United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
EPA/600/3-86/008
March 1986
Research and Development
£EPA
A Toxicity
Evaluation of Lower
Fox River Water and
Sediments
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EPA/600/3-86/003
March 1986
A Toxicity Evaluation of Lower Fox River Water and Sediments
By
Gregory J. Lien, Kenneth E. Biesinger, Leroy E. Anderson,
Edward N. Leonard, and Michael A. Gibbons
ERL-DUL-1043
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, MN 55804
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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 endorsement or recommendation for use.
11
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Abstract
Many persistent, xenobiotic compounds have been identified from Lower
Fox River water, biota, sediment, and effluent discharges; some of which are
suspected of causing adverse effects to aquatic organisms.
Water and sediment were collected as grab samples from the Lower Fox
River in late January, in mid-March, and in late April, 1985. Samples were
transported to the Environmental Research Laboratory-Duluth (ERL-D) and a
determination of their potential toxicity was accomplished through laboratory
bioassays using four freshwater invertebrates and one freshwater vertebrate.
Results from the present toxicity evaluation of Lower Fox River water
and sediment indicate a general absence of lethal effects as defined by the
bioassays used and within the framework of the study. Significant sublethal
effects were recorded in the form of reduced growth or fewer progeny,
however, the effects were not observed for more than one species or testing
period and no pattern was evident from this analysis.
111
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Contents
Page
Abstract iii
List of Figures vi
List of Tables vii
Acknowledgments viii
I Introduction 1
II Materials and Methods 2
Site Selection
Sampling Protocol
Chemical Analysis
Bioassay Methods
Statistical Analysis
Quality Assurance
III Results 15
Physical/Chemical Conditions
Biological Effects
IV Summary 24
V Discussion 25
VI References 26
v
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Number
Figures
Map of Study Area and Locations of Sampling
Stations for Toxicity Evaluation of Lower Fox
River Water and Sediments.
VI
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Tables
Number Page
1 Summary of Test Conditions for Bioassay Experiments. 8
2 Physical and Chemical Data for Water Collected from 16
the Lower Fox River at Various Locations and Dates.
3 Mean Percent Survival of Daphnia magna Exposed to Lower 18
Fox River Water for 48 hrs.
4 Survival and Growth of Pimephales promelas Exposed to 19
Lower Fox River Water for Seven Days.
5 Survival and Reproduction of Ceriodaphnia dubia Exposed 20
to Lower Fox River Water.
6 Survival and Reproduction of Daphnia magna in Elutriate 21
Tests.
7 Mean Percent Survival of Daphnia magna, Hyalella azteca 22
and Ephemerella sp. in Solid Phase Tests.
8 Reproduction of Daphnia magna in Solid Phase Tests. 23
VI1
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Acknowledgements
The authors wish to acknowledge the assistance of Floyd Boettcher in
collection of field samples, the drafting of figures by Barbara Halligan, the
identification of Ephemerella by Douglas Jensen, and the review of earlier
drafts by Kenneth Hokanson.
Vlll
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INTRODUCTION
The Lower Fox River in northeastern Wisconsin is approximately 64 km
(40 miles) long and extends from the outlet of Lake Winnebago to Green Bay.
This river is a major tributary to Green Bay, draining an area of 11,752 km^
(~ 7% of the total drainage area of Lake Michigan). The fall of the Lower
Fox River from its headwaters at Lake Winnebago to its mouth at Green Bay
averages 1.3 meters per mile-*- and its flow is controlled by 11 dams, with the
last dam occurring 11.7 km upstream from the mouth. The river is navigable
through a series of locks.
The Lower Fox River is one of the most industrialized in the country re-
ceiving input from 14 pulp and/or paper mills, an electric generating
facility and 8 municipal wastewater treatment plants serving approximately
250,000 people. Lake Winnebago, a hypereuthropic lake, is a major contri-
butor of nutrients entering the river.
Until recently the Lower Fox River was one of the 10 most polluted
rivers in the United States. Concerted efforts by the Fox Valley Water
Quality Planning Agency and the Wisconsin Department of Natural Resources
(WDNR) to clean up the river have reduced the organic wastes by 90%; the
final cleanup to meet the standards set by Section 208 of the Clean Water Act
is underway. Persistent, xenobiotic compounds continue to be a problem,
however. A total of 105 organic compounds have been identified from biota,
water, sediment, and effluent discharges^. Many of these chemicals are known
to be toxic and/or bioaccumulate and are suspected of causing adverse effects
to aquatic organisms. Toxics (via suspended solids, biota, and water) from
the Lower Fox River enter Green Bay at Green Bay, Wisconsin. The zone of
impact on the water quality of Green Bay extends as far at 15.5 km into the
bay 3.
1
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A previous study was conducted by personnel of the ERL-D to measure the
total toxicity of industrial and municipal wastewater treatment effluents and
their receiving waters from the Lower Fox River^. They could not attribute
the toxicity they found to any one effluent. The present study was made to
determine if there was instream toxicity and if bottom sediments from the
Lower Fox River were toxic. Water and sediment samples were collected from
several stations (Fig. 1) in the Lower Fox River and transported to ERL-D for
testing.
Acute tests were performed using Daphnia magna on river aliquots and
elutriate water. A 10 day I), magna chronic test was conducted on elutriate
waters. A seven day Ceriodaphnia dubia life cycle test and a seven day fat-
head minnow (Pimephales promelas) subchronic test were conducted on river
aliquots. Ten day tests for solid phase sediment toxicity were conducted
using I), magna, Hyalella azteca and Ephemerella sp.
MATERIALS AND METHODS
Site Selection
Sampling sites on the Lower Fox River were selected in an effort to ob-
tain an indication of instream toxicity, avoiding effluent plumes and immed-
iate mixing zones as much as possible. Consideration was also given to the
accessibility of the possible sites under both winter and spring conditions
and the availability of sediment in the immediate area of the water sampling
site. A station in Lake Winnebago (M) or a station in the river system up-
stream of any known point source discharges (L) were selected for "reference
stations" for these experiments.
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mi
5 10
I. RKM39.9
J. RKM 45.4 .
K. RKM 52.8
A. Mouth.
C- RKM 2.6.
D. RKM 4.a
F. RKM 10.5.
G. RKM II.I
L. RKM 63.2
8. RKM 1.0
E. RKM 7.6
H. RKM 28.3
.M. Lake W
Figure 1. Map of Study Area and Locations of Sampling Stations for Toxi-
city Evaluation of Lower Fox River Water and Sediments (RKM =
river kilometers).
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Sampling Protocol
Water and sediment for bioassays and chemical analyses were collected as
grab samples from the Lower Fox River, Wisconsin, and from Lake Winnebago in
late January, in mid-March, and again in late April, 1985. Samples were
obtained through holes drilled in the ice for the January sampling period.
Holes were hand drilled to avoid possible contamination from power auger
equipment.
All glassware used for collection, transportation and storage of water
samples was washed with detergent, rinsed with tap water, distilled water,
acetone, and hexane and then drained before being rinsed twice with one
gallon aliquots of river water from the respective stations. Water samples
were obtained from ~ 2 feet below the surface using a 1 gallon glass "jug-on-
a-stick" and were then transferred immediately to a 19 liter glass bottle.
All bottles were filled to exclude air and the covers were lined with alumi-
num foil. All containers were labeled at the time of collection.
A sample of water from each station was poured into a pre-cleaned 250 ml
polyethylene bottle which contained 0.5 ml of 2N zinc acetate for sulfide
analysis. Three liter water samples from each site were poured into
calibrated, solvent rinsed glass bottles each of which contained 100 ml of
(1:1) hexane/methylene chloride mixture. These samples were stored for
possible future organic analysis.
Within 36 hours after collection all samples were transported to the
laboratory and placed in a constant-temperature room which was maintained at
4° C. During transport from the field collection site to the laboratory, all
samples were maintained at temperatures above freezing but below final test
temperature.
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Sediment samples were obtained from 11 locations (A-G, I-L) in the Lower
Fox River and 1 station in Lake Winnebago (M) as grab samples using a stan-
dard size Ekman dredge. The nature of the bottom (i.e., hard, rocky) and
accessibility precluded sampling for sediment at some of the sites. The
depth of the river varied from 0.9 to 7.6 m. at the sites the sediments were
obtained. Bottom material from each site was transferred from the dredge
into pre-cleaned one gallon wide-mouth glass containers with foil lined
covers. Sediment samples were then transported and stored in the same manner
as the water samples.
Upon return to the laboratory the following sample splits were made: A)
250 ml subsamples of test water were poured into clean polyethylene bottles
and acidified to 0.2% with HNO-j in order to preserve the samples for metal
analysis; B) 250 ml subsamples of test water were poured into clean polyethy-
lene bottles and kept in the dark at 4° C for analysis of nitrate, nitrite,
phosphate, chloride and sulfate. Upon completion of the anion analysis, the
remainder of the sample was frozen and stored for future ammonia analysis; C)
250 ml subsamples of test water were poured into clean polyethylene bottles
for pH, alkalinity, hardness and conductivity measurements.
Chemical Analysis
The pH was measured at 20° C according to EPA Method 150.15 using a
Beckman model <5 70 pH meter standardized with pH 7.0 and 10.0 buffers before
use. The conductivity was measured at 20° C according to EPA Method
120.1-*, using a YSI model 31 conductivity meter. Water hardness was
measured according to EPA Method 130.2^. One hundred ml aliquots of
samples were titrated with 0.01 M EDTA to a blue endpoint at pH 10 using
Eriochrome Black T as an indicator. Alkalinity was measured according to
EPA Method 310.1^. One hundred ml aliquots of samples were titrated with
5
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0.02 N sulfuric acid to pH 4.4 endpoint using a Beckman model 5 70 pH meter
which was standardized with pH 7.0 and 10.0 buffers before use. Dissolved
oxygen was measured using a Beckman 0260 Oxygen Analyzer according to the
manufacturer's instructions", Method 6.1.4.2, which specifies air-saturated
water to calibrate the instrument.
Sulfides were analyzed according to EPA Method 376.1^. Iodine solution
(0.025N) was added to 200 ml aliquots of water samples preserved with zinc
acetate which were then titrated to a blue endpoint with 0.025 N PAO using
starch as an indicator.
— — — 3 — — 9
Anions (CL , N0£ , PO^ , NO 3 , SO^ ) were measured according to EPA
Method 300.0^ employing a model 12 Dionex (R) ion chromatography system with
a Gilson (R) automatic sample changer. Five ml aliquots were placed in 12
X 75 mm plastic test tubes, capped with aluminum foil and placed in the auto-
matic sample changer. All standard conditions were as specified in the EPA
method with the conductivity detector set at the 300 umho range, pump volume
at 2.5 ml/min. and sample loop volume at 1000 microliters.
Ammonia was measured on the ion chromatography system also; the manu-
facturers recommendations" for column types, eluent, and regenerant were
followed. Operating conditions for ammonia analysis were as follows: Columns
- 4 x 50 mm cation precolumn (Dionex P/N 030830), 4 X 250 mm cation separator
column (Dionex P/N 030831), cation fiber suppressor (Dionex P/N 036179);
detector - conductivity at 30 pmho scale; eluent - 0.003 N HCL; regenerant -
0.040 M TMAOH (Tetramethylammoniumhydroxide pentahydrate) at 3.1 ml/min.;
sample loop - 500 microliters; pump volume - 2.5 ml/min.
At least one in ten samples were analyzed in duplicate for both anion
and ammonia (cation) analysis as a check on the precision of the procedures.
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Environmental Monitoring and Support Laboratory (EMSL) quality assurance (QA)
samples were analyzed in each analytical run for all ions, except nitrite, as
a check for the accuracy of the methods. There was no known QA sample avail-
able for nitrite at the time of analysis. A separate nitrite standard was
analyzed in each analytical run as a check for any oxidation of nitrite to
nitrate in the mixed standards. No oxidation was observed in this standard.
At least one river sample for each analytical run was spiked with an anion or
ammonia standard to insure that no matrix effects were present.
Bioassay Methods
A determination of the potential toxicity of water and sediments from
the Lower Fox River was accomplished using four freshwater invertebrates and
one freshwater vertebrate in the following laboratory bioassays: a Daphnia
magna acute toxicity test; a Daphnia magna life cycle toxicity test; a sub-
chronic fathead minnow (Pimephales promelas) toxicity test; a life cycle
toxicity test using Ceriodaphnia; and a Hyalella and Ephemerella 10 day test.
The species tested, stage of development, medium, type of test, measured
response, duration, renewal, test solution volume, number of animals per
replicate, number of replicates per treatment, food added, and the tempera-
ture for these tests are given in Table 1.
All animals used in the bioassays were obtained from ERL-D culture stock
with the exception of the Ephemerella which were collected from the Sucker
River in northeastern Minnesota. Test organisms were cultured at the same
temperature as the test temperature with the exception of the Ephemerella,
which were acclimated over a period of 7 days from an initial temperature of
4° C to the eventual test temperature of 20° C. The photoperiod and the
feeding regimes were identical for both the culturing and the testing period.
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Acute tests were chosen as an initial screening process. In addition,
sub-chronic, partial life cycle, and life cycle tests were conducted to
determine possible effects on survival, growth, or reproduction.
Test temperatures were maintained by partially submerging the chambers
containing the water and/or sediment and the test organisms into thermostati-
cally controlled water baths. A photoperiod of 16 hours was maintained for
both the culture and exposure of test organisms. Water quality was routinely
monitored to ensure it's adequacy for the test organisms. Test chambers for
the ]). magna acute tests, Ceriodaphnia life cycle tests and the elutriate
bioassays were covered with glass to minimize evaporation.
All containers and other equipment coming in contact with test water
and/or sediment used in these bioassays were constructed of glass or stain-
less steel. The cleaning protocol for all containers and equipment used in
the laboratory for these tests included a detergent wash followed by three
tap water rinses, three distilled water rinses, an acid (10% HNO^) rinse
followed by another three distilled water rinses, an acetone rinse, and
finally by three distilled water rinses.
Individual bioassay procedures are described below for laboratory bio-
assays conducted with river water, liquid phase elutriate and solid phase.
A) Lower Fox River Water
Tests were conducted using Lower Fox River and Lake Winnebago water that
had been stored at 4° C for not more than 7 days. Prior to being utilized
for the bioassays described, a portion of the test water was rapidly warmed
each day to test temperature and the water was then aerated briefly to stabi-
lize dissolved gas concentrations.
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1) Daphnia magna Acute Test
An evaluation of the acute toxicity of Lower Fox River water was con-
ducted using the protocol established by EPA". Culturing, handling and
glassware cleaning procedures as outlined by EPA were followed with the
exception of a dilution water rinse, due to the small amount of test water
transported to and stored at the laboratory. The test solution was not
renewed over the 48 hour period.
At the end of 24 and 48 hours the number of living daphnids were counted
and recorded.
2) Fathead Minnow (Pimephales promelas) Sub-chronic Test.
The method described by Norberg and Mount 1" for measuring growth and sur-
vival of newly-hatched fathead minnow larvae was used for evaluating the sub-
chronic effects of toxicants. Test chambers were modified to provide sepa-
rate chambers for each of the replicates. All chambers were placed randomly
in a temperature controlled bath. The test chambers measured 6 cm x 18 cm x
9 cm high and were filled to a volume of 0.5 liters. A stainless steel
screen separated the chambers to form a 6 cm x 2.5 cm x 9 cm high sump on one
end. This sump was useful for removing "old" test solution during the re-
newal process.
During the daily renewal process the animals were first counted and any
dead fish were removed and recorded. Then waste products and dead brine
shrimp nauplii were removed using a siphon tube similar to the one described
by Norberg and Mountl^. After the waste material and most of the "old" test
solution was removed to a depth of 1 cm, the animals were again counted to
assure that none had been accidentally removed by the siphoning before adding
0.5 liters of "new" test solution. Dissolved oxygen concentrations were
measured periodically during the test, both on the "old" and the "new" test
solutions.
10
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The larval fish were fed 3 times per day at 5 hour intervals during the
simulated daylight period with 0.05-0.1 ml of concentrated live brine shrimp
nauplii (incubated 24 hours @ 28° C) that had been rinsed with distilled
water.
At test termination, surviving fish were removed, counted, recorded, and
dried to a constant weight (20-22 hrs @ 60° C). Final net dry weights for a
composite of the surviving fish from each replicate chamber for each treat-
ment were obtained utilizing an analytical balance (with an accuracy of .01
mg). Initial dry weights were obtained at the start of the test on four
groups of ten fish « 24 hrs old) by the same method.
3) Ceriodaphnia dubia Life Cycle Test
A life cycle test using Ceriodaphnia dubia and the method developed by
Mount and Norberg^ was conducted using Lower Fox River and Lake Winnebago
waters. One animal < 10 hours old was placed into each of the 30 ml glass
beakers filled with 15 ml of test water. This test water was warmed and
aerated in the manner described above and dispensed using a 30 cc Manostat
pipette.
The animals were fed 0.1 ml of YCTF (a suspension of yeast, a Cerophyl®
extract, and a commercially formulated trout food) each day of the test.
The test solutions were renewed on day 3 and day 5 by filling cleaned,
rinsed and labeled beakers with 15 ml of the "new" solution plus the food and
then transferring the adult Ceriodaphnia from the "old" to the "new" solution
with a pipette.
Survival of the original animals and the number of young produced were
recorded on renewal days as well as on day seven. The number of young produced
11
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in each brood was enumerated; differentiating broods by the relative size
of the offspring. The measured responses were the number of original animals
surviving beyond the third brood and the number of offspring produced in the
first three broods.
B) Liquid Phase Elutriate
The techniques developed for evaluating toxicity of dredge samples^ were
employed in an effort to obtain an index of chemical toxicity for contami-
nants solubilized from the sediments into the water column. These tests may
closely simulate the potential hazards encountered by aquatic organisms at
the sediment-water interface. Daphnia magna was used as the test organism
because it is very sensitive to industrial effluents and wastewater treatment
discharges in the liquid phase^.
The method employed in preparation of the liquid phase elutriate test
solution consisted of: homogenizing the sediment grab sample by mechanical
stirring; proportioning sediment to water at a 1:4 ratio (by volume); mecha-
nical agitation of the sediment water combination for 1/2 hr; settling of the
larger suspended solids for a minimum of 16 hrs; and finally, removal of a
majority of all suspended solids by centrifuging for 45 min. to 1 hr at 2600
rpm. Reconstituted hard water^ was used for the dilution water for the test
conducted during the January 1985 period; river water from the respective
stations was used for dilution water for the March and April testing periods.
The leachate from this process was then decanted from centrifuge bottles and
placed into test chambers; the solids were discarded.
1) Daphnia magna Acute Test
The Daphnia magna acute and chronic tests for the liquid phase elutriate
experiments were conducted simultaneously in the same chamber (an acute test
12
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was not conducted during the April testing period). At the start, 10 (5 pr 6
day old) animals were placed into each beaker. No food was added to any of
the containers during the acute phase of this test. Survivors were counted
and recorded after 24 and 48 hours.
2) Daphnia magna Chronic Test
In conjunction with the above described acute test and after 48 hours had
elapsed, the animals in each container were reduced to 5. Food consisting of
1 ing (oven dry weight) commercial trout food in a suspension and 2 x 10'
cells Selanastrum was added to each beaker every 2-3 days after the second
day. The chronic test ran for a total of 10 days after which time the number
of surviving original test organisms and the number of offspring produced
were counted and recorded.
C) Solid Phase
To allow the test organisms more direct access to all phases of chemicals
in a system (solubilized, bound to suspended solids, and those incorporated
in the sediments) a solid phase bioassay adapted from the method described by
Nebeker et al. -* was utilized.
Wet sediment was stored at 4° C for not more than 10 days before the
solid phase bioassays began. From a homogenized sample of sediment from each
station, 200 ml of sediment was subsampled and placed into each of 2 or 3
replicate 2 liter battery jars. Eight hundred ml of water from the respec-
tive station was then gently poured into each battery jar, bringing the total
volume to 1,000 ml. These systems were left undisturbed overnight to allow
the particulate matter to settle and to allow for exchange between the water
and sediments. Before test organisms were introduced, aeration was provided
for 1/2 hour through a glass Pasteur pipette with the tip submerged 2-3 cm
below the surface of the water.
13
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The test started when the animals were introduced and continued for a
total of 10 days. Ten 5 or 6 day old Daphnia; 5 adult Hyalella; and for the
March testing period, 5 larval Ephemerella were placed in each of 2 or 3
replicate chambers for each treatment. Stainless steel mesh bent into a U
shape and measuring 6.5 cm x 6.5 cm was provided as a substrate for the
Ephemerella. The test chambers were left uncovered and were aerated through-
out the test. Deionized, distilled water was used to replace losses due to
evaporation. Food was added to the systems every 2 to 3 days in the form of
5 rag (oven dry weight) commercial trout food in a suspension and 10^
Selanastrum cells per chamber.
The test was terminated after 10 days by first counting and recording the
number of surviving test organisms contained in the overlying water and then
by screening the sediments (using a sieve with 500 micron openings) to re-
cover the remainder. The number of original test animals surviving to test
termination and the total number of daphnids produced in each chamber were
recorded.
Statistical Analysis
All biological effects data were analyzed using a one-way analysis of
variance and a two sided Dunnett's test (p = 0.05).
Quality Assurance
Sampling sites for this investigation were selected using the criteria
outlined in the Description of Study Area section. The collection, identi-
fication, transportation, and storage of environmental samples were under
the direct supervision of the first author. The first and second authors
directly supervised all biological measurements and procedures and assigned
14
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chemical samples to co-authors for analysis. Statistical analysis was con-
ducted by the first author.
RESULTS
Physical and chemical measurements of the pH, alkalinity, conductivity,
hardness, chloride, nitrite, nitrate, phosphate, sulfate, ammonia (total as
NH, and unionized) and sulfide for all stations sampled on Lake Winnebago
and the Lower Fox River are given in Table 2. The hydrogen ion activity was
elevated in April compared to the sampling times in January or March by as
much as a ten-fold increase. Alkalinity and hardness values were relatively
constant over the sampling period. Conductivity measurements were consist-
ently higher at station A (mouth of the river) than at stations upstream.
Chloride concentrations were as great or greater at the mouth of the river
when compared to stations upstream. Nitrite, phosphate and sulfide were
below detection limits of the methods used. Nitrate concentrations were much
higher at all Lower Fox River stations in March than in either January or
April. For samples collected in April the sulfate concentrations and the
total ammonia as NH,+ were considerably higher at station A than for other
stations and times tested.
The values for the water quality parameters measured during the bio-
assays appeared adequate to meet the requirements of the test organisms.''^
The dissolved oxygen content of the water the fathead minnows (Pimephales
promelas) were exposed to was _>. 5.9 mg/1. Measurements of the dissolved
oxygen content of the elutriate water revealed values 2. 8.0 mg/1. The solid
phase test systems were continually aerated. The pH ranged from 7.6-9.3,
alkalinity from 134 to 172 mg/1 as CaC03, and hardness from 158 to 199 mg/1
as CaCOg for the Daphnia experiments.
15
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Table 2. Physical and Chemical Data for Water Collected From the Lower Fox River at Various Locations and Dates
Sampling Station^
Jan. 30-31, 1985
pH
alkal mg/1
cond ps/cm
hardness rag/1
Cl mg/1
N02 mg/1
N03 mg/1
P04 mg/1
S04 mg/1
NH * mg/1 (total)
NHj-N mg/12
sulfides mg/1
March 13-14, 1985
temp °C3
PH
alkal mg/1
cond ps/cm
hardness mg/1
Cl mg/1
N02 mg/1
N03 mg/1
P04 mg/1
S04 mg/1
NH,+ mg/1 (total)
NH3-N mg/12
sulfides mg/1
April 30, 1985
temp °C3
PH
alkal mg/1
cond ps/cm
hardness mg/1
Cl mg/1
N02 mg/1
N03 mg/1
P04 mg/1
S04 mg/1
NH,+ mg/1 (total)
NH3-N mg/12
sulfides mg/1
A
7.96
164.8
397
193.9
18
<0.2
0.9
<0.2
22.7
0.56
0.015
<1.0
3
7.63
133.6
440
164.9
20
<0.05
4.0
<0.15
22.5
1.07
0.014
<1.0
18
8.38
153.7
419
188.1
31.3
<0.05
1.1
<0.10
36.0
1.93
0.102
<1.0
B
7.97
163.8
361
188.0
10
<0.2
1.4
<0.2
19.7
0.24
0.007
<1.0
3
7.80
146.4
380
175.5
16
<0.05
4.0
<0.15
19.0
0.40
0.008
<1.0
17
8.90
141.8
300
159.1
13.4
<0.05
1.1
<0.10
16.0
0.26
0.048
<1.0
C
8.00
163.4
309
187.6
14
<0.2
1.4
<0.2
19.7
0.26
0.008
<1.0
3
7.87
145.7
348
178.0
16
<0.05
4.0
<0.15
19.0
0.44
0.010
<1.0
17
8.75
141.3
315
159.2
13.6
<0.05
1.1
<0.10
16.0
0.24
0.035
<1.0
D
8.05
163.6
318
189.4
14
<0.2
1.4
<0.2
20.2
0.23
0.008
<1.0
3
7.78
132.7
379
166.5
20
<0.05
4.4
<0.15
19.5
0.64
0.012
<1.0
17
8.65
144.8
315
162.7
16.4
<0.05
1.2
<0.10
18.0
0.33
0.039
<1.0
E F G
8.
168.
360
195.
13
<0.
1.
<0.
19.
0.
- 0.
- <1.
3
7.
158.
355
180.
14
- <0.
- 3.
<0.
18.
- 0.
- 0.
<1.
18 19 19
8.95 8.93 8.
138.3 140.4 140.
273 310 285
158.7 159.6 160.
11.4 11.4 11.
<0.05 <0.05 <0.
1.0 0.4 0.
<0.10 <0.10 <0.
15.0 16.0 15.
0.28 0.40 0.
0.057 0.078 0.
<1.0 <1.0 <1.
08
9
1
2
5
2
7
23
008
0
94
5
4
05
8
15
5
24
006
0
98
4
2
6
05
9
10
0
26
055
0
H
8.02
162.9
362
188.4
13
<0.2
1.4
<0.2
18.3
0.16
0.005
<1.0
_
7.94
160.9
412
189.6
13
<0.05
3.5
<0.15
18.5
0.18
0.005
<1.0
-
-
-
-
-
-
-
-
-
-
-
-
I
-
-
-
-
-
-
-
-
-
-
3
7.94
165.8
383
198.9
13
<0.05
3.4
<0.15
17.7
0.13
0.003
<1.0
18
9.01
140.8
300
158.4
11.4
<0.05
0.8
<0.10
15.0
0.30
0.066
<1.0
J
-
-
-
-
-
-
-
-
-
-
3
7.83
166.3
399
194.9
13
<0.05
4.0
<0.15
17.0
0.16
0.003
<1.0
18
9.3
140.4
282
159.6
11.4
<0.05
0.8
<0.10
15.0
0.33
0.115
<1.0
K L
7.99
166.6
328
188.4
13
<0.2
1.3
<0.2
14.8
0.05
0.002
<1.0
3
7.75 7.67
164.0 168.2
415 395
194.9 192.9
17 10
<0.05 <0.05
3.7 3.0
<0.15 <0.15
19.2 14.5
0.28 0.10
0.005 0.002
<1.0 <1.0
18 17
9.06 9.27
148.4 139.0
290 275
162.3 164.2
13.1 9.5
<0.05 <0.05
0.6 0.4
<0.10 <0.10
16.0 13.0
0.28 0.16
0.069 0.051
<1.0 <1.0
M
8.19
172.4
367
197.4
11
<0.2
1.3
<0.2
16.8
0.34
0.015
<1.0
_
7.61
155.3
378
187.4
10
<0.05
0.8
<0.15
13.5
0.05
0.001
<1.0
-
-
-
-
-
-
-
-
-
-
—
-
1 See Figure 1
Unionized ammonia calculated from total ammonia as NH4* using the table in Appendix A-l
from Thurston et al. 197916. , ,
3 At time of collection J-"
-------
There was no significant adverse effect on survival of Daphnia, fathead
minnow, or Ceriodaphnia exposed to Lower Fox River water over the entire
testing period and for the various lengths of exposures (Tables 3, 4 and 5).
There was, however, a significant adverse effect on growth of fathead minnows
exposed to water from station D collected in January (Table 4), and water
collected in March significantly reduced Ceriodaphnia reproduction for all
test stations except L.
The elutriate water was not toxic to Daphnia in the acute tests (Table 6).
Survival was >_ 93% in the 10 day elutriate tests with the exception of sta-
tion K in April when there was total mortality in 2 of 3 replicates. Elu-
triate water from station G in March and station K in April significantly re-
duced young production; however, Daphnia exposed to elutriate water from
several stations had greater young production than those in reference waters.
Survival of Daphnia in the reference units for the January and March
solid phase test was unsatisfactory (70% & 43%, respectively); however,
survival was >^ 80% for all other stations (Table 7). There was no signifi-
cant adverse effect on survival of Daphnia in the solid phase test in April.
Survival of Hyalella in the reference units of the solid phase test was _>. 80%
and there was no significant adverse effect on survival of Hyalella in the
treatments over the entire testing period. Survival of Ephemerella exposed
to the solid phase during the March testing period was less than satisfactory
(73%) in the reference units and, therefore, no significance can be attri-
buted to the response in the treatments. Reproduction of Daphnia was consis-
tently lower in the reference units of the solid phase tests than in the
treatments (Table 8).
17
-------
Table 3. Mean Percent Survival of Daphnia magna Exposed to Lower Fox River
Water For
Stations
A
B
C
D
E
F
G
H
I
J
K
L
M
48 hrs.
January
100
100
100
100
-
-
100
90
-
-
100
-
100+
March
100
100
100
100
-
-
100
100
100
100
100
100
100+
April
100
100
100
100
100
100
100
-
100
100
100
100+
_
+ Reference station
18
-------
Table 4. Survival and Growth of Pimephales promelas Exposed to Lower Fox
Stations
A
B
C
D
E
F
G
H
I
J
K
L
M
River Water For
Seven
Mean Percent
January
97
83
80
90
-
-
90
97
-
-
87
-
93+
Days.
Survival
March April
93
93
87
98
-
-
90
97
93
90
93
93
97+
90
97
100
87
100
97
97
-
87
93
97
93+
"
Specific
January
22.9
23.0
25.8
20.7*
-
-
23.2
22.9
-
-
23.4
-
26. 6+
Growth Rate
March
28.7
29.9
29.5
28.4
-
-
29.6
28.8
28.3
28.4
27.5
29.6
29. 2+
(%/Day)
April
29.7
30.7
30.6
29.7
30.2
30.5
30.2
-
28.9
30.2
30.4
29. 7+
™
* Significantly less (p = 0.05) than reference station
+ Reference station
19
-------
Table 5. Survival and Reproduction of Ceriodaphnia dubia Exposed to Lower
Fox River Water.
Stations Mean Percent Survival
A
B
C
D
E
F
G
H
I
J
K
L
M
Jan Mar
90 100
100 100
100 100
80 100
-
-
100 100
90 100
100
100
90 100
100
100+ 100+
Apr
100
100
100
80
100
100
70
-
100
90
80
90+
-
No. of young in 3 broods
per surviving female + SD
Jan Mar
25 _+ 5 22
24 +_ 4 21
21+7 20
24+4 21
-
-
21 _+ 9 21
23+5 22
23
23
22+9 22
28
22 + 6+ 28
_+ 2*
+_ 4*
± 3*
± 3*
-
-
± 3*
+ 2*
_+ 5*
± 3*
+ 2*
_+ 3
+ 3+
Apr
29 +
25 _+
25 _+
26 _+
25 +_
27 _+
27 _+
-
25 +_
27 +
24 _+
26 +_
—
3
2
3
2
3
3
3
4
2
4
2+
* Significantly less (p = 0.05) than reference station
+ Reference station
20
-------
Table 6. Survival and Reproduction of Daphnia magna in Elutriate Tests.
Survival in 48 hrs Survival in 10 days Young production/chamber
Stations (mean percent) (mean percent) (mean + S.D.)
Jan Mar Apr Jan Mar Apr Jan Mar Apr
119 +_ 13 43 +_ 6 93 _+ 6
94 ^ 12 34 _+ 5 78 _+ 8
95 _+ 14 29 _+ 10 75 JK 5
125 _+ 16 49 _+ 6 95 4- 18
72 ± 5
68 _+ 3
101 +_ 8 21 _+ 3* 81 +_ 8
29 +_ 3 55 JK 14
24 +_ 4 89 _+ 7
24 _+ 4 22 _+ 38*
35 jf 5+ 74 _+ 7+
73 _+ 23+
* Significantly less (p = 0.05) than reference station
+ Reference station
A
B
C
D
E
F
G
H
I
J
K
L
M
100 100
100 100
100 100
100 100
_
- - -
100 100
_
100
100
100
100+
100+
100 100
93 100
100 100
100 100
-
_
100 100
-
100
100
100
100+
100+
100
100
100
100
100
100
100
-
100
100
33*
100+
-
21
-------
Table 7. Mean Percent Survival of Daphnia magna, Hyalella azteca and
Stations
A
B
C
D
E
F
G
H
I
J
K
L
M
Ephemerella sp.
Daphnia
Jan Mar
93 97
97 83
100 100
97 100
-
-
97 87
-
100
100
90
43+
70+
in Solid
Apr
100
100
95
100
100
60
100
-
90
100
100
95+
_
Phase Tests.
Hyalella
Jan Mar
67 80
87 87
100 93
80 100
-
-
87 93
-
67
73
87
80+
90+
Apr
90
100
90
90
80
50
80
-
100
80
100
90+
_
Ephemerella
Mar
47
60
40
67
-
-
73
-
53
87
67
73+
_
+ Reference station
22
-------
Table 8. Reproduction of Daphnia magna in Solid Phase Tests.
Station
A
B
C
D
E
F
G
H
I
J
K
L
M
+ Reference station
Daphnia magna reproduction
(number of
January
335 _+ 49
313 _+ 30
326 + 30
315 +_ 5
-
-
317 + 21
-
-
-
_
young/chamber + S.D.)
March
371 +_
325 _+
312 _+
332 _+
-
-
280 +_
377 _+
226 +_
369 _+
93 +
91
73
25
40
80
35
63
53
60+
April
393 + 52
421 + 91
418 +_ 14
291 +_ 10
327 +_ 33
270 +_ 147
405 + 105
382 + 212
223 + 27
482 +_ 146
268 + 1+
197 + 50+
23
-------
SUMMARY
Physical and inorganic chemical measurements made on Lower Fox River
water show few abnormal values. Unionized ammonia (NI^-N) levels were higher
in April due to the influence of an increase in pH during that period on the
relative percentage of unionized to total ammonia. Nitrate concentrations
were elevated in March compared to the January or April study periods.
Conductivity, ammonia, chloride, and sulfate values all measured higher at
station A compared to the other stations. Dissolved oxygen measurements
obtained from the WDNR^' for the Lower Fox River range from 12.0 - 17.9 mg/1
for January, 12.7 - 18.0 mg/1 for February, 9.9 - 17.4 mg/1 for March, and 7.5
-17.9 mg/1 for April, 1985.
Results from the present study of Lower Fox River water indicate a
general absence of lethal effects as defined by the bioassays used and within
the temporal and spatial framework of the study. Significant sublethal
effects resulting from exposure to Lower Fox River water included reduced
growth of fathead minnows for station D in January and fewer Ceriodaphnia
progeny for stations A-K in March.
The liquid phase elutriate test showed few lethal effects with the
exception of total mortality of Daphnia magna in two of three replicates for
station K in April; the third had 100% survival. Production of young Daphnia
varied greatly in the elutriate test with two stations (G in March and K in
April) producing significantly fewer young than the reference station (L) and
three stations (A and D in January and D in March) producing significantly
more young than the reference station.
Survival and reproduction of Daphnia magna in the solid phase test was
often less for the reference stations than for the other stations. The solid
24
-------
phase was not toxic to Hyalella in the 10 day tests. Ephemerella were not
well suited for the static environment of the solid phase test.
DISCUSSION
Laboratory bioassays using Lower Fox River water and sediment failed to
reveal consistent lethal effects. A pathogen may have been the cause for the
total mortality of Daphnia observed in two of three replicates for Station K
in the elutriate tests in April. The observed presence of indigenous zoo-
plankton (Crustacea) in water collected for these experiments further subs-
tantiates the lack of mortality recorded in the bioassays. Consequently, an
analysis of contaminants in Lower Fox River water was not undertaken.
No pattern was evident for the sublethal effects observed and the ef-
fects were not observed for more than one species or testing period. The
cause-effeet relationship for the significantly fewer Ceriodaphnia pro-
duced in March is not known. In the elutriate tests, nutrients present in
the water and those released from the sediment may have been more of a
causative agent for the variability observed in young production than a toxic
influence.
Cairns et al.^° reported acute toxicity to Daphnia magna exposed to liquid
phase elutriate from two stations on the Lower Fox River (mouth and 0.8 km
upstream) collected in 1982. Their measurements of suspected toxicants
showed all were below the acutely toxic level. A second testing yielded no
significant mortality.
A previous investigation by personnel of ERL-D^ also indicated no toxi-
city to fathead minnows (Pimephales promelas) or Ceriodaphnia in ambient
tests conducted in 1983. The investigators did, however, record adverse
25
-------
effects with dilution or receiving water used in conjunction with effluent
toxicity testing. In a personal communication, D. McCauley^ reported a
lethal effect on fathead minnows (Pimephales promelas) under test conditions
similar to the ones described herein with water collected near the DePere Dam
(in the vicinity of Station G) in April, 1984. Toxicity events in the Lower
Fox River appear to be episodic in nature.
Several limitations of the present investigation's ability to accurately
assess potential instream toxicity are evident. The limitations of a grab
sample at particular points in time are inherent. The process(es) occurring
during prolonged storage and the resulting effects on the integrity of water
samples are not well defined. It is likely that aeration used to stabilize
dissolved gases for the bioassays may have altered the environmental samples
and their potential toxicity. Also, the influence of laboratory bioassay
temperature conditions on potential toxicity is open to discussion. In situ
bioassays and instream measurements could more accurately evaluate potential
instream toxicity in an environment such as the Lower Fox River.
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Wisconsin, 1970-1980: An Historical Perspective. IPC Technical Paper
Series, Number 105. Institute of Paper Chemistry, Appleton, Wisconsin.
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2. Wisconsin Department of Natural Resources. 1978. Investigation of
Chlorinated and Nonchlorinated Compounds in the Lower Fox River Water-
shed. EPA-905/3-78-004. U.S. Environmental Protection Agency, Chicago,
IL. 229 p.
26
-------
3. Sullivan, J.R. and J.J. Delfino. 1982. A Select Inventory of Chemicals
Used in Wisconsin's Lower Fox River Basin. University of Wisconsin Sea
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4. Anon. 1985. The Toxicity of Some Industrial Effluents and Their Effects
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00
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