Determination
of Point and Nonpoint Source Toxicity in the
Clark Fork River Basin Using the Daphnid, Ceriodaphnia Dubia
DelWayne
R. Nimmo
Water Resources Division
National Park Service
Affiliated with Colorado State University
Ft. Collins, CO 80523
Joseph
C. Greene
Environmental
Research Laboratory
Corvallis/ORD
Corvallis, OR 97333
Loys
P. Parrish, Tom Willingham and Glenn J. Rodriguez
U.S.
Environmental Protection Agency
999 18th Street
Denver, CO 80202
Mark
A. Kerr
Montana Department of Health
and Environmental Sciences
Water Quality Bureau
Helena, MT 59620
Glenn
R. Phillips
Department
of Fish, Wildlife and Parks
Capitol Station
Helena, MT 59601
Abstract—Ceriodaphnia
dubia, a small planktonic daphnid was used to biomonitor point sources of
toxicity in wastewater and nonpoint source toxicity in stream samples obtained
from the Clark Fork River Basin, MT. Brief descriptions, results and
discussions are presented for studies of wastewater from a kraft mill near
Frenchtown, MT and potential toxicity of water samples from 19 sites along the
Clark Fork River in 1985. In 1987, dilutions of Missoula, MT municipal
wastewater fortified with ammonia were tested, as was the wastewater before
and after chlorination. Potential toxicity of water samples from eight sites
along the upper Clark Fork River were also tested. All studies were
cooperative efforts with the Montana Departments of Health and Environmental
Sciences and Fish, Wildlife and Parks.
Ceriodaphnia appear to be indicators of toxicity in a
variety of test conditions such as ammonia in wastewater and metals from past
mining activities. The daphnids indicated toxicity from other substances in
the wastewater or perhaps the influence of characteristics of the wastewater
that increased ammonia toxicity. All example of nonpoint source effects was
toxicity in samples from Silver Bow Creek MT, where impaired conditions to
aquatic life resulting from the presence of metals have been reported for
years. During some of the tests with wastewater, toxicological endpoints were
observed using the actual number of daphnids that reproduced in a test, not
the average number of young. There was circumstantial evidence in 1985 that
copper alone was responsible for the toxicity in Silver Bow Creek. However,
the later studies performed under different hydrological conditions found
toxicity was probably due to a combination of metals, some of which had not
been measurable earlier. For well-defined "control" of
"standard" conditions during testing, there are indications that
waters to be used as reference media for Ceriodaphnia need further research.
Nevertheless, the use of daphnids to test the ambient conditions described in
this paper should encourage environmental managers to consider approaches with
this or similar species in the future.
INTRODUCTION
In 1985 and 1987, a planktonic daphnid,
Ceriodaphnia dubia, was used by the Environmental Protection Agency (EPA)
Region VIII Denver, CO, to determine toxic conditions in various Montana
waters. All of the testing was conducted in the Clark Fork River Basin, which
has documented water quality problems resulting from historic mining
activities and pollution by municipal wastewaters and industries such as pulp
processing (Anonymous, 1986). The approach used the daphnid as an
environmental “sensor” or biomonitor in much the same way as a pH meter
"senses” the hydrogen ion concentration, except that the organisms
integrate the toxicity of all constituents and their interactions within the
matrix of the chemistry of the sample.
Results from some of these studies have been considered in
management decisions regarding permit limits, treatment technologies etc., but
all results were used for the development of, or the validation of testing
methods using Ceriodaphnia in field situations. The demonstration of the
procedures to various state and federal agency personnel, training of agency
personnel, technical assistance to state and Local officials as well as
obtaining experience in waters of differing characteristics.
The purposes of this paper are (1) to demonstrate how
daphnids were used in biomonitoring, (2) to note some important observations
and discoveries made during the testing, and (3) to include various (baseline)
toxicological and chemical parameters measured during the testing so
comparison may be made in successive studies in the Clark Fork River Basin.
MATERIALS AND METHODS
Location and Test Condition
Mobile laboratories were located at Frenchtown. MT on property owned by a
kraft liner board mill (currently Stone Container). May through June 1985 and
at the Missoula Wastewater Treatment Plant. May through June 1987. Locations
of ambient sampling stations along the Clark Fork River and mobile laboratory
sites are shown in Figure 1.
Two
studies were conducted in 1985 (Table
1):
1.
A chronic 7 -day static renewal test of kraft mill wastewater,
Frenchtown, MT; and
2.
A chronic 7 -day static renewal test of 19 ambient samples obtained
(daily) along the Clark Fork River from Butte to Noxon Dam, MT. Two studies
were conducted in 1987 (Table 1):
3.
48-hour acute and chronic 7 -day static renewal test of Missoula, MT,
wastewater fortified with ammonia singly, and chlorine singly; the latter as
part of standard treatment measures at the wastewater treatment facility; and
4.
A chronic 7-day static renewal test of eight ambient samples obtained on four
separate days from the upper Clark Fork River, including Silver Bow Creek
(Butte to Missoula, MT).
Sample Collection. Preservation and Analysis
For the studies of wastewater from both the kraft mill at Frenchtown (1985)
and the city of Missoula (1987) grab samples were diluted with Clark Fork
River water. It also served as a reference water. The kraft mill samples were
unaltered except for preparation in a dilution series of 4.0. 2.0 1.5. 1.12.
0.64. 0.36. 0.2 and 0% wastewater (0% being 100% river water).
For the Missoula wastewater, two series of test dilutions
were prepared, both fortified with ammonia using the methods and procedures of
Nimmo et al., (1989). One series of dilutions was wastewater plus ammonia
diluted with river water for a range of 100, 75, 56, 32, 18, 10 and 0% (the
latter being river water without ammonia). The other dilutions were river
water and river water plus ammonia in a similar series; 100,75,56,32, 18, 10
and 0% (the latter also river water without ammonia). The reason for this
design was to conduct the tests within the matrices of wastewater dilutions
versus Clark Fork River water to provide information on the potential effects
of other constituents in wastewater compared to the effects of ammonia. A
comparison of results from the two series of dilutions showed them to be a
fair simulation of conditions in the mixing zone of the Clark Fork River below
the wastewater treatment plant. Because of the volatility of ammonia, the
dilutions prepared were immediately dispensed into test containers and results
of the testing are reported as milligrams-per-liter (mg/l;ppm) un-ionized
(NH3). For the pre- and post-chlorinated tests, grab samples of wastewater
were obtained from the treatment plant immediately before chlorination and at
the discharge point. Both were diluted with Clark Fork River water in a series
3;8 above then dispensed into the test containers.
For ambient tests, grab samples of Clark
Fork River water were collected for seven consecutive days, May 10-16, 1985
and in 1987, collected four times; May 25,27,29, and June I. In 1985, samples
were collected from 19 sites; in 1987, eight sites. Upon arrival at the mobile
laboratories each sample was mixed with dilution water and split into two
samples, with one used without alteration for the daphnid test, the other used
for chemical analysis and analysis of water characteristics.
In 1985, a portion of the unaltered samples were composited,
with 1125 mls of test water added daily to an autoclavable polypropylene
shipping bottle. The bottles were maintained at ambient river temperature (I
0-12OC) by floating in a covered tank in which river water was pumped
continuously. On the last (7th) day of sampling, the bottles were shipped via
ground transportation to the U.S. EPA Laboratory, Corvallis, OR. Elemental
chemical analysis was performed on filtered samples using an inductively
coupled plasma atomic emission spectrophotometer (ICAP) using the methods of
U.S. EPA (1979, 1982). In 1987, uncomposited and unaltered samples destined
for analysis of metals were maintained on ice at 4oC and transported to
Missoula, then repacked and shipped on ice to the U.S. EPA Laboratory,
Corvallis, OR, where they were filtered and analyzed as above. Because Silver
Bow Creek water was acutely toxic to the daphnids, samples were diluted with
local reference water believed to be free from the influence of metals, and
tested separately using a series of dilutions as described above with the
waste water tests. The various reference waters used in these tests are
discussed in the results section.
For 7-day chronic tests, methods of
Mount and Norberg (1984) and U.S. EPA (1985a) were used and involved exposure
of the daphnids through a life cycle. Endpoints considered were lethality,
number of young produced per female, and in the case of wastewater from
Missoula where some unusual findings were discovered, the number of adults
that produced young-regardless of how many young were produced. Under
controlled conditions where no toxicity is involved, the parthenogenetic
females begin producing young on the fourth day and within three more days two
additional broods are produced, for a total of 20 to 40 neonates per female.
Chronic tests were begun by placing one
neonate, less than 24 hours old, in each of ten 30-ml beakers filled with test
sample, for a total of 10 test organisms per dilution. Every day for seven
consecutive days, organisms were transferred into renewed test solutions. A
diet of commercially prepared dried cereal leaves, trout chow and bakers yeast
supplemented with algae (Selenastrum capricornutuml was fed daily to each
neonate. Test temperature was 25o+1oC and conditions of the animals
(living or dead) number of young per female or any unusual behavior were
recorded daily.
Lethal concentrations (LC50s), or
effective concentrations (EC50s) and their corresponding 95% confidence
intervals for acute tests were computed by the trimmed Spearman Karber method
(Hamilton et ai, 1977). For the ambient tests of stream water, significance of
survival was analyzed by Student's "t" test Data from reproduction
(number of young per female) were first tested for homogeneity of variance,
then analyzed by the Analysis of Variance (ANOV A) procedure. In the case of
unequal numbers of replicates (survivors in the seven-day tests), a
"t" test with Bonferroni's Adjustment was used to identify
differences in reproduction of test animals among sampling sites compared to
those from a control site. When there was an equal number of replicates,
Dunneu's procedure was performed. If the data were not nom1ally distributed, a
nonparametric test such as the Wilcoxon's Rank Sum Test with Bonferonni
Adjustment was used. (Florence Kessler, U.S. EP A, personal communication).
RESULTS AND DISCUSSION
Kraft
Mill Wastewater Study. Frenchtown. MT. 1985
Due to public concern about water quality in the middle and lower Clark Fork
River, in particular the release of treated wastewater from the kraft mill, a
chronic toxicity test was conducted. Results of this effort were considered
along with other relevant data for modification of a discharge permit
originally issued by the state of Montana, April 1984. Ceriodaphnia were
tested for seven days on dilutions of wastewater ranging from 4% wastewater to
control (Clark Fork River water) as previously discussed. To provide a range
of dilutions bracketing discharge volumes, the 4% dilution was chosen as a
doubling of the 2% dilution, the latter being a permit limit for the facility
based on low now conditions in the receiving stream.
Daphnids produced significantly fewer young in the 4%
dilution (4% waste- water, 96% river water) but there was no significant
effect of wastewater on survival of daphnids (Table
2) at a dilution of 2%.
Analysis of priority pollutants were conducted on two different occasions on
both the wastewater and river water, but they did not reveal any chemical
constituents that appeared responsible f or chronic toxicity at 4% (Nimmo et
al., 1985).
Missoula. MT Wastewater Study. 1987
For a number of years there were concerns about operational problems at the
Missoula Wastewater Treatment Plant, particularly about dairy wastes and the
potential for providing treatment of sewage from outlying areas. Data
collected by the Montana Water Quality Bureau in 1984 and 1985 indicated the
possibility of ammonia toxicity in the mixing zone of the Clark Fork River,
especially during low flows. There was also concern about chlorine, which was
seasonally added as a disinfectant beginning in June and ending in September
of each year. The limitations on chlorine and ammonia were part of a permit
renewal in September of 1987 after substantial expansion of the plant had been
completed May. 1987.
Results of the 48-hour Ceriodaphnia test in
ammonia-fortified dilutions of wastewater mixed with Clark Fork River water
and ammonia-fortified Clark Fork River water are shown in Table
3.
Significantly more daphnids died in dilutions of wastewater than in river
water (LC50 1.93 mg/l ammonia in wastewater vs. an LC50 > 1.95 mg/l in
river water). The latter value refers to insufficient lethality at a measured
1.95 mg/l in river water to calculate a lethal concentration; and. that
increased toxicity other than can be attributed to ammonia is occurring in the
wastewater. Another possibility is that the characteristics of wastewater
mixed with the river water compared to river water alone, enhance the toxicity
of ammonia.
Compared to the acute tests above, results
of chronic tests in Missoula’s ammonia-fortified wastewater and
ammonia-fortified Clark Fork River water were not significantly different,
with a seven-day LC50 in wastewater of 1.68 mg/1 ammonia and in river water,
2.36mg/J (Table 4). However, survival and reproduction (average number of
daphnids produced per female) of daphnids in wastewater was significantly
different at 1.33-mg/l ammonia compared to 1.74 mg/1 in river water. It was
also noted that the number of daphnids that did not reproduce in seven days in
the river water was directly correlated with the concentration of ammonia;
therefore, using this endpoint, the effective concentration or EC50 was 0.44
mg/l. A similar endpoint was not observed in the tests in wastewater.
Results of the pre- and
post-chlorination study where ammonia was not added, showed the LC50 for
unchlorinated wastewater was approximately 68% wastewater; whereas the EC50
based on reproductive success was 79% wastewater (Table
5). Results of the
post -chlorinated test showed the LC50 and EC50 to be about the same, both
greater than 75% wastewater (Table 6). In these tests the number of young
produced per female daphnid was between 32 and 35 in reference waters or
significantly greater than in reference waters from previous studies at the
kraft mill (Table 2) and in Missoula wastewater
(Table 4).
Ambient Testing of the Clark Fork River, 1985
The
purpose of this study was to determine and document nonpoint sources of
toxicity from metals or other constituents in the Clark Fork River. Results of
toxicity testing and chemical constituents are shown in Table 7 and suggest
the following:
1. Using survival of daphnids as a criterion, all stations on Silver Bow Creek were acutely toxic.
2.
Analysis of Variance (ANOV A) of the reproduction data (average number
of young produced per female in water from each site) showed all stations were
significantly less than the Taylor Creek control except the station at Huson.
However, based on the 95% confidence interval around the mean, daphnids
reproduced as well in water from the confluence of tile Little Blackfoot and
Clark Fork Rivers, as in Taylor Creek water used as a reference.
3.
When Silver Bow Creek samples were diluted with Taylor Creek water and tested
for seven days the toxicity of Silver Bow Creek water decreased downstream (Table
8). For instance, the effect/no effect dilution at the Colorado
Tailings was between 18% and 10% ambient water; at Ram say, it was 32% and
18%; finally, just above Warm Springs Ponds, 75% and 56% Silver Bow Creek
water (Fig. 1).
4.
An association appears between toxicity of the dilutions discussed in (3)
above and copper concentrations. At the effect/no effect dilutions, estimated
concentrations of copper measured in 100% Silver Bow water ranged from
geometric means of 7.2 to 11.2 ug/l, or a fairly restricted range. By
contrast, the geometric mean of zinc ranged from 65.3 ug/l at Colorado
tailings to 48.9 ug/l at Ram say; but was only 10.2 ug/l just above Warm
Springs Ponds (Fig. 1). It is possible that the interaction of the copper and
zinc or other constituents of the water influenced toxicity.
Ambient Testing of the Clark Fork River. 1987
Some observations from the 1985 ambient testing of the upper Clark Fork River prompted a repeat study in 1987. In 1985, only Silver Bow Creek samples were acutely toxic to the daphnids and their reproduction in samples from the remaining stations was less than in the Taylor Creek control water (Table 4) except at Huson and perhaps in water from the Little Blackfoot River. Surprisingly, lethality was not found in water from some upper stations of the Clark Fork River such as the confluence below Warm Springs Creek, Deer Lodge and stations below the Little Blackfoot River confluence (Fig.1). It was hypothesized that due to unusually dry conditions of the season, metals in the watershed were not mobilized. No precipitation occurred immediately prior to or during the period of study.