EPA-600/3-76-096
November 1976
Ecological Research Series
                         CADMIUM  AND  ZINC  TOXICITY
                              TO  JORDANELLA FLORIDAE
                                          Environmental Research Laboratory
                                         Office of Research and Development
                                        U.S. Environmental Protection Agency
                                               Duluth, Minnes

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                 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.

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                                              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

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                                 DISCLAIMER
     This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
                                      ii

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                                 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

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                                  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 yg/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.
                                      iv

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                             CONTENTS

                                                                     Page
Foreword	±±±
Abstract	^v
Acknowledgments	v^_

   1.  Introduction	    -j_
   2.  Conclusions  	    2
   3.  Recommendations	^	    3
   4.  Materials and Methods  	    4
            Water characteristics,  	    4
            Exposure system	    4
            Toxicant solution	    4
            Tissue analysis	    8
            Biological procedures	    8
            Statistical analysis  	    9
   5.  Results	10
            Cadmium toxicity  	   10
            Zinc toxicity	12
            Resulting test values and application  factors	12
   6.  Discussion	14

References	16
Appendices

   A.  Recommended bioassay procedure for Jordanella  floridae
          (Goode and Bean) chronic tests	19
   B.  Cadmium  concentrations  in whole  fish  tissue  after  30
          and 100 days of  exposure	32
   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

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                               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.
                                         VI

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                                  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.

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                                  SECTION 2

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

2.   Significant cadmium uptake occurred in fish exposed to concentrations of
     1.7 yg/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.

3.   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.

4.   Significant zinc uptake occurred in fish exposed to concentrations of
     47 yg/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.

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

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

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                                  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.

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                                   SECTION  4

                            MATERIALS AND  METHODS
WATER CHARACTERISTICS

     Untreated Lake  Superior  water was  used  in  all  tests  at  25+2  C.
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  (rag/liter):
Dissolved oxygen, 8.3  (7.2-9.5); hardness, 44 as CaC03  (41-46);  alkalinity,
42 as CaC03  (39-44); and  acidity, 2.4 as  CaC03  (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  CdCl2
and ZnSOit'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.

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


Survival (%)C
Mean total length (mm)

Males/females
at termination
Mean total length (mm)
Male
Female
Mean spawn ings /female
Total embryos produced
Mean hatchability (%)
Measured cadmium concentration (pg/liter)
31 + 5.6a
Ab

70
16 + 1.5a
Bb

10
A 	
16 + 2.9
A

77
19 + 1.6
B

77
18 + 2.3
8.1 + 2.0
A
31
77
18 + 0.7
B
days
97
20 + 1.7
4.1 + 0.81
A

87
20 + 1.9
B

93
18 + 2.3
1.7 + 0.52
A

97
18 + 3.5
B

100
17 + 2.1
0.11 + 0.07
(control )
A

97
20 + 1.0
B

97
19 + 1.7
100^ days
0/0

0/1

31 (l)d*
0 *
0 *
0
0
0/3

0/1

33 + 2.1 (4)*
1.7 * 1.0
163 •* 35
1
68
2/5

2/5

49 + 7.8 (4)
43 + 3.2 (10)
5.2 * 3.0
1,560
(>
' 1,270
6
2/5

2/5

53 + 1.7 (4)
43 + 3.1 (10)
10.4
3,320
7
8.6
3,280
3
2/5

52 + 1
43 + 2
11.0
4,240
6
2/5

.7 (4)
.2 (10)
11 .4
3,440
6
2/5

2/5

55 + 3.3 (4)
45 + 2.9 (10)
8.8
3,220
6(
8.4
3,430
Mean + standard deviation.




Duplicate chamber.




Thirty fish per chamber.




Number of fish analyzed.




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

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


                                 AT  SEVERAL CONCENTRATIONS OF ZINC (test 1)


Survival (%)c
Mean total length (nun)

Males/females
at termination
Mean total length (mm)
Male
Female
Mean spawnings/female
Total embryos produced
Mean hatchability
Measured zinc concentration (pg/liter)
267 + 28a
A"

10
—

0/0

—
—
—
-
Bb

0


0/0

—
—
—

139 + 18
A

70
18 + 2.2a

0/4

B

80
18 + 0.9

2/5

45 + 5.7 (2)d*
40 + 6.0 (9)
0.3
130
7
1.4
197
;
75 + 11
A

100
18 + 2.0

2/5

51 + 4
43 + 2
4.0
1,310
5
B
30 days
97
18 + 1.4
100 days
2/5

5 (4)
5 (10)
4.6
1,650
3
47 + 11
A

90
19 + 1.7

2/5

55 + 2
43 + 1
9.4
3,230
7,
B

100
19 + 1.8

2/5

7 (4)
9 (10)
3.4
995

28 + 11
A

83
18 + 2.7

2/5

56 + 3
43 + 2
4.8
1,560
7f
E

90
19 + 2.0

2/5

h (4)
4 (10)
4.4
1,240
>
10 + 13
(control)
A

83
18 + 2.5

2/5

B

97
19 + 2.0

2/5

54 + 3.4 (4)
43 + 2.2 (10)
10.8
3,650
7
4.8
1,190
}
^lean + standard deviation.


 Duplicate chamber,


CThirty fish per chamber.


 Number of fish analyzed.

it
 Significantly different from control according to Dunnt*tt's procedure (P =  0.05).

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




                          AT  SEVERAL CONCENTRATIONS  OF ZINC  (test  2)


Survival (%)c
Mean to ta 1 leng t h (mm )
Measured zinc concentrations (pg/liter)
26? + 28a
Ab

0
-
Bb

k 0
-
139 + 18
A

B

0*0
-
-
85 + 11
A

B
30
20 * 23
—
—
51+9
A
days
82
22 + 2.33
B

97
21 + 3.5
26 + 8
A

93
21 + 1.7
B

93
22 + 1.8
(control)
A

93
23 + 2.4
B

87
22 + 2.4
100 days
Males/females
at termination
Mean total length (mm)
Male
Female
Mean spawn ings / i ema 1 e
Total embryos produced
Mean hatchability (%)
0/0

-
-
-

0/0

-
-
-

0/0

-
-
-

0/0

-
-
-

2/3

3/4

52 + 8.4 (5)^
36 +4.6 (7)
4.0
322
f
0.3
11
8
2/5

53 + 2
38 + 5
2.2
272
8
2/5

9 (4)
5 (10)
6.6
2,440
)
2/5

53 + 1
40 + 3
5.4
1,050
8
2/5

7 (4)
5 (10)
3.2
348

2/5

54 + 1
43 + 2
9.4
2,320
6
2/5

S (4)
.5 (10)
4.6
1,360
J
Mean + standard deviation.




Duplicate chamber.




Thirty fish per chamber.




Number of fish analyzed.




Significantly different from control .i<
cording tii Dunnett's procedure  (P = 0.05).

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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 yg/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  (Fj) 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

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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.

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                                  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  pg/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 ug/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 ug/liter (Table 1).   No  significant differences
in survival, growth, or reproduction were observed  below 8.1  yg/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 Fj 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 ug/liter was less than  20%.  No adverse effects were
seen when control larvae were transferred to  16 ug/liter and  below for
this period.

     The 96-hr LC50 value calculated for  4-5 week  old juveniles  was
2,500 yg 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 Mg/liter.  Concentrations of cadmium
(yg/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

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100
                                                          ,—— •
                                                          lOOdoys
                                                          30 days  \
                                                          lOOdays
                                                           30 days
                              CADMIUM
                             — P9^
                              — pg/fish
                  8      12      16     20      24
                  EXPOSURE CONCENTRATION  (pg/l)
28
32
    Figure 1.  Log cadmium content measured from whole body tissue of
              flagfish exposed  to several cadmium concentrations for
              30 and 100 days.
                                 11

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ZINC TOXICITY

Test 1

     Survival of  larvae previously  exposed  to  zinc  as  embryos was reduced at
267 yg/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 yg/liter).  Mean
spawnings/female  and embryo  production appeared to  be  reduced at 139  yg/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 yg/liter  (Table 3).   No larvae survived
at the two highest concentrations (139 and  267 yg/liter).  By the end of the
test, female growth was significantly lower than  the controls at 85 and
51 yg/liter.  Mean spawnings/female and  embryo production appeared to be
reduced at 85 yg/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  yg/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 (Fj) 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 yg/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 yg 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

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 1000
  100
UJ
   10
                                                     100 days
                                                     • M» • £
                                                       ^.«


                      .                    ^^--—* 3O days
                    ^••^ •*->«.            ——•*
                ^-"       --*.    ---*
               fc^             ^^»^
                                           ZINC


                                          — jjg/fc

                                          — pg/fish
            20     40     60      80     100     120     140     160

                   EXPOSURE  CONCENTRATION  (pg/l)
  Figure 2.  Log zinc content measured  from whole body tissue of flagfish

             exposed to several zinc  concentrations for 30  and 100 days.
                                  13

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                                  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 Cast (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 zinc 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 yg/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

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concentration (pg/gram) and total uptake  (yg/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

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                                 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. R., 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., Jr., 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

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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, B.C.

Pickering, Q. H., and M. H. Cast.  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. Contain. 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 Zn65 in the  coho salmon
     egg, Oncorhynchus  kistuch.  Comp. Biochem. Physiol. 26: 271-279.
                                    17

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                            APPENDICES
Recommended Bioassay Procedures for Jordanella floridae
  (Goode and Bean) Chronic Tests  	 19

Cadmium Concentrations in Whole Fish Tissue After 30
  and 100 Days of Exposure	32

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

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                                   APPENDIX A

                       RECOMMENDED BIDASSAY PROCEDURE FOR
               JORDANELLA FLORIDAE (GOODE AND BEAN) CHRONIC TESTS
A.   Physical system
     1.   Diluter:  Proportional diluters (Mount and Brungs, 1967) should
          be employed for all long-term exposures.  Check the operation
          of the diluter daily, either directly or through measurement of
          toxicant concentrations.  A minimum of five toxicant concentrations
          and one control should be used for each test with a dilution
          factor of not less than 0.30.  An automatically triggered emergency
          aeration and alarm system must be installed to alert staff in case
          of diluter, temperature-control, or water-supply failure.

     2.   Toxicant mixing:  A container to promote mixing of toxicant-
          bearing and w-cell water should be used between diluter and tanks.
          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.

     3.   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.
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4.   Flow rate:  The flow rate of each tank  (spawning or progeny)
     should be equal to 6-10 tank volumes/24 hr.

5.   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 60%.  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.

6.   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.

7.   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.

8.   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.
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 9.    Light;  The Duro-Test Vita-lite1*2 lights used should simulate
      sunlight as nearly as possible.  Fluorescent tubes have proved
      satisfactory at the NWQL.

10.    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.

11.    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.

12.    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.

13.    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,
            •^Mention of trade names does not constitute endorsement.
            2Duro-Test, Inc., Hammond, Ind.
                                     21

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      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.

 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 Fj 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

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3.   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.

4.   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.

5.   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.

6.   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.

7.   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

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     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

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10.   Larval exposure;   From early spawnings in each duplicate tank,
      use the larvae hatched in the egg incubator cups (Step B.8. above)
      for 30-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.

11.   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.

12.   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.

13.   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

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          b.   Mortality during the test;

          c.   Number of spawns and eggs;

          d.   Hatchability; and

          e.   Larval survival, growth, and deformities.

C.   Chemical system
     1.   Preparing a stock solution:  If a toxicant cannot be introduced
          into the test water as is, a stock solution should be prepared
          by dissolving the toxicant in water or an organic solvent.
          Acetone has been the most widely used solvent, but dimethylformanide
          (DMF) and triethylene glycol may be preferred in many cases.   If
          none of these solvents 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,
          OPE10) (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

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     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/1. 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/1. 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.

2.    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.

3.    Measurement of other variables;  Temperature must be recorded
     continuously (See A.11.).
                                   27

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     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 for
     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

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          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.

D.   Statistics
     1.   Duplicates;  Use true duplicates for each level of toxic agent,
          i.e., no water connections between duplicate tanks.

     2.   Distribution of tanks;  The tanks should be assigned to locations
          by stratified random assignment (random assignment of one tank
          for each level of toxic agent in a row followed by random
          assignment of the second tank for each level of toxic agent in
          another or an extension of the same row).

     3.   Distribution of test organisms:  The test organisms should be
          assigned to tanks by stratified random assignment  (random
          assignment of one test organism to each tank, random assignment
          of a second test organism to each tank, etc.).

E.   Miscellaneous
     1.   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

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     eggs 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.

2.   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.

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

4.   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

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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

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                 APPENDIX B




CADMIUM  CONCENTRATIONS  IN WHOLE FISH TISSUE




     AFTER 30 AND 100 DAYS OF EXPOSURE
Measured
cadmium
concentration
in water
(UR/1.)

31
16
8.1
4.1
1.7
0.11
(Control)
31
16
8.1
4.1
1.7
0.11
(Control)
31
16
8.1
4.1
1.7
0.11
(Control)
Number
of
samples
30-day
6
10
10
10
10
10
UR/8
Mean
+ 1
standard
deviation
exposure
74.0+29.4
70.8+18.6
39.3+11.0
26.9+9.8
16.9+6.0
1.7+0.6
100-day exposure (adult)
1
4
10
10
10
10
34.1
21.1+8.5
10.4+2.4
6.0+1.6
4.1+1.3
0.2+0.1
30-day exposure (F,)
-
10
10
10
10
10
-
30.0+5.2
26.7+7.4
18.4+4.1
' 9.9+2.3
_a
Total ug/fish
Mean
+ 1
standard
deviation

1.1+0.2
2.0+1.0
1.2+0.5
0.9+0.5
0.5+0.2
0.1+0.01
4.4
3.7+1.3
6.5+2.0
4.5+1.6
2.3+0.5
0.2+0.01

0.8+0.2
1.3+1.6
0.6+0.2
0.4+0.1
a
   detection of cadmium in fish tissue.
                      32

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                          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)
Measured
zinc
concentration
in water
(UB/1.)

267
139
75
47
28
10
(Control)
Test 1
ug/g
Mean +b
standard
deviation
30-day
a
293+55.3
211+32.4
196+34.0
16O+26.9
116+29.1
Total UK/fish
Mean +
standard
deviation
exposure

8.3+4.0
4.9+1.5
6.2+3.0
4.4+2.2
3.5+1.5
Measured
zinc
concentration
in water
(ug/1.)

267
139
85
51
26
0
(Control)
Test 2
"g/g
h
Mean +
standard
deviation
30-day
_a
-

228+13.0
178+22.7
147+19.4
Total UK/fish
Mean +
standard
deviation
exposure



11.1+5.2
7.1+2.4
8.6+4.4
           100-day exposure (adult)
                                          100-day exposure (adult)
267
139
75
47
28
10
(Control)
267
139
75
47
2S
10
(Control)

324+18.7
214+41.3
212+26.6
167+29,2
134+29.4
-
137+87.5
137+52.6
128+47.7
94+23.2
75+30.5
30-day exposure (Fi)

272+34.9
220+58.7
205+55.9
1.S7+24.1
115+7.5

12.5+5.7
8.5+7.8
5.2+1.6
3.7+0.8
'3.5+1.3
267
139
85
51
26
0
(Control)
267
139
85
51
26
0
(Control)
-

235+13.7
211+20.7
174+28.9
117+6.1


155+87.9
110+29.9
86.6+42.4
56.8+19.6
30-day exposure (F])


200+10.0
178+12.5
161+15.0
145+15.5


4.4+1.2
6.4+1.6
4.4+1.7
4.9+1.7
 **No fish were analyzed because of high mortality.




 Five fish were analyzed per concentration.
                              33

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                                   TECHNICAL REPORT DATA
                            (Mease read luamctions on the reverse before completing)
 . REPORT NO.
  EPA-600/3-76-096
                                    3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
  CADMIUM AND ZINC  TOXICITY TO JORDANELLA FLORIDAE
                                                           5. REPORT DATE
                                                            November 1976
                                                     (Issuing date)
                                                           6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)
                                                           8. PERFORMING ORGANIZATION REPORT NO.
  Robert L. Spehar
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  Environmental Research Laboratory-Duluth
  U.S. Environmental  Protection Agency
  6201 Congdon Boulevard
  Duluth, Minnesota   55804
                                     10. PROGRAM ELEMENT NO.
                                       1BA608
                                     11. CONTRACT/GRANT NO.

                                       In-house
 12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental Research Laboratory-Duluth
  Office of Research  and Development
  U.S.  Environmental  Protection Agency
  Duluth, Minnesota   55804
                                     13. TYPE OF REPORT AND PERIOD COVERED
                                     14. SPONSORING AGENCY CODE

                                                EPA-ORD
 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
  pg/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 yg/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	
      1974.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                                                    COSATI Field/Group
    Water pollution
    Metals
    Toxicity
    Contamination
    Fishes,
Effluents
Fresh water
Fresh water  fishes
Cadmium
Zinc
Heavy metals
Flagfish
Toxicity tests
Acute effects
Chronic effects
Flow-through
6A
6F
 3. DISTRIBUTION STATEMENT

  RELEASE  TO PUBLIC
                       19. SECURITY CLASS (This Report)
                        UNCLASSIFIED
                                                                           . NO. OF P,
                       20. SECURITY CLASS (This page)
                        UNCLASSIFIED
                                                  22. PRICE
                          _40_
EPA Form 2220-1 (9-73)
                                             34
                                            £ U.S. GOVERNMEKT PRINTING OFFICE: 1976-757-056/5436

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