RESULTS OF ALGAL ASSAYS PERFORMED ON WATERS
COLLECTED FROM THE LOWER CLARK FORK RIVER SYSTEM
AT STATIONS BELOW MILLTOWN DAM TO BELOW NOXON DAM
Report Ills August 6-8, 1985 Sampling
by
Joseph C. Greene1, Michael Long3, Cathy Lee Bartelsa
and Julius U. Nwosu2
*Corvallis Environmental Research Laboratory
Hazardous Waste and Water Branch
Hazardous Waste Assessment Team
3Northrop Services, Inc.
200 S.W. 35th Street
Corvallis, Oregon 97333
INTRODUCTION
A large amount of public concern has been expressed over the
general health of the lower Clark Fork River system. The proposed
modification of the existing wastewater discharge permit for the
Champion International paper mill at Frenchtown has generated much
of this concern. Other sources of wastewater, namely the City of
Missoula wastewater treatment plant and historic metals deposits
(primarily copper and zinc) originating upstream from Milltown
Dam, have also been mentioned as possible sources of stress on the
river (Montana, 1984).
The Selenastrum capricornuturn algal assays performed during this
study were undertaken to fulfill a request for technical
assistance from the Montana Water Quality Bureau through the US
EPA Region VIII staff in Denver, CO. The Montana Department of
Health and Environmental Services has a Lower Clark Fork River
Monitoring Plan. Data gathered from these assays will be used in
conjunction with other data collected under this monitoring plan
to define existing conditions in the Lower Clark Fork River
system.
METHODS
Water was collected as surface grab samples, along the lower Clark
Fork River (Table I) by Mr. Gary Ingman a biologist working for

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the Montana Department of Health and Environmental Sciences.
River water collected at each site was collected into a large
plastic container and mixed well. Water -from the large container
was then subsampled -for algal assay by the US EPA Corvallis
Environmental Research Laboratory, OR (CERL) and chemical analysis
by the State of Montana. The samples were stored in the dark at
4»C. The samples were pretreated by autoclaving -followed by
¦filtration (AF). The samples were autoclaved in their
polypropylene shipping bottles. Prior to filtration they were
cooled to room temperature and re-equi1ibrated to their original
pH by bubbling with 17. C0» in air.
The assays, performed with three replicate flasks for each
nutrient or chelator addition, were inoculated with a final
concentration of 1000 cells/ml Ss_ capricornutum and cultured for
14 days. Yields were reported as mg/1 dry weight (Miller, Greene
and Shiroyama, 1978).
Analyses of all nitrogen and phosphorus species were performed
according to the USEPA (1979) methods for chemical analysis of
^wastes ancJ\water\.
Elemental chemical analysis was performed on an inductively
coupled plasma atomic emission spectrometric instrument (ICPAES)
according to the USEPA (1983) method for trace element analysis of
water and wastes.
RESULTS AND DISCUSSION
notential results for each set of
^Vf^asks^wfth^heir associated standard deviation and
test flasks with tneir «	=hown in Table II. We were unable to
coefficient of ^i»i*arowth potential or limiting nutrients of the
determine the algg	(below Huson) because of toxic
sample collected fro	solution. Analysis of the heavy metal
constituents P^^^.^^^riolution identified zinc to be
concentrations in the Site #13 tes^	^ ^
the toxic agent limiting «19 9 ^ x (Miller et.al., 1985).
of zinc for £L_ gapncorni	"adequate to cause inhibition of
The .457 mg/1 zinc was more than adequate
growth.
,	,-nllection was unseasonally low.
River flow at the time of wa	_b nQt believed to be from a
Therefore, the source ° oriainating at the upstream Superfund
slug of contaminated wa	suspect that particulate matter
sites at Silver	C ^	We	h/ve bBen plcked up
containing . high	th. Mo„tan, Water Quality
during sampling..	indicate that the sample collected from
«£"# 12 at harper Bridge contained "some sloughed periphyton in
Site #1-* at Harper	yA1 the site #13 sample collected at
Huson1contained "considerable amounts of sloughed periphyton in

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TABLE I. Identification of Clark Fork River
sampling stations showing both Montana and
EPA site identification codes.
*********************************************
***** CLARK FORK RIVER *****
*** SITE IDENTIFICATION ***
**ses
MT
s s s ss s
EPA
SSSSSBSBBSSSSSZSSSBSSBSSSSSaSSSS**
4
10
Below Hi 11town Dam
6
11
Above Missoula WWTP
11
12
At Harper Bridge (above Champion)
15
18
At Huson (below Champion)
20
14
At Superior
22
15
Above Flathead River confluence
25
16
Ab6ve Thompson Falls Reservoir
27
17
Below Thompson Falls Reservoir
29
18
Below Noxon Dam
*********************************************

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TABLE II.
Algal growth potential results froa 14-day tests performed on autodaved and filt-
ered (AF) waters collected fro* the lower Clark Fork River during August 1985.
fHHHHHHHHIiHHHfHHIHHilHilHHHHHHHHHHIHtHitHHIHiHHHI
CHEK.	1.00 0.05	1.00
SITE ID	ag/L «g/L	ag/L	Liait.
No.
(63) Stats.
Control
N
P
H+P
EDTA
H+E
P+E
N+P+E Nutr.
10.
32800 Hean
0.61
1.27
1.84
32.39
0.09
0.08
1.57
28.80 p/n

SD
0.42
0.24
0.25
0.43
0.04
0.01
0.16
0.86

CV (Z)
69
19
14
1
44
16
10
3
11.
32801 Hean
0.95
0.88
3.71
34.49
0.12
0.10
3.30
35.29 p/n

SD
0.78
0.78
0.21
2.24
0.02
0.03
0.23
0.95

CV (Z)
82
89
5
7
18
31
7
3
12.
32802 Hean
2.67
1.51
4.16
30.38
0.52
0.81
3.88
38.54 p/n

SD
0.41
1.39
0.59
2.57
0.1B
0.33
0.63
1.88

CV (Z)
15
92
14
8
35
41
16
5
13.
32803 Hean
0.17
0.15
0.10
0.15
0.13
0.13
0.19
0.17 T

SD
0.06
0.02
<0.01
0.069
0.05
0.03
0.03
<0.01

CV (Z)
37
11
7
45
35
25
14
4
14.
32804 Hean
0.11
0.06
0.07
0.08
0.15
0.24
18.42
28.17 a/p/n

SD
0.01
0.03
0.04
0.03
0.02
0.03
0.08
1.42

CV (Z)
13
48
56
38
13
11
< 1
5
15.
32805 Hean
0.66
0.69
2.39
28.49
0.17
0.08
3.05
31.06 a/p/n

SD
0.46
0.59
0.93
0.12
0.10
0.02
0.39
3.75

CV (Z)
70
85
39
<1
57
30
13
12
16.
32806 Hean
1.02
1.00
1.6B
28.56
0.09
0.07
3.11
34.69 a/p/n

SD
0.81
0.36
1.46
2.70
0.04
0.01
0.24
0.67

CV (Z)
80
36
87
9
41
18
8
2
17.
32807 Hean
0.25
0.54
1.69
33.05
0.13
0.12
3.93
38.09 a/p/n

SD
0.29
0.77
1.67
0.00
0.04
0.03
0.38
1.84

CV (Z)
118
143
99
0
31
23
10
5
18.
32808 Hean
0.13
0.62
0.44
0.85
1.23
1.17
4.26
31.82 a/p/n

SD
0.06
0.90
0.40
0.87
0.02
0.31
0.19
2.37

CV (Z)
45
145
91
102
2
26
5
7
¦ - Indicates that the saaple Has growth liaited by trace aetals. This conclusion
is aade when the yield in EDTA spiked flasks is = or > 201 of the growth in
siailar flasks without EDTA.
CV- The standard deviation divided by the aean tiaes 100.
T = Toxic concentration of heavy aetals present in solution preventing growth.

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TABLE III.
Analytical results of aetal concentrations found in waters collected fro* the loner
Clark Fork River systea during August 1985.
HHHHHHHHHHHHHHIIHHHHHIfHHHHIilHiHIIHHHHHHHilHHHH
Chea. ag / liter
SITE ID 	
No. (6336) Zn Cu Cd Ni Ca Hg Cr As Pb A1 Nn S
10
800
<.002 <.002 <.002 <.006
20.2
11
801
<.002 <.002 <.002 <.006
18.8
12
802
<.002 <.002 <.002 <.006
17.2
13
803
0.457 <.007 <.002 0.031
21.9
14
804
<.003 <.002 <.002 <.006
20.2
15
805
<.003 <.002 <.002 <.006
16.4
16
806
0.009 <.002 <.002 <.006
17.8
17
807
<.002 <.002 <.002 <.008
14.7
18
808
<.002 <.002 <.002 <.006
20.1
11.3 <.005 <.015 <.025 <.025 <.001 9.797
8.7	<.004 <.015 <.025 <.025 <.001 5.812
9.2 <.006 <.015 <.025 <.025 1.036 6.553
8.8	<.004 <.015 <.025 <.025 <.001 5.532
8.2 <.004 <.015 <.025 <.025 <.001 5.053
7.1 <.005 <.015 <.025 <.025 <.001 2.405
7.0 <.004 <.015 <.025 <.025 <.001 2.204
6.9	<.004 <.015 <.025 <.025 <.001 1.706

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river and sample". These solids are perhaps the explanation for
the high concentration of zinc. However, we were surprised to
also -find in this sample concentrations o-f N0a+N03+NH3 in excess
o-f 1.00 mg/1 (Table IV). Chemical analyses of nutrients in the
Site #13 sample indicate that the test water would have been
primarily P limited and secondarily limited by N if it had not
contained toxic concentrations of heavy metal.
Algal growth in all of the water samples collected from Sites 14
through 18 were primarily limited from further growth by trace
nutrients. Iron is the trace nutrient that often causes plant
growth limitation. Although the iron is present in the test
solution it is not biologically available. Chelated iron is
biologically available and essential for plant growth. We believe
that our addition of EDTA chelated the iron in these test waters
and made the iron biologically available to S^. capricornutum.
Increased growth did indeed occur with the addition of EDTA. This
is best illustrated when one compares the yields of samples spiked
with phosphorus to those spiked with phosphorus plus EDTA. For
example, the phosphorus spiked test water collected from Site #18
produced a maximum yield of 0.44 mg/1 dry weight whereas the
phosphorus plus EDTA spiked culture produced nearly 10-times more
biomass (4.26 mg/1 dry weight). In this set of tests, water
samples not spiked with P were generally too low in yield to
clearly determine these differences. Once the requirement for
chelated iron was satisfied, phosphorus became the growth limiting
nutrient in all of the samples collected from Sites #14 through
#18. Furthermore, samples collected from Sites #10 through #12
were primarily phosphorus limited.
Analysis of the metals present in these autoclaved and filtered
test waters are shown in Table III. The potentially toxic heavy
metals that might originate at the upstream Silver Bow Creek sites
are below detection in all of the test waters except Site #13 and
#16. The concentration of zinc found in Site #16 test water is
considered within normal background levels and would not cause a
negative environmental effect. These analytical results are
supported by the bioassay test results in that no inhibition of
algal growth was demonstrated in any test water except that
collected from Site #13.
Results of chemical analysis for phosphorus and nitrogen are shown
in Table IV. Most of the ortho and total phosphorus values are
below detection limits. Except for Site samples #13 and 18, the
NOa+NO® were also below detection. These analytical results do
not lend themselves well to interpretation other than to suggest
that the system may be low in algal growth potential. In fact,
Site #11, 12 and 16 samples produced algal yields that were within
the moderately high productivity subgroup (0.81-6.00 mg/1 dry
weight) of Miller et. al. (1974). All other stations were within
the moderate productivity subgroup of 0.11-0.80 mg/1 dry weight.
A histogram illustrating the maximum algal yield in the control
cultures is displayed in Figure I. It is interesting to note that

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TABLE IV.
Results of nutrient analysis on Haters collected
troa the loner Clark Fork River during August 19B5
CHEH. PHOSPHORUS	NITROGEN
SITE < (63) ORTHO TOTAL N02+NQ3 NH3 0R6.
10.
32800
<0.005 0.006 <0.010
0.010
0.303
11.
32B01
<0.005 <0.010 <0.010
0.015
0.552
12.
32802
<0.005 <0.010 <0.010
0.034
0.655
13.
32803
<0.005 <0.010 0.924
0.368
0.752
14.
32S04
<0.005 <0.010 <0.010
0.017
0.576
IS.
32805
<0.005 0.006 <0.010
0.010
0.969
16.
32806
<0.005 <0.010 <0.010
<0.005
0.641
17.
32807
<0.005 <0.010 <0.010
0.013
0.597
IB.
32808
0.006 <0.010 0.054
0.006
0.299

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FIGURE 1. Productivity classification of the lower Clark Fork
River based on 14-day yields in the culture controls.

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z I I I I I I II I II I I I I I I I I I I I I :
HIGH
Moderately high
MODERATE
JL1
1
1
X
o
h
_L
LOVl m

to // /Z /3 t¥ ts" /*
SAMPLING SITES
// /8

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there are two subsets of information. Algal growth potential
increases from below Mi 11 town Dam at Site #10 to Site #12 at
Harper Bridge (perhaps also at Site #13 if it hadn't contained so
much zinc). The water collected at Site #14 at Superior, MT.
produced the lowest algal growth potential of all waters tested
that did not contain algal growth inhibitors. The growth
potentials then increased downstream from Superior until they
peaked at Site #16 above Thompson Falls Reservoir. The growth
potential decreased below Thompson Falls Reservoir and below Noxon
Dam as the water moved downstream towards Lake Pend Oreille.
Although the magnitude of algal yields changed, the pattern was
the same in the set of samples collected during December 1984
(Greene, Long and Bartels, 1985). These data indicate that there
is a continuing source of nutrients entering the Clark Fork River
below sampling Site #14 at Superior and upstream from sampling
Site #15 which was located above the confluence of the Clark Fork
River with the Flathead River.
SUMMARY AND CONCLUSIONS
Algal assays were performed to define the effects of heavy metals
or domestic and industrial effluents upon potential growth of
planktonic algae in the lower Clark Fork River. Results of these
assays led to the following conclusions:
(1)	Algal growth potential could not be defined for the Si^e #3^
sample collected below Huson, MT. because of the toxic levet~~6f
zinc (.457 mg/1) in the sample.
(2)	Relative to N and P, all of the 9 samples were primarily
phosphorus limited. This includes Site #13 that had growth
limited by zinc toxicity.
(3)	The highest algal growth potentials, which fell within the
moderately high productivity classification of 0.81 to 6.00 mg/1
dry weight, were found at Site #11 from above the Missoula
wastewater treatment plant, Site #12 collected at Harper's Bridge
(above Champion International) and Site #16 above Thompson Falls
Reservoir.
(3)	Sites #10, 14, 15, 17 and 18 produced algal biomass that
placed them in the moderate productivity classification which
produced biomass of 0.11 to 0.80 mg/1 dry weight.
(4)	We predict from chemical analysis that the Site #13 sample,
which contained toxic concentrations of zinc, was primarily
phosphorus limited. Our basis for this conclusion is that ortho
and total phosphorus are below analytical detection but the total
soluble inorganic nitrogen is 1.292 mg/1. A productivity
classification could not be established for this sample.

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<5) Algal growth in samples collected -from Sites #14 through #18
were primarily limited by iron. Iron was present but not
biologically available.
REFERENCES
Greene, J.C. , M. Long and C.L. Bartels. 1985. Results of algal
assays performed on waters collected from the lower Clark Fork
River system at stations below Mi 11town Dam to below Noxon Dam.
Report I: December 10-14, 1984 sampling. Report to the State of
Montana DHES, Water Quality Bureau. USEPA, Corvallis
Environmental Research Laboratory, DR.
Miller, W.E., J.C. Greene and T. Shiroyama. 1978. Selenastrum
capricornutum Printz Algal Assay Bottle Tests Experimental Design,
Application and Data Interpretation Protocol. U.S. Environmental
Protection Agency, Corvallis, Oregon. EPA-600/9-78-018.
Miller, W.E., T.E. Maloney and J.C. Greene. 1974. Algal
productivity in 49 lake waters as determined by algal assays.
Water Res. 8s667—679.
Miller, W.E., S.A. Peterson, J.C. Greene and C.A. Callahan. 1985.
Comparative toxicology of laboratory organisms for assessing
hazardous waste sites. J. Environ. Dual. 14(4)t569-574.
Montana Department of Health and Environmental Sciences. Water
Quality Bureau. 1984. Lower Clark Fork River Monitoring Plan 1984
- 1985. Helena, MT.
US Environmental Protection Agency. 1979. Methods for Chemical
Analysis of Water and Wastes. Environmental Monitoring and
Support Laboratory, Cincinnati, OH. EPA-600/4-79-020.
US Environmental Protection Agency. 1983. Inductively coupled
plasma-atomic emission spectrometric method for trace element
analysis of water and waste — Method 200.7. Environmental
Monitoring and Support Laboratory, Cincinnati, OH 45268.

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