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6201 CONGDON BOULEVARD
DULUTH, MN 55804
ANNUAL REPORT
1973
ASS0CIATE LABORATORY OF
NERC-C orval1i s. OR
#*aSTA\
# n \
ISB/
Office of Research £~ Monitoring

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CONTENTS
Forward
Research Objective Achievement Plans (ROAP's)
16AAD Effects of Heavy Metals on Aquatic Life
16AAK Effects of Hazardous Synthetic Organics in
the Freshwater Environment
16ABI Temperature and Dissolved Oxygen Requirements
21AKG Effects of Nitrogen (Air) Supersaturation on
Columbia River Fishes
25AIC Effect of Oil(s) on Freshwater Fish, Other
Freshwater Life, and Wildlife
16AAJ Short Cut Methods to Determine Toxicant Criteria
16AAI Development of Biological Test Methods
16AA0 Complex Effluent Criteria
16AAF Evaluation of Laboratory Chronic Bioassay
Methodology by Continuous Chronic Exposure of a
Natural Ecosystem
05AAD Tolerance Levels of Ammonia for Aquatic Life
21AKH Tolerance Limits of Hydrogen Sulfide for Aquatic
Life
16AAE Bioassay Technical Support
1BA021 Technical Assistance Plan
Publications by Staff Members for 1973
Page
ii
11
23
33
34
36
47
55
55
55
56
57
59
68

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FOREWORD
This is the first, annual report prepared by the National
Water Quality Laboratory. We have tried to provide an overview
of the activities and highlights of the findings of the
various studies so that those in the scientific community can learn
of our present work and contact staff working on projects of
especial interest. All data should be treated as tentative and
subject to minor change. We decided that with this provision we
could include much more very recently completed work.
Three events in 1973 made this a significant year to us. In
June we announced that part of the mineral we had been using to
trace tailings in Lake Superior (and in Duluth drinking water)
was asbestos and we alerted EPA that there might be serious health
concerns. At the time of this writing, the trial is nearly complete
and the U. S. District Court has said that in its judgment there is
primafacie evidence of a "significant health hazard". Phil Cook,
Gary Glass, and Jim Tucker deserve special mention for thi.se findings.
The second event was the official opening of our Monticello
Field Station located at Monticello, Minnesota. At this facility
we have eight 1,700 foot long channels in which self-sustaining
fish populations will be maintained. Heat can be added to provide
an initial At of up to 20° C. In this facility we will be able to
simulate, on a rather large experimental scale, the effects of a
thermal plume in a river and determine its ecological consequences.
We also plan use of toxic materials in future research after the
initial work with heat. We feel that only after validating in such
ways can models of biologic response be considered established.
Finally, the new permanent laboratory building for our Western
Fish Toxicology Field Station in Corvallis, Oregon, was ready for
occupancy. For the first time in about six years, the staff has a
permanent home! Work there is concentrated on problems important in
the Pacific Northwest, especially those related to the salmon fishery.
Both for the dogged persistence in seeing the building to completion
and for the development of the only adult salmon bioassay facility,
the staff of Western Fish Toxicology Station deserves special mention.
We would welcome your suggestions on this report, and we
encourage you to inquire if you desire further information on ai*y
project. We also hope that you will let us know if you do not receive
our quarterly newsletter, or if you wish to receive reprints of
published work.
Donald I. Mount, Ph.D.
Director

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ANNUAL REPORT
1973
National Water Quality Laboratory
Duluth, Minnesota 55804
In 1971 the Office of Research and Development of the Environmental
Protection Agency initiated a procedure for research planning. This
procedure uses an Environmental Research Objective Statement (EROS)
prepared by staff members at Headquarters to relate what research
should be performed and the available resources to perform that research.
Upon receipt of the EROS by the laboratory, Research Objective
Achievement Plans (ROAP) are prepared utilizing the resources as defined
in the EROS to plan research programs which will satisfy the objectives
as outlined in the EROS.
This report will be divided into the research, both intramural
and extramural, performed by the staff to meet the stated objectives
of these ROAP's.
ROAP 16AAD - Effects of Heavy Metals Upon Freshwater Aquatic Life
I. An approach to the trace metal analysis of environmental samples
utilizing flameless atomic absorption spectroscopy with the graphite
tube furnace was outlined in a preliminary NWQL report (December
1973). The method has several advantages, but because it is a
relatively new and unproven technique, there can be certain associated
dangers and difficulties with respect to the number it produces.
The report focuses on some possible problems, but more important it
attempts to outline a practical systematic approach which is of aid
to NWQL analysts involved in developing methods. The proposed
approach consists of three general steps namely, (1) necessary
measures to obtain reproducible atomization; (2) varying operating
conditions for the purpose of optimization and diagnosing possible
problems; and (3) establishing validity, showing agreement of
analyses by the flameless technique to those obtained by another
established method and analyzing appropriate standard samples. In
the report, each of these considerations is expanded to the extent
of suggesting specific steps which are oriented toward Perkin Elmer
instrumentation. Such considerations as proper charring temperature,
atomization temperature, program times, preparation of standard
sample alteration techniques and use of background correction are
covered. At NWQL the method has been employed to effectively measure
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cadmium, lead, copper, managanese, and iron In various samples,
which include Lake Superior interstitial waters and overlying
waters, bioassay systems and sewage effluent. Comparisons of results
to those obtained by flame atomic absorption, anodic stripping
voltammetry and/or standard colorimetric methods have been quite
favorable.
A minor amount of work has also been done with o.ther metals
such as chromium, cobalt, nickel, arsenic, selenium, molybdenum,
and silver. (John g. Poldoski and James T. Fiandt).
A method has been developed for preparing mercury film electrodes
for use in trace metal analysis by anodic stripping voltammetry.
This helped to fulfill NWQL needs for ultrasensitive analytical
methods in monitoring cadmium, lead, and copper. Briefly, the
preparation scheme consists of plating mercury onto an appropriately
prepared surface of wax impregnated graphite rod. The basic
electrode construction is accomplished by machining the graphite
rod to fit into a tubular teflon electrode body. The overall
electrode dimensions are roughly 6mm in diameter by 90 mm long.
In normal use the electrode requires periodic reconditioning,
which is easily and reproducibly accomplished in about 15-20
minutes. Typical reproducibility of peak currents resulting from
several scans of a single solution containing 10 yg/1 each of cadmium,
lead, and copper is about 1-2 percent. The sensitivity is characterized
by the fact that a five minute plating time is sufficient to
determine cadmium, lead, and copper in acidified Lake Superior water,
which typically measures 0*02 yg/1, 0.1 yg/1, and 0.5 yg/l,
respectively. Measurements made on other sample types such as
bioassay waters, well waters, fish tissue, and sewage effluent
agree well with the corresponding measurements made by atomic absorption.
The bioassay water was monitored routinely over a period of several
months. The main advantages compared to other available film
electrodes appears to be the ease and reliability of electrode
reconditioning, the ability to analyze in highly acidic media (pH =1),
and the high sensitivity required to analyze metal ions in the
sub-parts yg/1 range. (John E. Poldoski and Edward N. Leonard).
II. The determination of the relative susceptibility of
anadromous salmonids to metals is being studied. Swim-up stage
(ca. 1 month-old) chinook salmon (Oncorhynchus tshawytscha) were
determined to be considerable more susceptible to the lethal effects
of cadmium, copper, and zinc than were the newly hatched fry. Data
obtained to date are summarized in the following table:
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96-hr. LC50 values
At Hatch
Swim-up
4 mo.-old
Chinook Salmon Cd > 25 yg/1	1.9 yg/1
(0. tschawytscha) Cu	31 yg/1	18 yg/1
Zn > 700 yg/1	103 yg/1
Steelhead Trout Cd
(Salmo gairdneri) Cu
Zn
0.95 yg/1
20 yg/1
100 yg/1
A 21-month exposure of sockeye salmon to zinc was terminated.
Adult sockeye salmon (0. nerka) were held in zinc for 3 months
prior to artificial spawning; the subsequent egg-to-smolt zinc
exposure continued for an additional 18 months. No adverse effects
were observed at the highest concentrations tested (120 ppb
adults through smolts and 240 ppb eggs through smolts). Smolts
from all zinc concentrations appeared normal as evidenced by
survival at 30 ppt seawater for 96 hours. (A zinc concentration
of 240 yg/1 is about 1/4 of the 96-hour LC50 for sockeye
salmon, but. is much higher than the 100 yg-/l 96-hour LC50 for the
more susceptible chinook salmon and the'steelhead trout.)
Because results reported by the Western Fish Toxicology Station
indicate very low levels of cadmium, copper, and zinc to be lethal
to salmonids, quarterly water samples were collected and analyzed
from most major salmonid streams in Western Oregon. The purpose of
this program was three-fold: 1) to determine if levels of these
metals occurred which were near or above those concentrations
reported as lethal (i.e. Cd, 1 yg/1; Cu, 20 yg/1; Zn, 100 yg/1).
Existence of such levels would either indicate an area of problem
pollution or possibly an unexplained research finding; 2)
to check the general acceptability of existing STORET water
quality data for these streams; and 3) to collect heavy
metal data for these streams using more sensitive analytical methods
than those available in the past.
The results of the survey are summarized briefly in the
following table:
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Summary of_6 water quality parameters from Western Oregon streams
Alkalinity (nig/1 CaCO^)
No. Occurrences
Hardness (mb/1 CaCO^)
No. Occurrences
pH	6.5-7.0
No. Occurrences	47
Cadmium (ppb)
No. Occurrences
Copper (ppb)
No. Occurrences
Zinc (ppb)
No. Occurrences
10-30
31-60
61-100
91
20
3
10-30
31-60
61-100
85
23
3
7.01-7.5
7.51-8.0
8.01-8.5
58
12
3
<0.01
0.01-0.1
0.11-0.22
81
18
7
<0.1
0.1-1.0
1.1-5.3
65
15
28
<0.1
0.1-1.0
1.1-11
35
35
40
On the basis of these data it was concluded that: 1) barring
exceptional circumstances, lethal or near—lethal levels of cadmium,
copper, or zinc probably do not occur in Western Oregon streams;
2) existing STORET water quality data are generally adequate, although
data are included which in most cases should be regarded as aberrant
due to sample contamination or analytical or mathematical errors;
and 3) a typical Western Oregon stream has alkalinity and hardness
below 30 mg/1 as CaC03, a near neutral pH, no detectable cadmium
or copper, and less than 10 yg/1 of zinc.
A grant to the Oregon State Game Commission was awarded in October
1973 for the study of the Effects of Copper and Zinc on the Seawater
Adaptation of Juvenile Coho Salmon. The results of this research
will be a major first step in determining if this critical life
history phase is susceptible to adverse sub-lethal effects of toxic
chemicals. In addition, the study will provide specific data on the
effects of copper and zinc, as well as develop and test an array
of criteria for testing toxic effects from the level of the whole
organism to sub-cellular levels. (Gary A. Chapman).
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III. The effects of cadmium and lead on brook trout (Salvelinus
fontinalis) was studied. The purpose was to determine the maximum
acceptable toxicant concentration (MATC) for brook trout exposed
to cadmium and lead in Lake Superior water. Survival, growth,
and reproduction were used to measure adverse toxic effects upon
the fish. The tests were also designed to evaluate the use of
cadmium and lead application factors for brook trout. Both tests
involve the exposure of first generation trout (juvenile through
adults) and the offspring or second generation trout (egg through
maturity). Nominal water concentrations ranged from 6.0 to 0.4
yg Cd/liter and 500 to 31 yg Pb/liter. Analysis of each test
showed that approximately 80% of the total cadmium and 70% of the
total lead was available as dissolved cadmium and lead (Cd, Pb
passing through a 0.10 y filter). Anodic stripping voltammetry
(ASV) was used as a comparison with atomic absorption on some
samples (10); the methods were found in good agreement.
Several attempts to calculate a 96-hour TL50 for brook trout
and cadmium failed because the fish always died at approximately
the same rate in all concentrations tested (400 to 12 yg Cd/liter)
and this produced an essentially flat mortality-concentration curve.
Two months after the first generation exposure of brook trout
to cadmium was initiated all trout at 6 yg Cd/liter began to
experience periods of severe hypertensive activity in which the fish
would frantically swim around the tank with totally uncontrollable
movements. All fish at this concentration died prior to spawning.
During the spawning period all males and several females at 3 yg
Cd/liter also became very hyperactive and died. Males seemed most
sensitive to cadmium during courting activity, especially when
the females began digging in the gravel substrates. Adult survival,
spawning, egg liatchability, and 90-day second generation alevin
survival and growth were unaffected at 1.5 yg Cd/liter and below.
The second generation exposure was continued beyond the 90-day
period in order to study second generation effects on adult survival
and reproduction. Results of this exposure are not yet available.
The 96-hour TL50 for brook trout to lead was 4.5 mg Pb/liter.
During the first generation exposure of brook trout to lead,
most trout at 500 yg Pb/liter became somewhat hypertensive and
some were subject to periodic involuntary muscle spasms. Viable
eggs spawned, egg hatchability and 90-day survival of second generation
offspring were also affected at 500 yg Pb/liter. Significant
numbers of second generation alevins with deformed spines (scoliosis)
were noted at hatch in 250 and 500 yg Pb/liter. Three to four
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months after hatch the caudal peduncle of normal looking alevlns
at 250 and 500 yg Pb/litor began to turn black and each fish
eventually suffered from severe scoliosis. Six months after hatch,
black tails and spinal deformities also began occurring to a lesser
extent in second generation trout exposed to 125 yg Pb/liter. Hie
second generation exposure is currently being continued in order
to study the effects on reproduction. Complete results of this
exposure are not yet available. (Duane A. Benoit and Edward N. Leonard)
IV.	The acute and chronic toxicity of methylmercurie chloride
to brook trout (Salvelinus fontinalls) was determined. Nominal water
concentrations were 3.0, 1.0, 0.3, 0.1, 0.03yg/liter mercury and
controls. All measured concentrations were slightly less than
nominal concentrations. Water analyses indicated that 90-95% of
the mercury in the test water was organic mercury.
Two 96-hour acute toxicity tests were run—one on yearling brook
trout (200 grains each) and one on five-month old juvenile brook
trout (12.0 grams each). The calculated TL50's were 65.0 yg/1 for
the 200 gram trout, and 84 yg/1 for the 12 gram trout.
A brook trout partial life-cycle chronic with methylmercuric
chloride was completed in March 1972. The offspring of these trout
were carried on, under exposure, to investigate second generation
effects. The second generation study will terminate in March 19 74.
In the first generation study (partial chronic), all trout exposed
to 3.0 yg/1 of mercury died prior to spawning. Spawning was
good at all other mercury concentrations except 1.0 yg/1
where egg mortality, fry survival and growth were affected. Second
generation data on spawning is not yet available. These preliminary
first generation exposure data suggest that 1.0 yg/1 mercury is
definitely unsafe for brook trout. (James M. McKim)
V.	Acute and chronic tests to determine the effects of cadmium
on growth, reproduction, and survival of Jordanella floridae,
(flagfish) were completed
Fourteen-and 30-day acute tests were run. Median tolerance
limits (TL50) were similar for adult fish and one-day old fry. Values
ranged from 18.4 to 25.5 yg Cd/1.
Two chronic tests approximately 16-weeks in length were completed
to determine effects on growth and reproduction. The second test
was done at higher concentrations to obtain effects not apparent
in the first chronic test. All significant effects noted
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were calculated by using an analysis of variance and Dunnetts' Test,
(P = .05). Concentrations in Test 1 ranged from 6.6 yg/1 to 0.06
yg/1 with a .5 reduction between concentrations. There was no
effect at any level on growth or reproduction. Only the number
of spawnings was significantly different from control at 6 yg/1.
Cadmium concentrations in chronic Test 2 ranged from 31 yg/1
(high) to 0,11 yg/1 (control). At these concentrations there was
no significant effect on growth in initial 30-day tests and 30-day
second generation tests at concentrations less than 3.1 yg/1. At
31 yg/1 there were no eggs produced and thus no second generation.
Growth of adult fish was significantly (P = .05) decreased at 16
and 31 yg/1. All males at these levels died and females showed
a marked decrease in length and weight. Both males and females
also became highly convulsive for short periods of time at these
concentrations.
Reproduction studies were based on spawning results, eggs
produced, and hatchability. There were significant effects on all
aspects of spawning and egg production at concentrations of 8.1 yg/1
and above. There was a thirty percent reduction in the total number
of eggs produced at 8.1 yg/1. The two higher levels, however, severely
affected growth and caused mortalities leading to significant
decreases in spawning or no spawning at all. Fish in the two
high concentrations showed more convulsive activity when they began
spawning indicating an additional stress especially in the males.
There were no detrimental effects on hatchability at any
cadmium concentration. There was, however, an increase in hatchability
at higher concentrations. The highest hatchability was found when
control eggs were transferred to the high concentration for incubation.
Percentages ranged from 65 percent in the control to 83.1 percent
for eggs transferred to the high concentration (31.1 yg/1). Since
a concentration of 8 yg/1 affected reproductive activity in chronic
test 2 an effect at 6 yg/1 in chronic Test 1 would indicate the
lowest possible effect level of cadmium to this species. However,
only the number of spawnings were affected, not egg production in
general. (Robert L. Spehar)
VI. A method was developed to measure total mercury in water
at concentrations below 0.2 yg/1. During the past 3 years total
mercury has been routinely measured in experimental waters containing
less than 0.2 yg of Hg/liter, which is the lowest limit for many
analytical laboratories. To achieve detection limits of 0.012-0.020
yg/liter.r, chemicals are used from those manufacturers which yield
the lowest amount of mercury contamination. A sample of 150 ml is
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analyzed in a 250 ml flat bottom boiling flask, which has been
cleaned with hot nitric acid. To the sample is added 2 ml
concentrated HNO3, 2 ml concentrated IlpSOit, 1 ml 6% KHnO[f, and
2 ml 5% K2S2O85 This mixture is heated just to boiling, cooled to
room temperature and then reduced to the. elemental mercury with 1 ml
sodium chloride-hydroxylamine sulfate solution and 5 ml stannous
sulfate. By aerating the solution using a Neptune Dyna-Pump, the
mercury vapors are released into a closed circulating system for
measurement of the concentration in the sample. The vapor passes
through a magnesium perchlorate drying tube, into an absorption
cell of 2.0 cm O.D. X 18 cm. which is aligned in the light beam
of a Perkin-Elmer 403 Atomic Absorption Spectrophotometer and continues
to circulate until a maximum absorbance value is reached. The
absorbance values are expanded 10 times by the concentration mode
on the instrument. A minimal of 1/16 inch I.D. tygon tubing is
used for all the connections in the apparatus.
Using this procedure to analyze water samples from a fathead
minnow bioasnay with methyl mercuric chloride, the following table
summarizes the analyses for 37 weeks.
Nominal Concentration
as yg He/1 in Test Water
0.015
0.030
0.060
0.120
0.240
Measured Concentration
	,	 yg Hg/1	
Mean	95% Confidence Interval
0.018	.015 - .021
0.036	.031 - .041
0.063	.055 - .071
0.114	.102 - .126
0.247	.225 - .269
Mercury analyses at these concentrations in natural waters
are important b ecause there is significant uptake by aquatic
organism, especially fish. The total body residues of the fish
from this exposure ranged from 1.5 yg of Hg/gram in the 0.018 yg/liter
concentration to 10.9 yg/gram in the fish from the 0.247 yg/liter.
The control fish from water less than 0.01 yg/liter contained only
0.2 yg/gram total body residues. (Gayle F. Olson)
VII. A chronic bioassay with fathead minnows exposed to
mercuric chloride has been running for about 8 months. The nominal
concentrations are 12.0, 6.0,.3.0, 1.5, 0.75, and 0 yg
Hg/liter, All fish were dead in the 12.0 yg/1 concentration after
8 days. Only 4 stunted and deformed fish survived at 6.0 yg/liter.
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The incidence of deformities appears to increase with concentration
among the adult fish. Thirty and sixty day growth and survival
effects have not been completed on the hatched fry. After
termination of exposure, residue analyses will be run on the adult
fish for total and methyl mercury (G'ayle F. Olson)
Two chronic tests with Jordanella and methyl mercury were
completed. In the first test, concentrations tested x^ere .6,
1.25, 2.5, 6.0, 15.0 yg/1 Hg and controls. At the completion of the
test all fish were dead at concentrations of 2.5 yg/1 and above.
There was 70% mortality at 1.25 yg/1 and 20% mortality at .6 yg/1.
There was no spawning at 2.5 yg/1 and a nearly 50% reduction of
spawnings at the low concentration, of .6 yg/1.
In the second test concentrations tested were 1.5, .75, .375,
.187 yg/1 Hg, and controls. At 16 weeks there was a 70% mortality
at 1.5 yg/1 and 25% mortality at .75 yg/1. Spawning was inhibited
at a concentration of .375 yg/1 and above and no spawning occurred
at 1.5 yg/1. In a 30-day growth study with fry at these
concentrations, all levels of Hg, including the low concentration
of .187 ppb, inhibited growth. Adult fish at 16 weeks from the
same concentrations were found to be of normal length and weight
at the three low concentrations tested.
Jordanella exposed to Hg and containing Hg in their tissues
were placed~in clean water in a continuous flow system. The fish
were sampled monthly for tissue analysis. At six months there was
no appreciable loss of Hg from tissues.
A mercury uptake study designed to show the relative significance
of Hg uptake by fish from food as compared to that from water using
bluegills was completed. The Hg test water contained .25 yg/1 Hg.
The food source was clam tissue containing 2.62 mg/kg Hg. Ten fish
were maintained for 30 days under each of four test conditions, the
fish tissues were then analyzed for Hg. In clean water and receiving
clean food the fish tissue contained 0.32 mg/kg Hg. In clean water
and receiving Hg food the fish contained 2.9 mg/kg Hg. In Hg water
with clean food the fish contained 1.6 mg/kg Hg. In Hg water with
Hg food the fish contained 4.4 mg/kg Hg. A concentration of .187 ppb
would permit maintenance of normal populations of Jordanella and
.375 ppb would have a definite inhibitory effect. (Wesley E. Smith)
VIII. Bionomics, Inc., Warehara, Massachusetts, was awarded a
contract to determine the maximum acceptable toxicant concentrations
of lead (Pb(M03)2)» cadmium (CdCL?), chromium (Na2CrOy), and copper
(CuS0,() on the egg or fry of nine species of fish exposed for 60
days post-hatch.
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Exposures of lake and rainbow trout to load in soft water have
been completed. Based on 60-day fry survival, the estimated MA.TC
for rainbow trout eggs and fry in soft water lies between 61 and 131
Hg Pb/liter. Whole body lead residues were dose related and ranged
up to 57 nig/kg in the rainbow trout after 60 days exposure
to 2 7 yg Pb/liter and 56 mg/kg in the lake trout after 60 days
exposure to 22 yg Pb/liter.
Channel catfish exposures in both hard and soft water have also
been recently completed and all six tests indicated a safe and
unsafe level; however, the data have not been finalized and are not
yet available. (Duane A. Benoit)
IX. In toxicity tests with the "heavy" or transition metals
in various natural waters, gross differences in toxicity are observed
due to the formation of both soluble and insoluble hydroxide, carbonate,
phosphate, and other complexes. In addition, toxicity is moderated
by the physiological effects of calcium, magnesium, sodium and changes
in pll in such waters. Although variations in metal toxicity have
long been recognized as generally related to water quality, few
concerted efforts have been made to determine the chemical relationships
causing such differences. Attempts were made through acute toxicity
tests of copper to Daphnia magna to elucidate the chemical-toxicity
relationships that may be applicable to many of the heavy metals
in natural waters. The overall objectives were to determine the
chemical factors regulating toxicity and the presence of toxic
aqueous forms of the metal.
The effects investigated were primarily the effects of the
naturally occurring anions: hydroxide, carbonate, bicarbonate, chloride,
sulfate, and orthophosphate on ionic copper distributions and resultant
toxicity. Tests included varying concentrations of pyrophosphate
as a means of determining the mathematical relationship between
complex formation and toxicity. Comparative tests with varying
soditim, potassium, and pH were also conducted, since experimentally
the anions above are added as the appropriate sodium, potassium or
acidic form.
Results of analytical measurements and chemical equilibrium
calculations indicate that only a small percentage of the copper
added even to very soft water (Lake Superior total alkalinity ^ 45 mg/1)
remains as the cupric (Cu"1"1") form at moderate pll's. The proportion
of the cupric form present decreases markedly with increasing pH
and/or carbonate alkalinity, and decreases in proportion to the
concentration of added complexing agents and the stability constant
of the copper complexes formed.
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The principal result of the toxicity tests was the finding that
toxicity at a given pH is linearly related to the concentrations of
the soluble cationic copper forms (Cu^Jand CuOII+). Additional
toxicity tests in waters with widely varying carbonate alkalinities
have shown, that copper toxicity is inversely related to the total
carbonate (CO^ + HC03-) concentration, and the degree of copper
carbonate formation. All anions tested to date, including chloride,
sulfate, orthophosphate, pyrophosphate, carbonate, and bicarbonate
reduce cationic copper concentrations (and toxicity) in proportion
to the stability of the cupric complex formed. Cupric ion
concentrations may be calculated directly from published stability
constants and measurements of pH, carbonate alkalinity, etc., and
copper toxicity is predictable under conditions of fixed pH, sodium,
and calcium concentrations.
The second major finding of the toxicity tests was the
indication that toxicity of the cupric ion varied as a logarithmic
function of both pl-l and log sodium (pNa) concentrations (i.e., the
slope of toxicity curves varies as a combined function of pH and
pNa). Cupric ion toxicity increases approximately tenfold
between pH 6.0 and pH 8.0 at low sodium levels, and decreases
approximately tenfold with an increase in sodium concentration
from 10" M to 10~2 M at pH 8. The effects of pH and sodium are
interdependent however, with sodium having less effect at pll 6, and
pH having less effect at high sodium concentrations.
The relative toxicity of the cupric ion at various pH's and
in the presence of varying sodium appears to be entirely dependent
on physiological mechanisms and it is expected that a similar
interdependent effect of calcium will be observed. The question of
the relative toxicity of the cupric and copper hydoxy ions cannot
be answered at the present time, since concentrations of these
ions cannot be varied independently of pH. Since interchange of
these two ions at the site of toxic action involves only a single
proton,
CuOH+ + H- < Cu44" + H20
they are probably about equally toxic. (Robert W. Andrew)
ROAP 16AAK - Effects of Selected Hazardous Synthetic Organics
in the Freshwater Environment
I. A study was conducted to determine the relationship
between the toxicity of DDT to fathead minnows when present in water,
in food, and in water plus food. Fish were exposed to each of these
conditions for 266 days starting with 45-day old fry through a
reproductive period of their life. The contribution of DDT from each
source was monitored by tissue residue analysis through both gas
chromatographic and liquid scintillation techniques. Two DDT water
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concentrations (2.0 yg/1 and .5 yg/1) and one DDT concentration
for the food (50 yg/g) were used. The food consisted of clams,
half of them exposed to Cl4 labeled DDT at the same water
concentration as the high DDT fathead water exposure, and the
other half were used as a "clean" food containing no DDT.
Residue data agree closely with mortality data, the results
indicating higher tissue residues with slightly greater mortality
for all fish exposed to DDT in water plus contaiminated food when
compared to fish in corresponding DDT water exposures alone.
Mortality and residues among fish only fed DDT w re intermediate
between those of the groups fed and not fed DDT in the low water
concentration. Statistical analysis of the mortality data shows
that the presence of DDT in food caused a significant reduction in
the probability of survival. Two separate mortality periods
occurred, indicating increased susceptibility to DDT at both the
fry stage up to 80 days of age, and at spawning time when highly
colored males were most susceptible. The percentage of total
tissue residues attributable to the DDT food source remained
relatively constant after 28 days exposure at about 35% for fish
exposed to 2.0 yg/1 DDT water plus DDT food, and 60% for fish
exposed to 0.5 yg/1 DDT in water plus food.
There was no survival of fry at 2.0 yg/l DDT in the water both
with and without food containing DDT. All fry died within 5 days
of hatch. Groups of fry (40 each) spawned from adult fathead
minnows exposed to 2.0 yg/1 DDT in the water and 2.0 yg/1 DDT
in the water plus DDT in the food were transferred to control water
for 30 days. Fry from the adults exposed to DDT in both food and
water experienced about two times greater mortality than those
from adults exposed to DDT in the water only. These data agree
with egg residue results that indicate almost two times greater
residue levels (40.9 vs 24.0 yg/g) for eggs from adults exposed to
2.0 yg/1 DDT in the water plus DDT in the food as compared to those
of adults exposed to DDT in the water alone.
Elimination rates were determined for fish exposed to 0.5 yg/l
DDT in water and 0.5 pg/1 DDT in water plus DDT in food. Initial
DDT residue levels were 25.0 and 70.0 yg/g respectively. Essentially,
there was zero DDT elimination for the 0.5 yg/1 DDT water exposed
fish up to 56 days, whereas the fish exposed to 0.5 yg/1 DDT in
water plus DDT in food had a rapid elimination within the first
28 days followed by a slower elimination. At 56 days, greater than
50% of the total tissue residues were lost and the body burden was
equal to that of the non-DDT in food exposed fish. (Alfred W. Jarvinen)
12

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II. Diazinon is an organophosphorus insecticide first
introduced in 1952. Initial use was limited but has increased
greatly in recent years. It has been recommended as an alternative
to several organochlorine insecticides for application to
agricultural crops. Although it is considered a "non persistent"
insecticide, recent studies have indicated that it may have a half
life of several months in water under some conditions. Throughout
1972 and 19 73 a number of flow-through bioassays were conducted to
determine the effects of diazinon on fishes. Tests in the coming
year will begin to establish the relationship between these
constant concentration diazinon exposures and long-term exposure
to fluctuating concentrations.
Fathead minnows, Pimephales promelas Rafinesque, were exposed to
diazinon from hatch through spawning and the progeny for two months.
The most pronounced chronic effect on this species was the incidence
of spinal deformity (scoliosis and/or lordosis). Percentages of
surviving minnows crippled after four months of exposure at various
measured concentrations are as follows: 1099 yg/1 (87%), 511 yg/1
(83%)k,229Mg/l (89%)» 119 yg/1 (89%), 59 yg/1 (67%), 27 yg/1 (44%),
14 yg/1 (40%), 6.8 yg/1 (26%), 3.2 yg/1 (29%), Control (15%). When
disturbed many fish had tetanic convulsions in concentrations as
low as 14 yg/1. There was no reproduction among fathead minnows
exposed to concentrations of 14 yg/1 or higher. Survival of eggs
to hatch was reduced in the lowest concentration tested (3.2 yg/1).
From acute tests, a 96-hr LC50 of 7.0 mg/1 was estimated.
Brook trout, Salvelinus fontinalis (Mitchill) were exposed for
six months prior to and during spawning, and progeny for two months
as eggs and three months after hatch. After six months adult fish
exposed to 10 yg/1 and 5 yg/1 had an incidence of spinal deformity
(scoliosis and/or lordosis) of 33% and 12%, respectively. Mortality
was 25% at 10 yg/1 and 4% at both 5 yg/1 and 2.5 yg/1. During the
first three months fish in 10 yg/1 lost 4% in weight, those in 5 yg/1
gained 11% and the others in lower concentrations and the diazonon-free
control gained 40% to 50%. During the subsequent three months, fish
exposed to 10 yg/1 gained only 32% in weight compared to 47% to
58% for fish in lower concentrations. Data are not yet available
for the possible effects on reproduction. Acute tests provided an
estimated 96-hr LC50 of 0,6 mg/1.
The estimated 96-hr LC50 for Florida flagfish, Jordanella floridae
(Goode and Bean) is 1.5 mg/1. For bluegills, Lepomis macrochirus
Rafinesque the estimated 96—hr LC50 is 0.5 mg/1. (Donald T. Allison)
13

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III.	The toxic effects of captan on survival, growth, and
reproduction of fathead minnows (Pimephales promelas) and on the
survival of bluegills (Lepomis macrochirus) and brook trout
(Salvelinus fontinalls) were determined in a flow-through
system. In a 45-week exposure of fathead minnows, survival and
growth were adversely affected at 39.5 yg/1. Adverse effects on
spawning were suspected but not statistically demonstrated at 39.5
and 16.5 yg/1. The maximum acceptable toxicant concentration
(MATC), based on survival and growth, lies between 39.5 and 16.5
yg/1. The lethal threshold concentration (LTC) derived from acute
exposures was 64 yg/liter, resulting in an application factor
(MATC/LTC) between 0.26 and 0.62. LTC values for the bluegill and
brook trout were 72 and 29 yg/liter, respectively. Hie estimated
MATC is between 44.6 and 18.7 yg/liter for the bluegill and
between 18.0 and 7.5 yg/liter for the brook trout. The half-life
of captan in Lake Superior water with a pH of 7.6 is about 7 hours
at 12° C. and about 1 hour at 25c C. Breakdown products from an
initial 550 yg/liter of captan were not lethal to 3-month-old
fathead minnows.
Acute toxicity tests have been completed in a study of the
acute and chronic toxicity of endrin and malathion mixtures to
flagfish (Jordanella floridae). The data hasn't been completely
analyzed, but it appears that the acutely toxic effects of the
mixtures are additive. Tests are in progress to determine if the
chronic effects are additive. (Roger 0. Hermanutz)
IV.	A research contract with Union Carbide Corp., under Miss
Anne Spacie as principal investigator, is now nearing completion.
The following preliminary results have been obtained from chronic
and acute exposures of three fish and three invertebrate species to
parathion.
Adult bluegills were crippled (scoliosis) or developed tumors
in the throat area after several months exposure to 0.34 yg/1, and
accumulated 30 to 60 times this amount in their tissues. In a test
still in progress, fathead minnow growth was reduced at 3.2 yg/1,
and crippling occurred at 15 yg/1. Brook trout were less sensitive,
being unaffected after several months exposure to 470 yg/1, although
egg hatchability was reduced at 32 yg/1. Surprisingly, the trout
accumulated 200 times this amount in blood and 400 times this amount
in their muscle tissue. Ninety-six hr. TL50 values for the three
species were 0.51 mg/1 for bluegills, 2.5 mg/1 for fatheads, and 1.8
mg/1 for brook trout.
Only TL50 data are available for the Invertebrates, and
some of these are reported below:
14

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Daphnia	4 day
7 day
3 wk.
Gammarus	4 day
Midge	4 day
7 day
0.63	yg/1 parathion
0.4	Ug/1 parathion
0.08	yg/1 parathion
0.4	yg/1 parathion
31	yg/1 parathion
3.1	yg/1 parathion
(John G. Eaton)
V. The atrazine and lindane research contracts with Bionomics,
Inc. under the direction of Dr. Ken Macek are also nearing completion.
These contracts, like the one with parathion, involve exposure of
three fishes and three invertebrates in order to determine acute and
chronic toxicity.
Considering atrazine first, there was no observable adverse
effects of 0.25 mg/1 on fathead minnow adults' after several months
or on their progeny after 30 days. The acute TL50 concentration
was 0.83 mg/1. Bluegills were unaffected by chronic exposure to
0.25 mg/1 atrazine but suffered 15% mortality acutely at 0.36 mg/1.
Brook trout experienced reductions in growth of adults and fry at
concentrations of 0.25 mg/1 and above. The atrazine median tolerance
limit of brook trout was 4.9 mg/1, so application factors for
fatheads and bluegills would appear to be similar to one another
but different from the one for trout. Among the invertebrates, the
chronic safe concentration of atrazine for the midge was 0.12 mg/1
with adult emergence being decreased at 0,25 mg/1. Fecundity was
reduced in Daphnia at 0.3 m.g/1 and in Gammarus at 0.14 mg/1. Acute
TL50's were 6.9 mg/1 for Daphnia magna (96 hr), 5.7 mg/1 for
Gammarus (48 hr), and 0.72 mg/1 for midges (48 hr).
On the lindane contract, 25 yg/1 was found to be safe chronically
for bluegills, 49 yg/1 for fatheads, and 16 yg/1 for brook trout.
Acute TL50's for the three were found to be 30 yg/1, 54.5 yg/l, and
25.8 yg/1 indicating application factors are similar for all three
species. For the invertebrates when exposed to lindane, the lowest
chronic effect levels were 6.2 yg/1 for the midge, 25 yg/1 for
Daphnia magna, and 2.2 yg/1 for Gammarus. Forty-eight hour^TL50^
concentrations for the three invertebrates were 1,6 mg/1, 48 yg/1,
and 31 yg/1, respectively. (John G. Eaton)
VI. A contract with Envirogenics Co,, El Monte, California,
Rick D. Cardwell as principal Investigator, is also desinged to
investigate the acute and chronic toxicity of chlordane to fathead
minnows, bluegills, brook trout and three aquatic invertebrates
(Daphnia magna, Gammarus sp., and a midge) , Acute and
of the fish are almost completed and testing of the invertebrates
has just begun.
15

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Estimated 96-hr LC50's for fathead minnow, bluegill, and brook
trout are 40, 65, and 100 yg/1, respectively. Chronic mortality of
fathead minnows exposed from the larval stage was not greatly affected
by chlordane concentrations ranging from 6 yg/1 to zero until the
fish began to spawn. Subsequently, mortality was high in
concentrations of 1.4 yg/1 and above. Mortality of adult brook trout
during an exposure lasting six months prior to spawning was as
follows: 6.7 yg/1 (100%), 3.3 yg/1 (50%), 1.7 ug/1 (21%), 0.8 ug/1
(12%), 0.4 ug/1 (12%), control (4%). Control trout were transferred
to the 6.7 ug/1 concentration at the beginning of the spawning period.
To date, after two weeks of spawning activity in the lower
concentrations, no fish have spawned in chlordane levels of 6.7 or
3.3 yg/1. In the partial chronic using adult bluegills the only
excessive mortality prior to spawning occurred in the highest
concentration (6.7 yg/1) where 45% of the fish died. However,
during spawning bluegill mortality was as follows: 6.7 yg/1 (30%),
3.3 yg/1 (15%), 1.7 yg/1 (5%), 0.8 yg/1 (5%), 0.4 yg/1 (0%),
control (0%). (Donald T. Allison)
VII. Another contract with Bionomics, Inc. is designed to provide
information on the chronic and acute effects of treflan, heptachlor,
endosulfan, and acrolein on fathead minnows and Daphnia magna. The
fathead minnow chronics with treflan and endosulfan will be
terminated this month. The heptachlor and acrolein fathead chronics
are at the spawning stage. A 6-month time extension with no
additional funding was approved for this contract in order to
complete-the last 2 pesticide chronics and remaining Daphnia work.
Seven days after starting, 65-80% mortality and 20-35% loss of
equilibrium was observed in the fish at 40 yg/1 of treflan. All
fish chronically exposed to 20 yg/1 died within the first 49 days
and all of the fish at 10 yg/1 died within 174 days. These fish
exhibited hemorrhaging, scoliosis, or loss of equilibrium. Some
mortalities occurred at 5 yg/1 between 174 and 348 days exposure
indicating an accumulative toxic effect that eliminated only the
least resistant fish at that concentration. Daphnia magna acute
values (mg/1) for treflan are: 24-hour TL50 0.320 (range 0.211-
0.411); 48-hour TL50 0.193 (range 0.110-0.321). A treflan Daphnia
magna chronic test was completed with production of young being reduced
at 0.10, 0.05, and 0.025 mg/1.
The endosulfan fathead minnow acute incipient TL50 was 0.86 yg/1.
High mortality was observed at .60 yg/1 and all fish died within
236 days. Control eggs transferred to this concentration died prior
to or at hatch and transferred control fry died in one day. Daphnia
magna acute values (mg/1) for endosulfan are: 24-hour TL50 0.213
(range 0.153-0.299); 48-hour TL50 0.156 (range 0.049-0.494).
16

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A 10-day heptachlor acute TL50 for fathead minnows was 7.02 yg/1
(range 5.76-8.54). During the heptachlor chronic all fish died
at 4.0 yg/1 between test days 34-38.
Preliminary static bioassays with acrolein indicate an acute
toxicity of 0.05-0.10 mg/1 for fathead minnows. Mean growth increments
for 30 to 60 days chronic exposure were slightly greater in all
experimental groups than controls. Daphnia magna acute values (mg/1)
for acrolein are: 24-hour TL50 0.061 (range 0.021-0.260); 48-hour
TL50 0.057 (range 0.012-0.210). (Alfred W. Jarvinen)
VIII.	In a contract on	the chronic and acute toxicities to
baygon, baytex, propachlor,	and methomyl to fathead minnows and
Daphnia magna, with Kenneth	Macek, Bionomics, as princiapl investigator,
time independent TL50's and	confidence limits have been established
for propachlor at 0.52 mg/1 (0.43-0.63) and methomyl at 1.75 mg/1
(0.90-3.5). Chronic exposures will begin soon. (Roger 0. Hermanutz)
IX.	Yearling brook trout (Salvelinus fontinalis) were exposed
through a reproductive period to five concentrations (39-502 ng/1)
of the insecticide toxaphene for seven months on an Interagency
Agreement with the Fish Pesticide Research Laboratory, Columbia, MO.
All concentrations except the lowest reduced egg viability. The
maximum acceptable toxicant concentration (MATC) for brook trout
exposed to toxaphene in water lies between 39 and 68 yg/1 which
results in an application factor (MATC/96-hr LC50) of 0.0037-0.0065.
Analytical methodology has been developed to quantitatively
identify toxaphene in water at concentrations as low as 0.025 pg/1.
This sensitivity is achieved by extracting 20 liters of water with
polyurethane foam.
Toxaphene residue levels in the brook trout exposed to 502 ng/1
were 8 mg/kg, a concentration factor of 16,000. Elimination of one-
half of the total toxaphene residues in adult brook trout removed
from the high exposure occurred after about 50 days. The toxaphene
components in the tissues during uptake were only 2 to 18% different
from those in the standard. After elimination of half the residues,
however, a 2 to 44% greater change was observed in early eluting
peaks as compared to later ones. (Leonard H. Mueller)
X.	Since 1962, mirex has been used extensively as a control
agent for the imported fire ant (Solenopsis nevissiuma richteri Forel).
The insecticide is usually distributed as a bait consisting of
corncob grits, soybean oil, and mirex. The effect of mirex on marsh
and estuary species has been established, but little work has been
done with fresh-water animals until recently. During the last year
tests with both the bait and technical mirex have been conducted
using Daphnia magna and aquatic insects.
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Daphnia magna were exposed to different levels of bait from
very low to far in excess of what would be spread in the
environment according to present Department of Agriculture regulations,
Mirex leached from a 0.15% commercial bait formulation into the test
water to concentrations of 0.34 yg/1 to 1.21 yg/1. There were
no significant effects on _D. magna survival and reproduction at these
levels.
Since mirex is soluble in water only to the above mentioned
concentrations, acetone was used a s carrier to introduce mirex
into test water at higher concentrations. Statistical analysis
has yet to be completed on the results of three tests, but an
apparent effect on both survival and reproduction occurs between
nominal concentrations of 40 and 80 yg/1. Further tests are planned
in order to more firmly establish this chronic effect level. Also,
residue data will be determined from the exposed adult D. magna.
Experiments with the midge Tanytarsus dissimilis Joh. using
bait as the insecticide source have shown no effect at water
saturation concentrations of mirex. These experiments are being
repeated for verification. Further tests are planned to determine
the effects of 30-day exposure of mirex to stream insects and to
determine the importance of ingestion to mirex toxicity. (Richard
L. Anderson and Richard W. Carlson)
XI.	The chronic toxicity of Sevin, Malathion and Diazinon to
Daphnia magna was studied in three-week constant flow tests.
Concentrations in yg/liter causing no significant impairment to
reproduction were 8.7 for Sevin, 0.16 for Diazinon and 0.57 for
Malathion. (Kenneth E. Biesinger)
XII.	Continuous-flow and static bioassays were conducted at 18° C,
with survival and reproduction as measures of relative toxicity of
eight PCB's Aroclor 1221 (A-1221), 1232, 1242, 1248, 1254, 1260,
1262, and 1268. Three PCB-mixtur.e bioassays were also conducted.
Aroclor 1248 was the most toxic to Daphnia magna of the eight
Aroclors tested in static tests; the 3-week LC50 was 25 yg/1.
Aroclor 1254 was the most toxic PCB to Daphnia under continuous-flow
conditions with a 3-wk LC50 of 1.3 yg/1. Ninety-six hour LC50 values
for 1242 and 1248 on Gammarus pseudolimnaeus in continuous-flow
tests were 73 and 20 yg/1. Survival after 60 days was 52% at 8.7
yg/liter 1242 and 53% at 5.1 yg/liter 1248. Reproduction and survival
of young were normal at 2.8 yg/liter 1242 and 2.2 yg/liter 1248.
The midge Tanytarsus dissimilis, in continuous-flow tests, did not
emerge in abundance above 5.1 yg/liter 1248 or 3.5 yg/liter 1254.
The 3-wk LC50 for Aroclor 1254 was 0.65 yg/liter for larvae and 0.45
y^/liter for pupae. Tissue residues in Gammarus pseudolimnaeus
ranged from 4.0 yg/gram 1254 in control animals to 552 yg/gram 1248
in scuds held for 60 days in water containing 5.1 yg/liter 1248.
A1 Nebeker)
18

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XIII.	Two 9-raonth continuous-flow bioassays and several
intermediate length continuous-flow tests were conducted to determine
safe levels of Aroclor 1242, 124 8, and 1954 for the fathead minnow
Pimephales promelas) and Aroclor 1248 for the flagfish, Jordanella
floridae. Calculated 96-hour LC50 values for newly hatched fathead
minnows were 7.7 yg/liter for Aroclor 1254 and 15 jig/liter for 1242.
Three-month old fatheads had a 96-hour LC50 of 300 yg/liter for
1242. Reproduction occurred at and below 1.8 yg/liter 1254 and
at and below 5.4 yg/liter 1242. Newly hatched young were the
most sensitive life stage. Growth of young fatheads was also
affected above 2.2 yg/liter 1248, and none survived above 5.1 yg/liter
after 30 days. Young flagfish did not survive at 1248 concentrations
above 5.1 yg/liter and did not grow well above 2.2 yg/liter. (A1 Nebeker)
Fathead minnow fry less than 24 hours old at the start of the
test were exposed for 30 days to Aroclors 1248 §nd 1260. Median
toxicity values of 4.35 yg/1 for 1248 and 2.5 ys/1 f°£ 1260 were
obtained. Total mortality was observed at 8.0 y^/1 and 7.0 yg/1,
respectively. Chronic tests have recently been completed with these
Aroclors at concentrations of 3 yg/1 and below. While the data
have not been analyzed statistically, there were no readily apparent
effects on survival, growth, or reproduction. (Dave DeFoe)
XIV.	The fathead minnows exposed to Aroclor 1248 and 1260
accumulated about 80 percent of the residue present after the 250
day exposure within the first 90 days. Aroclor 1248 was accumulated
in lipids up to concentrations approximately 1.3 x 10s times that
in the water. Previous work at NWQL measured bioaccumulation factors
for Aroclor 1242 and 1254, and the data from these PCB studies
indicate a consistent direct relationship between the percentage
of chlorine in the PCB and the bioaccumulation factor (inverse
relationship with water solubility).
The female fathead minnows in both exposures contained about
two times more PCB/s on wet weight basis than the males and were
more variable. Terminal males exposed to 3.5 yg/1 Aroclor 1248
contained 194 + 12 yg/gm on a wet weight basis. Since these
variations can~be accounted for largely by the greater percentage
lipids in the females, the data show that the residues cannot be
adequately described throughout the bioassay unless the sex of the
fish is included in the analysis.
The fathead minnows which were thinned after 66 days of exposure
were transferred to Lake Superior water, and the residues and growth
in composite samples were measured four times over a 60-day period.
Approximately 30 percent of the PCB residue (as total yg) was
eliminated and/or metabolized in 60 days. (Oilman D. Veith)
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XV.	Brook trout were exposed to sublethal water concentrations
(1.0, 0.33, 0.11, 0.04, 0.01, and 0.0 yg/1, nominal concentration)
of a commercial polychlorinated biphenyl, Aroclor 1254, to determine
its effect on growth and reproduction and to determine the uptake
of PCB's by the trout. The adult fish were terminated after 71
weeks of exposure with no effects noted on growth or survival.
Reproduction occurred successfully in October and November at all
toxicant concentrations. Exposure of the eggs and fry produced will
continue until April 1974.
Residue samples were taken from fish after 14, 27, 36, 41,
48, 55, 60, and 71 weeks exposure. Analyses completed thus far
indicate that after 55 weeks of exposure the brook trout accumulate
from 0.5 to 12.0 yg/1 PCB (on a wet weight basis) in the whole body
and from less than detectable (<0.20 mg/1) to 2.0 mg/1 in the fillet
from nominal water concentrations of 0.01 to 1.0 yg/1. (Virginia
M. Snarski)
Oceans Systems, Tarrytown, New York, was awarded a contract
which now has been underway for just over a year and deals with the
acute and chronic toxicity and tissue accumulation of the two most
common phthalate esters found in the environment—di-n-butyl phthalate
(DBP) and di-2-ethyl-hexanoyl phthalate (DEHP)—in both fish and
invertebrate animals. Three phthalate chronic exposures of fish
are underway at this time. One involves the exposure of bluegills
to DEHP at concentrations of 2.09, 0.77, 0.38, 0.16, 0.07 mg/1 and
controls. The other two chronics are both with brook trout, one
involving exposure to DEHP (4.21, 1.48, 0.53, 0.11, 0.03 mg/1 and
controls), and the other to DBP (2.28, 0.68, 0.18, 0.05, 0.01 mg/1
and controls). No DEHP effects were noted on blueglll or brook
trout survival, growth or reproduction at any of the concentrations
tested. All brook trout exposed to 2.28 mg/1 DBP died after one
month, and all trout exposed to 0.68 mg/1 DBP died following four
months exposure. Studies on spawning success, and growth and
survival of young, for both DEHP and DBP are underway and will be
complete in four to five months. (James M. HcKiiu)
XVI.	In a contract with Envirogenics Company of El Monte,
California, to determine the acute toxicity of acetone, triethylene
glycol (TEG), dimethylformamide (DMF), and Triton X-100 (a product
of Rohm and Haas Company) to three species of fish and the chronic
toxicity of the last three to fathead minnows and brook trout,
the following approximate 7-day LC50 values (in mg/per liter) and
have been determined:
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Chemical
Fathead Minnovr	Bluefti.ll
Brook trout
TEG
92,500
56,500
67/100
Acetone
7,100
7,500
6,000
DMF
7,000
6,200
8,400
Triton X-100
5
14
11
In the chronic tests with brook trout, the fish are. beginning to spam,
and so far individual levels of up to 2 rag Triton X-100, 100 nig DMF,
and 640 rag TEG per liter have produced no significant effects. In
the first month of the chronic tests with fathead minnows, 2 mg
Triton X-100 per liter killed 95% of the fry exposed to it, 640 mg
TEG per liter killed 70% of the fry exposed to it, but 100 mg DMF
per liter had no significant effect. (Charles E. Stephan)
XVII. In a malathion degradation study at room temperature in
Lake Superior water, the half-life at pH 6 was found to be 3 days,
as compared to 9 weeks in distilled water. The solubility of endrin
in Lake Superior water was found to be near 90 yg/1 at room
temperature. A total of about 3,300 water and tissue analyses
were run in direct support of the fish acute and chronic pesticide
bioassays in the past 12 months. The acquisition of a new GLC
system, including automatic sampler, integrator, and recorder, along
with a programmable calculator and plotter, has reduced by about
one-half the time spent by three persons on gas chromatographic
determinations and calculations. (Leonard H. Mueller)
Lake Michigan fishes representing 13 species from 11 regions were
captured in the fall of 1971 and analyzed for chlorobiphenyls (PCB's)
and DDT and analogs. The major PCB's in Lake Michigan fishes are
the tetra-, penta-, hexa-, and heptachlorobiphenyls, although the
lesser chlorinated PCB's present in fishes (expressed as Aroclor
1254 - wet weight) ranged from 2.7 yg/gm in smelt to 15 yg/gm
in lake trout. Essentially all trout and salmon greater than 12
inches in length contained PCB's of concentrations greater than the
FDA tolerance level of 5 yg/gm. The concentrations of total DDT
(sum of analogs) ranged from less than 1 yg/gm in suckers to
approximately 16 yg/gm in large lake trout.
The presence of PCB's was routinely confirmed by perchlorination
to decachlorobiphenyl. Composite samples from selected regions of
the lake were also qualitatively analyzed by GC/MS. The major
components (based on weight) identified include Ci?_, C13, and Ci7
hydrocarbons in addition to hydrocarbon mixtures resembling fuel oil;
21

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the six p»p"* and o,p'* isomers of DDT, DDD, and DDE; approximately
15 PCB's containing 2 to 7 chlroine atoms per molecule; nonochlor;
diotyladipate, dibutyl-, dihexyl-, and dioctylphthlates; hexachloro-
benzene, and dieldrin. The "heptachlor epoxide" commonly reported
in Lake Michigan was identified as dichlorobenzophenone and no evidence
of heptachlor epoxide was found. Other components of the Lake
Michigan fishes included a series of polychlorinated cyclodiene
compounds, DD MU, cis- and trans-chlordane, kelthane, polynuclear
aromatic, hydrocarbons, and chlorinated polynuclear compounds.
(Gilman D, Veith and Douglas W. Kuehl)
XVIII. The need to characterize complex mixtures of potentially
hazardous organic chemicals and their metabolites in fish tissue
relies heavily on gas chromatography/mass spectrometry as a primary
analytical tool. An evaluation of present residue cleanup techniques
has demonstrated that the development of cleanup techniques for GC/MS
analyses have not kept pace with developments¦in instrumentation.
Techniques which are suitable for quantitative analyses via GLC
generally do not provide adequate cleanup capabilities for explicit
confirmation via GC/HS when the concentration of the chemicals is
less than 0.5 yg/gm in the wet tissue. Moreover, attempts to
scale-up procedures increase the likelihood of contaminating the
sample with reagent impurities.
A procedure for isolating greater quantities of pesticides and
related chemicals which may be present at concentrations less than
0.1 yg/gm for subsequent GC/MS analysis has been evaluated and submitted
for publication. After exhaustive extraction of the trace chemicals
and lipids from the tissue, the trace chemicals are removed from
the bulk of the co-extracted lipids through preferential desorption
from Micro Cel-E, a sorbant with a 490 weight-percent capacity for
oil. Briefly, large quantities of lipid residue are mixed with
Micro Cel-E to form a dry powder from which the pesticides and
related chemicals are preferentially partitioned into an organic
solvent mixture. The trace chemicals are subsequently isolated from
the remaining co-extractants by gel permeation chromatography.
Although the basic principles of the individual steps are methods
existing in the literature, neither is sufficient when used alone,
and the outlined procedure combines the high capacity of Micro Cel-E
with the high efficiency of gel permeation chromatography. The
procedure permits the semi-quantitative isolation of trace chemicals
from 0.5 Kg of fish tissue into 100 yl of hexane, sample preparation.
(Gilman D. Veith and Douglas W. Kuehl)
22

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XIX. The need for identification of specific chemical compounds
responsible for environmental pollution has resulted in the
development of new instrumental techniques which are much more sensitive
and specific than nuclear magnetic resonance or infrared spectroscopy.
Because of the complexity and trace amounts in environmental samples,
the most valuable technique to date has been shown to be computerized
gas chromatography-mass spectrometry. The GC/MS computer system
now in use at the National Water Quality Laboratory consists of a
Varian 1700 gas chroinatograph interfaced to a Varian CH~5 series III
single focusing mass spectrometer and equipped with a Varian Spectro
System 100 MS an on-line 12K computer. The computer makes
possible efficient use of the data generated by the mass spectrometer.
The computer peripherals include a 240 thousand word magnetic drum
and two magnetic tape drives for program and data storage, a display
scope, line printer, punch and plotter for output devices and a
data phone coupler for library search.
During the first eleven months of 1973, 120 GC/HS or direct
probe/MS run samples were analyzed at NV7QL for a total of
approximately 36,000 spectra. Sample types include standards,
environmental extracts of water, fish and lake sediment, extracts
from laboratory bioassays, extracts of waste oil samples, new potentially
hazardous synthetic organic compounds, and special samples done at
the request of laboratories other than NWQL. (Gary E. Glass)
XX. Recently total DDT analogs in our fish food supply were observed
to be approximately twice what they had been previously. It was
found that rising costs necessitated a reduction in the amount of
fish meal and the subsequent switch from soy bean oil to fish oil
to satisfy fatty-acid requirements. Determinations of pesticide
concentrations in other commercially available fish foods have
revealed total DDT analogs to routinely run from about 0.2 to 0.3
mg/KG. Because of the. desire to hold pesticide residues resulting
from the food to a minimum in fish used for toxicity research,
negotiations are underway to have a private source prepare fish
food to our specifications using ingredients known to have low
pesticide content. (Virginia M. Snarski)
ROAP 16ABI - Temperature and Dissolved Oxygen Requirements for Fresh-
water Organisms
Oxygen
I. Effects of reduced concentrations on embryos and larvae of
freshwater fishes.
Tests on the effects of reduced dissolved oxygen concentrations
on the embryos and larvae of the mountain whitefish, largemouth bass,
white bass, and lake trout were completed.
23

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Mountain whitefish were tested at oxygen concentrations of
100, 50, 35, 25, 20, and 12.5% saturation at 4 C, and 100, 50, and
25% saturation at 7 C. A reduction of the oxygen concentration to
50% saturation at both 4 C and 7 C did not affect survival. At
4 C a moderate reduction in survival occurred at 35% saturation,
and marked reductions at saturations of 25% and below. No hatching
occurred at 12.5% saturation. At 7 C survival was similar between
the controls and 50% saturation, and this survival was also similar
to those at 4 C with the same oxygen concentration. Few fish hatched
at 25% saturation and 7 C, and those died within 24 hours after
hatch. Development (incubation time, growth, and first-feeding)
was inhibited with each successive lowering of the dissolved oxygen
at both temperatures.
Largemouth bass were tested at oxygen concentrations of 100,
70, 50, 35, and 20% saturation at 20 C and 23 C. At oxygen concentrations
as low as 35% saturation, survival of largemouth bass was adequate.
However, even at the highest reduced oxygen saturation (70%) growth
was inhibited, and at saturations of 50% and below, hatching was
premature and first feeding was delayed.
White bass were tested at oxygen concentrations of 100, 70, 50,
35, and 20% saturation at 16 C and 19 C. At 16 C white b ass showed
a decrease in survival with each consecutive reduction in the oxygen
concentration at 7 days after hatching. This trend was only slight
from 100% saturation to 35%, but a marked reduction in survival took
place at 20%. There were no differences in average fish length at
different oxygen concentrations at this time. At 35% saturation
and above, larvae were first observed in a horizontal sustained
swimming position on the fourth day after hatching; at 20% on the
following day. At 19 C survival at hatch was similar to and
development faster than at 16 C and the same respective oxygen
concentration. All larvae at 20% saturation and 19 C died before
reaching the horizontal swimming stage, and after 10 days of exposure a
high mortality was observed at all of the higher oxygen concentrations.
This mortality was probably due to starvation, although plankton
was offered to the fish. It is apparent that 20% saturation of
dissolved oxygen is not adequate for young white bass survival, and
concentrations of 35% saturation inhibit yolk sac absorption.
Lake trout were tested at oxygen concentrations of 100, 50,
35, 20, and 15% saturation at 7 C, and 100, 50, 40, 30, and 20%
saturation at 10 C Lake trout development (at both temperatures)
from fertilization through first feeding was inhibited at all reduced
oxygen saturations tested (50% and lower). Survival was adversely
affected at all reduced concentrations although only slightly at
50% saturation at 7 C.
24

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A test to determine the effects of reduced dissolved oxygen
concentration on the spawning behavior of the largemouth bass is
underway. Mature fish are being exposed to oxygen concentrations
of 100, 80, 65, 50, and 35% saturation and a "natural" temperature
regime and photoperiod.
Bluegills are being held in 3-foot and 4-foot circular tanks
and are being exposed to a "natural" temperature regime and photoperiod.
This test will determine if the bluegill will successfully spawn in
these tanks, and will be suitable for spawning behavior testing at
reduced oxygen concentrations. (Richard E. Siefert and Anthony R.
Carlson)
Consumption during embryonic and larval stages
II.	Measurements of the oxygen consumption during embryonic
and larval stages of brook trout, lake trout and coho salmon were
made at 7 to 8 C. Brook trout and lake trout were similar in rate
of oxygen consumption; the coho salmon consumed oxygen at a much
higher rate. In each species the consumption rate increased markedly
after hatching, and again when feeding was started. By feeding
some and witholding food from others of the same age, it was proved
that the second abrupt increase was caused by feeding and not by
the developmental stage.
Oxygen consumption rates increased with the age of the embryo.
The mean rates at hatch in ug per fish per minute were: brook trout,
.075; lake trout, .072 and coho salmon, .124. The rates at first
feeding were brook trout, .160; lake trout, .235 and coho salmon,
.707. The rates two weeks after first feeding were brook trout,
.281; lake trout, .429 (one week after); and coho salmon, .891.
The oxygen consumption of brook trout embryos and larvae during
their first 13 weeks of incubation was measured at dissolved oxygen
concentrations of 20, 35, 50, and 80% saturation. The rates of oxygen
consumption and time of hatching were practically the same at 50
and 80% saturation; the consumption rate was less and hatching was
delayed by a day at 35%. At 20% saturation the consumption rate
was about a fifth of those at 50 and 80%, hatching was delayed by
5 days, and nearly 90% had died by the twelfth week of incubation.
(William A. Spoor)
Effects of low concentrations for periods varying in time of onset
and duration on embryonic and larval stages
III.	Studies to learn the effects on respiration and survival
of exposing embryonic and larval stages of freshwater fish to low
concentrations of oxygen for periods varying in time of onset and
in duration were conducted with northern pike and largemouth bass.
25

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It seems well established (to a considerable extent by work
done at the NWQL) that embryos and larvae can develop and survive
through the start of feeding during continuous exposure to oxygen
concentrations as low as 25% saturation, but the literature contains
very little useful information about the effects of temporary exposure
to reduced oxygen concentrations, especially to concentration ranges
likely to be encountered by the fish. This lack of information
is unfortunate because the oxygen concentrations during the spawning
seasons of warmwater fish seem more likely to undergo temporary
depressions lasting for a few hours (e.g., a pre-dawn sag) or for
a few days than to remain at low concentrations throughout the entire
embryonic and larval periods. Further, judging by the results of
experiments conducted so far, if the oxygen concentration is adequate
for normal development most of the time, but subject to a sag, the
fish are in greater danger than they would be at a permanent low
concentration.
The study with northern pike was a preliminary study undertaken
primarily to test the equipment and experimental design. This work
showed that a reduction in the dissolved oxygen concentration from
near saturation to 25% saturation two or three days before hatching,
and during hatching, is more damaging than a change from high to
low several days after hatching or remaining at 25% saturation
throughout the embryonic and larval periods. Change from low to high
concentrations improved survival, and embryos changed from low to
high two days before hatching developed as well as those kept at
the higher concentrations throughout.
The work on largemouth bass was the first stage of the study of
this species. In this work one set of 4 groups of embryos (each
group starting with 200 fertilized eggs) was started at 90 to
100% oxygen saturation and another set of 4 groups was started at
25% saturation. One group of each set was changed to the alternative
concentration on the second day of incubation, another group on
the fourth, and a third group on the sixth (hatching occurred on the
4th or 5th day). The fourth group of each set was maintained at the
original oxygen concentration throughout. At 25% saturation 47%
survived through the start of feeding and, as with the pike, changing
from low saturation to high improved survival. The reciprocal change
was detrimental, that from high to low on the sixth day causing
total mortality within 24 hours. At 90% saturation 86% survived
through the start of feeding.
The information obtained from the study of northern pike and
largemouth bass suggests the importance of such variables as (1)
the decree to which the oxygen concentration is lowered, (2) the duration
of exposure to the reduced concentration, and (3) the developmental
stage at which the reduction is made. Earlier work at this laboratory
26

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has shown that the (4) temperature and (5) water flow rate
must be taken into account. Other variables that may be
significant are (6) the rate of change in concentration and (7)
the effect of changing from an intermediate to a low concentration.
Because the flagfish, green sunfish and fathead minnow will spawn
in the laboratory throughout the year, they were tested as substitutes
for use in studying these variables when embryos from seasonally
spawning fish are not available. The fathead minnow looks the most
promising because it provides large numbers of fertilized eggs of
known age. (William A. Spoor)
IV. A research grant under the guidance of John J. Peterka,
North Dakota State University, Fargo, North Dakota, was awarded to
determine the dissolved oxygen concentrations in natural spawning
habitats of selected freshwater fishes.
Major effort from February to April was spent toward developing
and assembling equipment for measuring dissolved oxygen in the field.
From April to May measurements of dissolved oxygen, water, and air
to temperatures were made in a marsh used- by spawning northern pike.
From May to July measurements of dissolved oxygen, water, and air
temperatures were made in spawning beds of largemouth bass,
pumpkinseeds, and bluegills.
Large fluctuations in dissolved oxygen were found in the
northern pike spawning site at a depth of .01 m from the bottom
(near the embryos). For example, from April 18 to May 9, the maximum
recorded dissolved oxygen concentration was 16.4 mg/liter and the
minimum was 1.9 mg/liter. Temperatures at this depth ranged from
12.4 C to 5.0 C. Bluegill embryos in a natural nest were exposed to
an average daily change of 4.8 mg/liter of dissolved oxygen and a 3.3 C
temperature change. (Richard E. Siefert)
Temperature
I. Largemouth Bass Spawning. Largemouth bass were tested to
quantify reproductive success at constant winter temperatures. The
test is one of a series to determine if the usual length winter chill
period and the associated low temperatures at the latitude at which
a species is abundant are needed for successful reproduction. The
data add to temperature criteria information for the species and
broaden the data base for estimating criteria for other species.
Information from field studies indicates that largemouth bass
reproduce successfully when exposed to winter temperature regimens
ranging from (1) annual temperatures in Silver Springs, Florida,
which are constant the year around at 22,8 C, with spawning occurring
07

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primarily in April through June, to (2) a few to several months at
temperatures under ice in northern areas with spring rise in temperature
triggering spawning in April through J vine when water temperature has
exceeded 15.6 C, to (3) high seasonal temperatures in subtropical
areas which decrease to about the minimum spawning temperature in
fall, and then increase to trigger spawning between November and Hay
at 15.6 - 26.7 C with the peak in February at 21.1 C. Reproductive
success under these temperature regimens has not been determined
quantitatively.
Largemouth bass were exposed to nominal constant temperatures
of 15, 18, 21, 24, and 27 C. Twelve fish were held at these
temperatures from October 27 to March 22 when four fish (two pairs)
were placed in duplicate 4-foot diameter tanks for spawning. Two
spawning substrates were placed in each tank. These consisted of
gravel-covered nylon mesh mats in 14 x 17" plastic pans.
Nesting activity, as indicated by gravel swept from the center
of the mat, was highest at 18 and 21 C, but occurred to some extent
in all tanks,
Spawning occurred only at 21 C between April 18 and June 10.
Eleven spawnings from both duplicates produced a mean number of
eggs per spawning of 7,000 and 2.5 spawnings per female. The mean
fertility of the spawnings at 21 C, measured at formation of the
neural keel, was 96%. (Bernard R. Jones)
II. Yellow Perch Growth. Growth of juvenile yellow perch was
determined in an eight-week study conducted at constant temperatures
at 2 C intervals from 8 - 34 C. Growth was maximum at 28 C with no
statistically distinguishable reduction at 26 or 30 C.
Rate of growth increased with increase in temperature up to 30 C.
Rates at 26, 28, and 30 C, at which best growth occurred, were 3.5,
3.8, and 3.5 percent increase in weight per day, respectively. At
32 C the rate dropped to 2.8 percent per day and was accompanied
by a high percentage of deformed individuals. Little or no growth
occurred at 8 C and complete mortality occurred in less than 7 days
at 34 C. These relationships, particularly the closeness of the
temperature of optimum growth to the lethal temperature, are
illustrated in the following figure'.
28

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u 3"
CL
In.
IC»-
in
i a
I B
temperrture: c.
Rainbow Trout Behavior. A study of the behavior of 3-4 inch
rainbow trout in relation to different thermal barriers is being
conducted in cooperation with the University of Minnesota, Duluth.
The work, which is being conducted by B. Monson a graduate student,
is designed to determine the thermal barriers that fish acclimated
to temperatures of 5, 10, and 15 C will penetrate to obtain food and
the effect on the fish of penetrating high temperature barriers.
The laboratory work is scheduled for completion in January 1974.
(J . Howard McCormick)
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III.	Acontract was awarded to Bayshore Systems Corporation,
Springfield, Virginia. Under the direction of Joseph R, Jahoda,
to develop two types of remote sensing prototypes: an acoustic
tag to identify position and temperature of individual large
fish and an activity detector to locate offspring from natural
reproduction. Effort has been concentrated on finishing the design
of the acoustic fish tag to be field tested at the Mont:ic.ello
Field Station. The Bayshore Systems approach is to use a sufficient
number of hydrophones to decrease the r*ange of the fish tag transmitters
and thereby increase the life of the batteries. By limiting the
effective range of the fish tag to about 75 feet and receiving
a signal once an hour, a life of 1 year and a size of 0.60 X 1.7
inches long (7 grams in water) is feasible. To obtain the
temperature of each fish, two possible techniques are available
(a) a thermistor is included in the fish tag and it controls the
repetition rate of the pulsed signal, (b) thermistor elements are
placed in the hydrophones and the temperature of the immediate
area is monitored. One correlates the temperature of the
hydrophone with the closest fish. A tag approximately 1.3" long
and 5 grams in water can be achieved with this approach. A broadband
receiver and amplifier system will locate up to 20 fish tags per
channel operating at individual frequencies of 50-250 KH^.
Field tests have been delayed because of delays in installation
of conduit, however one test was made on site in November. Hi is
test identified significant interference problems with the transducer
and receiver in the water which will require some form of electro-
magnetic shielding to reduce the background "noise level". This
problem lias not yet been solved and may result in further cost over
runs to complete this contract. A time over run to September 1974
was requested during the past quarter. (Kenneth E. F. Hokanson)
IV.	A grant was awarded to the University of Minnesota, St.
Paul, Minnesota, with Lloyd L. Smith, Principal Investigator, to
determine temperature requirements of egg, larvae, and juvenile
walleye and sauger. A grant extension was awarded this year to
complete work on the sauger which have been difficult to collect for
experimentation. Juvenile sauger were collected from the Mississippi
River and growth tests were initiated this summer. The optimum
temperature for growth, fed excessive amounts of minnows, was 22° C.
The upper incipient lethal temperature of juvenile sauger was 27° C
when acclimated to 10° C and reached a maximum of 31° C when acclimated
to 26° C. Sauger thermal requirements are very similar to
walleye. These data support the assumptions used in establishing
temperature criteria for the Ohio River. All experimental work
has been completed and the final report will be submitted next
quarter on schedule. (Kenneth E. F. Hokanson)
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V.	A contract was awarded to Eco-Logic Analytical Consultants,
San Diego, California with C. R. Bernick as Program Manager, to
complete work started by Plessey Environmental Systems involving
historical temperature records as related to fish population
dynamics. The data have been encoded on computer tapes and
catalogued as to the type and amount of information available.
All information with the exception of the computer tapes and a
conversion program has been received. Analyses of the data base
are now being made to determine the best way of presenting the
encoded information. Planned use of the encoded data includes
analyses of the data and making the data base available to federal,
state, academic and industrial personnel. (Kenneth E. Biesinger)
VI.	A grant was awarded to Waterloo Lutheran University,
Waterloo, Ontario, Canada, with Robert McCauley as Principal
Investigator, to describe the seasonal changes in preferred
temperatures of adult yellow perch. Initial efforts concentrated
on a literature review of publications in the'field of temperature
preference and correspondence and consultation with workers active
in the field. Consideration of the types of temperature selection
devices suitable for yellow perch resulted in the decision to work
with horizontal gradient tanks following the design of Zahn, an
eight-foot deep vertical gradient tank, and a behavioral
thermoregulatory device modified after the original design of Neal
and Magnuson. Three rooms allotted by their university were painted,
electrical circuits and fish holding tanks were installed. Problems
were encountered in establishing a stable horizontal gradient, but
this has been solved. Adult perch have been collected from Lake
Erie and Lake St. Clair and will be acclimated to temperatures
5-20° C prior to temperature selection studies.
The principal investigator has received another grant to study
the effects of the immediate thermal history on temperature selection
of rainbow trout. This grant has provided an additional technician
part-time for the perch grant. Rainbow trout are fed a small radio
tag with a thermistor the size of a pill. Preliminary results suggest
that rainbow trout maintain a constant internal body temperature
while they move over a wider range of external water temperature.
(Kenneth E. F. Hokanson)
VII.	In order to determine the influence of turbidity on fish
species abundance in the western arm of Lake Superior, a grant was
awarded to the University of Wisconsin, Superior, Wisconsin, under
the direction of William A. Swenson.
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Hours to Median Mortality at 10° C in Supersaturated Water —^
Species
Coho salmon
(Oncorhynchus kisutch)
a.	juvenile (parr)
b.	hold-over smolts
c.	jacks (premature
adult males)
d.	adult jmales
e.	adult males
Chinook Salmon, spring
COnc.orhynchns tshawytscha)
a.	juyenile (parr)
b.	smolts
c.	adult females
d.	adult males
1.30 atm 1.25 atm 1.20 atm
1.15 atm
7.8
8.8
43; 73 131; 157; 264 0 in 13 days
11.5; 13.0 29.5; 38.5
40; 45
56; 63
19
21
20
17.5
16.3
45; 51
74; 86
36
46; 80
40; 68.5
303
15% in 13 days
166
264
No mort. in 268
526
Species
Sockeye salmon
(Oncorhynchus nerka)
a.	juvenile (parr)
b.	smolts
c.	adults
Steelhead trout» summer
(Salrno gairdrieri)
a.	yearlings
b.	adults
1.30 atm 1.25 atm 1.20 atm
37.5 63; 127
21; 23 58; 58
<18	82
18.5; 18.5 46
31	118
1.15 atm
303
30% in 8 days
0% in 268 hours.
Steeinead, winter
(Salmo gairdneri)
a.	parr
b.	adult females
c.	adult males
Rainbow trout
(Salmo gairdneri)
a.	parr
b.	1.5 year old
Largemouth bass
(HI crop terns salmoides)
a.	Juvenile
b.	adults
1/
32; 38
26.5
31.5
78; 87
92
76
51
46
280
10% in 350 hrs,
30% in 350 hrs,
30% in 120 hrs,
193 5% in 10 days 0% in 10 days
138 10% in 10 days 0% in 10 days
Sample size was at least 10 fish per level except for adult summer
steelhead
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Preliminary analysis of data from the first field collecting
season indicates that temperatures during the warm water period
tend to separate fish into different size groups and predator from
prey species. A second factor, turbidity, tends to continue this
process by driving trout farther offshore out of the turbid zone,
leaving walleye pike as the prime large predator in the in-shore
zone during turbid periods. The walleye does not feed as actively
at Lake Superior temperatures as do trout. Turbidity is believed,
through a species specific avoidance mechanism, to reduce predation
on smelt below that expected in clear water. On the other hand,
smelt under turbid conditions tend to move in-shore and off the
bottom, where they enter the habitat of mid-water larval lake
herring.
It is believed that during these periods, when the predacious
smelt move into the water column position occupied by the herring,
that they exert their most adverse influence on the year-class-strength
of the herring stocks.
The fact that smelt yearling feed avidly on newly hatched lake
herring has been confirmed by laboratory observation at the NWQL.
Field evidence of this predator-prey relationship is, however, only
circumstantial in that smelt feeding on larval fish was confirmed.
The implications of these interactions between environmental
factors and interspecific relationships between fishes suggest
that turbidity is an important factor contributing to the decline
of the lake herring of Western Lake Superior. However, this
decline is not independent or separate from the introduction of the
smelt or over-exploitation by the fishery. (J. Howard McCormick)
RAOP 21AKG - Effects of Nitrogen (Air) Supersaturation on Columbia
River Fishes
A system has been developed to supply water supersaturated with
air to fish testing chambers by injecting air into water under
pressure. The system is inexpensive, easy to control, and provides
water of uniform quality. Further, a simple method for measuring
the total dissolved air pressure was devised and used in all
testing to monitor the levels of supersaturation. Tests were
conducted at several levels of supersaturation ranging
from 1.10 to 1.30 atmospheres of total dissolved gas pressure and
the time to 50 percent mortality was determined for several life stages
of coho salmon (Oncorhynchns kisutch), spring chinook salmon
(Oncorhynchns tshawytscha), sockeye salmon (Oncorhynchus nerka),
summer and winter steelhead (Salmo gairdnerl), and largemouth bass
(Micropterus salmoldes). These are listed below:
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Considerable differences were noted in survival time and
at least eight factors were identified which influence the
tolerance of fishes to air supersaturated water. These are species,
subspecies or race, sex, life stage, acclimation and test
temperatures, swimming activity and solar radiation.
Spring chinook smolts, yearling summer steelhead, yearling
rainbow trout and adult female coho were the four most sensitive
animals tested; largemouth bass and carp were the least sensitive
fish when tested at 10° C and judged by time to median mortality.
Aquatic insects and crayfish were more resistant than fish to
supersaturation, but did develop gas emboli in their blood, gills,
and tissues at higher levels of supersaturation. Supersaturated
water is acutely lethal to at least one life stage of several
Pacific salmonids at levels at or above 1.15 atm TDGP (115 mm Hg
hyperbaric), A level of 1.10 atm total dissolved gas pressure
(TDGP 110 percent of atmospheric pressure or ca 76 mm Hg hyperbaric)
was not acutely lethal and is probably safe for most aquatic
environments except in fish farms, hatcheries, or possible heated
effluents where prolonged exposure to 1.10 atra TDGP would cause
mortality and ill health.
The Weiss Saturometer as modified by Western Fish Toxicology
Station staff is a suitable means of measuring total dissolved gas
pressure and has an accuracy of + 1 mm Hg.
The external signs of gas bubble disease vary between species
and become most severe at moderate to low levels of supersaturation.
Positive identification of air emboli in the blood is necessary to
confirm gas bubble disease. Gross pathobiology of gas bubble disease
has been described relative to these experiments. These results
provide a baseline for developing a rank order of susceptibility to
gas bubble disease and for developing application factors for
estimating effects between species in later chronic tests. Smolts
of spring chinook salmon were found to be most sensitive and
largemouth bass were least sensitive to the test conditions. It is
recommended that the maximum allowable level of total dissolved
gas pressure be limited on an interim basis to 1.10 atm (110
percent of atmospheric pressure). (Ronald R. Garton)
ROAP 25AIC - Effect of Oil(s) on Freshwater Fish, Other Freshwater
Life, and Wildlife
Research undertaken in 1973 on the effects of oils on the fresh-
water environment concentrated on meeting the requirements of
the Federal Water Pollution Control Act of 1972. This legislation
34

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required that EPA determine the lonp-term chronic biological
effects of the disposal of waste oil. A number of acute and chronic
exposures of various forms of used crankcase oil to selected
freshwater organisms were therefore conducted. In addition,
extensiv e chemical analyses were undertaken to characterize
the various chemical components and structure of the oils used.
Biologleal Tests
I. Flow-through 7-day acute toxicity tests were performed by
exposing a top minnow, Jordanella floridac, and the fathead minnow,
Pimephales proroclas, to soluble components of waste oil. The stock
toxicant solutions were prepared by pumping 238 liters of Lake
Superior water through 26 liters of oil for 18 hours and then allowing
the mixture to separate for 6 hours. The aqueous phase was removed
and used as 100% toxicant. This stock solution was diluted with a
proportional diluter to the desired concentrations. Twenty 30 day-
old fish in 40 liters of water at 25° C, and with a turnover rate
of 6 tanks/day, were used on the Jordaaella tests. Fathead tests
were run at 20° C with fifteen 45 day-old fish in 14 liters of water
and with a turnover rate of 10 tank volumes per day. The 96-hr
LC50's were estimated by graphical interpolation to be 1.0% by
volume dissolved oil for Jordanella and 1.6% for fatheads. Additional
flow-through tests with these species are presently underway.
Static tests with oil layers were performed with fatheads.
Fifteen fish were placed in tanks containing 20 liters of aerated
Lake Superior water at a temperature of 18° C. The 96-hr LC50
was determined by graphical interpolation to be 1.1% by volume.
Preliminary tests with oil-water emulsions indicate that the
LC50 for fatheads is approximately 0.042! by volume oil.
A test to determine the effects of chronic exposures of up to
14 dissolved waste oil on the growth, survival, and reproduction
of Jordanella floridae is presently underway. This test began with
eggs which subsequently hatched and survived in all tested
concentrations. However, 1 day-old fry hatched in control water and
transferred to 1% and 0.4% dissolved oil were dead in 48 hours. In
addition, 1 day-old fry transferred from 0.064% to 1.00% and from
0.026% to 0.40% did not survive. Apparently some acclimation to the
toxicant occurs during the egg stage which allows the fry to survive
in concentrations which are toxic to fry unexposed as eggs.
(Steve F. Hedtke)
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Chemical Analysis of Waste Oil Toxicauts
II. Chemical studies at the National Water Quality Laboratory
have centered on characterizing the waste oil and the dissolved oil
stock solutions. Studies of the residues found in the exposed fish
will be completed in the near future.
The waste oil used in this study was analyzed and found to
consist of 76 percent saturated hydrocarbons; 4 percent aromatic
hydrocarbons including C2 to Cg alkylbenzenes, Ci to C3 allcylnapthalenes,
Ci and C2 alkylindenes, methylbiphenyl, Ci and C2 alkylfluorenes and
alkylphenonthrenes; 3 percent polar compounds; 15 percent solid
material; 2 percent volatile components, predominantly C7 through
Cm hydrocarbons; and the following metals:
A1 - 15 mg/1, Cu = 18 mg/1, Fe = 220 mg/1, Pb « 18,500 rag/1,
Si - 17 mg/1, Sn = 6 mg/1, Na = 59 mg/1, Ca = 688 mg/1,
Zn ® 1359 mg/1, and Mg = 410 mg/1.
The dissolved oil toxicant is being characterized by atomic
absorption spectroscopy and through the use of XAD nonionic exchange
resins in conjunction with GC/MS. The stock solution of water has
been shown to contain the following metals: Pb = 4.4 + 0.2 mg/1,
Zn <= 16 + 1 mg/1, Cd* = 60+2 yg/1, Hg <0.1 yg/1, Ag*~<0.08 yg/1,
Mo* <0.2 mg/1, Co <0.8 yg/1, As*	mg/1, Cr <0.14 mg/1, Se* <0.1,
mg/1, Ni <0.1 mg/1, Fe <0.1 mg/1, Mn <0.1 mg/1, and Cu <0.15 mg/1.
Particulate Pb or Zn did not exist in any significant amounts. Rather,
studies with XAD-2 resins indicate that most of the Pb in the water
is in an absorbable form, possibly an organometallic form which
may be more readily accumulated by fish. Fish tissue samples have
been collected in an effort to determine the metal residues and relate
the observed levels of those from other bioassays with metals.
Studies have shown that the hydrocarbon fractions of the stock
toxicant are similar to those in the waste oil, with the exception
of a decrease in the polynuclear hydrocarbons on a relative bases.
More detailed studies of the water, as well as the exposed fish
tissue, are underway (Gilman D. Veith, Douglas W. Kuehl, and John E.
Poldoski)
*Excluding possible volatile forms.
ROAP 16AAJ - Short Cut Methods to Determine Toxicant Criteria
I. Investigations were carried out to gather more information
on the validity of the application factor approach (ratio between
the "no effect" toxicant concentration and the 96-hour TL50) used
to predict the chronic toxicity of pollutants in varying environmental
situations. This experiment consisted of investigating the effect
of water quality and spleces type on the level of the mortality
found with zinc. If the level of death response could be predicted
for a species in a given water from results obtained on another species
in a different water, then more confidence could be placed in the
predictive value of this approach.
36

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To test this hypothesis, rainbow and brook trout were exposed
simultaneously to zinc in two different water hardnesses. In order
for the prediction to be valid, it was a necessary condition that
the slope of the response curves be the same. The results of
these experiments are shown in the following table.
Table 1. Number of Observed Deaths Out of 50 Fish Exposed for Ten
Days to Specified Measured Ayerage Total Zinc Concentrations
Hard Water (150 mg/1 CaCO^)
Soft Water (45 mg/1 CaCO^)
Brook Trout
Rainbow Trout
Brook Trout
Run 1
Measured

Measured

Measured

Measured
Average

Average

Average

Average
Zn

Zn

Zn

Zn
Concen-
Number
Concen-
Number
Concen-
Number
Concen-
tration
Dead
tration
Dead
tration
Dead
tration
7.69
39
6.87
50
2.58
42
2.81
4.79
6
4.50
50
1.73
36
1.88
3.09
5
3.14
45
1.14
23
1.23
2.27
1
2.00
8
.77
8
.85
1.44
1
1.42
5
.54
1
.61
Control
1
Control
0
Control
0
Control
"Rainbow Trout
Number
Dead
50
49
50
44
38
1
Run 2
9.06
48
8.63
50
2.50
27
2.38
50
5.84
15
5.91
50
1.64
23
1.60
50
3.65
2
3.54
49
1.04
3
1.05
45
2.33
0
2.20
4
.69
4
.65
34
1.49
0
1.49
0
.42
0
.44
17
Control
0
Control
0
Control
0
Control
0
The preliminary analysis of these data indicated that the
assumption of equal slopes held between species but not for waters
at different hardness. In addition, on the basis of the components
of zinc present that were measured, it was not possible to explain
the difference in the waters to a changing ratio of "toxic metal
(Zn) species". Essentially, all zinc in the bioassay solutions
appears to be "dissolved". The observed differences of the toxicity
of zinc have not yet been fully explained. More chemical
experiments are required to determine the extent of complexatiori.
Additional bioassays are required to determine the effect of ions
such as calcium on the organism. (Todd W. Thorslund, John E.
Poldoski, and James M. McKim)
37

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II, The importance of early detection of lead poisoning for
the prevention of death and serious morbidity has been demonstrated.
The enzyme, Aamino levulinic acid dehydratase (MAI)) , is a
component in the biosynthesis of heme, where it catalyses
the formation of porphobilinogen.
Lead, even at very low concentrations, severely inhibits
this enzyme. The present study of the activity of this enzyme in
the red blood cells of brook trout was initiated to investigate the
use of this enzyme in predicting chronic lead poisoning long before
other physiological and biochemical changes might occur.
An improved method for the assay of ALAD (Burch & Siegal, Clin.
Chem. 17: (10) 1971) was adapted for use with fish. Accuracy
and reproducibility of the procedure was checked on human blood
samples. The values fell within the stated normal limits for
humans not exposed to lead. These results are presented in Table
2, along with the corresponding blood lead levels as assayed by the
Delves Micro Cup Method (H. Delves, Analyst, 95; 431, 1970).
Table 2. Control ALAD and Blood Lend Values


Units


Number
ALAD
Pb it (yg/nl)

Sampled
(mean)
(mean)
Human
5
120
15.3
Brook Trout
19
333
0.06
Catfish
5
246

Rainbow Trout
1
307
—
As seen in Table 2, brook trout ALAD was found to be higher than
human controls. A mean value of 19 brook trout controls was 333 units
of ALAD. A rainbow trout control value was 307 and the catfish control
value was 246 units. Enzyme analyses were performed on brook trout
exposed to sublethal concentrations of cadmium and mercury for one
month, and no effect on ALAD activity was seen. A two-month exposure of
brook trout to lead at water concentrations of 250 and 500 yg lead/liter
caused a marked decrease in enzyme activity. Blood lead levels
ranged from 6-8 yg/g at the two water concentrations tested. In
trout exposed continuously for 22 months to water lead concentrations
of 0, 30, 60, and 125 yg lead/liter, marked decreases in enzyme
activity occurred even at the lowest Pb levels.
38

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These preliminary data show a uniform decrease in enzyme
activity as the lead concentration increases and suggests that
this enzyma may be of importance in detecting subtle Pb poisoning
in fish populations. (E. Hunt and J. McKim).
III.	Carbonic anhydrase (CA, carbonate hydro-lyase, E.G. A.2.1.1)
is of special concern in environmental biochemical studies because
it is active in gill tissue and therefore is particularly vulnerable
to water soluble toxicants. This enzyme is important both in ion
regulation and respiration process In fish, and plays an important
role in the function of many organs in addition to the gill. A
primary function of this enzyme, in gill tissue is to catalyze the
interaction of water and carbon dioxide with carbonic acid, which
evidently is the rate limiting sequence for the several_interrelated
reactions involving H20, C02» H2C03, H+, HC03~, and C03".
In the current investigation 55 compounds and 2 mixtures, which
generally have relevance to water pollution and water quality problems,
were studied for their effect on CA from red blood cells of the
catfish, Ictalurus punctatus. This enzyme was inhibited strongly by
heavy metal cations, less strongly by metallo-organic ionic compounds,
some anions and some pesticides, and weakly inhibited by light metal
cations, metallo-organic non-ionic compounds and some test organic
compounds.
Certain physical-chemical properties of this enzyme were also
determined: activity vs/pH, activity vs/incubation temperature
(incubation temperature of enzyme varied; reaction carried out at
1° C), activity vs/reaction temperature, activity vs/enzyme
concentration, and activity vs/substrate concentration. (James H. Tucker
and Glenn M, Christensen)
IV.	Many ions and complexes of heavy metals are significant water
pollutants, with compounds of mercury and cadmium being among the
most toxic agents to aquatic animals.
Most biochemical oriented studies of water toxicants are carried
out with mature animals although there is a clear need also to study
pre-adult life stages. This has a particular concern in environmental
protection activities because some research evidence indicates that
specific pre-adult life stages may be more affected by some water
contaminants and other environmental stressors than adults. This
evidence indicates that permissible concentrations of water
contaminants must be based upon experiments which include exposure
studies of pre-adult stages.
39

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In the current study embryos and alevins from control and
exposed brook trout were homogenized and subjected to analysis for
ATP (adenosine-S'-triphosphate), protein (total), GOT (L-aspartate:
2-oxoglutanate aminotransferase, E.C. 2.6.1.1), ACH (acetylcholine
acetyl-hydrolase, E.C. 3.1.1.7), and ALP (orthophosphoric monoester
phosphohydrolase, E.C. 3.1.3.1). In addition, the weight of each
organism and the thickness of the chorion of each embryo were determined.
ATP, being a very ubiquitous reactant in living matter, was
studied as it seeins likely that most metabolic malfunctions would
affect the biochemistry of this tissue component. Also, it has
been shown that oxidative-metabolism increases in fish embryos
after fertilization, which would involve ATP mobilization and reactivity.
Prior investigations at this laboratory showed that the total
protein level and GOT activity changed in the blood plasma of adult
fish upon exposure to metal cations. It has also been reported
that certain metal cations affect the activity of fish brain ACH.
Cholinesterase activity can first be detected in eggs of Salmo gairdnerii
ten days after fertilization, which indicates"an important stage in the
development of the nervous system. In another study it was found that
the activity of ALP (with acid, phosphatase and pyrophosphatase)
increased during development of the embryos of rainbow trout, particularly
during the "eyed" stage.
The results of this study showed that the enzymes ACH and ALP
in brook trout embryos are either absent or are very inactive.
Therefore, it is likely that a toxicant which impairs the activity of
these enzymes-—as is the case with chlorinated hydrocarbons and
carbamates—would not show a high toxicity during this stage of
development. Methylmercury(II) appeared to have some effect upon
these factors in embryos at 0.1 to 1.0 yg/liter; Cd (II) may have a
slight effect upon the parameters measured, whereas Pb (II) has no
significant effect. With regard to fry, the three ions all appear
to elicit biochemical changes. The weight of the animals decreased;
there were only subtle effects upon the protein content; the
activity of GOT and ALP increased. The activity of ACH increased
for Pb (II) and decreased for MeHg (II) and Cd (II). (Glenn M.
Christensen).
V. The uptake and distribution of Hg, Cd, and Pb in 8 tissues
(gill, liver, kidney, blood, muscle, spleen, gonad, and brain) of
the brook trout were measured over a 38-week exposure period.
After 38 weeks exposure of first generation trout to Cd, tissue
cadmium residues in the kidney showed the greatest increase,
followed by the gill and liver (Table 1). Slight increases were
also noted in the spleen and gonads; however, no increase was
detected in the muscle tissue. The average percent recovery of
cadmium from spiked samples was 99.6 + 6.5.

-------
All brook trout tissues sampled showed increases in lead residues
(Table 2) following 38 weeks of Pb exposure. The kidney showed the
greatest increase, followed by the gill and liver. Preliminary
results of the lead residues in tissues of 44-week old second generation
offspring showed a similar relationship (Table 3). The gill, liver,
and kidney of second generation trout at 500 ug Pb/liter, however,
contained much larger concentrations of lead than the first
generation fish. The average percent recovery of lead from spiked
samples was 95.0 + 14.4, (Duane A. Benoit, Edward N. Leonard, and
James T. Fiandt)
Table 1
. First Generation Brook Trout After 38 Weeks Of
to Cadmium (10 fish sampled per concentration)
Exposure
Mean
Cadmium Residues lig/g;
ram of
Brook
Trout Tissues
(Dry tft.)
Cadmium Exposure
Cone, yg/liter
^0.04
0.3
0.7
1.5 3.0
6.0
Gill

0.5
2.5
3.2
3.7 5.0
9.3-f
Liver

0.2
2.4
4.1
8.2 9.8
5.9
Kidney

1.6
11.4
21.4
28.4 49.4
45.2
Muscle

0.1
0.2
0.2
0.2 0.1
0.1
Spleen

0.6
0.8
1.0
1.4 1.8
1.6
Gonad

0.3
1.4
2.6
3.8 3.8
2.8
a/
Since there were no fish alive in the 6.0 yg/liter concentration
after 38 weeks, the data for the "20 weeks" fish was included in
the Table.
41

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Table 2. First Generation Yearling Brook Trout After 38 Weeks of
Exposure to Lead (10 Fish Sampled Per Concentration)
Lead Residues yg/gram of Brook Trout Tissues (Dry Wt.)
Lead Exposure
Cone, pg/liter
0.4
31
62
125
250
500
Gill

5.7
34.8
48.6
117.0
206.9
326.0
Liver

1.7
20.8
32.2
67.8
73.2
83.4
Kidney

4.5
43.6
66.5
216.2
332.8
476.6
Muscle

3.4
5.1
8.0
6.6
3.4
4.4
Spleen

3.6
12.4
8.2
18.0 '
29.0
59.6
Gonad

3.0
9.5
4.4
12.8
16.3
13.9
Table 3
. Second Generation Br^ok Trout Offspring After 44 Weeks
of Exposure to Lead—

Lead Residues pg/gram of Brook Trout Tissues CDry Wt
.)
Lead Exposure
Cone, -pg/liter






0.4
31
62
125
250
500
Gill

6.2
54.5
47.0
89.2
266.0
499.4
Liver

3.1
20.7
14.4
35.5
134.0
235.4
Kidney

2.0
17.0
22.7
101.8
326.2
824.4
Muscle

1.8
3.4
2.4
3.1
4.8
6.6
Spleen

2.0
1.2
(7.3)
C5.2)
(32.2)
(30.3)
Gonad

2.4
2.4
3.4
5.6
7.2
13.1
Brain

1.6
3.4
(3.8)
(1.0)
(10.1)
(13.0)
—^ All numbers are the average of ten (10) fish sampled. Exceptions are
the numbers in parentheses which are the average of 2-5 fish sampled.
42

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The concentration of mercury in the tissues of brook trout
exposed to methylmercury chloride in the water for 39 weeks is
presented in Figure 1. The uptake from the water was extremely
rapid, and even at the lowest water concentrations tested, the
residues exceeded FDA's limit of 0.5 ppm for mercury in foods for
human consumption. Half-life studies were run on fish removed from
all the mercury water concentrations tested. After 4 months in
mercury-free water there was no significant drop in the total
mercury levels in these previously exposed animals.
These tissue data indicate that, depending upon the length of
exposure, mercury may eventually accumulate to toxic levels when
they are exposed to low levels of methylmercury chloride. With
regard to human consumption of mercury-exposed fish, none of the
water concentrations tested produced fish acceptable for human
consumption. (James M. McKim).
Figure 1
1
MU5CLE
3 u E/L
] uE/L
.3 u E/L
.03 uG/L
FDR.
CONTROL
WEEKS
43

-------
VI. Channel catfish, Ictalurus punctatus, were exposed to
methylmercurie chloride at nominal water concentrations of 4.0,
1.0, 0.25, and 0.06 yg/liter for six months in a flow-through system
at 25° C,
The experimental findings indicate that (1) at 25° C
me thy liner curie chloride at water concentrations of 4 and 1 yg/liter
is lethal to the channel catfish in thirty and ninety days, respectively,
with obvious histopathologic changes to the gill, kidney, gut,
and liver; (2) tissue uptake data for the lower non-lethal
concentrations indicated toxicant accumulation which may reach toxic
levels in the tissues upon longer exposure; (3) based on the FDA's
"interim guideline" level of 0.5 ppra in food, none of the
concentrations tested resulted in muscle residue levels acceptable
for human consumption. (James H. Tucker)
VII* The purpose of this study was to evaluate changes in
cough frequency as a possible short-term indicator of the long-term
effects of various toxicants on brook trout. All tests were conducted
using Lake Superior water and the fish were studied individually
in electrode chambers. (William A. Spoor, Timothy M. Neiheisel,
Robert A. Drummond 1971).
Changes in cough frequency, locomotor activity, and feeding
behavior of yearling brook trout appeared within 2-24 hours at
copper concentrations as low as 6-15 ug/liter. Each of these responses
appears to be useful for predicting the concentration range of
copper likely to have no long-term effects on the species. Cough
frequency was the best of the three indicators for showing stress
according to the copper concentrations tested. (Robert A. Drummond,
William A. Spoor, and Gayle F. Olson 1973).
The lowest concentration of methylmercuric chloride (MMC) and
mercuric chloride added to Lake Superior water that caused a
significant increase in cough frequency in brook trout was 3 yg/liter.
Cough frequency is a good short-term indicator of the long-term
effects of MMC. Increases in cough frequency were proportional to
the concentration (from 3 to 12 yg Hg/liter) of both compounds at
pH 7.5. The fish were more responsive to MMC when the pH. of the test
water was lowered to 6.0; response to mercuric chloride was not
changed by lowered pH. Fish exposed to MMC at pH 6.0 contained more
total mercury in their gills and red blood cells than fish tested
at pH 9.0. The uptake of mercury by brook trout exposed to mercuric
chloride did not differ significantly at pH 6.0 and 9.0 (Robert A.
Drummond, Gayle F. Olson, and Allen R. Batterman 1974).
44

-------
The lowest concentration of cadmium to cause a significant
increase in cough frequency was 6 yg Cd/liter. No additional tests
are planned. A manuscript—some factors which affect the toxicity
of cadmium to brook trout—is currently being prepared for possible
publication.
Lead nitrate at a concentration of 125 yg Pb/liter was the
lowest concentration of lead tested thus far to cause a significant
increase in cough frequency. Additional tests are planned to
determine if brook trout will respond to concentrations less than
125 yg Pb/liter.
A definitive increase in cough frequency has not been demonstrated
when brook trout are exposed to sodium dichromate and zinc sulfate
for periods lasting several days. Although some fish exhibited an
increase in cough frequency, the results are considered inconclusive.
The following concentrations of malathion, lindane, and diazinon
were found to cause a significant increase in cough frequency among
brook trout:
Lowest
concentration
tested
thus far	
12.2+1.4 yg^l
5.3+1.0 yg/1
24.6+4.4 yg/1
Atrazine
and/or	Not tested yet
Parathion
*A11 eight fish lost virtually all of their natural body coloration.
They were light-tan in color when examined at the end of the test.
All compounds will be retested at concentrations equal, or less
than, the above reported values because several control fish responded
to the acetone (260 ppm) carrier. We have modified our delivery
system so that brook trout are not subjected to a concentration of
acetone greater than 125 ppm. Preliminary tests Indicate that brook
trout do not respond to 128 ppm of acetone by increasing their cough
frequency.
Compound
tested
Malathion
Lindane
Diazinon
No. fish
tested
8
8
8*
No. fish,
exhibiting
a significant
increase in
coufih. frequency
8
8
4
45

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Several pilot tests were conducted to determine if fish other
than trout and salmon would respond to toxicants by increasing their
cough frequency. Largemouth bass, northern pike, bluegill sunfish,
black crappie and channel catfish were found to respond to copper
by increasing their cough frequency. Yellow perch, black crappie
and channel catfish (other species not tested) exhibited an increase
in cough frequency when exposed to methylmercuric chloride. In
general, the increase in cough frequency was directly proportional
to the concentration of copper and methylmercuric chloride as found
for brook trout.
An instrument for indirectly monitoring specific movements or
physiological events of fish, such as the frequency of opercular movements,
"coughing'1, locomotor activity, and heart rate, was developed at the
National Water Quality Laboratory. The previous instrumentation for
indirectly monitoring these movements (locomotor activity excepted)
required a set of inexpensive electrodes and an expensive EKG-EEG-
EMG polygraph, consisting of a preamplifier, amplifier, and strip-
chart recorder. We have now developed (Drummond and Dawson 1974)
an inexpensive solid-state amplifier that can replace the expensive
preamplifier and amplifier and is compatible for use with a variety
of recording equipment. The solid-state amplifier should be
particularly useful to researchers and students interested in
instrumentation of this type but who cannot afford the necessary
equipment costing several thousands of dollars. (Robert A. Drummond)
VIII. Concomitant with exposure of aquatic organisms to toxic
chemicals is the need to measure the toxicant in the test chamber.
For many discrete organic chemicals, the present analytical methodology
is adequate to measure the chemicals at concentrations
of a few ng/l. However, for toxicants which are complex mixtures
such as the PCB mixtures, the determinable limit of present techniques is
often 20 to 50 ng/l which precludes analytical support data for exposures
at 10 ng/l.
To increase the sensitivity of PCB analyses in chronic exposures
of trout to Aroclor 1254 and to assess the possibility of PCBs in Lake
Superior water, a perchlorination technique using SbCl5 was refined
and evaluated. The greater sensitivity is realized due to the
conversion of all chlorinated biphenyls to a single chlorocarbon,
decachlorobiphenyl, and to the inherent greater electron capture
response of the more highly chlorinated product. Equally important
is the fact that the water extract can be taken to dryness and
perchlorinated directly allowing the decachlorobiphenyl to be cleaned
up for GLC analysis without interference from trace PCB contamination
by reagents and glassware.
46

-------
Analyses of PCBs In Lake Superior water and water from control
tanks in the exposure laboratory were conducted by passing 20 St of
water through a column of polyurethane foam, extracting the adsorbed
PCBs from the column with hexane and acetone, perchlorinating the
extract residue, and quantitating by GLC. Confirmations of the
perchlorination products were made using direct-probe mass
spectrometry. The concentration of PCBs in the control tank water
was approximately 3.4 ng/1 (as Aroclor 1254 determined by analysis
of fish residues) and the lake water contained less than 1,5 ng/1
PCBs. The detection limit of the perchlorination method for
PCB analysis is approximately 1 ng/1 in water, and is largely
dependent on contaminants in the SbCl5 and the hexane extraction
solvent. The technique also provides a confirmation technique for
PCB in tissue residue analyses where Gc/MS confirmation is not
available. (Gilman D. Veith)
F-OAP 16AAI - Develpment of Biological Test Methods
I. Research emphasis for this ROAP is placed on the development
of techniques and procedures which will provide a broader base of
test organisms for both acute and chronic bioassay testing. Laboratory
procedures for successful rearing and propagation of a species must
be developed prior to ascertaining if the species can be "stressed".
If this cannot be accomplished, then the organism cannot be classified
as a candidate for use in future exposures to develop water quality
criteria for aquatic life.
Bioassay techniques for the parthenogenic chironomid, Tanytarsus
dissimilis Joh. were developed. These techniques include both
static and flowing full life cycle exposures and acute bioassays of
selected stages of the life cycles. The procedures are now being
prepared for presentation to the National Water Quality Laboratory
Aquatic Bioassay Committee for their adoption as recommended
procedures. Copies should be available in early 1974. (Richard L.
Anderson)
Art Buikema, Virginia Polytechnic Institute has established
laboratory colonies of the planktonic rotifer Polyarthra vulgaris
using natural water and a food consisting of mixed protozoans
enriched with a vitamin and trace mineral mixture. Colony densities
of up to 10,000 rotifers per liter have been achieved.
First experiments were directed toward establishing basic
biological information and were centered on determining photoperiod
and temperature requirements. Other experiments are examining the
population dynamics of this rotifer. A bioassay procedure is being
developed. (Richard L. Anderson)
47

-------
II. Acute toxicity work with salmon in FY 73 was directed
toward determination of the effects of various experimental conditions
on the LC50 data produced from the standard 96-hour flow-through
bioassay.
A continuous flow serial dilution apparatus was designed and
two preliminary tests were run at five concentration levels of zinc
to determine toxicity of zinc to juvenile sockeye salmon. The 96-hour
LC50 was determined to be approximately 1000 pg/1 for fish averaging
6 cm in length and weighing 1.8 gms. The diluter system was then
modified to deliver three concentrations of zinc solution, one
at the 96-hour LC50 level, one at 130% of that and another at 65%
of that level. Subsequent experiments were conducted at
these concentrations with sockeye to determine the effect of changing
experimental variables, such as acclimation time and feeding.
Two sets of experiments were run in which one-half of the test
fish were fed Oregon moist pellets and the other fish were not fed.
For the first 48 hours, the fed fish were more sensitive to zinc
poisoning, but after about 72 hours, the fed fish were less sensitive
than those which were not fed. This phenomenon has not been adequately
explained, but it does demonstrate that bioassay results between fed
and non-fed fish are not directly comparable, and this factor can be
an important source of variability in toxicity data.
Three sets of experiments were run to compare the effect of
acclimation time upon LC50 data. The experiments compared one week
acclimation to none, two weeks versus three days, and one week versus
two weeks. In each case, acclimation was done in the test aquaria with
test dilution water. In each set of experiments, the fish with the
longest acclimation time were more resistant to zinc poisoning.
At lethal concentrations of zinc, sockeye become very skittish
after about 48 hours of exposure. When disturbed, they rush about the
aquaria in a panic which results in the fish rolling over to belly-up
position, where it remains quivering and often with gills and gill
covers flared and extended. This sometimes leads to death, but the
fish often recover to die later in the experiment. Since some fail
to recover, any disturbance may cause a change in death rate of the
fish, and thus a change in LC50.
Experimental results to date emphasize that toxicity data are
strongly influenced by conditions of the test. The final report of
this research project will be a proposal for a standard bioassay
technique to be used in toxicity tests with juvenile Pacific salmon.
(Ronald R. Garton)
48

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III,	the National Water Quality Laboratory has continued to
support the Committee on Methods for Toxicity Tests with Aquatic
Organisms in its effort to obtain uniformity in methodology and
nomenclature. Other organizations cooperating in the work of the
committee include two Department of the Interior laboratories,
EPA Office of Pesticides Programs, two other EPA research laboratories,
three industry associations, some EPA enforcement offices, several
individual companies, and some state and university programs. Over
200 other interested people are being contacted through a mailing
list. The committee plans to publish four methods for acute tests
with fish and macroinvertebrates, encourage support of activities
of the American Society for Testing and Materials in the area of
toxicity tests with aquatic organisms, and disband in 1974. NWQL's
input to such activities is funneled through the National Water Quality
Laboratory Committee on Aquatic Bioassays, which is responsible for
establishing methods for acute and chronic tests with fish and
macroinvertebrates. (Charles E, Stephan)
IV,	A series of observations were made in the lbaoratory on
seven small native fish species to determine the advisability of
their use in chronic (life-cycle) toxicity studies. Specific
requirements essential for completing a life cycle in the laboratory
were studied for each species.
Notropis lutrensis, the red shiner, has matured and spawned
in the laboratory in 16 weeks from hatch at 22-25 C and a 16-hour
light photoperiod. Under these conditions they have continued spawning
semi-continuously for more than one year. The species is easily
maintained. Adult fish are fed trout food granules and frozen brine
shrimp while larval fish are started readily with brine shrimp
nauplii.
Notropis hypselopterus, the sailfin shiner, has matured and
spawned in the laboratory in 20 weeks from hatch at 25 C and 16-hour
light photoperiod. This species spawns semi-continuously for more
than one year under these conditions. These fish require a water
depth of about 14 inches for spawning. Feeding is as reported for
the red shiner,
Johnny darters were satisfactorily reared from eggs but although
various combinations of temperature and photoperiod manipulation were
used, no spawning occurred in 17 months from hatch. It is believed
that this species is probably a second year spawner even under
optimum conditions, and work was suspended on it for that reason.
49

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Brook stickleback. This species was obtained front the Duluth
area as adults. Spawnings were obtained readily from each of five
tanks containing two males and three females. No problem was
experienced in hatching the eggs, and young were raised easily on
brine shrimp nauplii. It was discovered, however, that the young fish
would not tolerate any handling of any kind, apparently dying from
shock. This means that it would be a practical impossibility to
use the fish as a test animal in the laboratory, so work on the
species was discontinued.
Jordanella florlda. Procedures and test methods involving
the use of this species as a test organism have been published. The
species shows tremendous promise as a test animal because of the
relatively short period from egg to egg (approximately 8 weeks).
It is a prolific egg producer with numerous spawnings, and is quite
easy to rear and maintain under laboratory conditions.
Green sunfish. Some work with this species has been done. They
have been spawned and the Fx fish matured and spawned in one year.
From hatched eggs Fj fish are being raised in an attempt to speed
growth and reduce the age of maturity. At present, the prospects
are encouraging and it is believed that this species would make a
satisfactory laboratory test fish.
Longear sunfish. Work with this fish is in progress. There is
no problem in spawning the species, and feeding of adults and young
is accomplished with ease. Current efforts include an attempt to
grow and mature the F^ fish in the least possible time. (Wesley E.
Smith)
V. A rearing study with Gammarus pseudolimnaeus, a freshwater
amphipod, was completed this year. Objectives were to determine
the effects of food, light, and test duration on the fecundity of
this animal. Results showed that this species feeds equally well on
soaked aspen or birch leaves, but does poorly with maple leaves.
A light intensity of greater than 20-foot candles is needed for
satisfactory reproduction. Light intensities of 500-foot candles
visually increased the algal growths in the test tank waters and
appeared to hinder the swimming of the amphipods. General Electric
daylight fluorescent bulbs were apparently as satisfactory for
rearing this species as a combination of Durotest Optima and wide-
spectrum Grolux bulbs. No marked differences were found in the
reproductive behavior between animals in tanks under a constant
photoperiod and with a variable photoperiod. After 13 weeks at 17° c
the production of young started. Young production continued throughout
the duration of the test (a period of 38 weeks). (Jack W. Arthur)
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VI.	The copepod, Eucyclops agilis, was cultured In 1 L. battery
jars at a temperature of 18 + 1° C on a cycle of 12 hours darkness/12
hours light of an intensity of ^ 13-foot candles at the water surface.
Cultures were initiated by the introduction of ten gravid female
copepods into a battery jar nearly filled with lake water
filtered through #250 mesh screening. To this is added 3 ml
of prepared copepod food*. It is possible to maintain a population
adequate for the initiation of a flow-through bioassay by initiating
at least two cultures per week. A flow-through chronic bioassay
involving survival, growth and reproduction has been developed for
these copepods and is being evaluated prior to a formal methods
write-up.
*Food preparation: Blend for five minutes at high speed 5 grams of
trout starter granules, 225 grams of Cerophyll, and 125 ml of lake
water. Pour blended mixture through #250 mesh screening, rinsing
the remaining portion from the blender with 25 ml lake water and
also pour this rinse through the screening. Stoi-e in the refrigerator.
(Rita R. Le Due)
VII.	Dan Martin, Bureau of Sport Fisheries and Wildlife,
Yankton, South Dakota, has maintained stock cultures of Cyclops
in Missouri River water and Lake Superior water for eight months, and
it appears that permanent cultures have now been established. No
differences between populations of each culture were noted.
Observations on life history using live food cultures have been
completed, and observations on life history using dried foods are
in progress. Stock cultures of Cyclops using dried foods are underway.
All efforts to culture Bosmina under controlled, reproducible
conditions have failed. "Air-locking" appears to be the principal
cause of mortality and no method of preventing this has been found.
Consequently, cultures of Chydorus, a similar sized cladoceran, are
being maintained and future emphasis will be placed on the study of
this organism. (Richard E. Seifert)
Grant 8022j0 - An Evaluation of the Sensitivity of Cladocerans
to Copper, Chromium and Zinc. Miami University, Miami, Ohio. Charles
W. Winner - Project Investigator. Timothy W. Niehiesel - Project
Officer.
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This research grant awarded to Miami University, Miami, under
the direction of Charles W. Winner is designed (1.) to devise acute
and chronic bioassay techniques for use with cladocerans, taking
into account adequate nutrition, the effects of various food
types, and the size of containers in which bioassays are conducted;
(2) to determine how much_variation exists between various species
of cladocerans in their sensitivity to heavy metals; and (3) to
evaluate the application-factor-concept as it applies to acute
and chronic toxicities of heavy metals to cladocerans.
Two quarters of work have been completed, with most of the
work being directed toward the development of a rearing technique
that will ensure reproducible results in both short- and long-term
toxicity testing. It was found that: (1) vitamin-enriched algal
food was superior to either yeast or fish pellets, particularly
in the standard reconstituted water, (2) standard soft reconstituted
water reduced longevity when compared to pond water, however,
longevity and reproduction seem to be consistent for animals reared
in it; (3) that increasing the salt con entration of the standard
soft water to produce a medium hard water increases the longevity
beyond that obtained in pond water; (4) daily transfer of animals
to new media reduced longevity when compared to transfer every three
days; and (5) species sensitivity to toxicants with short-term
tests indicate D. amibgua was more sensitive to copper than
_D.. magna.
Several acute toxicity tests have been completed with Daphnia
mapnn using standard soft water, and algae as food. The LC50 values
for Zn and Cu are intermediate between the fed and unfed LC50
values found by Biesinger and Christensen (1972) in Lake Superior
water. (Timothy M. Neiheisel)
VIII. Under the direction of Lloyd L. Smith, Jr., University
of Minnesota, St. Paul, Minnesota, five lines of experiments are
incorporated in the project plans to evaluate the goldfish and
fathead minnows as possible standard bioassay animals for both
acute and chronic testing: (1) acute lethal bioassays; (2) non-toxic
chronic bioassays (for development of methodology); (3) toxic chronic
bioassays; (4) investigation of a reference toxicant; (5) tests to
determine the effects of temperature and temperature acclimation on
acute bioassays.
Four and one-half of the planned nine series of acute bioassays
have been completed.
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Table 1. Mean LC50 Values for Replicate (N=5) Acute Bioassays of
Four Toxicants to Goldfish and Fathead Minnows (in jag/1).
Toxicant
LC50
NaCl
PCP*
r "*"6
Cr
Guthion
Fathead Minnow




24-hr
7711
.201
136
5.45
96-hr
7547
.197
51
1.81
Asymptotic
7538
.197
17.5
0.72
Goldfish




24-hr
9613
.220
2.65
7.59
96-hr
7086
.201
118
2.35
Asymptotic
7086
.201
30.1
0.95
*PentacliloroT>henol
The pattern of mortality differs widely among the different
toxicants. The onset of mortality is rapid with both sodium
chloride and penta-chlorophenol (PCP). By 48 hours, mortality usually
ceases and the defined asymptotic LC50 is attained by 96 hours. With
hexavalent chromium, the onset of mortality is slow, particularly
with goldfish where there is frequently insufficient mortality by
24 hours to report an LC50. However, mortality then continues to
occur for the entire 11-day test. Mortality from Guthion is somewhat
unusual. The fish react immediately to the addition of Guthion,
and some mortality occurs from 2 minutes to a few hours after
introduction of the toxicant. This mortality appears relatively
independent of toxicant concentration such that equal percentages
of fish may die at low, intermediate, or high concentrations. There
is then a period of approximately 12 to 24 hours when no mortality
occurs, and then fish begin to die again. This resumption of mortality
does correspond to toxicant concentrations. Like chromium, the
defined asymptotic LC50 is not attained by 11 days, although this
level is approached.
An abnormal stock of fish was detected by either a different
pattern of response as with PCP or by decreased 96-hr and asymptotic
LC50's as with sodium chloride and Guthion. Only the hexavalent
chromium did not reveal a difference in response.
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A 23 factorial chronic bioassay of temperature, tank size, and
food was set up with goldfish. The purpose of this experiment
is to develop techniques for chronic bioassays of goldfish, particularly
with the intent of obtaining reproduction in less than one year.
The bioassay has been running for approximately one year.
Table 2. Mean Weight (g) of Goldfish After 11 Months Growth Under
Given Experimental Conditions

20°
C


25°
C

Oregon
diet
moist
Mixed
diet
Oregon moist
diet
Mixed
diet
large
tank
small
tank
large
tank
small
tank
large
tank
small
tank
large
tank
small
tank
82.51
78.01
72.14
1/
112.67
93.72
89.62
80.62
90.07
79.09
87.56
66.27
114.67
97.96
81.48
82.46
Although all fish in the experiment have attained a size and
reached an age at which spawning is known to occur, no reproductive
behavior or secondary sexual characteristics have appeared. Gonads
of fish that have died during the past few months have been undeveloped.
Since it is not desirable for such a chronic bioassay to be conducted
for more than one year, this experiment has not revealed satisfactory
conditions to achieve reproduction.
A chronic exposure of fathead minnows to Guthion has also been
initiated. Initial results Indicate that eggs and fry survival is
satisfactory at concentrations below approximately 2.5 yg/1. (Robert
W. Andrew)
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ROAP 16MO - Complex Effluent Criteria.
The contract to determine suitable sites for evaluating
the biological testing of aqueous effluents was completed. The
final report for this contract listed eighteen possible industrial or
municipal sites acceptable for testing. (Charles E. Stephan)
ROAP 16AAF - The Evaluation of Laboratory Chronic Bioassay Methodology
by Continuous Chronic Exposure of a Natural Ecosystem.
Construction and completion of the test facility on Shayler Run
to validate laboratory findings in a natural stream were completed
in 1968. The first year of testing was designed to accumulate
baseline data prior to beginning a two-year exposure to Cu at a
constant concentration of 120 ppb. The Cu++ exposure was continued
for 35 months with data accumulated over the entire exposure period.
This five-year study was completed in mid 1973, with the details of
the study being written in five parts.
Part I is the introduction to the project dealing with the
background and rationale, the physical aspects of the project,
copper data analyses and the stream water quality. Part II encompasses
the biological aspects of the study, handling j:he fish species
native to the stream and the effects of the Cu exposure to these
fishes. Part III encompasses the effects of Cu*"*" on the invertebrates
of the stream. Part IV addresses itself to the acute and chronic
toxicity tests conducted throughout the study. Finally, Part V
will be a Summary Report for the completed study. Preliminary
results from the data accumulated during this study can be obtained
from the Chief of the Newtown Fish Toxicology Station. (William B.
Horning)
ROAP 05ADD - Tolerance Levels of Ammonia for Aquatic Life
This research grant was awarded to Dr. C. J. D. Brown, Montana
State University, in the fall of 1970 to determine the acute and
chronic toxicity of ammonia to the rainbow trout and the fathead
minnow. During the first year grant period, because of several
set-backs not directly related to the exposure research, very little
was accomplished. However, upon final completion of the physical
testing facility, research was started and is now progressing quite
well. Dr. Brown has retired since the grant was initiated and Dr.
Vance Thurston is now the principal investigator.
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To date, 38 rainbow trout bioassays have been completed using
ammonia, nitrite, copper or ammonia/copper complexes as the
toxicant. The water quality conditions for these bioassays were pH
7.9, temperature 9° C and total alkalinity of 180 mg/1 expressed
as CaC03 . Results from these bioassays show that the lethal
threshold concentration for ammonia using adult rainbows (340 gram
average weight) is 13.5 mg/1; that the lethal threshold
concentration for ammonia to juvenile rainbows (17 gram average
weight) is 26.8 mg/1; that the lethal threshold concentration
for rainbow fry is between the values for adults and juveniles; that
the lethal threshold concentration for copper to rainbows in the
1-4 gram range averaged 0.16 mg/1; that the lethal threshold
concentration for nitrite to rainbows is 0.14 to 0.22 mg/1
(expressed as total nitrogen). When bioassays with copper and ammonia
combined were conducted, the toxicity of the mixture was greater
than the toxicity of copper and ammonia separately. Field studies
are now being conducted to verify these results under natural
conditions. Present projections indicate that the grant will be
successfully completed by December 1974. (Kenneth E. F. Hokanson)
ROAP 21AKH - Tolerance Limits of Hydrogen Sulfide for Aquatic Life
Grant R800992 - Influence of Hydrogen Sulfide on Fish and
Arthropods. This grant was awarded to the University of Minnesota,
St. Paul, Minnesota, under the direction ot Lloyd L. Smith to continue
work on chronic effects of hydrogen sulfide on several fishes and
important fish food organisms. Experimental work continues only
with Hexagenia and Louisiana crayfish as this 5-year grant
nears completion. "No effect" levels for growth of juvenile bluegill
and brook trout were 0.002 mg/1 undissociated hydrogen sulfide in
recently completed bioassays. Progress continues on development of
an analytical technique for measurement of undissociated hydrogen
sulfide in aqueous solutions. A vapor phase equilibration technique
has been developed in which sulfide in water is displaced by nitrogen.
Recovery of 100% sulfide has been achieved by collection on glass
bead columns coated with 0.1 N zinc acetate. Calibration curves are
being developed in order to determine ionization constants at several
temperatures and pH conditions. This procedure is being developed
to improve the accuracy of the current standard methods for total
sulfide measurements and to correct the reported bioassay results
if necessary. The principal investigator has requested a time
extension to prepare the final report since project personnel have
now been shifted to a new Environmental Protection Agency grant to
develop hydrogen cyanide criteria. (Kenneth E. F. Hokanson)
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ROAP 16AAE - Bioassay Technical Support
I.	Organic Compounds in Industrial Wastewaters. In response
to a memorandum from William Donaldson last December, a work plan
was developed to assess the short-term toxicity of approximately
30 organic compounds identified in industrial wastewaters. Static
96-hour tests have now been completed for nine compounds (Styrene,
acetophenone, furfural, cresol, camphor, ethanol, benzyl alcohol,
eugenol and butanol) using one .to two month old fathead minnow larvae at
a test temperature of 20° C. Preliminary LC50 values showed that
P-cresol was the most toxic (19.8 mg/1) with ethanol the least
toxic (13,480 mg/1). All tests were run in natural Lake Superior
water without the addition of any other solvent to aid in solubilization
of the test compound. Two compounds were also tested in soft
reconstituted water according to a formula recommended by the
Committee on Methods for Toxicity Tests for Aquatic Organisms.
Almost identical toxicity results were obtained with this diluent
water when compared to that obtained in Lake Superior water. (Vincent
R. Mattson and Charles T. Walbridge)
II.	Toxicity of a Disinfected Sewage Effluent. The purpose of
this study was to establish short-term lethal levels and no effect
concentrations of a chlorinated, dechlorinated, ozonated and
nondisinfected secondary sewage effluent to selected macroinvertebrates
and fish. A draft of the report has been completed and is undergoing
internal review. This report will be submitted for publication in
the Environmental Protection Agency's Ecological Research Series.
Source of the secondary sewage effluent was from Silver Bay, Minnesota.
Lake Superior water served as the diluent source for the tests. The
7-day exposure tests were conducted with 7 fish and 6 invertebrate
species. The chlorinated effluent was appreciably more toxic than
the other three effluents with short-term animal survival affected
in chlorinated sewage concentrations as low as 6-12 percent. The
corresponding 7-day TL50 values of total residual chlorine to the
fish and invertebrates ranged from 0.08 to 0.26 and 0.21 to >0.81
mg/1, respectively. More than 50 percent of all the animals tested
survived in 100 percent concentrations of dechlorinated and ozonated
effluent and 11 of 13 species in 100 percent concentration of
nondisinfected effluent. With the chlorinated effluent most fish
mortality occurred in the first 24 hours whereas longer exposure
periods were needed to cause invertebrate mortality.
The three species used for the long-term generation cycle tests
were fathead minnows, the amphipod, Gammarus psuedolimnaeus, and
the water flea, Daphnia magna. For these tests the highest nominal
sewage concentrations were 20 percent. Survival and reproduction
of these animals were affected mainly in the chlorinated effluent.
For the fathead minnows, survival and spawning was affected in mean
57

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total residual chlorine levels of 110 and 42 Ug/1. Survival of
the next generation progeny after 30 days was reduced in mean residual
chlorine concentrations of >21 yg/l. The amphipods failed to survive
in the highest mean test chlorine level (123 y gg/l) and did not .
reproduce in mean residual chlorine concentrations > 53 pg/1.
Reproduction at mean residual chlorine concentrations of 19 Pg/l
were lower than the corresponding controls. For the daphnia tests,
yeast and trout chowcerophyll was continuously added to the diluent
water to maintain satisfactory survival and reproduction in the control
tanks. Approximately 10 ug/1 total residual chlorine eliminated their
[survival and reproduction. Statistical analysis of all chronic data
will be performed to further elucidate the levels of residual chlorine
havingno-effeet for all three test species. (John W. Arthur)
III.	Acute Toxicity of Selected Toxicants to Six Fish Species -
Envlrognelcs Company. Purpose is to determine the acute toxicity of
seven compounds to six fish species. The following is a summary
of the results gathered so far and reported in terms of measured
concentrations. The threshold LC50 values for selenium dioxide
to bluegills, goldfish, and fathead minnows were about 18, 9, and 3
mg/1, respectively. The threshold LC50 values for sodium cyanide
to goldfish, fathead minnows, and brook trout were about 0.5, 0.2,
and 0.2 mg/1, respectively. The threshold LC50 values for sodium
arsenite to bluegills and goldfish were about 32 and 33 mg/1,
respectively. The threshold LC50 values for beryllium sulfate to
goldfish was about 60 mg/1 and that for sodium pentachlorophenate
to goldfish about 0.2 mg/1. However, 500 mg/1 lead chloride did
not kill fathead minnows in a four-day static exposure. A range finding
test has been conducted with goldfish and 0-xylene. (Charles E.
Stephan)
IV.	Literature Research on Water Quality Requirements and Toxicology
of Fishes (18050 FQW) - Oregon State University, Peter Doudoroff.
The purpose of this project is to prepare critical reviews of the
world's literature on the toxicology of cyanides, sulfides, ammonia,
and ammonia salts, carbon dioxide, and dissolved oxygen to fish. In
addition, Soviet literature has been collected dealing with fish
toxicology and lengthy English summaries have been prepared.
To date 24 Russian articles on fish toxicology have been
translated into English with lengthy summaries and comments.
(Mary J. Lewis)
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V.	Effects of Chlorinated Effluents In Lake Michigan (18050
HPM) - University of Wisconsin, Alfred Beeton. The purpose is to
determine the effects of chlorinating a large municipal effluent on
Lake Michigan biota and possible corrective measures. Chemical
surveys of the Milwaukee harbor around the Jones Island sewage
outfall showed that active chlorine species were limited to a small
area within 100 M of the outfall. Monochloramine concentrations
of 0.133 mg/1 were measured at the outfall, 0.03 mg/1 70 M from the
outfall and no chloramines measurable 200 M from the outfall in
the surface waters. At a depth of 6 M, no chloramines were detectable
at sampling points surrounding the outfall. The 96-hour TL50 values
of sodium hypochlorite and monochloramine to Cyclops bicuspedata
was determined to be 0.005 - 0.1 and approximately 0.09 mg/1,
respectively. (Donald T. Olson)
VI.	Environmental Impact of Products from the Chlorination and
Ozonation of Municipal and Industrial Waste (R800675), University
of Minnesota, Carlson. The purpose of this grant has been the
determination of chemical and toxicological changes in organic
compounds due to disinfection practices such as chlorination and
ozonation.
It has been shown that chlorine is incorporated into organic
molecules by lowering the pH. Determinations are also being made
of the structural components necessary for toxicity using the Hansch
approach. With chlorinated phenols, toxicity increased to Daphnia
magna with increasing molecular chlorine content. All compounds
that have been investigated have been oxidizable with ozone without
significant toxicity. During this oxidation with ozone, hydrogen
peroxide was produced and is of concern because of its known high
toxicity of 182 compounds to Daphnia magna. (Gary E. Glass)
VII.	Effect of Cyanide on Freshwater Fish and Invertebrates,
University of Minnesota, Smith. Purpose of this project is to
determine the effects of temperature, pH, and oxygen on cyanide
toxicity, the additive or synergistic relationships between heavy
metals and cyanide, evaluate the life history stages most sensitive,
and determine application factors that might predict safe levels of
cyanide or cyanide complexes. This project is scheduled to start
this month, (Robert A. Drummond)
ROAP 1BA021 - Technical Assistance Plan
In 1973 the National Water Quality Laboratory responded to
requests by various organizations to such an extent that 1,979 man-days
59

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(7.6 man-years) of effort were expended. The following Table
demonstrates the relative distribution of these requests in man-days:
USEPA Office of Enforcement (Reserve
1,449
Mining Company Case)
- 94
USEPA Regions
Other USEPA (mostly Headquarters)
213
States, Municipalities and Universities
45
Other Federal Agencies
15
Literature Review for Journal of the
105
Water Pollution Control Federation

Miscellaneous
58
Total
1,979
The most significant responses involved the Reserve Mining
Company litigation, preparation of the freshwater temperature criteria
for aquatic life, and the annual literature review, "Effects of
Pollution on Freshwater Fish", published in the Journal of the Water
Pollution Control Federation (June 1973, 45:1370-1407).
The USEPA's Division of Water Quality and Non-Point Source
Control requested that the staff of NWQL prepare a document for
temperature criteria to be used as their response to Section 304(a)(1)
of PL92-500. The NWQL staff, utilizing the National Academy of
Science revision of Water Quality Criteria, 1968, developed maximum
weekly average temperatures for growth and reproduction, for heated
plume limitations to protect against low temperature shock, and
short-term maximum temperatures to protect against mortality
during the seasons of growth and reproduction for 24 fish species.
These criteria were included in the Proposed Criteria for Water
Quality, Volume I, USEPA, October 1973.
The annual literature review on pollution effects on freshwater
fish involved the acquisition, evaluation, and summarization of 375
publications. This effort will be repeated for the 1973 literature
and is an excellent means for the staff to be aware of current research
being conducted on pollution effects to the aquatic environment.
The Reserve Mining Company case is currently under litigation
with the effort now being expanded to provide technical case
coordination for the Justice Department, USEPA and State attorneys
who are prosecuting the case. During 1973, field and laboratory
studies were completed in order to assess the effects of the
Reserve Mining Company discharge into Lake Superior at Silver Bay,
Minnesota. Reports on the results of these studies were compiled and
submitted to the U. S. District Court, Minneapolis, Minnesota. The
summary given to the Courts of each study in which staff at the
NWQL played a major role is given below.
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Characterization of the North Shore Surface Waters of Lake Superior.
The taconite processing operations at Silver Bay, Minnesota,
are contributing to an increase in turbidity in Lake Superior,
particularly in the area southwest of Silver Bay, but also to a lesser
extent in other nearby areas. Suspended solids in the same area
have been increased as the result of the ore-processing operations.
Material from the tailings discharge is being transported to all
parts of the study area, and current data indicate that this being
the case, the tailings fines are at least being distributed throughout
the Duluth arm of Lake Superior.
Many episodes of green water seen along the North Shore of
Lake Superior are caused by particulate matter from the tailings
operation.
A Study of Western Lake Superior: Surface Sediments, Interstitial
Water and Exchange of Dissolved Components Across the Water-Sediment
Interface
A study of western Lake Superior during the summer of 1972 has
demonstrated that lake sediments contribute dissolved silica, calcium,
magnesium, potassium, manganese and ortho phosphate to the overlying
lake water.
The surface sediments in the area of Silver Bay, Minnesota,
were found to be mainly composed of taconite tailings covering an
area greater than 110 square miles. This layer was found to be more
than 8 cm thick 1-3 miles off shore at Beaver Bay and Split Rock
sampling stations.
The composition of lake water in the area of Silver Bay,
Minnesota, when compared to an area at Hovland, Minnesota (70 miles
up current, NE), showed higher concentrations of potassium (+10%),
manganese (+800%), suspended solids (+800%), and turbidity (+500%).
Suspended solids in the Silver Bay area are mainly composed of taconite
tailings.
During the second cruise period, September 15-24, 1972, current
meter data showed a large river of water (3.6 trillion gallons/day)
flowing past RMC's delta in a southwesterly direction toward Duluth.
Calculations based on Lake current data and increases in concentrations
of measured components show that 4,500 tons of suspended taconite
tailings, 5,100 pounds of dissolved manganese, and 200,000 pounds
of dissolved potassium were being transported "downstream" in the
bottom 30 meters of Lake water per day.
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Interstitial water of lake sediments is much higher in
concentrations of dissolved components. Higher concentrations of
silica (+30%), magnesium (+50%), and copper (+200%) were found
to occur in taconite tailings sediment compared to lake sediments.
Samples of lake water collected at different depths indicate
the lake was generally well mixed. However, measurable concentration
gradients of silica, potassium, manganese, copper and oxygen were
observed at several sampling stations, with metals increasing
and oxygen decreasing close to the water-sediment interface.
Any material which is deposited on the lake bottom and is
subsequently dissolved in the interstitial water is available for
further chemical and biological interaction in the lake system.
Lake currents and turbulence effectively mix the bulk water, eliminating
extensive concentration gradients. Undisturbed lake water-sediment
cores stored for two months at lake conditions, showed distinct
concentration profiles of dissolved substances exchanging from
higher concentrations in the interstitial water to the interface
bulk water. Earlier studies demonstrated that taconite tailings
dissolved under lake conditions in the laboratory. These findings
have now been documented in Lake Superior. Lake sedimented taconite
tailings continue to contribute dissolved components to the
interstitial and bulk water of the lake.
Distribution of Taconite Tailings in Lake Superior and Public Water
Supplies
This data report includes results of the analysis of Lake
Superior water used in the drinking water supplies of Duluth, Two
Harbors, Beaver Bay, Silver Bay, and Cloquet, Minnesota, where
tailings were found to be present. Grand Marais, Minnesota, Superior,
Wisconsin, wellwater and Ashland, Wisconsin, water samples did not
show tailings by X-ray diffraction. Analysis of sediment from all
streams and rivers tributary to Western Lake Superior showed only one
sample with a trace of cummingtonite-grunerite. Water samples saved
at the Lakewood Pumping Station of the Duluth water supply from the
periods 1939-1940 and 1949-1950 did not contain cummingtonite-
grunerite. Samples from 1964-1965, after the Reserve Kining Co.
discharge had begun, contained large amounts of this mineral. These
results confirm the validity of using the amphibole mineral cummingtonite-
grunerite, which makes up a large % of the Reserve Mining Company
discharge, as a valid tracer for the discharge. The asbestiform
morphology of taconite tailings amphibole minerals is described and date
are presented showing the characteristic acicular or fibrous habit.
Stomach and Residue Analysis of Fourhorn and Slimy Sculpins from Two
Areas Along the North Shore of Lake Superior
1. Sculpins living in the vicinity of the taconite plant at
Silver Bay eat fewer Pontoporeia than sculpins living away from it,
indicating a reduction in Pontoporeia in the areas sampled near the
plant.
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2.	At least at certain times, sculpins living near the taconite
plant eat more fish eggs, perhaps due to a reduced abundance of
Pontoporeia.
3.	The results from the first cruise, while less informative,
support the results of the second cruise in regard to the regional
abundances of Pontoporeia,
This part of the study was initiated to determine if measurable
changes in the concentrations of trace metals and organic residues
occur in sculpins which live in the areas of Lake Superior that are
covered with taconite tailings. The work was undertaken with full
realization that the content of some metals such as mercury in fish
may not be determined by the concentration of the metal in the
habitat. Rather, the observed residue may be the result of more
subtle transport interactions of the animal with the environment
and food supply. Moreover, whereas mercury concentrations within
a population may be correlated with size, the concentration of other
metals, such as Fe, Cu, Mn, and Zn, appear to be regulated by
species-specific physiological processes. Furthermore,
it was found that there was no accumulation of copper in the
opercie, red blood cells, and blood plasma in fish exposed to
lethal and sublethal concentrations of copper. Increases in the
concentration of copper in the liver and gill tissue were not
observed at low concentrations of copper until the fish were exposed
to 27 yg/l in water. Thus, the lack of residues does not preclude
a stress on the animal.
The data have shown that discernible trends do not exist in the
concentrations of metals in sculpins along the north shore of
Lake Superior. The absence of any pronounced differences may be due
to insufficient sample size from the population, analytical variations
which are significant at the low concentrations encluntered, unelucidated
physiological processes alluded to above, similarities in the
composition and availability of the metals in the two environments,
or a combination of these effects. Of particular interest is the
fact that, although trends are not apparent along the north shore,
the data presented consistently show that both fourhorn and slimy
sculpins from the north shore contain approximately 1.5 to 2 times
more mercury than do the sculpins from the Apostle Islands or
Keweenaw Bay area. It will be important to determine whether this
variation is a result of different geochemical environments in the
respective watersheds, or if the Apostle Islands and Keweenaw Bay
area are more representative of the natural Lake Superior environment
and the entire north shore region of Lake Superior has been
contaminated.
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Periphyton Growth on Artificial Substrates in Lake Superior
1.	Mean water transparency measurements at two transects
southwest from Reserve Mining Company were less than transects
sampled northeast from the plant,
2.	The predominant current direction measured in the surface
water layers in the study area was in a southwesterly direction,
3.	Diatoms comprised the largest portion of the periphyton
forms counted from both substrates. Summarizing the periphyton forms
by phyla showed no consistent differences at the transect locations
used during each sampling phase.
4.	Significant correlations were found at each depth between
total cells and chlorophyll, and dry weight and solids from the
two-week nylon net determinations.
5.	Higher periphyton growth and solids concentrations were
determined from the most inshore nylon nets as opposed to those
suspended offshore. Mean tailings levels were more equally distributed
except at Split Rock where higher levels were found closet inshore.
6.	Detectable levels of tailings were measured from nets suspended
in the North Shore waters as far as 61 miles northeast and 22 miles
southwest from the plant.
7.	.Statistical considerations in this report neither confirm
nor deny an association between periphyton growth and taconite
tailings.
Effect of Taconite Tailings Upon Lake Superior Periphyton Under
Controlled Conditions
Periphyton growth was monitored during this study by chlorophyll
and cell count measurements and indirectly by oxidizable organic
material. Growth was found to be significantly stimulated in tanks
having tailings concentrations of greater than or equal to 5 mg/1.
Transfer of Elements Associated with Taconite Tailings to the Liver
and Kidney of Rainbow Trout
Liver and kidney of rainbow trout accumulated several radioactive
elements previously associated with neutron-activated taconite
tailings. Livers contained gamma-emitting radionuclides of sodium
(24Na) , potassium (^K), bromine (8ZBr), iron (59Fe) , cesium (l5ltCs),
cobalt (60Co), and rubidium (8t5Rb) from the tailings; the kidneys
were not analyzed for the short-lived materials (2t+Nn, J+2K, and 82Br)
but did contain the other radioactive elements found in the livers.
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Since contamination of the biological samples was avoided,
the transfer of these elements from taconite tailings to internal
organs of fish indicates that at least these elements in tailings
are biologically available.
The Toxicity of Two Flocculants and One Flotation Reagent to
Selected Aquatic Animals in Lake Superior Water
Superfloc 330, Calgon M-500, and Azamine A-3 were found to be
toxic to aquatic life. Twelve-day constant flow tests with lake
and rainbow trout showed that 0.3 mg/liter Superfloc 330 killed
half of the animals tested. Mysis relicta were killed at
concentrations of <0.06 mg/1 Superfloc 330 and a concentration
of 0.9 ing/1 Azamine A-3 in 14 days.
Analyses and Laboratory Experiments with Taconite Tailings
The chemical characteristics of taconite' tailings are complex
and vary with the physical characteristics of the sample, such as
particle size distribution, as well as chemical conditions. Tailings
have been shown by spark source mass spectrometry, neutron activations,
and atomic absorption and emission spectroscopic techniques, to
contain a large number of the elements. Many of these metals can
also be found in water associated with tailings and in larger
concentrations in acid leachates from tailings. Among these
elements are Hg, Cu, Mn, As, Zn, Mg, Se, and Co.
Gas chromatographic and mass spectrometric analysis of organic
residues extracted from composite tailings samples demonstrates
the presence of hydrocarbon oils associated with the tailings
patticles.
X-ray diffraction and electron micrographs of RMC taconite
tailings show the presence of asbestlform minerals. Preliminary
data show 1 x 10H (100 million) fibers of asbestiform minerals
(cummingtonite-grunerite) per milligram of <2 micron size fraction.
Hie total number of particles per milligram of <5u size composite
of taconite tailings was found to be 8.8 X 108 (800 million) by
Coulter Counter.
The presence of an organic complexing agent in an aqueous suspension
of taconite tailings gives the predictable increase in dissolution
of tailings due to the stabilization of metal ions going into solution.
Such complex formation is known to influence metals concentrations
in natural waters.
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Analysis of the overlying and interstitial waters from 64-day-old
settled tailings samples, kept cold and dark, shows large increases
in concentrations of Si02j Ca, Mg, Na, K, Cu, Mn, and Fe from the
original lake water values. These increases are also evident in
measurements of the water just over the sediment interface,
indicating the transport of these dissolved elements into the water
column. A large increase in specific conductance is observed as
the interface is approached and oxygen levels are depressed.
No differences in solubility of taconite tailings were found
due to neutron-activation followed by leaching for the measured
parameters Si02, Ca Mg, Na, K, Ca, Mn, Fe, pH, and conductance.
A Summary of the Studies Regarding the Effect of the Reserve Mining
Company Discharge on Lake Superior
1.	Cummingtonite is an accurate tracer for measuring tailings
in Lake Superior. It is not found in tributaries.
2.	Tailings are deposited on the bottom over most of the
western part of Lake Superior.
3.	Tailings are a major component of the suspended solids of
the western part of the Lake.
4.	Tailings are a major factor causing green water at least
along the Minnesota shore.
5.	Tailings reduce water clarity 25% or more over an area of
at least 600 square miles.
6.	Tailings have been found as a major component of the
suspended solids in the City of Buluth and National Water Quality
Laboratory intakes in every sample that has been analyzed during
1972-1973.
7.	In 500 days, tailings dissolved approximately twice as
fast as a natural lake sediment and to the extent of .8 to 1.0%.
8.	Calcium, magnesium, sodium, alkalinity, manganese and silica
are major constituents comprising the soluble portion.
9.	The discharge contributes at least 160,000 pounds per day
of dissolved solids to the Lake water, not including contribution
from suspended tailings, tailings on the Lake bottom, and those in
contact with the interstitial water.
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10.	A 100 square mile test: area of the lake having high
tailings deposition contained higher potassium and manganese
concentrations compared to a similar area of low tailings deposition.
The interstitial water had higher silica, magnesium, copper, calcium,
and manganese, but lower organic carbon and hydrogen and reactive
phosphate concentrations,
11.	The discharge usually has higher counts of bacteria than
the intake, but coliform counts are relatively low.
12.	Bacteria associated with the tailings are stimulated to
grow or survive longer in lake water with tailings present.
13.	Tailings as low as 4 ppm have a mild stimulatory effect on
phytoplankton growth under some conditions. The manganese content
of tailings may be an important contributing factor.
14.	Results from incomplete experiments suggest tailings may
have a strong stimulatory effect on algal periphyton.
15.	Pontoporeia, an important food species of lake trout and
herring, limited to a few lakes in the U. S., are reduced in numbers
over an area at least some 30-40 miles southwest of the plant. There
is an increase in midges and oligochaetes.
16.	This reduction in Pontoporeia is reflected in altered food
habits of a fish, the sculpin, living in the area of reduced
Pontoporeia populations.
17.	Tailings do not appear to be directly toxic to most
organisms.
18.	Changes in organism populations would have to approach 50%
before they would be detected in the Lake.
19.	Tailings are chemically and biologically active.
The final summary was prepared by Dr. Donald I. Mount, Director,
NWQL as an overview for all the summary reports.
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Publications by Staff Members in 1973
1.	Benoit, D. A., and F. A. Puglisi. 1973. A simplified
flow-splitting chamber and siphon for proportional diluters. Water
Research 7:1915-1916.
2.	Bouck, G. R. 1972. Effects of diurnal hypoxia on
electrophoretic protein fractions and other health parameters of rock
bass (Ambloplites rupestris). Trans. Amer. Fish. Soc-. 101(3):
488-493.
3.	Brungs, W. A. 1973. Continuous-flow bioassays with
aquatic organisms: Procedures and Applications. Biological Methods
for the Assessment of Water Quality, ASTM STP 528, American Society
for Testing and Materials, pp. 117-126.
4.	Brungs, W. A. 19 73. Effects of residual chlorine on
aquatic life. J. Water Pollution Control Fed. 45(10):2180-2193.
5.	Druimuond, R. A., W. A. Spoor, and G. F. Olson. 1973.
Some short-term indicators of sublethal effects of copper on brook
trout, Salvelinus fontinalis. J. Fish. Res. Board Can. 30(5):
698-701.
6.	Eaton, J. G. 1973. Recent developments in the use of
laboratory bioassays to determine "safe"levels of toxicants for fish.
Bioassay Techniques and Environmental Chemistry, Ann Arbor Science
Publishers, Inc., Ann Arbor, MI. pp. 107-115.
7.	Eaton, J. G. 1973. Chronic toxicity of a copper, cadmium
and zinc mixture to the fathead minnow (Pinvephales promelas
RafInesque). Water Research 7:1723-1736.
8.	Gast, M. and Brungs, W. A. 1973. A procedure for separating
eggs of the fathead minnow. Prog. Fish-Cult. 35(1):54.
9.	Glass, G. E. ed. 1973. Bioassay Techniques and Environmental
Chemistry, Ann Arbor Science Publishers, Inc., Ann Arbor, MI. 499 p.
10.	Hale, J. G. 1972. White sucker spawning and culture of
young in the laboratory. Amer. Fish Farmer 4(1): 14-15.
11.	Hermanutz, R. 0., L. H. Mueller, and K. D. Kempfert. 1973.
Captan toxicity to fathead minnows, bluegills, and brook trout.
J. Fish. Res. Board Can. 30(12):1811-1817.
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12.	Hokanson, K. E. F., J. H. HcCormick, and B. R. Jones.
1973. Temperature requirements for embryos and larvae of the
northern pike, Esox lucius (Linnaeus). Trans. Amer Fish. Soc.
102:89-100.
13.	Hokanson, K. E. F., J. H. HcCormick, B. R. Jones, and
J. H. Tucker. 1973. Thermal requirements for maturation, spawning
and embryo survival of the brook trout, Salvelinus fontinalis (Mitchill),
J. Fish. Res. Board Can. 30(7):975-984 .
14.	Horning, W. B. II, and R. E. Pearson. 1973. Growth
temperature requirements and lower lethal temperatures for juvenile
smallmouth bass (Micropertus dolomieui). J. Fish. Res. Board Can.
30(8)-.1226-1230 .
15.	Imlay, M. J. 1973. Effects of potassium on survival
and distribution of freshwater mussels. Malacologia 12(1):97-113.
16.	Lewis, Mary J. 1973. Book Review: Current Awareness
and the Chemist. Elizabeth E. Duncan. Scarecrow Press, Inc.,
Metuchen, N. J. 1972. 150 p. Special Libraries 64(5/6)5261-262.
17.	McKim, J. M., G. M. Christensen, J. H. Tucker, D. A. Benoit,
and M. J. Lewis. 1973. Literature Review: Effects of pollution on
freshwater fish. J. Water Pollution Control Fed. 45(6):1370-1407.
18.	Mount, D. I. 1973. Chronic effect of low pH on fathead
minnow survival, growth and reproduction. Water Research 7:987-993,
19.	Nebeker, A. V. 1973. Temperature requirements and life
cycle of the midge Tanytarsus dissimilis (Diptera: Chironomidae).
J. Kansas Ent. Soc. 46(2):160-165.
20.	Siefert, R. E., W. A. Spoor, and R. F. Syrett. 1973.
Effects of reduced oxygen concentrations on northern pike (Esox lucius)
embryos and larvae. J. Fish. Res. Board Can. 30(6):849-852. •
21.	Smith, W. E. 1973. A cyprinodontid fish, Jordanella
floridae, as reference animal for rapid chronic bioassays. J. Fish.
Res. Bd. Canada 30(2) :329-330. .
22.	Smith, W. E. 1973. Thermal tolerance of two species of
Gammarus. Tran. Amer. Fish Soc. 102(2):431-433.
23.	Stephan, C. E. 1973. Chemistry and fish toxicology.
Bioassay Techniques and Environmental Chemistry, Ann Arbor Science
Publishers, Inc., Ann Arbor, MI. pp. 97-105.
24.	Stephan, C. E., and D. I. Mount. 1973. Use of Toxicity
Tests with Fish in Water Pollution Control. Biological Methods for
the Assessment of Water Quality, ASTM STP 528, American Society for
Testing and Materials, pp. 164-177.
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