Ecological Research Series
IIC TOXICITY OF CHLORDANE,
TRIFLURALIN, AND PENTACHLOROPHENOL
TO SHEEPSKEAD MINNOWS
ICYPRINODON VARIEGATUSI
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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CHRONIC TOXICITY OF CHLORDANE, TRIFLURALIN, AND
PENTACHLOROPHENOL TO SHEEPSHEAD MINNOWS (Cyprinodon variegatus)
by
Patrick R. Parrish, Elizabeth E. Dyar,
Joanna M. Enos, and William G. Wilson
EG&G, Bionomics
Marine Research Laboratory
Pensacola, Florida 32507
Contract No. 68-03-2069
Project Officer
David J. Hansen
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmen-
tal Research Laboratory, U.S. Environmental Protection Agency
(EPA), and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of
the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or re-
commendation for use.
11
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FOREWORD
The protection of our estuarine and coastal areas from dam-
age caused by toxic organic pollutants requires that regulations
restricting the introduction of these compounds into the environ-
ment be formulated on a sound scientific basis. Accurate infor-
mation describing concentration-response relationships for organ-
isms and ecosystems under varying conditions is required. The
Environmental Research Laboratory, Gulf Breeze, contributes to
this information through research programs aimed at determining:
The effects of toxic organic pollutants on individual
species and communities of organisms;
The effects of toxic organics on ecosystem processes
and components;
The significance of chemical carcinogens in the estu-
arine and marine environments.
This report describes effects of three chemicals in full
life-cycle tests with an estuarine fish, the sheepshead minnow.
The data will be useful in establishing estuarine water quality
criteria, and limiting effluents containing chlordane, triflura-
lin, or pentachlorophenol.
Thomas W. Duke
Director
Environmental Research
Laboratory
111
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ABSTRACT
Sheepshead minnows (Cyprinodon variegatus) were exposed to
each of three chemicals--chlordane, trifluralin, or pentachloro-
phenol—in flowing, natural seawater to determine acute and chronic
(full life-cycle) effects. The calculated 96-hour LCSO's and 95%
confidence limits, based on measured concentrations, were: chlor-
dane, 12.5 micrograms per liter (yg/£), 3.4-45.9 yg/&; trifluralin,
190 yg/£, 128-282 yg/£; and pentachlorophenol, 442 yg/£/ 308-
635 yg/£.
In a chronic test, sheepshead minnows were exposed to mean
measured concentrations of chlordane (0.5-18.0 yg/£) for 189 days.
Exposure to concentrations >2.8 yg/£ caused significant (P<0.5)
mortality of parental fish. Exposure to chlordane concentrations
>0.8 yg/£ significantly reduced hatch of embryos spawned by paren-
tal fish and exposure to concentrations >1.7 yg/£ caused signifi-
cant mortality of second generation fish. The estimated maximum
acceptable toxicant concentration (MATC) of chlordane for sheeps-
head minnows was >0.5<0.8 yg/£; the application factor (AF) limits
were 0.04-0.06.
Sheepshead minnows were exposed to mean measured concentra-
tions of trifluralin (1.3-34.1 yg/£) for 166 days. Exposure to
concentrations >17.7 yg/£ caused significant mortality of parental
fish. Exposure to trifluralin concentrations >9.6 yg/£ signifi-
cantly reduced growth of parental fish and exposure to concentra-
tions >4.8 yg/£ significantly reduced fecundity of parental fish.
Exposure to concentrations >9.6 yg/£ significantly reduced hatch
of embryos spawned by parental fish, and survival and growth of
second generation fish. The estimated MATC of trifluralin for
sheepshead minnows was >1.3<4.8 yg/£; the AF limits were 0.007-
0.025.
Sheepshead minnows were exposed to mean measured concentra-
tions of pentachlorophenol (18-389 yg/£) for 151 days. Exposure
to concentrations >88 yg/£ caused significant mortality of parental
fish. Exposure to pentachlorophenol concentrations >195 yg/£ sig-
nificantly reduced hatch of embryos spawned by parental fish and
survival of second generation fish. The estimated MATC of penta-
chlorophenol for sheepshead minnows was >47<88 yg/£; the AF limits
were 0.11-0.20.
This report was submitted by EG&G, Bionomics, in fulfillment of
Contract No. 68-03-2069, sponsored by the U.S. Environmental Protec-
tion Agency- The report covers efforts provided between June 1974
and May 1977.
iv
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CONTENTS
Foreword iii
Abstract iv
Tables vi
Acknowledgment x
1. Introduction 1
2. Conclusions 4
3. Recommendations 5
4. Materials and Methods
Test materials 6
Test water 6
Test animals 7
Test methods 7
Chemical analyses 9
Statistical analyses 18
5. Results and Discussion
Chemical analyses 19
Acute toxic ity 22
Chronic toxicity 24
Bioaccumulation 35
Application factors 37
Summary 39
References 41
Appendix 46
v
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TABLES
Number
Nominal and Measured Concentrations of Chlordane
During Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing, Nat-
ural Seawater 19
Nominal and Measured Concentrations of Trifluralin
During Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing, Nat-
ural Seawater 20
Nominal and Measured Concentrations of Pentachlor-
ophenol During Acute and Chronic Exposures of
Sheepshead Minnows (Cyprinodon variegatus) in
Flowing, Natural Seawater 21
Toxicity of Three Chemicals to Sheepshead Minnows
(Cyprinodon variegatus) Exposed for 96 Hours in
Flowing, Natural Seawater 22
Calculated 96-Hour LCSO's for Three Chemicals
and Sheepshead Minnows (Cyprinodon variegatus) . . . .23
Cumulative Mortality (%) of Parental Sheepshead
Minnows (Cyprinodon variegatus) Exposed to Chlor-
dane in Flowing, Natural Seawater 25
Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) During Three 10-Day
Spawning Periods; Fish Were Exposed to Chlordane
in Flowing, Natural Seawater for 189 Days 26
Number of Eggs Spawned per Day per Female Sheeps-
head Minnow (Cyprinodon variegatus) During Three
10-Day Spawning Periods; Fish Were Exposed to
Chlordane in Flowing, Natural Seawater for 189
Days 27
Hatching Success of Juveniles from Eggs Spawned
by Sheepshead Minnows (Cyprinodon variegatus)
Exposed to Chlordane in Flowing, Natural Seawater. . .27
VI
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Number Pa9e
10 Percentage Mortality, Average Standard Length,
and Average Weight (Determined in Water) of 28-
Day Old Sheepshead Minnow (Cyprinodon variegatus)
Juveniles Hatched from Eggs Spawned by Fish Ex-
posed to Chlordane for 88-91 Days (Group 1) and
139-148 Days (Group 2) 28
11 Cumulative Mortality (%) of Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Trifluralin
in Flowing, Natural Seawater for 166 Days 30
12 Growth of Sheepshead Minnows (Cyprinodon
variegatus) Exposed for 166 Days to Trifluralin
in Flowing, Natural Seawater 30
13 Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) During Three 10-Day
Spawning Periods; Fish Were Exposed to Triflur-
alin in Flowing, Natural Seawater for 166 Days .... 31
14 Number of Eggs Spawned per Day per Female Sheeps-
head Minnow (Cyprinodon variegatus) During Three
10-Day Spawning Periods; Fish Were Exposed to
Trifluralin in Flowing, Natural Seawater for 166
Days 32
15 Hatching Success of Juveniles from Eggs Spawned
by Sheepshead Minnows (Cyprinodon variegatus)
Exposed to Trifluralin in Flowing, Natural Sea-
water 32
16 Percentage Mortality, Average Standard Length,
and Weight (Determined in Water) of 28-Day Old
Sheepshead Minnow (Cyprinodon variegatus) Juve-
niles Hatched from Eggs Produced by Fish Exposed
to Trifluralin for 113-122 Days 33
17 Cumulative Mortality (%) of Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Pentachlorophenol
in Flowing, Natural Seawater 34
18 Hatching Success of Juveniles from Eggs Spawned by
Sheepshead Minnows (Cyprinodon variegatus) Exposed
to Pentachlorophenol in Flowing, Natural Seawater. . . 34
19 Percentage Mortality, Average Standard Length, and
Weight (Determined in Water) of 28-Day Old Sheeps-
head Minnow (Cyprinodon variegatus) Juveniles which
Were Hatched from Eggs Spawned by Fish Exposed to
Pentachlorophenol for 133-142 Days in Flowing,
vii
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Number
Natural Seawater.
20 Accumulation of Chemicals by Sheepshead
Minnows (Cyprinodon yariegatus) 36
21 Concentrations (\ig/H) of Three Chemicals Toxic
to Sheepshead Minnows (Cyprinodon variegatus) in
Acute and Chronic Tests, and the Relationship
of Acute Toxicity to Chronic Toxicity 37
22 Comparison of Application Factors for Freshwater
and Saltwater Fishes 38
23 Summary of Significant Effects of Chlordane,
Trifluralin, and Pentachlorophenol on Sheepshead
Minnows (Cyprinodon variegatus) During Chronic
(Full Life-Cycle) Exposures in Flowing, Natural
Seawater 40
A-l Growth of Sheepshead Minnows (Cyprinodon
variegatus) Exposed for 189 Days to Chlordane
in Flowing, Natural Seawater 46
A-2 Growth of Sheepshead Minnows (Cyprinodon
variegatus) Exposed for 151 Days to Pentachlor-
ophenol in Flowing, Natural Seawater 47
A-3 Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) During Two 10-Day Spawn-
ing Periods; Fish Were Exposed to Pentachloro-
phenol in Flowing, Natural Seawater for 166
Days 48
A-4 Number of Eggs Spawned per Day per Female
Sheepshead Minnow (Cyprinodon variegatus) Dur-
ing Two 10-Day Spawning Periods; Fish Were Ex-
posed to Pentachlorophenol in Flowing, Natural
Seawater for 166 Days 49
A-5 Concentrations of Chlordane in Surviving Adult
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed for 189 Days 49
A-6 Concentrations of Chlordane in Surviving 28-Day
Old Juvenile Sheepshead Minnows (Cvprinodon
variegatus) 50
A-7 Concentrations of Chlordane in Eggs/Embryos Pro-
duced by Adult Sheepshead Minnows (Cvprinodon
variegatus) Exposed for 171-181 Days 50
Vlll
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Number Page
A-8 Concentrations of Trifluralin in Surviving Adult
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed for 166 Days 51
A-9 Concentrations of Trifluralin in Surviving 28-
Day Old Juvenile Sheepshead Minnows (Cyprinodon
variegatus) 51
A-10 Concentrations of Pentachlorophenol in Surviving
Adult Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 151 Days 52
A-ll Concentrations of Pentachlorophenol in Eggs/Embryos
Produced by Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 133-142 Days 52
A-12 Concentrations of Pentachlorophenol in Surviving
28-Day Old Juvenile Sheepshead Minnows (Cyprinodon
variegatus) 53
IX
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ACKNOWLEDGMENTS
We thank the Project Officer, Mr. David J. Hansen, for his
interest and guidance. Thanks to Messrs. Terry A. Hollister and
G. Scott Ward, EG&G, Bionomics Marine Research Laboratory, for
their help with statistical analyses; Mr. George C. Cramm for his
technical assistance; and Ms. Susan Walker for typing the manu-
script. The assistance of Mr. Kenneth S. Buxton, EG&G, Bionomics
Analytical Chemistry Laboratory, is also greatly appreciated, as
is the review of the manuscript by Kenneth J. Macek, Ph.D., and
Sam R. Petrocelli, Ph.D., EG&G, Bionomics.
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SECTION I
INTRODUCTION
Methods for chronic (full life-cycle) toxicity tests with
freshwater fishes are well established. In contrast, until re-
cently little was known about the effects of toxicants on com-
plete life cycles of saltwater fishes. The use of sheepshead
minnows (Cyprinodon variegatus) for life-cycle tests was proposed
by Schimmel and Hansen (1975) and a tentative method was de-
scribed by Hansen and Schimmel (1975) . Results of several par-
tial and full life-cycle tests with sheepshead minnows were re-
ported more recently (Hansen and Parrish, 1977; Hansen et al.,
1977; and Parrish et al., 1977). Our studies were undertaken.to
gain information essential to the determination of marine water
quality criteria. Laboratory toxicity tests were conducted, the
results of which allowed estimation of "safe" and "unsafe" con-
centrations of chlordane, trifluralin, and pentachlorophenol for
survival, growth, and reproduction of sheepshead minnows.
Chlordane is an organochlorine pesticide which has been
widely used, especially for the control of soil pests (U.S. De-
partment of Health, Education, and Welfare, 1969). Although
chlordane is relatively insoluble in water (6-9 micrograms per
liter; yg/£) and does not migrate readily through soil (National
Research Council of Canada, 1975), measurable concentrations of
chlordane have been found in major U.S. river basins, in the
Great Lakes, and in southeastern U.S. estuaries (Henderson et
al., 1969; Henderson et al., 1971; and Bugg et al., 1967).
That technical chlordane is acutely toxic to nontarget or-
ganisms has been shown in tests with freshwater invertebrates
and fishes (Cardwell et al., 1977) and saltwater invertebrates
and fishes (Parrish et al., 1976). The waterflea, Daphnia magna,
was the most sensitive freshwater organism; the 96-hour LC50
(concentration estimated to be lethal to 50% of the test orga-
nisms) was 28 yg/& (with 95% confidence limits of 25-32 yg/£).
Cardwell et al. (op. cit.) also reported that freshwater fish
were nearly as sensitive as Daphnia, with 96-hour LCSO's (and 95%
confidence limits) of 37 yg/X. (33-41 yg/£) for fathead minnows
(Pimephales promelas); 47 yg/& (no confidence limits) for brook
trout (Salvelinus fontinalis) ; and 59 yg/2. (50-71 yg/£) for blue-
gill (LepomisL macrochirus) . Parrish et al. (op. cit.) found one
saltwater invertebrate to be much more acutely sensitive to chlor-
dane than were the freshwater invertebrates or fishes. Two other
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saltwater invertebrates and two fishes were slightly more sensitive
than were the freshwater species. The 96-hour LC50 (and the 95%
confidence limits) for pink shrimp (Penaeus duorarum) was 0.4
yg/£ (0.3-0.6 yg/£); the 96-hour EC50(concentration estimated to
be effective in reducing shell growth by 50%) for eastern oysters
was 6 yg/£ (5-8 yg/£); and the 96-hour LC50 for grass shrimp
(Palaemonetes pugio) was 5 yg/£ (4-6 yg/£). The 96-hour LCSO's
for saltwater fishes were 24 yg/£ (20-29 yg/£) for sheepshead
minnows and 6 yg/£ (5-7 yg/£) for pinfish (Lagodon rhomboides) .
(All the median effect concentrations in both studies were based
on measured concentrations of chlordane.)
In chronic tests with freshwater fishes, Cardwell et al.
(op. cit.) reported "...the lowest concentrations of technical
chlordane found to cause major chronic effects were 0.32 yg/£ for
brook trout, 1.22 yg/£ for bluegill...." Neither a maximum ac-
ceptable toxicant concentration (MATC) nor application factor
limits (Mount and Stephan, 1967) were reported for either fish
species, however, because of experimental difficulties.
Trifluralin is a herbicide used extensively in agriculture.
Sanders (1970) reported 48-hour LCSO's for freshwater inverte-
brates in static tests as follows: waterflea, 560 yg/£; seed
shrimp (Cypridopis vidua) , 250 yg/£; scud (Gammarus fasciatus) ,
1,800 yg/£; sowbug (Asellus brevicaudus), 2,000 yg/£; grass
shrimp (Palaemonetes kadiakensis), 1,200 yg/£; and crayfish
(Orconectes sp.), 50,000 yg/£. Similar acute toxicities have
been reported for other freshwater invertebrates (Sanders and
Cope, 1966; 1968) . Macek et al. (1969) reported that the acute
toxicity of trifluralin increased with increased temperature.
The 96-hour LC50 (and 95% confidence limits) for bluegills was
190 yg/£ (160-230 yg/£) at 12.7 degrees Celsius (°C) and 47 yg/£
(40-55 yg/£) at 23.8°C; the 96-hour LC50 for rainbow trout (Salmo
gairdneri) was 210 yg/£ (182-240 yg/£) at 1.6°C and 42 yg/£ (38-
46 yg/£) at 12.7°C, based on static tests.
Recently, Macek et al. (1976) reported the results of acute
and chronic toxicity tests with trifluralin and waterfleas and
fathead minnows. The 48-hour LC50 (and 95% confidence limits) for
waterfleas was 193 yg/£ (155-327 yg/£). The • MATC was >2.4<7.4 yg/£
and the application factor (AP) limits were 0.012-0.037. For fat-
head minnows, the incipient LC50 (estimated after 12 days of ex-
posure in a flow-through system) was 115 yg/£ with 95% confidence
limits of 45-211 yg/£. The MATC was >1.9<5.1 yg/£ and the AF lim-
its (calculated by using the incipient LC50) were 0.017-0.044.
Pentachlorophenol is an organochlorine which is used exten-
sively in this country and abroad in agricultural and industrial
applications, particularly in wood processing (Bevenue and
Beckman, 1967) . Acute and sublethal effects of pentachlorophenol
or its sodium salt on fresh- and saltwater invertebrates and
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fishes have been reported (Crandall and Goodnight, 1962; Bandt and
Nehring, 1962; Tomiyama and Kawbe, 1962; Tomiyama et al., 1962;
Alderdice, 1963; Alabaster, 1969; Norup, 1972; Webb and Brett,
1973; and Kaila and Saarikoski, 1977). Except for the relatively
resistant guppy (Poecilia reticulata) studies by Crandall and
Goodnight (op. cit.)and the crayfish (Astacus fluviatilis) studied
by Kaila and Saarikoski (op. cit.), acute median effect concentra-
tions were from 170-800 yg/fc. Tagatz et al. (1977) found that
pentachlorophenol affected the composition of communities of estu-
arine organisms at lower concentrations. Significantly fewer in-
dividuals and species were found in mean measured pentachlorophenol
concentrations >76 yg/&. Webb and Brett (op. cit.) reported sub-
lethal effects in sockeye salmon (Oncorhynchus nerka) exposed to
sodium pentachlorophenate. Growth rate and conversion efficiency
were similarly affected, the median effect concentrations being
1.74 and 1.80 ug/&, respectively- We know of no chronic fish
studies with pentachlorophenol.
Our chronic tests were conducted during the following periods:
chlordane, 6 September 1975-12 March 1976; trifluralin, 16 Febru-
ary-23 July 1976; and pentachlorophenol, 12 December 1975-8 June
1976.
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SECTION 2
CONCLUSIONS
Sheepshead minnows (Cyprinodon variegatus) are suitable test
animals for full life-cycle toxicity tests. Effects of chemicals
on the reproductive portion of the life cycle and the critical
life stages (embryo and juvenile) of the successive generation
are readily determined.
Tests with this species of saltwater fish are practical means
of determining maximum acceptable toxicant concentrations and ap-
plication factors because of (a) the amenability of sheepshead
minnows to laboratory culture and (b) the relatively short period
of time required to reach sexual maturity and complete the full
life cycle.
Results of chronic toxicity tests showed that sheepshead min-
nows were adversely affected by exposure to (a) 0.8 pg/£ chlordane;
(b) 4.8 yg/£ trifluralin; or (c) 88 yg/Ji pentachlorophenol. There-
fore, exposure to concentrations equal to or greater than these
would adversely affect the production of this saltwater fish spe-
cies.
Measured concentrations of technical chlordane and triflura-
lin were consistently less than nominal, even though low concen-
trations of the solvents, acetone or triethylene glycol, were
employed to aid dissolution and the toxicant solutions were con-
tinuously replenished. This indicates that toxicity tests with
these insecticides would not be valid unless based upon measured
concentrations of the chemicals. Measured concentrations of pen-
tachlorophenol were near nominal.
The application factor limits derived for sheepshead minnows
exposed to trifluralin were similar to those derived for a fresh-
water fish. Application factors derived for sheepshead minnows
exposed to other pesticides have also been similar to those de-
rived for freshwater fishes. This suggests that application fac-
tors, at least for pesticides, can be applied similarly to fresh-
water or saltwater fishes.
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SECTION 3
RECOMMENDATIONS
Spawning groups comprising five sheepshead minnows in the
ratio of 2 male fish:3 female fish are satisfactory to evaluate
effects of a chemical on fecundity during a chronic exposure.
A 10-day spawning period for a spawning group is sufficient
to monitor spawning success; at least two spawning periods should
be monitored for all treatments if possible.
Survival of juvenile fish hatched from embryos from two dif-
ferent spawning periods should be monitored to assess potential
cumulative effects of the toxicant.
Studies should be conducted with sheepshead minnows and other
toxicants in full life-cycle tests because tests with this salt-
water fish appear to provide accurate estimates of maximum accept-
able toxicant concentrations in a shorter time and with less ef-
fort than do tests with most freshwater fishes.
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SECTION 4
MATERIALS AND METHODS
TEST MATERIALS
The chlordane used in this study was a brown liquid, techni-
cal grade, 100% active ingredient and was obtained from Velsicol
Chemical Corporation, 341 East Ohio Street, Chicago, Illinois
60611.
The trifluralin used was an orange powder, technical grade,
99% active ingredient (lot number 5GB70 X-14788) and was obtained
from Eli Lilly and Company, Greenfield, Indiana.
The pentachlorophenol was purchased from J.T. Baker Chemical
Company, Phillipsburg, New Jersey 08865, and was a fine, white
powder, BAKER GRADE (lot number 416801).
Concentrations of each chemical are reported here as yg of
the technical material described above per a of seawater.
Stock solutions of each chemical were prepared on a weight:
volume basis by adding appropriate amounts of chlordane to re-
agent grade acetone or trifluralin or pentachlorophenol to tri-
ethylene glycol. These l-Jl stock solutions were placed in amber
glass bottles and stored in the laboratory. New stock solutions
were prepared as required.
TEST WATER
All water used for holding, acclimation, and testing was
natural seawater which was pumped from Big Lagoon into the labo-
ratory. The pump intake was 85 meters (m) offshore at a depth of
approximately 3 m. Water was pumped by a #316 stainless steel
pump through hard polyvinylchloride (PVC) pipes, through a fiber-
glass, sand-filled filter, and through a 5-micrometer (ym) poly-
propylene bag filter into an elevated fiberglass reservoir. Wa-
ter was continuously and vigorously aerated in the reservoir and
flowed by gravity through PVC pipes to the diluters.
No attempt was made to alter the salinity of the water, but
temperature was maintained at 30±1°C by heating the incoming sea-
water in the elevated reservoir with Teflon® tube-bundle heat ex-
changers and in small fiberglass-coated plywood boxes above the
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diluters with electric quartz heaters. Test aquaria were also
placed in constant-temperature water baths.
TEST ANIMALS
All sheepshead minnows used in these studies were collected
from Big Lagoon, near Bionomics Marine Research Laboratory. They
were held in sand-banked ponds on the laboratory grounds and in
fiberglass or glass tanks in the laboratory. All fish were accli-
mated to test conditions for 14 days before testing according to
the requirements of The Committee on Methods for Toxicity Tests
with Aquatic Organisms (1975). Mortality was <3% during acclima-
tion. During holding and acclimation, fish were fed frozen adult
or live nauplii of Artemia salina (San Francisco Bay Brand) or
flaked commercial fish food (BiOrell® and Tetramir®) which con-
tained no detectable concentrations (<0.1 ug/g) of chlorinated
hydrocarbon pesticides or polychlorinated biphenyls as determined
by our electron-capture gas chromatograph analyses.
Size of sheepshead minnows for the acute tests was as fol-
lows: chlordane—3.8-5.8 centimeters (cm) standard length (SL) ;
trifluralin—1.0-1.5 cm SL; and pentachlorophenol—1.0-2.0 cm SL.
TEST METHODS
Acute Tests
All procedures followed methods of The Committee on Methods
for Toxicity Tests with Aquatic Organisms (op. cit.) except as
stated. The 96-hour tests were conducted in an intermittent-flow
system by using a proportional diluter (Mount and Brungs, 1967)
constructed to deliver 1 a/cycle at a dilution ratio of 75%. The
average number of cycles was 5/hour, providing approximately 99%
replacement of water in the aquaria within 24 hours (Sprague,
1969). A mechanical injector (manufactured by George Frazer,
Duluth, MN) equipped with a 50-milliliter (ma) glass syringe and
stainless steel needle injected the appropriate volume of each
chemical stock solution through polyethylene tubing to the mixing
cell. Test containers were 30 x 30 x 61-cm glass aquaria. Each
contained 20 fish and approximately 82 £ of water.
By continuing the acute exposure, a 21-day test was conducted
with trifluralin in order to determine an incipient or threshold
LC50 (Sprague, op. cit.)
Chronic Tests
Chronic tests were conducted as described above except that
the proportional diluter was constructed for 50% dilution, test
containers were duplicated for each treatment (separate aquaria
were placed in an upper and lower water bath), and the diluter
was modified (Schimmel et al., 1975) to include a solvent control
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wherein the same volume of solvent/carrier (acetone or triethylene
glycol) was added to chlordane-, trifluralin-, or pentachloro-
phenol-free seawater as was added to the highest chemical concen-
tration. One injector, equipped with a 30-m£ glass syringe and a
stainless steel needle, metered the respective stock solutions
through polyethylene tubing into the mixing chamber. A second
injector, equipped with a 50-m£ glass syringe with stainless steel
needle, metered acetone or triethylene glycol to each solvent con-
trol. Maximum solvent concentration was 29 microliters (y£) per
a (parts per million, ppm).
To begin each test, eggs were stripped from adult fish whose
egg production was enhanced by two injections of human chorionic
gonadotrophin hormone (Schimmel et al, 1974). Testes were ex-
cised from males, macerated in seawater, and the resultant sus-
pension was mixed with the eggs. Within 1 hour after visual con-
firmation of fertilization, a group of 50 embryos (two groups per
duplicate treatment) was placed in an embryo incubator cup (a
100-m£ glass jar with the bottom cut off and 480-pm square mesh
nylon screen attached with silicone sealant). The incubator cups
were suspended from a rocker-arm apparatus (Mount, 1968) which
gently oscillated them in test aquaria. Embryos were removed
from each cup by pipette daily, counted, and the cups were washed
with bursts of fresh water to clean the screens. This procedure
was repeated until all living embryos had hatched. Survival of
embryos, length of time required for the embryos to hatch, and
number of live fry were recorded. Then, 40 fry were impartially
selected from each duplicate treatment and were placed in glass
chambers (14-cm wide x 20.5-cm high x 26-cm long with 381-pm
square mesh #316 stainless steel screen over one end). Fish were
fed live brine shrimp nauplii or flaked commercial fish food ad
libitum. Salinity and dissolved oxygen were measured daily.
Light was provided by two 3.7-m fluorescent bulbs suspended 46 cm
above the test containers, providing approximately 1,100 lux in-
cident to the water surface. The photoperiod was 16 hours light
and 8 hours dark. Survival was monitored daily by visually in-
specting each test container, and any physical or behavioral
changes were recorded. Growth was monitored biweekly according
to the photographic method of McKim and Benoit (1971), and average
weight was determined monthly by weighing each group in water.
When the fish were approximately 28 days old, the population
was reduced to 20 fish per aquarium and released from the cham-
bers into the aquaria. This number was chosen to ensure that
sufficient fish would grow and mature for at least two spawning
groups (each of which was comprised of five fish in the ratio of
2 males to 3 females) in each treatment.
The effects of each chemical on spawning were determined af-
ter fish began to exhibit signs of sexual maturity. Monitoring
of spawning activity was begun on day 93 of the chlordane test,
on day 113 of the trifluralin test, and on day 111 of the penta-
-------
chlorophenol test. Spawning chambers were constructed by lacing
pieces of 6.5-millimeter (mm) square mesh #316 stainless steel
screen together with #316 stainless steel wire. The chambers
were 30.6-cm wide x 30.5-cm long x 25.5-cm high, and were sup-
ported by 5-cm high extensions of the screen ends. Beneath each
spawning chamber, a 29.5-cm square x 4.7-cm high collection tray
was placed to retain the demersal eggs that sank through the bot-
tom of the spawning chamber. The tray was constructed of plate
glass and silicone sealant, with a 4-cm wide strip of 480-ym
square mesh nylon screen along one side of the bottom to facili-
tate consolidation of eggs. Spawning groups, which consisted of
two males and three females, were placed in the spawning chambers
for a 10-day period. All possible 2:3 ratios in each aquarium
were spawned once and extra, unspawned fish from each duplicate
aquarium of a treatment were combined whenever possible to form
additional spawning groups. Each day, one end of each spawning
chamber was lifted slightly and the collection tray was removed
from the aquarium. The eggs spawned and fertilized during the
previous 24 hours were washed with seawater, transferred by large-
bore glass pipette into glass Petri dishes, counted, and sepa-
rated into groups of 50 embryos. Each 50-embryo group was then
placed in an incubator cup as described above and placed in the
same aquarium as the spawning group which produced it. The pro-
cedure described for initiating the tests was followed to monitor
hatching and survival of second-generation juveniles. At least
two groups of juveniles per duplicate from each treatment were
monitored, except in the higher concentrations where toxicant-
induced mortality made it impossible to obtain spawning groups
and subsequent embryos and juveniles.
CHEMICAL ANALYSES
Seawater
'For the chlordane and pentachlorophenol acute tests, water
was collected from each aquarium at the beginning and end of the
96-hour exposure and for the acute trifluralin test, on days 1
and 21. Water was collected from alternate duplicate aquaria
weekly during the chronic tests. Water samples were prepared and
analyzed under the conditions described below.
Chlordane—
Unfiltered 800-mX, seawater samples were extracted twice with
50-m£ portions of Nanograde© dichloromethane. The combined ex-
tracts were dried with anhydrous sodium sulfate (previously heated
at 500°C for 24 hours) and concentrated to approximately 1 m£ in
a Kuderna-Danish evaporative concentrator. The concentrated ex-
tracts were quantitatively transferred to a 15-m2, centrifuge tube
and evaporated to <0.5 m£ by a gentle stream of clean, dry air.
The samples were then diluted to appropriate volumes with Nano-
grade hexane, and an aliquot was withdrawn for analysis by gas
liquid chromatography under the following conditions:
-------
Instrument—Tracer Model MT-550 equipped with a Ni63
electron capture detector
Column—2-m x 2-millimeters (ram) (ID) Pyrex®, packed
with 3% OV-101 on 100/120 mesh Supelcoport
Recorder—Corning Model 841 strip chart; 0-1 mV full
scale
Temperatures (°C)—Column: 200
Inlet: 230
Outlet: 245
Detector: 305
Gas flows (cubic centimeters [cc] of nitrogen/min
[min]—Carrier: 40
Make up: 48
Chart speed—0.5 cm/min
Attenuation—8 x 100
Retention times—4.1, 4.6, 5.5, 6.6, 8.2 (main peak),
9.1, 9.5 (shoulder), and 14.2 min
Response—Half-scale chart deflection at 8.2 min. for
3.0 nanograms (ng) of chlordane
Calibration curves were obtained by plotting the sum of the
peak heights (in mm) at 4.1, 4.6, 5.5, 6.6, 8.2, 9.1, 9.5, and
14.2 min against the weight (ng) of chlordane analyzed. Accord-
ing to the National Research Council of Canada (1975), quantita-
tion of chlordane GLC profiles by peak height summation or by
peak area summation produces comparable accuracy. A tangent skim
correction was applied to all samples in which peaks eluted on
the tail of the solvent.
Trifluralin—
Unfiltered 500-m£ seawater samples were extracted twice with
50-mJl of Nanograde dichloromethane. The extracts were dried with
anhydrous sodium sulfate (previously heated at 500°C for 24
hours), filtered, combined into a Kuderna-Danish evaporative con-
centrator, and concentrated to approximately 3 ma. The concen-
trated extracts were quantitatively transferred to a 10-m£ grad-
uated mixing tube and evaporated to <0.5 m£ by a gentle stream
of clean, dry air. The samples were then diluted to 10.0 ml with
Nanograde hexane. Approximately 1 mJl was removed from each sam-
ple and sealed in a septum vial for gas chromatographic analysis
under the following conditions:
Instrument—Hewlett-Packard 5840A gas chromatograph
equipped with a Ni63 electron capture detector and
a Model 7671A automatic sampler
10
-------
Column—2 m x 2 mm (ID) Pyrex packed with 5% OV-17
on 100/120 mesh Chromosorb G.H.P.
Temperatures (°C)—Column: 240
Inlet: 260
Detector: 330
Gas flow—Carrier: 30 cc nitrogen/min
Make-up: 20 cc 95% argon:5% methane/min
Set points—Slope sensitivity: 0.75-0.20
Area rejection: 50,000 counts
Cycle time: 8.0 min
Calibration method: external standard
Percent retention window: 5.0
Retention time—3.71-3.76 min
Minimum detectable concentration—0.36 yg/£ when using a
500-m£. water sample
Daily instrument calibration was performed by using 0.5,
1.0, and 1.5 yg/m£ trifluralin-in-hexane standards, respectively,
dependent upon the expected concentration range of samples.
Standards were checked every eighth sample during an automatic
sampling sequence.
Extraction and analysis efficiency were determined as fol-
lows: samples were produced by adding 1.0 m£ of a 5.0 and 0.5
vig/mfc trifluralin-in-acetone solution to 500-m£ volumes of syn-
thetic seawater to obtain concentrations of 10.0 yg/& and 1.0 yg/
trifluralin, respectively. Unfortified samples of synthetic sea-
water (500 m£) were used as blanks. All samples were extracted
and analyzed according to the above procedure with the following
results.
Trifluralin Trifluralin Percentage
added (pg/fc) recovered (yg/£) recovery
10.0
10.0
10.0
1.0
1.0
1.0
0.0
0.0
0.0
11
12
14
1.0
1.1
1.2
<0.36
<0.36
<0.36
110
120
140
100
110
120
Pentachlorophenol—
The methodology for the analysis of pentachlorophenol in
seawater was modified from an EPA (1974) procedure for the
11
-------
analysis of pentachlorophenol in urine and water.
Unfiltered 100-m£ seawater samples were measured volumetri-
cally, transferred to a 250-m£ separatory funnel, acidified with
4 ma of reagent grade concentrated sulfuric acid, and extracted
with 30 ma of Nanograde hexane by vigorously shaking the contents
of the funnel for approximately two minutes. The seawater was
discarded and the hexane extract was transferred into a small
amber glass bottle equipped with a Teflon-lined screw cap.
An ethereal solution of diazomethane (N-methyl-N-nitroso-p-
toluenesulfonamide) was prepared according to Aldrich Chemical
Company's Bulletin Z10, 025-0. This solution was added to a po-
tassium hydroxide-water-ethanol mixture; the ethyl ether was dis-
tilled and collected at -60°C until the distillate was colorless.
The ethereal distillate contained approximatley 3 g of diazome-
thane in approximately 200 ma of ethyl ether. All batches of
diazomethane were stored at -60°C in a high-draft air exhaust
hood throughout the analyses.
A 0.5-m£ aliquot of the 30-mjl hexane extract was added by
pipette to a 15-m£ centrifuge tube equipped with a Teflon-lined
screw cap. Then, 0.1 ma of the diazomethane solution was added.
The tube was capped immediately, the contents were mixed for one
minute by using a Vortex® Genie mixer, and the tube and contents
stored at room temperature for twenty minutes. After methyla-
tion, 1 ma of 20% distilled water-in-Nanograde methanol was added
and the tube was shaken by the Vortex mixer for two minutes to
destroy the excess diazomethane. The phase separation between
hexane (top layer) and the water-methanol (bottom layer) was im-
mediate and no emulsification occurred. An aliquot of the hexane
layer was removed and analyzed for pentachlorophenol by gas-
liquid chromatography (GLC).
The methylated pentachlorophenol-hexane extracts were di-
luted with Nanograde hexane prior to GLC analysis as follows:
Nominal
concentration of Concentration of Expected
pentachlorophenol pentachlorophenol Extract Hexane Dilution pentachlorophenol
in seawater in hexane extract volume added factor concentration
(yg/£) (yg/m&) (ml.) (m£) (yg/m£)
400
200
100
50
25
Control
1.33
0.667
0.333
0.167
0.0833
-1 •— '
0.5
0.5
0.5
0.5
0.5
0.5
39.5
19.5
9.5
4.5
2.0
0
80
40
20
10
5
.__
0.0167
0.0167
0.0167
0.0167
0.0167
All sample and standard injections were identical (2
12
-------
throughout the analytical period. In this manner, the effect of
varying volumes of solvent upon the chromatogram of pentachloro-
phenol was eliminated, and the analyses of all seawater (except
control samples) were performed with identical GLC instrumental
conditions.
The GLC operating conditions were as follows:
Instrument—Perkin-Elmer Model 3920 equipped with a
1-m x 2-mm (ID) Pyrex column packed with 5% (by weight)
OV-101 on 100/120 mesh Chromasorb W, HP
Detector—Electron capture with 15 mC Ni63
Temperatures (°C)—Inlet: 220 Outlet: 240
Column: 150 Detector: 300
Gas flow—Carrier: 28 cc nitrogen/min
Make-up: 55 cc nitrogen/min
Recorder—0-1 mV sensitivity, 0.677 cm/min chart speed
Response—10 picograms of pentachlorophenol gave 86% and
15% of full-scale recorder response with an electro-
meter attenuation of 16X and 64X, respectively. Penta-
chlorophenol eluted in 1.55 minutes.
Nanograde hexane (up to 6 y£) gave no pentachlorophenol re-
sponse. In addition, a series of reagent blanks prepared by me-
thylating 0.5 ma of hexane produced a consistent pentachlorophenol
response equivalent to 0.013 ug pentachlorophenol/£ of seawater.
This background of pentachlorophenol contamination was subtracted
only from the control seawater samples.
According to EPA (1974), the hexane extraction of acidified
urine removes at least 90% of the dissolved pentachlorophenol;
the methylation reaction is complete over large concentration
ranges of both pentachlorophenol and diazomethane; and finally,
the methanol/water rinse does not extract methylated pentachloro-
phenol from the hexane layer. In view of the expected high re-
covery of pentachlorophenol by the above method, we prepared 100-
mji portions of synthetic seawater containing known concentrations
of pentachlorophenol. The seawater was extracted, methylated, and
diluted with hexane to provide the pentachlorophenol standards
which were used to calibrate the response of the chromatograph to
unknown pentachlorophenol concentrations. A graph of the GLC peak
height versus the concentration of pentachlorophenol in the stan-
dard synthetic seawater was constructed and all sample concentra-
tions were obtained by interpolating the sample peak heights to
the corresponding pentachlorophenol concentration.
Extraction efficiency and mean recovery for the analytical
13
-------
methods were 86.5±8.0% for chlordane and 116.3±11.2% for triflura-
lin. Data from chlordane analyses have been corrected for re-
covery; trifluralin data were based on 100% recovery.
Fish Tissue
Fish were collected for residue analyses as follows:
a. adults alive at the end of the respective exposure;
b. juveniles alive at the end of the 28-day growth period; and
c. eggs and embryos randomly collected during the spawning pe-
riods .
Chlordane—
Tissue samples were prepared for gas chromatographic analysis
by blending the sample in methylene chloride for 30 seconds using
a Polytron® homogenizer. The extract was filtered through anhy-
drous sodium sulfate into a Kuderna-Danish evaporative concentra-
tor and concentrated to approximately 3 mJl. The extract was eva-
porated to dryness using a gentle stream of clean, dry air and
immediately diluted with a 1.0 ran of Nanograde hexane for florisil
column adsorption chromatography.
The concentrate was quantitatively transferred with hexane
to a 10 x 300-mm Pyrex chromatographic column containing a 10.2-cm
layer of activated (130°C) florisil 60/100 mesh with a 2.5-cm
layer of anhydrous sodium sulfate above it. The column was pre-
washed with petroleum ether and the solvent was adjusted to within
1 cm above the upper sodium sulfate layer before sample addition.
Chlordane was eluted from the column with 100 m£ of 6% peroxide-
free ethyl ether (EPA, 1974) in petroleum ether with a flow rate
of 5 m£ per minute.
The eluate was concentrated in a Kuderna-Danish evaporative
concentrator to approximately 3 m£ . The extract was transferred
to a 15-m£ centrifuge tube and evaporated to dryness with air.
The residue was then dissolved in hexane for analysis by gas li-
quid chromatography with the following instrumental conditions:
Instrument—Tracer Model MT-550 equipped with a Ni63
electron capture detector
Column—2-m x 2-mm (ID) Pyrex packed with 3% OV-101
on 100/120 mesh Supelcoport
Recorder—Corning Model 841 strip chart; 0-1 mV full-
scale
Temperatures (°C)—Column: 200 Outlet: 245
Inlet: 230 Detector: 305
Gas flows—Carrier: 40 cc nitrogen/min
Make-up: 48 cc nitrogen/min
14
-------
Chart speed—0.5 cm/min
Attenuation—8 x 100
Retention times—4.1, 4.6, 5.5, 6.6, 8.2 (main peak),
9.1, 9.5 (shoulder), 14.2 min
Response—Half scale chart deflection at 8.2 min for
3.0 ng chlordane
Calibration curves were obtained by plotting the sum of the
peak heights at 4.1, 4.6, 5.5, 6.6, 8.2, 9.1, 9.5, and 14.2 min-
utes against the weight (ng) of chlordane injected. According
to The National Research Council of Canada (1975), quantitation
of chlordane GLC profiles by peak height summation or by peak
area summation produces comparable accuracy. A tangent skim cor-
rection was applied to all samples in which peaks eluted on the
tail of the solvent.
To determine extraction and analysis efficiency, three sam-
ples were produced by adding 1.0 m£ of a 1.0 ppm solution of
chlordane in acetone to each tissue sample. The samples were ana-
lyzed by the above method with the following results.
Tissue weight Chlordane Chlordane Percentage
(g) added (yg/g) recovered (yg/g) recovery
0.6343
0.7905
1.1228
1.6
1.3
0.89
1.5
1.2
0.81
94
92
91
The minimum detectable concentration of chlordane in tissue was
0.1 yg/g when using a 1-g sample.
Trifluralin—
Weighed groups of whole fish (total weight <10 g) were
homogenized with a 25-m£ volume of 1:1 petroleum ether-diethyl
ether by using a PT-10 Willems Polytron homogenizer. The homo-
genate was centrifuged and the mixed ether phase was decanted and
saved. An additional 25-m£ volume of 1:1 petroleum ether-diethyl
ether was added to the homogenate and the extraction repeated.
The two ether extracts were combined, transferred to a Kuderna-
Danish evaporative concentrator, and concentrated to approximately
3 ml. The extract was transferred with hexane to a separatory
funnel, the volume was diluted to 15 m£ of hexane, and the mixture
was extracted 4 times with 30-m£ volumes of acetonitrile. The
acetonitrile phases were combined and transferred to a separatory
funnel containing a 650-mA volume of distilled water and a 40-m2,
volume of saturated sodium chloride solution. The mixture was
extracted twice with 100-nU volumes of hexane. The hexane extracts
were combined in a separatory funnel, and the hexane was washed
15
-------
twice with 100-mx, volumes of distilled water. The water was dis-
carded and the hexane was transferred to a column containing an 8-
to 10-cm layer of anhydrous sodium sulfate. The separatory funnel
and column were rinsed three times with 10-m£ volumes of hexane.
The hexane was combined and collected in a Kuderna-Danish evapora-
tive concentrator, and concentrated to approximately 3 ma. The ex-
tract was quantitatively diluted to an appropriate volume for gas
chromatographic analysis under the following instrumental condi-
tions :
Instrument—Hewlett-Packard 5840A gas chromatograph
equipped with a 15 mC Ni63 electron capture detector
and a Model 7671A automatic sampler
Column—3-m x 2-mm (ID) Pyrex, packed with 5% OV-17
on 100/120 mesh Chromosorb G.H.P.
Temperatures (°C)—Column: 240
Inlet: 260
Detector: 330
Gas flow—Carrier: 30 cc nitrogen/min
Make-up: 20 cc 95% argon:5% methane/min
Set points—Slope sensitivity: 200
Area rejection: 10,000 counts
Cycle time: 6.0 min
Calibration method: External standard
Percent retention window: 5.0
Retention time—3.85 min.
Daily instrument calibration was performed with 0.1 and 1.0
yg/m£ trifluralin in hexane standards. Standards were checked after
every eighth sample during an automatic sampling sequence.
To determine extraction and analysis efficiency, samples were
produced by adding 5 m£ of a 0.5 yg/m£ or 5 m£ of a 5.0 yg/m£
trifluralin-in-acetone standard to 5.0 g of uncontaminated fish
samples. The fortified fish samples were extracted and analyzed
according to the above procedure with the following results.
Trifluralin Trifluralin Percentage
added (yg) recovered (yg) recovery
0
0
0
2.5
2.5
2.5
25.0
25.0
25.0
<0.4
<0.4
<0.4
1.62
1.76
1.84
18.1
15.7
16.3
65
70
73
72
63
65
16
-------
The minimum detectable concentration of trifluralin in tissue was
0.4 yg.
Pentachlorophenol—
Wet fish tissue was weighed, transferred with a 0.4 normal
sodium hydroxide solution to an appropriate vessel for grinding,
and homogenized with a Polytron PT-10/20. The homogenizer blades
were rinsed with a portion of distilled water, and the rinse was
added to the sample. Samples weighing <2 g were extracted with
5 mi of 0.4 normal sodium hydroxide, and the blades were rinsed
with 5 m£ of distilled water. Samples weighing 2-7 g were ex-
tracted with 12.5 m£ of 0.4 normal sodium hydroxide and the blades
were rinsed with 12.5 ml of distilled water.
Two nU of the basic aqueous extract containing pentachloro-
phenol were extracted with 5 m£ of Nanograde hexane to remove the
majority of fats and oils, and the hexane was discarded. The
aqueous phase was acidified to pH 2 by adding concentrated hydro-
chloric acid and extracted with an additional 2 m£ of Nanograde
hexane by a Vortex Genie tube shaker. The stable emulsion formed
between hexane and the aqueous acid extract was separated by cen-
trifuging at 3,600 rpm for 20 minutes in a 20-cm diameter centri-
fuge head.
After phase separation, 0.5 m£ of the hexane was transferred
to a clean, Teflon-capped tube and methylated with diazomethane,
according to EPA (1974). Solutions containing known weights of
pentachlorophenol (Chemical Service Company, Catalog Number 7)
were methylated and used to standardize the gas chromatographic
response to known pentachlorophenol weights. Aliquots of the
methylated pentachlorophenol in hexane were analyzed by gas
chromatography using the identical instrumental conditions pre-
viously described for the analysis of pentachlorophenol in water.
The percentage recovery of pentachlorophenol from fish tissue
was determined by adding 0.5 pg of pentachlorophenol to each of
three 2-g tissue samples. The pentachlorophenol was extracted,
methylated, and analyzed as described above with the following
results.
Pentachlorophenol Pentachlorophenol Percentage
added (yg/g) recovered (yg/g) recovery
0.25 0.19 76
0.25 0.19 76
0.25 0.22 88
Due to the prevalence of pentachlorophenol contamination in an
analytical chemistry laboratory environment, three reagent blanks
were analyzed and found to contain 2.1 ng of pentachlorophenol
per blank. The pentachlorophenol due to reagents and solvents
17
-------
was subtracted from all tissue samples reported.
Extraction efficiency and mean recovery for the analytical
methods were 92.3±1.0% for chlordane; 68.2±4.5% for trifluralin;
and 80.0±6.9% for pentachlorophenol. Data reported have been
corrected for recovery.
STATISTICAL ANALYSES
In the acute tests, the LCSO's and 95% confidence limits
were calculated by linear regression analysis after probit trans-
formation (Finney, 1971).
In the chronic tests, differences between treatments were
determined by analysis of variance (Sokol and Rohlf, 1973) . Dif-
ferences weje considered significant at the 95% (P<0.05) confi-
dence level. Post-hoc tests were conducted on treatment means by
using Dunnett's procedure (Steel and Torrie, 1960).
The control was compared to all other treatments (including
the solvent control) in all tests. In two instances there was a
significant difference between the control and the solvent con-
trol—see the chlordane and trifluralin results and discussion
sections for details.
18
-------
SECTION 5
RESULTS AND DISCUSSION
CHEMICAL ANALYSES
Mean measured concentrations of chlordane in seawater were
from 10-15% of nominal during the acute test and from 19-60% of
nominal during the chronic test (TABLE 1), reflecting the low
solubility of this chlorinated hydrocarbon pesticide in water.
TABLE 1. NOMINAL AND MEASURED CONCENTRATIONS OF CHLORDANE DURING
ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING, NATURAL SEAWATER.
Concentration
Test Nominal
ACUTE Control
18
23
31
42
56
75
100
Measured
0 hour
NDa
1.3
2.6
3.8
6.6
_a
12.0
12.0
96 hour
ND
2.4
2.7
4.3
5.9
6.7
3.4
18.0
Avg.
1.8
2.6
4.0
6.2
6.7
7.7
15.0
% of nom.
10
11
13
15
12
10
15
Mean
S.D.
Range % of nom. # samples
CHRONIC Control
Sol. control
1.9
3.8
7.5
15.0
30.0
NDa
_b
0.5
0.8
1.7
2.8
18.0
_
±0.4
±0.6
±1.1
±1.8
0.0
—
ND-1.7
ND-2.3
0.4-4.4
0.9-8.0
—
26
21
23
19
60
18
—
13
14
17
16
2
aNot detectable; <0.23
analyzed.
19
-------
Mean measured concentrations of trifluralin in seawater were
from 47-56% of nominal during the acute test and from 21-38% of
nominal during the chronic test (TABLE 2).
TABLE 2. NOMINAL AND MEASURED CONCENTRATIONS OF TRIFLURALIN DUR-
ING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING, NATURAL SEAWATER.
Concentration (yg/£)
Test Nominal
ACUTE Control
107
142
190
253
337
450
600
CHRONIC Control
Sol. control
6.3
12.5
25.0
50.0
100.0
Measured
Day l
0.48
60
86
124
162
216
250
368
Mean
NDa
_b
1.3
4.8
9.6
17.7
34.1
Day 21
0.78
48
66
86
100
162
176
268
S.D.
—
±0.6
±2.3
±6.1
±6.5
±15.2
Avq.
0.63
54
76
105
131
189
213
318
Range
-
ND-3.1
1.2-10.2
4.1-32.0
6.6-31.0
13.0-65.0
% of nom.
..
50
54
55
52
56
47
53
% of nom.
-
21
38
38
35
34
# samples
23
-
21
23
23
23
16
aNot detectable; <0.36
analyzed.
20
-------
Mean measured concentrations of pentachlorophenol were from
84-110% of nominal during the acute test and from 72-98% of nomi-
nal during the chronic test (TABLE 3).
TABLE 3. NOMINAL AND MEASURED CONCENTRATIONS OF PENTACHLOROPHENOL
DURING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING, NATURAL SEAWATER.
Concentration (pq/a)
Test Nominal
ACUTE Control
142
190
253
337
450
600
CHRONIC Control
Sol. control
25
50
100
200
400
0 hour
ND3
JD
-k
290
370
480
460
Mean
0.2
_b
18
47
88
195
389
Mea
96 hour
0.04
—
240
_a
490
545
S.D.
±0.3
±6
±10
±21
±42
±78
sured
Avg.
-
265
370
485
502
Range
ND-0.9
9-36
23-65
42-136
80-270
290-510
% of nom.
-
105
110
108
84
% of nom.
72
94
88
98
97
# samples
24
22
24
23
24
7
aNot detectable; <0.04 pg/fc.
kftfot analyzed.
A special analysis of the pentachlorophenol was conducted in
order to identify the presence of certain impurities. The analy-
ses was by mass spectroscopy and revealed the following:
Impurity
Tetrachlorophenol
Hexachlorodibenzodioxin
Octachlorodibenzodioxin
Concentration (pg/g; parts per million)
3,000
<1
1
Not enough of the pentachlorophenol sample remained for analyses
for other impurities (personal communication, Dean R. Bransona).
aDean R. Branson, Ph.D., personal communication, December 1977,
Dow Chemical Company, Midland, MI 48640.
21
-------
ACUTE TOXICITY
The acute toxicity of trifluralin and pentachlorophenol to
sheepshead minnows was similar; chlordane was one order of mag-
nitude more toxic (TABLES 4-5).
TABLE 4. TOXICITY OF THREE CHEMICALS TO SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED FOR 96 HOURS IN FLOW-
ING, NATURAL SEAWATER.
Chemical
Measured
concentration
(yg/ft)
Mortality
Chlordanea
Trifluralin*3
Pentachlorophenolc
Control
1.8
2.6
4.0
6.2
6.7
7.7
15.0
Control
54
76
105
131
189
213
318
Control
142d
190d
265
370
485
502
0
0
0
0
0
0
20
70
0
0
5
15
15
30
50
95
0
0
0
10
10
40
90
aSalinity was 26 °/oo and temperature was 28°C.
^Salinity was 21 °/oo and temperature was 29°C.
GSalinity was 24 °/oo and temperature was 29°C.
^Nominal concentration.
22
-------
TABLE 5. CALCULATED 96-HOUR LCSO's FOR THREE CHEMICALS AND
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS). CALCULA-
TIONS WERE BASED ON MEASURED CONCENTRATIONS OF EACH
CHEMICAL.
Chemical 96-hour 95% confidence
LC50 (yg/£) limits (yg/&)
Chlordane 12.5 3.4-45.9
Trifluralin 190 128-282
Pentachlorophenol 442 308-635
The acute toxicity of chlordane to sheepshead minnows tested
under dynamic conditions was similar to that reported for other
estuarine fishes tested under dynamic conditions (Parrish et al . ,
1976) and slightly greater than the toxicity reported for fresh-
water fishes tested under dynamic conditions (Cardwell et al.,
1977) or static conditions (Henderson et al . , 1959). The 96-hour
LCSO's for two saltwater fishes were 24.5 and 6.4 yg/£, and the
range of LCSO's for seven freshwater fishes was 36.9-190 yg/£.
Sheepshead minnows were not as sensitive to trifluralin in
acute tests as were two freshwater fishes. Macek et al. (1969)
reported a 96-hour LC50 of 47 pg/X. for bluegill tested at 23.8°C
and a 96-hour LC50 of 42 yg/& for rainbow trout tested at 12.7°C.
The incipient or threshold LC50 of trifluralin to sheepshead
minnows was estimated after 10 days of exposure and was 84 yg/£ ,
with 95% confidence limits of 48-145 yg/£. Macek et al . (1976)
reported the incipient LC50 of trifluralin to fathead minnows
(estimated after 12 days of exposure) to be 115 yg/Jl, with 95%
confidence limits of 45-211
The susceptibility of sheepshead minnows to pentachlorophenol
cannot be related to the susceptibility of other fishes to this
chemical because few references exist in the literature. Data
exist concerning the effects of sodium pentachlorophenate on
fishes, but a comparison of the toxicity of the two chemicals may
not be valid because of the differing chemical characteristics,
particularly the effect of pH on toxicity (Kaila and Saarikoski,
1977). However, Davis and Hoos (1975) reported 96-hour LCSO's
for sodium pentachlorophenate and rainbow trout, coho salmon
(Oncorhynchus kisutch) , and sockeye (0. nerka) ranging from 37-
130 yg/fc. The static tests were conducted in freshwater, initial
pH 5.7-9.0. Schimmel et al . (in press) reported the 96-hour LC50
of sodium pentachlorophenate for three saltwater fishes: longnose
killifish (Fundulus similis) >306 yg/&; pinfish, 53 yg/£; and
23
-------
striped mullet (Mugil cephalus), 112 yg/£. The tests were con-
tinuous flow and the concentrations were measured.
CHRONIC TOXICITY
Chlordane
Exposure to chlordane significantly reduced survival of pa-
rental fish during the 189-day study. All fish exposed to 18 yg/£
died within 30 days, and mortality of fish exposed to 2.8 yg/£ was
significantly greater than mortality of control fish at the end of
the study (TABLE 6).
Growth of parental fish after 34, 94, and 189 days of expo-
sure to chlordane was not significantly different from growth of
control fish (TABLE A-l).
The total number of eggs spawned by parental fish in the sol-
vent control and all chlordane concentrations was significantly
different from the control. The number of eggs spawned by fish
in the solvent control and fish exposed to 0.5 yg/£ of chlordane
was significantly greater than the number of eggs spawned by con-
trol fish, but the number of eggs spawned by fish exposed to chlor-
dane concentrations >0.8 yg/£ was significantly less than the num-
ber of eggs spawned by the control or solvent control fish (TABLE
7). Because female fish died or were killed by male fish in spawn-
ing chambers in the control and three concentrations, we calculated
eggs per female spawning day. These values were obtained by di-
viding the number of eggs obtained from a spawning chamber during
a 24-hour period by the number of live female fish in the spawning
chamber during the same period. The same pattern of effect ob-
served for total eggs spawned was evident: more eggs were spawned
per day per female in the solvent control and 0.5 yg/£ of chlordane
than were spawned in the control; fewer eggs were spawned in con-
centrations >0.8 yg/£ but the differences were not significant
(TABLE 8). Therefore, fecundity was not considered to be signi-
ficantly affected by exposure to chlordane concentrations <2.8 yg/£.
Hatching success of juveniles from embryos produced by fish
exposed to chlordane decreased as exposure duration of parental
fish increased. For the first and second spawning periods, hatch-
ing success in only one concentration (2.8 yg/£) was affected.
For the last spawning period, however, hatching success in two
lower concentrations (0.8 and 1.7 yg/Ji) was significantly less
than in the control (TABLE 9).
Similarly, juvenile mortality during 28 days posthatch in-
creased with increased duration of exposure of parental fish to
chlordane. Mortality of exposed juveniles from the first spawning
period was not significantly greater than mortality of control
juveniles in any concentration, but mortality of juveniles from
the second spawning period was significantly greater in two concen-
24
-------
trations (1.7 and 2.8 \ig/H). Growth of surviving juveniles was
not affected in any concentration, however (TABLE 10).
TABLE 6. CUMULATIVE MORTALITY (%) OF PARENTAL SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO CHLORDANE IN FLOWING,
NATURAL SEAWATER. MORTALITY IS THE AVERAGE FROM DUPLI-
CATE AQUARIA AND DOES NOT INCLUDE DEATHS WHICH OCCURRED
IN THE SPAWNING CHAMBERS.
Mean measured concentration (yg/£)
Solvent
Day Control control 0.5 0.8 1.7 2.8 18.0
1-30 0 0 0006 100
31-60 0 0 0000
61-90 0 0 0000
91-120 0 0 0008
121-150 0 0 2222
151-189 0 0 0 2 8 18
TOTAL 0 0 2 4 10 34a 100a
aSignificantly greater than the control.
25
-------
TABLE 7. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) DURING
THREE 10-DAY SPAWNING PERIODS; FISH WERE EXPOSED TO CHLORDANE IN FLOWING, NA-
TURAL SEAWATER FOR 189 DAYS. FIVE FISH (2 MALES AND 3 FEMALES) WERE PLACED
IN A SPAWNING CHAMBER IN DUPLICATE AQUARIA, A AND B.
Mean measured concentration
Day
93
to
102
Control
A
B
264 182'
Solvent
Control
0.5
0.8
1.7
2.8
A
B
A
385 398 443
B
B A
B
B
454 176 259 244 76a 67 524
K>
139
to
148
412a 372
338 287 103'
387 327a 167a 462 88 247 135
177
to
186
127 269 404 691 392 655 167a 310a 208 121
a b
28
SUB-
TOTAL
803 823 1,127 1,376 938 1,496 670 736 914 285 314 687
TOTAL 1,626
2,503C
2,434(
l,406d l,199d l,001d
aDeath
^No spawning chamber; all surviving fish had been spawned.
cSignificantly greater than the control.
^Significantly less than the control.
-------
TABLE 8. NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) DURING THREE 10-DAY
SPAWNING PERIODS; FISH WERE EXPOSED TO CHLORDANE IN
FLOWING, NATURAL SEAWATER FOR 189 DAYS. FIVE UNSPAWNED
FISH (2 MALES AND 3 FEMALES) WERE PLACED IN A SPAWNING
CHAMBER IN DUPLICATE AQUARIA, A AND B.
Day
Mean measured concentration
Solvent
Control Control
0.5
0.8
B
B
B
B
1.7
A B
2.8
Mean and S.D.
of treatment
B
93-102
139-148
177-186
Mean of
duplicate
9-
16
4
10
10
12
9
10
13
11
14
13
13
10
23
15
15
5
13
11
15
14
22
17
6
11
6
8
9
6
11
9
8
15
7
10
3
3
4
3
3
8
_a
6
17
4
21
7
10±4
14±5
14±5
8±2
7±5
7±6
No spawning chamber; all surviving fish had been spawned.
TABLE 9. HATCHING SUCCESS OF JUVENILES FROM EGGS SPAWNED BY
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO
CHLORDANE IN FLOWING, NATURAL SEAWATER. PERCENTAGE
HATCH AND STANDARD DEVIATION REPRESENT POOLED DATA
FROM DUPLICATE AQUARIA DURING THREE 10-DAY SPAWNING
PERIODS.
Mean measured
concentration
Percentage
hatch and S.D.
Number of
(yg/£)
Control
Sol. control
0.5
0.8
1.7
2.8
1st
99 ±1
99 + 1
98±2
96±3
98±0
90±2a
2nd
99 + 1
98±3
97±1
98±3
99±1
81±10a
3rd
99±1
99±1
97±3
74±21a
72±32a
83b
Mean
99 + 1
99±2
98±2
85±19
84±26
85±8
embryos examined
638
1,089
1,151
596
565
472
aSignificantly less than the control.
^Insufficient data for statistical analysis; only one group
of embryos.
27
-------
TABLE 10. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND AVE-RAGE WEIGHT (DETERMINED
IN WATER) OF 28-DAY OLD SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) JUVENILES
to
oo
HATCHED FROM EGGS SPAWNED BY FISH EXPOSED TO CHLORDANE
1) AND 139-148 DAYS (GROUP 2) .
Mean measured
concentration
(ygA)
Control
Sol. control
0.5
0.8
1.7
2.8
GROUP 1
Number
of juv.
80
120
160
80
80
80
Mortality
(%)
4
4
3
4
1
12
Length and
S.D. (cm)
1.20±0.13
1.22±0.16
1.17±0.14
1.12±0.13
1.18±0.24
1.24±0.14
Weight
(g)
0.05
0.05
0.05
0.05
0.05
0.05
Number
of juv.
120
80
40
80
80
80
FOR 88-91
DAYS ('
GROUP 2
Mortality
(%)
0
1
0
10
22a
29a
Length and
S.D. (on)
1.12+0.16
1.0.910.12
1.19+0.12
1.1910.16
1.09+0.19
1.0910.17
Weight
(g)
0.03
0.03
0.03
0.04
0.03
0.03
Significantly greater than the control.
-------
Trifluralin
Exposure to trifluralin significantly decreased survival of
parental fish during the 166-day study. All fish exposed to
34.1 yg/Jt died and 66% of the fish exposed to 17.7 yg/£ died, a
significantly greater percentage than control mortality (TABLE 11).
Several trifluralin induced effects were observed. As early
as day 14 posthatch, juvenile fish in concentrations >17-7 yg/J,
were in poor condition. They exhibited loss of equilibrium and
lay on the bottom of the aquaria with opercular movement only.
Fish in two lower concentrations (4.8 and 9.6 yg/£) had darkened
coloration in the caudal peduncle area. On day 37, fish in
17.7 yg/& were noticeably "fatter" than control fish. This edem-
atous condition persisted throughout the exposure. On day 53,
approximately one-fourth of the body of surviving fish in 17-7 yg/£
was darkened, the fish were extremely sluggish, and they exhibited
loss of equilibrium, occasionally swimming upside down. Fish still
alive in 34.1 yg/& exhibited complete loss of equilibrium. On day
129 (12 days posthatch), F1 juveniles in 17.7 yg/Jl were sluggish
and exhibited loss of equilibrium.
Exposure to trifluralin caused decreased growth of parental
fish. Surviving fish in 34.1 yg/2. were less than half the length
of control fish on day 37, for example, and fish in 9.6 and
17.7 yg/£ were significantly smaller than control fish at the end
of the exposure (TABLE 12).
The total number of eggs spawned by parental fish in the sol-
vent control and all trifluralin concentrations was significantly
less than the number of eggs spawned by parental control fish
(TABLE 13). Because female fish died or were killed by aggressive
males during spawning in all treatments except 17.7 yg/X,, we cal-
culated eggs per female spawning day. The same pattern of effect
observed for total eggs spawned was evident: fewer eggs were
spawned per day per female in all treatments than were spawned
in the control. However, significantly fewer eggs were spawned
only in trifluralin concentrations >4.8 yg/£ (TABLE 14). There-
fore, fecundity (eggs per female per spawning day) was considered
to be significantly affected by exposure to trifluralin concentra-
tions >4.8 yg/£.
Exposure to trifluralin decreased hatching success of juve-
niles from embryos produced by parental fish. Further, hatching
success decreased as exposure duration of parental fish increased.
For example, the mean hatch of juveniles in 9.6 yg/£ was 98% for
the first spawning period. Hatch decreased to 54% for the second
spawning period, however, and no juveniles hatched from embryos
produced during the third spawning period (TABLE 15).
29
-------
Trifluralin also adversely affected juvenile survival and
growth. Mortality increased as concentration increased, with max-
imum mortality of 67% in 17.7 yg/£ compared to 14% in the control.
Length of surviving juveniles in 9.6 yg/£ was significantly less
than length of control juveniles (TABLE 16).
TABLE 11. CUMULATIVE MORTALITY (%) OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO TRIFLURALIN IN FLOW-
ING, NATURAL SEAWATER FOR 166 DAYS. MORTALITY IS THE
AVERAGE FROM DUPLICATE AQUARIA AND DOES NOT INCLUDE
DEATHS WHICH OCCURRED IN THE SPAWNING CHAMBERS.
Mean measured concentration (yg/£)
Solvent
Day Control Control 1.3 4.8 9.6 17.7 34.1
1-40
41-80
81-120
121-166
TOTAL
9
0
2
0
11
5
0
0
0
6
0
0
0
28
0
0
0
28
26
0
0
0
26
34
0
30
2
76
20
2
2
66a 100a
aSignificantly greater than the control.
TABLE 12. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 166 DAYS TO TRIFLURALIN IN FLOWING, NATURAL
SEAWATER. MEAN STANDARD LENGTH AND STANDARD DEVIATION
ARE GIVEN IN CENTIMETERS AND WERE DETERMINED PHOTO-
GRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN GRAMS AND WAS
DETERMINED IN WATER.
Mean measured
concentration Day 37 Day 94 Day 166
(yg/&) Length Wt. Length Wt.
Length Wt.
Control
1.3±0.3
Sol. control 1.1±0.3
1.3
4.8
9.6
17.7
34.1
1.3±0.2
1.2±0.3
1.1±0.2
0.9±0.3a
0.6±0.2a
0.08
0.07
0.08
0.08
0.07
0.08
0.01
3.0±0.2
2.8±0.3
3.0±0.3
2.6±0.7
2.6±0.4
2.2±0.4a
1.5±0.3a
0.8
0.7
0.8
0.7
0.8
0.7
0.2
3.4±0.3
3.4±0.3
3.5±0.4
3.2±0.4
2.9±0.4a
2.7±0.4a
_b
1.1
1.0
1.2
1.0
0.9
0.9
aSignificantly less than the control,
bAll fish had died.
30
-------
TABLE 13. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) DURING
THREE 10-DAY SPAWNING PERIODS; FISH WERE EXPOSED TO TRIFLURALIN IN FLOWING,
NATURAL SEAWATER FOR 166 DAYS. FIVE FISH (2 MALES AND 3 FEMALES) WERE PLACED
IN A SPAWNING CHAMBER IN DUPLICATE AQUARIA, A AND B.
Mean measured concentration
Solvent
Day Control Control 1.3 4.8 9.6 17.7
A B AB ABABABAB
113
to 1,118 559 172 484 232 522 453 410 690 588 62 244
122
136
to 428 940 593 823 115a 122° 181° 264b 86 68 39 23
145
to -c 489k 807 561b 459 572b 230 618 56 31b -c -c
166
1,546 1,988 1,572 1,868 806 1,816 864 1,292 832 687 101 267
TOTAL 3,534 3,440d 2,622d 2,156d l,519d 368d
aOnly one female fish in spawning chamber.
"Death(s) occurred in spawning chamber.
°No spawning chamber because of lack of surviving fish in appropriate sex ratio.
dSiginficantly less than the control.
-------
TABLE 14. NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) DURING THREE 10-DAY
SPAWNING PERIODS; FISH WERE EXPOSED TO TRIFLURALIN IN
FLOWING, NATURAL SEAWATER FOR 166 DAYS. FIVE FISH (2
MALES AND 3 FEMALES) WERE PLACED IN A SPAWNING CHAMBER
IN DUPLICATE AQUARIA, A AND B.
Day
Mean measured concentration
Control
A B
Solvent
control
A B
1.3
4.8
9.6
B
B
B
Mean and S.D.
of treatment
25±9
20±8
17.7
A B
113-122
136-145
157-166
Mean of
duplicate
37
14
_a
26
19
31
24
25
6
20
27
18
16
27
22
22
8
12
15
12
17
24
23
21
15
6
8
10
14
9
21
15
23
3
2
9
20
2
1
8
2
1
_a
2
8
1
_a
4
16±6 12±6b 8±10b 3±3b
aNo spawning chamber because of lack of surviving fish in the
appropriate sex ratio.
"Significantly less than the control.
TABLE 15. HATCHING SUCCESS OF JUVENILES FROM EGGS SPAWNED BY
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO
TRIFLURALIN IN FLOWING, NATURAL SEAWATER. PERCENTAGE
HATCH AND STANDARD DEVIATION REPRESENT POOLED DATA
FROM DUPLICATE AQUARIA DURING THREE 10-DAY SPAWNING
PERIODS.
Mean measured
concentration
Percentage
hatch and S.D.
1st
2nd
3rd Mean
Number of
embryos examined
Control
Sol.
control
1.3
4.8
9.6
17.7
99±1
99±2
99±1
99 + 2
98±3
12±10b
99±1
99±1
84±10
65 + 28
54a
Ob
97±2
98±2
97±3
94±3
Ob
_c
99±1
98±2
95±8
90±18
91±17
12±10
828
864
671
687
337
136
Insufficient data for statistical analysis; only one group of
embryos.
^Significantly less than the control.
cNo spawning chamber; all surviving fish had been spawned.
32
-------
TABLE 16. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND
WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) JUVENILES HATCHED FROM
EGGS PRODUCED BY FISH EXPOSED TO TRIFLURALIN FOR 113-
122 DAYS.
Mean measured
concentration
(yg/a)
Control
Sol. control
1.3
4.8
9.6
17.7
Number
of juv.
160
160
160
160
160
12
Mortality
(%)
14
15
12
26
44a
67a
Length
(cm)
1.10
1.11
1.01
1.00,
0.85b
1.08C
Weight
(g)
0.03
0.06
0.05
0.05
0.05
0.10
aSignificantly greater than the control.
'-'Significantly less than the control.
cNot included in statistical analyses; only four sur-
viving fish.
Pentachlorophenol
Exposure to pentachlorophenol significantly reduced survival
of parental fish during the 151-day study. All fish exposed to
389 yg/£ died within 60 days and mortality of fish exposed to 88
and 195 yg/Jl was significantly greater than mortality of control
fish at the end of the exposure (TABLE 17). A diluter malfunction
on day 94 caused low dissolved oxygen concentrations and random
mortality. Those deaths were not included in TABLE 17 because
they were not considered to be toxicant related.
Pentachlorophenol did not significantly affect growth of sur-
viving parental fish or their fecundity (TABLES A-2—A-4).
Hatching success of juveniles from embryos produced by fish
exposed to 195 yg/£ during the second spawning period was signif-
icantly less than hatching success of control juveniles (TABLE
18) .
Mortality of juveniles hatched from embryos produced by fish
exposed to 195 yg/fc was significantly greater than mortality of
control juveniles. Growth of surviving juveniles in all concen-
trations was not affected, however (TABLE 19).
33
-------
TABLE 17. CUMULATIVE MORTALITY (%) OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO PENTACHLOROPHENOL
IN FLOWING, NATURAL SEAWATER. MORTALITY IS THE AVER-
AGE FROM DUPLICATE AQUARIA AND DOES NOT INCLUDE DEATHS
WHICH OCCURRED IN THE SPAWNING CHAMBERS.
Mean measured concentration (yg/£)
Solvent
Day Control control 18 47 88 195 389
1-30
31-60
61-90
91-120
121-151
TOTAL
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
2
0
0
10
0
0
2
0
10
0
12
6
96
4
4 12a 28a 100a
Significantly greater than the control.
TABLE 18. HATCHING SUCCESS OF JUVENILES FROM EGGS SPAWNED BY
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO
PENTACHLOROPHENOL IN FLOWING, NATURAL SEAWATER. PER-
CENTAGE HATCH AND STANDARD DEVIATION REPRESENT POOLED
DATA FROM DUPLICATE AQUARIA DURING TWO 10-DAY SPAWNING
PERIODS.
Mean measured
concentration
Percentage
hatch and S.D.
Number of
(ygA)
Control
Sol.
control
18
47
88
195
1st
99±2
100±1
98±3
95±4
91±1
87±10
2nd
98±2
97±3
99±2
99±2
95±6
64±14a
Mean embryos examined
98±2
99±2
98±2
97±3
93±5
77±16
600
732
722
608
350
634
Significantly less than the control,
34
-------
TABLE 19. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND
WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) JUVENILES WHICH WERE
HATCHED FROM EGGS SPAWNED BY FISH EXPOSED TO PENTA-
CHLOROPHENOL FOR 133-142 DAYS IN FLOWING, NATURAL SEA-
WATER.
Mean measured
concentration
(yg/A)
Control
Sol. control
18
47
88
195
Number
of juv.
160
160
120
120
120
57
Mortality
(%)
7
5
3
7
1
28a
Length
(cm)
1.12
1.12
1.17
1.07
1.00
1.07
Weight
(g)
0.04
0.03
0.04
0.04
0.03
0.05
aSignificantly greater than the control.
BIOACCUMULATION
Chlordane was accumulated by parental fish during the 189-day
exposure. Chlordane was also accumulated in eggs/embryos produced
by these fish and in 28-day old FI juveniles. Concentration fac-
tors (based on measured water and tissue concentrations) for pa-
rental fish ranged from 13,000-22,OOOX and were similar to those
reported for sheepshead minnows exposed for a much shorter period
(Parrish et al., 1976). Concentration factors for juveniles
ranged from 6,500-26,800X (TABLES A-5—A-6). No concentration
factors are reported for eggs/embryos because the small sample
weight caused considerable variability in analytical results
(TABLE A-7).
Trifluralin was accumulated by parental fish during the 166-
day exposure and by 28-day old FI juveniles. Concentration fac-
tors (based on measured concentrations) for parental fish ranged
from 4,500X-11,500X and concentration factors for juveniles ranged
from 1,500X-11,500X (TABLES A-8—A-9). No eggs/embryos were ana-
lyzed.
Concentration factors for trifluralin were an order of mag-
nitude greater than those reported for a freshwater fish. Macek
et al. (1976) found a maximum concentration factor of 1,333X in
fathead minnows. However, tissue concentrations were measured in
"eviscerated carcass." Interestingly, in our study the same con-
centration factor was observed in adult fish that had been exposed
for 166 days and in second generation, 28-day old juveniles.
Pentachlorophenol was accumulated by parental fish in the
151-day exposure; pentachlorophenol was also accumulated in eggs/
35
-------
embryos produced by these fish and in 28-day old FI juveniles.
Concentration factors for parental fish ranged from 5-27X; for
eggs/embryos, 13-26X; and for juveniles, 16-48X (TABLES A-10—
A-12). Schimmel et al. (in press) also found that pentachloro-
phenol was not appreciably accumulated by marine fishes. The max-
imum concentration factor for longnose killifish was 41X and that
for striped mullet, 79X after a 4-day exposure. (The chemical
used in the study was sodium pentachlorophenate but tissue concen-
trations were reported as pentachlorophenol).
TABLE 20. ACCUMULATION OF CHEMICALS BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS). CONCENTRATION FACTORS WERE
DERIVED BY DIVIDING MEASURED CONCENTRATIONS IN FISH
(WHOLE-BODY, WET-WEIGHT) BY MEASURED CONCENTRATIONS
IN TEST WATER.
Chemical
Aroclor® 1016
Aroclor® 1254
Chlordane
Dieldrin
Endrin
Heptachlor
Hexachlorobenzene
Methoxychlor
Kepone®
Lindane
Pentachlorophenol
Toxaphene
Trifluralin
Concentration Exposure
factor duration
(range) (days)
6,900-73,000
25,000-55,000
8,500-12,300
6,500-26,800
13,000-22,000
3,500-7,300
3,300-4,800
7,400-21,300
85-6,700
110-260
3,600-20,000
340-730
16-48
5-27
3,100-20,600
1,500-11,500
4,500-11,500
28
21
28
28
189
4
33
4
4
140
36
4
28
151
4
28
166
Source
Hansen et al . ,
Schimmel et al
Parrish et al.
This study.
Parrish et al.
Schimmel et al
Schimmel et al
Parrish et al.
Parrish et al.
Hansen et al . ,
Schimmel et al
This study.
Schimmel et al
This study.
1975
., 1974
, 1976
, 1974
., 1975
., 1976
, 1975
, 1977
1977
., 1977a
., 1977b
36
-------
APPLICATION FACTORS
Application factors were calculated for each of the chemicals
based on the results of the acute and chronic toxicity tests
(TABLE 21).
TABLE 21. CONCENTRATIONS (yg/fc) OF THREE CHEMICALS TOXIC TO
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) IN ACUTE
AND CHRONIC TESTS, AND THE RELATIONSHIP OF ACUTE TOXI-
CITY TO CHRONIC TOXICITY.
96-hour LC50
(95% confidence MATC Application
limits) limits factor limits5
Chemical
Chlordane
Trifluralin
Pentachlorohenol
12.5 >0.5<0.8 0.04-0.06
(3.4-45.9)
190
(128-282)
442
(308-635)
>1.3<4.8 0.007-0.025
>47<88 0.11-0.20
aDerived by dividing the Maximum Acceptable Toxicant Concen-
tration limits by the 96-hour LC50.
The application factor limits derived for sheepshead minnows
and trifluralin were similar to the application factor limits de-
rived for a freshwater fish. We know of no application factors
for freshwater fishes and chlordane or pentachlorophenol. However,
the similarity of results of life-cycle tests with other chemicals
and marine and freshwater fishes support the validity of using
sheepshead minnows for conducting life-cycle tests and deriving
application factors which can be used to assess the impact of
chemicals, particularly pesticides, on freshwa'ter or saltwater
environments (TABLE 22).
37
-------
TABLE 22. COMPARISON OF APPLICATION FACTORS FOR FRESHWATER AND SALTWATER FISHES.
APPLICATION FACTOR LIMITS WERE DERIVED BY DIVIDING THE MEASURED WATER CON-
CENTRATION IN WHICH NO EFFECT WAS OBSERVED AND THE LOWEST MEASURED WATER
CONCENTRATION IN WHICH AN EFFECT WAS OBSERVED BY THE 96-HOUR OR INCIPIENT LC50,
u>
00
Chemical
Carbofuran
Chlordane
Endrin
Heptachlor
Malathion
Methoxychlor
Pentachlorophenol
Trifluralin
C.
C.
C.
J.
C.
P.
C.
J.
L.
P.
C.
C.
C.
P.
Species
variegatus
variegatus
variegatus
floridae
variegatus
promelas
variegatus
floridae
macrochirus
promelas
variegatus
variegatus
variegatus
promelas
Habitat
Saltwater
Saltwater
Saltwater
Freshwater
Saltwater
Freshwater
Saltwater
Freshwater
Freshwater
Freshwater
Saltwater
Saltwater
Saltwater
Freshwater
Application
factor limits
0.04-0.06
0.04-0.06
0.35-0.91
0.25-0.35
0.09-0.18
0.07-0.15
0.08-0.18
0.02-0.03
0.04-0.06
0.02-0.06
0.24-0.47
0.11-0.20
0.007-0.025
0.017-0.044
Source
Parrish et al. , 1977
This study.
Hansen and Parrish, 1977
Hermanutz, in press.
Hansen and Parrish, 1977
Macek et al. , 1976
Parrish et al., 1977
Hermanutz, in press.
Eaton, 1970
Mount and Stephan, 1967
Parrish et al. , 1977
This study.
This study.
Macek et al. , 1976
-------
SUMMARY
1. Chlordane was more acutely toxic to sheepshead minnows than
was trifluralin or pentachlorophenol. The estimated 96-hour
LCSO's, based on measured water concentrations, were 12.5 yg/£,
190 ygA, and 442 yg/£, respectively.
2. Exposure to chlordane concentrations >2.8 pgA caused signifi-
cant (P<0.5) mortality of parental fish. Exposure to concen-
trations >0.8 pgA significantly reduced hatch of embryos
spawned by parental fish and exposure to concentrations
>1.7 pgA caused significant mortality of second generation
fish. The estimated MATC of chlordane for sheepshead minnows
was >0.5<0.8 pgA; the AF limits were 0.04-0.06.
3. Exposure to trifluralin concentrations >17.7 pgA caused sig-
nificant mortality of parental fish. Exposure to concentra-
tions >9.6 pgA significantly reduced growth of parental fish
and exposure to concentrations >4.8 pgA significantly reduced
fecundity of parental fish. Exposure to concentrations
>9.6 pgA significantly reduced hatch of embryos spawned by
parental fish, and survival and growth of second generation
fish. The estimated MATC of trifluralin for sheepshead min-
nows was >1.3<4.8 pgA; the AF limits were 0.007-0.025.
4. Exposure to pentachlorophenol concentrations >88 pgA caused
significant mortality of parental fish. Exposure to penta-
chlorophenol concentrations >195 pgA significantly reduced
hatch of embryos spawned by parental fish and survival of se-
cond generation fish. The estimated MATC of pentachlorophenol
for sheepshead minnows was >47<88 pgA;. the AF limits were
0.11-0.20.
5. The relationship of acute and chronic toxicity for sheepshead
minnows exposed to trifluralin (as expressed by an application
factor) was similar to that for a freshwater fish exposed to
trifluralin.
6. Sheepshead minnows are a suitable saltwater fish for chronic
(full life-cycle) toxicity tests, and data from this study
and from other studies suggest that application factors de-
rived from tests with pesticides and this fish can be used
for freshwater or saltwater fishes.
39
-------
TABLE 23. SUMMARY OF SIGNIFICANT EFFECTS OF CHLORDANE, TRIFLURA-
LIN, AND PENTACHLOROPHENOL ON SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) DURING CHRONIC (FULL LIFE-
CYCLE) EXPOSURES IN FLOWING, NATURAL SEAWATER.
]
Chemical Generation Life stage Effect t
Chlordane Parental Adult Increased
mortality
F1 Embryo Decreased
hatch
Juvenile Increased
mortality
Trifluralin Parental Adult Increased
mortality
Decreased
growth
Decreased
fecundity
F1 Embryo Decreased
hatch
Juvenile Increased
mortality
Decreased
growth
Pentachlorophenol Parental Adult Increased
mortality
Fj Embryo Decreased
hatch
Juvenile Increased
mortality
yiean measured
concentration
(ugA)
>2.8
>0.8
>1.7
>17.7
>9.6
>4.8
>9.6
>9.6
>9.6
>88
>195
>195
40
-------
REFERENCES
Alabaster, J.S. 1969. Survival of fish in 164 herbicides, in-
secticides, fungicides, wetting agents and miscellaneous
substances. Int. Pest. Cont. 7(l):29-35.
Alderdice, D.F. 1963. Some effects of simultaneous variation
in salinity, temperature, and dissolved oxygen on the re-
sistance of young Coho salmon to a toxic substance. J.
Fish. Res. Bd. Can., 20:525-550.
Bandt, H.J. and D. Nehring. 1962. Die toxikilogische wirkung
von pentachlorophenolnatrium and hexachlorophen auf fische
und niedere wassertiere. Z. Fisch., 10:543-548.
Bevenue, A. and H. Beckman. 1967. Pentachlorophenol: a dis-
cussion of its properties and its occurrence as a residue
in human and animal tissue. Residue Rev., 19:83-126.
Bugg, J.C., Jr., J.E. Higgins, and E.A. Robertson, Jr. 1967.
Chlorinated pesticide levels in the eastern oyster
(Crassostrea virginica) from selected areas of the South
Atlantic and Gulf of Mexico. Pestic. Monit. J., 1(3):9-12.
Cardwell, R.D., D.G. Foreman, T.R. Payne, and D.J. Wilbur. 1977,
Acute and chronic toxicity of chlordane to fish and inverte-
brates. EPA-600/3-77-019, U.S. EPA, Duluth, Minnesota.
125 pp.
The Committee on Methods for Toxicity Tests with Aquatic Organ-
isms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. EPA-600/3-75-009, U.S.
EPA, Gulf Breeze, FL. 61 pp.
Crandall, C.A. and C.T. Goodnight. 1962. Effects of sublethal
concentration of several toxicants on growth of the common
guppy. Limnol. & Ocean., 7:233-239.
Davis, J.C. and R.A.W. Hoos. 1975. Use of sodium pentachloro-
phenate and dehydroabietic acid as reference toxicants for
salmonid bioassays. J. Fish. Res. Bd. Can., 32 (3) :411-416.
Eaton, J.G. 1970. Chronic malathion toxicity to the bluegill
(Lepomis macrochirus). Water Res., 4(1):673-684.
41
-------
Finney, D.J. 1971. Probit Analysis. Cambridge University
Press, London. 333 pp.
Hansen, D.J. and S.C. Schimmel. 1975. An entire life-cycle bio-
assay using sheepshead minnows (Cyprinodon variegatus).
Fed. Reg., 40(123)11:26,904-26,905.
Hansen, D.J., S.C. Schimmel, and J. Forester. 1975. Effects of
Aroclor® 1016 on embryos, fry, juveniles, and adults of
sheepshead minnows (Cyprinodon variegatus). Trans. Am.
Fish. Soc., 104(3):584-588.
Hansen, D.J. and P.R. Parrish. 1977. Suitability of sheepshead
minnows (Cyprinodon variegatus) for life-cycle toxicity tests
Pages 117-126. IN: Aquatic Toxicology and Hazard Evaluation.
ASTM STP 634. F.L. Mayer and J.M. Hamelink, Eds. American
Society for Testing and Materials, Philadelphia, PA.
Hansen, D.J., S.C. Schimmel, and J. Forester. 1977. Endrin:
effects on the entire life cycle of a saltwater fish,
Cyprinodon variegatus. J. Toxicol. Environ. Health., 3:721-
733.
Henderson, D., Q.H. Pickering, and C.M. Tarzwell. 1959. Rela-
tive toxicity of ten chlorinated hydrocarbon insecticides
to four species of fish. Trans. Am. Fish. Soc., 88:23-32.
Henderson, D., W.L. Johnson, and A. Inglis. 1969. Organochlo-
rine insecticide residues in fish. Pestic. Monit. J., 3(3):
145-171.
Henderson, D., W.L. Johnson, and A. Inglis. 1971. Residues in
fish, wildlife and estuaries. Pest. Monit. J., 5(1):1-11.
Hermanutz, R.O. IN PRESS. Endrin and malathion toxicity to
flagfish (Jordanella floridae) . Arch. Environ. Contain. &
Toxicol.
Kaila, K. and J. Saarikoski. 1977. Toxicity of pentachloro-
phenol and 2,3,6-trichlorophenol to the crayfish (Astacus
fluviatilis L.). Environ. Pollut., 12:119-123.
Macek, K.J., C. Hutchinson, and O.B. Cope. 1969. The effects
of temperature on the susceptibility of bluegills and rain-
bow trout to selected pesticides. Bull. Environ. Contam.
Toxicol., 4(3):174-183.
Macek, K.J., M.A. Lindberg, S. Sauter, K.S. Buxton, and P.A.
Costa. 1976. Toxicity of four pesticides to water fleas
and fathead minnows. EPA-600/3-76-099. 58 pp.
McKim, J.M. and D.A. Benoit. 1971. Effects of long term expo-
42
-------
sure to copper on survival, growth, and reproduction of
brook trout, Salvelinus fontinalis. J. Fish. Res. Bd. Can.,
28(5):655-662.
Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparat-
us for fish toxicological studies. Water Res., 1:21-29.
Mount, D.I. and C.E. Stephan. 1967. A method for establishing
acceptable toxicant limits for fish - malathion and the
butoxyethanol ester of 2-4-D. Trans. Am. Fish. Soc., 96(2):
185-193.
Mount, D.I. 1968. Chronic toxicity of copper to fathead minnows
(Pimephales promelas, Rafinesque). Water Res., 2:215-223.
National Research Council of Canada. 1975. Chlordane: its ef-
fects on Canadian ecosystems and its chemistry. Subcommittee
on Pesticides on Related Compounds, Subcommittee Report No.
2. 189 pp.
Norup, B. 1972. Toxicity of chemicals in paper factor effluents.
Water Res., 6:1,585-1,588.
Parrish, P.R., J.A. Couch, J. Forester, J.M. Patrick, Jr., and
G.H. Cook. 1974. Dieldrin: effects on several estuarine
organisms. Proc. 27th Annu. Conf. Southeast. Assoc. Game
Fish Comm., pp. 427-434.
Parrish, P.R., G.H. Cook, and J.M. Patrick, Jr. 1975. Hexa-
chlorobenzene: effects on several estuarine animals. Pro.
28th Annu. Conf. Southeast. Assoc. Game Fish Comm., pp. 179-
187.
Parrish, P.R., S.C. Schimmel, D.J. Hansen, J.M. Patrick, Jr., and
J. Forester. 1976. Chlordane: effects on several estuarine
organisms. J. Toxicol. Environ. Health, 1:485-494.
Parrish, P.R., E.E. Dyar, M.A. Lindbergh, C.M. Shanika, and J.M.
Enos. 1977. Chronic toxicity of methoxychlor, malathion,
and carbofuran to sheepshead minnows (Cyprinodon variegatus).
EPA-600/3-77-059. 36 pp.
Sanders, H.O. and O.B. Cope. 1966. Toxicities of several pesti-
cides to two species of cladocerans. Trans. Am. Fish. Soc.,
95:165-169.
Sanders, H.O. and O.B. Cope. 1968. The relative toxicity of
several pesticides to naiads of three species of stoneflies.
Limnol. & Ocean., 13 (1) :112-117.
Sanders, H.O. 1970. Toxicities of some herbicides to six spe-
cies of freshwater crustaceans. J. Water Pollut. Control
43
-------
Fed., 42 (8) : 1, 544-1,550.
Schimmel, S.C., D.J. Hansen, and J. Forester. 1974. Effects of
Aroclor® 1254 on laboratory-reared embryos and fry of
sheepshead minnows (Cyprinodon variegatus). Trans. Am.
Fish. Soc., 103:582-586.
Schimmel, S.C. and D.J. Hansen. 1975. Sheepshead minnows
(Cyprinodon variegatus): an estuarine fish suitable for
chronic (entire life-cycle) bioassays. Proc. 28th Annu.
Conf. Southeast. Assoc. Game Fish Comm., pp. 392-398.
Schimmel, S.C., P.R. Parrish, D.J. Hansen, J.M. Patrick, Jr., and
J. Forester. 1975. Endrin: effects on several estuarine
organisms. Proc. 28th Annu. Conf. Southeast. Assoc. Game
Fish Comm., pp. 187-194.
Schimmel, S.C., J.M. Patrick, Jr., and J. Forester. 1976. Hep-
tachlor: uptake, depuration, retention, and metabolism by
spot, Leiostomus xanthurus. J. Toxicol. Environ. Health,
2:169-178.
Schimmel, S.C., J.M. Patrick, Jr., and L.F. Faas. IN PRESS. Ef-
fects of sodium pentachlorophenate on several estuarine ani-
mals: toxicity, uptake, and depuration. Pages 147-155. IN
Pentachlorophenol: Chemistry, Pharmacology and Environmental
Toxicology- Vol. 12. K. Ranga Rao, Ed. Plenum Press, New
York.
Schimmel, S.C., J.M. Patrick, Jr., and J. Forester. 1977a.
Toxicity and bioconcentration of BHC and lindane in selected
estuarine animals. Arch. Environ. Contam. Toxicol., 6:355-
363.
Schimmel, S.C., J.M. Patrick, Jr., and J. Forester. 1977b. Up-
take and toxicity of toxaphene in several estuarine organ-
isms. Bull. Environ. Contam. Toxicol., 5:353-367.
Sokal, R.R. and F.J. Rohlf. 1973. Introduction to Biostatistics.
W.H. Freeman & Company, San Francisco, CA. 368 pp.
Sprague, J.B. 1969. I. Review paper: measurement of pollutant
toxicity to fish. I. bioassay methods for acute toxicity.
Water Res., 3 (11) :793-821.
Steel, R.G.D. and J.H. Torrie. 1960. Principles and Procedures
of Statistics. McGraw-Hill Book Company, Inc., New York,
NY. 481 pp.
Tagatz, M.E., J.M. Ivey, J.C. Moore, and M. Tobia. 1977- Ef-
fects of pentachlorophenol on the development of estuarine
communities. J. Toxicol. Environ. Health, 3:501-506.
44
-------
Tomiyama, T. and K. Kawabe. 1962. The toxic effect of penta-
chlorophenate, a herbicide, on fishery organisms in coastal
waters. I. The effect on certain fishes and a shrimp. Tap.
Soc. Sci. Fish. Bull., 28:379-382.
Tomiyama, T., K. Kobayashi, and K. Kawabe. 1962. The toxic ef-
fect of pentachlorophenate, a herbicide, on fishery organ-
isms in coastal waters. III. The effect on Venerupis
philippinarum. Tap. Soc. Sci. Fish. Bull., 28:417-421.
U.S. Department of Health, Education, and Welfare. 1969. Report
of the Secretary's Commission on Pesticides and Their Re-
lationship to Environmental Health. Washington, D.C. 677
pp.
U.S. EPA. 1974. Manual of Analytical Methods for the Analysis
of Pesticide Residues in Human and Environmental Samples.
Pesticides and Toxic Substances Effects Laboratory, National
Environmental Research Center, Triangle Park, NC, Section
5A940, (a).
Webb, P.W. and J.R. Brett. 1973. Effects of sublethal concen-
trations of sodium pentachlorophenate on growth rate, food
conversion efficiency, and swimming performance in under-
yearling sockeye salmon (Oncorhynchus nerka). J. Fish. Res.
Bd. Can., 30:499-507.
45
-------
TABLE A-l.
APPENDIX
GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS) EXPOSED FOR 189 DAYS TO CHLORDANE
IN FLOWING, NATURAL SEAWATER. MEAN STANDARD
LENGTH AND STANDARD DEVIATION ARE GIVEN IN
CENTIMETERS AND WERE DETERMINED PHOTOGRAPHI-
CALLY. AVERAGE WEIGHT is GIVEN IN GRAMS AND
WAS DETERMINED IN WATER.
Mean measured
concentration
( y g/ £ )
Control
Sol. control
0.5
0.8
1.7
2.8
18.0
Day 35
Length
(cm)
1.3±0.2
1.3±0.2
1.3±0.2
1.310.2
1.3+0.2
1.3+0.3
_a
Wt.
(g)
0.1
0.1
0.1
O.l
0.1
0.1
Day 94
Length
(cm)
2.9±0.4
2.9+0.2
3.010.3
2.810.4
2.8+0.3
2.9+0.4
-
Wt.
(q)
0.8
0.8
0.8
0.7
0.7
0.8
Day 189
Length
(cm)
3.6+0.4
3.7+0.3
3.710.3
3.610.4
3.510.3
3.610.4
—
Wt.
(q)
1.3
1.3
1.4
1.1
1.1
1.6
aAll fish had died.
46
-------
TABLE A-2.
GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS) EXPOSED FOR 151 DAYS TO PENTA-
CHLOROPHENOL IN FLOWING, NATURAL SEAWATER.
MEAN STANDARD LENGTH AND STANDARD DEVIATION
ARE GIVEN IN CENTIMETERS AND WERE DETERMINE
PHOTOGRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN
GRAMS AND WAS DETERMINED IN WATER .
Mean measured
concentration
Sol
(ygA)
Control
. control
18
47
88
195
389
Day 34
Length
(cm)
i.
1.
1.
1.
1.
1.
1.
2 + 0.3
2±0.3
3±0.3
2±0.3
2±0.3
3±0.3
5±0.1
Wt.
(g)
0.06
0.08
0.06
0.07
0.06
0.06
0.07
Day 91
Length
(cm)
2.
2.
2.
2.
2.
2.
7±0.7
8±0.4
7±0.4
6±0.3
8±0.4
6±0.4
_a
Wt.
(g)
0.7
0.7
0.7
0.7
0.7
0.6
-
Day
151
Length
3
3
3
3
3
3
(cm)
.1±0.
.1±0.
.1±0.
.0±0.
.1±0.
.1±0.
-
2
3
3
2
3
2
Wt.
(g)
0.8
1.0
0.9
0.9
0.9
0.9
—
aAll fish had died.
47
-------
CO
TABLE A-3. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
DURING TWO 10-DAY SPAWNING PERIODS; FISH WERE EXPOSED TO PENTACHLORO-
PHENOL IN FLOWING, NATURAL SEAWATER FOR 166 DAYS. FIVE FISH (2 MALES
AND 3 FEMALES) WERE PLACED IN A SPAWNING CHAMBER IN DUPLICATE AQUARIA,
A AND B.
Day
111
to
120
133
to
142
Mean measured concentration (pg/£)
Solvent
Control control 18 47 88 195
AB A BABABA B AB
206 233 378 499 411 348 171 206 114 90a 228 531
144 358 242a 391 497 482 369 667 643 119a 215 327
SUB~ 350 591 620 890 908 830 540 873 757 209 443 858
TOTAL 941 1,510 1,738 1,413 966 1,301
aDeath(s) occurred in spawning chamber.
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TABLE A-4.
NUMBER OF EGGS SPAWNED PER DAY PER FEMALE
SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) DUR-
ING TWO 10-DAY SPAWNING PERIODS; FISH WERE EX-
POSED TO PENTACHLOROPHENOL IN FLOWING, NATURAL
SEAWATER FOR 166 DAYS. FIVE FISH (2 MALES AND
3 FEMALES) WERE PLACED IN A SPAWNING CHAMBER
IN DUPLICATE AQUARIA, A AND B.
Mean measured concentration (yg/£)
Solvent
Day Control control 18 47 88 195
111-120
133-142
A
8
5
B
8
12
A
13
8
B
17
13
A
14
17
B
12
16
A
6
12
B
7
22
A
4
21
B
4
6
A
8
7
B
18
11
Average of
duplicate
10
10 15 16 14
14 12
8 14
Mean and S.D.
of treatment
8±3
13±4
15±2
12±7
9±8
11±5
TABLE A-5.
CONCENTRATIONS OF CHLORDANE IN SURVIVING ADULT
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EX-
POSED FOR 189 DAYS. MEAN (AND STANDARD DEVI-
ATION) TISSUE RESIDUES ARE WHOLE-BODY, WET-
WEIGHT. DUPLICATE ANALYSES OF EACH POOLED
SAMPLE (AT LEAST 2 FISH PER SAMPLE) WERE PER-
FORMED.
Mean measured
concentration
Water
(yg/M
Control
Sol. control
0.5
0.8
1.7
2.8
Tissue
(yg/g)
NDa
NDa
6.7±3.6
10.6+3.4
26.4±15.6
61.5±33.6
Concentration
factor
_ .
13,400
13,250
15,529
21,964
Number
of analyses
2
2
6
6
6
6
*Not detectable; <0.1 yg/g.
49
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TABLE A-6.
CONCENTRATIONS OF CHLORDANE IN SURVIVING 28-
DAY OLD JUVENILE SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS). THE FISH HATCHED FROM EMBRYOS
PRODUCED DURING THE FIRST TWO SPAWNING PERIODS
(DAYS 93-102 AND 139-148). MEAN (AND STANDARD
DEVIATION) TISSUE RESIDUES ARE WHOLE-BODY, WET-
WEIGHT. DUPLICATE ANALYSES OF EACH POOLED
SAMPLE FROM DUPLICATE AQUARIA A AND B WERE PER-
FORMED.
Mean measured
concentration
Water Tissue
(ug/O (yg/g)
Group 1 Group 2
Control -a 0.26±0.15
Sol. control -a 0.30±0.01
0.5 11±1 4±0
0.8 15±3 8±1
1.7 33±10 11±0
2.8 75±3 31±15
Concentration
factor
Group 1 Group 2
22,000 8,000
18,750 10,000
19,412 6,471
26,786 11,071
*No analyses.
TABLE A-7.
CONCENTRATIONS OF CHLORDANE IN EGGS/EMBRYOS
PRODUCED BY ADULT SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED FOR 171-181
DAYS. TISSUE RESIDUES ARE WET-WEIGHT. DUP-
LICATE ANALYSES OF EACH POOLED SAMPLE FROM
DUPLICATE AQUARIA A AND B WERE PERFORMED.
Measured concentration
Water
(yg/M
Control
Sol. control
0.5
0.8
1.7
2.8
Tissue
(yg/g)
.a
a
5.5, 5.6, 2.4, 2.8
83, 84, 31, 32
46, 47, 413, 403
23, 19
No analyses.
50
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TABLE A-8. CONCENTRATIONS OF TRIFLURALIN IN SURVIVING
ADULT SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 166 DAYS. AVERAGE TISSUE RESIDUES
ARE WHOLE-BODY, WET-WEIGHT. DUPLICATE ANALYSES
OF A POOLED SAMPLE FROM EACH TREATMENT WERE
PERFORMED.
Measured concentration
Water
(vgA)
Control
Sol. control
1.3
4.8
9.6
17.7
Tissue
(yg/g)
0.5
2.6
15
34
46a
79a
Concentration
factor
11,538
7,093
4,792
4,463
*0nly one analysis.
TABLE A-9. CONCENTRATIONS OF TRIFLURALIN IN SURVIVING 28-
DAY OLD JUVENILE SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS). THE FISH HATCHED FROM EMBRYOS
PRODUCED DURING THE FIRST SPAWNING PERIOD (DAYS
113-122). MEAN (AND STANDARD DEVIATION) TISSUE
RESIDUES ARE WHOLE-BODY, WET-WEIGHT. DUPLICATE
ANALYSES OF EACH POOLED SAMPLE FROM DUPLICATE
AQUARIA A AND B WERE PERFORMED.
Mean measured
concentration
Water Tissue Concentration Number
(yg/£) (yg/g) factor of analyses
Control 0.9±0.3 4
Sol. control 16+11 4
1.3 15±14 11,538 4
4.8 14+7 2,917 4
9.6 14±4 1,458 4
17-7 75 4,237 1
51
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TABLE A-10. CONCENTRATIONS OF PENTACHLOROPHENOL IN SUR-
VIVING ADULT SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS) EXPOSED FOR 151 DAYS.TISSUE
RESIDUES ARE WHOLE-BODY, WET-WEIGHT. ANALYSES
OF POOLED SAMPLES FROM DUPLICATE AQUARIA A
AND B WERE PERFORMED.
Mean measured
concentration
Water
(yg/ft)
Control
Sol. control
18
47
88
195
Tissue
(yg/g)
NDa
0.006
0.48
0.54
0.80
0.97
Concentration
factor
_
27
11
9
5
Number
of analyses
4
4
3
3
4
3
aNot detectable; <0.005 pg/g.
TABLE A-ll. CONCENTRATIONS OF PENTACHLOROPHENOL IN EGGS/
EMBRYOS PRODUCED BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED FOR 133-142
DAYS. TISSUE RESIDUES ARE WET-WEIGHT. ANAL-
YSES OF EACH POOLED SAMPLE WERE PERFORMED.
Measured concentration
Water
(yg/O
Control
Sol. control
18
47
88
195
Tissue
(yg/g)
NDa
NDa
0.24
0.88
2.3
4.3
Concentration
factor
,_ „_ —
13
19
26
22
aNot detectable; <0.1 yg/g.
52
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TABLE A-12. CONCENTRATIONS OF PENTACHLOROPHENOL IN SUR-
VIVING 28-DAY OLD JUVENILE SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS). THE FISH HATCHED
FROM EMBRYOS PRODUCED DURING THE FIRST SPAWN-
ING PERIOD (DAYS 111-120). TISSUE RESIDUES
ARE WHOLE-BODY, WET-WEIGHT. ANALYSES OF A
POOLED SAMPLE FROM DUPLICATE AQUARIA A AND B
WERE PERFORMED
Mean measured
concentration
Water
(yg/A)
Control
Sol. control
18
47
88
195
Tissue
(yg/g)
NDa
NDa
0.86
1.6
1.4
7.4
Concentration
factor
__«
48
34
16
38
Number
of analyses
4
4
3
3
4
3
aNot detectable; <0.01 yg/g.
53
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-010
4. TITLE ANDSUBTITLE
Chronic Toxicity of Chlordane, Trifluralin,
and Pentachlorophenol to Sheepshead Minnows
(Cyprinodon variegatus)
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
January 1978 date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Patrick R. Parrish, Elizabeth E. Dyar, Joanna
M. Enos, and William G. Wilson
8. PERFORMING ORGANIZATION REPORT NO.
BP-78-1-006
9. PERFORMING ORGANIZATION NAME AND ADDRESS
EG&G, Bionomics
Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 32507
10. PROGRAM ELEMENT NO.
1EA714
11. CONTRACT/GRANT NO.
68-03-2069
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT Sheepshead minnows (Cyprinodon variegatus) were exposed to three
chemicals—chlordane, trifluralin, or pentachlorophenol—in flowing, na-
tural seawater to determine acute and chronic (full life-cycle effects).
Mortality of parental fish exposed to mean measured chlordane concen-
trations >2.8 yg/£ was significantly greater than that of control fish.
Hatch of juveniles from embryos of parental fish exposed to >0.8 yg/£ was
significantly less than hatch of control juveniles. The estimated maxi-
mum acceptable toxicant concentration (MATC) was >0.5<0.8 yg/£ and the
application factor (AF) limits, 0.04-0.06.
Exposure to mean measured trifluralin concentrations >9.6 yg/£ sig-
nificantly decreased growth of parental fish. Fecundity of parental fish
exposed to concentrations >4.8 yg/fc was significantly less than that of
control fish. Survival and growth of second generation fish were signi-
ficantly less than the control in concentrations >9.6 yg/£. The esti-
mated MATC was >1.3<4.8 yg/& and the AF limits, 0.007-0.025.
Mortality of parental Sheepshead minnows exposed to mean measured
pentachlorophenol concentrations >88 yg/& was significantly greater than
mortality of control fish. The estimated MATC was >47<88 yg/& and the
AF limits, 0.11-0.20.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Toxicity
Fish
Saltwater
Chemicals
Chlordane
Trifluralin
Pentachlorophenol
Chronic toxicity
Flowing seawater
Application factor
Life cycle
18. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
53
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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