Ecological Research Series
 TOXICITY  OF  COPPER TO DAPHNIDS  IN
RECONSTITUTED AND  NATURAL  WATERS
                           Environmental Research Laboratory
                          Office of Research and Development
                          U.S. Environmental Protection Agency
                                Duluth, Minnesota 55804

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

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                                     EPA-600/3-76-051
                                     May 1976
    TOXICITY OF COPPER TO DAPHNIDS IN

    RECONSTITUTED AND NATURAL WATERS
                   by

            Robert W. Winner
            Miami  University
           Oxford, Ohio  45056
            Grant No.  R802210
             Project Officer

          Timothy W. Neiheisel
     Newtown Fish Toxicology Station
Environmental  Research Laboratory-Duluth
         Cincinnati, Ohio  45244
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
   OFFICE OF RESEARCH AND DEVELOPMENT
    ENVIRONMENTAL RESEARCH LABORATORY
        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.  Approval does not signify that the contents neces-
sarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
                                   ii

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                                ABSTRACT
        The toxicity of copper was compared for Daphnia magna cultured in
reconstituted versus pond water and fed on trout-pellet versus vitamin-
enriched, algal foods.  Effects of a chronic copper stress were highly
variable when animals were tested in reconstituted waters.  This varia-
bility is thought to be due to variability in the quality of the
distilled-water matrix.  The vitamin-enriched algal food was found to be
superior to the trout-granule food in culturing D_. magna.  Control ani-
mals lived much longer and test animals were less sensitive to a chronic
copper stress.  The acute and chronic toxicity of copper was also com-
pared for four species of Daphnia.  When tested in pond water and fed
vitamin-enriched algae, the two largest species (Eh magna and D_. pulex)
were significantly less sensitive to an acute copper stress than the two
smallest species (£. parvula and D_. ambigua).  There was, however, no
significant difference in sensitivity to a chronic copper stress when re-
duced longevity was used as the index.  Application factors for the four
species varied from 0.47 to 0.62 and were not significantly different.

        This report was submitted in fulfillment of Grant No. R802210 be-
tween the Environmental Protection Agency and Miami University.  Work was
completed as of September 30, 1975.
                                    iii

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                                CONTENTS
Section                                                             Page
        ABSTRACT	111
        FIGURES	   vi
        TABLES	viii
        ACKNOWLEDGMENTS 	    x
  I.    CONCLUSIONS 	    1
 II.    RECOMMENDATIONS 	    4
III.    INTRODUCTION  	    5
 IV.    METHODS	    7
          Culture Techniques  	  .    7
          Toxicant Information	,  10
          Techniques for Monitoring the Copper Stress  	   10
  V.    RESULTS AND DISCUSSION	   12
          Chemical  Characteristics of Test  Waters  	   12
          Toxicant Concentrations in Test Vessels  	   12
          Reconstituted Waters as Culture Media for  Daphnids   ...   14
          Acute Toxicity of Copper  	   22
          Chronic Toxicity of Copper  	  .   25
          Effect of a Chronic Copper Stress on the Instantaneous
            Rate of Population Growth 	   59
 VI.    REFERENCES	   67

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                                FIGURES
No.                                                                 Page

 1.    Survival of Daphm'a magna fed algae in unaerated
       filtered, standard water	   15

 2.    Survival of Daphm'a magna in unfiltered aerated versus
       unaerated standard water 	   17

 3.    Survival of Daphm'a magna in unfiltered versus aerated ultra-
       pure standard water  	   18

 4.    Survival of Daphnia magna fed algae in filtered, unaerated
       medium water 	   20

 5.    Comparative survival and reproduction of Daphnia magna in
       filtered, unaerated standard, medium, and pond water ....   21

 6.    Survival of Daphnia magna exposed to chronic copper stresses
       in the pond-water, algal system (Experiments 1 and 2)  ...   26

 7.    Survival of Daphnia ambigua exposed to chronic copper stresses
       in the pond-water, algal system (Experiments 1 and 2)  ...   27

 8.    Survival of Daphm'a pulex exposed to chronic copper stresses
       in the pond-water, algal system (Experiments 1 and 2)  ...   28

 9.    Survival of Daphm'a parvula exposed to chronic copper stresses
       in the pond-water, algal system (Experiments 1 and 2)  ...   29

10.    Combined survivorship curves (Experiments 1 and 2) for
       Daphnia magna exposed to chronic copper stresses in the pond-
       water, algal system	   32

11.    Combined survivorship curves (Experiments 1 and 2) for
       Daphnia ambigua exposed to chronic copper stresses in the
       pond-water, algal system 	   33

12.    Combined survivorship curves (Experiments 1 and 2) for
       Daphnia parvula exposed to chronic copper stresses in the
       pond-water, algal system 	   34

13.    Combined survivorship curves (Experiments 1 and 2) for
       Daphnia pulex exposed to chronic copper stresses in the pond-
       water, algal system	   35

                                   vi

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                                 FIGURES

                               (Continued)

No-                                                                 Page

14.
Survival of Daphnia magna fed algae and exposed to chronic
copper stresses in filtered, unaerated standard water  .  .
                                                                      37
15.    Survival of Daphnia magna fed algae and exposed to chronic
       copper stresses in unfiltered, aerated standard water  ...   38

16.    Survival of Daphnia magna fed algae and exposed to chronic
       copper stresses in unfiltered, unaerated standard water  . .   39

17.    Survival of Daphnia magna fed algae and exposed to chronic
       copper stresses in medium water  	   41

18.    Effect of trout-granule food on the sensitivity of Daphnia
       magna to chronic copper stresses in pond water	   42

19-    A comparison of longevity in Daphnia magna fed algae versus
       trout-granule food in pond water 	   44

20.    Temporal changes in the mean brood size of Daphnia magna
       exposed to chronic copper dosages  	   45

21.    Temporal changes in the mean brood size of Daphnia put ex
       exposed to chronic copper dosages  	   46

22.    Temporal changes in mean brood sizes of Daphnia parvula
       exposed to chronic copper dosages  	   47

23.    Temporal changes in mean brood sizes of Daphnia ambigua
       exposed to chronic copper dosages  	   48

24.    Effect of food type on age at reproductive maturity of
       Daphnia magna in pond water  	   60
                                  Vll

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                                  TABLES
 No.                                                                  Page

  1.     Composition  of  the  medium used  in  culturing  Chiamydomonas
        reinhardii  	     8

  2.     General  chemical  characteristics of the  three  test media  .  .     9

  3.     Copper concentrations  in  test beakers  after  72 hours  of
        incubation	    13

  4.     Acute toxicity  (72-hr  LC50  in ppb)  of  copper to four  species
        of Daphnia  in the pond-water, algal  system 	    23

  5.     Acute toxicity  of copper  and  chromium  to j).  magna and £.
        ambigua  in unaerated,  filtered  standard  water   	    24

  6.     Acute toxicity  of copper  to algal-fed  ID. magna in differ-
        ent waters  ..... 	    24

  7.     Acute toxicity  of chromium  to four  species of  Daphnia in
        the pond-water, algal  system  	    25

  8.     Vital statistics  for three  chronic  copper  toxicity tests
        with D. magna in  the pond-water, algal system   	    31

  9.     Relationship between food and soluble  copper concentration
        remaining in test beakers after 72  hrs of  incubation  in
        pond water	    40

10.     Analyses of cumulative mean brood sizes  for J).  magna  exposed
        to five copper  concentrations in the pond-water, algal
        system	    49

11.     Analyses of cumulative mean brood sizes  for D_.  pulex  exposed
        to five copper  concentrations in the pond-water, algal
        system	    50

12.     Analyses of cumulative mean brood sizes  for J).  parvula ex-
        posed to five copper concentrations in the pond-water,
        algal system	    51

13.    Analyses of cumulative mean brood sizes for j).  ambigua ex-
        posed to four copper concentrations in the pond-water,
        algal system	    52

                                   viii

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                                 TABLES

                               (Continued)


Np_.                                                                 Page

14.    Total young and mean brood size for parental-, FI- and F2-
       generations of D_. magna on a 28-day chronic-copper stress
       in the pond-water, algal system  	   53

15.    Per cent survival of parental, FI, and Fa generations of
       £. magna after 28 days of copper stress in the pond-water,
       algal system	   53

16.    The relationship between mean brood size and copper con-
       centration for ]D. magna in reconstituted waters	   55

17.    Effect of food type on survival and reproduction of Daphnia
       magna in pond water	   57

18.    Analyses of cumulative mean brood size for j). magna in five
       copper concentrations when fed trout-granule food in pond
       water	   58

19.    Mean age at reproductive maturity of 13. magna as affected
       by food type and copper concentration in pond water  ....   59

20.    Instantaneous rate of population growth (r) as affected by
       copper for four species of Daphnia in the pond-water, algal
       system	   61

21.    Effect of a copper stress on the instantaneous rate of
       population growth of D_. magna in the trout-granule, pond-
       water system	   62

22.    Maximum allowable toxicant concentration of copper (ppb) for
       four species of Daphnia, based on longevity, brood size and
       r_	   63

23.    Application factors of copper for four species of Daphnia
       in the pond-water, algal system  	   64

24.    Effect of water and food type on copper application factors
       for Daphnia magna  	   65
                                   ix

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                             ACKNOWLEDGMENTS


        This research has benefitted from the dedicated assistance of a
number of individuals.  Robert Debelak and William Randall  were respon-
sible for maintaining stock cultures of animals and algae and  for most of
the daily tedium of checking experimental animals.  Theodore Keeling and
Robert Yeager collected the data on the sensitivity of trout-granule-fed
animals to copper.  Michael Parrel 1 provided invaluable assistance in
statistical analysis and in computer programming.

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                               SECTION I

                              CONCLUSIONS
1.  Survival of Daphnia magna was highly variable in Marking's (1969)
    reconstituted, standard water and medium waters.  Most of the varia-
    bility is thought to be due to variations in the quality of the dis-
    tilled water.  Since longevity was improved by vigorous, overnight
    aeration of the distilled water, the variable quality of the water
    was probably due to one or more volatile toxicants.

2.  J). magna reared in reconstituted, standard water lost the biramous
    portions of their swimming antennae about 24 days after birth.  Nei-
    ther the use of an ultrapure standard water, nor vigorous aeration
    of the distilled water base, prevented the development of this an-
    tenna! damage.  The onset of antennal damage was, however, delayed
    from day 24 to day 57 by doubling the calcium concentration of the
    standard-water formula.  Antennal damage was never observed in the
    pond-water system.

3.  Unless a methodology can be developed for producing a distilled
    water of consistently high quality in laboratories whose raw-water
    supply is of highly variable quality, bioassay research involving
    the use of reconstituted waters is likely to produce results which
    are as, or more, variable than results which are produced using
    natural waters.

4.  The vitamin-enriched algal food of Murphy (1970) proved excellent
    for the maintenance of six species of Daphnia (D_. magna, ]). ambigua,
    ID. parvula, D_. pulex, D_. pulicaria, and I), galeata-mendotae).  When
    animals were fed this food, and maintained in pond water, we had no
    trouble with population depressions or die-offs in our stock cul-
    tures over a 2-year period.

5.  Survival and reproduction of animals was highly reproducible in both
    algal, pond-water and trout-granule, pond-water systems under both
    control and experimental conditions.

6.  The two largest species (D_. magna and D_. pulex) were significantly
    less sensitive (P = 0.05) to an acute copper stress than were the
    two smallest species (J). parvula and ]3. ambigua).

7.  The 72-hr LC50 values for copper were very close, and not signifi-
    cantly different (P = 0.05) for D_. magna tested on algal- and trout-
    granule foods in pond water.

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 8.   Interspecies response to an acute chromium stress was much more
     variable than to an acute copper stress, with D_. ambigua being most
     sensitive and ]D_. pulicaria being least sensitive.

 9.   The MATC was estimated to be 40 ppb copper for D_. magna, D_. ambigua.,
     ]D. parvula, and D_. pulex in the pond-water, algal system when lon-
     gevity was used as the criterion.  However, the MATC was estimated
     to be 10 ppb copper for D_. magna tested in the trout-granule, pond-
     water system.

10.   A 3-week time interval is not sufficient to determine the effect of
     a chronic copper stress on survival of D_. magna in pond water.  The
     effect of a chronic copper stress on the three smallest species (D_.
     pulex, D_. parvula, and |). ambigua), however, can be evaluated within
     a 3-week time period.

11.   Fj and F2 generations of D. magna were no more susceptible, or re-
     sistant, to a chronic copper stress than was the parental generation.

12.   The MATC, based on longevity, was estimated to be between 5.0 and
     7.5 ppb copper for D_- magna tested in unaerated, standard water and
     between 15.0 and 20.0 ppb in aerated, standard water.

13.   JD. magna exhibited a highly variable response to a chronic copper
     stress in medium water, with the MATC varying between 10 and 60 ppb.
     Differences are thought to be due to variations in distilled-water
     quality.

14-   Reproductive response, of four species,  to a chronic copper stress
     was more variable than was longevity in the pond-water, algal sys-
     tem.  EL magna did not exhibit a reduction in mean brood size at any
     concentration in which animals lived long enough to reproduce and
     did exhibit a stimulation of reproduction up to a concentration of
     60 ppb.  There was never a stimulatory effect of copper on brood
     sizes in J3. pulex, JD. parvula, or D_- ambigua.  ]3. pulex exhibited a
     significant reduction in mean brood size at 80 ppb, D_. parvula at
     60 ppb, and J). ambigua at 40 ppb (P = 0.05)

15.   In JD. magna. J). pulex and JL  parvula, survival was a more sensitive
     index of a chronic, copper stress than was reproduction.  In D_. am-
     bigua, survival and reproduction were equally sensitive as indices
     of a chronic, copper stress.

16.   When fed the trout-granule food, D_. magna exhibited a reduction in
     mean brood size at all copper concentrations greater than 10 ppb.

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     There was, also, a delay in attaining reproductive maturity at high-
     er copper concentration which did not occur in algal-fed animals.

17.   The trout-granule food was definitely inferior to the algal food.
     Animals reared on the trout-granule food exhibited reduced longevity
     under control conditions and greater sensitivity to copper as mea-
     sured by either mean brood size or longevity.

18.   Daphnia parvula, D_. pulex, and]), ambigua all exhibited reductions
     in the instantaneous rate of population growth (r.) at copper concen-
     trations greater than 40 ppb.  Because of the stimulatory effect of
     copper on brood size in D_. magna, jr did not decline in this species
     until the chronic copper stress exceeded 60 ppb.

19.   Application factors for P.. magna, D_. pulex, D_. parvula and D_. am-
     bigua were 0.47* 0.57, 0.62, and 0.59, respectively.

20.   Application factors determined for daphnids (= 0.5) were much larger
     than literature values of copper application factors for fish (= 0.1)

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                               SECTION II

                             RECOMMENDATIONS


1.  Reconstituted waters should not be recommended for routine bioassay
    work unless, or until, a methodology can be developed for producing a
    distilled-water base of uniformly high quality.

2.  More work is needed to adequately define the dissolved mineral  re-
    quirements of daphnids before we can confidently recommend a mineral
    composition for reconstituted waters.

3.  The nutritional adequacy of a food should be demonstrated before it
    is used in routine bioassay work.  It may well be that the same food
    will not be adequate for different species of cladocerans or even for
    different species within the genus Daphm'a.

4.  An application factor of 0.5, based on a 72-hr LCso and an MATC cal-
    culated from reductions in longevity, should predict a safe concen-
    tration of copper for daphnids which are not exposed to other
    stresses.

5.  A reduction in longevity should be used as the most sensitive index
    of a chronic copper stress in daphnids.

6.  Although D_. magna's sensitivity to copper was not significantly dif-  .
    ferent from that of the other species tested, further research  is
    needed to determine whether this is true in relation to other toxi-
    cants.

7.  Concerted effort should be devoted to determining whether safe  con-
    centrations based on chronic bioassay experiments with single species
    under laboratory conditions are adequate to protect the important
    components of communities.  Laboratory-derived safe concentrations
    should be tested on more complex systems, such as experimental  ponds
    or streams.

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                               SECTION III

                              INTRODUCTION
        Laboratory studies on the toxicity of chemicals to aquatic organ-
isms may be carried out for a variety of reasons.  One of these is to
produce information that will contribute to predicting the effects of
chemicals on the biota of aquatic ecosystems.  This is an extremely dif-
ficult objective to achieve because of the inordinately complex nature of
ecosystems.  Both the abiotic and the biotic components are not only very
complex, but they are also highly variable in space and time.   If the in-
tegrity of an ecosystem is to be protected, concentrations of chemicals1
must not be allowed to exceed the tolerance of the most sensitive species
under those environmental conditions and at those times when it is most
sensitive.  The difficult task is that of predicting a safe concentration
for a large number of species exposed to fluctuating, physical-chemical
conditions.  Even if one pursues the more limited objective of protecting
only certain "important" or "valuable" species, the task is still  exceed-
ingly difficult.

        Most of the literature dealing with the acute and chronic effects
of chemicals on aquatic life is concerned with effects on a few species
of fish and on a very few species of invertebrates.  Virtually all of the
work on planktonic invertebrates (zooplankton) has dealt with  a single
species, Daphnia magna (Kemp et al_., 1971).  J). magna has a very limited
geographic range in N. America and even within this range it is confined
to small bodies of water (Brooks, 1957).  Probably, very few aquatic bio-
logists in the U. S. have ever seen a wild specimen of D_. magna.  Since ID.
magna is not a frequent component of zooplankton communities,  continued
use of this species in research designed to formulate water-quality stand-
ards can be justified only if it can be demonstrated that D_. magna has a
sensitivity comparable to that of more widespread, important members of
zooplankton communities.  One of the objectives of this study  was to com-
pare the copper sensitivity of D_. magna with that of other, more common,
species of Daphnia.

        The ability to predict safe concentrations for chemicals is com-
plicated by the necessity to take into account the sensitivity of the
species throughout its life cycle.  Safe concentrations, ideally, should
be based on tests of the sensitivity of a species throughout several gen-
erations.   This ideal is continuously compromised because of the immedi-
ate need for setting water-quality standards and the difficulty in main-
taining many species under laboratory conditions.  It has been suggested
that the ratio between chronic and acute toxicity of a toxicant (the ap-
plication factor of Mount and Stephan, 1969) is relatively constant for
closely-related species or in waters of differing chemical composition.

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If this concept were to prove valid, the factor could be used  to extrapo-
late from the acute response of a species to its chronic response.   Al-
though there are some published data on application factors in fish,
there are no such data available for daphnids.  A second objective  of the
present study was to evaluate the application-factor concept as it  applies
to daphnids.

        Finally, much of the variability in bioassays of heavy metals is
known to be a consequence of differences in the chemistry of test waters.
It has been proposed (e.g., Marking, 1969;  Stephen, 1975) that this vari-
ability can be eliminated, or greatly reduced, by using reconstituted
waters of known chemical composition in bioassay research.   There are no
published data, however, to indicate that reconstituted waters are  physi-
ologically adequate for the long-term maintenance of any invertebrate
animal.  The third objective of this grant  was to evaluate the adequacy
of reconstituted waters as culture media for daphnids.

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                               SECTION IV

                                 METHODS
CULTURE TECHNIQUES

General

        Both acute and chronic testing were initiated with new-born
daphnids (1-12 hrs old).  Each animal was maintained, individually, in 40
ml of medium in a 50-ml, Pyrex or Kimax glass beaker.  All animals were
maintained in environmental chambers at a temperature of 20*C, on a 15-hr
photoperiod, at approximately 300 foot candles of illumination.

        Each animal was fed and examined daily; in chronic experiments
this was continued until all animals were dead (i.e., exhibited no move-
ment).  Daily records were maintained for each animal to indicate:  molts,
age at reproductive maturity, and whether the animal was gravid.   Off-
spring were counted and removed daily.  Any abnormalities in appearance
were also noted.

        Each animal was transferred to fresh medium, in a clean beaker,
by eye dropper at 3-day intervals.  Tips of eye droppers were cut off to
prevent mechanical damage to the larger daphnids.  All glassware was
cleaned by soaking in 7% nitric acid for at least 24 hrs.

Foods

        All stock animals, and animals in most of the experiments, were
fed vitamin-enriched algae (Ch1amydomonas re1nh ard i1).  The chemical com-
position of the medium in which the algae were cultured is given in Table
1.  Algae were cultured in Corning 4422 wide-bottomed culture flasks.
Each flask contained 1 L of medium and was incubated under the same light-
temperature regime as that described for the daphnids.  Vitamins were
added to the cultures at the concentrations suggested by Murphy (1970)..
Air, enriched to 10% with C02. was bubbled through the cultures,  and the
cultures shaken, several times each day.

        The algae were concentrated from the culture medium by centrifug-
ing through a non-metallic, continuous-flow centrifuge (6. M. Mfg. Corp.,
El Cajon, CA).  The algal concentration was adjusted to a volume such
that it gave a reading of 1.5 absorbance units, at a wavelength of 665
millimicrons, in a spectrophotometer.  Three drops of this concentrate
were fed to each animal daily.  This averaged 0.34 mg, dry weight, or
0.31 mg, ash-free dry weight, of algae per feeding.

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TABLE 1.   COMPOSITION (MG/LITER) OF THE MEDIUM
               USED IN CULTURING
           Chlamydomonas reinhardii
  Constituent               Concentration (mg/1)
NaN03
CaCl2.2H20
MgSOiL.7H20
K2HPOit
KH2PO^
NaCl
EDTA
FeSOi,
H3B03
ZnS04.7H20
MnCl2.4H20
Mo03
CuSoit.5H20
Co(N03)2.6H20
Calcium pantothenate
B12
Thiamin
Riboflavin
Nicotinamide
Folic acid
Biotin
Choline
Putrescine
5.0
0.25
0.75
0.75
1.75
0.25
0.05
0.005
0.012
0.009
0.001
0.0007
0.0016
0.0005
7.0
0.0008
0.6
0.4
1.3
3.3
0.3
5.0
0.3

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        One series of chronic tests was carried out using the trout-granule,
dried-grass, food developed at the Duluth National Water Quality Labora-
tory (Biesinger and Christensen, 1972).  Glencoe trout-starter granules
and the dried grass, which was actually a commercially-available health
food called Cerophyl (Cerophyl Lab. Inc., Kansas City, MO), were kindly
provided by K. B. Biesinger.  The trout granules were ground in a mortar
and pestle and sieved, dry, through #20 plankton netting to remove the
larger particles.  The food suspension contained 0.4 g of trout granule
and 0.02 g of Cerophyll per liter of pond water, was made up fresh weekly
and kept in a refrigerator.  Each animal was fed 114 microliters of the
mixture daily.  This averaged 0.42 mg, dry weight, and 0.31 mg, ash-free
dry weight, of food per feeding.  By coincidence, the ash-free dry weights
of algae and trout granule foods, per feeding, were identical.

Culture Media

        Daphnids were reared in three different kinds of water at various
times during the study.  Most of the work, both acute and chronic, was
done in a local pond water.  Considerable time and effort, however, was
expended in trying to develop a "reconstituted" water that would be
satisfactory for the rearing of daphnids.  These reconstituted waters
were created by dissolving reagent-grade salts in double-, glass-distilled
water.  The salt concentrations are those originally suggested by Marking
(1969) for waters that he designated as "standard" and "medium" water.
Chemical characteristics of the pond and the two reconstituted waters are
presented in Table 2.  Reconstituted waters were also made up from double-
distilled water that had been further processed by filtration.  Both a
Barnstead, D-8904, Organic Removal Filter and an Aqua-Pure, P50-1, Taste-
and-Odor-Removal Filter (Cuno Eng. Corp., Meriden, Conn.) were tested to
see whether or not they would improve the quality of the reconstituted
waters.  Finally, an ultrapure water, kindly provided by Dr. William
Hoyle of the Miami University Chemistry Department, was tested in an at-
tempt to improve the quality of the reconstituted water.  This ultrapure


   TABLE 2.  GENERAL CHEMICAL CHARACTERISTICS OF THE THREE TEST MEDIA
Water
pH range
Total
Hardness
Total
Alkalinity
Standard               6.8-7.9              40-48                30-35
Medium-Hard            8.2-9.5             160-180              110-120
Local Pond             8.2-9,5             130-160              100-118

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water was produced by processing water from a still through, in sequence,
a mono-bed deionizer, an alkaline permanganate distillation and two glass
stills.
TOXICANT INFORMATION

        Copper was added to the culture medium as reagent grade
A fresh stock solution of 100 ppm Cu was made up in double, glass-
distilled water on each day of use.  Copper content of the double-
distilled  water was less than 1.0 ppb and was determined with a Model
453 Instrumentation Laboratory atomic-absoprtion spectrophotometer using
an APCD-MIBK extraction (Analytical Qual. Control Lab., 1971) and the
method of standard additions (Christian and Feldman, 1970).  Test concen-
trations were prepared by transferring appropriate aliquots of the 100
ppm stock solution to the test media with a microliter pipette.  Concen-
trations of copper in the test media were monitored by atomic-absorption
spectroscopy.


TECHNIQUES FOR MONITORING THE COPPER STRESS

Effect of Copper on Longevity

        Differences in longevity were determined by following each cohort
until all individuals had died.  Differences in survival  curves were ana-
lyzed by chi-square analysis (Mantel, 1966) in a computer program devel-
oped by, and available from, Carl Hacker, Department of Public Health,
University of Texas, Austin, Texas.

Effect of Copper on Reproduction

        In daphnids, not exposed to a toxic stress,  reproductive perform-
ance is largely determined by temperature and food resources.  Tempera-
ture determines age at sexual maturity, longevity, and frequency of re-
production; food resources determine brood sizes and vitality of the
young.  Temperature and food resources were the same for all  animals in
the present study, therefore differences in reproductive performance can
be attributed to genetic differences or to the effects of exposure to
differing copper concentrations.

        Reproductive output can be affected directly by a toxicant (caus-
ing a change in brood size or frequency)  or, indirectly, by shortening
the life-span of the female (fewer broods) or changing the age at repro-
ductive maturity.   Studies which do not entail frequent examination of
individual  animals do not provide information which will  allow the
                                    10

-------
investigator to discriminate between direct and indirect effects of the
toxicant on reproduction.

        The impact of copper on brood sizes was evaluated by a randomized,
complete-block design, analysis of variance and Duncan's new multiple
range test (Duncan, 1955).  These analyses were run, consecutively, from
the second brood, through each brood, until the last brood was produced
in the experiment.  The technique permits an evaluation of the impact of
copper on cumulative mean brood size at any point in time throughout the
entire chronic bioassay.

Effects of Copper on the Instantaneous Rate of Population Growth

        Another way to evaluate the impact of a stress on a species is to
calculate its effect on the rate at which the species can increase in
numbers.  This can be estimated by calculating jr, the instantaneous rate
of population growth, which is equal to the birth rate minus the death
rate.  If one plots the population density, on a logarithmic scale,
against time, on an arithmetic scale, the slope of the line is jr.  This
instantaneous rate is affected by both reproductive performance and lon-
gevity.  I have calculated _r for each cohort in each chronic study, using
a life-table computer program (C. Hacker, Dept. of Public Health, Univer-
sity of Texas, Austin, TX) by solving for r in the equation:

                           1 = sP-j  • mi • e^n'

where e is the base of the natural logarithm, jr is the instantaneous rate
of population growth, P-j is the proportion of female survivors of age i,
m\ is the mean number of progeny per female of age i, and i is in days.
Significant differences between r-values were determined by randomized,
complete-block design, analysis of variance and Duncan's new multiple
range test.
                                    11

-------
                                SECTION V

                         RESULTS AND DISCUSSION
CHEMICAL CHARACTERISTICS OF TEST WATERS

Reconstituted Waters

        Reconstituted-water cultures were monitored over 3-day intervals
to document extremes in pH and dissolved oxygen.  In standard water, pH
varied between 6.8 and 7.9.  Oxygen concentrations varied between 7.5 and
8.3 ppm (83 to 90% saturated).  In medium water, pH varied between 8.2
and 9.5 and oxygen between 7.8 and 13.5 ppm (85 to 145% saturated).  As
would be expected, due to the interaction between photosynthesis and
respiration, highest pH and 02 values occurred at the end of the light
period and lowest values at the end of the dark period.  The standard
water had a total hardness of 40-48 and a total alkalinity of 30-35 ppm,
as CaCOs-  The medium water had four times the salt concentrations of
standard water (Table 2) and, therefore, the hardness and alkalinity was
four times greater.

Pond Water

        Pond water was similar in alkalinity and hardness to the recon-
stituted medium water, having a total alkalinity ranging between 110-120
ppm and a total hardness ranging between 130-160 ppm, as CaC03.   Oxygen
concentrations varied between 8.7 and 11.4 ppm (95-125% saturated) and pH
varied between 8.2 and 9.5 in the p&nd-water,  algal system.   Variation
was less in the trout-pellet, pond-water system.  Oxygen extremes were
7.3 to 7.9 (90-98% saturated) and pH varied from 7.9 to 8.0.  The differ-
ences between the two systems are due to the presence or absence of sig-
nificant numbers of algae.
TOXIC CONCENTRATIONS IN TEST VESSELS

        The concentration of a toxicant gradually decreases in static
test systems.  The 72-hr renewal of culture medium in the present study
was designed to minimize this reduction in toxicant concentration.  Table
3 presents data on measured concentrations of total copper in test beak-
ers after 72 hrs of incubation under test conditions (i.e., each beaker
contained a test animal which was fed daily).  Each value is for a single
beaker when nominal concentrations were above 10 ppb.  The 10-ppb concen-
trations were extracted into MIBK and each value in the table is for a
                                    12

-------
TABLE 3.  COPPER CONCENTRATIONS IN TEST BEAKERS AFTER 72 HOURS OF INCUBATION
Nominal Copper
Concentration (ppb)
10
- 20
30
50
60
100
5.9
15.8
27.0
19.3
49.7
46.0
47.8
76.8
Measured Copper Concentrations
(ppb)
3.6
15.4
27.0
16.4
44.7
47.6
50.2
88.0
8.3

33.7
17.6
43.8
49.0
51.1
79.5
7.6

29.3
18.1
47.6
45.3
55.2
87.0
8.4

31.0
22.8
50.2
52.7
51.7



29.0
22.1
52.9
51.0
53.3

Mean
(ppb)
6.8
15.6
24.4
48.4
51.6
82.8
V
10
Loss
32
22
19
3
24
17

-------
100-ml aliquot taken from a composite of three test beakers.  Copper loss
over the 72 hrs ranged from a mean of 32% for the 10 ppb concentration to
a mean of only 3.2% for the 50-ppb concentration.  Losses were higher
from concentrations both above, and below, 50 ppb.
RECONSTITUTED WATERS AS CULTURE MEDIA FOR DAPHNIDS

Standard Water
              i
        Our initial experiments with standard water were very encouraging.
Figure 1 illustrates survival curves for J). magna reared in standard
water that hacf been reconstituted from double-distilled water processed
through a Barnstead Organic-Removal Filter.  Animals were fed the algal
diet and each curve is for a cohort of ten, newborn animals.  The first
three experiments  (6/8, 7/4, and 8/12) produced very similar results,
with mean longevities of 30.5, 30.9, and 30.2 days.  Subsequent to these
experiments, however, we have had highly variable results in attempting
to maintain |D. magna in standard water.  Figure 1 depicts this variabil-
ity in survival.

        The original standard water was made from double-distilled water
that had been processed through a "used" Barnstead filter.  When the
problem of reduced longevity developed, it seemed probable that the cart-
ridge had become saturated and was leaching some toxic substance back in-
to the water.  Upon ordering new cartridges, we discovered that they were
saturated with formalin to inhibit the growth of microorganisms.  Barn-
stead then provided us with cartridges to which no formalin had been add-
ed.  Water filtered through these cartridges was even more toxic to daph-
nids (Fig. 1), mean longevity dropped to 9.5 days.  Obviously such cart-
ridges would have to be conditioned by passing water through them for
some unknown time period if they were to be used for our purposes.  Water
processed through an Aqua-Pure filter was more compatible with daphnids,
but such water caused a precipitation of copper from our 100 ppm stock
solutions.  Furthermore, water passed through an Aqua-Pure filter exhi-
bited a five-fold increase in the concentration of phthallate esters ac-
cording to an analysis performed for us by the Organic Analysis Section,
Methods Development and Quality Assurance Research Laboratory, E. P. A.,
Cincinnati,  Ohio.  Therefore, we did.not attempt any further research
with this filter.  We also tried a Barnstead, standard deionizing filter;
daphnids died within a few hours in standard water made up from water
filtered through such a cartridge.  It was becoming obvious that cart-
ridges,  or filters, not only remove substances from water but that they
are also likely to add substances to the water and that some of these are
toxic to daphnids.  This defeats one of the main objectives of using re-
constituted  water, i.e., having a reproducible, chemically-defined medium.
                                    14

-------
100
                                                    6/8
        NEW
      BARNSTEAD
        FILTER —
                                    20
                                        DAYS
30
40
       Figure 1. Survival of Daphnia magna fed algae in unaerated filtered, standard
               water

-------
        It seemed probable that any toxic substance, which was passing
through two distillations, was volatile.  Therefore, we vigorously ae-
rated our unfiltered, double-distilled water overnight before adding the
salts.  Daphnids did live longer in this aerated, standard water.  This
is evident in Fig. 2, which shows survival curves for pairs of cohorts of
D_. magna which were cultured simultaneously in standard water made up
from aerated, and unaerated, distilled water.  The difference is not due
to 02 or pH; the salts are stirred into the distilled water with a mag-
netic stirrer and this is sufficient to create 02 and pH values compar-
able to the overnight aeration.  The initial 02 for both waters was at
saturation and the pH was 7.4.  Apparently, however, the vigorous, over-
night aeration does reduce the concentration of some chemical  or chemicals
which decrease the life span of D_. magna.

        A new problem, however, developed during this phase of the recon-
stituted water study.  Daphnids reared in standard water, made from un-
filtered, aerated, double-distilled water, lost the biramous portions of
their swimming antennae about 24 days after birth.  Loss of the antennae
was preceded by the appearance of a dark spot at the base of the biramous
portion of the antennae.  During the next molt, the antennae broke and
were missing throughout the remainder of the animal's life.  This did not
occur in any of the earlier experiments when water had been filtered
through Barnstead, organic-removal cartridges.   Possibly, the cartridges
removed some toxicant from the water, or, leached something beneficial
into the water.

        In an attempt to eliminate the antennal damage,  "ultra-pure"
water was used as a base for standard water.  Animals maintained in stand-
ard water made up from ultra-pure water did not live longer than animals
in standard water made from aerated, double-distilled water (Fig. 3).
Furthermore, they also lost the terminal portions of their antennae about
25 days after birth.

        Murphy (1970) reported that he successfully reared daphnids in
water that had only calcium acetate in solution.   The animals  obtained
all other mineral requirements from their algal food.  Since Murphy's
culture water contains about twice as much calcium as does Marking's
standard water, I doubled the calcium sulfate content of standard water
to determine whether that might prevent the antennal damage.  Doubling
the calcium concentration does not prevent antennal  damage, but it does
delay it from about day 24 to day 57.   Doubling the calcium content of
standard water may also markedly increase longevity.  In fact either,
aeration or increasing calcium, increases longevity (Figs. 2 and 3).

        Murphy (1970) does not mention any antennal  damage in  his cul-
tures.   He says (personal  communication) that,  under the "best" conditions,
                                    16

-------
   100
 >
'>
 1_
 3
o
 i_
 0)
O.
          o AERATED

          x UNAERATED
               10
                      20
                                  DAYS
    Figure 2. Survival of Daphnia magna in unf iltered aerated versus unaerated
           standard water

-------
oo
                    100
                     50-
               >	q
       AERATED
   STANDARD WATER
o
k.
0)
Q.
                     50
  "ULTRAPURE"
STANDARD  WATER
                               10
             20
                              30      40
                                 DAYS
80
                       Figure 3. Survival of Daphnia magna in unfiltered versus aerated ultrapure
                              standard water

-------
|D. magna has a life span of about two months at 20°C.   It may be, then,
that his animals die before the antenna! deformity can  develop.

Medium Water

        Efforts to culture JD. magna in medium water followed the same
trend as described for standard water.  Figure 4 illustrates the progres-
sive reduction in longevity that we observed in our medium-water cul-
tures.  Animals reared in filtered, unaerated medium water live consi-
derably longer than animals reared in filtered, unaerated standard water
and, under the best condition, may live as long as animals in pond water.
Figure 5 depicts survival curves for 3 cohorts of ten animals initiated
on the same day in filtered, unaerated standard, medium, and pond water.
All were fed vitamin-enriched algae.  It should be pointed out that the
survival curves for the reconstituted waters depicted in Fig. 5 are the
best that were observed and that the pond-water survivorship curve was
the poorest of all observed during this study.  The reason for greater
longevity in medium water, compared to standard water,  is unknown.   There
are, at least, three possible causes:  (1) the low salt concentration, in
some way, directly reduces longevity, (2) additional trace elements are
added, as contaminants, with the reagent-grade salts in the medium water,
or (3) the higher salt concentrations in the medium water may complex
some toxic substance or substances present in the distilled water.   Pos-
sibility number 2 seems improbable, since the animals are fed algae which
have been cultured in a medium containing trace elements (Table 1).  If
possibility number 1 is true, one would expect a more rapid turnover of
daphnid populations in soft-water lakes than in hard-water lakes.  To the
best of my knowledge, this possibility has never been suggested, or
tested.  The fact that aeration increases longevity in  standard water
suggests that possibility number 3 may be the explanation.   Allen Maki,
Environmental Water Quality Research Department, Proctor and Gamble,
Cincinnati, Ohio, states (personal communication) that they have discon-
tinued the use of standard water in their bioassay work because of poor
survival and reproduction of the daphnids in standard water.

Summary

        We have found it difficult to maintain viable stock cultures of
Daphm'a in reconstituted waters.  We have had to replenish our stock
animals in reconstituted water, from time to time, with animals from
our pond-water stocks.  At no time, over a 2-year period, did we have
any difficulty in maintaining daphnids in our pond-water, algal  system.
I suspect that life-history statistics for daphnids reared in reconsti-
tuted waters made up in different laboratories, or at different times in
the same laboratory, may be as, or more, variable than in different,
natural waters.  I believe that an adequate, reproducible,  reconstituted
                                    19

-------
to
o
                        100
                     
                     Q>
                     O
                                   10
20
40      50

  DAYS
60
                                                                                         .- 8/12
70
80
90
                             Figure 4. Survival of Daphnia magna fed algae in filtered, unaerated medium

                                     water

-------
i Rn
13U
3
2 50
c
0)
0
0)
a.
OJ
'

\ \
\ 	
WATER MEAN
TYPE LONGEVITY
POND 62.3
MEDIUM 72.6
STANDARD 30.2

10 20

\f n v
X OX
» 1 v
V. 1 U v
4^ V 	 *
k 	 4 bo x- — MEDIUM
\ \ >
1 \ .V
MEAN i-^ POND 	 »-oo x 	 x
BROOD SIZE1, V xx
	 •• \ o-o \
17-6 '.-• 	 STANDARD V k
13.9 M 9 \
14.5 v._. I H0
" VT
\ » \
* xb
30 40 50 60 70 80 90
DAYS
Figure 5. Comparative survival and reproduction of Daphnia magna in fil-
         tered, unaerated standard, medium, and pond water

-------
water can be developed if a consistently high-quality distilled water can
be produced.  I suspect, however, that, given the quality of distilled
water available to most laboratories, reconstituted waters are likely to
create more problems in acute chronic toxicity studies than they solve.
In any event, much more effort must be spent in the development and test-
ing of reconstituted waters before they can be recommended for routine
bioassays.


ACUTE TOXICITY OF COPPER

Relative Sensitivity of Four Species in the Pond-Mater, Algal System

        On day 3 of each chronic bioassay, a 72-hr LC50 value was calcu-
lated by probit potency analysis (Finney, 1971).  In each experiment, co-
horts of 10 newborn animals were started at copper concentrations of 0,
20, 40, 60, 80, 100, 120 and 140 ppb.  Three such experiments, started at
different times, were run for D_. magna and two such experiments were run
for each of the other three species.  Inter- and intraspecific comparisons
of LC50 values were made only when the respective concentration-response
curves were demonstrated to be parallel (Finney, 1971).

        Data from the three experiments with D_. magna, and the two tests
for D_. ambigua, were combined to give a single best estimate of an LCso
value for each species since all of the concentration-response curves were
parallel.  In the case of D_. pulex and ]3. parvula, concentration-response
curves for the two experiments were not parallel.  One concentration-
response curve for each of these species was, however, parallel to the
concentration-response curves of j). magna and D_. pulex.  Potency analysis
was used to compare the LC5p values from those concentration-response
curves which were parallel (Table 4).  The LC50 values were not signifi-
cantly different (P = 0.05) for the two largest species (D_.  magna and J).
pulex) nor for the two smallest species (]3. parvula and D_. ambigua).  The
two largest species were, however, significantly less sensitive to an
acute copper stress than were the two smallest species (Table 4).

        The acute toxicity of copper to D_. magna and D_. ambigua was also
tested in filtered, unaerated, standard water (Table 5).   Variability was
quite low, concentration-response curves were parallel, and the mean 72-hr
LCso values were not significantly different (P = 0.05).
                                    22

-------
   TABLE 4.   ACUTE TOXICITY (72-HR LC50 IN PPB)  OF COPPER
              TO FOUR SPECIES OF Daphnia IN THE

£. magna
86. 5a

POND-WATER,
D. pulex
86.0
54. QC
ALGAL SYSTEM
£• parvula
72.0
57. Oc

D. ambigua
67. 7b

     values sharing an underline are not significantly  dif-
ferent.

Calculated from the pooled data of 3 experiments.

^Calculated from the pooled data of 2 experiments.

CLC50 values from non-parallel  dose-response curves  which
 were not used in the analyses  for significant differences.
                            23

-------
        TABLE 5.  ACUTE TOXICITY OF COPPER AND CHROMIUM TO CL magna
                   AND D. ambigua IN UNAERATED FILTERED,
                              STANDARD WATER
Species
n Metal
Mean 72-Hr LC50
(ppm)
Standard
Deviation
|3. magna
JL ambigua
J3. magna
D. ambigua
4
3
5
6
Cu
Cu
Cr
Cr
13.5
12.4
 5.2
 1.7
2.72
1.46
2.80
1.20
Toxicity to D. magna in the Three Test Waters

        Table 6 illustrates the effect of changes in water chemistry on
the acute toxicity of copper to J). magna.  The expected, inverse relation-
ship between toxicity and alkalinity is apparent.  It is also obvious that
subtle, unidentified changes in water chemistry can markedly affect toxi-
city.  The 72-hr LC50 values of animals tested in filtered, unaerated-
unfiltered and aerated-unfiltered standard water were 13.5, 25.2 and 31.6
copper, respectively.  Acute toxicity of copper in medium water was more
variable than in pond water, but the means were not significantly differ-
ent (P = 0 05).
              TABLE 6.  ACUTE TOXICITY OF COPPER TO ALGAL-FED
                       D. magna IN DIFFERENT WATERS
Water
Mean 72-Hr LC50
n (ppm)
Range
(ppm)
Standard
Deviation
Unaerated, Filtered
   Standard             4
Unaerated, Unfiltered
   Standard             2
Aerated, Unfiltered
   Standard             1
Medium                  7
Pond                    3
               13.5

               25.2

               31.6
               78.2
               85.1
                    10.1-16.5

                    24.9-25.5
                    56.8-101.4
                    81.6-88.8
                    2.72

                    0.42
                   16.60
                    3.63
                                     24

-------
Effect of Food Type on Sensitivity of D. maqna

        Contrary to expectation, the two foods tested did not affect the
acute toxicity of copper to D_. magna in pond water.  The mean 72-hr LC50
for animals fed algae daily was 85.1 ppb (n = 3; SD = 3.6) and for animals
fed the trout-granule diet it was 83.4 ppb (n = 3; SD = 2.8).  These were
not significantly different (P = 0.05).

Acute Toxicity of Chromium

        Sensitivity to an acute chromium stress was much more variable
than to an acute copper stress (Table 7).  However, all of the concentration-
response curves were parallel, both intra- and interspecifically.  D_. am-
bigua was much more sensitive to chromium in pond water than were the
other three species.  It was also more sensitive than D_. magna in filtered,
standard water (Table 6).  Apparently, the similarity of response by vari-
ous species of Daphm'a to copper stress does not necessarily hold for
other stresses.
           TABLE 7.  ACUTE TOXICITY OF CHROMIUM TO FOUR SPECIES
                OF Daphnia IN THE POND-WATER, ALGAL SYSTEM
Species
x 72-hr LC50
n (ppm)
Standard
Deviation
                          2                 42.1                    8.49
D_. ambigua                2                  7.7                    1.34
D_. galeata                2                 65.6                    1.75
£. pulicaria              2                110.8                   30.10


CHRONIC TOXICITY OF COPPER

Effect of a Chronic Copper Stress on Longevity

Comparative Sensitivity of the Four Species--
        Survival curves for each chronic test on each species are presented
in Figs. 6 through 9.  All of these tests were run in the pond-water, algal
system.  The results of chi-square analyses of these survivorship curves
are encouragingly consistent.  Survivorship curves at 20 and 40 ppb were
never significantly different (P = 0.05) from those of control animals.
With one exception, all survival curves at concentrations of 60 ppb, or
above, were significantly different from those of control animals.  The
                                     25

-------
                       100
to
Oi
        Oppb
     x 20ppb
       40ppb
                                   eoppb
                                   SOppb
                                  lOOppb
                                   20
60       80
  DAYS
100
120
140
                           Figure 6.  Survival of Daphnia tnagna exposed to chronic copper stresses in
                                   the pond-water, algal system (Experiments 1 and 2)

-------
                       100
to
-3
                                                20        30

                                                    DAYS
50
                           Figure 7. Survival of Daphnia ambigua exposed to chronic copper stresses in
                                   the pond-water, alaal system (Experiments 1 and 2)

-------
                         100
to
00
                                                                          o   Oppb
                                                                          x 20ppb
                                        10
                                                     DAYS
                          Figure 8. Survival of Daphnia pulex exposed to chronic copper stresses in
                                  the pond-water, algal system (Experiments 1 and 2}

-------
to
CO
                                                              '--X--X--X--X-X--X,
                                        10        20        30        40
                                                           DAYS
 i     r
50
 T*
60
70
                             Figure 9.  Survival of Daphnia parvula exposed to chronic copper stresses in
                                      the pond-water, algal system (Experiments 1 and 2}

-------
one exception is the second chronic test with D_.. ambigua where survival
differed from that of control animals only at 80 and 100 ppb.  It should
be pointed out that the longevity of all animals, including the controls,
was considerably reduced in experiment number 2 with ID. ambigua (Fig. 7).
This disparity between experiments did not occur with the other 3 species.

       The reproducibility of life-history statistics is especially evi-
dent in the three chronic toxicity experiments with J3. magna.  This is
evident from the data in Table 8.  The third chronic experiment was per-
formed at a later date to check on the toxicity of a new batch of copper
sulfate.  This reproducibility implies both a rather uniform gene pool in
the experimental animals (relative to copper sensitivity) and good repro-
ducibility in the test system. This being the case, I feel that it is
reasonable to assume that the somewhat greater variability in the response
of the other 3 species reflects greater genetic variability in the test
populations rather than variability in the physical-chemical characteris-
tics of the test system.  The probability that most of the variability is
in the organisms is enhanced by the fact that I), ambigua. CL parvula, and
D_. pulex were collected locally from wild populations, whereas D_. magna
was obtained from a culture that had been maintained under laboratory
conditions for many years and was probably started from a very few par-
thenogenetic females.

       If most of the variability is in individual-animal response, ra-
ther than in test conditions, one gets a better impression of population
response by combining the survivorship curves from the separate experi-
ments for each species into combined curves.  This is done for each of
the four species in Figs. 10 through 13.  In these curves, the more rapid
die-off of animals in the 60 ppb copper solution is clearly evident.   It
is obvious that there is a significant increase in toxicity between 40
and 60 ppb.  If current hypothesis is correct (Stiff, 1971; Pagenkopf et
al., 1974), there must have been a saturation of the complexing capacity
of the pond water within this range and a significant increase in the
concentration of cupric ions.

       It is evident, from these experiments, that the impact of a chronic
copper stress on survival is not significantly different for these 4 spe-
cies of Daphnia.  If, however, the chronic experiments had been termi-
nated at the end of 3 weeks, as done by Biesinger and Christensen (1972),
D.. magna would have appeared to be less sensitive since the die-off at 60
ppb did not begin until the experiment had progressed beyond day 30 (Fig.
10).  Obviously, daphnids very rarely, or never, attain their physiologi-
cal  life expectancy in nature and one could speculate that it is really
not worth the effort to carry chronic experiments through to the death of
the last animal.  It is clear, however, that j). magna was under stress in
these experiments at a copper concentration of 60 ppb, and that this
                                    30

-------
TABLE 8.  VITAL STATISTICS FOR THREE CHRONIC COPPER TOXICITY TESTS
           WITH D. magna IN THE POND-WATER, ALGAL SYSTEM
Copper
Cone (ppb)
0
20
40
60
80
100
x Longevity
Ex 1
79.1
99.6
94.2
45.3
22.2
7.5
Ex 2
93.2
99.7
82.7
48.8
11.0
2.7
(days)
Ex 3
96.7
91.7
106.0
69.0
20.6
3.0
Total Young
Ex 1
2314
3180
3329
1689
464
0
Ex 2
3223
3863
3394
2150
364
26
Ex 3
3287
2850
3110
2328
696
14
x Brood Size
Ex 1
10.6
12.5
13.3
13.7
10.8
-
Ex 2
13.1
14.6
15.2
16.5
14.0
13.0
Ex 3
12.3
12.9
13.1
13.2
14.2
7.0

-------
                   100
co
TO 75-ft H
_>
'>
£
^50^
c
0>
O
i.
i •
\
\
\
^
v
i
i
i
i
                  o
                  X
-X-X--X-X--X--X—X--X-X  A
                X--X
 Oppb  o  60ppb
20ppb
40ppb
                                                                            SOppb
                                                                         "lOOppb
                       0   10   20  30  40   50  60  70   80  90  100 110  120 130 140

                                                    DAYS
                       Figure 10. Combined survivorship curves (Experiments 1 and 2) for Daphnia
                                magna exposed to chronic copper stresses in the pond-water, algal
                                system

-------
100
         o  Oppb
         x 20ppb
   ^     A 40ppb
   w
GOppb
SOppb
              10
20        30
  DAYS
       50
 Figure 11. Combined survivorship curves (Experiments 1 and 2) for Daphnia
         ambigua exposed to chronic copper stresses in the pond-water,
         algal system

-------
co
                      100
        o  Oppb
        * 20ppb
        A40ppb
                              60ppb
                              SOppb
                                   10
20
          r
30       40
   DAYS
 r
60
T
70
                          Figure 12. Combined survivorship curves (Experiments 1 and 2) for Daphnia
                                   parvula exposed to chronic copper stresses in the pond-water,
                                   algal system

-------
oo
CJl
                         0
                                                            o  Oppb   a  60ppb
                                                            x 20ppb   A  SOppb
                                                            *40ppb
                           o
10
20       30
    DAYS
40
50
                        Figure 13. Combined survivorship curves (Experiments 1 and 2) for Daphnia
                                 pulex exposed to chronic copper stresses in the pond-water, algal
                                 system

-------
would not have been detected in a 3-week chronic bioassay.  Given the
added stresses of a real world, it would seem possible that a copper con-
centration of 60 ppb would tip the scales sufficiently to eliminate the
species.  The problem of extrapolating from laboratory experiments to
more complex, natural systems is, of course, the crucial problem.  Sooner
or later, if we want to protect species, as they exist in complex commu-
nities, we shall have to study stressed animals as components of more
complex systems.

Reconstituted-Water, Algal Systems

Standard water—
       Survival curves for two experiments run on D_. magna in standard
water made up from unaerated, Barnstead-filtered, double-distilled water
are illustrated in Fig. 14.  Chi-square analysis of survival  curves indi-
cate that survival differed significantly (P = 0.05) from that of con-
trol animals at copper concentrations of 5.0 ppb, or higher,  for the 1/23
experiment.  Survival was significantly different from that of control
animals at a copper concentration of 7.5 ppb,  or higher, in the 2/14 ex-
periment.  These experiments were run after we had noticed a  reduction in
longevity of animals in our filtered, standard water and it is probable
that the animals were subjected to some chemical stress in addition to
the copper stress.

       Figure 15 illustrates survival curves for two experiments to de-
termine the effect of a chronic copper stress  on £. magna in  standard
water made up from unfiltered, aerated, double-distilled water.  Survival
was reduced significantly from that of control animals at a copper con-
centration of 15 ppb, or greater, in the 3/5 experiment and at concentra-
tions of 20 ppb, or greater, in the 3/25 experiment (P = 0.05).

       Survival curves, for a single experiment, in which £.  magna was
reared at different copper concentrations in unfiltered, unaerated stand-
ard water are presented in Fig. 16.  In this experiment, survival was
significantly less (P = 0.05) than that of control animals at copper con-
centrations of 5.0 ppb, or greater.

       Although the longevity of control animals was somewhat variable,
and the animals were probably exposed to other chemical stresses, the
lowest concentration that reduced longevity varied only from  5.0 to 7.5
ppb in the 3 unaerated experiments and from 15.0 to 20.0 ppb  in the 2
aerated experiments.  Aeration of the distilled-water base appears not
only to increase the life-span of control animals, but also to decrease
the sensitivity of the animals to a chronic copper stress.
                                   36

-------
O5
                        100
                   _     50
 o>
O
                                 b-
            o    0 ppb
            A  2.5 ppb
            x  5.0 ppb
            D  7.5 ppb
                   +-   100
                         SO-
                                                                           I/23
                                                                         I  I VI I  I
                                                                           2/14
                                                          DAYS
                        Figure 14. Survival of Daphnia magna fed algae and exposed to chronic
                                 copper stresses in filtered, unaerated standard water

-------
CO
QO
                          100
V\   A
                  0 ppb
                10 ppb
                15 ppb
                20 ppb
                25 ppb
                30 ppb
                                                 20       30
                                                   DAYS
                 40
50
                         Figure 15. Survival of Daphnia magna fed algae and exposed to chronic
                                 copper stresses in unf iltered, aerated standard water

-------
                      100
                    (O
                    >
co
eo
 0)
O
 v.
 0)
O.
                                                           0 ppb
                                                           5 ppb
                                                          10 ppb
                                                          15 ppb
                                                          20 ppb
                                                                                               35
                            Figure 16. Survival of Daphnia magna fed  algae and exposed to chronic
                                      copper stresses in unfiltered, unaerated standard water.

-------
Medium water--
       Only 2 chronic copper bioassays were run in medium water.  These
resulted in quite different results (Fig. 17).  In the first experiment,
all copper concentrations of 20 ppb, or more, caused a significant reduc-
tion in longevity (P = 0.05).  In the second experiment, only copper con-
centrations of 80 and 100 ppb reduced longevity significantly (P = 0.05)
below that of control animals.  There was also a significant difference in
longevity of control animals, with those of experiment 1 living much
longer.

Pond-Water. Trout-Granule System—
       Animals fed the trout-granule food were much more sensitive to
copper than were animals fed algae (Figs. 6 and 18).  Algal-fed animals
did not exhibit any reduction in longevity at concentrations of 40 ppb,
or less.  Animals reared on the trout-granule food, however, had survival
reduced significantly below that of control animals at copper concentra-
tions greater than 20 ppb (P = 0.05).  Initially,  I suspected that the
two foods differentially removed copper from solution and, thereby, al-
tered effective concentration.  To test this hypothesis, beakers contain-
ing 20 and 80 ppb copper, but no animals, were incubated under the same
conditions used in the chronic toxicity experiments.  The beakers were
"fed," each day, the same ration of each food used in the chronic testing.
The same batch of copper stock was used for both foods.   After 72 hours,
the contents were filtered through 0.45 micron filters (HA Mi Hi pore) and
the filtrates analyzed for copper by atomic-absorption spectroscopy.   The
results (Table 9) show that the soluble copper concentrations were not
different.  It is, of course, possible that the concentrations of cupric
ions did differ, but the fact that the 72-hour LC50 values were almost
identical for the two systems (see section on acute toxicity of copper),


         TABLE 9.  RELATIONSHIP BETWEEN FOOD AND SOLUBLE COPPER
                 CONCENTRATION REMAINING IN TEST BEAKERS
                AFTER 72 HRS OF. INCUBATION IN POND WATER
                            Nominal  Copper      Measured Copper
                            Concentration        Concentration
             Food               (ppb)                 (ppb)
Trout-Granule
Algae
Trout-Granule
Algae
Trout-Granule
Algae
20
20
80
80
80
80
15.8
15.4
46.5
40.5
49.2
48.0
                                    40

-------
    100
g>
'>
 CD
O
 501



  0

100
      50-
   0 ppb
x 10 ppb
A 20 ppb
a 30 ppb
A 40 ppb
• 50 ppb
• 60 ppb
             5   10  15  20  25  30  35  40  45  50  55
                                DAYS
     Figure 17. Survival of Daphnia magna fed algae and exposed to chronic
            copper stresses in medium water

-------
                       100
to
o  Oppb
x 20ppb
  40ppb
a 60ppb
  SOppb
                                                               r
                                                     30       40
                                                       DAYS
                           Figure 18. Effect of trout-granule food on the sensitivity of Daphnia magna
                                    to chronic copper stresses in pond water

-------
strongly suggests that animals fed on the two foods were exposed to com-
parable concentrations of the toxic species of copper.  If this is so,
the greater sensitivity to copper of animals fed the trout-granule food
must reflect a nutritional stress imposed in addition to the copper
stress.  The fact that control animals fed trout-granule food survived
only about one-half as long as animals fed algae (Fig. 19) supports the
conclusion that the trout-granule food is nutritionally inferior to the
algal food.  This nutritional inadequacy may not apply to all species of
Daphnia.  Dewey Bunting (personal communication) states that, although he
cannot maintain continuous cultures of I), magna on trout food, he has no
trouble in maintaining Jh pulex on trout pellets.

Effect of a Chronic Copper Stress on Reproduction

Comparative Sensitivity of the Four Species—
        The reproductive response of the four species to a chronic copper
stress was somewhat more variable than was survival.  Temporal changes in
mean brood size, at each copper concentration, are illustrated in Figs. 20
through 23 for the four species.  Although brood sizes oscillate, there
is no evidence that any of the species approached a reproductive senility
in old age.  The oscillatory pattern does suggest that the results of an
analysis of the impact of copper on brood size might vary, according to
when the analysis was made during the life-span of the various cohorts.
The question, thus, arises:  how long should a chronic toxicity test be
run, if effects of a stress on brood size are to be evaluated?  To answer
this question for a copper stress, sequential analyses of variance and
Duncan's multiple-range tests were run on mean cumulative brood sizes
from brood 1, through each consecutive brood, until the final brood had
been incorporated into the analyses.  In other words, the experiment "was
stopped," after each brood had been produced, to determine what differ-
ence, if any, existed in mean brood size at the different copper concen-
trations.  These sequential analyses for the four species are presented
in Tables 10 through 13.  It is obvious from Table 10 that D_. magna is,
reproductively, relatively insensitive to copper in the pond-water, algal
system.  At no point in time, from brood 1 through brood 33, was cumula-
tive mean brood size at 80 ppb significantly different from that of con-
trol animals.  Furthermore, copper was stimulatory up through a concen-
tration of 60 ppb, i.e., mean brood sizes were always larger than the
controls and, almost always, significantly so (Table 10).  In D_. magna,
total reproductive output descreased at copper concentrations greater
than 40 ppb (Table 9) only because the adults have a shorter life-span,
and, therefore, produce fewer broods.

        The young JD. magna which developed in copper concentrations of
20, 40, 60 and 80 ppb were viable and cohorts of 10 were carried through
28-day FI and F2 generations in their respective concentrations (Tables
                                    43

-------
   TROUT-GRANULES
    10  20  30  40  50  60  70  80  90  100 110 120 130 140
                               DAYS
Figure 19. A comparison of longevity in Daphnia magna fed algae versus
         trout-granule food in pond water

-------
                       10      15      20
                         BROOD  NUMBER
25
Figure 20. Temporal changes in the mean brood size of Daphnia magn£ ex-
         posed to chronic copper dosages
                              45

-------
      32

      16

       0
      32
   LU
   Sl6
   O
   £32
      16-
       0
      32
      16-
40 ppb  Cu
                  ppb Cu
       VI   III '"""fT 1 ™T J^ T -ju -ni-iy-.
                 5        10      15       20
                    BROOD  NUMBER
Figure 21. Temporal changes in the mean brood size of Daphnia pulex ex-
        posed to chronic copper dosages
                            46

-------
       16H
        8
   0
  16


   8
                 CONTROL
    N
    O
    o
    o o
    en 16
LU 8
S  •

   0
  16


   8
       O+-T
                     40 ppb Cu
          80 ppb  Cu
                            -•-60 ppb
                               Cu
                 5      10      15      20      25
                       BROOD  NUMBER
Figure 22. Temporal changes in mean brood sizes of Daphnia parvula ex-
         posed to chronic copper dosages
                             47

-------
         8-
         0

         16

      LU
      N

      
-------
TABLE 10.  ANALYSES OF CUMULATIVE MEAN BROOD
  SIZES FOR D. magna EXPOSED TO FIVE COPPER
      CONCENTRATIONS IN THE POND-WATER,
                ALGAL SYSTEM3
Through
Cumulative
Broods
1-4
5-7
8-11
12
13-14
15-24
25-27
28-30
31-33
Nominal
Copper Concentrations
(ppb)
40
60
60
60
60
60
60
60
60

60
40

40
40

40
40
40
40
40

0
20

20
80

80

20

20
20

20

20
80
80
20
20

80

0
80

80


80
0
0
0
0
0
80
0
0

      sizes were significantly different (P =
0.05) in any two concentrations not mutually
underlined.  Brood sizes decrease in concen-
tration, as sequenced, from left to right.
                     49

-------
 TABLE 11.  ANALYSES OF CUMULATIVE MEAN BROOD
   SIZES FOR D_. pulex EXPOSED TO FIVE COPPER
       CONCENTRATIONS IN THE POND-WATER,
                 ALGAL SYSTEM9
Through
Cumulative
Broods
1
2
3
4-10
11
12-15
16
17
Nominal
Copper Concentrations
(ppb)
20
20
20
20
20
40
40
40

0
0

40
40
40
20
20

60


40
40

0
0
60
60
60

20

60
60

60

60
0
0
0
0

80
80
80
80
80
80
80
80
       sizes were significantly different (P =
0.05) in any two concentrations not mutually
underlined.   Brood sizes decrease in concen-
trations, as sequenced,  from left to right.
                     50

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 TABLE 12.   ANALYSES OF CUMULATIVE MEAN BROOD
      SIZE  FOR D.  parvula IN FIVE COPPER
       CONCENTRATIONS IN THE POND-WATER,
                 ALGAL SYSTEM3
Through
Cumulative
Broods
1
2
3-4
5
6-8
9-14
15-16
17
18-19
20-25
26-32
Nominal
Copper Concentrations
(ppb)
0
0
0
0
0
0
20
20
60
60
60

20

20
20
20
20

20
0
60
20
0
0

40


40

40
40

40

40
60
0
0
20
40

60

60

60
60
60
60
40
40
40
40
20

80
80
80
80
80
80
80
80
80
80
80
aBrood sizes were significantly different (P
 = 0.05) in any two concentrations not mutual-
 ly underlined.  Brood sizes decrease in con-
 centrations, as sequenced, from left to right.
                       51

-------
   TABLE 13.  ANALYSES OF CUMULATIVE
    MEAN BROOD SIZE FOR D_. ambigua
     IN FOUR COPPER CONCENTRATIONS
    IN THE POND-WATER, ALGAL SYSTEMa
Through
Cumulative
Broods
1
2-3
4
5
6
7-9
10-11
12
13-17
Nominal
Copper Concentrations
(ppb)
20
20
20
20
20
20
20
20
20

0

0
40
0
0
0
0
0
0

40

40
0
40
40
40

40
40
40

60
60
60
60
60
60
60
60
60

aBrood sizes were significantly differ-
 ent (P = 0.05) in any two concentra-
 tions not mutually underlined.  Brood
 sizes decrease in concentrations, as
 sequenced, from left to right.
                   52

-------
14 and 15.  There is no evidence for either reduced viability or reduced
fecundity in the F! and F2 individuals when compared to the parental gen-
eration.  This contradicts the conclusion of Hueck and Adema (1968) that
copper is more toxic to second-generation daphnids than to their parents.


     TABLE 14.  TOTAL YOUNG AND MEAN BROOD SIZE FOR PARENTAL-, Fx-
               AND F2-6ENERATIONS OF ]). magna ON A 28-DAY
          CHRONIC-COPPER STRESS IN THE POND-WATER, ALGAL SYSTEM
Parental
Copper
(ppb)
0
20
40
60
80
Total
Young
951
1001
1218
1300
332
x Brood
Size
11.1
13.3
16.9
17.8
13.3
Fi F?
Total
Young
839
976
1028
674
128
x Brood
Size
14.2
16.0
17.1
18.7
10.7
Total
Young
375
937
814
1438
788
x Brood
Size
15.0
16.4
23.9
20.8
15.8
Based on 10, individually-reared, newborn animals started at each concen-
tration and exmined daily for 28 days.
            TABLE 15.  PER CENT SURVIVAL OF PARENTAL, FI AND F2
                 GENERATIONS OF I), magna AFTER 28 DAYS OF
              COPPER STRESS IN THE POND-WATER, ALGAL SYSTEM
Copper
(ppb)
0
20
40
60
80
Parental
90
90
100
70
40
F!
100
90
90
100
10
F2
100
90
90
100
40
     Each cohort consisted of 10, individually-reared-newborn animals

        j). pulex consistently exhibited a significant reduction (P = 0.05)
in mean brood size at copper concentrations of 80 ppb (Table 11).  There
were never significant differences  in mean brood sizes at copper concen-
                                    53

-------
trations of 0, 20, 40 or 60 ppb.  Copper stimulated fecundity at concen-
trations of 20, 40, and 60 ppb but these were usually not significantly
larger than brood sizes of control animals (Table 11).  Cumulative mean
brood sizes through broods 16 and 17, however, were significantly larger
at 40 ppb than for controls.

        D_. parvula exhibited a reduction in cumulative mean brood size
only at copper concentrations of 80 ppb, or more, if the analysis was
made subsequent to brood 8 (Table 12).  If analyzed prior to brood 9,
cumulative mean brood sizes in both 60 and 80 ppb were significantly
smaller than those of control animals.  Although cumulative mean brood
sizes of animals reared at 20 and 60 ppb were frequently larger than
those of control animals, the differences were never significant (P =
0.05).

        Table 13 shows the analyses of the reproductive response of I).
ambigua to copper stresses.  Except for cumulative broods 1 and 6, the
mean brood size was always significantly smaller (P = 0.05) at 60 ppb
than at lower copper concentrations.  Analyses of cumulative mean brood
sizes from brood 10 through the last brood (17) indicate that mean brood
sizes at 40 ppb are significantly smaller than those of control animals.
Only the 20 ppb concentration caused a slight increase in brood size in
D_. ambigua, but this difference was never significant.  Prior to brood
9, one would conclude that copper concentrations greater than 40 ppb
caused a decrease in brood size of J3. ambigua; after brood 9, one would
conclude that copper concentrations in excess of 20 ppb resulted in a
decrease in brood size.

        In summary, D_. magna was, reproductively, least sensitive to a
chronic copper stress.  Even at a copper concentration of 100 ppb, where
only 4 broods were produced in three chronic experiments, the mean brood
size, of those animals which had broods, was 10 (Table 9).  Mean brood
sizes at 40 and 60 ppb were usually significantly larger than those of 0
ppb.  ]). pulex was second least sensitive to copper.  Only at a copper
concentration of 80 ppb was there a significant reduction in brood size.
D_. parvula was third least sensitive; if analyzed after brood 8, there
was a significant reduction in brood size at a copper concentration of
60 ppb.  D_. ambigua was most sensitive; mean brood sizes were, in all
cases, significantly smaller at 60 ppb and at 40 ppb if analyzed after
brood 10.

Sensitivity of D. magna in Reconstituted Waters

Standard Mater—
        The reproductive response of D_. magna to a copper stress is more
variable in filtered, unaerated standard water (Table 16)  than is it in
                                   54

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                          TABLE 16.  THE RELATIONSHIP BETWEEN MEAN BROOD SIZE AND COPPER
                                CONCENTRATION FOR D. magna IN RECONSTITUTED WATERS
en
01
Experiment
No.



1





2





3


Unaerated Standard
Water
Copper
(ppb)
0
2.5
5.0
7.5
10.0

0
2.5
5.0
7.5
10.0

0
5.0
10.0
15.0
20.0
Mean
Brood Size
11.7
10.73
6.9
3.0
3.5

9.9
10.2
10.0
7.8
9.9

14.6
8.5
7.3
9.0
4.5
Aerated Standard
Water
Copper
(ppb)
0
5.0
10.0
15.0
20.0
25.0
0
5.0
10.0
15.0
20.0
25.0





Mean
Brood Size
11.0
11.0
12.9
15.9
15.5
16.8
10.2
11.5
12.4
13.8
11.1
7.8





Unaerated Medium
Water
Copper
(ppb)
0
10
20
30
40
50
0
20
40
60
80






Mean
Brood Size
12.9
16.1
16.5
16.7
16.0
13.1
18.1
18.3
14.4
10.9
5.4






              Horizontal  lines  indicate that mean brood sizes above and below the line are significant-
               ly different  (P = 0.05).

-------
pond water (Table 8).  In experiment 1, there was a significant reduc-
tion (P = 0.05) in mean brood size at copper concentrations above 2.5
ppb.  There was a significant reduction in longevity at copper concen-
trations greater than 5.0 ppb (Fig. 14).  In experiment 2, there were no
significant differences in mean brood size at any of the copper concen-
trations, although survival was reduced at copper concentrations of 7.5
ppb, or greater (Fig. 14).  In experiment 3, there was a significant re-
duction in mean brood size at copper concentrations greater than 15.0
ppb, but survival was significantly reduced at 5.0 ppb (Fig. 16).  The
stimulatory effect of copper on brood size that was so obvious in the
pond-water experiments did not occur in unaerated standard water and re-
production was more sensitive than survival in 2 of the 3 experiments.

        The relationship between copper, longevity and brood size in the
unfiltered, aerated, standard-water bioassays is more similar to the re-
sults observed in the pond-water experiments (Table 16).  In experiment
1, there was never a significant reduction in mean brood size at any
copper concentration and mean brood sizes were significantly larger (P =
0.05) at 15, 20, and 25 ppb than at 0 ppb.  Again, survival  was more
sensitive, being significantly reduced at concentrations greater than 10
ppb  (Fig. 15).

        In the second experiment, mean brood size was not significantly
reduced (P = 0.05) at any copper concentration.   Mean brood sizes were
slightly larger at concentrations of 10 and 15 ppb than in 0 ppb, but
never significantly so.  Survival was significantly reduced at concen-
trations greater than 15 ppb (Fig. 15).

Medium Mater—
        Mean brood size for £. magna cultured in the medium-water,  algal
system are presented in Table 16.  In the first experiment,  copper had a
slight stimulatory effect on mean brood size up to a concentration of 40
ppb but brood sizes were significantly larger (P = 0.05) than the con-
trols only at 10 and 20 ppb.  Mean brood size was not significantly
smaller at 50 ppb copper than in the control.  Since survival  was sig-
nificantly reduced at 20 ppb (Fig. 17), the results of this experiment
agree with the pond-water experiments;  i.e., survival is more sensitive
to a copper stress than is reproduction.

        Copper had no significant effect on mean brood size in the second
experiment (Table 16).   The sequential  analyses of variance and multiple-
range tests indicated that there were never significant differences in
mean brood size at any point in the chronic experiment; survival, how-
ever, was significantly reduced at 80 ppb copper (Fig. 17).
                                   56

-------
Effect of Food-Type on Reproductive Sensitivity of D. magna in Pond Water

        The trout-granule food not only reduced the life-span of 1). magna,
at all copper concentrations, it also increased the reproductive sensi-
tivity of the species (Table 17).  Tables 10 and 18 present the results
of the sequential analyses of cumulative mean brood sizes in different
copper concentrations for the algal- and the trout-granule foods.  As
discussed previously, copper did not cause a reduction in brood sizes in
those algal-fed animals which lived long enough to reproduce.  Table 18
clearly shows, however, that animals fed the trout-granule food exhibited
significant reductions in mean brood size at copper concentrations of 20
ppb or more (P = 0.05).  Animals, on this diet, are more sensitive to
copper, using either survival or reproduction as an index.  Also, the
two indices agree that the maximum allowable concentration is less than
20 ppb for D_. magna on the trout-granule diet.  It should also be noted
that, unlike the algal-fed animals, there was no stimulatory effect of
copper on reproduction when the trout-granule food was used.
       TABLE  17.   EFFECT OF  FOOD TYPE ON SURVIVAL AND REPRODUCTION
                     OF Daphnia magna IN POND WATERS
Copper
(ppb)

0
20
40
60
80
100
Mean Longevity (Days)
Algal Food Trout Food

86.2
99.7
88.5
46.7
16.6
5.1

50.5
31.3
30.0
31.2
13.6
1.5
Mean
Algal Food

11.9
13.6
14.3
15.1
12.4
13. Ob
Brood Size
Trout Food

21.4
14.8
11.7
9.6
8.2
— —
aEach mean  is  calculated  from  a  cohort of 20 animals.

bBased on only two  broods of 13  each.

         Biesinger and  Christensen  (1972) evaluated the impact of a chron-
ic copper stress  on ]3.  magna using  the same trout-granule food.  They
state that  there  was a  WTeproductive  impairment at a copper concen-
tration  of  20  ppb in Lake Superior  water, which has a total alkalinity
about one-fourth  that  of  our pond water.  However, they maintained their
daphnids in groups  of  five and young were removed, and counted, only
weekly.  It was not possible,  therefore, for them to differentiate be-
tween a  direct impact  of  copper  on  reproduction and an indirect impact
                                    57

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   TABLE 18.  ANALYSES OF CUMULATIVE MEAN
      BROOD SIZE FOR D. magna IN FIVE
       COPPER CONCENTRATIONS WHEN FED
      TROUT-GRANULE FOOD IN POND WATER9
Through
Cumulative
Broods
1
2
3
4-5
6-20
Copper Concentrations (ppb)
0
0
0
0
0
20

20

20
20
20

80

40
40

40
40


40
60
60
60
60

60
80
80
80
80

aBrood sizes were significantly different (P
 = 0.05)  in any two concentrations not mutu-
 ally underlined.  Brood sizes decrease in
 concentrations,  as sequenced, from left to
 right.
                   58

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through a reduction  in  longevity and  fewer  broods.  This made it impos-
sible to determine what effect, if any,  copper had on brood sizes.  In
the present study, for  example, the total reproductive output from ani-
mals maintained at 60 ppb Was considerably  less than for control animals
(Table 8) in spite of the fact that animals  in the 60 ppb concentration
had significantly larger broods than  did control animals.  The differ-
ence in reproductive output has nothing  directly to do with reproduction,
it simply reflects the  shortened life span of animals maintained at 60
ppb.   Biesinger and Christensen (1972)  ran  their chronic tests for only
three weeks and they used a three-week LC50  as their estimate of the
highest concentration that did not affect longevity.  Use of both a three-
week time limit and  of  LC50 values seem  questionable as tools for deter-
mining the maximum concentration that does not significantly reduce sur-
vival.  This,coupled with a lack of data showing the effect of copper on
brood size, casts some  doubt on their statement, "...  reproductive im-
pairment was found to be a more sensitive measure of toxicity than sur-
vival ."

        In the present  study, animals reared on the trout-granule food
also exhibited a retardation in reproductive development when cultivated
in copper concentrations greater than 20 ppb.  This was not true for
animals fed the algal food (Table 19,«Fig. 24).  This is yet another in-
dication that that trout-granule food makes  the animals more sensitive
to a copper stress.


        TABLE 19.  MEAN AGE AT REPRODUCTIVE MATURITY OF D_.  magna
                   AS AFFECTED BY FOOD-TYPE AND COPPER
                        CONCENTRATION  IN  POND WATER
                                                 Copper (ppb)
Experiment            Food             0         20        40         60
I
II
III
IV
V
Algae
Algae
Algae
Trout-Granule
Trout-Granule
5.3
4.7
6.5
5.6
6.2
5.3
4.5
6.1
7.5
6.6
5.1
4.4
6.5
8.6
7.7
5.4
6.0
6.6
13.4
11.1
EFFECT OF A CHRONIC COPPER STRESS ON THE
INSTANTANEOUS RATE OF POPULATION GROWTH

        The instantaneous rate of population growth, _r, takes into ac-
count longevity and all facets of reproduction (e.g., age at reproductive
                                    59

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        lOO-i
                                      ° Algal Food
                                       Trout-granule  Food
               I           I
                           10       15       20      25
                           Age in  Days
Figure 24.  Effect of food type on age at reproductive maturity of Daphnia
          magna in pond water
                                60

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maturity, age at reproductive  senility, frequency of reproduction, and
number of offspring per  brood).  Although other  stresses may have dif-
ferent effects, in the present study, jr-values,  within a species, were
largely determined by the  interaction between  longevity and brood size;
copper did not affect age  at reproductive maturity or frequency of re-
production in the pond-water,  algal  system.  Differences between species
were also related to species differences in frequency of reproduction
and age at reproductive  maturity.

Comparative Response of  the Four Species

        Species differences in the  instantaneous rate of population
growth are presented in  Table  20.  J). magna had  the smallest r-values
under control conditions.  This is a reflection  of:  (1) longer matura-
tion (]). magna produce their first young at an age of about 7 days, ID.
pulex at an age of 6 days, J3.  parvula and ID. ambigua at an age of 5
days); (2) longer period of embryological development (3 days for J).
magna and 2 days for the other species); and (3) ]D. pulex had larger
broods than did JD. magna.  The early maturation  and short period of em-
bryological development  of J).  ambigua was somewhat negated by its pro-
ducing the smallest broods of  any of the four  species.
          TABLE  20.   INSTANTANEOUS  RATE  OF  POPULATION GROWTH  (r)
                  AS  AFFECTED BY  COPPER  FOR FOUR SPECIES
                OF Daphnia IN THE POND-WATER, ALGAL  SYSTEM
Capper
(ppb)
0
20
40
60
80
100
D. maqna

0.373
0.405
0.409
0.325 a
0.287
*
D. pulex

0.498
0.469
0.453
0.230
*
*
D. parvula

0.423
0.424
0.390
*
*
*
D. ambigua

0.459
0.454
0.432
0.233
*
*
      Horizontal  lines indicate that the r-vales  above and  below
       the line are significantly different (P = 0.05).

      *No reproduction occurred at these concentrations.
                                   61

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        Table 20 shows that, except for ]3. magna, there is a significant
reduction in jr at concentrations above 40 ppb.  This is due to both an
increase in mortality and a reduction in brood size.  In fact, reproduc-
tion is shut off completely for the three smaller species at a copper
concentration of 80 ppb.

Effect of Food-Type on r

        As was true for longevity and reproduction, £ was affected at
lower copper concentrations in the trout-granule system than in the algal
system (Table 21).  Algal-fed ]D. magna did not exhibit a reduction in jr
at copper concentrations less than 80 ppb, whereas D_. magna fed on the
trout-granule diet exhibited a reduction in _r at 60 ppb (Tables 20 and
21).  Under all conditions, in the present study, £ was a less sensitive
index of copper stress than longevity.  This may, however, not be true
for other stresses.  It seems possible that conditions could exist where
_r was more sensitive than either longevity or brood size.  Such a situa-
tion might involve stresses that affect age at reproductive maturity or
frequency of reproduction.


     TABLE 21.  EFFECT OF A COPPER STRESS ON THE INSTANTANEOUS RATE
                 OF POPULATION GROWTH OF D.. magna IN THE
                    TROUT-GRANULE, POND-WATER SYSTEM
Copper (ppb)          Experiment 1          Experiment 2           Mean
0
10
20
40
60
70
80
90
0.384

0.318
0.237
0.211 a

0.070

0.331
0.339
0.291
0.259
0.211
0.191
0.162
0.067
0.358

0.305
0.248
0.211

0.116

aHorizontal lines indicate that r-values above and below the line are
significantly different (P = 0.05).
A Comparison of Longevity, Brood Size, and r
as Indices of Copper Stress

        Table 22 compares the maximum allowable toxicant concentration
for copper that would be permitted in the pond-water, algal system using
                                   62

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         TABLE 22.  MAXIMUM ALLOWABLE TOXICANT CONCENTRATION OF
                    COPPER (PPB) FOR FOUR SPECIES OF
                      Daphnia BASED ON LONGEVITY,
                            BROOD SIZE AND R
                          	MATC Based on
        Species           Longevity         Brood Size
D. magna
D. pulex
D. parvula
D. ambigua
40
40
40
40
80+
60
60
40
60
40
40
40
longevity, brood size, and jr as indices.  Longevity and _r both predict
that the three smaller species would be adversely affected at copper
concentrations greater than 40 ppb.  For J3. magna, however, longevity is
a more sensitive index than is jr.  This reflects the stimulatory effect
of copper on brood size in D_. magna, which more than offsets the reduced
longevities at 60 ppb.  Brood-size response to a chronic copper stress is
more variable than either £ or longevity.  In J3. magna. 13. pulex, and 13.
parvula. brood size is less sensitive to a copper stress than either r_
or longevity.  For D_. magna, use of brood size as an index would predict
an MATC of copper twice that predicted by longevity.  For J). ambigua.
the three indices are in agreement in predicting the MATC to be 40 ppb
copper.

An Evaluation of the Application-Factor Concept

Comparison of Interspecific Application Factors—
        Application factors, using only those 72-hr LC50 values from
parallel concentration-response curves (Table 4) and MATC values calcu-
lated from chi-square analyses of combined survival curves for two chron-
ic tests for each species, are presented in Table 23.  Survival was used
as the best estimate of MATC because it was as, or more, sensitive than
brood size or £ for each of the four species.  These application factors
vary only from 0.46 for D_. magna to 0.59 for D_. ambigua.

        Application factors which have been published for fish (e.g.,
Mount, 1968; Mount and Stephan, 1969; and McKim and Benoit, 1971) have
been calculated using the mean of several, independently-determined LC5o
values.  The statistical validity of this is questionable (Finney, 1971),
since in none of the publications is there any evidence that the LC50
values are from parallel concentration-response curves.
                                    63

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        TABLE 23.  APPLICATION FACTORS OF COPPER FOR FOUR SPECIES
               OF Daphnia IN THE POND-WATER, ALGAL SYSTEM
Species
]D. magna
D_. pulex
D. parvula
D. ambigua

MATC
(Longevity)
40
40
40
40
72-Hr
LC50
85.1
86.0
72.0
67.7
Application
Factor
0.46
0.47
0.56
0.59
        The use of mean LCso values for calculating application factors
 in the present study does not, however, significantly change the results.
 Using mean,  72-hr LC50 values for each species, the factors still vary
 only from 0.46 for D_. magna to 0.62 for J3. parvula.  A one-way, complete
 random design ANOVA indicates that these values are not significantly
 different (P = 0.05).

        I can find no references in the literature to application  factors
 for Daphnia.  Copper application factors, as reported in the literature
 for fish, are much smaller than those we have observed for Daphnia.  For
 example, in a study of brook trout, the application factor for copper
 was estimated to lie between 0.10 and 0.17 (McKim and Benoit, 1971).
 Mount (1968) and Mount and Stephan (1969), working with fathead minnows,
 estimated the application factor for copper to be between 0.03 and 0.07
 in hard water and between 0.14 and 0.24 in soft water.  The larger ap-
 plication factors for the daphnids may be due to the fact that an acute
 test comprises a much greater fraction of the total life cycle of daph-
 nids than of the life cycle of fishes.

 Effects of Water Chemistry on the Application Factor--
        The acute and chronic toxicity of copper was more variable in re-
 constituted waters than in pond water (Table 24) and, therefore, applica-
 tion factors were more variable for D_. magna.  This greater variability
was probably caused by several  factors.  In standard water, for example,
 the very low test concentrations, differing by only 2.5 to 5.0 ppb, are
 probably,  in themselves,  contributing to the variability.  In the two
 filtered-water experiments a shift of the MATC, from only 2.5 to 5.0 ppb,
 doubled the application-factor estimate.  Also, as previously pointed
out, there was a secondary,  and variable, chemical stress in some, or
all, of the reconstituted-water experiments.  A reproducibly-adequate,
distilled-water base should  reduce the test variability.  There is a
                                   64

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suggestion of this in the two experiments with aerated standard water
(Table 24).  Not only are the application factors more similar for these
two experiments, they are also much closer to the application factors
calculated for D_. magna in pond water.


           TABLE 24.  EFFECT OF WATER- AND FOOD-TYPE ON COPPER
                  APPLICATION FACTORS FOR Daphnia magna
Water
Pond
Pond
Pond
Pond
Pond
Filt. Std.
Filt. Std.
Food
Algae
Algae
Algae
Trout-Pellet
Trout- Pel let
Algae
Algae
72-hr LC50
88.8
85.0
81.5
81.4
85.3
13.0
13.0
MATC
40
40
40
10
10
2.5
5.0
Application
Factor
0.45
0.47
0.49
0.12
0.12
0.19
0.38
Unfilt. Std.      Algae                25.5         2.5         0.10

Aer. Std.         Algae                31.6         10          0.32
Aer. Std.         Algae                31.6         15          0.47
Medi urn
Medium
Algae
Algae
101.4
88.1
10
60
0.10
0.68
        The greatest disparity in results occurred in the two experiments
with reconstituted medium water.  Estimates of both 72-hr LC50 and MATC
values were very different for the two experiments.  Again, I feel that
this is due to variability in the quality of our distilled water.

Effect of Food-Type on the Application Factor—
        The trout-granule food greatly increased the sensitivity of ID.
magna to a chronic copper stress, but did not significantly alter the
LC50 values from those obtained with the algal food (Table 24).  As a
consequence, the difference between LC50 and MATC was increased and the
application factor was much smaller for animals on the trout-granule
food.  The two tests with the trout-granule food, like the three tests
with algal food, were very consistent.  The smaller application factor,
like those observed in some reconstituted-water experiments, may reflect
                                    65

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an added stress, in this case, a nutritional  stress.   It is also inter-
esting to speculate, since the chronic response exhibited a striking
change and the acute response did not, that the mode  of action of copper
may be different in acute and chronic situations.
                                   66

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                               SECTION VI

                               REFERENCES


Analytical Qual. Control Lab.  1971.  Methods for Chemical Analysis of
        Water and Wastes.  U. S. E. P. A., Cinn., OH.  312 pp.

Biesinger, K. A. and G. M. Christensen.  1972.  Effects of various
        metals  on survival, growth, reproduction, and metabolism of
        Daphnia magna.  J. Fish. Res. Bd. Canada.  29:1691-1700.

Brooks, 0. L.   1957.  The Systematics of »®rth American Daphnia.  Mem.
        Conn. Acad. Arts Sci. XIII.  Yale U. Press, New Haven, Conn.
        180 pp.

Christian, G. D. and F. J. Feldman.  1970.  Atomic Absorption Spectros-
        copy.   Applications in Agriculture, Biology, and Medicine.
        Wiley-Interscience, New York, N. Y.  490 pp.

Duncan, D. B.   1955.  Multiple range and multiple F tests.  Biometrics.
        11:1-42.

Finney, D. J.   1971.  Probit Analysis.  Cambridge U. Press, London.  333
        PP-

Hueck, H. J. and D. M. Adema.  1968.  Toxicological investigations in an
        artificial ecosystem.  A progress report on copper toxicity to-
        wards algae and daphniae.  Helgolander wiss. Meersunters.  17:
        188-199.

Kemp, H. T., J. P. Abrams, and R. C. Overbeek.  1971.  Effects of chemi-
        cals on aquatic life:  Water quality criteria data book.  Vol. 3.
        U. S. E. P. A. Water Poll. Contr. Res. Serv.  18050 GWV05/71.
        U. S. Govt. Print. Off., Washington, D. C.  528 pp.

Mantel, N.  1966.  Evaluation of survival data and two new rank order
        statistics arising in its consideration.  Cancer Chemother.
        Repts.  50:163-170.

Marking, Leif L.  1969.  Toxicity of quinaldine to selected fishes.  In-
        vest. Fish Control No. 23.  Bur. Sport Fish. Wild!., Washington,
        D. C.   10 pp.

McKim, J. M. and D. A. Benoit.  1971.  Effects of long-term exposures to
        copper on survival, growth, and reproduction of brook trout
        (Salvelinus fontinalis).  J. Fish. Res. Bd. Canada.  28:655-662.

                                    67

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

Mount, D. I. and C. E. Stephan.  1969.  Chronic toxicity of copper to
        the fathead minnow (Pimephalas promelas) in soft water.  J. Fish.
        Res. Bd. Canada.  26:2449-2457.

Murphy, J. S.  1970.  A general method for the monoxenic cultivation of
        the Daphnidae.  Biol. Bull. 139:321-332.

Pagenkopf, 6. K., R. C. Russo, and R. V. Thurston.  1974.  Effect of
        complexation on toxicity of copper to fishes.  J. Fish. Res. Bd.
        Canada.  31:462-465.

Stephan, C. E. (Ed.).  1975.  Methods for acute toxicity tests with
        fish, macroinvertebrates, and amphibians.  Ecol. Res.  Ser,   No.
        EPA-660/3-75-009.  U. S. Environmental  Protection Agency,
        Duluth, Minn.  61 pp.

Stiff, M. J.  1971.  Copper/bicarbonate equilibria in solutions of bi-
        carbonate ion at concentrations similar to those found in natural
        waters.  Water Res.  5:171-176.
                                   68

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                                   TECHNICAL REPORT DATA
                            (Please read lasO-uctions on the reverse before completing)
 REPORT NO.
 EPA-60Q/3-76-051
2.
 TITLE AND SUBTITLE
 Toxicity of Copper to Daphnids in Reconstituted
 and Natural Waters
                                                            5. REPORT DATE
                                                            May 1976  (Issuing Date)
                              6. PERFORMING ORGANIZATION CODE
                              3. RECIPIENT'S ACCESSIONING.
 AUTHOR(S)

 Robert W.  Winner
                              8. PERFORMING ORGANIZATION REPORT NO.
 PERFORMING ORGANIZATION NAME AND ADDRESS
 Miami University
 Oxford,  Ohio  45056
                                                            10. PROGRAM ELEMENT NO.

                                                                 1BA608
                              11. CONTRACT/GRANT NO.

                               Grant  R802210
12. SPONSORING AGENCY NAME AND ADDRESS
 U.S. Environmental Protection Agency
 Office of Research and Development
 Environmental Research Laboratory
 Duluth, Minnesota  55804
                              13. TYPE OF REPORT AND PERIOD COVERED
                               Final	
                              14. SPONSORING AGENCY CODE

                                 EPA-ORD
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
       The toxicity of  copper was compared for Daphnia magna cultured in
  reconstituted versus  pond water and fed  on trout-pellet versus vitamin-
  enriched, algal foods.   Effects of a chronic copper stress were highly
  variable when animals were tested in reconstituted waters.  This varia-
  bility is thought to  be due to variability in the quality of the distilled-
  water matrix.  The vitamin-enriched algal food was found to be superior
  to the trout-granule  food in culturing D_.  magna.   Control animals  lived
  much longer and test  animals were less sensitive to a chronic copper
  stress.   The acute and  chronic toxicity  of copper was also compared for
  four species of Daphnia.  When tested in pond water and fed vitamin-
  enriched algae, the two largest species  (D_.  magna and p_. pulex) were
  significantly less sensitive to an acute copper stress than the two
  smallest species  (D.  parvula and D_. ambigua).  There was, however,  no
  significant difference  in sensitivity to a chronic copper stress when
  reduced longevity was used as the index.   Application factors for  the four
  species varied from 0.47 to 0.62 and were not significantly different.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                                              COS AT I Field/Group
 Daphnia,  Copper, Bioassay
 Animal nutrition
                  Application
                 Daphnia mac
                                                            factor
 06T
                 Daphnia pulex
                                              Daphraa parvula
                                              Daphnia ambiqua
                                              Toxicity test
                                               Reconstituted water
 3. DISTRIBUTION STATEMENT

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