNH(n-C3H7)
CXCXCXCXCXCCCXCXCXCXCX
X = C1 or H
CHCHCHCHCHCC1
Company
J.T. Baker Chemical Co.
Phllllpsburg, N.J.
ICN Pharmaceuticals, Inc.
Plainvlew, N.Y.
Monsanto Co.
St. Louis, Mo.
J.T. Baker Chemical Co.
Chloroform
2,4-Dichlorophenol
CHC1,
CC1CHCC1CHCHCOH
2,4-Oichloro- : C(OCH2COOH)CC1CKCC1CHCH
phenoxyacetic acid ' '
Dioctyl phthalate C(CQOC8H17)C(CQOC8H17)CHCHCHCH
Mai athion
Trisodium nitrilo-
triacetic acid
Phenol
(CH30)2PSSCHCCOOC2H5)CH2(COOC2H5)
CHCHCHCHCHCOH
Fisher Scientific Co.
Fair Lawn, N.J.
AldHch Chemical Co.
Milwaukee, Us.
Aldrich Chemical Co.
Milwaukee, Ws.
U.S. Steel Chemicals Div.
Pittsburgh, Pa.
American Cyanamid Co.
Princeton, N.J.
ICN Pharmaceuticals, Inc.
Plainview, N.Y.
J.T. Baker Chemical Co.
Phillipsfaurg, N.J.
Trademark of the Monsanto Co., St. Louis, Missouri
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CONCLUSIONS
A continuous flow system was developed for testing insoluble and volatile
organic compounds on embryo-larval stages of fish. Use of a closed test system,
devoid of an air-water interface greatly reduced volatility as a test variable.
Fluctuations in exposure concentrations were no greater for chloroform than for
non-volatile compounds. Mechanical homogenization proved highly effective in
suspending hydrophobic compounds in influent water. Continuous, moderate
agitation in the test chamber and regulation of detention time further precluded
the need for carrier solvents. Fish eggs and larvae were easily maintained in
the closed system, and there was no evidence that this procedure altered test
responses.
Numerous classes of organic compounds were found to be highly toxic and
teratogenic to developmental stages of fish. Of eleven compounds tested, those
which proved most toxic to eggs, embryos, and early larvae included chlorobenzene,
2,4-dichlorophenol, phenol, and polychlorinated biphenyl (Capacitor 21). Chloro-
benzene at 90 yg/1 produced complete lethality of trout eggs, and LC 's
ranged from 8 to 33 ug/1 in tests with the largemouth bass and goldfish.
The three other compounds gave log probit LC50's of 2 to 70 ug/1 when trout
stages were exposed in hard water, and LC-t's were 0.3, 1.0, and 1.7 pg/1 for
phenol, Capacitor 21, and 2,4-dichlorophenol. Phenol was le-s toxic to develop-
mental stages of the goldfish and bluegill. The LCgg's wer 0.34 and 1.69 mg/1
when tests were conducted in hard water, and the LC,'s varied from 2.0 to 8.8
yg/1. Depending on water hardness, LC,'s (ug/1) determined with the rainbow
trout ranged from 4.9 to 6.2 for chloroform, 21.9 to 32.5 for 2,4-D, and 29.0
to 77.2 for atrazine. Though not tested on the trout, LC.'s determined with
the goldfish ranged from 143.2 to 215.0 yg/1 for aniline and 141.1 to 439.6 yg/1
for malathion. The least toxic compounds included NTA and OOP. In tests with
trout developmental stages, the LC^'s varied from 90.5 to 114.0 and 139.5 to
149.2 mg/1, respectively. Though phenol was somewhat more toxic in hard water,
hardness was not an appreciable factor in most tests.
On the basis of these data, It was evident that chlorinated aromatic
hydrocarbons were among the most toxic compounds. These findings are consistent
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with other studies which also have shown that numerous chlorinated aromatic
hydrocarbons exert marked effects on fish reproduction, often accumulating to
high levels in eggs and tissues (Birge, et al_., 1979b). Only chloroform and
phenol exhibited comparable effects on fish embryo-larval stages. Chloroform,
a solvent of high lipid solubilityi is a narcotizing agent, and phenol, a
widely used germicide, is an effective protein denaturant. Of three monosub-
stituted benzene compounds tested (i.e.-, aniline, chlorobenzene, phenol),
toxicity varied with the different substitution groups, generally increasing
in the order NH2, OH, and Cl.
It was further concluded that log probit analysis could be successfully
applied to dose-response data to determine threshold concentrations (LC,) at
which organic compounds become lethal or teratogenic to embryo-larval stages.
In addition, when exposure was maintained from fertilization through 4 days
posthatching and responses for lethality and teratogenesis were combined, LC,'s
provided a close approximation to maximum acceptable toxicant concentrations
(MATC) determined in partial and complete life-cycle tests (Table 20).
Developmental stages of the different fish species usually exhibited
differential sensitivity to the various organic toxicants. Though the order
of species sensitivity varied somewhat with different compounds, trout embryos
and alevins generally exhibited the least tolerance. Differences between 1C,
values determined for the trout and other species frequently exceeded one and
sometimes two orders of magnitude. Less variation occurred among the five
remaining species, and the goldfish often was the most tolerant.
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RECOMMENDATIONS
Consideration should be given to revising the protocol for embryo-larval
tests, to provide technological Improvements which will ensure 1) more adequate
regulation of exposure concentrations of volatile toxicants, and 2) testing of
hydrophoblc compounds under conditions which minimize or preclude the need for
carrier solvents. Additional study is recommended to modify the new procedure
described herein to accommodate 1} testing of organlcs which exist in the gaseous
state at ambient temperatures, and 2) use of a wider variety of test organisms
(£•&•» Daphnia, juvenile fish). Several halomethanes included among the 129 prior-
ity toxicants listed by EPA have boiling points which range from -29 to +4.5°C
(£.£., methyl chloride, methyl bromide, dichlorodifluoromethane). Consequently,
such compounds cannot be stabilized adequately in conventional open aquatic test
systems. However, the closed flow-through procedure described in the present
study could be further adapted to facilitate such testing. Gases would be dis-
persed in influent water using the mixing assembly, and test water would be per-
fused continuously through the closed exposure chamber. Moderate.agitation in
the exposure chamber and regulation of detention time would further augment homo-
geneous dispersal of toxicant in test water. The new procedure also could be
effectively modified to permit use of Daphnia and other aquatic species in tests
with volatile and hydrophobic compounds which are difficult to stabilize using
conventional procedures.
Convincing evidence has been presented that fish embryo-larval tests which
extend beyond hatching by 30 days or more yield responses comparable to those
produced in chronic life-cycle studies (McKim, 1977). However, considering the
many contaminants which actually or potentially may enter aquatic ecosystems,
still shorter and more cost-feasible tests are needed for environmental assessments,
In the present investigation, when embryo-larval tests were carried through 4 days
posthatching and frequencies of lethality and teratogenesis were combined, log
probit LC,'s were in good agreement with MATC's developed in chronic life-cycle
studies. These data Indicated that embryo-larval tests of shorter duration than
those presently recommended (U.S. EPA, 1978) may prove valuable in estimating
long-term effects of aquatic toxicants. Accordingly, it may be appropriate to
consider revising exposure periods specified in the present protocols for embryo-
larval testing (U.S. EPA, 1978).
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In addition, further attention should be given to use of LC, values in
determining water quality criteria. Unlike the MATC which generally is expressed
as the range between the lowest toxic and highest no-effect concentrations, the
LCj represents a discrete value for which reliable confidence limits can be
established. Furthermore, it may prove useful to calculate LC.Q'S which could
be used in conjunction with LC, values to characterize slope of the threshold
X
end of the dose-response curve and to provide an additional reference pofnt for
establishing regulatory criteria.
Provided that the number of exposure concentrations is sufficient to
delineate an adequate dose-response curve, LC-, values can be calculated with
present log probit programs. However, as existing probit methods were designed
primarily for calculating LC5« values (Finney9 1971; Stephan, 1977), attention
should be given to the development of new regression procedures formulated
especially to delineate near-threshold concentrations (e_.£., LC.Q, LC^}.
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DEVELOPMENT OF TEST SYSTEM AND PROCEDURES
Materials and Methods
Selection of animal, species. Fish used In this study Included the bluegill
sunfish (Lepomls macrochlrus), channel catfish (rctalurus punctatusj. goldfish
(Carassius auratus), largemouth bass (Micropterus salmoides), rainbow trout
(Salmo gairdnerl), and redear sunflsh (Lepomls microlophus). Species were
chosen for economic importance, seasonal availability, suitable egg production,
and for variations in ecological and geographic distribution, including warm
and cold water habitats. This selection also included species with different
patterns of reproduction, involving a number of developmental variables which
may respond differentially to organic toxicants (e_.£., yolk quantity, hatching
time, spawning habits).
Gravid rainbow trout were provided by the Erwin National Fish Hatchery,
Erwln, Tennessee. Eggs and sperm were obtained by artificial spawning and
milking procedures of Leitritz and Lewis (1976). Fertilization was accomplished
by mixing eggs and milt for 20 min. Freshly fertilized eggs from bass, bluegill,
goldfish, and redear sunfish were collected locally from the Frankfort National
Fish Hatchery, Frankfort, Kentucky. Channel catfish spawn was obtained from
either the Frankfort Hatchery or the Senecaville National Fish Hatchery,
Senecaville, Ohio.
Selection of organic toxicants. Toxicity tests were conducted with aniline,
atrazine, Capacitor 21, chlorobenzene, chloroform, 2,4-dichlorophenol, 2,4-di-
chlorophenoxyacetic acid, dioctyl phthalate, malathion, trisodium nitrilotri-
acetlc acid, and phenol. All analytical and toxicity data were expressed as
concentrations of the pure compounds, except for atrazine which was reported
as the wettable powder (80% pure). These compounds were selected to provide
varying combinations of volatility and water solubility and included aromatic
hydrocarbons, aromatic amines, chlorinated hydrocarbons, organophosphates, and
phthalates. This choice permitted adequate evaluation of aquatic test procedures
for organic compounds and provided fish embryo-larval toxicity data for a number
of important classes of organic trace contaminants. Chemical formulae and
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Table 2. Toxlcity tests performed on embryo-larval stages of fish.
Organic Compound F1sh Species
Aniline Largemouth Bass, Chanel* Catfish, Goldfish
Atrazine Channel Catfish, Rainbow Trout
Capacitor 21 Largeroouth Bass, Redear Sunfish, Rainbow Trout
Chlorobenzene Largemouth Bass, Goldfish, Rainbow Trout
Chloroform Rainbow Trout
2,4-Dichlorophenol Channel Catfish, Goldfish, Rainbow Trout
2,4-Diehloro- Largemouth Bass, Goldfish, Rainbow Trout
phenoxyacetlc acid
Dioctyl phthalate Largemouth Bass, Goldfish, Rainbow Trout
Malathion Goldfish
Trisodium nitrilp- Channel Catfish, Goldfish, Rainbow Trout
triacetic acid*
Phenol Bluegill Sunfish, Goldfish, Rainbow Trout
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sources of the selected organic compounds are given 1n Table 1. A summary, of
the toxiclty tests performed in this Investigation 1s presented In Table 2.
Test conditions and expression of data. Each organic compound was tested
using four or more concentrations at each of two water hardness levels (10 and
200 mg/1 CaC03). Exposure was Initiated 20 win after fertilization in trout,
1 to 2 hr postspawning for bass, bluegill, goldfish, and redear sunfish, and
2 to 12 hr after spawning for channel catfish. Average hatching times were 23,
4.5, 4, 3.S» 3.5, and 2.5 days for trout, catfish, goldfish, redear, bass, and
bluegill, respectively. Toxicity tests were performed in temperature-regulated
environmental rooms. Test water was monitored at regular intervals for tempera-
ture, dissolved oxygen, specific conductivity, water hardness, and pH, using a
YSI tele-thermometer with thermocouple (model 42SC), YSI oxygen meter (model
51A}» Radiometer conductivity meter (model DCM 2e), Orion divalent cation
electrode (model 93-32), and a Corning digital pH meter (model 110). Flow rates
from peristaltic and syringe pumps were monitored twice daily. Temperature
varied from 12.5 to 14.5°C for trout, 25.9 to 29.6°C for catfish, and 18.2 to
25.8°C for the remaining species. Dissolved oxyqen levels at the above tempera-
ture ranges were 9.1 to 10.5, 5.8 to 6.8, and 6.5 to 8.9 mg/1, respectively.
Monitoring data for pH, hardness, conductivity, and flow rates are summarized
in Table 3. Although routine assays were not conducted for suspended solids,
sample measurements ranged from 4.0 to 15.0 mg/1 (American Society for Testing
and Materials, 1977).
Control eggs were cultured simultaneously with experimentals and under
Identical conditions, except for omission of the toxicants. Eggs were examined
dally to gauge extent of development and to remove dead specimens. Sample size
ranged from 100 to 150 eggs per exposure concentration. Percent survival,
expressed as the frequency in experimental populations/controls, was determined
at hatching and 4 days after hatching. In all instances, survival frequencies
were based on accumulative test responses Incurred from onset of treatment.
Although about 50% of the tests were extended through 8 days posthatching,
larval lethality usually was insignificant after the first 4 days. Significant
lethality occurred after 4 days only in tests with aniline and 2,4-0, and these
results are discussed in .the text. Hatchability included all embryos which
survived to complete the hatching process. Teratogenesis was determined at
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10
hatching and expressed as the percent of survivors affected by gross,-debili-
tating abnormalities likely to result In eventual lethality (B1rge and Black,
1977a). Normal survivors were defined as those animals free from teratic
defects. Teratic organisms were seldom encountered 1n control populations and
never exceeded 1%. Counting teratic larvae as lethals, log probit analysis
(Flnney, 1971) was used to compute control-adjusted LC5Q and LC, values with
95Z confidence limits. The LC.'s were used to estimate toxicant concentrations
which produced 1% impairment of test populations. All probability (P) levels
were determined using analysis of variance.
Test water. Considerable attention was given to the development of a
reconstituted water suitable for toxicity testing. Reconstituted water usually
provides more stable test conditions than natural water, as the latter may be
subject to substantial seasonal fluctuations in composition (e..g_., total
dissolved solids, hardness, pH). Also, problems with background contaminants
generally are minimized when prepared water Is used. However, it is essential
to use a formulation which gives chemical and physical characteristics similar
to natural water. The test water described below has been used extensively
during the past four years, and has given toxicity responses with metals (e_.£.,
Cd, Cir, Hg, Zn) that compare closely with results obtained using natural water
of high quality. Considering access, quality control, and other factors, use
of reconstituted water did not increase cost.
Reconstituted water was prepared by the addition of reagent-grade calcium,
magnesium, sodium, and potassium salts to distilled, double deionized water.
Physicochemical characteristics are given in Table 4. Concentrations of cations
and anlons were within ranges published for freshwater resources in Arizona
(Dutt and HcCreary, 1970), Kentucky (U.S. Geological Survey, 1970), and other
areas of the U.S. (McKee and Wolf, 1963; Mount, 1968). Total chloride content,
total dissolved solids, and the concentration of sodium plus potassium were
under maximum levels of 170 mg/1, 400 mg/1, and 85 mg/1 observed for 95% of
U.S. waters found to support a good, mixed fish fauna (Hart, e£a]_., 1945).
Specific conductivity compared -favorably with values of 150-500 gmhos/cm recom-
mended for fish propagation (McKee and Wolf, 1963), and osmolarity was well
under the maximum limit of 50 mOsm/Kg water suggested for U.S. freshwaters
(National Technical Advisory Committee, 1968). Total alkalinity and pH also
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Table 3. Water quality characteristics observed during tox1 city tests with organic compounds.
Embryo-Larval
Compound
Aniline
Atrazlne
Capacitor 21
Chlorobenzene
Chloroform,
2,4-Dichlorophenol
Bioassays
Designated
Mater Hardness
(mg/1 CaC03)
50
200
50
200
50
200
50
200
50
200
50
200
2,4-Dichloro- 50
phenoxyacetic add 200
Dioctyl phthalate
Malathion
Tri sodium nitrllo-
triacetic acid
Phenol
50
200
50
200
50
200
50
200
Observed
Water Hardness
(mg/1 CaC03)
46.9 ± 3.4
195.3 ± 14.3
51.3 ± 1.1
201.9 ± 0.2
49.5 ± 0.2
204.0 ± 1.7
51.2 ± 1.2
2Q3.4 ± 3.3
48.8 ± 0.7
210.2 ± 1.2
50.0 ± 0.9
200.0 ± 2.2
50.8 ± 1.5
200.1 ± 2.9
53.3 ± 0.9
205.2 ± 2.7
54.3 * 1.3
196.6 ± 3.6
51.8 ± 3.1
187.1 ± 18.6
50.9 ± 1.2
199.2 ± 1.0
Test Parameters
pH
7.7 ± 0.1
7.7 ± 0.1
8.0 1 0.1
7.9 ± 0.1
7.8 ± 0.1
7.6 ± 0.1
7.6 ± 0.0
7.6 ± 0.1
7.3 ± 0.0
7.3 ± 0.0
7.8 ± 0.1
7.8 ± 0.1
7.8 i 0.2
7.7 ± 0.1
7.5 ± 0.2
7.4 ± 0.1
7.7 ± 0.1
7.6 ± 0.1
8.1 ± 0,1
7.9 ± 0.1
7.9 ± 0.1
7.8 ± 0.1
(Mean ± Standard
Conductivity
(umhos/cm)
121.3 ± 2.0
257.7 ± 17.5
106.2 ± 0.8
240.7 ± 4.4
109.9 ± 1.0
240.8 ± 4.9
104.2 ± 1.9
260.3 i 13.9
91.8 ± 1.4
223.4 i 1.1
124.2 ± 13.0
263.1 ± 19.6
122.4 ± 1.3
259.2 ± 4.4
101.6 ± 2.1
235.5 ± 4.1
106.6 ± 0.7
220.0 ± 5.0
140.0 ± 4.1
290.0 ± 5.1
123.3 ± 11.9
258.5 ± 15.6
Error)
Flow Rate
(ml/hr)
206.7 ± 13.7
207.8 ± 7,7
179.5 ± 10.2
178.8 ± 16.8
200.4 ± 11.0
204.0 ± 5.6
216.2 ± 4.0
213.3 ± 5.9
191.8 ± 1.8
190.7 t 1.5
193.9 ± 2.3
191.8 ± 4.2
185.4 ± 6.2 .
170.0 ± 11.0
186.1 ± 3.7
187.1 ± 6.6
181.6 ± 2.0
179.6 ± 1.0
195.6 ± 1.2
194.9 ± 0.1
193.5 ± 2.5
194.3 ±4.2
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12
were within optimum ranges for aquatic habttat (Baas Becking, et^aK, I960;
McKte and Wolf, 1963; fjTAC, 1968). As maintained In the test system described
below, dissolved oxygen ranged from 9.1 to 10.5 mg/1 at temperatures of 12.5
to 14.5°C used for trout embryos. A minimum of 7 mg/1 has been recommended
for trout and salmon spawning waters (NTAC, 1968).
Embryo-larval test system. Toxlcity tests were conducted using the flow-
through system illustrated in Figures 1 and 2. Using graduated flow from a
syringe pump, toxicant was administered to a mixing chamber which was situated
ahead of each egg exposure chamber. Test water was delivered to the mixing
chamber by regulated flow from a peristaltic pump. Continuous aeration was
supplied to the peristaltic pump reservoirs. Solutions from the two pump
channels were mixed by mechanical stirring or homogenization, and delivered
from the mixing unit to the test chamber under positive pressure. Toxicant
exposure level was regulated by adjusting the mixing ratio between pumping
units and/or by varying the concentration of toxicant delivered from the syringe
pump. Flow rates from syringe and peristaltic pumps were monitored using
Silmont micro and no. 12 liquid flow meters, respectively. Flow rate was set
at 200 ml/hr for SOQ-ml test chambers, giving a detention time of 2.5 hr. The
flow-through system was operated using Brinkmann (model 131900} and Gil son
(model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355),
Sage pumps were fitted with modified syringe holders, as noted previously by
Birge, et al_. (1979a), and each unit was operated using up to six double-ground
glass syringes. Syringe capacity varied from 1 wl to 100 ml, depending upon
the toxicant.
To preclude loss of organic toxicants of high volatility, a closed exposure
chamber devoid of an air-water interface was designed for use with fish embryo-
larval stages. Test chambers were constructed from 3" Pyrex pipe joints,
provided with clamp-locking 0-ring seals. Using standard glass-blowing tech-
niques, the pipe was cut and sealed to give a capacity of 0.5 liter (Figure 3).
An outlet tube was annealed to the cover, with an inlet positioned near the
bottom of the chamber. A stainless steel inlet screen was positioned 3 cm
above the bottom of the dish, dividing the chamber into an upper egg compartment
and a lower stirring compartment. Fish eggs were supported on the inlet screen,
and a Teflon-coated magnetic stirring bar was used in the lower compartment to
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13
Table 4. Reconstituted test water.
Components and Characteristics
Dissolved Salts1, mg/1
CaCl2
MgSQ4-?H2Q
NaHC03
KC1
Chemical Composition, mg/1
Ca
Mg
Na
K
Cl
HC03
so4
2
Phy s i cochemi ca 1 Character! s ti cs
Hardness, as mg/1 CaC03
PH
Total alkalinity, as mg/1 CaC03
Conductivity, pmhos/cm
Osmolarity, mOsm/Kg HgO
Total dissolved solids, mg/1
Dissolved oxygen, mg/1 at 13.5°C
Hardness
50
37.5
37.5
100
5
13.6
3.7
27.4
2.6
26.3
72.6
14.6
53.3 ± 1.3
7.84 ± 0.02
66.7 ± 0.4
133.6 ± 1.4
8.9 ± 0.2
121.4 ± 4.4
9.9 ± 0.2
(mg/1 CaCQa)
200
150
150
100
5
54.2
14.8
27.4
2.6
98.2
72.6
58.5
197.5 ± 5
7.78 ± 0
65.3 ± 0
282.0 ± 1
12.7 ± 0
336.7 ± 7
10.1 ± 0
.8
.02
.6
.9
.4
.8
.2
Prepared in distilled, deionized water with a specific conductivity of
0,25 ymhos or less.
p
Measurements made at 25°C except where noted. Mean with standard error
determined for 10 replicates.
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14
provide moderate, continuous agitation of test water. An upper outlet screen
was used to retain test organisms. The outlet screen was held in place by a
Pyrex pedestal, and the Inlet screen was supported on the constricted upper
wall of the stirring compartment (Figure 3). Access to test organisms was
obtained by opening the watertight joint and removing the chamber cover. Prior
to opening the chamber, a rapid-disconnect was used to remove the inlet line and
drain the fluid level down to the 0-ring seal. When perfused with a continuous
flow of oxygen-saturated water, the sealed chamber was essentially free of
standing air space.
As noted above, toxicant and test water were blended by either mechanical
mixing or homogenization, using mixing chambers. A stoppered 250-ml side-arm
flask, operated with a magnetic stirrer (Hagnestir, model S829Q), was adequate
for maintaining stable concentrations of water-soluble organic compounds
(Figure 2). However, high speed homogenization was required to suspend hydro-
phobic organics in test water. This was accomplished with an Oster homogenizer,
equipped with a 400-ml glass container. The latter was provided with terminal
inlets for syringe and peristaltic pump lines and a side outlet for supply of
water-toxicant homogenate to the test chamber (Figures 3.1, 3.2). Pyrex tubing
%
(3 mm O.D.} was used to extend pump inlet lines to a depth of 3 cm above the
stirring blades. Though homogenization initially was maintained continuously,
intermittent operation generally proved adequate. Blending time was regulated
with an electronic timer and varied for different organic compounds, depending
on the stability of their aqueous suspensions. In addition, moderate agitation
supplied to the exposure chamber and regulation of flow rate were used to
prevent immiscible organics from partitioning out of test water.
Analy tica1 prgcedures. Exposure concentrations for all organic toxicants
were confirmed by daily analyses of test water, using either gas chromatography
(SLC) or spectrophotometric methods.* Aniline, chlorobenzene, dioctyl phthalate,
and malathion were analyzed on a Packard gas chromatograph (model 7400} with a
flame ionization detector (FID). Capacitor 21 was analyzed with the same
Instrument, using an electron capture detector (unmodified tritium foil}.
Quantification of 2,4-dichlorophenoxyacetic acid was accomplished with an FID,
using a Hewlett Packard gas chromatograph (model 5838A). Chloroform concentra-
tions were determined by direct sampling, using the Hewlett Packard GLC equipped
-------
Figure 1
Embryo-Larval Test System
DIRECTION OF FLOW
EGG
COMPARTMENT
STIRRING
COMPARTMENT
MIXING
CHAMBER
HOMOGENIZER
FLOW
METER
TEST
WATER
PERISTALTIC
PUMP
in
TEST CHAMBER
Test water and toxicant were supplied to the mixing chamber using peristaltic and
syringe pumps. Insoluble toxicants were suspended 1n test water by mechanical
homogenization and a magnetic stlrrer was used to provide additional agitation
In the stirring compartment of the test chamber.
FLOW
METER
SYRINGE
PUMP
TOXICANT
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16
with a Purge and Trap system (model 7675A) and a flame iom'zation detector.
Pre-purified nitrogen served as the carrier gas for all GLC determinations and
as the purge gas for the chloroform analyses. Column packings were obtained
from Supelco, Inc., except for 10% Carbowax 20H on 80/100 Anakrom U which was
prepared in our laboratory. External standards were used for quantification
unless otherwise indicated. Atrazine, 2,4-dicnlorophenol, trisodium m'trilo-
triacetic acid, and phenol were analyzed on a Varian-Techtron 635 spectropho-
tometer. Standard curves were prepared from authentic samples of toxicants in
the appropriate solvents.
Aniline was extracted from 0.25 to 1.0 litsr water samples with reagent-
grade benzene. The extracts were dried with anhydrous sodium sulfate and
concentrated using an air stream. Aniline concentrations were determined using
a glass column (2 m X 2 mm I.D.). The stationary phase was 1.51 OV-17/1.958
QF-1 on 80/100 Chromosorb W HP. Oven, inlet, and detector temperatures were
75°, 190°, and 2QO°C, respectively, and the nitrogen flow rate was 40 ml/min.
The detection limit for aniline in water was 40 ug/1. Aniline standard
solutions-were prepared in distilled water, extracted, and analyzed in the
same manner as test water samples.
Atrazine was determined employing a modification of a previously reported
procedure (White, et aj_., 1967). A 100-ml test water sample was extracted with
chloroform. Carbon tetrachloride (5 ml) and 50% sulfuric acid (2 ml) were added
to the chloroform layer, and this mixture was shaken for 30 sec at 15-min
intervals over a 2-hr period. The solution was transferred to a 125-ml erlen-
meyer flask, mechanically mixed for 15 min with 20 ml of water, and allowed
to stand for 2 hr. Atrazine in the water layer was analyzed spectrophotomet-
rically at 225, 240, and 255 nm, and the detection limit was 10 yg/1.
Capacitor 21 was extracted from 0.5 to 2,0 liter test water samples,
using multiple aliquots of reagent-grade chloroform. The combined extracts were
dried with anhydrous sodium sulfate, concentrated to near dryness with an air
stream, and quantitatively reconstituted in ethyl acetate. Capacitor 21 concen-
trations were determined on a 2 m X 2 mm I.D. glass column. The stationary
phase was 3% Dexsil 300 GC on 80/100 Chromosorb W HP. Oven, inlet, and detector
temperatures were 230°, 250°, and 260°C, respectively, and the carrier gas flow
rate was 55 ml/min. The 4-chlorobiphenyl component of Capacitor 21 was used as
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17
Figure 2
Multichannel Assembly of Toxicity Test Units
2.1 Components included an electronic timer (A), liquid flow meters (B}»
mixing chambers used for insoluble (C) and soluble (D) toxicants,
peristaltic pump (E), and exposure chambers (F). Syringe pumps
were mounted outside the environmental room to avoid effects of low
temperature and high humidity on operation.
2.2 View of magnetic stirrers (F) situated beneath the drainboard used
to support exposure chambers (E).
2.3 A bank of 10 exposure chambers housed in a 6' X 10' environmental
room. Inlet lines from mixing chambers (A) were attached with rapid
disconnects (black arrow). The watertight drainboard (8) contained
spillage. Test chamber outlet lines (white arrow) were connected
to waste receptacles (C).
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-------
18
a quantitative "marker" for this multi-component toxicant. Capacitor 21 in
reagent-grade ethyl acetate was used to prepare standards for quantification,
and the detection Hmit was 0.1 ug/1.
Chlorobenzene was extracted from 0.1 to 1.0 liter water samples using
ether or chloroform. The extracts were dried with anhydrous sodium sulfate and
concentrated with an air stream. Chlorobenzene was analyzed on the same column
used for aniline determinations. Oven, inlet, and detector temperatures were
80°, 115°, and 230°C, and the carrier gas flow rate was 37 ml/min. Chlorobenzene
in benzene or ethyl acetate was used to prepare the standard curve, and the
detection limit was 5.0 ug/1.
Chloroform was analyzed directly from 1 to 15 ml samples of test water,
using the purge and trap system described above. Each sample was purged with
dry, pre-purified nitrogen (10 ml/rain). Chloroform was adsorbed on a Tenax SC
trap at ambient temperature, desorbed at 200°C, and analyzed at programmed
temperatures of 70 to 105°C on a 2 m X 2 mm I.0. glass column. The stationary
phase was 10% Carbowax 20M on 80/100 Anakrora U, and the detector temperature
was 250°C. The carrier gas flow rate was 19 ml/min, and the detection limit
was 0.1 ng/l.
Samples of 2,4-dichlorophenol were analyzed using a modification of the
phenol analysis procedures (American Public Health Association, 1975). Added
to each 0.25 liter sample of test water were 2 ml of ammonium chloride (50 g/1),
5 ml of 0.5 N ammonium hydroxide, 2 ml of 4 aminoantipyrine (12 g/1), and 2 ml
of potassium ferricyam'de (48 g/1). After standing 0.5 to 1.0 hr» the solution
was extracted with chloroform and dried over anhydrous sodium sulfate. The
samples were quantified at 470 nm, and the detection limit was 1.0 pg/1.
Test water samples of 2,4-diehlorophenoxyacetic acid (2,4-D as the
potassium salt) were collected in 0.05 to 0.5 liter volumes, diluted to 0.5 1
with distilled water where necessary, and acidified with 5 ml of concentrated
hydrochloric acid. The 2»4-D was extracted with multiple aliquots of reagent-
grade chloroform. The extracts were evaporated to dryness with a stream of
air and reacted at 60°C with diazomethane in ether for 10 min. Several ml of
ethyl acetate were added, and the subsequent mixture was concentrated by evapor-
ation with an air stream. Samples of the 2,4-D methyl ester were analyzed on
a 2 m X 2 mm I.D. glass column at programed temperatures of 160 to 240°C. The
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19
Figure 3
Exposure Chamber
3.1 Disassembled chamber, including cover (A), egg compartment (B),
stirring compartment (C), screen support (D), and 0-ring with
inlet and outlet screens (E).
3.2 Assembled test chamber, showing outlet from egg compartment (A),
locking clamp (B), and stirring compartment inlet (C).
-------
-------
20
stationary phase was 10% Carbowax 20M on 50/60 Anakrom U. Inlet and detector
temperatures were 250° and 265°C, respectively. Standards for 2,4-0 were
prepared 1n ethyl acetate, and the detection limit was 50 yg/1.
Dloctyl phthalate (OOP) was extracted from 0.1 to 1.0 liter test water
samples with multiple aliquots of reagent-grade chloroform. The combined extracts
were dried with anhydrous sodium sulfate and concentrated to near dryness with
an lir stream. OOP was reconstituted in ethyl acetate and quantified using a
0.5 m X 2 mm I.D. glass column. The stationary phase was 1.5% OV-17/1.95%
QF-1 on 80/100 Chromosorb W HP. Oven, inlet, and detector temperatures were
235°» 250°, and 260°C, respectively, and the carrier gas flow rate was 50 ml/min.
OOP in reagent-grade ethyl acetate was used to prepare the standard curve, and
the detection limit was 25 jjg/1.
Malathion was extracted from 0.1 to 2.0 liter test water samples with
several aliquots of chloroform. The combined extracts were dried with anhydrous
sodium sulfate and evaporated to near dryness with air. Malathion, reconstituted
in ethyl acetate, was quantified with the same column used for analyses of
dioctyl phthalate. Oven, inlet, and detector temperatures were 210°, 230°,
and 250°C, respectively. The carrier gas flow,rate was 45 ml/min. Malathion
standards were prepared in ethyl acetate, and the detection limit was 50 jig/1.
Trisodium nitrilotriacetic acid (NTA) was analyzed by the zinc-zincon
method (U.S. EPA, 1974). To prevent interference with calcium and magnesium
ions, NTA samples were batch-treated with ion exchange resin (Dowex 50W-X8,
50-100 mesh). Prepared samples were quantified at 620 nm, and the detection
limit was 0.5 mg/1.
Phenol concentrations were determined using the 4-aminoantipyrine proce-
dure with chloroform extraction as described in Standard Methods (American
Public Health Association, 1975). Samples were quantified at 450 nm, and the
detection limit was 1.0 yg/1. *
Initial Performance Evaluation
Sudan IV dye in chlorobenzene (10 g/1) was injected into the test
system at rates calculated to give dye concentrations which ranged from
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21
0.7S to 1.84 mg/1, using peristaltic/syringe pump dilution ratios of 13,300 to
5,440, respectively. The dye-chlorobenzene was quantified at a wavelength of
540 pm, using a Model 635 Varian-Techtron spectrophotometer. Actual dye concen-
trations, determined on samples of test water pipetted at random from the egg
compartment, were in close agreement with calculated values (Table 5). The
flow rate for test water was 200 ml/hr and collection intervals varied from 5
to iO min for operating periods of 0.5 to 5.0 hr. Visual inspection of the
flow pattern also revealed highly uniform distribution of the insoluble Sudan IV-
chlorobenzene.
Subsequent to this initial evaluation, the system continued to provide
good reproducibility of exposure concentrations in actual toxicity tests.
Results summarized in Table 6 include analytical data for chlorobenzene and
dioctyl phthalate, two compounds of low water solubility, and chloroform, a
highly volatile organic. Variations in exposure levels were no greater than
for soluble compounds of low volatility,such as NTA (Table 18) and phenol
(Table 19). In addition, toxicant concentrations were regulated with precision
down to 1 yg/1 or less (Tables 11, 19). For example, using a calculated concen-
tration of 1 wg/1 phenol, actual concentrations (mean ± standard error) in four
tests were 0.7 ± 0.2, 1.2 ± 0.3, 1.3 ± 0.3, and 1.5 ± 0.3.
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22
Table S. Regulation of Sudan IV-Chlorobenzene in continuous flow tests.
Dye
Dilution
Factor
13300
8980
7900
6670
6670
GQQG
6000
5830
5630
5440
Concentration (rag/1 )1
Calculated
0.75
1.11
1.27
1.50
1.50
1.67
1.67
1.72
1.78
1.84
Actual, Mean ± S.E.
0.73 t 0.03
1.10 ± 0.02
1.36 ± 0.04
1.60 ± 0.03
1.58 ± 0.03
1.64 ± O.OD
1.72 ± 0.06
1.77 ± 0.04
1.73 ± 0.04
1.83 ± 0.04
Operating Time
(minutes)
150
60
60
300
150
30
30
30
60
60
Sampling Interval
(mi nutes }
30
5
5
60
30
5
5
5
5
5
Sudan IV dye in chlorobenzene (10 g/1) was delivered to exposure chambers by a
Sage syringe pump (model 355), and the flow of dilution water was regulated with
a multichannel Silson peristaltic pump (model HP8).
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23
Table 6, Regulation of organic compounds in continuous flow toxicity tests with fish
embryo-larval stages.
Compound uWa!er
/c_,,_j__* Hardness
(Species) (mg/1 CaCQ3
Chlorobenzene 50
(Largemouth Bass)
200
Chloroform 50
(Rilnbow Trout)
200
Dioctyl phthalate 50
(Largemouth Bass)
200
Actual
Concentrati on
i Mtan ± S.E.
' (mg/1 )
0.013 ± 0.002
0.038 ±0.003
0.16 ± 0.01
2.55 i 0.28
27.3 ± 1.4
0.009 ± 0.001
0.040 t 0.006
0.15 ± 0.02
3.10 ± 0.34
23.2 ± 1.8
0.004 * 0.001
0.008 ±0.001
0.059 4 0.006
0-69 ± 0.03
10.1 ± 0.7
0.003 ± 0.001
0.010 ± 0.001
0.056 ±0.004
0.63 ± 0.02
10.6 ± 0.4
0.055 ± 0.006
0.30 ± 0.03
46.3 ± 4.0
66.9 ± 3.3
149 ± 15
0.065 ± 0.012
0.30 ± 0.03
35.5 ± 3.1
60.6 ± 4.3
146 ± 16
Percent , 7-
Hatchability1^
91(2)
86
75(11)
27(55)
4(100)
100
93(2)
72(13)
25(42)
4(100)
95
92
89(3)
73(4)
36(37)
106
88(1)
83(3)
72(3)
23(40)
97
93
74
39(1)
13(4)
97
91(1)
71
39(1)
16(3)
Percent
Hatching
89
86
67
12
0
100
91
63
15
0
95
92
86
70
23
106
87
80
70
14
97
93
74
39
12
97
90
71
39
16
Normal Survival
4 Days
Posthatching
80
60
24
0
0
93
64
14
0
0
95
92
86
70
23
106
87
80
70
14
95
91
67
26
2
96
90
64
30
7
Frequency determined as survival in experimental population/control.
Frequency of teratic survivors in hatched population is given parenthetically.
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24
APPLICATION OF TEST SYSTEM
Embryo-Larval Toxicity Tests
Toxieity tests were performed on the eleven organic compounds listed in
Table 2, using two levels of water hardness (50 and 200 mg/1 CaCOj). In all
cases, survival data for experimental populations were control-adjusted. Control
survival ranged from 88 to 99% except in chloroform tests with trout, where it
averaged 72%. Log probit values for the organic toxicants appear in Tables 7
and 8, and dose-response data are summarized in Tables 9 through 19.
Aniline. Tests were conducted on developmental stages of largemouth bass,
channel catfish, and goldfish, and survival data are shown in Table 9. Using
soft water, aniline LC5Q values at hatching were 5.5, 9.3, and 32.7 mg/1 for
catfish, goldfish, and bass (Table 7). Embryonic sensitivity to LC5Q and higher
concentrations of aniline increased with treatment times to hatching, which were
2.5, 3.5, and 4.5 days for bass, goldfish, and catfish, respectively (Figure 4}.
This was in agreement with previous results for embryo-larval tests with mercury
(Birge, et al_., 1979c), When exposure-was extended beyond hatching, the only
significant change in median lethal concentrations occurred in tests with bass.
The aniline LC5Q decreased markedly to 11.8 and 5.4 mg/1 at 4 and 8 days pust-
hatching, and these values differed significantly from those observed at
hatching (P < 0.05). More moderate reductions to 5.5 and 5.1 mg/1 were observed
in tests with goldfish. In contrast, aniline LC^'s for catfish showed virtu-
ally no change from hatching through 8 days posthatching. Although bass eggs
exhibited the greatest tolerance to aniline, early posthatched lethality was
highest for this species. It appeared that while embryonic sensitivity to
aniline increased with hatching time, an inverse relationship existed between
hatching time and sensitivity of early larval stages. Water hardness did not
exert appreciable effects on LCg^'s, but low concentrations of aniline appeared
somewhat more toxic in hard water. In tests with goldfish and catfish, the
respective LC,'s at 4 days posthatching were 143 and 249 pg/1 aniline in hard
water, and 215 and 648 yg/1 in soft water (Table 8). Aniline produced substan-
tial teratogenic impairment only at the higher exposure concentrations, and
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I
\00
80
60
40
20
0
• -BASS
o- GOLDFISH
A- CATFISH
WATER HARDNESS-50 mg/l CaCOg
0-1
I 10 100
ANILINE CONCENTRATION (mg/l)
Figure 4. Toxicity of aniline to fish eggs. Aniline exposure was
maintained through hatching, giving treatment times of 2.5, 3.5, and
4.5 days for bass, goldfish, and catfish.
rsi
in
1000
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26
the greatest incidence of teratic defects was observed for goldfish. At 14,
34, and 100 mg/1, aniline administered In hard water produced abnormalities in
hatched populations at frequencies of 2%, 22%, and 43f .
The LCgg values for aniline calculated for the three fish species fell
at or somewhat below the range of 10 to 100 mg/1 reported in most 96-hr tests
on aquatic biota (Cnristensen, 1976). One investigation showed aniline to be
approximately ten times more toxic to trout than to goldfish (McKee and Wolf,
1963). In the present study, a 5 to 6-fold difference in sensitivity was
observed between embryo-larval stages of bass and catfish when IC^'s were
taken at hatching. However, when treatment was extended through 8 days post-
hatching, no significant heterogeneity of response could be demonstrated,
Toxicity tests with atrazine were performed on channel catfish
and rainbow trout (Table 10). As stated previously, concentrations were
reported for a commercially available wettable powder which contained 80%
atrazine. When trout eggs were treated in hard water, the LCgn at hatching and
4 days posthatching was 1.1 mg/1 (Table 7). In tests with the catfish, LC^'s
were 0.31 and 0.24 mg/1 at hatching and at 4 days. These values were 10 to
100 times lower than those reported for the fry of Coregpnus fera (Gunkel
and Kausch, 1976). Atrazine LC,'s calculated' for the rainbow trout were 29.0
and 77.2 pg/1 in soft and hard water, respectively (Table 8) and these values
were in reasonable agreement with a maximum acceptable toxicant concentration
(MATC) range of 65 to 120 ng/1 reported in partial life-cycle studies with
brook trout (Macek, et_al_. , 1976). Catfish embryos appeared more sensitive
than trout stages, but reliable LC, values could not be calculated. Posthatched
lethality was infrequent for both trout and catfish, and water hardness did not
appreciably alter toxicity. Atrazine was highly teratogenic in all tests. For
example, terata occurred at frequencies of 16%, 471, and 86£ in hatched catfish
populations exposed in hard water to atrazine at 0.42, 4,81, and,46.7 mg/1,
respectively.
Capacitor 21. Capacitor 21, a polychlorinated biphenyl, was administered
to embryos and larvae of largemouth bass, redear sunfish, and rainbow trout
(Table 11). Redear sunfish and bass were exposed at both levels of water hard-
ness and trout was tested only in hard water. Trout and bass stages were about
equally sensitive to Capacitor 21, and LCgg values varied from 2.0 to 2.7 yg/1
-------
27
at hitching and from 1.5 to 2.0 pg/1 at 4 days posthatching (Table 7). Develop-
mental stages of the redear sunfish were somewhat more tolerant, and the 4-day
LCg^'s ranged from 8.0 to 13.0 pg/1. The LC,'s calculated at 4 days varied
from 1.3 to 3.5 yg/1 for the redear and 0.5 to 1.0 pg/1 for bass and trout
(Table 8). The 1C, values, which gave an overall range of 0.5 to 3.5 ug/1
for three species of fish, were close to MATC's of 1.1 to 3.0, 1.8 to 4.6, and
2.1 to 4.0 pg/1 determined In life-cycle studies with fathead minnows exposed
to Aroclors 1248, 1254, and 1260, respectively (Nebeker, e£ al_.» 1974; McKim,
1977; DeFoe, et^al_., 1978). Similarly, Aroclor 1254 at concentrations as low
as 0.48 yg/1 produced 16% reproductive impairment in Daphm'a magna (Nebeker
and Puglisi, 1974).
Capacitor 21 proved to be the most toxic of all eleven compounds tested.
Embryonic lethality was the most sensitive test response, and teratogenesis
was observed only at concentrations which substantially reduced hatchability.
Water hardness exerted no appreciable effects on toxicity.
Chlorobenzene. Largemouth bass, goldfish, and rainbow trout eggs were
treated with Chlorobenzene, and as noted previously the procedure of toxicant
injection provided accurate regulation of exposure concentrations for this
insoluble compound (Table 12). Trout eggs were exposed for 16 days in soft
water to mean concentrations (± S.E.) of 0.09 ± 0.02, 0.31 ± 0,04, 1.60 ± 0.19,
4,27 ± O.iO, and 32.0 t 3.7 mg/1 Chlorobenzene. Complete lethality occurred
in all instances, and tests performed in hard water produced similar results.
Embryo-larval stages of the other piscine species were more tolerant to Chloro-
benzene. The LCgg's at 4 days posthatching ranged from 0.05 to 0.06 mg/1 with
bass and from 0.88 to 1.04 mg/1 with goldfish (Table 7). Lethality in experi-
mental larval populations was highly significant for bass in both hard and
soft water (P < 0.01).
In tests conducted with goldfish, the LC, values at 4 days posthatching
were 10 pg/1 in soft water and 33 wg/1 in hard water (Table 8). Frequencies
of teratic bass larvae did not exceed 22 at or below an exposure concentration
of 0.04 mg/1, but ranged from 11% at 0.2 mg/1 to 100% at 27.3 mg/1. Fewer
abnormalities occurred with the goldfish. While limited data exist concerning
the effects of Chlorobenzene on other aquatic species, high potentials for
toxicity and bioconcentration (Metcalf, 1977; Branson, 1978) further stress
-------
28
the importance of developing an acceptable freshwater criterion for this haz-
ardous compound.
Chloroform. This was the most volatile compound tested, and it was admin-
istered to embryo-larval stages of rainbow trout (Table 13). As noted above,
the enclosed test chamber minimized evaporative loss of the toxicant and provided
good regulation of exposure concentrations. The LC5Q values at hatching were
2.03 and 1.24 mg/1 in soft and hard water, respectively, and the larval stages
were unaffected through 4 days posthatching {Table 7). Depending on water
hardness, the LC,'s for chloroform ranged from 4.9 to 6.2 ug/1 (Table 8).
Occurrences of teratogenesis were concentration dependent, and reached 4056 in
hatched populations at 10.6 mg/1. Chloroform LCgQ's, determined in 96-hr tests
on aquatic organisms, were reported by NIOSH (Christensen, 1976) to range from
approximately 10 to 100 mg/1. However, these high values could be due in part to
loss of this highly volatile toxicant from open exposure systems. The chronic
value recently cited for freshwater invertebrates was 500 yg/1 (U.S. EPA, 1979),
but no details were given concerning specific test conditions. If open test
chambers were employed, particularly if used with static or static-renewal
procedures, variations in exposure levels of chloroform due to evaporative loss
could have been appreciable.
2,4-Dichlgrpphenol (DCP). Aquatic toxicity tests were performed with OCP,
using embryo-"larval stages of channel catfish, goldfish, and rainbow trout
(Table 14). When trout were exposed at the two water hardness levels, the LCcg's
at.hatching ranged from 0.07 to 0.08 mg/1, with no further change after 4 days
(Table 7). Catfish and goldfish were considerably less sensitive, as LC5Q's
at 4 days posthatching varied from 0.26 to 1.35 mg/1. Exposure time through
hatching was 24 days for trout and 4 days for catfish and goldfish, correlating
well with the higher lethality observed for trout. During the posthatched
period, high concentrations of DCP produced significant lethality of goldfish
(P < 0.05). Consequently, the LC,-0 values determined with trout and goldfish
tended to converge with time from hatching, and this is consistent with results
for aniline. However, LC^g's indicated that trout at 4 days were still about
4 to 5 times more sensitive to DCP than were goldfish. Catfish larvae, like
those of trout, suffered little lethality. While DCP was not highly teratogenic,
frequencies of teratic goldfish larvae ranged up to 24% and were observed at
-------
29
concentrations as low as 1 yg/1 in tests with catfish and trout (Table 14).
Variations in water hardness appeared to have no substantial effects on toxicity.
At low exposure concentrations, catfish and trout were equally susceptible
to DCP, and the LC,'s calculated for these species varied between .1,6 and 2.8
ug/1 (Table 8). While no freshwater criterion has been established for this
compound, the LC.'s were comparable to the fish flesh tainting threshold of
0.1 to 15 ug/1 determined for o-chlorophenol, p-chlorophenol, and 2,4-dichloro-
phenol (U.S. EPA, 1976). In embryo-larval tests with the goldfish, LC,'s were
considerably higher, ranging from 39.8 to 48.1 pg/1.
2,4-01chlorophenoxyacetic acid (2,4-0). Embryo-larval stages of
largemouth bass, goldfish, and rainbow trout were exposed to 2,4-0, which was
administered as the potassium salt (Table 15). With the rainbow trout, LC5Q's
at both hatching and 4 days posthatching were 11.0 mg/1 in soft water and 4.2
mg/1 in hard water (Table 7). Bass eggs were less sensitive than trout, and
LCgp's ranged frcm 161 to 165 mg/1 at hatching and 82 to 109 mg/1 at 4 days.
Goldfish eggs were extremely tolerant to 2,4-D. The LCgp's at hatching exceeded
the highest exposure levels administered (187-201 mg/1). However, goldfish
larvae were more sensitive and posthatched lethality was significant compared
to controls (P < 0.05). At 4 and 8 days posthatching, depending on water
hardness, LC^'s dropped to 119 to 133 rag/1 and 58 to 68 mg/1, respectively.
A similar effect on goldfish larval stages was noted in tests with DCP. The
LCgg's calculated with warm water species (i_.e_., goldfish, bass) approached
those reported in 96-hr tests using several species of freshwater fish (Brungs,
e_t aJL, 1978).
The LC, values for 2,4-D varied from 21.9 to 32.5 wg/1 when trout stages
were treated in hard and soft water (Table 8). In tests with goldfish and
bass, LC.'s were considerably higher (3.2-13.1 mg/1), and approached the no-
effect level of 10 mg/1 for 2,4-0 established in 96-hr static tests with juvenile
stages of salmon (McKim, et_a]_., 1975). It is of interest to note that the 1C.
values calculated for embryo-larval stages of the three test species encompassed
the MATC range of 0.3 to 1.5 mg/1 determined for 2,4-D (butoxyethanol ester) in
a 10-month chronic test with the fathead minnow (Mount and Stephan, 1967).
Mater hardness was not a major factor concerning the toxicity of 2,4-0
to goldffsh and bass. However, based on LCgg values, trout embryo-larval stages
-------
30
were about 2.5 times more sensitive to 2,4-0 when tests were conducted in hard
water. Not only were embryonic and larval survival reduced at the higher hard-
ness, but teratogenesis also was more frequent.
Dloctyl phthalate (POP). OOP was tested on largemouth bass, goldfish,
and rainbow trout (Table 16), and proved to be one of the least toxic compounds
evaluated. Bass was the most sensitive of the three species, and 4-day LC5Q
values were 56 and 45 mg/1 in soft and hard water, respectively (Table 7).
When trout stages were exposed through 4 days posthatching, the LC^'s were
139 mg/1 in soft water and 149 mg/1 in hard water. With goldfish, the high
survival of embryos and larvae precluded calculation of probit Lean's. However,
median lethal concentrations for OOP appeared to fall at or somewhat above 200 mg/1,
based on the posthatched survival frequencies of 57 to 67% at 186 to 191 mg/1,
While reliable probit LCj's could not be determined from the dose-response data,
detectable effects on fish embryo-larval stages occurred at concentrations as
low as 100 to 500 yg/1, depending on test species. Frequencies of teratic
larvae were low for all species and reached a maximum of.only 16% when trout
embryos were exposed to OOP at 148 mg/1.
The low toxicity observed with OOP was in agreement with results of a
previous study in which Sugawara (1974) compared effects of three phthalate
esters on eggs of the brine shrimp. While di-n-butyl phthalate (DBF) at 10.3
mg/1 significantly reduced hatchability, diethyl phthalate (DEP) exerted no
effects below 61.6 mg/1. Dimethyl phthalate (DMP), the least toxic of the
three phthalate esters, did not reduce survival at the highest concentration
tested (60 mg/1). In tests with adult fish, DBP was found to be somewhat more
toxic, as 96-hr TLcn's ranged from 0.7 to 6.5 rag/1 (Mayer, et a!., 1972).
3U ~"~ ~"~
However, it should be noted that the latter investigation employed acetone at
0.7 ml/1 to solubilize the toxicant, and the carrier solvent may have contri-
buted to greater toxicity.
Malathjon. Embryo-larval stages of goldfish were exposed to malathion
(Table 17), and LC50 values at hatching were 2.61 and 3.15 mg/1 in soft and hard
water, respectively. Due to substantial larval lethality at high concentrations,
4-day LC50's decreased to 1,20 mg/1 in soft water and 1.65 mg/1 in hard water
(Table 7). These values were similar to 96-hr J-C5Q's of 1.9, 1.2, and 1.1 mg/1
-------
31
reported in static tests with the carp, guppy, and white perch, respectively
(Rehwoldt, et a],., 1977). At 4 days posthatching, LC^s calculated for goldfish
varied from 141.1 to 439.6 pg/1 (Table 8). This range was close to the MATC
of 200 to 580 yg/1 determined for the fathead minnow exposed in a 10-month
chronic test (Mount and Stephen, 1967). It appears from these values that
goldfish and fathead minnow are more tolerant to malathion than several other
fish species (U.S. EPA, 1976; Parrish, et al_-> 197*7). For example, in a life-
cycle reproductive study with the flagfish, the MATC was observed to fall within
a range of 8.6 to 10.9 pg/1 (Hermanutz, 1978). The selective toxicity of
malathion for different piscine species was discussed previously 1n the study
by Mount and Stephen (1967).
Teratogenesis was observed only at malathion concentrations which produced
embryonic lethality. In soft water, abnormalities were present in 2%, 6%, and
25% of hatched populations when developmental stages were exposed to 0.6, 2.0,
and 5.2 mg/1, respectively. Water hardness exerted no appreciable effects on
toxicity.
Trisod1 urn nitri1otriacetic acid (NTA). This compound was administered
to channel catfish, goldfish, and rainbow trout, and was relatively non-toxic
to both embryos and larvae (Table 18). At 4 days posthatching, NTA LC5Q values
were 91, 240, and 329 mg/1 for trout, goldfish, and catfish stages exposed in
soft water (Table 7). NTA was somewhat less toxic in hard water, with icon's
of 114, 243, and 385 mg/1. These values were comparable to the 96-hr LCgQ of
114 mg/1 calculated for the fathead minnow (Arthur, et..a]_-, 1974) and the
3-week LC50's of 145 to 650 mg/1 determined in tests with Daphnia (Biesinger,
et al_., 1974). Much higher values were observed in static tests with eleven
marine organisms, as 168-hr TL50's ranged from 1,800 to >10,000 mg/1 (Eisler,
et a]_., 1972).
In the present Investigation, LC,'s varied from 17 to 138 mg/1 in soft
water and 20 to 131 mg/1 in hard water (Table 8). These figures were in good
agreement with the MATC (54-114 mg/1) reported in 8-month life-cycle studies
with the fathead minnow (Arthur, et_a]_., 1974; McKim, 1977). In embryo-larval
tests with the catfish and trout, occurrences of teratogenesis were substantial
throughout the NTA concentration range. However, abnormalities were far less
frequent in hatched populations of goldfish.
-------
32
Phenol. Toxicity tests with phenol were performed on bluegill sunfish,
goldfish, and rainbow trout eggs, and survival data are summarized in Table 19.
When trout stages were treated in soft water, phenol LC50 values were 0.33 mg/1
at hatching and 0.31 mg/1 at 4 days posthatching (Table 7). The LC5Q for phenol
in hard water was 0.07 mg/1 at hatching, with no change at 4 days. In tests
with goldfish, LC50's for phenol in soft water were 1.22 and 0.84 mg/1 at
hatching and 4 days posthatching. When hard water was used, the LCj-fj's were
0.39 and 0.34 mg/1. Bluegill stages were the most tolerant, and the LCcn's were
3.34 and 2.43 mg/1 at hatching and 2.42 and 1.69 mg/1 at 4 days posthatching in
soft and hard water, respectively. The LC,'s for phenol to trout were 0.3 and
8.6 ug/1 in hard and soft water (Table 8). Phenol LCj's calculated at 4 days
posthatching for bluegill and goldfish ranged from 2.4 to 4.0 wg/1 in soft water
and from 2.0 to 8.8 vg/1 in hard water.
Frequencies of embryonic lethality increased with hatching times of the
three species, which were 2.5, 3,5, and 22 days for the bluegill, goldfish, and
trout, respectively (Table 19). Consistent with findings from several embryo-
larval tests discussed above, trout suffered less posthatched lethality than
did species with shorter developmental periods. However, considering the LCc0's
for phenol in hard water taken at 4 days posthatching, the trout was still
approximately 5 times and 24 times more sensitive than the goldfish and bluegill,
respectively. Corresponding LC. values also revealed phenol to be more toxic
to the trout.
Compared to findings for other organics, phenol toxicity was affected
more by variations in water hardness. Based on frequencies of hatchability of
trout eggs exposed at concentrations of 0.01 to 10 mg/1, phenol was approximately
ten times more toxic when administered in hard water (Figure 5), and hardness
was even a greater factor when LC, values were compared at 4 days posthatching.
However, median lethal concentrations for phenol were somewhat l«ess affected
by water hardness. This compound produced substantial numbers of teratic larvae
in trout and goldfish populations. For example, when trout eggs were exposed in
soft water, the frequencies of teratic survivors ranged as high as 73£, and
teratogenesis occurred at or below the limiting concentration for lethality.
Static and flow-through tests with phenol have been reported for a number
of fish species, and 48-hr and 96-hr values varied from 7 to 100 mg/1 (McKee
-------
100
80
§ 60
40
01
20
0
- o
o -
50mg/l CaC03
200 mg/l CaC03
0-001
0-01 0-1 I
PHENOL CONCENTRATION (mg/l)
Figure 5. Effect of water hardness on phenol toxiclty to trout
eggs. Phenol exposure was maintained from fertilization through
hatching.
C/J
10
-------
34
and Wolf, 1963; Brown, et al_., 1967; NAS-NAE Committee, 1973; Christensen, 1976;
U.S. EPA, 1976; Fogels and Sprague, 1977). A 24-hr LCgo of 5 mg/1 phenol was
given for trout embryos, and 2 mg/1 impaired development of oyster eggs (U.S.
EPA, 1976). Although no-effect concentrations of 0.1 and 1.0 rag/1 have been
reported for bluegill and trout (NAS-NAE Conmittee, 1973), phenol at 20 to 70
pg/1 has been shown to induce pathologic changes in gills and other tissues
(U.S. EPA, 1976). As shown in Table 19, a concentration of I pg/1 produced low
frequencies of lethality for embryo-larval stages of goldfish and trout. For
example, control-adjusted lethality averaged 10% when trout were exposed to
1 jig/1 phenol administered in hard water. Considering this and the probit LC,'s
of 0.3 to8.8jjg/l determined for fish embryos and larvae, the maximum concen-
tration of 0.1 mg/1 suggested for freshwater fish (NAS-NAE Committee, 1973)
may be inadequate for the protection of sensitive developmental stages. An EPA
criterion of 1 yg/1 has been established for domestic water supplies and for
protection against fish flesh tainting (U.S. EPA, 1976). It is of interest
that, as for 2,4-dichlorophenol, the ICJs for phenol were close to the organo-
leptic threshold.
Good agreement was obtained when MATC's developed in partial and complete
life-cycle studies were compared-with LC-, values determined in the embryo-larval
tests which extended through 4 days posthatching (Table 20). Consequently,
embryo-larval tests of shorter duration and higher cost feasibility than those
presently recommended (U.S. EPA, 1978) may prove useful in estimating long-term
biological effects of aquatic toxicants. The high sensitivity of the shorter
tests was due in part to the use of combined responses for lethality and terato-
genesis. The teratic larvae included in LC, determinations represented a sig-
nificant number of test organisms which ordinarily would have become actual
mortalities in longer tests. In addition, use of probit analysis gave greater
discrimination of dose-response data than usually obtained with the statistical
procedures (e_.ju, analysis of variance) traditionally applied to results of
chronic studies. However, reliability of 1C 's is dependent upon adequate
characterization of the dose-response. Generally, determinations are possible
when treatment is administered at 4 to 6 well-selected exposure levels, but
additional exposure concentrations may be required in certain instances.
Truncations of or significant internal discontinuities within the survival curves
-------
35
may skew LC, values or affect determinations of reliable confidence intervals,
Such problems were most apparent in tests with OOP (Table 16).
Teratogenesis in Fish Embryos
The debilitating effects of organic toxicants on fish development were
not limited to embryonic and larval lethality, as most test compounds produced
appreciable numbers of teratic larvae. The occurrence of embryonic anomalies
ranged from 0% to 12% at near-threshold concentrations and usually increased
with toxicant exposure level. Compounds producing the greatest frequencies of
terata included atrazine, chlorofaenzene, and NTA. There were several instances
in which teratogenesis occurred at or below the limiting concentration for
lethality (e_-£., phenol to trout). However, considering overall effects of
the eleven compounds, embryonic lethality usually was a more sensitive test
response.
The teratic defects observed in hatched populations did not differ
substantially from those described previously for boron and certain heavy metals
(Birge and Black, 1977a, b). -Defects of the vertebral column were the only
teratogenic responses observed in all toxicity tests. The most common types of
spinal defects included acute lordosis, scoliosis, kyphosis, and extreme rigid
coiling of the vertebral column. In the order named, overall frequencies for
these abnormalities in trout were 16%, 32%, 25%, and 17%, amounting to 90% of
the gross teratogenic impairments observed for this species. Among other
defects, there were less frequent occurrences of dwarfed bodies, microcephaly,
partial twinning, absent or reduced eyes and fins, amphiarthrodic jaws, and
pericardia! edema.
-------
36
SUWARY
Tests wtre conducted on eleven compounds selected to represent various
degrees and combinations of volatility and water solubility. The principal
objective was to develop a test system especially designed to accommodate
compounds difficult to stabilize using conventional procedures. The flow-
through test system which was developed provided adequate regulation of toxicant
injection, giving good reproducibility of exposure concentrations for volatile
and hydrophobic orgam'cs. Aeration supplied to peristaltic pump reservoirs
was adequate to meet oxygen demands of test organisms, precluding any signif-
icant air flow through the exposure chamber. Lack of direct aeration and
elimination of standing air space from the test chamber effectively minimized
volatility, and fish eggs and larvae were easily maintained in the closed test
system.
Comparing effects of the eleven organics on fish embryos and larvae, the
more toxic compounds included Capacitor 21, chlorobenzene, chloroform, dichloro-
phenol, and phenol, and the least toxic were OOP and NTA. Results supported
the view that numerous aromatic and chlorinated hydrocarbons present serious
hazards to developmental and juvenile stages of fish (Mebeker, et aj_., 1974;
Schimnel, et a]_., 1974; U.S. EPA, 1976; Birge, et a]_., 1978). For the most
part, water hardness did not substantially affect the toxicity of organic
compounds. However, in those instances where hardness was a factor, toxicity
generally was greater when organic compounds were administered in hard water.
This effect was best illustrated in testing phenol to trout and goldfish and
2,4-D to trout, and more often was due to increased effects on embryos rather
than larvae.
Of the six fish species tested, developmental stages of the rainbow trout
exhibited the greatest sensitivity to most organic compounds. Trout embryos
proved more susceptible than trout larvae to toxicant exposure, as survival
usually did not change significantly during the posthatched period. Although
fish with shorter developmental times usually displayed greater overall toler-
ance, larval stages of the warm water species (e_.£., bass, goldfish) were more
affected than trout alevins by certain toxicants, including chlorobenzene,
2,4-dichlorophenol, and 2,4-D.
-------
37
Environmental monitoring data are largely incomplete for many organics.
However, a number of compounds tested in the present study have occurred in
natural waters at concentrations which could prove hazardous to embryo-larval
stages of fish. Examples of such chemicals include atrazine (Richard, et alI.,
1975), chloroform (Metcalf, 1977}, malathion (Cook, et al_., 1976), PCB's
(Kartell, et a].., 1975; Nadeau and Davis, 1976; U..S, EPA, 1976), and phenols (NAS-
NAE Committee, 1973). Consequently, there is a need to delineate further the
freshwater criteria for such organics.
Embryo-larval stages present a broad spectrum of target sites for trace
contaminants, including mechanisms associated with fertilization, cellular dif-
ferentiation, proliferation and growth, basic metabolism and systemic functions,
as well as the hatching process and the initial accommodation to a free-living
existence. Due to their high sensitivity and simple maintenance requirements,
fish embryos and larvae are particularly suitable organisms for toxicity tests
with organic compounds. When frequencies of teratogenesis were combined with
embryonic and larval lethality, probit 1C, values determined in embryo-larval
tests generally provided good agreement with MATC's established in partial and
complete,life-cycle studies. The present study further confirms use of embryo-
larval tests in screening aquatic contaminants for toxic properties and developing
freshwater criteria (McKim, 1977; Birge, et al., 1979b).
-------
Table 7. Log problt LCgQ values for organic compounds.
Compound
Aniline1
Atrazine
Capacitor 21
Chlorobenzene*
Species
Largemouth
Bass
Goldfish
Channel
Catfish
Rainbow
Trout
Channel
Catfish
Redear
Sunfish
Largemouth
Bass
Rainbow
Trout
Galdfish
Largeraouth
Bass
Rainbow
Trout
Exposure
50 mg/1 CaCOa
Days Beyond , r
Hatching (Jj50}
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
32.7
11.8
9.3
5.5
5.5
5.0
0.92
0.87
0.34
0.22
0.019
0.013
0.0023
0.0015
-
3.48
, 0.88
0.34
0.05
< 0.09
95% Confidence
Limits
24.0
8.1
7.4
4.1
4.8
4.4
0.67
0.63
0.18
0.15
0.016
0.011
0.0021
0.0014
-
3.08
0.67
0.22
0.04
-
- 44.1
- 16.6
- 11.6
- 6,8
- 6.3
- 5.7
- 1.20
- 1.15
- 0.59
- 0.32
- 0.029
- 0.017
- 0.0027
- 0.0016
_
- 3.87
- 1.12
- 0.51
- 0.07
-
200 mg/1 CaCOa
(mg/f)
29.9
7.1
7.6
4.6
6.3
6.2
1.11
1.08
0.31
0.24
0.014
0.008
0.0027
0.0018
0.0020
0.0020
2.37
1.04
0.39
0.06
< 0.09
95% Confidence
Limits
20.8
4.8
6.0
3.6
5.4
5.3
0.86
0.84
0.20
0.16
0.011
0.007
0.0024
0.0017
0.0016
0.0017
1.96
0.86
0.25
0.04
-
- 42.5
- 10.3
- 9.5
- 5.7
- 7.3
- 7.3
- 1.39
- 1.35
- 0.48
- 0.35
- 0.080
- 0.011
- 0.0030
- 0.0019
- 0.0021
- 0.0021
- 2.86
- 1.25
- 0.58
- 0.08
-
CO
00
-------
Table 7 - continued.
Exposure
Compound Species Days Beyon
Hatching
Chloroform Rainbow
Trout
2,4-Dlchlorophenol Goldfish
Channel
Catfish_
Rainbow
Trout
2,4-Dichloro- Goldfish
phenoxyacetic acid
Largemouth
Bass
Rainbow
Trout
Dioctyl phthalate Goldfish
Rainbow
Trout
Largemouth
Bass
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
0
4
50 mg/1 CaC03
d LC50
(mg/1)
2.03
2.03
1.76
0.39
1.85
1.35
0.08
0.08
> 187
133.1
165.4
108.6
11.0
11.0
> 186
> 186
139.1
139.5
65.5
55.7
95% Confidence
Limits
0.95
0.95
1.26
0.29
1.25
0.94
0.07
0.07
108.6
130.6
92.5
7.8
7.8
-
122.8
123.2
59.2
50.5
- 3.75
- 3.75
- 2.48
- 0.62
- 2.82
- 2.02
- 0.10
- 0.10
- 174.8
- 274.1
- 138.4
- 15.1
- 15.1
-
- 164.9
- 165.2
- 71.9
- 60.6
200 mg/1 CaCOa
LC5Ov
(mg/1)
1.24
1.24
1.24
0.26
1.70
1.07
0.07
0.07
> 201
119.1
160.7
81.6
4.2
4.2
> 191
> 191
154.0
149.2
32.1
45.5
95% Confidence
Limits
0.62 -
0.62 -
0.78 -
0.17 -
1.08 -
0.68 -
0.05 -
0.06 -
98.5 -
122.9 -
64.8 -
2.8 -
2.8 -
;
127.0 -
125.8 -
21.5 -
39.6 -
2.16
2.16
1.85
0.97
2.65
1.66
0.09
0.09
150.6
230.6
103.5
5.9
5.9
-
216.0
203.8
46.4
51.1
-------
Table 7 - continued.
Compound
Malathion
Trisodium nitHlo-
triacetic acid
Phenol 1
Species
Goldfish
Channel
Catfish
Goldfish
Rainbow
Trout
Bluegill
Sunfish
Goldfish
Rainbow
Trout
Exposure
Days Beyond
Hatching
0
4
0
4
0
4
0
4
0
4 ,
0
4
0
4
50 ing/1 CaCOa
-Lc§n
(•9/1)
, 2.61
1.20
388.3
329.3
269.6
240.4
92.3
90.5
3.34
2.42
1.22
0.84
0.33
0.31
95% Confidence
Limits
2.25 -
1.06 -
362.7 -
308.8 -
232.3 -
206.9 -
80.0 -
78.2 -
2.17 -
1.51 -
0.58 -
0.50 -
0.26 -
0.23 -
3.08
1.35
415.5
352.6
309.6
276.1
104.4
102.6
5.46
4.06
2.02
1.32
0.40
0.41
200 mg/1 CaCOa
LC50
(mg/1)
3.15
1.65
393.5
384.7
257.0
243.4
120.1
114.0
2.43
1.69
0.39
0.34
0.07
0.07
95% Confidence
Limits
2.81
1.50
345.9
343.1
221.5
211.2
99.6
98.6
1.58
1.05
0.25
0.23
0.04
0.05
- 3.56
- 1.80
- 438.2
- 423.6
- 295.5
- 277.9
- 141.8
- 129.9
- 3.95
- 2.83
- 0.57
- 0.48
- 0.10
- 0.11
LC§Q values for aniline, chlorobenzene, and phenol differ somewhat from those previously published
(Birge, et a_L » 1979a). In the earlier investigation, teratlc larvae were not Included 1n probit
determinations.
-------
Table 8. Log problt 1C, values determined at 4 days posthatchlng for organic compounds.
50 mg/1 CaC03
Compound
Aniline*
Atrazlne
Capacitor 21
Chlorobenzene^
Chloroform
2,4-D1chlorophenol
Species
Largeniouth Bass
Goldfish
Channel Catfish
Rainbow Trout
Channel Catfish
Redear Sunfish
Largemouth Bass
Rainbow Trout
Goldfish
Largeniouth Bass
Rainbow Trout
Rainbow Trout
Goldfish
Channel Catfish
Rainbow Trout
LCi
(p9/D
-
215.0
647.6
29.0
-
3.5.
t).5
-
10.0
-
-
6.2
48.1
2.8
2.8
95% Confidence
Limits
_
125.1 -
412.6 -
11.0 -
-
1.6 -
0.4 -
-
5.0 -
-
-
0.2 -
20.6 -
1.0 -
1.4 -
-
333.5
892.2
56.9
-
5.1
0.7
-
17.0
-
-
34.9
75.5
5.9
4.8
200 mg/1 CaCQa
LCi
(,19/1)
-
143.2
249.3
77.2
-
1.3
0.9
1.0
33.0
8.0
-
4.9
39.8
1.6
1.7
95% Confidence
Limits
-
79.0 -
158.5 -
36.8 -
-
0.6 -
0.7 -
0.6 -
18.0 -
2.0 -
-
0.3 -
0.1 -
0.4 -
0.7 -
-
231.4
359.6
130.1
-
2.0
1.0
1.3
52.0
14.0
-
22.5
68.9
4.8
3.2
-------
Table 8 - continued.
50 mg/1 CaCOa
Compound
2,4-Dlchloro-
phenoxyacetic acid
MalatMon
Tri sodium nltrllo-
trlacetic acid
Phenol 1
Species
Goldfish
Largemouth Bass
Rainbow Trout
Goldfish
Channel Catfish
Goldfish
Rainbow Trout
BluegUl Sunfish
Goldfish
Rainbow Trout
LCi.
(ng/D
8,210
13,102
32.5
141.1
138,387
28,528
16,902
4.0
2.4
8.6
95% Confidence
Limits
2,677
4,418
8.9
98.8
117,087
17,410
10,864
0.5
0.3
4,2
- 15,027
- 21,886
- 83.6
- 186.6
- 156,791
- 40,835
- 23,223
- 12.1
- 8.7
- 14.8
200 mg/1 CaCOa
LCi
(pg/D
8,851
3,214
21.9
439.6
130,949
30,142
20,198
2.0
8.8
0.3
95% Confidence
Limits
3,835
1,218
6.2
326.2
90,740
18,925
12,054
0.3
2.5
0.1
- 14,637
- 5,989
- 55.4
- 546.6
- 166,682
^'42,387
- 28,566
- 5.5
- 20.1
- 0.8
1
LCj values for aniline, chlorobenzene, and phenol differ somewhat from those previously
published (Birge, et aj_., 1979a). In the earlier Investigation, teratlc larvae were not
Included 1n prob1t~3eterminations.
r\a
-------
43
Table 9. Toxicity of aniline to embryo-larval stages of fish.
Water
Species Hardness
{mg/1 CaC03)
Largemoutn 50
Bass
200
Goldfish 50
200
Channel 50
Catfish
200
Aniline
Concentration
Mean ± S.E.
(rag/1 )
0.051 ± 0.008
0.75 ± 0.08
7.75 ± 1.95
105 ± 12
160 ± 10
0.045 ± 0.011
0.51 ± 0.12
10.2 t 0.1
93.7 i 9.1
169 ± 7
*0.10
0.85 ± 0.08
10.5 ± 1.6
43.2 ± 8.6
126 ± 6
185 ± 35
S 0.10
1.13 + 0.18
13.6 t 2.8
33.7 ± 3.4
99.5 ± 9.0
189 ± 39
0.38 ± 0.21
1.11 ± 0.97
4.24 ± 0.58
5.06 ± 1.52
44.4 ± 5.4
106 ± 13
1.20 ± 0.95
2.27 ± 1.01
8.27 ± 2.46
27.8 ± 9.8
49.6 ± 2.3
114 ± 11
Percent 1 7-
Hatchability1'*
100-
95(1)
71(4)
43(13)
31(24)
100(1)
89(1)
65(3)
47(10)
30(25)
100
84(1)
61(4)
31(22)
13(35)
0
100
75
53(2)
29(22)
14(43)
0
98
95(1)
69(4)
55(1)
0
0
98(1}
62(2)
53(13)
37(13)
0
0
Percent
Hatching
100
94
68
37
24
99
88
63
42
22
100
83
59
25
8
0
100
75
52
23
8
0
98
94
66
54
0
0
97
61
46
32
0
0
Normal Survival*
4 Days
Posthatching
100
83
52
28
18
97
75
44
25
15
100
82
57
0
0
0
99
74
50
0
0
0
98
94
49
54
0
0
97
58
45
32
0
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 95, 99, and 96% for largemouth bass,
goldfish, and channel catfish, respectively.
2Frequency of teratic survivors in hatched population is given parenthetically.
-------
44
Table 10. Toxicity of atrazine to embryo-larval stages of fish,
Species
Rainbow
Trout
Channel
Catf i sh
Water Atrazine
Hardness CE"^nffJon
/__/i r,rn \ Mean ± S.E.
(rng/1 CaC03) (mg/1)
50 . 0.019 ± 0.003
0.054 ± 0.003
0.54 ± 0.02
5.02 ± 0.06
50.9 ± 0.4
200 0.017 ± 0.002
0.060 ± 0.003
0.52 ± 0.02
5.02 ± 0.06
49.7 ± 0.6
50 0.028 ± 0.008
0.059 ± 0.008
0.43 ± 0.04
4.83 ± 0.53
46.7 ± 4.3
200 0.033 ± 0.006
0.054 ± 0.005
0.42 ± 0.03
4.81 ± 0.62
46.7 ± 3.7
Percent i o
Hatchability *
94
91(3}
73(6}
26(62)
0
100(2}
93(3}
78(4}
25(65}
0
84(1}
72(4)
42(13)
. 43(69}
* 20(100}
90(1}
71(4)
47(16)
49(47)
23(86}
Percent Normal Survival1
Hatching
94
88
69
10
0
98
90
75
9
0
83
69
37
13
0
89
68
39
26
3
4 Days
Posthatching
94
88
68
10
0
98
90
74
9
0
83
69
37
13
0
88
67
39
19
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 92 and 95% for rainbow trout and
channel catfish, respectively.
7
Frequency of teratic survivors in hatched population is given parenthetically.
-------
45
Table-11. Toxiclty of Capacitor 21 to embryo-larval stages of fish.
Water
Species Hardness
(mg/1 CaC03)
Redear 50
Sunfish
200
Largemouth 50
Bass
200
Rainbow 200
Trout
Capacitor 21
Concentration
Mean ± S.E.
1.7 ± 0.5
2.1 ± 0.4
6.5 ± 2.7
17.1 ± 3.8
1.4 ± 0.3
2.2 ± 0.7
4.5 ± 1.7
11.0 ± 1.4
0.5 ± 0.2
0.8 ± 0.3
1.5 ± 0.5
2.4 ± 0.9
0-4 ± 0.1
0.5 ± 0.1
1.5 ± 0.2
2.9 ± 0.7
2.2 t 0.3
2.5 ± 0.7
4.2 ± 1.7
6-0 ± 1.1
Percent , 9
Hatcfiability1'^
96
96(1)
94
63(12)
98
96
91(5)
64(9)
98
93(1)
82(2)
48(8)
97
98(1)
85(1)
47(9)
41(20)
17(8)
6(100)
0
Percent
Hatching
96
95
94
55
98
96
86
58
98
92
80
44
97
97
84
43
33
16
0
0
Normal Survival^
4 Days
Posthatching
94
94
86
40
95
91
86
38
93
86
66
10
90
94
69
11
32
14
0
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 97, 98, and 881 for redear sunfish,
largemoutfi bass, and rainbow trout, respectively.
?
"Frequency of teratic survivors in hatched population is given parenthetically,
-------
46
Table 12. Toxicity of chlorobenzene to embryo-larval stages of fish.
Water
Species Hardness
(mg/1 CaC03)
Goldfish 50
200
Largemouth 50
Bass
"
200
Chlorobenzene
Concentration
Mean ± S.E.
(mg/D
0.007 ± 0.002
0.039 ± 0.004
0.14 ± 0.04
2.93 ± 0.43
10.2 ± 1.1
0.010 ± 0.002
0.058 ± 0.009
0.37 ± 0.08
2.00 ± 0.15
12.2 ± 2.8
0.013 ± 0.002
0.038 ± 0.003
0.16 ± 0.01
2.55 ± 0,28
27.3 ± 1.4
0.009 ± 0.001
0.040 ± 0.006
0.15 ± 0.02
3.10 ± 0.34
23.2 ± 1.8
Percent
Hatchability1*^
98
96(1}
98(4)
63(7}
22(78)
102(1)
99(1)
89(4}
66(6}
28(63)
91(2}
86
75(11}
27(55)
4(100}
100
93(2)
72(13)
25(42}
4(100}
Percent
Hatching
98
95
94
59
5
101
98
85
62
10
89
86
67
12
0
100
91
63
15
0
Normal Survival!
4 Oays
Posthatching
97
91
81
34
4
98
93
76
38
0
80
60
24
0
0
93
64
14
0
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 99 and 93% for goldfish and largemouth
bass, respectively.
2
Frequency of teratic survivors in hatched population is given parenthetically.
-------
47
Table 13. Toxicity of chloroform to embryo-larval stages of rainbow trout,
Mater
Hardness
(mg/1 CaC03)
Chloroform
Concentration
Mean ± S.E.
(mg/1)
Percent Normal Survival1
Ha'tchability1'2 Ha1.rh,nn 4 Days
Hatchn ng Posthat*h1ng
50 0.004 ± 0.001 95 95 95
0.008 ± 0.001 92 92 92
0.059 ± 0.006 89(3) 86 86
0.69 ± 0.03 73(4) 70 70
10.1 ± 0.7 36(37) 23 23
200
0.003 ± 0.001
0.010 ± 0.001
0.056 ±0.004
0.63 ± 0.02
10.6 ± 0.4
106
88(1)
83(3)
72(3)
23(40)
106
87
80
70
14
106
£7
80
70
14
Frequency determined as survival in experimental population/control
Control survival at 4 days posthatching averaged 72%.
n
Frequency of teratic survivors in hatched population is given
parenthetically.
-------
48
Table 14. Toxicity of 2,4-dichlorophenol (DCP) to embryo-larval stages of fish.
Species
Goldfish
Channel
Catfish
Rainbow
Trout
Mater DCP
Hardness Concentration
fmn/7 ^aiTi \ riean «• ^ . ii . •
\flly/ I LaLUn/ /-.— JT \
•j \'"9/ ' /
50 0.017 ± 0.005
0.036 ± 0.004
0,17 i 0.02
4.84 ± 0.74
27.5 ± 2.1
200 0.015 ± 0.004
0.025 ± 0.004
0.11 ± 0.02
4.24 ± 0.69
25.5 ± 3.3
50 * Q.ooi
0.008 ± 0.006
0.024 ± 0.011
0.24 ± 0.01
3.52 ± 0.18
25.9 ± 3.1
200 *> 0.001
0.008 ± 0.002
0.016 ± 0.006
0.082 ± 0.015
0.10 ± 0.03
3.05 ± 0.36
24.6 ± 3.6
50 0.026 ± 0.004
0.052 ± 0.002
0.072 ± 0.007
0.47 ± 0.01
0.86 ± 0.08
4,64 ± 0.59
27.4 i 4.7
200 0.024 ± 0,004
0,048 ± 0.002
0.071 ± O.Q06
0.51 ± 0.01
0.81 ± 0.07
6.35 ± 0.97
34.4 ± 5.0
Percent , 9-
Hatchability1*"
96
96
87
51(14)
0
97
97
89
43(24)
0
99(2)
99(4)
101(12)
89(10)
72(8)
0
98(1)
96(6)
93(6)
92(10)
88(9)
68(6)
0
83(1)
62(1)
65(1)
22(9)
0
0
0
85
51
61(1)
24(9)
0
0
0
Percent Normal Survival*
Hatching
96
96
87
47
0
97
97
89
33
0
97
95
89
80
66
0
97
90
87
82
80
62
0
82
61
64
20
0
0
0
85
51
60
22
0
0
0
4 Days
Posthatching
96
95
82
0
0
94
95
84
0
0
97
95
89
79
55
0
97
92
86
' 80
79
51
0
82
61
64
20
0
0
0
85
51
60
20
0
0
0
1
Frequency determined as survival In experimental population/control. Control
survival at 4 days posthatching averaged 96, 96, and 92% for goldfish, channel
catfish, and rainbow trout, respectively.
"Frequency of teratic survivors in hatched population is given parenthetically.
-------
49
Table 15. Toxtclty of 2,4-dichlorophenoxyacetic acid (2,4-0) to embryo-larval
stages of fish.
Mater
Species Hardness
(mg/1 CaC03)
Goldfish 50
200
Largemouth 50
Bass
4
200
Rainbow 50
Trout
200
2,4-D
Concentration
Mean ± S.E.
(ing/i)
0.20 ± 0.03
5.12 ± 0.56
37.5 ± 3.8
78,0 ± 6.0
187 ± 4
0.28 ± 0.08
4.95 ± 0.28
37.1 ± 4.4
61.3 ± 7.4
201 ± 8
- 1.00
2.59 ± 0.39
51.0 ± 3.0
119 ± 22
« 1.00
5,07 ± 0.84
35.6 ± 5.0
178 ± 61
* 0.05
0.32 ± 0,04
5.66 + 0.54
36.0 ± 2.3
82.2 ± 10.4
144 ± 21
< 0.05
0.63 ± 0.07
9.54 ± 0.94
47.9 ± 0.3
78.5 ± 8.5
134 ± 12
Percent , 9-
Hatchability1'*
100(1)
97(1)
93(4)
91(7)
76(11)
100
97
93(3)
91(10)
73(12)
100
98
89
65(3)
100(1)
97
88(1)
49(15)
96(1)
86
69(2)
45(4)
30(15)
0
91
72(2)
55(4)
33(24)
6(100)
0
Percent
Hatching
99
96
89
85
68
100
97
90
82
64
99
98
89
63
99
97
87
42
95
86
68
43
26
0
91
71
53
25
0
0
Normal Survival 1
4 Days
Posthatching
96
91
83
71
34
97
93
84
72
30
99
97
79
47
98
94
78
30
95
86
68
43
26
0
91
71
53
24
0
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 98, 99, and 89% for goldfish, largemouth
bass, and rainbow trout, respectively.
>
"Frequency of teratic survivors in hatched population is given parenthetically.
-------
50
Tablt 16. Toxicity of dloctyl phthalate (OOP) to embryo-larval stages of fish.
Water
Species Hardness
(ing/1 CaC03)
Goldfish 50
200
Rainbow 50
Trout
200
Largeeiouth 50
Bass
200
OOP
Concentration
Hem ± S.E.
(mi/1)
0.040 ± 0.004
0.099 ± 0.014
0.52 ± 0.10
28.1 ± 2.3
64.4 ± 2.9
186 ± 15
0.030 ± 0.004
0.080 ± 0.011
0.44 ± 0.05
40.0 ± 4.1
80.7 ± 5.1
191 ± 14
0.040 ± 0.004
0.10 ± 0.01
0.48 ± 0.03
55.3 ± 3.1
71.9 ± 3.2
148 ± 10
0.045 ± 0.006
0.10 ± 0.01
0.50 ± 0.03
48.9 ± 4.0
88.8 ± 8.0
142 ± 12
0.055 ± 0.006
0.30 ± 0.03
46.3 ± 4.0
66.9 ± 3.3
149 ± 15
0.065 ± 0.012
0.30 ± 0.03
35.5 ± 3.1
60.6 ± 4.3
146 ± 16
Percent , ,-
Hatehability1^
100
101(2}
101(2)
97(3)
96(3)
89(6)
99
99(1)
100(1)
96(3)
88(5)
77(11)
101
99(1)
95
94(3)
. 89(5)
% 53(16)
99(1)
97(1)
92(2)
87(3)
82(7)
54(12)
97
93
74
39(1)
13(4)
97
91(1)
71
39(1)
16(3)
Percent
Hatching
100
99
99
94
93
84
99
98
99
93
84
69
101
98
95
91
85
45
98
96
90
84
76
48
97
93
74
39
12
*
97
90
71
39
16
Normal Survival1
4 Days
Posthatching
101
99
99
86
80
67
98
93
94
79
64
57
101
98
95
91
85
45
96
96
90
84
76
47
95
91
67
26
2
96
90
64
30
7
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 9.5, 94, and 98% for goldfish, rainbow
trout, and largemouth bass, respectively.
j
Frequency of teratic survivors in hatched population is given parenthetically.
-------
51
Table 17. Toxicity of malathion to embryo-larval stages of goldfish.
Water Malathion Percent Normal Survival
»a^«QCC Concentration Percent •, <,-
Hardness „„,_ A <-. e u,<.-u,k,'i,-*,,1»^
W^»> H^-E- HatCh°"my ^-H. PosLtchln,
50 < 0.05 99 99 98
0.28 ± 0.12 97(1) 96 92
0.60 ± 0.15 87(2) 85 74
1.99 ± 0.33 73(6) 69 41
5.24 ± 0.65 31(25) 23 0
200
* 0.05
0.11 ± 0.07
1.02 ± 0.08
2.16 ± 0.21
5.50 ± 0.70
98
96(1)
90(1)
68(4)
33(20)
98
95
89
65
26
96
89
73
37
0
Frequency determined as survival in experimental population/control
Control survival at 4 days posthatching averaged 98%.
2
Frequency of teratic survivors in hatched population is given
parenthetically.
-------
52
Table 18. Toxicity of trisodium nitri"Iotriacetic acid (NTA) to embryo-larval
stages of fish.
Water "*
Species Hardness CS""nffJ0n
(mf,ii f»,rn ^ Mean ± S.E.
(rag/1 CaC03) (ng/1)
Channel 50 1.05 ± 0.23
Catfish 9.30 ± 0.81
48.9 ±5.5
96.7 ± 5,6
241 ± 23
512 i 36
741 ± 15
918 ± 63
1100 ± 200
200 1.21 ± 0.26
8.65 ± 0.45
47.0 ± 2.0
95.1 ± 6.3
226 ± 26
501 ± 15
736 ± 41
954 ± 49
1100 ± 200
Goldfish 50 10.6 ± 0.5
48.0 ± 4.1
110 ± 6
216 ± 15
503 ± 85
910 ± 30
200 8.73 ± 0.66
50.9 ± 4.1
109 ± 11
205 ± 12
498 ± 78
825 ± 55
Percent , «-
Hatchabilityit<:
100
98(3}
99(3}
92(2}
91(9)
47(19)
10(55}
0
0
100
100
98(1)
91
84(5}
58(15)
23(35)
21(50)
0
98
92
83(3}
65<6)
39(3)
0
98
90(1}
82
60(4}
42(4)
0
Percent Normal Survival
Hatching
100
95
96
90
83
38
5
0
0
100
100
97
91
80
49
15
11
0
98
92
81
61
38
0
98
89
82
58
40
0
4 Days
Pos thatching
100
94
94
89
74
16
0
0
0
100
98
96
91
79
42
0
0
0
97
90
76
56
28
0
98
87
80
54
30
0
-------
Table 18 - continued.
53
Species
Rainbow
Trout
Water , NTA
u,_j____ Concentration
Hardness Mpan -*•*?£
50 1.21 ± 0.10
9.94 ± 0.55
48.2 ± 2.2
92.0 ± 2.6
193 ± 9
317 t 17
455 ± 18
1000 i 82
200 1.05 ± 0.11
8.94 ± 1.59
47.6 ± 2.3
94.4 ± 3.7
177 ± 5
299 ± 20
461 ± 21
1135 ± 126
Percent 1 «-
Hatchability1'
100
85(4)
82(9)
59(19}
42(48)
4(75)
0
0
97(2}
88 2)
85 6)
65 14)
53(38)
25(23)
12(91)
0
Percent Normal Survival1
Hatching
100
82
75
48
22
1
0
0
95
86
80
56
33
19
1
0
4 Days
Posthatching
100
82
74
48
22
1
0
0
95
86
80
56
33
19
1
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 97, 96, and 91« for channel catfish,
goldfish, and rainbow trout, respectively.
"Frequency of teratic survivors in hatched population is given parenthetically.
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54
Table 19. Toxicity of phenol to embryo-larval stages of fish.
Water
Species Hardness
(mg/1 CaC03)
Bluegill 50
Sunfish
2QQ
Soldfish 50
200
Rainbow 50
Trout'
200
Phenol
Concentration
Mean ± S.E."
(flig/15
0.004 ± 0.001
0.010 ± 0,001
0.091 ±0.005
1.07 ± 0.05
10.2 ± 0.3
0.006 * O.C01
O.QOi ± 0.001
0.090 ± 0.005
0.95 ± 0.02
9.88 ± 0.42
0.0013 ± 0.0003
0.009 ± 0.002
0.072 ± 0.014
0.99 ± 0.11
10.0 ± 0.3
0.0007 ± 0.0002
0.008 ± 0.001
0.079 ± 0.010
0.88 ± 0.11
9.58 ± 0.61
0.0015 ± 0.0003
0.009 ± 0.001
0.068 ± 0.009
0.84 ± 0.07
8.79 ± 0.40
0.0012 ± 0.0003
0.010 ± 0.001
0.070 ± 0.009
0.91 ± 0.07
9.33 ± 1.23
Percent . 9-
Hatchability1'*
99
97
94
66(2)
39(15)
99
96
93
64(3)
35(14)
99
93
88(8)
64(15)
29(38)
99
99
80(3)
43(21)
18(72)
100 .
100(2)
92(6)
41(32)
9(73)
90
75(1)
58(4)
21(22)
0
Percent
Hatching
99
97
94
65
33
99
96
93
62
30
99
93
81
54
18
99
99
78
34
5
100
98
86
28
2
90
74
56
16
0
Normal Survival*
4 Days
Posthatchi ng
99
95
90
60
31
98
94
87
57
27
98
90
80
54
12
98
96
78
34
4
100
96
86
27
0
90
74
56
15
0
Frequency determined as survival in experimental population/control. Control
survival at 4 days posthatching averaged 98, 91, and B9% for bluegill sunfish,
goldfish, and rainbow trout, respectively.
j
frequency of teratic survivors in hatched population is given parenthetically.
-------
Table 20. Comparison of LCj's determined in embryo-larval tests with MATC's derived from life-cycle studies.
Organic
Compound
Atrazine (ug/1)
2,4-D2 (mg/1)
Malathion (ug/1
NTA (mg/1)
PCB (ug/1)
(Capacitor 21)
LC, values were
LC1
29.0-77.2
0.02-0.03
3.2-13.1
8.2-8.9
) 141.1-439.6
16.9-20.2
28.5-30.1
130.9-138.4
0.5-0.9
1.0
1.3-3.5
compared with
Embryo-Larval
Test Species
Rainbow Trout
Rainbow Trout
Largemouth Bass
Goldfish
Goldfish
Rainbow Trout
Goldfish
Channel Catfish
Largemouth Bass
Rainbow Trout
Redear Sunfish
MATC
65-120
0.3-1.5
200-580
54.0-114.0
1.8-4.6 (PCB 1254)
5.4-15.0 (PCB 1242)
1.1-3.0 (PCB 1248)
2.1-4.0 (PCB 1260)
MATC's from partial (P) and complete (C)
Life-Cycle
Test Species
Brook Trout
Fathead Minnow
Fathead Minnow
Fathead Minnow
Fathead Minnow
life-cycle tests.
Type of
Life-Cycle
Testl
P
P
P
P
C
References
Macek, et al.
(197B7~~
Mount & Stephan
(1967)
Mount & Stephan
(1967)
Arthur, et al .
(1974F™
Nebeker, et al.
(1974)
DeFoe, et al.
( 19787 ~~
tests, respectively.
life-cycle
01
en
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56
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