ACUTE AMD CHRONIC PARATMION TOXICITY
TO FISU AND INVERTEBRATES
By
Anne Spacie
Contract No. 68-01-0155
Project 18050 EQG
Program Element 1B1021
Project Officer
John G. Eaton
National Water Quality Laboratory
Environmental Protection Agency
6201 Congdon 3oulevard
Duluth, Minnesota 55804
Prepared for
OFFICE OF RESEARCH AN J) MCKI TORINO
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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ABSTRACT
Acute and chronic toxicities of parathion (0, 0-diethyl 0, p-r.itrophenyl
phosphorothionate) were found for bluegill, Lcpcmis macroc'nirus
(Rafmesque). brook trout, Salvelinus for.tir.ales (Mitchell), fathead minnow,
Pimephales r:roinelas O^afinesaue). water flea, Danhnia magna Strauss,
scud, Gam mar us fasciatus Say, ar.d midge, Chironomus tcr.tyis
Fabricius. The 96-hour LC50 values were: bluegill - 0.51 ir.g/1, trout -
1.76 mo/l, minnow 1.6 to C . 5 rr.g /l, P. magna - 0 .63 g/l, G. fasciatus -
0.4 /ig/l, and C. ter.tans - 31 jug/l. Deformities and depressed acetyl-
cholinesterase were found in bluegills above 0.17 pg/l after 23 months
exposure. Trout showed no ill effects at 7. 2 pg/l but trout eggs had reduced
hatchabihty at 3 2 ug/1. Minnows had reduced egg production ar.d deform-
ities at about 4 jig/1 in 8-1/2 months. Bluegills had parathion residues of 5
to 25 times the chronic water concentrations, trout had several hundred
tir.es, and minnows had residue levels intermediate to the two other species.
Chronically exposed fish appeared to be rrost sensitive to parathion at the
juvenile cr adult stages. The chronic no-ill-effect level for D. magna "was
0.08 ^g/1, for G. fasciatus less than 0.04 ug/1, and for C. tentans, less
than 3.1 jig/1.
iii
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV MATERIALS AND METHODS 7
Water Supply and Environmental Control 7
Fish Bioassays 12
Invertebrate Bioassays 17
Chemical Analysis 20
V RESULTS 25
Bluegill 25
Brook Trout 40
Fathead Minnow 57
Daphnia magna. 62
Gamrr'.arus fasciatus 64
Chironomus ten:ans 66
VI DISCUSSION 69
VII REFERENCES 73
VIII APPENDIXES 77
A. Bioassay Data 77
B. 3rown Trout Tests 101
C. Chlorinated Hydrocarbon 105
Pesticides in Fish Food
iv
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list or Figures
Figure Page
1. Concentration factors for parathion residues in 28
whole bluegills at various exposure times.
?.. Male bluegill exposed to 1-5 fj.g/1 parathion 31
for ?3 months.
3. Two adult bluegills exposed to 1.5 jig/1 33
parathion for 23 months.
4. Pararhion in brook trout blood vs. parathion 46
in water. Six month exposure.
5. ' Parathion in brook trout muscle vs. parathion 48
in water. Six month exposure.
6. Brook trou: brain acetylcholinesterase 50
activity vs. parathion in water.
Six month exposure.
7. Parathion in brook trout blood vs. 55
parathion in water. Nine month exposure.
Bl. Parathion uptake and washout in brown trout 104
muscle, 12°"C.
v
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LIST or TA3LES
Table Page
1 Analys-is of test water 8
2 Summary of chronic test conditions 10
3 Chronic test photoperiod and schedule
4 Bluegill acute test 26
5 Parathion residues in whole bluegills - 27
acute test
6 Incidence of deformities - bluegill chronic 30
test
7 Bluegill chronic exposure to parathion - 34
spawning records
8 Survival and lengths of bluegill larvae 37
9 Parathion residues in bluegills - 38
18 month exposure
10 Parathion in blueciils - ?.3 months exposure 39
11 Brook trout acute LC50 41
12 Farathion residues in brook trout chronic test 41
13 Parathion in brook trout blood - six morr.h 45
exposure
14 Parathion residues in brook trout tissue - 47
six month exposure
15 Brain acetylcholinesterase - brook trout - 49
six month exposure
16 Brook trout spawning record 51
17 Brook trout parathion residues and AChE - 52
nine months
18 Brook trout parathion residues and AChE - 56
recovery
19 Fathead minnow acute test 58
?0 Minnow adult weights and deformities - 59
8 . 5 months •
?A Minnow spawning record 60
22 Parathion residues in whole fathead minnows 61
23 Parathion :oxici:y to Daphnia magna 63
24 Parathion toxicity to Gammarus fasciatus 65
25 Parathion toxicity to_C_. •. en tans 67
vi
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LIST OF TABLES (CONT.)
Table . Pa ere
A1 Mean monthly analysis of parathion - 78
bluegill chronic test
A2 Routine water quality - bluegill chronic 80
test
A3 Mean monthly temperatures - bluegill 80
chronic test
A4 Bluegill survival - chronic test 81
A5 Brain acetylcholinesterase - bluegill 82
18 month exposure
A6 Brain acetylcholinesterase - bluegill 83
?3 mor.th exposure
A7 Brook trout acute test 84
A8 Routine water quality - brook trout chronic test 85
A9 Mean monthly temperatures - brook trout 85
chronic test
A10 Parathior. in water - brook trout chronic test 86
All Brook trout survival in chronic exposure 87
A12 Brook trout weights - chronic exposure 88
A13 Brook trout gonadadosorr.atic index - 89
six month exposure
A14 Brook trout gonadcsomatic index - 90
nine month exposure
A15 Summary of Residue results for blood and muscle cf 91
trout exposed to parathion for 6 and 9 months
A16 Routine water quality - fathead minnow 92
chronic test
A17 Mean monthly water temperature - 92
fathead minnow chronic test
A18 Mean monthly analysis of parathion - 93 .
fathead minnow chronic 'est
A19 Growth and survival of fathead minnows 94
in parathion
A20 IX. magna acute test 95
A2l HK magna chronic test 96
A?2 Gamrr.nrus acute test 97
A23 Gammarus sub-chronic test 98
A24 C_. ter.tans acute tes: 99
A?5 tontans sub-chronic test 100
B1 Brown trou: acute test 102
B2 Parathior: residues in brown trout muscle 103
CI Pesticide residues in fish food 106
vii
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ACKNOWLE DG EM ENT3
The author would like to thank the staff of Aquatic Environmental
Sciences, especially G. A. Cary, G. R. I wen, W. J. Kuc, R. H,
Sugatt, and A. G.. Vilkas for long hours of devoted work. Dr. G. F.
Doebbler of AES and E. Fritche, E. Yeh, and 3. Shields of the Central
Scientific Laboratory, Union Carbide Corporation, Tarrytown. New York,
performed the parathion analyses on water and tissue samples.
John G. Eaton and other members of the National Water Quality Laboratory,
Duluth, Minnesota, developed the chronic bioassay procedures used
throughout this project ana provided continuing advice and assistance.
viii
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SECTION I
CONCLUSIONS
1. Juvenile bluegills were found to have a 96-hour median lethal
parathion concentration (LC50) of 0.51 mg/l and a maximum
acceptable toxicant concentration (MATC) of 0.17 to 0.34^g/l
based on adult deformities.
2. Brook trout had a 96-hour LC50 of 1.76 mg./l. Chronic levels of
7.2 jig/l produced no harmful effects. However, levels of 32/u g/l
reduced halchability of embryos from untreated parents.
3. Fathead minnows had 96-hour LC50 values between 1.6 and 0.5 mg/l
and a chronic MATC of about 4 iu g/l based on egg production and
adult deformities.
4. Daphnia magna had a 96-hour LC50 o; 0.63 u g/l and MATC of
about 0. 08 /i g/l.
5. Ganmarus fasciatus had a 96-hour LC50 of about 0.4 u g/l ana a
MATC of much less than 0.04 iu g/l, which caused mortalities in one
month.
6. Chironomus tentans had a 96-hour LC50 of 3'1 /i g/l based on
parathion concentration in water, and a MATC of less than 3.1
li g/l, a lethal level in two weeks.
7. Bluegill tissue concentrated chronic parathion residues at 5 to
25 times the water concentration, brook trout several hundred times,
and minnows at rates intermediate to the-other two species. No
parathion above background levels was detected in D. magna.
8. Chronically exposed fish appeared to be most sensitive to parathion
during juvenile or adult stages. The egg and fry stages were not
particularly sensitive.
- 1 -
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Intentionally Blank Page
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SECTION II
RECOMMENDATIONS
1. Parathion applications to water bodies should be limited to levels
acutely tolerated by crustaceans such as Daobnia.
2. Trout and other species collected from para.hion-treated sites
should'be examined further for potentially harmful residue levels.
3. The selectivity, toxicity, and persistence of alternative insecticides
should be compared to parathion for possible substitution.
4. Food chain dynamics of parathion distribution in aquatic communities
should be examined.
5. Metabolic pathway studies of parathion degradation in fish should
relate effects of temperature, rates of reaction, and proportion of
major metabolites.
6. Further histological characterization of parathion damaged tissues
in fish would help to determine the mechanisms of such damage.
7. Further study is needed to insure bluegill spawning during chronic
tests.
8. Five month chronic tests on Gamrr.arus are not practical unless better
survival rates are achieved in culture.
S. More efficient techniques are needed for performing the egg
incubation and larval survival phases of chronic bioassays.
-3-
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Intentionally Blank Page
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section rri
INTRODUCTION
Among organophosphorous insecticides, parathion is one of the most
persistent and toxic.. About 15 million pounds of it are used annually
for control of insect pests on fruit trees, cotton, tobacco, and other
crops.1 A small portion reaches aquatic environments by direct
application (such as for mosquito control), land, runoff, leachinc, erosion,
irrigation, aerial spraying, and spills during manufacturing, formulation,
and application. Once in a water-soil system, its rate of degradation
is variable, depending on pH, organic content, and microbial activity.
Upon application to soil, parathion may persist for 3 months, ' 5
years, 3 or in an extreme case, 16 years.4 In distilled water of pH 7.4
the hydrolysis reaction has a half-life of about ICS days, decreasing
with greater pH and temperature.'' In contrast, natural river water
degrades 50°-- of parathion within a week.* In natural lake sediments
Graetz et_al7 found a half-life of 178 to 68 days.
Damage to aquatic fauna has been reported for parathion residues in water
and sediments. After spraying an orchard. Nicholson8 found reduced
populations of aquatic insects in nearby ponds containing 1,9 ppm parathion
in mud. Killifish and freshwater mussels exposed in a treated cranberry
bog showed uptake of parathion residues." Parathicn spills have also
caused kills of fish and amphibians.^
Like all organop'nosphates, parathion disrupts the central and peripheral
nervous systems of animals when enzymatically converted to its oxygen
analog, paraoxon. Paraoxon then combines covaler.tly with acetylcholi-
nesterase (AChE) to block the hydrolysis of acetycholine, the transmitter
substance in the synapse of motor and parasympathetic nerves. A full
account of this inhibition is given by O'Brien.11 The AChE-phosphate
complex is, at first, hydrolyzable. Animals may recover from acute doses
of the inhibitor. However, the AChE-complex can gradually dealkylate,
forming the phosphate anion which resists hydrolysis. Therefore,
regeneration of AChE after long-term exposures of parathion is extremely
slow.
Weiss demonstrated AChE activity in fish, lr and its susceptability to
paraoxon inhibition.13 Bluegills exposed to 0.1 mg/l parathion for 8
hours recovered only 50*>oof normal activity in 30 days. i-'athec>d minnows
had a somewhat quicker response and recovery. Carter1'"' found that
channel catfish with 50p,'> inhibition from met hylparathion recruired 20 to
-5-
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30 clays to regain SC^of normal activity. Studies of AChF. inhibition in
vitro have found fish'5' 16' 17 and lobsters18 to be less sensitive than
birds or mammals. Yet parathion is very toxic to both fish and crustaceans.
The two principal metabolic products in rats^ - paraoxon and diethyl
hydroqen phosphorothionate have also beer, found in rainbow trout. lc
The proportion of ".he two metabolites formed, and their rates of formation
vary with species. Metabolism can be slow enough to permit accumulation
of the parathion in tissues, as reported for trout. 16 brown bullheads,
mosquitofish, ,M fathead minnows, 22 and oysters.23
Literature values for the acute toxicity of parathion to aquatic species
have been summarized by Kempt, Abrams, and Overbeck.24 Only a few
? n
studies have considered long-term effects. Mount and Boyle found
convulsions in bullheads exposed to 0.03 mg/1 parathion for one month.
Matsue25 observed retarded growth of goldfish at 0.1 mg./l for one month.
Reduced growth rates of oyster larvae in 0.05 mg/1 were found by Davis
and Hide.26 After 40 days at 0.01 mg/l, male guppies tested by Billard27
showed reduced spermatogenesis. Jensen and Gaufin28 reported a
30-day LC50 of 2 . Z fjg/l for Pteronarcys califorr.ica and 0. 44 fj.g/1 for
Acroneuria pacifica.
Such work demonstrates that parathion is highly toxic to aquatic species,
may accumulate in tissues, and .may persist long enough to cause chronic
damage. To find ecologically "safe" levels allowable in aquatic systems,
the long-term effects of parathion must be studied further. The objective
of the present study was to find the greatest chronic levels of parathion
that produce no harmful effects to brook trout, bluecills, fathead
minnows, Daphnia magna. Gair.marus fasciatua and Chrionomus tentans.
For each species, the maximum acceptable toxicant concentration
(MATC), defined by Mount and Stephan, ' ^ was then related to the acute
LC50. Because standardized bioassay techniques were used, the results
of these tests may be correlated with those for other species and compounds.
The combined information will facilitate the prediction of chronic no-effect
levels in freshwater.
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SECTION IV
MATERIALS AND METHODS
WATER SUPPLY AND ENVIRONMENTAL CONTROL
Water Supply
Water for acute and chronic testing came directly from a 50 ft well
located on the Tarrytown, N. Y. laboratory site. An analysis of the
water composition is given in Table 1. Analytical methods for these
results followed Standard Methods. ^ Prom .the well, water was pumped
to the laboratory through black iron and PVC pipe. At the laboratory the
water was sterilized with ultraviolet light, and proportioned into lines
of ambient (11-1 3CC) ana heated water for distribution to testing stations.
When heated, the hard water precipitated carbonate solids in the piping
and steam heat exchanger. Therefore, part way through the program the
heated water was softened by exchange cf sodium for calcium. The
hardness dropped about 20% in the bluegill chronic exposure, the only
test underway at the time.
This system did not provide supplemental chilling. 1't was not installed
because ambient water temperature the previous winter was about 9°C,
the optimum for brock trout egg incubation. However, during the actual
brook trout test, the water temperature was as high as i3°C in winter.
Temperature Control
Lines of both hot and cold water fed each test. Proportional mixing of the
two flows was regulated by pneumatic temperature controller with sensing
element in the mixing zone of the pipe. The resulting temperature
variation was within ±1°C for instantaneous changes, and±?.°C for
daily trends. The actual temperature in at least one chamber per test
was continuously monitored by thermistor probe read by a 20-channel
electronic temperature monitor. Each channel had individual high and
low set points which activated an alarm if exceeded* Once per hour all
channels were recorded on punch paper tape and teletype.
Aeration
C
The well water coming directly from the piping was sometimes over or
under-saturated with gases. For this reason water was aerated by air-
stone as it entered the first proportional diluter cell. The adult fish
chambers, particularly trout, had supplemental aeration to insure dissolved
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Table 1 . ANALYSIS OF TEST WATER
(mg/'l)
Component Concentration I Component Concentration
Alkalinity (as CaC03)
147
1 Lead
t
<0. 000?
Aluminum
0.01
j Magnesium
27
Ammonia (as N)
0.15
' Manganese
0.005
Arsenic
<0.017
Mercury
< .0002
Barium
0.10
Nitrate (as N)
1. 7
Boron
0.02
¦ Nitrite (as N)
0. 007
Cadmium
0.0005
Phenol
<0. 0016
Calcium
76
i Phosphorus
0.005
Chloride
130
| Potassium
0.6
Chlorine
<0.001
; Silicon
6
Chromium
<0.006
Sodium
3.9
Copper
0.03
; Sulfate
39
Cyanide
<0.0001
! Strontium
0.6
Fluoride
0.02
1 Tin
0.01
Hardness (as CaC03)
297
i
i Titanium
0.01
Iron
•
<0.006
i
| Zinc
< .001
Taken 3/10/72 and 10/19/72
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oxygen levels greater than 60% saturation.
Toxicant Delivery
Reagent grade parathion (0, 0-diethyl 0-p-nitrophenyl phosphorothionate)
of 99% purity (Pfrjltz and Eauer) was used throughout. A stock solution
of parathion in water or acetone was injected into a proportional diluter
of the Mount and Brungs design.51 Depending on the test, several injection
devices were used: Sage 341 syringe pump, Mariotte bottle with constant
flow, mechanical syringe pump,31 or LKB Perplex peristaltic purr.p. The
latter two devices were most reliable and precise over long periods of
time. The peristaltic pump had the advantage of an unlimited stock
solution reservoir, and was equal in precision to the mechanical syringe
as long as the cycle time of the diluter remained constant. To prevent
accidental injections during low-flow conditions, the electrical pumps
were wired to stop if the diluter did not cycle.
All parts of the diluter in contact with test water were of glass, latex
or Tygon tubing, and silicone glue. The diluter produced five
concentrations of parathion, plus a control, each split into duplicate
flows by a mixing cell. Individual delivery tubes went to each duplicate
tank. Tanks were designated by the same number as the corresponding
diluter cell:31 "0" for the control, "1" for the highest toxicant concen-
tration used, "2" tor the second highest, up to "5" for the lowest.
Duplicate tanks were designated "A" or "B".
The tank sizes and flow rates "for each chronic test are summarized in
Table 2. Bluegill adult tanks were made of No. 306 stainless steel. Trout,
minnow and Gamrr.arus adult tanks were commercial aquaria of glass with
slate bottoms and outside stainless reinforcement. All other containers
were glass with silicone glue (Dow Corning) construction.
Lighting
All tests were illuminated by Duro-Test Optima fluorescent lamps.
These were set with a Tork timer to give the Evansville, Indiana,
photo-period shown in Table 3. The timer wiring was modified to give 30
minutes of gradual change at dawn and dusk.1' Daphr.ia and midges were
held on a constant 16-hour light cycle. Average light intensifies at the
test sites were: bluegill, 75 ft-c; brook trout 70 ft-c; minnow 82 ft-c;
and invertebrates 70 - 80 ft-c. The test photoperiods for bluegills and
trout corresponded to the actual calendar. The minnow and invertebrate
tests were begun on the dates shown in Table 3, but these did not
correspond to the calendar.
-9-
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Table 2. SUMMARY OF CHRONIC TEST CONDITIONS
Test
animal
Adult tank
volume, 1
Minimum flow
per tank, l/hr
Larval chamber
volume, 1
Temperature,
°C
Bluegill
Brook trout
Fathead minnow
Daphnia
Gammarus
Chironomus
170
85
28
3
15
3
40
30
10
0.8
0.5
0.8
25
15-9
28
18
18
25
a Two such chambers were used per adult tank.
Minnow chambers were not true duplicates. See page 9.
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Table. 3. CKRCXIC TEST PHOTGPERIOD AMD SCHEDULE
Period cf 1 iylit includes 30 min
of dawn and 30 min of dusk
Date
Light
period, hr:m:n
Event
Jan.
1
10:30
15
10:45
Feb.
1
11:15
-Begin Gammarus
15
11:45
Mar.
1
12:15
-Begin brook trout test
15
13:00 -
Apr.
1
13:30
15
14:15
—
Gammarus reproduction
May
1
14:45
—
15
15:15 —J
Fathead minnow spawning
June
I
15:30
_ |
15
15:45
July
1
. 15:45
a
Bluegill spawning
15
15:30
Aug.
1
15:00
15
14:30
Sept.
1
14:00
15
13:30
Oct.
1
12:45 .
-Begin bluegill test
15
12:15
Nov.
1
11:^0
-Brook trout spawning
15
11:00
ST
Dec.
1
10:45
-Begin fathead test
15
10:30
-11-
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riSH BIOASSAYS
Blueqill
One stock of immature bluegills 5 to 8 cm long was collected from a
portd in Duchess Co., N.Y., and acclimated in the laboratory for three
months prior to the chronic testing. .Other immature fish, taken from
the same pond one year later, were used for the acute studies.
Fish of both stocks had few internal parasites and a small number of
gill parasites which were eradicated with formalin.
For the acute tests, an aqueous emulsion of 180 c parathion and 9 g
Triton X-100 (Rohm and Haas) per li:er of water was sonicated, and then
injected into a proportional diluter by peristaltic pump. Each of the 8 5
liter test chambers received a flow of 30 1/hr. Acclimated test fish
were randomly distributed at the start. Mortalities were counted and
removed at regular intervals throughout the 96-hour tests. Nc aeration
or feeding took place. Direct analysis of parathion in water was made
at least once during each test.
The chronic exposure followed the "Recommended 3ioassay Procedure for
Bluegill Lepomis macrochirus (Rafinesque) Partial Chronic Tests" of the
National "Water Quality Laboratory, Duluth, Minn, (revised January 1972).
The tanks had a flow of 40 l/hr and supplemental aeration. A stock solution
of Z to 4 g parathion, and 0.Z4 g Triton X-100 per liter acetone was injected
by syringe pump. The nominal concentrations chosen for the test were
originally 8 . 0, 4.0, Z.0, 1.0, and 0.5 u g/1.
On Dec. ZZ, 1971, Z0 fish were randomly distributed to each tank.
During the first two months of exposure, the fish at 8. 0 y. g/1 showed
tremors and convulsions. Therefore after 64 days, that concentration
was removed while a 0. Z5 fjg/l level with new fish was added. Fish
were fed Oregon Moist Pellets and Silver Cup trout food ad libitum.
Lengths and weights were recorded at the start, at thinning, and at
termination of the adults.
Spawning did not occur the first summer, although the fish appeared
mature. In the second summer season the males developed territorial
behavior. Then each tank was thinned to 3 males and 7 females. All
discarded fish were frozen at -18°C for lator chemical analysis. A
cement substrate, Z7 cm square by 5 cm thick with a bowl-like depression,
was placed at each end of each tank. To reduce aggressive male behavior,
the tanks were divided into thirds by two black plastic curtains (53 cm
wide by 40 cm long). These did not restrict mobility but reduced
disturbance to the nests.
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During spawning, the nes's were checked for eggs daily. Spawned
eggs were removed from the cement by gentle brushing, and extra eggs
were siphoned from the tanks. Counting was done volumetrically. by
counting the eggs in one milliliter, then determining the total volume
of eggs. Two hundred eggs were incubated in an egg cup (4 oz. glass
jar with 40 mesh nylon screen across the open bo:tom) suspended from
a rocker arm in the larval chamber. The larval chamber received water
from the corresponding parental tank. After three days the number of
hatched larvae was counted, and 50 fry were retained in the cup. Feed-
ing with large amounts of newly hatched Arterr.ia salina (San Francisco
Bay Brand) 4 times daily began on the 4th day after the spawn. At 7 days
after spawn, larvae we re released to a 18 x 38 x 13 cm wide larval
chamber with a water depth of 13 cm. Mortalities were subsequently
checked weekly. Thirty, 60 and 90 days after spawning, the surviving
larvae were counted and measured photographically in an all-glass box
set over a lightbox with millimeter transparent grid.33
All remaining adults were removed after 23 months exposure, examined,
and frozen for residue analysis.
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Brook Trout
Yearling brook trout, Saivelinus fontineles {Mitchell} from a hatchery
'in Sullivan Co., N.Y., approximately 60 g each, were acclimated a:
12°C for three weeks prior to testing. Until the start of the chronic
and acute tests, they showed no symptoms of disease and were not
chemically treated. During the chronic test, fungus and furunculosis
were treated with 20 ppm formalin, 4 ppm Furpyrinol, or 40 ppm malachite
green.
The acute test method followed that for bluegills. An aqueous emulsion
of 0.05 g Triton X-10Q per gram of parathion was injected Into the diluter
by peristaltic pump. Water for parathion analysis was sampled at the
start, and at 2, 4, and 5 days. Selected fish were frozen at -18°C for
later residue analysis.
The chronic brook trout exposure followed the procedure given by the
National Water Quality Laboratory.34 On May 1. 1972, 14 trout were
randomly distributed to each of 12 tanks.- The diluter and flow were
identical to those in the acute test. A stock solution of 8.0 g parathion
and 0.24 g Triton X-100 per liter acetone was injected into the diluter by
syringe pump. The maximum theoretical acetone concentration was 1.5 mg-/l
in the highest concentration used.
Trout were fed Oregon Moist Pellets (Moore-Clark Co.) according to
the manufacturer's recommended ration. Weights and total length were taken
at the start, at 11? days, at 178 days (discards only), and at the end, 282
days. At 178 days, the number in each tank was reduced to ? males and 4
females. Secondary sexual characteristics made it possible to distinguish
the sexes of most but not all of the fish. Discards were dissected and
the tissues frozen for residue analyses.
On November 22, at 206 days, two polyethylene basins, 25 x 30 x 15 cm deep
were placed in each tank as spawning substrates. These had nylon screening
at each end to permit water circulation, and .5 cm of coarse gravel in the .
bottom. These substrates were difficult to handle and clean. Two weeks
later the gravel was replaced by a horizontal layer of stainless steel
screening about 5 cm off the bottom. Rocks were glued to the screen.
These artificial substrates were far easier to handle and eggs easier to
locate. Work by Benoit55 has since showed that "his type of substrate
will jeopardize the fertilization of the eggs unless a baffle is added to the
bottom to prevent eggs from rolling away from the spawning site.
Eggs were removed from the substrates each afternoon, and counted
manually. Fifty eggs from each spawn were hatched in egg cups
suspended in shaded larval chambers. The chambers received ?. 6 1/hr
directly from the mixing cells which supplied the parental tanks. .Other
eggs from each spawning were incubated for 12 days and examined for
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viability. Under low magnification the embryonic development could
be seen when the eggs were preserved in an aqueous solution of 5°k<
formalin, 4'f> glacial acetic acid, and 6% glycerin (V/V). Viability was
calculated as the percent of embryos with a neural keel.. During incubation,
the eggs were checked daily for mortalities and fungus. Insufficient larvae
were hatched to carry out the larval exposure planned for this part of the test.
On Feb. 6, after 2R2 days, the remaining adult trout were removed. All
but two from each tank were killed immediately and frozen at -4° to -1S°C.
The others were held in flow-through tanks of clean water for 2 to 7 days.
Parathion residues were measured to show whether chronic parathion uptake
was reversible.
To supplement the hatchability data from the chronic test, additional
brook trout embryos from the Nashua National Fish Hatchery, Nashua, N.H.
were exposed to parathion levels of 10, 3 2, and 100 ^ig/l with a control,
at 10°C. Toxicant solutions of these concentrations were delivered at
rates of 300 ml/hr to each of four larval chambers by constant drip Mariotte
bottles. Each chamber incubated two egg cups containing 100 19-day-old
embryos each. These were held until hatch. Every other day, the embryos
were treated with a 4 mg/1 solution of malachite green for one hour to prevent
fungus.
-15-
-------
Fathead Minnow
A slock of fathead minnows, Pimphales promelas (Rafinesque), originally
from the Newtown l'ish Toxicology Station,, Cincinnati, Ohio, v/ere cultured
for two years in :he laboratory and in an outside pond. All test fish were
taken from this stock. Acute tests v/ere performed as for biuegills. An
aqueous emulsion of 29.0 g parathion and 1.43 g Triton X-100 per liter water
was made by sonication and injected by peristaltic pump. The flow rate
to each 10 liter test tank was about Is l./hr in all acute tests.
The chronic exposure followed the procedure of the National "Water Quality
Laboratory.34 A total of 35 rr.innow larvae, 5 to 14 days old, were distributed
randomly to each tank on Aprii 1 (Dec. 1 test date). ror the first month they
were fed powdered fish food, mixed with green algae, and Artemia nauplii.
From then on, they were fed trout starter mash_ad libitum.
Nominal parathion concentrations of 30, 25, 12, 6, and 3 jug/1 were
produced from a stock solution cf 1.7 parathion and 0.1 g Triton X-100
per liter acetone.
At 30 and 60 days the minnow larvae were counted and photographically
measured.33 At 70 days each tank was thinned to 15 fish and three
artifical substrates v/ere added. These were made from 3-inch asbestos
and cement drain tiles cut in half longitudinally.
Spawning began on Oct. 22 (June 22, test date) and continued through
Dec. 19 (Aug. 19, test date) when the adults v/ere terminated. During
spawning, eggs were removed and counted daily. Fifty eggs from each
spawning were incubated irt an egg cup suspended in a larval chamber
receiving v/ater from the parent tank. Each larval chamber was 900 cm' by
15 cm deep divided ir.to two sections. These two halves v/ere not true
duplicates, since they received the same water, but they did separate
larvae spawned at different times. All egg cups were treated once with a
solution of 4 mg/'l malachite green.to prevent fungal problems. Larvae
were counted after hatching was completed, usually in 5 days- Of those
hatched, 40 we re released into the chamber for 30-day survival tests.
Because a large excess of eggs was produced in the controls, some of
these were also hatched at high parathion concentrations. At 30 days all
larvae were counted and measured photographically. 33 The adult
minnows were killed after 8-1/2 months of exposure, examined for
deformities, measured, and frozen at -18°C for residue analysis.
-16-
-------
INVERTEBRATE BIOASSAYS
Daphnia magna
A stock of Daphnia rnagna_ Strauss from the National Water Quality
Laboratory, Duluth, Minn., was continuously cultured throughout the
project. Groups of daphnids were held in 3 liter jars which had be-
come coated with diatoms and other aigae. No flow was reauired to
maintain the cultures. A temperature of 18 to 21°C was suitable for
reproduction. A food supply of 1.5 g C-erophyl, 2.0 g trout mash, and 1.0 g
yeast in 500 ml well water was given periodically. When culture jars
became too filled with detritus, about once a month, the daphnids were
transferred to clean jars.
Before the acute or chronic test, several dozen large adults were selected
and held in clean 250 ml beakers overnight. Within ?4 hours enough
first instar daphnids were usually produced to begin a test. These were
counted and randomly distributed to the test containers. In an acute test,
the number of surviving Da phnia were counted daily, and in a chronic,
weekly. Young produced during the longer tests were counted and
removed weekly. Size differences made it possible to distinquish the
original animals from their offspring. Da phnia in all tests were fed 5 ml
of the food mixture per tank daily.
Test jars had a U-shaped notch cut in the upper lip, covered with No. 405
Nytex screening to maintain a constant water level without losing test
animals. Jars were held in an 18CC water bath for temperature control.
Each jar received a separate stream of water (0.8 l/hr) from the toxicant
diluter.
Gammarus fasciatus
Adult and immature Gammarus were collected in coves and tributaries
of the Hudson River in an area above the salt wedge. The most successful
collection methods were wire cage traps weighted with rocks, and dip
nets. There are several very closely related Gammarus species in this
area. Subsamples of the population used for testing were identified as
G. fasciatus by Dr. E. L. Bousfield of the National Museum of Canada,
Ottawa, Ontario, and by Dr. S. Ristich of Boyce Thompson Inst., Yonkers,
N.Y. Because immature and female gammarids are extremely difficult to
identify, we cannot exclude the possibility that some other species were
included in the test population of G. fasciatus.
-17-
-------
Tests were started with young ganmarids collected from the field because
laboratory cultures did net produce sufficient young.. Gti.T.marids were raised
in the laboratory by confining two paired adults to a small, static container
until the offspring were produced. The adults were immediately removed
to avoid possible cannibalism of the young.
All tests were performed in 15-liter aquaria held at 18 to ?CTC Each tank
was supplied with several square centimeters of well-soaked maple leaves
for cover and food. During chronic tests and culturing the Gammarus were
fed small cubes of beef liver daily. This food source gave higher survival
rates than dried fish food, aquatic macrophytes, or maple leaves alone.
The proportional diluter was supplied with a 200 mg./l stock solution
of parathion in acetone. The maximum possible acetone concentration was
about 7. 5 mg/l in acute and chronic tests.
Originally a 5-month chronic exposure starting with newly hatched
gammarids was planned for this project. The chronic test was started
five times and each time encountered difficulties in survival or parathion
level.
However, two sub-chronic exposures of juvenile cammarids for 35 and 43
days duration were completed, with estimates of the LC50 levels for these
times.
-18-
-------
Chironomus tentans
A stock of Chironomus teritens Fabricius provided by Or. William Cooper
of Michigan State University was cultured according to his methods.
Several 2-liter aquaria with screen covers were filled halfway with
water. A substrate of Cerophyl, trout food, and toilet tissue was
blended together until lumpy and added in small amounts. It was
important to aerate gently -o avcide anaerobic conditions and yet leave
the substrate undisturbed. Tor culturing. a photopericd o: 16 hours of
light was used; a dawn and dusk period was not necessary. The static
culture tanks were held at 21 to 25°C. Once a day, the emerged adults
were transferred to a large screened cage which had shallow dishes of
water covering the bottom. After mating, the females deposited their
eggs in these pans where they were easily detected and removed. Egg
cases produced in this way were used to begin tests or returned to fresh
culture tanks.
Acute tests were performed with fourth instar larvae, the last stage of
development before pupation. Sub-chronic tests were started with second
instar larvae, about one week old. These were much easier to handle
and count than first mstars and did not suffer as many losses from
handling. All tests were conducted in a ?1°C water bath, in 3-liter
battery jars similar :o those used for Daphnia ¦ Two sub-chronic tests,
originally planned for one month each, were ended after two weeks cecause
of mortality at all parathion levels. The lack of response of larvae to
probing was interpreted as mortality.
-19-
-------
CHEMICAL ANALYSIS
Samples for routine wa.er quality measurements were taken by siphoning
or dipping water from the center of each tank, away from the sides, top,
or bottom. Dissolved oxygen samples were taken in BOD botlles and
measured with a Yellow Springs Instruments polarographic oxygen meter.
Hardness, alkalinity, and pH were determined by Standard Methods.
Water for parathion analysis in fish tanks was sampled in the same way.
For invertebrate tests, it was taken directly from the diluter and pooled,
to avoid trapping test animals with the water. During long term tests,
parathion analysis was made weekly for all tanks. Daily samples were
pooled weekly in the case of the minnow test. Analysis of samples stored
for one week in amber class bottles were not significantly different from
replicates analyzed immediately. Depending on parathion concentration
expected, from 90 to 900 rr.ls of water were extracted in volumetric flasks
with 5.0 ml pesticide-crade hexane by IS minutes of stirring. The hexane
layer was allowed to separate and was removed by pipette. Duplicate
alicuots were analyzed by gas chromatography. The gas chromatographic
parameters for the Hewlett-Packard 5700 or 7620 series instruments used
were:
Detector: Ni-63, Electron Capture, 265°C
Injection Port: 230UC
Column: 4 ft. x 1/4 in. ?>% OV - 17 on 80 - 100 mesh
Chromosorb W at 200°C
Carrier Gas: 10% methane in argon; flow: - 50 ml per
min through column, 50 ml per min, detector
purge
Injection Size: 10 microliters or suitable volume
Response Factors: Parathion - 45000 counts per nanogram
Paraoxon - 20000 counts per nanogram
System noise level (as parathion) based on hexane blanks:
0.001 to 0.02 ng of parathion on column
The same GC conditions were used for hexane extracts of water ar.d fish blood,
and fractions of tissue extracts after clean-up.
Fresh fish whole blood, drawn from the caudal vein, was extracted by shaking
with 5 ml of hexane per ml blood and analyzed by GC without clean-up. This
method produced some interferences, but its speed and simplicity recommended
it over a long clean-up procedure. All other biological tissues were preserved
at - 18°C until analysis. Two grams of the frozen tissue were blended with 60 g
anhydrous sodium sulfate, and extracted overnight with 50 ml pcsticide-grade
hexane. The sodium sulfate was filtered, washed, and the hexane washes
pooled (about 20 ml). The solvent was evaporated at 60 to 70°C just until
-20-
-------
dryness. The residue was then redissolved and transferred to a silicic acid
column (5 ml of 100 mesh Mallinkrodt chromatography grade) with 1 to Z ml
hexane. The column was washed with Z0 ml hexane, and then elated with
3C% v/v benzene in hexane. A Z5 ml benzene-hexane fraction was collected
beginning at the 30% benzene break through (~-4 ml) for low level parathion
residues (in the yg/kg range). A SO ml fraction was collected for higher
parathion levels. The effluent was diluted or concentrated, as required, and
analyzed by GC.
For fish tissues such as muscle, the reproducibility of analyses on replicate
aliqucts of the same fish was t5 to 7%. Recovery of spiked samples averaged
90.8 ±7.6% based on seven tests. The range of recoveries was 83.1 to 103%;
the coefficient of variation was 8.4%.
For acetylcholinesterase determinations, whole fish brains were dissected,
stored up tc Z months in liquid nitrogen (-196°C), thawed, homogenized in
phosphate buffer and assayed by the kinetic colorimetric method of Zllman
et al.28
-21-
-------
Statistical Methods
Whenever two or more partial kills occurred .in an acute bioassay, the
LC50 and 9 5% confidence limits were calculated by the grophjcal method
of Litchfield and Wilcoxon.Mortalities were plotted on a probit scale
against the log of toxicant concentration. A straight line was fitted to
the points by eye. and checked by the Chi-square test. The I.C84, LC50,
and LC16 points were rend from the line, and the total number of animals
used between the 16%; and 84%; points (N) were noted. Then the confidence
limits of LC50 were found as follows:
i
f = (f (LC'84/LCyO + LC50/l:i6) ) exp (2.77 / Ii)
~
LC5C x f - upper limit
LC50 / f - lever limit
Reproductive impairment of Daphnia (RI 50) was found in the same way,
after expressing the number of offspring produced at each concentration as
a percentage of the number produced in the control.
In the few cases when less than two partial kills occurred, LC50 values
were calculated by the moving average method, 37 which does not require
a straight line graph:
Rj_ = number of deaths at dosage i (= 1, 2P 3* • .!"-¦)
where 1 = lowest dosage
L = highest dosage
Sj_ = number of survivors at desage i
N = nuiber cf individuals per xank
? = geometric factor between desage levels (> 1)
t = Student's t for (? = 0.05) and (l\-l) (L-l). d.f.
G = ( (H(L-l)/2) - R1 - R2 - Rl_!) / (Rl - Pa)
M = log D + leg F ( ( (1,-2) / 2) + 3)
V = 1
rl - R1
(1-G)SR,S.., + R9Sq + + R- ,S, , +
£-1 -kj *L jLj JLmj i-j
LG50 - antilcg (M ± t ?v)
-------
To determine differences in mean weight, length, etc., between
experimental tanks, a one-way analysis cf variance was used, provided
the variances were homogeneous (Sartlett's Test). I: a significant F
value occurred, the differing tanks were found by Duncan s Multiple
Range Test. These tests are described in most general statistics texts.
Routine water quality data such as temperature and oxygen were computed
as monthly means for each tank of the chronic tests. All parathion
analyses, taken each week, were used to calculate a mean, range, and
standard deviation (SD) for each test tank.
-23-
-------
Intentionally Blank Page
-------
SECTION V
RESULTS
BLUEGILL
Acute Bioassay
Five preliminary and one final acute bioassays were performed at ZZ°C.
Table 4 gives the test results and calculated LC50 values for the final
test. The sequence of symptoms of acute poisoning were: lethargy,
dark, discoloration, hypersensitivity, tremors, coughing, convulsions,
extended pectoral fins and opercula, loss of equilibrium, scoliosis,
hemorrhages, tetanus, and death.
Whole fish from the acute test were analyzed for parathion residues
(Table 5). They had been exposed for various lengths of time depending
on mortality rate. Figure 1 shows that the increase in parathion
concentration in tissue was roughly linear.
-25-
-------
Table 4. BLUEGILL ACUTE TEST
Number Surviving Per Tank
Hour
OA
OB
1A
1 B
2A
2B
3A
3 B
4A
4 B
5A
5B
0
5
5
5
5
5
5
5
5
5
5
5
5
24
5
5
3
2
4
5
4
3
5
3
3
4
48
5
5
0
0
4
3
3
3
5
3
3
4
96
5
5
0
0
1
1
2
1
5
2
3
2
144
5
5
0
0
1
1
2
0
5
2
3
2
Analyzed Parathion Concentration, mg/l
0
nd
nd
2. 10
2. 19
1 . 24
1 . 24
0.63
0.63
0.50
0. 50
0.33
0.33
48
nd
nd
2.25
2. 25
1 . 25
1 . 25
0.64
0.64
0. 53
0.53
0.35
0.35
Median Lethal Concentrations, mg/l
Hour
LC 50
Upper CL
Lower CL
24
2. 25
4 .35
1. 16
48
1 . 15
1.92
0.68
96
0.51
0.72
0. 36
144
0.46
0.95
0. 22
-------
Table 5. PARATHION RESIDUES IN WHOLE BLUEGTLLS
ACUTE T2ST
Parathion in
water, mg/l
Parathion in
fish, mg/kg
Exposure,
hours
Concentration
factor
5A
0. 34
49. 9
18
145
5B
0.34
61.4
24
173
4B
0.51
50.7
12
98
4B
0.51
32.5
12
63
3A
0.64
294. I
72
462
3 B
0.64
197.8
70
311
2B
1 . 25
318.6
46
256
2B
1 . 25
311.6
' 46
250
1A
2.22
402.9
29
181
1A
2.22
430.0
29
193
IB
2.22
33E . 3
29
152
-27-
r
-------
Figure 1. Concentration factors for parathion residues
in whole bluegills at various exposure times.
500
duo
E
k.
Cnf
O
o
<
Ll_
200
z
o
1—
<
cr
i—
z
LU
100
o
Z
o
o
i 1
i i
i i i i
—
© y
©
f /
'a
§
© /
9
r = 0.946
Of
-
/ ©
-
1 1
i i
i i i i
0
20 40 60
EXPOSURE TIME, hours
80
-28-
-------
Bluoqill Chronic Test
The maximum no-ill-effcct level of chronic parathion exposure to
bluegills fell between 0.17 and 0.34 jig/l. based on deformations of
adults. Growth rate, spawning, and fry survival were no: clearly
affected by toxicant concentrations up to 3. 2 fig/1.
A summary of parathion analyses in water, water quality, and test
temperature are given in Tables Al, A?., and A3. Bluegill survival,
(Table A4) showed that test concentrations did not cause significant
mortalities. Some deaths of extremely deformed fish did occur. But the
majority of deaths followed handling, particularly at the time of thinning.
For this reason, weight measurements were not taken more freaucntly
during the test. There were no significant differences in weights among
tanks at the start of the test, at the thinning, or at the end. Final sizes
of fish were approximately 17 cm, 105 g for females, and ?1 cm, ?00 g
for males.
Within the first four months of testing, tremors, scoliosis, and extended
pectoral fins were observed ir. the two highest concentrations. In six
months, protrusions in the hyal or "throat" region were noticeable at
these levels. The same symptoms continued, and gradually increased
during the 23-month test. Table 6 lists the incidence of scoliosis and
"throat" protrusions. The percentages are somewhat different at 18 and
23 months because some very deformed fish were removed at thinning.
A typical protrusion in the hyal region is shown in Figure 2. It was a
semi-rigid, vascularized mass of fatty and connective tissue extending
from the branchiostegal and urohyal bone area. Scoliosis or lateral
bending of the spine, usually posterior to the dorsal fin, is illustrated
in Tigure 3. In bluegills, the bends were both right and left-handed.
Spawning proceeded successfully through the second summer. Egg
production, hatchability, and fry survival are summarized in Table 7.
Numbers per tank are listed chronologically downward. In general,
the hatchabilities improved as we reduced handling of the egg cups.
Likewise, 7-day fry survival improved as more Artemia were fed.
Spawning data cannot be correlated to parathion level.
The numbers of males and females present were sufficient to provide
good spawning:
-29-
-------
Table 6. INCIDENCE OF DEFORMITIES - BLUEGILL CHRONIC TEST
Tank
OA
OB
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
Mean parathion
conc., ug/l
0.06
0.06
3.23
3. 14
1.53
1.59
1.00
1.C0
0.34
0.34
0. 16
0. 17
18 months
Number of fish
10
15
17
13
20
17
16
15
9
13
10
9
Scoliosis, %
0
13
59
38
45
24
38
33
1 1
0
0
0
Throat, %
0
13
88
100
70
76
75
67
1 1
0
0
0
23 months
Number of fish
9
9
8
8
1 1
9
9
9
8
9
7
7
Scoliosis, %
0
11
25
50
18
33
22
22
0
0
0
0
Throat, %
0
0
62
88
64
100
89
89
38
56
0
0
-------
Figure I. Male bluegill exposed tc 1.6 pg/l parathion
for 2.3 months. Length, 2) cm. Arrow indicates
abnormal protrusion o: i'aVty and connective
tissue.
-------
Intentionally Blank Page
-------
X
'N,
r f
f
t
¦ v
> : V
?
-------
Figure 3. Two adult bluegills exposed to 1. 5 ug/l
parathicn for Z5 months, showing scoliosis.
Dorsal view.
-------
"33-
-------
Table 7. BLUEGILL CHRONIC EXPOSURE TO PARATHION - SPAWNING RECORDS
Tank
OA
OB
1A IB 2A 2B
3A
3 B 4A
4 B 5A
5 B
Nominal parathion
conc., ug/l
0
0
4.00 4.00 2.00 2.00
1.00
1.00 0.50
0.50 0.25
0.25
Hundreds of eggs
spawned
5 49
20
1
90
38
105
2
41
80
71
27
8
28
44 ,
10
2
9
209
15
66
9
25
15
2
167
45
64
103
49
14
271
10
48
26
112
38
229
Percent hatch,
3 days
72
46
44
49
40
80
37
57
83
57
46
36
4
59
35
77
80
74
49
62
61
45
9
32
79
84
86
78
76
10
62
64
82
16
18
18
Percent survival,
7 days, as % of
92
56
66
68
78
0
68
32
100
hatch
98
92
76
8
86
36
96
94
96
90
100
92
80
20
100
46
84
100
100
60
100
84
90
96
97
36
59
86
22
-------
Table 7. BLUEGILL CHRONIC EXPOSURE TO PARATHION - SPAWNING RECORDS (CONT.)
Tank OA OB 1A IB 2A ZB 3A 3B 4A 4B 5A 5B
a
Percent survival,
8
10
0
0
0
0
0
0
32
2
14 days
78
4
18
0
2
0
78
28
26
28
30
48
20
0
100
-
14
82
0
80
0
/
18
28
85
10
0
Percent survival, 3
6
10
-
—
—
-
—
_
28
0
2 1 days
76
0
16
-
2
-
66
28
20
22
16
46
20
-
84
-
2
68
46
18
28
81
0
g
Percent survival,
30 days
6
76
8
—
16
—
2
—
64
24
22
—
18
22
64
16
46
34
18
14
26
5
62
Q
Calculated as percent of larvae hatched.
-------
Tank
Males
Females
Tank
Males
Females
OA
2
7
3A
1
8
OB
1
8
3B
3
6
1A
4
4
4A
3
5
IB
4
4
4B
3
6
2A
3
8
5 A
I
3
2B
3
6
5 B
3
4
Growth rates of 30, 60, and 90-day-old bluecill fry were significantly
different, but not related to toxicant. Mean lengths and survival,
Table 8, correlated inversely with numbers per tank. Large quantities
of food were given four times daily. It appears that the larval chambers
were too small for 40 fry. Regardless of the starting number of fry,
each tank population diminished to a few individuals at the end of the
three months. At 90 days, one fish in tank 1A (3.4 /ig'l) had developed
scoliosis.
During the first two months of the chronic test fish at 8.0 fjg/l began to
show tremors. When these were removed after 64 days, their brain
AChE activity was 1.97 ± 0.40 jumcl/min/g wet wt. This is about 11% of
normal bluegill brain activity. Although the fish showed signs of
poisoning, they were not moribund. Tables AS and A6 show the extent
to which normal AChE activity was inhibited in the chronically exposed
fish. The slightly greater depression in Table'A6 was probably caused
by higher parathion levels in water during the month before termination
(Table Al). The majority of fish in all tanks except the lowest and
control appeared hypersensitive to stimuli. When startled, they swam
away or towards the signal, almost randomly. A disturbance could
trigger clonic (rhythmic) or alternating convulsions in some fish.
Parathion residues of bluegill tissue at 18 arrl P.3 months exposure are
reported in Tables 9 and 10. In muscle, the levels are no greater than
25 times the water concentration. Blood levels are similar or slightly
higher. The unusually high levels in Tank 3 (see Table 10) were affected
by accidentally high water concentrations for two weeks before the
sampling. The greatest residues were in ovaries which is reasonable
because of their high lipid content. Although the data are semi-
quantitative, the residues in spawned eggs are similar to those in
the ovaries.
-36-
-------
Table 8. SURVIVAL AND LENGTHS OF BLUEGILL LARVAE
30 day
60 day
90 day
Mean length,
Mean length,
Mean length
Tank
Number
mm
Number
mm
Number
mm
OA
40
10.1
3
19.7
2
23 .0
OA
9
1 1.3
7
14.9
3
Z0.7
OB
4
11.0
3
14.0
3
Z3.7
1A
3 1
10.5
13
13.Z
1 1
18.5
1A
11
13.Z
10
19.7
8
ZZ.8
2A
8
13.6
7
Z3 .4
5
3Z.4
ZA
8
15.1
8
18.Z
7
Z4. 9
ZB
Z3
10.7
6
16.8
6
ZZ. Z
3A
8
13.2
7
Z0.3
6
Z8 .7
3A
12
9.4
4
1Z.Z
3
18.0
4B
3 Z
9. 1
7
13.6
3
26.3
4B
3 Z
8.9
4
16 .2
4
Z0.5
5A
IZ
10.5
1 1
16.3
7
Z3 . 1
5A
1Z
11.5
10
14.8
4
Z8 .8
-------
Table 9. PARATHION RESIDUES IN BLUEGILLS
18 MONTH EXPOSURE
(yg/kg wet wt)
Tank
0
5 4
3
2
1
Nom. parathion
0
0.25 0.50
1.00
2.00
4.00
ug/l
•Muscle residue
5.4
9.8' 16.9
16.0
13. 1
107.0
27.8
9.6 6.6
14.2
16. 1
92.4
0.3
23.7
11.6
17.4
97.8
2.9
7 . 1
13.4
10. 1
90. 1
5.8
14.1
39.6
141.2
13.5
39 .9
128.4
12.3
Mean * SD 8 .4±11
.0 9.7 ±0.
1 14.6+10.013
.6 ±1.4 22
.7113.4
109r20.8
Blood residue
29.9
15.2 17.6
35.8
41.4
163
19.7 19.5
49 .9
1 IS .8
288
27 .0
Ovary residue
17.8
23.9
74
144
3436
21.2"
30.6
214
158
31.2
238
643
31.0
585
20.7
Egg residue
nil
73
552
179
813
-
nil
246
134
1089
429
Note:
Recovery of spiked muscle samples (equivalent to 100 j/g/kg) gave 88.4%
recovery in a single test.
Precision among triplicate samples gave a coeff. of variation of 13. 1%
All blocd residues except No. 1 and No. 2 sets should be considered
semiquantitative since levels are at or near detectable minimum in the
direct extraction samples. No evidence of paraoxen was found.
-38-
-------
Table 10. PARATHION IN BLUEGILLS
23 MONTHS EXPOSURE
( u 9/kg we-t; wt)
Tank
0
5
4
3
2
1
Nom. parathion,
0
0.25
0.50
O
O
cr
2.00
4.00
u 9/1
Muscle residue3
1 . 2
1.0
2. 1
30.2
5.4
26 . 1
0.9
3.7
1.2
2.3
14.6
12.1
0.8
3.6
11.6
32.6
14.2
12.8
0.2
1. 2
6.0
1. 1
8.3
31.2
0.9
4.5
0.8
14.4
25.3
10.6
1.1
3.1
4.4
23.7
4.7
10.9
Blood residue
0.9
0.6
6.8
253.0
37.5
133.7
1.2
1.8
9.4
118.5
42.7
159.6
2. 1
1.5
8.1
62.9
56.0
125.3
< 0.1
3.9
25.7
78.7
61.7
123 .7
< 0.1
3.8
3.7
104.3
51.0
131.4
2.0
4.0
8.5
97.8
67.2
164.2
< 0.1
4.1
157.3
•
9.4
111.0
These are all at or below reliably detectable levels (-Z5ug/kg ).
Precision at these low levels is estimated at about ±50%.
^Mean parathion concentration for last month of test was accidentally
high: 2.5ug/l (Table Al).
-39-
-------
3ROOK TROUT
Acute Test
Two preliminary "and one final acute test were completed at 1?.°C. The
96-houi 7.C50 for the final test was 1. 76 mg/l (Table 11). During the
test, re; ^ :i mortality occurred only at the highest concentration (Table A7).
Many trcut in the three highest concentrations showed symptcrr.s of
lethargy, irregular breathing, and light discoloration of the skin. No
visible scoliosis, convulsions, or hemorrhages appeared. There was
no evidence of disease during the acclimation period cr test.
The 96-hour result was normalized for 5% mortality in the control by
substituting 100 (R—5)/95 for each percent mortality, R. The calculated
48-hour value is higher then the ?.4-hour one although the mortality
remained the same. This .was a consequence of the higher parathion
analyses at 48 hour than at the start (Table A7).
Residues in trout muscle taken from the same test fish are shown in
Table 12. 3ecause :he trout were removed as they died, the exposure
times for these fish are not uniform. The concentration factors illustrate
the extent to which trout can concentrate parathion within a few days.
Further results of trout uptake studies are reported in Appendix B.
Brook Trout Chronic Test
At parathion levels o: up to 7 ug/1 adult brook trout showed no deleterious
effects over a nine month period. No significant differences in growth
rate, maturity, or spawning success were judged to be caused by the
parathion dosages. The brook trout exposed to these levels, however,
showed uptake of relatively large parathion concentrations in blood,
muscle, and other tissues.
Water quality parameters, temperature, and parathion analyses for the
test are given in Tables AS. A9, and A10. Most of the mortalities which
occurred during the test (Table All) can be attributed to disease, either
fungus or furunculosis, or to failures in the water delivery system. No
fish developed symptoms typical of acute organophosphate poisoning
such as scoliosis or convulsions. Weight measurements made at the
start, at 4 months, and at 9 months (Table Al?) indicate a uniform weight
increase at all exposure levels. No mean weights were found to be
significantly different at-the beginning or end of the test. Most of the
largest weight gains can be explained by lower numbers of fish in those
tanks.
-40-
-------
Table 11. BROOK TROUT ACUTE LC50
LC50,
Upper CL,
Lower CL,
Hour mg/l
mg/1
mg/l
24 2.25
2.58
1.96
48 2.57a
2.78a
2.38a
96 1.76
2. 10
1 .48
aEstimated because
cf change in parathion conc. See page 40,
Table 12. PARATHION
RESIDUES IN 3ROOK TROUT MUSCLE
Acute Test
Exposure, Mean water
Muscle
Concentration
hr conc., mg/l
conc., mg/kg wet wt
factor
8 3.18
217
68
8 3.18
346
109
114 1.86
165
89
114 1.86
215
116
144 0.53
121
228
144 0.53
83
157 .
140 0.27
70
259
140 0.27
93
344
each analysis from a separate fish
_ A 1 _
-------
Fish discarded from the test at the time of thinning, six months, were
analyzed for parathion residues in blood, muscle, and other tissues, as
well as acetylcholinesterase activity. The blood residues, in Table 13.
were plotted against the analyzed parathion levels in the water at the
time of removal (Figure 4).
A least-squares line fitted to the data has the equation-
Cb - 207 Cw - 39.
where is concentration in blood, and Cwthe concentration in water.
Thus the trout took up about 200 times as much parathion in the blood as
was found in the water.
The residue levels for muscle and other tissues show large concentration
factors as well (Table 14). The muscle residues were plotted against
water concentration in Figure 5 and fitted to a curve by eye. If treated
as a straight line, this has an equation of:
Cm = 553 Cw - 176
No paraoxon was detected in any of the analyses despite the use of
solvents to extract it from the clean-up columns. The residues
reported in Table 14 were the highest concentrations found in any chronic
exposure. Brain acetylcholinesterase activities of the same fish, Table 15,
were similarly plotted. Figure 6 shows that abou: 5.6 ^g/l of parathion
was required to depress the brain AChE activity to 50c/= of normal. However,
no gross observable symptoms of central nervous system poisoning were
detected in the fish in the highest dosage.
Brook Trout Spa'wr.inq
The brook trout spawned between Dec. 23 and Jan. 15, test dates. A
summary of the number of spawnings, egg production, and survival of
the embryos and larvae is given in Table 16. Only a very small percentage
of embryos were deformed, and these did not correlate to parathion concen-
tration. The uniformly low hatchability of the embryos is attributed to
handling damage and high temperatures at the time of spawning. The fact
that the test temperature (13°C) was greater than optimal (S°C) probably
caused the small number of spawnings and delayed development of the fish
As a check of maturation, the gonad weights of the discarded fish were taken at
the time of thinning (Table A13), and at the test termination - after spawning
(Table A 14). The gonad weights were divided by the fish's total weight to
arrive at a gonad-osomatic index expressed as a percent. The weights shown
-42-
-------
in Table A14 are somewhat biased since small and immature fish were selectively
discarded. For both males and females, the development was uneven and did not
appear to be related to toxicant concentration. The later gcnadoscmatic indexes
show that spawning was not complete. For example, female gonadcsomatic indexes
greater than 1.00 represent loose eggs in the body cavity which were never released.
Adult Bccok Trout Termination
The adult brook trout were terminated on February 6, three weeks after the last
spawning. Blood was obtained from 34 fish representing all concentration groups
and was analyzed for parathion residues. Ivluscle tissue from 31 fish was analyzed
for parathion. Residue analyses fcr parathion were also done on kidney, gill,
gonad and gastrointestinal tract samples from each of Z to 3 fish in each exposure
concentration group. Brain ACHase was determined on 39 fish.
Tissue residue results, including blood levels, are tabulated in Table 17. Blood
levels versus water concentrations of parathion analyzed at the time of removal
of the fish are plotted in Figure 7. Blood and tissue residue levels measured
after 9 months exposure were lower than corresponding values determined on fish
sacrificed after 6 months exposure.
Several of the adult trout from each parathion concentration groups were exposed to
flowing water without toxicant for 48 and 168 hours. Blood and muscle parathion
residue analyses on these fish are shown in Table 18. Brain ACHase determinations
were also done and results are given in Table 18. ACHase inhibition was essentially
irreversible over the time studied (168 hours). The blood showed a rapid loss of
parathion within 48 hours and a subsequent slower decrease. Muscle parathion
concentrations decreased more slowly than blood lc-vels. The limited samples avail-
able and the large variance among individual fish make it difficult to quantify
"wash-out" rates but the blood and tissue uptake appears to be readily reversible.
Some general conclusions may be drawn on the basis of the 6 and 9 month analyses.
Parathion residues in blood correlate with water concentrations at the time of
sampling; blood levels appear to rapidly reflect exposure levels in the water. Tissue
residues also correlate with water concentrations, however tissue levels will change
less rapidly with change in water concentrations. High correlation exists between
blood parathion concentrations and tissue parathion concentrations when measured
after chronic exposure (6 or 9 months); the correlation coefficient for muscle
parathion versus blood parathion was 0 .967 (p <0.001).
t
The high variance in water concentrations measured on a weekly basis probably
account for the differences in residue results observed at 6 and 9 months. Parathion
appears to be reversibly taken-up and released and residue levels reflect the
immediate past exposure conditions.
-43-
-------
Brain ACHase levels, as expected, exhibited an inverse dependence on water
concentration. The degree of inhibition was less marked than with bluegills.
Essentially the same ACIIase effects were found at 6 and at 9 months. Rela-
tively high variance within the concentration groups and especially the control
group was found. However the highest concentration group exhibited ACIIasc
levels significantly lower than the other experimental groups (excluding the
control group) (p= 0.05).
Ninetcen-day-old brook trout eggs were incubated at 10CC in three concentrations
of parathion and without parathion until hatched. The following percent survivals
to hatch were observed:
Eggs in the 32 and 100 ug /l solutions exhibited poor development and more trans-
parent chorions than controls. Some cf the eggs in both concentrations showed
cerebral development without corresponding spinal development. Part of the lower
embryo survival at the two highest levels resulted from premature hatching. Although
the hatchability at 10 ug/l was less than the control group, development appeared
to be normal.
control
77.5 %
10 ;/q/i
52.5%
3 2 „q/l
41. 5%
loo i/g/i
38.4%
-44-
-------
Table .13. PARATHION IN BROOK TROUT BLOOD
Six Month'Exposure, Individual Fish
(ug/1)
•Tank OA 1A 2B 3A 4A 4B 5A 5B
o
6.2
1007
737
647
113
153
68
79
63.7
1597
885
6 95
96
151
24
58
10.6
1139
726
96
61
69
40. 5
776
337
93
52
103
0.5
66 2
515
101
80
6.9
528
6 54
42
50
649
68
1 10
50
Mean 21
1302
788
603
100
152
58
78
SD 25
417
20*8
136
9
1
23
24
-45-
-------
Figure 4. Parathion in brook trout blood vs.
parathion in water, six month exposure.
1600 -
2 1200
oo
2 800
o
c_>
x 400
= 0.938
<
cr
<
CL
0
2
4
6
8
PARATHION CONCENTRATION IN WATER, pg//
-46-
-------
Table 14. PARATHION RESIDUES IN BROOK TROUT TISSUE
Six Month Exposure
(yg/kg wet wt)
Tank
OA
1A
2B
3A
4 B
5A
5B
Water cone, tig/1
0.03
6 .7
4.0
2.6
1.4
0.6
0.6
Muscle
nil
18
12
nil
4454a
3229a
1S58
1820®
1874
1258h
14 11
92 1
998
1124
17 6 a
474a
606
244
130
17 l3
139
Kidney
nil
125
2942
556
998
815
3332
1245
1051
123
nil
nil
277
Gill
nil
26
5954
4090
2503
2430
3294
1932
2659
492
516
-
nil
160
Gonad
nil-
16
2208
1344
2136
414
579
2050
1284
175
11
nil
176
^Average of duplicate samples
Average of triplicate samples
All others are single samples
Reagent Blank = 0 . 3t/g/kc
Recovery % at 1000 ^g/kg level averaged 90.8 ±7.6% (coeff. variation = 8.47c);
range 83. 1 to 103% for seven tests.
All values uncorrected for % recovery.
-47-
-------
Figure 5. Parathion in brook trout muscle vs.
parathion in water, six month exposure
5000
4000
op
a)
5
+-»
^ 3000
QO
a.
o
CO
2000
<
cn
<
Q.
1000 -
A
2 4 6
PARATHION IN WATER,
-48-
-------
Table 15. BRAIN ACETYLCHOLINSSTE.IASE ACTIVITY OF BROOK TROUT
Six Month Exposure, Individual Fish
01 mol/min./g)
Tank
OA
1A
2 B
3A
4A
4B
5A
5B
-
6.60
3 .79
5.22
4.19
4.96
4.28
5.51
6.72
6.49
1.88
3.44
4.42
4.65
4.59
4.96
6.49
6.63
5.34
5.85
5.85
6.66
4. 16
6 .03
6.72
5.85
5.74
6 . 57
6.49
4.71
Mean
6 .47
2.84
4. 54
4.30
5.37
4.44
5.71
6.60
SD
0.31
1.35
0.90
0. 16
0.67
0.29
0.74
0. 16
C3
-49-
-------
Figure 6. Brook trout brain acetylcholinesterase activity vs.
parathion concentration in water, six month exposure.
PARATHION IN WATER, »g/J
-------
Table 16. BROOK TROUT SPAWNING RECORD
Tank
joA
V
IB
2B
3A
3 B
4A
43
5A
5B
Number spawnings
1
j
2
3
2
2
2
3
7
1
Total eggs spawned
1 360
1249
787
716
9 24
929
1607
479
48
12 day viability,
% of spawn
85
42
2
76
56
15
100
78
0
24
Total hatchability,
% of spawn
6
12
20
12
0
6
22
4
0
30 day fry survival,
% of hatch
0
0
10
67
0
67
18
0
0
-51-
-------
Table 17. BROOK TROUT PARATHION RESIDUES AND AChE - 9 MONTHS
Tank
Parathion
in water
U9/1
Blood
Muscle
Gill
Kidney
Gonad
G. I.
Tract
AChE,
H mol/min/g
OA
0.01
23
0.8
3 . 97
42
0.6
3. 1
0:2
<0.01
0.7
5.04
94
1.9
5. 12
23
1.0
3.01
OB
0.01
58
0.6
1 .4
0.1
-
28
4.49
i
33
5.77
29
0. 1
4.62
20
4. 19
1A
8.30
733
1, 220
3, 172
400
1,8 12
8, 989
3 .44
1,021
399b
2.79
990
4, 157
! 2.15
I
IB
[
J 8.72
625
1,623
i
| 3.17
)
!
743
876
1, 727
217
939
160
1 4.39
i
720
2, 18 Ia
1, 143
614
1, 780
11,598
| 3.86
2A
5.53
361
297
.1
} 6.24
355
j 4.45
1
1
!
671
2, 358
311
1, 054
5, 666C
| 5.91
2B
1
4. 24
337
3 18
1, 483
374
705
200
t
j 4.46
446
-
J 5.21
304
a
j 6.17
413
1, 085
78
519
352
! 4.71
-------
Table 17. BROOK TROUT PARATHION RESIDUES AND AChE - 9 MONTHS (CONT.)
Tank
Parathion
in water
\B/l
Blood
Muscle
Gill
Kidney
Gonad
G. I.
Tract
AChE,
umol/min/g
3A
2.86
184
270
4.30
252
189a
703
192
6
1, 672
4.8 1
482
4.35
166
3 19a
5.44
3 B
2.76
232a
272
959
183
407
1, 105
4.42
217a
•511°
1, 083C
774
15, 048°
1, 210
4.76
4A
1 .45
1 14
7
179
1 1
36°
106
6 . 24
1 11
1 18
6.06
4 B
1.26
141
5.85
87
44
265
53
-
1 10
6.00
82
34
4 .36
5A
0.53
41
33
a
138
16
69
135°
5.63
40
58
5.73
52
5.27
5B
0.44
39
36
6.51
33
5.65
36
73
96
63°
38
28
5. 56
63
5.45
^duplicate samples average,
analysis done immediately after fish was sacrificed; all other samples were stored at - 18°C for 2-1/2 months
Q
probable interference contribution to parathion value
-------
Intentionally Blank Page
-------
Figure 7. Parathion in brook trout blood vs.
parathion in water, nine month exposure.
1000
x 400
<
en
<
Q_
r = 0.946
200
9—
6
8
0
10
2
4
PARATHION IN WATER, jjg//
-55-
-------
Table 18. BROOK TROUT PARATHION RESIDUES AND AChE - RECOVERY
48 HOUR ELUTION IN FRESH WATER
168 HOUR ELUTION IN FRESH WATER
Tank
Blood |
residue,
v g/1
Muscle !
residue, j
M g/kg j
AChE
u mol/min/g
OA
Z7
35
5.4Z
OB
Z 5
41
4.99
1A.
507
587
4.45
IB
3 96
634
i
'
4.08
ZA
ZZ3
478
4.09
ZB
ZZ9
161
o
o
3A
18Z
187
4.48
3 B
364
Z90
4.30
4A
94
9Z
5.77
4B
144
' 74
8.48
5A
1Z3
85
6.11
5B
57
75
3.84
i
i
i
Tank
!
Blood )
residue, j
u g/i j
Muscle
residue,
ug/kg |
AChE,
H mol/min/g
< CD
O O
1
». i
— t
j
i
4.31
5.74
1A
IB
¦
Z93 ]
4ZZ !
94 Z '
1, 1Z4 •
*
4.78
3.6Z
2A i
ZB
196
1Z7
365
46Z
4.91
3A
3 B
65
40
ZZ3
58
4. Z3
5.4 1
4A
4 B
35
84
60
186
4. 59
4.0 Z
5A
5 B
Z1
Z3
6Z
128
9.08
4.88
-------
FATHEAD MINNOW
Acute Test
One preliminary static test using aduit minnows at ?2°C gave the
following results based on measured initial concentrations:
LC50 - 24 hr = 2.28 (2.92 - 1.77) mg/l
48 hr = 2.02 (2.64 - 1:55) mg/l
96 hr = 1.60 (?. 05 - 1.25) mg/l
A final flow-through test at 24°C using adults (approx. ?.2 g each) gave
the results reported in Table 19.
Fathead Minnow Chronic Exposure
The fathead minnow chronic exposure gave a maximum acceptable parathion
concentration of about 4 yg/l based on adult deformities and reproductive
impairment. Water quality, temperature, and parathion levels for the tes:
are summarized in Tables A16, A17, and A18.
For the firs: 30 days of the test, minnow larvae showed cood survival and
growth (Table A19). There were no significant weight differences between
tanks. At 60 days, the mean length in tank 3B was significantly less
(P = 0.05) than tanks 3A, 5A, -and 5B, but not different from any others
(Table A19). During.the third month of exposure, symptoms of poisoning
began to appear in several of the higher aosaces. Convulsions and
scoliosis were observed occasionally. By the time of spawning, there
were deformities (including scoliosis, lordosis. 3nd abnormally shaped
heads) in almost half of the fish in the 50 jug/1 tanks (Table ?0). Weights,
however, were not significantly different between tanks, as calculated
by analysis of variance.
Spawning occurred first in the controls, and in the other tanks several
days later. Egg production (Table 21) and the number of spawns per
female were highest in the two ccntrol tanks. The hatchabihty rate
did not appear to be related to parathion; the 30-day larval growth and survival
were too sparse to judge toxicant effect.
Some whole minnows were analyzed for parathion during the test but
because of their small size, the fish were pooled. Fish taken at 70 days
exposure (the thinning), had lower residue levels than fish sampled
later (Table 22). The maximum concentration factors for the chronically
exposed minnows were about 100 to 170, which was less than trout, but
greater than bluegills.
-57-
-------
Table 19. FATHEAD MINNOW ACUTE TEST
24°C
Number Surviving
Tank
OA
OB
1A
IB
2A
2 B
3A
3 B
4A
4B
Start
10
10
10
10
10
10
10
10
10
10
24 hr
10
10
0
0
0
0
9
6
9
8
48 hr
10
10
0
0
0
0
7
1
6
4
96 hr
10
10
0
0
0
0
6
1
6
4
Parathiona ,
mg/l ••
nil
nil
5.31
3.88
1.92
2.24
1.04
1 .04
0.53
0.49
Means cf four analyses.
Hour
LC 50
mg/l
Upper CL,
mg/l
Lower CL,
ma/l
24
2.20
3.90
1 .24
48
0.50
1.03
0.24
96
0.50
1.03
0.24
-58-
-------
Table 20. MINNOW ADULT WEIGHTS AND DEFORMITIES -8.5 MONTHS
Tank j OA
?
OB
1A
IB
2A
2B
3A
3 B
4A
4 B
5A
5 B
No. females
8
9
1 1
9
7
5
6
8
9
7
8
5
Mean wt, g
1.15
0.
98
1.
05
1 .
16
1.29
1.
32
1 . 25
1.
07
0.
94
1 .
09 '
1 .
22
1 .
23
No. males
4
4
I
2
3
2
2
4
4
1
5
8
Mean wt, g ¦
2.30
1.
88
1.
92
1.
38
1 .78
1 .
46
1 .88
1 .
99
1.
73
1 .
75
1 .
99
1 .
90
% deformed
o
o
0.
0
46
.2
45
.4
30.0
28
. 6
0.0
50
.0
15
.4
25
.0
7
.7
0
.0
-------
Table 21. MINNOW SPAWNING RECORD
£*
Tank
OA
OB
1A
IB 2A
2B
3A
3B 4A
4B
5A
5B
Total eggs j
spawned
j
2096
3591
463
51 251
979
103
397 2205
0
570
399
Number of j
spawnings j
14
21
8
1 2
7
3
5 12
0
5
4
Spawns per j
female j 1.75
X
Mean%hatch j 72.4
i
2.33
0.73
0.11 0.29
1 .40
0.50
0.62 1.33
0.00
0.62
>
0.80
88.2
65. 5a
42.0
44.4
-
5 I.2 74 . 3
-
75.0
65.5
Spawns hatched
9
19
3
2 0
5
0
6 14
0
6
4
30 day survival,
percent
70.0
42.5
40.0
12.5
Mean length, cm
0.98
1 .49
1.20
1 .68
Four sets of eggs spawned in OB were hatched in 1A - 70% mean hatchability
-------
Table 22, PARATHION RESIDUES IN WHOLE FATHEAD MINNOWS
(iu g Ag)
Water
parathion
conc. ug/*
0.15
49.0 21.7
15.5
9.0
4.2
70 days
exposure
1,040 (20) 615 (2)
251 (19)
214 (22)
256 (8)
62-138
days
exposure
_
9, 975 ( 3) 4, 400 (5)
2, 256 (3)
1,796 (1)
1,411 (4)
260 days
exposure
14 (2)
8,300 (2) 2,270 (2)
510 (2)
601 (2)
846 (2)
Numbers in parentheses are numbers of fish pooled
-6 1-
-------
DAPHNIA MAGNA
The acute and chronic toxicities of parathior. to Daphrna rn.§9!i?l are
summarized in Table 23. Representative test data are in Tables A?0
and A21. The daphnids responded to parathion poisoning within narrow
limits. Thus, the MATC of 0.08 ug/1 was very near the 3 week chronic
LC50 of 0 .14 '1.
The concentration needed to reduce reproduction by 50£, RI50, was greater
than the chronic LC50. This occurred because young were produced during
the third week of exposure at concentrations that later killed the adults
(Table A2l.)
Symptoms of toxicity during the acute exposures wore erratic swimming
in a circular motion, and later, inactivity and immobility.
The MATC of 0.08 ^g/1 (Table A2.1) was 0.08 times the 48-hour LC50 of
1.00 and 0.13 times the 96-hour acute value. The MATC was considered
to be the highest concentration tested in which the number of young
produced did not differ significantly from controls, as determined by
analysis of variance.
il
For residue analyses, 0.4 to 20 mg collections of treated Daphr.ia were
filtered and air dried. Hexa.ne extracts were cleaned with a silicic acid
column and analyzed by GC. No evidence of significant parathion residues
could be detected above the GC background noise fcr any sublethal exposure
levels. There is a possibility of decomposition of parathior. during the
air drying process.
-62-
-------
Table 23, PARATHION TOXICITY TO DAPHNIA MAGNA
Acute LC50, /jg/l
24 hr
48 hr
96 hr
2.70 (5.61 - 1.30)
1.00 (1.73 - 0.58)
0.62 (0.90- 0.43)
3.21 (5.10 - 2.02)a
1.27 (1.63 - 1 .09)a
0.65 (0.88-0.48)a
Chronic LC50, [£)/1
1 week
2 week
3 week
RI50
0.28 (0.29 - 0.26)
0.25 (0.22 - 0.28)
0. 14 (0. 16 - 0.13)
0.24 (0.3 2
- 0. 17)
0. 56 (0.85 - 0.37)
0.38 (0.70 - 0.21)
0. 19 (0.30 - 0. 12)
0.48 (0.87
- 0.26)
0.35 (0.47 - 0.26)
0. 37 (0.74 - 0. 18)
0.45 (0.55 - 0. 37)
MATC - 0.08 iig/l aStatic test at 2Z°C
All other tests flow-through at 18± 1°C
-------
GAMMARUS FASCIATUS
Results of the parathion acute and chronic tests to G. fascistus are
reported in Table 24. Two long-term tests were terminated when
survival in the lowest concentration became less than 50'?> of the
survival in the controls. Table shows one of these chronic tests
in which the controls and low levels began reproducing.
-64-
-------
Table 24. PARATHION TOXICITY TO GAMMARUS FASCIATUS
LC50, /ig/1
24 hr
48 hr
96 hr
Temp.,°C
2.1 (3.8 - 1.2)
0.62
0.43 (0.65
- 0. 29)
20
1.27 (1.62 - 0
99)
0.82 (0.9S
- 0.68)
0.62 (1. 16
-0.33)
19
2.3
0.86 (1.23
- 0.60)
0.26 (1.00
- 0.07)
20
-
-
0.25 (0.29
-0.21)
20
35 da>s - 0.09
43 days -0.07
(0.69
(0.50
- 0.01)
- 0.C1)
19
20
MATC <0.04 jig/1
-65-
-------
CHIRONOMUS TENTANS
The C. tentans results summarized in Table 2.5 show a striking change
in the LC50 with length of exposure. - Between acute and chronic periods
the lethal concentration varied over three orders of magnitude. Preliminary
static acute tests using the tissue paper substrate were discarded when
we discovered thai parathion was removed from the water in several days.
The flow-thrcuch tests eliminated this problem of decreasing water
concentration.
-66 -
-------
Table 25. PARATHICrJ TOXICITY TO C° TENTANS
Exposure, days
LC5Q, ug/1
Instar at start
1
660 (1040 - 420 )
4
2
135 (703 - 26)
4.
4
31.0 (43.4 - 22. 1)
4
5
7.3 (11.8- 4.6)
2
8
2.2. (3 .2 - 1.5)
2
14 .
2,3 (3.4 - 1.6)
2
14
2.9 (3.7 - 2.3)
2
-67-
-------
Intentionally Blank Page
-------
SECTION VI
DISCUSSION
Of the fish species tested, bluegills were most sensitive to parathion
poisoning. The 96-hour LC50 of 0.51 (0.72 - 0.36) mg/'l found for
juveniles agrees with the 0.58 ppm value reported by Pickering,
Henderson, and Lemke"^ for large bluegills in soft water. They also
reported a much lower level, 0.095 ppm, for small bluegills under static
conditions.
Yearling brook trout exhibited a 96-hour LC50 o: 1,76 (?.10 - 1.48) mg/1.
This is somewhat greater than the 7?-'nour LC50 cf 0.9? mg/l given by
Leland16 for rainbow trout. Trout responded over a wider range of parathion
levels than either bluegill or minnow.
Adult fathead minnows were killed by concentrations within the range of
bluegill and trout response. A static test gave a 96-hour LC50 of 1.60 (?.05 - l.?5)
mg/1 while a flow-through test gave 0.50 (1.03 - 0.24) mg/1 in the same
time period. The static result agrees with literature LC50 96-hour values:
1.3 mg/14®, 1.4 mg/l41' and 1.6 mg/l4\ By comparison, the
flow-through result seems low. Test results were confirmed in preliminary
testing, fish were handled carefully to avoid stress, and parathion
concentrations were checked by independent analysts.
Chronic effects to bluegills, including scoliosis, abnormal protrusion
of tissue, and depressed acetylcholinesterase activity, were found at
parathion concentrations above 0.17 to 0.34 /ig/l." Scoliosis was found
under similar conditions to involve calcium deposition, and fusion of
two to four vertebrae (Carter14). The protrusion in the "throat" area of
many test fish was unexpected. Affected fish had shallow, rapid breathing
and flaired gill covers - presumably because of nerve damage. The
protrusion may result from mechanical damage to the branchiostegal area •
indirectly caused by spasmodic buccal pumping. Further histological
characterization would help to determine the mechanism of such parathion-
related tissue damage.
At 3. 2 fig/1 parathion. bluegills were able to survive with 167o of normal
AChE activity. In a tank under optimum conditions and little disturbance,
they could function, marginally. In nature, their ability to feed and
escape predators would be doubtful.
-69-
-------
Brook trout exposed to 7. 2 ug/l parathicn for 9 months developed no
harmful effects, as Judged by weight gain; external appearance, AChF
activity, and production of embryos The da.ta are not conclusive, however,
about the sensitivity of the embryos ond larval stages. A level of 3E jig/1
reduced hatchability of embryos from untreated parents, while a level of
10 jig/1 did r.ot.
Fathead minnows showed deformities and reduced egg production in chronic
parathion concentrations above 4 uc /1. No reduction in growth rate,
hatchability or fry survival were demonstrated at the higher levels. Thus
in both the bluegill and minnow tests, the juvenile and adult stages seemed
more sensitive to parathion than their eggs or larvae. This could simply be
a function of length of exposure. However. Lewallen43 found that 2.0mg/l
parathion had no effect on one-week-old rainbow trout sac fry, while 7 0%
of one-month-old fry were killed at the same level in 74 hours Because
parathion toxicity depends on activation, it is possible that older animals
metabolize parathion more efficiently than young ones.
The retention of parathion in blood, muscle, and other tissues was demon-
strated for all three fish species. Bluegill ond trout showed concentration
factors of several hundred in acute tests. In chronic tests, where residues
probably reach a steady state, brook trout had the highest parathion in
tissues, including muscle with 553 times the level in water. Whole
minnows had concentration factors of about ?00, while bluegills had. at
the most, about 25 times the level in water. This suggests that bluegills
metabolize parathion more readily than the other two fish, which could
explain their greater sensitivity. We cannot rule out the possibility that
this is a temperature as well as species effect. Test temperatures
increased from trout, to minnow, to biuegill It is possible that bluegills
held at lower temperatures would metabolize less, and store more parathion.
Further metabolic pathway studies in fish or in other cold-blooded animals
should relate effects of temperature, rate of metabolism, and proportions
of major metabolites.
The two crustaceans tested, magna and GL fasciatus. were more sen- •
sitive to parathion than either the insect. C. tentans. or the fish. For
Daphnig. the maximum acceptable chronic level, about. 0.08 ug/l was
0.13 times the acute level of 0.63 jig'!. The acute value is close to
0.8 jig/l reported by Pnester'1'1 (50 hours) and Boyd45 (26 hr.). Gammarus
had a chronic effect level of much less than 0.04 ug/l - a lethal level
in one month. At such low effect levels (which approach the chromato-
graphic background for routine work) it became impractical to carry out
meaningful tests for more than one month Since crustaceans such as
Daphnia and Gammarus are typical foods for many small fish, parathion
levels in.natural waters should be limited to less than 1 jig/1. In terms of
protecting food chain organisms, and hence fish, this would not be an
overly conservative level.
-70-
-------
The midge, C. tentans. had a wide range of sensitivities to parathion,
depending on flow rare, larval stage, and exposure time. It gave a 96-
hour LC50 of 31 ug/1 and chronic effect level of less than 3 fig/1, a
lethal concentration in ? weeks. The midge was the only species tested
that is closely associated with sediment. The adsorption properties of
parathior. on the organic material used in the tests was probably the
dominant influence on toxicity. Certainly the midge larvae could tolerate
a relatively high single dose of parathion if it were adsorbed rapidly by
the substrate.
A few problems stand out in the methods for chronic toxicity testing
of aquatic species. For seasonally applied pesticides, a one or two
year chronic test of constant concentrations in water may not be real-
istic. Only the shorter-lived invertebrates would normally be exposed
for chronic time periods. Bluegills are not practical species for chronic
tests unless a method is devised to insure spawning during the first
summer in the laboratory. ?or all fish, egg handling, incubation, and
larvae.rearing techniques used were cumbersome and required crca:
labor. In many cases the harmful effects of handling outweighted -he
effect of the toxicant under study. Among the invertebrates, Daphnia
and Chironomus proved to be convenient test organisms. In long-term
tests, Gammarus had too poor survival using present culture techniques.
Tinally, before accepting the results of such tests at face value, further
understanding is needed of the pesticide in a natural system. Mechanisms
of direct uptake and trophic concentration should be considered, as well
as microbial degradation, and adsorption on sediments. The effect on
fish productivity of a reduction of invertebrates by pesticides should be
examined.
-71-
-------
Intentionally Blank Page
-------
SECTION VII
REFERENCES
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Potential in Pesticide Manufacturing. EPA Tech Series Report. T3-
00-72-04. 1972. ?50 p.
2. Lichtenstein, E.P. and K.R. Schulz. Effects of Moisture and Micro-
organisms on the Persistence and Metabolism of Some Organophosphorus
insecticides in Soils, with Special Emphasis on Parathion. J Econ
Entomol 57-618-627. 1954.
3. Wolfe, H. R., D. C. Staiff, J. F. Armstrong, and S. W. Comer.
Persistence of Parathion in Soil. Bull Envir Contam Toxicol
10:1-9. 1972.
4. Stewart, D. K. R., D. Chisholm, and M. T. II. Ragab. Long Term
Persistence of Parathion in Soil. Nature. 229:47. 197 1.
5. . Gomaa, H. M.# and S. D. Faust. Chemical Hydrolysis and Oxidation
of Parathion and Paraoxon in Aquatic Environments, in Fate of Organic
Pesticides in the Aquatic Environment, Washington, D. C., American
Chemical Society. 1972. p. 189-209.
6. Eichelberger, J. W., and J. J. Lichtenberg. Persistence of Pesticides
in River Water. Environ Sci and Tech. 5:541-544. 1971.
7. Graetz, D. A., G. Chesters, T. C. Daniel, L. W. Newland, and G. B.
Lee. Parathion Degradation in Lake Sediments. J Water Poll Contr
Fed. 2 (pt. 2) :R76-R94. 1970.
8. Nicholson, H. P., et al. Insecticide Contamination in a Farm Pond.
Trans Am Fish Soc. 91:213-222. 1962.
9. Miller, C. W., B. M. Zuckerman, and A. J. Charig. Water Trans-
location of Diazinon-C14 and Parathion-S"*5 off a Model Cranberry
Bog. Trans Am Fish Soc. 95:345-349. 1966.
10. Baker, R. A. Pesticide Usage and its Impact on the Southeast. EPA
Pesticide Study Series 8. 1972. 411 p.
11. O'Brien, R. D. Insecticides: Action and Metabolism. New York,
Academic Press, 1967. p. 32-82.
-73-
-------
12. Weiss, C. M. Tfie Determination of Cholinesterase in the Brain Tissue
of Three Species of Freshwater Fish and its Inactivation in Vivo.
Ecology. 39:194-199. 1958.
13. . Physiological Effect of Organic Phosphorus Insecticides
on Several Species of Fish. Trans Am Fish Soc. 90:143-152. 1961,
14. Carter, F. L. In Vivo Studies of the Brain Acetylcholinesterase
Inhibition of Orgar.ophcsphate and Carbamate Insecticides in Fish.
Ph.D. Thesis, Louisiana State U. 20?.p. 1970.
15. Hitchcock, M,, and S. D. Murphy. Activation of Parathion and Guthion
by Mammalian, Avian, and Piscine Liver Homogenates and Cell
Fractions. Toxicol Appl Pharmacol. 19:37-45. 1971.
16. Leland, H. V., II, Biochemical Factors Affecting Toxicity of Parathion
and Selected Analogs to Fishes. Ph.D. Thesis, U. of Michigan.
123o. 1968.
17. Potter, J. L., and R. D. O Brien. Parathion Activation by Livers of
Aquatic and Terrestrial Vertebrates . Science. 144:55-56. 1964.
18. Carlson, G. P., Comparison of the Metabolism of Parathion by Lobsters
and Rats. Bull Environ Contair. Toxicol. 9:296-300. 1973.
19. Nakatsugawa, T., N.M. Tolman, and P. A. Dahm. Degradation of
Parathion in the Rat. Biochem Pharmacol. 18:1 103-11 14. 1969.
20. Mount, D. I., and H. W. Boyle, Parathion - Use of Blood Concentration
to Diagnose Mortality "of Fish. Environ Sci and Technol. 3: 1 183-1 185.
1969.
21. Mulla, M. S., J. O. Keith, and F. A. Gunther. J Econ Entomol.
59: 1085-1090. 1966.
22. Solon, J. M., J. L. Lincer, and J. H. Nair III. Effect of Sublethal
Concentrations of LAS on the Acute Toxicity of Various Insecticides
to the Fathead Minnow. Water Res. 3: 767-755. 1969.
23. Lowe, J. I., P. D. Wilson, and B. Davison. Laboratory Bioassays.
Prog Rept. Bur Comm Fish Center for Estuarine and Menhaden Res.
Contr. No. 98. Circ 335. p. 20-22. 1970.
24. Kemp, PI. T., J. P. Abrams, ar.d R. C. Overbeck. Effect of Chemicals
on Aquatic Life. Water Quality:Criteria Data Book, Vol. 3, 5?.8 p.
May, 1971.
-74-
-------
25
26
27
28
29
30
31.
32
33
34
35
36
IvCatsue, Y., T. Endo, andKTabata. Effect of Perathion on Aquatic
Animals in Sublethal Concentrations. Nippon Suisangeku Kaishi
(Tokyo). 23:358-362. 1957-1958.
Davis, H. C., and H. Hidu. Effects of Pesticides on Embryonic
Development of Clams and Oysters, and on Survival and Growth of
the Larvae. US Tish and Wildl Serv, Fish Bull. 67:393-404. 1969.
Billard, R., end P. deKinkelin. Sterlisation E)es Testicules De
Guppies Par Des Doses Men Letales De Parathion. Ann Hydrobiol.
1:91-99. 1970.
Jensen, L. D., and A. R. Gaufin. Long-term Effects of Organic
Insecticides on Two Species of Stonefly Naiads. Trans Am Fish Soc.
93:357-363. 1964.
Mount, D. I. and C. E. Stephan. A Method for Establishing Accept-
able Toxicant Limits for Fish - Malathion and the Butoxyethanol
Ester of 2,4-D. Trans Amer Fish Soc. 96:185-193. 1967.
American Public Health Association. Standard Methods for the
Examination of Water and Wastewater, 13th ed. New York, APHA.
1971.
Mount, D. I. and W. A. Brungs. A Simplified Dosing Apparatus for
Fish Toxicology Studies. Water Res. 1:21-29. 1967.
Drummond, R. A. and W. F. Dawson. An Inexpensive Method for
Simulating Diel Patterns. Trans Am Fish Soc. 99(?):434-435. 1970.
McKim, J. M., and D. A. Benoit. Effect of Long-term Exposures to
Copper on Survival, Reproduction, and Growth of Brook Trout
Salvelinus fontinalis (Mitchell). J Fish Res Bd Canada. 28:655-66?.
1971.
Weber, C. I., ed., Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA -
670/4-73-001. July, 1973.
Benoit, D. A. Artificial Laboratory Spawning Substrate for Brook
Trout (Salvelinus fontinalis Mitchell). Trans Am Fish Soc: 103(1):
144-145. 1974.
Litchfield. J. T., and F. Wilcox'on. A Simplified Method of Eval-
uating Dose-Effect Experiments. J Pharmacol Exper Therap.
96:99-113. 1949.
-75-
-------
37. Thompson, W. R. Use of Moving Averages and Interpolation to
Estimate Median Effective Dose. Beet Rev. 1 1:115-145. 1947.
38. Ellman, G. L., K. D. Courtney, V. Andres Jr. , and R. M. Teather-
stone. A New and Rapid Cclorimetric Determination of Acetyl-
cholinesterase Activity. Biochem Pharmacol. 7:88-95. 196 1.
39. Hokanson, K. E. F., f. H. McCormick, 3. R. Jones, and J. H.
Tucker. Thermal Requirements for Maturation, Spawning, and
Embryo Survival of the Brook Trout. J fish Res Board Can.
30:975-984. 1973.
40. Pickering, Q. II., C. Henderson, and A. E. Lemke. The Toxicity
of Organic Phosphorus Insecticides to Different Species of V/arm-
water Fishes. Trans Am Fish Soc. 91:175-184. 296Z.
41. Henderson, C., Q. H. Pickering, and C. M. Tarzwell. The Toxicity
of Organic Phosphorus and Chlorinated Hydrocarbon Insecticides to
Fish. 1959 Sem 3iol Probl Water Poll., R. A. Taft San Engr Ctr
Tech Rpt W 60-3. 1960.
42. Tarzwell, C. M. Pollutional Effects of Organic Insecticides. Trans
24th Am Wildl Ccnf. p. 132-142. 1959.
43. Lewallen, L. L. and V/. H. Wilder. Toxicity of Certain Organo-
phosphorus and Carbamate Insecticides to Rainbow Trout.
Mosquito News. 22:369-372. 1962.
44. Priester, L. E., Jr. Accumulation and Metabolism of DDT, Parathion,
and Endrin by Aquatic Food-chain Organisms. Ph. D. Theisis.
Clem son U. 1965.
45. Boyd, J. E. The Use of Daphnia magna in the Microbioassay of
Insecticides. Ph. D. thesis, Penn State U. 1957.
46. Kadoum, A. M. Rapid New Method of Sample Cleanup for GC
Analysis of Insecticide Residues. Bull En.v Contam Toxicol.
2:264-273. 1967.
-76-
-------
SECTION VIII
APPENDIXES
APPENDIX A - BI OAS SAY DATA
-77-
-------
Table A1. MEAN MONTHLY ANALYSES OF PARATHION
BLUEGILL CHRONIC TEST
(ug/i)
Tank
Dec.
Jan.
Feb.
Mar.
Apr.
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Jan.
Feb.
Mar.
Apr.
OA OB
nil
0.01
0.01
0.02
0.03
0.01
0.01
0.02
0.01
0.06
0.07
0.04
0.02
0.01
0.04
0. 10
0. 17
nil
0.01
0.01
0.02
0.03
0.01
0.02
0.02
0.01
0.06
0.07
0.03
0.02
0.01
0.04
0. 10
0. 17
1A
4. 54
4.40
3.80
4.77
2. 93
2.38
3.72
2.08
3.03
2.27
4.96
2.39
2.44
3.80
1.81
0.97
3 . 29
IB
4.87
4. 10
3.70
4.74
2.50
2.51
3 . 52
1 .99
3.07
2. 27
4.96
2. 58
2.44
3 .80
1.81
0.97
3.29
2A
2.44
2.12
2.25
2.44
1 . 29
1.21
1 .83
0.99
1 .55
1 . 19
1 .74
1 .24
I .87
1 . 58
1 .26
0.37
1 .86
2B
2.60
2. 37
2.11
2.27
1 .24
1.12
1 . 93
1 .03
1 . 54
1.19
1 .74
1.15
I .87
1 .58
1 .26
0.37
1 .86
3A
I . 23
1 .06
0.94
1 .30
0.70
0.64
1.09
0. 57
0.75
0.50
0. 57
0.42
0.80
1.45
0.47
0. 19
1. 18
3 B
1 .00
1 .04
0.91
1.31
0.65
0.63
1.04
0.64
0.75
0. 50
0. 57
0.35
0.80
1.45
0.47
0.19
I . 18
4A
0. 50
0.45
0.36
0. 58
0.32
0. 32
0. 39
0. 25
0.44
0. 18
0. 22
0. 17
0. 27
0. 13
0. 15
0.11
0.31
4B
0. 57
0.40
0.37
0.61
0.34
0.34
0.48
0. 26
0.46
0. 18
0. 22
0.06
0. 27
0. 13
0. 15
0. 1 1
0.31
5A
(9. 19)
(9.50)
0. 14
0. 17
0.02
0.11
0. 19
0.12
0. 19
0. 12
0. 13
0.09
0.06
0.08
0.08
0. 17
0.40
5B
(9.63)
(9.21)
0. 14
0. 18
0. 10
0.13
0.20
0.12
0.23
0.12
0. 13
0.05
0.06
0.08
0.08
0.17
0.40
-------
Table A1. MEAN MONTHLY ANALYSES OF PARATHION (cont)
BLUEGILL CHRONIC TEST
(j/g/i)
Tank
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4 B
5A
5B
May
0. 12
0. 12
2.00
2.00
0.93
0.93
0.92
0.92
0.22
0.22
0. 14
0. 14
June
0.21
0.21
2.88
2.88
1.62
1.62
1.33
1 . 33
0.39
0.39
0. 29
0.29
July
0.04
0.04
4.72
4.72
2.07
2.07
1 .98
1 .98
0.49
0.49
0. 26
0.26
Aug.
0. 19
0.20
3 .77
2.78
2. 16
1 .97
1 .33
1. 33
0. 56
0. 56
0.38
0.38
Sept.
0. 12
0. 12
2.83
2.8 1
1 . 20
1 . 18
I .39
1.39
0.75
0.79
0. 17
0. 16
Oct.
0.03
0.03
5.04
5.03
2. 29
2.33
2. 53
2.55
0.44
0.44
0.32
0.31
Mean3
0.06
0, 06
3.23
3. 14
1 .53
1 .59
1 .00
1 .00
0. 34
0.34
0. 16
0. 17
SDa
0.09
0.09
1 .88
1 .83
0.98
1 .03
0.74
0.74
0.26
0. 26
0. 14
0. 14
High
0.39
0.39
10.59
10.59
4. 23
3.95
3.01
3.01
1 .03
1 .08
1 . 10
1 . 10
Low
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
3 based on weekly samples
-------
.Table A2. ROUTINE WATER QUALITY
Bluegill chronic test
i
V
1
N
Mean
Range
D.O., mg/l |
PH j
Alkalinity, mg/l y
Acidity, mg/l |
Hardness mg/l I
i
1008
2 52
252
252
252
6.1
7 .80
157
6.4
26 5
3.8-7.3
7.62-8.20
142-172
0.3-13. 1
158-309
Table A3 . MEAN MONTHLY TEMPERATURES
Bluegill chronic test, degrees C.
Month
T j Month
I
T
—-I
Month
T
Jan.
23.0
Sept.
26.4
i
May
25.0
Feb.
24.2
Oct.
27. 1
June
25. 1
Mar.
25.0
Nov.
26.0
July
27 . 2
Apr.
24.7
Dec.
26.9
Aug.
27 . 2
May
25.6
Jan.
24.7
Sept.
26 .6
June
26. 1
Feb.
24.4
Oct.
July
26.2
Mar.
24.4
Nov.
Aug.
26.6
Apr.
25.7
-80-
-------
Table A4. BLUEGILL SURVIVAL- CHRONIC TEST
Tank Number
Date
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4B
5A
5 B
71,
Dec. 1
20
20
20
20
20
20
20
20
20
20
-
-
72,
Mar. 1
20
20
17
13
20
17
17
17
20
20
49a
II3
May 1
19
20
17
12
20
17
17
17
19
20
34
10
O
o
r"t"
I—^
19
19
17
13
20
17
17
1 5
18
1 7
25
10
73,
Mar. 1
18
19
17
13
20
17
17
15
14
14
12
10
May 1
13
17
17
13
20
17
16
15
9
13
12
10
July lb
10
15
17
13
20
17
16
15
9
13
10
9
Nov. 1
9
9
8
8
1 1
9
9
9
8
9
7
7
aEleven of same stock started in Tank 5B, Feb. 25, 1972.
49 from a new stock started in Tank 5A, Feb. 25, 1972.
^Later thinned to 3 males, 7 females per tank.
-------
Table A5. BRAIN AC ETYCHOLIN ESTERASE -
DLUEGILL 18 MONTH EXPOSURE
(orncl/min/g wet wt)
Tank
1
2
3
4
5
0
1.26
3 . 14
3.03
6.34
7.35
12.97
1.36
3.47
2. 92
7.40
7 .43
7. 16
1.28
4. 07
3.22
5.88
5.07
1 .74
4. 22
3.19
6 . 74
5.35
1. 23
4. 98
2. 10
6.04
1 .66
4.35
2. 15
2.16
4. 23
2. 94
0.76
3 . 26
3.78
1 .22
4. 14
3. 17
1.03
2.32
2.51
2.46
Mean
1.37
3 . 35
2.86
6.59
7.39
7.32
°/c of Control
18.7
.45.8
39. 1
90.0
101
100
-82-
-------
Table A6 . BRAIN7 ACETYLCHOLINESTERASE -
BLUEGIT.I. 23-MONTH EXPOSURE
Oaniol/min./g wet vvt)
Tank
1
2
3
4
5
0
1 .09
2.07
1.21
3.64
8. 15
4. 53
1 . 15
1.41
1 .75
5.37
5.85
9. 76
1.95
2. 53
1 .38
3.07
7 .46
12.20
1.92
7.49
.Mean
1 .40
1.S8
1 .45
4.03
7.15
8.50
% of Control
16 . 5
23 .3
17.0
47 .4
94. 1
100
-83-
-------
Table A 7 . BROOK TROUT ACUTE TEST
Number Surviving Per Tank
Hour
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4 B
5A
5B
0
10
10
10
10
10
10
10
10
10
10
10
10
24
10
10
0
0
10
10
10
10
10
10
10
10
48
10
10
0
0
10
10
9
10
10
10
10
9
96
10
9
0
0
5
4
8
9
10
9
10
9
Analyzed Parathion Concentration,
mg/1
0
nd
nd.
3 .30
3.06
1. 59
1.59
0.88
0.91
0.43
0
.47
0
.21
0
. 25
48
nd
nd
4.84
4.60
1.96
2.11
1 .06
1.00
0.51
0
. 52
0
. 24
0
. 28
96
nd
nd
4.41
3.79
1.94
1.96
1 .04
0.86
0.43
0
.42
0
. 25
0
. 26
Mean Weight of Fish, g
81.1 83.8 86.0 78.2 83.6 89.6 73.1 8Z.4 68.4 87.7 74.3 82.9
-------
Table A8 . ROUTINE WATER QUALITY - BROOK TROUT CHRONIC
1 N
1
Mean
Range
D.O., mg/l
CD
8.2
6.7-10.5
pH j 280
7.81
i
30
O
Alkalinity, mg/l
312
154
1 50-183
Acidity, mg/l
00
6.0
0
1
o
o
Hardness, mg/l
284
295
256-342
Table A9. MEAN MONTHLY TEMPERATURES - BROOK TROUT CHRONIC
(±2CC)
Month
Month
Month
May
June
July
Aug.
14.0
13.8
14.2
15.8
Sept.
Oct.
N ov.
Dec.
16.7
15.0
13.4
13.3
Jan.
Feb.
March
13. 1
13.6
14.2
-85-
-------
Table A10. MEAN MONTHLY ANALYSIS OF PARATHION IN WATER
BROOK TROUT CHRONIC TEST
(jJg/1)
OA
OB 1A
IB
2A
2 B
3A
3 B
4A
4B
5A
5 B
0.07
0.04
0.04
0.02
0.02
0. 16
0.02
0.01
0.01
0.05
0.01
0.07
0.05
0.03
0.01
0.02
0. 16
0.02
0.01
0.01
0.07
0.01
9.50
6. 12
9.49
11 .86
1 ,
7 ,
3 .
3 .
6
9
8.40
6.40
9.,52
11 .70
32
19
68
77
62
30
1 .
7.
4.
3,
6.
9,
32
19
84
77
62
73
3.41
2.70
4.43
5.36
0.80
3.77
2. 16
2.15
3.06
4.96
12.34 12.34 6.03
3. 27
2. 57
4. 50
5.30
0.80
3.77
2.
2.
3.
4.
6.
54
15
06
53
02
1.84
1.59
2. 94
3.84
0.59
2. 56
0.94
0.72
, 13
, 25
,43
1 .83
1 .53
2.84
3.84
0.59
2. 56
0.89
0.72
2.13
3.26
4.43
0.86
0.63
0.76
0.91
0.3 1
1.11
0.83
0.42
1.12
1 .49
1 . 92
0.94
0.63
1.07
0.90
0.3 1
1.11
0.83
0.42
1.12
1 .47
1 .92
0.40
0.46
0.59
0. 36
0.11
0. 29
0.21
0. 1 1
0. 13
0.60
1 .07
0.46
0.44
0.62
0.42
0.11
0. 29
0.21
0,
0,
11
13
0.65
1 .07
Test mean 0.05
Number 45
SD 0.11
High 0.23
Low nil
0.05 7.12 7.17 3.38 3.35 2.12 2.10 0.88 0.92 0.34 0.36
45 45 45 45 45 45 45 45 45 45 45
0.1 1 4.34 4.17 1.99 1.98 1.44 1.41 0.62 0.60 0.31 0.32
0.23 16.90 14.00 6.74 6.82 5.01 5.01 2.30 2.30 1.06 1.14
nil 0.28 0.28 0.62 0.62 0.06 0.06 0.19 0.19 0.02 0.01
-------
Table All. BROOK TROUT SURVIVAL IN CHRONIC EXPOSURE
Tank
OA
OB
IA
IB
ZA
ZB
3A
3 B
4A
4B
5A
5 B
Date
May 1
14
14
14
14
14
14
14
14
14
14
14
14
June 1
14
6
10
, 7
14
13
oa
1Z
13
13
14
14
July 1
14
5
8 '
6
14b
!b
°b
1Z
13
13
14
13
July 19
14
5
8
6
15
9
1Z
1Z
13
13
14
13
Aug. 1
14
4
8
6
15
7
9
1Z
7
1Z
14
13
Sept. 1
14
4
8
6
15
7
9
1Z
7
1Z
14
13
Oct. 1
14
4
8
6
1Z
7
8
1Z
6
1Z
14
13
Oct. Z5°
14
4
8
6
1Z
6
7
1Z
6
1Z
14
13
Nov. 1
6
6
6
6
6
6
6
6
6
6
6
6
Dec. 1
6
6
6
6
6
6
6
4
5
6
6
6
Jan. 1
6
6
6
6
6
6
6
4
4
6
6
6
Teb. 1
6
6
5
6
5
6
6
4
4
5
6
6
Feb. 6
6
6
5
5
5
6
6
4
4
5
5
6
^Complete mortality from low flow and dissolved oxygen, May Z7.
One remaining fish from ZB moved to 2A on July 19.
Nine new fish added to ZB, twelve added to 3A.
c
Thinning. All tanks reduced to Z males, 4 females.
All fish from ZB and 3A removed. Six fish transferred from each corresponding duplicate.
-------
Table A12. BROOK TROUT WEIGHTS - CHRONIC EXPOSURE
Tank
OA
OB
1A
IB
2A
2 B
3A
3 B
4A
4B
5A
5B
May 1
Mean weight,
Number
SD
g'
63.9
14
12.8
62.8
14
11.1
60.6
14
8.8
62.6
14
13.6
60.6
14
6.9
67 .6
14
9.5
60.0
14
8.4
68.2
14
8.7
67.0
14
10.5
59.3
14
8. 1
59.'9
14
8.2
63.0
14
10.0
Aug. 25
Mean weight,
Number
SD
g
182.3
14
32.8
201.9
4
64.8
179.7
8
42.5
204.2
6
25.2
163.8
15
17.4
-
-
198.2
12
19.2
209.0
7
28.6
184 . 9
12
21. 5
172.4
14
20.4
183.3
13
31.9
Feb.
Mean weight,
Number
SD
g
331.7
6
80.7
439.7
6
78.3
347.6
5
72.8
436.2
5
78.6
370. 2
5
119.9
290. 5
6
47.7
394.5
6
100.6
320.8
4
17.3
358.8
4
149.7
337.8
5
63.7
351.2
5
45.9
416. 2
6
74 .6
-------
Table A 13 . BROOK TROUT GONADOSOViATIC INDZX
Six-Month Exposure
($
Tank
OA
1A
2B
3A
4A
4 B
5A
53
Female ' 0.21
0.28 1.27
3.87
0.30
0. 33
0.31
J 0 . 34
t
i.
0. 24
3.11
0.36
9.75
0.34
4. 25
7. 10
5.01
i
j
5.65
2.74
Male
¦ 0.78
1.08
0.96
1 .32
1.53
2.26
1 .74
? 0.53
1.90
2. 38
1 . 19
1 .29
7 . 16
0.39
: 0.37
1. 12
1. 24
0.29
2. 18
; 2.88
1.88
1. 25
1.73
2.51
-89-
-------
Table A 14 . BROOK TROUT GONADOSOMATIC INDEX
Nine-Month Exposure
(percent)
OA
OB
1A
IB
2A
2 B
3A
3B
4A
4 B
5A
5B
0.77
0.65
0.05
0.68
0.02
0.41
0.48
0.70
0.39
0.05
0.51
0.7;
0.51
0.48
0. 58
0,62
0.04
nil
0. 14
nil
0.49
nil
0.47
o. n
1 .26
nil
1 .49
nil
0.67
1.00
0.46
0. 53
0.70
0.33
0. 51
0.50
0.28
0.35
nil
0.43
0.63
0. 58
0. 57
0.50
0.4.
0.40
0.41
0. 26
3. 39
1.68
0.45
0.26
1.06
0.38
0. 18
2.36
0. 14
0. 3i
0.34
0.56
0.70
0.0;
-------
Tabl» A1 5. SUMMARY CP RESIDUE RESULTS FOR BLOOD AXD MUSCLE Or TROUT
EXPOSED TO PARATHION FOR 6 and 9 MONTHS.
Avg.
Water . , . Cone.
Tank Cone. No. of Months of -.~s_i ¦u-"~i Ratio
No. ppb Fish Exposure Blood(B) Tissue(T) B/'T
21 7.1 2 6 i 3 C 2 3842 0.34
5 9 805 1260 0.64
22 3.4 3 6 788 1884 0.42
5 9 361 435 0.83
23 2.1 5 6 603 1142 0.53
5 9 256 306 0.84
24 0.90 3 6 126 419 0.30
5 9 107 59 1.81
25 0.35 4 6 68 171 0.40
5 9 43 52 0.83
-91-
-------
Table A16 . ROUTINE WATER QUALITY
Fathead Minnow Chronic Test
N
Mean
Range
>
r
D.O., mg/l 1
384
7. 1
8.1-6.3
PH \
90
7 . 94
8.05-7.78
Alkalinity, mg/l ¦
90 '
153
168-135
Acidity, mg/l j
90
2.5
9.1-05
Hardness, mg/l j
1
}
90
238
261-194 •
Table A 17 . MEAN MONTHLY WATER TEMPERATURE
Fathead Minnow Chronic Test
April
00
Sept.
24. 2
May
23 . 9
Oct.
24.3
June
23.5
Ncv.
23.5
July
23.8
Dec.
24.3
Aug.
23.2
-92- .
-------
Table A 18. MEAN MONTHLY ANALYSIS OF PARATHION IN WATER
Fathead Minnow Chronic Test
(j/g/i)
Tank
OA
OB
1A
IB
2A
2 B
3A
3 B
4A
4 B
5A
5B
April
0.30
0. 24
50.76
53.47
20. 17
24. 30
23. 13
24. 00
12. 90
13.37
4.31
4.75
May
0.04
0.04
42.06
42.06
19.87
19.87
13. 10
13. 10
6.80
6.80
4.88
4.88
June
0. 12
0. 12
59.39
59.39 .
26.56
26.56
17.36
17.36
11.07
1 1 .07
4.82
4.82
July
0.07
0.06
53.88
46.33 '
20.86
21.41
15.11
10.82
8.99
8 . 26
3.53
3.60
Aug.
0.11
0.06
41.17
42.94 -
18.08
19.96
13.28
14. 57
8 .77
7.78
4. 98
5. 56
Sept.
0.34
0. 30
48.62
48 .68
20.06
17.75
14.48
13.00
8.39
7.48
4.43
4.05
Oct.
0.06
0.06
40. 25
39.06
18.82
18.3 1
10.85
10. 58
5. 37
5.39
3 . 39
3.41
Nov.
0.02
0.02
58.28
58. 12
28. 52
27. 99
16. 13
15.31
8 .44
6.83
2.64
3.85
Dec.
0.02
0.04
55 . 56
53.25
27.07
23.50
15.00
12.21
7.98
8. 18
4.25
3 . 97
Mean
0. 14
0.12
49.40
48.53
21.51
21 .94
15.61
15. 36
8.95
9.02
4.46
4.36
SD
0. 14
0. 18
18.08
15.06
8. 16
7. 18
7.55
7.40
4. 14
2.87
1.55
1.18
High
0.82
0.82
104.20
104.20
44.86
44.86
38.70
38.70
17.90
17.90
7.30
7.30
Low
nil
nil
8.61
16.81
3. 19
6.70
2.06
3.34
2.8 1
2.24
1 . 13
1 .60
-------
Table A 19. GROWTH AND SURVIVAL OF FATHEAD MINNOWS IN PARATHION
30 days
60 days
Number
Mean
Number
Mean
Mean
Tank
present
length, mm
present
length, mm
growth, mm
OA
20
14.7
19
22.6
7.9
OB
33
14.6
28
22.3
7.7
1A
27
13.8
23
20. 2
6 .4
IB
28
15.2
27
23.0
7.8
2A
24
14.9
24
19.7
4.8
2B
28
14. 1
27
19.6
5.5
3A
25
15.5
23
23 . 2
7.7
3 B
28-
13.8
26
19.3
5 . 5
4A
26
16.0
23
22.4
6.4
4B
31
14.5
29
20.4
5.9
5A
23
16.4
23
23 . 3
6.9
5 B
26
14.8
22
23.8
9.0
number at start - 35 per tank
-------
Table AZO. D. MAGNA ACUTE TEST
Flow-through, 18°C
Number Surviving
Tank
OA
OB
1A
IB
2A 2B
3A
3 B
4A
4B
Start
10
10
10
10
10 10
10
10
10
10
24 hr
10
10
7
7
10
7
7
10
9
9
48 hr
10
10
0
0
8
4
7
6
9
9
72 hr
10
10
0
0
8
3
7
5
9
9
96 hr
10
10
0
0
5
3
7
4
9
9
Psrathion,
M?/1
0.11
0. 10
1.56
1. 55
0.85 0
.86
0.45
0.44
0.23
0.24
LC50, ug/1 24
hr =
2.70 (5.61
- 1.30)
48
hr =
1.00
(1 .73
-0.58)
96
hr =
0.62
(O.SO
- 0.43)
-95-
-------
Table A 21. D. MAGNA CHRONIC TEST
Number Surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4B
5A
5B
Start
10
10
10
10
10
10
10
10
10
10
10
10
1 wk
10 A
9A
0
0
0
. 0
10
9
10
10
10
10
89 Y
34 Y
2 wk
8 A
6A
0
0
0
0
8A
8A
10 A
9A
10A
10 A
476 Y
589Y
27 1Y
233Y
387 Y
219Y
375Y
4 16 Y
3 wk
7 A
3A
0
0
0
0
OA
OA
9 A
8A
10A
9 A
205Y
5 1Y
103 Y
176Y
268 Y
280 Y
1 14Y
367 Y
Total young
770
674
—
—
—
—
374
409
655
499
489
783
Parathion Analysis, pg/\
t
Start
0.01
0.02
0.80
0.79
0.37
0. 38
0. 28
0.27
0. 16
0. 16
0.09
0.09
1 wk
0.02
0.02
0.72
0.70
0.31
0.30
0.20
0.20
0. 10
0. 10
0.08
0.08
2 wk
0. 02
0. 02
0.85
0.85
0.48
0.47
0. 23
0.23
0. 10
0. 10
0.08
0.07
LC50, fjtg/1
1 wk
CO
fM
O
11
(0
.29 -
0.26)
RI50 =
0.24 (0
.32 -
0. 17)
2 wk
= 0.25
(0
.22 -
0.28)
MATC
= 0.08 ug/1
3 wk
= 0. 14
(0
16 -
0. 13)
Adults - A
Young - Y
-------
Table A22. GAMMARUS ACUTE TEST
Number Surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4B
Start
10
10
10
10
10
10
10
10
10
10
24 hr
10
10
6
5
7
6
10
10
10
10
48 hr
10
10
0
0
0
0
9
10
10
10
7 2 hr
10
10
0
* 0
0
0
8
7
9
10
96 hr
10
10
0
0
0
0
5
5
8
10
Parathion,
/fi/l
0.08
0.09
2. 12
• 2.09
1. 13
1. 14
0.44
0.43
0. 19
0. 19
LC50, ug/I - 24 hr = 2. 1 (3.8 - 1 .2)*
48 hr = 0.62
96 hr = 0.43 (0.65 - 0.29)
for juveniles at 20°C
-------
Table A23. GAMMARUS SUB-CHRONIC TEST
20°C
Number Surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3B
4A
4 B
Start
20
20
20
20
20
20
20
20
20
20
8 days
20
20
0
14
16
17
16
15
19
18
23 days
16
19
0
0
1
0
4
7
15
13
30 days
15
15
0
0
0
0
0
1
6
7
36 days
13
14A3
0
0
0
0
0
1
6Aa
7A
8Y
5Y
4Y
43 days
13
14A
0
0
0
0
0
1
3A
7 A
Mean
parathion
cone.
i/g/i
0.03
0.02
1 .64
1.65
0.24
0.24
0. 15
0.15
0.04
0.05
LC50 - 43 days = 0.07 (0.50 - 0.01) pg/l aAdults - A
young - Y
-------
Table A24. C. TENTANS ACUTE TEST
Number Surviving -
Tank
OA
OB
1A
IB
2A
2B
3A
3 B
4A
4B
5A
5B
Start
10
10
10
1 0
10
10
10
10
10
10
10
10
24 hr
10
10
10
10
10
10
10
10
10
10
10
10
48 hr
10
10
5
8
6
9
10
10
10
10
10
10
7 2 hr
10
9
2
2
5
7
9
9
10
8
10
10
96 hr
10
9
0
0
5
4
9
8
9
8
10
10
Analyzed Parathion, yg/l
Start
0. 14
0. 14
66.3
66.3
33.6
33.6
17.6
17.6
5.7
5.7
5.8
5.8
72 hr
0.09
0.09
48 .6
48.6
33.0
33.0
17 . 2
17.2
6.2
6.2
4.0
4.0
LC50, ug/l 48 hr - 135 (703 - 26) for 4th instar larvae at 21°C
96 hr = 31.0 (43.4 - 22. 1)
-------
Table A25. C. TENTANS SUB-CHRONIC TEST
Number Surviving
¦ — r ¦
Tank i OA
1
OB
1A
IB
2A
2B
3A
3C
4A
4B
5A
5B
Start
10
10
10
10
10
10
10
10
10
10
10
10
4 days
10
10
6
8
10
8
10
9
9
10
9
9
7 days
10
0
4
8
6
7
9
8
6
8
7
14 days
10E
10E
0
0
0
0
3
4
3
1
3
4
Mean
parathion
cone.,
ug/l
0.3
0.3
5 2.9
52.9
23.6
23.6
11.0
11.0
5.0
5.1
3. 1
3. 1
E - emerged LC50 - 14 days = 2.3 (3.4 - 1.6) ug/l
-------
APPENDIX B - BROWN TROUT TESTS
Young brown trout, Snlmo trutta Linnaeus, 16 to 19 err; total length, were
acutely exposed to parathion in an LC50 test and also in a residue uptake
test. The acute LC50 test method was identical to that of the brook trout.
The LCoO results for 1.4 and 96 hours (Table Bl) show essential agreement
with those for brook trout. Symptoms of parathion poisoning v/ere also
similar.
In a separate test, brown trout of the same stock were exposed to the
two highest parathion levels which had caused no acute mortalities - 0.6 8
and 0.47 mg/1. A total of 45 fish were used in each of the two tanks. During
the parathion exposure period, three fish were removed from each tank at
selected intervals up to 64 hours. At 65 hours, the remaining fish v/ere
transferred to two duplicate tanks with uncontaminated flowing water. Again
three fish per tank v/ere removed at time intervals until all fish were gone
at 79 hours. Muscle samples dissected from the dorsal fin region were
preserved at -18°C and later analyzed for parathion residues. The residue
values shown in Table BZ are each the mean of true duplicates. Precision
for the clear.-up and chromatographic method was ±5%.
A simple model was applied to the data from the 0.47 mg/1 exposure and
plotted (rigure Bl). Assuming first order kinetics, the change in fish
residue, C, can be expressed as a function of Cw, the toxicant concentration
in water:
dc/dt = kjCw- kpC
c = k,cwA2 H
-------
Table Bl. BROWN TROUT ACUTE TEST
Number Surviving Per Tank
Hour
OA
OB
IA
IB 2A
2B
3A
3B
4A
4B
5A
5B
0
10
10
10
10 10
10
10
10
10
10
10
10
24
9
10
6
0 10
10
10
10
10
10
10
10
48
9
10
0
0 9
10
10
10
10
10
10
10
96
9
10
0
' 0 5
1
10
10
10
10
10
10
144
9
10
0
0 0
0
9
6
10
1
10
10
Analyzed Parathion Concentration, m
g/i
Pooled daily
nd
nd
2.40
2.40 1.63
1.63
1.04
1.04
0.68
0.68
0.47
0.47
Brown Trout Acute LC50
Hour
LC50,
Upper CL,
Lower CL,
mg/l
mg/l
mg/l
24
2. 15
2.34
1 .97
48
1 .93
2.01
1.85
96
1.51
1.65
1.38
144
1. 13
1.03
1 .24
-------
.Table B2. PARATHION RESIDUES IN BROWN TROUT MUSCLE
Parathion
in water, mg/l
0 .47
0.68
Uptake
2 hr
4.10
5.48
residues,
4. 53
10.61
mg/kg
4 hr
4.87
7.48
6 .48
8 hr
3.11
10.91
4.63
12.45
16 hr
9.08
7.98
11.95
16.66
32 hr
21.78
15.79
23.64
43.34
64 hr
22.88
r 46.49
34.80
57.99
Washout
2 hr
64.05
residues,
72.85
mg/kg
3 hr
22.74
42.21
4 hr
29.44
66.92
33.24
4.. 5 hr
8.27
27 .66
16 hr
22.38
41.78
33.94
44.79
35 hr
7.90
22.53
26 . 23
42.50
64 hr
23.14
21.29
25.60
55.74
77 hr
28. 13
22.76
44.52
78 hr
13.89
18.96
27 .38
48.35
79 hr
12.59
36 . 20
32.77
47.96
True duplicates analyzed at each time period.
Precision ± 5%
-103-
-------
Parathion uptake and washout in brown trout muscle, 12°C
-
.
<
bB,b0.0®»bd01
• .....
: s
"** 1" o ©
i© e **
• Jr
o
©
j&r
• 9
"• ff
fj
C=kiCw kiCvv -k2t-
k2 +(Co" k2 )e ¦
ki =1.00 hr"1
k2 =0.002 hr"1
a 0.5 mg/i Parathion Exposure—
Clean Water Exposure —»-
B 1 1 1 I 1 1
3 10 20 30 40 ^0 60 <*0 80
1 I I 1 1 1 1 1
10 30 50 70 90 110 130 150
ELAPSED TIME, hours
-------
APPENDIX C - CHLORINATED HYDROCARBON PESTICIDES IN FISH FOOD
Samples of fish food used for chronic tests were analyzed for chlorinated
hydrocarbon pesticides. Initial attempts to attain effective clean-up
using standard Fiorisil chromatography failed with these food samples.
We subsequently used the silicic acid chromatography procedure reported
by Kadoum.'16 rood samples were extracted with hexane in the presence
of Na?S04. Concentrated extracts were applied to columns of silicic acid
(Mallinkrodt 100 mesh, chromatography grade) and eluted with hexane and
2% 10%, 40%, and 70% benzene in hexane and benzene. Fractions were
pooled, evaporated and rechromatographed a second time with collection
of individual fractions: hexane, benzene, 10% benzene. 40% benzene,
70% benzene, and benzene. These were analyzed by GC on 3% OV - 17
on Chromosorb W at 195°C using electron capture detection. Standard
solutions of 13 chlorinated hydrocarbon pesticides were chromatographed
to provide retention times (RT) for identification purposes and peak areas
(as counts) for quantification. The conditions of elution from the silicic
acid and the GC RT's were used for tentative identifications. Good
matches, however, were not obtained for heptachlor epoxide and lindane
and nearest peaks were used; all peaks eluted in the DDT and DDT
degradation product area were summed and calculated as DDT + DDD + DDE.
These, therefore, are maximum values. From the blood samples, resolvable
peaks distinct from the interferences could not be obtained in the retention
time regions of dieldrin or endrin in the 70% benzene fraction. The
presence a: low level (ppb) or absence of these pesticides could not be
verified. The overall results are summarized in Table CI. In general
we conclude that chlorinated hydrocarbon pesticide levels were low in
each food source used and should not have significantly affected our
tests.
-105-
-------
Table CI. PESTICIDE RESIDUES IN FISH FOOD
(ugAg)
Food Sample
Aldrin
Heptachlor
DDE
DDD
DDT
I-Ieptachlcr
Epoxide
Lindane
(c)
Dieldrin
Endrm
Starter Mash < 1 ,
No. 3 Pellet nil
No. 6 Pellet nil
No. 5 Pellet (a)
<0. 1
<0.2
<0.1
<0.1
<0.5
<0.5
<0.5
<0.5
nil
nil
nil
<1
<0.3
<0.3
<0.6
< 1
ND
ND
ND
ND
(a) a GLC peak near the Retention time (RT) of aldrin, but probably net aldrin, gave
a value of 10-15 ppb calc. on the basis of aldrin.
(b) No true natch of R.T. with standard; nearest GLC peak taken as Heptachlcr epoxide
for calc. of No. 5 sample.
¦(c) No true match of R.T. with standard; nearest peak taken as lindane for calc. R.T.
was 256 sec. vs. 249 sec. for lindane stdz. Peak may bs a phthalaie ester,
(d) These pesticides could not be detected (ND) unless present at very high levels
because of broad peaks of relatively high level interferences present in the 70%
benzene fraction.
-106-
------- |