EPA-R3-73-011C ECOLOGICAL RESEARCH SERIES
FEBRUARY 1973
Effects of Chemical Variations
in Aquatic Environments
Vol. Ill
Lead Toxicity to Rainbow Trout
and Testing Application Factor Concept
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring* Environmental Protection Agency, have
been grouped into five series. These five broad
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development and application of environmental
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was consciously planned to foster technology
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EPA-R3-73-011c
February 1973
EFFECTS OF CHEMICAL VARIATIONS IN AQUATIC ENVIRONMENTS:
Volume III
Lead Toxicity to Rainbow Trout
and
Testing Application Factor Concept
By
Patrick H. Davies
W. Harry Everhart
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 $1.26 domestic postpaid or $1 QPO Bookstore
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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
Four chronic bioassays were conducted to determine the toxicity of
lead to rainbow trout. Results obtained from acute and chronic
bioassays in hard water (alkalinity 243.1 mg/liter) and soft water
(alkalinity 26.4 mg/liter) were used to test the application factor
approach as related to different water qualities. The toxicity of
lead to rainbow trout in hard water was determined on a total and
dissolved lead basis. The 96-hr TL5Q and "MATC" on a total lead
basis were 471 mg/liter and 0.12 to 0.36 mg/liter respectively, which
yielded an application factor of .0002 to .0008. Analysis of the
free or dissolved lead gave a 96-hr TL_Q of 1.38 mg/liter and a "MATC"
of 0.018 to 0.032 mg/liter, resulting in an application factor of
.0130 to .0232. Total and free lead were considered to be the same
in soft water. The 18-day TL™ and "MATC" obtained from the soft
water bioassays were 140 yg/Iiter and 6.0 to 11.9 pg/liter lead
respectively. Computations using the TL50 and "MATC" values gave a
soft water application factor of .0429 to .0850. The maximum ac-
ceptable toxicant concentration ("MATC") was determined in both hard
and soft water bioassays on the occurrence of abnormal black tails
caused by chronic lead exposure. The application factor approach as
related to different water qualities was found to be very promising
when lead analysis was limited to the free or dissolved metal and
failed when total hard water lead concentrations were used.
iii
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CONTENTS
Section
I
II
III
IV
V
VI
VII
VIII
IX
Conclusions
Recommendst ions
Introduction
Methods
Results
Acknowledgments
References
Publications
Appendices
Page
1
3
5
9
19
61
63
67
69
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FIGURES
No. Page
1. Fish no. R-0, H-l lateral view 27
2. Fish no. R-0, dorsal view 27
3. Fish no. R-0, X-ray - lateral view 28
4. Fish no. R-0, X-ray - ventral view 28
5. Fish no. L-10, H-l - lateral view 29
6. Fish no. R-l, H-l - lateral view 29
7. Fish no. L-10, X-ray - lateral view 30
8. Fish no. R-l, X-ray - lateral view 30
9. Control fish, X-ray - lateral view 31
10. Control fish, X-ray - ventral view 31
11. Lead toxicity to rainbow trout and 58
validity of application factor
12. Solubility and species distribution for 72
Pb (II) in hard water
13. Solubility and species distribution for 73
Pb (II) in soft water
14. Graphic interpretation of the nature of 80
lead in hard water when analyzed by
atomic absorption spectrophometry
VI
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TABLES
No. Page
1. Mean chemical analysis of hard and soft water supplies 20
2. Average water quality for hard water growth bioassay 21
3. Hard water lead analytical results 23
4. Percent of fish affected by physical abnormalities in 25
the final sampling of the hard water growth bioassay
5. Average water quality for soft water growth bioassay 36
6. Soft water growth bioassay—lead analytical results 37
7. Average water quality for reproduction bioassay 40
8. Soft water reproduction bioassay—lead analytical 41
results
9. Average water quality for soft water F- generation 45
reproduction bioassay
10. Soft water F- generation reproduction bioassay—lead 44
analytical results
11. Percent of fish affected by physical abnormalities 46
in the F. generation reproduction bioassay at
termination of experiment
12. Mortality results for F. generation reproduction 47
bioassay based on initial 250 fish per aquarium
13. Chemical results for static hard water acute bioassay #1 49
14. Lead analysis results of static hard water acute 50
bioassay #1
15. Lead analysis and 96-hr mortality results for static 51
hard water acute bioassay #2
16. Chemical results for static hard water acute 52
bioassay #3
VI1
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TABLES
No. Page
17. Lead analysis and 96-hr mortality results for hard 53
water acute bioassay #3
18. Lead analysis and mortality for soft water acute 54
bioassay
19. Summarized analytical data for lead in the hard water 55
chronic bioassay
20. Summarized analytical data for lead in the soft water 55
F, generation reproduction bioassay
21. Summary of application factor data 57
Vlll
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Section I
CONCLUSIONS
Hard Water Bioassay Findings
1. In hard water the mean hardness, M.O. alkalinity and pH was 353.5
rag/liter, 243.1 mg/liter and 8.02 respectively.
2. Lead analysis in hard water is reported as total and free lead.
3. A static acute bioassay gave a 96-hr (50$ tolerance limit) TL of
471 mg/liter total lead. The maximum acceptable toxicant concen-
tration ("MATC") from the hard water chronic bioassay was between
0.12 and 0.36 mg/liter total lead. The acute and chronic ("MATC")
total lead concentrations give an application factor between .0002
and .0008. The "MATC" is based on the occurrence of black tails
which were caused by lead. Lead-attributed mortalities in the
chronic bioassay occurred in the high concentration (3.24 mg/liter
total lead).
4. On a free lead basis, the static hard water acute bioassay gave a
96-hr (50% tolerance limit) TL5 of 1.38 mg/liter. The "MATC" of the
chronic bioassay based on black tails, was between 0.018 and 0.032
mg/liter free lead. The hard water free lead application factor
lies between .0130 and .0232. Lead-attributed mortalities occurred
at a free lead concentration of 0.064 mg/liter in the high concentra-
tion aquarium.
5. Physical abnormalities consisted of black tails and spinal curvatures
(lordoscoliosis).
Soft Water Bioassay Findings
6. In soft water the mean hardness, M. 0. alkalinity and pH were 27.2
mg/liter, 26.4 mg/liter and 6.88 respectively.
7. Lead analysis in soft water is considered as free lead. A flow-through
acute bioassay gave an 18-day (50% tolerance limit) TL^ of 140 jag/liter
lead. A "MATC" based on black tails, was found to be between 11.9 and
6.0 yg/liter lead. The soft water application factor for lead lies
between .0429 and .0859. Lead-attributed mortalities with fry and
fingerling fish occurred in the high concentration aquarium (95.2 yg/
liter).
8. Three-year-old brood fish of the soft water reproduction bioassay
yielded viable eggs and fry from all but the lowest toxicant concen-
tration. The high lead concentration of this experiment was 27.9
yg/liter.
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9. Two-year-old rainbow trout proved to be inadequate for use in
determining possible effects of lead on reproduction. Only 24%
of the 63 females examined were found to be reproductively mature.
10. No growth differences attributable to lead were found in either the
hard or soft water growth bioassays.
11. Hematocrit and hemoglobin determinations did not provide a consistent
statistical difference in either the hard or soft water growth bio-
assays. However, the tap water FI generation reproduction
bioassay in which the lead concentration was increased, gave a
statistically lower hematocrit value for the high concentration
(95.2 ug/liter Pb).
Testing of the Application Factor Approach as Applied to Water Qualities
12. Using free or dissolved lead analyses, the application factor
approach succeeded in estimating "MATC" levels in both hard and
soft water experiments. Therefore, it appears that the application
factor approach as applied to different water qualities does have
promise when the metal analysis is limited to that of measuring the
dissolved metal.
13. On a total (nominal) lead basis, multiplying the soft water appli-
cation factor by hard water, total lead TL,-0 concentration failed
to approximate the hard water "MATC". The converse relationship
of multiplying the hard water application factor by the soft water
TL5Q also failed to approximate the soft water "MATC". Therefore,
on the basis of total lead, the use of an application factor for
estimating the "MATC" of a water for which only acute tests can be
made, failed.
14. The method of analysis, free versus total metal analysis, appears to
pose a problem only where a particular metal becomes complexed or
suspended in a specific water quality or type.
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Section II
RECOMMENDATIONS
More information needs to be obtained to learn those factors that may
affect growth in chronic bioassays if toxicant effects are to be
properly assessed. Our studies indicate that four factors should re-
ceive particular concern. (1) Position effects: Aquaria should be
positioned in such a manner as to provide equivalent light to all tanks
because of plankton growths which could act as a supplemental food
source. The possibility of temperature difference between aquaria in
a particular experiment must be minimized. If aquaria are placed on
stands with one higher than another, stratified temperature zones in
the room cause different aquarium temperatures. Possible temperature
differences caused by heat registers, windows, doorways and electrical
motors in a room must be considered. Aquaria for a particular experi-
ment need to be positioned to minimize and provide equal exposure to
human activities. (2) Equal available space: The available space per
gram of fish in a set of experimental aquaria should be the same for
any particular bioassay. This may be accomplished through the use of
dividers or by adjusting water volumes by aquarium stand pipe height.
(3) Feeding rates adjusted daily: Feeding rates based on total weight
of fish per aquarium should be adjusted daily. The total weight can
be determined bimonthly and adjusted daily from a computerized growth
rate projection. (4) Individual marked fish: Toxicant related growth
effects can be assessed most reliably by following the growth of indi-
vidually marked fish. Individualized marking by cold branding was
preferred in this study.
Chronic bioassay beginning with eggs obtained from one female for
each concentration should be avoided. Size and genetic differences of
such eggs can easily mask any toxicant-related effects. The problem
is compounded in that eggs obtained from individual females will most
likely hatch on different dates, thus greatly complicating data collec-
tion and handling. These complications can be avoided by using eggs
collected from a large number of females that were spawned on the same
date. Experimental results also show that use of 2-year-old rainbow
trout as brood fish is impractical for studying toxicant-related effects
on reproduction. Only 24% of the rainbow trout females encountered in
this study were reproductively mature as 2-year-old fish.
Results from this study support the application factor concept as related
to different water qualities only where analysis of the soluble or free
metal was employed. Where heavy metals form complexes in natural waters,
some standardization of water sample collection and method of analysis
will be required if the application factor approach is to find widespread
use. A standardization of metal analysis for free metal ions will also
be required for acute and chronic bioassay experiments where the complex-
ing of a particular heavy metal occurs.
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Considerable research is needed on heavy metals, such as lead, which
become complexed and/or precipitated in water of different hardness
and/or alkalinity. The problems associated with such metals and
attempts to estimate "MA.TC" levels are numerous and mostly unresolved.
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Section III
INTRODUCTION
The effect of heavy metals and their accumulation in the various tropic
levels of our environment is a source of much concern today. Lead is
one metal that is obtaining increased prominence and a source of much
public concern (Hall, 1972). The atmosphere,coastal and inland waters
adjacent to highly populated and industrialized areas show an alarm-
ingly high rate in increased lead concentrations over the past 30 years
(Chow and Earl, 1970; Hall, 1972). Lead is a significant pollutant in
many of our nation's water-ways because of its indiscriminate release
in many industrial effluents.
Lead warrants particular interest in Colorado because of the activities
of several mining and milling operations which release it to the aquatic
environment in effluent waters. It is becoming quite apparent that the
relative solubility of some metals in "natural" waters has little rele-
vance regarding their possible toxic effects on the aquatic and terres-
trial environments. Lead is ametal previously thought to be of little
concern because of its relative insolubility in all but the softest
of "natural" waters. Toxic modes other than transference or absorption
across the gill membrane are greatly in need of investigation.
An abundance of toxicological data on a multitude of toxicants has been
collected and reported during the past century (McKee and Wolf, 1963).
Most of this data have been reported as the median tolerance limit
(TL or TL,-0). Many, by multiplying the TL_0 value by some arbitrary
fraction, have erroneously interpreted these findings as being those
concentrations at which fish life can survive and reproduce. Frequently
bioassay data is of little value due to the lack of accompanying water
quality information.
Little information has been available on the effect of long-term, sub-
lethal exposures of fish to toxic agents. The effect of toxicants on
reproduction is almost unknown except for a few elements. Information
on the sensitivity of different life stages of fish to a particular
toxicant is generally lacking. Consequently, estimates of safe concen-
trations based on acute toxicity studies are very difficult to make and
frequently erroneous. There exists a vital need for long-term or chronic
toxicity studies. During the past few years, some headway has been made
in obtaining such information (Arthur, 1970 - LAS detergent; Buhler,
Rasmusson and Shanks, 1969 - DDT; Brungs, 1969 - Zinc; McKim and Benoit,
1971 - Copper; Mount, 1962 - Endrin; Mount, 1968 - Copper; Mount and
Stephan, 1967a - Cadmium; Mount and Stephan, 1969 - Copper; and Picker-
ing and Thatcher, 1970 - LAS detergent). However, the problem regarding
methods by which laboratory toxicity finds might be applied to a
multitude of aquatic species and water types must be resolved.
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Henderson (1957) discussed a number of constituents involved in develop-
ing "application factors" whereby laboratory toxicity studies could be
used in determining permissible concentration of toxic substances in the
aquatic environment. Mount and Stephan (1967b) proposed the development
of an application factor based on dividing the "maximum acceptable
toxicant concentration" — MATC (i..,e. > that concentration obtained
from a chronic bioassay at which no inhibiting effect on growth or repro-
duction occurred) by the 48-hr or 96-hr TL,-0 concentration for a partic-
ular toxicant. The fraction or application factor so obtained is then
multiplied by a TLc« value found for fish and waters on which long-term
testing cannot be performed. The resulting concentration hypothetically
establishes the maximum permissible concentration of a particular toxi-
cant in other water quality types containing different fish species. It
has been proposed that such an application factor will hold true irres-
pective of fish species or water quality involved. The authenticity
of such factors needs to be established.
Brungs, 1969, further defined the "laboratory fish production index"
(LFPI) proposed by Mount and Stephan (1967b) as reflecting toxicant
effects on reproduction, growth, spawning behavior, egg viability and
fry survival. "The highest observed toxicant concentration that has no
effect on these biological factors based on a continuous chronic exposure
is termed the maximum acceptable concentration of that toxicant", (MATC).
The reproductively mature fish in the present study were artificially
spawned and therefore failed to adhere strictly to the criteria outlined
above. The further use of the MATC in this report will be in quotation
marks (i.e. "MATC") to signify the absence of spawning behavior data.
This study had two primary objectives. (1) to determine acute and
chronic toxicities of lead to rainbow trout and (2) to determine the
validity of the application factor concept using two distinctly differ-
ent water qualities. The two water quality types used are: (1) a hard
well water (353 mg/liter total hardness) from a 20 - ft well located at
the Colorado Game, Fish and Parks Research Center and (2) a soft dechlor-
inated tap water (carbon filtered) with a total hardness of 27 mg/liter.
Chronic toxicity of lead was conducted using two life stages of rainbow
trout. Three-inch rainbow fingerlings were used to measure growth effects
and mature, 2-year-old rainbow trout (spawned as 3-year-olds) were used
to measure the effect of chronic lead toxicity on reproduction, egg
viability and fry growth.
Application factors were calculated for both soft and hard waters. This
entailed dividing the "MATC" of the chronic bioassays by the 96-hr TL™
for each of the respective waters. The factors or fractions so calculated
were used to determine the validity of the application factor concept.
This was accomplished by multiplying the application factor obtained from
the acute and chronic bioassays for the hard water by the 96-hr TL,-0 value
obtained from the acute bioassay performed in dechlorinated soft water.
The resulting "MATC1 should equate or approximate the "MATC" value obtained
from the chronic bioassay in soft water. The converse relationship for
hard water should also occur if the application factor concept is upheld.
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Previous Lead Experimentation Applicable to the Present Study
Carpenter (1927 and 1930) found that one part lead in three million parts
of water (0.3 mg/liter) was lethal to minnows, sticklebacks and trout
in distilled water. The speed of the toxic reaction is dependent on the
total quantity of metallic ion present, as well as upon the actual con-
centration. Fish sensitivity to lead varies inversely to fish size and
weight, and varies directly, consistent with Van 't Hoff's Rule, to
water temperature. She further states that the toxic action to be purely
external in process, chemical in type, and mechanical in effect; ji..e.,
a colloidal substance forms on the gill epithelium and eventually causes
death by suffocation.
Dawson (1935) reported that the surface mucous, described by Carpenter,
did not constitute an effective barrier to the absorption of lead. In
his experiments, brown bullheads, Ameiurus nebulosus, survived 16-183
days in tap water renewed at 2-day intervals with 27.3 mg/liter of lead,
but observations on the peripheral blood demonstrated direct injury to
the erythrocytes followed by a mild regenerative response with the
eventual development of pronounced secondary anemia.
Jones (1938) with soft water static bioassays reported lethal concentra-
tion limits of 1 mg/liter lead (as lead nitrate) with the minnow,
Gasterosteus sp. Lower lethal concentrations were obtained by periodic-
ally renewing the lead solution. He also found that younger fish are
more sensitive to the toxic action of lead. The addition of calcium
salts (as nitrate or chloride) was found to reduce the toxicity of lead.
Lloyd (1965) reported a reduced toxicity of lead with increase in water
hardness. Rainbow trout were found to be more susceptible to the
toxicity of lead when the dissolved oxygen concentration of the water
was reduced (Lloyd, 1961 and 1965).
Lloyd (1965) has postulated that the acute toxicity of lead to fish
results from attaining a lethal threshold concentration of a heavy metal
at the gill surface. If the rate at which the heavy metal ions enter
the gill epithelium is greater than the rate at which they are removed
into the blood stream, then a buildup will occur, and the fish will die.
The fish will survive if the converse relationship exists. Haider (1964)
reported that acute toxicity causes death through erosion of the gill
epithelium and a resulting suffocation. He further states that chronic
lead poisoning amounts to a resorption of the toxicant, apparently
through the gills, slowly resulting in functional damage of the inner
organs.
Behavioral symptoms of fish chemically exposed to lead were described by
Haider (1964) These are: Fish were exposed to lead acetate in low con-
centrations which were increased over time. The fish showed a gradual
darkening over their whole body. This occurred very quickly (about 2 hr)
when fish were injected with lead. A sharply defined darkening occurred
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to the caudal part of the body when lead was injected into the top of
the ventral fins. Fish that were daily fed lead in their diet also
became completely dark colored. Irregularity in breathing was occa-
sionally observed but over time this occurred more frequently. It
became difficult for the fish to maintain their equilibrium and were
seen to tip over into a slanting position and move jerkily through the
aquarium. The fish were frequently observed lying on the aquarium
bottom with their fins spread out. From time to time wave-type
muscular movements occurred over the body. The fish declined food.
The slime secretions reported by Carpenter (1927 and 1930) were not ob-
served.
Dorfman and Whitworth (1969) reported on the effects of fluctuations of
lead, temperature and dissolved oxygen on the growth of brook trout,
Salvelinus fontinalis. All treatment combinations that included a
lead concentration of 25 mg/liter, administered once a day as a slug
dose, reduced growth. Treatment combinations that included 15 mg/liter
and 10 mg/liter of lead administered as a slug dose once a day showed
no apparent effect on growth.
Information on the toxicity of various lead salts have been summarized
by McKee and Wolf (1963). Concentrations of 0.1 to 240 mg/liter of
lead for different water qualities have been reported as acutely toxic
to fish as a 96-hr TL_0 concentration. Pickering and Henderson (1965)
conducted static bioassays in soft (alkalinity - 18 mg/liter, hardness -
20 mg/liter) and hard (alkalinity - 300 mg/liter, hardness - 360
mg/liter) waters. Using lead chloride in soft and hard waters, the
96-hr TL5Q for fathead minnows was 5.58 mg/liter of lead and 482 mg/liter
of lead respectively. Similar tests in soft and hard water on bluegills
give a 96-hr TL-^ of 25.9 and 442 mg/liter of lead respectively.
Henderson (unpublished data) compared TI,_0 toxicity concentrations for
static versus constant-flow bioassays. using lead chloride in soft
water (pH - 7.4, alkalinity - 18 mg/liter and hardness - 20 mg/liter)
with fathead minnows as the test fish, he determined the 96-hr static
TL50 to be 5.6 mg/liter of lead as compared to a 96-hr constant-flow
TL-0 of 0.97 mg/liter of lead. Using a constant-flow bioassay, Henderson
found TLcn concentrations at the indicated time intervals to be, in mg/
liter of lead: 1.1 (48 hr), 0.97 (96 hr), 0.75 (5 days), 0.40 (10 days),
0.31 (20 days) and 0.31 (35 days).
8
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Section IV
METHODS
EXPERIMENTAL CONDITIONS
Two different water qualities were used for the bioassay experiments.
Hard water with a mean hardness of 353.5 mg/liter and a mean M.O.
alkalinity of 243.1 mg/liter was obtained from a 20-ft well located at
the Research Center for the Colorado Division of Game, Fish and Parks.
Fort Collins city water with a mean hardness of 27.2 mg/liter and a
mean alkalinity of 26.4 mg/liter was dechlorinated by passage through a
Hayward Model S-40 Sand Filter (modified) containing 12 cubic ft of
activated carbon. The filter was a Maycor epoxy-lined carbon steel
vessel with PVC (polyvinylchloride) and stainless steel internal
components. Piping from the filter and within the chronic and acute
bioassay rooms was PVC.
The chronic bioassays were conducted at the ambient temperature of
Fort Collins city water which is obtained from the Cache La Poudre
River. The well water maintains a year-round temperature of 15°C and
was cooled to the ambient tap water temperature using a "Min-o-cool"
fiberglass tank with two 1-horsepower refrigeration units. Soft water
was cooled to the ambient hard water temperature when summer water
temperatures exceeded 15 C. Temperature adjustments were performed on
a weekly basis when required. Temperatures were monitored in the soft
and hard water control aquaria by Foxboro recording thermometers.
Chronic bioassays were performed in 325-liter, glass aquaria (142 cm x
47 cm x 50 cm) with plexiglass covers. Each aquarium was aerated with
two 6-inch airstones. An initial aquaria hygiene problem was alleviated
through the use of a settling trap, similar to an Imhoff cone, which
was mounted outside each aquarium, for removal of settleable solids.
Supernatant water from the settling trap was siphoned to another con-
tainer packed with a dacron polyester fiber through which the water
was pumped for removal of suspended matter and returned to the aquarium
(Citation in preparation). Aquaria walls were brushed two to three
times a week to remove algal growths of Achanthes sp. The toxicant
delivery system for each bioassay utilized a proportional diluter (Mount
and Brungs, 1967) that delivered 2 liters of test water per cycle or
approximately 1 liter per 2 min to each exposure aquarium. This rate
of delivery gives a 50% water replacement time of about 6.3 hr (Sprague,
1969). Lead nitrate (PbNO.,) comprised the stock solution that supplied
the diluters. Acute bioassays employed a similar toxicant delivery
system and used 35-liter aquaria with each test concentration duplicated.
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CHEMICAL METHODS
Water quality analyses were performed weekly on the chronic bioassays.
Parameters that were measured include: temperature, conductivity, pH,
dissolved oxygen, alkalinity and hardness. Chemical methods were made
in accordance with standard methods of the American Public Health
Association et al. (1965). Temperature determinations were made with
a TRI-R Electric Thermometer. A Beckman Model RB-2 Solu Bridge was
used for conductivity measurements. A Corning Model 12 pH Meter gave
readings to a hundredth of a pH unit. Dissolved oxygen, alkalinity
and hardness were determined by the azide modification method, pH
titration to a. 4.5 (M.O.) end point, and the EDTA titrimetric method,
respectively.
ANALYTICAL METHODS FOR LEAD
First and foremost, it is necessary that one understands that the
occurrence of lead in what might be called "natural" water constitutes
an extremely complex relationship of a multiplicity of various physical,
biological and chemical interactions. The resulting system is not one
that can be empirically described at this point in time, and is one
which will require an extensive amount of research before a compatible
understanding of the system is achieved. (See Appendix A for a more
detailed discussion on the complexities associated with analyzing the
character of lead in water).
Atomic Absorption
Atomic absorption analysis was performed on water samples collected from
all aquaria of the hard water growth bioassay. Analysis of soft water
growth and reproduction bioassay samples was limited to the high con-
centration aquaria because of the detection limit of the equipment.
Analyses were also performed on water taken from the two high concen-
trations of the F, generation reproduction bioassay. Weekly grab samples
were taken in new 140-ml Nalgene bottles, acidified with three to five
drops of "Suprapur" nitric acid (EM Laboratories, Inc.). Analysis was
conducted using a Perkin-Elmer 303 Atomic Absorption Spectrophotometer
set at a wave length of 2170.0 A or 2833.1 A. A Perkin-Elmer 10 mv
Recorder was also used with a noise suppression level setting of three
and 10X to 30X scale expansion. Unknown lead concentration levels were
determined from a calibration curve constructed from acidified standards
prepared in distilled water. (See Appendix B for a further discussion
of problems associated with the analysis of lead by atomic absorption
spectrophometry).
10
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Pulse Polarography
Water samples from hard water growth, soft water growth and reproduction
bioassays were analyzed by pulse polarography from 14 August 1970 to
31 December 1970. Lead concentrations were monitored weekly from com-
posite samples. Twenty-five milliliter water samples were collected
daily (Monday through Friday) and pooled in a 140-ml Nalgene bottle
which was sealed and returned to the bottom of the aquarium to prevent
changes due to C0_ absorption. Following Friday's sampling, the bottle
was stored in a "whirl Pack" filled with water from the experimental
aquarium where it remained until analysis. The time delay for this
analysis was a few hours to two months with an average delay of about
two weeks. Such delays did not create significant systematic error to
affect analytical results. However, changes (were they to occur) would
result primarily from pH changes due to loss or increase of carbon
dioxide, a consequence that was minimized by the method of sample
storage.
The pulse polarographic analyses were conducted using a Melabs Pulse
Polarographic Analyzer with an accompanying electrode stand. Because
of the availability of instrumentation, some analyses were done using
a PAR Model 170 Electrochemical System with a PAR Model 174 Drop Knocker
Assembly. The two systems showed no significant difference in analytical
results. The indicator electrode, used with both systems, was a dropping
mercury electrode from which the drops were mechanically dislodged at
3- to 5-sec intervals. A large mercury pool served as both reference
and counter electrode. Reagent grade chemicals were used without further
pur if icat ion.
Analysis procedure for lead concentrations above 10 yg/liter
Under the experimental conditions employed,using 100 mv pulse modulation
in the differential mode, the detection limit was approximately 10 yg/liter
lead. Solutions with nominal lead concentration above this value were
analyzed by first adding 25 ml of the sample to the cell, deaerating for
8 min with prepurified nitrogen, and running the polarogram. Polarograms
were similarly run with the addition of 0.1 ml saturated LiCl solution
(Ca 10 II), in an attempt to attain a lead signal not obtained directly.
Under these conditions the peak for lead reduction to the amalgam occurs
at approximately -420 mv. Of the samples analyzed by these two procedures,
approximately 60% gave no signal for lead.
Following direct analysis procedures which were largely unsuccessful in
obtaining a lead signal, the samples were acidified with a few drops of
concentrated HC1 (to about pH 2) and a polarogram run Immediately. This
procedure gave a lead signal for those samples where no signal was obtained
11
-------
by direct analysis. A small known amount of a lead standard solution
was then added to the acidified solutions in an amount sufficient to
increase the peak height by about 30%, and a second polarogram was run.
Sample concentrations were calculated in the normal manner from the two
polarograms using the added lead as an internal standard. Ultimately,
all samples with a nominal concentration above 10 yg/liter were acidified
immediately prior to analysis. (See Appendix C for a more detailed
discussion of the pH problems associated with the analysis of lead by pulse
polarography).
Analysis jprocedure for lead concentrations below 10 yg/liter
Solutions with nominal lead concentrations of less than 10 pg/liter were
extracted prior to analysis. The following was added to 50 ml of sample:
5 ml 1% sodium diethyldithiocarbamate, 2.5 ml 1 M sodium citrate,and
sufficient HC1 to adjust the pH to 11.0. The resulting solution was ex-
tracted with 10 ml MIBK (methyl isobutyl ketone). Five milliliters of
0.5 M LiCl in methanol was added to the MIBK extract and the solution
diluted to 25 ml with methanol. The methanol solution was then added to
the cell, deaerated for 10 min, and a differential pulse polarogram run.
Peak heights were compared with those obtained for standard lead solu-
tions subjected to the same procedure. Under these conditions the lead
peak occurs at about -370 mv. The detection limit is approximately
2 yg/liter. Some analyses were made by a similar procedure employing
an extraction of the 8-hydroxyquinolirie complex in NH./NH.C1 buffer.
Results for the two extraction procedures were comparable.
Metal Analysis of Feed
Analyses for zinc, copper and lead were made on the feeds used in the
lead bioassay experiments. Samples were dried for 24 hrs at 95 C. Sample
sizes of 0.5 g and 1.0 g were digested in 1 ml concentrated perchloric and
2 ml concentrated nitric acids using a wet, pressure digestion method
(Adrian, 1971). Amount of lead in feed was found to be less than 2.5
yg/g, (See Appendix D for more detailed results).
CHRONIC BIOASSAY METHODS
Hard and Soft Water Growth Bioassays
Fish and toxicant system
Two growth bioassays were initiated on 14 May 1970, one in filtered soft
water and the other in hard water. Seventy rainbow trout, averaging 83.3
12
-------
mm in length and 6.1 g in weight, were placed in each aquaria. These
fish were hatched and raised to fingerling size at Bellvue Research
Hatchery from eggs obtained from the McCleary rainbow trout strain at
Soap Lake, Washington. The fish were graded for size uniformity just
prior to being placed in the test aquaria. Fish were acclimated to
water quality and the aquarium environment for 2 weeks prior to the
introduction of toxicant. The proportional diluter for the soft water
bioassay was set at 33.3% dilution ratio with a high nominal concentra-
tion of 30 yg/liter lead. Nominal lead concentrations in yg/liter
for the soft water growth bioassay were: S-l (30.0), S-2 (20.0), S-3
(13.3), S-4 (8.9), S-5 (5.9) and S-C (0.0). For the hard water growth
bioassay the proportional diluter was set at a 66.6% dilution ratio
with resulting nominal lead concentrations in mg/liter of: H-l (3.24),
H-2 (1.08), H-3 (0.36), H-4 (0.12), H-5 (0.04) and H-C (0.00).
Feed and feeding rates
The growth bioassay fish were fed the standard Colorado diet at a feed-
ing rate and pellet size obtained from the modified Cortland feeding
chart (Deuel, Haskell and Tunison, 1937). The feeding rate ranged from
7% of body weight per day for 6-g fish to 1.0% for 325-g fish. After
6 months of feeding the Colorado diet, fish condition indicated the
possibility of nutritional deficiencies. Consequently, a supplemental
feeding of beef liver was initiated weekly (personal communication Dr.
Donald Horak, Nutritionist, Colorado Game, Fish and Parks). Feeding
rates were adjusted monthly for the first 8 months from the total weight
of fish per aquarium obtained using a triple beam balance. Following
this period, feeding rates were adjusted every 3 months.
Growth measurements
Ten fish of the initial 70 placed in each aquarium were fin clipped for
individual identification. Growth measurements consisting of total
lengths (mm) and wet weight (g) were determined on these individuals for
the first 3 months of the experiment. It was then decided to discontinue
the collecting of growth data from fin-clipped individuals because of
the questionable influence of handling on these selected individuals as
compared to the remaining unmarked fish in an aquarium. Subsequent to
this decision, a random sample of 10 fish was taken from each aquaria
for individual length and weight data. On 30 March 1971, the number of
fish per aquarium was reduced to 20 fish. These fish were individually
marked with a cold brand utilizing liquid nitrogen and branding tools
(Mighell, 1969). Growth rates of these individuals were followed during
subsequent measuring periods. Analysis of co-variance was used to
determine possible lead related growth differences.
13
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Sacrificing for hematological investigations
The hard and soft water fish were sacrificed periodically to adjust
number of fish per aquarium and as crowding was observed. Fish were
sacrificed at the 4th, 6th, 8th, llth, 15th and 19£h months of the
experiment. The number of fish sacrificed per sampling period aver-
aged about 10 and depended somewhat on the number of mortalities that
may have occurred. These fish were randomly selected, anesthetized with
0.2 g/gal MS-222, weighed and measured,
A variety of techniques have been described in the literature for col-
lecting blood from various size fish (McCay, 1929; Root, 1931; Dawson,
1935; Field, Elvehjem and Juday, 1943; Schiffman, 1959; Hesser, 1960;
Hunn, Schoettger and Whealdon, 1968; and Klontz et al., unpublished).
Of these, cardiac puncture and puncture of the dorsal aorta are difficult
for fish under 6 inches in length. For small fish, severance of the
caudal peduncle has proven to have the greatest utility.
After anesthetization in MS-222, the fish was wrapped in a large ab-
sorbent towel to remove excess water and allow an adequate means for
holding the fish. A stainless steel scalpel was used to sever the
caudal peduncle. Chemists at Colorado State University have found metal
contamination in tissues after using scalpels that were not stainless
steel (R. K. Skogerboe, personal communication). The caudal peduncle
was severed approximately midway between the adipose fin and the caudal
fin. The flowing blood was collected in capillary tubes that were filled
with heparin solution (1 ml - 1000 USP units) and blown empty for re-
moval of excess heparin. Regular heparinized capillary tubes have proven
inadequate in preventing clotting of rainbow trout blood.
Heparin was selected because: Wintrobe (1934) preferred heparin as an
anticoagulant. Hesser (1960) found heparin the most satisfactory with
crenation and/or lysis of the red blood cells minimal. He also stated
that EDTA (ethylenediamine tetracetic acid) salts, ammonium and potassium
oxalate and citrates gave fair results. Some crenation occurs with the
use of oxalates. Other anticoagulants have been used in fish hematology.
McCay (1929) used mineral oil on needles and syringes and oxalates and
citrates in test tubes. Duthie (1939) coated containers with wax. Field
et al. (1943) used 0.1 M sodium oxalate or heparin in a syringe.
Hematological methods
Three capillary tubes of heparinized blood were collected from each fish.
Blood from one tube was used in making a hemoglobin determination. The
second capillary tube was used in making hematocrit and plasma protein
determinations and the third was kept as a spare. The cyanmethemoglobin
method of determining hemoglobin (Seiverd, 1964) is preferred by this
laboratory and employs a Bausch and Lomb Spectronic 20. Klontz et_ al.
14
-------
(unpublished) has also recommended this method for standard use with
rainbow trout. Hesser (1960) preferred the acid hematin method to the
cyanmethemoglobin method because of the formation of a proteinecious
gel in the latter which he felt gives inaccurate readings by obtaining
solutions of varied densities.
It is believed that the cyanmethemoglobin method far outweighs other
disadvantages inherent in the acid hematin method, those of time and
temperature variations, stability of the hematin color and the effects
of lipids and plasma protein of color development. It was found that
by swirling the cuvette in the cyanmethemoglobin method that the gela-
tinous precipitate will normally float to the surface and will not
inhibit the passage of light in a colormetric determination. The
gelatinous precipitate can then readily be removed from the sample by
use of a wooden applicator.
Hejatocrits were determined by the micro-hematocrit method (Hesser,
1960; Seiverd, 1964). The capillary tube, following 10-min centrifuga-
tion using an International Model MB Micro Capillary Centrifuge, was
broken above the point of separation between the cell pack and plasma
fractions. Plasma protein levels were determined using American Optical
Company T. S. Meter, Model 10401 (a Goldberg Refractometer). Results
are reported in g/100 ml. The plasma was placed onto the refractometer
and the plasma protein concentration read off the protein scale.
Soft Water Reproduction Bioassay
Fish and toxicant system
The soft water reproduction bioassay was initiated on 21 August 1970
with a 2-year-old rainbow trout obtained from the Federal Genetic Re-
search Hatchery at Beulah, Wyoming. Two-year-old fish were selected
for spawning as 3-year-olds to alleviate problems associated with
variable reproductive maturity existing with 2-year-old rainbow females.
Preliminary observations indicated that four fish were the maximum
number per 325-liter aquarium permitting free movement and the mainte-
nance of good environmental conditions. The fish used in this test were
selected on the basis of compatibility and adaptation to aquarium con-
ditions. Two males (averaging 720 g) and two females (averaging 892 g)
were placed in each of the six test aquaria. A 33.3% dilution ratio
with a high nominal concentration of 30 yg/liter was also used in this
experiment.
Feed and feeding rate
Fish used in the reproduction bioassay were fed a maintenance ration of
Rangen 7/32" brood pellets at 1% of body weight per day. A weekly supple-
ment of beef liver was also fed.
15
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Spawning and hatching
Plastic pans (30 cm x 25 cm x 15 cm) were filled to an approximate
depth of 8 cm (6 kg) with gravel (15-30 mm diameter) and placed in the
reproduction bioassay aquaria in an attempt to obtain natural spawning.
With attempts unsuccessful in obtaining natural spawning,the fish were
anesthetized with MS-222 and spawned artificially. Fertilized eggs
were placed on nylon net egg trays in 35-liter aquaria of the acute
bioassay system. The eggs of each female were hatched in separate
aquaria. A 50% diluter with a nominal high concentration of 100 ng/liter
lead supplied the toxicant.
SQET WATER F. Generation Reproduction Bioassay
Fish and toxicant system
At an age of 1 month, 250 successfully hatched fry from each concentra-
tion were transferred from hatching aquaria to the large 325-liter
aquaria. As with the hatching aquaria system, a 50% diluter with a
nominal high concentration of 100 ug/liter supplied the toxicant. The
experiment was terminated 15 December 1971.
Feed and feeding rates
The feeding rates for the F.. reproduction bioassay were adjusted at 2-
week intervals and based on the total weight of fish per aquarium.
Rates concur with the modified Cortland feeding chart. A weekly beef
liver supplement is also fed. At the age of 6 months, the number of
fish per aquarium was reduced to 30. Subsequent to this time, the feed-
ing rate was adjusted daily based on a computerized growth rate projec-
tion and adjusted every 2 weeks from current weight data.
Growth measurements
Growth measurements of the F.. reproduction fish were collected every 2
weeks. Initially this consisted of a count and total weight measurement
of fish per aquarium. At an age of 6 months the fish were cropped,
leaving a total of 30 fish per aquarium. Dr. David Bowden, statistician
with Colorado State University, stressed the need for a large sample size
to reduce within sample variation where individually marked fish did not
exist. The total length in millimeters of individual fish sacrificed
was determined and will be used as the index for measuring growth differ-
ences between lead concentrations.
16
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The growth bioassay experiments indicated a possible effect of aquarium
space on growth where the number and size of fish varied from one con-
centration to another. Utilizing a proportional space-weight relation-
ship, aquaria were divided with a plexiglass divider. For example,
if the control aquarium has 1000 g of fish and the high concentration
has 500 g of fish, the available space in the control aquarium would be
considered at 100% and the high concentration aquarium with one-half
the weight of fish would be divided in half. Therefore, the available
space per gram of fish would be the same for both aquaria.
ACUTE BIOASSAY METHODS
Hard Water Acute Bioassays
Flow-through bioassay
One hard water acute bioassay was performed. A proportional diluter
calibrated for a 25% dilution ratio delivered a nominal high concentra-
tion of 10 mg/liter. Ten fish were placed in each of six 35-liter
aquaria. The test rainbows were pre-acclimated to hard water for at
least 30 days prior to being transferred to the acute bioassay aquaria.
Total lead concentrations in each aquaria were analyzed from nonacidified
samples by atomic absorption. Individual fish length and weight data
were collected.
Static bioassays
Three static well water bioassays were conducted with 10 rainbow trout
per concentration using 35-liter aquaria. The aquaria were aerated with
a large airstone to provide mixing and supply dissolved oxygen. Routine
water chemistries consisting of hardness, alkalinity, pH, temperature
and dissolved oxygen were performed. Lead concentrations were analyzed
from acidified samples by atomic absorption spectrophotometry, and by
pulse polarography in static bioassay #3. Individual fish length and
weight data was collected.
The first static bioassay was used to determine the approximate toxicity
of lead in hard water. The nominal lead concentrations were 50, 100, 300,
500, 600 and 1000 mg/liter. The second static bioassay was used to
"pinpoint" a TL,-0 concentration because of the inability of running a
flow-through bioassay which would have exceeded the solubility of lead in
the diluter stock solution. The nominal lead concentrations were 500,
520, 540, 560 and 580 mg/liter. The third static bioassay was run so that
pulse polarographic, free lead, data might be obtained. The determination
of TL,-n concentrations were made by log-probit analysis (Sprague, 1969).
17
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Soft Water Acute Bioassays
The initial soft water flow through acute bioassays employed a 25%
dilution ratio and failed to produce mortality differences between
high and low lead concentrations.
Subsequent soft water acute bioassays were performed using a 50% di-
lution ratio. Each of the test concentrations were duplicated in two
35-liter aquaria by dividing flows from the respective diluter reser-
voirs. Routine water chemistries and lead analyses were made on each
acute test. Chemical analyses were made for alkalinity, hardness, pH,
dissolved oxygen, temperature and conductivity. Fish were fed at an
appropriate feed rate in the soft water acute bioassays since the time
of exposure exceeded 10 days. The TL,-n concentration was determined by
a computerized log-probit analysis (Daum, 1969; and Daum and Killcreas,
1966).
18
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Section V
RESULTS
CHRONIC BIDASSAY RESULTS
Hard Water Growth Bloassay
Introduction
The hard water growth bioassay was initiated 14 May 1970 using 70
fingerling rainbow trout averaging 83.3 mm in length and 6.1 g in
weight. The purpose of this experiment was to determine lead-
attributed mortality, growth or other effects that could be used to
calculate an application factor, and to test the application factor
concept as it applies to two different water qualities. The experiment
was terminated 15 December 1971 after 19 months of chronic lead exposure.
Water analysis
Detailed chemical analyses were made on the hard water source (Table 1).
Of particular interest is a mean hardness of 353.5 mg/liter a methyl
orange alkalinity of 243.1 mg/liter, and a mean pH of 8.02. These
values were determined from the control aquarium while occupied by fish.
Metal analysis on the water source was performed by atomic absorption
spectrophotometry except for the base lead levels which were determined
by pulse polarography. Metal analyses of particular interest are lead,
zinc and copper which in hard water were found to be 0.003, 0.03 and
0.005 mg/liter respectively.
Table 2 gives the chemical results from weekly water analyses over dura-
tion of experiment for dissolved oxygen, pH, conductivity, alkalinity,
hardness and temperature. The mean, standard deviation and range of a
particular analysis are reported. The dissolved oxygen in the six ex-
perimental aquaria was 7.015 + 0.405 mg/liter. An analysis of variance
accompanied with a least significant difference test (Sokal and Rohlf,
1969) showed that the dissolved oxygen levels in aquarium C (control)
were significantly different from the other aquaria (Table 2); however,
this was not considered biologically significant. The higher dissolved
oxygen levels of the control aquarium probably result from fewer number
of fish in this aquarium due to a large number of mortalities that occur-
red early in the experiment. Statistical analysis on pH, conductivity,
alkalinity, hardness and temperature revealed no significant difference
19
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Table 1. Mean chemical analysis of hard and soft water supplies
(concentrations in nig/liter).
Analysis
Alkalinity M.O. (as CaCO_)
Alkalinity phth (as CaC03)
Hardness (EDTA)
Chloride
Nitrate-nitrite nitrogen
Dissolved silica (molybdate-reactive)
Sulfate
IDS (total dissolved solids)
Specific conductance (micromhos/cm)
Dissolved oxygen
pH
Cadmium
Calcium
Chromium
Copper
Iron
Lead
Magnesium
Manganese
Molybdenum
Nickel
Potassium
Silver
Sodium
Zinc
Hard water
243.1
0.0
353.5
23.65
25.52
16.4
103.2
584
1456
7.42
8.02
0.00
75.9
0.00
0.005
0.05
0.003
29.68
0.00
0.0
0.00
1.20
0.00
14.72
0.03
Soft water
26.4
0.0
27.2
3.45
0.95
8.7
2.4
62
170
7.04
6.88
0.00
10.8
0.00
0.005
0.05
0.003
2.71
0.00
0.0
0.00
1.35
0.00
9.40
0.02
20
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Table 2. Average water quality for hard water growth bioassay (May 1970-December 1971).
ro
Tank
Dissolved oxygen (mg/1
Standard Deviation
Range
PH
Standard deviation
Range
Conductivity
Standard deviation
Range
Alkalinity mg/1 (CaCC>3)
Standard deviation
Range
Hardness mg/1 (CaCO-)
Standard deviation
Range
Temperature ( C)
Standard deviation
Range
H-l
7.01
0.92
4.2-9.2
7.86
0.17
7.49-8.27
1450.2
114.87
1200-1800
242.3
17.20
210-290
353.5
24.98
276-400
13.84
3.41
5.0-20.0
H-2
6.61
1.01
3.0-8.3
7.88
0.19
7.48-8.26
1476.6
107.97
1225-1900
242.7
17.36
210-290
352.1
26.42
276-400
14.96
2.72
9.7-20.5
H-3
6.57
1.09
4.0-10.4
7.88
0.18
7.46-8.28
1463.9
116.06
1200-1900
243.0
17.78
210-290
353.2
24.90
276-400
14.90
2.68
9.7-21.0
H-4
7.02
0.90
4.8-9.0
7.92
0.17
7.59-8.20
1448.0
117.11
1125-1650
242.6
17.26
210-290
353.6
24.52
276-400
13.99
3.29
7.5-19.5
H-5
6.98
1.05
3.6-11.5
7.92
0.19
7.66-8.29
1450.0
118.96
1150-1800
242.8
17.42
210-290
353.4
24.78
276-400
14.03
3.21
7.5-20.0
H-Control
7.42
0.80
4.6-8.8
8.02
0.15
7.64-8.25
1456.5
112.07
1200-1750
243.1
18.21
210-298
353.5
24.70
276-400
15.12
2.76
9.0-21.0
-------
between the test aquaria. Chemical results for the six hard water
aquaria were pH (7.94 + 0.08), conductivity (1462.3 + 1.43), M.O. alka-
linity (242.7 + 0.4 mg/liter), hardness (352.85 + 0.75 mg/liter) and
temperature (14.48 + 0.64°C).
Lead analysis
Average values in the six hard water aquaria for lead concentrations
determined by pulse polarography and atomic absorption are given in
Table 3. Equilibrium calculations show that the total solubility of
lead in hard water at a pH of 8.0 is 30 ug/liter (Figure 12 in appendices).
However, as seen from Table 3b, pulse polarographic analysis for free lead,
aquaria #1, #2 and #3 exceeds the calculated solubility. The difference
between analyzed concentrations and the calculated solubility are not sur-
prising, considering the uncertainties of the equilibrium calculations
because of the inaccuracies obtained in defining the experimental con-
ditions from which the calculations were made. The nominal or added
concentrations of lead are much higher than the calculated solubility
(Table 3). Therefore, only a fraction of the total lead added would exist
as soluble species in this water, with the remaining lead existing as
colloidal and precipitated forms.
Comparing pulse polarography and atomic absorption results, one can see
that the lead concentrations found by atomic absorption are much higher
than the corresponding values obtained by pulse polarography (Table 3).
These results are qualitatively those to be expected, because lead (II)
precipitates are generally slow to form and aggregate. This means that
substantial amounts of colloidal material can be present, suspended in the
samples. The atomic absorption analysis method will analyze such material,
whereas the pulse polarography method will not. Therefore, one can roughly
say that analysis of water samples by atomic absorption gives total lead,
consisting of dissociated and suspended forms but not that which has pre-
cipitated to the bottom of an aquarium. Conversely, pulse polarography
will measure only that lead which is dissociated or free in solution.
(See Appendix C for further discussion of problems associated with analyz-
ing lead by atomic absorption).
Analysis of variance and least significant difference tests at a 95% con-
fidence interval on the hard water lead data revealed a significant
difference between the atomic absorption analyzed lead concentrations for
H-l (2.03 mg/liter), H-2 (0.78 mg/liter) and H-3 (0.37 mg/liter). However,
nonsignificance was found between H-3, H-4 (0.20 mg/liter) and H-5 (0.10
mg/liter) and is believed to be the result of analytical problems assoc-
iated with analysis of these lower lead levels by atomic absorption and the
inability of knowing what lead species are present in any particular sample.
For this reason, the computed concentrations obtained from the analyzed H-l
concentration of 2.03 mg/liter lead might be considered to be the most
reliable. However, any atomic absorption analysis of lead in hard water
gives relatively meaningless results because of the various types of com-
plexes, suspended or precipitated, which may or not be present in
22
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Table 3. Hard water lead analytical results (concentrations in mg/liter).
to
UJ
a. Hard water growth bioassay
analyzed by atomic absorption
Nominal concentration*
Analyzed concentration
Standard deviation
Range
Computed concentration from
high analyzed concentration
b. Hard water growth bioassay
analyzed by pulse polarography
Nominal concentration
Analyzed concentration*
Standard deviation
Range
Computed concentration from
high analyzed concentration
•H-l
3.24
2.03
0.76
0.5-3.4
2.03
3.24
.064
.008
.005-. 087
.064
H-2
1.08
0.78
0.26
0.3-1.2
**
0.68
1.08
.044
.031
.03 2-. 090 .
.021**
Aquariun
H-3
0.36
0.37
0.13
0.1-06
**
0.23
0.36
.032
.005
021-.043
.007**
i #
H-4
0.12
0.20
0.07
0.1-03
**
0.07
0.12
.018
.004
.010-. 027
.002**
H-5
0.04
0.10
0.03
0.05-0.20
**
0.03
0.04
.011
.002
.010-. 015
.001**
H-C
0.00
0.00
—
0.0
.003
.001
.002-. 004
—
* Values used in report.
** Computed from analyzed high concentration.
-------
proportion to their presence in an aquarium at the time a sample is taken.
Therefore, stating that atomic absorption analysis represents total lead
is not completely correct because of the absence of precipitated lead
species which may not be collected in a grab sample.
Because of problems associated with hard water total lead analyses by
atomic absorption spectrophotometry, the nominal concentrations, that is,
the amount of lead added to any particular aquarium, are considered to
be the most reliable. The actual amount of lead added to each aquarium
can readily be determined by knowing the concentration of the stock solu-
tion and the dilution ratio of the proportional diluter. The nominal
concentrations, in mg/liter, are: H-l (3.24), H-2 (1.08), H-3 (0.36),
H-4 (0.12), H-5 (0.04) and H-control (0.00). The analyzed lead concen-
trations determined by pulse polarography represent free lead values.
These are, in mg/liter: H-l (.064), H-2 (.044), H-3 (.032), H-4 (.018)
and H-5 (.011). The pulse polarographic concentration for lead in the
hard water control aquarium, H-C (0.003 mg/liter), will be used as the
base lead level for hard water.
Growth results
The growth data for the fish in H-C (the hard water control aquaria) was
found to be statistically larger than the remaining five aquaria. This
growth difference is attributed to drastic reduction in the total number
of fish in the aquaria 4 months after initiation of the experiment. An
error in the feeding rate for these fish also contributed to their
greater growth. No statistical growth difference was found for the re-
maining five aquaria, H-l (3.24 mg/liter Pb) through H-5 (0.04 mg/liter
Pb). Condition coefficients, K = 10 weight/(length) , were determined
and found to be nonsignificant between the six test aquaria.
Occurrence of physical abnormalities
Black tails were first noted in the high hard water concentration,
aquarium H-l (3.24 mg/liter Pb), 6 months after the initiation of the
experiment. One month later in December of 1970, some fish started ex-
hibiting eroded caudal fins and spinal curvatures. By March of 1971, the
"blacktail" effect was observed to be 100% in H-l, 80% in H-2 (1.08 mg/
liter Pb), and 1% in H-3 (0.36 mg/liter Pb). Aquaria H-4 (0.12 mg/liter
Pb), H-5 (0.04 mg/liter Pb) and the control H-C (0.003 mg/liter Pb), did
not demonstrate this effect. Upon terminating the experiment in
December of 1971, the percentage of black tails, spinal curvatures
(lordoscoliosis), and eroded caudal fins was determined for the final 10
fish removed from each of the six aquaria (Table 4). Black tails were
not observed in the lower level concentration aquaria (H-4, H-5 and
control) at any time during the experiment. From these data the maximum
acceptable toxicant concentration ("MATC") in hard water was found to
occur between the nominal concentration of 0.12 mg/liter (aquarium H-4)
24
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and 0.36 rag/liter (aquarium H-3). Table 4 gives the percent of affected
fish for both free and total lead concentrations.
Table 4. Percent of fish affected by physical abnormalities in the
final sampling of the hard water growth bioassay (based on
10 fish).
Abnormality H-l H-2 H-3 H-4 H-5 H-C
Black tail 100 90 70 0 0 0
Lordoscoliosis 100 60 10 0 0 0
Eroded caudal fin 30 20 10 0 0 0
Total Pb (mg/liter) 3.24 1.08 0.36 0.12 0.04 0.00
Free Pb (mg/liter) .064 .044 .032 .018 .011 .003
Discussion relating the effects of lead to various physical
abnormalities
The blacktail phenomenon entails a process whereby the entire caudal region
just posterior to the dorsal fin is blackened (Figures 1, 5 and 6).
Haider (1964) created caudal region blackening by injecting lead into the
top of the ventral fins. This occurs at or posterior to the first caudal
vertebra (or 32nd vertebra). Black tails preceded occurrences of spinal
curvature by at least a month and they were not found to begin simultan-
eously—at least not by observing the exterior morphology of the fish.
Hoffman in his work with whirling disease attributed black tails to pres-
sure exerted on the sympathetic nerves which control the caudal pigment
cells (Hoffman, Dunbar and Bradford, 1962). The pressure arises from a
spinal curvature in the region of the 26th vertebra from which the sym-
pathetic nerves originate. In the lead work it is possible that vertebral
cartilage damage, with a resulting effect on the sympathetic nerves,
occurred prior to an observable flexure in the spinal column, and that the
blacktail phenomenon arises subsequent to vertebral damage.
The spinal curvatures found in the fish exposed to lead were of two types:
scoliosis (bilateral spinal flexures - see Figures 2 and 4, and/or lord-
osis (dorso-ventral spinal flexures - see Figures 1, 3, 5, 6, 7, and 8).
25
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The lead-exposed fish which exhibited spinal curvatures usually demon-
strated both types, in which case the curvature would be described as
lordoscoliosis. The spinal curvature usually started at about the 26th
vertebra and extended posteriorly. Figures 9 and 10 are X-rays of a
normal fish.
Accompanying the two conditions of black tails and lordoscoliosis, 40%
of the affected fish have also exhibited extensive erosion of the caudal
fin with an associated hemorrhagic area at its base, possible again as
a result of neural damage, associated bacterial infection, and/or bio-
chemical blockage (Figures 1 and 3). Also observed and not previously
reported in association with lordoscoliosis spinal abnormalities is the
total or nearly complete paralysis of the entire caudal region and flex-
ured portion of the fish. A progressive muscular atrophy of the caudal
region was observed with or subsequent to paralysis. This is a chronic
disorder marked by progressive wasting away of the muscles with paralysis.
Observations of the affected lead-exposed fish revealed that swimming was
accomplished by motion or bending anterior to the caudal and flexed
portions of the fish. The affected fish were normally lethargic, lying
on the aquarium bottom, but on occasion would dart around the aquarium.
They exhibited difficulty in maintaining position or equilibrium and
would swim tilted to one side. Quivers or trembling movements were
observed periodically as a radiating wave through the caudal region.
Haider (1964) also reported symptoms of lying on bottom, darting movements,
tilted swimming and wave-type muscular movements over the body.
It would appear very doubtful that the fish exhibiting pronounced lordos-
coliosis would be able to spawn under natural stream conditions because
of the paralysis and flexure of nearly half of the fish. One gravid
female existed in the H-l aquarium upon termination of the experiment
15 December 1971. This fish was artificially spawned and the eggs fer-
tilized by milt from two ripe males of the H-l aquarium. The fertilized
eggs, which had very pale coloration, were placed in a hatching aquarium
with the flowing water temperature adjusted to 11°C. Of the 1000 eggs col-
lected, 100% mortality occurred within 14 days. A definite statement
cannot be made regarding possible inhibition of viable egg reproduction at
this concentration of 3.24 mg/liter lead, because of a sample size of one
fish.
Comparison of similar disease related abnormalities with those
found in the lead experiments
As previously stated, Hoffman reported similar occurrences of black tails
and spinal curvatures as being symptomatic of whirling disease (Hoffman,
Dunbar and Bradford, 1962). The lead-exposed fish were examined by our
fish pathologist for the possible parasitic infection of Myxosome cerebralis,
the protozoan that causes whirling disease. The fish were found to be free
of the protozoan.
26
-------
Figure 1. Fish no. R-0, H-l (.064 mg/liter Pb) - lateral view.
Note severe caudal fin erosion and cold brand.
Figure 2. Fish no. R-0, dorsal view. Note extreme scoliosis.
27
-------
Figure 3. Fish no. R-0, X-ray - lateral view.
flexures and caudal fin erosion.
Note spinal column
Figure 4. Fish no. R-0, X-ray - ventral view.
scoliosis of spinal column.
Note extreme
28
-------
Figure 5. Fish no. L-10, H-l (.064 mg/liter Pb) - lateral view.
Note ventral lordosis and cold brand.
Figure 6. Fish no. R-l, H-l (.064 mg/liter Pb) - lateral view.
Note dorsal lordosis.
29
-------
Figure 7. Fish no. L-10, X-ray - lateral view.
lordosis of spinal column.
Note ventral
Figure 8. Fish no. R-l, X-ray - lateral view.
lordosis.
Note severe dorsal
30
-------
Figure 9. Control fish, X-ray - lateral view. Note no flexing of
spinal column.
Figure 10. Control fish, X-ray - ventral view. Note no flexing of
spinal column.
-------
Reichenback-Klinke and Elkan have reported that abnormal spinal curva-
tures have been seen in: (a) hormonal imbalance; (b) faulty genetic
factors; and (c) chronic parasitic infections with Icthyosporidium hoferi,
mycobacteria and Acamthocephala (Reichenback-Klinke and Elkan, 1965).
Icthyosporidium hoferi (Icthyophonus hoferi) is a fungal parasite found to
infect trout and most other fish species (Reichenback-Klinke and Elkan,
1965). The organism has been found in skin lesions, gills, kidney, liver,
spleen, intestine and brain tissues (Erickson, 1965). The effect of the
organism on replacement of brain tissue may cause pressure or damage to
the motor nerves which govern the myomeric musculature, resulting in a
characteristic sigmoid flexure of the spinal column. The lead-exposed fish
in which spinal curvatures developed were examined for Icthyosporidium
spheres and were found to be free of the infective agent.
Nutritional deficiencies of ascorbic acid (vitamin C) have also been re-
ported to cause acute scoliosis and/or lordosis in trout (Poston, 1967).
Neuhaus and Halver reported a twisting in cartilage in gill filaments
long before acute damage was observed in the spinal column, gill cover
or eye support cartilage (Halver, 1970 and Neuhaus and Halver, 1969).
Another nutritional deficiency was reported by Shanks who found pro-
nounced scoliosis in rainbow trout fed diets deficient in the amino acid
tryptophan (Shanks, Gahimer and Halver, 1962). Halver (1970) reported
profound scoliosis and lordosis occurring within 4 weeks in rainbow
trout in which tryptophan was deleted from the diet.
A question might be raised regarding a possible inhibitory effect of
lead on the biochemical metabolism of vitamin C and tryptophan. de Bruin,
1971, reported (on research performed by Gontea and associates with guinea
pigs) that lead decreases the normal vitamin C content of the adrenal gland
(Gontea, 1964). It has also been suggested that lead interferes in some
manner with the enzymatic degradation of tryptophan (Fati, 1961, as
reported by de Bruin, 1971).
At the level of cellular metabolism, the best known adverse effect of lead
is its inhibition of enzymes dependent upon the presence of free sulfhy-
dryl (SH) groups for their activity (Chisolm, 1971). This effect is most
clearly manifested in the biosynthesis of heme. Specifically, lead in-
hibits the enzymatic metabolism of delta-aminolevulinic acid (ALA) and
the final formation of heme from iron and protoporphyrin (de Bruin, 1971).
The enzyme that catalyzes ALA metabolism is ALA-dehydrase. This enzyme
is extremely sensitive to the inhibitory effect of lead.
All cells synthesize their own heme-containing enzymes. Therefore, ALA-
dehydrase is widely distributed in tissues. Millar and his colleagues
(as reported by Chisolm, 1971) found that ALA-dehydrase activity was
inhibited in the brain tissue of heavily lead-poisoned laboratory rats at
about the same rate as it is in the blood suggesting that the enzyme may
be implicated in brain damage. The toxic effect of lead in the central
nervous system is little understood. Two different mechanisms appear to
be involved in lead-caused brain damage (encephalopathy). (1) Edema:
the walls of the blood vessels are somehow affected so that the capillar-
ies become too permeable, causing a swelling of the brain tissue. Severe
32
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swelling within the enclosed cavity can cause a destruction of brain
tissue. (2) Direct injury to nerve cells: it appears that certain
brain cells may be directly injured, or their function inhibited, by
lead (Chisolm,1971).
In conclusion, it would appear that lead might exert its effect on the
occurrence of black tails and spinal curvatures through two possible
chanels: (1) by a biochemical inhibition in metabolism, or (2) by some
direct or indirect influence on the central nervous system whereby
motor nerves controlling myomere musculature and sympathetic nerve
control of caudal pigment cells are affected. However, nothing con-
clusively can be stated at this time regarding the mode by which lead
exerts an effect. It is possible that both channels could operate
simultaneously. The conditions (l^.je., black tails, spinal curvatures
and paralysis) are highly suggestive of possible neurological damage to
fish chronically exposed to lead. Chisolm, 1971, reports that another
result of chronic overexposure to lead is peripheral nerve disease,
affecting primarily the motor nerves of the extremities. The tissue
damage appears to be in the myelin sheath of the nerve fiber. Spe-
cifically, the mitochondria of the Schwann cells, which synthesize the
sheath, seem to be affected. The paralysis of nerves occurs mainly as
radialis and fibularis paralysis. Direct lead damage to the central
nervous system in humans can give rise to delirium, coma, epilepsy and
lead eclampsia (Haider, 1965).
Mortality results
Four mortalities, believed to be lead induced, occurred in H-l, the
high concentration of hard water aquarium having a nominal lead concen-
tration of 3.24 mg/liter. These mortalities occurred on 15 June 1971,
11 August 1971, 10 September 1971, and 12 October,1971. The fish exhibit-
ed black tails from the caudal fin to the dorsal fin, acute lordosco-
liosis, and complete erosion of the caudal fin occurred in three of the
mortalities. A hemorrhagic area at the base of the caudal fin occurred.
Prior to death these fish exhibited similar behavior: (1) showed no
interest in food (anorexia), (2) had a great deal of difficulty in
maintaining equilibrium or otherwise holding their positions in the
water, (3) moved horizontally through the water with the head at a steep
downward incline, and (4) remained largely inactive lying on the
aquarium bottom. This behavior would continue as long as a month before
death. Other mortalities would have undoubtedly resulted had the ex-
periment not been terminated. Upon terminating the experiment 15
December 1971, two of the six remaining fish demonstrated the stressed
behavior just described.
No lead-attributed mortalities occurred in any of the remaining five
aquaria. On 3 and 7 September 1970 the control aquarium (H-C) had two
periods of severe stress from low dissolved oxygen due to malfunctions
with the diluter. A total of six mortalities occurred on these two dates.
33
-------
Subsequent to this, however, was a continuous die-off which continued
until 28 September with a total mortality of 51 fish. Examination after
death showed no gross abnormalities other than a slight erosion of the
gill lamellae. The fish were also examined at the U.S. Bureau of Sport
Fisheries and Wildlife Hatchery at Springville, Utah, and found to be
free of IPN (infectious pancreatic necrosis) and IHN (infectious hema-
topoetic necrosis). It would appear that the stress due to low dissolved
oxygen may have caused irreversible damage to the fish which resulted in
an extended period of mortality. The five fish that remained in this
aquarium were very healthy for the remainder of the experiment.
Hematological results
Fish from the hard water growth bioassay were sacrificed on seven
occasions during the experiment. An attempt was made to sacrifice 10
individuals each sampling period, but this number varied somewhat be-
cause of mortality adjustments that were made to maintain an equal
number of fish per aquarium. The control aquarium H-C was sampled on
only the first and last sacrifices because of the large number of mor-
talities that occured during September of 1970. The fish sampling
periods were: 14 August 1970, 15 October 1970, 16 December 1970, 25
March 1970, 8 July 1971, 11 November 1971, and termination of 15 December
1971.
Analysis of variance at a 95% confidence interval was performed on the
hematological parameters investigated (hematocrit, hemoglobin, and plasma
protein) for the first five sampling periods, 14 August 1970, through 8
July 1971. Analysis revealed no consistent, significant difference be-
tween the experimental aquaria attributable to lead. Analysis was limited
to these five occasions because the necessity of deleting results ob-
tained from precocious males which have high hematocrit and hemoglobin
levels. Analysis could not be performed on data obtained during those
periods in which lead-attributed mortalities occurred again because of
the necessity to delete data obtained from reproductively mature fish.
As would be expected, fish with eroded caudal fins showed significantly
reduced hematocrit and hemoglobin levels and a drastic reduction in the
plasma protein level. These conditions are indicative of post hemorrhagic
anemia due to blood losses from the hemorrhaged area at the base of the
caudal fin.
Soft Water Growth Bioassay
Introduction
The soft water growth bioassay was initiated 14 May 1970 with 70 hatchery-
reared, fingerling rainbow trout averaging 83.3 mm and weighing 6.1 g. The
purpose of the experiment was to determine a soft water application factor
34
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for lead and to test the application factor concept regarding its use
for different water quality types. The experiment was terminated after
19 months of chronic lead exposure.
Water analysis
Table 1 gives a detailed chemical analysis on the tap (soft) water source.
Noteworthy analyses are hardness (27.2 mg/liter), M.O. alkalinity (26.4
mg/liter) and pH (6.88). These values were determined from the control
soft water growth aquarium. Metal analysis of the source water gave levels
for lead, zinc and copper of 0.003, 0.02 and 0.005 mg/liter respectively.
Chemical results from weekly water analysis of the experimental aquaria
are given in Table 5. The mean, standard deviation and range for a par-
ticular analysis are reported. The range for mean value in the six test
aquaria are: dissolved oxygen (6.94 +0.17 mg/liter), pH (6.875 + 0.045),
conductivity (176.2 + 1.2), methyl orange alkalinity (26.25 + 0^25 mg/liter),
hardness (27.05 + 0.15 mg/liter), and temperature (14.40 + 0.46 C).
Analysis of variance on these parameters gave a nonsignificant F value
between the six aquaria.
Lead analysis^
Average lead concentrations determined by pulse polarography and atomic
absorption are given in Table 6. Equilibrium calculations show that the
total solubility of lead in soft water at a pH of 7.0 is approximately
500 yg/liter (Figure 13 in appendices) . The nominal concentrations of
lead in soft water are considerably lower than the corresponding calcu-
lated solubility (Table 6). Consequently, most of the lead added would
exist in the soluble form. The pulse polarography results for lead in
soft water are approximately the same as the nominal values (Table 6) .
These results are to be expected because of the higher calculated solu-
bility (500 yg/liter) versus the amount actually added.
35
-------
Table 5. Average water quality for soft water growth bioassay (May 1970 - December 1971).
Tank #
Dissolved oxygen (mg/1)
Standard deviation
Range
PH
Standard deviation
Range
Conductivity
Standard deviation
Range
Alkalinity mg/1 (CaC03)
Standard deviation
Range
Hardness mg/1 (CaCOj)
Standard deviation
Range
Temperature ( C)
Standard deviation
Range
S-l
7.11
0.75
4.6-8.5
6.92
0.26
6.00-7.55
174.1
31.77
115-270
26.0
5.95
10-42
26.9
5.41
18-40
14.47
2.78
8.5-19.0
S-2
7.11
0.80
5.2-8.5
6.91
0.28
6.32-7.60
173.1
35.90
120-320
26.3
5.96
10-42
27.1
5.23
18-40
13.94
3.26
7.5-20.5
S-3
6.77
1.02
3.6-8.4
6.88
0.27
6.21-7.80
176.8
33.00
110-280
26.4
5.88
10-42
27.2
5.15
18-40
14.85
2.97
8.5-21.3
S-4
6.82
0.97
4.0-8.4
6.83
0.24
6.34-7.70
172.7
33.83
115-300
26.5
5.83
10-42
27.2
5.20
18-40
13.94
3.29
7.5-20.8
S-5
6.97
0.97
4.8-8.4
6.86
0.28
6.34-7.90
177.4
30.80
115-260
26.5
5.88
10-42
27.2
5.22
18-40
14.82
2.99
8.5-22.2
S-Control
7.04
0.94
4.2-8.4
6.88
0.29
6.29-7.90
170.5
30.08
120-290
26.4
5.84
10-42
27.2
5.26
18-40
14.07
3.23
7.5-21.0
-------
Table 6. Soft water growth bioassay—lead analytical results (concentra-
tions in yg/liter).
Aquarium
S-l S-2 S-3 S-4 S-5 S-C
Analyzed by pulse polarography
Nominal concentration 30.0 20.0 13.3 8.9 5.9 0.0
Analyzed concentration* 25.6 19.5 12.2 8.3 5.4 3.1
Standard deviation 3.5 2.6 2.0 1.4 1.0 1.0
Range 20-32 12-24 10-17 6-10 4-7 2-4
Computed concentration from
high analyzed concentration 25.6 17.0** 11.3** 7.6** 5.0** —
Analyzed by atomic absorption** 52.5 35.0** 23.3** 15.5** 10.3** —
Standard deviation 27.0
Range 5-110
* Values used in report
** Computed from analyzed high concentration
Atomic absorption analyses were performed on the highest concentration
aquarium (S-l) and gave an average lead level of 52.5 ug/liter. Calcu-
lated values for the other aquaria were obtained by multiplying the
analyzed high concentration by a factor of 66.6%, a value corresponding
to the 33.3% dilution ratio set by the diluter (Mount and Brungs, 1967).
These concentrations are considerably higher than both the nominal and
pulse polarography results. Such an occurrence might result from exceed-
ing the analytical capability of the procedure and equipment used. Another
possible or accompanying explanation could be a resulting accumulation of
complexed lead in excess of that added to the aquaria due to the adherence
of various lead forms to particulate matter in the tanks. Aquarium clean-
liness was a problem requiring bi-weekly scrubbing of aquarium walls and
resulting in large amounts of particulate matter partially suspended in
the water. Water was removed from an aquarium via an overflow pipe which
tends to decant clearer water from the surface with a concomitant buildup
of denser particulate matter below the surface. This material tends to
remain in suspension because of a circulating action caused by the aera-
tion and filtration systems. Removal of particulate matter by filtration
was a slow process. Atomic absorption analysis of acidified water samples
containing such material would undoubtedly reflect higher than nominal
lead concentrations, whereas pulse polarography would not.
37
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The pulse polarography results for analysis of lead in soft water are
considered to be the most reliable. These results yielded the following,
in yg/liter: S-l (25.6), S-2 (19.5), S-3 (12.2), S-4 (8.3), S-5 (5.4)
and S-C (3.1). Analysis of variance and least significant difference
tests showed significant difference between all concentrations at a 95%
confidence interval.
Growth results
The growth data collected from the soft water growth bioassay showed no
statistical difference in growth between the six experimental aquaria _
at a 95% confidence interval. Coefficient of condition factors, K - 10
weight/(length) , and analyses of variance were determined on fish in
the six aquaria, and revealed no statistical difference in condition
at the same confidence level.
Occurrence of physical abnormalities
Fish with black tails were observed in the two high concentrations, S-l
(25.6 yg/liter Pb) and S-2 (19.5 yg/liter Pb). However, no black tails
were found in the final sample in December 1971, because of the random
sacrificing of these individuals for other experimental purposes. No
physical abnormalities were observed in S-3 (12.2 vg/liter Pb), S-4
8.3 V g/liter Pb), S-5 (5.4 yg/liter Pb) or the control aquarium (3.1
y g/liter Pb). From these data it would appear that the "MATC" for lead
in soft water would exist somewhere around 12 p g/liter. Results from
the F. generation reproduction bioassay gave a pronounced "MATC" for
soft water. These will be discussed later.
Mortality results
No lead-attributed mortalities occurred in the soft water growth bio-
assay. Ten fish died in the control aquarium on 15 April 1971 due to
low dissolved oxygen because of an accidental shut down of the aera-
tion system. The mortalities were replaced with marked fish of the
same size that had been held in similar water quality.
Hematological results
Fish from the soft water growth bioassay were sacrificed on 18 August
1970, 14 October 1970, 16 December 1970, 25 March 1971, 12 July 1971,
11 November, and 15 December 1971. An attempt was made to sample 10
38
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fish from each aquarium at each sampling period. However, in order to
obtain equal numbers of fish in each aquaria, it was necessary to adjust
the number of fish sacrificed to compensate for fish losses. The hema-
tological parameters tested were hematocrit, hemoglobin, and plasma
protein. Analysis of variance at a 95% confidence interval revealed no
consistent significant difference attributable to lead in the various
experimental aquaria. As was the case in the hard water hematological
findings, only that data collected between August 1970 through July
1971, five sampling periods, were compatible for a reliable statistical
analysis because of the necessity of deleting data collected from pre-
cocious males. A nonsignificant effect of lead on the hematological
parameters tested could be due to the low lead concentration of this
experiment.
Water Temperatures in Soft and Hard Water Growth Bioassays
The hard (well) water with a year-round temperature of 15 C was cooled
to the ambient soft (tap) water temperature except when soft water
temperatures exceeded 16 C, in which case, the soft water was cooled to
the hard water temperature. A t-statistic was used to analyze possible
temperature difference between the hard and soft water growth bioassays
and revealed no statistical difference.
Soft Water Reproduction Bioassay
Introduction
This experiment was complementary to the soft water growth bioassay with
the purpose of determining the effect of lead on rainbow trout repro-
duction. The bioassay was initiated 21 August 1970 with 2-year-old fish,
two males and two females, and terminated following spawning after 8
months of chronic lead exposure.
Water Analysis
Chemical results from weekly water analysis are reported in Table 7. These
results from the six reproduction aquaria are dissolved oxygen (7.14+
0.21 mg/liter), pH (6.855 + 0.055), conductivity (179.0 + 3.1), M. 0.
alkalinity (29.85 +0.05 mg/liter), hardness (28.4+0.3 mg/liter), and
temperature 12.17 + 0.58°C). Analysis of variance and a least signifi-
cant difference test at a 95% confidence interval showed no significant
difference in the chemical parameters tested.
39
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Table 7. Average water quality for reproduction bioassay in soft water (August 1970 - May 1970).
Tank
Dissolved oxygen (mg/1)
Standard deviation
Range
PH
Standard deviation
Range
Conductivity
Standard deviation
Range
Alkalinity mg/1 (CaC03)
Standard deviation
Range
Hardness mg/1 (CaCO-)
Standard deviation
Range
Temperature (°C)
Standard deviation
Range
R-l
7.35
0.96
5.4-9.0
6.91
0.20
6.45-7.33
175.9
21.70
145-230
29.9
4.92
18-40
28.1
6.58
22-40
11.59
3.00
7.2-18.7
R-2
7.12
0.92
4.6-8.4
6.90
0.14
6.62-7.18
182.1
26.55
135-265
29.9
4.92
18-40
28.7
4.65
26-40
12.72
2.74
9.0-19.8
R-3
7.16
1.09
4.6-9.0
6.83
0.14
6.48-7.09
178.3
20.53
135-230
29.8
5.03
18-40
28.6
4.82
20-40
11.64
3.08
7.2-18.8
R-4
6.93
1.06
4.2-8.4
6.83
0.14
6.55-7.20
181.5
22.96
135-235
29.8
5.03
18-40
28.6
4.72
20-40
12.75
2.85
9.2-20.5
R-5
6.93
0.89
4.2-8.2
6.85
0.15
6.62-7.20
181.1
24.84
135-250
29.9
4.92
18-40
28.6
4.72
20-40
12.62
2.91
7.2-20.2
R-Control
6.93
0.90
5.4-8.8
6.80
0.13
6.53-7.08
179.7
23.58
140-250
29.8
5.03
18-40
28.7
4.65
20-40
11.68
3.13
7.2-19.0
-------
Lead analysis
Lead results determined by pulse polarography and atomic absorption are
reported in Table 8. As was previously found to occur in the soft water
growth bioassay, the pulse polarography results closely approximated the
nominal lead levels, whereas, atomic absorption findings computed from
analysis of the high concentration were significantly higher than the nom-
inal and pulse polarography lead levels. (Explanations for this were
postulated under the soft water growth lead findings). Limited data and
within group variance made statistical analysis impractical for pulse polar-
ographic lead findings. For this reason the computed lead levels determined
from the pulse polarographic analysis of the high concentration are considered
to be the most reliable results. The lead concentrations in yg/liter are:
R-l (27.0), R-2 (18.0), R-3 (12.0), R-4 (8.0), R-5 (5.3) and R-C (2.6 -
analyzed value.
Table 8. Soft water reproduction bioassay—lead analytical results
(concentrations in yg/liter).
Aquarium//
R-l R-2 R-3 R-4 R-5 R-C
Analyzed by pulse polarography
Nominal concentration 30.0 20.0 13.3 8.9 5.9 0.0
Analyzed concentration 27.0 17.5 10.4 7.7 6.0 2.6
Standard deviation 5.3 3.3 2.1 1.6 1.6 0.8
Range 21-34 10-23 8-15 6-10 4-8 2-4
Computed concentration from *
high analyzed concentration 27.0 18.0** 12.0** 8.0** 5.3**
Analyzed by atomic absorption 58.5 39.0** 25.9** 17.3** 11.5**
* Values used in report
** Computed from analyzed high concentration
Occurrence of physical abnormalities
At termination the 3-year-old brood fish of the soft water reproduction bio-
assay did not demonstrate any of the physical abnormalities (i.e., black tails,
lordoscoliosis or eroded caudal fins) as seen in some of the growth bioassay
fish. With lead concentrations the same as in the soft water growth bioassay,
which showed only a slight effect due to lead, the noneffect would probably
be expected. One fish died in the soft water reproduction bioassay prior to
spawning. This was a female in the lowest concentration aquaria (R-5 with
5.3 yg/liter Pb) that refused to eat and soon starved to death.
AT
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Spawning
Plastic pans (30 cm x 25 cm x 15 cm) were placed in each of the six test
aquaria on 4 March 1970. Each pan contained 6 kg gravel (15-30 mm diameter)
which yielded a depth of approximately 8 cm. Attempts at making a redd
occurred in aquaria #2 (18.0 yg/liter Pb) and #5 (5.3 yg/liter Pb) and a
few dead eggs were observed outside of the spawning pans in these aquaria
on 24 March. Natural spawning did not occur in any of the aquaria, pos-
sibly as a result of competition or inadequate spawning pan size.
All fish were anesthetized in MS-222 on 31 March 1971 and attempts at
artificial spawning were made. Females that were not gravid at this time
were examined weekly until spawning occurred. Artificial spawning was com-
pleted 7 May 1971. All reproductive fish were numbered in accordance with
the aquarium in which they resided. Thus, the fish in Aquarium #1 (highest
lead concentration) became females //la and //lb and males //la and //lb.
Fertilized eggs were placed on nylon net egg trays in 35-liter aquaria of the
acute bioassay system. The hatching aquaria identification number coincided
with the females from which the eggs were obtained with one aquarium used
for hatching the eggs of a particular female. A 50% diluter with a nominal
high concentration of 100 yg/liter supplied the toxicant. This concentra-
tion is considerably higher than that set for the parent generation (nominal
high concentration of 30 yg/liter) in an attempt to obtain lead-caused
mortalities in the high concentration.
Hatching and separation to large F.. aquaria
Atomic absorption analysis of the high concentration for lead in the hatching
aquaria (la and lb) was 101.8 yg/liter. Concentrations for the other aquaria
were computed from the analyzed high concentration using the 50% diluter
ratio. Successful hatching occurred in aquaria: //la(101.8 yg/liter Pb),
#2a(50.9 yg/liter Pb), #2b(50.9 yg/liter Pb), #3b(25.4 yg/liter Pb),
#4a(12.7 yg/liter Pb), Ca(control, 3.1 yg/liter Pb, base level). Aquaria
#lb, #3a, $4b, $5a, #5b, and Cb suffered 100% egg mortality. Surviving eggs
took 29 to 30 days to hatch at a mean temperature of 12.66 C. The fry
hatched from aquarium #2a exhibited 80% deformity. An explanation for the
100% egg mortalities at designated concentrations and for the deformed fry in
aquarium #2a cannot be given. The extent to which lead-affected egg mor-
talities cannot be stated since mortalities occured in high and low concen-
trations alike. Weekly chemical data from the hatching aquaria showed a
mean dissolved oxygen of 8.88 mg/liter (range 8.0-10.6) and a mean tempera-
ture of 12.66°C (range 10.0-14.0), which represents a mean saturation of 100%.
Viable fry were collected from at least one of the two reproduction females
for all concentrations except aquarium #5 (parental lead concentration of 5.3
yg/liter or F., progeny lead concentration of 6.4 yg/liter). Two hundred
and fifty fry were transferred from the hatching aquaria (//la, #2b, #3b,
#4a and Ca) to the large bioassay aquaria that their parents previously
42
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occupied. With 100% egg mortality in aquaria #5a and #5b (6.4 ug/liter Pb),
an additional 250 fry were transferred from the hatching aquarium /Ma
(12.7 ug/liter Pb) to the large chronic bioassay aquarium, #5. Tanks //la
and #4a completed hatching on 3 May and were separated on 3 June to the
large aquaria (250 fish from hatching aquarium #la to F.. reproduction
aquarium #1, and 500 fish from hatching aquarium #4a with 250 allocated to
FI reproduction aquaria #4 and #5, respectively). Aquaria #2b, #3b and
Ca/hatched on 31 May and 250 fish were transferred to the respective FI re-
production aquaria on 28 June 1971.
Use of 2-Year-Old Rainbow Trout as Spawning Stock to Study Toxicant
Effects on Reproduction
The 2-year-old fish of the hard and soft water growth studies were to be
used as brood fish. Examination of all remaining fish during the November
1971 sampling period, indicated a low percentage of reproductively mature
females. Because of reproductive immaturity, the thought of using 2-year-
old rainbow trout as brood fish was discontinued.
Data collected from the hard and soft water growth bioassays during the No-
vember and December sampling periods substantiated our decision to terminate
the experiment. These fish were approximately 2 years old. Of the 51 fish
remaining in the hard water growth bioassay, 27 were females of which 29.6%
or eight were gravid. The remainder would not have produced eggs until next
year. Fifty-seven fish remained in the soft water growth bioassay. Of
these, 36 were females of which only 7 or 19.4% were gravid. The 29 other
females would not have produced eggs until next year. Both experiments had
one aquarium (H-3 and S-3) in which there were three gravid females. Aquaria
H-4, H-5 and S-C had no reproductively mature females. Obviously, the per-
centage of reproductively mature females as 2-year-old rainbow trout is too
low for providing a reliable brood stock and valid statistical results for
studying effects of toxicants on reproduction,
Soft Water F. Generation Reproduction Bioassay
Introduction
This experiment was conducted on the progeny obtained from the 3-year-old
brood stock of the soft water reproduction bioassay. The experiment ran for
approximately 7 months from the date of hatching to 15 December 1971, when
it was terminated. Lead levels were increased above those of the parent
generation in an attempt to obtain lead-caused mortalities in the higher
concentration aquaria.
43
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Water analysis
Weekly water analyses were made on the six test aquaria (Table 9). The
mean, standard deviation and range of the various determinations are reported.
The range for the mean values are: dissolved oxygen (7.29 +_ 0.15 mg/liter),
pH (6.87 + 0.04), conductivity (148.6 + 2.2), methyl orange alkalinity
(23.0 + 0.3 mg/liter), hardness (23.95 +0.15 mg/liter) and temperature (16.31
+ 0.51~~°C). An analysis of variance and least significant difference tests
at a 95% confidence interval revealed no statistical difference between the
six experimental aquaria for the chemical parameters analyzed.
Lead jma lysis
The average lead concentration for the two high concentration aquaria was
determined by atomic absorption spectrophotometry. Lead levels in the other
aquaria, F,#3 through F-C, could not be reliably determined because of
detection problems associated with atomic absorption analysis. Aquarium F.^2
reflected a higher than nominal lead concentration (Table 10) and probably
corresponds to a lead concentration at which analytical problems with atomic
absorption become apparent. For this reason, lead concentrations for aquaria
through F^/5 were computed using the 50% dilution ratio set by the
uter. Therefore, the reported lead concentrations for F, generation re-
production bioassay are, in tig/liter: F #1 (95.2), F #2 (47.6), FI #3 (23.8),
Fx#4 (11.9), F^/5 (6.0) and V^C (3.1) (Table 10).
Table 10. Soft water FI generation reproduction bioassay — lead analytical
results (concentrations in yg/liter) »
Aquarium
"l-1
Fl"2
V3
V4
V5
Frc
Analyzed by atomic absorption
Nominal concentration 100.0 50.0 25.0 12.5 6.2 0.0
Analyzed concentration 95.2 52.6
Standard deviation 16.8 15.6
Range 60-130 20-90
Computed concentration from*'
high analyzed concentration 95.2 47.6** 23.8** 11.9** 6.0** 3.1***
* Values used in report
** Computed from analyzed high concentration
*** Base level of lead
44
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Table 9. Average water quality for soft water F- generation reproduction bioassay May 1970-December 1971)
Tank #
Dissolved oxygen (mg/1)
Standard deviation
Range
pH
Standard deviation
Range
Conductivity
Standard deviation
Range
Alkalinity mg/1 (CaCCL)
Standard deviation
Range
Hardness mg/1 (CaCOO
Standard deviation
Range
Temperature (°C)
Standard deviation
Range
V1
7.44
0.73
6.2-9.0
6.91
0.24
6.40-7.26
146.4
11.57
123-165
22.8
5.70
16-34
24.0
4.92
18-34
15.85
1.17
12.8-17.5
V2
7.26
0.96
4.0-8.2
6.87
0.22
6.40-7.24
147.8
14.58
120-170
22.8
5.66
16-34
24.1
4.92
18-3'
16.63
0.82
15.0-18.0
Fr3
7.37
0.76
5.8-8.2
6.85
0.20
6.46-7.14
146.9
13.92
120-170
23.3
5.63
16-34
24.1
4.92
18-34
15.80
1.24
12.4^-18.0
F,-*
7.19
0.61
6.0-7.8
6.89
0.30
6.10-7.25
147.9
13.67
120-170
22.7
5.70
16-34
24.1
4.92
18-34
16.80
1.05
14.2-18.5
V5
7.15
0.87
6.0-9.6
6.87
0.30
6.05-7.16
150.8
15.92
120-175
22.7
5.70
16-34
24.1
4.92
18-34
16.82
1.04
13.9-18.0
F--Control
7.13
1.04
5.0-10.0
6.83
0.24
6.25-7.18
147.9
12.65
123-170
23.1
5.82
16-34
23.8
4.84
18-34
16.10
1.08
12.8-19.0
-------
F.#l were found to be statistically larger than fish
. F_#2 was different from aquaria F #1, F^#3 and F-^C.
nce existed in growth between F^/3, F-^M, F^/5 and
Growth results
Six months after hatching, 30 fish from each aquaria were analyzed for
possible growth differences. Total length in millimeters was selected as
the parameter for measuring growth. Since the fish in aquaria F^#l, F.#4
and F.#5 hatched 1 month prior to the hatching of fish in aquaria F-#2,
F. #3 and F.. C (control), the growth measurements were taken 1 month apart
(at 6 months from the two hatching dates). Aquarium F..#l (95.2 yg/liter Pb)
had a mean length of 113.87 mm, F #2 (47.6 yg/liter PB) 99.13 mm, F.^/3
(6.0 yg/liter Pb) 92.67 mm, and F,C (3.1 pg/liter Pb) had a mean length of
90.08 mm. Analysis of variance and a least significant difference test at
a 95% confidence interval revealed that there was a significant difference
in growth. Fish from F,#l were found to be statistically larger than fish
from all other aquaria.
No statistical difference
These differences are not thought to be lead caused. The egg sources for
each aquarium came from individual females of the reproduction bioassay. Eggs
that were obtained from these females were of different sizes. The eggs ob-
tained from high concentration reproduction bioassay R-l were larger than
those collected from any of the other brood females. Egg size, associated
with possible genetic differences of the individual brood stock, are thought
to cause the growth differences observed in the fingerling progeny. Because
of these differences and problems associated with handling such data and
data collection of fish with different hatching dates, it was decided to
terminate the experiment.
Occurrence of physical abnormalities
Black tails were first observed in the F reproduction bioassay 11/2 months
after hatching. Table 11 gives the total number of fish per aquarium,
percent black tails, eroded caudal fins and spinal curvatures. Initially,
250 fry were placed in each of the six experimental aquaria. This experiment
was terminated on 15 December 1971.
Table 11. Percent of fish affected by physical abnormalities in the F^
generation reproduction bioassay at termination of experiment.
F #1 F #2 F #3 F #4 F_#5 F-C
Number of fish 76 207 193 204 238 192
Abnormality:
Black tail 100.0 91.3 42.0 2.0 0.0 0.0
Lordoscoliosis 3.9 0.0 0.0 0.0 0.0 0.0
Eroded caudal fin 7.9 1.4 0.5 0.0 0.0 0.0
46
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From these data the maximum acceptable toxic concentration ("MATC") exists
between the lead concentrations of F #4 (11.9 yg/liter) and F #5 (6.0 yg/
liter). These concentrations are in close agreement with the apparent
"MATC" value of 12.2 yg/liter lead obtained in the soft water growth bio-
assay. The 2.0% black tails reported for aquarium FI#4 represents four
fish. Therefore, the true "MATC" value for lead in soft water is probably
very close to the 11.9 yg/liter lead concentration. As witnessed in
the hard water growth bioassay, one might expect the occurrence of lord-
oscoliosis to become much more pronounced as these fish increase in size.
Mortality results
Initially, 250 fish were transferred to each of the test aquaria from
the hatching tanks. Fish were placed in aquaria F-#1 (95.2 yg/liter Pb),
F../M (11.9 yg/liter Pb) and F #5 (6.0 yg/liter PB) on 3 June, 1971, 1
month after hatching. Aquaria F #2 (47.6 y g/liter Pb), F #3 (23.8
yg/liter Pb) and F..C (control with 3.1 yg/liter Pb) received fish on
28 June, 1971, 1 month following egg hatching.
The experiment was terminated 15 December 1971. No mortalities occurred
in aquaria F #1, F../M and F-#5 during the last 25 days of this period.
Therefore, trie mortality data presented in Table 12 are indicative of
total mortality figures with equal time exposure to lead for all aquaria.
Mortalities of 66.8% in aquarium F.#l clearly show a lea'd-caused effect.
Mortalities in the lower concentrations are probably not attributable
to lead. Table 12 gives the percent of fish unaccounted for. Cannibal-
ism was observed, and the number of missing fish probably reflect the
extent to which this occurred in each aquaria.
Table 12. Mortality results for F- generation reproduction bioassay
based on initial 250 fish per aquaria.
Total no. fish accounted for
No. mortalities
% mortality
% of fish unaccounted for
Fj*!
229
153
66.8
8.4
F.^2
238
29
12.2
11.6
F1#3
200
7
3.5
17.6
F1#4
208
4
1.9
16.8
F.^/5
241
3
1.2
3.6
F1C
218
26
11.9
12.8
Hematological results
Fish sampled from aquaria F,//l, F..//4, and F.^/5 were one month older than
fish in the remaining aquaria. Hematocrit values for fish from aquaria
F,#l (95.2 yg/liter Pb) were significantly lower at a 95% confidence
interval than hematocrit values obtained from fish of aquaria F^#4 and
FT#5. No statistical difference was found between fish of aquaria F-^/4
and F1#5. Anemia, signified by low hematocrit and hemoglobin levels,
would normally be expected where chronic lead poisoning has occurred
47
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(Dawson; 1935, Farrelly and Pybus, 1969; and de Bruin, 1971). Fish
sampled from aquaria F.#2, F-#3 and F.C were one month younger than fish
from the other three aquaria because of the difference in spawning and
hatching dates. Analysis of variance and least significant difference
tests showed no statistical difference existing in the hematocrit
,#3 and F-C aquaria. The nonsignificance
""probably indicative of the younger aged fish,
which normally exhibit increase in hematocrit percentages with increased
size and age, especially in the initial year of growth.
percentages for fish from F..#2, F-#S ~«« *.»
of the hematocrit values is probabl\ '
ACUTE BIOASSAY RESULTS
Hard Water Acute Bioassays
Flqw-_thr_o_ugh_b_ioas say
A hard water flow-through acute bioassay was conducted with a nominal
high concentration of 10 mg/liter lead using rainbow trout with mean
length and weight of 152 ran and 35.2 g respectively. The solution in the
high concentration was milk white due to the complexation of lead as a
carbonate. After 3 days one mortality occurred in the low concentration
with a nominal lead concentration of 3.16 mg/liter. Upon examination,
the fish was found to be diseased. After a total of 9 days no additional
mortalities occurred and the test was terminated. A decision was made to
perform an acute static test to determine the approximate toxicity of
lead in hard water.
Static bioassay
A hard water static acute bioassay was conducted using six 35-liter aquaria.
Ten rainbow trout with a mean length of 93 mm and weighing 8.2 g were
placed in each concentration and a control aquarium. The nominal lead con-
centrations were, in mg/liter: 1000, 600, 500, 300, 100 and 50. One
hundred percent mortality occurred within 1 hr at a concentration of 1000
mg/liter lead. The pH of this water was 4.55 and lies within a tolerable
pH range for rainbow trout as stated by McKee and Wolf, 1963. Total mor-
tality occurred in the 600 mg/liter lead aquarium at 18 hr. No mortalities
occurred in the 500, 300, 100 and 50 mg/liter aquaria or the control after
8 days of exposure.
Table 13 gives water chemistry findings determined from each of the aquaria.
It can be noted that the hardness of about 385 exists for all aquaria,
whereas, the alkalinity and pH decrease as the lead concentration increases.
This results from the precipitation of carbonates as lead carbonate. One
can readily see why the alkalinity (a measurement of the carbonate system)
is reduced with the precipitation of carbonates from solution. The decrease
in pH results from the release of hvdrogen ion which was tied up in the
bicarbonate buffering system as HCO«.
48
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Table 13. Chemical results for static hard water acute bioassay #1.
Pb nominal
concentration 1000 600 500 300 100 50 Control
Hardness
(ing/liter) 386 384 382 388 386 384 384
Alkalinity
(mg/liter) 2 5 30 118 218 240 267
pH 4.55 6.11 7.12 7.78 7.99 8.01 8.15
Temperature (°C) 12.0 11.0 11.5 12.0 11.5 11.0 11.0
Dissolved oxygen
(mg/liter) 9.2 8.6 8.6 9.4 9.4 9.2 9.0
It is rather obvious that acute lead toxicity occurs from the amount of
free lead to which fish are exposed and not the complexed or precipitated
lead species as demonstrated by no mortality occurring in 500 mg/liter
lead and 100% mortality with 600 mg/liter. Atomic absorption analysis of
the various lead concentrations showed a tremendous variation from one
sampling period to another. A large portion of this variation undoubtedly
resulted from a disproportionate sampling of free, colloidal and precipi-
tated species of lead in the agitated aquaria. This variance was reduced
by collecting water samples in beakers which would stand for 45 minutes
to allow the precipitated forms to settle out of solution, at which time
10 ml of water was pipetted off the surface, acidified and analyzed by
atomic absorption. Table 14 gives the results of three consecutive
sampling periods collected in this manner. Note the daily decrease in
the observed lead concentration with time. This could largely result
from the aggregation of colloidal lead forms within the aquaria over time
to form precipitates which settle out of solution. As stated previously,
formation of lead precipitates is a slow process. This occurrence could
be visualized in the aquaria themselves. Initially, upon the addition of
lead nitrate, all the aquaria were very milky. The 500, 600 and 1000
mg/liter aquaria became quite clear within 3 hr with a large amount of
lead carbonate precipitated on the aquaria bottoms. The aquaria with lead
concentrations of 50, 100 and 300 mg/liter remained cloudy during the
8 days of the experiment but the cloudiness diminished daily.
49
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Table 14. Lead analysis results (in ing/liter) of static hard water
acute bioassay #1. I/
Aquarium no. - Nominal Pb concentration
Samples 1000 600 500 300 100 50
1 220 17.0 1.0
2 0.50
3 0.27
15.7
8.52
2.05
23.3
6.17
1.15
3.87
2.60
0.70
I/ Analyzed by atomic absorption and therefore does not represent free
lead values.
Static bioassay #2
On the basis of the first hard water static bioassay, it would appear
that a flow-through acute bioassay would require a high concentration of
600 mg/liter lead. The stock solution concentration was computed to be
1256 g/liter at a 50% dilution ratio and 1666 g/liter at a 33% dilution
ratio with our proportional diluter. These stock solution concentrations
greatly exceed the solubility of lead nitrate in water (376.5 g/liter at
0 C). Running a flow-through acute bioassay in hard water would be totally
impractical both from the standpoint of having to redesign our proportional
diluter in order to achieve the desired high concentration but also because
of the wide range of concentrations obtained with either a 50% or 33%
dilution ratio.
Due to the above problem, it was decided that a narrow-range static bio-
assay would be much more practical and also serve to delimit the con-
centration range at which mortalities occur. It is recognized that the
lead concentration mortality levels would be somewhat less with a flow-
through bioassay than with a static bioassay but considering the wide con-
centration range of the flow-through bioassay, the latter is considered to
provide the most useful information. The experiment consisted of five
35-liter aquaria with lead concentrations of 500, 520, 540, 560 and 580
mg/liter and a control aquarium. Ten rainbow trout with a mean length
and weight of 85.9 mm and 5.9 g respectively, were acclimated to hard
water for 8-days and placed in each of the experimental aquaria.
Water chemistry data was similar to that of the previous test. Hardness
(385 mg/liter) was essentially the same for all aquaria. The M.O. alkalin-
ity decreased from 25 mg/liter (500 mg/liter Pb) to 6 mg/liter (580 mg/
liter Pb). The pH decreased from 7.17 to 6.23 with increased lead
50
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concentration. The temperature ranged from 13.8°C to 14.2°C, and dis-
solved oxygen ranged from 8.4 to 9.0 ing/liter. The acute bioassay ran
for 96-hr at which time it was terminated because of no additional
mortalities occurring during the last 24-hr (Eaton, 1970). The 96-hr
mortality distribution was as follows: 500 mg/liter lead (0%), 520
mg/liter lead (0%), 540 mg/liter lead (30%), 560 mg/liter lead (100%)
and 580 mg/liter lead (100%) (Table 15) . A TL5Q (96-hr) value of 542
mg/liter was determined by a log-probit method {Sprague, 1969).
Table 15. Lead analysis (in mg/liter) and 96-hr mortality results for
static hard water acute bioassay #2.
Aquarium no. = Nominal Pb concentration
Samples 500 520 540 560 580
(Days post sampling)
1 1.28
2 1.53
3 0.54
4 0.52
X 0.97
0.85
0.56
0.36
0.17
0.48
1.13
1.05
0.50
0.47
0.79
6.42
6.32
4.26
4.17
5.29
8.75
8.42
4.64
4.45
6.54
% Mortality 0 0 30 100 100
Table 15 gives the analytical results for lead as determined by atomic
absorption spectrophotometry. As seen previously, the concentration de-
creases daily, probably as a result of the slow precipitation of lead.
These results are assumed to approach free lead values for the aquaria
containing nominal lead concentrations of 520, 540, 560, and 580 mg/
liter. As was stated previously, atomic absorption results for lead in
soft water give essentially free lead results because of the high solu-
bility of lead in this water. In a general sense, the analysis for lead
in hard water in which the carbonate buffering capacity has been elimin-
ated by the precipitation of lead carbonate, is analogous to atomic
absorption lead analysis in soft water. However, such analyses cannot
be construed as actually free lead, since pulse polarographic analysis
was not performed to substatiate this. The mean concentrations for the
four sampling periods is given in Table 15. Using these data, a log-
probit plot (Sprague, 1969), gave a TL,-0 concentration of 1.00 ppm lead
in 96-hrs. A nominal TL,-0 lead concentration of 542 ppm was determined
by the same procedure.
(See Appendix E for discussion on the nature of lead in hard water as
analyzed by atomic absorption spectrophotometry).
51
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Static bioassay #3
A third hard water static bioassay was performed for the purpose of
obtaining and comparing pulse polarographic free lead results with
analytical results obtained by atomic absorption spectrophotometry.
The bioassay was run in 35-liter aquaria placed in a Min-o-cool tank
which served as a water jacket for maintaining a temperature of 7 C.
The soft water acute bioassay (yet to be reported) was conducted at a
temperature of 7 C. Therefore, both experiments were run at comparable
temperatures. Ten rainbow trout with a mean length and weight of 132
mm and 24 g respectively, were placed into each of six static aquaria
eight hours after the introduction of the toxicant. This allowed for
an initial equilibrium-free lead level to be achieved and minimized
toxicant shock to the fish. The nominal lead concentrations tested
were 500, 490, 480, 470, 460 rag/liter lead and a control aquarium.
Water chemistry data is presented in Table 16. It will be noted that
the hardness (290 mg/liter) and alkalinity (228 mg/liter) in the control
aquarium is lower than that in the static hard water bioassays 1 and 2,
in which the hardness was 384 mg/liter and the alkalinity was 267
mg/liter. These differences lie within the natural seasonal range
found in the hard water source as shown in Table 2. As was found in
the previous two static bioassays, the alkalinity and pH decrease with
increased lead concentrations and the hardness remains relatively un-
changed. As would be expected, it can also be seen that conductivity
increases with increased lead concentrations (Table 16).
Table 16. Chemical results for static hard water acute bioassay #3
Pb nominal
concentration 500 490 480 470 460 Control
Hardness
(mg/liter) 300 300 300 300 300 290
Alkalinity
(mg/liter)
phth 0000 09
M.O. 7 9 10 20 21 228
pH 6.89 6.91 6.97 7.08 7.26 8.78
Conductivity 1480 1460 1440 1420 1400 1080
Temperature (°C) 7.0 7.0 7.0 7.0 7.0 7.0
Dissolved oxygen
(mg/liter) 9.8 9.4 9.8 9.4 9.2 9.8
52
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Water samples for lead analysis were collected daily in 4 oz Nalgene
bottles which were sealed, placed in a plastic bag filled with experi-
mental water, and maintained at the 7°C bioassay temperature. Upon
termination of the experiment, the samples for the four day sampling
period were allowed to stand undisturbed to permit settling of lead
carbonate precipitate at which time aliquot samples were pipetted off
the surface and pooled for pulse polarographic and atomic absorption
analysis. The pooled samples were analyzed by four procedures: 1)
pulse polarography, 2) atomic absorption spectrophotometry: a) un-
filtered, b) filtered 0.45y (Millipore filter), and c) filtered 0.025y
(Millipore filter). The analytical results for lead are given in
Table 17.
Table 17. Lead analysis (in rag/liter) and 96-hr mortality results for
static hard water acute bioassay #3.
Aquarium no. = Nominal Pb concentration
460 470 480 490 500
Analytical procedure
1. Pulse polarography 0.29 1.26 2.77 4.79 7.18
2. Atomic absorption
a.
b.
c.
unfiltered 0.25
0.45y filtered 0.103*
0.025y filtered 0.005*
1.30 2.85
0.545*
0.805*
5.05
0.380*
0.465*
7.56
—
••••
% Mortality 0 30 100 100 100
* Atomic absorption analysis utilizing a modified APDC - MIBK extraction
technique (Fishman and Midgett, 1968).
It can be noted from the analytical data (Table 17) that in the analysis
of lead in hard water, where the carbonate buffering capacity of the water
has been eliminated by the addition of lead, pulse polarography and atomic
absorption results are quite comparable. It is also evident that the fil-
tration of water sample through either 0.45y or 0.025y filters removes
free lead from the water and gives inconsistent and erratic results.
Marvin, Proctor and Neal, 1970, reported similar finding in analyzing for
copper in filtered fresh and seawater samples.
The bioassay ran for a period of 96 hours at which time the test was ter-
minated because of no additional mortalities occurring during the last
24 hours of the experiment (Eaton, 1970). The mortality distribution is
53
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given in Table 17. The nominal lead concentration giving 50% mortality
occurred between 470 ppm lead with 30% mortality and 480 ppm lead with
100% mortality. A log-probit method of analysis (Bprague, 1969), gave a
nominal 96-hr TLqn of 471 mg/liter lead and a free lead 96-hr TL,.n of
1.38 mg/liter. DU 50
Soft Water Acute Bioassay
A soft water flow-through acute bioassay was conducted using a 50% dilu-
tion ratio with a high nominal concentration of .50 mg/liter lead.
Rainbow trout with a mean length and weight of 161 mm and 43.0 g respec-
tively, were acclimated to soft water with a mean hardness of 30 mg/liter
for 2 weeks. Ten fish were acclimated in duplicate 35-liter aquaria for
each concentration of lead for 3 days prior to the addition of toxicant.
Water chemistry results gave the following mean values: hardness (30.0
mg/liter), alkalinity (29.3 mg/liter), pH (6.85), conductivity (165
yhos/cm), and temperature (7.0 C). The mean dissolved oxygen was
8.36 mg/liter and ranged from 6.4 to 9.8. Table 18 gives the mean lead
concentrations analyzed by atomic absorption and percent mortality for
each duplicate concentration. The TL,.- value was calculated utilizing a
computerized log-probit method of analysis from the lethal threshold con-
centration and was found to be .14 mg/liter over an 18-day period(Daum,
1969; Daum and Killcreas, 1966). The fish were fed an appropriate ration
during the experiment because of the extended time period over a routine
96-hr acute.
Table 18. Lead analysis in mg/liter and mortality results for soft
water acute bioassay.
Aquaria
la Ib 2a 2b 3a 3b 4a 4b 5a 5b Ca
Mean lead
concentration .55 .52 .26 .28 .16 .13 .077 .067 .1 .03 0
% mortality 100 100 70 90 40 27 0 20 000
APPLICATION FACTORS FOR HARD AND SOFT WATER BIOASSAYS
Hard Water Application Factor
As explained previously, the lead analyses in hard water by atomic absorp-
tion spectrophotometry are relatively meaningless because of the inability
54
-------
of knowing what the nature (free, colloidal or precipitated) of lead .
measured in a sample actually is. This situation leaves two avenues
by which the application factor can be approached. These are: (1) we
know the total amount of lead added (i.e_., the nominal concentration),
and (2) the pulse polarography analysis of free lead in the chronic
hard water growth bioassay. Table 19 summarizes this analytical data
for lead that is of particular concern for calculating the hard water
application factor.
Table 19. Summarized analytical data for lead in the hard water chronic
bioassay.
Lead concentration Aquaria
(mg/liter) H-l H-2 H-3 H-4 H-5 H-C
Nominal concentration 3.24 1.08 0.36 0.12 0.04 0.00
Free lead concentration
(pulse polarography) .064 .044 .032 .018 .011 .003
The application factor is determined by dividing the maximum acceptable
toxicant concentration ("MATC") of the hard water chronic bioassay by
the TLc concentration of the acute bioassay (Mount and Stephan, 1967b) .
Nominal lead (NL) concentration application factor
The "MATC" of the hard water chronic bioassay was found to occur between
aquarium H-4, with a no effect nominal concentration level of 0.12 mg/liter,
and aquarium H-3 (0.36 mg/liter Pb) in which fish exhibited the "blacktail"
effect. The 96-hr TL,-0 nominal concentration at the lethal threshold in
the static acute bioassay #3 was 471 mg/liter lead. The hard water nominal
lead application factor is calculated to lie between .0002 and .0008.
"MATP"
Hard Water Application Factor (NL) = -^ (96-hr) =
0.12 mg/liter . 0.36 mg/liter. or from .0002 to .0008
471 mg/liter an 471 mg/liter
Free lead (FL) concentration application factor
The "MATC" on a free lead basis lies between aquarium H-4 (.018 mg/liter)
and H-3 (.032 mg/liter). The free lead 96-hr TL5Q concentration was
55
-------
1.38 mg/liter. Therefore, the hard water, free lead application factor
is calculated to lie between .0130 and .0232.
"MATP" =
Hard Water Application Factor (FL)
50
0.018 mg/liter and 0.032 me/liter, or from .0130
1.38 mg/liter to .0232
Soft Water Application Factor
Table 20 summarizes the analytical data for lead in the soft water F-
generation reproduction bioassay. Since we consider the nominal and free
lead concentration to be the same for all practical purposes, in soft
water, the "MATC" occurred between aquaria S-5 (6 yg/liter), no effect
level, and S-4 (11.9 yg/liter) in which four fish exhibited the "black-
tail" effect. An 18-day soft water flow-through acute bioassay gave a
lethal threshold TL5Q value of 140 yg/liter.
Table 20. Summarized analytical data for lead in the soft water F.
generation reproduction bioassay.
Lead concentration Aquaria
( yg/liter) 1234
Nominal concentration 100.0 50.0 25.0 12.5 6.2 0.0
Free lead concentration
(atomic absorption) 95.2 47.6* 23.8* 11.9* 6.0* 3.1**
* Computed from analyzed high concentration.
** Determined by pulse polarography.
Free lead (FL) concentration application factor
The soft water free lead application factor is calculated to lie between
.0429 and .0850.
Soft Water Application Factor (FL) = - /VQ—j \
TL^0 (18 day)
6 yg/liter and 11.9 yg/liter _ .._. fc .___
140 yg/liter 140 yg/liter ' or from '°429 to -0850'
-------
TESTING VALIDITY OF APPLICATION FACTOR APPROACH AS APPLIED TO FISH IN
DIFFERENT WATER QUALITIES
Table 21 summarizes elements of the application factor data needed to
test the applicability of the application factor concept to toxicity
in different water qualities.
Table 21. Summary of application factor data,
Hard water
Nominal lead
Concentration
Free lead
concentration
Soft Water
"MATC"
^50
(lethal threshold)
Application factor
0.12 to
0.36 mg/liter
471 mg/liter
.0002-. 0008
.018 to
.032 mg/liter
1.38 mg/liter
.0130-. 0232
6.0 to
11.9 yg/liter
140 yg/liter
.0429-. 0850
Figure 11 gives the schematic for testing the application factor concept
which would permit chronic data developed for one water quality to be
used to approximate the maximum acceptable toxicant concentration of
another quality of water for which only acute toxicity data are available.
Test of Application Factor Approach as Applied to Nominal Lead Concentration
in Hard and Soft Water _____
... Soft Water __ (NL)TL ^ (NL) "MATC" For
U' Application Factor Hard Water Acute ^ Hard Water (0.12-0.36
mg/liter)
.0429- .0850 x 471 mg/liter = 20.21 - 40.04 mg/liter
Test: INVALID (Computed "MATC" range, off by a factor of 56 to 334)
(2) Hard Water x TL ^ "MATC" for
Application Factor (NL) Soft Water Acute ^ Soft Water (6.0-11.9
yg/liter)
.0002 - .0008 x 140 yg/liter - .03 - .11 yg/liter
Test: INVALID (Computed "MATC" range, off by a factor of 54 to 397)
57
-------
Figure 1L Lead toxlcity to rainbow trout and validity of application factor.
HAED WATER BIOASSAYS
SOFT WATER BIOASSAYS
Acute toxicity
50% tolerance limit
TL5Q (static)
oo
Chronic toxicity
(MATC)
(flow through)
Chronic toxicity
(MATC)
(flow through)
Application
factor
Acute toxicity
50% tolerance limit
TL (flow through)
Application
factor
/
Application factor
X
TLcfl acute bioassay
in soft water
Application factor
X
TL,-0 acute bioassay
In hard water
-------
Test of Application Factor Approach as Applied to Free Lead Concentration
in Hard and Soft Water
(1) Soft Water x (FL ) TL £ (FL) "MATC" for
Application Factor Hard Water Acute Hard Water (.018-. 032
rag/liter)
.0429 - .0850 x 1.38 mg/liter = .059 - .117 mg/liter
Test; VALID -"MATC" approximated (Computed "MATC" range off by a
factor of 1.8 to 6.5)
(2) Hard Water x TL5Q £ "MATC" for Soft Water
Application Factor (FL) Soft Water Acute (6.0-11.9 yg/liter)
.0130 - .0232 x 140 yg/liter = 1.8 - 3.3 yg/liter
Test: VALID - "MATC" approximated (Computed "MATC" range off by a
factor of 1.8 to 6.6)
Discussion of the Application Factor Approach as Applied to Different
Water Qualities
The application factor concept appears to be functional with heavy metals,
such as lead, which exhibit a complexing behavior in different water
qualities only if analysis for the free or soluble metal is employed.
This analytical limitation poses a serious and unresolved problem if the
application factor concept is to find widespread use. The determination
of "MATC" and acute TLc0 concentrations would require analysis of the
dissolved metal. In addition, water samples collected to monitor pollu-
tant concentrations of a particular water, would necessitate the deter-
mination of free metal ions if such results were to be compared to
previously determined "MATC" values.
Most agencies monitoring heavy metal stream pollution problems use atomic
absorption spectrophotome try as the primary instrument for metal analysis.
Sample filtration does nor, appear to be a reliable method for determining
dissolved metals by atomic absorption spectrophotometry. Filtration as
a method for approximating free metal analysis will require experimental
verification with many complexing metals before the procedure could be
considered reliable. Until that time, atomic absorption spec trophotometry
should not be expected to provide data useful in deriving or implementing
application factor data where the complexing of heavy metals occurs.
59
-------
Section VI
ACKNOWLEDGMENTS
Dr. W. Harry Everhart, Chairman Fishery Major of the Department of
Fishery and Wildlife Biology at Colorado State University, was Project
Director. Colorado Division of Game, Fish and Parks provided personnel
(John Goettl, James Sinley, and Norwin Smith), and the bioassay
facilities at the Fort Collins Research Center. Dr. Janet Osteryoung,
Quantitative Chemist with Colorado State University, performed the
pulse polarographic analysis of the lead water samples and constructed
the solubility diagrams for lead in soft and hard water.
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.
61
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Section VII
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3. Arthur, J. W. 1970. Chronic effects of linear alkylate sulfonate
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4. Brungs, W. A. 1969. Chronic toxicity of zinc to the fathead minnow,
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5. Buhler, D. R., M. E. Rasmusson, and W. E. Shanks. 1969. Chronic oral
DDT toxicity in juvenile coho and chinook salmon. Toxicol. and Appl.
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6. Carpenter, K. E. 1927. The lethal action of soluble mettalic salts
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7. Carpenter, K. E. 1930. Further researches on the action of metallic
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8. Chisolm, Jr., J. J. 1971. Lead poisoning. Scientific American
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9. Chow, T. J., and J. L. Earl. 1970. Lead aerosols in the atmosphere:
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10. Daum, R. J. 1969. Revision of two computer programs for probit
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11. Daum, R. J., and W. Killcreas. 1966. Two computer programs for
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12. Dawson, A. B. 1935. The hemopoietic response in the catfish, Ameirus
nebulosus, to chronic lead poisoning. Biol. Bull. 68(3):335-346.
13. de Bruin, A. 1971. Certain biological effects of lead upon the
animal organism. Arch. Environ. Health 23(4):249-264.
14. Deuel, C. R., D. C. Haskell, and A. V. Tunison. 1937. The New York
State Fish Hatchery feeding chart. Fish. Res. Bull. No. 3. State
of New York Cons. Dept., Albany, N. Y. 29 p.
63
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15. Dorfman, D., and W. R. Whitworth. 1969. Effects of fluctuations
of lead, temperature, and dissolved oxygen on the growth of brook
trout. J. Fish. Res. Bd. Can. 26:2493-2501.
16. Duthie, E. S. 1939, The origin, development and function of blood
cells in certain marine teleosts. Pt. I—Morphology. J. Anat.
73(3):396-412.
17. Eaton, J. G. 1970. Chronic malathion toxicity to the bluegill
(Lepomis macrochirus Rafinesque). Water Res. Pergamon Press., Great
Brit. 4:673-684.
18. Erickson, J. D. 1965. Report on the problem of Ichthyosporidium
in rainbow trout. Prog. Fish-Cult. 27(4):179-184.
19. Farrelly, R. 0., and J. Pybus. 1969. Measurement of lead in blood
and urine by atomic absorption spectrophotometry. Clin. Chem.
15(7)-.566-574.
20. Fati, S. 1961. The metabolism of tryptophan in experimental lead
toxicity. Biochiin Appl. 8:280-293.
21. Field, J. B., C. A. Elvehjem, and C. Juday. 1943. A study of the
blood constituents of carp and trout. J. Biol. Chem. 148(2):261-269.
22. Fishman, M. J., and M. R. Midgett. 1968. Extraction techniques
for the determination of cobalt, nickel, and lead by atomic absorp-
tion in Trace inorganics in water: Am. Chem. Soc., Advances in
Chemistry Ser., no. 73: 230-235.
23. Gontea, I., et al. 1964. Vitamin C in lead poisoning in the guinea
pig. Igiena 13: 501-509.
24. Haider, G. 1964. Heavy metal poisoning in fish. I. Demonstration
of lead poisoning in rainbow trout (Salmo gairdneri Rich.) (Transl.
from German). Z. Angew. Zool. 51:347-368.
25. Hall, S. K. 1972. Pollution and poisoning: High lead levels are
dangerous to man, and ambient concentrations are presently rising.
Envir. Sci. and Tech. 6(1): 31-35.
26. Halver, J. E. 1969. Vitamin requirements, p. 209-232. In 0. W.
Neuhaus, and J. E. Halver (ed.). Fish in research. Academic Press,
New York and London. 311 p.
27. Halver, J. E. 1970. Nutrition in marine aquiculture. p. 75-102.
In W. J. McNeil (ed.). Marine Aquiculture. Oregon State Univ. Press.
Corvallis, Oregon. 102 p.
28. Henderson, C. 1957. Application factors to be applied to bioassays
for the safe disposal of toxic wastes. Trans. Sem. Biol. Prob.
Water Poll., R. A. Taft San. Engin. Center, 1956.
61;
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29. Hesser, E. F. 1960. Methods for routine fish hematology. Prog.
Fish-Cult. 22(4):164-171.
30. Hoffman, C. L., C. E. Dunbar, and A. Bradford. 1962. Whirling
disease of trouts caused by Myxosoma cerebralis in the United States.
U. S. Dep. Int., Bur. Sport Fish, and Wildl. Spec. Sci. Rep.—
Fisheries No. 427. 15 p.
31. Hunn, J. B., R. A. Schoettger, E. W. Whealdon. 1968. Observations
on the handling and maintenance of bioassay fish. Prog. Fish-Cult.
30 (3):164-167.
32. Jones, J. R. E. 1968. The relative toxicity of salts'of lead, zinc,
and copper to the stickleback (Gasterosteus aculeatus L.) and the
effect of calcium on the toxicity of lead and zinc salts. J. Exp.
Biol. 15:394-407.
33. Lloyd, R. 1961. Effect of dissolved oxygen concentrations on the
toxicity of several poisons to rainbow trout (Salmo gairdneri
Richardson). Exp. Biol. 38:447-455.
34. Lloyd, R. 1965. Factors that affect the tolerance of fish to
heavy metal poisoning. Biol. Prob. in Water Poll., PHS Pub. No.
999-WP-25.
35. Marvin, K. T., R. R. Proctor, Jr., and R. A. Neal. .1970. Some
effects of filtration on the determination of copper in freshwater
and seawater. Limnol. Oceanog. 15(2):320-325.
36. McCay, C. M. 1929. Studies on fish blood and its relation to water
pollution. In A biological survey of the Erie-Niagara system.
New York Cons. Dep., Albany, p. 140-149.
37. McKee, J. E., and W. W. Wolf. 1963. Water quality criteria.
Calif. Water Quality Control Bd. Pub. 3-A. 548 p.
38. McKim, J. M., and D. A. Benoit. 1971. Effects of long-term exposures
to copper on survival, growth, and reproduction of brook trout
(Salvelinus fontinalis). J. Fish. Res. Bd. Can. 28 (5):655-662.
39. Mighell, J. L. 1969. Rapid cold-branding of salmon and trout with
liquid nitrogen. J. Fish. Res. Bd. Can. 26(10) -.2765-2769,
40. Mount, D. I. 1962. Chronic effects of endrin on bluntnose minnows
and guppies. USDI, Bur. Sport Fish and Wildl. Pub. 58.
41. Mount, D. I. 1968. Chronic toxicity of copper to fathead minnows
(Pimephales promelas, Rafinesque). Water. Res. 2:215-223.
42. Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicological studies. Water Res. 1:21-29.
-------
43. Mount, D. I., and C. E. Stephan. 1967a. A method for determining
cadmium poisoning in fish, J. Wildl. Manage. 31:168-172.
44. Mount, E. I., and C. E. Stephan. 1967b. A method for establishing
acceptable toxicant limits for fish—Malathion and the butoxyethanol
ester of 2,4-D. Trans. Amer. Fish. Soc. 96:185-193.
45. Mount, D, I., and C. E. Stephan. 1969. Chronic toxicity of
copper to the fathead minnow (Pimephales promelas) in soft water.
J. Fish. Res. Bd. Can. 26:2449-2457.
46. Pickering, Q. H., and C. Henderson. 1965. The acute toxicity of
some heavy metals to different species of warm water fishes. Proc.
19th Ind. Waste Conf., Purdue Univ. p. 578-591.
47. Pickering, Q. H., and T. 0. Thatcher. 1970. The chronic toxicity
of linear alkylate sulfonate (LAS) to Pimephales promelas Rafinesque.
J. Water Poll. Control Fed. 42(2):243-254.
48. Poston, H. A. 1967. Effect of dietary L-ascorbic acid on immature
brook trout. Cortland Hatchery Rep. No. 35 for the year 1966.
Fish. Res. Bull. No. 5. State of New York Cons. Dep. Albany, N. Y.
p. 46-51.
49. Reichenback-Klinke, H., and E. Elkan. 1965. The principal diseases
of lower vertebrates. Academic Press, London and New York. 600 p.
50. Root, R. W. 1931. The respiratory function of the blood of marine
fishes. Biol. Bull. 61(3):151-153.
51. Schiffman, R. H. 1959. Methods of repeated sampling of trout blood.
Prog. Fish-Cult. 21(4):151-153.
52. Seivard, C. E. 1964. Hematology for medical technologists. 3rd
ed. Lea and Fegiger, Philadelphia. 643 p., illus.
53. Shanks, W. E., G. D. Gahimer, and J. E. Halver. 1962. The indispen-
sible amino acids for rainbow trout. Prog. Fish-Cult. 24(2):68-73.
54. Sprague, J, B. 1969. Measurement of pollutant toxicity to fish.
I. Bioassay methods for acute toxicity. Water Res. 3:793-821.
55. Stumm, W., and J. J. Morgan. 1970. Aquatic chemistry. An intro-
duction emphasizing chemical equilibria in natural waters. Wiley
Interscience, New York. 583 p.
56. Wintrobe, M. M. 1934. Variations in the size and hemoglobin content
of erythrocytes in the blood of various vertebrates. Folia Haematol.
51:32-49.
66
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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.
Everhart, W. Harry and Robert A. Freeman
Toxicity of aluminum hydroxide complexes in neutral and
basic media to eggs and early life history stages of
rainbow trout. (In preparation).
Davies, Patrick H., John P. Goettl, James R. Sinley, and Norwin Smith.
Toxicity of lead to rainbow trout. (In preparation).
Davies, Patrick H. Testing the application factor approach as applied
to different water qualities. (In preparation).
67
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Section IX
APPENDICES
69
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CONTENTS OF APPENDICES
Appendix A.
Appendix B.
Appendix C.
Appendix D.
Appendix E.
Discussion of the complexities associated with analyzing
the character of lead in water.
Discussion of the problems associated with the analysis
of lead by atomic absorption spectrophotometry.
Discussion of the pH problems associated with the analysis
of lead by pulse polarography.
Atomic absorption analysis of feed.
Discussion on the nature of lead in hard water as
analyzed by atomic absorption spectrophotometry.
70
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APPENDIX A
DISCUSSION OF THE COMPLEXITIES ASSOCIATED WITH ANALYZING THE CHARACTER
OF LEAD IN WATER
(personal communications, Dr. Janet Osteryoung)
Equilibrium calculations show that the total solubility of lead in hard
water is about 30 ug/liter and 500 ug/liter in soft water (Figures 12
and 13). The solubilities and distribution diagrams for the different
lead species in hard and soft water are intended to be suggestive rather
than definitive. In each figure the arrow indicates the pH of the
water in the test aquaria (used data from H-l, hard water high concen-
tration, and S-l, soft water high concentration). The curve, C , gives
the solubility of lead in solution. Soluble complexes such as PbNO^, not
shown, were omitted because they were found to be unimportant in that
they represent an insignificant fraction of the total species present.
All values are computed at 25 C because of the general lack of equi-
librium constant data at the different bioassay temperatures which
exhibited large seasonal fluctuations, ranging from 5°C to 21°C. By
reason of the resulting inaccuracies existing inherently by assuming a
temperature of 25 C, no activity corrections were made. All equilibrium
constants are thermodynamic values. In any case, should such correction
have been made, they would be small due to the ionic strengths in soft
and hard water which are only 1-2 and 10-20 mM, respectively.
In soft water with a pH _^ 5.4, PbSO, is present and limits the lead con-
centration in solution (Figure 13). Above pH 5.4, a situation character-
ized in the test aquaria, PbCO-(s) and Pb«(OH)~CO,j (s) are present and
limit the lead concentration. In hard water a pH > 6.0 is the level at
which PbCO~(s) and Pb2(OH)2CO,(s) are present and cause a corresponding
limitation (Figure 12). Under experimental conditions, the most
important factor determining lead solubility in both these waters is the
carbonate concentration which in turn depends on the partial pressure of
C02(g) and the pH. Both pH and C02(g) concentrations are subject to large
local fluctuations due to fish respiration, resulting in an extremely
sluggish C02(g) £ C02(aq) equilibrium. In addition, the equilibria
involving Pb (II) precipitation and dissolution are very slow. Consequently,
the equilibrium calculations only indicate permissible ranges and are not
necessarily an accurate description of the system, even if they could be
done completely and accurately.
The large standard deviations that exist for both analytical methods used
in determining lead concentration (pulse polarography and atomic absorp-
tion) are believed not to be due to the methods of analysis, and probably
not due to the diluter system other than on those occasions when a diluter
malfunction occurred. They probably arise through local changes in solution
conditions in the aquaria which cause precipitation of lead or solution of
suspended material. One factor which would greatly influence this is
temperature. Since temperature was not held constant and fluctuated
71
-------
15 -
13
PH
Figure 12. Solubility and species distribution for Pb (II) in hard
water. Arrow indicates average pH in aquarium H-2(Stumm
and Morgan, 1970).
72
-------
SOFT WATER
15
13
PH
Figure 13., Solubility and species distribution for Pb (II) in soft
water. Arrow indicates average pH in aquarium S-2(Stumm
and Morgan, 1970).
73
-------
seasonally with a range of 5 C to 21 C, the rate of colloid and pre-
cipitate formation would most certainly be affected. This situation
would exert the greater influence on atomic absorption results where
analysis was performed soon after sampling. Another factor which might
contribute to the variance of lead results is the effect of metabolic
byproducts, particulate matter, and algal growths on the various lead
species in the test solutions.
-------
APPENDIX B
DISCUSSION OF THE PROBLEMS ASSOCIATED WITH THE ANALYSIS OF LEAD BY
ATOMIC ABSORPTION SPECTROPHOTOMETRY
Relatively speaking, atomic absorption analysis of lead in soft water
approximates total lead present, and in this case also the amount of
free lead because of the high solubility (500 yg/liter) of lead in
soft water. It is somewhat erroneous to speak of lead analysis being
a total analysis when dealing with a highly alkaline or hard water,
such as the well water used in this study. This assumes that such an
analysis would measure free, colloidal and precipitated forms of lead
in an amount proportional to their actual existence in an aquarium.
Such an assumption is particularly invalid with regard to precipitated
lead forms which would tend generally to accumulate on the bottom of an
aquarium and not remain uniformly distributed in the water. In addition,
with the proportional dilutor system continuously supplying new solutions
of lead, there would be a continuous buildup in the aquarium of pre-
cipitated lead forms which would greatly exceed that encountered in a
static situation.
The above factors are further complicated by respiratory and metabolic
by-products, and the effect of particulate matter and algal growth in
the aquarium on the character of lead in such solutions. Analytical
results by atomic absorption in hard water are relatively meaningless
because of the inability to define the nature and character of the lead
species sampled. For these reasons, pulse polarographic analysis which
measures free lead, and the nominal lead concentrations, the known
added amount of lead, provides the only definable knowledge we have of
the hard-water lead system.
75
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APPENDIX C
DISCUSSION OF THE PH PROBLEMS ASSOCIATED WITH THE ANALYSIS OF LEAD BY
PULSE POLAROGRAPHY
(personal communications, Dr. Janet Osteryoung)
As previously stated, about 60% of the samples analyzed by direct
analysis or by the addition of LiCl gave no signal for lead. A lead
signal was obtained upon acidification of these-samples. Several tests
of standard lead solutions in the range 2 x_10 to 1 x 10~ M (0.04 -
2 mg/liter) containing 1 x 10"J to 4 x 10 M KN03> NaN$3, LiCl, or
KC1, showed that the pulse polarography signal (ip) in "neutral" solu-
tions is at least a factor of two less than in acid solutions (pH _£ 4).
The decrease in signal is apparently most pronounced at lower lead
levels. For example, "neutral" solutions containing 0.105 mg/liter
lead give no signal, but acid solutions give a clearly defined signal,
which was a factor of six above the limiting instrument sensitivity.
Experiments employing HC10,, HC1, and HNO, as the acid, convincingly
demonstrate that pH rather than the choice of anion controls this
effect. However, it is not possible to investigate this phenomenon in
buffered solutions because of the effect of the ionic strength and the
complexing behavior of the anions present on the signal. Heiewe are
talking about the anions associated with a buffered system and not those
associated with the particular acid used. Of the acids used, the CIO,
and NO- anions would exhibit no complexing behavior, and the effect of
Cl would be only slight compared to that of a buffered system. There-
fore, it appears almost impossible to do a reliable study on the effect
of pH.
The question of the effect of acidity on the magnitude of the signal is
serious and unresolved. Experiments on standard solutions show con-
clusively that the problem does not occur because of changes in the
rate of lead reduction with changes in medium, for the same phenomenon
is observed both with differential and normal pulse polarography. Lower
signals in an unbuffered neutral solution are due in part to simultaneous
reduction of residual oxygen which consumes hydrogen ion and produces a
local region of high pH near the indicator electrode. This in turn,
can cause local precipitation of Pb(OH)2» However, this effect should
be much more pronounced in the differential than in the normal pulse
mode. Since the observed signal decrease is nearly the same in the two
modes, local pH changes cannot be entirely responsible.
In normal pulse polarography, the magnitude of the diffusion current
depends only on the concentration and the diffusion coefficient of the
substance being reduced. Equilibrium calculations show, for the standard
solutions investigated, that there should be no problem with insolubility
in the bulk solutions, and therefore the concentration should not vary
as a function of pH. This leaves the diffusion coefficient as the only
-------
factor capable of influencing the magnitude of the diffusion current.
When+changin^_pH from 7 to 4, the predominant lead species change from
PbOH to Pb respectively; it is surprising that the respective dif-
fusion coefficients should vary by a factor of four (i, is proportional
to D 1/2) or that the factor should increase with dilution. This, however,
seems to be the most reasonable hypothesis in the absence of further
data.
A question that legitimately could be asked is: Why, with acidification
of samples, are pulse polarography results reported as soluble and not
total lead? The samples were acidified immediately prior to the running
of a polarogram. It would appear that insufficient time existed for the
conversion of the undissociated (colloidal and precipitated forms) to
dissociated or soluble species of lead. Yet,rwith the resulting lower
pH, the diffusion coefficients of the soluble species present were in-
creased by changing the predominant lead species from PbOH to Pb ,
thus giving an adequate lead signal.
77
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APPENDIX D
ATOMIC ABSORPTION ANALYSIS OF FEED
Feed size
Zn (yg/g)
Cu (yg/g)
Pb (pg/g)
#0 crumbles
#1 crumbles
#2 crumbles
#3 crumbles
#4 cmmbles
1/8" pellets
7/32" pellets
75.60
91.59
106.34
131.81
114.35
96.85
100.48
11.15
15.25
18.16
17.96
16.67
23.57
23.45
<2.5
<2.5
<2.5
<2.5
<2.5
<2.5
<2.5
78
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APPENDIX E
DISCUSSION ON THE NATURE OF LEAD IN HARD WATER AS ANALYZED BY ATOMIC
ABSORPTION SPECTROPHOTOMETRY
As seen from Figure 14, the observed lead concentration when analyzed
by atomic absorption spectrophotometry is mostly of a colloidal nature
(in a hard water with an alkalinity of 267 mg/liter) for nominal lead
additions from 0 to 500 mg/liter. The graph as drawn would represent
some point in time, let us say 6 to 24 hr, after lead is added to the
hard water. Lead would exist mostly free in solution above that point
in which the added lead eliminates the carbonate system, at about
520 mg/liter of added lead. This is also the point corresponding to
100% precipitation of lead; above this point lead additions would go
freely into solution. The graph is intended to be descriptive and not
represent what definitely occurs in the lead hard-water system. The
graph does not, nor is it intended to depict possible changes that
might occur in the whole anion and cation system resulting from decrease
in pH due to the addition of lead which releases hydrogen ions from the
bicarbonate system, or from absorption of atmospheric carbon dioxide by
the water, which would tend to replenish carbonates.
79
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Figure 14. Graphic interpretation of the nature of lead in well water
when analyzed by atomic absorption spectrophotometry.
14
13
12
11
o
•Mostly colloidal lead
Free lead
100
200
300
400
500 40 80 600
Nominal Pb Concentration (mg/1)
80
0 U. S. GOVERNMENT PRINTING OFFICE : 1973—!
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1
Accession Number
w
5
2
Subject Field & Group
05C
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Department of Fishery and Wildlife Biology
Title
EFFECTS OF CHEMICAL VARIATIONS IN AQUATIC ENVIRONMENTS: Lead toxicity
to rainbow trout and testing application factor concept.
10
Authors)
Davies, PatrickH., and
W. Harry Everhart
16
21
Project Designation
EPA WQO 18050-3DYC
Note
22
Citation
Environmental Protection Agency report
number, EPA-R3-73-011c, February 1973.
23
Descriptors (Starred First)
*Rainbow trout, *lead, *toxicity
25
Identifiers (Starred First)
*Rainbow trout, *lead,
application factor
*toxicity
27
Abstract
Four chronic bioassays were conducted to determine the toxicity of lead to rainbow
trout. Results obtained from acute and chronic bioassays in hard water (alkalinity
2*6 1 ing/liter) and soft water (alkalinity 26.^ ing/liter) were used to test the
application factor approach as related to different water qualities. The toxicity
of lead to rainbow trout in hard water was determined on a total and dissolved lead
basis. The 96-hr TL^ and "MATC" on a total lead basis were U?l ing/liter and 0.12
to 0.36 ing/liter respectively, which yielded an application factor of .0002 to .0005.
Analysis of the free or dissolved lead gave a 96-hr TL^o of 1.38 mg/liter and a
"MATC" of 0.018 to 0.032 mg/liter, resulting in an application factor of ,,0130 to
0232 Total and free lead were considered to be the same in soft-water. The
18-day TLcro and "MATC" obtained from the soft water bioassays were 1UO yg/liter ^
and 6.0 to 11.9 jig/liter lead respectively. Computations using the TL5(? and MATC
values gave a soft water application factor of .OU29 to .0850. The maximum accept-
able toxicant concentration ("MATC") was determined in both hard and soft water
bioassays on the occurrence of abnormal black tails caused by chronic lead exposure.
The application factor approach as related to different water qualities was found to
be very promising when lead analysis was limited to the free or dissolved metal and
failed when total hard water lead concentrations were used.
Harry Everhart
Institution
Cornell University
WR:IOZ (REV. JULY 1969)
WRSIC
SEND. W,TH COPY OF DOCUMENT. TO: WAT M RESOURCES^ MINT
WASHINGTON. D. C. 20240
* 6PO: 1970-384-930
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