PB86-122579
    Acute and Chronic Effects  of"  Water
    Quality Criteria Based Metal.  Mixtures  on
    Three S.^uatic  Species
    (U.S.) Environmental Research Lab.-Duluth,  MM
    Nov 8 5
       psgggagsg^^

  SS2S3Z!aS^<3S3B?E2HBffiBS3a«S^ig3^^
y.S. Derfeenl oJ

-------
                                             EPA/600/3-85/074
                                             November? 1985
ACUTE AND.CHRONIC EFFECTS OF WATER QUALITY CRITERIA


   BASED METAL MIXTURES ON THREE AQUATIC SPECIES
            R.L. Spehar and J.T. Fiandt
         ENVIRONMENTAL RESEARCH LABORATORY
         OFFICE OF RESEARCH AND DEVELOPMENT
        U.S.  ENVIRONMENTAL PROTECTION AGENCY
                 DULUTH, MN 55804

-------
TECHNICAL REPORT DATA
IFlease read Instructions on the rtverse before completing)
i. REPOST NO.
EPA/6CO/3-85/074
4. TITLE ANDSUBTITLE
Acute and Chronic Effects of
Metal Mixtures on Three Aqua
7. AUTHORISt
R.L. Spehar and J.T. Fiandt
9. PERFORMING ORGANIZATION NAME Ah
U.S. Environmental Protectio
Environmental Research Labor
6201 Congdon Boulevard
Duluth, .MN 55804
2. 3. RECIPIENT'S ACCESSION NO.
; PB8 1> 1 2 2 5 7 9 /AS
5. REPORT DATE
Water Oualitv Criteria P-^H November 1935
Water Quality Criteria B^sed6 PEnFORM1NG ORGANIZATION CODE
tic Species
8. PERFORMING ORGANIZATION REPORT NC.
4D ADDRESS 10. PROGRAM ELEMENT NO.
n Agency
itory-uuluth 11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
Environmental Research Laboratory
Office of Research and Development 14. SPONSORING AGENCY CODE
U.S. Environmental Protection Agency
Duluth, MN 55804 EPA-600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT Acute and chronic I
combined •• mixture
nultiplea of Che LC
speciei. These i :u
water quality crite
Arsenic, cadnium, c
concentration! caui
(C. dubia) during a
thia or two times tt
value cauaed 15 to
concentrat ione aign
growth after 7 and
rainbow trout were
average concentrati(
Acute teata with me
action waa more that
daphnide baaed on t(
mixture. Chronic t<
fathead minnows but
octal interactiona i
effects were obaervi
minnows and daphnid
below no effect com
a chronic baaia. Tt
the type and degree
quality criteria ma;
preaent concurrently
17.
a. DESCRIPTORS
axicity teata were conducted to determine the effecta of metals
i at proposed water quality criteria concentration and at
'0 and MATC obtained from tests on six metals with three aquatic
lies were the first part of a larger reaearch effort to derive
ria for combined pollutants by the U.S. EPA.
iroaium, copper, mercury, and lead combined at criterion maximum
:d nearly 100 percent mortality to rainbow trout and daphnids
:ute exp^aur.e. Fathead minnows were not adversely affected at
lia concentration, although a mixture of A to 8 tinea the maximum
bO percent mortality. Metals combined at the. criterion average
ficantly reduced daphnid young production and fathead minnow
i2 days, respectively. Embryo hatchability and survival of
reduced at 4 times thia criterion but not at the criterion
jn.
ala mixed tt multiples of the LC50 indicated that their joint
i additive to fathead minnoua and nearly strictly additive to
)xic units calculated from the individual components of the
ists showed that the joint action was lesa than additive to
nearly strictly additive to daphnids indicating that long term
Day be different in fish than in lower invertebrates. Adverse
;d at mixture concentrations of 1/2 to 1/3 of the MATC on fathead
i, respectively, suggesting that components of mixtures at or
:entrations nay contribute significantly to mixture toxicity on
ese resulti point out the need for additional (tudies to determine
of interaction of toxicants becauae tingle chemical water
r not sufficiently protect some apeciea when other toxicants are
r .
KEY WORDS AND DOCUMENT ANALYSIS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group

18. 3ISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (Tni! Rtportl 21. NO. OF PAGES
Unclassified 54
20. SECURITY CLASS iThis pagti 22. PRICE
Unclassified
EPA Form 2220-1 (R»v. 4-77)    PREVIOUS EDITION is OBSOLETE

-------
                      NOTICE

This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication.  Mention of trade names
or commercial products does not constitute endorse-
ment, or recommendation for use.
                       11

-------
                                 ABSTRACT




     Acute and chronic toxicity tests were conducted to determine Ithe




effects of metals combined as mixtures at proposed water quality criteria




concentrations and at multiples of the LC50 and MATC obtained from tests




on six metals with three aquatic species.  These studies were the first




part of a larger research effort to derive water quality criteria for.




combined pollutants by the U.S. EPA.




     Arsenic, cadmium, chromium, copper, mercury, and lead combined at




criterion maximum concentrations caused nearly 100 percent mortality to




rainbow trout and daphnids (C_. dubia) during acute exposure.  Fathead




minnows were not adversely affected at this or two times this concentration,




although a mixture of 4 to 8 times the maximum value caused 15 to 60




percent mortality.  Metals combined at the criterion average concentrations




significantly reduced daphnid young production and fathead minnow growth




after 7 and 32 days, respectively.  Embryo hatchability and survival of




rainbow trout were reduced at 4 times this criterion but not at the criterion




average concentration.




     Acute tests with metals mixed at multiples of the LC50 indicated that




their joint action was more than additive to fathead minnows and nearly




strictly additive to daphnids based on toxic units calculated from the




individual components of the mixture.  Chronic tests showed that the joint




action was less than additive to fathead minnows but nearly strictly additive




to daphnids indicating that long term metal interactions may be different




in fish than in lower invertebrates.  Adverse effects were observed at  mixture




concentrations of 1/2 to 1/3 of the MATC on fathead minnows and daphnids,




respectively, suggesting that components of mixtures at or below no effect




concentrations may contribute significantly to mixture toxicity on a




chronic basis.  These results point out the need for additional studies





                                   1 i i

-------
to determine the type and degree of interaction of toxicants because

single chemical water quality cri.teria may not sufficiently protect some

species when other toxicants are ;present concurrently.
Keywords:   Mixtures, Metals, Fish, Invertebrates, Acute toxicity, Chronic
           toxicity.

-------
                             ACKNOWLEDGMENTS







     The authors would like to express their appreciation to D.J. Ruppe




and E.N. Leonard for providing analytical'assistance for this study.  We




greatfully acknowledge J.R. Amato for his invaluable cooperation and




technical assistance in the laboratory.  We also wish to thank S.J.




Broderius for valuable suggestions and helpful discussions regarding the




study of chemical mixtures.

-------
                               INTRODUCTION


     Much of the information in the literature on the toxicity of chemicals
                                                                     i

to aquatic life deals with studies involving single toxicants.  Numerous


toxicity tests have been conducted to determine the acute and chronic effects


of toxicants to provide data for the derivation of water quality criteria.


To date, existing water quality criteria have been derived for single


toxicants, yet it is rare to find natural waters in which only single


toxicants are present.  Aquatic organisms are usually exposed to a wide


variety of toxicants from exposure to direct effluent discharges or from


non-point source pollution due to chemical runoff.  For this reason,  the


utility of water quality criteria on single toxicants is often questioned.


     In an effort to establish more effective water quality criteria  and


hazard assessment programs, several mathematical models have been developed


to predict the effect of mixtures of chemicals on aqjatic organisms [1-6].


The application of these methods, however, has generally applied to acute
                                       *
lethality tests [7] and little work has.been done to investigate the


effects of mixtures on aquatic organisms on a chronic basis at sublethal


concentrations [8-13].  Results from these tests appear to be somewhat contra-


dictory and show no clear trend as to how chemicals interact as mixtures


during acute and chronic exposure.


     Due to the lack of adequate information, especially on the chronic


effects of mixtures, little guidance .has been given for setting water


quality standards based on chemical mixtures.  An early, tentative approach


for evaluating joint toxicity has been to assume that there is an additive


action between diverse toxicants [14].  A similar approach has more recently


been taken by the European countries [7].  Based on their review of the


literature on the effects of mixtures on freshwater fish and other aquatic

-------
organisms, it was proposed that for pollution control purposes, the

concentration addition model is adequate to describe the acutely lethal
                                 I

joini. effect of commonly occurring constituents of sewage and industrial

wastes.  This proposal is based on the rationale that the joint acute

lethal toxicity of. chemicals to fish can be predicted assuming simple

addition of the proportional contribution from each toxicant, but that

toxicity based on concentrations approaching no-effect are less than

additive and probably do not contribute to the chronic toxicity of mixtures.

It was concluded, however, that more empirical studies are needed on the

long-terra joint effect of mixtures of toxicants, especially, to determine

the contribution of small fractions of the toxic units of the individual

components.

     The first objective of this research was to determine if the single

chemical water ouality criteria proposed by the U.S. Environmental

Protection Agency (EPA) in 1984 [20)1 for selected inorganic chemicals were

sufficient to protect selected aquatic species when they were present as

mixtures.  These criteria are not specific for individual species but

are based on several species from a variety of aquatic families.  The present

studies would not show thu type and degree of interaction of chemicals in

these mixtures but would indicate their effect at proposed criteria

concentrations.

     The second objective was to measure the contribution of fractions of

toxic units of mixtures by using acute (LC50) and chronic (MATC) values

obtained from tests on individual chemicals in mixture tests at, above,

and below these concentrations.  In addition to the MATC, an estimate of
1  The numerical national water quality criteria proposed in 1984 [20],
guidelines for deriving these values [26], and the corresponding terminology
used in this text have been changed slightly since the completion of this
research project due to the inclusion of new research data and public comments.
For the most recent information on this subject see [48].

-------
the level of 50 percent reduction in growth and reproduction was used as




the toxic unit in chronic tests.  Results from these tests would be species-




specific and would indicate the possibility of concentration addition.




     Inorganic chemicals, specifically arsenic, cadmium, chromium,




copper, mercury and lead were selected for this study because of their




importance to EPA in deriving individual chemical water quality criteria




and because these chemicals are found together as mixtures in commonly




occurring sewage and industrial wastes [15].  Tests were conducted with fathead




minnows, rainbow trout, and daphnids.  Fathead minnows and rainbow trout




are important forage and game fish species, respectively, and are




representatives of both warm and cold water aquatic organisms.  Daphnids




were chosen for this study because they are among the most sensitive




aquatic organisms to most of the selected chemicals.

-------
                          METHODS AND MATERIALS




Test Water




     Tests were conducted in Lake Superior water that was filtered




through sand and heated to 25 +_ 3° C for tests with fathead minnows or




cooled to 10 _+ 3° C for tests with rainbow trout .  Tests with daphnids




were conducted at 25 _+ 2° C with reconstituted hard water [16] and water from




the Lester River located adjacent to the Environmental Research Laboratory




in Duluth, MN.  All organisms were cultured in the respective water before




they were tested.  Most of the tests conducted with daphnids were done in




Lester River water because better survival and reproduction results were




observed in this water than in either Lake Superior or reconstituted




water.  Lester River water was collected from just below the water surface




and stored in polyethylene f- gallon jugs at 6° C prior to testing.  River




water was filtered twice through 45 cm mesh screening and vigorously




aerated before all daphnid tests.  Routine chemical characteristics of




all test waters were measured according to procedures described by the




American Public Health Association et al. [17] and are shown in Table




1 for each species.  Dissolved oxygen concentrations in the test chambers




for all waters were at or above 70 percent saturation.




Exposure Systems and Toxicant Solutions




     Flow-through tests with fathead minnows and rainbow trout were




conducted using a dilution system [18] that delivered five concentrations at a




0.5 dilution factor and a control to up to 4 replicate chambers per




treatment.  Lake Superior water was fed from stainless steel headboxea to




the diluter after it was vigorously aerated to remove excess dissolved




gases.  All toxicant solutions were delivered to the diluter mixing cell




via FMI pumps (Fluid Metering Inc., Oyster Bay, N.Y. 11771) from 19 liter

-------
glass stock bottles containing either individual or mixtures of stock




solutions.




     Glass test chambers measured 7 cm wide x 18 cm long x 9 cm high with




a water depth of 6.4 - 7.0 cm.  The flow rate to each chamber was -15 j*




1 ral/min.  Wide spectrum fluorescent bulbs provided a light intensity of




30-45 and 110-320 lux for tests with rainbow trout and fathead minnows,




respectively, at the water surface during a 16 hr photoperiod.  This




included a 30 rain, gradual brightening and dimming period with incandescent




lights to simulate dawn and dusk [19].




     Static renewal tests with daphnids were conducted in 30 ml plastic




disposable beakers (Plastics, Inc., Minneapolis, ,-flO containing 15 ml of




solution.  Replicate test beakers (10 per concentration) were used in all




tests for each of 6 toxicant concentrations and a control, utilizing a




0.5 dilution factor scheme.  Test beakers were located in a water bath to




provide the necessary temperature control.  Light intensify at the water




surface was 25-100 lux for the 16 hr automatically controlled photoperiod.




     Stock solutions-for all tests were prepared by dissolving reagent




grade sodium arsenite (Na2As04-7H20), cadmium nitrate (Cd(N03)2-H20),




sodium dichromate (Na2Cr207.f^O), cupric nitrate (Cu(N03)2.3H20),




mercuric nitrate (Hg(N03>2.H20) and lead nitrate (Pb(N03>2) in distilled




water.  For mixture tests, stock solutions of arsenic and chromium were




kept separate from each other and from the other chemicals because of the




formation of insoluble compounds at high concentrations.  Although




several valence states of arsenic and chromium exist in natural waters,




trivalent arsenic and hexavalent chromium were chosen for our studies




because numerous toxicological studies with aquatic organisms on these




forms were available in the literature and because their chemical properties

-------
were suitable for mixture testing.  Both forms, however, are easily!




converted to different valence states depending upon water quality (i.e.,




reversible reactions occur between trivalent and pentavalent arsenic and




hexavalent and trivalent chromium [20]).  Therefore, arsenic and chromium




species were analyzed by methods described by Ficklin [21] and Martin and




Riley 1982 [22], respectively, to determine the species present under our test




conditions.  Trivalent arsenic was found to stay in this form when arsenic




was tested individually, however, peutavalent arsenic was measured periodically




in the presence of the other chemicals.  The ratio of trivalent to pentavalent




arsenic ranged from 0 to 100 percent in chambers containing mixtures.




Hexavalent chromium, essentially stayed in this form ( > 95 percent) in all




tests.  Nitrates of copper, cadmium, mercury and lead were used in this study




because of their solubility properties and stability in these mixtures.




Quality Control




     Samples of all test solutions were taken from the test .chamber or




mixing flasks and analyzed according to a monitoring program that




characterized the test pattern.  Procedures for metal water analyses




were those described by the U.S. EPA [23].  Measurements of all of the




metals except for mercury are expressed as total acid exchangeable metal.




Mercury measurements are reported as total recoverable metal.  Detection




limits for these procedures are included in Tables 2 and 3.  To verify




the accuracy of the method of analyses, known amounts of metal were added




to control water to obtain percentage recoveries each time water samples were




taken.  Mean, standard deviation and number of analyses in parentheses of




percentage recoveries for arsenic, cadmium, chromium, copper, mercury,




and lead in all tests were 100^6 (61), 98jf6 (58), 100_+5 (61), 59^7 (65),




97+6 (59), 92j*9 (60), respectively.  Quality control samples from the




Environmental Monitoring and Support Laboratory, Cincinnati, OH were also

-------
obtained for use as checks on our quality control.
All values were
similar to those stated above for spiked recoveries.
In addition, samples,
except for mercury, were periodically filtered through a 0.45 ~m Millipore
filter to measure the portion of dissolved metal.
Mean, standard deviation
and number of analyses for dissolved measurements (in percent) of total
acid exchangeable arsenic, cadmium, chromium, copper and lead were 101~8 (11),
93+9 (11), 102~3 (11), 92~6 (11), and 75~14 (16), respectively.
Because
mercury loss is high during the filtration procedure, samples were differentiated
instead as to their inorganic and organic components to characterize the
metal in solution [23).
Mercury samples were, ganeral1y, found to be >
90 percent inorganic mercury in our test water.
All concentrations are
expressed as the metal, not as the compound tested.
Biological Procedure~
The test animals used in this study were fathead minnows (Pimephales
promel~), rainbow trout (Salmo .sairdneri) and ceriodaphnids (Ceriodaphnia
dubia).
The name cp.riodaphnids will .hereafter be used in the text as daphnids
for simplicity.
Rainbow trout embryos « 24 hr old after fertilization)
were obtained frem the Minnesota Department of Natural Resources cold
water fish hatchery located at the mouth of the French River, near Duluth.
Minnesota and were incubated and reared at our laboratory at 100 C prior
to testing.
Both fathead mlnnows and daphnids were obtained from existing
cultures at our laboratory.
Procedures for conducting acute lethality
tests to dete~ine LC50 values closely followed those described by the
American Society for Testing and Materials (ASTM) [16].
Acute tests with
fish were continuous flow through tests and were initiated by randomly
distributing 10 rainbow. trout (- 90 day old, ~ 1.5 g) and fathead minnows
(- 30 day old, - 0.15 g) to each duplicate test chamber per treatment.
Tests .with fish were for 96 hr.
Acute (48 hr) static tests with daphnids
7

-------
were initiated l'y randomly distributing five « 24 hr) daphnids to each
duplicate exposure chamber.
Procedures for conducting flow-through
chronic tests with fish were similar to those described by ASTM [24] and
renewal tests with daphnids were done according to Mount and Norberg
( 25].
Survival, growth and/or YOung production were used as the respons~
variables in these chronic tests.
Testing Design
The first part of this study began in May of 1983 by exposing each
species to a mixture of the six metals at water quality criteria
concentrations proposed by the U.S. EPA [20] (Table 4).
Water quality
criteria concentrations for aquati.c 1i fe are expressed as two numbers, a
maximum and average concentration.
The definition and guidelines for
deriving these concentrations a~e glven by Stephan et al. [26].
Measured
water concentrations in our tests correlating to these criteria are shown
in Tables 2 and 3.
Tests included concentrations at, above, below and in
proportion to the criteria for each metal.
Acute lethality tests were
used to determine effects at criterion maximum concentrations and chronic
tests were utilized in tests at criterion average concentrations [26].
Criteria concentrations of cadmium, copper and lead were adjusted for
water hardness according to guidelines for deriving national water quality
criteria
[26J.
The second part of this study was initiated by exp.1sing fathead
minnows and daphnids to each metal to determine respective LC50 aud
chronic (MATC) values.
Subsequently, each species was exposed to mixtures
at, above, and below these concentrations to determine possible additive
interactions of the six selected metals.
Rainbow trout were not used in
these studies hecause tests with this species were too long (up to 90
days) for the number of tests required.
(3

-------
Statistical Procedures




     Forty-eight and 96 hr LC50 values were determined with a computerized,




modified trimmed Spearman-Karber method described by Hamilton et al. [27].




Daily mortality data from replicate exposure tanks vere combined before




LC50 values were calculated.




     For early life-stage tests with rainbow trout and fathead minnows,




survival, embryo hatchability, and larval deformity data were transformed




to arcsin % [28] for variance stabilization.  Individual weights of fish in




replicate chambers were pooled before data were subjected to Dunnett's




one-sided comparison of treatment means to control means (P = 0.05) [29].




Survival and young production data for life cycle daphnid tests were




analyzed using the procedure of Hamilton [30] as modified by Rogers [31].

-------
RESULTS AND DISCUSSION
Criteria Exposures
Survival .of species exposed to metals mixed at their res~ective
criterion maximum coocentrations is shown in Table 5.
Metal mixtures at
this criterion concentration reduced the survival of rainbow trout by 95%
and killed all of the daphnids in acute tests but caused no significant
effect on fathead minnow survival after 96 hours.
Concentrations between
4 and 8 times this criterion reduced survival of fathead minnows from 15
to 60 percent and killed all of the rainbow trout.
A mixture of one half
of the maximum concentration had no significant effect on any of the
three species.
The sensitivities of these species appear to be directly
related to their sensitivities to each of the individual metals.
Both
rainbow trout and daphnirls are in families that were consistently the most
sensitive to each of these metals when compared to other aquatic species
[20).
.Conversely, fathp'~j minOows were usually found to be more tolerant
than trout to all of the metals except mercury, where their sensitivity ranked
between that of daphnids and rainbow trout.
Survival, growth and young production of SpeClp.s exposed to metals
mixed at their respective criterion average concentrations are presented
in Tables 6 and 7.
Concentrations of 8 and 16 times this value reduced
the survival of developing rainbow trout embryos and fathead minnow larvae
hy nearly 100 percent (Table 6).
A lower concentration of 4 times this
criterion also significantly reduced survival of trout sac larvae after
approximately 45 days of exposure and that of early juvenile fathead
minnows after a 32-day t~st.
A further reduction in the survival of
juvenile trout was observed at this concentration by the end of the 90-
day teat, however, due to fungal disease in two of the four replicate
control chambers during ~he la~t three week<' of the test, significant
10

-------
differences were not ob~erved.
Growth determi~ations of trout eKposed for
a 70-day period were a190 hampered by control t~mFeratures that were - 1° C
lower than the other treatmentg for app~oximately 21 days during the
90-day eKposure.
This resulted in:slightly smaller control fish by the
end of the test.
Although fish appeared smaller in chambers at 4 times
the criterion.average compared to those in lower concentrations, statistical
decreases ingrowth were not observed at ~his concentration dfter 70
days.
Growth determinations on f~thead minnows, however, indicated
dramatic decteages in weight. at lower miKture concentrations including a
significant 30 percent reduction at the criterion average concentration
(Table 6).
Visual decreases in growth were noted at this concentration
as early as 3 weeks after the test started.
Significant adverse effects
of miKtures at the criterion average concent=ation were also observed in
tests with daphnids which caused approximately 80 percent reduction in
young production after a 7-day test (Table 7).
Although survival of
daphnids was decreased by 40 percent by this concentration, the di~ferences
were not statistically significant.
The tolerance of rainbow trout to the criterion average mixtures was
surprising due to their normally sensitive nature to these individual meta19'.
However, t~is response may have been due to the luck of statistical dif-

ferences in the data as a result of the decreased growth of fish in the
controls, as waa previously stated.
Although trout appp.ared healthy in a
mixture at the criterion average concentration, it is likely that this
concentration could have been an effect concentration if control growth
was normal.
This hypothesis is supported by the fact that this concentration
caused adverse effects on both daphnids and fathead minnows; species
which, generally, have been shown to be as sensitive or less sensitive,
respectively, than trout to individual metals (20).
In contrast to the
11

-------
observed response for rainbow trout, the h~J~ sensitivity of fathead
present mixtures is not known,
TIle reason for their sensitivity to tle
1
although copper could have b~en th~ mJjor
minnow growth was unexpected.
factor contributing to the decrease in growth.
Copper was shown to
decre~se growth of this species at very low levels in individual me~al
tests conducted as part. of this.8tudy (see the following text) and in
recent studies conducted by Benoit [32] in Lake Superior water.
r;opper
concentrations causing effects were nearly the same as the critel'ia
concentrations in our mixtures.
The above results suggest that all three test species may not be
sufficiently protected if the selected metals were present in ~ater as
mixtures at proposed water quality criteria r.oncentrations.
These data
are particularily meaningful because rainbow trout are an economically
important species and were advers~ly affected at least at tte criterion
maximum concentrations.
Althou~h pre~ently proposed criteria do not
attempt to protect all species, the chr0nic adverse effect" of metal
mixtures observed in this study on daphnids and fathead ~inpows als?
indicate that important forage organisms may not be protected which could
ultimately cause a decrease in more desirable fish populations.
Individual Metal and Mixture Exposures
Acute LC50 values for fathead minnows and daphnidn exposed to individual
metals are shown in Tables 8 and 9.
r.omparison of the results inJicate
that daphnids were, generally, more sensitive to thes~metals than were
fathead minnows even though daphnids were tested in Lester River water
which was higher in hardness, alkalinity and organic content than tnat of
Lake Superior water.
These water quality character~stics usually decrease
.the toxicity of metals by decreasing their bioavailahility by complexation
in water [33-35].
Both species were less sensitive to arsenic, chromium,
12

-------
and lead than to mercury, cadmium and copper but their sensitivity to
these metals was not the same after the respective exposur~ periods.
Fathead minnows were most sensitfve to cadmium then copper 3nd mercury,
wl,creas daphnids were most sensit ive to mercury then cadmium and copper.
Sensitivity differences in these tests were also probably related to water
quality differences since some water quality parameters may effect some
metals differently.
For example, water hardness has been found to effect
the toxicity of cadmium, copper and lead more than that of arsenic, mer.cury
and chromium
[201.
However, the acute values obtained for both species
in these .tests were similar to those observed by other investigators [201.
The LC50 values calculated for these metals in mixture tests for
, .
each species are included in Tables 8 and .9 along with the fractions of
,toxic units (LC50s) calculated for each metal.
The toxic unit approach for
calculating the combined effects of mixtures of toxicants was reviewed
by Sprague [21.
In this method, 'fractions of toxic units of the individual
toxicant in the mixture adding u~ to a total of 1.0 indicate a strictly
additive joint action, < 1.0 more than additive and> 1.0 less than
;additive.
TIle sum of the fractions of .toxic units calculated .for fathead
cminnows from Table.8 was 0.53 intlicatingthat thes.e metals wer.e more than
additive in their acute joint action.
Conversely, the value calculated
for daphnids was 1.47 (Table 9) suggesting a .nearlystrictly additive
doint action..
Differences in the joint action correlate t~ the sensitivity
Aifferenc,es. of,.these species to the indblidua~ metals as was noted above.
,Qifferences 'in the behavior of ,metals in mixtun:s. have also, been rep.o~te<.1
iin ~ review of 'the literatur~ [7] on effects of mixtures on aquatic
,organisms. :'.This. review showed that the same metals may be ..~dditive, more
:than additive_o.r. less than 'additi~e ,depending upon the :I!pec~es;' combination
,of. metals, or water quality present.
.Ande.rson, and ~eber [3] suggested
13

-------
that discrepancies found between additive .action and mor~ than additive
actions of metals in organisms may be due to water quality characteristics
such as water har~ness, which m~y alter metallic forffis.
They discussed
that more than additive responses were shown in tests with copper and
Zlnc using soft water and additive responses were observed fr~m exposures
with hard water.
More recent studies by Anderson and others [36, 37J
indicate that the reduction in toxicity of a metal with increasing hardness,
however, may not be great when other metals are present concurrently.
Although additional .work is needed to make correlations between water
quality characteristics ~nd the reaction of chemicals in mixturp.s, water
quality effects on toxicity may also be a plausible explanation fo: the
slight differences observed in.this study since water quality parameters
in these tests were different.
The chronic effects of individual metals on fathead mlnnows are
~h0wn in
Table 10.
Growth in these tests was the most or among the most
sensitive measure of response to all metals.
A significant value obtained
in the arsenic test at 1,340 ug/1 was not used in the calculation of the
HATC because si~nificant adverse effects were not observed in the next
higher concentration.
The MATC's or chronic values (calculated as the
geo~etric mean :of the highest no-effect and lowest effect concenttations from
Table 10) for this species and each metal are included in Table 11.
~~TC's
calculated for this species were similar to chronic values observed for
the respective metals by other investigators [20].
Comparison of the
above MATCs for fathead minnows showed that species sensitivity to
the metals differed slightly in chronic tests compared to that from
acute tests.
Cadmium was the most toxic metal in acute tests followed by
copper, mercury, lead, arsenic and chromium; whereas mercury was much
more toxic on a chronic basis followed by copper, cadmium, lead, chromium,
14

-------
and arsenic.  Acute to chronic ratios for each metal arc also included in




Table 11.  Tests with mercury showed the highest ratr'.o (193) for fathead




minnows indicating that it is much more toxic on a chronic basis.than on




an acute basis to this species than the. other metals.




     The effects of these metals on the survival and young production of




daphnids are presented in Table 12.  Both mortality and young production




were used to determine the overall toxicity comparison (scaled T-statistic)




[30, 31] in these tests.  Young production was the most sensitive parameter




measured with all the metals except chromium for which survival was




slightly more sensitive.  The high sensitivity of daphnid young production




to metals and other chemicals including several industrial effluents has




also been observed by Mount [38].  The MATC's for each of the mp.talr. and




daphnids are shown in Table 11.  As with the acute tests, chronic values




obtained for daphnids were generally lower than those calculated for




fathead minnows indicating their high chronic sensitivity to these metals.




Comparisons of the above values for daphnids to these metals showed




that their sensitivity differed slightly on an acute versus chronic basis




as it did with fathead minnows.  Mercury was the. most toxic metal to




daphnids in acute tests but cadmium was more toxic on a chronic basis.




This was the opposite of that observed for fathead minnows.  The ranking




of acute to chronic ratios for these two metals and daphnids was also




opposite that observed for fathead minnows with the highest ratio being




for cadmium (12.4) and lowest for mercury (0.73) (Table 11).  Generally,




acute to chronic ratios were lower for daphnids than fathead minnows,




especially for mercury.  This difference may be attributed to the greater




influence of food on metal toxicity in chronic tests with daphnids which




could have decreased biological availability and/or toxicity of these




chemicals.  Biesinger and Christensen [39] and Lima et al. [40] showed





                                    15

-------
that the presence of food decreases the toxicity of metals to these
types of organisms.
Measured water concentrations and results of chronic mixture tests
for fathead minnows and daphnids are: presented in Tables 13-17.
Two 32-
day flow-through tests with fathead minnows were conducted to determine
the effects of mixture concentrations at 0.5 dilution ratios of the MATC's.
Results from these tests (Table 16) show that metals mixed at MATC, and 4/3
MATC concentrations caused nearly 100 percent mortality and decreased
growth of juvenile fish after 32 days of exposure.
All larvae that
hatched at these c,oncentrations were deformed and all but a few fish di.ed
only one week after hatching.
Metals combined at 1/2 and 2/3 of the MATC
values al~o caused 40 to 50 percent mortality, respectively, although
s,tatistical differences at P = 0.05 were just on the border of not being
significant.
The lack of statistical significant difference here appears
to be attributed to the fact that mortalities in duplicate chambers at
these concentrations were more dissimilar than usual.
The authors, however,
feel that these are real differences because deformitie~ and abnormal
behavior of newly hatched larvae were observed at these concentrations
early in each experiment at hatch.
In addition, growth of juvenile fish
exposed to mixtures of 2/3 of the MATC were reduced by as much ~~ 25
percent by the end of the test.
The effects of mixture concentrations on survival and young production
of daphnids are shown in Table 17.
Metals com~ined at 4/3 MATC, MATC,
and 2/3 MATC concentrations all caused 100 percent mortality.
The next
lower concentration of metals mixed at 1/3 of the MATC did not adversely
effect survival but significantly reduced young production by approximately
60 percent.
The lower effect level observed for daphnids compared to
that for fathead minnows agrees with the findings from individual metal
16

-------
may be similar to those in mixtures.
tests and suggests that the mechanism of toxic action of individual metals

. i
The adverse effects of mlxtur~s at
i
fractions of the MATC also indicate that 'chronic joint action may occur
in these species at levels presumed acceptable based o~ tests with in-
dividual metals.
Other ~tudies on the effects of metal mixtures on fish
[8, 41J and more recent ones on invertebrates [42) .have also indicated
the adverse effects of mixtures occur at concentrations having no significant
effect on an individual basis.
The literature on the sublethal effects
of combinations of metals on aquatic organisms indicate that joint action
may occur at sublethal levels, but that there is no clear trend as to the
degree of response [7).
As in acute tests, chronic responses vary in the
literature [7] from less than additive to more than additive depending
upon the chemical or species tested~
Because the MATC is not a point estimate level, the degree of
sublethal joint action based on this endpoint in the present chronic
tests cannot be calculated precisely.. In order to determine the type and
degree of joint action, an estimate of the value causing a 50 percent
. reduction in weight and the number of young produced per female for
fathead minnows and daphnids, respectively, exposed to individual metals
and metal mixtures, was obtained from response curves relating these
endpoints to exposure concentration.
Figures 1 and 2 show that these
response relationships were curvilinear for the metals tested on an
individual a8 well as mixture basis, respectively.
This pattern was
similar for both species and all metals in both types of tests and was
characterized by a gradual decrease in the curve followed by a rapid
decline as exposure concentration increased.
Slopes for the steep part of
the curves were, generally, greater for metals in the mixture than for
metals tested individually indicating that the observed joint action
17

-------
caused greater toxicity than that caused by each metal during the same
, exposure period.
The simi12rity';in the slopes of the response curves for
individual as well as combined m~tals suggest that modes of dction of
metals may also be similar under these conditions and not changed but
merely enhanced by metal interactions in some fashion.
This has been
postulated previously for lethal tests with metals and binary metal
mixtures by Hewitt [43].
All values below the 50 percent response level in' Figures 1 and 2
were
significantly less than the control (p = 0.05) and are shown by the
open symbols.
Howeve,r, some values above this level were also statistically
significant for both species exposed to the individual metals (Figure 1)
suggesting 'that the 50 percent response is probably too high a level to
estimate chronic no effect concentrattons.
In general, no effect concentrations
(i.e, MATC) are estimated from chronic endpoints that are significantly
~irEerent from the control.
These endpoints are not estimates of the
degree of adverse effe~t and ~ay or may not be biologically significant.
Additional chronic studies are needed to define meaningful point estimates
which would better correlate to biological effect concentrations.
Chronic concentrations and fractions of this toxic unit based on the
response curves for each species exposed to individual metals and to the
mixture are shown in Table 18.
Sums of the fractions of toxic units of
3.31 and 1.08 indicate that the chronic joint action of these metals was
less than additive with fathead minnows and nearly strictly additive with
daphnids, respectively.
Comparisons of these results with the acute
joint action determined above 'for these species show that the interaction
of metals may be different on an acute and chronic basis and between
different classes of organisms.
The joint action of the selected metals
was more than adrlitive to fathead minnows at acutely lethal levels but
18

-------
was less than strictly additive at sublethal concentrations as determined
by effects on growth.
The joint action displayed on daphnids was nearly
strictly additive on .both an .acute and chronic basis.
These differences
agree with tentative concl~sions made on the joint action of m~tal mixtures
on fish and invertebrates by the European countries [7J.
Generally, the
few sublethal mixture tests reviewed using growth as the endpoint showed
. that chemical interactiops were less than additive than the corresponding
effect on survival.
The data available for aquatic invertebrates showed
a generally additive joint action.
However, the review reported that one
study [44J indicated that sublethal metal mixtures may be more than
additive on fish reproduction and suggested that this work be extended to
include other commonly occurring toxicants.
TIle ~eason for differences
in the joint action of metals on the present test species is not .clear but
it may be linked to the species sensitivity differences noted above and
thus attributed to differences in metabolic defense mechanisms of these
. different types of organisms.
These mechanisms are primitive in daphnids,
and Inay be the same in this species during both acute and chronic exposure.
On the other hand~ defense mechanisms in fish are more specialized than
daphnids which may allow fish to become more tolerant to metal toxicity
during long-term exposures as they develop resulting in a lesser joint acLlon
of the toxicant.
Such mechanisms of detoxification may be related to the
binding of metals to special proteins (i.e., metailothioneins) as suggested
in the literature [45].
However, further study of the mechanisms of
toxic action of chemicals are needed and may be the key to understanding
long-term chemical interactions.
The mode of action of metals in fish is obviously different on 6n
acute basis than on a chronic basis.
It is generally considered that
metals cause acute toxi.city by destruction of the gill membranes.
Studies
19

-------
by Hewitt [43] indicate that the gills of zebra fish (Brachydanio rerio)
were the primary sit-e of sigrti ficant metal accumul ation during acut.e
exposure and that the relative hioconcentration of metals was significantly'
. greater in gill tissue of fish exposed to me~dl mixtures than to individual
metal solutions.
These results may explain the more than additive response
obt~ined in our acute tests with fathead minnows.
Knowledge of metal
concentrations in fish exposed to mixtures on a long-term basi3 would
help to explain the difference in metal interactions observed between
acute and chronic tests.
Although it appears from the present studies that mixtures ~f metals
may be less than additive to fish in long-term exposures, the adverse
effect observed on survival of this species at concentrations of 2/3 to
1/2 of the MATC are low enough to cause concern that these organisms may
not be protected by criteria that are based on MATC concentrations derived
for individual metals.
Chronic adverse effects on daphnid reproduction
caused by metals mixed as low as 1/3 of the ~~TC can be correlated to
the nearly strictly additive effect shown above, and suggests that these
organisms may be more susceptible to metal interactions than fish.
Recent studies by Biesinger et al. [42] hAve also indicated that metal
interactions may be nearly additive on daphnid reproduction based on
complete 1i fe-cycle studies. . Similarly, Hermans et a1. [12] suggested
that. although the joint toxicity at sublethal levels is lower than at
lethal levels to daphnids, the toxicity of mixtures containing primarily
organic chemicals remains much higher than that of individual chemicals and
may be near concentration addition.
Still other studies on the effects
of mixtures on fish [8, 43] and algae [46] indicate that some metals as
well as other chemicals such as pesticides [37] may be even more than
additive on a chronic basis.
20

-------
     The effects of metals mixed at fractions of both acute and chronic


values on the>selected species correspond with the effects noted in the first


part of this study from mixtures based on proposed water quality criteria
                                 i

concentrations.  Water quality criteria are based on data from tests with


individual chemicals on several families of aquatic organisms and are not


species-specific.  However, endpoints such as the LC50 and MATC are species-


specific and are used to determine the criteria for individual chemicals.


Thus, the adverse effecto caused by metal mixtures at proposed water quality


criteria concentrations appear to be due to the joint action of the selected


metals at concentrations having no significant effect on an individual basis,


and suggest that components of mixtures at or below no observable effect


concentrations may contribute significantly to the toxicity of these mixtures.


The effects of mixtures at sublethal levels are of particular importance


since these concentrations would be allowed to exist continuously throughout


the year as criteria and may not be sufficient to protect some aquatic


species.  These effects may also be magnified in waters having a low buffering


capacity and pH which may increase the bioavailability and/or toxicity of


metals.  Recently completed studies by Hutchinson and Sprague [47] show


that reproductive failure of fish was complete when fish were exposed to


trace metal mixtures in water cf low pH (pH 5.8).  This level of pH alone


caused no effects on fish reproduction in very soft (6.0 mg/1 as €3003) water.


     The results of the present tests.suggest that further study is


needed to determine the type and degree of interaction of toxicants on


both an acute and chronic basis and to determine the possible effects of


water quality characteristics on these interactions.  Results obtained


from these tests would help identify general characteristics of certain


mixtures and may provide data for new methods and possibly the rationale


for deriving water quality criteria for combined pollutants.



                                    21

-------
                                REFERENCES



1.  Lloyd, R.  1961.  The toxicity of mixtures of zinc and copper sulphates

         to rainbow trout (Salmo gairdneri Richardson).   Ann.  Appl.  Biol.

         49: 535-538.
                                «
2.  Sprague, J.B.  1970.  Measurement of pollutant toxicity to fish. 11.

         Utilizing and applying bioassay results.  Water Research 4: 3-32.

3.  Anderson, P.O., and L.J. Weber.  1975.  The toxicity to aquatic

         populations of mixtures containing certain heavy metals.  Inter-

         national Conference on Heavy Metals in the Environment, Toronto,

         Ontario, Canada, October 27-31, 933-954.

4.  Marking, L.L.  1977.  Method for assessing additive toxicity of

         chemical mixtures, aquatic toxicology and hazard evaluation,

         ASTM STP 634, F.L. Mayer and J.L. Hamelink, Eds.  American

         Society for Testing and Materials, 99-108.

5.  Durkin, P.R.  1981.  Approach to the analysis of toxicant  interactions

         in the aquatic environment, aquatic toxicology and hazard assess-

         ment: Fourth Conference, ASTM STP 737, D.R. Branson and K.L.

         Dickson, Eds.  American Society for Testing and Materials,

         388-401.

6.  Konemann, H.  1981.  Fish toxicity tests with mixtures of  more than two

         chemicals; a proposal for a quantitative approach and experimental

         results.  Toxicology 19: 229-238.

7.  European Inland Fisheries Advisory Commission.  1980.  Report on

         combined effects on freshwater fish and other aquatic life of

         mixtures of toxicants in water.  EIFAC Technical Paper No.  37. •

         Food and Agriculture Organization of the United Nations, Rome.

         49 pp.



                                    22

-------
 8.   Eaton,  J.G.   1973.   Chronic  toxicity  of  a  copper,  cadmium,  and  zinc




          mixture  to the  fathead  minnow (Pimephales  promelas  Rafinesque).




          Water Research  7:  1723-1736.




 9.   Muska,  C.F.,  and L.J. Weber.   1977.   An  approach  for  studying the




          effects  of mixtures  of  environmental  toxicants on whole organism




          performances.   In:  Recent Advances  in Fish Toxicology, A Symposium.




          R.A.  Tubb, Ed.   EPA-600/3-77-085, Ecological  Research  Series,




          Environmental  Research  Laboratory-Corvallis,  Oregon.   pp.  77-87.




10.   Kiokemeister, E.  1979.   The effects  of  multiple  toxicants  on the




          growth of the  guppy  (Poecilia reticulata).  Masters Thesis,




          Oregon State University,  Corvallis, OR.




11.   Broderius, S.J., and L.L.  Smith,  Jr.   1979.   Lethal and  sub-lethal




          effects  of binary  mixtures of cyanide and  hexavalent  chromium,




          zinc, or ammonia to  the fathead  minnow (Pimephales  promelas)




          and rainbow trout  (Salmo  gairdneri).   J. Fish. Res. Board  Can.




          36: 164-172.




12.   Hermans, J.,  H. Canton,  N.  Steyger, and  R. Wegman.   1984.   Joint




         effects of a mixture  of  14 chemicals on mortality and  inhibition




         of  reproduction  of  Daphnia magna. Aquatic  Toxicology  5:  315-322.




13.   Hennanutz, R.O., J.G. Eaton,  and L.H. Mueller.   1985. Toxicity of




          Endrin and Malathion mixtures to flagfish  (Jordanella  floridae).




          Arch. Environ. Contain.  Toxicol.  14: 307-314.




14.   National Academy of Science,  National Academy of  Engineering.   1973.




          Water Quality  Criteria 1972.   U.S.  Environmental Protection




          Agency,  Ecological  Research Series, EPA-R3-73-033.   March, 1973.




          594 p.
                                     23

-------
15.  U.S. EPA.   1980.   Treatability Manual,  Vol II.   Industrial Descriptions.




          U.S.  EPA, Cincinnati,  OH.




16.  American Society for Testing and Materials.   1980.   Standard practice




          for conducting acute toxicity
-------
25.   Mount, D.I.,  and T.J.  Norberg.   1984.   A seven-day life cycle cladoceran




          toxicity test.   Environ.  Tox.  and Chem.,  Vol. 3:  425-43^]




26.   Stephan, C.E., D.I.  Mount,  D.J.  Hansen, J.H.  Gentile,  G.A.  Chapman,




           and W.A. Brungs.   1983.   Guidelines: for  deriving numerical




           national water quality criteria  for the  protection of aquatic




           life and its uses.   U.S.  EPA, Duluth,  MN.   July  5.




27.   Hamilton, M.A.,  R.C. Russo  and  R.V. Thurston.   1977.   trimmed Spearman-




          Karber method for estimating median lethal  concentrations-toxicity




          bioassays.   Environ.  Sci.  Technol. 7:  714-719.   Correction 12:




          417 (1973).




28.   Dixon, W.J.,  and F.J.  Massey,  Jr.  J957.  Introduction to Statistical




          Analysis, 2nd Ed.   McGraw-Hill Book Co.,  New York, N.Y.




29.   Steel, R.G.D., and J.H. Torrie.   1960.  Principals and Procedures of




          Statistics.  McGraw-Hill  Book Co., New York, N.Y.




30.   Hamilton, M.A.  1984.   Statistical  analysis  of the seven-day Ceriodaphnia




          reticulata reproductivity  toxicity test.   U.S.  EPA, Duluth, MN.




          Contract Order No. J3905NASX-1.  16 January.  48  pp.




31.   Rogers, J.  1984.   University  of Wisconsin at  Superior, Wisconsin,




          and U.S. EPA, Environmental Research Laboratory at Duluth, MN.




          July.  (Personal  Communication).




32.   Benoit, D.A.   1985.   U.S.  EPA,  Environmental  Research  Laboratory at




          Duluth,  MN.  July.  (Personal Communication).




33.   Andrew, R.W.   1976.   Toxicity  relationships  to copper  forms in natural




          waters.   In:  Toxicity to  biota of metal  forms in  natural waters,




          R.W. Andrew,  P.V.  Hodson  and D.E. Konasewich, Eds.  (Windsor,




          Ontario, Great Lakes  Research Advisory Board, International




          Joint Commission, 1976).   pp.  127-143.
                                      25

-------
34.  Giesy, J.P., Jr., G.J. Leversee, and D.R. Williams.  1977.  Effects


          of naturally occurring aquatic organic fractions on cadmium

                            'l
          toxicity to Simoceptuilus serrulatus (Daphnidae) and Gambusi a


          affinis (Poeciliidae) .  Water Res. 11: 1013-1020.


35.  Nelson, H.P., R.J. Er.ickson, D.A. Benoic, V.R. Maccson and J.R.


          Lindberg.   1985.  The effects of variable hardness, pH, alkalinity,


          suspended clay,  and humics on the chemical speciar.ion and  aquatic


          toxicity of copper.  EPA Contract No. 68-01-6388, U.S. EPA, Duiuth,


          MM, and Science  Applications International Corporation (formerly


          JRB Associates,  McLean, VA).


36.  Anderson, P.D., H. Horovitch, and N.C. Weinstein.   1979.   Pollutant


          mixtures in the aquatic environment: A complex problem in toxic


          hazard assessment.  Proc.  Fifth Annual Aquatic Toxicity Workshop,


          Hamilton,  Ontario, Nov. 7-9, 1978.  Fish. Mar. Serv. Tech. Rep.


          862, pp. 100-114.


37.  Anderson, P.O.   1981.  Paradigms in multiple toxicity in management


          of toxic substances in our environment, Ed. B.W. Cornaby.  Ann


          Arbor Science Publ. Inc.,  p. 75-100.


38.  Mount, D.I.  1985.  U.S. Environmental Research Laboratory, Duiuth, MN.


          July.   (Personal Communication).


39.  Biesinger,  K.E.,  and G.M. Christensen.  1972.   Effects  of various


          metals on survival, growth, reproduction and  metabolism of Daphnia


                  J. Fish. Res. Board. Can. 29:  1691-1700.
40.  Lira?  A.R., C. Curtis, D.E. Hammer meister ,  T.P. Markee ,  C.E. Northcott,


          and L.T. Brooke. • 1984.  Acute and chronic toxicities of drsenic


          III to fathead minnows, flagfish, daphnids and an amphipod.  Arch.


          Environ. Contain. Toxicol. 13: 595-601.
                                      26

-------
41.  Spehar, R.L., E.N. Leonard,  and D.L.  DeFoe.   1978.   Chronic effects




          of cadmium and zinc mixtures on  fiagfish (Jordanella floridae).




          Trans. Am. Fish. Soc.  107: 354-360.




42.  Biesinger, K.E., G.M. Christensen, and J.T.  Fiandt.   1985.   Effects




          of metal salt mixtures  on Daphnia magna reproduction.   Manuscript,




          Environmental Research  Laboratory, Duluth,  MN  55804.




43.  Hewitt, L.A.  1980.  Dose and time related response  patterns in




          test populations of Brachydonio  rerio exposed  to copper,  cadmium




          and mercuvy in pure solutions and in binary mixtures.   Master's




          Thesis, 'Joncordia University, Montreal, Quebec, Canada.  123 pp.




44.  Weinstein, N.L.  1978.  Multiple toxicity assessment for mixtures of




          aquatic pollutants.  Master's Degree, Concordia University, Montreal,




          Quebec, Canada.  116 pp.




45.  Friberg, L., M. Piscator, and G. Nordberg.  1971.  Cadmium in the




          environment.  CRC Press.  A division of the Chemical Rubber




          Co., Cleveland, Ohio.   166 pp.




46.  Wong, P.T.S., Y.K. Chan, and D. Patel.  1982.  Physiological and bio-




          chemical responses of several freshwater algae  to a mixture of




          metals.  Chemosphere,  11: 367-376.




47.  Hutchinson, N.J., and J.B.  Sprague.  1985.  Toxicity of Trace Metal




           Mixtures to American Flagfish in Soft  Acid Water and Implications




           for Cultural Acidification.  Can. J. Fish. Aquatic. Sci. 42: (In




           press).




48.  U.S. EPA.  1985.  W;£er quality criteria; availability of documents.




           Federal Register, 50:30784-30796.
                                        27

-------
   Table  1.  Measured water quality characteristics  for metal  tests  conducted  in  Lake  Superior,
             reconstituted and Lester  River water.
        Test
       Species
  Hardness
  as CaC03
   (mg/l)
Alkalinity
as CaCC>3
 (mg/l)
pHa
   Total
Organic Carbon    Conductivity
   (mg/l )	(pmhos)
                                              Lake Superior Water

    Rainbow trout     45.3+0.8 (23)b    42.7+_1.3 (24)    7.7  (7.2-7.9)

    Fathead minnow    43.9+1.0 (90)     42.4+1.9 (90)    7.4  (6-0 3.1)
                                                       2.0+1.4 (2)
                                                      91+1.4 (2)
ro
CO
    C. dubia
    C. dubia
                        Reconstituted Waterc

165+4.0 (2)       112+4.0 (2)      8.1,(7.8-8.3)
                                              Lester River Water
100+7.9 (11)      97+9.3 (22)
                8.2 (8.0-8.5)
                                    1.6 (1)
                 7.1+0.7 (9)
                                   451+1.4 (2)
                    194+20 (9)
   a Mean (range)

   b Mean +_ standard deviation (number of samples)

   c Reconstituted hard water [16]

-------
Table 2.   Measured water concentrations for tests  with  fathead  minnows,  rainbow
                               t
       trout, and daphnids  exposed  to metal mixtures  at  criterion  maximum

                                concentrations.
Treatment (pg/1)
Metal

As
Cd
Cr
Cu
Hg
Pb

As
Cd
Cr
Cu
Hg
Pb
Cont rol

<1.0b
<0.25
<1.0
1.0d
<0.05
<0.5

<1.0
<0.25
<1.0
1.0d
<0.05
. <0.5
1/2 Max

69+0. 4C
0.7+0.1
6.8+0.6
4.4+0.1
0.6+0.0
9.9^1.4

81+3.4
0.8+0.0
7.3+0.2
6.1+1.1
0.5+0.1
11+0.5
Maxa
Fathead
143+1.4
1.5+0.2
15+4.8
7.9+0.7
1.3+0.0
23±4 -7
Rainbow
148+9.2
1.6+0.0
14+1.3
9.2+1.6
0.9+0.1
. 17+1.6
2x Max
Minnow
262+3.5
2.9+0.1
26+2.4
15+0.2
2.3+0.2
40_+4 . 0
Trout
288+18.4
3.2+0.1
26+1.8
17+0.9
1.9+0.2
36+0.5
4x Max

556+4.2
6.0+0.0
50+1.6
30+1.0
4.2+0.0
79^0.9

536+42
5.7+0.4
47+0.2
30+0.6
3.6+0.4
74+6.4
8x Max

1,092+93
13+0.0
90+0.8
58+1.6
7.9+0.6
161+13.4

1,075+122
12+1.1
90+6.8
57+1.8
7.2+0.0
157+17.0
Ceriodaphnia dubia"
As
Cd
Cr
Cu
Hg
Pb
<1.0
<0.25
<1.0
1.0d
<0.05
<0.5
72
3.4
4.2
8.5
0.34
64
157
7.0
10.2
19.0
0.9
143
308
11. 1
21.7
39.1
1.8
284
645
34.4
42.4
81.5
4.2
611
_e
-
-
-
-

a Criterion maximum concentrations

b Detection limit

c Mean ^ standard deviation

d One measurement was made at che beginning of the test

e Daphnids were not tested at this level.
                                         29

-------
 Table 3.  Measured wacer concentrations for tests with fathead minnows,  rainbow




       trout, and daphnids exposed to metal mixtures at criterion average




                                concentrations.
Treatment (pg/D
Metal
Control
Avga
2x Avg
4x Avg
8x Avg
16x Avg
• . Fathead Minnow
As
Cd
Cr
Cu
Hg
Pb
<1.0b
<0.25
<1 .0
1.0
<0.05
<0.5
66+9. 3C
1.4+0.2
6.4+1.8
5.7+0.7
0.2+0.1
0.9+0.2
139+14.7
3.2+0.6
13+1.9
11+0.4
0.3+0.1
1.7+0.4
275+26
6.9+0.6
26+2.1
19+0.7
0.6+0.1
2.9+0.3
548+45
14+1.6
64+1.6
39+1.6
1.3+0.2
7.1+1.7
1,225+266
31+5.6
130+27
101+42
2.3+0.0
16+5.8
Rainbow Trout
As
Cd
Cr
Cu
Hg
Pb

As
Cd
Cr
Cu
Hg
Pb
<1.0
<0.25
<1.0
J.O
<0.05
<0.5

<1.0
<0.25
<1 .0
1.0
<0.05
<0.5
79+9.6
1.7+0.3
8.4+1.7
7.3+1.0
0.2+0.1
1 . 1+0 . 3

68+2.3
6.7+0.3
7.0+0.3
13+0.6
0.13+0.01
3.6+0.5
154+1.8
3.8+0.4
15+2.8
13+1.2
0.4+0.1
1.8+0.4
Ceriodaphnia
137+7.5
13+1.3
14+0.4
28+1.4
0.27+0.01
8.3+0.6
306+40
7.5+0.7
29+3.1
23+2.2
0.7+0.2
3.4+0.7
dubia
286+14
28+2.5
29+0.6
58+2.6
0.57+0.04
16+0.4
571+49
14+1.1
56+6.4
43+6.1
1.5+0.2
6.0+0.7

575+32
5.6+6.2
59+0.5
120+5.5
1.3+0.02
33+3.9
1,260+128
31+2.0
122+9.0
86+4.3
2.9+0.5
12J+1.4

1,152+26
117+15.6
121+1.5
248+12.7
3.4+0.2
70+6.3
a Criterion average concentration




b Detection limit




c Mean + standard deviation
                                       30

-------
Table 4.  Proposed National Water Quality Criteria (1984) for selected




          metals and freshwater aquatic life.
Criteria (jjg/1)
Metal
Arsenic"1'-'
Cadmium'1'2
Chromium"1^
Copper'1'2
Mercury'*'2
Lead*2
Max. Cone.
140
1.8a, 8.2b
11
7.-6a, 25. lb
1.1
22a, 128b
Avg
72
1.
7.
5.
0.
0.
. Cone .

8a, 8.2b
2
2a, 17. 3b
2
9a, 5.1b
a Adjusted for hardness of Lake Superior water (45 rag/1 as CaCO^) [26]




  Adjusted for hardness of reconstituted water (168 mg/1 as CaCO^) [26]
                                     31

-------
Table 5.  Survival of fathead minnows, rainbow trout, and daphnids

exposed Co metal mixture.-at multiples of criterion maximum

concentrations.         i
Treatment3
Control
1/2 Max
Max
2x Max
4x Max
8x Max
Fathead winnow"
100+0. Od
100+0.0
1004-0.0
100+0.0
85^7.1
40+0.0
Rai ibow trout
100+0:0
100+0.0
5MO
0+0.0
0+0.0
0+0.0
C. dubiac
90+32
100^0.0
0+_0.0
0+^0.0
0^0.0
o+p.o
a Treatment number corresponds to measured concentrations in
  Table 2.

° Test's conducted in Lake Superior water for 96 hr.

c .Test conducted in reconstituted water [16] for 48 hr.

^ Mean + standard deviation.
                               32

-------
 Table 6.  Survival and growth of fathead minnows and rainbow troiit




    exposed to metal mixtures at multiples of criterion average




          concentrations for 32 and 90 days, respectively




                     . in Lake Superior water.
Treatment3

Control
Avg
2x Avg
4x Avg
8x Avg
16x Avg

Control
Avg
2x Avg
4x Avg
8x Avg
16x Avg
Embryo
Hatchability
U)

100+0. Ob
100+0.0
100+0.0
. 100+0.0
97+5.0
97+5.0

96+2.3
92+2.3
95+1.2
88+3.5*
8+5.3*
0+0.0*
Normal
Larvae at
Hatch (%)
Fathead Minnow
100+0.0
100+0.0
100+0.0
54+9.2
iO+4.2*
0+0.0*
Rainbow Trout
99+1.0
99+1.0
99+1.0
98+2.9
0+0.0*
0+0.0*
Survival
(%)

87+19
93+0.0
94+9.2
43+14*
4+5.0*
0+0 . 0*

96+2. 3C
92+2.8
95+1.2
89+3.5*
8+5.3*
0+0-0*
Weight
(nig)

174+46
129+36*
44+29*
6.5+2.5*
2.0+0.0*
0+0.0*

12l+42d
195+85
185+62
150_+48
-
~
a Treatment corresponds to measured concentrations in Table 3




" Mean +_ standard deviation




c Based on 45-day exposure period (see text)




" Based on 70-day exposure period (see text)




* Significant decrease from control (P = 0.05)

-------
 le 7.   Survival and  number  of  young  per  female  of  C_.  dubia exposed to

        metal mixtures  at multiples of  criterion average  concentrations after

        7 days  in  reconstituted  water.
•eatment3
mtrol
/2x Avg
LVg
!x Avg
>x Avg
!x Avg
. 6x Avg
Survival
(%)
90 + 32b
80 _* 42
60 +_ 52
0 _* 0.0*
0 _+ 0.0*
0 + 0.0*
0 jf 0.0*
Young per
Female
15.6 jf 1.0
12.5 _+ 1.1
3.3 +_ 1.0*
_d
_d
_d
_d
Scaled
T-statistic of
Overall Comparison
_c
0.81
3.37*
2.08*
2.08*
2.08*
2.08*
Treatment  corresponds  to measured  concentrations  in Table 3 except for
L/2x  Avg concentration (not  shown  in  Tabls  3)

lean  _+  standard  deviation

to  value because other values  are  compared  to  the control

Jo  value because all animals died

>ignificant  decrease  from  control  (P  = 0.05)
                                    34

-------
   Table 8.   Ninety-six hour LC50 (tjg/l)  and fractions of this toxic unit

             for 30-d old fathead minnows exposed to individual metals and to
                                    i
             a metal  mixture in .Lake Superior water.
    As	        Cd	Cr	Cu	Hg	Pb	

                             Individual Metal Tests

   12,600          13.2           43,300          96         172         2,100
(9,900-15,900)a (10.9-15.9)   (36,600-51,300)   (83-111)     86-347)   (1,100-4,000)

                               Metal Mixture Test

    1,200           1.2            4,550          7.8        13.9         125
 (1,000-1,500)    (1.0-1.5)    (3,710-5,590)     (6.6-9.2)  (11.4-17.0)  (104-149)

                              Fraction of Toxic Unitb

   0.10              0.09          0.12          0.08        0.08         0.06


 a Ninety-five  percent confidence limits

 b Mixture  LC50 divided by individual metal  LC50 (sum of fractions = toxic unit
   of 0.53)
                                      35

-------
Table 9.  Forty-eighi hour LC50 (^ig/1) and fractions of this toxic unit for

          < 24-hr old £. dubia exposed to individual metals and to a metal

          mixture in Lester River water.
As
1,448
1, 214-1.727)3
344
(303-390)
Cd
27.3
(21.9-34.1)
6.1
(5.3-7.0)
Cr
Individual Metal
144
(110-189)
Metal Mixture
34.7
(30.6-39.4)
Cu
Tests
66
(55-81)
Test
16.8
(14.7-19.1)
Hg
8.8
(6.6-11.8)
2.3
(2.0-2.6)
Pb
248
(212-290)
60.7
(53.0-69.4)
Fraction of Toxic Unitb
0.24
0.22
0.24
0.26
0.26
0.25
a Ninety-five perce.it confidence limits

b Mixture LC50 divided by individual metal LC5C (sura of  fractions = toxic unit
  of 1.47)
                                          36

-------
  Table 10.  Survival ind growth of fathead minnows exposed to individual




                 .net a is for 32 days in Lake Superior water.
Measured
Cone enc rac ion >
(ug/l) '
Emhrvo
Mac chab i 1 i cy
Norma 1 Larvae
ac Hacch
3urv ival
(':>
Wei?h:
Arsenic
<1.0a (control)
l,340*79b
2,520*78
4,400-159
8,340*428
16,410*791
Cadraium
<0.1a (con:rol)
l.d»0.1b
3.8*0.3
7.6-0.6
15.6*1.4
29.5j^2.3
Chromiuia
<20a (control)
220+18°
435*20
863*48
1.630*72
3,170*130
Copper
<2.0a (control) '
4.8*0.3b
8.0*1.3
16.0") .9
31.072.8
65.0*5.0
Mercury
<0.05a (control)
0. 23+0.03'
0.36*0.05
0.65*0.07
l.il»U.Ut>
2.24*0.08
Lead
<1.0a (control)
69+5b
123+10
232*20
466*65
946*145
100*0.0
1 00~0 . 0
100*0.0
100*0.0
100*0.0
ioojo.0

100*0.0
100*0.0
100*0.0
100*0.0
100*0.0
97*4.9

100*0.0
100*0.0
100*0.0
100*0.0
100*0.0
100*0.0

100*0.0
100*0.0
100~0.0
100*0.0
94.9.0
100*0.0

100*0.0
100*0.0
100*0.0
94*9.0
1UU+U.O
100+0.0

100*0.0
100~0.0
100*0.0
100*0.0
100~0.0
100*0.0
;oo+o.o
100+0.0
ioo*~o.o
100~0.0
100~0.0
97»_5.0
100+0.0
lPO+"o.O
100*0.0
97+4.9
0*
0*
97+5.0
100+0.0
97+5.0
94*9.0
100*0.0
100*0.0
100*0.0
100*0.0
100*0.0
100+0.0
0*
0*
100+0.0
100+0.0
100+0.0
100+0.0
1UU+U.U
70_»0.04*
100+0.0
100+0.0
100+0.0
100+0.0
0*
0*
84+5.0
100+0.0
87+0.0
77*14
84*5.0
0*
93*0.0
97*5.0
97*5.0
77*14
2.0+4.0*
0*
94+9.0
97+5.0
97+5.0
94+9.0
100+0.0
90+_4.0
90+4.0
100+0.0
63+14*
47+19*
2.0+4.0*
0*
100+0.0
94+9.0
93*0.0
80*0.0
y«**.u
79+.11
86+9
90+14
94+9
64+23
0*
0*
109+3.0
91~2.0*
100*1.0
68+8.0*
48*5.0*
Td
96*1.0
94*2.0
93*3.0
107*13
44C*
_d
123*6.0
113*4.0
113*8.0
115*6.0
105+1.0
86+J1*
128+1.0
112+16
74+5.0*
27+10*
9C*
-d
124+1.0
121*13
123+1.0
120*_5.0
eu+t.u*
28+3.0*
122+13
110+10
104*14
123+26
Td
_d
3 Deteccion limit



b Mean + standard deviation




c Weight of one surviving  fish



<* No value bccaus?  all  animals died




* Significant decr^js"  frori the control  (P " 0.05)



                               37

-------
Table 11.  MATCs and acute to chronic ratios for fathead minnows and daphnids




           exposed to individual metals.
Test
Species

Fathead minnow3
C. dubiab
As Cd Cr
MATCs
3,330 10.9 2,270
1,140 2.2 63
Cu Hg
(ug/1)
6.2 0.89
45 12
Pb

329
52
Acute/Chronic Ratios0
Fathead minnow
C. dubia
3.8 1.2 19
1.3 12.4 2.3
16 193
1.5 0.73
6.4
4.8
a Tests were conducted for 32-d in Lake Superior water.




^ Tests were conducted for 7-d in Lester River water.




c Acute LC50 (individual values, tables 8 and 9) divided by the MATCs.
                                      38

-------
   Table 12.  Survival  and number of young per female of £. dubia exposed Co
              individual metals after 7 davs in Lester River water.
Measured
Concent rat ion
(ug/1)
Arsenic
C 1.0* (control)
102+6C
1 88+~6
404*15
793+52
1636"+58
3250+60
Cadmium
< 0.25a (control)
0.6+0.1=
1.5+0.1
3.2+0.2
6.6+0.3
13.4+1.0
25.6V2.1
Chromium
< l.0a (control)
10.3+l.lc
21.0+1.0
41.4+2.4
94.8+5.7
171+11
345+2
Copper
3.4+0.3a (control)
9.9+0.3c
16.7+0.3
31.6+2.6
63.9+2.5
122+7
237M
Mercury
< 0.0ia (control)
0.5i+0.02c
1.05+0.01
2'.077o.03
4.36+0.10
8.69+0.24
16.9J+O.I5
Lead
< 1.0a (control)
36+lc
7473
165"+9
32l77
608+22
1284+3
Survival
X

80+42
100+0.0
90+32
100+0.0
100+0.0
50+53
0+0.0*

100+0.0
100+0.0
90+32
100+0.0
100+0.0
90+32
q+o.o*

100+0.0
90+32
100+0.0
90+32
20+42*
0+0.0*
oTo.o*

90+32
90+32
100+0.0
'100+0.0
80+42
60+52
0+0.0*

100+0.0
100+0.0
100+0.0
80+42
100+0.0
100+0.0
20j+42*

90+32
100+0.0
10Q+0.0
30+48*
30+48*
0+0.0*
0+0.0*
Young per
Female

19.8+1.5
19.8+1.4
14.8+1.5
14.6+1.8
18.3+1.2
6.2+0.5*
- _d

25.4+1.6
22.6~+1.5
24.2+1.4
18.8+1.7*
11.3+1.0*
5.3+"l.2*
7 . 5^0 . 9*

13.3+1.1
17.2+0.9
17.3+2.2
18.9+1.2
14. 2+0. fi
5.5+1.9*
- _d

29.0+0.8
26.2+1.4
28.2+0.8
27.5+1.9
10.0+3.0*
1.5+0.5*
~ _d

29.6+2.0
25.9+1.0
27.8+1 .0
29.9+1.6
28.5+1.5
27.7+1.7
6.7O.9*

19.3+0.6
21.3+0.7
16.6+0.8*
16. 071.3*
8.0+2.4*
~ _d
_d
. Scaled
T-^tatisei: of
Overall Comparison

.b
0.44
0.97
1.00
0.54
3.36*
2.03*

_b
0.50
0.50
1.15*
2.90*
4.04*
4.14*

-b
0.53
0.16
0.15
1.20*
_e
2.47*

J>
0.70
0.53
0.53
2.28*
11.86*
2\08*

-b
0.66
0.32
0.45
0.18
0.29
_e

_b
0.98
1.16*
1.55*
_e
2.10*
2.10*
a Detection limit
b No value because other values are compared to Che control
c Mean _+ standard deviation
<* No value because all animals died
e Too few degrees of freedom to calculate value
* Significant decrease from the control (P » 0.05)
                               39

-------
Table 13.  Measured water concentrations for a test with fathead minnows




           exposed to metal mixtures at multiples of the MATC in Lake Superior




           water (Test 1).
Treatment (jJg/O
Metal
As
Cd
Cr
Cu
Hg
Pb
Control
< 1.0a
< 0.1
< 1.0
1.0
< 0.05
< 1.0
1/16 MATC
199+6b
-0.6^0.1
135+9
1.3^0.4
0.05^0.01
16.3+0.9
1/8 MATC
403jf21
1.3^0.1
272^18
1.7+0.3
O.U0.02
34J+1.4
1/4 MATC
805^54
2.6+0.3
557^30
2.4jf0.3
0.2^0.03
70^2.3
1/2 MATC
1,545^64
5.3+0.5
1,137_+30
3.8+0.4
0.4_+0.03
143^6
MATC
3,203^158
10.8+0.3
2,237^61
6 . 7+^0 . 7
0.7^0.1
237j+91
a Detection limit.




" Mean + standard deviation.
                                      40

-------
Table 14.  Measured water concentrations for a test with Eathead minnows




           exposed to metal mixtures at multiples of the MATC in Lake




           Superior water (Test 2).
Treatment (pg/D
Metal
As
C
-------
    Table  15.  Measured water concentrations  for  a  test  with  C_.  dubia exposed  to metal  mixtures  at




               multiples of  the MATC  in  Lester  River  water.
Treatment  Mean + standard deviation.

-------
Table 16.  Survival and growth of fathead minnows exposed to ir.etal mixtures

           at multiples of the MATC for 32-days in Lake Superior water.
Treatment3
Control
1/16 MATC
1/8 MATC
1/4 MATC
1/2 MATC
MATC

Control
1/12 MATC
1/6 MATC
1/3 MATC
2/3 MATC
4/3 MATC
Embryo
Hatchability
(%)
10(5+0. Ob
100+0.0
100+0.0
1 00+0 . 0
lOOj+0.0
100^0.0

100+0.0
100+p.O
lOOj+0.0
1 00+0.0
100+0.0
97+4.9
: Normal
Larvae at
Hatch (%)
Test 1
100+p.O
100+0.0
100+0.0
100+0.0
100+0.0
0+0.0*
Test 2
100^0.0
100+0.0
. 100+0.0
100+0.0
100+p.O
0+0 . 0*
Survival
(%)
87+9
86^10
74+_9
83^14
63+^14
10^4*

84+_5
90j+14
87j+0.0
87+p.O
50+_24
3.5+4.9*
We i gh t
(
-------
Table 17.  Survival and number of you;ig per female of C. dubia exposed to




           metal mixtures at multiples of the MATC for 7 days in Lester




           River water.
Treatment3
Control
1/12 MATC
1/6 MATC
1/3 MATC
2/3 MATC
MATC
4/3 MATC
Survival
(%)
1 00+0. Ob
90^32
90+32
90+32
0+0.0
0+0.0
0+0.0
Young per
Female
17.8+1.5
18.0^2.1
19.6^1.8
6.9^1.5*
_d
_d
_d
Scaled
T-statistic of
Overall Comparison
_c
0.03
0.53
2.16*
2.47*
2.47*
2.47*
a Treatment corresponds to measured water concentration in Table 15.




b Mean +_ standard deviation.




c No value because other values are compared to the control.




d No value because all animals died.




* Significant decrease from the control (P = 0.05).
                                    44

-------
Table 18.  Chronic concentrations3 (|jg/l) and fractions of this toxic unit
                                                                      i
           based on a 50 percent reduction in weight and the number of|
                                                                      i
           young per female for fathead minnows and C. dubia, respectively,

           exposed to individual metals and a metal mixture.
 Species
   As
 Cd
 Cr
Cu
 Hg
Pb
Fathead minnow
C. dubia
7,079
1,259
                          Individual Metal Tests
14.5
 6.0
3,467
  132
11
56
 1.4
12.6
331
26',
Fathead minnow    2,630
C. dubia            275
                           Metal Mixture Test
            8.9
            0.6
          1,998
             17
            5.9
           18
          0.7
          3.2
           234
            15
                         Fraction of Toxic
Fathead minnow
C. dubia

0.37
0.22
0.61
0.10
0.58
0.13
0.54
0.32
0.50
0.25
0.71
0.06
a Concentrations are recalculated  from log units at the  50  percent value
  (Figures 1 and 2).

b Mixture value divided by individual metal value  (sum of  fractions  =  toxic
  units of 3.31 and 1.08 for fathead minnows and daphnids,  respectively).
                                    45

-------
Figure  1 .
     Percent reduction (50%) in weight and number of young  per female

     of  fathead minnows and C. dubia,  respectively,  exposed  to individual

     metals for 32  and 7 days.. Open symbols =  significant  decrease from

     control (P = 0.05), X = total  fathead mortality, S = steep slope.
100



80



60



40



20
          UJ
          o
                      S-2.01,
S—1.3
                    As
S-I.Od
                  Cu
             Control 2.0  2.8  3.6   4.4
I
I
I
I
I

iS—1.5
           Control -0.2   0.6   1.4   2.2
                                *    * Rjtheod minnow
                                •—-« C.di-'bia
             Control  1.4  2.2  3.0  3.8

               LOG CONCENTRATION (jig/I)
           Control  1.0    1.8   26   3.2

             LOG CONCENTRATION (pg/l)
                                         46

-------
Figure 2.
     Percent  reduction (50%)  in  weight and number  of young per  fenale
     of  fathead minnows and £. dubia, respectively,  exposed to  a mixture
     of  metals for 32 and 7 days.   Open symbols  =  significant decrease
     from  controls (P = 0,05), S = steep slop:?.
            Control -1.0 -0.2  0.6   1.4
Control -1.2  -0.4  0.4   1.2
         o
         oe
100
BO
60
20
0
C
e
	 ••
i
l
i
1 i
1
• S— 2.4\
Cr
. < ., i 	
.
S-2.21
1.
ontroi 0.8 1.6 2.4 3.2
LOG CONCENTRATION (pq/l)
                                         Control  0.6   1.4  2.2   3.0
                                           LOG CONCENTRATION

-------

-------