EPA-R3-73-046
September 1973
Ecological Research Series
i **s""K** ^
Office of Research and Development
U.S. Environmental Protection Agency
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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
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
1. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
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EPA-R3-73-046
September 1973
THE EFFECTS OF METHOXYCHLOR
ON AQUATIC BIOTA
By
James W. Merna
Institute for Fisheries Research
Michigan Department of Natural Resources
Ann Arbor, Michigan 48104
and
Paul J. Eisele
The University of Michigan
School of Public Health
Ann Arbor, Michigan 48104
Project 18050 DLO U.S. Environment:-.: FY tvrtion Agency
Project Officer
- .-•• • - % \1
Chicr-so, i;:r.2:S \rj-j. »4
Dr. W. Brungs
National Water Quality Laboratory
Environmental Protection Agency
6201 Congdon Boulevard
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 95 cents
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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
recommendation for use.
U.S. Esr-'i/cr:-. .''••: '^r-^^on Agency
11
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ABSTRACT
Methoxychlor inhibited phytoplankton production at a concentration of
50 ng/l, however the effect may have been overestimated due to
inhibitory effects of ethanol used as a solvent.
Sate of breakdown of methoxychlor in water varied considerably with
the biological activity in the water. Methoxychlor was rapidly
incorporated in the particulate fraction of the water by adsorption
or metabolism.
Continuous-flow bioassays yielded 96-hour TL,50 values for
invertebrates ranging from 0. 61 fJ.g/1 for Gammarus pseudolimnaetus.
to 7. 05 Mg/1 for Orconectes virilis. Fathead minnows (Pimephales
promelas) and yellow perch (Perca flavescens) had 96-hour
values of 8. 63 and 22. 2 ng/1 respectively.
Hatching of fathead minnow eggs was inhibited at all levels of
exposure tested between 1.0 and 0. 125 jug/1- There was no spawning
at 2 Aig/1.
Growth of yellow perch was retarded at all levels tested between
5. 0 and 0. 625 ng/l. All perch died at 10 /ug/1 during the growth
study.
Perch which had been subjected to long-term exposure to 5 ^ug/1 of
methoxychlor had an abnormally high oxygen demand when held in
a respirometer with a water velocity of 0. 6 foot per second.
This report was submitted in fulfillment of research grant 18050-DLO
under the sponsorship of the Office of Research and Monitoring,
Environmental Protection Agency.
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CONTENTS
Section
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Methods 7
Primary production 7
Breakdown studies 10
Acute fish toxicity 10
Chronic fish toxicity 11
Respiration studies 12
Invertebrate toxicity 13
V Results and Discussion 17
Primary production 17
Breakdown studies 29
Acute fish toxicity 34
Chronic fish toxicity 37
Respiration studies 43
Invertebrate toxicity 44
VI Stream Construction 53
VII Acknowledgments 55
VIII References 57
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FIGURES
No. Page
1. Constant current aquarium used for insect
toxicity studies 14
2. Methoxychlor concentrations in test water measured
during 7-day productivity study 26
3. Methoxychlor concentrations in filtered and unfiltered
water samples measured during 10-day productivity
study 27
4. Temperature profiles in Third Sister Lake during
productivity studies 28
5. Concentration of methoxychior in replicated samples
of buffered distilled water at pH 7 and pH 9 during
220 days of hydrolysis 30
6. Concentration of methoxychior in replicated samples
of aged Ann Arbor tap water, which had previously
held fish, during 18 days of breakdown 31
7. Concentration of methoxychior in replicated samples
of Koch Warner Creek water during 20 days of
breakdown 3 2
8. Concentration of methoxychior in filtered and
unfiltered samples of Third Sister Lake water during
10 days of breakdown 33
9. Changes in median tolerance limit of methoxychior for
fathead minnows with time of exposure 35
10. Percent survivorship and recovery of Chironomidae
larvae from 28-day chronic methoxychior bioassay 49
11. Percent emergence of Stenonema and pupation of
Cheumatopsyche during 28-day chronic methoxychior
bioassay 50
12. Growth of Stenonema, as expressed by number of
exuyia per individual, during 28-day chronic
methoxychior bioassay 51
13. Photographs of experimental streams during
construction and test operation 54
VI
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TABLES
No. Page
1. Primary production data for 9 July 1970 expressed as
counts per minute 18
2. Primary production data for 10 July 1970 expressed'as
counts per minute 19
3. Primary production data for 4 August 1970 expressed
as counts per minute 20
4. Primary production data for 6 August 1970 expressed
as counts per minute 21
5. Primary production data for 8 August 1970 expressed
as counts per minute 22
6. Primary production data for 10 August 1970 expressed
as counts per minute 23
7. Summary of primary production data from two studies
of the effects of ethanol, Triton, and methoxychlor 24
8. Chemical characteristics of test waters 29
9. Measured concentrations of methoxychlor in 30 liters
of water during the exposure of five yellow perch
(approximately 50 g total weight) for 96 hours 34
10. Concentrations of methoxychlor and number of yellow
perch that died during 96-hour continuous-flow toxicity
study (eight perch per tank were used in Test I, and
ten in Test H) 36
11. Summary of dose response of fathead minnows and
yellow perch to 96-hour continuous flow methoxychlor
bioassay interpreted through logistic and log-probit
analysis 3 7
12. Growth and survival of fathead minnows exposed to
methoxychlor at various levels of concentration 38
13. Growth and survival of fathead minnows exposed to
methoxychlor at various levels of concentration in
test unit D 39
14. Specific growth rates of fathead minnows subjected to
various concentrations of methoxychlor 39
vii
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No. Page
15. Average weight in grams of yellow perch during
exposure to methoxychlor, and average gain in weight
during test periods in unit C 40
16. Specific growth rates of yellow perch subjected to
various concentrations of methoxychlor 41
17. Spawning and hatching success of fathead minnows at
methoxychlor levels of 2 jug/1 and less 42
18. Oxygen consumption in milligrams per gram of fish
per hour (mg/g/hr), by yellow perch after long-term
exposure to methoxychlor, measured at two rates of
swimming speed 43
19. Results of 96-hour continuous-flow invertebrate bioassays,
with recorded values of water temperature and mean
dissolved oxygen, interpreted through logistic and
log-probit analyses 45
20. Dissolved oxygen levels in the Stenonema sp. and
Chironomus tentans continuous-flow 96-hour bioassay
(Test I) 46
21. Results of chronic continuous-flow invertebrate bioassay
interpreted at 2-week intervals through logistic and
log-probit analyses 47
22. Growth in milligrams of all insect larvae tested during
chronic continuous-flow methoxychlor bioassay 52
Vlll
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SECTION I
CONCLUSIONS
Methoxychlor is highly toxic to aquatic organisms, and continuous
exposure to concentrations less than 1.0 jug/1 can cause serious sub-
lethal effects. Breakdown is rapid in productive waters, but could be
slow enough to result in extended exposure periods in very unproductive
waters. Gammarus, an important fish food organism, would probably
be the most seriously damaged organism in an aquatic system.
Continuous exposure of fish to sublethal levels will reduce growth,
hatching success of eggs, and the ability to withstand stress.
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SECTION II
RECOMMENDATIONS
The study of sublethal effects of methoxychlor should be extended to
include field studies in natural habitats. This is being started now in
the artificial streams constructed as part of this project.
In the future, we should watch for the accumulation of significant
background levels in areas where insect control programs require
frequent repeated applications of methoxychlor. Levels sufficient to
cause sublethal effects are especially apt to persist in unproductive
waters.
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SECTION III
INTRODUCTION
The purpose of this study was to determine effects of methoxychlor on
the aquatic system with emphasis on the sublethal effects of long-term
exposure on fish and invertebrates.
Numerous studies in the last several years (Burdick et al., 1964;
Peakall, 1970; Reinert, 1969; and Wurster, 1968) have shown a variety
of environmental problems attributable to the widespread use of DDT.
Awareness of these studies, by an aroused public, has resulted in the
complete banning of DDT. Methoxychlor was one compound recommended
to replace DDT in many control programs especially those for the
European bark beetle and black fly larvae. Methoxychlor was recom-
mended because it is considered to be persistent but biodegradable, and
readily metabolized when ingested by living organisms. Consequently
it would not concentrate in the food chain.
Current literature agrees that the rate of breakdown of methoxychlor is
rapid in natural waters. Burdick et al. (1968) found that ponds treated
with 5 Mg/1 of methoxychlor contained undetectable concentrations when
sampled 36 days later. Wallner et al. (1969) concluded that a helicopter
application for European elm bark beetles was probably not hazardous to
aquatic life since fallout on a stream was quickly diluted and moved
downstream with little storage in the bottom sediments. Approximately
1 Mg/kg of methoxychlor persisted in the bottom silt from spring through
fall.
Rheinbold et al. (1971), Kennedy et al. (1970), and Metcalf et al. (1971)
all found methoxychlor to be much less concentrated than DDT in tissues
of fishes and invertebrates. The only noted exception was snails which
concentrated twice as much methoxychlor as DDT (Rheinbold et al.,
1971). However, Kruzynski and Leduc (1972) documented fish mortality
and tissue concentrations as high'as 2.65 mg/kg from spray applications
of as little as 0. 2 pound per acre methoxychlor. They also found in
their feeding experiments (0. 01 to 2. 0 mg/kg/day) that brook trout retained
about 80% of the ingested insecticide resulting in concentrations up to
45 mg/kg in whole fish tissue. This concentration caused anemia and
severe tissue damage in the liver and kidney. These authors indicated
tissue damage was synonymous with concentration, however I do not
believe anyone has successfully demonstrated that concentration
necessarily precedes sublethal effects.
All studies to date have consisted of low-level feeding experiments or
short-duration exposure studies. Most exposure studies have been
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based on a single application which, because of rapid breakdown,
results in very short exposure time. Kennedy et al. (1970) conducted
a chronic study with bluegills in ponds, however it was a single-
treatment method, and he did not monitor methoxychlor concentrations.
Despite the rapid breakdown rate of methoxychlor, any use that demands
frequent repeated applications (such as fruit orchards) will result in
maintaining significant background levels in our natural waters. For
this reason we instigated the present study to determine the effects of
long-term exposure of aquatic organisms to sublethal levels. The
study has consisted of the following segments:
1. Effects of methoxychlor in phytoplankton production in Third
Sister Lake, Washtenaw County, Michigan, as measured in experiments
with Cl4 in bottles.
2. Rate of breakdown of methoxychlor in waters varying in pH and
hardness.
3. Acute toxicity of methoxychlor to fathead minnows and yellow
perch in both static- and continuous-flow bioassays.
4. Chronic toxicity to fatheads and perch as interpreted by effects on
growth, hatching success of fathead eggs and respiration requirements
of perch.
5. Acute and chronic continuous-flow bioassays conducted on various
stream- and pond-dwelling aquatic invertebrates. Acute bioassays
were run for 96 hours on Stenonema interpunctatum (group) (mayfly),
Chironomus tentans (midge), Cheumatopsyche sp. (caddisfly), Gammarus
pseudolimnaeus (scud) and Orconectes virilis (crayfish). Chronic
bioassays were run for 28 days on Stenonema terminatum (group),
SK interpunctatum (group), and C. tentans. Chronic bioassays were
run for 42 days for Cheumatopsyche sp. and_G_. pseudolimnaeus.
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SECTION IV
METHODS
Primary production. -- The procedure for determining the effect of
methoxychlor on primary production consisted of dosing 5-gallon Pyrex
jugs of lake water from Third Sister Lake with different combinations
and concentrations of alcohol and Triton X-100 solutions of methoxychlor,
and incubating the jugs in the lake at a depth of 1 meter. The jugs were
then periodically sampled for productivity determinations and
methoxychlor analysis.
Water was pumped from the lake at a depth of 1 meter into a 150-gallon
capacity polyethylene drum. The jugs, previously acid-cleaned and
rinsed with distilled water, were filled randomly in three "slugs" by
gravity flow from the drum. The water in the drum was continuously
mixed with a plastic plunger during the jug-filling period. Upon
completion of the filling procedure the jugs were covered with black
polyethylene bags to minimize light shock to the algae in the water.
The jugs were treated with methoxychlor in alcohol and Triton X-100
solution. One milliliter of solution was used in all cases. The
treatment was as follows:
a. Control--no additive.
b. ETOH--1 ml of 95% ethanol.
c. Triton X-100--1 ml of a solution composed of 8 drops
of Triton X-100 in 100 ml of distilled water.
d. ETOH/Triton X-100--1 ml of a solution of 95% ethanol
plus 1 ml of the Triton noted above.
e. 1 Mg/1 methoxychlor--1 ml of a solution containing 0.02 mg
technical grade methoxychlor, in 95% ethanol with 8 drops
of Triton X-100 per 100 ml of solution.
f. 10 jug/1 methoxychlor--1 ml of a solution containing 0. 2 mg
technical grade methoxychlor in 95% ethanol with 8 drops
of Triton X-100 per 100 ml of solution.
g. 50 jug/1 methoxychlor--1 ml of a solution containing 1 mg
of technical grade methoxychlor in 95% ethanol with 8 drops
of Triton X-100 per 100 ml of solution.
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Each of the above treatments was done in duplicate. The lake was also
sampled along with the treatments using a Van Dorn bottle to show the
effect of containment.
The jugs were suspended and incubated in the lake at a depth of 1 m.
This was accomplished by suspending the jugs from a series of floats
anchored in the middle of the lake in the form of a square approximately
50 feet on a side. The jugs were secured to the floats by means of
snap clips and nylon lines affixed to the jug harness. The jugs were,
in this manner, incubated at a depth of 1 m for the entire period of the
experiment.
The jugs for the first run, 8-13 July, were dosed with the required
solutions after being suspended in the lake. This was accomplished
by volumetric ally pipetting the above mentioned solutions into the jugs
while mixing with a plastic plunger. The jugs for the second run,
3-13 August,were dosed prior to suspending them in the lake.
Finally, the jugs were sampled by the use of a vacuum pump arranged
to evacuate the sample bottles by pulling the water from the jugs into
the sample containers. The volume withdrawn for the various analyses
was as follows:
a. Productivity--125-ml aliquot
b. Methoxychlor--two, 250-ml aliquots
To determine the productivity, the jugs were sampled in the manner
described above. Three 125-ml Pyrex bottles were filled from the
jug. One of the bottles was covered with aluminum foil. Each bottle
was then injected with 1 ml of a 0. 5-mc solution of NaH^cC^. A
scintillation standard was prepared for each run. The bottles were
then placed horizontally on a rack and suspended at a depth of 1 m
in the lake.
For the first run, the dark bottles (aluminum-foil covered) were
incubated on the rack with the light bottles. For the second run, the
dark bottles were suspended in a doubled black plastic bag at a depth
of 1 m. This was done because it was felt that the reflection from
the foil might influence the light-bottle productivity, and because of
the possibility of small holes in the foil resulting in increased
respiration rates. Each light bottle had a bottle lying next to it on
the rack. The terminal bottles were blanks or ballast bottles such
that all of the experimental bottles were exposed to the same light
conditions.
After 4 hours incubation in the rack, the bottles were taken to the
laboratory where the samples were vacuum -filtered through 0.45;u
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pore size membrane filters. These filters were air dried and glued
to planchettes.
The filters were counted using a proportional beta counter for
10 minutes each to determine the amount of carbon- 14 fixed by the
plankton.
These counts were then used to determine the productivity of the
samples according to the formula:
Primary productivity c r/R (C) (f)
where: r = counts per minute from the jug. This is the jug
Light bottle + light bottle _ dark
2
R = (scintillation count) (0. 25) (0. 838) (2. 22 X 106)/40, 290
0. 25 = counter efficiency
0.838 a membrane adsorption
2. 22 X 106 = disintegrations/ minute /£ic
40, 290 = scintillation count / minute //uc
C = 20.4 X 103 mg of available carbon per M3 of the
lakewater
f = 1.06 (isotope correction factor)
A scintillation standard was prepared with each run. The average of the
scintillation counts per minute was used for the productivity calculations
for each run. These were as follows:
Run No. 1 19, 885. 7 cpm
Run No. 2 18, 545. 3 cpm
Quantitative determinations of methoxychlor in these, and all subsequent,
studies were made on a gas chromatograph equipped with an electron-
capture detector and a 1/8-inch by 6-foot stainless steel column packed
with 5% QF1 on Varaport 30. Methoxychlor was extracted from water
samples with hexane, using an extraction impeller driven by a magnetic
stirrer. The hexane extracts were dried with sodium sulfate before
injecting into the chromatograph. Area of peak was determined by
multiplying the peak height times the width at half height.
Methoxychlor determinations for the productivity studies were made as
follows. Two 300-ml samples were withdrawn from the 50 yg/1 jugs.
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One sample was filtered through a 0.45/u membrane filter before
extraction, and the other was analyzed in its original form. The
difference between the two values should represent the fraction of
methoxychlor adsorbed to, or incorporated in, biotic forms.
Temperature profiles were run daily during Run No. 1 and on the
first day of Run No. 2 with an electric thermometer. The tempera-
ture of the water was measured at half-meter increments from 0 to
5 m and in 1-m increments from 5 to 14 m.
Secchi disc readings were only taken during Run No. 1 to obtain an
estimate of the water transparency. The apparatus was lowered
into the water until it just disappeared from sight. The depth was
recorded in meters.
Breakdown studies. --Several authors (including Henderson et al.,
1959) have indicated a lack of influence of water quality parameters,
e.g., pH, alkalinity, and hardness, on the toxicity of chlorinated
hydrocarbons to fishes. However, the breakdown rate of many
insecticides has been found to be dependent on factors such a pH
(Muhlmann and Schrader, 1957) and the presence of suitable micro-
organisms (Mendel et al., 1967). Therefore, our experiments on
breakdown were conducted using a variety of test waters:
(1) distilled water buffered with phosphate to pH 7 and 9; (2) water
from Koch Warner Creek at Saline, Michigan, which serves as the
water source for our experiments on chronic toxicity; (3) aged Ann
Arbor tap water which had previously held fish; and (4) water from
Third Sister Lake which contained plankton.
In all experiments the methoxychlor was added to the test water from
stock solutions containing ethanol and sufficient Triton X-100 to
assure solution of the insecticide in the water. Highest concentrations
were 500 mg/1 ethanol and 0. 10 mg/1 Triton.
Acute fish toxicity. --Four replicated 96-hour static bioassay tests
were conducted; two with fathead minnows (Pimephales promelas)
and two with perch (Perca flay esc ens). All tests were run in 10-gallon
aquaria containing 30 liters of water. Ten fatheads or five perch were
used in each aquarium. The perch weighed approximately 5 g each and
the fatheads about 1 g. All static tests were conducted with aged Ann
Arbor tap water. Continuous-flow 96-hour studies were run on fatheads
in Ann Arbor tap water, and on perch in water from Koch Warner Creek.
The dosing apparatus utilized in these studies was a modification of the
unit described by Mount and Brungs (1967).
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The results were analyzed statistically by probit analysis and logistic
analysis on a computer.
Chronic fish toxicity. -- Chronic studies involved the use of four
continuous -flow bioassay units, with fathead minnows and yellow perch
as test fish. Fatheads were used in three units dosed at 2.0, 1. 0, 0. 5,
0. 25, and 0. 125 Mg/1- The unit containing perch was dosed at 10.0, 5.0,
2. 5, 1. 25, and 0. 625 Mg/1. Each unit also had one control tank. The
methoxychlor was dissolved in methanol, with Triton X-100 as a wetting
agent, and injected into the units by means of a syringe injector. The
injector was designed and built by a number of the staff of the Institute
for Fisheries Research and proved to be very satisfactory. The final
test solutions contained a range of approximately 0.4 mg/1 Triton X-100
and 40 mg/1 methanol in the high methoxychlor levels, to 0.026 mg/1
Triton X-100 and 2. 6 mg/1 methanol in the low levels. The control tanks
received no solvent solution. All aquaria were aerated throughout the
study. Fatheads, in the chronic studies, were evaluated for dose
response to growth and egg hatching success. Perch were evaluated
for growth and respiratory effects.
Two bioassay units containing fatheads were dosed for 6 months,
during which time we maintained water temperatures below 70 F
and a photoperiod of 12 hours in order to retard spawning. At the
end of 6 months we increased the temperature to 74 F and the light
period to 16 hours, after which spawning commenced in about 6 weeks.
Pieces of 2. 5-inch plastic pipe were put in the aquaria for spawning
receptacles. Ten pieces of pipe were cut in half lengthwise and
stacked in the rear of each aquarium. The pieces of pipe were
inspected daily for eggs. All eggs were removed from the spawning
receptacles and put in hatching containers made from short pieces of
1-inch plastic pipe. A cover was made from one-half of a 1-inch
plastic pipe nipple. Both the nipple and bottom of the pipe were
covered with fine mesh Nitex. These hatching containers were
returned to the aquarium and kept in motion by suspending them
from an off-set revolving rod (approximately four revolutions per
minute). A separate egg container was used for each lot of eggs,
and a record was kept of the hatching success of each lot.
Two feeding plans were used to determine the amount of food given
to the fish as a daily ration. The fathead minnows in units A and B
were fed all of the frozen brine shrimp they would eat three times
per day. When we started dosing these two units we observed that
the minnows in the two highest levels of methoxychlor had exceptionally
good appetites and probably ate more brine shrimp than did the fish in
controls or lower levels. If the high-level fish were eating more, they
could possibly compensate for any adverse effects of the methoxychlor
and maintain a normal rate of growth. Consequently, to avoid this
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possibility in unit D we changed the plan of feeding to 20% of the total
body weight of the fish each day. A feeding of 20% of body weight is
an exceptionally heavy feeding. However, the frozen brine shrimp had
a high water content, and it became obvious that 20% of body weight
was not supplying an adequate diet. We thus later fed the fish in unit D
all they would eat, the same as in units A and B.
We started feeding brine shrimp to the yellow perch in unit C at a rate
of 10% of their body weight. We found that brine shrimp is not a good food
item for perch, and changed to feeding My sis relict a at 10% of body
weight. This resulted in a tremendous improvement in the growth rate
of the perch.
All fish were weighed periodically, with no definite schedule. We
avoided weighing the fatheads in units A and B while they were spawning
to prevent possible handling mortality. The fish were weighed in a
beaker of water after they had been carefully blotted dry with paper
towel. All fish from an aquarium were weighed together, and the
average weight per individual determined from the total weight of fish.
Respiration studies. --Perch from the long-range bioassays were used
to determine the effect of continuous exposure of methoxychlor on rate
of respiration. Five perch surviving from the highest level (5. 0 jug/1)
of treatment, five from a low level (1. 25 /ug/1), and five from the
controls, were monitored each as a group in a tunnel respirometer
similar to the one described by Brett (1962). The perch, which were
dosed at 10 jug/1, died during the first month of exposure, and those
dosed at 0. 625 and 2. 5 yg/1, died as a result of an aerator failure in
the bioassay unit before they could be included in the respiration study.
The tunnel respirometer allows for the measurement of oxygen
consumption at any desired temperature and current velocity. Care
was taken to assure that each lot of fish was handled in precisely the
same manner throughout the respiration study.
All respiration measurements were made at 65 F. Each group of fish
was assayed at a resting velocity (0. 25 foot per second) and a working
velocity (0. 6 foot per second). The fish were in the respirometer for
1 week at each velocity. Velocity measurements were made with a
Gurley current meter, and oxygen concentrations were monitored with
a YSI meter. Fish were always put in the test chamber on Monday
morning and remained there for the entire week. A determination of
oxygen consumption was made once each day during the week. Each
day the oxygen content was adjusted to a range of 8.0 to 9.0 mg/1.
The concentration could be readily increased by bubbling oxygen into
the open chamber or decreased by bubbling nitrogen. The chamber
was closed and sealed each day at approximately 4:00 p.m. Oxygen
12
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consumption was measured and the chamber was reopened the following
morning after approximately 19 hours (11:00 a.m.). On Friday after-
noon the fish were removed, oxygen was adjusted, and the respirometer
was sealed to determine background respiration by bacteria and other
microorganisms over the weekend. The background respiration values
for two weekends were averaged and thus used as the mean value for the
intervening week. This background value was subsequently subtracted
from each daily respiration determination, in order to arrive at the rate
of oxygen consumption by fish expressed as milligrams of oxygen per
gram of fish per hour (mgO2/g fish/hr).
Several respiration values were discarded when it was discovered that
air bubbles had inadvertently been trapped in the respirometer. The
fish were not fed while in the respirometer nor while being held over
the weekend. Since each lot was used for two consecutive weeks (one
week at each velocity), they were thus without food for that period of
time. Each group of perch was assayed at the resting velocity during
the first week, and at the faster velocity the second week.
Invertebrate toxicity. -- All bioassays were by continuous flow, using
the serial dilution system and injector described earlier. Specially
designed aquaria (Fig. 1) were used for the stream-dwelling insects
Stenonema and Cheumatopsyche. Although a paddle-wheel system was
available to produce a flowing water current in these aquaria, it was
not used, as the current was maintained by the dosing rate. Pond- or
pool-dwelling invertebrates were maintained in appropriate sized
aquaria, e.g., midges in 1-gallon aquaria; crayfish in 5-gallon aquaria;
and scuds in screened containers used for the fish eggs in the fish
section of the study.
Test organisms were collected by Hester-Dendy multiple-plate
samplers or dip nets, and care was taken to assure a minimum of
handling.
During all bioassays the methoxychlor was added as a slurry with
methanol and sufficient Triton X-100 to assure solution of the
insecticide in test water. In all cases the dilution systems were
operated at a flow rate of 5 liters per hour. The concentrations of
methoxychlor in the aquaria were monitored by electron capture gas
chromatography. Dissolved oxygen levels and water temperature were
also monitored.
Most acute bioassays were run in replicate with the exception of those
involving the crayfish. Mortality was measured daily for a period of
4 days. All TL^Q values and 95% fiducial limits were calculated by
both logistic and log-probit analyses. Log-probit equations were also
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Outfall Tul
tatw Inlet
2.5" Channel
H.lght
9"Widfh
Figure 1. --Constant current aquarium, used for insect toxicity studies.
14
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generated to indicate the slope of the dose-response curve. Both types
of analyses were run using computer programs by R. Hartung (personal
communication) and R. Daum (1969). The data from the replicate
bioassays were combined to calculate the TL^Q'g. Mean measured
pesticide concentrations were used in these dose-response analyses.
All chronic bioassays were run in replicate. All organisms were fed
either by natural periphyton build up in the aquaria or leaf fragments
in the case of the scuds.
Measurements of growth, mortality, and emergence were recorded
during the 1-month period of the bioassays. Growth was measured
both by counting the number of exuvia daily in the case of the mayflies,
and by changes in the mean length of all species, exclusive of the scuds,
over the 28-day period. Length, and diameter at midpoint, were
measured by a modified approach to that used by Martin (1967) to
measure juvenile salmon. Ectachrome slides were taken of each test
group of insects initially and at 2-week intervals. The insects were
photographed in a petri dish placed on a millimeter grid paper above
a light source. These slides presented a permanent record of insects
of each test chamber, and when projected on a screen they provided
relative measurements with greater precision. Not all the necessary
corrections as to the thickness of the petri dish and the depth of the
water in the dishes were made, but these conditions plus the focal
length of the camera were kept constant. Corrections were made for
the differences in the projection measuring.
There were, of course, losses of all insect species from initial
stocked populations due to mortality and emergence. A cutoff length
value was therefore arbitrarily assigned to indicate a size beyond
which the organism would pupate or emerge, to prevent consistent
negative growth through loss of larger organisms. The length of
organisms beyond this species specific length were not counted in the
growth measurements. Losses due to mortality were not considered
except in the cases of the higher dose levels (2. 0 and 1. 0 ^g/1) where
the initial population size was greatly reduced through mortality.
Growth was not considered at these dosages because of the marked
loss in sample size.
The differences in mean length were converted to wet weight in
milligrams using a similar formula to that of Hynes and Coleman
(1968).- For the purpose of calculating volumes, Cheumatopsyche
and C. tentans were considered cylindrically shaped, and Stenonema
was considered to be shaped like a cylinder halved longitudinally.
The measured radius at midpoint was also used instead of the assumed
proportion of the length.
15
-------
Screens were placed over all aquaria to collect emerging adults.
However, some adults did escape, presumably through the hole cut
for the dosed water supply.
Emergence, pupation, and mortality were recorded daily as were
the number of exuvia. The accuracy of counting exuvia decreased
as the test continued due to a buildup of an iron precipitate from the
well water. Although the aquaria were cleaned thoroughly midway
through the experiment, the heavy precipitate prevented adequate
identification and collection of exuvia after the initial 2-week period.
This also caused a clogging of the midge aquaria outflows which
resulted in some of the aquaria overflowing, occasionally washing
out adults that may have emerged.
16
-------
SECTION V
RESULTS AND DISCUSSION
Primary production. --Two experiments were run using the carbon-14
technique outlined in the methods section. Run No. 1 was made on
9 and 10 July 1970, and Run No. 2 was made on 4 August, 6 August,
8 August and 10 August 1970. The data from Run No. 1 are in Table 1
and Table 2 for 9 and 10 July, respectively. It will be noted that for
this run there is no duplicate of the Ethanol (ETOH) jug or the Ethanol
+ Triton X-100 (ETOH + Triton) jug. This was an inadvertent error
made during the dosing of the jugs after they had been placed in the lake.
Also, there is no production indicated for jugs 7 and 8 on 9 July. The
counts for these two jugs indicate that the series of subsamples from
these jugs were not injected with carbon-14 through operator error.
Due to problems with counting, this run was terminated early. The
information obtained was used to aid in the design of Run No. 2.
Data from Run No. 1 indicate an effect from the ETOH treatment on
both days. However, there was only one jug with this treatment
because of a dosing error. Although there seems to be a dose effect
from the methoxychlor, no effect was present that could not be assigned
to the ETOH.
Run No. 2 consisted of two experiments: a 2 X 2 design to test the effect
of the ETOH and Triton, and a one-way design to test the effect of
methoxychlor with the ETOH + Triton as a control.
The data from Run No. 2 are presented in Tables .3-6 for 4, 6, 8, and
10 August, respectively, and a summary of all data is given in Table 7.
In the 2 X 2 design with the ETOH, Triton, and ETOH + Triton combina-
tions, an analysis of variance was run to see if any of the combinations
significantly depressed productivity because visual inspection of the data
lead one to believe that the ethanol had a depressing effect on productivity.
The analysis of variance supported the above hypothesis on 8 August 1970
(Table 5) and on 10 August 1970 (Table 6) as follows: on 8 August 1970,
the mean of the experimental units that did not receive ETOH was
64. 2500 as compared to the mean of the experimental units that did
receive ETOH of 49.8000. On 10 August 1970, this comparison of
means was 45. 0500 to 17. 8250. The analysis of variance gave an F
value of 8. 0678 (0. 95 significance) for 8 August 1970, and an F value
of 24.6866 (0.99 significance) was obtained for 10 August 1970. No
explanation can be given for the lack of effect on the runs of 4 and 6
August 1970. It will be recalled that the effect was noted after 24 hours
during Run No. 1. A possibility could be a different community of
organisms during the two runs.
17
-------
Table a.. —Primary production data for 9 July 1970 expressed as counts
per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50 ng/1]
Jug
No.
11
12
2
5
6
8
3
9
4
7
1
10
Lake
Treatment
Control
11
ETOH
ETOH + Triton
Triton
ii
Meth-1
11
Meth- 10
it
Meth -50
11
• • •
Light
bottle
141.6
133.5
81.3
249. 1
142.0
2.4
94.8
171.9
96.5
2.3
55.4
113.4
690.4
Light
bottle
244.0
297.7
90.7
225.2
167.6
2.9
81.0
181.5
113.6
2.5
97.4
169.0
758.3
Dark
bottle
20.2
23.3
27.5
31.0
17.0
2.8
25.2
20.0
21.4
3.9
40. 2
19.2
63.7
Factor^
r
172.6
192.2
58.5
206.2
137.8
• • •
64.7
156.7
83.7
36.2
122.0
660.7
Primary
pr oduc -
tion \2/
16.2
18. 1
5.5
19.4
12.9
• • •
6.1
14.7
7.9
• • •
3.4
11.5
62.0
\/ r = the mean of the two light bottles, minus the value for the dark
bottle.
The formula for primary production is
Y
Prim. Prod. = — Cf , where Primary Production is measured
o
as mg carbon/M /4 hours
(Scintillation count)(0. 25)(0. 838)(2. 22 X 106)
40,290
C = 20.4 X 103
f = 1.06
For tests on 9 and 10 July, Scintillation count was 19, 885. 7 cpm.
For tests on 4, 6, 8 and 10 Aug (Tables 3-6) Scintillation count
was 18,545.3 cpm.
18
-------
Table 2. --Primary production data for 10 July 1970 expressed as counts
per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50 Mg/1]
Jug
No.
11
12
2
5
6
8
3
9
4
7
1
10
Lake
Treatment
Control
"
ETOH
ETOH + Triton
Triton
M
Meth-1
M
Meth -10
it
Meth -50
ii
Light
bottle
62.6
54.9
71.4
94.5
128.7
154.9
56.6
146.3
62.4
104.7
55.9
68.9
327.3
Light
bottle
176.9
151.2
48.8
107.4
142.7
150. 1
68.8
134.9
76.0
105.2
41.7
95.4
294.9
Dark
bottle
29.3
49.3
23. 1
18.0
28. 1
24.1
21.2
22.3
26.2
25.2
22.7
21.4
61.2
Factor^
r
90.5
53.8
37.0
82.9
107.6
128.4
41.5
118.3
43.0
79.7
26. 1
60.8
249.9
Primary
produc -
tion >?/
8.5
5.1
3.5
7.8
10.0
12.1
3.9
11.2
4.1
7.5
2.5
5.7
23.5
See footnotes to Table 1.
19
-------
Table 3. --Primary production data for 4 August 1970 expressed as
counts per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50
Jug
No.
4
7
5
11
3
13
1
12
2
10
6
9
8
14
Lake
Treatment
Control
"
ETOH
it
Triton
"
ETOH + Triton
it
Meth-1
ti
Meth- 10
11
Meth -50
11
Light
bottle
226.3
332.7
311.6
320.4
249. 1
394.4
142.6
435. 1
195.6
386.5
317.3
306.0
243.0
284.0
706.5
Light
bottle
261.7
372.2
305.0.
364.9
194. 1
427.0
115.2
470.0
188.8
369.5
339.0
247.1
175.6
347.9
667.6
Dark
bottle
11.0
16.8
16.9
16.3
15. 1
22.6
8.1
26.6
11.1
29.3
18.2
3.6
20.8
18.3
69.9
Factor^
r
232.5
335.7
291.4
326.4
206.5
388. 1
120.8
426.0
181. 1
347.7
310.0
273.0
188.5
297.7
617.2
Primary
produc -
23.5
33.9
29.4
32.9
20.8
39. 2
12. 2
43.0
18.3
35.1
31.3
27.5
19.0
30.0
62.3
See footnotes to Table 1.
20
-------
Table 4. --Primary production data for 6 August 1970 expressed as
counts per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50
Jug
No.
4
7
5
11
3
13
1
12
2
10
6
9
8
14
Lake
Treatment
Control
ii
ETOH
it
Triton
it
ETOH + Triton
ti
Meth- 1
it
Meth -10
n
Meth -50
M
• • •
Light
bottle
273.9
295. 1
338.5
460.6
320.4
437.1
235.3
355.7
268.7
409.6
224.7
327. 1
254.6
168.8
647.9
Light
bottle
257.3
333.5
354.0
419.2
390.2
474.3
314.9
411.7
180.7
400.6
217.0
289. 1
231.9
271.5
647.3
Dark
bottle
19.4
15.7
14.3
34.9
6.3
33.0
12.5
26.6
21.8
19.9
21.5
20.0
20.7
25.8
78.6
Factor^
r
246.2
298.6
332.0
405.0
349.0
422. 7
262.6
362.1
200.4
385.2
199.4
288. 1
222.6
194.4
569.0
Primary
produc-
tion fy
24.8
30. 1
33.5
40.9
35.2
42.7
26.5
36.5
20.2
38.9
20. 1
29. 1
22.5
19.6
57.4
See footnotes to Table 1.
21
-------
Table 5. --Primary production data for 8 August 1970 expressed as
counts per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50
Jug
No.
4
7
5
11
3
13
1
12
2
10
6
9
8
14
Lake
Treatment
Control
ii
ETOH
it
Triton
"
ETOH + Triton
1!
Meth-1
ii
Meth- 10
it
Meth -50
ii
Light
bottle
663.8
691.4
497. 2
501.4
599.8
718.7
439.8
619.8
488.0
608.2
517.4
598.2
340.4
384.0
723.2
Light
bottle
556.9
668.8
564.2
549.8
602. 1
702.3
408.4
571.5
502.8
595.9
576.9
605.3
282.8
373.0
717.5
Dark
bottle
20.8
23. 1
23.8
29.2
18.2
27.4
26.9
22.2
19. 1
•21.5
28. 1
18. 1
25.6
29.4
71.3
FactorN^
r
608.3
657.0
506.9
496.6
599. 1
683. 1
397.2
573.4
475.7
599.9
519.1
583.6
286.0
349. 1
649. 1
Primary
produc -
tion^
61.4
66.3
51.1
50.1
60.4
68.9
40. 1
57.9
48.0
58.6
52.4
58.9
28.9
35.2
65.5
See footnotes to Table 1.
22
-------
Table 6. --Primary production data for 10 August 1970 expressed as
counts per minute
[Treatments were: Control, ETOH, Triton, and
Methoxychlor (Meth) at 1, 10, and 50 Mg/1]
Jug
No.
4
7
5
11
3
13
1
12
2
10
6
9
8
14
Lake
Treatment
Control
ii
ETOH
n
Triton
n
ETOH + Triton
n
Meth-1
it
Meth -10
ii
Meth -50
ti
• • *
Light
bottle
469.4
519.0
199.8
203.2
370.3
551.4
151.4
272.2
234.6
299.9
259.5
293.0
250.5
300.8
683.6
Light
bottle
393.8
492.9
180.3
210.4
374.5
543.9
132.7
251. 1
251.7
310.3
278.8
283.3
243.0
303.8
680.1
Dark
bottle
16.7
15.5
19.2
27.6
17.3
21.7
17.9
28.6
17.6
30.4
22.4
17.7
19.1
25.0
75.6
Factor^
r
414.9
490.4
170.8
179.2
355. 1
526.0
124.2
233.0
225.6
274.7
246.8
270.4
227.6
277.3
681.8
Primary
produc-
tion N2/
41.8
49.5
17.2
18.1
35.8
53.1
12.5
23.5
22.8
27.7
24.9
27.3
23.0
28.0
68.8
1 2
V V See footnotes to Table 1.
23
-------
Table 7. —Summary of primary production data from two studies of the effects
of ethanol, Triton, and methoxychlor
[Treatments were Control, ETOH, Triton, methoxychlor
at 1, 10 and 50 ng/1, and lake]
Date,
1970
Run #1
9 July
10 July
Mean
Run #2
4 Aug
6 Aug
8 Aug
10 Aug
Treatments
Con-
trol
17.2
6.8
12.0
28.7
27.5
63.9
45.7
ETOH
5.5
3.5
4.0
31.2
37.2
50.6
17.7
Triton
12.
11.
12.
30.
39.
64.
44.
9
1
0
0
0
7
5
ETOH +
Triton
19.4
7.8
13.6
27.6
31.5
49.0
18.0
Meth
-1
10.4
7.6
9.0
26.7
29.6
53.3
25.3
Meth
-10
7
5
6
29
24
55
26
.9
.8
.9
.4
.6
.7
. 1
Meth
-50
7.
4.
5.
24.
21.
32.
25.
5
1
8
5
1
1
5
Lake
62.0
23.5
42.8
62.3
57.4
65.5
68.8
Mean 41.5
34.2
44.6
31.5
33.7
34.0
25.8
63.5
24
-------
The production figures seem to indicate an initial depressing effect
due to the methoxychlor at the 50 /ug/1 level on 4, 6, and 8 August,
with slight stimulation of productivity on 10 August. The stimulatory
effect is noted at the 1- and 10-/jg/l levels in the data for 8 and 10
August.
The summary of the productivity data is presented in Table 7.
Run No. 1 shows a short-term dose effect due to the methoxychlor
when compared to the ETOH + Triton control. Run No. 2 shows an
initial depressing effect at the 50-/ug/l level lasting for 5 days,
followed by a stimulatory effect in 7 days.
Methoxychlor levels, monitored throughout the productivity studies,
tended to give differing results for the two runs. In Run No. 1
(Fig. 2) the unfiltered sample remained virtually unchanged for
2 days, then dropped rapidly to approximately one-half of the initial
value by the 4th day. The concentration then changed little throughout
the remainder of the experiment. The filtered samples decreased
more consistently than did the unfiltered samples. The half life of
the methoxychlor is approximately 6. 5 days for the unfiltered
material and approximately 4. 6 days for the filtered material.
Figure 3 presents the data from Run No. 2, and illustrates the lack
of an induction period prior to the start of degradation of the methoxy-
chlor. The decay of the pesticide in the unfiltered material, while
starting more rapidly than in Run No. 1, does not decay to the extent
that was evident in the first run. The filtered samples are different
from Run No. 1, showing an increase in concentration after an initial
drop in concentration. This could indicate early metabolism, or
adsorption, by the biota followed by death, decay and release of the
methoxychlor.
It appears that the level of methoxychlor stabilized after a period of
7 days, in both filtered and unfiltered samples, at a level approximately
75% of the initial concentration. If'the line for the unfiltered material
in Figure 8 was projected, it would indicate a half life of about 18 days.
The data for the filtered samples indicate an initial drop with a half life
of approximately 4-5 days followed by an increase from the lower level.
Temperature profiles conducted during the productivity studies are
shown in Figure 4. The lake was thermally stratified with the thermo-
cline starting at approximately 2.5m and extending to a depth of 8 m.
The range of temperatures during the study is indicated in Figure 3 by
the cross-hatched area. The temperature at the incubation depth (1 m)
ranged from 23. 5 C to 26. 5 C during the period studied.
25
-------
Jug 1
Jug 10
= 30
o>
2
.s
i
5 20-
V
o
u
x
o
•
V
•
\
Unfilt*r«d Samples
Filtered Samples
4 6 8 10
Time In Days
Figure 2. —Methoxychlor concentrations in test water measured
during 7-day productivity study
26
-------
45-
40-
2
e
w
§
U
X
o
10-
, Jug 8
#——+- Jug 14
Unfiltered Samples
Filtered Samples
*
2 4 6 8 10
Time in days
Figure 3. —Methoxychlor concentrations in filtered and unfiltered
water samples measured during 10-day productivity
study
27
-------
IA
I.
0
3
o.
«
o
0 5
°-hsH
i-
2-
3-
4-
5-
6-
7-
8
9
10
11
12
13
14
Temperature °C
10 15
20
25
Figure 4.—Temperature profiles in Third Sister Lake during
productivity studies
28
-------
Breakdown studies. --Table 8 contains the chemical data from the
various water supplies used for breakdown studies.
Figure 5 shows the rate of breakdown of methoxychlor in distilled
water in relation to pH. As can be seen from the figure, the half
life of the compound had not been attained in 220 days. The half
life estimated from these data is 270 days. Hydrogen ion concentra-
tion within the range studied (pH 7-9) had no effect on the breakdown
rate. Figure 6 depicts the rate of breakdown in aged Ann Arbor tap
water which had previously held fish. The half life in this case was
8 days. The results from two experiments conducted with water
from Koch Warner Creek are shown in Figure 7; here, in each test,
two different loss rates are apparent in the data. The initial high
rate was presumably due to the adsorption of methoxychlor on
particles which settled and were missed in the sampling procedure.
This hypothesis is supported by the study (Fig. 8) conducted with
Third Sister Lake water, where in two experiments the majority
of the methoxychlor was located in the particulate fraction. Measured
half life was different in the two experiments; it was 7 days in the
June experiment, and (by extrapolation) 18 days in the July trial.
Table 8. --Chemical characteristics of test waters
Water source
Alka-
pH linity
(mg/1)
Hard-
ness
(mg/1)
Tempera-
ture
(°C)
Koch Warner Creek 8.2 180 400 20 ± 2
Third Sister Lake 8.5 60 80 24 ± 1
Distilled 7 and 9 1 1 20 ± 2
/
Ann Arbor tap water, .aged 7.0 40 60 20 ± 2
29
-------
100
50
o>
20
J
g
i TO
I
* t
Is
*
k. *
*.*
*pH 7
• pH 9
40 80 120 160 200 240
Days
Figure 5. --Concentration of methoxychlor in replicated samples
of buffered distilled water at pH 7 and pH 9 during
220 days of hydrolysis
30
-------
50
W
2
w
'§
20
o 10
^
u
§
U
I 5
*
*
* Aquarium 1
• Aquarium 2
TO 12 14 16 18 20
Days
Figure 6. --Concentration of methoxychlor in replicated
samples of aged Ann Arbor tap water, which
had previously held fish, during 18 days of
breakdown
31
-------
50
0)
e
V
I
I
1
c 10 - • • Aquarium 1
•j * Aquarium 2
i
2 4 6 8 10 12 14 16 18 20
Days
Figure 7. --Concentration of methoxychlor in replicated
samples of Koch Warner Qreek water during
20 days of breakdown
32
-------
50
June
$ 20 |- UnWered Samples
0)
8
£
S 10
w
e
o
5
e
Filtered Samples
2 4 6 8 K> 12 14 16 18 20
Days
Figure 8. --Concentration of methoxychlor in filtered and
unfiltered samples of Third Sister Lake water
during 10 days of breakdown
33
-------
Acute fish toxicity. --The static 96 -hour TLso values obtained during
the experiments were 7. 5 ng/l for fatheads, and 30. 0 ng/l for perch.
There are sizable differences in susceptibility of fish species to
hydrocarbon insecticides. However, the results from static tests
particularly with larger fish are often suspect. Our static tests on
perch substantiate the weakness of these tests, as shown in Table 9,
where the nominal dose and the measured concentrations of methoxy-
chlor are given. It is obvious that the perch were exposed to the
nominal concentration for a very short period of time.
Figure 9 shows the change in TL^Q with time during the continuous -
flow test for the fathead minnow. These results are averages of two
replicate tests, involving two test chambers with ten fish per chamber
at each concentration. Spot checks for methoxychlor, run on the
high-dose and low-dose levels at the start and end of the experiment,
indicated that the nominal doses were within ± 10% of the measured
concentrations .
Replicated continuous -flow tests were also run using yellow perch
with 8 fish per tank in Test No. I and 10 per tank in Test No. II.
Table 10 gives a summary of the nominal doses, the measured
concentrations, and the accumulative mortality during the 96 -hour
tests.
The mortality of test fish in the continuous -flow studies was
interpreted by logistic and log-probit (Daum, 1969) analysis. The
replicated tests were lumped to give one analysis for both the fat-
head minnows and yellow perch. Table 11 gives a summary of the
analysis. The TL5Q values were 8. 54 jug/1 for fatheads by logistic
analysis and 8. 63 /ug /I by log probit. Corresponding values for
perch were 20. 12 ngfl and 22. 20 jug/1. The perch had a much steeper
slope (12. 351) of dose response than the fathead minnows (4. 696)
indicating a broader range of toxicity to fatheads .
Table 9. --Measured concentrations of methoxychlor in
30 liters of water during the exposure of five yellow
perch (approximately 50 g total weight) for 96 hours
Time
(hours)
4
24
48
96
Nominal concentrations of methoxychlor
i..~ /-n
40
35.4
15.4
5.1
2.0
\H6 / •«•/
30
21.4
12.3
2. 1
1.3
20
15.5
8.3
2.2
1.0
34
-------
12
K>
V •
.- 0
M
i
I •
• •
• ^^«
• •
8 4
§
o
• •
7 9
Days
11
13
15
Figure 9. --Changes in median tolerance limit of methoxychlor
for fathead minnows with time of exposure
35
-------
Table 10. —Concentrations of methoxychlor and number of yellow perch
that died during 96-hour continuous-flow toxicity study (eight perch per
tank were used in Test I, and ten in Test II)
_,. Nominal concentrations of methoxychlor (jug/1)
,, 6. Test I Test II
(hours)
40 20 10 5 30 15 7.5
24 Measured 41.4 20.7 9.6 4.6 26.9 12.9 7.1
cone.
Accum. 7000500
mortality
48 Measured 32.0 19.0 9.4 4.7 26.6 12.5 6.3
cone.
Accum. 8000900
mortality
72 Measured 39.6 21.4 9.0 2.7 27.1 11.4 6.5
cone.
Accum. 8200900
mortality
96 Measured '28.4 11.5 5.8
cone.
Accum. 8600900
mortality
36
-------
Table 11. --Summary of dose response of fathead minnows and yellow
perch to 96-hour continuous flow methoxychlor bioassay interpreted
through logistic and log-probit analysis
Species
TL50 dug/1)
with 95%
fiducial limits
Log-probit
equation
Fathead minnows
*
Logistic 5.30 - 8.54 - 13.77
Log-probit 6.42- 8.63 - 10.62 Y - 0. 604 + 4. 696 X
Yellow perch
Logistic 15.95 - 20. 12 - 25.38
Log-probit 19.69 - 22.20 - 25.59 Y = -11. 630 + 12.351 X
_
TL5Q values underlined.
The difference in susceptibility of the two species is also reflected in
the various TL values obtained from the probit analysis. Values for
fatheads ranged from TLgo of 4. 60 to TLio of 19. 33 M£/l- Values for
the perch were TLg0 of 17.49 to TL10 of 34. 26 jug/1.
Chronic fish toxicity. --Table 12 contains data on survival and growth
of fathead minnows in units A and B during 4 months of exposure to
methoxychlor. The fish were reweighed on 1 April 1971, following
spawning. However, all surviving fish had reached maximum size
and their weight was not considered indicative of dose response. All
tanks contained 14 fish at the start of the assay. There was some
fish mortality in all of the aquaria; however, we are not sure that it
was related to the levels of concentration of methoxychlor. In unit A
there was considerable mortality at the lower levels, but almost none
at the highest concentration (2 Mg/D- This indiscriminate mortality in
unit A made it difficult to interpret changes in the mean weight of the
fish. We can show no relationship between growth of the fatheads and
concentration of methoxychlor in unit A.
There was less mortality in unit B; however both growth and mortality
of the fish appeared to be more closely related to methoxychlor levels.
The average gain in weight of the fatheads in the two highest levels of
37
-------
Table 12. --Growth and survival of fathead minnows exposed to
methoxychlor at various levels of concentration
Unit
A-6
A-5
A-l
A-2
A-3
A-4
B-6
B-5
B-l
B-2
B-3
B-4
Methoxy-
chlor
concentra-
tion (/ug/1)
Control
2.0
1.0
0.5
0.25
0.125
Control
2.0
1.0
0.5
0.25
0. 125
Number
of fish
alive
8/5/70
11
13
5
4
8
6
13
7
8
9
10
12
Average
weight
(g)
4/13/70
0.83
0.84
0.91
0.67
0.76
0.89
0; 66
0.85
0.84
0.82
0.72
0.79
Average
weight
(g)
8/5/70
1.44
1.82
2. 14
1.20
2.00
1.83
1.46
1.31
1.31
1.66
1.42
1.59
Average
weight
gain
(g)
0.61
0.98
1.23
0.53
1.24
0.94
0.84
0.46
0.47
0.84
0.72
0.80
methoxychlor (1 jug/1 and 2 jug/1) was only one-half of the gain at the
three lower levels and among the control fish.
Table 13 contains a summary of the growth and mortality of fathead
minnows in unit D. These data show no relationship between growth
and methoxychlor concentration.
Fish growth was converted to specific growth rates (Brown, 1957)
which express growth as percent growth per unit of time. This analysis
is recommended when initial sizes are not identical since growth tends
to be logarithmically related to size. Table 14 contains specific growth
rates of fathead minnows subjected to various concentrations of
methoxychlor in units A and B. There is no indication that methoxychlor
has retarded growth of any of the test fish.
Table 15 contains a summary of the growth and survival of yellow perch
(unit C) subjected to various levels of methoxychlor from 11 August 1970
to 30 March 1971. All perch died at 10 A
-------
Table 13.--Growth and survival of fathead minnows exposed to
methoxychlor at various levels of concentration in test unit D
Concentration
methoxy-
fog/D
0. 125
0. 25
0.50
1.0
2.0
Control
Duration
18 Aug 1970
Number Aver-
of fish age
at weight
start (g)
20 0.99
20 0.87
20 0.96
20 0.95
20 0.92
20 1.00
of exposure
5 Jan 1971
Number Aver-
of fish age
surviv- weight
ing (g)
20 1.51
18 1.17
20 1.34
19 1.33
15 1.23
20 1.40
Average
weight
gain (g)
18 Aug 1970-
5 Jan 1971
0.52
0.30
0.38
0.38
0.31
0.40
Table 14.--Specific growth rates of fathead minnows subjected to
various concentrations of methoxychlor
Cone entration
fog/D
Unit A
Control
0. 125
0.25
0.50
1.00
2.00
Unit B
Control
0. 125
0. 25
0.50
1.00
2.00
Growth
13 April to
5 Aug 1970
0.481
0.634
0.848
0.511
0.749
0.679
0.695
0.612
0.603
0.616
0.390
0.378
interval
5 Aug 1970 to
1 April 1971
0. 190
0.317
0.211
0.446
0.319
0. 261
0. 180
0.203
0.322
0.203
0.291
0.354
Total
13 April 1970
to 1 April 1971
0.277
0.419
0.416
0.467
0.457
0.395
0.347
0.336
0.413
0.337
0.322
0.363
39
-------
Table 15. --Average weight in grains of yellow perchv during exposure
to methoxychlor, and average gain in weight during test periods in unit C
Concen- At these dates
tration of 11 Aug 1970 29 Dec 1970 17 Feb 1971 30 March 1971
methoxy- Num.- Weight Num.- Weight Num.- Weight Num- Weight
chlor ber ber ber ber
(jug/1) fish fish fish fish
Control
0.625
1.25
2.50
5.00
10.00
6
9
9
9
9
9
8.33
7.56
7.00
7.00
5.89
7.56
6
9
8
9
7
0
11.80
9. 11
8.75
8.00
7.28
• • •
5
9
8
9
7
0
19.20
14.44
13.75
12.67
11.15
• • •
5
9
8
9
7
0
25.20
19.44
18.00
16.56
14.57
• • •
Gain in weight during periods
Control
0.625
1. 25
2.50
5.00
11 Aug
to
29 Dec
3.47
1.55
1.75
1.00
1.39
29 Dec
to
17 Feb
7.40
5.33
5.00
4.67
3.87
17 Feb
to
30 March
6.00
5.00
4. 25
3.89
3.42
11 Aug 1970
to March 30
1971
16.87
11.88
11.00
9.56
8.68
\/Perch were fed frozen Artemia prior to 29 December, and frozen
Mysis relicta for the remainder of the study.
40
-------
My sis. Prior to 29 December, growth seemed to be retarded equally
at all levels of methoxychlor. After we improved the diet of the perch,
however, there was a considerable range of effect at various levels of
exposure.
The weight gains of the perch were also converted to specific growth
rates which are given in Table 16. The rates for the control fish are
significantly higher than any of the test fish, however the effect is not
linear with dose within the levels tested.
Table 17 contains data on hatching success of fathead minnow eggs
as influenced by various levels of methoxychlor. There was no
spawning at 2 vg/1, and only two lots of eggs at 1 fjig/l. The only
enigma here is the 92% hatching success of one of these lots of eggs.
Totals from tests A and B give a 66% hatching success for eggs from
control fish, and a range of 29 to 37% success for eggs from treated
fish. Hatching percentage was not linear with dose.
An analysis of variance indicated no significant difference in the
numbers of eggs deposited per individual spawning. We had no way
of knowing how many females actually spawned, or whether some
spawned more than once.
We selected one lot of eggs from the control tank of unit A, put
one-half of the eggs in an aquarium dosed at 2 vgll, and left the
other half in the control tank. None of the eggs hatched at 2
whereas 81% of those left in the control tank hatched.
Table 16. --Specific growth rates of yellow perch subjected to various
concentrations of methoxychlor
Growth interval
Concentration
(iug/D
Control
0.625
1.25
2.50
5.00
10.00*
1 1 Aug. to
29 Dec, 1970
0.249
0. 133
0.159
0.095
0.151
• • •
29 Dec. 1970
to 17 Feb.
1971
0.974
0.921
0.904
0.920
0.853
• • •
17 Feb. to
30 March
1971
0.647
0.708
0.641
0.638
0.637
• * •
Total
11 Aug.
1970 to
30 March
1971
0.477
0.407
0.407
0.371
0.390
• • •
All fish died at 10 /ug/1.
41
-------
Table 17. --Spawning and hatching success of fathead minnows at methoxy-
chlor levels of 2 |Ug/l and less
Data given are number of eggs (E) and percentage hatched (%H)
Concentration of
Unit 1.0 0
E %H E
A 0 0 12
89
58
90
33
144
...... 78
42
Total 0 0 546
B 174 0 59
89 92 11
407
• •• •• •••
• •• • • - •••
• •• •• •••
• •• •• • • •
Total 263 31 477
Total
A + B 263 31 1,023
.5
%H
100
11
0
0
9
36
100
83
35
0
91
27
f t
..
• •
• •
25
31
methoxychlor
0.
E
109
232
37'
• • •
• • •
• • •
* • •
378
152
141
94
67
28
262
29
105
22
37
937
1,315
25
%H
11
13
95
• •
t B
• •
t m
20
15
27
11
90
75
5
66
64
96
95
33
29
Gug/Dvi'
0.
E
269
84
221
• • •
• • *
• • •
* • •
574
42
88
174
264
20
22
212
157
23
1,002
1,576
125
%H
31
0
58
• •
9 9
• •
• •
37
71
17
66
13
85
90
36
43
74
39
37
Control
E
51
50
55
5
57
17
5
240
159
364
31
39
190
46
32
70
181
1, 112
1,352
%H
0
0
82
100
94
82
80
51
69
85
87
85
68
100
31
94
22
69
66
v
No eggs were spawned at a concentration of 2.0
42
-------
Respiration studies. --Values of oxygen consumption in the respirometer
by the control perch and by those exposed to two different levels of
methoxychlor, are given in Table 18. A two-way analysis of variance
indicated the only significant differences within these sets of data were
that the fish from high level methoxychlor (5. 0 jUg/D tested at the high
velocity give significantly high values of oxygen consumption. The
val ues are significantly higher (at 95% level of confidence) than those
for any other combination of methoxychlor and velocity. Exposure to
5 jug/1 of methoxychlor apparently caused physiological damage to the
perch, resulting in a high oxygen demand when subjected to continual
physical exertion.
Table 18. --Oxygen consumption in milligrams per
gram of fish per hour (mg/g/hr), by yellow perch
after long-term exposure to methoxychlor,
measured at two rates of swimming speed
Exposure
to
methoxychlor
Swimming speed
Low velocity High velocity
(0.25 ft/sec) (0.6 ft/sec)
Control
1.25/ug/l
5.0 Atg/1
0.1758
0.1717
0.1872
0.1406
0.1187
0.0943
0.1792
0.1570
0.1682
0.1524
0.1475
0.1733
0.1842
0.1346
0.3836
0.2179
0.1821
0.1556
43
-------
Invertebrate toxicity. --The 96-hour TL50r , fiducial limits, log-probit
equation, water temperature, and mean dissolved oxygen are given in
Table 19. The TLso value is flanked by its 95% fiducial limits. In some
cases a non-significant regression was obtained using probit analysis.
This implies that the data were not a good fit to the log-probit analysis
nor the logistic analysis as both are similar. A TLso value was given
through the logistic analysis to approximate the true value. Because
the logistic and log-probit analyses are based on similar assumptions,
they yield similar TLso values as is evident here. In all cases the
96-hour TL5Q values were in the low micrograms -per -liter range.
The most susceptible organism was the adult scud while the crayfish
was least susceptible with 96-hour TLso values of 0. 75 and 7.05,
respectively. The crayfish were second -year -class adults (mean
carapace length, 3.3 cm) and the mayflies, midges, and caddisflies
were late larval instars.
The steep slopes of the midge and crayfish log-probit line indicate
that the toxicity range is narrow and thus there may be a threshold
pesticide level beyond which toxicity is great. The effect of oxygen
level on toxicity in crayfish is apparent as the toxicity at low
dissolved oxygen levels (unaerated aquaria) is three times that of
normal dissolved oxygen levels (aerated aquaria). This distinction
was made as it was found that the dissolved oxygen levels were greatly
depressed during the first bioassay, therefore a second bioassay was
conducted, artificially aerating the aquaria. This oxygen effect was
noted in other bioassays (Table 20) although there was no significant
difference between dosed and control aquaria. Jensen (1965) observed
increased oxygen uptake of two stonefly species under DDT stress and
it has been generally accepted that this is the case.
Although it appears that age may be a factor in scud susceptibility to
methoxychlor as the 96 -hour TLso for the young is less than that of
the adult, this may be more a case of different water temperatures
involved in both bioassays.
The chronic bioassay TLso'g , fiducial limits, log-probit equations,
temperature, and mean dissolved oxygen are given for 2-week periods
in Table 21. The duration of chronic tests varied from 2 weeks to
42 days depending on the length of time the various species could be
maintained in good condition. For most invertebrates the mortality
decreased greatly after the second week of dosing. Insect resistance
or vigor tolerance to pesticides has been reported especially for
Stenonema (Grant and Brown, 1967). Although organisms had
different 96 -hour TLso's > they all had similar chronic TLso's
for as long as the respective bioassays were conducted. Replicate
bioassays were combined for the dose response analyses.
44
-------
Table 19.--Results of 96-hour continuous-flow invertebrate bioassays, with
recorded values of water temperature and mean dissolved oxygen, interpreted
through logistic and log-probit analyses
Test organism
Water Mean
temp D.O.
(° F) (ppm)
96-hr TLso^ Mg/1
(with 95% fiducial Log probit equation
limits)
Stenonema
interpunctatum
Larva
Logistic
Log probit
Chironomus
62±4 8.2 1.09 1.96 3.11
NS regression
Y = 4.013 + 3.950 X
tentans
Larva
Logistic 62±4 8.2
Log probit
Cheumatopsyche sp.
Larva
Logistic 54 ±4 6.1
Log probit
Gammarus
pseudolimnaeus
Young
Logistic 54±4 6.1
Log probit
Adult
Logistic 37±3 7.5
Log probit
Orconectes
virilis
Adult (unaerated)
Logistic 60±4 1.7
Log probit
Adult (aerated)
Logistic 60 ±4 5.5
Log probit
1.19
1.41
2.89
2.42
0.84
NS
0.47
0.41
0.62
0.41
3.22
NS
1.62
1.59
3. 24
3.26
2.30
1.81
3.63
4.34
1. 14 1.54
regression
0.75
0.61
2. 15
2.15
1.22
0.98
3.79
10.61
7.05 15.42
regression
Y = 3.450 + 7.716 X
Y - 3.554 + 2.814 X
Y = 4.873 + 1. 298 X
Y = 5.579 + 2.698 X
Y = 3. 131 + 5.626 X
None
values are underlined.
45
-------
Table 20. --Dissolved oxygen levels in the Stenonema sp. and Chironomus
tentans continuous-flow 96-hour bioassay (Test I)
Replicate A
Concentration
Methoxychlor 9.62 ±1.47 1.23 ±.99 0.92 ±.40 0
Gug/D
Oxygen
(mg/1) 7.71± .52 8.35 ±.64 8.35 ±.21 8.41 ±.19
Replicate B
High cone. Low cone. Control
Methoxychlor 8.0 ±1.35 0.55 ±.064 0
kg/1)
Oxygen 8.47 ±1.02 8.49 ±.16 8. 74 ± . 172
(mg/1)
46
-------
Table 21. --Results of chronic continuous-flow invertebrate bioassay interpreted
at 2-week intervals through logistic and log-probit analyses
Water Mean
Test organism^ temp D.O.
(°F) (ppm)
Stenonema
terminatum
Larva
Logistic 68±4 6.0
Log probit
Stenonema
interpunctatum
Larva
Logistic 6 8 ±4 6.0
Log probit
Logistic 68±4 6.0
Log probit
Cheumatopsyche sp.
Larva
Logistic 54±4 6.5
Log probit
Logistic 54±4 6 . 5
Log probit
Logistic 54±4 6.5
Log probit
Gammarus
pseudolimnaeus
Young
Logistic 54±4 6.5
Log probit
Logistic 54±4 6.5
Log probit
Logistic 54±4 6.5
Log probit
Inter- TLjj) 'in/ug/1
val (with 95% fiducial
(weeks) limits)
2 0.32 0.41 0.51
0.002 0.41 1.44
2 0.38 0.47 0.58
0.25 0.47 0.74
4 0. 13 0.22 0.38
NS regression
2 0.60 0.71 0.85
0.44 0.72 1.37
4 0.42 0.49 0.57
NS regression
6 0.17 0.21 0.26
0.07 0.21 0.33
2 0.26 0.30 0.34
0.28 0.33 0.36
4 0.23 0.29 0.36
NS regression
6 0.20 0.22 0. 24
NS regression
Log-probit equation
Y = 5.515 + 1.321 X
Y = 5.431 + 1.302 X
Y = 6. 148 + 2.068 X
Y = 5.246 + 1. 753 X
Y = 6.645 + 2. 127 X
Y - 6.926 + 2.877 X
Y = 8. 104 + 3.402 X
Y = 11.76 + 19. 203 X
None
*
TLcjQ values are underlined.
47
-------
The chronic midge bioassay, while mentioned, has no data presented
in Table 21 because of seemingly high mortality in the controls due to
low recovery of initial test organisms (Fig. 10). This loss negates
standard dose response analyses. Mulla (1969) indicates that high
mortality in her bioassay controls was due to cannibalism, and the
same may be true here. All recovered insects were alive in all dosed
aquaria except at the two highest dose levels, 2.0 and 1.0 jug/1- It
was assumed that the low recovery in these aquaria was due to
pesticide induced mortality while low recovery in the control was
due to cannibalism. Highest recovery of initial test organisms was
in a low dosage (0. 25 ng/l) aquaria.
Emergence is expressed as emergence only for Stenonema, and
pupation only for Cheumatopsyche (Fig. 11), as no caddisflies
emerged. Pupation and emergence are lumped for C_. tentans
(Fig. 10). The total emergence for a 28-day period is plotted and
the two mayfly species groups are combined as are all replicate
bioassays.
Interestingly the low dosage levels (0. 125 or 0. 25 /ug/1) experienced
higher, or comparable, emergence than the controls in all cases.
Although the curves appear to be skewed, if dose levels were trans-
formed to logarithmic scale as they are in standard dose response
analyses, the curves would approach normal or Gaussian distributions.
Growth as expressed for Stenonema by number of exuvia per 28-day
bioassay is plotted in Figure 12. As can be seen the numbers of
exuvia per individual are generally dose related, as there were
fewer exuvia in the highest dosage aquaria with a general linear
increase as the dosing level was decreased.
Table 22 indicates the calculated growth for all species in milligrams
per individual using the length measurements made at 2-week intervals.
Again, as in the emergence data, the mayflies and midges seemingly
have faster growth at the low pesticide levels (0. 125 and 0. 25 jug/1)
than in the control. The growth 'of Cheumatopsyche is greater in the
control than in all dosed samples.
In all cases there is markedly reduced growth at the 0. 5 jug/1
pesticide level indicating that there may be a threshold beyond which
lower pesticide exposures are not inhibitory with regard to growth.
Dissolved oxygen levels were measured weekly in all mayfly aquaria
after it was established that the midge aquaria maintained the same
levels as their source mayfly aquaria. The mean dissolved oxygen
levels corresponding to the pesticide levels ranged from 5. 7 mg/1
to 7.35 mg/1 with a low and high individual value of 4. 9 mg/1 and
48
-------
ao-i
20-
60-
I
40-
Survivorship and Recovery
Recovered from Aquaria
Survivorship from Number
at Start
Pupation and or Emergence
* Replicate 1
v Replicate 2
• Mean
Methoxychlor Level
Figure 10. --Percent survivorship and recovery of Chironomidae
larvae from 28-day chronic methoxychlor bioassay
49
-------
St»nontmo
(•nwrgwice)
* S.. InterpunctotuHi
v S.. terming turn
• Mean
60-1
40 -
S
20 1
0 .125 .25
Cheumatopsyche
(pupation)
1.0
Mcthoxychlor Uv«l
Figure 11. --Percent emergence of Stenonema and pupation of
Cheumatopsyche during 28-day chronic methoxy-
chlor bioassay
50
-------
2.0
"5 1.5
.2 1.0
>
x
I 05
Growth
int»rpunctofu«i
• MUan
.25 05 10
M«thoxychlor
2JO
Figure 12. --Growth of Stenonema, as expressed by number of
exuvia per individual, during 28-day chronic
methoxychlor bioassay
51
-------
Table 22. --Growth in milligrams of all insect larvae tested during chronic
continuous-flow methoxychlor bioassay
Organism
Time
(weeks)
Concentration of
methoxychlor
0.5
0.25 0.125
Control
Stenonema interpunctatum
group
S^. terminatum group
Mean
2
2
-0.200 1.087 0.057 0.369
0.646 1.291 2.432 2.224
0.223 1.189 1.244 1.296
Chironomus tentans
(Replicate 1)
(Replicate 2)
Mean
Cheumatopsyche sp.
Mean
2 0.487
2 0.155
0.321
2 0.347
4 -0.057
0.145
0.932
0.843
0.888
0.601
0.521
0.561
0.773
0.838
0.806
0.579
0.507
0.543
0.600
0.201
0.400
1.306
1.532
1.419
8. 1 mg/1, respectively. A one-way analysis of variance (ANOVA)
showed the means to be significantly different at the 95% level.
Paired Scheffe contrasts at the 95% level showed the mean dissolved
oxygen level of the second highest pesticide level tank (5. 7 mg/1) to
be significantly different from that of the control tank (7. 35 mg/1).
52
-------
SECTION VI
STREAM CONSTRUCTION
One phase of this study consisted of the construction of six experimental
streams that will constitute a permanent facility for conducting long-term
sublethal pollution studies. The streams are of such size and design
that fish and other aquatic fauna can be raised in a nearly natural habitat.
The streams were constructed within a millrace at the Saline Research
Station of the Institute for Fisheries Research, Michigan Department of
Natural Resources. About 400 feet of the millrace was divided length-
wise by a cement wall to form two streams each 18 feet wide. These
streams were further divided crosswise to form six stream segments
each 120 feet long. Each stream segment has a shallow riffle with a
depth of about 1 foot at the head end, and then a gradual slope into a
pool with a depth of 3 1/2 feet at the foot end. The crosswise dividers
are cement boxes 8 feet wide and 20 feet long, and in addition to
dividing the stream they serve as mixing chambers for the methoxychlor,
and collection boxes for fish when draining the streams.
An electric butterfly valve controls the flow of water from a small
impoundment to the streams, and excess flow can be diverted into a
bypass creek.
The streams will be operated with a total of 1 cfs of water (0. 5 cfs for
each streanst segment). The design of the streams dictates that the flow
be continuous through the length of the experimental area rather than
each stream having an individual water supply. This fact dictates
the experimental design of the study. The two upstream sections will
be control streams, the two middle sections low-level treatment, and
the two downstream sections high-level treatment. Charcoal will be
used to filter out the methoxychlor below the experimental sections.
An underestimate of the cost of construction of the streams resulted
in extensive delays in completion. The streams were completed in
the fall of 1972, and will be used for the first experiments during the
summer of 1973. Figure 13 shows photographs of the streams during
construction and in testing operation during the past winter.
53
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Figure 13
Stages in construction of the experimental streams at Saline:
Upper left--stream bed and footings for dividing wall and mixing boxes.
Upper right--cement forms for water supply box at head of streams.
Lower right--completed water supply box at head of streams.
Lower left--completed facility, from downstream end, during first
winter of operation.
54
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SECTION VII
ACKNO WLEDGMEN TS
The author gratefully acknowledges the following invaluable contributions
to the project.
Dr. W. Brungs, National Water Quality Laboratory, Duluth, Minnesota,
served as project officer and as such gave generously his time and
talents.
Dr. M. E. Bender, Virginia Institute of Marine Science, Gloucester
Point, Virginia, assisted in planning the project and supervision during
early stages of the study.
Dr. R. Hartung, University of Michigan, School of Public Health, Ann
Arbor, Michigan, and Dr. W. C. Latta, Institute for Fisheries
Research, Michigan Department of Natural Resources, Ann Arbor,
Michigan, served as project directors and advisors throughout the
study.
Mr. R. C. Barber supervised construction of the experimental streams.
The contribution of his entire crew is gratefully acknowledged.
Mr. J. R. Novy assisted with the bioassays and construction of
equipment.
Mr. Merna's time on this study was supported in part by Dingell-
Johnson Federal Aid under Project F-28-R Michigan.
55
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SECTION VIII
REFERENCES
1. Brett, J. R., "The Design of a New Fish Respirometer, "
Biological Problems in Water Pollution, Third Seminar,
U.S. Department of Health, Education and Welfare,
pp 312-314 (1962)
2. Brown, Margaret E., "Physiology of Fishes, " Academic Press Inc.,
New York, Volume 1, 447 pp (1957)
3. Burdick, G. E., E. J. Harris, H. J. Dean, T. M. Walker,
Jack Skea, and David Colby, "The Accumulation of DDT in Lake
Trout and the Effect on Reproduction, " Transactions of the
American Fisheries Society, Volume 93, pp 127-136 (1964)
4. Burdick, G. E., H. J. Dean, E. J. Harris, J. Skea, C. Frisa,
and C. Sweeney, "Methoxychlor as a Blackfly Larvicide,
Persistence of its Residues in Fish and its Effect on Stream
Arthropods, " New York Fish and Game Journal, Volume 15,
No. 2, pp 121-142 (1968)
5. Daum, R. J., "A Revision of Two Computer Programs for
Probit Analysis, " Entomology Society of America, -Bulletin 16,
No. 1, pp 10-15 (1969)
6. Grant, C. D., and A. W. A. Brown, "Development of DDT
Resistance in Certain Mayflies in New Brunswick, "
Canadian Entomologist, Volume 99, pp 1040-1050 (1967)
7. Henderson, C. , Q. H. Pickering, and C. M. Tarzwell,
"The Toxicity of Organic Phosphorus and Chlorinated Hydrocarbon
Insecticides to Fish, " Biological Problems in Water Pollution,
Technical Report No. W60-3, pp 1-76, U.S. Department of Health,
Education and Welfare (1959)
8. Hynes, H. B. N., and M. Coleman, "A Simple Method of Assessing
the Annual Production of Stream Benthos, " Limnology and
Oceanography, Volume 13, pp 569-573 (1968)
9. Jensen, L. D. , "Acute and Long Term Effects of Organic Insecticides
on Two Species of Stonefly Naiads, " PhD thesis, University of Utah,
101 pp (1965)
57
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10. Kennedy, Harry D., L. L. Eller, and D. F. Walsh, "Chronic
Effects of Methoxychlor on Bluegills and Aquatic Invertebrates, "
U.S. Department of the Interior, Bureau of Sport Fisheries and
Wildlife, Technical Paper No. 53, pp 1-18 (1970)
11. Kruzynski, George M., and G. Leduc, "Methoxychlor, a New
Threat to the Atlantic Salmon, " Atlantic Salmon Journal No. 1,
pp 1-5 (1972)
12. Martin, J. W., "A Method of Measuring Length of Juvenile Salmon
From Photographs, " Progressive Fish-Culturist, Volume 29,
pp 238-240 (1967)
13. Mendel, J. L., A. K. Klein, J. T. Chen, and M. S. Walton,
"Metabolism of DDT and Some Other Chlorinated Organic
Compounds by Aerobacter aerogenes, " Journal Association of
Official Analytical Chemists, Volume 50, pp 897-903 (1967)
14. Metcalf, Robert L., G. K. Sangha, and I. P. Kapoor, "Model
Ecosystem for the Evaluation of Pesticide Biodegradability and
Ecological Magnification, " Environmental Science and Technology,
Volume 5, No. 8, pp 709-713 (1971)
15. Mount, D. I., and W. A. Brungs, "A Simplified Dosing Apparatus
for Fish Toxicology Studies, " Water Research, Volume 1,
pp 21-29 (1967)
16. Muhlmann, V. R., and G. Schrader, "Hydrolyse der Ivsektiziden
Phosphorsaureester, " Zeitschrift Fuer Naturforschung, Volume 12,
pp 196-208 (1957)
17. Mulla, M. S., and A. M. Khasawinah, "Laboratory and Field
Evaluation of Larvicides Against Chironomid Midges, "
Journal of Economic Entomology, Volume 62, pp 37-41 (1969)
18. Peakall, D. B. , "Pesticides and the Reproduction of Birds, 11
Scientific American, No. 222, pp 73-78 (1970)
19. Rheinbold, Keturah A., I. P. Kapoor, W. F. Childers, W. N. Bruce,
and R. L. Metcalf, "Comparative Uptake and Biodegradability of DDT
and Methoxychlor by Aquatic Organisms, " Illinois Natural History
Survey Bulletin, Volume 30, No. 6, pp 405-417 (1971)
20. Reinert, Robert E., "insecticides and the Great Lakes, "
LIMNOS, Volume 2, No. 3, pp 3-9 (1969)
58
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21. Wallner, W. E. , N. C. Leeling, and M. J. Zabik, "The Fate of
Methoxychlor Applied by Helicopter for Smaller European Elm
Bark Beetle Control, " Journal of Economic Entomology,
Volume 62, No. 5, pp 1039-1042 (1969)
22. Wurster, Charles F. , Jr., HDDT Reduces Photosynthesis by
Marine Phytoplankton, 1( Science, Volume 159, No. 3822,
pp 1474-1475 (1968)
•( S ("..OWKNMKNT "HINTING OF FK 1 1973 — ^6-312/136
59
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Jf. Report $g.
w
The Effects of Methoxychlor on Aquatic Biota
s,
6.
8; fe.fs
James W. Merna
and
Paul J. Eisele
Inst. Fisheries Research
Mich. Dep. Nat. Resources
Museums Annex, Ann Arbor, Mi. 48104
University or Michigan
Sch. Public Health
Ann Arbor, Michigan
48104
18050-DLO
Type .. I Repcii and
Period Covered
Environmental Protection Agency report number,
EPA-R3-73-046, September 1973.
Continuous -flow bioassays yielded 96-hour TL^o values for invertebrates
ranging from 0.61 jug/1 for Gammarus pseudolimnaeus to 7. 05 Mg/1 for
Orconectes virilis . Fathead minnows (Pimephales promelas) and yellow
perch (Perca flavescens) had 96-hour TLso values of 8. 63 and 22. 2
respectively. Hatching of fathead minnow eggs was inhibited at all levels
of exposure tested between 1. 0 and 0. 125 jug/1. There was no spawning
at 2 jug/1. Growth of yellow perch was retarded at all levels tested
between 5. 0 and 0. 625 /ug/1. All perch died at 10 /ug/1 during the growth
study. Perch which had been subjected to long-term exposure to 5 ug/1
of methoxychlor had an abnormally high oxygen demand when held in a
respirometer with a water velocity of 0.6 foot per second.
17a. Descriptors
Pesticide toxicity, pesticides, bioassay
fib. Identifiers
Pesticide toxicity, lethal limits, phytotoxicity, fish toxicity, fish reproductioi
, J*c arfflr Cl-ss,
it.
Pages
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON. D C 2O24O
James W. Merna
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