U.S. DEPARTMENT OF COMMERCE
                              National Technical Information Service

                              PB-289 939
The Acute  Toxicity of Zinc to
Rainbow and  Brook Trout
Comparisons in  Hard and Soft Water

(U.S.) Environmental Research Lab, Duluth, MN
Oct 78

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United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
Brook  Trout

Comparisons in
Hard  and  Soft
Water
EPA-600/3-78-094
October 1978

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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 nine series. These nine broad cate-
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      1.  Environmental  Health Effects Research
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      3,  Ecological Research
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      5,  Socioeconomic Environmental Studies
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      7,  Interagency  Energy-Environment Research and Development
      8.  "Special" Reports
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This report has been assigned to the  ECOLOGICAL RESEARCH series. This series
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This document is available to the public through the National Technical Informa-
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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
  EPA-600/3-78-094
                              2.
4. TITLE AND SUBTITLE
  THE  ACUTE TOXICITY OF  ZINC TO RAINBOW AND BROOK TROUT
  Comparisons in Hard and  Soft Water
                                                             3.
             5. R
               October  .1978  issuing date
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

  Gary  W.  Holcombe and Robert  W.  Andrew
                                                             8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
   SAME AS BELOW
              10. PROGRAM ELEMENT NO.

                 1BA608
              11. CONTRACT/GRANT NO.
                  In-house
12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental  Research  Laboratory - Duluth,  MN
  Office of Research and  Development
  U.S. Environmental Protection Agency
  6201 Congdon,  Blvd.,  Duluth, MN  55804
                                                             13. TYPE OF REPORT AND PERIOD COVERED
              14. SPONSORING AGENCY CODE
                  EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
 The means  and ranges of  the  96-hour LCSO's  derived from  three tests were  0.55 (0.37 to
 0.76) and  2.5 (1.9 to 3.0) mg Zn/liter  for  rainbow and 2.0  (1.6 to 2.4) and 6.0 (5.0 to
 7.0) mg  Zn/liter for brook trout in soft and  hard water,  respectively.  Based on overall
 means, brook trout were  approximately 2.7 times more resistant than rainbow trout.  Zinc
 toxicity to both species  increased with increasing pH, and  decreased with increasing
 hardness and alkalinity.
17.
a.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
 Freshwater biology
 Trout
 Zinc sulfates
 Bioassay
 Toxicology
 Fishes
18. DISTRIBUTION STATEMENT

 RELEASE TO  PUBLIC
I).IDENTIFIERS/OPEN ENDED TERMS

 Z.IIK-  poisoning
 Brook  trout
 Rainbow  trout
 96-hour  LC50
 Acute  toxicity
19. SECURITY CLASS (This Report)
  UNCLASSIFIED
20. SECURITY CLASS (Thilpage)
  UNCLASSIFIED
c.  COSATl l-'icld/Croup
    06/C.T
                                                                            21. f
                            22. PRICE
EPA Form 2220-1 (R«v. 4-77)   PREVIOUS EDI TION la OBSOLETE
                                                                        «us cwttMnimmworna iin—ts?.060/IMl

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                                                EPA-600/3-78-094
                                                October 1978
THE ACUTE TOXICITY OF ZINC TO RAINBOW AND BROOK TROUT

         Comparisons in Hard and Soft Water
                         by
       Gary W. Holcombe and Robert W. Andrew
         Environmental Research Laboratory
              Duluth, Minnesota 55804
         ENVIRONMENTAL RESEARCH LABORATORY
        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 U.S. Environmental Protection
Agency, Environmental Research Laboratory-Duluth, 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
     Zinc is a ubiquitous pollutant and occurs in water from natural sources
as well.  Regulatory agencies face increasing difficulty trying to establish
acceptable limits for such pollutants and at the same time not setting limits
far below ambient concentrations which seemingly cause little harm to the
aquatic environment.

     Several important factors are known that make zinc biologically less
active and therefore less toxic.  Complexes of zinc in mineral form or with
organic materials may reduce its availability.  Inherent water quality
characteristics such as pH, hardness, and alkalinity also change the
biological activity of zinc.

     The work described in this report further elucidates the subtleties
of these latter effects.  Such information should make the task of
specifying acceptable limits easier for a variety of natural waters.
                                       Donald I. Mount, Ph.D.
                                       Director
                                       Environmental Research Laboratory-
                                       Duluth
                                     iii

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                                  ABSTRACT
     The means and ranges of the 96-hour LCSO's derived from three tests were
0.55 (0.37 to 0.76) and 2.5 (1.9 to 3.0) mg Zn/liter for rainbow and 2.0 (1.6
to 2.4) and 6.0 (5.0 to 7.0) mg Zn/liter for brook trout in soft and hard
water, respectively.  Based on overall means, brook trout were approximately
2.7 times more resistant than rainbow trout.  Zinc toxicity to both species
increased with increasing pH, and decreased with increasing hardness and
alkalinity.
                                     iv

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                                   CONTENTS
Foreword	iii
Abstract	iv
Tables	vi
Acknowledgments  	 vii

     1.  Introduction  	   1
     2.  Conclusions 	   2
     3.  Recommendations 	   3
     A.  Materials and Methods 	   A
              Exposure system  	   A
              Water characteristics  .	   A
              Toxicant solutions 	   A
              Biological methods 	   6
              Statistics 	   6
     5.  Results	   9
     6.  Discussion	11

References	1A

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                                    TABLES
Number
       Exposure Dates, Water Quality Characteristics, and Test
         Conditions of the Three Acute Zinc Exposures 	
       Measured Zinc Concentrations in the Soft and Hard Water
         Exposures During Each of the Three Tests, and Percent
         Mortality of Rainbow and Brook Trout Occurring at Each
         Concentration in 96-Hours  	
       Zinc Toxicity (LC50) and Slope of the Probit Mortality
         Curve in Soft and Hard Water in the Three Acute Tests  	    8

       Correlation Coefficients of Experimental Variables, LC50,
         and Slope of Mortality Curves for Rainbow (Upper Right)
         and Brook Trout (Lower Left), Respectively (N=6) 	   10
                                      vi

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                               ACKNOWLEDGMENTS
     We thank E.  N.  Leonard  and J.  Poldoski  for  water quality  analyses and
zinc measurements;  and other members  of the  Environmental  Research Laboratory-
Dulutli staff for  advice and  critical  review  of this  manuscript.
                                     vii

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                                  SECTION 1

                                INTRODUCTION
     Zinc is widely used in industry and is a common water pollutant; thus,
knowledge of its toxicity to aquatic organisms is important.  Numerous studies
have documented the acute toxicity of zinc to various species cf fish tested
under conditions of stable water quality.  Several of these studies include
work on salmonid species (1-5).  Another study by Nehring and Goett.1  (6)
compared the acute toxicity of zinc to four different salmonid species.  Acute
tests comparing the toxicity of zinc to rainbow trout under conditions where
water quality parameters were varied have also been conducted (7-9).  A study
has been conducted comparing the effects of varying pH and hardness on the
toxicity of zinc to fathead minnows (Pimephales promelas) (10).  Cairns et al.
(11) studied the effects of variable pH, temperature and zinc solubility in a
test on the bluegill sunfish (Lepomis macrochirus).  Both Skidraore  (12) and
Pagenkopf (13) have reviewed the current state of knowledge of the  toxicity
of zinc to fish and the relationship to water quality.

     This study included three separate exposures of rainbow trout  (Salmo
gairdneri) and brook trout .(Salveli.""5 fontinalis) to zinc (as the  sulfate) in
hard and soft water to determine acute lethality, to compare toxicity differ-
ences between the two species, and to determine the effects of water quality
changes on the toxicity of zinc to each species.  The concentration of zinc
which was lethal to 50 percent'of the fish exposed at that concentration in
96 hours (96 hour LC50) was calculated for each species of fish in  both hard
and soft water for each of the three tests.  Tests were conducted using the
same nominal zinc levels and with the same stock of fish at different ages
to yield information on the effects of size of the fish on susceptibility to
zinc.  In addition, two methods of artificially hardening water were used to
compare resulting changes in zinc toxicity.  Information gained from this
study was used to compare and supplement data obtained by other investigators,
and to estimate the effect of differing water quality conditions on the
determination of an application factor (14) for zinc.

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                                 SECTION 2

                                CONCLUSIONS
     Results of these acute tests indicated that rainbow trout were approximately
3.7 and 2.4 times more sensitive to zinc than were brook trout in soft and hard
water, respectively.  Based on overall means, rainbow trout were approximately
2.7 times more sensitive to zinc than brook trout under the same experimental
conditions.

     Increasing the mean water hardness from approximately 44 to 170 mg/1 (as
CaC03) using Ca(N03)2*4H20 did not change alkalinity, but did decrease the
toxicity of zinc by a factor of 2.5 and 2.1 for rainbow and brook trout,
respectively.

     Increasing both hardness (from 47 to 179 mg/1 as CaC03) and alkalinity
(from 42 to 170 mg/1 as CaCQ-$) using a water hardener, decreased zinc toxicity
by a factor of 5.7 to 6.8 and 3.3 to 3.9 for rainbow and brook trout,
respectively.

     Both the LC50's and the slopes of the probit mortality curves were
significantly correlated with hardness, alkalinity and pH.

     Susceptibility to zinc appeared to decrease slightly with an increase in
the size of exposed fish but there was no significant correlation of LC50's
or slopes of the mortality curves with weight of the fish.

     Zinc was slightly more toxic to rainbow and brook trout in water hardened
with Ca(N03)2*4H20 than in water hardened to the same degree with a water
hardener (18).   It appeared that this difference was correlated with changes
in alkalinity and possibly with zinc complexation or precipitation by
carbonate.

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                                  SECTION 3

                               RECOMMENDATIONS
     It appears that if a source of hard water  is  unavailable,  a  water
hardener (18)  should be used to artificially harden  water  for  use in toxicity
tests rather than Ca(N03)2*4H20.  The  latter method  does not correspondingly
increase the alkalinity with increasing hardness as  usually occurs in natural
waters.

     When fish that are being held in  soft  water are used  in a hard water
toxicity test, the fish should be acclimated to the  hard water conditions  for
at least one week or until they are actively feeding to prevent placing a
double stress on the fish.

     Better control and more accurate  measurement  of variables such as  pH,
hardness, and alkalinity, coupled with measurements  of complexation or
precipitation, are needed to adequately interpret  such metal toxicity tests
in the future.

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                                   SECTION 4

                             MATERIALS  AND METHODS
EXPOSURE  SYSTEM

     The  exposure  systems  for  these  tests  consisted  of two,  two liter/cycle,
proportional  diluters  (15)  with  dilution factors  of  0.65  and adjusted  to  cycle
almost  simultaneously.   Each diluter had a series of flow splitting chambers
(16) which divided each  of  the six concentrations equally into  two  duplicate
stainless steel tanks.   One diluter  operated  with soft water and one with
artificially  hardened water.   Both rainbow and  brook trout were exposed during
each test, each species  in  one set of duplicate tanks in  each system so that
exposure  conditions would be identical between  species for each type of
water.  The tanks  measured  92  by 36  by 41  cm  deep with a  31  cm  standpipe  which
yielded a Lank  volume of 100 liters.   Flow rates  for both diluters  averaged
650 ml/minute with a 90  percent  replacement time  of  six hours (17).  Tanks
were covered with  fiberglass mesh screens  and were surrounded by curtains to
minimize  disturbance of  the fish.  A constant 12  hour photoperiod was
used during all tests and a combination of Sylvania  Gro-Lux  and Duro-Test
Vita Lite fluorescent lamps was  the  source of illumination.

WATER CHARACTERISTICS

     Water for  all tests was obtained directly  from  Lake  Superior,  passed
through an ultraviolet sterilizer to kill  pathogens,  and  heated to  15^1°C
(Table  1) by a  hot water heat exchange system.  Water used in the three soft
water exposures was unaltered Lake Superior water.   The hard  water  used in
tests //I and #2 (Table 1) was produced by  passing Lake Superior water  through
a water hardener (18).   For test #3,  Lake  Superior water  was  hardened  by
dripping calcium nitrate (Ca(N03)2'4H20) dissolved in distilled water  into
the diluter headbox from a  Mariotte  bottle.   In this  way,  calcium hardness was
increased without  increasing the alkalinity.  Hardness, alkalinity,  specific
conductance, and pll measurements were made daily  on  the test  water  in  one or
more tanks of each diluter  system and in most cases  measurements were  made on
samples from nil tanks on one day during each test.   Water chemistry information
from all tests  Is  contained in Table  1.  Dissolved oxygen  measurements were
made twice during  each test in each  diluter and ranged from  9.4 to  10.2 mg/
liter.  All water  characteristics were measured using methods described by the
American Public Health Association (19).

TOXICANT SOLUTIONS

     Zinc sulfate  (granular, hydrated ZnSOl|-7H20,  reagent  grade), manufactured
by Mallinckrodt Chemical Company, was dissolved in distilled  water  in  a

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              TABLE 1.  EXPOSURE DATES, WATER QUALITY CHARACTERISTICS, AND TEST CONDITIONS Or THE THREE  ACUTE  ZIHC EXPOSURES
Ui
Test number
and Water
exposure dates type
#1
6-28-73 Soft
through
7-2-73
Hard I

\n
8-1-73 Soft
through
8-5-73
HardS

#3
12-17-73 Soft
through
12-21-73
Hard//

pH

7.63 (7.53-7.69)*
(14) t
7.41 (7.32-7.46)
(14)

7.58 (7.39-7.70)
(14)
7.17 (6.9A-7.A5)
(14)

7.38 (7.19-7.50)
(15)
7.31 (7. 14-7. A3)
(15)
Hardness
(ing/liter as CaC03)

46.8+l«4t
(12)1
177.6+4.6
(2A)

47.0+1.5
(12)
179.0+5.0)
(12)

44.4+0,5
(A)
169.7+4.0
(9)
Alkalinity
(mg/liter as CaC03)

41.8+0.6t
(12)?
170.2+0.7
(24)

42.8+0.2
(12)
170.1+1.3
(14)

42.5+0.7
(A)
43.0+0.3
(9)
Specific conductance
(micromhos)

72.4 (64.9-80.5)*
(14)?
261.8 (248.0-269.0).
(14)

74.3 (65.4-93.0)
(14)
256. A (227.0-280.0)
(14)

68.8 (65.5-73.1)
(15)
274.5 (256.0-312.0)
(15)
Temperature
(°C)

14.9 (14.7-15.0)*
(5)1
1A.9 (14.4-15.4)
(5)

15.2 (14.8-15.5)
(5)
15.7 (15.3-16.5)
(5)

15.0 (14.9-15.2)
(5)
14. 8 (14.6-14.9)
(5)
                - Mean and range.

                - Mean and standard deviation.

                — Number of measurements.
               §
                — Water hardened with a water hardener  (Lezike  IB) .

              * - Water hardened using Ca(H03)2i4H20.

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 Mariotte bottle and introduced to the diluters by a toxicant metering device
 (20).   Nominal zinc concentrations were different for the soft and hard water
 diluters, but were kept the same throughout the three tests for each water
 type.   Measured zinc concentrations (Table 2) varied somewhat due to slight
 changes in the toxicant metering device.  At the start of each test, after
 fish had been placed in them, the tanks were spiked with a calculated volume
 of solution from the Mariotte bottle to bring the zinc concentrations up to
 approximate nominal levels.  Daily water samples were taken from one set of
 duplicate tanks in both diluters and analyzed for zinc daily during each test.
 Zinc concentrations in the test solutions were measured by direct aspiration
 of acidified samples (10~?-K HCIO^) into a Perkin-Elmer 403 Atomic Absorption
 Spectrophotometer-with an air-acetylene flame (21).  On several occasions zinc
 concentrations were also measured by differential pulse polarography using a
 PAR Model 174 Polarographic analyzer with a dropping mercury electrode to
 determine an approximation of "dissolved" zinc.   Calibration curves constructed
 by the  method of known additions of zinc to control water were used in both
 methods of zinc analysis.  Comparisons of results from the two methods of
 analysis showed that between 90 to 100 percent of the total zinc present in
 the test water was dissolved.

 BIOLOGICAL METHODS

     The juvenile rainbow and brook trout used in the three tests were
 obtained from Cedar Bend Trout Farm in Scandia,  Minnesota.  The species were
 kept in separate fiberglass holding tanks in unaltered Lake Superior water
 maintained at 15°C prior to being transferred to the test tanks.   Fifty trout
 of each species were placed by random assignment in the respective duplicate
 tanks of both diluter systems for tests //I and #2.   In test #3,  only 30 brook
 trout were used per tank in one set of duplicates in both diluters to avoid
 overcrowding;  only 11 -rainbows were used per tank in the other sets of
 duplicates because of an inadequate supply of fish.   Exposure  dates and the
 approximate size of each species  of trout at the time of exposure are given
 in Tables  1 and 3,  respectively.   For  all tests,  the fish were allowed to
 acclimate  to test water conditions before zinc exposure was started.   Acclimation
 was  considered adequate when all  fish  were actively feeding in the test tanks,
 which required one week in test //I and three weeks  in tests #2 and //3.   Feeding
 was  discontinued 48 hours prior to zinc  exposure and fish were not fed  during
 the  tests.   Mortality was the effect  used throughout this series  of exposures.
 Procedures  used regarding aeration,  tank cleaning,  and checking for and
 removing dead  fish followed the basic  flow-through  acute toxicity test  method
 described  by the Committee on Methods  for Toxicity  Tests with  Aquatic Organisms
 (22).

 STATISTICS

     The 96 hour LCSO's and the slope  of the  percent mortality vs.  concentra-
 tion curves for each  species  in each  test  were determined using  the  probit
mtsthod  (23).   The  computer program used  (24)  fits a  linear curve  to  the probit
of the  percent  mortality  vs.  log  zinc  concentration  by iteration  of  maximum
likelihood.   Stepwise multiple regression (25) of LC50 and the slope  of the
mortality  curves  vs.  the  experimental  variables:  species,  weight  of  fish,  pH,
hardness and  alkalinity were  also  performed.   For comparative  purposes  similar
calculations were performed  using  data  from Mount (10).
                                        6

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TABLE 2.  MEASURED ZINC CONCENTRATIONS  IN  THE  SOFT AND  HARD  WATEE EXPOSURES DURING EACH OF THE THREE TESTS, AND  PERCENT  MORTALITY  OF RAINBOW AND BROOK TROUT OCCURRING AT EACH CONCENTRATION  IN  96-HOURS
Test
Soft
Ztnc cone. 96-hr 96-hr
in ralnbov brook
(Z) (!)
2.84+0.17* lOOt 78t
1.90+0.14 98 66
1.25+0.10 96 44
0.84+0.10 80 16
0.56+0.12 74 2
<0.01 2 0
(control)
«1
Hard
Zinc cone. 96-hr 96-hr
in rainbow brook
CO (I)
7.39+0.42" lOOt 78t
4.80+0.24 100 12
3.21+0.30 90 10
2.21+0.17 16 0
1.47+0.15 6 -2
<0.01 0 2
(control)
Test ti
Soft
Zinc cone. 96-hr 96-hr
in rainbow brook
nig/liter mortality mortality
(I) (I)
2.45+0.14* 96t 52t
1.55+0.11 90 46
1.00+0.05 82 6
0.64+0.03 68 8
0.41+0.02 32 0
£0.01 0 0
(control)
Hard
Zinc cone. 96-hr 96-hr
in rainbow brook
m (i)
9.64+0.07* lOOt 96t
6.58+0.18 100 28
3.83+0.08 94 4
2.34+0.15 8 0
1.31+0.03 0 0
<0.01 0 0
(control)
Test
Soft
Zinc cone. 96-hr 96-hr
in rainbou brook
m m
2.93+0.10* lOOr 73. 3i
1.83+0.10 100 20
1.32+0.09 90.9 0
0.86+0.16 36.4 0
0.53+0.04 36.4 0
<0.01 0 0
(control)
«
Hard
Zinc cone. 96-hr 96-hr
»•> (Z)
7.86+0.28* 10oT 96. 7S
4.84+0.44 100 36.6
3.31+0.26 100 10
2.19+0.14 54.5 3.3
1.49+0.15 27.3 0
<0.01 0 0
(control)
  Percent mortality of 50 fish exposed.




^ Percent mortality of 11 fish exposed.




  Percent mortality of 30 fish exposed.

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TABLE 3.  ZINC TOXICITY (LC50) AMD SLOPE OF THE PROBIT MORTALITY  CURVE IN SOFT




          AND HARD WATER IN THE THREE ACUTE TESTS '
Species Teat
tested no.
Rainbow trout 1
Rainbow trout 2
Rainbow trout 3
Brook trout 1

Brook trout 2
Brook trout 3
Rainbow trout 1
Rainbow trout 2
Rainbow trout 3r
Brook trout 1
Brook trout 2
Brook trout 3?
Mean
Fish
wt.*
(g.)
3.9
4.9
28.4
3.0
3.9
19.0
3.9
4.9
28.4
3.0
3.9
19.0
LC50
(mg/D
Soft water
0.370
(0.195-0.494)+
0.517
(0.410-0.610)
0.756
(0.538-0.944)
1.55
(1.38-1.75)
2.12
(1.41-14.4)
2.42
(2.20-2.72)
Hard water
2.51
(1.65-3.71)
2.96
(2.77-3.15)
1,91
(1.53-2.28)
6.14
6,98
(3.87-9.97)
4.98
(4.49-5,58)
Slope
(probit/log cone.)
2.98
2.87
4.66
3.68
3.22
8.15
9.42
13.84
7.37
5.21
9.08
6.90
* Mean live weight of 50 fish weighed in aggregate.




t 95 percent confidence (fiducial) limits.




T Water hardened using Ca(N03)2-4H20.

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                                  SECTION 5

                                   RESULTS
     The percent mortality resulting at all zinc concentrations  in  96 hours
for both species of trout in the three tests is shown in Table 2.   A sumnary
of the 96-hr LCSO's calculated from the mortality data, the slope of the
mortality curves (probit vs. log cone.), and size of test fish is shown in
Table 3.  The means and ranges of the 96-hr LCSO's derived from  the three
tests were 0.55 (0.37 to 0.76) and 2.5 (1.9 to 3.0) mg Zn/liter  for rainbow and
2.0 (1.6 to 2.4) and 6.0 (5.0 to 7.0) mg Zn/liter for brook trout in soft and
hard water, respectively.

     Based on overall means, brook trout were approximately 2.7  times more
resistant to zinc toxicity (higher LC50) than rainbow trout, under  the
same experimental conditions.  Linear correlations of LC50 and the  slope
of mortality curves with the experimental variables (Table 4), showed
significant correlations with the covariants; pH, hardness, and  alkalinity.
No correlation was found with fish weight.  Zinc toxicity in both species
increased (decreasing LC50) with increasing pH, and decreased with  increasing
hardness and alkalinity.  Slope of the mortality curve for both  species
decreased with increasing pH, and for rainbow trout increased with  increasing
hardness and alkalinity.  With brook trout the relationship of slope to
hardness and alkalinity was non-significant.  On the basis of these correlations,
multiple regression techniques (least squares) were used to fit  equations for
each species and the two species combined, relating LC50 to the  significant
variables pH, hardness, and alkalinity:

     Rainbow Trout LC50 (mg Zn/1)  =  12.39 - 1.65 (pH) + 0.0081
                                      (hardness) + 0.0056 (alkalinity)

     Brook Trout LC50 (mg Zn/1)    =  26.50 - 3.42 (pH) + 0.018
                                      (hardness) + 0.010 (alkalinity)
     Combined LC50* (mR Zn/1)      =  11.16 - 1.47  (pH) -I- 0.0074
                                      (hardness) -I- 0.0047 (alkalinity)
* In order to combine LCSO's from both species into a single  regression
  equation, brook trout LCSO's were divided by 2.7 which is the  ratio  of
  the mean LCSO's for the two species.

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TABLE 4.  CORRELATION COEFFICIENTS OF EXPERIMENTAL VARIABLES, LC50, AND SLOPE

          OF MORTALITY CURVES FOR RAINBOW (UPPER RIGHT) AND BROOK TROUT (LOWER

          LEFT), RESPECTIVELY (N»6)
                  LC50    Slope
                             Fish
pH      Hardness  Alkalinity  wt.
LC50 - 0.97 -0.83 0.96
Slope N.S. - -0.87 0.87
pH -0.83 -0.93 - N.S.
Total hardness 0.96 N.S. N.S.
Total alkalinity 0.85 N.S. N.S. N.S.
Fish wt. N.S, N.S. N.S. N.S.
Brook
trout
0.86 N.S.* Rainbow
trout
0.87 N.S.
N.S. N.S.
N.S. N.S.
N.S.
N.S.


* Not significant, P=0.05, to be significant at P=0.05 with N=6, r must be
  greater than 0.73.
                                       10

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                                   SECTION 6

                                  DISCUSSION
     Previously published zinc LC50's for rainbow trout exposed in soft water
agree closely, even in static exposures,  with the range of LCSO's observed
for rainbow trout (0.37 to 0.76 mg Zn/liter)  in the present study.  In a series
of soft water (hardness = 25 mg/liter as  CaC03) exposures, Goettl et al. (9)
observed 96-hr LCSO's for rainbow trout ranging from 0.24 to 0.83 mg Zn/liter
depending on size of fisli and test conditions.   Chapman (5) reported 96-hr
LCSO's for steelhead trout in soft water  (hardness * 25 mg/liter as CaC03)
ranging from 0.093 to 0.82 mg Zn/liter depending on which life stage was
exposed.  Nehring and Goettl (6) reported a 14-day LC50 of 0.41 mg Zn/liter
for rainbow trout in soft water (hardness = 40 mg/liter as CaC03).  Herbert
and Shurben (8) found the 48-hr LC50 for  rainbow trout in soft water (hardness
= 44 mg/liter as CaCO^) to be 0.91 mg Zn/liter in a static bioassay.  Soft
water LC50 values obtained from zinc exposures of salmonid species other
than rainbow trout were also within the range of those obtained for rainbows
in the present study.  Sprague (4) obtained a 24-hr LC50 of 0.65 mg Zn/liter
exposing Atlantic salmon (Salmo salar), Rabe  and Sappington (3) derived a
24-hr LC50 of 0.42 mg Zn/liter using cutthroat trout (Salmo clarki), Chapman
(26) found a 96-hr LC50 of 0.7 mg Zn/liter with sockeye salmon (Oncorhynchus
nerka), and Nehring and Goettl (6) calculated 14-day LC50s of 0.67 and 0.64
mg Zn/liter for cutthroat and brown trout (Salmo trutta), respectively.

     The hard water LCSO's for rainbow trout  (1.9 to 3.0 mg Zn/liter) in the
present study were gc'iuirjlly lower than those derived by other investigators.
Herbert and Sliurben (8) nnd Brown (2) reported 48-hr LCSO's of 3.86 and 3.5
mg Zn/liter, respectively in water with a total hardness of 320 mg/liter as
CaC03.  Solbe (1) found a 48-hr LC50 of 4.76  mg Zn/liter in very hard water
(hardness = 500 mg/liter as CaC03),  and Ball  (27) obtained a 120-hr LC50 of
4.6 mg Zn/liter in hard water.  Goettl et al. (9) derived 96-hr LCSO's
between 1.19 and 7.21 mg Zn/liter in water with a total hardness of 350
mg/liter as CaC03 depending on size of fish and test conditions.  Differences
in toxicity of zinc to rainbow trout between  the present study and those found
by other investigators are believed due mainly to differences in total hardness
and slight variations in pH between exposures.

     The range of 96-hr LCSO's for brook  trout from the present study was
1.6 to 2.4 mg Zn/liter in soft water, while Nehring and Goettl (6) observed a
14-day LC50 of 0.96 mg Zn/liter for brook trout in soft water (hardness =
40 mg/liter as CaC03).   Considering the longer exposure in the earlier work,
these results are in reasonable agreement.
                                       11

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      The relationship of  zinc  toxicity to  carbonate  alkalinity  lias  not  been
 reported previously,  probably  because  this parameter has  not  been varied
 independently of calcium  hardness.   Use of the  water hardener (18)  and
 additions of calcium  (as  the nitrate)  indicate  that  calcium hardness  and
 carbonate alkalinity  are  equally  important factors governing  the toxicity
 of zinc to fish.   Except  for the  work  of Mount  (10)  and Cairns  et al.  (11),
 pH likewise has  not been  widely recognized as having such a large effect
 on zinc toxicity.

      These tests present  an ideal situation for comparison of toxicity
 differences between species since both species  were  exposed in  the  same
 d.lluter system under  identical experimental conditions for both hard  and
 soft  water.   Rainbow  were found to be  more sensitive to zinc  than brook trout.
 in both hard and soft water.   The mean ratio of LCSO's for the  two  species
 was 2.7.   A series of acute tests comparing zinc  toxicity differences
 between species  was also  conducted by  Nehring and Goettl  (6).   They found
 rainbows to be 2.5 times  more  sensitive to zinc in soft water than were
 brook trout.   The  inverse relationship of  decreased  zinc  toxicity with
 increased water  hardness  found for both species of fish in the  present
 study agreed with  previous reports of  Cairns and  Scheier  (28) and Pickering
 and Henderson (29) on bluegills (Lepomis macrochirus), Sinley et al.  (30)
 on rainbow trout,  and Mount (10)  using fathead  minnows.   Zitko  and Carson
 (31)  also observed decreased zinc toxicity with increased water hardness on
 Atlantic  salmon  and concluded  this was caused by  a competition  for active
 sites between magnesium and zinc.

      When comparing the 96-hr  LC50 values  with  the size of fish exposed
 (Table 3)  for both species in  both soft and  hard  water (disregarding  test
 //3 in hard  water), it appears  that zinc toxicity  decreased as size increased.
 Although  not  statistically significant (p  >_ 0.05), there  appeared to  be a
 slight trend  toward decreased  zinc_toxicity  with  increased fish size.  This
 trend,  however, more  closely correlated with a  decrease in pH,  probably as
 a  result  of  increased C02 excretion  from the larger  fish.   Coodman (32),
 Goettl et al.  (9), and Chapman (5) have also found that,   in general, smaller
 rainbow trout  are  more susceptible to  zinc  than larger ones.

      Mount  (10) also  found that zinc was more toxic  to fathead  minnows with
 increasing pH  and  decreasing hardness.   Using Mount's data, a significant
 correlation of LCSO's with hardness and  pH were found.  The relationship
 with  alkalinity was non-significant,  since alkalinity was  not varied
 independently  in his  tests.  The multiple regression equation calculated from
 Mount's data was:  Fathead minnow LC50  (mg Zn/1) = 47.87  - 6.04 (pH) + 0.072
 (hardness).  This  indicated that  the toxicity of  zinc to   fathead minnows
 (LC50)  fluctuated more widely with pH or hardness changes  than  it did with
 either  of the  salmonid species tested  in the present study.  The LCSO's for
 fathead minnows however,  were much higher because a  higher proportion of the
 zinc was in a precipitated or other non-available form (13).

     Because of the effects of pH, hardness, and alkalinity on LC50 values for
 zinc,   considerable caution should be  exercised when comparing the sensitivities
of species tested in different  waters.   Slight differences in experimental
 conditions, especially pH, result  in  changes in LC50 values that make direct

                                       12

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comparisons of relntivo  acute sensitivities  impractical,  unless the  effect
of water quality on the  LC50 has been  previously  determined.   For example,  at
a constant hardness and  alkalinity the LC50  for trout  varies  by a factor of
approximately two for each unit  change in pH.   This  also  indicates that  when
an application factor is determined for zinc,  the water quality must be  the
same in the acute and chronic tests.
                                     13

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                                  REFERENCES
 1.  Solbe, J. F., D. E. L. G.  The Toxicicy of Zinc Sulphate to Rainbow
     Trout in Very Hard Water.  Water Res., 8:389-391, 1974.

 2.  Brown, V. M.  The Calculation of the Acute Toxicity of Mixtures of
     Poisons to Rainbow Trout.  Water Res., 2:723-733, 1968.

 3.  Rabe, F. W., and C. W. Sappington.  The Acute Toxicity of Zinc to
     Cutthroat Trout (Salmo clarki).  In:  Biological Productivity of the
     Coeur d' Alene River as Related to Water Quality.  1970.  Completion
     Rept. Water Resources Res. Inst. Univ. Idaho, Moscow, Idaho,  pp. 1-16.

 4.  Sprague, J. B.  Lethal Concentrations of Copper and Zinc for Young
     Atlantic Salmon.  J. Fish. Res. Board Can., 21(l):17-26, 1964.

 5.  Chapman, G. A.  Toxicities of Cadmium, Copper, and Zinc to Four Juvenile
     Stages of Chinook Salmon and Steelhead.  Trans. Am. Fish.  Soc. 107.   In
     press.

 6.  Nehring, R. B.,  and J. P. Goettl,  Jr.  Acute Toxicity of a Zinc Polluted
     Stream to Species of Salmonids.  Bull. Environ. Contain. Toxicol., 12(4):
     464-469, 1974.

 7.  Lloyd, R.   The Toxicity of Zinc Sulphate to Rainbow Trout.   Ann.  Appl.
     Biol., 48
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12.  Skidmore,  J.  F.   Toxicity of Zinc Compounds to Aquatic Animals with Special
     Reference  to  Fish.   Q.  Rev.  Biol.,  39:227-248, 1964.

13.  Pagenkopf, G. K.,   Zinc  Speciation and Toxicity to Fishes.   In:  Toxicity
     to Biota of Metal Forms in Natural Water.   Proc.  of workshop held at
     Duluth,  MM, Oct.  7-8,  1975.   Publ.  International  Joint Commission, Windsor,
     Ontario, 1976.   pp.  77-91.

14.  Mount, D.  I., and C. E. Stephan.   A Method for Establishing Acceptable
     Toxicant Limits  for  Fish—Malathion and the Butoxyethanol  Ester of
     2,4-D.  Trans. Am. Fish. Soc.,  96:185-193, 1967.

15.  Mount, D.  I., and W, A, Brungs.   A Simplified Dosing Apparatus for Fish
     Toxicology Studies.   Water Res.,  I;21-29,  1967.

16.  Benoit,  D. A., and F.  A. Puglisi.  A Simplified Flow-Splitting Chamber
     and Siphon for Proportional Diluters.  Water Res.,  7:1915-1916, 1973.

17.  Sprague, J. G.   Measurement of  Pollutant Toxicity to Fish.   I.  Bioassay
     methods for acute toxicity.   Water Res,, 3:793-821, 1969,

1-8. —Lamkej Ar  E.   A  Water Hardener  for-Experimental Use.   J. Am. Water Works
     Assoc.,  61(8) :415-416,  1969.

19.  American Public  Health  Association, American Water  Works Association,
     and Water  Pollution  Control Federation.  Standard Methods  for the
     Examination of Water and Waste  Water.  13th Ed.  New York,  N.Y., 1971.
     874 pp.

20.  McAllister, W. A., W.  L. Mauck,  and F.  L.  Mayer.  A Simplified Device  for
     Metering Chemicals in Intermittent-Flow Bioassays.   Trans.  Am. Fish,
     Soc.,  101:555, 1972.

21.  Perkin-Elmer. Analytical Methods for Atomic Absorption Spectrophotometry.
     September, 1976.   2  pp.

22.  The Committee on Methods for Toxicity Tests with  Aquatic Organisms.
     Methods for Acute Toxicity Tests  with ?ish, Macroinvertebrates, and
     Amphibians.  EPA-660/3-75-009,  U.S. Environmental Protection Agency,
     Duluth,  MM, 1975. 67 pp.

23.  Finney,  D. J. Probit Analysis.   3rd Ed.,  Cambridge Univ.  Press.   1971.
     333 pp.

24.  Stephan, C. E.,  K. A. Busch, R.  Smith,  I.  Burke,  and R. W.  Andrew.
     Computer Program for Calculating  an LC.50.,   In press.

25.  Efroynisen, M. A. Edited by A. Ralston and H. S. Wilf.  Multiple Regression
     Analysis,  Mathematical  Methods  for Digital Computers,  Part  V,  (77),  1960.
     464 pp.
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26.  Chapman, G. A.  Effects of Continuous Zinc Exposure on Sockeye Salmon
     During Adult-to-Smolt Freshwater Residency.  Trans. Am. Fish. Soc., 107,
     In press.

27.  Ball, I. R.  The Relative Susceptibilities of Some Species of Freshwater-
     Fish to Ppisons - II.  Zinc.  Water Res.,  1:777-783, 1967.

28.  Cairns, J., Jr., and A. Scheier.  The Effects of Temperature and Hardness
     of Water Upon the Toxicity of Zinc to the  Common Bluegill (Lepomis
     macrochirus Raf.).  Notul. Nat., 299, 1957.  12 pp.

29.  Pickering,  Q. H., and C. Henderson.  The Acute Toxicity of Some Heavy
     Metals to Different Species of Warm-Water  Fishes.  Air Water Pollut.
     Int. J., 10:453-463, 1966.

30.  Sinley, J.  R., J. P. Goettl, Jr., and P. H. Davies.  The Effects of
     Zinc on Rainbow Trout (Saline gairdneri)  in Hard and Soft Water.  Bull.
     Environ. Contam. Toxicol., 12(2):193-20l,  1974.

31.  Zitko, V.,  and W. G. Carson.  A Mechanism  of the Effects of Water Hardness
     on the Lethality of Heavy Metals to Fish.   Chemosphere, 5:299-303, 1976.

32.  Goodman, J.  R.  Toxicity of Zinc for Rainbow Trout (Salmo gairdnerii).
     Calif. Fish and Game, 37(2):191-194, 1951.
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