PB83-243436
Evaluation of a Fathead Minnow
"Pimephales promelas' Embryo-Larval Test
Guideline Using Acenaphthene and Isophorone
(U.S.) Environmental Research Lab.-Duluth, MN
Jul 83
s^
U.S. Dcȣrtmsnt of Commsrce
Flafei?.! Tocfiaica! Information Service
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TECHNICAL REPORT DATA
(Please rsad Instructions on the reverse before completing)
1, REPORT NO.
EPA-GOO/3-83-062
A, TITLti AND SUBTITLE
Eval.nation of a Fathead Minnow lllmc^h£l_es_ 1H£HLCLL;L;2. Embryo-
Larva]. Test Guideline Using Acenaphthent! and 'Isophorone
S. REPORT DATE
July 1983
6. PERFORMING ORGANIZATION CODi
3, RECIPIENT'S ACCESSION NO.
2 A 3 4 3 6
7. AUTHOB(S)
8. PERFORMING ORGANIZATION REPORT NO.
A. E. Lcmke, E. Durhan, and T. Fclhabcr
9, PERFORMING ORGANIZATION NAME AND AODBESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Dul.uth
6201 Congdon Boulevard
Duluth, UN 5ri804
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGI5NCV NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, MN 55804
13, TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/03:
16, SUPPLEMENTARY NOTES
10. ABSTRACT
A set of 4 embryo-larval bioas.says (2 each with isophorone and acenaphthene,
respectively, were conducted with the fathead minnow, Piniephales prome.las. The
objective of the study was to evaluate a specific method for this type of test.
The no effect levels when compared to the controls were 0.208 and 0.226 mg/1
acenaphthene and 19.5 and 6.89 mg/1 isophorone, respectively. The only problem'
encountered was in the feeding regime which may have a possibility .for improvement
as control weights varied..
17.
KEY WORDS AND DOCUMENT ANAtYSIS
DESCRIPTOHS
18. DISTRIBUTION STATEMENT
REI.KASK TO PUBLIC
b.lOENTIFIERS/OPEN ENDED TEHMS
19. SECURITY CLASS ('fhll Report/
UNCLASSIFIED
2O. SECURITY CLASS (Thit'
UNCLASSIF] ICO
e. COSATI Field/Group
21. NO. OF PAGES
29
EPA Form 3220-1 (Rev.
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NOTICE
Tills document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
1.1
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EPA-600/3-83-062
July 1983
Evaluation of a Fathead Minnow Piinephalcs promelas Embryo-Larval Test
Guideline Using Acenaphtbene and Isophorone
Arraond E. Lemke, Elizabeth Durban, and Taryl Felhaber
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
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Evaluation of a Fathead Minnow Plinophales promelas Embryo-Larval Test
Guideline Using Acenaphthcnie and Isophorone
An;1-:-: ,1 E , Lemke
Eli i-.'ibeth Durban
Taryl Felhaber
Toxicity testing in biological aquatic systems lias been used for more
than 20 years. Various kinds of tests have been conducted including static,
flow-through, short-term, long-terra, and static renewal tests. Species
tested include most of the common species of fish and many species of
invertebrates. A large bulk of the testing has been on Daphnia magna and the
fathead minnow. There is a general consensus that much of the testing is
imprecise and perhaps inaccurate. Determination of expected accuracy and
precision of such testing was necessary before the use o£ such tests could be
required by EPA.
The testing plan being reported here was to provide each of several
laboratories with two chemicals and a set of guidelines, ask them to perform
two tests with each chemical, and report as the primary criteria the
no-effect level of each chemical on the early life-history stages of the
fathead minnow. A description of this criteria is in Appendix .1. Partici-
pating laboratories were required to provide all other necessary items for
following the guidelines, including water, chemical expertise, fish and
testing personnel. An absolute minimum of consultation assistance was
provided by the test set manager. It was decided that the guidelines must
stand by themselves eventually and this was best determined by minimizing any
assistance Crotn the' project chief.
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Participants were asked to provide a detailed description of all the
.activities. Included in this do':ail was to be any other endpoints such as 96
or 30-day LCSOs which were gathered as part of determining the no-effect
level. Each participant was asked to also include a thorough discussion of
interpretation and/or technique problp.ni;> which were encountered during the
conduct of the four required tests. The laboratory operator:; were also
encourageo to make suggestions for rectifying any of the problems which they
encountered. This report describes the participation of the Environmental
Research Laboratory-Duluth in this interl.nboratory'comparison test.
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METHODS AND MATERIALS
The water supply was sand filtered Lake Superior water; hardness 45-47
mg/1 as CaCO-j, alkalinity 40-42 tng/1 as CaC03, pH 7,8. Other chemical
parameters of. the water are as described in Bicsinger and Christensen (1972).
All water was heated in the head box by stainless st'.'.c:l immersion coil and
aerated vigorously to bring all gases into equilibrium with the atmosphere.
Water was delivered to the diluter . through a rigid PVC pipe and controlled by
a stainless steel and anodized aluminum liolcnoid valve. Experimental water
temperatures were monitored continuously in three randomly assigned chambers
and taken twice weekly in all chambers with a calibrated thermcmeter.
Reported temperatures are for the calibrated thermometer readings.
The diluter (DeFoe, 1975) utilized in this work provided useful
' flexibility in making up the required solutions on a continuing basis. Toxic
materials can be added directly by changing syringe sizes or by making
different" concentrations in solvent of the desired materials. This equipment
was constructed of glass with silicons glue joints and teflon tubing. Teflon
has been shown to be i-iuch less adsorptive than other plastics and does not
contain plasticizets which may leak out and cause toxicity.
Test chambers ware of glass with a minimum of silicone glass and ceramic
glue. Chamber size was 46 cm x 16 cm x 18 cm containing 10 cm water with an
8 nn frpphoarH. W^it^r v;olum.(^ was approximately S*25 liters. Cycle tiiue of
the diluter was about 420 cycles in 24 hours and during each cycle, 0,5
liters was delivered to each chamber resulting in a 25 fold turnover in eacli
chamber per 24 hours.
Toxic Solutions
Acenaptliene was dissolved in dimethylformamide (CMF) at the required
amount to add 16 .11! of solvent-toxicant solution to each liter of water. At
3
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the highest concentrations some acenapthene was noted coning out of solution
at the injection point. This floating material was not sampled when the
analysis for active ingredient was accomplished.
Isophoronc was added directly into the dilutor mixing chambers at eac'.i
concentration. It is fairly water soluble and no residues were noted at any
test concentrations.
Test Organisms
Embryos of the fathead minnow Pimephales promelas were removed from the
cement asbestos spawning tile by gentle rolling (Cast and Brungs, 1973). A
dissecting microscope was utilized to pick embryos that were undergoing cell
division. Embryos were assigned in groups of five in a stratified random
fashion to the screen bottomed glass embryo cup. The cups were set in petri
dishes containing sufficient dilution water to keep the embryos covered
during the distribution. Transfers were made by carefu.l manipulation using
an eye dropper with an enlarged opening. Embryo numbers varied from 15-35
per cup but were equal for any single test. Embryo numbers were varied by
embryo availability and by choice to test the effect of this parameter.
After all embryos were transferred, the incubation cups were moved to
the test chambers with minimum air exposure (less than 10 seconds) and hung
on an oscillating rockerarm apparatus. This equipment cycled the embryo cups
vertically causing gentle movement of the test water and maintained all test
parameters in close proximity to the embryos. The fish in these tests were
fed beginning the first day of hatching. Tills was done to prevent starvation
of the earliest hatchllngs. The first feeding was put directly i.ito the
embryo cups. On the fourth day of exposure all live embryos were released
into the test chambers by unhooking the incubation cups from the aeration
apparatus and then submerging by tipping into the chamber. Larvae were
A
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allowed Co swim out of their own volition and the cups were removed. 24 hours
later. This procedure minimized handling which is very stressful on newly
hatched larvae. However, this procedure resulted in varying numbers of
larvae in each chamber, as hatching success vaiied .
Feeding wr.s accomplished by putting an aliquot of. settled brine shrimp
nauplii Artomia sal ina (Jungle brand) into each flow splitter of the diluter
assuring equal distribution to each duplicate. Feeding was done at the
beginning, middle, and end of an 8 hour period 5 days per week with two
feedings approximately 1 hour opart 2 days on weekends. Sufficient shrimp
were added so at least some were not eaten. No siphoning of tanks was done
the first week after hatching, thus preventing handling injury.and allowing
any micro organisms in the water to grow. After 1 week approximately 5 grams
per day of a very fine trout starter was added daily to each tank and tanks
were siphoned every other day. Fish were killed and individually we'.ghed to
~"\
10 grams on the 28th day post hatch. All weights were recorded and an
analysis of variance and Dunnetts test were performed to determine difference
from the regular and solvent control with the acenaphthene and with only the
regular control in the isophorone tests.
Ch emiea1 Analysis
Chemical analyse,1; for the toxicants were performed on all chambers
initially (1st day) and finally (last day) for each test. Also twice weekly
analyses w,?.re performed with duplicates alternating. All results reported
were as active ingredient analyzed. Dissolved oxygen analysis was performed
once a week at each concentration with duplicates alternating. Hardness,
alkalinity, and pit analysis were performed twice during each test.
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Analysis for aeenaphthene was accomplished by using a Balrd AtomLc Model
SFR 100 spectrofluorlineter. The fluorlmeter was chosen as the instrument
because it is very sensitive to the aromatic rings of the aeenaphthcne
molecule. This structure fluorcsces readily and no concentration or cleanup
of the samples was needed. Water samples were taken and mixed at a ratio of
75°7 test solution, 25% isopropanol. This mixture was alloweu to equilibrate
until all of the air bubbles were gone (2-16 hours). Appropriate amounts of
this solution were analyzed and the results recorded.
Standards were made by adding weighed amounts of acenaplit'icnc to
dr'.methyl formaroi.de solvent and injecting appropriate aliquots into clean room
temperature lake water. A standard curve was produced and used as a
comparison. Spiked samples were prepared similarly from control water
obtained from the experimental equipment.
Operating parameters for the fluoriraeter:
Excitation Wavelength: 290 nm
Emission Wavelength; 336 nm
Excitation Slit Width: 10 nm
Emission Slit Width: 20 nm
The isophorone was analyzed by gas chromatography. Weighed amounts of
isophorone were added to hexane and used for standards. The procedure for
analysis was as follows: 50 ..1! of isophorone water solution was sampled from
the test tanks and added to 50 ml redistilled haxaue in 100 nil volumetric
flasks. Test samples and spiked recovery samples were extracted by stirring
for 1.5 hours on an electric stirrer with a teflon stirring bar, A 5 pi
aliquot of the hexane layer was injected onto the GC column by automatic
sampler. The mean retention .time was 5.4 minutes under the following
operating conditions:
6
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Instrument: Hewlett Packard 571DA gas ehromatograph with a KID detector
Column: 6 ft. x 2 mm ID glass column packed with H)% carbowax 20 M on 80/100
Gas Chrom Q
Carrier ftas: Nitrogen
Detector Temp.: 250° 0
Injr.ctor Temp.: 7.50° C
0» en Temp,: 140° C 150 thermal.
All samples were injected twice and the mean of the two infections was
reported. The precision of the instrument was periodically checked by
duplicate analyses.
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RESULTS •
General Parameters
Temperature of the test chambers was maintained between 2<'t.2°C and
25.6°C at all times. No excursions beyond these limits were noted in any of
the four tests. Mean temperature was 25.1*0. Dissolved oxygen was always
maintained at plus 90% of saturation, mainly because of the, high turnover
rate in the test chambers. Hardness was always between 47-48 mg/1 as CaCOj
and alkalinity at 38-40 mg/1 as CaC03, and pH was 7.5-7,8,
The chemical analysis for acenaphthene tests 1 and 2 are found in Table
1. !">at a presented are analyzed concentrations in mg/1 of material. There
were no large excursions from the tesc parameters due to equipment'.
malfunction during the acenaphthene tests.
The isophorone chemical analyses for tests 1 and 2, respectively, are
presented in Table ?.. All concentrations are in rag/1, Concentration 4 in
test 2 had a complete failure of the toxicant airiition equipment during a 16
hour period in the middle of the test which i.s reflected in the mean and
standard deviation of test 2,
Reprodiicibi 1 i t y
The spilt sample precision was 98,4% _+ l.ITi n ~ 8 for the acenaphthene,
and 95.3% -t- 6.9% n = 8 for the isophorone. Spike recoveries were 99% +• 5%
for acenaphthcae and 99.5% + 7.2% for isophorone tent 1 and 106,8% t 7. IX for
isophorone test 1 and 2, respectively. All reported data on .the tests were
adjusted for recoveries prior to running the statistical evaluations for the
test data. No data were discarded.
The test concentrations all were nominally at a 0.5 factor from each
other with the exception of: isophorone test 1 in which the difference between
the tor two concentrations was only 0.33%.
R
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Biolog i c_n 1 Re s u 11 a
The no-effect level for acenaphthmie when compared to the solvent
control was between 0.133 and 0.2fj3 mg/1. for test 1 and between 0.146 and
0.285 mg/1 for test 2 (Steele and Torrie, 1960).
The no—effect levels when compared against the normal control were 0.133
to 0.263 mg/1 for test 1 and between 0.593 and 1,02 mg/1 for test 2. In both
tests with acenupthene the solvent control fish were the largest. Also, the
two lowest concentrations were, larger than the normal, control fish. This
appears to be a usual occurrence in hioassay testing.
The no-effect levels for isophorone hased on growth were between 15.6
and 22.7 mg/1 for test 1 and between 4.2 «-md 8.8 mg/1 for test 2, The total
growth of the test fish was also higher in test 2 with the controls in test 1
averaging 0.141 grams and those in test ''. averaging 0.202 grains.
The effect of egg numbers was also tested as part of the work. The
final low mean weight in the second acenaphthene test and the first
isophorone test was at first thought to he caused by the larger number of
fish used as opposed to the first acenaphthene test. To test this theory a
test was conducted using 35 embryos per hatching cup (70 per concentration).
The test was begun so that the larvae began to feed on Tuesday allowing 4
jays of full feed before the weekend. Th?.s(? fish weiru. thp heaviest of the
four tests.
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DISCUSSION
No apparent difference in the testing procedures were found. All
procedures and practices were as similar as possible. Water temperatures,
flow rates, food sources, food rearing practices, feeding rates, source of
fi.sh and all similar factors were very similar if not identical. One
procedure not noted in the protocol was that of feeding dttv.iriR the first 48
hours post hatch. In test 1 the eggs hatched on Saturday and Sunday so they
were only fed twice. In test 2 they hatched mid-week and were fed three
times a day during the first 2 days of life. The'presence of a wider
variation in size in test 2 perhaps reflects this as the standard deviation
was almost double 0.026 vs. 0.017 in tests 2 and 1, respectively. This
variation in growth rate was already noted by observation after about 2 weeks
of testing. The guidelines called for at least one concentration to be equal
to the control in growth of the test animals and at least one concentration
to be significantly lower in growth, if growth was used as an end point.
This requirement was wet in all four of the reported tests. Variation in
mean growth between tests made the use of a control absolutely necessary.
One cannot compare weights of two separate tests'but each test must be
compared to its own control.
If solvents are used, more reproducibi1ity is obtained when toxicnc
effects are compared to solvent controls rather than to the normal controls.
The acenaphthene tests are nearly identical if iud$;erf against the solvent
control and about three times different when iud»eu against the normal
control. The extra growth usually found with usable solvents may make the
results appear less toxic if only comparison to a normal control is made.
The difference noted results from the anparent stimulation of growth by the
solvent DMF which at low levels acts us a nutrient source for bacteria. This
10
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in turn nourished microorganisms which the newly hatched fry used as food.
This throe-fold difference is also found in the isophorone test. Numbers of
fish as noted previously are not n factor if all life sustaining parameters
are maintained at a high level.
Feeding of the newly hatched fry still, could be improved. If natural
waters are used, certain unknown factors may interfere. The two lowest
growth tests were adjacent to each other in time., but none of the usual test
parameters were different in any of the tests.
The protocol as written in the guideline document appears quite easy to
follow and has a limited amount of "art". This.is particularly true if no
sorting or thinning of the hatched fry is done. If any further work is to be
done, it should be in the area of feeding during the first week post hatch
because the growth differences noted took place during this time span.
11
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REFERENCES
Biesinger, K. K., nntl G. M, Christiansen. 1972. Effects of various metals on
survival, growth, reproduction and metabolism of Dnphnia magna. J.
Fish. Res. Hoard Can. 29: 1691-1700.
DeFoe, D. L. 1975. Multichannel toxicant injection system for flow-through
bioassays. J. Fish. Res. Board Can. 32: 544-546.
Cast, M., and W. A. Brungs. 1973. A procedure for separating eggs cC the
fathead minnow. Prog. Fish-Cult. 35: 54.
Steel, R. G. D., and J. H. Torric. 1960. Principles and procedures of
statistics with special reference to the biological sciences. New York:
McGraw Hill.
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Table 1
Aeenaphthetie Chemical Analysis
Mean and Standard Deviation
Test 1
Test 2
Control
Not
Found
Not
Found
Solvent
Control
Not
Found
Not
Found
0
+ 0
0
+0
#1
.069
.009
.070
.005
Conccntrat
#2
0
+0
0
+0
.133
.004
.146
.016
ion mg/1
#3
0.263
+0.061
0.285
+0.032
Anal
0
+ 0
0
+ 0
yzcd
r?4
.474
.120
.593
.179
1
+0
1
+0
«
.029
.200
.022
.529
N =
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Table 2
Isophorone Chemical Analysis
Mean and Standard Deviation
Test 1
Test 2
Control
Not
Found
Mot
Found
2
+0
2
±°
#1
.14
.26
.18
.17
Concentration ing/1
#2 «
4
±°
4
+0
.18
.23
.15
.22
8
+0
8
+ 2
.29
.34
.78
.81
Analyzed
ff4
15.61
+ 0.92
14.51
+ 1 . 44
22
+0
27
+5
«
.66
.87
.63
.41
N = 8
J4
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Table 3
Toxicity Results
Weights of Surviving Fish and Comparison with Controls
Aeenaphthene
1
Aeenaphthene
2
Isophorone
I
Isophorone
2
X
SO
N
3c
SD
N
X
SD
N
X
SD
H
Control
0.1-%
0.032
31
0.122
0,042
45
0.141
0.047
54
0.202
0.041
68
Solvent Cone.
Control 1
0.218 0
0.034 0
33
0.180 0
0.048 0
50
None 0
0
None 0
0
.200
.037
40
.167
.054
35
.145
.066
52
.239
•.046
69
Cone.
2
0.200
0.034
39
0.176
0.056
49
0.144
0.060
62
0.204
• 0.0 ';7
63
Cone.
3
0.152ab
0.030
50,
0.155b
0.039
40
0.143
0.052
36
0.1793
0.045
71
Cone.
4
0.140ab
0.028
35
0.126b
0.033
52
0,140
0.040
48
0.162®
0.039
71
Cone .
5
0.073ab
0.023
31
0.079ab
0.010
29
0.1153
0.031
63
0.171a
0.041
59
a = 1 tail test different froa normal control 0.99% level
b = i tail test different from solvent control 0.99% level
c = 1 tail test different from solvent control 0.95% level
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Appendix 1
Guidelines for Conducting Flow-Through Karly Life Stage Toxicity Tests with
Fathead Minnows for Use in the USE PA, OTS-01U) Round Robin Tost
1. In an Early L^fe Stage Toxicity Tost with fathead minnows, organisms1, are
exposed to toxicant during part of the embryonic stage, all of the
larval stage and part of the juvenile stage.' The organisms are examined
for statistically significant reductions in survival and weight in order
to determine lower and upper chronic endpoints.
A lower chronic endpoint is the highest tested concentration (a) in an
acceptable chronic test, (b) which did not cause the occurrence (which
wes statistically significantly different from the control at the 95%
level) of any specified adverse effect, and (c) below which no tested
concentration caused such an occurrence.
An upper chronic endpoint is the lowest tested concentration (a) in an
acceptable chronic, test, (b) which caused the occurrence (which was
statistically significantly different from the control at the 95% level)
of any specified adverse effect and (c) above which all tested
concentrations caused such an occurrence,
2, Not enough information is currently available concerning early life
stage tests with fathead minnows to allow precise specification of
details for all. aspects of the test. Enough such tests have been
conducted ~nd enough aspects have been studied, howuver, to indicate
that these Guidelines are appropriate. A prudent course of action for
anyone planning to conduct such, tests would be to initially conduct a
16- . • •
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test with no toxicant to gain experience nut) to determine if the
requirements of sections 10, 11, 19, 20, 26 and 27 can bo mot: using the.
planned water, food, procedures, etc. If a solvent may bo: used In the
preparation of a stock solution, it would also be prudent to test: one or
more concentrations of one or more solvents at the same time (sec
Section A), General infc""mat i.on on such things as apparatus, dilution
wj.i~.er, toxicant, randomization of test chatrbers and organisms, and
methods for chemical analyses, can be found in Draft #10 of the proposed
ASTM Standard Practice for Conducting Acute Toxicity Tests with Fishes,
Macroinvertehrates, and Amphibians.
3. Tests should he conducted with at least five toxicant co?icentrat ions in
a geometric series and at least one control treatment. The
concentration of toxicant in each treatment, except for highest
concentration and the control treatment, should usually be 50 percent of
that, in the next higher one.
4. If a solvent other than water is used to prepare test solutions, a
solvent control (at the highest solvent concentration present in any
other treatment) using twice as many test organisms and test chambers as
the other treatments is required in addition to the regular control,
unless such a control has already been tested in the same water vith the
same species of fish, food, and test procedure and the water quality has
not changed significantly, A concentration of solvent is acceptable
only if it is (or has been) shown that concentration or a higher one
does not cause an increase or decrwase in survival or weight at the end
of the test that is statistically significantly different from the.
control at the 95% level using a two-tailed test.
17
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*5 . For each treatment (toxicant concentration and control) there1, must: be at
least two replicate test chambers each containing one or more embryo
cups with at least 60 embryos divided equally between the embryo cups at
the beginning of the test.
6, Two test chambers have been used routinely:
a. Twenty fish have been tested in -3 ehftmber which is 16 cm x 44 cm x
18 cm high with a 16 cm x 18 cm 40-inesh stainless steel screen ft cm
from one end, with a water depth of 12.8 cm and with a flow rate of
190 ml/minute,
b. Fifteen fish have been tested in a chamber which is 6,5 cm x 18.0 cm
x 9.0 cm high with a 6.5 cm x 9.0 era 40~mesh stainless steel screen
2.5 cm from one end, with n water depth of 4.5 cm and with a flow
rate of 15 ml/minute.
All of the above are inside dimensions. In both test chambers the water
depth is controlled by a standnipe located in the smaller screened
compartment with the test solution entering at the other end of the test
chamber.
7. Embryo cups should be glass cylinders about 4.5 cm inside diameter and
about 7 cm high with 40-mesh nylon or stainless steel screen glued to
the botton. The embryo cups must be suspended in the test chamber in
such a way as to insure that the organisms are plways submerged and that
test solution regularly flows into and out of the cup without agitating
the organisms too vigorously, A rocker arm apparatus dtivtni by a 2
r.p.ra. motor and having a vertical-travel distance of 2.5 - 4.0 cm has
been successful ly used, as have self-starting siphons that cause the
level o£ solution in the test chamber to rise and fall.
. 18
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8. Any water in which fathead minnows will survive, grow, and reproduce
sat isfactorily should be an acceptable dilution water for early life
,'itage toxicity tests with fathead minnows.
9, A 16-hr light and 8-hr dark photoperiod should be provided. A 15- to
30-mimite transition period at "lights on" and "lights off" may be
desirable. Light intensities from 10 to 100 lumens at the water surface
have been used successfully, but the intensity should be about the same
for all test chambers. Lights should he provided by wide—spectrum
(color Rendering Incex > 90) fluorescent lamps.
10. Tests should be conducted at 25° C. The temperature in each test
chamber should be between 24 and 26° C at all times and must be between
20 and 28° C at all times. If the water is heated, precautions should
be taken to assure that supersaturation of dissolved gases is avoided
and total dissolved gases should be measured at least once during the
tekt in the water entering the control treatment.
£
11. The dissolved oxygen concentration should be between 75 percent and 100
percent saturation at all times in all test chambers. At no time during
the test should one test chamber have a dissolved oxygen concentration
that is more than 1.1 times the dissolved oxygen concent rat ion occurring
in another chamber at the same time,
12. The flow rate of test solution through the test chambers must be great
enough to maintain the dissolved oxygon concentration (sec- sections 11
and 22) and to insure that the toxicant concentrations are not decreased
significantly due to uptake by test organisms and material on the sides
aiifi bottoms of the chambers .
' ' 19
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13. A test begins when embryos in embryo cups are placed in test solution
and ends 3?, days later,
14. Embryos and Fish should not be treated Co cure or prevent disease or
fungus before or during a test.
15. Embryos should be obtained From a fatbend minnow stock culture
maintained at 25° C and a dissolved oxygen concentration between 75
percent and 100 percent saturation with a 16-hr light: and 3-hr dark
photoperiod. Frozen adult brine shrimp has been success fully used as a
food for adult fathead minnows. The maximum production of embryos by
fathead minnows has been obtained in a 30 cm x 60 cm x 30 cm deep
chamber with a water depth of 15 cm when 15 cm x 30 cm quadrants are
formed with stainless steel screen and one male, one female and one or
two substrates are placed in each quadrant. Half-round spawning
substrates (Benoit and Carlson, 1977) with an inside diameter of 7.5 cm
and a length of 7.5 cm have been used successfully.
16. The afternoon before a test -is to begin, all of the substrates should be
removed from an appropriate number of tanks in the stock culture unit
and should be replaced about the time the lights are turned on the next
morning. Enough (at least three) substrates with embryos on them should
be I'eitiuved six hours later and soaked in dilution water for two hours.
For each individual substrate the ^-nbryos should be gently separated
(Cast and Hrungs, 1973) and removed ;;nd visually examined using a
dissecting scope or a magnifying viewer. Empty shells and undeveloped
and onaque embryos should be discarded. If less than 50 percent of the
embryos from a substrate appear to he healthy and ft?rtile, all the
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embryos from that .substrate should be discarded. Single embryos with no
fungus or partial shells attached are preferable, although embryos with
partial shells attached and clumps of two or throe embryos (with or
without separation) have been used success fully, An approximately equal
number of acceptable embryos from one substrate should be impartially
distributed to each embryo cup and the process repeated for at least two
more substrates until the proper number of embryos have been placed in
each cup to give at least 60 embryos per treatment. The embryo cups
should be standing in dilution water when the embryos are being
distributed and then the cups should be randomly placed in the test
chambers.
17. Twenty to 7.4 hours after the.y are placed in the embryo cups, the embryos
should be visually examined under a dissecting scope or magnifying
viewer and all dead embryos should be counted and discarded. Embryos
that are alive but heavily fungused should also be counted and
discarded. Forty to 48 hours after the start of the exposure all dead
, and heavily fuugused embryos should be counted and removed. The
remaining healthy, fertile embryos should be impartially reduced to the
desired number of test organisms (,-tt least 30 par treatment). IE more
than about 35 percent of embryos in the control treatment arc discarded
within the firnt 48 hours of the test because they are dead or heavily
fungused, it will probably be cost-eftective to restart the test. In
addition, if toxicant related effect;, are seen at 48 hours, it will
probably be cost effective to restart the test since all of the toxicant
concentrations will probably cause adverse effects. Each day thereafter
dead embryos should be counted and discarded.
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18. In each treatment, when hatching is about 90 percent complete or 48 hr
after first hatch In tli.it treatment, the live young fish ahould.be
counted and the live fish that arc visibly (without the use of a
dissecting scope or magnifying viowfir) lethargic or grossly abnormal in
either swimming behavior or physical appearance should be counted. All
of the normal and abnormal live fish should he released into the test
chambers. Unhatched embryos should be left in the cups'and released
into the test chamber when they hatch. The ra: £9 of time-to-hatch (to
the nearest day) in each cup should be recorded.
19. A test should be terminated if t'-c average percent of embryos (based on
the number of: embryos after thinning) that produce live fry for release
into test chambers in any control treatment is less than 50 peicent or
if the percent hatch in any control embryo cup is more than 1.6 times
that in another control embryo cup,
20. The flow rate, size of the test chamber and the amount of food added
should be such that the average weight of the control fish at the end o£
the test would not be significantly greater if only half as many fish
were tested per test chamber.
21, Each test chamber containing live fish over two days old must be fed
live newly hatched brine sliLintp at least two times a day at least six
hrs apart (or three times a day aboui. four hours apart) on days 2-5
after hatch and at least five days a week thereafter. They must be fed
at least once a day on all other days. Other food mny. als\t be provided
in addition to the above. The amount of food provided to each chanihor
may be proportional to the mi'sber an.d size; of fish in the chamber, but
each chamber wist be treated in a comparable manner. Quantifying the
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amount of live newly hatched brine shrimp to be fed is difficult, but
the fish should not be excessively overfed or underfed. A largo buildup
of food on the. bottom of the charnbaer i.a a sign of Recessive
overfeeding, A sign of not feeding enough of the light kind of food is
that in a sideview the abdomen does not protrude.
22. Test chambers should be clc.ined often enough to maintain the dissolved
oxygen concentration (see sections 11 and 12) and to insure that the
toxicant concentrations are not decreased significantly due to sorption
by matter on !:he bottom and sides. In most tests if the organisms are
not ove.rfe:! too much and the flow rate is not too low, removing debris
from the bottom once or twice a week should he adequate. With some
toxicants that promote growth of bacteria the sides and bottoms should
be cleaned more often. Debris can be removed with a large pipette and
rubber bulb or by siphoning into a white bucket. A dark tip on the
pipette or siphon should help fish avoid being sucked up, but the
pi "•(•• te or bucket should be examined Co insure that no live fish is
d iscarded.
23. Temperatures should bs recorded in till test chambers once at the
beginning of the test and once near the middle of the test. In
addition, temper ature should he recorded at least hourly in one test
chamber throughout the test. The dissolved oxygen concentration should
be measured in each treatment nt least once a week during the test.
Hardness, pH, alkalinity, and acidity should be measured once a week in
the control treatment and one P. in the highest toxicant concentration.
The concentration of toxicant: should be measured at least twice a week
in each treatment.
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24. Dead fish should be removed and recorded when observed, Ac a minimum
11, 18, 25 and 32 days aft«r Che beginning of the test, the live fish
should be counted and the fish that are visibly (without the use of a
dissecting scope or magnifying viewer) lethargic and grossly abnormal in
cither swimming behavior or physical appearance should be counted,
25. The fish should not be fed for the last 24 hours prior to terinination on
day 32. At terminal'inn the weight (wet, blotted dry) of each fish that
was alive at the end of the test should he determined. If the fish
exposed to toxicant appear to ba edematous compared to control fish,
determination of dry, rather than wet, weight is desirable.
26. A test is not acceptable if the average survival of the controls at the .
end of the test is less than 80 percent or if survival in any control
chamber is less than 70 percent.
27.' A test is not acceptable if the relative standard deviation (RSD = 100
times the standard deviation divided by the mean) of the weights of the
fish that were alive at the end of the test in any control test chamber
is greater than 40 percent.
28. Data to be statistical!'/ analyzed are:
(A) percent of healthy, fertile embryos at /sO-'iS hours
(H) percent of embryos that produce live fry for release into test
chambers
(C) percent of embryos that produce live, normal .fry for release into
test chambers
(D) percent of embryos that produce life fish at end of test
(E) percent of "mbryos that produce live, normal fish at end of test
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(F) weights of individual fish thnt were alive at end of test.
Although item A is based on the number of embryos initially placed in
embryo cups, items B, C, D, and K are based on the number of embryos
after thinning.
29. Dichotomous data (live-dead, normal-abnormal) should be analyzed using
contingency tables (Sokal and Kohl, f, Biometry, 1969, p. 587) or log
linear techniques. For weight data the individual fish a;.: used as the
replicates unless a two-tailed V test indicates that differences between
replicate test chambers are not negligible. Weight data may be analyzed
using (Steel and Torrie, Principles end Procedures of Statistics, 1960,
p. Ill) should be used to identify treatments producing weights that are
statistically significantly lower than those of the controls at the 95
percent level.
30. Although the results of -the analyses of all six types of. data in section
28 should be reported, the lower and upper chronic endpoints are only
baaed on statistically significant reductions in survival and weight at
the end of the test (items D and F), Item A is apparently relatively
insensitive and item B is included in item D. In addition, abnormal
fish seem to weigh less than normal fish and so will be covered in item
F. A1.no, since The c'.ist incL ion between normal and abnormal is
subjective, this kind of data is expected to be less reproducible from
one investigator to another than the other kinds oC data. Although
items 1) and F are considered primary, the other items are included
because, tlioy may provide useful in format i.on.
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References
Benoit, D. A., and H. W. Carlson, 1977. Spawning Success of Fathead Minnows
on Selected Artificial Substrates. Prog. Kish-Cult. 39: 67-69.
Flickinger,, S. A. 1969. Determinations of Sexes in the Fathead Minnow,
Trans. Am. Fish. Soc. 98: 526-527.
Cast, M. H. , and W. A. Brungs. 1973. A Procedure for Separating figgs oE the
Fathead Minnow. Prog. Fish-Cult. 35: 54;
May, R. C. 1970. Feeding Larval Marine Fishes in the Laboratory: A Review.
Calif. Mar. Res. Comm., California Cooperative Oceanic Fisheries
Investigations Report 14: 76-83.
This tentative procedure was written by Clisrl.es Stephan with the help of many
members of the staCf of the Environmental Research Laboratory in Dul.uth,
Minnesota,
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