Ecological Research Series
TOXICITY OF DIAZINON TO BROOK TROUT AND
FATHEAD MINNOWS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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EPA-600/3-77-060
May 1977
TOXICITY OF DIAZINON TO BROOK TROUT
AND FATHEAD MINNOWS
by
Donald T. Allison
Roger 0. Hermanutz
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory—
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
11
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FOREWORD
Our nation’s fresh waters are vital for all animals and plants, yet
our diverse uses of water———for recreation, food, energy, transportation,
and industry———physically and chemically alter lakes, rivers, and streams.
Such alterations threaten terrestrial organisms, as well as those living in
water. The Environmental Research Laboratory in Duluth, Minnesota, develops
methods, conducts laboratory and field studies, and extrapolates research
findings
——to determine how physical and chemical pollution affects
aquatic life;
——to assess the effects of ecosystems on pollutants;
——to predict effects of pollutants on large lakes through
use of models; and
——to measure bioaccumulation of pollutants in aquatic
organisms that are consumed by other animals, including
man.
This report describes the acute and chronic effects of the
organophosphate insecticide diazinon on two species of freshwater
fishes.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
111
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ABSTRACT
Average 96—hr LC5O’s for diazinon under flow—through conditions were
7.8, 1.6, 0.77, and 0.46 mg/i, respectively, for fathead minnows, flagfish,
brook trout, and biuegills
The chronic effects of diazinon on fathead minnows and brook trout were
determined in flow—through systems with constant toxicant concentrations.
Fathead minnows exposed to the lowest concentration tested (3.2 pg/i) from
5 days after hatch through spawning had a significantly higher incidence of
scoliosis than the control (P = 0.05). Hatch of their progeny was reduced
by 30% at this concentration 0 Yearling brook trout exposed to 4.8 pg/l
and above began developing scoliosis and lordosis within a few weeks. Growth
of brook trout was substantially inhibited during the first 3 months at
4.8 pg/i and above. Neurological symptoms were evident in brook trout at
2.4 pg/i and above early in the tests, but were rarely observed after 4 or
5 months of exposure. Exposure of mature brook trout for 6 — 8 months to
concentrations ranging from 9.6 pg/i to the lowest tested (0.55 pg/i)
resulted in equally reduced growth rates for their progeny. Transfer of
progeny between concentrations indicated that effects noted for progeny of
both species at lower concentrations were the result of parental exposure
alone and not the exposure of progeny following fertilization.
iv
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Page
Foreword
Abstract
Acknowledgments
5. Results
Acute toxicity tests
Fathead minnow chronic toxicity tests
Brook trout preliminary test
Brook trout partial—chronic toxicity test.
Diazinon accumulation in brook trout tissues
6. Discussion
References . . . 29
Appendices
A. Recommended bioassay procedures for fathead minnow Pimephales
promelas Rafinesque chronic tests
B. Recommended bioassay procedure for brook trout Salvelinus
fontinalis (Mitchill) partial chronic tests
C. Test conditions during acute and chronic exposures (six tables)
D. Accumulation of diazinon in tissues of adult brook trout.
CONTENTS
iv
vi
1. Introduction
2. Conclusions
3. Recommendations . .
4. Materials and Methods
Physical systems
Chemical systems
Biological systems
Acute toxicity tests
Fathead minnow chronic toxicity tests
Brook trout preliminary test
Brook trout partial—chronic toxicity test
Statistical analysis
1
3
4
5
5
5
6
6
6
6
6
7
8
8
8
• 14
14
25
26
32
47
61
67
V
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TABLES
Number Page
1 Acute Toxicities of Diazinon to Fathead Minnows ( Pimephales Promelas) ,
Bluegills ( Lepomis Macrochirus) , Brook Trout ( Salvelinus Fontinalis) ,
and Flagfish ( Jordanella Floridae ) 9
2 Survival and Growth of Parental Stock of Fathead Minnows ( Pimephales
Promelas ) Continuously Exposed to Diazinon After 30 and 61 Days . . . 10
3 Incidence of Scoliosis in Parental Fathead Minnows ( Pimephales Promelas )
Continuously Exposed to Diazinon After Hatch 11
4 Survival and Growth of Parental Stock of Fathead Minnows ( Pimephales
Promelas ) Continuously Exposed to Diazinon After 31, 64, 97, 135,
167,and274Days(Test#2) 12
5 Spawning and Hatchability of Eggs from Fathead Minnows ( Pimephales
Promelas ) Continuously Exposed to Diazinon (Test #2) 15
6 Survival and Growth after 30 and 60 days for Progeny of Fathead
Minnows ( Pimephales Promelas ) Continuously Exposed to Diazinon . . . 17
7 Survival, Growth, and Incidence of Scoliosis and Lordosis for Parental
Brook Trout ( Salvelinus Fontinalis ) Continuously Exposed to Diazinon
for 91 and 173 Days 20
8 Spawning, Viability, and Hatchability of Eggs from Brook Trout ( Salvelinus
Fontinalis ) Continuously Exposed to Diazinon for 6 — 8 Months . . . . 22
9 Survival and Growth to 122 days for Progeny of Brook Trout ( Salvelinus
Fontinalis ) Continuously Exposed to Diazinon 23
vi
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ACKNOWLEDGMENTS
The authors wish to thank Helen E. Herrmann for daily maintenance
and monitoring of the test systems; Leonard H. Mueller for supervision
of diazinon analysis; Mary J. Hoffman for diazinon analyses; and Alfred
W. Jarvinen, John G 0 Eaton, and other members of the Environmental Research
Laboratory—Duluth for advice, assistance, and critical review of the
manuscript.
vii
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SECTION 1
INTRODUCT ION
The organophosphate insecticide diazinon [ O,O—diethyl—O—(2—isoprophyl—
6 methyl—4—pyrimidinyl) phosphorothioatell has been used on agricultural
crops since 1954 (Bartsch, 1974). Diazinon usage has increased in recent
years because of the world—wide trend towards intensified agricultural
production. Records for a representative corn—producing region in Kansas
where irrigation was introduced in 1963 indicate that diazinon was first
used in 1964. Three years later 12 times as much diazinon was being
applied annually (Knutsonetal., 1971). Actual or notential restriction
on the use of organochlorine pesticides has increased interest in the use of
organophosphates. Diazinon has been considered as one of two logical
substitutes for chlordane, which was applied at an annual rate of 600,000
pounds circa 1972 (Anonymous, 1972).
Diazinon is used throughout the world on rice crops where application
every 20 days has proved effective (Sethunathan and Pathak, 1972). Currently,
in the United States diazinon is one of the insecticides most frequently
applied to onions and sweet potatoes (Bartsch, 1974). It is recommended
for pest control on most of the major vegetable, fruit, and nut crops, for
grasshopper control on rangeland, for control of lawn and household pests,
and as a livestock spray (Farm Chemicals Handbook, 1974).
The probability of water contamination by diazinon and the subsequent
persistence of the chemical appear to be influenced by a complex of physical
and biological factors which are poorly understood at present. Munson
(1970) observed that field—applied diazinon remained in the top few inches
of soil until degraded or removed by runoff. This study indicates a low
probability for direct contamination of ground water. Suett (1971) observed
that field—applied diazinon persisted longer in peaty loam than in sandy
loam but he notes that laboratory studies by other workers have produced
conflicting results. Rate of hydrolysis is greatly influenced by pH, but
diazinon has an apparent potential for persisting many months in non—acid
bodies of water. Cowart et al. (1971) recorded a half—life of less than
2 weeks at pH 6.0. Gomaaetal.. (1969) reported half—life values of about
6 and 4 months at pH 7 4 and 9.0, respectively. Aerobic conditions accelerate
degradation, and suspended soil particles inhibit degradation in water
(Sethunathan and Pathak, 1972). Sethunathan and co—workers have found that
at least two species of naturally occurring bacteria are able to rapidly
degrade diazinon in water and flooded soil. Large populations of these
bacteria develop following repeated application of diazinon. These
1
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bacterial populations do not develop in the presence of structurally
related organophosphates or affect their rates of degradation (Sethunathan
and Pathak, 1972). No reports were found of general monitoring of natural
waters for the presence of diazinon.
Studies of the toxicity of diazinon to fishes have been limited to a
few acute exposures (Weiss, 1961; Coppage, 1972). The present study was
undertaken to discover the effects of prolonged exposure to diazinon on
freshwater fishes and to estimate the highest concentration which would not
be detrimental to their populations. We used the laboratory fish—production
index (LFPI) defined by Mount and Stephan (1967) and their concept that
the highest concentration of a toxicant producing no measurable effects on
production rates of adults and progeny in a laboratory environment is
termed the maximum acceptable toxicant concentration (NATC). An “application
factor” is then derived for a given toxicant and a given species by dividing
the MATC by the acute toxicity (LC5O) value for that combination. Mount
and Stephan (1967) proposed that, if a number of application factors for
several species subjected to one toxicant fell within a narrow range, the
“safe” chronic concentrations for similar species could be estimated by
multiplying their acute toxicity LC5O’s by this application factor. An
ancillary purpose of the present test was to determine if the application—
factor concept could be applied to diazinon and freshwater fishes. Fathead
minnows ( Pimephales promelas ) and brook trout ( Salvelinus fontinalis ) were
selected for the chronic exposures as representatives of warm— and cold—
water fishes that could be reared under laboratory conditibns. Acute
toxicity values (96—hr LC5O) were obtained for these species as well as
bluegills ( Lepomis macrochirus ) and flagfish ( Jordanella floridae) .
2
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SECTION 2
CONCLUS IONS
Although a long—term no—effect concentration was not determined with
fathead minnows, 3.2 pg/i appeared to be close to a maximum acceptable
toxicant concentration (MATC) as defined by Mount and Stephan (1967). The
application factor (NATC/96—hr LC5O) for diazinon with this fish is
probably 1O .
Most chronic effects noted for brook trout were minimal or no longer
apparent at the lower concentrations. However growth inhibition of
progeny was the same at all tested concentrations (9.6 — 0.55 pg/i).
Therefore, it is probable that the MATC was considerably less than 0.55
pg/i, which indicates an application factor for brook trout of 10 or lower.
Diazinon—related effects observed at lower concentrations for progeny
of both species appear to have been caused by the exposure of their parents
and not by exposure following fertilization (indicated by transfer of
progeny between concentrations). Progeny exposed to diazinon throughout
embryonic, larval, and juvenile stages did not develop the neurological
symptoms and spinal deformities observed in their parents 0 Results following
exposure of control progeny to diazinon indicate that this phenomenon was
not due to selection of resistant parental stock.
An initial exposure of brook trout to diazinon coinciding with the
spawning period might cause a greater reduction in reproductive potential
than that observed in the present test.
Whole—body levels of diazinon in fishes will probably not exceed 100
times the ambient water concentration even after exposure for many months.
3
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SECTION 3
RECONNENDATIONS
The estimated application factors for diazinon reported in this
paper should not be used to extrapolate MATC’s for other species of fishes
from their 96—hr LC5O’s 0 A just—completed chronic exposure of another fish
indicates that diazinon may elicit a wide range of species-related
application factors.
The variance in application factors plus the occurrence at low
concentratious of effects potentially harmful to population survival suggest
that chronic—exposure studies should be conducted with other species of
fishes.
Definitive studies should be made to determine if progeny of diazinon—
exposed fish are more resistant than their parents or if young fish in
general are more resistant.
The effects of diazinon on survival—related behavior should be
studied.
The chronic toxicity of diazinon to invertebrates should be investigated.
4
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SECTION 4
MATERIALS AND METHODS
The acute toxicity tests met the requirements outlined by the American
Public Health Association (1971). The designs of the chronic and partial—
chronic toxicity tests followed the procedures recommended by the committee
on aquatic bioassays, Environmental Research Laboratory—Duluth, Minnesota
(Appendixes A and B), except as noted below.
PHYSICAL SYSTEMS
All of the tests described in this paper were conducted under flow—through
conditions. Proportional diluters (Mount and Brungs, 1967) delivered five
toxicant concentrations plus control water to duplicate exposure chambers
in all tests. Lake Superior water was delivered at a rate of 10 to 14
test—chamber volumes p r day except as noted in Appendix C, Table 1, during
the fathead minnow acute tests. Water quality values for the acute tests,
other than temperature and dissolved oxygen, fell within the ranges given
for the chronic and partial chronic tests in Appendix C, Tables 2 and 3.
The physical characteristics of the test chambers are given in Appendix C,
Table 4.
The normal Duluth, Minnesota, photoperiod was used during the brook trout
partial—chronic test instead of the Evansville, Indiana, photoperiod given in
Appendix B. Modified spawning substrates described by Benoit (1974) were
substituted for pans of loose gravel.
CHEMICAL SYSTEMS
Technical grade diazinon (92,5% purity) was introduced by diluter—
operated syringe injectors in all tests except the brook trout partial chronic.
The brook trout partial chronic utilized a Mariotte bottle and metering device
similar to that described by Mount and Brungs (l967) Stock solutions of
diazinon were dissolved in acetone. n all tests except those with bluegills,
Triton X—100 was added as a surfactant at 3% of the diazinon concentration.
Therefore, within individual test systems the nominal concentrations of
Triton X—100 and acetone were directly proportional to nominal diazinon
concentrations in that system. The maximum nominal amount of acetone
concurrent with the highest concentration of diazinon was 24 mg/i in the
acute tests, less than 2 rng/l in the fathead minnow chronic test, and less
than 0.1 mg/i in the brook trout partial—chronic test.
Water concentrations of diazinon were measured three to six times
during each 96—hr acute toxicity test (Appendix C, Table l) During the
chronic, preliminary, and partial—chronic tests alternate chambers from each
5
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treatment level were measured at least once a week whenever fish were
present. The concentrations in larval chambers were measured separately
(Appendix C, Tables 5 and 6).
All determinations for diazinon concentration were made by gas
chromatography (GC) and electron capture detector (sensitivity limit = 50
picograms). At least one water sample in each set was run in duplicate,
and spiked control water was used to monitor recovery rate.
The methods used to determine diazinon residues in brook trout tissues
are outlined in Appendix D.
BIOLOGICAL SYSTEMS
Acute Toxicity Tests
Species, sources, ages, and numbers of fishes used are presented in
Appendix C, Table 1
Fathead Minnow Chronic Toxicity Tests
Fifty 4—day—old fathead minnows from laboratory stock were randomly
assigned to each test chamber on September 14, 1971. Survivors were
counted and measured after 30 and 60 days by the photographic method of
McKim and Benoit (l97l) All chambers were randomly thinned to 15 fish
at 61 days. After 91 days of exposure it was decided that all diazinon
concentrations (1,100 — 69 pg/i) were too high, and the test was terminated.
A second test was begun on January 5, 1972, with 50 5—day—old larvae
per chamber and average measured concentrations of diazinon between 6O 3
and 3.2 pg/i. Counts and lengths of survivors were determined by photography
at 31, 64, and 97 days 0 Random thinning of each test chamber to 15 fish
was delayed until the 167th day of exposure because larger numbers were
desired to measure the development of scoliosis 0 Five spawning substrates
were placed in each test chamber at thinning. As there appeared to be no
subsequent competition between males for spawning territories, no additional
fish were removed. Adult exposures were terminated after 274 days when no
spawning was observed in any group for one week. Larvae from the initial
hatches were exposed 30 days and then replaced by newly hatched larvae
which were exposed for 60 days 0
Brook Trout Preliminary Test
Beginning, January 2, 1973, duplicate 2—month exposures of six yearling
brook trout were conducted at average measured concentrations of 56, ii, 2 6
and 0 pg/i. These fish were taken from the same stock reserved for the
partial—chronic test, and results from the preliminary exposures were used
to determine concentrations to be used in the partial chronic 0
Brook Trout Partial—Chronic Toxicity Test
Yearling brook trout were obtained from a commercial hatchery and
acclitninated 4 months to laboratory conditions 0 Twelve fish were randomly
6
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assigned to each adult exposure chamber on April 3, 1973. Lengths and
weights of individual fish anethetized with Ethyl—m—Aminobenzoate Methanosul—
fonate were taken at the start of the test and after 91 and 173 days. At 173
days each exposure chamber was thinned to a nominal of two males and four
females. Only fish that appeared healthy, undeformed, and mature were kept. Two
spawning substrates were placed in each adult exposure chamber at this time
(September 24, 1973).
All spawnings of 10 or more eggs were incubated at least 11 days to
determine viability. Samples of 50 eggs from spawnings of this size or larger
were incubated until completion of hatch or the death of all eggs. Larvae
were inventoried by photograph 2 days after completion of hatch, randomly
thinned to 25, and returned to their hatching containers. Feeding was initiated
25 days after the median hatch date. Thirty days after the median hatch date
larvae were inventoried by photograph and transferred to available larval
chambers. Excess groups of larvae were killed and inventoried at this age.
Fish reared in the larval chambers were photographed at 60 and 90 days. Sur-
vivors were killed and inventoried 122 days after hatch.
STATISTICAL ANALYSIS
The 96—hr LC5O’s for the acute toxicity tests were derived by the method
described by Litchfield and Wilcoxon (1949).
Data from the chronic and partial—chronic toxicity tests were subjected
to one—way analysis of variance (P = 0.05) and Dunnett’s procedure for
comparison of treatment means to control mean, P = 0.05 (Steel and Torrie,
1960). Percentage data (e.g. survival) were transformed to arc sin prior
to analysis. Other statistical analyses were used as noted in the section on
results.
7
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SECTION 5
RESULTS
ACUTE TOXICITY TESTS
Bluegills were most sensitive to acutely toxic concentrations of diazinon.
They were followed in order by brook trout, flagfish, and fathead minnows.
Results of individual tests and average 96—hr LC5O’s are presented in Table 1.
FATHEAD MINNOW CHRONIC TOXICITY TESTS
Except for two apparently random occurrences, fathead minnows exposed to
diazinon at average concentrations ranging trom 69 to 1,100 g/l had higher
survival than either control during the first 30 days. During the next 31
days there was some evidence of toxicant—related :deaths, but the differences
were not statistically significant. Growth measurements at 30 and bl days
showed a non—significant concentration—related decrease in average length of
survivors. However, analysis of variance of the instantaneous growth rates
( [ in (iength 2 /length 1 )]idays) between 30 and 61 days demonstrated a signi-
ficant decrease for fish exposed to 229 i ig/l. and above (Table 2). After 13
weeks the incidence of scoliosis in diazinon—exposed groups ranged from 60 to
88% versus 7% in the controls (Table 3). The first test was terminated at
this time.
During the second fathead minnow chronic test the fish were exposed to a
series of average concentrations between 3.2 and 60.3 .ig/1. Survival as
lower in the controls than in treated groups during the first 167 days of the
test. There was a non—significant increase in mortality rate for fish exposed
to 60.3 i tg/l. between 97 and 167 days (Table 4). During the remainder of the
test (107 days), which encompassed spawning, adult fathead minnows exposed to
60.3 pg/l. suffered 50% mortality compared to 7% in the control. Mortality
in lower concentrations did not differ significantly from that in the control
during this period.
There were indications of diazinon—related inhibition of growth between
64 and 97 days (Table 4) , but none of the growth data for exposed parental
fish were significantly different from that of the control throughout the
entire second test.
The incidence of scoliosis followed the same pattern observed in the
first test and generally declined with decreasing concentrations (Table 3).
Although the incidence of deformed fish in the cOntrols during the
second test was higher, analysis of variance indicated significant differences
between the controls and parental fish exposed to all concentrations except
6.9 tg/1.
8
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TABLE 1. ACUTE TOXICITIES OF DIAZINON TO FATHEAD MINNOWS
( PINEPH.ALES PRONELAS) , BLUEGILLS ( I PoMIS MACROCHIRUS) ,
BROOK TROUT ( SALVELINUS FONTINALIS) , AND FLAGFISH
( JORDANELLA FLORIDAE )
Species
Test
96—hr
(mg
LCSOa
/1.)
Slope
Average
96-hr LC5O
(mg/i.)
Fathead minnows 1 6.8 1.8
(5.4 - 8.5)
2 6.6 1.7
(5.1 — 8.6)
3 10.0 2.4 7.8
(6.7 - 15.0)
Bluegilis 1 D. 48 2.2
(0.34 — 0.67)
2 0.44 1.9
(0.31 — 0.62)
Brook trout 1 0.80 1.8
(0.44 — 1.14)
2 0.45 2.1
(0.32 — 0.63)
3 1.05 2.5 0.77
(0.72 — 1.52)
F lagfish 1 1.5 2.2
(1.2 - 1.9)
2 1.8 i.6
(1.6 — 2.0)
aCll d by the method of Litc1hfie] d. and Wilcoxon (1949).
The 95% confidence interval is given in parenthesis.
9
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TABLE 2. SURVIVAL AND GROWTH OF PARENTAL STOCKa OF FATHEAD MINNOWS ( PINEPHALES PRONELAS ) CONTINUOUSLY
EXPOSED TO DIAZINON AFTER 30 AND 61 DAYS (TEST #1)
It n
Measured diazinon concentration in water ( g/1.)
1.100
511
229
118
69
Cont
rol
A
B
A
B
A B
30 days
A
B
A
B
A
B
Survival (%)b
70
78
814
80
146
82
714
0 C
82
70
58
36
Average total
length (mm)
8.6
8.7
10.14
10.7
10.9
11.9
11.2
—
11.5
11.3
11.9
10.3
(Standard
deviation)
(1.6)
(1.3)
(2.1)
(1.9)
(1.9)
(2.0)
(1.6)
—
(2.0)
(2.0)
(2.0)
(2.0)
61 days
Survival (%)b
38
8
68
66
l li
70
62
0’
60
142
148
28
Average total
length (mm)
11.9
12.6
114.9
114.8
16.1
16.5
17.7
—
19.14
19.7
19.9
19.7
(Standard
deviation)
(1.8)
(1.9)
(2.6)
(2.14)
(2.14)
(3.2)
(2.6)
—
(3.14)
(3.14)
(3.7)
(3.14)
Instantaneousd
growth rate
105
119
116
105
126
105
147
169
179
166
209
x 10,000
112*
110*
116*
—
174
188
aThIS test discontinued before spawning because of high incidence of scoliosis at all diazinon concentratic’ns.
bEach group started with fifty 4—day—old larvae.
of the larvae in this test chamber died suddenly 2 weeks after start of test from unexplained cause.
*
I- . .
0
days
Significantly different from control (P 0.05).
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TABLE 3. INCIDENCE OF SCOLIOSIS IN PARENTAL FATHEAD MINNOWS ( PINEPHALES PROMELAS ) CONTINUOUSLY
EXPOSED TO DIAZINON AFTER HATCH (TESTS #1 AND #2)
Item
Average measured_diazinon_concentration in water (ugh.)
1.100
511
229
ii8
69
Control
60.3
28.0
13.5
6.9
3.2
Control
Total number of
fish
Scoliosis at 13
weeks ( 7 )a
Scoliosis at 19
weeEs (%)b
Scoliosis at 2 4
weeEs (%)C
23
b3*
2 1 u
bb
29
b6*
21
u3*
30
6o*
30
7
63
61 *
10*
11
*
51 *
81
4Q *
41 *
5 4
26
26
6
29
314*
1 8
19
21
agesults at termination. Each group had previously been thinned randomly to 30 fish.
bhesmltS for survivors of 100 fish per group at start of test.
CR1t include deaths between 19 and 24 weeks..
*
Significantly different from control (P — 0.05).
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TABLE 4. SURVIVAL AND GROWTH OF PARENTAL STOCK OF FATHEAD NINNOWS ( PrMEPHALES PROMELAS ) CONTINUOUSLY
EXPOSED TO DIAZINON AFTER 31, 64, 97, 135, 167, AND 274 DAYS (TEST #2)
Item
Average
“ easured diazinon corentratiori in water ( g 1.)
13.5 6.9 3.2 Control
A 1 3 A B A B A B
31 dayr
60.3
28.0
A B
A
B
Survival
68 3 31 84
83 86
9? 66 86 80 76 70 72
Average total
length (nun)
13.9 10.2 14.6
14.5 15.2
14.5 114.4 10.0 13.9 114.2 13.7 12.9
(Standard deviation)
(1.7) (1.7) (1.8)
(1.7) (1.14)
(1.8) (2.2) (2.3) (2.7) (2.2) (2.6) (2,8)
614 days
Survival (9)”
(31
112
80
80
(34
93)
62
74
72
68
58
60
Average total
length (inn)
19.6
20.1
22.)
22.0
22.2
21.1
21.5
1(1,6
20.1
19.7
19.0
19.0
(Standard deviatIon)
(2.9)
(3.6)
(14.1)
(3.2)
(3.5)
(14.2)
(5.1)
(1.8)
(4.9)
(3.5)
( .3)
(6,4)
Instantaneous
growth rate 1 ’
X 10,000
00
05
134
23 i
115
II I
121
86
112
99
99
117
Continued On page 13
-------
TABLE 4 (Continued). SURVIVAL AND GROWTH OF PARENTAL STOCK OF FATHEAD NINNOWS ( PINEPHALES PRONELAS )
CONTINUOUSLY EXPOSED TO DIAZINON AFTER 31, 64, 97,
135, 167, AND 274 DAYS (TEST #2)
Item
Average measured diazinon conce”ration in water_(pg/U)
60.3
28.0
13.5
6.9
3.2
Control
A B
A B
A B
A B
A B
A B
97 day
jurvival ( 5 )a
72
78
10
7 ?
02
90
58
66
68
68
52
52
Average total
length (mm)
2 5.
25.2
20.8
27.1
21.14
25.6
26.6
24.1
26.2
25.5
25.7
25.8
(Itandard deviation)
(5.5)
(6.5)
(6.9)
(6.2)
(5.3)
(6.9)
(7.6)
(7.5)
(7.1)
(5.8)
(6.2)
(8.6)
Instantaneous B
growth rate
64
69
72
63
64
59
65
79
80
66
92
81
x 10,000
jurvival ( )°
135 dayls
Eu 76 ‘ 2 70 76 86 56 52 65 66 50 56
durvival ( )5
161 days
50 65 70 62 71 02 56 50 60 66 50 56
107 uays—274 days
40 60 80 80 93 93 87 80 [ 00 100 93 93
5grViV . C
I I I I I
5Q* 30 93 83 100 93
°Each group started with dfty
b ( length 2 /length 1 )
days
5—day—old larvae.
CEach group randomly thinned to 15 fish at 167 days.
*
Significantly different from control (1’ 0.05).
-------
No spawning was observed in 60.3 pg/i, and spawning was very limited in
28.0, 13.5, and 6.9 pg/i. However, observed spawning in one of the duplicate
control groups was limited to ii eggs, although egg production from the other
control duplicate was roughly equivalent to the total egg production from
both groups exposed to 3.2 jig/i (Table 5). More than 500 eggs from the 13.5
pg/i concentration were incubated, but all died within 2 days. Three egg
samples (50 eggs each) from 6.9 pg/i had a hatch success of 76%. Thirty egg
samples from 3.2 pg/i had an average hatch success of 65% versus 92% for 20
samples from the one control group that spawned regularly. It was observed
that hatch success was very variable in 3.2 pg/i and quite uniform in the
control. Bartlett’s test for homogeneity of variance indicated a significant
difference (Snedecor, 1956; Steel and Torrie, 1960). Application of Cochran
and Cox’s approximate test for unpaired observations and unequal sample
variances indicated that the average hatch success in 3.2 pg/i was
significantly lower than that in the control (Snedecor, 1956; Steel and
Torrie, 1960). Hatch success of paired egg samples from parents exposed to
3.2 pg/i was not improved by incubation in control water.
Hatch success of control eggs was not affected by being transferred to
either 60.3 or 28.0 pg/i shortly after fertilization (Table 5). Diazinon
had no apparent effect on incubation time between fertilization and hatch
The growth and survival of progeny up to 60 days after hatch were not
affected by 3.3 pg/i (3.2 pg/i as eggs incubated in adult chambers). The
one group of progeny reared at 6.8 pg/i had lower survival but greater
growth than the controi (Table 6). Control progeny transferred to 62.6 and
28.0 pg/i at spawning and reared at these concentrations for 60 days after
hatch weighed less than controls and had an average survival rate only two—
thirds that of the control. There was no evidence of scoliosis in the
progeny after 2 months of exposure to diazinon.
BROOK TROUT PRELIMINARY TEST
In 56 pg/i diazinon brook trout showed signs of distress (lethargy and
loss of equilibrium) within 1 week and ceased feeding by the second week. The
same symptoms were observed after 3 weeks in 11 pg/i. Both groups began eating
in the second month of exposure, but tetanic convulsions were common when the
fish were disturbed. One fish died in 56 pg/i during the 2—month exposure.
None of the trout developed permanent deformities, but temporary flexing of
the spine resembling scoliosis and lordosis was observed. No distress was
observed in 2.6 pg/i.
BROOK TROUT PARTIAL-CHRONIC TOXICITY TEST
Reduced feeding, lethargy when undistrubed, and hyperactivity followed by
tetanic convulsions when disturbed were common symptoms at exposures of 9 6
and 4.5 pg/I after 1 and 4 weeks, respectively. Some tetany was observed
in 2.4 pg/i during the second and third month. Fish from these three concen-
trations were inactivated very rapidly by Ethyi—m—Aminobenzoate Methanesulfonate
used as an anesthetic during inventory at 91 days. Between the third and sixth
month none of these symptoms were observed, and even those fish with severe
spinal deformities began to eat normally.
14
-------
TABLE 5. SPAWNING AND HATCHABILITY OF EGGS FROM FATHEAD MINNOWS ( PIMEPHALES PROMELAS ) CONTINUOUSLY
EXPOSED TO DIAZINON (TEST #2)
Item
Average me
‘ ured
di ‘‘jnco concentration to water (pg/ i.)
13. 6.9 3.2
B A
60.3
28.0
A
B
A
B
A
B
A
Number of’ mature
females at
termination 1
lumber of spawn ago
Total number of eggs A
Eggs/spawning -
Eggs/female 0
Hat000bliitya 93 b
(0 rundard dcvi ation) (5.0)
(A) (IA)
6
7
B
- I
0
0
0
896
6
0
0
0
936
(4.1))
(9)
5 1 2 3 5 7
5 3 0 02 25
767 0 551 0 3,630 2,000
153 — 110 — 86 83
153 0 275 0 726 285
— ‘r6 — 68 6i
65*
— — (8.0) — (73.5) (19.5)
: J ±1L
8
53
5,767
109
720
92
(4.3> (6.6)
6
11
11
2
9
(7.0) —
(22> —
Continues Ofl pare 16.
-------
0 ’
TABLE 5 (Continued). SPAWNING AND HATCHABILITY OF EGGS FROM FATHEAD MINNOWS
( PIMEPHALES PROMELAS ) CONTINUOUSLY EXPOSED TO DIAZINON (TEST #2)
al tchability samples contained 50 r,ggs.
bliatellability of eggs transferred from r-t,uitrol A at spawning.
C_Includes deaths dcrlmf spawsiog period. t i-h prose vtcrtrd with 15 fish.
*
Significantla different from control (P — 0.05) usIng approximate test for
unequal simple variance.
It n
Average
measured diasinon con
ntratiOfl ix water (ia/l.)
6.9 3.2
Control
60.3 28.0
A B A B
l3.
A B
A B
A j B
A B
atimated larvae/
famale
Mature males
Maturefasales
Mature males and
females U.)
reproductive potential.
0 1 0 84 304 380
Gocad 1 develo nemt
3 1 5 5 5 7 3 1 6
1 7 1 7 5 7 2 3 5 7 8 6
27 60 93 73 67 7 1 i7 53 80 67 80 30
-------
-4
TABLE 6. SURVIVAL AND GROWTH AFTER 30 AND 60 DAYS FOR PROGENY OF FATHEAD NINNOWS
( PINEPHALES PROMELAS ) CONTINUOUSLY EXPOSED TO DIAZINON (TEST #2)
Item
Average measured rliazinon rowentratic,s in water
(i’gJ .)
62.6
28.0 6.8
— A B A 4 A
30 Days
3.3
B A
Control
A B
B
Survival ( )
33 a
19 a
5 5S
18a
,
78
70
60
73 a
(Standard deviation)
(13)
(8)
(42)
(9)
—
(10)
(18)
(19)
(114)
Average total length
( I B m)
13 a
ii6
l3.2 ’
11 a
16.0
—
13.0
13.0
03.7
1 - 4 a
(Standard error of mean)
(0.5)
(1.2)
(1.0)
(0.4)
—
(0.4)
(0.6)
(0.2)
(0.4)
)A)b
(4)
(4)
(3)
(4)
( 1)
(4)
(4)
(6)
(4)
Average weight (mg)
28 a
40 a
iha
13 a
22
18
28
ham
(Standard error of mean)
(3)
)i)
(3)
(5)
(2)
(2)
(6)
( 8 )b
(2)
(2)
(1)
(2)
(2)
(2)
(4)
(2)
continued on page 18.
-------
TABLE 6 (ContInued). SURVIVAL AND GR,OWTU AFTER, 30 AND 60 DAYS FOR PROGENY OF FATHEAD MINNOWS
( PIMEPHALES PRONELAS CONTINUOUSLY EXPOSED TO DIAZINON (TEST #2)
lt n
Average measure
diazjnon coricentrati
i in water (ugh.)
62.6
28.0
6.8
3.3
Control
A B
A B
A B
A B
A B
60
Days
Survival (%)
4j
45 5
4 4 a
52
—
59
74
61
72 a
(Standard deviation)
(i)
(6)
—
(6)
—
(1)
(23)
(16)
(15)
Average total length
(m i s)
l9.l
l80
219°
;T.3
26.7
—
19.4
21.9
20.8
22 1 a
(Standard error of mean)
(0.1)
(1.9)
—
(1.3)
—
(0.2)
(0.6)
(1.5)
(0.3)
Average weight mg)
92 a
80°
ii6
63
259
—
102
131
119
134 a
(Standard error of mean)
(N)b
(8)
(2)
(21)
(2)
—
(1)
(j 7)
(2)
(1)
(4)
(2)
(4)
(2)
(18)
(2)
(2)
(2)
a gg 5 transferred from ontro1 A at spawning.
bNumber of larval groups initially composed of 40 lurvue from single hutch of eggs.
-------
During the first 173 days mortality in 9.6, 4.8, and 2.4 pg/i was 25%,
4%, and 4%, respectively (Table 7). The first death was observed (in 9.6
pg/i) after 24 days, but more than half of the deaths occurred between 163
and 173 days. Although all fish retained at thinning were apparently healthy,
there was an additional 8% mortality during the spawning period in both 9.6
and 4.8 pg/i.
During the first 91 days brook trout exposed to 9.6 pg/i lost 4% of
their initial weight 0 Fish in 4.8 pg/i gained 12%. Weight gain in lower
concentrations and the control ranged from 39% to 44% (Table 7). Reduced
growth in the higher concentrations was a temporary phenomenon. During the
next 82 days percentage gain in weight was equivalent in all concentrations
except one sublot in 9.6 pg/i. This latter group also gained weight but at
about one—half the general rate. Analysis of variance of instantaneous growth
rates indicated significant inhibition of growth in 9.6 and 4.8 Pg/i only
during the first 91 days. Irregular feeding and shedding of eggs during
spawning precluded growth—rate analyses after 173 days.
Some of the brook trout exposed to 9.6 and 4.8 pg/i developed incipient
scoiiosis and lordosis within a few weeks. After 173 days the incidence
of spinal deformities was 33% and 12%,. respectively, at these concentrations
(Table 7). Although all deformed fish were discarued at thinning, one tish
in 4.8 pg/i subsequently developed iordosis.
Incorrect sex ratios and high within—treatment variance in egg viability
prevented any accurate analysis of egg production, egg viability, or hatch.
There was no indication that diazinon adversely affected these indices of
reproduction (Table 8). The incubation time of eggs was not affected by
diazinon. All females in the control and lower concentrations spawned, but
both the 9.6 and 4.8 pg/i concentrations had one female with immature ovaries
20 days after all other females had ceased spawning and another female in 9.6
pg/i had not spawned by this time although her body cavity was filled with
loose eggs.
There was no correlation between diazinon concentration and incidence
of embryonic deformity or survival of progeny from hatch to 122 days (Table 9).
The average total length of larvae from parents exposed to 9.6 pg/i
(eggs exposed to 11.1 pg/i) was significantly less than that of control
larvae two days after hatch. After 122 days all progeny groups from parents
exposed to diazinon were significantly smaller than the controls. Analysis
of variance of instantaneous growth rate to 122 days indicated that growth was
significantly inhibited in all treated groups except those reared in 2.7 pg/i
(Table 9). Cross transfers of paired egg samples between the control and
11.1 pg/i indicated that the growth of progeny during the larval stage was
not affected by the presence or absence of diazinon. Progeny from parents
exposed to diazinon showed no increase in growth when reared to 30 days after
hatch in control water. Exposure of progeny from control parents to 11.1 pg/i
for 122 days after hatch caused no decrease in growth.
19
-------
0
TABLE 7. SURVIVAL, GROWTH, AND INCIDENCE OF SCOLIOSIS AND LORDOSIS FOR PARENTAL BROOK TROUT
( SALVELINUS FONTINALIS ) CONTINUOUSLY EXPOSED TO DIAZINON FOR 91 AND 173 DAYS
t Ier.
Averag’ measured dianinon concentration in water (pg_/I.)
9.6 4.8 2.4 1.1 0.55
A A I I f A ji A 8
Control
B
Start
Average total length
(mm)
225 227 232 232 231
230 230 224 237 234 230 228
(Standard deviation)
(17) (13) (17) (18) (21)
(17) (21) (15) (23) (21) (19) (15)
Average weight (g)
112 121 126 128 124
123 121 112 135 129 120 138
(Standard deviation)
(21) (31) (34j (36) (37)
(29) (32) (28) (41) (39) (33) (23)
Survival (%)a
92 92
92*
100 100
100
100
91 Days
100 100 100 100 100 100 100
100 100 100 100
Average total length
(m m)
224
223
234
236
250
249
253
247
258
258
251
254
(Standard deviation)
(17)
(15)
(17)
(15)
(24)
(17)
(20)
(19)
(19)
(16)
(23)
(17)
Average weight (g)
108
116
138
146
180
173
175
168
192
189
177
180
(Standard deviation)a
(40)
(34)
(43)
(29)
(51)
(33)
(36)
(46)
(45)
(37)
(43)
(35)
Instantaneous 6
growth rate
X 10,000
—4
—
—5
a
10
12*
13
41
37
39
41
45
43
39
42
40
43
46
44
Scoliosis and/or
lordosis (2)
17
17
17
0
0
0
0
0
0
0
0
0
Cortinued on page 21.
-------
TABLE 7 (ContInued). SURVIVAL, GROWTH, AND INCIDENCE OF SCOLIOSIS AND LORDOSIS FOR PARENTAL BROOK TROUT
( SALVELINUS FONTINALIS ) CONTINUOUSLY EXPOSED TO DIAZINON FOR 91 AND 173 DAYS
aEOCh group started with
b 1 (weight 2 lweightj )
days
CLeniths unavailable for
*ltgmlficantly different
N.)
9.6
Item A B
Survival ( 1 )b 83 67
75*
Average total length
(mm) 236 C
(Standard deviation) (24) —
Average weight (g) 158 137
(Standard deviation) (85) (70)
Instantaneousb
growth rate 46 20
)( 10,000
Soliosis and/or
lordosis (1) 17 50
Avera
A B
92 100
96
262
— (17)
e Co’ .oc!a!4on wat!. control
— A B IA BL IJ A B
173 Days
92 100 100 100 100 100 100 100
96 100 100 100
289 272 293 283 288 296 287 284
(28) (42) (22) (30) (23) (18) (32) (26)
270 262 280 264 269 291 272 260
202
(81)
46
25
220
(49)
52
0
(89)
49
0
(70)
51
0
(59)
57
0
(98)
55
0
(73)
41
0
(54)
53
0
(109)
52
0
(79)
45
0
12 yearling fish.
some fish because of extreme deformities.
from control (P 0.05).
-------
TABLE 8. SPAWNING, VIABILITY, AND HATCHABILITY OF EGGS FROM BROOK TROUT ( SALVELINUS FONTINALIS )
CONTINUOUSLY EXPOSED TO DIAZINON FOR 6 — 8 MONTHS
Item
A
u.l)a
It
1 T
A
Avern c nc so re1 di as! cas a t rat, sri
1 • 1 ( 1 1 4)a
B A B A I B
O.55(0.80 ( a
A B
Control
A B
Number f females
spawning
1
1
1
2
1
2
1
3
1 4
1
2
I i
Total number of eggs
spawned
&67
215
5149
1,060
6142
,7 o
2,914 1
,893
,‘?, 678
1 ,0014
1 ,037
518
1,905
1476
Number eggs/female
b67
215
5149
530
321
865
982
63!
670
i,OOB
B
Viable spawns
1, 1
Number
5
0
1
1
3
7
3
3
387
Total number eggs
6147
—
-
253
268
868
2,338
728
855 957
3142
Number viable eggs
620
—
—
1142
33)
Sir
1,8149
6314
763 972
730
88
Percent viability
96
—
—
56
51
914
78
87
89 98
(Standard deviatIon)
(5)
-
-
(2)
(29)
(7)
(12) (a)
-
Percent hatchC
914
—
59
35
‘To
68
78
8 914
67
(6)
147
(Standard deviation)
(5)
—
—
—
-
(25)
(30)
(13)
(10)
Total
nuimbernsales
5
3
14
14
Gon im141 development
3 3
2 s
44
2
14 4
Total
percent mature males
60
66
50
50
50
25
66 100
100 440
25
100
Total
number females
1
3
2
2
2
2
3 3
14 t
2
44
‘rotal
percent nature
Temales
100
d
66
50
100
101
100
401) 10(1
100 101)
100
100
tt Values in purentliesi are coneentrati on at win ci eggs Were incubated f’s I lswi 51) syr Ws In’.
b; 5 11 vii I it canto! 5’ :’) me viable eggs.
CHatcB ,I ( i , y ‘imp i’ancl,;jine’nj 50 eggs tram cacti viable spawn
done rip 1 ,,: Ic nd not, spawn
-------
(A
TABLE 9. SURVIVAL AND GROWTH TO 122 DAYS FOR PROGENY OF BROOK TROUT ( SALVELINUS FONTINALIS )
CONTINUOUSLY EXPOSED TO DIAZINON
Item
A
11.1
B
A
5.6
1
Avcr 8 e meaoured diazinon concentration
2.7 1.1
0 A j B I I
in water
B
(p/fl)
A
0.80
Control
B A B
n
Average total length
(nm)
11.9*
15.1
15.2 5.5
15.2
15.8
(Standard error 01
mean)
(N)a
(0.1)
( )
_
(1)
(0.2) (0.3)
( I) (li)
(0.1)
( 5)
(0.3)
(4)
30 deys
Survival )))
105
—— ——
90 91
100 99
100 100
100 99
100
(Stanthoru deviation)
(0.0)
—-
—— ——
(0.0) (1.9)
(0.0) (0.0)
(0.0) (2.3)
(u)*
(3)
(0) (0)
(i) )i)
(3) (7)
(3) (2)
(3) ( )
(1)
Average total ien ;1h
(mm)
20.2*
10.6
22.8
21.8
21.2*
22.5
(Otmodard error of
mearO
( l4( *
(0.1)
( )
U)
(0.2)
(5)
(0.5)
( 6)
(0.1)
( 5)
(0.2)
(5)
I nstaat.çsroouu South
rate °
x1o,000
)9*
11
29
122
9
—
26
Corit flueS on page 24.
-------
TABLE 9 (Continued). SURVIVAL AND GROWTH TO 122 DAYS OR ‘ROGENY or BROOK TROUT ( SALVELINUS FONTINALIS )
CONTINUOUSLY EXPOSED TO DIAZINON
Average total length
(cr )
(Standard error of
mean)
(N (a
Instantaneous growth
rate b
x’o,ooo
55 .5*
(0.1.)
(3)
143*
1.69*
(0.05)
59.0*
(0.2)
(1.)
147
7 7 77 *
(0.01.)
55.2 *
(3.5)
(4)
I 38*
1.63*
(0.29)
55.1
(‘.3)
( I .)
140*
0.66*
(0.11)
155
a ndom samples 01 25 larvae (less when hatch was less than 50%) takes 2 days after hatch from isdividual hatchability samples of 50 eggs.
4 jL (lengths/].ength1 )
days
*Slgnificantly different from control (P 0.05).
Survival (%)
(Standard .leviation)
(N) a
83
(9.2)
(3)
122 daya
(0)
914
(0)
( I)
96
.0)
(3)
60
(5.i)
(2)
86
94
(2.8)
(2)
67
(15.1)
(3)
61
96
(1.0)
(3)
(1)
Average weight (g)
65.8
(0.8)
(1 .)
2.76
(0. IT)
-------
There was no evidence of scoliosis or lordosis among surviving progeny
122 days after hatch.
DIAZINON ACCUMULATION IN BROOK TROUT TISSUES
Accumulation data are presented in Appendix U, Table 1.
25
-------
SECTION 6
DISCUSSION
The purpose of this study was the determination of chronic “no—effect”
concentrations and subsequent calculation of “application factors” by
comparison with acute data.
Reductions in production rates were statistically significant for both
fathead minnows and brook trout at the lowest diazinon concentrations tested
(3.2 and 0.55 ig/l, respectively). Therefore, maximum acceptable toxicant
concentrations (MATC), as defined by Mount and Stephan (1967) cannot be
postulated for these species. However, subjective examination of the data
suggests that rough approximations of no—effect levels may be determined.
In the fathead minnow chronic test it appeared that effects that could
influence population success became progressively less as concentrations
decreased. Probably 3.2 j ig/l is not too much higher than the concentration
at which measurable effects would not be observed. The brook trout chronic
test produced mixed results. Concentrations of 4.8 ugh and above were
obviously detrimental to the fish, but the apparent toxicant—related
stunting of progeny was just as pronounced at 0.55 ug/l as at concentrations
as high as 9.6 i.ig/l. Therefore, it was not possible to estimate a
concentration at which all detrimental effects could no longer be measured,
but overall effects on population survival would probably be minimal below
0.55 ug/l. In summation, these chronic exposures indicated application
factors (MATC/96—hr LC5O) in the order of magnitude of 10 or lower for
both brook trout and fathead minnows. These values are about 2 to 3 orders
of magnitude lower than the actual or tentative application factors reported
for other pesticides tested against one or both of these species (Mount and
Stephan, 1967; Carison, 1972; Herinanutzetal., 1973; several unpublished
studies performed by or supervised by the Environmental Research Laboratory—
Duluth). On the other hand, a recent chronic diazinon exposure of flagfish
( Jordanella floridae ) by Allison (unpublished data) produced an application
factor with this species of the order of magnitude of 102. At this time
we cannot justify the application of a single factor to acute toxicity
results to estimate the chronic toxicity of diazinon to other species of fishes.
The generally higher early survival of fathead minnow larvae in the
presence of diazinon may have been an artifact resulting from the use of
acetone in the diazinon stock solutions. Increased growth of microorganisms
in the test chambers was correlated with the amounts of acetone introduced as
well as diazinon concentrations present. These organisms may have contributed
substantially to the food of larval fish during a period when a continuous
supply was needed.
26
-------
The negative results reported during brook trout spawning should be
viewed with some reservation. Inaccurate determination of sex at thinning
resulted in undesirable sex ratios in most groups during spawning. Reproductive
success in some concentrations was based on the productivity of only four
females rather than eight as planned. In addition, most of the spawnings
throughout the system were apparently not fertilized as no embryonic development
was observed in these eggs. A few spawnings with high viability rates occurred
in all concentrations usually early in the spawning period. It is believed
that some defect in the design of the modified spawning substrates was
responsible for this phenomenon as it was concurrently observed in other
tests in which this equipment was used in this laboratory and elsewhere.
Mortality and inhibition of ovarian development or spawning response
of female brook trout indicate that, under similar conditions approximately
one—quarter and one—half of the females in a population would make no
contribution to reproduction in 4.8 and 9.6 pg/i, respectively. However,
the small number of fish used in this test should be taken into consideration
if extrapolation to larger populations is attempted. The neurological
symptoms prevalent early in the test were not observed during the reproductive
period. If the initial exposure to diazinon had occurred shortly before the
spawning period, reproduction might have been inhibited to a greater extent
in some or all of the concentrations tested.
A number of investigators have reported spinal deformities in fishes
exposed to pesticides. MaCann and Jasper (1972) made an extensive study of
crippling in juvenile bluegills following acute exposure to several pesticides.
Fractures of the caudal vertebrae and localized hemorrhaging often occurred
within a few hours. Dislocation of the spine was permanent after the fish
were removed from the toxicant. Fathead minnows and brook trout in our
chronic exposures developed scoliosis and lordosis over periods of weeks or
months. Individual response was variable and ranged from small kinks in the
caudal peduncle to extensive spinal displacement approaching 1800. The acutal
effects observed in fathead minnows were greater than the reported data
indicate. In diazinon—exposed fish the majority of these deformities were
gross, whereas most deformities in the controls were relatively minor and
did not appear to affect swimming ability. No spinal deformities were observed
in progeny of either species although these fish were exposed for relatively
long periods to concentrations that produced scoiiosis and lordosis in the
parental stock. It seemed possible that genetic adaptation was involved as
abnormal brook trout were culled before spawning, perhaps leaving more
resistant fish. However, progeny from unexposed parents in the control
groups also failed to develop spinal deformities when reared in the presence
of diazinon. This appears to preclude selection of resistant progeny as the
reason for this phenomenon. Brook trout used in the preliminary test did not
develop permanent deformities although the highest concentration was almost
six times the maximum used in the chronic test. Fish used in the preliminary
test were approximately 4 months younger, which may indicate that age has some
bearing on this syndrome.
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Freshwater fish populations could be directly damaged by prolonged
exposure to diazinon at concentrations several thousand times lower than
those causing acute mortality. Diazinon may persist many months in some
freshwater environments although comprehensive field data are not available.
There is evidence that the introduction of diazinon into the aquatic
environment and its subsequent persistence are affected by a great number
of poorly understood physical, chemical, and biological factors. Uniform
concentrations of an organophosphate insecticide, as employed in these
tests, could probably occur only in exceptional circumstances (such as point
discharge from a manufacturing operation). Further studies investigating
the chronic effects of fluctuating and intermittent exposures of fishes and
invertebrates to diazinon and other organophosphates are needed to aid in the
establishment of environmentally safe concentrations for these “non—persistent”
pesticides.
28
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REFERENCES
American Public Health Association. 1971. Standard methods for the
examination of water and wastewater. 13th ed. American Public
Health Association, New York, NY. 874 p.
Anonymous. 1972. Concentrates. Chem. Eng. News. 50:7.
Bartsch, E. 1974. Diazinon. II. Residues in plants, soil, and water.
Residue Rev. 51: 37—68.
Benoit, D. A. 1974. Artificial laboratory spawning substrates for brook trout
( Salvelinus fontinalis Nitchill). Trans. Am. Fish. Soc. 103: 144—145.
Carison, A. R. 1972. Effects of long—term exposure to carbaryl (Sevin) on
survival, growth, and reproduction of the fathead minnow ( Pimephales
promelas) . J. Fish. Res. Board Can. 29: 583—587.
Coppage, D. L. 1972. Organophosphate pesticides: Specific level of brain
AChE inhibition related to death in sheepshead minnows. Trans. Am. Fish.
Soc. 101: 534—536.
Cowart, R. P., F. L. onner, and E. A. Epps. 1971. Rate of hydrolysis of
seven organophosphate pesticides. Bull. Environ. Contam. Toxicol. 6:
23 1—234.
Gomaa, H. N., I. H. Suffet, and S. D. Faust. 1969. Kinetics of hydrolysis of
diazinon and diazoxon. Residue Rev. 29: 171—190.
Hermanutz, R. 0., L. H. Mueller, and K. D. Kempfert. 1973. Captan toxicity
to fathead minnows ( Pimephales promelas) , bluegills ( Lepomis macrochirus) ,
and brook trout ( Salvelinus fontinalis) . J 0 Fish. Res. Board Can.
30: 1811—1817.
Knutson, H.., A. M. Kadoum, T. L. Hopkins, G. F. Swoyer, and T. L. Harvey.
1971. Insecticide usage and residues in a newly developed Great Plains
irrigation district. Pesticides in Soil. 5: 17—27.
Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified method of
evaluating dose—effect experiments. J. Pharm. Exp. Ther. 97: 99—113.
McCann, J. A., and R. L. Jasper. 1972. Vertebral damage to bluegills exposed
to acutely toxic levels of pesticides. Trans. Am. Fish. Soc. 101:
3 17—322.
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McKim, J. M. and D. A. Benoit. 1971. Effects of long—term exposures to copper
on survival, reproduction, and growth of brook trout ( Salvelinus fontinalis
Mitchill). J. Fish. Res. Board Can. 28: 655—662.
Meister Publishing Co., Farm chemicals handbook. 1974. Willoughby, OH 484 p.
Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus for fish
toxicology studies 0 Water Res. 1:
Mount, D. I., and C. E. Stephan. 1967. A method of establishing acceptable
toxicant limits for fish — malathion and the butoxyethanol ester of
2,4—D. Trans. Am 0 Fish. Soc. 96: 185—193.
Munson, J. D. 1970. Insecticidal control of corn rootworm larvae in
Nebraska. Dissertation Abs. 31: 2040—B.
Snedecor. G. W. 1956. Statistical Methods, 5th ed 0 Iowa State Univ.
Press, Ames, IA. 534 p.
Sethunathan, N., and M. D. Pathak. 1972. Increased biological hydrolysis
of diazinon after repeated application in rice paddies. J. Agr. Food
Chem. 20: 586—589.
Steel, R. G. D., and J. H. Torrie. 1960. Principles and Procedures of
Statistics. McGraw—Hill Book Co., Inc., New York, NY. 481 p.
Suett, D. L. 1971. Persistence and degradation of chlorfenvinphos, diazinon,
fonofos, and phorate in soils and their uptake by carrots. Pest. Sci.
2: 105—112.
Weiss, C. M. 1961. Physiological effects of organic phosphorous insecticides
on several species of fish. Trans. Am. Fish. Soc. 90: 143—152.
30
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APPENDICES
Page
A Recommended Bioassay Procedures for Fathead Minnow Pimephales
p romelas Rafinesque Chronic Tests 32
B Recommended Bioassay Procedure for Brook Trout Salvelinus
fontinalls (Mitchill) Partial Chronic Tests 47
C Test Conditions During Acute and Chronic Exposures (six tables) . . 61
D Accumulation of Diazinon in Tissues of Adult Brook Trout 67
31
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APPENDIX A
RECOMMENDED BIOASSAY PROCEDURE FOR
FATHEAD MINNOW PIMEPHALES PROMELAS RAFINESQUE CHRONIC TESTS
by
Environmental Research Laboratory—Duluth
(formerly National Water Quality Laboratory)
6201 Congdon Boulevard
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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RECO €NDED BIOASSAY PR(EEDURES
Preface
Recommended Bioassay Procedures are established by the approval of both
the Committee on Aquatic Bioassays and the Director of the National
Water Quality Laboratory. The main reasons for establishing them are:
(i) to permit direct comparison of test results, (2) to encourage
the use of the best procedures available, and (3) to encourage
uniformity. These procedures should be used by National Water Quality
Laboratory personnel whenever possible, unless there is a good reason
for using some other procedure.
Recommended Bioassay Procedures consider the basic elements that are
believed to be important in obtaining reliable and reproducible
results in laboratory bioassays. An attempt has been made to adopt
the best acceptable procedures based on current evidence and opinion,
although it is recognized that alternative procedures may be adequate.
Improvements in the procedures are being considered and tested, and
revisions wiU be made when necessary. Comments and suggestions are
encouraged.
Director, National Water Quality Lab, (I MQL)
Committee on Aquatic Bioassays, MQL
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Recommended Bioassay Procedure for
Fathead Minnow Pimephales promelas Rafinesque Chronic Tests
April, 1971
(Revised January, 1972)
A. Physical system
1. Diluter : Proportional diluters (Mount and Brungs, 1967) should
be employed for all long—term exposures. Check the operation
of the diluter daily, either directly or through
measurement of toxicant concentrations. A minimum of five
toxicant concentrations and one control should be used for
each test with a dilution factor of not less than 0.30. An
automatically triggered emergency aeration and alarm system
must be installed to alert staff in case of diluter, temperature
control or water supply failure.
2. Toxicant mixing : A container to promote mixing of toxicant
bearing and w—cell water should be used between diluter and
tanks for each concentration. Separate delivery tubes
should run from this container to each duplicate tank.
Check at least once every month to see that the intended
amounts of water are going to each duplicate tank or chamber.
3. Tank : Two arrangements of test tanks (glass, or stainless
steel with glass ends) can be utilized:
a. Duplicate spawning tanks measuring 1 x 1 x 3 ft. long
with a one sq. ft. portion at one end screened off
and divided in half for the progeny. Test water is
to be delivered separately to the larval and spawning
chambers of each tank, with about one—third the water
volume going to the former chamber as to the latter.
b. Duplicate spawning tanks measuring 1 x 1 x 2 ft. long
with a separate duplicate progeny tank for each
spawning tank. The larval tank for each spawning
tank should be a minimum of 1 Cu. ft. dimensionally
and divided to form two separate larval chambers with
separate standpipes, or separate 1/2 sq. ft. tanks
may be used. Test water is to be supplied by delivery
tubes from the mixing cells described in Step 2 above.
Test water depth in tanks and chambers for both a & b
above should be 6 inches.
4. Flow rate : The flow rate to each chamber (larval or adult)
should be equal to 6 to 10 tank volumes/24 hr.
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5. Aeration : Total dissolved oxygen levels should never be allowed
to drop below 60% of saturation, and flow rates must be increased
if oxygen levels do drop below 60%. As a first alternative flow
rates can be increased above those specified in A.4. Only
aerate (with oil free air) if testing a non—volatile toxic agent,
and then as a last resort to maintain dissolved oxygen at 60%
of saturation.
6. Cleaning : All adult tanks, and larvae tanks and chambers after
larvae swim—up, must be siphoned a minimum of 2 times weekly
and brushed or scraped when algal or fungus growth becomes
excessive.
7. Spawning substrate : Use spawning substrates made from inverted
cement and asbestos halved, 3—inch ID drain tile, or the equiva-
lent, each of these being 3 inches long.
8. Egg incubation cups are made from either 3—inch
sections of 2—inch OD Ci 1/2—inch ID) polyethylene water hose
or 4—oz., 2—inch OD round glass jars with the bottoms cut off.
One end of the jar or hose sections is covered with stainless
steel or nylon screen (with a minimum of 40 meshes per inch).
Cups are oscillated in the test water by means of a rocker arm
apparatus driven by a 2 r.p.m. electric motor (Mount, 1968).
The vertical—travel distance of the eips should be 1 to 1 1/2
inches.
9. Light : The lights used should simulate sunlight as nearly as
possible. A combination of Durotest (Optima PS) 1 ’ 2 and wide
spectrum Grow—lux 3 fluorescent tubes has proved satisfactory at
the NWQL.
10. Photoperiod : The photoperiods to be used (Appendix A) simulate
the dawn to dusk times of Evansville, Indiana. Adjustments in
day—length are to be made on the first and fifteenth day of
every Evansville test month. The table is arranged so that
adjustments need be made only in the dusk times. Regardless
of the actual date that the experiment is started, the Evansville
test photoperiod should be adjusted so that the mean or estimated
hatching date of the fish used to start the experiment corresponds
to the Evansville test day—length for December first. Also,
the dawn and dusk times listed in the table need not correspond
to the actual times where the experiment is being conducted. To
illustrate these points, an experiment started with 5—day—old
larvae in Duluth, Minnesota, on August 28 (actual date), would
require use of a December 5 Evansville test photoperiod, and
the lights could go on anytime on that day just so long as they
remained on for 10 hours and 45 minutes. Ten days later (Sept. 7
actual date, Dec. 15 Evansville test date) the day—length
Mention of trade-names does not constitute endorsement
Duro—Test, Inc., } aminond, md.
3 Sylvania, Inc., New York, NY
35
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would be changed to 10 hours and 30 minutes. Gradual changes
in light intensity at dawn and dusk (Drununond and Dawson, 1970),
if desired, should be included within the day—lengths shown,
and should not last for more than 1/2 hour from full on to full
off and vice versa.
11. Temperature : Temperature should not deviate instantaneously
from 25° C by more than 2° C and should not remain outside the
range of 24 to 26° C for more than 48 hours at a time. Temperature
should be recorded continuously.
12. Disturbance : Adults and larvae should be shielded from
disturbances such as people continually walking past the
chambers, or from extraneous lights that might alter the
intended photoperiod.
13. Construction materials : Construction materials which contact
the diluent water should not contain leachable substances and
should not sorb significant amounts of substances from the water.
Stainless steel is probably the preferred construction material.
Glass absorbs some trace organics significantly. Rubber should
not be used. Plastic containing fillers, additives, stabilizers,
plasticizers, etc., should not be used. Teflon, nylon, and
their equivalents should not contain leachable materials and
should not sorb significant amounts of most substances. Un—
plasticized polyethylene and polypropylene should not contain
leachable substances, but may sorb very significant amounts of
trace organic compounds.
14. Water : The water used should be from a well or spring if at
all possible, or alternatively from a surface water source.
Only as a last resort should water from a chlorinated municipal
water supply be used. If it is thought that the water supply
could be conceivably contaminated with fish pathogens, the
water should be passed through an ultraviolet or similar ster-
ilizer immediately before it enters the test system.
B. Biological ystem
1. Test animals : If possible, use stocks of fathead minnows from
the Na ional Water Quality Laboratory in Duluth, Minnesota or
the Fish Toxicology Laboratory in Newtown, Ohio. Groups of
starting fish should contain a mixture of approximately equal
numbers of eggs or larvae from at least three different females.
Set aside enough eggs or larvae at the start of the test to
supply an adequate number of fish for the acute mortality
bioassays used in determining application factors.
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2. Beginning test : In beginning the test, distribute 40 to
50 eggs or 1 to 5—day—old larvae per duplicate tank using a
stratified random assignment (see D.3). All acute mortality
tests should be conducted when the fish are 2 to 3 months old.
If eggs or 1 to 5—d y—o1d larvae are not available, fish up to
30 days of age may be used to start the test. If fish
between 20 and 60 days old are used, the exposure should
be designated a partial chronic test. Extra test animals
may be added at the beginning so that fish can be removed
periodically for special examinations (see B.l2.) or for
residue analysis (see C.4.).
3. Food : Feed the fish a frozen trout food (e.g., Oregon
Moist). A mThimum of once daily fish should be fed ad
libitum the largest pellet they will take. Diets should
be supplemented ceekly with live or frozen—live food
(e.g., Daphnia , hop ed earthworms, fresh or frozen brine
shrimp, etc.). Larvae should be fed a fine trout starter
a minimum of 2 times daily, ad libituu; one feeding each
day of live young zooplankton from mixed cultures of
small copepods, rotifers, and protozoans is highly
recommended. Live food is especially important when
larvae are just beginning to feed, or about 8 to 10 days
after egg deposition. Each batch of food should be
checked for pesticides (including DDT, TDE, dieldrin,
lindane, methoxychlor, endrin, aldrIn, BHC, chiordane,
toxaphene, 2,4—D, and PCBs), and the kinds and amounts
should be reported to the project officer or recorded.
4. Disease : Handle disease outbreaks according to their
nature, with all tanks receiving the same treatment
whether there seems to be sick fish in all of them or
not. The frequency of treatment should be held to a
minimum.
5. Measuring fish : Measure total lengths of all starting fish
at 30 and 60 days by the phonographic method used by McKim
and Benoit (1971). Larvae or juveniles are transferred
to a glass box containing 1 inch of test water. Fish
should be moved to and from this box in a water—filled
container, rather than by netting them. The glass box
is placed on a translucent millimeter grid over a
fluorescent light platform to provide background
illumination. Photos are then taken of the fish over
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the millimeter grid and are enlarged into 8 by 10 inch
prints. The length of each fish is subsequently
determined by comparing it to the grid. Keep lengths of
discarded fish separate from those of fish that are to be
kept.
6. Thinning : When the starting fish are sixty (± 1 or 2) days
old, impartially reduce the number of surviving fish in
each tank to 15. Obviously injured or crippled individuals
may be discarded before the selection so long as the number
is not reduced below 15; be sure to record the number of
deformed fish discarded from each tank. As a last resort in
obtaining 15 fish per tank, 1 or 2 fish may be selected for
transfer from one duplicate to the other. Place five spawning
tiles in each duplicate tank, separated fairly widely to reduce
interactions between male fish guarding them. One should
also be able to look under tiles from the end of the tanks.
During the spawning period, sexually maturing males must be
removed at weekly intervals so there are no more than four
per tank. An effort should be made not to remove those
males having well established territories under tiles where
recent spawnings have occurred.
7. Remov4 g eggs : Remove eggs from spawning tiles starting at
12:00 noon Evansville test cime (Appendix A) each day.
As indicated in Step A.9., the test time need not correspond
to the actual time where the test is being conducted. Eggs
are loosened from the spawning tiles and at the same time
separated from one another by lightly placing a finger on
the egg mass and moving it in a circular pattern with
increasing pressure until the eggs being to roll. The
groups of eggs should then be washed into separate,
appropriately marked containers and subsequently handled
(counted, selected for incubation, or discarded) as soon as
possible after all eggs have been removed and the spawning
tiles put back into the test tanks. All egg batches must
be checked initially for different stages of development.
If it is determined that there is more than one distinct
stage of development present, then each stage must be
considered as one spawning and handled separately as
described in Step B.8.
8. incubation and larval selection : Impartially select
50 unbroken eggs from spawnings of 50 eggs or more and
place them in an egg incubator cup for determining
viability and hatchability. Count the remaining eggs and
discard them. Viability and hatchability determinations
must be made on each spawning (>49 eggs) until the number
of spawnings (>49 eggs) in each duplicate tank equals the
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number of females in that tank. Subsequently, only eggs
from every third spawning (>49 eggs) and none of those
obtained on weekends need be set up to determine hatch—
ability; however, weekend spawns must still be removed from
tiles and the eggs counted. If unforseen problems are
encountered in determining egg viability and hatchability,
additional spawnings should be sampled before switching to
the setting up of eggs from every third spawning. Every
day record the live and dead eggs in the incubator cups,
remove the dead ones, and clean the cup screens. Total
numbers of eggs accounted for should always add up to
within two of 50 or the entire batch is to be discarded.
When larvae begin to hatch, generally after 4 to 6 days,
they should not be handled again or removed from the egg—
cups until all have hatched. Then, if enough are still
alive, 40 of these are eligible to be transferred
immediately to a larval test chamber. Those individuals
selected out to bring the number kept to 40 should be
chosen impartially. Entire egg—cup—groups not used for
survival and growth studies should be counted and
discarded.
9. Progeny transfer : Additional important information on
hatchability and larval survival is to be gained by
transferring control eggs immediately after spawning to
concentrations where spawning is reduced or absent, or
to where an affect is seen on survival of eggs or larvae,
and by transferring eggs from these concentrations to
the control tanks. One larval chamber in, or corresponding
to, each adult tank should always be reserved for eggs
produced in that tank.
10. Larval exposure : From early spawnings in each duplicate
tank, use the larvae hatched in the egg incubator cups
(Step B.8. above) for 30 or 60 day growth and survival
exposures in the larval chambers. Plan ahead in setting
up eggs for hatchability so that a new group of larvae is
ready to be tested for 30 or 60 days as soon as possible
after the previously tested group comes out of the larval
chambers. Record mortalities, and measure total lengths
of larvae at 30 and, if they are kept, 60 days post—
hatch. At the time the larval test is terminated they
should also be weighed. No fish (larvae, juveniles, or
adults) should be fed within 24 hrs. of when they are to
be weighed.
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11. Parental termination : Parental fish testing should be
terminated when, during the receding day—length photo—
period, a one week period passes in which no spawning
occurs in any of the tanks. Measure total lengths and
weights of parental fish; check sex and condition of
gonads. The gonads of most parental fish will have
begun to regress from the spawning condition, and thus
the differences between the sexes will be less distinct
now than previously. Males and females that are readily
distinguishable from one another because of their
external characteristics should be selected initially for
determining how to differentiate between testes and
ovaries. One of the more obvious external characteristics
of females that have spawned is an extended, transparent
anal canal (urogenital papilla). The gonads of both
sexes will be located just ventral to the kidneys. The
ovaries of the females at this time will appear transparent,
but perhaps containing some yellow pigment, coarsely
granular, and larger than testes. The testes of males
will appear as slender, slightly milkly, and very finely
granular strands. Fish must not be frozen before making
these examinations.
12. Special examinations : Fish and eggs obtained from the test
should be considered for physiological, biochemical, histo-
logical and other examinations which may indicate certain
toxicant related effects.
13. Necessary data : Data that must be reported for each tank
of a chronic test are:
a. Number and individual total length of normal and deformed
fish at 30 and 60 days; total length, weight and number
of either sex, both normal and deformed, at end of test.
b. Mortality during the test.
c. Number of spawns and eggs.
d. Hatchability.
e. Fry survival, growth, and deformities.
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C. Chemical system
1. Preparing a stock solution : If a toxicant cannot be introduced
into the test water as is, a stock solution should he prepared
by dissolving the toxicant in water or an organic solvent.
Acetone has been the most widely used solvent, but dimethylformanide
(DMF) and triethylene glycol may be preferred in many cases.
If none of these solvents are acceptable, other water—miscible
solvents such as methanol, ethanol, isopropanol, acetonitrile,
dimethylacetamide (DNAC), 2—ethoxyechanol, glyme (dimethylether
of ethylene glycol, diglyme (dimethyl ether of diethylene glycol)
and propylene glycol should be considered. However, dirnethyl
sulfoxide (DMSO) should not be used if at all possible because
of its biological properties.
Problems of rate of solubilization or solubility limit should be
solved by mechanical means if at all possible. Solvents, or as
a last resort, surfactants, can be used for this purposo, only
after they have been proven to be necessar in the actual test
system. The suggested surfactant is p_tert—octylphenoxynOflaethOXY
ethanol (p—i, 1, 3, 3_tetramethylbutylphenOXYnOflaeth0XYethafl0) ,
OPE 0 ) (Triton X—lOO, a product of the Rohm and Haas Company, or
equivalent).
The use of solvents, surfactants, or other additives should be
avoided whenever possible. If an additive is necessary, reagent
grade or better should be used. The amount of an additive used
should be kept to a minimum, but the calculated concentration of
a solvent to which any test organisms are exposed must never exceed
one one—thousandth of the 96—hr. TLSO for test species under the
test conditions and must never exceed one gram per liter of water.
The calculated concentration of surfactant or other additive to
which any test organisms are exposed must never exceed one-twentieth
of the concentration of the toxicant and must never exceed one-tenth
gram per liter of water. If any additive is used, two sets cf
controls must be used, one exposed to no additives and one exposed
to the highest level of additives to which any other organisms
in the test are exposed.
2. 4easurement of toxicant concentration: As a minimum the
concentration of toxicant must be n easured in one tank at each
toxicant concentration every week for each set of duplicate
tanks, alternating tanks at each concentration from week to
week. Water samples should be taken about midway between the
top and bottom and the sides of the tank and should not include
any surface scum or material stirred up from the bottom or sides
of the tank. Equivolume daily grab samples can be composited
for a week if it has been shown that the results of the analysis
are not affected by storage of the sample.
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Enough grouped grab samples should be analyzed periodically
throughout the test to determine whether or not the concentration
of toxicant is reasonably constant from day to day in one tank
and from one tank to its duplicate. If not, enough samples must
be analyzed weekly throughout the test to show the variability
of the toxicant concentration.
3. Measurement of other variables : Temperature must be recorded
continuously (see A.lO.).
Dissolved oxygen must be measured In the tanks daily, at least
five days a week on an alternating basis, so that each tank is
analyzed once each week. However, if the toxicant or an additive
causes a depression in dissolved oxygen, the toxicant concentration
with the lowest dissolved oxygen concentration must be analyzed
daily in addition to the above requirement.
A control and one test concentration must be analyzed weekly for
p11, alkalinity, hardness, acidity, and conductance or more often,
if necessary, to show the variability in the test water. However,
if any of these characteristics are affected by the toxicant
the tanks must be analyzed for that characteristic daily, at
least five days a week, on an alternating basis so that each
tank is analyzed once every other week.
At a minimum, the test water must be analyzed at the beginning
and near the middle of the test for calcium, magnesium, sodium,
potassium, chloride, sulfate, total solids, and total dissolved
solids.
4. Residue analysis : When possible and deemed necessary, mature
fish, and possibly eggs, larvae, and juveniles, obtained from
the test, should be analyzed for toxicant residues. For fish,
muscle should be analyzed, and gill, blood, brain, liver, bone,
kidney, CI tract, gonad, and skin should be considered for
analysis. Analyses of whole organisms may be done in addition
to, but should not be done in place of, analyses of individual
tissues, especially muscle.
5. Methods : When they will provide the desired information with
acceptable precision and accuracy, methods described in Methods
for Chemical Analysis of Water and Wastes (EPA, 1971) should be
used unless there is another method which requires much less time
and can provide the desired information with the same or better
precision and accuracy. At a minimum, accuracy should be measured
using the method of known additions for all analytical methods
for toxicants. If available, reference samples should be
analyzed periodically for each analytical method.
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D. Statistics
1. Duplicates : Use true duplicates for each level of toxic agent,
i.e., no water connections between duplicate tanks.
2. Distribution of tanks : The tanks should be assigned to locations
by stratified random assignment (random assignment of one tank
for each level of toxic agent in a row followed by random assign-
ment of the second tank for each level of toxic agent in another
or an extension of the same row).
3. Distribution of test organisms : The test organisms should be
assigned to tanks by stratified random assignment (random assignment
of one test organism to each tank, random assignment of a second
test organism to each tank, etc.).
E. Miscellaneous
1. Additional information : All routine bioassay flow through methods
not covered in this procedure (e.g., physical and chemical
determinations, handling of fish) should be followed as
described in Standard Nethods for the Examination of Water and
Wastewater, (American Public Health Association, 1971), or
information requested from appropriate persons at Duluth or
Newtown.
2. Acknowledgments : These procedures for the fathead minnow
were compiled by John Eaton for the Committee on Aquatic
Bioassays. The participating members of this committee are:
Robert Andrew, John Arthur, Duane Benoit, Gerald Bouck,
William Brungs, Gary Chapman, John Eaton, John Hale,
Kenneth Hokanson, James McKim, Quentin Pickering, Wesley
Smith, Charles Stephan, and James Tucker.
3. References : For additional information concerning flow
through bioassays with fathead minnows, the following
references are listed:
American Public Health Association. 1971. Standard
methods for the examination of water and wastewater.
13th ed. APHA. New York.
Brungs, William A. 1969. Chronic toxicity of zinc to the
fathead minnow, Pimephales promelas Rafinesque. Trans. Amer.
Fish. Soc., 98(2): 272—279.
Brungs, William A. 1971. Chronic effects of low dissolved
oxygen concentrations on the fathead minnow ( Pimephales promelas) .
3. Fish. Res. Bd. Canada, 28(8): 1119—1123.
43
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Brungs, William A. 1971. Chronic effects of constant elevated
temperature on the fathead minnow. ( Pimephales promelas) . Trans.
Amer. Fish. Soc. 100(4): 659-664.
Carlson, Dale R. 1967. Fathead minnow, Pimephales promelas
Rafinesque, in the Des Moines River, Boone County, Iowa, and
the Skunk River drainage, Hamilton and Story Counties, Iowa.
Iowa State Journal of Science, 41(3): 363—374.
Drummond, Robert A., and Walter F. Dawson. 1970. An
inexpensive method for simulating Diel patterns of lighting
in the laboratory. Trans. Amer. Fish. Soc., 99(2): 434—435.
Isaak, Daniel. 1961. The ecological life history of the
fathead minnow, Pimephales promelas (Rafinesque). Ph.D.
Thesis, Library, Univ. of Minnesota.
Markus, Henry C. 1934. Life history of the fathead minnow
( Pimephales promelas) . Copeia, (3): 116—122.
McKim, J. M., and D. A. Benoit. 1971. Effect of long—term
exposures to copper on survival, reproduction, and growth
of brook trout Salvelinus fontinalis (Mitchill). J. Fish.
Res. Bd. Canada, 28: 655—662.
Mount, Donald I. 1968. Chronic toxicity of copper to
fathead minnows ( Pimephales promelas , Rafinesque). Water
Research, 2: 215—223.
Mount, Donald I., and William Brungs. 1967. A simplified
dosing apparatus for fish toxicology studies. Water Research,
1: 21—29.
Mount, Donald I., and Charles E. Stephan. 1967. A method
for establishing acceptable toxicant limits for fish —
malathion and the butoxyethanol ester of 2,4—D. Trans.
Amer. Fish. Soc., 96(2): 185—193.
Mount, Donald I., and Charles E. Stephan. 1969. Chronic
toxicity of copper to the fathead minnow ( Pimephales promelas )
in soft water. J. Fish. Res. Bd. Canada, 26(9): 2449—2457.
Mount, Donald I., and Richard E. Warner. 1965. A serial—
dilution apparatus for continuous delivery of various
concentrations of materials in water. PHS Pubi. No. 999—
WP—23. 16 pp.
Pickering, Quentin H., and Thomas 0. Thatcher. 1970. The
chronic toxicity of linear alkylate sulfonate (LAS) to
Pimephales promelas , Rafinesque. Jour. Water Poll. Cant.
Fed., 42(2): 243—254.
44
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Pickering, Quentin H., and William N. Vigor. 1965. The
acute toxicity of zinc to eggs and fry of the fathead
minnow. Progressive Fish—Culturist, 27(3); 153—157.
Verma, Prabha. 1969. Normal stages in the development
of Cyprinus carpio var. cominunis L. Acta biol. Acad. Sd.
Hung., 21(2): 207—218.
Approved by the Committee
on Aquatic ioassayS, NWQL
Approved by the Director, NWQL
45
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Appendix A
Test (Evansville, Indiana) Photoperiod
For Fathead Minnow Chronic
Dawn to Dusk
Time Date Day—length (hour and minute )
6:00 — 4:45) DEC. 1 10:45)
6:00 — 4:30) 15 10:30)
6:00 — 4:30) JAN. 1 10:30)
6:00 — 4:45) 15 10:45)
)
6:00 — 5:15) FEB. 1 11:15) 5—month pre—
6:00 — 5:45) 15 11:45) spawning growth
) period
6:00 — 6:15) MAR. 1 12:15)
6:00 — 7:00) 15 13:00)
)
6:00 — 7:30) PR. 1 13:30)
6:00 — 8:15) 15 14:15)
6:00 — 8:45) MAY 1 14:45)
6:00 — 9:15) 15 15:15)
)
6:00 — 9:30) JUNE 1 15:30)
6:00 — 9:45) 15 15:45) 4—month spawning
) period
6:00 — 9:45) JULY 3. 15:45)
6:00 — 9:30) 15 15:30)
)
6:00 — 9:00) AUG. 1 15:00)
6:00 — 8:30) 15 14:30)
6:00 — 8:00) SEPT. 1 14:00)
6:00 — 7:30) 15 13:30)
)
6:00 — 6:45) OCT. 1 12:45) post spawning period
6:00 — 6:15) 15 12:15)
)
6:00 — 5:30) NOV. 1 11:30)
6:00 — 5:00) 15 11:00)
46
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APPENDIX B
RECOMMENDED BIOASSAY PROCEDURE FOR
BROOK TROUT SALVELINTJS FONTINALIS (MITCHILL) PARTIAL CHRONIC TESTS
47
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RECOI*(ENDED BIOASSAY PROCEDURES
Preface
Rec ended Bioassay Procedures are established by the approval
of both the Co ittee on Aquatic Bioassays and the Director of the
National Water Quality Laboratory. The main reasons for establishing
them are: (1) to permit direct comparison of test results,
(2) to encourage the use of the best procedures available, and
(3) to encourage uniformity. These procedures should be used by
National Water Quality Laboratory personnel whenever possible,
unless there is a good reason for using some other procedure.
Reco ended Bioassay Procedures consider the basic elements that
are believed to be important in obtaining reliable and reproducible
results in laboratory bioassays. An attempt has been made to adopt
the best acceptable procedures based on current evidence and opinion,
although it is recognized that alternative procedures may be adequate.
Improvements in the procedures are being considered and tested, and
revisions will be made when necessary. Coents and suggestions are
encouraged.
Director, National Water Quality Lab (NWQL)
Committee on Aquatic Bioassays, NWQL
48
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Recommended Bioassay Procedure for
Brook Trout Salvelinus fontinales (Mitchill) Partial Chronic Tests
April, 1971
(Revised January, 1972)
A. ca1 system
1. Diluter : Proportional diluters (Mount and Brungs, 1961) should
he employed for all long—term exposures. Check the operation
of the diluter daily, either directly or through the measure—
nent. of toxicant concentrations. A minimum of five toxicant concen-
trations and one control should be used for each test with a dilution
factor of not less than 0.30. An automatically triggered emergency
aeration and alarm system must be installed to alert staff in case of
diluter, temperature control or water supply failure.
rT
oxicant mixing : A container to promote mixing of toxicant
hearing and w—cell water should be used between diluter and
tanks for each concentration. Separate delivery tubes should
run from this container to each duplicate tank. Check to see
that the same amount of water goes to duplicate tanks and
that the toxicant concentration is the same in both.
3. Tank : Each duplicate spawning tank (preferably stainless steel)
should measure 1.3 X 3 X 1 ft. wide with a water depth of 1 foot
and alevin—juvenile growth chambers (glass or stainless steel with
glass bottom) 7 X 15 X 5 in. wide with a water depth of 5 inches.
Growth chambers can be supplied test water by either separate
delivery tubes from the mixing cells described in Step 2 above
or from test water delivered from the mixing cell to each
duplicate spawning tank. In the second choice, test water must
always flow through growth chambers before entering the spawning
tank. Each growth chamber should be designed so that the test
water can be drained down to 1 inch and the chamber transferred
over a fluorescent light box for photographing the fish (see
B.10).
. Flow rate : Flow rates for each duplicate spawning tank and growth
chamber should be 6—10 tank vclumes/2 hr.
5. Aeration : Brook trout tanks and growth chambers must be aerated
with oil free air unless there are no flow limitations and 60%
of saturation can be maintained. Total dissolved oxygen levels
should never be allowed to drop below 60% of saturation.
6. Cleanin& : All tanks and chambers must be siphoned daily and
brushed at least once per week. When spawning commences,
gravel baskets must be removed and cleaned daily.
49
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7. Spawning substrates : Use two spawning substrates per duplicate
made of plastic or stainless steel which measure at least
6 X 10 X 12 in. with 2 inches of .25 to .50 inch stream gravel
covering the bottom and 20 mesh stainless steel or nylon screen
attached to the ends for circulation of water.
8. Egg incubation cups are made from —oz. 2—inch 01
round glass jars with the bottoms cut off and replaced with
stainless steel or nylon screen (1 o meshes per inch). Cups
are oscillated in the test water by means of a rocker arm
apparatus driven by a 2 r.p.m. electric motor (Mount, 1968).
9. Light : The lights used should simulate sunlight as nearly
as possible. A ombination of Duro—Test (Optima FS) 1 ’ 2 and wide
spectrum Gro—lux- fluorescent tubes has proved satisfactory at the
NWQL.
10. Photoperiod : The photoperiods to be used (Appendix A) simulate
the dawn to dusk times of Evansville, Indiana. Evansville dates
must correspond to actual dates in order to avoid putting natural
reproductive cycles out of phase. Adjustments in photoperiod
are to be made on the first and fifteenth of every Evansville
test month. The table is arranged so that adjustments need be
made only in the dusk times. The dawn and dusk times listed in
the table (Evansville test time) need not correspond to the
actual test times where the test is being conducted. To illustrate
this point, a test started on March first would require the use
of the photoperiod for Evansville test date March first, and the
lights could go on any time on that day just so long as they
remained on for twelve hours and fifteen minutes. Fifteen days
later bhe photoperiod would be changed to thirteen hours.
Gradual changes in light intensity at dawn and dusk (Druinmond
and Dawson, 1970), may be included within the photoperiods shown,
and should not last for more than 1/2 hour from full on to full
off and vice versa.
11. Temperature : Utilize the attached temperature regime (see Appendix
B). Temperatures should not deviate instantaneously from the
specified test temperature by more than 2 e C and should not remain
outside the specified temperature ±10 C for more than L 8 hours at
a time.
12. Disturbance : Spawning tanks and growth chambers must be covered
with a screen to confine the fish and concealed in such a way
that the fish will not be disturbed by persons continually walking
1 Mention of trade names does not constitute endorsement.
2 Duro—Test, Inc., Hammond, md.
3 Sylvania, Inc., New York, N. Y.
50
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past the system. Tanks and chambers must also be shielded
from extraneous light which can affect the intended photoperiod
or dami ge light sensitive eggs arid alevins.
13. Construction materials : Construction materials which contact the
diluent water should not contain le .able substances and
should not sorb significant amounts cf substances from the
water. Stainless steel is probably the preferred construction material.
Glass absorbs some trace organics significantly. Pubber should not
be used. Plastic containing fillers, additives, stabilizers,
plasticizers, etc., should not be used. Teflon, nylon, and
their equivalents should not contain leachable materials and
should not sorb significant amounts of most substances. Un—
plasticized polyethylene and çoly ropylerie should not contain
leachable substances, but may sorb very significant amounts of
trace organic compounds.
1 1 e. Water : The water used should be from a well or spring if at all
possible, or alternatively from a surface water source. Only
as a last resort should water from a chlorinated municipal water
supply be used. If it is thought that the water supply could be
conceivably contaminated with fish pathogens, the water should be
passed through an ultraviolet or similar sterilizer immediately before
it enters the test system.
B. Biological system
1. Test animals : Yearling fish should be collected no later than March 1
and acclimated in the laboratory to test temperature and water quality
for at least one month before the test is initiated. Suitability of’
fish for testing should be judged on the basis of acceptance of food,
apparent lack of diseases, and 2% or less mortality during acclimation
with no mortality two weeks prior to test. Set aside enough fish to
supply an adequate number for short-term bioassay exposures used in
determining application factors.
2. Beginning. test : Begin exposure no later than April 1 by distributing 12
acclimated yearling brook trout per duplicate using a stratified random
assignment (see D.3). This allows about a four month exposure to the
toxicant before the onset of secondary or rapid growth phase of
the gonads.
Extra test animals may be added at the beginning so that fish can
be removed periodically for special examinations (see B.12),
or for residue analysis (see C.li).
3. Food : Use a good frozen trout food (e.g., Oregon Moist). Fish should
be fed the largest pellet they will take a minimum of two times
daily. The amount should be based on a reliable hatchery feeding
schedule. Alevins and early juveniles should be fed. trout starter
a minimum of five times daily. Each batch of prepared food should be
checked for pesticides (including DDT, TDE, dieldrin, endrin, aidrin,
51
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BBC, chiordane, toxaphene, 2, 1 4—D, and PCBs), and the kinds and amounts
should be reported to the project officer or recorded.
14 Disease : Handle disease outbreaks according to their nature,
with all tanks receiving the same treatment whether there seems
to be sick fish in all of them or not. The frequency of treatment should
be held to a minimum.
5. Measuring fish : Record mortalities daily, and measure fish
directly at initiation of test, after three months and at
thinning (see B.6) (total length and weight). Fish should not
be fed 2 hours before weighing and lightly anesthetized with
.E—222 to facilitate measuring (100 mg 1S—222/liter water).
6. Thinning : When secondary sexual characteristics are well
developed (approximately two weeks prior to expected spawning),
separate males, females and undeveloped fish in each duplicate
and randomly reduce sexually mature fish (see D. )to the desired
number of 2 males and females, and discard undeveloped fish
after examination. Place two spawning substrates (described
earlier) in each duplicate. Record the number of mature, immature,
deformed and injured males and females in each tank and the number
from each category discarded. Measure total length and weight of all
fish in each category before any are discarded and note which ones
were discarded (see C.14).
7. Removing eggs : Remove eggs from the redd at a fixed time
each day (preferably after 1:00 p.m. Evansville time, so the
fish are not disturbed during the morning).
8. incubation and viability : Impartially select 50 eggs from
the first eight spawnings of 50 eggs or more in each duplicate
and place them in an egg incubator cup for hatch. The remaining
eggs from the first eight spawnings (>50 eggs) and all subsequent
eggs from spawnings should be counted and placed in separate egg
incubator cups for determining viability (formation of neural keel
after 11—12 days at 90 C). The number of dead eggs from each spawn
removed from the nest should be recorded and discarded. Never place
more than 250 eggs in one egg incubator cup. All eggs incubated
for viability are discarded after 12 days. Discarded eggs can be
used for residue analysis and physiological measurements of toxicant
related effects .
9. Progeny transfer : Additional important information on hatchability
and alevin survival can be gained by transferring control eggs
immediately after spawning to concentrations where spawning is
reduced or absent, or to where an affect is seen on survival of
eggs or alevin, and by transferring eggs from these concentrations
to the control tanks. Two growth chambers for each duplicate
spawning tank should always be reserved for eggs produced in that
tank.
52
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10. Hatch and alevin thinning: Remove dead eggs daily xom the
hatchability cups described in Step 8 above. When hatching
commences, record the number hatched daily in each cup. Upon
completion of hatch in any cup, randomly (see 0.4) select 25 alevins
from that cup. Dead or deformed alevins must not be included
in the random selection but should be counted as being dead or
deformed upon hatch. Measure total lengths of the 25 selected
and discarded alevins. Total lengths are measured by the
photographic method used by McKim and Benoit (1971). The fish
are transferred to a glass box containing 1 inch of test water.
They should be moved to and from this box in a water filled
container, rather than by netting them. The glass box is
placed on a translucent millimeter grid over a fluorescent light
box which provides background illumination. Photos are then
taken of the fish over the millimeter grid and are enlarged into
8 X 10 inch prints. The length of each fish is subsequnetly de-
termined by comparing it to the grid. Keep lengths of discarded
alevins separate from those which are kept. Place the 25 selected
alevins back into the incubator cup and preserve the discarded
ones for initial weights.
11. Alevin—juvenile exposure : Randomly (see D.4) select from the incubation
cups two groups of 25 alevins each per duplicate for 90—day
growth and survival exposures in the growth chambers. Hatching
from one spawn may be spread out over a 3 to 6 day period;
therefore, the median—hatch date should be used to establish
the 90—day growth and survival period for each of the two
groups of alevin. If it is determined that the median—hatch
dates for the five groups per duplicate will be more than three
weeks apart, then the two groups of 25 alevin must be selected
from those which are less than three weeks old. The remaining
groups in the duplicate which do not hatch during the three
week period are used only for hatchability results and then
photographed for lengths and preserved for initial weights. In
order to equalize the effects of the incubation cups on growth,
all groups selected for the 90—day exposure must remain in the
incubation cups three weeks before they are released into the
growth chambers. Each of the two groups selected per duplicate
must be kept separate during the 90—day period. Record
mortalities daily, along with total lengths 30 and 60 days
post—hatch and total length and weig1 t at 90 days post—hatch.
Alevins and early juveniles should not be fed 24 hours before
weighing. Total lengths are measured by transferring the growth
chambers described earlier to a translucent millimeter grid
over a fluorescent light box for photographing as described in
Step 10 above. Survival and growth studies should be terminated
after three months. Terminated fish can be used for tissue
residue analysis and physiological measurements of toxicant
related effects.
53
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12. Parental termination : All parental fish should be terminated
when a three week period passes in which no spawning occurs
in any of the spawning tanks. Record mortality and weigh
and measure total length of parental fish, check sex and
condition of gonads (e.g., reabsorption, degree of maturation,
spent ovaries, etc.) (see C.4).
13. p cial examinations : Fish and eggs obtained from the
test should be considered for physiological, biochemical, and
histological investigations which may indicate certain toxicant
related effects.
14. Necessary data : Data that must be reported for each tank of
a chronic test are:
a. Number and individual weights and total lengths of
normal, deformed, and injured mature and immature
males and females at initiation of test, three months
after test commences, at thinning and at the end of
test.
b. Mortality during the test.
c. Number of spawns and eggs. A mean incubation time
should be calculated using date of spawning and the
median hatch dates.
d. Hatchability.
e. Fry survival, growth and deformities.
C. Chemical system
1. Preparing a stock solution : If a toxicant cannot be introduced
into the test water as is, a stock solution should be prepared
by dissolving the toxicant in water or an organic solvent. Acetone
has been the most widely used solvent, but dimethylformanide (DMF)
and triethylene glycol may be preferred in many cases. If none
of these solvents are acceptable, other water—miscible solvents
such as methanol, ethanol, isopropanol, acetonitrile, dimethyl—
acetamide (Dt4AC), 2—ethoxyethanol, glyme (dimethylether of
ethylene glycol, diglyme (dimethyl ether of diethylene glycol)
and propylene glycol should be considered. However, dimethyl
sulfoxide (DMSO) should not be used if at all possible because
of its biological properties.
Problems of rate of solubilization or solubility limit should be
solved by mechanical means if at all possible. Solvents, or as
a last resort, surfactants, can be used for this purpose, only
after they have been proven to be necessary in the actual test
54
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system. The suggested surfactant is p_tert—octylphenoxynonaethoxY
ethanol (p—i, 1, 3, 3 tetramethylbutylphenoxynonaethoxYethanOl,
0PE 10 ) (Triton X—lOO, a product. of the Rohm and Haas Company,
or equivalent).
The use of solvents, surfactants, or other additives should be
avoided whenever possible. If an additive is necessary, reagent
grade or better should be used. The amount of an additive used
should be kept to a minimum, but the calculated concentration
of a solvent to which any test organisms are exposed must never
exceed one one—thousandth of the 96—hr. TL5O for test species
under the test conditions and must never exceed one gram per liter
of water. The calculated concentration of surfactant or other
additive to which any test organisms are exposed must never
exceed one—twentieth of the concentration of the toxicant and
must never exceed one—tenth gram per liter of water. If any
additive is used, two sets of controls must be used, one exposed
to no additives and one exposed to the highest level of additives
to which any other organisms in the test are exposed.
Measurement of toxicant concentration : As a minimum the con-
centration of toxicant must be measured in one tank at each
toxicant concentration every week for each set of duplicate tanks,
alternating tanks at each concentration from week to week. Water
samples should be taken about midway between the top and bottom
and the sides of the tank and should not include any surface scum
or material stirred up from the bottom or sides of the tank.
Equivolume daily grab samples can be composited for a week if it
has been shown that the results of the analysis are not affected
by storage of the sample.
Enough grouped grab samples should be analyzed periodically
throughout the test to determine whether or not the concentation
of toxicant is reasonably constant from day to day in one tank
and from one tank to its duplicate. If not, enough samples must
be analyzed weekly throughout the test to show the variability
of the toxicant concentration.
3. Measurement of other variables : Temperature must be recorded
continuously (see A.l1).
Dissolved oxygen must be measured in the tanks daily at least five
days a week on an alternating basis, so that each tank is analyzed
once each week. However, if the toxicant or an additive causes
a depression in dissolved oxygen, the toxicant concentration with
the lowest dissolved oxygen concentration must be analyzed daily in
addition to the above requirement.
A control and one test concentration must be analyzed weekly for
pH, alkalinity, hardness, acidity, and conductance or more often,
55
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if necessary, to show the variability in the test water. However,
if any of these characteristics are affected by the toxicant,
the tanks must be analyzed for that characteristic daily, at
least five days a week, on an alternating basis, so that each
tank is analyzed once every other week.
At a minimum, the test water must be analyzed at the beginning
and near the middle of the chronic test for calcium, magnesium,
sodium, potassium, chloride, sulfate, conductance, total solid,
and total dissolved solids.
4. Residue analysis : When possible and deemed necessary, mature
fish, and possibly eggs, larvae, and juveniles, obtained from
the test, should be analyzed for toxicant residues. For fish,
muscle should be analyzed, and gill, blood, brain, liver, bone
kidney, GI tract, gonad, and skin should be considered for
analysis. Analyses of whole organisms may be done in addition
to, but should not be done in place of, analyses of individual
tissues, especially muscle.
S. Methods : When they will provide the desired information with
acceptable precision and accuracy, methods described in Methods
for Chemical Analysis of Water and Wastes (EPA, 1971) should be
used unless there is another method which requires much less
time and can provide the desired information with the same
or better precision and accuracy. At a minimum, accuracy should
be measured using the method of known additions for all analytical
methods for toxicants. If available, reference samples should be
analyzed periodically for each analytical method.
D. Statistics
1. Duplicates : Use true duplicates for each level of the toxic
agent, i.e., no water connections between duplicate tanks.
2. Distribution of tanks : The tanks should be assigned to locations
by stratified random assignment (random assignment of one tank
for each level of the toxic agent in a row followed by random
assignment of the second tank for each level of the toxic agent
in another or an extension of the same row).
3. Distribution of test organisms : The test organisms should be
assigned to tanks by stratified random assignment (random assign-
ment of one test organism to each tank, random assignment of a
second test organism to each tank, etc.).
4. Selection and tninnin test organisms : At time of selection or
thinning of test organisms the choice must be random (random, as
defined statistically).
S I;
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E. Miscellaneous
1. Additional information : All routine bioassay flow through
methods not covered in this procedure (e.g., physical and
chemical determinations, handling of fish) should be followed
as described in Standard Methods for the Examination of Water
and Wastewater (American Public Health Association, 1971).
2. Acknowledgments : These procedures for the brook trout were
compiled by 3. M. McKim and D. A. Benoit for the Committee on
Aquatic Bioassays. The participating members of this committee
are: Robert Andrew, John Arthur, Duane Benoit, Gerald Bouck,
William Brungs, Gary Chapman, John Eaton, John Hale, Kenneth
Hokarison, James McKim, Quentin Pickering, Wesley Smith, Charles
Stephan, and James Tucker.
3. References : For additional information concerning flow through
bioassay tests with brook trout, the following references are
listed:
Allison, L. N. 1951. Delay of spawning in eastern brook trout
by means of artificially prolonged light intervals. Progressive
Fish—Culturist, 13: 111—116.
American Public Health Association. 1971. Standard methods
for the examination of water and wastewater. 13th ed. APHA,
New York.
Carson, B. W. 1955. Four years progress in the use of
artificially controlled light to induce early spawning of brook
trout. Progressive Fish—CulturiSt, 17: 99—102.
Drummond, Robert A., and Walter F. Dawson. 1970. An inexpensive
method for simulating Diel patterns of lighting in the laboratory.
Trans. Amer. Fish. Soc., 99(2): 434—435.
Environmental Protection Agency. 1971. Methods for Chemical
Analysis of Water and Wastes. Analytical Quality Control Laboratory,
Cincinnati, Ohio.
Fabricius, E. 1953. Aquarium observations on the spawning
behavior of the char, Salmo alpinus . Rep. Inst. Freshwater Res.
Drottingholm, 34: 14—48.
Hale, 3. G. 1968. Observations on brook trout, Salvelinus
fontinalis spawning in 10—gallon aquaria. Trans. Amer. Fish.
Soc.. 97: 299—301.
57
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Henderson, N. E. 1962. The annual cycle in the testis of
the eastern brook trout, Salvelinus fontinalis (Mitchill)
Canadian Jour. Zool., 40: 631—645.
Henderson, N. E. 1963. Influence of light and temperature
on the reproductive cycle of the eastern brook trout
Sa1veli us fontinalis (Mitchill). J. Fish. Res. Bd. Canada,
20(4): 859—897.
Hoover, E. E., and H. E. Hubbard. 1937. Modification of
the sexual cycle in trout by control of light. Copeia,
4: 206—210.
MacFadden, J. 1961. A population study of the brook trout
Salvelinus fontinalis (Mitchi].l). Wildlife Soc. Pub. No. 7.
McKim, J. M., and D. A. Benoit. 1971. Effect of long—term
exposures to copper on survival, reproduction, and growth
of brook trout Salvelinus fontinalis (Mitchill). J. Fish. Res.
Bd. Canada, 28: 655—662.
? unt, Donald I. 1968. Chronic toxicity of copper to fathead
minnows ( Pimephales p ome1as , Rafinesque). Water Research,
2: 215—223.
W unt, Donald I., and William Brungs. 1967. A simplified
dosing apparatus for fish toxicology studies. Water Research,
1: 21—29.
Pyle, E. A. 1969. The effect of constant light or constant
darkness on the growth and sexual maturity of brook trout.
Fish. Res. Bull. No. 31. The nutrition of trout, Cortland
Hatchery Report No. 36, pages 13—19.
Wydoski R. S., and E. L. Cooper. 1966. Maturation and
fecundity of brook trout from infertile streams. J. Fish.
Res. Bd. Canada, 23(5): 623—649.
Approved by the Committee on
Aquatic Bioassays, NWQL
Approved by the Director, NWQL
58
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____________ Date _______________________________
6:00 - 6:15) MAR. 1
6:00 - 7:00) 15
6:00 - 7:30) APR. 1
6:00 — 8:15) 15
6:00 — 8:45) MAY 1
6:00 - 9:15) 15
6:00 - 9:30) JUNE 1
6:00 — 9:45) 15
6:00 - 9:45) JULY 1
6:00 - 9:30) 15
6:00 - 9:00) AUG. 1
6:00 - 8:30) 15
6:00 8:00) SEPT. 1
6:00 7:30) 15
- 6:45) OCT. 1 12:45)
— 6:15) 15 12:15)
) Spawning and
— 5:30) NOV. 1 11:30) egg incubation
— 5:00 15 11:00)
6:00 - 4:45) DEC. 1 10:45)
6:00 - 4:30) 15 10:30)
)
6:00 - 4:30) JAN. 1 10:30)
6:00 - 4:45) 15 10:45)
)
6:00 — 5:15) FEB. 1 11:15)
6:00 — 5:45) 15 11:45)
Appendix A
Test (Evansville, Indiana) Photoperiod
For Brook Trout Partial Chronic
Dawn to Dusk
Ti me
Day-length (hour and minute )
12:15)
1 3:00
1 3:30)
14:15)
)
14:45)
15:15)
)
15:30) Juvenile-
15:45 adult exposure
15:45
15:30
)
1 5 :00)
14:30)
)
14:00)
13:30)
6:00
6:00
6:00
6:00
Alevin-juvenile
exposure
59
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Appendix B
Temperature Regime for Brook Trout Partial Chronic
Months Temperature ° C
Mar.
Apr. 12
May 14
Juvenile-
June adult 15
exposure
July 15
Aug. 15
Sept. 12
Oct. [ 9 1
Spawning A constant temperature
Nov. and 9
egg incubation
Dec. ri i must be established just
prior to spawning and egg
throughout the 3—month
Alevin-
Feb. juvenfle I alevin-juvenhle exposure.
Jan. I incubation, and maintained
exposure
Mar. 9
60
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APPENDIX C
TABLE 1. TEST CONDITIONS DURING 96-HR ACUTE EXPOSURES OF FATHEAD MINNOWS ( PIMEPHALES PROMELAS) ,
BLUEGILLS ( LEPONIS MACROCHIRUS) , BROOK TROUT ( SALVELINUS FONTINALIS )
AND FLAGFISH ( JORDANELLA FLORIDAE ) TO DIAZINON
F ti ad_minnow __________ Blue ill ____________ Brook trout ___________ F lagfish
1 2i _____ 1 _______ ______ _______ 3 - 2
ERL_Da ERL_Da ERL_Da Federal Federal Commercial Commercial Commercial ERL_Da ERL_Da
$ource culture culture culture hatchery hatchery hatchery hatchery hatchery culture culture
Age 15—week 20—week 13—week 1—year 1—year 1—year 1—year 1—year 6—week 7—week
Average length
(mm) 3 0 30 35 50 56.5 l90 220 170 i8.i 17.8
Number fish!
concentration 20 20 20 10 20 20 20 20 10 10
kater volume
(liters) 19 19 57 19 19 57 57 57 27 27
Flow (tank
volume/day) 5 10 8 10 10 10 10 10 11 11
Temperature (°c) 25+1 25+1 25+1 25+0.5 25+0.5 12+0.5 12+0.5 12+0.5 25+0.5 25+0.5
Dissolved oxyger JOS 96 101 100 98 65 75 86 105 103
(9 saturation) (95-1.15) (87-101) (100-108) (93-103) (88-103) (63-106) (58-107) (78-95) (103-107) 102-105)
Measured
concentratiun 11.7 10.6 7.9 0.89 0.80 0.92 0.76 2.3 3.1 3.0
(mg/1.)b (11.0-12.6) (8. i2.3) (7.6-8.6) (0.86-0.93) (0.69-0.88) (0.76-1.2) (0.68-0.82) (1.9-2.6) (2.9-3.3) (2.9-3.2)
6.0 1,9 4.1 0.66 oil 0.39 0.35 0.93 1.6 2.1
(5.6-6.5) (1%.3_5.9) (3.6-1.7) (0.63_0.145) (0.38-0.11) (0.36-0.17) (0.28-0.39) (o.8 1.0) (i. i.8) (1.8-2.2)
3.6 3, 6 3.0 0.22 0,22 0.16 0.16 0.51 0.82 1.3
(3.2-3.1) (2.9-3.8) (2.6-3.1) (0.21-0.23) (0.21-0.21) (0.11-0.18) (0.12-0.1.6) (0.66-0.57) (0.76-0.85) )l.2-l.i)
2.1 1.9 2.3 0.08 0.10 0.08 o.o6 0.23 0.36 0.92
(1.9—2.3) (1.7-2.1) (2.1-2.6) (0.07-0.09) (1.39-0.11) (0.07-0.10) (0.05-0.07) (o.2 0.26) (0.35-0.38) (0.91—0.95)
1.1 1.1 1.7 0 . 0.06 u.ui 0.03 0.20 0.68
(1.0-1.1) (0.9— .2) (1.6-1.9) (0.02-0.06) (0.01-0.05) (o.03-0. ) (0.03—0.06) (0.17-0.22) (o. —o.77)
0 0 0 0 0 0 0 0 0 0
(N)/concentratior4 (3) . (3) (5) (i) (3) (3) )1) ( 1) (6) (6)
aElnEal Research Lahoratoryhuluth
bhange in parentheses.
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TABLE 2. QUALITY OF TEST WATER DURING CHRONIC EXPOSURES OF
FP THEAL MINNOWS ( PINEPHIALES PROMELAS ) ‘10 DIAZINON
(TESTS #1 AND #2)
Item
Average
Range
Temperature (°c)
Adult chambers
25.0
214.0—26.0
Larval chambers
25.5
24.5—26.5
Dissolved oxygen
(% saturation)
85
T 4 —lOT
11
75 a
7.2—7.8
Hardness
44
42—47
Alkalinity
42
39—44
Acidity
2.9
1.2—14.8
aMd
62
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TABLE 3. QUALITY OF TEST WATER DURING PARTIAL—CHRONIC EXPOSURES
OF BROOK TROUT ( sALvELINus FONTINALIS ) TO DIAZINON
Item
Average
Range
Temperature (°c)
—
+ 1° from recommended
temperature for date
Dissolved oxygen
(% saturation)
Adult cham1 ers
86
54—103
Larval chambers
101
88—109
pH
73 a
7.o—y.6
Hardness
45
42—47
Alkalinity
42
40—47
Acidity
3.4
1.2-11.3
aMd
63
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a’
TABLE 4. PHYSICAL CHARACTERISTICS OF TEST CHAMBERS USED FOR LONG—TERM EXPOSURES OF FATHEAD
MINNOWS ( PIMEPHALES PROMELAS ) AND BROOK TROUT ( SALVELINUS FONTINALIS ) TO DIAZINON
Species
Exposure
Material
Dimensions (cm)
(hxlxw)
Water depth (cm)
Volume
(liters)
Fathead
minnows
adult
glass
30x53x30
18
28.6
larval
glass
32x30xl3
18
7.0
Brook trout
preliminary
glass
30x90x30
15
i40.5
adult
stainless
steel
4Ox9Ox3O
30
81.0
larval
glass
l8xl Oxl2
13
6.2
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U I
TABLE 5. NONINAL AND MEASURED DIAZINON CONCENTRATIONS (pg/i.) IN WATER DURING CHRONIC
EXPOSURES OF FATHEAD MINNOWS ( PIMEPHALES PRONELAS )
a
Number ol abservatlons.
Larvae rearel at t mb c mentratim,n
1 t e
Ad 1L L mb:rmm (tvmmL #m )
Mutt v (m hvrv )(em .t
Larval vhaabers (teat #2)
Nmmrm rmai cmovvrmtrrmtit)
‘12)
252
115 (2.5
2
£ 2.a
1 .2 5.6
i,L)
3.2 t
51.5
31.2
15.6
7.2
1.9
0
Aver a e Ifleammu ret
eo avertrativr m
) 0)9 Ill
219
IlL
69
2
62.3
21.2
13.5
69
21
(5
62
33
0
)ltan3ard ivvia(io)
38) (52)
LI))
(17)
(5)
— 19.3)
).5
)s.I
L.5)
1.71
—
L.6(
fl.c)
-
(.6)
)o,; )
-
(N)a
(I 1 ) (16)
(12) (jL)
I
(15)
(16) (57) ( 21)
(Si)
(27)
(69
( (ml)
(iS)
-
(27)
-
(0)
-
(13)
(21) (15)
,
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TABLE 6. NOMINAL AND MEASURED DIAZINON CONCENTRATIONS (pg/i.) IN WATER DURING
PARTIAL—CHRONIC EXPOSURES OF BROOK TROUT ( SALVELINUS FONTINALIS )
Item
Adult chambers
Larval chambers
Nominal
concentration
12.0
6.0
3.0
1.5
0.15
0
12.0
6.0
3.0
1.5
0.75
0
Average measured
concentration
9.6
L .8
2.1
1.1
0.55
0
11.1
5.6
2.7
1. 4
0.80
0
(Standard
deviation)
(2.2)
(1.2)
(0.6)
(° .L )
(0.19)
—
(1.2)
(o.6)
(0.3)
(o. )
(0.18)
—
(N) a
(29)
(29)
(29)
(29)
(29)
(29)
(3k)
(36)
( 31k)
(3 )
(35)
(35)
0’
0 .
aNb of observations.
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APPENDIX D
ACCUMULATION OF DIAZINON IN TISSUES OF ADULT BROOK TROUT
It was expected that diazinon would not be concentrated in fishes to the
extent common for the organochlorine pesticides. The exposure of brook trout
for extended periods of time during this study provided material to test this
hypothesis. Brook trout removed for thinning and at termination were analyzed
for diazinon.
Tissue concentrations were determined by gas chromatography with the
same equipment used to monitor water concentrations. Most of the tissue—
residue data were rejected because it was subsequently found that diazinon
decomposed rapidly even in frozen tissues. A few determinations made by the
methods outlined below were deemed acceptable.
Extraction from muscle tissue and eggs was begun within 30 mm. of
death. Three extractions were made with hexane in a stainless steel blender;
arthydrous sodium sulphate was used as a drying agent. Samples were cleaned on
20—g activated florisil columns.
Blood was drawn in heparinized syringes and placed in glass vials with
Teflon—lined lids. Three milliliters of hexane and glass beads were added
immediately, and the samples were shaken vigorously. Blood samples were
extracted four times with hexane and dried with anhydrous sodium sulphate.
Results are presented in Table 1 (Appendix D). Although the data are
limited, the accumulation factor for this organophosphate apparently is low
compared to that of most organochiorine pesticides. Tissue concentration
was directly proportional to water concentration.
67
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TABLE 1, ACCUMULATION FACTORSa FOR DIAZINON IN TISSUES OF ADULT
Itm
Adult exposed 6 months
item
Adults exposed 8 moxthp (spawning completed)
Exposure (water
concentration)
4.8 pg/I.
1.1 pg/i.
Exposure (water
concentration
9.6 pg/i.
4.8 pg/i.
2. pg/i.
1.1 pg/i.
0.55 pg/I.
Mature
males
Immature
males
Spawned
females
Mio od
13x
lix
Muscle
lbx
2 1 1 x
51x
l9x
35x
25x
25x
(Standard deviation) 0
(9)
(26)
(6)
(N)b
(II)
U2)
(N)b
(ii)
(l)C
(6)c
(3)C
(12)
(12)
(12)
Eggs
iSix
(M)b
(1)
5 Tissue concentration (ng/g)/water concentration (pg/i.)
bNembor ol tissue samples. Pooled samples unless otherwise noted.
CAnalyzed individually.
BROOK TROUT ( SALVELINUS FONTINALIS )
a’
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TECHNICAL REPORT DATA
(Please read Inwuctions on the reverse before completing)
L REPORT NO.
EPA—600/3—77—060
3. RECiPIENTS ACCESSIONNO.
4. TITLE AND SUBTITLE
TOXICITY OF DIAZINON TO BROOK TROUT AND FATHEAD
MINNOWS
5. REPORT DATE
May 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Donald T. Allison and Roger 0. Hermanutz
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory—Duluth,
Off ice of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1BA6 08
ii. CONTRACT/GRANT NO.
In—house
12. SPONSORING AGENCY NAME AND ADDRESS
SAME AS ABOVE
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Fathead minnows exposed to diazinon from 5 days through 24 weeks post hatch
developed severe scoliosis. The incidence and degree of spinal deformity
correlated to exposure level. Fish in 3.2 igJl (the lowest concentration
tested) had 6O / more deformities than controls (P=O.05). Hatch of eggs from
fathead minnows exposed to 3.2 ugh was 3O lower than the controls.
Yearling brook trout exposed to 4.8 ug/l and above developed scoliosis and
lordosis within a few weeks. Growth was substantially inhibited (P—0.05)
during the first 3 months of exoosure at 4.8 ug/l and above. Exposure to
2.4 ugh and above caused frequently observed neurological symptoms for the
first 4 to 5 months. Progeny of parents exposed 6 to 8 months to all levels
tested (0.55 to 9.6 pg/l) were smaller than controls at 122 days post hatch
(P=O. 05).
Acute toxicity tests with diazinon yielded 96—hr LC5O’s of 7.8, 1.6, 0.77
and 0.46 mg/i respectively for fathead minnows, flagfish, brook trout and
bluegills.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
C. COSATI Field/Group
Pesticides
Organic phosphates
Diazinon
Toxicity
Bioassay
Fresh water fishes
Trout
Chronic toxicity
Acute toxicity
6F
7C
Minnows
18. DISTRIBUTION STATEMENT
.
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
20. SECURITY CLASS (This page)
-
21. NO. OF PAGES
22. PRICE
Unclassified
EPA Form 2220.1 (9-73) *U.SG0V NMEN PRlMTI GOFF%C [ ign_757-056 (56Z
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