EPA-R3-73-OHb ECOLOGICAL RESEARCH SERIES
FEBRUARY 1973
Effects of Chemical Variations
in Aquatic Environments
Vol. II
Toxic Effects of
Aqueous Aluminum to Rainbow Trout
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
-------
RESEARCH REPORTING SERIES
Research reports of the office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include format!en,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
-------
EPA-R3-73-011b
February 1973
EFFECTS OF CHEMICAL VARIATIONS IN AQUATIC ENVIRONMENTS:
Volume II
Toxic Effects of Aqueous Aluminum to Rainbow Trout
By
W. Harry Everhart
Robert A. Freeman
Colorado State University, Fort Collins, CO
Project 18050 DYC
Project Officer
J. Howard McCormick
National Water Quality Laboratory
6201 Congdon Blvd.
Duluth, Minnesota 55804
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price 75 cents domestic postpaid or SO cents GPO Bookstore
-------
EPA Review Notice
This report has been reviewed by the Environmental Protection
Agency and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of
the Environmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or recommenda-
tion for use.
ii
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ABSTRACT
Fertilized eggs, fry, and fingerlings were exposed to aqueous aluminum
complexes in neutral and basic media under constantly flowing, controlled
conditions of aluminum concentration, pH, and temperature. Toxicities of
various concentrations were highly pH dependent. Dissolved concentrations
over 1.5 ppm aluminum caused physiological and behavioral aberrations as
well as acute mortality. Toxic effects of suspended aluminum, though
greater at lower concentrations, do not increase as much as the effects of
dissolved aluminum with higher concentrations. Growth of trout exposed
to high dosages of aluminum was reduced only as long as or slightly longer
than the exposure continued.
Egg and fry bioassays were conducted with exposures in trays and simulated
natural redds. Fertilization was not affected by any concentrations
tested, and most mortalities occurred during hatching and in the post-
swim-up stage. Trends in toxicity were similar to those found with finger-
lings indicating dissolved aluminum to be more toxic than equivalent
suspended amounts.
This report was submitted in fulfillment of Grant Number WP-01398-01,
under (partial) sponsorship of the Water Quality Office, Environmental
Protection Agency.
iii
-------
CONTENTS
Section
I
II
III
IV
V
VI
VII
VIII
Conclusions
Recommendations
Introduction
Methods
Results
Acknowledgment s
References
Publications
1
3
5
9
21
37
39
hi
-------
FIGURES
1. Solubility of aluminum hydroxide complexes (Modified from
Hem, 1967).
Page
2. Schematic diagram of experimental apparatus. 10
3. Arrangement of artificial redds (A,A) and tray divided 18
into two sections (B,B) in troughs.
k. Growth of fingerlings associated with aluminum exposures 23
at pH 8.0.
5. Growth of fingerlings associated with aluminum exposures 25
at pH 8.5 and 9-0.
6. Growth of fingerlings associated with aluminum exposures 28
. at pH 7.0.
7. Growth rates of fingerlings recovering from exposure to 31
5.2 ppm Al at pH 8.5 and 9-0.
8. Growth rates of fingerlings recovering from exposure to 32
9-52 and 5.2 ppm Al at pH 7-0.
vi
-------
TABLES
Mo.
1. Summary of water supply analyses. 15
2. Summary of recovery growth rates 30
3. Comparison of growth rates, H: Slope = Slope 33
X SS U
k. Fertilization and early life history with controls and 35
treatments at pH 9-0.
5. Fertilization and early life history with controls at 36
pH 8.0 (troughs 1 and 2), 0-52 ppm Al at pH 7.0 (troughs
3 and U), 5.2 ppm Al at 7.0 (troughs 5 and 6), and 5.2
ppm Al at pH 8.0 (troughs 7 and 8).
VII
-------
SECTION I
CONCLUSIONS
1. Dissolved and suspended forms of aqueous aluminum are toxic to
rainbow trout,
2. Debilitated condition of fish after only a week's exposure to 5.2
ppm aluminum at any pH tested would have made it difficult for them to
withstand a flow rate characteristic of a trout stream or river during
such activities as migration, feeding, or escaping from predators.
3. Extremely acute mortalities occurred at 5.2 ppm dissolved aluminum
while mortality rates were more chronic with equivalent suspended amounts.
Effects on activity and coloration were more pronounced in dissolved con-
centrations. Both forms advanced gill hyperplasia to the point of near
total fusion of filaments.
4. At 0.52 ppm the occurrence of first symptoms of distress did not ap-
pear until after three to six weeks of exposure. Eventual mortalities
were much higher in number at pH 7.0 (44%) than at 8.0 (8%) where all
aluminum was soluble.
5. At 0.05 ppm of dissolved aluminum, growth was excellent and behavior
normal. It is evident that this concentration can be tolerated without
any obvious effects on growth or behavior. This amount represents sat-
uration at pH 7.0 and is totally soluble at all higher pH's.
6. Mortality rates at 5.2 ppm at pH 7, 0.52 ppm at pH 7, and 5.2 ppm at
pH 8 were all similar indicating toxic effects begin at lower concentra-
tions of suspended aluminum than dissolved aluminum. The concentration
dependency of suspended aluminum toxicity is of very low order while sol-
uble toxicities are strongly concentration dependent.
7. Loss of appetite, greatly decreased activity and gill hyperplasia
initiate sooner at 5.2 ppm than 0.52 ppm (pH 7 and 8), but after six weeks
there was little difference between the fish of the two groups.
8. Neither brief nor extended contact with lethal amounts of both soluble
and/or insoluble aluminum produces irreversible physiological damage as
far as growth is concerned to young trout surviving the exposure.
Fertilization and Early Life History
9. Comparison of mortalities from egg lots fertilized in the aluminum con-
centrations and in the control water did not show any significant advantages
or disadvantages to either.
-------
10. At pH 9.0 only the 5.2 ppm aluminum concentration demonstrated a
higher mortality than the control.
11, Aluminum concentration of 5.2 ppm at pH 8.0 demonstrated the highest
overall mortality. Aluminum at 5.2 ppm and pH 7.0 was next toxic.
Twenty-nine percent of the pH 8.0 mortality occurred at hatching when
young rainbows died just as the embryo was breaking through the shell.
At pH 7.0 eight percent of the mortality occurred just as the head of the
embryo emerged from the egg shell.
-------
SECTION II
RECOMMENDATIONS
General
Basic chemical research is needed to produce more accurate methods of
chemical analysis in natural waters. Much is lost with instruments
capable of precise determinations if we don't know the form of the chem-
ical or if natural complexes interfere with analysis.
The problem of recommending safe concentrations for aluminum is much more
difficult than for most other metals. Methods of determining aluminum
in solution are commonly insensitive to colloidal precipitates. The
aluminum methods proposed by Standard Methods are additionally somewhat
insensitive to polymeric aluminum hydroxide ions. Atomic absorption
methods are less sensitive to aluminum than other common metals.
In the practical sense, the enforcement of regulations regarding permis-
sible aluminum concentrations will be very different since toxic effects
occur at concentrations that will be difficult to analyze in unknown
waters where interference also adds to detection problems. It is certain,
however, that the old rule of permitting 1/10 of the 96 hour TLcQ concen-
tration will not hold. Under some conditions 1.5 ppm aluminum would be
more toxic than 5 ppm under other conditions. The effect of complexation
by ligands other than hydroxide is, as yet, another total unknown.
If hydroxide complexed aluminum is present only as anionic and neutral or
near neutral precipitated forms, a condition that should hold for most
natural waters with pH greater than 5.5, tolerable concentrations of
either form probably should not exceed 0.10 ppm if rainbow trout are to
survive and grow normally.
-------
SECTION III
INTRODUCTION
A real need in the management of our water resources is basic informa-
tion on which to base recommendations for establishing water standards
or criteria for pollutants. The importance of our sport and commercial
fisheries emphasizes the need for detailed studies of the effects of
toxicants on the fish populations. Among the objectives of this grant
were to investigate the toxicity to rainbow trout (Salmo gairdneri) of
soluble anionic species and neutral precipitates of aluminum hydroxide
and lead nitrate complexes under known conditions of pH and concentra-
tion. The aluminum studies were divided into effects of aluminum on
fingerling rainbows and effects of aluminum on fertilization and early
life history.
Aluminum is abundant in the earth's crust, but its natural occurrence
is limited to highly insoluble complex minerals. As a result, the con-
centration of aluminum in natural waters is minute. The importance of
aluminum as a pollutant has been reemphasized by the impending develop-
ment of oil shale mining in several Rocky Mountain States. The
liberation of aluminum from Dawsonite (NaAl(OH),. C0_, which is present in
some oil shale ore, is a biproduct of some oil shale mining processes.
Behavior of aluminum in aqueous solution is extremely complex; it forms
a variety of complexes with water, hydroxide, fluoride, silicate, and
sulfate. The free aluminum ion is rare occurring only in very minute
amounts. Possible forms exhibit vastly different structures and ac-
tivities and will certainly be found to produce different effects on
living organisms.
The chemical nature of aluminum in dilute solution is controversial. Slow
reaction rates, the formation of metastable solids and gels, colloidal and
subcolloidal suspensions, and marginal analytical techniques add to the
problems of determining water associated structures. The reports of Hem
and Roberson (1967), Hem (1968) and Roberson and Hem (1969) provide lucid
explanations of the behavior of aluminum in natural waters.
Solubility of aluminum under conditions where hydroxide complexes dominate
(the vast majority of natural waters) is, briefly, a direct function of pH,
decreasing toward 5.5 and increasing toward both extremes of the pH scale
(Figure 1). Soluble forms in acidic environments are polymeric and cation-
ic while monomeric anions are present in basic media. The critical
importance of pH is exemplified by its control of complexation, polymeriza-
tion, hydrolysis, and solubility.
In acidic conditions, monomeric hydrolysis of the hexaquo complex is fol-
lowed by the joining of two such monomers to form a dimer, sharing the
two OH ions on a common edge and eliminating two water molecules (Hem, 1968).
-------
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
CATIONIC
POLMERS
i r
NEUTRAL
POLYMERIC
PRECIPITATES
ANIONIC
MONOMERS
Test Conditions Investigated
J__ L
3.0 4.0
5.0 6.0 7.0 8.0 9-0
FIG. I SOLUBILITY OF ALUMINUM HYDROXIDE
COMPLEXES (Modified from Hem, 1967)
-------
This shared edge is the basic building linkage in the continuing pro-
cess of polymerization with no apparent limit on polymer size. As
the polymer enlarges, chains and then six membered planar rings of
complexes are formed. The ring systems become less soluble as the poly-
mers enlarge. The basic complexes retain a monomeric configuration but
hydrolize at four sites to produce the anion, (Al (OH),(H_0)- .
Previous investigations into aluminum toxicity have shown little regard
for aluminum's complex chemistry, resulting in widely divergent results.
For example, Jones (1964) reported that 0.07 ppm was the limiting safe
concentration of aluminum to sticklebacks whereas Schaut (1939) stated
that as long as aluminum did not result in critically low pH's (via
hydrolysis) it should have no toxic effects on river fish. Many studies
have utilized conditions which exceeded the solubility of aluminum
with no account of the precipitates. Others have investigated conditions
where pH could have been a lethal factor.
Previous studies with fish include Thomas (1915), Ebeling (1928), Oshima
(1931), Ellis (1937), Sanborn (1945), Minkina (1946), Pulley (1950),
Wallen et al (1957), and Jones (1964).
T
-------
IV METHODS
The "bioassay apparatus was of constant flowing design. Principal com-
ponents were eight troughs , three aluminum administering devices , a
hydroxide diluter, heating and cooling units, and a pressure regulating
reservoir (Figure 2).
Water for the system was tapped directly from a 10" municipal main.
All piping for the system was either rigid or flexible polyvinyl chloride
except for one section of steel pipe carrying high temperature water from
the heaters to a temperature control valve. It was also necessary to use
brass gate valves in this section (See Table l) . All other valves and
fittings were plastic or polyethylene.
Two heaters connected in series with 110,000 BTU/hr were used to heat
a portion of the water to about 60 C for eventual mixing to attain a
temperature of 13 C (55 F) for the experiments. Mixing was achieved with
a Powers UliO photopanel temperature control valve. To prevent supersat-
uration of the heated water, the input to the pressure regulating reservoir
was splashed vigorously and directly so as to keep the water swirling and
liberate excess gases. When ambient water temperature exceeded 55 F, the
entire flow was passed through two 1 H.P. Min-o-cool refrigeration units.
All water was passed through two activated carbon filters connected in
series for dechlorination. The piping of the dechlorinators was arranged
to permit the removal and refilling of one unit without interrupting flow
to the system.
The pressure regulating reservoir was equipped with a 1 1/2" diameter
standpipe which was constantly just overflowing. This assured a constant
head to the system and constant flow through the hydroxide diluter and to
the troughs .
At full scale operation, the system required about 22 liters per minute:
Two liters per minute per trough, a small portion of which came from the
hydroxide diluter which required 3.5 liters per minute, and one liter
per minute diverted from between the dechlorinators to a 3-gallon tank con-
taining several f ingerlings . These fish were used to monitor the perform-
ance of the first dechlorinator . As long as there was no mortality in this
tank, the first dechlorinator was effectively removing chlorine and the
second dechlorinator was not being consumed. The remainder of the 22 liters
overflowed the standpipe of the pressure regulating reservoir.
The aluminum stock solution was dosed to the system by three constant flow
devices, one per set of duplicate conditions, described by Freeman (1971).
A constant flow was metered into the hydroxide diluter where it was mixed
with filtered concentrated WaOH solution metered from a. 2-liter Mariotte
-------
TEMPERATURE
A
MAIN
CO »-
HEATING
\
-1
&
^l
».
\\ 1
diib
\
CO
w
vt
DECHLORINATORS
ALTERNATE
INPUTS
PRESSURE
REGULATING
RESERVOIR
EXPERIMENTAL
TROUGHS
©
REFRIGERATION UNITS
\
HYDROXIDE DILUTER
/ Al.
RECORDING
THERMOMETER
FIG. 2. SCHEMATIC DIAGRAM OF EXPERIMENTAL APPARATUS
-------
bottle. The diluter was constructed of 6.35 mm (1/4") plexiglass,
forming a rectangular trough about 35 cm x 6.2 cm x 7.5 cm. Standpipes
of soft glass tubing inserted in rubber stoppers were placed in appro-
priate holes in the bottom of the trough of the diluter. The effluent
from each standpipe was collected in a glass funnel suspended below the
diluter and carried via rubber tubing to the appropriate trough. Two
baffles at the head of the diluter induced turbulence to assure uniform
mixing of the concentrated NaOH. The flow from the standpipes could be
regulated by diameter of the tube and depth of placement. Enough water
was passed through the diluter to maintain a constant level in its trough
at all settings of the standpipes. At times, small plexiglass filter
boxes packed with glass wool were placed over the smaller standpipes to
filter particles of iron hydroxide precipitated from the water supply
(Table 1) by the high pH of the diluter. This prevented adhesion of the
iron hydroxide to the inside surfaces of the standpipes and the resulting
disruption of desired constant flow rate.
The reagents were mixed and diluted in large orifice boxes at the head
of each set of duplicate troughs. Each box was fitted with a perforated
vertical and sloped horizontal baffle to induce turbulence and insure
uniform dispersion of the reagents. Holes on opposite sides of the
boxes delivered equal amounts of test solution to each of the duplicate
troughs. Each box was calibrated to insure maintenance of correct dilu-
tion and flow rate. The remainder of the 2 liters per minute per trough
was drawn directly from a manifold connected to the pressure regulating
reservoir. Turnover time for the troughs was 69 minutes.
The fiberglass troughs measured 5' x 1' x 1' (152.4 cm x 30.5 cm x 30.5
cm). A mean depth of 29.7 cm was maintained by a standpipe at the end
of each trough. A perforated weir blocked the standpipe from the rest
of the trough, and 1/4" mesh screens were placed over the troughs to pre-
vent fish from jumping out.
A sensor of a Foxboro recording thermometer was placed midway in one
trough to monitor the temperature of the system.
A Corning Model 12 pH meter, graduated to 0.005 units, was used to
monitor pH and a Gallet 7 jewel stopwatch graduated to 1/10 second was
used to monitor the various flows of the system.
The NaOH was a purified flake from J. T. Baker and Co. The aluminum for
the system was supplied by anhydrous aluminum chloride from Allied Chem-
ical Co.
This study was designed to examine the toxic effects of aluminum at dif-
ferent pH's and concentrations. Concentrations chosen were significant
in terms of possible solubility within the range of pH to be investigated,
The range of conditions investigated and corresponding behavior of the
aluminum were:
11
-------
Total Aluminum
Added
5.2 ppm
0 . 52 ppm
.052 ppm
PH
7.0
1% dis
99% sus
10% dis
90% sus
100% dis
PH
8.0
10% dis
90% sus
100% dis
100% dis
PH
8.5
32.% dis
68% sus
100% dis
100% dis
PH
9.0
100% dis
100% dis
100% dis
dis - dissolved
sus - suspended
The mean solubility product prescribed by Hem (1967), K^ = 1.95+ 1 x lO-1?,
was used to calculate the amounts of aluminum necessary to be added to
achieve desired conditions, as well as those forms present under observed
conditions. The rapid turnover time of the experimental tanks plus the
movement of the fingerlings were sufficient to prevent significant settling
of the precipitates at supersaturated conditions. When settling was ob-
served it occurred behind the perforated weirs at the lower end of the
troughs.
The procedure supplied by R. W. Smith and J. D. Hem (M.S.) involving the
kinetic complexation of aluminum with ferron was employed to determine the
various forms in which the aluminum species exists in aqueous media. The
three forms described by Smith and Hem are the monomeric form, Al , des-
cribed as Al , (A1(OH)_ or Al(OH),-, the polynuclear form, Al , described
as a six-membered aluminum hydroxide ring, and the colloidal form Al ,
described as Al(OH) , aluminum hydroxide.
The only significant deviation from the procedure outlined by Smith and Hem
was that anhydrous aluminum chloride was used to prepare the aluminum
standard stock solution because of the difficulty encountered in preparing
the solution from aluminum metal. This should not in any way affect the
results and may in this case actually be desirable since the source of
aluminum in the toxicity experiment was also anhydrous aluminum chloride.
Using this procedure on fresh solutions containing 5.2 ppm aluminum, the
curves similar in shape to those described by Smith and Hem were obtained.
The following data were then derived from the curves and the calibration
curve using the methods described by Smith and Hem.
In general, this method entails extrapolation of the flat portion of the
curve to zero time and estimating the amount of Al + Al from the absorb-
ance. The amount of Al is determined by the extrapolation of the curved
portion of the curve to zero time. Al is determined by subtraction of Al
+ Al from the total aluminum in solution, in this case 5.2 ppm.
12
-------
Initial pH A1H Alb Al° %A1 diss*
6.0 1.1 2.2 1.9 21
7.0 1.3 2.4 1.5 25
8.0 1.8 2.4 1.0 35
9.0 4.0 0.8 0.4 77
*% Al dissolved - Ala /Al total X 100%
Although they do not agree absolutely, the values for the percent dissolved
aluminum as a function of pH follow the order of the calculated values.
Fluoride ion was periodically measured using a fluoride specific ion elec-
trode and was consistently found to be very near 1.0 ppm, the value the
City of Fort Collins claims to maintain. The method did not work at 0.52
ppm. Smith and Hem note that fluoride ion interferes with the analysis
at low aluminum concentrations.
An interesting experimental result was observed by allowing the ferron to
react completely with the aluminum species. In every case, regardless of
pH, if the ferron-aluminum complex absorbance was measured 12-24 hours after
the beginning of the experiment, all solutions were observed to have a final
absorbance reading equivalent to a concentration of 5.1;K1 ppm. This
measurement is representative of the total concentration of aluminum species
in solution and agrees with the atomic absorption spectroscopy measurements
in which the total aluminum concentration of solutions was found to be
5.0ppm+%.
Considering the difficulty in establishing steady state conditions in the
troughs and the slow approach to equilibrium of the aluminum distribution,
the agreement between calculated and experimentally determined aluminum dis-
tributions is better than might be expected. Either set of values illus-
trates that experimental conditions were chosen to span the range of
distribution from dissolved to suspended aluminum.
Aluminum chloride was dissolved in tap water in the reservoirs of the
constant flow devices. These devices were stopped for about 10 minutes
for the refilling process. The hydrolysis of A1C1., results in highly acidic
stock solutions. About 20 ml of concentrated hydrochloric acid was added
per 200 liters of stock solution to insure rapid and total solution of the
aluminum chloride when the reservoirs were refilled. Concentrations were
adjusted so that either 18, 19, or 20 ml per minute, depending on which
device was used, would supply the correct concentrations when diluted to
two liters in the headboxes. Flows of these devices were monitored at
three or four times daily during the experiment. The mean and variance of
the concentrations of the experiments were calculated from these data.
This was possible since all factors influencing test concentrations, namely
concentrations of the stock solutions, delivery rates, and dilution rates
were known.
13
-------
Ambient pH of the input water plus the addition of various concentrations
of the acidic aluminum solutions required that a basic additive be intro-
duced to bring the test pH to the range desired and convert the cationic
aluminum to its anionic form.
The pH of the system was controlled by dilute NaOH solution from the
hydroxide diluter. A concentrated solution of 9.8 N NaOH was delivered
at 1.4 ml per minute into 3.5 liters of water at the head of the diluter.
Various amounts of sodium hydroxide were diluted via the diluter standpipes
to bring pH's to test levels. The pH of the system was recorded three or
four times daily and used to calculate the pH mean and variance of the
system.
Data were taken from the continuous trace of the recording thermometer
at three-four intervals to calculate mean and variance of temperature.
During the course of the tests, water samples were collected at about two
week intervals for analysis of dissolved solids, dissolved oxygen, alkalin-
ity, chloride, nitrate, silicate and sulfate (Table 1). Determinations
were made by the procedures described in Standard Methods for the Examina-
tion of Water and Wastewater (1965). Metallic cations were determined by
atomic absorption and emission spectroscopy.
Fingerlings
Limitations in laboratory size and available water made it necessary to
raise the fingerlings in a water supply other than that used for the
toxicity tests. Trout were hatched and reared to test size in well water
and exposed to aluminum, after acclimation, in tap water (Table 1). All
trout used in these experiments were hatched in the laboratory. Eyed eggs,
spawned from infectious-pancreatic-necrosis-free brood stock, were super-
ficially disinfected with Merthiolate and hatched in tray incubators. The
eggs were hatched and fry raised to test size in well water. All fish used
in the experiments had a recorded disease free history. Minor conditions
of gill hyperplasia were prevalent when fish were held in well water. This
condition rapidly disappeared when the trout were transferred to tap water
for acclimation. Troughs holding the experimental subjects were cleaned
daily. The well water supplied 10' x 1' x I1 (305 cm x 30.4cm) troughs
at mean depth of 27.2 cm at a rate of 8 liters per minute. Fry and
fingerlings were fed at all times according to a feeding chart modified
from Deuel et al (1942). Clark's pelletized commercial trout chow was the
food source.
Fry up to about a month old were fed six times per day. Older fry and
fingerlings both before, during, and after testing were fed four times per
day. Growth of all test animals was recorded at approximately two-week
intervals and feeding rates adjusted according to growth.
Pretest weights were determined by weighing a sample of the pretest finger-
lings. Weights of test animals during the test and during recovery were
determined by weighing the entire group.
-------
Table 1. Summary of water supply analyses
This water supply was used for the aluminum toxicity determinations. Concentrations are in mg/1,
Date
1/21/70
2/2/70
2/21/70
3/25/70
4/13/70
4/27/70
5/7/70
5/23/70
6/16/70
Dissolved
Oxygen
10.6
9.6
10.0
10.0
9.0
8.6
8.8
8.4
9.2
Total
Alkalinity3
40.3
40.9
40.9
46.2
50.6
51.0
39.3
17.1
13.6
£*£
7.65
7.50
7.60
7.72
7.75
7.62
7.44
6.85
6.90
Temp. (°C)a
5.0
4.4
3.3
3.4
7.2
8.3
11.7
12.5
12.9
Cl
2.65
7.75
3.50
0.75
2.50
1.50
3.00
NO., SO, S:00
""" j "^4 ^
0.25 2.88 12.75 ^ H
0.30 *** 11.40
0.13 *** 10.80
0.16 *** 10.53
0.20 *** 12,30
~
0.22 *** 12.20
0.23 *** 10.20
cu ro
en
00 rt
CO
O
pi
rt
oo H
LD CO
rt
&> CO
"O P)
rt
O
SB
H
en
rt
^J CO
O P
rt
T)
CS
Not determined.
a Before adjustment.
* Less than 0.05 ppm.
** Less than 0.01 ppm.
*** Not detectable.
-------
Continuation of Table 1
H
ON
Date
1/21/70
2/2/70
2/21/70
3/25/70
5/7/70
5/25/70
Ca (2)
16
20
10
11
22
22
Mg (10)
5.0
1.0
0.2
0.2
0.4
0.4
Na (10)a
2.9
1.0
2.0
1.1
4.5
4.5
K (1)
4.4
*
*
*
*
A
Al (l)a
A
*
*
*
A
A
Fe (10) Zn (1)
0.20 **
0.20 **
0.15 **
0.45 **
0.45 **
CU (1)
A
A
A
A
Parentheses indicate confidence factors for data, i.e. the true value lies between that reported
multiplied by or divided by the factor in parenthesis.
-------
Test animals were transferred without tempering from the well water to
the tap water for pretest acclimation. Fish for all tests, except at
pH 7.0, were acclimated to tap water and pH for 8 days prior to intro-
duction to test conditions. At pH 7.0 acclimation was limited to 3 days.
In all cases, fish adapted to the change in water without any noticeable
behavioral or physiological problems. Mortalities during the acclimation
never exceeded 1%. During this time the pH was raised at the rate of 0.2
units per day to the test pH. At the end of the acclimation, healthy
trout were selected from this group and placed directly into the various
test conditions.
Behavior, growth, and mortality were recorded. Controls were weighed
once or twice during the test to adjust feeding rates. The rates for all
trout were then adjusted according to the growth of the controls rather
than weighing the test fish and possibly altering mortality rates. Test
fish were fed the prescribed food amount or as much as they would consume
daily, whichever amount was smaller.
At the end of the exposures all groups were weighed. Some recovery sub-
jects were transferred directly back to well water while others remained
for a time in dechlorinated tap water. Growth of these fish was deter-
mined at about 10-day intervals by weighing the entire group. From these
data the long term effects of aluminum on growth could be determined.
Time of death, weight of fish at death, and trough number for each mor-
tality were recorded. From these data it was possible to determine the
time of mortality of 50% of the group. Averages from the duplicates pro-
vided the reported TL50 time. Smirnov's statistic (Birnbaum, 1962), a
non-parametric statistic used to compare two unknown distribution functions,
was used to compare the empirical distribution functions of mortality
rates between sets of duplicate conditions. The suspected dependence of
survival time on size was also investigated by a simple regression; however,
no significant relationship was discovered.
Fish were held prior to testing and for recovery in well water of the fol-
lowing analysis: D. 0., 5.2-5.8 ppm; total alkalinity, 301-335 ppm; pH,
7.2-7.5; temperature, 16.5-16.7 C; chloride, 11-20 ppm; nitrate 2.9-3.2
ppm; sulfate, 62-86 ppm; silicate 14-17 ppm; calcium, 200-220 ppm; sodium,
125 ppm; potassium, 0.4-1.0 ppm; aluminum, iron, zinc and copper all less
than 0.05 ppm.
Fertilization and very early life history
Eggs and sperm were collected from IPN certified free brood stock and fer-
tilized in control water and in the aluminum concentrations. Fertilizing
in control and treatment waters gave a chance to observe the effects of
the aluminum on eggs, sperm, and the fertilized egg. In a further effort
to approach natural conditions half of the eggs were reared in artificial
redds of gravel and the other half in trays (Figure 3). An effort was
17
-------
FIG. 3. ARRANGEMENT OF ARTIFICIAL REDDS (A,A) AND TRAY
DIVIDED INTO TWO SECTIONS (B, B) IN TROUGHS.
18
-------
made to deposit 200 eggs in each redd and in each tray. However, there
was some variation in the actual number which was determined volumetrieally
to reduce any handling stress. Concern over the hatching success in the
artificial redds dictated the use of trays. Dead eggs could not be removed
from the eggs in the gravel and fungus could well have been the cause of
loss rather than the aluminum. Eggs in the trays were examined daily and
dead eggs removed.
In the first run, controls and experimentals were all at pH 9-0 with
treatments of 0.052, 0.52, and 5-2 ppm aluminum. Replicates of each were
provided. Half of the eggs were fertilized directly in the treatment water
and the other half of the eggs were fertilized in the control water and
then transferred to the treatment tanks, but separated in redds and trays.
In the second run controls were carried at pH 8.0, and the treatment tanks
at pH T-0 with aluminum concentration of 0.52 ppm, pH 7-0 and aluminum con-
centration of 5-2 ppm, and pH 8.0 and aluminum concentration of 5.2 ppm.
19
-------
V RESULTS
Fingerlings
Aluminum Toxicity at pH 8.0
Duplicate concentrations of 0.05 ppm, 0.52 ppm, and 5.2 ppm aluminum
were tested. Age of test fish was approximately 6 months at the start
of the test. All exposures in this experiment were conducted for 45
days (1080 hours).
The control troughs received approximately.^. 39 mg NaOH per liter to
maintain a mean pH of 8.02 (S_ = 8.4 x 10 ). No mortality occurred
among the control fish for the duration of the test. These fish ap-
peared healthy and active throughout the test.
At pH 8.0, 90% of the 5.2 ppm aluminum was insoluble. The insoluble
aluminum hydroxide remained suspended in the troughs giving a slight
whitish irridescent cast to the water. The coloration of slight accu-
mulations of settled precipitate behind the weir at the downstream end
of the troughs indicated that some iron was being coprecipitated by the
aluminum hydroxide. The mean concentration of total aluminum added was
5.23 ppm (s = 0.051). The mean pH for the test was 8.02 (s = 8.9 x
10 ), thus a mean concentration of 0.55 ppm dissolved aluminum was
maintained.
Within 24 hours an overall slowdown in activity was observed in the 5.2
ppm troughs, and almost all interest in feeding had disappeared. On no
day of the test was more than half of the prescribed amount of food con-
sumed. After five days gill hyperplasia was evident in many of the trout
in both troughs. By the sixth day, many fish were experiencing momentary
equilibrium problems or swimming on their backs breaking the surface,
increasing in severity until the fish completely lost equilibrium and
remained upside down for a period of from 3 to 24 hours preceding death.
The coloration of these fish was very dark, many nearly black. During
the second week of exposure, periods of increased activity and feeding
occurred for durations of up to one day. After the fourteenth day, how-
ever, no increases in activity were ever observed. Overall physical
condition steadily deteriorated with deaths occurring regularly. Within
30 days these fish lost all negative photoaxis to sunlight and all fright
reaction to approach to the troughs by humans. Late in the test, trout
could be prodded without attempting to flee. Respiratory rates were con-
siderably higher than any other of the tests at pH 8.0.
After 45 days, weights of the 12 survivors averaged 30.2% of the control
weight as opposed to averaging 106.5% of the control weight at the start
of the test (Figure 4). Prior to release in well water, for recovery,
experimental fish were tempered to it for a half hour. From this
21
-------
additional stress, however, 11 of the 12 died within 24 hours. The one
survivor had returned to normal activity and feeding after six days.
2 _3
A mean concentration of 0 .514 ppm aluminum (s = 5.8 x 10 ) was main-
tained at a pH of 8.03 (s2 = 7.2 x 10~ Representing total saturation
with soluble aluminum. Under these conditions, fish were slightly less
active than the controls after a single day. After three days, a definite
intermediate condition between the controls and the 0.52 ppm test existed.
By the sixth day, consumption of food was considerably reduced. These fish
consumed their total prescribed diet on about one half of the test days,
feeding little if at all after 30 days. Darker coloration was evident in
this test also, although not to the degree of the 5.2 ppm test. After 10
days exposure, there was a noticeable decrease in the fright reaction of
the fish in these troughs. Periodic fluctuations in overall activity
were observed throughout the test. Gill hyperplasia was evident in about
half of the fish at 21 days. At the conclusion of the test, low power
microscopic examination revealed that nearly all fish were showing hyper-
plasia to some degree.
Although only two deaths occurred very late in these tests, symptoms
preceding mortality were identical to those exhibited by fish in the higher
concentrations. Although these fish showed a net weight increase the aver-
age increase was 38% of the control increase. Their weights averaged 58.1%
of the control weight following the exposures as opposed to 95.9% of the
control weight at the start of the test (Figure 4). Continued exposure
under these conditions would undoubtedly produce high mortalities.
At the conclusion of the test, five fish from each trough were placed
without tempering into well water for recovery. Within 48 hours all symp-
toms of the exposure except the gill hyperplasia had disappeared. In the
following 16 days, these fish showed extremely rapid weight increase
comparable to the controls. No additional mortalities occurred and at the
conclusion of the recovery period the fish were normal in all appearances.
A set of duplicate concentrations were also run at a mean concentration of
0.0516 ppm aluminum (s « 6.0 x 10 ) at pH 8.04 (s = 3.3 x 10 ). These
conditions required the addition of 2.55 mg NaOH per liter to maintain pH.
This amount of aluminum is the saturation point at pH 7.0 and is 100%
soluble at all higher pH's.
These fish were healthy and active throughout the test, showing fast growth
and normal behavior at all times. They averaged 101% of the control weight
after the test as opposed to 97.2% of the control weight at the start of
the test. Further testing of this concentration was not conducted since
in the range of our investigations the form of this amount of aluminum
would be unchanged.
Aluminum Toxicity at pH 8.5
At pH 8.5, about 3.19 mg NaOH per liter was necessary to maintain the control
pH, while the test troughs required about 27.0 mg per liter. Fifty finger-
lings were placed in each trough. Of the.mean 5.14 ppm aluminum (s = 8.8 x
) added at mean pH 8.48 (s = 8.0 x 10 ) a mean of 1.57 ppm aluminum
22
-------
UJ
UJ
8/27 9/10 9/24 10/8 10/22 11/22
I/I
1/8
1/50
2/21
3/8
DAYS
FIG. 4. GROWTH OF FINGERLIIMGS ASSOCIATED WITH ALUMINUM EXPOSURES
AT pH 8.0.
-------
was in solution. Trout used in these tests were about 6 weeks»old at
the beginning. Mean temperature for these tests was 12.6 C (s = 0.14)
On introduction to the test conditions, fish showed immediate stress,
darkening in coloration, and a total loss of interest in food. Mortal-
ities began to occur almost immediately with all the symptoms previously
described. In addition, fecal casts were obvious from all fish. This
test was terminated after 222 hours (9 days 6 hours). Aluminum addi-
tions were stopped and pH was allowed to return to ambient, 7.7. Within
12 hours, about half of the fish had returned to normal coloration and
began taking small amounts of food. After 72 hours, however, there was
still no fright reaction in these fish. Overall normality seemed to have
returned after five days, except for persisting gill hyperplasia. On
the 8th day these fish were transferred without tempering into well
water. For the next 10 days they showed no weight increase. After 18
days recovery, weighings indicated a near normal growth rate had resumed.
At this time the weights of these fish averaged 55.2% of the control
weight as opposed to averaging 106.9% of the control weight at the start
of the test (Figure 5). After 49 days recovery they averaged 63.9% of
the control weight. Mortalities during the first 60 days of recovery
were 3% as opposed to 5% among controls. In the next 80 days there were
no additional mortalities. After 140 days recovery, test fish averaged
82.3% of the control weight.
Aluminum Toxicity at pH 9.0
At pH= 9.0 approximately 5.07 mg NaOH per liter was added to maintain con-
trol pH while 35 mg per liter was required to maintain the test pH. The
mean control pH,was 8.99 (s = 5.5 x 10~ ). The mean test pH was 8.99
(s? = 8.1 x 10 ) with a mean aluminum concentration of 5.2 mg per liter
(s = 8.0 x 10 ). Under these conditions the mean concentration of
soluble aluminum was 5.5 ppm (97.15%) and 0.15 ppm suspended (2.85%).
Six week old fingerlings were used for this test. Mean temperature for
the test was 12.6°C (s = 0.18).
These fish showed immediate shock and heavy mortality within 48 hours.
Fecal casts were wide spread and gill hyperplasia was universal within
three days. No fish ever accepted food. Coloration was very dark and ac-
tivity was minimal. Test conditions were terminated after 113 hours (4
days, 17 hours) by stopping the aluminum addition and allowing pH to
return to ambient. Within 36 hours some fish began feeding and about one-
half showed lightening of coloration. No fright reaction to approach ap-
peared until after 5 days. By the sixth day, however, activity and
feeding were near normal. On the eighth day some fish showed an apparent
relapse as some test symptoms reappeared. On the tenth day the entire
group was transferred to well water.
These fish also showed a loss in weight for the first two weeks of recovery
resuming normal growth after 30 days recovery. Before testing, their
weights averaged 116.75% that of the controls, but this dropped to 50.0%
2k
-------
ro
9.0 CONTROL
8.5 CONTROL
FIG. 5. GROWTH OF FINGERLINGS ASSOCIATED WITH ALUMINUM EXPOSURES
AT pH 8.5 AND 9.0.
-------
before climbing to 76.8% after 60 days recovery (Figure 5). During the
first 140 days of recovery there were no mortalities among the recovering
fish. After 140 days recovery, test fish averaged 85% of the control
weight.
Aluminum Toxicity at pH 7.0
A final series of tests was carried out at a nominal pH of 7.0 At the
beginning of these tests, the ambient input pH to the system was about
7.4. Since the pH controlling mechanism is suitable only for controlling
pH's by either raising or lowering at a given time, not both simultaneously,
it was decided to run the control and 0.52 ppm test without hydroxide
addition and control only the 5.2 ppm test. (The addition of 0.52 ppm alum-
inum did not reduce the pH of the system enough to permit control by
hydroxide addition. At 5.2 ppm approximately 13.4 mg NaOH was added per
liter for pH control. Solubility of aluminum in the pH range from 6.5 -
7.0 is practically constant so the absolute control of pH in this range
is not nearly as critical as at a higher pH.
Complications were encountered when the water flow to the entire system
became reduced for some unknown reason. As a result, the flow to the
control troughs was reduced to 1.4 liters per minute for the test. All
flow rates were in excess of that needed to support the quantities of fish
used. No stress reactions were observed among the controls as a result of
this adjustment.
On the seventh day of these tests, hydrated aluminum sulfate was introduced
to the municipal water supply to precipitate the increased turbidity due
to heavy snow melt run off in the Cache la Poudre River. These additions
were continued for the duration of the test. According to treatment plant
personnel, the alum was added empirically, but averaged 250 Ibs. per
million gallons of water. Such additions resulted in a total concentration
of about 2.4 ppm aluminum with about 0.04 ppm soluble. Since the plant
utilized large settling basins and is situated about 25 miles from the
University, it is assumed that only the soluble portion was present in the
input to the system. Such an amount is negligible in comparison to the
additions of the tests themselves. Some curious behavior on the part of
the control trout, namely refusal to feed, was noted on about 5 days of
the test. The alum additions may be accountable for this. As a result of
these additions, the input pH to the system fluctuated far more than
normally. The pH of the 5.2 ppm test was difficult to regulate on this
account.
The pH of the input water eventually became reduced to levels where the
hydroxide diluter could have been used to raise the pH of the controls and
0.52 ppm tests. The solubility of aluminum is nearly constant in the
region of the test pH. Sudden increases in the pH of the tests would ex-
pose the fish to conditions unlike those being tested if the input pH
should arise sharply and hydroxide additions were continued, so no hydroxide
was added. For the same reason, the pH of the 5.2 ppm test was held below
26
-------
7.0. This precaution proved to be well founded as on several occasions
the input pH increased to well above 7.0, and fish would have been ex-
posed to acutely toxic conditions invalidating the test, had hydroxide
additions been in progress.
The trout were eleven weeks old at the start of the test. All exposures
were conducted for 45 days (1080 hours). The mean temperature for the test
was 12.8°C (s = 0.28). Twenty-five fish were used in each trough. The
mean pH for the controls was_2.44 (s = 2.9 x 10 ) for the first seven
days and 6.85 (s = 4.4 x 10 ) thereafter.
2 -3
At a mean concentration of 0.513 ppm aluminum added (s = 6.3 x 10 ),
the pH was 1.27 (s = 2.8 x 10~ ) for the first seven days of exposure
and 6.52 (s = 4.0 x 10 ) thereafter.
Under these conditions, fish showed a darkening of coloration and reduced
feeding activity beginning on the seventh day. Gill hyperplasia was ob-
served on some individuals. These symptoms gradually increased in incidence
and severity for the first five weeks of exposure. Mortalities began to
occur on the twenty-third day and occurred regularly thereafter. At the
conclusion of the exposure (45 days) these tests had not achieved 50% mor-
tality. The average TL n value reported for these conditions is the
average of times projected by linear regression of the mortality rates. At
the conclusion of the test these trout were feeding very erratically. Their
average weight was 32.5% of the control average as opposed to 94.1% of the
control average at the start of the tests (Figure 4). After 74 days recov-
ery the average weight of test fish remains only 43.1% of the control
average.
Another pair of-duplicate exposures received a mean 5^14 ppm aluminum
(s = 8.1 x 10 ) at a mean pH of 6.80 (s = 6.8 x 10 ). Under these con-
ditions feeding stopped after four hours exposure. After four days, ac-
tivity was drastically reduced and coloration was very dark. Gill hyper-
plasia was observed on all individuals within seven days. These trout
exhibited a small degree of fright reaction throughout the test and began
feeding very lightly for the last three weeks of exposure. Mortalities
occurred at a regular rate throughout the test again preceded by a total
loss of equilibrium. No fecal casts were observed.
Survivors of the test were extremely emaciated, averaging only 24.1% of the
control weight as opposed to 91.8% of the control average at the beginning
of the test (Figure 6). A single mortality occurred among the controls
during this test. Resumption of growth among these fish proceeded very
slowly as they averaged 29.5% of the control weight after 74 days recovery.
Smirnov's statistic was used to verify the agreement of mortality rates in
sets of duplicate conditions. These tests indicated no significant differ-
ence among mortality rates of any set of duplicates (a = .10) except for
the test at pH 9.0. Although by the conclusion of that study mortality
-------
ro
CO
FIG. 6. GROWTH OF FINGERLINGS ASSOCIATED WITH ALUMINUM EXPOSURES
AT PH 7.0.
-------
rates were identical, early differences caused the tests to be sig-
nificantly different (a =.01). The severity and short duration of this
exposure are undoubtedly causing these variations.
As a result of noting the significant reduction in growth rates of the
fingerling rainbows during the actual bioassay determinations experimental
fish were maintained for several months following exposures of aluminum
(Table 2).
Statistical analysis comparing the slopes of the recovery growth plots
(Figures 7 and 8), as suggested by Dixon and Massey (1969) reveal no sig-
nificant differences (a = .10) between growth rates of any group of test
fish when compared to controls at the same weight (Table 3). The immediate
implications of these findings suggest that neither brief or extended con-
tact with lethal amounts of both soluble and/or insoluble aluminum will
produce irreversible physiological damage as far as growth is concerned,
to young trout surviving the exposure. Mortality rates returned to the
control rate in all cases except for the test at pH 8, 5.2 ppm aluminum.
In these conditions, 11 of 12 survivors succumbed during the first 24 hours
of recovery, presumably due to their extremely debilitated condition, stress
of handling, weighing and changing the water supply. Effects of maturation
and fertility were not studied and may be significant.
Thus, the growth of trout exposed to high dosages of aluminum under these
conditions will be reduced only as long as or slightly longer than the
exposure is continued. In the case of a 45 day exposure, this means that
the survivors are essentially 45-60 days behind in their development. In
young fish this may easily account for a 100% weight difference even after
an extensive recovery period. Applying this to natural situations, the
size differential and subsequent delay in development could be important
factors in susceptibility to predation, procuring available food, negotiat-
ing currents and spawning.
Fertilization and early life history
The research to study the effects of aluminum on the fertilization and early
life history was designed to determine the toxicity by exposing eggs in
simulated natural redds and in submerged screen trays (Figure 3) .
Culturing the eggs in the simulated natural redds was unsuccessful as very
heavy mortalities (80% to complete) indicated eggs and sac fry were addi-
tionally stressed by conditions (likely poor water flow through the gravel)
in the redd along with the aluminum exposure. The few eggs that did develop
to hatching lagged behind the eggs in the trays. Simulated natural conditions
precluded disturbing the gravel for accurate counts at the eyed and hatching
stages. Dead eggs could not be removed for the same reason, and fungus
developed in all redds to provide an additional source of mortality. Likely
a more sophisticated system of forcing water through the gravel would have
improved the conditions.
29
-------
Table 2. Summary of Recovery Growth Rates
Tests
pH 8.0
Concentration control
Exposure time
(days)
*Average weight
start bioassay (gins)
*Average weight
end bioassay (gms)
*Mortality during
bioassay (%)
Average weight
end recovery (gms)
Recovery time (days)
^-Mortality during
recovery (%)
45.0
7.4
21.6
.0
30.0
16.0
8.0 8.0 8.5
.52 5.2 ppm control
45
7.
12.
8.
17.
16.
45.0
1 7.9
5 6.5
0 77.0
5
0 1.0
0 83
9.25
2.0
3.0
.0
73.5
161.0
5
8.5 9.0
5.2 ppm control
9.25
2.2
1.5
51.0
58.3
161.0
3
4.
1.
3.
t
73.
165.
2
7
9
5
0
5
0
9.0 7.0
5.2 ppm control
4.7
2.2
2.2
67.0
65.3
165.0
2
45.0
3.4
9.6
2.0
205.9
290.0
3
7.0
0.52 ppm
45.0
3.2
3.1
44.0
109.0
290.0
5
7.2
5 . 2 ppm
45.0
3.1
2.3
58.0
96.4
290.0
16
* Average of 2 replicates
+ Adjusted to allow for periodic random thinning of groups to prevent overcrowding
-------
pH 8.5 CONTROL
pH 8.5 TEST
pH 9.0 CONTROL
pH 9.0 TEST
0
40 80 120 160
DAYS RECOVERY
200
FIG. 7. Growth Rates of Fingerlings
Recovering from Exposure to 5.2ppm
Al at pH 8.5 and 9.0.
-------
6.0
CONTROL
0.52 ppm
5.2 ppm
0 60 120 180 240 300
DAYS RECOVERY
FIG. 8. Growth Rates of Fingerlings
Recovering from Exposure to 0.52 and
5.2 ppm Al at pH 7.0.
32
-------
Table 3
Comparison of Growth Rates During Recovery Periods
Hypothesis: Slope = Slope
Test Control
Al ppm
Slope
Control
Test
Control
Test
Control
Test
Test
8.5
8.5 5.2
9.0
9.0 5.2
7.0
7.0 0.52
7.0 5.2
.0167
.0212
.0163
.0158
.0145
.0137
.0160
.932
.973 .890 n.s.
.930
.894 .097 n.s.
.955
.973 .176 n.s.
.971 .330 n.s.
33
-------
Eggs and fry exposed in the submerged trays were studied in two experimental
series: the first was made with controls and treatments at pH 9.0, and the
second was made with controls at pH 8.0, with the 0.52 ppm Al at pH 7.0,
and with the 5.2 ppm Al replicated at pH 7.0 and pH 8.0. Of first concern
was the possible effects of aluminum on the fertilization. In both series
all the control eggs were fertilized in the control water. One tray in
each treatment trough was supplied eggs fertilized in control water and
one tray was supplied with eggs fertilized in the appropriate aluminum con-
centration. Tables 4 and 5 present the data on egg mortality to the eyed
stage for both series. None of the data would support any claim that alum-
inum toxicity reduced fertilization. In fact, in the first series the
mortality to the eyed stage is lower for the eggs fertilized in the aluminum
concentrations. The data in Tables 4 and 5 also confirm the high fertility
of the eggs.
Although the figures for the egg and fry survival would seem to encourage
the application of statistical comparisons, problems with the rearing and
with maintaining constant pH's reduce the power of the early life history
data. Consequently only general observations seem warranted. For example,
total mortality from fertilization to termination for series one at standard
pH 9.0 indicate a mortality of 74% for the controls, 60% for the 0.052 ppm
Al, 55% for the 0.52 ppm Al, and 78% for the 5.20 ppm Al.
In the second series of the early life history exposures, total mortality
from fertilization to termination was 50% for controls, 58% for the 0.520
ppm Al at pH 7.0, 68% for the 5.2 ppm Al at pH 7.0, and 92% for 5.2 ppm
Al at pH 8.0.
A general conclusion is that dissolved aluminum is more toxic as evidenced
by higher mortality of 5.2 ppm at pH 8 and pH 9, but that high concentra-
tions of suspended aluminum (5.2 ppm, pH 7.0) are also nearly as toxic.
The trend is the same as for the fingerling rainbows.
-------
Table 4. Fertilization and early life history with controls and treatments at pH 9.0.
u>
VJI
Trough
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
A =
C =
A =
Treatment
Control
Control
Control
Control
0.052 *
0.052
0.052
0.052
0.520
0.520
0.520
0.520
5.200
5.200
5.200
5.200
pptn. aluminum concentration
fertilized in control water pH 9.0
fertilized in appropriate aluminum
Total Eggs at
Start each
Tray
C 188
C 201
C 165
C 190
A 276
C 196
A 211
C 183
A 337
C 171
A 301
C 174
A 226
C 220
A 335
C 198
concentration pH 9.0
Number Egg
Mortality to
Eyed Stage
36
36
26
42
8
21
2
35
3
32
0
22
12
35
1
29
Percent
Mortality
to Eyed Stage
19
17
16
22
3
11
1
19
1
19
0
13
5
16
1
15
-------
Table 5. Fertilization and early life history with controls at pH 8.0 (troughs 1 and 2), 0.52
ppm Al at pH 7.0 (troughs 3 and 4), 5.2 ppm Al at pH 7.0 (troughs 5 and 6), and 5.2
ppm Al at pH 8.0 (troughs 7 and 8).
Trough
Treatment
LO
cr\
Total Eggs at
Start each
Tray
* = ppm. aluminum concentration
C « fertilized in control water pH 8.0
A - fertilized in appropriate aluminum concentration and pH
Number Egg
Mortality to
Eyed Stage
Percent
Mortality
to Eyed Stage
1 Control
1 Control
2 Control
2 Control
3 0.520*
3 0.520
4 0.520
4 0.520
5 5.200
5 5.200
6 5.200
6 5 . 200
7 5.200
7 5.200
8 5.200
8 5.200
C 299
C 244
C 274
C 245
A 289
C 225
A 230
C 262
A 276
C 214
A 216
C 245
A 257
C 212
A 152
C 232
37
31
34
29
24
22
14
20
20
20
12
36
29
25
15
31
12
13
12
12
8
10
6
8
7
9
6
15
11
11
10
13
-------
SECTION VI
ACKNOWLEDGMENTS
The Department of Fishery and Wildlife Biology, Colorado State University,
initiated the grant proposal, provided the facilities for the aluminum
studies, and handled the administration. Dr. W. Harry Everhart, Professor
and Chairman Fishery Major, was Project Director.
Mr. Robert Freeman, Graduate Student, designed the test facility, con-
ducted the fingerling studies, and worked toward solving the aluminum
chemistry problems.
Dr. Janet Osteryoung, Quantitative Chemist, was technical consultant for
chemical problems. Mr. Kenneth Olsen, Graduate Student, Department of
Chemistry, monitored the pH and prepared aluminum concentrations for the
fry portion of the aluminum studies.
The support of the project by the Water Quality Office, Environmental
Protection Agency, and assistance from Mr. J. Howard McCormick, Grant
Project Officer, are acknowledged.
37
-------
SECTION VII
REFERENCES
1. American Public Health Association et al. 1965. Standard Methods
for the Examination of Water and Wastewater. 12th ed. American
Public Health Association, N. Y., N. Y. 769 pp.
2. Birnbaum, Z. W. 1962. "Introduction to Probability and Mathematical
Statistics", Harper & Bros., N. Y., N. Y., 325 pp.
3. Deuel, C. R., D. C. Haskell and A. U. Tunnison. 1942. The New York
State Fish Hatchery Feeding Chart. Fisheries Research Bulletin No.
3, New York State Conservation Department.
4. Dixon, W. J. and F. J. Massey, Jr. 1969. Introduction to Statistical
Analysis. McGraw-Hill Book Co., N. Y., N. Y. 638 pp.
5. Ebeling, G. 1928. Uber die gifligkeit einiger schwermetallsalze
an Hand eines Falles aus der Prasix. Zeits, Fisherei 26:49-61.
Abstract in J.A.W.W.A. 23:1626 (1931).
6. Ellis, M. M. 1937. Detection and measurement of stream pollution.
Bur. Fish. Bull. 22:365-437.
7. Freeman, R. A. 1971. A constant flow delivery device for chronic
bioassay. Trans. Am. Fish. Soc. 100(1):135-136.
8. Hem, J. D. 1968. Graphical methods for the study of aqueous aluminum
hydroxide, fluoride and sulfate complexes. U.S.G.S. Water Supply
Paper 1827-B.
9. Hem, J. D. and C. E. Roberson. 1967. Form and stability of aluminum
hydroxide complexes in dilute solution. U.S.G.S. Water Supply Paper
1827-A.
10. Jones, J.R.E. 1964. "Fish and River Pollution". Butterworth Scientific
Publications, Washington, D. C. 203 pp.
11. Minkina, A. L. 1946. On the action of iron and aluminum on fish.
Trudy Moskov. Zooparka 3:23-26. (In Russian). Quoted in: Doudoroff,
D. and M. Katz, 1953. Critical review of literature on to toxicity
of industrial wastes and their components to fish. II. The metals, as
salts. Sewage and Ind. Wastes, 25, 7: 802-839.
12. Oshima, S. 1931. On the toxic action of dissolved salts and their
ions upon young eels (Angilla japonica). J. Imp. Fish. Exp. Sta.
2:1939.
39
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13. Pulley, T. E. 1950. The effects of aluminum chloride in small con-
centration on various marine organisms. Texas J. of Science 2:405-411.
14. Roberson, C. E. and J. D. Hem. 1969. Solubility of aluminum in the
presence of hydroxide, fluoride and sulfate. U.S.G.S. Water Supply
Paper, 1827-C.
15. Sanborn, N. H. 1945. The lethal effect of certain chemicals on
fresh water fish. Canning Trade 67(49):10-12.
16. Schaut, G. G. 1939. Fish catastrophes during droughts. J.A.W.W.A.
31:771-822.
17. Snedecor, G. W. 1966. Statistical Methods. The Iowa State Univ.
Press, Ames, Iowa. 534 pp.
18. Thomas, A. 1915. Effects of certain metallic salts upon fishes.
Trans. Amer. Fish. Soc. 44(1):120-124.
19. Wallen, I. E., W. C. Greer and R. Lasater. 1957. Toxicity to Gambusia
affinis of certain pure chemicals in turbid waters. Sewage and Ind.
Wastes, 29:695-711.
1*0
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SECTION VIII
PUBLICATIONS
Freeman, Robert A. and W. Harry Everhart
1971 Toxicity of aluminum hydroxide complexes in neutral and basic
media to rainbow trout. Trans. Amer. Fish. Soc., 100(4):644-658,
Freeman, Robert A.
1971. A constant flow delivery device for chronic bioassay. Trans.
Amer. Fish. Soc., 100(1) -.135-136.
Freeman, Robert A.
1972. Recovery of rainbow trout from aluminum poisoning. (Submitted
to Trans. Amer. Fish. Soc.).
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1
Accession Number
w
5
Organization
2
Department of
Subject Field & Group
05C
Fishery
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
and Wildlife Biology
Fort Collins, Colorado 80521
Title
EFFECTS OF CHEMICAL VARIATIONS IN AQUATIC ENVIRONMENTS:
Toxic effects of aqueous aluminum to rainbow trout.
10
A u thorns)
Everhart ,
Robert A.
W. Harry and
Freeman
16
21
Project Designation
18050-2DYC
TVoJe
22
Citation
Environmental Protection Agency report
number, EPA-R3-73-011b, February 1973.
Descriptors (Starred First)
*Rainbow trout, ^aluminum, *toxicity , *pH
25
Identifiers (Starred First)
*Rainbow trout, ^aluminum, *toxicity
27
Abstract
Fertilized eggs, fry, and fingerlings were exposed to aqueous aluminum complexes
in neutral and basic media under constantly flowing, controlled conditions of aluminum
concentration, pH, and temperature. Toxicities of various concentrations were highly
pH dependent. Dissolved concentrations over 1.5 ppm aluminum caused physiological and
behavioral aberrations as well as acute mortality. Toxic effects of suspended aluminum,
though greater at lower concentrations, do not increase as much as the effects of
dissolved aluminum with higher concentrations. Growth of trout exposed to high dosages
of aluminum was reduced only as long as or slightly longer than the exposure continued.
Egg and fry bioassays were conducted with exposures in trays and simulated natural
redds. Fertilization was not affected by any concentrations tested, and most mortalities
occurred during hatching and in the post-swim-up stage. Trends in toxicity were similar
to those found with fingerlings indicating dissolved aluminum to be more toxic than
equivalent suspended amounts.
Abstractor
Harry Everhart
institution Cornen University
WRSIC
(REV. JULY 1969)
SEND. WITH COPY OF DOCUMENT. TO: *ATER RESOURCES SC JENT |F 1C
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
«U.S. GOVERNMENT PRINTING OFFICE: 197.3-514-154. 264-1-3
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