United States	Environmental Monitoring	AMD 82050C
Environmental Protection	Systems Laboratory	March 1983
Agency	P.O. Box 15027
Las Vegas NV 89114
Research and Development	
Use of Effluent Toxicity
Tests in Predicting the
Effects of Metals on
Receiving Streams on
Invertebrate Community

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EFFLUENT BIOASSAYS AND STREAM INVERTEBRATE COMMUNITIES
1 ley proof to:
Dr. Thomas W. La Point
Department of Biological Sciences
University of Nevada, Las Vegas
Las Vegas, Nevada 89154
702-798-4761

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USE OF EFFLUENT TOXICITY TESTS
IN PREDICTING THE EFFECT OF METALS
ON RECEIVING STREAM INVERTEBRATE COMMUNITIES
Theron G. Mi 11 er^
Advanced Monitoring Systems Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
and
Susan M. Melancon and Thomas W. La Point
Department of Biological Sciences
University of Nevada, Las Vegas
Las Vegas, NV 89154
^Present address, JRB Associates, 476 Prospect, La Jolla, California

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3
ABSTRACT
Intensive biological surveys were conducted during 1980 and 1981 on
Prickly Pear Creek, Montana, a stream receiving metal-rich runoff from aban-
doned mines. The surveys characterized the macroinvertebrate benthic com-
munity and its physical/chemical environment. During 1981, static and flow-
through bioassays were conducted using whole effluent and reconstituted fresh-
water. Total number of individuals and number of benthic taxa were more sen-
sitive indicators of community change than other biological indices such as
Shannon's diversity, evenness, and dominance. Stepwise multiple regression
and principal components analyses showed changes in inacroinvertebrate species
richness and relative abundance to be linear negative functions of ambient
metal concentratiions. Effluent bioassays showed potential for use in pre-
dicting in-stream biological effects from metal discharges. However, results
of bioassays with brook trout showed a large discrepancy in tolerance between
fish previously exposed to metals (acclimated) and those not exposed (nonacclim-
ated). Avoidance behavior to sublethal concentrations may determine the pres-
ence of sensitive members of the ecosystem. Therefore, safe effluent criteria
for toxicity should be based on presence of sensitive or desirable species in
the receiving system, rather than LC50s alone. The concept of water effect
(effluent: recovery zone) ratios merits further development.
Key words: onsite bioassays; mining; macroinvertebrates; water effect
ratio; pollution impact.

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4
INTRODUCTION
During 1980, the U.S. Environmental Protection Agency's (EPA) Office of
Water Regulations and Standards issued a directive requiring documentation of
water and biological quality in selected streams receiving mining, industrial,
or municipal sewage treatment plant discharges. In response to this directive,
a toxic metals study was designed to quantify four main objectives: 1) to doc-
ument the concentration and distribution of toxic metals in selected streams
receiving discharges from publicly owned treatment works (POTWs), mining activ-
ities, or industrial wastes; 2) to determine the biological state of receiving
waters where the aquatic life criteria for toxic metals are exceeded, including
sampling and analyzing fish, benthic invertebrates, and periphyton communities;
3) to report the extent to which criteria levels were observed to be exceeded;
and 4) to develop explanatory hypotheses when healthy biota existed where
criteria were exceeded.
Fifteen streams were originally sampled to provide a broad geographical
representation and range of watershed types and uses, pollution sources, water
quality characteristics, biota, and habitats. Results from the 1980 study in-
dicate that, in some cases, known sensitive species of fish and invertebrates
exist where EPA's acute and chronic criteria are exceeded. Analysis of these
data led us to propose two hypotheses. First, metals can be chelated by
organic and inorganic compounds in effluents and receiving streams, and are
thus rendered biologically nonavai1 able. Second, fish are able to acclimate
to sublethal metal concentrations which allow them to tolerate potentially
toxic ambient levels.

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5
To test these hypotheses, we conducted an intensive survey during 1980 and
1981 on Prickly Pear Creek, Montana. By the early 1860's, gold mining had
begun near the upper reaches of Prickly Pear Creek, in the Corbin and Spring
Creek drainages. Tailings and settling ponds from these long abandoned mines
are a prominent feature of these drainages; the Montana Water Quality Bureau
[1] reports that over 75% of Prickly Pear Creek was subject to streamback
modifications and dredging during the mining process. Drainage containing
high metal concentrations, including copper, zinc, silver, arsenic, and cadmium,
is released from a mine adit and oxidized tailings, and then carried into
Prickly Pear by Spring Creek. This paper discusses the importance of metal
partitioning and biological acclimation in ameliorating metal toxicity.
METHODS
Study Area
Prickly Pear Creek forms its headwaters in the Elkhorn Mountains approxi-
mately 32 km southeast of Helena, Montana (Figure 1). The stream flows north
for 64 km before entering Lake Helena and the Missouri River. Annual discharge
of Prickly Pear Creek downstream from the confluence of Spring Creek during
3	3
the 1979-80 water year ranged from 0.2 m /s to 1.2 m /s, with a mean of 0.5
3
m/s [1]. The study reach is generally characterized by continuous riffle
flow interspersed with a few distinct pools. The substrate is primarily
cobble and gravel throughout.
Five stations with similar riffle substrate and flow conditions were estab-
lished in Prickly Pear Creek (Figure 1). One station was located in the control

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6
zone (Oil), and two stations (013 and 014) were located in the impacted zone
0.2 km and 1.6 km downstream from Spring Creek. One site (017) was located in
the recovery zone approximately 8.5 km downstream from station 014. Station
018, located 2.4 km upstream from Montana City, was sampled only during 1981
(Figure 1).
Biological and Chemical Parameters
During 1980, five replicate box samples [2] were taken at each station
2
(0.1 m areal coverage) in Prickly Pear Creek. Collected	invertebrate species
were keyed to the lowest level possible, usually species.	Oligochaetes were
keyed only to class; chironomids were keyed to the family	level during 1980
and to subfamily during 1981. Native fish were collected	from each station
using a seine and by electroshocking a 100 m reach at each station. Fish used
in the bioassays were only those collected by seining.
Nutrients and metals sampled included all those listed in Table 1, although
of primary importance to this study were those metals in excess of recommended
criteria. Temporal variation was measured by integrating samples (ISC0 model
1680) over a three-hour period continuously for 24 hours. Hence, at each site,
there were eight 3-hour composite samples. In addition, one 24-hour composite
was taken at each site. All nutrients were analyzed with a Technicon Auto
Analyzer; the metals were analyzed using inductively coupled argon plasma emis-
sion spectroscopy (ICAP).
Static 24-hour and 96-hour flow-through bioassays were conducted at Prickly
Pear Creek inside a mobile laboratory using a modified Mount and Brungs [3]

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diluter. The bioassays were run using a) Prickly Pear Creek water collected
upstream from Spring Creek, b) Spring Creek water as a "whole" effluent, and
c) laboratory reconstituted fresh water. All test chambers were placed in a
water bath through which stream water at ambient temperature was circulated.
One fish species (Salvelinus fontinalis), and one invertebrate species,
Ephemerella grandis (Insecta), were used in the bioasay tests. The ji. grandis
were captured upstream from the confluence of Prickly Pear and Spring Creeks
where they were readily available near shore. Stream brook trout weighing 7
to 12 g were collected from the control station (Oil). Although this station
served as a control, trace amounts of metals were present. Hatchery trout
weighing 5 to 8 g were collected from the Bozeman National Fishery Research
Laboratory. Nonacclimated (nonmetal-exposed) hatchery trout were placed in
Prickly Pear Creek control water in the presence of equimolar concentrations
of EDTA for two days prior to testing. The hatchery trout were acclimated to
ambient Prickly Pear Creek metal concentrations by placing them into cages at
the control site for ten days prior to testing.
Bioassays were conducted to determine the response variability between
the two unrelated taxa, each with a known sensitivity to aquatic pollution.
Specific tests included whole effluent (e.g., Spring Creek water) flow-through
bioassays, using both stream and hatchery brook trout, _E. grandis, and hatchery
brook trout exposed to ambient metal concentrations in Prickly Pear Creek
upstream from Spring Creek. The static bioassays used ji. grandis indivi-
duals in Spring Creek water diluted with upstream Prickly Pear water, Spring
Creek water diluted with reconstituted laboratory water, and reconstituted
water spiked with copper.

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8
RESULTS AND DISCUSSION
Chemistry
During 1980, mean concentrations of total copper, zinc, arsenic, silver,
and cadmium in the impact zone (Table 2) were 3 to 5 times above recommended
EPA acute criteria [4]. One-way analyses of variance (ANOVA) and Student-
Newman-Keuls (SNK) multiple range tests [5] were used to determine patterns
of difference between stations." Sample variance homogeneity was determined
using Bartlett's test for homogeneous variances [5]. Concentrations of the
five key metals examined, with the exception of arsenic, were significantly
(a=0.05) greater in the impact zone than in either the control or recovery
zones.
During 1981, mean total copper, silver, and zinc were 2 to 4 times in
excess of the recommended criteria. Arsenic and cadmium levels did not exceed
established national criteria levels. In general, metal concentrations during
1981 were considerably lower than those measured the previous year (Table 2).
Macro-invertebrates
During 1980 there were a total of 47 distinct macroinvertebrate taxa col-
lected in five replicate box samples at four sites; only 8 of these were in
concentrations greater than 5% of the total population (Table 3). The control
zone site contained significantly higher (a=0.05) species richness and greater
total numbers than did the impact zone stations. Total invertebrate numbers
dropped an order of magnitude immediately downstream from the confluence of

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9
Prickly Pear and Sprinq Creeks, and total number of taxa decreased to a third
the upstream population (Figure 2). No significant differences among stations
were observed with respect to Shannon's diversity, evenness, and dominance.
As metal concentrations decreased further downstream, species richness and
total numbers increased. However, stations in the recovery zone were not
statistically different from those in the impact zone. Two possible reasons
for this may be: 1) sample variances are sufficiently great to preclude
parametric distinctions between the two sites; and 2) samples may not have
been collected sufficiently far downstream (near Station 017) to reflect a
true state of recovery. Total counts and number of taxa in the recovery zone
never regained levels comparable to the control. This may be partly due to
elevated zinc concentrations which at Station 017 were still in excess of
recommended criteria and seven times greater than concentrations in the con-
trol zone.
During 1981, there were 61 distinct taxa collected at the four Prickly
Pear sites (Table 3); of these only 9 were in concentrations greater than 5
percent of the total population. Species richness and mean counts per replicate
at the impact site (014) were one-half those found in the control zone. Species
richness at Station Oil was significantly fc=0.05) higher than at the other
three sites; however, there were no statistical differences in relative abun-
dances among stations. Species diversity and dominance were significantly
lower at 014 than at any other site. Impact to the benthic community down-
stream from the confluence with Spring Creek was, in general, substantially
less than that observed during 1980 (Figure 2), probably as a result of lower
metal concentrations in 1981.

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10
Forward stepwise multiple regression and factor analyses [6, 7, 8] were
used to determine patterns of relationships between metal concentrations and
macroinvertebrate populations in Prickly Pear Creek. Biological variables
entering into the analyses included total number of individuals, total number
of species, Shannon-Wiener diversity, species evenness, and Simpson's dominance.
The five metals (both dissolved and total) reported in excess of recommended
criteria during 1980 were used to develop statistically significant linear
combinations which could account for the observed variance in measures of
community structure.
Statistically significant regression equations emerged only for two
community structural parameters: total number of individuals per sample
(Totlind) and species richness (Nspecie) (Table 4). In the resulting regres-
sion equations, zinc and arsenic (both dissolved and total) were consistently
the most important metals accounting for a major proportion of the variance
in both total numbers and in species richness. Using dissolved metals only,
zinc, arsenic, and copper combined to consistently explain over 90% of the
observed variance in the two community parameters. The regression analyses
were unable to explain at the ct=0.05 level the observed variance for
diversity, evenness, and dominance.
Principal components analysis supports the results of the stepwise
regressions and aids in determining how the benthic community responds to
ambient metal concentrations. Both the dissolved and total forms of cadmium,
copper, silver, and zinc load positively on the first principal component
(Table 5). Both species richness and diversity (Hprime) load negatively
on the first component, which alone accounts for 60.2% of the observed var-

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11
iance in the variables. The stronger, negative, loading of diversity and
the high, positive, metal loadings implies a possible reduction in the species
intolerant of the range of metals present. Further sampling, particularly
behavioral drift in response to metal input, is necessary to verify this
hypothesi s.
The only metal loading strongly on the second principal component (Table 5)
is arsenic, both the total and dissolved form. Total arsenic has a particu-
larly high positive loading; the strong, negative, loadings for total number
of individuals and species richness is understood in terms of the highly
toxic nature of arsenic [9]. The positive loading for species' evenness (Even)
indicates a "selective inhibition of crucial enzymes" [10], perhaps reducing
several species' populations to low levels. The second principal component
explains a further 31.9% of the observed variance among the variables; hence,
a total of 92.1% of the observed variance is explained by both principal com-
ponents.
Bioassays
Onsite bioassay tests during 1981 emphasized whole effluent (Spring
Creek water) toxicities at each site. Consequently, in this study, most tests
were conducted using a serial dilution of Spring Creek water with either
Prickly Pear Creek water or reconstituted fresh water. Results of static bio-
assays using E_. grandis and serial dilutions of Spring Creek water with either
Prickly Pear control zone water or reconstituted fresh water are shown in
Figure 3. In both tests, the LC50 was at concentrations slightly less than

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12
100 percent effluent. The test using reconstituted fresh water as the diluent
demonstrated slightly greater toxicity although it was not statistically (p=0.05)
different. A flow-through bioassay (Figure 4) using _E. grandis resulted in a
very similar LC50 value to the static tests; the static LC50 to flow-through
LC50 ratio was 1.0.
The final test using grandis was conducted with copper spiked in recon-
stituted fresh water (Figure 5). The calculated LC50 was similar to that when
copper was spiked in the effluent tests. This provides some evidence that
Prickly Pear Creek water contains no factors capable of reducing metal toxicity.
Total organic carbon and suspended solids were at low concentrations (TOC 2.8
to 7.8 mg/1; total residues 26 to 299 mg/1; total nonfiltrable residues 11 to 67
mg/1). Therefore, we would further expect that Prickly Pear Creek should not
have toxicity-reducing characteristics [Cf. 11].
The initial test using brook trout was conducted with hatchery fish ac-
climated to Prickly Pear water for two days. EDTA was added to the medium
to prevent exposure to background metal concentrations. Simultaneously,
resident fingerling trout were collected from, and isolated in, control zone
water without EDTA. Bioassay results (Table 7) indicate hatchery fish are
more sensitive to metals than resident fish; even 100 percent concentrations
of Spring Creek water did not produce mortality in resident stream fish.
A second test was conducted after hatchery fish were allowed to acclimate
for ten days in Prickly Pear Creek control zone water. Subsequent tests were
conducted with whole "effluent" spiked with copper and zinc. No mortality was

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observed in either group of fish even at the highest concentrations. It
appears short term acclimation results in at least a 3-fold increase in metal
tolerance. This phenomenon provides some insight into the apparent anomaly
of observing fish where water quality acute criteria are exceeded. For
example, substantial numbers of brook trout were observed throughout the con-
trol, impact, and recovery zones of Prickly Pear Creek. Ultimately, bioavail-
ability of toxic metals to the stream biota appears to be a function of the
effluent metal chelating capacity or that of the receiving stream water, as
well as the extent of previous exposure to metals, and traditionally acknowl-
edged water chemistry parameters such as hardness, alkalinity, and pH.
S. fontinalis and E. grandis are common in Prickly Pear Creek and rep-
resent sensitive species within the resident fauna. Furthermore, other
members of their respective families are among the most sensitive species
listed in EPA's criteria documents. Assuming metal concentrations under which
these species live can be directly compared to national criteria, unacclimated
hatchery trout demonstrate an LC50 value notably greater than those published
for national criteria values. For example, the copper concentration was twice
the national acute criterion, and the zinc concentration was three times
greater than the national criterion. This discrepancy may be due to popula-
tion differences, or perhaps some acclimation was occurring despite the addi-
tion of EDTA. After acclimation, brook trout LC50 values for copper and zinc
were at least four times in excess of their national criteria values.
Mayfly LC50 values were similar to nonacclimated brook trout values,
even though the potential for acclimation existed. Consequently, E. grandis

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may be much more sensitive to metal pollution than trout. Ultimately, this
information strongly indicates that species selected for bioassays and their
history of exposure to sublethal metal concentrations will critically influ-
ence bioassay results. This phenomenon must be considered when evaluating
EPA1s proposed protocols for criteria modification.
Relationship of Instream Biological Data to Bioassay Data
t
Benthic community structure will ultimately reflect changes at both the
population and individual level. For this Prickly Pear Creek analysis,
species richness and the total number of individuals per sample were found
to be inversely related to concentrations of five metals found to be in excess
of criteria levels. Stepwise regression and principal components analyses
indicate arsenic and zinc to be particularly important in explaining the
observed variance in the number of species found and in the total number of
individuals present.
The importance of understanding yearly (and seasonal) variation in com-
munity structure prior to establishing site specific criteria (EPA draft pro-
tocol , 1982) is demonstrated by studying the responses in species richness and
total counts to 1980 and 1981 metal concentrations. Although trends were sim-
ilar both years, the relationship between biological parameters and metal con-
centrations in 1981 was weaker than in 1980. It will be necessary to define
how seasonal variation in flow, temperature, rainfall (and snowfall) affect
the interrelationships between the physical/chemical milieu and the resident
biota. Once these are known for a site, "critical seasons" can be established
during which it may be reasonable to stop (or increase) effluent release. For

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example, in the case of Spring Creek and Prickly Pear, there may be low flow
periods when the relative importance of metals in Spring Creek, as they influ-
ence benthic macroinvertebrates, increases. When Prickly Pear Creek is sub-
ject to greater volumes of water (e.g., summer storm events, spring snowmelt),
metals may be flushed from the Spring Creek sources and ultimately ambient
levels would decrease, although the short-term episodic consequence would be
just reverse. These chemical considerations must also be considered in light
of scouring impact to the benthic community during episodic events. Further
seasonal testing will more clearly define these relationships.
Flow-through and static bioassays on Prickly Pear Creek using Spring
Creek water as the effluent indicate strongly that acclimation can occur
in resident vertebrate species, even after only five to ten days. It demon-
strates that species used in bioassays should be chosen with care to determine
site-specific metal criteria and further, that resident biota may reflect
changes in ambient metal levels with greater sensitivity than top level
carnivores in the system.
Theoretically, a site-specific application factor relating LC50 concentra-
tions to recovery zone metal concentrations can be developed for potentially
toxic metals. An example of how this might be applied is demonstrated in the
following manner:
Table 7 lists the measured LC50 concentrations for two key metals in the
whole effluent flow-through test with £. grandis (Figure 4). Each value is
divided by its respective ambient total metal concentrations in the recovery

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zone, i.e., at station 018 (Table 2). This ratio yields a site specific
application factor. To obtain ambient impact zone levels which cause no
significant reduction of the Prickly Pear Creek invertebrate community, the
concentration of each metal would have to be reduced by the appropriate
application factor. For example, zinc concentrations in the impact zone
would be reduced by a factor of 5.4.
Ultimately, a site-specific application can be related to "effluent"
concentration by incorporating .a factor which accounts for the approximate
effluent LC50 dilution for the flow-through bioassay with _E. grandis (Figure
4), in this example, 0.9. For example, the effluent concentration of zinc
would have to be reduced by a factor of 4.86 (=5.4 * 0.9).
Part of the reduced metal toxicity in a receiving stream results from
dilution. As evidenced by the Ephemerella flow-through bioassay, during low
flows in late summer there is a 1:1 dilution of Spring Creek water with
Prickly Pear water. Therefore, in our example, to compute a seasonal site-
specific application factor, the effluent concentration for zinc would have
to be reduced by a factor of 5.4 * 0.9 * 0.5 = 2.34 where: 0.9 = effluent LC50
percent dilution; 5.4 = LC50 concentration to recovery zone ambient concen-
tration "application factor"; and 0.5 = level of effluent (Spring Creek)
seasonal dilution.
If each metal concentration presently exceeding acute aquatic life cri-
teria were to be appropriately adjusted, the recovery zone resident benthic
community theoretically could be expected to shift upstream to the point of

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complete mixing of Spring Creek with Prickly Pear Creek. Use of this appli-
cation factor concept would be further validated if we incorporate information
on avoidance behavior as opposed to strictly mortality data [12] into our pre-
dictions concerning the presence and absence of sensitive species.
Some general conclusions, then, can be drawn from this study. Total
numbers of individuals and total number of taxa in Prickly Pear Creek appear
to be sensitive indicators of community change more so than other biological
indices such as Shannon's diversity, evenness, or dominance. Changes in macro-
invertebrate species richness and relative abundance in Prickly Pear were
found to be linear functions of ambient metal concentrations.
Effluent bioassays may have potential for use in predicting in-stream
biological impacts from metal discharges. Sensitivity of the test species,
seasonality, and variation in community structural response all need to be
taken into account. Finally, onsite effluent bioassays with brook trout
indicate a large discrepancy between groups of fish previously exposed to
metals (acclimated) and groups of fish not exposed to metals (nonacclimated).
Variable or unknown histories of test organism exposure to metals can lead
to misleadingly high LC50 results. However, using acclimated animals to
develop criteria may not be appropriate for two reasons. First, intermittent
discharges may not allow acclimation. Second, acute to chronic application
factors may not be realistic, i.e., although short-term adult acclimation can
occur, changes in metabolic functions may impair juvenile growth and subsequent
reproduction.

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RECOMMENDATIONS
1.	Additional experimentation needs to be done on a site-specific basis to
tighten statistical replicability with invertebrate field collections,
if these are to be used in definition of control, impact, and recovery
zones in receiving streams. This could be done by increasing the number
of field replicates or by using alternative standardized samplers such
as artificial substrates.
2.	To evaluate the impact of a particular effluent on a receiving "control
zone," organisms which demonstrate a sensitive reaction to the effluent
should be used. Using a native organism will ensure acclimation to
background water quality characteristics of each stream.
3.	The validity of water effect ratios in site-specific criteria modification
needs to be tested at additional locations using a variety of effluent
types and quality of receiving waters (for example, with those having a
higher TOC or suspended solid content). Further, this technique should
be tested with other pollutants which are less stable or volatile (e.g.,
ammonia and organic compounds). The possibility of using this approach
for all types of complex discharges should be evaluated.
4.	Standard LC50 values do not predict "no effect" acute criteria values.
Presence or absence of sensitive members of the aquatic ecosystem may
stem from avoidance behavior to sublethal concentrations, since signifi-
cant impact can occur at concentrations much less than the LC50 values.

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18
RECOMMENDATIONS
1.	Additional experimentation needs to be done on a site-specific basis to
tighten statistical replicabi 1 ity with invertebrate field collections,
if these are to be used in definition of control, impact, and recovery
zones in receiving streams. This could be done by increasing the number
of field replicates or by using alternative standardized samplers such
as artificial substrates.
2.	To evaluate the impact of a particular effluent on a receiving "control
zone," organisms which demonstrate a sensitive reaction to the effluent
should be used. Using a native organism will ensure acclimation to
background water quality character!sties of each stream.
3.	The validity of water effect ratios in site-specific criteria modification
needs to be tested at additional locations using a variety of effluent
<
types and quality of rec©iw5 waters (for example, with those having a
higher TOC or suspended solid content). Further, this technique should
be tested with other pollutants which are less stable or volatile (e.g.,
ammonia and organic compounds). The possibility of using this approach
for all types of complex discharges should be evaluated.
4.	Standard LC50 values do not predict "no effect" acute criteria values.
Presence or absence of sensitive members of the aquatic ecosystem may
stem from avoidance behavior to sublethal concentrations, since signifi-
cant impact can occur at concentrations much less than the LC50 values.

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19
Thus, safe effluent criteria or standards for acute toxicity should be
based on the presence or absence of sensitive or desirable species in
the receiving system, rather than standard LC50s alone.
ACKNOWLEDGEMENTS
The study was supported as a cooperative effort between EPA's Environ-
mental Monitoring Systems Laboratory, Las Vegas, Nevada (EMSL-LV), and the
Environmental Research Laboratories at Corvallis, Oregon, and Duluth, Minnesota.
EMSL-LV designed and supervised the project; the field investigation and sub-
sequent biological laboratory analyses were performed by the UNLV-EPA Cooper-
ative Research Laboratory at the University of Nevada, Las Vegas.

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LITERATURE CITED
Montana Water Quality Bureau. 1981 Prickly Pear Creek: A report on man's
debilitating impacts. WQB Report No. 81-2. Department of Health and
Environmental Sciences, Helena, Montana. 157 pp.
Ellis-Rutter Associates. 1973. Brochure: Portable Invertebrate Box
Sampler. Douglasville, Pennsylvania. 4pp.
Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicol ogical studies. Wat. Res. 1:21-39.
U.S. Environmental Protection Agency. 1980. Water quality criteria
documents: availability. Federal Register 45:79318-79379.
Sokal , R. F. and F. J. Rohlf. 1981. Biometry. 2nd edition. W. H. Freeman
and Co., San Francisco.
Draper, N. R. and H. Smith. 1966. Applied Regression Analysis. J. Wiley
and Sons, New York.
Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner and D. H. Bent.
1975. Statistical Package for the Social Sciences (SPSS). 2nd edition.
McGraw-Hill Book Company.

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21
8.	Harmon, H. H. 1976. Modern Factor Analysis. University of Chicago Press,
Chicago, Illinois.
9.	Whitton, B. A. and P. J. Say. 1975. Heavy metals, In B. A. Whitton, ed.,
River Ecology. Blackwell Science Publishers, Oxford, pp. 286-311.
10.	Moss, B. 1980. Ecology of Freshwaters. Blackwell Science Publishers,
Oxfo rd.
11.	Brown, V. M., T. L. Shaw and D. G. Shurber. 1974. Aspects of water quality
and the toxicity of copper to rainbow trout. Wat. Res. 8:797-803.
12.
DeGraeve, G. M., R. L. Elder, D. C. Woods and H. L. Bergman. Effects of
naphthalene and benzene on fathead minnows and rainbow trout. Submitted
to Arc. Envir. Contam. Toxic.

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22
Table 1. Chemical parameters collected in Prickly Pear Creek.
A.	Technicon Auto Analyzer (mg/1) C.
Total phosphate
Orthophosphate
Hydrolysable phosphate
Kjeldahl nitrogen
Total ammonia (NH.)
Nitrates + nitrites
Total alkalinity	D.
B.	Additional Parameters (mg/1)
Total Ca + Mg hardness
Total organic carbon (carbon
analyzer)
Total residues
Suspended residues
Total sulfate
Metals - ICAP*
Cu, Cd, Zn, As, Ni, Ag, Cr, Se,
Ca, Mg, A1 , Pb (pg/1)
Total recoverable
Filtered through 0.45 ym
Sediments
In-Situ Parameters
Dissolved oxygen (mg/1)
pH (SU)
Conductivity (umho)
Temperature (°C)
Turbidity (NTU)
* ICAP = Inductively Coupled Argon Plasma Emission Spectroscopy.

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23
Table 2. Comparison of mean total concentrations (pg/1) of selected metals
versus calculated acute and chronic water quality criteria (U.S.
EPA 1980) for aquatic life, Prickly Pear Creek, Montana. Dashes in-
dicate no chronic criterion has been established.
Stations
Control	Impact	Recovery
Oil	013	014 017 018
1980 1981 1980 1981 1980 1980 1981
Hardness (mg/1)	57	65	160	124	124	117	133
Total Arsernc
actual (x)	400.2 ¦ 90.3	631.0	61.2	654.3	728.3	60.4
acute criterion	440	440	440	440	440	440	440
chronic criterion	------
Total Cadmijjm
actual (x)	4.5	3.2	24.5	3.4	15.5	3.5	3.4
acute criterion	2	2	5	4	4	4	4
chronic criterion	0.01	0.02	0.04	0.03	0.03	0.03	0.02
Total Copper
actual (x)	13.1	15.7*	109.1	112.0	34.1	18.7	27.2
acute criterion	13	15	35	27	27	26	2y
chronic criterion	5.6	5.6	5.6	5.6	5.6	5.6	5.6
Total Silver
actual (x)	23.1	6.7	43.4	10.4	25.0	5.2	15.8
acute criterion	2	2	9	6	6	5	7
chronic criterion	-------
Total Zinc_
actual (x)	65.9	13.2	1963.8	1000.4	840.1	464. 7	348.3
acute criterion	202	224	475	385	385	365	408
chronic criterion	47	47	47	47	47	47	47
* Dissolved copper: total measurements not available.

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Table 3. Percent composition of common (>5%) invertebrate taxa collected in box samples in Prickly Pear
Creek, Montana, 1980-81. Blanks indicate organism was not collected that station and year in
greater than 5% relative abundance. Extended species list is available from the authors.
Control
Oil
Percent Composition
1980
Impact Recovery	Control
013 014 017
011
1981
Impact
013
Recovery
014 017
Ephemeroptera
Baeti s tricaudatus
Baetis sp.
PIecoptera
Pteronarcel1 a badia
T richoptera
Arctopsyche grandi s
Brachycentrus sp.
Stactobiel la sp.
Di ptera
Chironomidae
Orthocladi inae
Di amesinae
Simul i iurn sp.
Atherix variegata
Coleoptera
Optioservus quadrimaculatus
25
39
26
6
44.
21
26
30
24
16
17
36
18
45
14
10
15
8
67
26
32
12
11
Hydracarina
Sperchon sp.
6	6

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25
Table 4. Stepwise multiple regression equations and results for Prickly
Pear Creek ambient metal concentrations and benthic community
structural parameters.
Overal 1
Equation	r^	F
All metals
Totlind = 456.0 - 0.342 Astotl - 1.448 Zndiss	0.962	0.003
-0.071 Asdi ss
Nspecie = 27.6 - 0. 150 Zndiss - 0.013 Astotl	0.945	0.006
+ 0.813 Cutotl
Dissolved metals only
Totlind = 292.3 - 4.764 Zndiss - 0.226 Asdiss	0.952	0.004
+ 10.866 Cudi ss
Nspecie = 23.0 - 0.220 Zndiss - 0.826 Asdiss	0.983	0.006
+0.263 Cudiss + 0.150 Agdiss
Total metals only
Totlind = 466.6 - 0.447 Astotl - 3.992 Agtotl	0.906	0.003
Nspecie = 26.1 - 0.550 Cdtotl - 0.012 Astotl	0.783	0.022

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26
Table 5. Principal components analysis using varimax rotation of biological
and metal (total and dissolved) data from Prickly Pear Creek, Montana.
Factor loadings less than 0.5 are not shown.
Variable	Factor Loadings
Name	I	2	3	Communal ity
Totli nd

-0.934

0.999
NSpeci e
-0.593
-0.776

0.954
Hprime
-0.863


0.825
Even

0.616
-0.784
1.002
Domi n


0.943
0.970
AsDi ss

0.655

0.684
AsTotl

0.906

0.928
CdDi ss
0.944


0.966
CdTotl
0.930


0.978
CuDi ss
0.815


0.795
CuTotl
0.987


0.990
AgDi ss
0.690


0.497
AgTotl
0.906


0.831
ZnDi ss
0.925


0.976
ZnTotl
0.924


0.939
Factor	12	3
Eigenvalue
% of Variance
Cumulative %
8.025
60.2
60.2
4.256
31.9
92.1
1.054
7.9
100.0

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27
Table 6. Bioassay results using acclimated and no.n-accl imated brook trout,
Prickly Pear Creek, Montana.
Test
Metal Concentrations (ng/1)
Zn	Cu
LC50
Hatchery Trout
Nonacclimated
Acclimated
1.45
2.13
0.088
0.188
75% of effluent*
>100% of effluent
Native Stream Trout
"Nonacclimated"
"Acclimated"
1.88
2.13
0.155
0.188
>100% of effluent
>100% of effluent
*Ef fl uent
= Spring Creek water.

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28
Table 7. Copper and zinc effluent LC50 concentration: recovery zone ambient
concentration ratios for Prickly Pear Creek, Montana. LC50 concen-
trations are found on Fig. 4; ambient metal recovery zone concentra-
tions are reported in Table 2.
LC50/Recovery zone concentrations (mg/1 ) Application factor
Copper
Zi nc
0.111/0.027
1.890/0.348
4.1
5.4

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29
FIG. 1. Station locations on Prickly Pear Creek, Montana.
FIG. 2. Total number of macroinvertebrate taxa and mean count per replicate
at control (Oil), impact (013, 014), and recovery (017) zone sites, Prickly
Pear Creek, Montana. For clarity, error bars are ommitted.
FIG. 3. Results of static bioassays with Ephemerella grandis in Prickly Pear
Creek, Montana. Tests using reconstituted fresh water and stream (control
zone) water are both shown. The horizontal bar is one standard deviation
around the mean.
FIG. 4. Results of a flow-through bioassay with Ephemerella grandis in Prickly
Pear Creek, Montana. The horizontal bar is one standard deviation around
the mean.
FIG. 5. Results of bioassays using Ephemerella grandis in reconstituted
freshwater spiked with copper, Prickly Pear Creek, Montana. The horizontal
bar is one standard deviation around the mean.

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Prickly Pear Creek, Montana
Montana City
Clancy
Alhambra
Direction
Flow
Mine Drainage, ^—014
Point f
Source /
•013
Jefferson City
011
Miles
' | '
0	12	3	4
letena
Kilometes

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a) 400"
*•>
to
o
"5.
0)
cc
aJ 300-
a
JZ
o
*¦«
(U
O
® 200H
to
n

~ 100-
c
ra

JQ
E
z
20-
10-
011 013 014 017
1980
Station and Year
011 013 014 017
1981

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90
80
70
60
50
40
30
20
10
5
2 ¦
®/a/
Sq /©Diluted with Reconstituted Water
/	LC50 = 78% Effluent
A /	Cu = 0.096 mg/l
/	Zn = 1.530 mg/l
I	ADiluted with Prickly Pear Stream Water
/	LC50 = 87% Effluent
/	Cu = 0.105 mg/l
r	Zn = 1.790 mg/l
n	1	1—i—i i i i
30 40 50 70 100
% of Spring Creek

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90-
80-
70-
60
50
10-
5-
2-
LC50 = 90% Effluent
Cu = 0.111 mg/l
Zn = 1.890 mg/l
20 30 40 50 70 100
% of Spring Creek

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LC50 = 0.097 mg/l
Hardness = 145
i i	r—i—\—|—r—i
30 40 50 70 100
Copper Concentration

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