ZINC TOXICITY

Test 1

Survival of larvae previously exposed to zinc as embryos was reduced at
267 ug/liter after 30 days (Table 2). After 100 days of exposure, all fish
had died at this concentration and growth of males was less than those of
the controls at the next lower concentration (139 ug/liter). Mean
spawnings/female and embryo production appeared to be reduced at 139 ug/liter
but significant differences could not be shown due to the variable response.

Test 2

After 30 days, survival of larvae not previously exposed to zinc as
embryos was adversely affected at 85 wg/liter (Table 3). No larvae survived
at the two highest concentrations (139 and 267 uwg/liter). By the end of the
test, female growth was significantly lower than the controls at 85 and
51 g/liter. Mean spawnings/female and embryo production appeared to be
reduced at 85 ug/liter but were not statistically different from the controls.

Embryo hatchability (Tables 2 and 3) and survival and growth of F)
larvae from exposed parents at 139 ug/liter and below were not significantly
different from those of control larvae.

The 96-hr LC50 value calculated for 4-5 week old juveniles was 1,500 yg
Zn/liter.

Fish exposed for 30 days to concentrations of 47 yg Zn/liter and
above contained significantly higher amounts of zinc than those in the
controls (Figure 2). Additionally, zinc uptake increased with increasing
exposure concentrations. Concentrations of zinc (yg/gram) in fish exposed
for 30 and 100 days were similar but older fish contained a significantly
higher total body content (yg/fish). Zinc concentrations were also
similar in 30-day-old exposed parental fish and progeny (F,) and in fish
previously exposed as embryos (test 1) and those not previously exposed
(test 2) after 100 days (Appendix C).

RESULTING TEST VALUES AND APPLICATION FACTORS

The results described above for chronic effects of cadmium and zinc
on survival, growth, and reproduction establish the maximum acceptable
toxicant concentration [(MATC) as described by Mount and Stephan (1967b)]
between 4.1 and 8.1 ug/liter cadmium and 26 and 51 yg/liter zinc. The
application factor (MATC/96-hr LC50) based on the 96-hr LC50 values
of 2,500 ug Cd/liter and 1,500 yg Zn/liter was between 0.0016 and 0.0032
for cadmium and 0.017 and 0.034 for zinc.

12


APPENDIX C
ZINC CONCENTRATIONS IN WHOLE FISH TISSUE AFTER 30 AND
100 DAYS OF EXPOSURE (test 1 begun with larvae exposed

as embryos; test 2 begun with unexposed larvae)

Test 1 Test 2
Measured ug/g Total ug/fish Measured e/g Total vg/fish
cmnetSaton | ean # cmeetacton | wean 2? | nemn ¢
in water standard in water tandard standard
Cug/2.) deviation deviation ug/l.) deviation deviation.
30-day exposure 30-day exposure
267 “= 267
139 293455.3 8.34.0 139 -
78 2132.4 4941.5 85
47 196434.0 | 6,243.0 32 220413.0 | 11,245.2
28 160426.9 4642.2 26 178422.7 Teg
10 116429.1 3.541.5 ° 167419.4 8.64
(Control) (Control)
100- oaure (adult
267 - 267 -
139 324¢18.7 137487.5 139
75 21dg41.3 137452.6 85 235413.7 155487.9
“7 212426.6 128447.7 s1 211420.7 110429.
28 167429.2 9423.2 26 17agen.9 | 86.6442.4
10 134429.4 75830.5 o 1746.1 56.8419.6
(Control) (Control)
30-day exposure (F1) 30-day exposure (F1)
267 267
139 272434.9 | 12.545.7 139
15 220458.7 8,547.8 35 200H10.0 | 4441.2
47 | 20se55.9 5,241.6 31 178412.5 6.44106
28 * ys7426.1 3740.8 26 | 161415.0 4 UH1.7
0 1547.5 “3.51.3 0 145415.5 4,981.7
(Contro1) Coneron) |

“No fish were analyzed because of high mortality.

Five fish were analyzed per concentration.

33


Protection Agency, 1971) should be used unless another method
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 by using the method of known additions
for all analytical methods for toxicants. If available, reference

samples should be analyzed periodically for each analytical method.

De Statistics

1.

Duplicates: Use true duplicates for each level of toxic agent,

i.e., no water connections between duplicate tanks.

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
assignment of the second tank for each level of toxic agent in

another or an extension of the same row).

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.

Fungusing of eggs: If fungusing of viable eggs occurs during
incubation it may be necessary to use a chemical fungicide. One
successful treatment with no apparent ill effects has been a
daily 5-min dip of eggs within each egg cup in Malachite green.
Prepare a stock solution of 2 Malachite green per liter of water
and retain this for use throughout the test. For treatment of
the eggs, add 0.5 ml of stock solution to 250 ml water at test
temperature and immerse each egg cup in the dip for a period of 5

min and then replace on the rocker. Opaque-white and fungused

29


TABLE 2. SURVIVAL, GROWTH, AND REPRODUCTION OF FLAGFISH (initially exposed as embryos)

AT SEVERAL CONCENTRATIONS OF ZINC (test 1)

Measured zinc concentration (ug/liter

+3
267 + 287 139 +18 +11 ae. 2 +11 (control)
we 8 A B A B A B A B A B
30_days
Survival we 10 * 0 70 80 100 7 90 100 83 90 83 7
Mean total length (mm) | -~ - 18+ 2.2% | 1840.9 |18+2.0| 18 +1.4 | 1941.7] 1941.8 | 1842.7] 1942.0 fie4 2.5] 19 + 2.0
100 days
Males/females 0/0 | 0/0 0/4 2/5 2s 2/5 2/5 2/5 2/5 2s 2/5 2/5
at termination
Mean total length (mm)
Male — |-- 4545.7 (2 5144.5 (4) 55 + 2.7 (4) 564+ 3.4 (4) 54+ 3.0 (4)
Fenale - | 40 ¥ 6.0 (9) 43 $2.5 (10) 43 $1.9 (9) 43 ¥ 2.4 (10) 43 $2.2 (10)
Mean spawnings/female - - 0.3 1.4 4.0 4.6 9.4 3.4 4.8 4A 10.8 4.8
Total embryos produced -_ _ 130 197 1,310 1,650 3,230 995 1,560 1,240 3,650 1,190
Mean hatchability _ 73 59 72 76 70

‘Mean + standard deviation.
*buplicate chamber.
“thirty fish per chamber.
4

Number of fish analyzed.

«
Significantly different from control according to Dunnett's procedure (P = 0.05).


REFERENCES

American Public Health Association, American Water Works Association, and
Water Pollution Control Federation. 1971. Standard Methods for the
Examination of Water and Wastewater. 13th ed. American Public
Health Association, New York, N.Y. 874 p.

Benoit, D. A., E. N. Leonard, G. M. Christensen, and J. T. Fiandt.
Toxic effects of cadmium on three generations of brook trout
(Salvelinus fontinalis),. Trans. Am. Fish. Soc. In press.

Brungs, W. A. 1969. Chronic toxicity of zinc to the fathead minnow,
Pimephales promelas Rafinesque. Trans. Am. Fish. Soc. 98: 272-279.

Cearley, J. E. 1971. Toxicity and bioconcentration of cadmium, chromium,
and silver in Micropterus salmoides and Lepomis macrochirus. Ph.D.
Thesis Univ. of Oklahoma, Oklahoma City.

Coleman, R. L., and J. E. Cearley. 1974. Silver toxicity and accumulation
in largemouth bass and bluegill. Bull. Environ. Contam. Toxicol.
12: 53-61.

Eaton, J. G. 1974. Chronic cadmium toxicity to the bluegill (Lepomis
macrochirus Rafinesque). Trans. Am. Fish. Soc. 103: 729-735.

Foster; N. R., J. Cairns, and R. L. Kaesler. 1969. The flagfish,
Jordanella floridae, as a laboratory animal for behavioral bioassay
studies. Proc. Acad. Natural Sci. Phila. 121: 129-152.

Foster, N. Re» A. Scheier, and J. Cairns, Jr. 1966. Effects of ABS
on feeding behavior of flagfish, Jordanella floridae. Trans. Am.
Fish. Soc. 95: 109-110.

Leonard, E. N. 1971. The determination of copper in fish tissues by
atomic absorption spectrophotometry. Atomic Abs. Newsletter. 10: 84-85.

Lingane, J. J. 1966. Analytical Chemistry of Selected Metallic Elements.
Reinhold Publ. Co., New York, N.Y. 143 p.

McAllister, W. A., Jv., W. L. Mauck, and F. L. Mayer, Jr. 1972. A

simplified device for metering chemicals in intermittent~flow bioassays.
Trans. Am. Fish. Soc. 101: 555-557.

16


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}

1. REPORT NO. 2. 3. RECIPIENT'S ACCESSION. NO.
EPA-600/3-76-096

4. TITLE AND SUBTITLE 5, REPORT DATE
CADMIUM AND ZINC TOXICITY TO JORDANELLA FLORIDAE a eaember 3076 issuing date)

7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO,

Robert L. Spehar

9. PERFORMING ORGANIZATION NAME AND ADDRESS: 10. PROGRAM ELEMENT NO.
Environmental Research Laboratory—Duluth 1BA608
U.S. Environmental Protection Agency 11, CONTRACT/GRANT NO,
6201 Congdon Boulevard
Duluth, Minnesota 55804 In-house
12. SPONSORING AGENCY NAME AND ADDRESS 13, TYPE OF REPORT AND PERIOD COVERED

Environmental Research Laboratory-Duluth

Office of Research and Development 14. SPONSORING AGENCY CODE

U.S. Environmental Protection Agency EPA-ORD
Duluth, Minnesota 55804

15. SUPPLEMENTARY NOTES

16, ABSTRACT
Cadmium and zinc toxicity to the flagfish (Jordanella floridae) was determined on the

basis of 96-hr median lethal concentrations (LC50) and significant decreases (P = 0.05
in survival, growth, and reproduction over the complete life cycle of the fish. The
96-hr LC50 values for cadmium and zinc to juvenile flagfish were 2,500 and 1,500
yg/liter, respectively. In chronic tests, reproduction was the most sensitive
indicator of cadmium toxicity and was inhibited at 8.1 yg/liter. Tissue-concentration
analysis showed that fish exposed to concentrations of 1.7 g/liter and above
accumulated significantly greater amounts of cadmium than those in the controls. In
zinc tests, survival of larvae (not exposed as embryos) and growth of females were the
most sensitive measure of zinc toxicity and were reduced at respective concentrations
of 85 and 51 yg/liter. Significant uptake of zinc occurred in fish exposed to
concentrations of 47 yg/liter and above. The lowest cadmium and zinc concentrations
causing adverse effects to the flagfish were similar to those affecting other fish
species. Application factors for both metals were similar to those reported for
cadmium exposed bluegills (Lepomis macrochirus) and zinc exposed fathead minnows

(Pimephales promelas) in hard water.

This report was submitted in partial fulfillment of Task Number 07E and ROAP Number
16-AAD by the Environmental Research Laboratory-Duluth. Work was completed as of

}May 1974. ‘ KEY WORDS AND DOCUMENT ANALYSIS
ja. DESCRIPTORS lb. IDENTIFIERS/OPEN ENDED TERMS |c. COSATI licld/Group
Water pollution eee ee er Heavy metals 6A
Metals tech * ter fishes Flagfish oF
Toxicity Presh wa Toxicity tests
Contamination Cadmium Acute effects
Fishes Zine Chronic effects
Flow-through
43. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES
UNCLASSIFIED
RELEASE TO PUBLIC 20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED
EPA Form 2220-1 (9-73) 34 ‘tr US. GOVERNMENT PRINTING OFFICE: 1976 757-056 /5436



FOREWORD

Our nation's freshwaters 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

--to measure bioaccumulation of pollutants in aquatic

organisms that are consumed by other animals, including
man

This report deals with two chemical pollutants, cadmium and zinc, and
their effects on the flagfish (Jordanella floridae). These chemicals were
utilized because they are virtually ubiquitous in the environment and because
of their observed toxic effect to some aquatic life. The flagfish was chosen
for study because of its short life cycle (6-8 wk) and for comparing its
sensitivity with other fish species having longer life cycles.

Donald I. Mount, Ph.D.

Director

Environmental Research Laboratory
Duluth, Minnesota

iii


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 TL50 for test species under the
test conditions and must never exceed 1 g/l. 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 0.1 g/l. 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

concentration 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 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.

Measurement of other variables: Temperature must be recorded

continuously (See A.11.).

27


WHOLE BODY CONTENT

T T T T T T TT T
r 100 days
:
om days
——_s
—
“A eee en nen ee LOO ys
: eer
oor - oo 4
a
lor 4
--°
arr" 30 days
oo, --
t we wwe eon”
a
oo"
e
ZINC
— 94
lr —-— 10/fish
1 1 1 1 1 1 1
20 40 60 80 100 120 140 160
EXPOSURE CONCENTRATION (y9/1)
Figure 2. Log zinc content measured from whole body tissue of flagfish

exposed to several zinc concentrations for 30 and 100 days.

13


APPENDIX A

RECOMMENDED BIOASSAY PROCEDURE FOR

JORDANELLA FLORIDAE (GOODE AND BEAN) CHRONIC TESTS

A. Physical system

1.

3.

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.

Toxicant mixing: A container to promote mixing of toxicant-

bearing and w-cell water should be used between diluter and tanks.
Separate delivery tubes should run from this container to each of
the duplicate spawning and corresponding progeny tanks for a total

of four delivery tubes for each concentration and the control.

Tank: Duplicate spawning tanks should be made of glass or a
combination of glass and stainless steel. They should measure

30- x 60- x 30-em. Separate progeny tanks (one corresponding to each
spawning tank) are of the same kind and size. Test water is to

be supplied by delivery tubes from the mixing cells described

in step 2 above.

Water depth in tanks should be 23-cm.

19


concentration (ug/gram) and total uptake (ug/fish) basis (Figure 1) show
that cadmium accumulated with age, but at a rate that was slower than the
growth of the fish. Similar results were indicated for the uptake of zinc
(Figure 2). The absence of significant additional metal uptake on a
concentration basis was probably related to mechanisms affecting rates of
uptake and elimination as was proposed by Coleman and Cearley (1974) for
silver uptake in largemouth bass (Micropterus salmoides) and bluegills.
Additionally, tissue concentration did not differ significantly between
fish exposed to zinc as embryos and those not previously exposed. This
was attributed to the inability of zinc to penetrate the egg chorion to a
large extent as was discussed by Wedemeyer (1968). Similar tissue
concentrations in parents and progeny were probably the result of the same
phenomenon. Experiments involving embryo and larval analyses would help
to clarify mechanisms of metal uptake.

Abnormal behavior of exposed fish occurred at cadmium and zinc
concentrations that caused death and inhibited reproduction. Cadmium
especially had this effect on male fish during spawning. Eaton (1974)
and Benoit et al. (in press) saw similar behavior in cadmium-exposed
bluegills and brook trout. Abnormal behavior of bass and bluegills
(uncontrolled swimming movements, convulsions, loss of equilibrium, and
apparent coma) was also observed by Cearley (1971) when these fish were
exposed to cadmium and silver. This type of behavior was attributed to the
inhibition of acetylcholinesterase, causing death by paralysis of the
muscles of respiration and/or depression of the respiratory center.

The flagfish application factors reported here for cadmium and zinc
were similar to those reported by Eaton (1974) and Brungs (1969) for
cadmium-exposed bluegills and zinc-exposed fathead minnows, respectively.
Since both bluegill and fathead minnow tests were conducted in hard water
compared to the soft water of this test, it would appear that water
hardness, at least for these tests would not affect the estimation of
chronic safe concentrations for these metals on the basis of calculated
application factors.

15


Dissolved oxygen must be measured in the tanks daily, at least

5 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 tor
pH, 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 5 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, glood, 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.
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 (U.S. Environmental

28


SECTION 5

RESULTS

CADMIUM TOXICITY

In 30 days, the highest concentration (31 ug/liter) retarded growth
and appeared to reduce survival (Table 1). Survival also appeared reduced
at the next lower concentration (16 yg/liter). However, due to the
variability of response in duplicate chambers, statistical significant
differences (P = 0.05) in survival could not be shown in any of the
concentrations. Additional mortality occurred at 16 and 31 pug/liter after
this period and by the 60th day, survival was less than 35% and growth of
fish at 16 ug/liter appeared to be reduced. Cadmium-induced mortalities of
fish were always preceded by periods of involuntary muscle spasms. At the
end of the test (approximately 100 days) all males were dead in the two
highest concentrations and growth of five surviving females was
significantly reduced. Mean spawnings/female and embryo production was
adversely affected at 8.1 ywg/liter (Table 1). No significant differences
in survival, growth, or reproduction were observed below 8.1 ug/liter.

Embryos from exposed parents incubated at 16 yg/liter and below
hatched as well as those in the control (Table 1). Survival and growth
of F, larvae were not adversely affected at these concentrations after 30
days of exposure; however, survival of larvae transferred at hatch from
control water to 31 yg/liter was less than 20%. No adverse effects were
seen when control larvae were transferred to 16 g/liter and below for
this period.

The 96-hr LC50 value calculated for 4-5 week old juveniles was
2,500 ng Cd/liter.

Fish exposed for 30 days to concentrations of 1.7 yg Cd/liter and above
contained significantly higher amounts of cadmium than did those in the
controls (Figure 1). Cadmium uptake increased with increasing exposure
concentrations but leveled off at 16 ug/liter. Concentrations of cadmium
(ug/gram) were lower in fish exposed for 100 days than those exposed
for 30 days but older fish contained a significantly higher total body
content (yg/fish). In addition, cadmium concentrations in 30-day-old
exposed parental fish and progeny (Fi) were similar (Appendix B).

10


SECTION 2

CONCLUSIONS

Reproduction was the most sensitive indicator of cadmium toxicity and
was inhibited at 8.1 ug/liter.

Significant cadmium uptake occurred in fish exposed to concentrations of
1.7 ug/liter and above. Uptake increased with increasing exposure
concentrations but leveled off at 16 yg/liter indicating an equilibrium
between tissue and water concentrations. Uptake also increased with time
but at a rate that was slower than the growth of the fish.

Survival of larvae (not exposed as embryos) and growth of females were
the most sensitive measure of zinc toxicity and were reduced at respective
concentrations of 85 and 51 yg/liter.

Significant zinc uptake occurred in fish exposed to concentrations of
47 we/liter and above. Zinc uptake increased with increasing exposure
concentrations and increased with time, but at a rate that was slower
than the growth of the fish.

The lowest cadmium and zinc concentrations causing adverse effects to the
flagfish were similar to those affecting other fish species.

Flagfish application factors for both metals were similar to those
reported for cadmium exposed bluegills and zinc exposed fathead minnows
in hard water.


10.

ll.

12.

13.

Larval exposure: From early spawnings in each duplicate tank,

use the larvae hatched in the egg incubator cups (Step B.8. above)
for 30-day growth and survival exposures in the progeny tanks.

At least one group of progeny from each adult tank should be
reared for 30 days in the corresponding progeny tank, but another
30-day growth study can be conducted with larvae transferred

from a different, non-corresponding spawning tank. Other
alternatives are to conduct only a single 30-day exposure in each
progeny tank, or to conduct two consecutive exposures with progeny
from the corresponding spawning tank. Plan ahead in setting up
eggs for hatchability so that a second group of larvae is ready
to be tested for 30 days as soon as possible after the previously
tested group comes out of the progeny tanks. Record mortalities
and total lengths and weights of larvae at 30 days. No fish
(larvae, juveniles, or adults) should be fed for 24 hr before

weighing.

Parental termination: Parental fish testing should be terminated
at the end of the 45th day or the beginning of the 46th day after
the first production in any tank of 30 or more eggs on one
spawning substrate. Record sex, total length, and total weight
of each fish.

Special examinations: Fish and eggs obtained from the test
should be subjected to physiological, biochemical, histological,
and other examinations that may indicate certain toxicant-related
effects.

Necessary data: The following data must be reported for each

tank of a chronic test:

a. Number of normal and deformed fish at 30 days, and total

length, weight, and number of either sex at termination of test;

25


stabilizers, plasticizers, etc., should not be used. Teflon,
nylon, and their equivalents do not contain leachable materials
and do not sorb significant amounts of most substances.
Unplasticized polyethylene and polypropylene do 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 the water supply is 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: Obtain original stock of flagfish from commercial

Florida supplier. The original fish should not be used as test
animals but only to initiate a laboratory stock. Use only F, or
later generations for testing. 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.

2. Beginning test: Distribute 40-50 eggs or twenty 5- to 7-day-old
larvae per duplicate tank by using a stratified random assignment
(see D.3.). Extra test animals may be added at the beginning so
that fish can be removed periodically for special examinations

or for residue analysis.

22


CONTENTS

Page
Foreword «6 1 6 eee ee ee ee ee ee FE
Abstract... ke ee eee ee iV
Acknowledgments, , ... .

1. Introduction, , ,
2. Conclusions |... lt tell lle ell
3. Recommendations , , ,
4, Materials and Methods... ........ eee eee eee
Water characteristics, .... 2... ee ee ee eee
Exposure system, . 2. 6 ee ew ee we ee ee ee
Toxicant solution, .. . 1 1 eee ee ee ee ee ee
Tissue analysis, . 2. 1 ee ee ee ee ee ee ee
Biological procedures, .. . 1 2 ee ee eee ee ee
Statistical analysis... 1. ee ee ee ee ee
5. Results... ee ee ee ee ee ee ee ee ee ee ee (10
Cadmium toxicity... 6. eee ee ee ee eee ee 10
Zinc toxicity... . 1. ee ee ee ee ee ee 12
Resulting test values and application factors. ..... 12
6. Discussion, . 1... ee ee ee ee ee ee ee te ee ee 14

WKOHORARERWNHE

References . . 1 1 ee eee ee ee ee ee ee ee ee ee 16
Appendices

A. Recommended bioassay procedure for Jordanella floridae

(Goode and Bean) chronic tests. . 2... ee eee ee eee 19
B. Cadmium concentrations in whole fish tissue after 30

and 100 days of exposure. . 2. ee ee ee ee ee ee ee 82
C. Zinc concentrations in whole fish tissue after 30 and

100 days of exposure (test 1 begun with larvae exposed

as embryos; test 2 begun with unexposed larvae) -..... 33


ABSTRACT

Cadmium and zinc toxicity to the flagfish (Jordanella floridae) was
determined on the basis of 96-hr median lethal concentrations (LC50) and
significant decreases (P = 0.05) in survival, growth, and reproduction
over the complete life cycle of the fish. The 96-hr LC50 values for
cadmium and zinc to juvenile flagfish were 2,500 and 1,500 ug/liter,
respectively. In chronic tests, reproduction was the most sensitive
indicator of cadmium toxicity and was inhibited at 8.1 yg/liter. Tissue-
concentration analysis showed that fish exposed to concentrations of
1.7 g/liter and above accumulated significantly greater amounts of
cadmium than those in the controls. In zinc tests, survival of larvae
(not exposed as embryos) and growth of females were the most sensitive
measure of zinc toxicity and were reduced at respective concentrations
of 85 and 51 wg/liter. Significant uptake of zinc occurred in fish
exposed to concentrations of 47 ug/liter and above. The lowest cadmium
and zinc concentrations causing adverse effects to the flagfish were
similar to those affecting other fish species. Application factors for
both metals were similar to those reported for cadmium exposed bluegills
(Lepomis macroc! irus) and zinc exposed fathead minnows (Pimephales promelas)
in hard water.

This report was submitted in partial fulfillment of Task Number 07E

and ROAP Number 16-AAD by the Environmental Research Laboratory-Duluth,
Work was completed as of May 1974.

iv


WHOLE BODY CONTENT

Figure 1.

ote
7S ~-----*
Tee eee 77777100 days
On
wo —— sen
-" wee,
o- me
30 days
CADMIUM
— 194
=--— y9/fish
8 2 16 20 24° «28 32

EXPOSURE CONCENTRATION (19/1)

Log cadmium content measured from whole body tissue of

flagfish exposed to several cadmium concentrations for
30 and 100 days.

11



SECTION 1

INTRODUCTION

Cadmium and zinc occur simultaneously in the environment (Lingane 1966;
Schroeder et al. 1967) and are toxic to fish (National Academy of Sciences
1973). Although most toxicity tests with fish have been short-term
acute exposures, an effort is being made at the Environmental Research
Laboratory-Duluth to determine more sensitive effects of toxicants through
chronic exposures of fish over their complete life cycle. Commonly used
species, such as fathead minnows (Pimephales promelas), bluegills (Lepomis
macrochirus), brook trout (Salvelinus fontinalis), and rainbow trout
(Salmo gairdneri), however, require long testing periods (5 months to
3 years) to obtain complete life cycle data. Smith (1973) has proposed
the use of the flagfish (Jordanella floridae) for rapid chronic bioassays
because of its short generation time (6~8 weeks) and other unique features.
Little information has been shown on the effects of toxicants on this
species in the literature (Foster et al. 1966, 1969).

The purpose of this work was to determine the toxicity of cadmium
and zinc to the flagfish by studying the effects of these metals on all
developmental stages of the life cycle. In addition, this study was
designed to evaluate the application factor concept (Mount and Stephan
1967b) with a species not previously tested.


Mount, D. I. 1968. Chronic toxicity of copper to fathead minnows
(Pimephales promelas, Rafinesque). Water Res. 2: 215-223.

Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicology studies. Water Res. 1: 21-29.

Mount, D. I., and C. E, Stephan. 1967a. A method for detecting cadmium
poisoning in fish. J. Wildlife Manage. 31: 168-172.

Mount, D. I., and C. E. Stephan. 1967b. A method for establishing
acceptable toxicant limits for fish - malathion and the
butoxylethanol ester of 2,4-D. Trans. Am. Fish. Soc. 96: 185-193.

National Academy of Sciences. 1973. Water Quality Criteria 1972. A
report of the Committee on Water Quality Criteria Environmental
Studies Board National Academy of Engineering, Washington, D.C.

Pickering, Q. H., and M. H. Gast. 1972. Acute and chronic toxicity of
cadmium to the fathead minnow Pimephales promelas. J. Fish. Res.
Board Can. 29: 1099-1106.

Schroeder, H. A., A. P. Nason, I. H. Tipton, and J. J. Balassa. 1967.
Essential trace elements in man: Zinc. Relation to environmental
cadmium. J. Chronic Dis. 20: 179.

Sinley, J. R., J. P. Goettl, Jr., and P. H. Davies. 1974. The effects
of zinc on rainbow trout Salmo gairdneri in hard and soft water.
Bull. Environ. Contam. Toxicol. 12: 193-201.

Smith, W. E. 1973. A cyprinodontid fish, Jordanella floridae, as a
laboratory animal for rapid chronic bioassays. J. Fish. Res. Board
Can. 30: 329-330.

Sprague, J. B. 1969. Measurements of pollutant toxicity to fish. 1.
Bioassay methods for acute toxicity. Water Res. 3: 793-821.

Steel, R. G. D., and J. H. Torrie. 1960. Principles and Procedures of
Statistics with Special Reference to the Biological Sciences.
McGraw-Hill Book Company, Inc., New York, N.Y. 481 p.

Wedemeyer, G. 1968. Uptake and distribution of Zn®5 in the coho salmon
egg, Oncorhynchus kistuch. Comp. Biochem. Physiol. 26: 271-279.

17




SECTION 4

MATERIALS AND METHODS

WATER CHARACTERISTICS

Untreated Lake Superior water was used in all tests at 25 +20.
Chemical characteristics of the test water were analyzed weekly according to
methods described by the American Public Health Association et al. (1971).
The means and ranges (in parentheses) for these measurements were (mg/liter):
Dissolved oxygen, 8.3 (7.2-9.5); hardness, 44 as CaCO3 (41-46); alkalinity,
42 as CaCO3 (39-44); and acidity, 2.4 as ca€o3 (1.5-3.4). The pH ranged
from 7.1 to 7.8.

EXPOSURE SYSTEM .

The exposure system consisted of a proportional diluter (Mount and
Brungs 1967) which delivered five toxicant concentrations and a control
to duplicate exposure chambers. Spawning chambers were glass aquaria,
30- x 60- x 30-cm, with a water volume of 43 liters. Larval chambers
were 15~ x 53- x 22-cm and contained 22 liters of water. Flow rate to
each spawning chamber was 15 liter/hr providing 95% replacement of test
water every 9 hr (Sprague 1969).

A combination of Duro-Test (Optima FS) and wide spectrum Gro-Lux
fluorescent bulbs provided a light intensity of 50 lumens at the water
surface. An automatically controlled 16 hr photoperiod was used.

TOXICANT SOLUTION

The stock solutions were prepared by dissolving reagent-grade CdCl,
and ZnSO,°7H20 in 15 liters of distilled water and were introduced from a
Mariotte bottle to the diluter by a chemical metering device (McAllister
et al. 1972). Weekly composite samples of the test water were analyzed
by a Perkin Elmer Model 403 atomic absorption spectrophotometer. Measurements
for cadmium concentrations in the water are included in Table 1 and those
for two zinc tests are shown in Tables 2 and 3. Analysis of variance
showed that zinc measurements were the same for the two tests. The method
of known additions of cadmium and zinc to Lake Superior (control) water was
used to construct calibration curves. The mean percent recovery and
standard deviation for 16 spiked cadmium and zinc samples were 91 + 0.8

and 100 + 2.0, respectively.


SECTION 3

RECOMMENDATIONS

Concentrations of cadmium and zinc not exceeding 4 and 26 ug/liter,
respectively, appear "safe" for the flagfish and are suggested as maximum
permissible levels under the conditions tested. However, since significant
cadmium uptake occurred in fish exposed to 1.7 yg/liter, maximum permissible
levels for cadmium may be lower in longer term exposures than were conducted
in this study. Additional tests in waters of various qualities are also
needed to more fully define the no-effect levels for these metals in natural

waters,

Further testing with this species and other toxic substances is
recommended for the determination of water quality criteria.


strand may be checked for eggs which are removed with a
propipettor. Eggs should be counted and then either retained

for incubation or discarded.

8. Egg 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 are made on the first four spawnings
and then on alternate spawnings until termination of this portion
of the study, 45 days following the first spawning of 30 or more
eggs in any tank on any one substrate. If fewer than 50 eggs are
present on any substrate 3 days after the first production of 30
or more eggs on one substrate, it is advisable to hatch those

that are present.

When larvae begin to hatch, on the 4th day or later depending
upon toxicant effect, they should not be handled again or removed
from the cups until all have hatched. At this time transfer 20
larvae to each corresponding progeny tank. If fewer than 20
larvae are present in any tank, transfer the number present.
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.

24


6.

7.

Food: All fish over approximately 2.5 cm long should be fed a
basic diet of frozen adult brine shrimp ad libitum at least

twice daily supplemented by one daily feeding with a high quality
fine granule dry trout food. Check for pesticides in each lot.
It is recommended that rapidly growing fish, between the ages of
1 week and 8 weeks, be fed as much as they will eat 6-8 times
daily. For 2-4 days following hatching, the larvae should be fed
a flour-fine dry food. If zooplankton is available it is a
superior ration. At 2-4 days of age newly hatched brine shrimp

nauplii should be given as a basis of the larval diet.

Disease: Handle disease outbreaks according to their nature; all
tanks should receive the same treatment whether all contain sick

fish or not. Hold the frequency of treatment to a minimum.

Measuring fish: Record the length and weight of individual fish
discarded at 30 days as a result of thinning (see B.6.) and of

growth-study fish at termination time of 30 days.

Thinning: When the starting fish are 30 days old, randomly reduce
the number of surviving fish in each tank to 15. Record the
number of discarded fish per tank. When fish are 5 weeks old,
Place a spawning substrate at each end of the tank. When a male
becomes territorial over each substrate in a tank remove the

extra males and randomly remove all females but five.

Removing eggs: Remove eggs from spawning substrates starting at
the same time each day in mid-afternoon. When a substrate is
removed from a tank it should immediately be replaced with a clean
substitute. Upon removal from a tank the substrate is immersed
in a shallow glass or stainless steel pan in water dipped from the

source tank. With a strong light over the work area, each yarn

23


ACKNOWLEDGMENTS

The author wishes to thank Dr. Kenneth E. Biesinger, Dr. Richard L. Anderson,
Mr. Edward N. Leonard, and Mr. James T. Fiandt for their instructive criticism
and assistance throughout the work. Sincere appreciation is extended for
Ms. Shirley L. Forseth for typing this manuscript.


SECTION 6

DISCUSSION

The response of this fish to sublethal concentrations of cadmium
and zinc was different from those of other tested species. Pickering
and Gast (1972) and Eaton (1974) found that embryos of fathead minnows
and larvae of bluegills were the stages most sensitive to cadmium
exposure. However, in the present study, spawning and embryo production
was the most sensitive measure of effect. For zinc tests, survival of
flagfish larvae and adult growth was adversely affected at lower
concentrations than those affecting reproduction. This finding was
different than the results of fathead minnow zinc exposures conducted by
Brungs (1969). He found that reproduction was the most sensitive measure
of zine toxicity. Although the response of flagfish to cadmium and zinc
was different from that of exposed fathead minnows and bluegills, the present
results indicate that flagfish sensitivity was similar to that of cadmium
exposed brook trout (Benoit, in press) and zinc exposed rainbow trout
(Sinley et al. 1974) tested in soft water.

In this test, larvae previously exposed to cadmium and zinc as
embryos were more tolerant than those not previously exposed. This effect
was much more pronounced in zinc tests as indicated by results shown in
Tables 2 and 3. Sinley et al. (1974) reported that rainbow trout not
exposed to zinc as embryos may be as much as four times more susceptible
to zinc than fish previously exposed as embryos. Although the mechanism
for this phenomenon is poorly understood, Wedemeyer (1968) showed that
coho salmon (Oncorhynchus kisutch) eggs accumulate zinc to the highest
proportion in the chorion (70%), but that the developing embryos only
accumulate a small amount (1%). Wedemeyer also pointed out that malachite
green concentrations greater than 5 ppm increased zinc permeability of the
vitelline membrane, thereby increasing the uptake of zinc in the yolk.
Although the malachite green concentrations used to control fungal
growth in the present tests were lower than those increasing zinc permeability,
the danger exists that the toxicity of zinc was affected to some extent.

No attempt was made to study the effects of this dye on zinc uptake in this
test. The known action of malachite green on zinc and possibly other
pollutants suggests, however, that it not be used in toxicity studies.

Relationships between cadmium tissue uptake and water concentration
in this test were similar to those observed by Mount and Stephan (1967a)
for cadmium-exposed bluegills. The leveling off of cadmium uptake at
16 ug/liter indicate that a possible equilibrium was reached between
cadmium concentrations in the water and in the tissues. Comparisons
between 30- and 100-day exposed fish and accumulations expressed on a

14


behavioral bioassay studies. Proc. Acad. Nat. Sci. Phila.
121:129-152.

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.

Mertz, J. C., and G. W. Barlow. 1966. On the reproductive
behavior of Jordanella floridae (Pisces: Cyprinodontidae) with
special reference to a quantitative analysis of parental fanning.

Z. Tierpsychol. 23:537-554.

Mount, Donald I, 1968. Chronic toxicity of copper to fathead

minnows (Pimephales promelas Rafinesque). Water Res. 2:215-223.

Mount, Donald I., and William Brungs. 1967. A simplified dosing
apparatus for fish toxicology studies. Water Res. 1:21-29.

Smith, W. E. 1972. A cyprinodontid fish, Jordanella floridae,
as a reference animal for rapid chronic bioassays. J. Fish. Res.
Bd. Canada 30:329-330.

U.S. Environmental Protection Agency. 1971. Methods for chemical
analysis of water and wastes. Analytical Quality Control

Laboratory, Cincinnati. 312 p.

Approved by the Committee
on Aquatic Bioassays, NWQL

Approved by the Director, NWQL

31


APPENDIX B

CADMIUM CONCENTRATIONS IN WHOLE FISH TISSUE

AFTER 30 AND 100 DAYS OF EXPOSURE

Measured Total_ug/fish
cadmium Mean
concentration Number #1
in water of standard
ug/l.) samples deviation deviation
30-day exposure
a 6 74.0429.4 1,140.2
16 10 70. 8418.6 2,041.0
8.1 10 39.3411.0 1,240.5
41 10 26.949.8 0,940.5
17 10 16.946.0 0.540.2
0.11 10 1,740.6 0.140.01
(Control)
100d ure (adult;
aL 1 34.1 404
16 4 21.148.5 3.74163
8.1 10 10. 442.4 6,542.0
41 10 6,041.6 4,541.6
7 10 4,141.3 2.30.5
0.12 10 0,240.1 0,240.01
(Control)
30-da; sure (F,
31 - - -
16 10 30.045.2 0,840.2
8.1 10 26.747 64 1,341.6
41 10 18,444.1 0,640.2
1.7 10 + 9.942.3 0.44062
0.11 10 - -
(Control)

*No detection of

cadmium in fish tissue.

32


Flow rate: The flow rate of each tank (spawning or progeny)
should be equal to 6-19 tank volumes/24 hr.

Aeration: Total dissolved oxygen levels should never be allowed
to drop below 60% saturation, and flow rates must be increased if
oxygen levels to drop below 69%. As a first alternative flow
rates may 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.

Cleaning: All spawning tanks and progeny tanks after larvae
swim up must be siphoned a minimum of two times weekly and

brushed or scraped when algal or fungus growth becomes excessive.

Spawning substrate: Substrates are made of orlon yarn, preboiled
to remove excess dye, strung on a 10- x 15-cm frame of stainless
steel. The yarn is strung so that the strands are parallel with
little or no space between strands. Any dark-colored yarn in
satisfactory. White and bright colors, such as yellow and orange,
are not accepted by the fish. Smooth-surfaced line, such as
braided nylon or monofilament, induces egg eating by adults and

is therefore not recommended.

Egg cup: Egg-incubation cups are made from 4-oz, 5-cm OD
round glass jars with the bottoms cut off. One end of the jar
is covered with stainless steel or nylon screen (with a minimum
of 16 meshes per cm). Cups are oscillated in the test water
by means of a rocker-arm apparatus driven by a 4-5 rpm electric
motor (Mount, 1968). The vertical-travel distance of the cups

should be 2,5-3.9-cm,

20


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.

ii


TABLE 3. SURVIVAL, GROWTH, AND REPRODUCTION OF FLAGFISH (not exposed

AT SEVERAL CONCENTRATIONS OF ZINC (test 2)

as embryos)

Measured zinc concentrations (ug/liter)

267 + 287 | 139 + 18 85401 sl+9 2648 (eoatron)
mys tala] a 5 A B 4 8 A 8
30 days
Survival (%)° ox 0 oF 0 20 * 23 82 97 93 93 2 87
Mean cotal length (mm) - - - - -- -- |o24 2.3 | ar4 3.5] 2rea.7 | 2241.8] 2342.4] 22424
Males/females 0/0} 0/0 | 0/0 | O/0 2/3 Us. 2s 2S 2/5 2s
at termination
Mean total length (mm)

Male - | - | - | - | sze ae od 5342.9 (4) y 53 41.7 (4) 54+ 1.8 (4)
Female - - - = | 36 # 406 (7) 38 + 5.5 (10) 40 # 3.5 (10) 43% 25 (10)
Mean spawnings/female - - - - 4.0 0.3 2.2 6.6 5.4 3,2 D4 4.6
Total embryos produced > - - - 322 i 272 2,440 1,050 348 2,320 1,360
Mean hatchability (%) 88 83 83 69

1 1 1 _

Ayean + standard deviation.
*buplicate chamber.
“thirty fish per chamber.

“number of fish analyzed.

*Significantly different from contro} according to Dunnett's procedure (P = 0.05).


graphical interpolation and were used in the determination of application
factors (maximum acceptable toxicant concentration (MATC)/96-hr LC50)
(Mount and Stephan 1967b) for both metals.

STATISTICAL ANALYSIS

For statistical evaluation, survival, growth, reproduction, and
cadmium tissue accumulation data were subjected to an analysis of
variance (P = 0.05) and Dunnett's two tailed comparison of treatment
means to control means (P = 0.05) (Steel and Torrie 1960). A two way
analysis of variance was applied to zinc accumulation data for
comparisons between tests.


EPA-600/3-76-096
November 1976

CADMIUM AND ZINC TOXICITY TO JORDANELLA FLORIDAE

by

Robert L. Spehar
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804

Program Element - 1BA608

ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804


3.

eges should be removed daily from each egg cup. Should algal
growth or detritus foul the egg-cup screen, preventing free flow
of water during the rocking cycle, the screens must be cleaned
taking care not to damage the eggs. If flow is free, do not

disturb the eggs.

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), or information may

be requested from appropriate persons at Duluth or Newtown.

Acknowledgments: These procedures for Jordanella floridae were
compiled by Wesley Smith for the Committee on Aquatic Bioassays.

References: For additional information concerning the flagfish
and continuous-flow bioassay testing, the following references

are listed:

American Public Health Association. 1971. Standard methods for

the examination of water and wastewater. 13th ed. New York. 874 p.
Drummond, Robert A., and Walter F. Dawson. 1970. An inexpensive
method for simulating diel patterns of lighting in the laboratory.

Trans. Amer. Fish. Soc. 99:434-435.

Eaton, John G. 1970. Chronic malathion toxicity to the bluegill,
Lepomis macrochirus. Water Res. 4:673-684.

Foster, Neal R., J. Cairns, Jr., and R. L. Kaesler. 1969. The

flagfish, Jordanella floridae, as a laboratory animal for

30


A

B

c

APPENDICES

Recommended Bioassay Procedures for Jordanella floridae
(Goode and Bean) Chronic Tests. ..... rare ©)

Cadmium Concentrations in Whole Fish Tissue After 30
and 100 Days of Exposure ......

Zinc Concentrations in Whole Fish Tissue After 30 and
100 Days of Exposure (test 1 begun with larvae exposed
as embryos; test 2 begun with unexposed larvae). ........ . 33

18


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:

1. Environmental Health Effects Research

2. Environmental Protection Technology

3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical

basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.


Cc.

1.

b. Mortality during the test;

Cc. Number of spawns and eggs;

d. Hatchability; and

e. Larval survival, growth, and deformities.

Chemical system

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 is acceptable, other water-miscible solvents,
such as methanol, ethanol, isopropoanol, acetonitrile,
dimethylacetamide (DMAC), 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 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
system. The suggested surfactant is p-tert-octylphenoxynonaethoxy—
ethanol (p~1, 1, 3, 3-tetramethylburylphenoxynonaethoxyethanol,
OPE,9) (Triton X-100, 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

26


9.

10.

11.

12.

13.

Light: The Duro-Test Vita~lite!,?

lights used should simulate
sunlight as nearly as possible. Fluorescent tubes have proved

satisfactory at the NWQL.

Photoperiod: A constant photoperiod of 16 hr light and 8 hr
darkness should be maintained throughout the entire test.
Gradual changes in light intensity at dawn and dusk (Drummond
and Dawson, 1970) are desirable as a sudden flood of light is a
shock to the fish. Any gradual changes of light intensity should
be included in the photoperiod and should not last for more than

1/2 hr from full on to full off and vice versa.

Temperature: Temperature should not deviate instantaneously
from 25° C by more than 2° C and should not remain outside the
range of 24°-26° C for more than 48 hr at a time. Temperature

should be recorded continuously.

Disturbance: Adults and larvae should be shielded from disturbances,
such as people continually walking past the tanks, or from extraneous

lights that might alter the intended photoperiod.

Construction materials: Construction materials that 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 organic compounds significantly. Rubber

should not be used. Plastic containing fillers, additives,

IMention of trade names does not constitute endorsement.

2Duro-Test, Inc., Hammond, Ind.

21


TABLE 1. SURVIVAL, GROWTH, AND REPRODUCTION OF FLAGFISH EXPOSED TO SEVERAL CONCENTRATIONS OF CADMIUM

Measured cadmium concentration (ug/liter)

a 0.11 + 0.07
31 + 5.6 16 + 2.9 8.1 + 2.0 4.1 + 0.81 1.7 + 0.52 (control)
A BP A B A B A 8 A B A B
30 days
Survival (%)° 70 10 7 ” 7 7 87 3 ” 100 7 7
Mean total length (mm) ]1641.5°*-- | 1941.6} 18+ 2.3] 1840.7 | 2041.7 | 2041.9] 18+ 2.3 | 1843.5] 17+ 2.1 | 2042.0] 194 1,7
|
100 days
Males/females oso oft 0/3 oft 215 us 2/5 2U5 215 25 215 215
at termination
Mean total length (mm)

Male ~ - 49 + 7.8 (4) S34 1.7 (4) 5241.7 (4) 55 + 3.3 (4)
Female a ay 334 2.1 (4) 43% 3.2 (10) 43% 3.1 (10) 43 2.2 (10) 45 ¥ 2.9 (10)
Mean spawnings/fenale o «| 0 ee) 5.2 * 3,0 10.4 8.6 11.0 ee) 8.8 8.4
Total embryos produced o *} 0 163+ 35 1,560 * 1,270 3,320 3,280 4,240 3,440 3,220 3,430

1 1
Mean hatchability (%) = 68 66 3 66 66

Mean + standard deviation.

Pbupticate chamber.

“thirty fish per chamber.

Number of fish analyzed.

“significantly different from control according to Dunnett's procedure (P = 0.05).


TISSUE ANALYSIS

Whole fish from each concentration were analyzed for cadmium and zinc
after 30 days of exposure and at the end of the test (approximately 100
days). For the cadmium test, 10 fish were analyzed per concentration
except at 16 and 31 ug/liter where only one and four fish survived,
respectively. Five fish were analyzed per concentration in each of
the two zinc tests. A 95 to 100% recovery of the metals for 35 spiked
tissue samples was obtained using a wet digestion, atomic absorption
spectrophotometric analysis (Leonard 1971).

BIOLOGICAL PROCEDURES

In chronic tests, enough embryos were incubated in each exposure
concentration (cadmium test, zinc test 1) or in the controls (zinc
test 2) to provide 30 1-day-old larvae to each chamber for the start of
the tests. Both exposed larvae and larvae not previously exposed as
embryos were utilized for zinc exposures to determine the effect of
embryo acclimation on larval sensitivity. All embryos were treated with
metal~free malachite green (4 mg/liter) for 10 min during the first 3 days
of incubation to control fungus.

Larvae were fed brine shrimp nauplii four times a day for the first
30 days. After this time, all fish were randomly reduced to 15 per
chamber and were fed a diet of frozen brine shrimp twice a day and
salmon starter granules once a day. The number of fish per chamber
was further reduced to 2 males and 5 females after males began displaying
territorial behavior, about the 60th day of the test.

Reproduction studies were started after 30 or more embryos appeared
on any one spawning substrate and were continued for a period of 45
days. Spawning substrates were approximately 13-cm long by 8-cm wide and
were made from 20 mesh stainless steel screen that was corregated and
wrapped in dark orlon yarn. After spawning, 30 to 50 embryos were
removed from the substrates, placed in oscillating egg cups (Mount 1968)
and incubated in the test water. All embryos not incubated for
hatchability determinations were counted and discarded.

Twenty newly hatched (F,) larvae from each toxicant concentration
were randomly selected and transferred to duplicate larval chambers for
30 day growth and survival exposure. In the cadmium test, control larvae
were also transferred to concentrations causing adverse effects to
parental fish, for 30 days, Additional procedures were done in accordance
with the bioassay procedures for Jordanella floridae recommended by the
Environmental Research Laboratory-Duluth (U.S. Environmental Protection
Agency 1972) (Appendix A).

Flow-through 96-hr acute toxicity tests were conducted in the chronic
test diluter system with juvenile flagfish 4-5 weeks old, according to
methods described by the American Public Health Association et al. (1971).
The 96-hr LC50 (median lethal concentration) values were derived by