ACUTE AND CHRONIC PARATHION TOXICITY
TO FISH AND INVERTEBRATES
by
Anne Spacie, Algirdas G. Vilkas, Gerald F. Doebbler,
William J. Kuc and Gerald R. Iwan
Union Carbide Corporation Environmental Services
Contract No. 68-01-0155
Project Officer
John G. Eaton
National Water Quality Laboratory
Duluth MN 55804
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON DC 20460
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DISCLAIMER
This report has been reviewed by the Office of Research and Monitoring,
U.S. Environmental Protection Agency, and approved for publication. Approv-
al does not signify that the contents necessarily reflect the views and pol-
icies of the U.S. Environmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or recommendation for
use.
ii
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FOREWORD
t
This report constitutes the last in a series describing research
projects intended to; I) generate additional freshwater aquatic life
toxicity data useful for evaluating environmental hazards of commonly used
pesticides; 2) advance the state-of-the-art of chronically testing the
species involved; 3) evaluate the ability of relatively inexperienced
investigators to successfully accomplish chronic studies with these species
and thus the advisability of recommending them for routine tests through such
documents as the Pesticide Registration Guidelines; and 4) provide data
required to evaluate the validity of'the application factor hypothesis.
These studies have been useful in the above regards and have therefore
contributed importantly to both basic and applied toxicology.
John G. Eaton, Project Officer
Environmental Research Laboratory-Duluth
Lii
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ABSTRACT
Acute and chronic aquatic bioassays were conducted with a variety of
aquatic organisms using parathion [0,0-diethyl Q-(p-nitrophenyl) phosphoro-
thioate] as the challenge compound. The bioassay organisms challenged in
this study and their resultant 96 hour LC50 values were: bluegill sunfish,
Lepomis macrochirus Rafinesque, 0.51 mg/1; brook trout, Salvelinus fontinal-
is (Mitchill), 2.00 mg/1; fathead minnow, Pimephales promelas Rafinesque,
1.6 to 0.5 tng/1; water flea, Daphnia magna Straus, 0.63 ug/1; scud, Gammarus
fasciatus Say, 0.4 ug/1; and midge, Chironomus tentans (Fabricius), 31.0
ug/1. Non-lethal effects such as deformities and acetylcholinesterase de-
pression were documented in bluegill chronically exposed for 23 months to
parathion concentrations in excess of 0.17 ug/1. Adult brook trout exhibit-
ed no toxic symptoms at 7.2 ug/1 parathion exposure but egg hatchability was
reduced at an exposure concentration of 32 ug/1. Chronic exposure of the
fathead minnow to a parathion concentration of 4 ug/1 for 8-1/2 months re-
sulted in adult deformities and decreased egg production. Parathion resi-
dues detected in exposed bluegills were 5 to 25 times that of the exposure
water while trout exhibited a bioconcentration of several hundred times that
of exposure water. Parathion residues in the fathead minnow were detected
as intermediate between bluegill and trout. Juvenile and adult stages of
chronically exposed fish appeared to be the most sensitive to parathion.
The chronic no-ill-effect concentration for D. magna was 0.08 ug/1, for G.
fasciatus it was less than 0.04 ug/1 and for C. tentans it was less than 3.1
ug/1. Mean LC50 concentrations for D. magna (48 hour) and G. fasciatus (96
hour) were 1.13 ug/1 (n = 2) and 0.39 ug/1 (n = 4) respectively.
iv
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgments ix
I. Introduction 1
II. Conclusions 3
III. Recommendations 4
IV. Materials and Methods 5
Water supply and environmental control 5
Fish bioassays 7
Invertebrate bioassays 12
Chemical analysis and statistical methods 14
V. Results 17
Bluegill 17
Brook trout 24
Fathead minnow 36
Daphnia magna 40
Gammarus fasciatus 41
Chironomus tentans 41
VI. Discussion 43
References 46
Appendixes
A. Bioassay data 50
B. Brown trout tests 64
C. Chlorinated hydrocarbon pesticides in fish food 68
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FIGURES
Number Page
1 Concentration factors for parathion residues in whole
bLuegills at various exposure times 19
2 Male bluegill exposed to 1.5 ug/1 parathion for 23 months 21
3 Two adult bluegills exposed to 1.5 ug/1 parathion for 23 months. .22
4 Parathion in brook, trout blood vs. parathion in water, six
month exposure 29
5 Parathion in brook trout muscle vs. parathion in water, six
month exposure 31
6 Brook trout brain acetylcholinesterase activity vs. parathion
in water, six month exposure 33
7 Parathion in brook trout blood vs. parathion in water, nine
month exposure 37
B1 Parathion uptake and washout in brown trout muscle, 12°C 67
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TABLES
Table Page
1 Analysis of Test Water 5
2 Summary of Chronic Test Conditions 7
3 Chronic Test Photoperiod and Schedule 8
4 Bluegill Acute Test 17
5 Parathion Residues In Whole BLuegills, Acute Test 18
6 Incidence of Deformities, Bluegill Chronic Test 20
7 Bluegill Chronic Exposure to Parathion, Spawning Records . . . .23
8 Survival and Lengths of Bluegill Larvae 25
9 Parathion Residues in Bluegills, 18 Months Exposure 26
10 Parathion Residues in Bluegills, 23 Months Exposure. ...... .27
11 Brook Trout 96 Hour Acute LC50 .27
12 Parathion Residues in Brook Trout Muscle, Acute Test . .28
13 Parathion in Brook Trout Blood, Six Month Exposure . .28
14 Parathion Residues in Brook Trout Tissue, Six Month Exposure . . .30
15 Brain AChE Activity of Brook Trout, Six Month Exposure ...... 32
16 Brook Trout Spawning Record 34
17 Brook Trout Parathion Residues and AChE, Nine Months 35
18 Brook Trout Parathion Residues and AChE, Recovery 38
19 Fathead Minnow Acute Test, 24°C 38
20 Minnow Adult Weights and Deformities, 8.5 Months 39
21 Minnow Spawning Record 40
22 Parathion Residues in Whole Fathead Minnows 40
23 Parathion Toxicity to Daphnia magna 41
24 Parathion Toxicity to Gammarus fasciatus .42
25 Parathion Toxicity to Chironomus tentans 42
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TABLES, APPENDIX A
Number Page
Ai Mean Monthly Analyses of Parathion, Bluegill Chronic Test 51
A2 Routine Water Quality, Bluegill Chronic Test 52
A3 Mean Monthly Temperatures, Bluegill Chronic Test 52
A4 Bluegill Survival, Chronic Test 52
A5 Brain AChE, Bluegill, 18 Month Exposure .53
A6 Brain AChE, Bluegill, 23 Month Exposure .53
A7 Brook Trout Acute Test 54
A8 Routine Water Quality Brook Trout Chronic 54
A9 Mean Monthly Temperatures, Brook Trout Chronic 54
A10 Mean Monthly Parathion in Water, Brook Trout Chronic 55
All Brook Trout Survival in Chronic Exposure 56
A12 Brook Trout Weights, Chronic Exposure. ..............57
A13 Brook Trout Gonadosomatic Index, Six Month Exposure 57
A14 Brook Trout Gonadosomatic Index, Nine Month Exposure 57
A15 Blood and Muscle Residues, Trout, Six and Nine Month Exposure. . .58
A16 Routine Water Quality, Fathead Minnow Chronic Test 58
A17 Mean Monthly Water Temperature, Fathead Minnow Chronic Test. . . .58
A18 Monthly Analysis of Parathion in Water, Fathead Minnow Chronic . .59
A19 Growth and Survival of Fathead Minnows in Parathion. ...... .59
^20 D. magna Acute Test, Flow-Through, 18°C 60
A21 D. magna Chronic Test 61
A22 Ganroarus Acute Test 62
A23 Gammarus Sub-Chronic Test, 20°C, Number Surviving 62
£• tentans Acute Test 63
A25 C. tentans Sub-Chronic Test. 63
TABLES, APPENDIX B
Bl Brown Trout Acute Test 64
B2 Parathion Residues in Brown Trout Muscle 66
TABLE, APPENDIX C
CI Pesticide Residues in Fish Food. 69
viii
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ACKNOWLEDGMENTS
The authors would like to thank the staff of Aquatic Environmental Sci-
ences, especially G, A. Gary, E. C. Hintze and R. H. Sugatt, for long hours
of devoted work. E. Fritsche, E. Yeh and 1. Shields of the Central Scien-
tific 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, devel-
oped the chronic bioassay procedures used throughout this project and provi-
ded continuing advice and assistance.
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SECTION I
INTRODUCTION
Among organophosphorous insecticides, parathion is one of Che most per-
sistent and toxic. About 15 million pounds of it are used annually for con-
trol 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, leaching, erosion, irrigation, aerial
spraying and spills during manufacturing, formulation, and application.
Once in a water-soil system, its rate of degradation is variable and depen-
dent on pH, organic control, and microbial activity. Upon application to
soil, parathion may persist for three months (2), five 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 108 days, decreasing with increasing pH
and temperature (5). In contrast, natural river water degrades 50 percent
of parathion within a week (6). In natural lake sediments Graetz et al (7)
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, Nicholson (8) found reduced
populations of aquatic insects in nearby ponds containing 1.9 ppm in mud.
Killifish and freshwater mussels exposed on a treated cranberry bog showed
uptake of parathion residues (9). Parathion spills have also caused kills
of fish and amphibians. (10).
Like all organophosphates, parathion disrupts the central and peripher-
al nervous systems of animals when enzymatically converted to its oxygen
analog, paraoxon. Paraoxon then combines covalently with acetylcholinester-
ase (AChE) to block Che hydrolysis of acetylcholine, the transmitter sub-
stance in the synapses 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 inhib-
itor. However, the AChE-complex can gradually dealkylate, forming the phos-
phate anion which resists hydrolysis. Therefore, regeneration of AChE after
long-term exposure of parathion is extremely slow.
Weiss demonstrated AChE activity in fish (12), and its susceptibility
to paraoxon inhibition (13). Bluegills exposed to 0.1 mg/1 parathion for
ei ght hours recovered only 50 percent of normal activity in 30 days. Fat-
head minnows had a somewhat quicker response and recovery. Carter (14)
found that channel catfish with 50 percent inhibition from methylparathion
required 20 to 30 days Co regain 80 percent of normal activity. Studies of
AChE inhibition in vitro have found fish (15, 16, 17) and lobsters (18) to
be less sensitive than birds or mammals. Yet parathion is very toxic to
both fish and crustaceans. The two principal metabolite products in rats
1
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(19), paraoxon and diethyl hydrogen phosphorothioate, have also been found
in rainbow trout (16), The proportion of the two metabolites formed, and
their rates of formation, vary with species. Metabolism can be slow enough
to permit accumulation of parathion in tissues, as reported for trout (16),
brown bullheads (20), mosquitofish (21), fathead minnows (22) and oysters
(23).
Literature values for the acute toxicity of parathion to aquatic spe-
cies have been summarized by Kempt, Abrams and Overbeck (24). Only a few
studies have considered long-term effects. Mount and Boyle (20) found con-
vulsions in bullheads exposed to 0.03 mg/1 parathion for one month. Matsue
(25) observed retarded growth of goldfish at 0.1 mg/1 for one month. Re-
duced growth rates of oyster larvae in 0.05 mg/1 were found by Davis and
Hidu (26). After 40 days at 0.01 mg/1, male guppies tested by Billard (27)
showed reduced spermatogenesis. Jensen and Gaufin (28) reported a 309-day
LC50 of 2.2 ug/l for Pteronarcys californica and 0.44 ug/1 for Acroneuria
pacifica,
Past research 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 highest chronic concentration
of parathion that produced no harmful effects to brook trout, bluegills,
fathead minnows, Daphnia magna, Gammarus fasciatus and Chironomus tentans.
For each species, the maximum acceptable toxicant concentration (MATC), de-
fined by Mount and Stephan (29), was then related to the acute LC50. Be-
cause 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
fresh water.
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SECTION II
CONCLUSIONS
1. Juvenile bluegills were found Co have a 96-hour median lethal para-
Chion concentration (LC50) of 0.51 mg/1. A maximum acceptable toxicant con-
centration (MATC) between 0.17 and 0.34 ug/1 was established based on adult
deformities.
2. Brook trout had a 96-hour LC50 of 2.00 mg/1. Chronic exposure to a
concentration of 7.2 ug/1 produced no harmful effects. However, levels of
32 ug/1 reduced hatchability of embryos from previously unexposed parents.
3. Fathead minnows exhibited 96-hour LC50 values between 1.6 and 0.5
mg/1 and a MATC of approximately 4 ug/1 based on egg production data and
incidence of adult deformities.
4. Daphnia magna exhibited a mean 48-hour LC50 of 1.13 ug/1 and a MATC
of approximately 0.08 ug/1.
5. Gammarus fasciatus had a mean 96-hour LC50 of 0.39 ug/1. The MATC
was determined to be lower than 0.04 ug/1, a concentration which caused mor-
talities in one month.
6. Chironomus tentans had a 96-hour LC50 of 31 ug/1 based on a measured
parathion concentration in water. The MATC was determined to be less than
3.1 ug/1, a concentration which produced lethality in two weeks.
7. Under chronic conditions bluegill tissue concentrated parathion res-
idues at 5 to 25 times the exposed water concentration. Brook trout concen-
trated parathion several hundred times and minnows at rates intermediate to
the other two species. No parathion above background levels was detected in
D. magna.
8. Juvenile and adult developmental stages of chronically exposed fish
appeared relatively more sensitive to parathion induced effects than egg and
fry stages.
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SECTION III
RECOMMENDATIONS
1. The concentration of parathion applied to bodies of water should be
limited to levels acutely tolerated by crustaceans such as Daphnia.
2. Trout and other fish species collected from parathion-treated sites
should be examined further for the presence of potentially harmful parathion
res idue levels .
3. Other insecticides should be evaluated as possible alternatives to
parathion in regard to their selectivity, toxicity and persistence.
4. Food chain dynamics of parathion distribution in aquatic communities
should be investigated.
5. Metabolic pathway studies of parathion degradation in fish should
integrate the effects of temperature, rates of reaction, and proportion of
major metabolites.
6. Further histological characterization of parathion damaged fish tis-
sues would help to determine the mechanisms of such damage.
7. Further culture information is needed to ensure bluegill spawning
during chronic tests if such studies are to be continued with this species.
8. Five month chronic tests using Gammarus as the bioassay organism are
not practical unless better survival rates can be achieved in culture situa-
tions .
9. More efficient techniques are needed for conducting the egg incuba-
tion and larval survival phases of chronic bioassays.
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SECTION IV
MATERIALS AND METHODS
WATER SUPPLY AND ENVIRONMENTAL CONTROL
Water Supply
Water used for conducting the acute and chronic tests of the present
study was supplied from a 50-foot well located on the Tarrytown, New York,
laboratory site. Results of a chemical analysis of ambient temperature well
water according to the methodology of Standard Methods (30) are given in
Table 1. From the well, water was pumped to the testing laboratory through
cast iron and PVC pipe. At the laboratory the water was sterilized with
ultraviolet light and proportioned into lines of ambient (11-13°) and heated
water for distribution to each of the testing stations. Heated water uti-
lized for conducting relatively warm water tests was softened by exchange of
sodium for calcium. This "softening procedure" consequently resulted in a
20 percent hardness reduction in the bluegill chronic exposure.
TABLE 1. ANALYSIS OF TEST WATER
Concentration, Concentration,
Component mg/1 Component mg/1
Alkalinity
(as CaC03)
147
Lead
<0.0002
Aluminum
0.01
Magnesium
27
Ammonia (as
N)
0.15
Manganese
0.005
Arsenic
<0.017
Mercury
<0.0002
Barium
0.10
Nitrate (as
N)
1.7
Boron
0.02
Nitrite (as
N)
0.007
Cadmium
0.005
Phenol
<0.0016
Calc ium
76
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
Tin
0.01
Hardness (as CaC03)
297
Titanium
0.01
Iron
<0.0006
Zinc
<0.001
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Temperature Control
Separate lines of both hot and cold water supplied each test system.
Proportional mixing of the two thermal flows was regulated by a pneumatic
temperature controller with a sensing element in the mixing zone of the test
system supply pipe. The resulting test water temperature variation was with-
in +1°C for instantaneous changes, and +2°C for daily trends. The ambient
temperature in at least one exposure chamber per test system was continuously
monitored by a thermistor probe read by a 20-channel electronic temperature
monitor. All channels monitoring test systems had individual high and low
temperature set point designations which activated an alarm if exceeded.
Each hour, the temperatures at all channels were recorded on punch paper tape
and teletype.
Aeration
To diminish the possibility of well water supersaturated with gases from
entering the test systems and inducing variable mortality, water was aerated
by air-stone as it entered the first proportional diluter cell. In addition,
adult fish exposure chambers had supplemental aeration to ensure dissolved
oxygen concentrations greater than 60 percent saturation.
Toxicant Delivery
Reagent grade (99 percent pure) parathion [0,0-diethyl 0-(p-nitropheny1)
phosphorothioate) obtained from Pfaltz and Bauer was used in the preparation
of al 1 test stock solutions. Stock solutions of parathion in either water or
acetone were injected into proportional diluters of the Mount and Brungs de-
sign (31). Depending on test type, various injection devices were used: Sage
341 syringe pump, a Mariotte bottle with constant flow, mechanical syringe
pump (31), or LKB Perpex peristaltic pump. To prevent accidental injections
during diluter malfunctions, the electrical pumps were wired to operate only
during normal diluter cycles.
All components of the diluter in contact with exposure water were either
of glass, latex or Tygon tubing and silicone adhesive. The diluter produced
five concentrations of parathion, plus a control, each split into individual
duplicate flows by a mixing cell. Tanks were designated by the same number
as the corresponding diluter cell (31): "0" for the control, "1" for the
highest toxicant concentration used, "2" for the second highest, up to "5"
for the lowest. Duplicate tanks were individually identified by "A" or "B"
designations.
Exposure tank sizes and flow rates for each of the chronic test systems
are summarized in Table 2. The bluegill adult exposure tanks were construct-
ed of No. 306 stainless steel. Trout, minnow and Gammarus adult exposure
tanks were commercial glass aquaria with slate bottoms and outside stainless
steel reinforcement. All other test containers were of glass and silicone
adhesive (Dow Corning) construction.
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TABLE 2. SUMMARY OF CHRONIC TEST CONDITIONS
Adult tank Minimum flow
volume, per tank, Larval chamber Temperature
Test animal liters liters/hour volume, liters deg C
Bluegill
170
40
6*
25
Brook trout
85
30
6*
9-15
Fathead minnow
28
10
7 -k
28
Daphnia
3
0.8
18
Gammarus
15
0.5
18
Chironomus 3
*Two such chambers were used per adult tank. Minnow chambers were not true
duplicates. See page 12.
Lighting
All tests were illuminated by Duro-Test Optima fluorescent lamps. These
were regulated by Tork industrial timers to provide appropriate Evansville,
Indiana, photoperiods as shown in Table 3. The timer wiring was modified to
permit 30 minutes of gradual photointensity change at dawn and dusk (32).
All Daphnia and midge tests were conducted under a 16-hour light, 8-hour dark
photoperiod. Average light intensities at the test exposure systems were:
bluegil 1, 75 ft-c; brook trout, 70 ft-c; minnow, 82 ft-c*, and invertebrates,
70-80 ft-c. While photoperiods for bluegill and brook trout exposures cor-
responded to actual calendar dates, the minnow and invertebrate tests were
begun on the Evansville, Indiana, dates shown in Table 3.
FISH BIOASSAYS
Bluegill Sunfish (Lepomis macrochirus) Parathion Acute and Chronic Exposures
Bluegill sunfish (Lepomis macrochirus) used in the parathion chronic
bioassay were collected as 5-8 cm long (total length) juveniles from a natur-
ally occurring pond population located in Dutchess County, New York. Prior
to their use in the chronic exposure, these bluegills were acclimated to lab-
oratory conditions at the Union Carbide facilities in Tarrytown, New York,
for a period of three months. A second stock of immature bluegill sunfish,
taken from the same pond one year later, were isolated and acclimated at the
Union Carbide facilities and used for the flow-through acute studies. All
bluegills selected for testing were relatively free of internal parasites but
initially harbored gill ectoparasites which were subsequently eradicated dur-
ing the acclimation periods by therapeutic formalin treatments.
Bluegill parathion acute bioassays consisted of five (four preliminary
and one definitive) tests conducted at 22°C under flow-through conditions.
The acute definitive bioassay was performed using five challenge concentra-
tions of parathion plus a control in duplicate over a 144-hour exposure per-
iod in 85-liter glass aquaria.
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TABLE 3. CHRONIC TEST PHOTOPERIOD AND SCHEDULE
Lighc period,
Date hours:minutes* Event
Jan
1
10:30
15
10:45
Feb
1
11:15
15
11:45
Mar
1
12:15 —
15
13:00
Apr
1
13:30
15
14:15
May
1
14:45
15
15:15
June
1
15:30
15
15:45
July
1
15:45
15
15:30
Aug
1
15:00
15
14:30
Sept
1
14:00
15
13:30
Oct
1
12:45 -
15
12:15
Nov
1
11:30
15
11:00
Dec
1
10:45
15
10:30 J
•Begin Gammarus test
-Begin brook trout test
Gammarus reproduction
Fathead minnow spawning
-Biuegill spawning
-Begin biuegill test
¦Begin fathead test
-Brook trout spawning
^Includes 30 minutes of dawn and 30 minutes of dusk.
Parathion stock solutions for the flow-through acute tests were pre-
pared as aqueous sonicated emulsions consisting of 180 gm parathion and 8 gra
Triton X-100R (Rohm and Haas) per liter of water. Five challenge concen-
trations were prepared by volumetrically dispensing appropriate amounts of
parathion stock solution via peristaltic pump into a proportional diluter
which provided a flow of 30 liters per hour to each of the 35-liter test
aquaria .
Direct analyses of the parathion challenge concentrations in each of the
definitive test exposure aquaria were made at test initiation and at 48
hours. The definitive acute bioassay was cons idered started when stock
bluegills, acclimated to test conditions without feeding, were randomly
distributed to each of the duplicate challenge concentrations at a rate of
five per aquarium. At regular time intervals, during the 144-hour exposure
period, each of the exposure aquaria was examined for biuegill mortalities or
signs of toxicant related stress. At no time during the definitive test were
fish fed or was aeration provided to exposure aquaria.
The parathion biuegill sunfish chronic bioassay was conducted following
procedures from the "Recommended Bioassay Procedure for Biuegill Sunfish Le-
pomis macrochirus (Rafinesque) Partial Chronic Tests" of the National Water
Quality Laboratory, Duluth, Minnesota (revised January, 1972). A flow rate
8
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of 40 liters per hour was accomplished in each of Che exposure canks by use
of a Mount and Brungs type proportional diluter calibrated to provide nomi-
nal parathion challenge concentrations of 8.0, 4,0, 2.0, 1.0 and 0.5 ug/1.
Stock parathion was injected by syringe pump to the diluter apparatus as a
solution of 2 to 4 gm parathion and 0.24 gm Triton X-100 per liter of
acetone.
The parathion bluegill chronic exposure commenced on December 22, 1971,
with the random distribution of 20 bluegills into each of the exposure
tanks. The photoperiod was adjusted at the start to correspond to that of
Evansville, Indiana, in October (Table 3), All fish were fed Oregon Moist
Pellets and Silver Cup trout food ad libitum and had their lengths and
weights recorded at the beginning of testing, at thinning, and at the test's
termination.
During the first two months of exposure, bluegills exposed to the 8.0
ug/1 exposure concentration experienced tremors and convulsions. This ne-
cessitated termination of the exposure series after 64 days and a new 0.25
ug/1 exposure concentration was added to the chronic as a replacement.
Development of normal male territorial aggression and spawning behavior
during testing did not occur until the second summer. At that time all ex-
posure tanks had their bluegill populations thinned to 3 males and 7 fe-
males. Surplus fish from each exposure concentration were frozen at -18°C
for subsequent chemical analyses. To simulate spawning beds, a cement sub-
strate, 27 cm square by 5 cm thick with a central shallow depression, was
placed at the end of each exposure tank. Two black plastic curtains (55 x
40 cm) serving as visual barriers were also partially submerged wihin each
exposure tank to diminish aggression between spawning males.
During the spawning period, all substrates were checked daily for eggs.
Eggs from all individual spawns were collected and counted by volumetricallv
enumerating the number of eggs in one milliliter and multiplying by the to-
tal volume of eggs spawned. Two hundred eggs from each spawn were incubated
in egg cups constructed from bottomless 4-oz glass jars with 40 mesh nylon
screen glued to the open bottoms. All egg cups were suspended by an oscil-
lating rocker arm assembly in larval chambers corresponding to the exposure
tank in which the incubating eggs were spawned. The 6-liter larval chambers
were 13 cm wide, 35 cm long and 18 cm high and contained a 13 cm depth of
exposure water which was received directly from their corresponding adult
exposure canks via a continuous siphon system. After a three-day incubation
the hatched fry were counted and their numbers recorded and 50 live fry were
retained suspended in the larval chambers. The fry were retained for seven
days in the cups and fed newly hatched Artemia salina four times daily. At
seven days post hatch, fry were released into the larval chambers and mor-
talities were recorded weekly. Surviving fry in each exposure larval cham-
ber were counted and measured at 30 and 60 days post hatch. Measurements
were done by placing surviving fry from each larval exposure chamber in an
all glass chamber set over a light box having a millimeter transparent grid
and taking a photograph relative to the grid image. The test was terminated
after 23 months of parathion exposure and all remaining adult bluegills were
examined and frozen for residue analysis.
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Brook Trout
Yearling brook trout, Salvelinus fontinalis (Mitchill), for use in the
parathion acute and chronic exposures were obtained from Sullivan County,
New York. The trout, approximately 60 gm each in weight, were acclimated at
12°C for three weeks prior to chronic testing. Trout used in both the acute
and chronic tests showed no disease symptoms prior to testing. During
chronic testing, therapeutic treatment for fungus and furunculosis was ac-
complished using 20 ppm formalin, 4 ppm Furpyrinol or 40 ppm malachite green
dips. Two preliminary and one definitive flow-through acute brook trout
bioassays were conducted following the bluegill acute bioassay methodology
but at 12°C. Toxicant was supplied to the acute exposure tanks as an aque-
ous emulsion of 0.05 gm Triton X-100 per gram of parathion. This was injec-
ted into the diluter by peristaltic pump. Water for parathion analysis was
sampled at the initiation of testing and at 2, 4 and 5 days of test expo-
sure. Selected fish were frozen at -18°C for later residue analysis.
The flow-through parathion chronic brook trout exposure followed the
procedure given by the National Water Quality Laboratory (34). On May 1,
1972, fourteen fish were randomly distributed to each of the 12 exposure
tanks. A stock solution of parathion was prepared using 8 gm of parathion
and 0.24 gm Triton X-100 per liter of acetone and was dispensed into the
chronic diluter system by syringe pump. The chronic physical system was
identical to that used in the flow-through acute exposures (flow of 30 li-
ters per hour in 85-liter aquaria) and provided a maximum theoretical ace-
tone concentration of 1.5 mg/1 in the highest exposure concentration series
used.
Trout in the chronic exposure were fed Silver Cup trout food according
to the manufacturer's recommended daily ration. Weights and total lengths
of chronically challenged trout were recorded at the start of testing, after
117 days exposure, at 178 days exposure (thinning, discards only) and after
282 days exposure (termination). Trout in the chronic exposure were thinned
to two males and four females per tank at 178 days in anticipation of spawn-
ing. Secondary sexual characteristics made it possible to distinguish the
sexes of most, but not all, of the fish. Surplus trout were dissected and
the tissues frozen for residue analysis.
After thinning of trout from the chronic system (day 206 of exposure,
November 22, 1971), two polyethylene basins, 25 cm wide, 30 cm long and 15
cm deep, were placed in each tank as artificial spawning substrates. A hor-
izontal layer of stainless steel screening with small rocks glued to the
surface was then placed about 5 cm from the bottom of the substrate. Work
by Benoit (35) indicates that this type of substrate necessitated a system
of baffles to prevent eggs from experiencing incomplete fertilization.
Eggs were removed from the substrate each afternoon, and counted manu-
ally. Fifty eggs from each spawn were incubated in egg cups suspended in
shaded larval chambers. The larval chambers in both chronics received expo-
sure water at a rate of 2.6 liters per hour directly from the mixing cells
of the diluter. Additional eggs from each spawn were incubated for 12 days
and examined for viability. Under low magnification the embryonic develop-
10
-------
merit could be seen when Che eggs were preserved in an aqueous solution of 5
percent formalin, 4 percent glacidal acid, and 6 percent glycerin (v/v).
Viability was calculated as the percent of embryos with a neural keel. Dur-
ing incubation, the eggs were checked daily for mortalities and fungus. 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, 32 and 100 ug/1 with a control at 10°C.
Toxicant solutions of these concentrations were delivered at rates of 200
ml/hr to each of four larval chambers by constant drip Mariotte bottles.
Each chamber incubated two egg cups containing 100 nineteen-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.
On exposure day 282, all but two of the surviving adult brook trout
from each exposure aquarium, of the parathion chronic exposure were sacri-
ficed. The sacrificed fish were immediately frozen at -4°C to -18°C while
the live fish were held in tanks of flowing clean water from two to seven
days. Tissue samples taken from fish held in the flowing water conditions
were analyzed for parathion residues.
Fathead Minnows
Fathead minnows, Pimephales promelas Rafinesque, used for bioassay
tests were obtained from the Newtown Fish Toxicology Station, Cincinnati,
Ohio, and cultured for two years at the Aquatic Environmental Sciences labo-
ratory. A preliminary static acute test and a definitive flow-through acute
using adult fathead minnows were conducted at 22°C and 24°C respectively.
The definitive flow-through acute test was conducted by the same procedure
used for the bluegill acute bioassays. Parathion stock was made as an aque-
ous sonicated emulsion of 29.0 gm parathion and 1.45 gm Triton X-100 per
liter of water and injected into the diluter apparatus by peristaltic pump.
The flow rate to each 19-liter test tank was approximately 15 liters per
hour in all acute tests.
The chronic exposure followed the procedure of the National Water Qual-
ity Laboratory (34). Thirty-five minnow larvae, 5 to 14 days old, were ran-
domly distributed to each of the exposure tanks on April 1, 1972 (Evansville
date, December 1). Minnow fry were fed a mixture of powdered fish food,
mixed with green algae, and Artemia nauplii for the first 30 days. After 30
days minnows were fed a commercial trout starter mash ad libitum each day.
The nominal parathion exposure concentrations selected for testing were
50, 25, 12, 6 and 3 ug/1. These exposure concentrations were produced from
a stock solution of 1.7 gm parathion and 0.1 gm Triton X-100 per liter ace-
tone injected into a proportional diluter by peristaltic pump.
At 30 and 60 days exposure, minnow fry from each replicate exposure
concentration tank were counted and photographically measured (33). At 70
days exposure the minnows in each tank were thinned to 15 fish and three
artificial spawning substrates were added. Substrates were made from 4-inch
sections of 3-inch diameter asbestos and cement drain pipe, bisected longi-
11
-------
tudinal1y.
Spawning began on October 11, 1972 (June 22, Evansviile test date) and
continued through December 19 (August 19, Evansviile test date) at which
time the test was terminated. During the spawning period, substrates were
checked daily for eggs. When spawns were encountered, eggs were counted and
fifty eggs from each spawn were incubated in an egg cup suspended in a lar-
val chamber receiving water from the corresponding parent tank. Each larval
chamber was 30 cm square by 15 cm deep, divided into two sections. Although
these two sections physically separated distinct groups of spawned fry, they
were not true duplicates since each received the same water.
Prior to incubation, all eggs were trea ted once with a solution of 4
mg/1 malachite green to prevent fungal infection. Larvae were counted after
hatching was completed, usually in five days. Of those hatched, 40 were
released into the larval 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). After 8 months of parathion exposure, the
adult minnows were sacrificed, examined for deformities, measured, and fro-
zen at -18°C for residue analysis.
INVERTEBRATE BIOASSAYS
Daphnia magna
A stock of Daphnia magna Straus originally obtained from the National
Water Quality Laboratory, Duluth, Minnesota, was continuously cultured
throughout the project in 3-liter jars which had become coated with diatoms
and other algae. Cultures were maintained under static conditions at tem-
peratures of 18 to 21°C, conditions which proved adequate for reproduction.
A stock food supply of 1.5 gm Cerophyl, 2.0 gm trout mash, and 1.0 gm yeast
in 500 ml well water was prepared and a few ml of this mixture were added
periodically to each culture tank. Cultured daphnids were transferred to
clean culture jars monthly as a precaution against excess waste accumulation.
In preparation of acute or chronic bioassays, several dozen gravid fe-
males were selected from culture and held in clean 250-ml beakers over-
night. Within a 24-hour period enough first instar daphnids were usually
produced to initiate testing. Instars were counted and randomly distributed
to each of the exposure test vessels. In an acute bioassay the number of
surviving Daphnia were counted daily and in chronic studies survivors were
counted weekly. Progeny produced during chronic tests were counted and dis-
carded each week. Size differences between adults and progeny enabled these
two life stages to be distinguished. Daphnia in all tests were fed a daily
ration of 5 ml per test container of the food mixture.
All Daphnia bioassays were conducted using 3-liter glass jars as the
exposure vessels. Each exposure vessel had a U-shaped notch cut into the
upper lip which was covered with No. 405 Nytex screen. This configuration
permitted establishing a constant water volume in the test vessels without
loss of test organisms. Exposure temperature was maintained constant by
12
-------
partially submerging all exposure vessels in a constant 18°C water bath, A
flow of exposure water at the rate of 0.8 liter per hour was provided to all
test vessels via a proportional diluter.
Gammarus fasciatus
Adult and immature Gammarus of several closely related species were
collected in coves and tributaries of the Hudson River in an area above the
salt wedge. Collection methods included wire cage traps weighted with rocks
and dip nets, 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 the Boyce Thompson Institute, Yon-
kers, New York, Because immature and female garamarids are extremely diffi-
cult to identify, the possibility cannot be excluded that some other species
were included in the test population of G. fasciatus.
Tests were started with juvenile gammarids obtained from field collec-
tions because laboratory cultures did not produce sufficient young.
All tests (acute and chronic) were performed in 15-liter aquaria main-
tained at 18 to 20°C. Each exposure tank was supplied with several square
centimeters of well-soaked maple leaves for cover and food. During the
chronic exposure tests and in culture, 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.
Bioassays were conducted using a proportional diluter supplied with a
200 mg/l stock solution of parathion in acetone. The maximum possible ace-
tone concentration achieved at this stock mix was about 7.5 mg/l in both the
acute and chronic tests.
Although a full 5-month chronic exposure starting with newly hatched
gammarids was originally planned, it was started five times but each time
encountered difficulties in organism survival or parathion level. However,
two chronic exposures of juvenile gammarids for 35 and 43 days were complet-
ed and estimates made of the LC50 levels for these exposure periods.
Chironomus tentans
A stock of Chironomus tentans Fabricius provided by Dr. William Cooper
of Michigan State University was cultured according to his methods. Several
2-liter aquaria with screen covers were filled with one liter of well wa-
ter. A substrate of Cerophyl, trout food, and toilet tissue was blended
together until lumpy and added to each aquarium in small amounts.
In the culture, a very slight aeration was used to prevent anaerobic
conditions, yet leave the substrate undisturbed. The static culture was
held at 21 to 25°C and had a 16-hour light photoperiod. Once a day, the
emerged adults were transferred to a large screened cage which had shallow
pans 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 designated for testing or were returned to
13
-------
fresh culture tanks.
Acute and subchronic Chironomus assays were performed with fourth in-
star larvae and second instars respectively. The second instars were easier
to handle and count than first instars and did not suffer from handling mor-
tality. All tests were conducted in a 21°C water bath using 3-liter jar
exposure chambers similar to those used for Daphnia. Two one-month sub-
chronics were terminated after two weeks of exposure due to mortality at all
parathion concentrations tested. In all tests, lack of larval response to
probing was interpreted as mortality.
CHEMICAL ANALYSIS AND STATISTICAL METHODS
Samples for routine water quality measurements were taken by siphoning
or dipping water from the center of each tank at mid depth. Dissolved oxy-
gen samples were takn in BOD bottles and measured with a Yellow Springs In-
struments polarographic oxygen meter. Hardness, alkalinity and pH were de-
termined by procedures contained in Standard Methods (30).
Water for parathion analyses in fish tanks was sampled in the same man-
ner as for water quality determinations. For invertebrate tests, to avoid
capture of test organisms within samples, water samples were taken directly
from the diluter mixing cells and pooled. During long term tests, analyses
were made weekly for all tanks with the exception of the fathead minnow
chronic exposure where samples were collected daily and pooled for a weekly
analysis. Analyses of samples stored for one week in amber glass bottles
were not significantly different from replicates analyzed immediately. Par-
athion was extracted from 90 to 900 ml water samples (sample size was con-
centration related) in volumetric flasks by stirring with 5.0 ml pesticide-
grade hexane for 15 minutes. The hexane layer was allowed to separate and
was removed by pipette. Duplicate hexane aliquots were analyzed for phthal-
ate content by gas chromatography (GC). The gas chromatographic parameters
for the Hewlett-Packard 5700 or 76720 series instruments used for all analy-
ses were:
Detector: Ni-63, electron capture, 265°C
Injection port: 230°C
Column: 4 ft x 1/2 in, 3% OV - 17 on 80-100 mesh
Chromosorb W at 200°C
Carrier gas: 10% methane in argon; flow: 50 ml per min
detector purge
Injection size: 10 microliters or suitable volume
Response factors: Parathion - 45,000 counts per nanogram
Paraoxon - 20,000 counts per nanogram
System noise level (as parathion) based on hexane blanks: 0.001 to
0.02 ng of parathion on column
Identical GC conditions were used for analyses of hexane extracts of
water and fish blood, and fractions of tissue extracts after clean-up.
Whole fish blood, drawn from the caudal vein, was extracted by shaking
with 5 ml hexane per ml blood and analyzed by GLC after concentration to 0.5
14
-------
ml without clean-up.
All other tissues were frozen at -18°C until analyzed. In the general
tissue analytical procedure employed, two to three grains of tissue (wet
wei ght) were blended with 60 gm anhydrous sodium sulfate and extracted over-
night with pesticide-grade hexane (50 ml).
The sodium sulfate was filtered, washed, the hexane washes pooled (ca
20 ml) and evaporated at 60°C just to dryness. The residue was re-dissolved
in hexane (ca I ml) and transferred to a 1-cm diameter silicic acid column
(5 ml 100 mesh Mallinckrodt chromatography grade). The column was washed
with 20 ml hexane and eluted with a 30 percent (v/v) benzene in hexane solu-
tion. A 25-ml benzene-hexane fraction was collected beginning at the 30
percent benzene break through ( I) for low level parathion residues (in the
ug/kg range). A 50-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 aliquots of the same fish was +5 to 7 percent. Recovery of spiked
samples averaged 90.8+7.6 percent based on seven tests. The range of recov-
eries was 83.1 to 103 percent; the coefficient of variation was 8.4 percent.
For acetylcholinesterase determinations, whole fish brains were dis-
sected, stored up to two months in liquid nitrogen (-1969C), thawed, homo-
genized in phosphate buffer, and assayed by the kinetic colorimetric method
of Ellman et al (38).
Statistical Methods
Whenever two or more partial kills occurred in an acute bioassay, the
LC50 and 95 percent confidence limits were calculated by the graphical meth-
od of Litchfield and Wilcoxon (37). Mortalities were plotted on a probit
scale against the log of toxicant concentration. A straight line was fitted
to the points by the eye, and checked by the cni-square test. The LC84,
LC50 and LC16 points were read from the line, and the total number of ani-
mals used between the 16 percent and 84 percent points (N) was noted. Then
the confidence limits of LC50 were found as follows:
f = (1/2 (LC84/LC50 + LC50/LCl6)exp(2.77/N1/2))
LC5Q x f = upper limit
LC50/f = lower limit
Reproductive impairment of Daphnia (RI50) 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 fewer than two partial kills occurred, LC50 val-
ues were calculated by the moving average method which does not require a
straight line graph:
15
-------
R^ - number of deaths at dosage i (= k, 2, 3, . . . L)
where k = lowest dosage, L = highest dosage
= number of survivors at dosage i
N = number of individuals per tank
F = geometric factor between dosage levels (>1)
t = Student's t for (P = 0.05) and (N-1)(L-1) d.f.
G = ((N(L-l)/2) - Rk - R2 - ... RL_L)/(RL-Rk)
M = log D + log F (((L-2)/2) + G)
1/2
LC50 = antilog (M _+ tV log F)
To determine differences in mean weight, length, etc., between experi-
mental tanks, one-way analysis of variance was used, provided the variances
were homoscedastic (Bartlett's Test). If 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 com-
puted as monthly means for each tank of the chronic tests. All weekly para-
thion analyses were used to calculate a mean, range and standard deviation
(SD) of the compound concentration in each exposure tank.
V =
(l-G)2RjtSk + R2S2 +...Rl-iSl-1 + G2RlSl
rL " Rk
N - l
16
-------
SECTION V
RESULTS
BLUEGILL
Acute Bioassay
Five preliminary and one definitive acute bioassays were performed at
22°C under previously described flow-through test parameters. The 96-hour
LC50 for the definitive test was 0.51 mg/1. LC50 values for 24, 48 and 144
hour exposures were also calculated and are presented in Table 4. The se-
quence of toxic symptoms of acute parathion toxicity were: lethargy, dark
discoloration, hypersensitivity, tremors, coughing, convulsions, extended
pectoral fins and opercula, loss of equilibrium, scoliosis, hemorrhages,
tetanus, and death.
Certain individual fish which succumbed during the acute test were
analyzed for parathion whole body residues and the results are presented in
Table 5. Incidence of lethality and time to death of analyzed bluegill var-
ied as a function of concentration. Concentration factors calculated by
dividing measured tissue parathion concentration (ug/kg) by exposure water
parathion concentrations (mg/1) (Table 4) were plotted against exposure time
at mortality. As shown in Figure 1, concentration factors exhibited a line-
ar increase with exposure time indicating increased parathion bioconcentra-
tion as a function of exposure period regardless of exposure concentration.
TABLE 4. BLUEGILL ACUTE TEST
a. Number surviving per tank
Hour
OA
0B
1A
IB
2A
2B
3A
3B
4A
4B
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
*8
b. Analyzed parathion concentration, mg/1
nd
nd
nd
nd
2.10 2.19 1.24 1.24 0.63 0.63 0.50 0.50 0.33 0.33
2.25 2.25 1.25 1.25 0.64 0.64 0.53 0.53 0.35 0.35
nd: not detectable.
continued
17
-------
TABLE 4, continued
c. Median lethal concentrations, mg/1
Hour
LC50 Upper CL
Lower CL
24
2.25 4.35
1.16
48
1.15 1.92
0.58
96
0.51 0.72
0.36
144
0.46 0.95
0.22
TABLE 5. PARATHION
RESIDUES IN WHOLE 3LUEGILLS, ACUTE
TEST*
Parathion in
Parathion in
Exposure,
Concentration
Tank
water, mg/1
fish, mg/kg
hours§
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
294.1
72
462
3B
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
338.3
29
152
*See Table 4 for actual lethality data.
SRepresents actual time to death.
Bluegill Chronic Test
The maximum no-ill-effect level determined for bluegill sun fish chroni-
cally exposed to parathion was between 0.17 and 0.34 ug/1 based on morpho-
logical deformities evident in adults. Growth rate, spawning and fry survi-
val were not clearly affected by parathion exposure concentrations tested up
to 3.2 ug/1.
A summary of parathion analyses in water, water quality, and test tem-
perature are given in Tables A1, A2 and A3, Bluegill survival data (Table
A4) showed that parathion concentrations tested did not cause significant
mortalities. Some deaths of extremely deformed fish however did occur with
the majority of deaths following handling, particularly at the time of thin-
ning. For this reason, weight measurements were not taken more frequently
during the test. There were no significant differences in bluegill weights
among tanks at the start of the test, at the thinning or at the end.
Lengths and weights of fish at termination were approximately 17 cm, 105 gm
for females, and 21 cm, 200 gm for males.
18
-------
EXPOSURE TIME, hours
Figure 1. Concentration factors for parathion residues in whole
bluegills at various exposure times. Selected whole bluegills as
listed in Table 5. Exposure time is actual time to death.
19
-------
Within Che first four months of testing, tremors, scoliosis and extend-
ed pectoral fins were observed in the two highest exposure concentrations.
In six months, protrusions in the hyal or "throat" region were noticeable at
these same exposure levels. The same symptoms continued, and gradually in-
creased in severity during the 23-month test. Table 6 lists the incidence
of scoliosis and "throat" protrusions. The percentages vary to some extent
between the 18 and 23 month examinations because some very deformed fish
were sacrificed at thinning.
TABLE 6. INCIDENCE OF DEFORMITIES, BLUEGILL CHRONIC TEST
Tank
OA OB
1A IB
2A 2B
3A 3B
4A 4B
5A 5B
Mean para-
thion cone,
Ug/l 0
.06
0.06
3.23
3.14
1.53
1.59
1.00
1.00
0.34
0.34
0.16
o. i;
18 months
No. of fish
10
15
17
13
20
17
16
15
9
13
10
9
Scoliosis, %
0
13
59
38
45
24
38
33
11
0
0
0
Throat, Z
0
13
88
100
70
76
75
67
11
0
0
0
23 months
No. of fish
9
9
8
8
11
9
9
9
8
9
7
7
Scoliosis, %
0
11
25
50
18
33
22
22
0
0
0
0
throat, X
0
0
62
88*
64
100
89
89
38
56
0
0
*Loss of symptoms not explainable.
Typical protrusions in the hyal region as shown in Figure 2 were serai-
rigid, vascularized masses of fatty and connective tissues extending from
the branchiostegal and urohyal bone area. Scoliosis or lateral bending of
the spine was usually noted posterior to the dorsal fin as illustrated in
Figure 3. In bluegills, the torsion observed was both right and left later-
ally oriented.
Successful bluegill spawning occurred throughout the second summer.
Egg production, hatchability and fry survival data are summarized in Table
7. In general, the hatchabilities improved as handling of the egg cups was
reduced and the 7-day fry survival improved as more Artemia were fed per egg
cup.
Although the numbers of males and females present per exposure aquarium
were sufficient to provide good spawning, as illustrated below (page 24),
the spawning data could not be statistically related to exposure concentra-
tion.
Growth rates of bluegill fry measured at 30, 60 and 90-day exposures
were significantly different, but not related to toxicant concentration.
Mean lengths and survival, Table 8, correlated inversely with numbers of fry
per tank although feeding was four times daily. Regardless of the starting
number of fry, each tank population fell to a few individuals at the end of
20
-------
Figure 2. Hale bluegill exposed to 1.5 ug/l parathion for 23 months. Length 21 cm.
Arrow indicates abnormal protrusion of fatty and connective tissue.
-------
-------
TABLE 7. BLUEGILL CHRONIC EXPOSURE TO PARATHION, SPAWNING RECORDS
Tank
OA
OB
1A IB
2A
2B
3A
3B
4A 4B
5A
5B
Nominal
parathion
cone, ug/1
0
0
4.00 4.00
2.00
2.00
1.00
1.00
0.50 0.50
0.25
0.25
Hundred
5
49
20
1
90
38
105
271
26
229
of eggs
2
41
80
71
27
8
10
112
spawned
28
44
10
2
9
209
48
38
15
66
9
25
15
2
167
45
64
103
49
14
Percent
72
46
44
49
40
80
37
57
83
86
hatch,
57
46
36
4
59
35
77
80
3 days
74
49
62
61
45
9
32
79
84
86
76
10
64
16
78
62
82
18
18
Percent
92
56
66
68
78
0
68
32
100
22
survival,
98
92
76
8
86
36
96
94
7 days*
96
90
100
92
80
20
100
46
84
100
60
100
90
97
100
84
96
36
59
Percent
a
10
0
0
0
0
0
0
32
2
survival,
78
4
18
0
2
0
78
28
14 days*
26
28
30
48
20
0
100
• •
14
82
0
80
18
85
0
28
10
0
Percent
6
10
• •
• •
• «
28
0
survival,
76
0
16
• c
2
• a
66
28
21 days*
20
22
16
46
20
• ¦
84
» .
2
68
46
18
81
t
28
0
Percent 6 8 .. .. ., .. .. .... 24
survival* 76 .. 16 2 64 22
30 days* 18 22 16 46 18 62
64 34 14 5
26
^Calculated as percent of larvae hatched.
23
-------
Tank
OA 0B
1A IB
2A
2B
3A
3B
4A
41 5A 5B
Males
2 1
4 4
3
3
1
3
3
3 2 3
Females
7 8
4 4
8
6
8
6
5
6 3 4
the three
developed
month exposure,
scoliosis.
At 90 days
, one
fish
in
tank
1A (3.
4 ug/1) had
During Che first two months of the chronic, bluegilL exposed to 8.0
ug/1 began to show tremors. When the bluegill were removed after 64 days
exposure, their brain AChE activity was determined at 1.97+0.40 uraol min/gm
wet weight. This is about 11 percent of normal bluegill brain activity.
Although bluegill showed signs of toxification, they were not moribund.
Tables A5 and A6 show the extent to which normal AChE activity was inhibited
in chronically exposed bluegill. The slightly greater AChE depression shown
in Table A6 was probably caused by elevated parathion levels in exposure
water during the month preceding termination (Table Al). The majority of
fish in all exposure concentrations except the lowest and control appeared
hypersensitive to stimuli. When startled, directional swimming response to
a stimulus was almost random. A disturbance could trigger clonic (rhythmic)
or alternating convulsions in some specimens.
Parathion residues in bluegill tissues exposed for 18 and 23 months are
reported in Tables 9 and 10. In muscle tissue, residue levels were no
greater than 25 times the exposure water concentration. Blood levels were
similar or slightly higher. The unusually high tissue concentrations in
Tank 3 (see Table 10) were affected by accidentally high water concentra-
tions for two weeks before the sampling. The greatest residues were discov-
ered in ovaries which is probably related to their high lipid content. Al-
though the data are semiquantitative, the residues measured in spawned eggs
are similar to those concentrations measured in the ovaries.
BROOK TROUT
Acute Test
Two preliminary and one definitive flow-through brook trout acute tests
were completed at 120C. The 96-hour LC50 for the definitive test was 2.00
rag/1 (Table 11). During the first 24 hours exposure of the bioassay test,
total mortality occurred at the highest exposure concentration (Table A7).
Many trout in the three highest exposure concentrations exhibited symptoms
of lethargy, irregular breathing, and light discoloration of the skin. No
symptoms of scoliosis, convulsions, or hemorrhages were visible in fish from
any of the test concentrations.
The brook trout parathion 96 hour LC50 (Table 11) calculation for the
definitive acute bioassay was based on mean measured exposure concentrations
(Table A7) of 4.00 mg/1, 1.86 mg/1, 0./96 mg/1, 0.47 mg/1 and 0.25 mg/1 par-
24
-------
TABLE 8. SURVIVAL AND LENGTHS OF BLU1GILL LARVAE
Tank
30 day
Mean
Length,
Number mm
60 day
Number
Mean
length,
nun
90 day
Mean
length,
Number mm
OA
OA
OB
40
9
4
10.1
11.3
11.0
3
7
3
19.7
14.9
14.0
2
3
3
23.0
20.7
23.7
1A
1A
31
11
10.5
13.2
13
10
13.2
19.7
11
8
18.5
22.8
2A
2A
2B
8
23
13.6
15.1
10.7
23.4
18.2
16.8
32.4
24.9
22.2
3A
3A
8
12
13.2
9.4
7
4
20.3
12.2
6
3
28.7
18.0
4B
4B
32
32
9.1
8.9
7
4
13.6
16.2
3
4
26.3
20.5
5A
5A
12
12
10.5
11.5
11
10
16.3
14.8
7
4
23.1
28.8
25
-------
TABLE 9. PARATHION RESIDUES IN BLUEGILLS, 18 MONTHS EXPOSURE
Tank no. [nominal cone. parat'nion, ug/lj
0
5
4
3
2
1
[0]
[0.25]
[0,501
[1.00]
[2.00]
1 [4.00]
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
8.4
9.7
14.6
13.6
22.7
109.0
SD
11.0
0.1
10.0
1.4
13.4
20.8
Blood residue*
41.4
163
119.8
288
Ovary residue*
23.9
74
144
3436
30.6
214
158
238
643
585
Egg residue*
nil
73
552
179
813
« « •
• • •
nil
246
134
1089
429
*In ug/kg wet wt.
Minimum
detectable
concentration 25
ug/kg; see
"Methods."
Notes:
Recovery of spiked muscle samples (equivalent to 100 ug/kg) gave 88.4%
recovery in a single test.
Precision among triplicate samples gave a coefficient of variation of
13.1%.
All blood residues except No. 1 and No. 2 sets were considered semi-
quantitative since levels are at or near detectable minimum in the direct
extraction samples. No evidence of paraoxon was found.
26
-------
TABLE 10. PARATHION RESIDUES IN BLUEGILLS, 23 MONTHS EXPOSURE
Tank no. [nominal conc. parathion, ug/1]
0
5
4
3
2
1
[0]
[0.25]
[0.50]
[1
.00]*
[2.00]
[4.00]
Muscle residue
See
note
¦kit
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
*Mean parathion concentration for last month of test was accidentally high:
2.5 ug/1 (Table Al).
**Muscle residue values all at or below reliably detectable levels (=25
ug/kg). Precision at these low levels is estimated at about +50%.
TABLE 11. BROOK TROUT 96 HOUR ACUTE LC50, rag/1
LC50 Upper CL Lower CL
2.00 3.03 1.32
Determined by Litchfield and Wilcoxon method from
data in Table A7.
athion. Use of the Litchfield and Wilcoxon method resulted in a 96 hour
LC50 for brook trout of 2.00 mg/1 parathion.
Trout muscle parathion residues determined for mortalities from the
definitive acute bioassay are presented in Table 12. Incidence of lethality
and time to death varied as a function of concentration. The concentration
factors presented in Table 12 are generally a function of exposure period
(time to death) and indicate a slight increase in residue concentration fac-
tor to 140 hours exposure followed by a decline (144 hours). Actual tissue
concentrations (mg/kg) were seen directly proportional to exposure concen-
tration (mg/1). Appendix B presents details of separate investigations con-
cerning the toxicity and bioconcentration characteristics of parathion to
the brown trout (Salmo trutta). Results indicate a substantial agreement to
data obtained for brook trout.
Brook Trout Chronic Test
Over a nine month exposure period, adult brook trout chronically ex-
posed to various parathion concentrations as high as 7 ug/1 showed no sig-
nificant differences in growth rate, maturity, or spawning success. How-
ever, relatively high residue concentrations of parathion were measured in
the blood, muscle and other tissues of brook trout chronically exposed to
the parathion concentrations tested.
27
-------
TABLE 12.
PARATHION RESIDUES IN
BROOK TROUT MUSCLE,
ACUTE TEST
Exposure
Muscle cone,
time to
Mean water
mg/kg wet
Concentration
death, hr
cone, mg/1
weight*
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 an individual fish.
Water quality parameters, temperature and parathion analyses data £or
the chronic exposure are given in Tables A8, A9 and A10. Most mortalities
which occurred during the test (Table All) were attributable to disease
(fungus or furunculosis) or to accidental test system mechanical failure.
At no time during chronic exposure did trout exhibit symptoms typical of
acute organophosphate poisoning such as tetany or convulsions. Fish weight
measurements made at the start of testing and after 4 and 9 months of expo-
sure (Table A12) indicate uniform weight gains for trout in all exposure
concentrations. Although no significant differences between mean weights
were found, weight variability between individuals was attributable to bio-
mass (density) differences between exposure tanks.
Fish discarded from the chronic at the time of thinning (six months
exposure) were analyzed for parathion residues in blood, muscle, and other
tissues and for acetylcholinesterase activity. The blood residues (Table
13) were plotted against the analyzed parathion exposure concentration in
water at the time of removal (Figure 4).
TABLE 13. PARATHION IN BROOK TROUT BLOOD, SIX MONTH EXPOSURE,
INDIVIDUAL FISH, ug/l
Tank
OA
1A
2B
3A
4A
4B
5A
5B
6.2
1007
737
647
113
153
68
79
63.7
1597
885
695
96
151
24
58
10.6
1139
726
96
61
69
40.5
776
337
93
52
103
0.5
662
515
101
80
6.9
528
654
42
50
649
68
110
50
Mean
21
1302
788
603
100
152
58
78
SD
21
417
208
136
9
1
23
24
28
-------
PARATH I ON CONCENTRATION IN WATER, yg/Z
Figure k. Parathion in brook trout blood vs.
parathion in water, six month exposure.
29
-------
A least-squares line fitted to the data has the equation;
(1 = 207 C - 39
b w
where is parathion concentration in blood and Cw the parathion concentra-
tion in water. Results indicated that trout concentrated parathion approxi-
mately 200 times over exposure water concentrations.
The parathion residue levels for inuscle and other tissues show equally
large concentration factors (Table 14). Muscle residue was 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:
C = 553C - 176
m w
No paraoxon was detected in any of the analyses despite the use of sol-
vents to extract it from the clean-up columns. The parathion residues re-
ported in Table 14 were the highest concentrations found in any chronic ex-
posure. Brain acetylcholinesterase activities of the same fish (Table 15)
were plotted against exposure concentration. Figure 6 shows that about 5.6
ug/1 of parathion was required to depress trout brain AChE activity to a 50
percent of normal level. However, no gross observable symptoms of central
nervous system toxification were noted in fish exposed to the highest
parathion concentration tested.
TABLE 14. PARATHION RESIDUES IN BROOK TROUT TISSUE, SIX MONTH
EXPOSURE, ug/kg WET WEIGHT
Tank no. [water cone , ug/1]
OA
[0.03]
1A
[6.7]
2B
[4.0]
3A
[2.6]
4B
[1.4]
5A
[0.6]
5B
[0.6]
Muscle
nil
18
12
nil
4454a
3229a
1958
1820a
1874
1258b
14llb
921
998
1124
176a
474^
606
244
130
171*
139
Kidney
nil
125
2942
556
998
815
3332
1245
1051
L23
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
All single samples except (a) average of duplicate samples and (b) average
of triplicate samples. Reagent blank = 3.0 ug/kg. Recovery at 1000 ug/kg
level averaged 9.08+7.6% (coefficient of variation = 8.4%); range 83.1 to
103% for seven tests. All values uncorrected for percent recovery.
30
-------
5000
4000
op
4)
?
3000
00
o
CO
13
2
Z 2000
<
or
<
Q_
1000
2 4 6
PARATHION IN WATER, ^g/1
Figure 5. Parathion in brook trout muscle
vs. parathion in water, six month exposure.
31
-------
TABLE 15. BRAIN ACETYLCHOLINESTERASE ACTIVITY OF BROOK TROUT
SIX MONTH EXPOSURE, INDIVIDUAL FISH, mnol/min/gm
Tank
OA 1A 2B 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 5.60
SD 0.31 1.35 0.90 0.16 0.67 0.29 0.74 0.16
Brook Trout Spawning
Brook trout in the chronic exposure spawned between December 23 and
January 15 test dates. A summary of the number of spawnings, egg production
and survival of embryos and larvae is given in Table 16, Only a very small
percentage of embryos were deformed and these numbers could not be correlat-
ed to exposure concentrations. Egg hatchability was uniformly poor but was
felt a function of handling damage and elevated water temperature (13°C)
during spawning. Exposure water temperature in excess of the optimal 9°C
was felt to have delayed maturation and inhibited spawning activity (39).
To document maturation, the gonad weights of the discarded fish were taken
at the time of thinning (Table A13) and at the test termination (Table
A14). The gonad weights divided by the fish's total weight produced a go-
nadosomatic index expressed as a percent. The weights shown in Table A14
are somewhat biased since small and immature fish were selectively discard-
ed. For both males and females, the development was variable and appeared
unrelated to toxicant concentration. The gonadosomatic indexes determined
at termination show that spawning was not complete. Female gonadosomatic
indexes greater than 1.00 represent loose eggs in the body cavity which were
never released (Table A14),
To supplement the chronic hatchability data, nineteen-day-old brook
trout eggs were incubated at 10°C in three concentrations of parathion until
hatched. The following percent survivals to hatch were observed:
Control 10 ug/1 32 ug/1 100 ug/l
77.5% 52.5% 41.5% 38.4%
Eggs in the 32 and 100 ug/1 solutions exhibited poor development and
more transparent chorions than controls. Some of the eggs in both concen-
trations showed cerebral development without corresponding spinal develop-
ment. The lower embryo survival at the two highest levels partly resulted
from premature hatching. Although the hatchability at 10 ug/1 was less than
the control group, development appeared to be normal.
32
-------
ao
c
E
o
-------
TABLE 16, BROOK TROUT SPAWNING RECORD
Tank
OA
IB
2B
3A
3B
4A
4B
5A
5B
Number spawnings
1
2
3
2
2
2
3
7
1
Total eggs spawned
360
1249
787
716
924
929
1607
479
48
12 day viability,
percent of spawn
26
85
42
2
76
56
15
100
78
0
24
Total hatchability,
percent of spawn
6
12
20
12
0
6
22
4
0
30 day fry survival,
percent of hatch
0
0
10
67
0
67
18
0
0
Note: overall spawning and hatchability variability attributable to external
variables of handling stress and temperature, not to parathion concentration.
Adult Brook Trout Termination
Chronically exposed adult brook trout were sacrificed on February 6,
three weeks after the last spawning. Blood obtained from 34 trout repre-
senting all concentration groups was analyzed for parathion residues as was
muscle tissue from 31 exposed fish. Residue analyses for parathion were
also done on kidney, gill, gonad and gastrointestinal tract samples from
each of two to three fish in each exposure concentration. Brain AChE was
determined for 39 fish.
Tissue residue analyses results, including blood levels, are tabulated
in Table 17. Blood parathion concentrations versus exposure water concen-
trations are plotted in Figure 7. Trout blood and tissue parathion residue
levels measured after nine months exposure were lower than corresponding
values determined on fish sacrificed after six months exposure.
Several of the adult trout from each parathion concentration group 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 AChE determinations were also performed and results are given in Table
18. Data indicate that AChE 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 rate of decrease while muscle para-
thion concentrations decreased more slowly than blood. The limited samples
available and the large variances among individual fish make it difficult to
quantify "wash-out" rates but the blood and tissue parathion uptake concen-
tration appears to be readily reversible.
Some general conclusions may be drawn on the basis of the six and nine
month analyses. Parathion levels in blood correlate with water concentra-
tions at the time of sampling. Blood levels appear to rapidly reflect expo-
sure levels in the water. Tissue residues also correlate with water concen-
trations; however, tissue levels will change less rapidly with change in
water concentrations. High correlation also exists between blood parathion
34
-------
TABLE 17. BROOK TROUT PARATHION RESIDUES AND AChE, 9 MONTHS
Parathion
in water.
Tank. ug/L
Paraehion, ug/kg wet weight
Blood Muscle Gill Kidney Gonad
GI
tract
AChE,
umol/min/g
OA
0.01
23
0.8
42
0.6
94
1.9
23
1.0
0B
0.01
58
0.6
33
29
0.1
20
1A
8.30
733
1220
1021
399
990
4157*
IB
8.72
625
1623
743
876
720
2181**
2A
5.53
361
297
355
671
2B
4.24
337
318
446
» • a
304
• • •
• ~ •
413*
3A
2.86
184
270
252
189**
482
166
319**
3B
2.76
232**
272
217**
5115
4A
i .45
114
7
111
118
4B
1.26
141
87
44
82
34
5A
0.53
41
33
40
58**
52
3.1
1.4
3172
1727
1143
2358
1483
1085
703
959
10835
179
265
138
0.2
0.1
400
217
614
311
374
78
192
183
774
11
53
16
<0.01
1812
939
1780
1054
705
519
6
407
150481
36 §
69
stored at -18°C for 2-1/2 months.
**Duplicate samples average.
§Probable interference contribution to parathion value.
3.97
0.7
5.04
5.12
3.01
28
4.49
5,77
4.62
4.19
8989
3.44
2.79
2.15
3.17
160
4.39
11598
3.86
6.24
4.45
56665
5.91
200
4.46
5.21
6.17
352
4.71
4.30
1672
4.81
4.35
5.44
1105
4.42
1210
4.76
106
6.24
6.06
5.85
110
6.00
4.36
135§
5.63
5.73
5.27
other
samples were
continued
35
-------
TABLE 17, continued
Parathion Parathion, ug/kg wet weight
in water, GI AChE,
Tank ug/l Blood Muscle Gill Kidney Gonad tract umol/rain/g
5B 0.44 39 36 6.51
33 5.65
36 73 96 63 § 38 28 5.56
63 5.45
^Analysis done immediately after fish was sacrificed; all other samples were
stored at -18°C for 2-1/2 months.
^Duplicate samples average.
SProbable interference contribution to parathion value.
concentrations and tissue parathion concentrations when measured after chron-
ic exposure (six or nine months); the correlation coefficient for muscle par-
athion versus blood parathion was 0.967 (p < 0.001).
The high variance in parathion water concentrations measured on a week-
ly basis during chronic exposure probably accounts for the differences in
tissue residue concentrations observed at six and nine months. Since para-
thion appears to be reversibly taken up and released, measured residue lev-
els reflect the immediate past exposure conditions.
Brain AChE levels, as expected, exhibited an inverse dependence on wa-
ter concentration. The degree of inhibition was less marked than with blue-
gills. Essentially the same AChE effects were found at six and nine months.
Relatively high variance within the concentration groups and especially the
control group was found. However, the highest concentration group exhibited
AChE levels significantly lower than the other experimental groups (exclud-
ing the control group) (p = 0.05)
FATHEAD MINNOW
Acute Test
A preliminary static acute test conducted at 22°C using adult minnows
gave the following results based on measured initial parathion concentra-
tions :
LC50 - 24 hr = 2.28 (2.92 - 1.77) mg/1
48 hr = 2.02 (2.64 - 1.55) mg/1
96 hr = 1.60 (2.05 1.25) mg/1
A definitive flow-through acute bioassay was conducted at 24°C using
adults (approx. 2.2 gm each). The results of this bioassay are reported in
Table 19.
36
-------
1000
^ 800
00
3*
Q
O
O
_i
CD
600
o
x 400
H-
<
QC
<
0_
200
10
PARATHION IN WATER, jjg/i'
Figure 7- Parath ion in brook trout blood vs.
parathion in water, nine month exposure.
37
-------
TABLE 18. BROOK TROUT PARATHION RESIDUES AND AChE, RECOVERY
48 hour elution in fresh water 168 hour elution in fresh water
Tank
Blood
residue,
ug/1
Muscle
residue,
ug/kg
AChE,
umol/min/g
Tank
Blood
residue,
ug/1
Muscle
res idue,
ug/kg
AChE,
umol/min/g
OA
27
35
5.42
OA
« • •
17
4.31
OB
25
41
4.99
OB
19
• « •
5.74
1A
507
587
4.45
1A
293
942
4.78
IB
396
634
4.08
IB
422
1124
3.62
2A
223
478
4.09
2A
196
365
4.91
2B
229
161
4.00
2B
127
462
3A
182
187
4.48
3A
65
223
4.23
3B
364
290
4.30
3B
40
58
5.41
4A
94
92
5.77
4A
35
60
4.59
4B
144
74
8.48
4B
84
186
4.02
5A
123
85
6.11
5A
21
62
9.08
5B
57
75
3.84
5B
23
128
4.88
TABLE 19. FATHEAD MINNOW ACUTE TEST, 24°C
a. Number surviving
Tank
OA
0B
1A
IB
2A 2B
3A
3B
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
Para-
thion*
nil
nil
5.31
5
.88
1.92 2.24
1.04
1.04
0.53
0.49
*Mg/l,
means
of
four
analyses.
b.
LC50
values, mg/1
Hour
LC50
Upper CI
Lower
CI
24
2
.20
3.90
1.24
48
0
.50
1.03
0.24
96
0
.50
1.03
0.24
38
-------
Fathead Minnow Chronic Exposure
The fathead minnow chronic exposure resulted in a maximum acceptable
parathion concentration of approximately 4 ug/1. This value was defined on
exposure concentration related incidence of adult deformities and reproduc-
tive impairment. Data on water quality, temperature and parathion concen-
trations for the chronic exposure are summarized in Tables A15, A17 and A18.
For the first 30 days of parathion exposure, minnow fry showed good
survival and growth (Table A19). There were no significant weight differ-
ences between exposure 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 toxification began to become evident in several of the higher exposure
concentrations with convulsions and scoliosis observed occasionally. By
time of spawning, there were deformities (including scoliosis, lordosis and
abnormally shaped heads) in almost half of the fish in the 50 ug/1 tanks
(Table 20). Weights, however, were not significantly different between
tanks, as determined by analysis of variance.
TABLE 20. MINNOW ADULT WEIGHTS AND DEFORMITIES, 8.5 MONTHS
Females Males Parathion exposure cone,
ug/1
Mean Mean Percent
Tank
Number
wt, gm
Number
wt
gm
de formed
Mean
SD
High
Low
OA
8
1.15
4
2
30
0.0
0.14
0.14
0.82
nil
0B
9
0.98
4
1
88
0.0
0.12
0.18
0.82
nil
1A
11
1.05
2
1
92
46.2
49.40
18.08
104.20
8.61
IB
9
1.16
2
1
38
45.4
48.53
15.06
104.20
16.81
2A
7
1.29
3
1
78
30.0
21.51
8.16
44.86
3.19
2B
5
1.32
2
1
46
28.6
21.94
7.18
44.86
6.70
3A
6
1.25
2
1
88
0.0
15.61
7.55
38.70
2.06
3B
8
1.07
4
1
99
50.0
15.36
7.40
38.70
3.34
4A
9
0.94
4
1
73
15.4
8.95
4.14
17.90
2.81
4B
7
1.09
1
1
75
25.0
9.02
2.87
17.90
2.24
5A
8
1.22
5
1
99
7.7
4.46
1.55
7.30
1.13
5B
5
1.23
8
I
90
0.0
4.36
1.18
7.30
1.60
Spawning first occurred in the controls followed by spawning in the
other exposure concentrations several days later. Total egg production
(Table 21) and the number of spawns per female during the reproductive phase
of the chronic exposure were highest in the two control tanks. In relation
to the control (normal) series, all parathion exposure concentrations tested
exhibited reduced or impaired reproductive capacity. Egg hatchability and
30-day fry growth and survival data could not be related to parathion expo-
sure concentration.
39
-------
TABLE 21. MINNOW SPAWNING RECORD
Total
30-day
eggs
No. of
spawns per
Mean
Spawns
survival,
Mean
spawned
spawnings
female <
I hatch
hatched
percent
length,
OA
2096
14
1.75
72.4
9
70.0
0.98
0B
3591
21
2.33
88.2
19
42.5
1.49
1A
463
8
0.73
65.5*
3
IB
51
1
0.11
42.0
2
40.0
1.20
2A
251
2
0.29
• a •
0
2B
979
7
1.40
44.4
5
3A
103
3
0.50
¦ • «
0
3B
397
5
0.62
51.2
6
4A
2205
12
1.33
74.3
14
4B
0
0
0.00
• • •
0
5A
570
5
0.62
75.0
6
5B
399
4
0.80
65.5
4
12.5
1.68
*Four sets of eggs spawned in OB were hatched in LA - 70% mean hatchability.
Whole minnows were analyzed for parathion during the test but because
of their small size were pooled. Fish taken at 70 days exposure (thinning)
had lower residue concentrations than fish samples at later sample points
(Table 22). The mean concentration factor for 260-day chronically exposed
minnows was approximately 115—less than trout but higher than bluegills.
TABLE 22. PARATHION RESIDUES IN WHOLE FATHEAD MINNOWS, ug/kg
Water parathion concentration, ug/1
Days
exposure 0.15 49.0 21.7 15.5 9.0 4.2
70 ... 1040(20) 615(2) 251(19) 214(22) 256(8)
82-138 ... 9975 (3) 4400(5) 2256 (3) 1796 (1) 1411(4)
260 14(2) 8300 (2) 2270(2) 510 (2) 601 (2) 846(2)
Numbers in parentheses are numbers of fish pooled.
DAPHNIA MAGNA
The acute and chronic LC50 values for parathion determined by exposure
of Daphnia magna are summarized in Table 23. Representative test data are
presented in Tables A20 and A21. The lethal response of daphnids to para-
thion occurred within narrow test concentration limits. Thus, the MATC of
0.08 ug/l was very near the three-week chronic LC50 of 0.14 ug/1. Symptoms
of parathion toxicity observed in daphnids during the acute exposures were
ati erratic circular swimming motion, and later, inactivity and immobility.
The parathion concentration needed to reduce reproduction by 50 percent
(RI50) was greater than the chronic LC50 value. This occurred because young
were produced during the third week of exposure at concentrations that later
killed the adults (Table A21).
40
-------
TABLE 23. PARATHION TOXICITY TO Daphnia magna
a. Acute LC50, ug/1
24 hour 48 hour 96 hour
1.00 (1.73 - 0.58) 0.62 (0.90 - 0.43)
1.27 (1.63 - 1.09)* 0.65 (0.88 - 0.48)*
b. Chronic LC50, ug/I
I week 2 week 3 week RI50
0.28 (0.29-0.26)
0.56 (0.85-0.37)
0.35 (0.47-0.26)
0.37 (0.74-0.18)
0.45 (0.55-0.37)
*Static test at 22°C; all other tests flow-through at 18+°C.
MATC = 0.08 ug/1.
The MATC of 0.08 ug/l (Table A21) was 0.08 times the 18-hour LC50 of
1.00 (Table 23) and 0.13 times the 96-hour acute value (Table 23). 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.
For residue analyses, 0.4 to 20 mg of parathion-exposed Daphnia were
collected, filtered and air dried. Hexane extracts were cleaned with a sil-
icic acid column and analyzed by GC. No evidence of significant parathion
residues could be detected above the GC background noise for any of the sub-
lethal exposure levels. The possibility of decomposition of parathion dur-
ing the air drying process may have contributed to the low residues detected.
GAMMARUS FASCIATUS
Results of parathion acute and chronic exposure using G. fasciatus as
the bioassay organism are reported in Table 24. Two long-term (subchronic)
tests were terminated when survival in the lowest concentration fell below
50 percent of the survival in the controls. Table A23 shows one of these
chronic tests in which the controls and low levels began reproducing. Table
A22 presents data on acute parathion exposure of G^_ fasciatus for a 96-hour
period. The mean of all acute tests resulted in a 96-hour LC50 of 0.39 ug/1.
CHIRONOMUS TENTANS
The C. tentans results summarized in Table 25 show a striking change in
LC50 with length of exposure. Between acute and chronic periods the lethal
concentration varied over three orders of magnitude. Preliminary static
2.70 (5.61 - 1.30)
3.21 (5.10 - 2.02)*
0.25 (0.22-0.28)
0.38 (0.70-0.21)
0.14 (0.16-0.13)
0.19 (0.30-0.12)
0.24 (0.32-0.17)
0.48 (0.87-0.26)
41
-------
acute tests using tissue paper substrate were discarded when it was discov-
ered that parathion was removed by such substrates from the water in several
days. The flow-through tests eliminated this problem of decreasing parathion
concentration. Data related to C. tentans acute and subchronic parathion
exposures are presented in Tables A24 and A25 respectively.
TABLE 24. PARATHION TOXICITY TO Gammarus fasciatus
LC50, ug/1
24 hour
48 hour
96
hour
Temp,
deg C
2.1 (3.8-1.2)
0.62
0.43
(0.65-0.29)
20
1.27 (1.62-0.99)
0.82 (0.99-0.68)
0.62
(1.16-0.33)
19
2.3
0.86 (1.23-0.60)
0.26
(1.00-0.07)
20
» • t
0.25
(0.29-0.21)
20
MATC probably greater
than 0.04 ug/1.
TABLE 25.
PARATHION TOXICITY TO Chironomus tentans
Exposure, days
LC50, ug/1
Instar
¦ at
start
1
660 (1040 - 420)
4 th
2
135 (703 - 26)
4 th
4
31.0 (43.4 - 22.1)
4 th
5
7.3 (11.8 - 4.6)
2nd
8
2.2 (3.2 - 1.5)
2nd
14
2.3 (3.4 - 1.6)
2nd
14
2.9 (3.7 - 2.3)
2nd
42
-------
SECTION VI
DISCUSSION
Of those fish species tested, bluegills were most sensitive to parathi-
on toxicity. The 96-hour flow-through acute LC50 of 0.51 (0.72-0.36) rag/1
determined for juvenile bluegills agrees with the 0.58 ppra value reported by
Pickering, Henderson and Lemke (40) for large bluegills in soft water.
These authors also reported a much lower parathion LC50 concentration of
0.095 ppm for small bluegills under static conditions.
Yearling brook trout exhibited a 96-hour LC50 of 2.00 (3.03-1.32) mg/1
under flow-through conditions. This is, however, somewhat greater than the
72-hour LC50 of 0.92 mg/1 given by Leland (16) for rainbow trout. It was
noted that trout responded over a wider range of parathion concentrations
than either bluegill or minnow.
Adult fathead minnows were killed by parathion concentrations within
the range of concentrations also lethal to bluegill and trout. A prelimin-
ary static acute test produced a 96-hour LC50 of L.60 (2.05-1.25) mg/l,
while a flow-through acute LC50 of 0.50 (1.03-0.24) rag/1 was realized in 96
hours. The preliminary static acute result (1.64 mg/1) agrees with litera-
ture 96-hour LC50 values of 1.3 tng/1 (40), 1.4 mg/1 (22, 41) and 1.6 mg/1
(42). By comparison, the flow-through results may seem low; however, all
test results were confirmed in preliminary testing, fish were handled care-
fully to avoid stress and parathion concentrations were checked by indepen-
dent analysis.
Chronic parathion effects on bluegill sunfish, including scoliosis,
abnormal protrusion of branchiostegal tissue and depressed acetylcholines-
terase activity, were found at parathion concentrations above 0.17 ug/1.
Scoliosis was found under similar conditions to involve calcium deposition,
and fusion of two to four vertebrae (Carter, 14). The protrusion in the
"throat" area of many parathion-exposed bluegills was unexpected. Affected
fish had shallow rapid breathing and flared gill covers—presumably due to
parathion-related nerve damage. The observed protrusions may have resulted
from mechanical damage to the fish's branchiostegal area as a result of such
spasmodic buccal pumping. Further histological characterization would help
to determine the cause of such parathion-related tissue abnormalities.
At a 3,2 ug/1 parathion concentration bluegills were able to live hav-
ing only 16 percent of normal AChE activity. Under optimum laboratory con-
ditions and little disturbance, affected fish could function marginally. In
nature, however, the ability of these same affected fish to feed and escape
predators would be diminished due to natural stress.
43
-------
Brook trout chronically exposed to 7.2 ug/l parathion for 9 months ex-
hibited no parathion-related toxicity symptoms, as judged by weight gain,
external appearance, AChE activity and production of embryos. The data ob-
tained for egg and fry survival during the chronic study are not conclusive
although a level of 32 ug/l parathion reduced hatchability of embryos from
untreated parents, while a level of 10 ug/l did not.
Fathead minnows exhibited deformities and a reduced egg production dur-
ing chronic exposure to parathion concentrations above 4 ug/l. No reduc-
tions in growth rate, hatchability or fry survival were demonstrated at the
higher concentrations tested. Thus, in both the bluegill and minnow tests,
the juvenile and adult stages seemed more sensitive to parathion than either
eggs or larvae. Although this could simply be a function of length of expo-
sure, Lewallen (43) found that 2.0 mg/1 parathion had no effect on one-week-
old rainbow trout sac fry, while 70 percent of one-month-old fry were killed
at the same level in 24 hours. Because parathion toxicity depends on activ-
ation, it is possible that older animals metabolize parathion more effici-
ently than young ones, increasing its toxic effect.
The retention of parathion in blood, muscle and other tissues was dem-
onstrated for all three fish species tested. Bluegill and trout acutely
exposed to parathion had residues several hundred times greater than expo-
sure water concentrations. In chronic tests, where residues probably reach
a steady state, brook trout demonstrated the highest parathion tissue resi-
dues, including muscle tissue with a concentration factor of 533. Whole
minnows had concentration factors of about 200 while bluegills had a maximum
parathion concentration of approximately 25. This suggests that bluegills
metabolize parathion more readily than either trout or fathead minnows which
could explain their greater parathion sensitivity. We cannot rule out the
possibility that the toxicity effect might be temperature as well as species
dependent. Temperatures during exposures were increased from trout, to min-
now, to bluegill. It is possible that bluegills held at lower temperatures
might metabolize less, and store more parathion. Further metabolic pathway
studies in fish or in other cold-blooded animals should be designed to re-
late effects of temperature, rate of metabolism and proportions of major
metabolites.
The two crustaceans tested, D. magna and G. fasciatus, were more sensi-
tive to parathion than either the insect, C. tentans, or the fish species
tested. For Daphnia, the maximum acceptable chronic level, about 0.08 ug/l,
was 0.13 times the 96-hour acute level of 0.63 ug/l. The acute value is
close to 0.8 ug/l reported by Priester (44) (50 hours) and Boyd (45) (26
hours). Gammarus, with a mean 96-hour LC50 of 0.39 ug/l, had a chronic ef-
fect concentration much less than 0.04 ug/l, a lethal level in one month.
At such low effect levels (which approach the chromatographic 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 lim-
ited to considerably less than 1 ug/l. In terms of protecting food chain
organisms, and hence fish, this would not be an overly conservative level.
44
-------
The midge, C. tentans, had a wide range of sensitivities to parathion
depending on flow rate, larval stage, and exposure time. It gave a 96-hour
LC50 of 31 ug/l and chronic effect level of less than 3 ug/1, a lethal con-
centration in two weeks. The midge was the only species tested that is
closely associated with sediment. The adsorption properties of parathion 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 application 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 a realistic ap-
proach, Only the shorter-lived invertebrates would under real conditions
normally be exposed for chronic time periods. Bluegills are not practical
species for presently conducted chronic bioassays unless a method is devised
to ensure spawning during the first summer of laboratory exposure. For all
fish bioassays, the egg handling, incubation and larvae rearing techniques
recommended and used were cumbersome and required considerable labor. In
many cases the harmful effects of handling outweighed the effect of the tox-
icant under invesigation. Among the invertebrates, Daphnia and Chironomus
proved to be convenient test organisms. In long-term tests, Gammarus had
inadequate survival using present cui ture techniques.
Finally, before accepting the results of such laboratory tests at face
value, an additional understanding of pesticide effect in a natural system
is needed. Mechanisms of direct uptake and trophic concentration should be
considered, as well as microbial degradation, and adsorption on sediments.
The effect on fish productivity and of reduction of invertebrates by pesti-
cides should also be investigated.
45
-------
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49
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APPENDIX A
BIOASSAY DATA
50
-------
TABLE Al. MEAN MONTHLY ANALYSES OF PARATHION, BLUEGILL CHRONIC TEST, ug/L
Tank
OA OB
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
Dec
Jan
Feb
Mar
Apr
May
June
July
Aug
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
June
July
Aug
Sept
Oct
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
0.12
0.21
0.04
0.19
0.12
0.03
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
0.12
0.21
0.04
0.20
0.L2
0.03
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
2.00
2.88
4.72
3.77
2.83
5.04
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
2.00
2.88
4.72
2.78
2.81
5.03
2.44
2.12
2.25
2.44
1.29
1.21
1.83
0.99
1.55
1.19
1.74
1.24
1.87
1.58
1.26
0.37
1.86
0.93
1.62
2.07
2.16
1.20
2.29
2.60
2.37
2.11
2.27
1.24
1.12
1.93
1.03
1.54
1.19
1.74
1.15
1.87
1.58
1.26
0.37
1.86
0.93
1.62
2.07
1.97
1.18
2.33
1.23
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
0.92
1.33
1.98
1.33
1.39
2.53
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
1.18
0.92
1.33
1.98
1.33
1.39
2.55
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
0.22
0.39
0.49
0.56
0.75
0.44
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
0.11
0.31
0.22
0.39
0.49
0.56
0.79
0.44
<9.19)(9.63)
(9.50){9.21)
0.14 0.14
0.17 0.18
0.02 0.10
0.11
0.19
0.13
0.20
0.12 0.12
0.19 0.23
0.12 0.12
0.13 0.13
0.09 0.05
0.06 0.06
0.08 0.08
0.08 0.08
0.17 0.17
0.40 0.40
0.14 0.14
0.29 0.29
0.26 0.26
0.38 0.38
0.17 0.16
0.32 0.31
Mean* 0.06 0.06 3.23 3.14 1.53
SD* 0.09 0.09 1.88 1.83 0.98
High 0.39 0.39 10.59 10.59 4.23
Low nd nd nd nd nd
1.59
1.03
3.95
nd
1.00
0.74
3.01
nd
1.00
0.74
3.01
nd
0.34
0.26
1.03
nd
0.34
0.26
1.08
nd
0.16 0.17
0.14 0.14
1.10 1.10
nd nd
*Based on weekly samples,
nd: not detectable.
51
-------
TABLE A2. ROUTINE WATER QUALITY, BLUEGILL CHRONIC TEST
N Mean Range
D.O., mg/l 1008 6,1 3.8-7.3
pH 252 7.80 7.62-8.20
Alkalinity, mg/l 252 157 142-172
Acidity, mg/l 252 6.4 0,3-13.1
Hardness, mg/l 252 265 158-309
¦ r i i ¦ m ¦ a—n—wnts—¦¦ a ¦sacarsac^^^^^^^^sacs i mmammammmmmmmammmmKmm
TABLE A3.
MEAN MONTHLY TEMPERATURES, BLUEGILL
CHRONIC TEST
Month
Deg C
Month
Deg C
Month
Deg C
Jan
23.0
Sept
26.4
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
TABLE A4. BLUEGILL SURVIVAL, CHRONIC TEST
Tank
Date
OA
0B
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
1971
Dec
1
20
20
20
20
20
20
20
20
20
20
1972
Mar
1
20
20
17
13
20
17
17
17
20
20
49*
11*
May
1
19
20
L7
12
20
17
17
17
19
20
34
10
Oct
1
L9
19
17
13
20
17
17
15
18
17
25
10
1973
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
Jul
11
10
15
17
13
20
17
16
15
9
13
10
9
Nov
1
9
9
8
8
11
9
9
9
8
9
7
7
*Eleven of same stock started in Tank 5B Feb 25, 1972; 49 from new stock
started in Tank 5A Feb 25, 1972.
§Later thinned to 3 males, 7 females per tank.
52
-------
TABLE A5. BRAIN ACETYLCHOLINESTERASE, BLUEGILLL, 18 MONTH
EXPOSURE, umol/min/g wee weight
Tank
1
2
3
4
5
1.26
3.14
3.03
6.34
7.35
1.36
3.47
2.92
7.40
7.43
1.28
4.07
3.22
5.88
1.74
4.22
3.19
6.74
1.23
4.98
2.10
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
1.37
3.35
2.86
6.59
7.39
L2.97
7.16
5.07
5.35
6.04
Mean 1.37 3.35 2.86 6.59 7.39 7.32
Percent of
control 18.7 45.8 39.1 90.0 101 100
TABLE A6. BRAIN ACETYLCHOLINESTERASE, BLUEGILL 23 MONTH
EXPOSURE, umol/min/g wet weight
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 1.98 1.45 4.03 7.15 8.50
Percent of
control 16.5 23.3 17.0 47.4 94.1 100
53
-------
TABLE A7. BROOK TROUT ACUTE TEST
a. Number surviving per tank
Tank
Hour
OA
OB
1A
IB
2A
2B
3A
3B
4A
4B
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
b. 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
4.18
3.82
1.83
1.89
0.99
0.92
0.46
0.47
0.23
0.26
SD
0.79
0.77
0.21
0.27
0.10
0.07
0.05
0.05
0.02
0.02
nd: not
detectable
•
c. Mean weight of
fish,
gm
81.1
83.8
86.0
78.2
83.6
89.6
73.1
82.4
68.4
87.7
74.3
82.9
TABLE A8. ROUTINE WATER QUALITY, BROOK TROUT CHRONIC
N Mean Range
D.O., mg/1 504 8.2 6.7-10.5
pH 280 7.81 7.47-8.06
Alkalinity, mg/1 312 154 150-183
Acidity, mg/1 284 6.0 4.0-10.0
Hardness, mg/1 284 295 256-342
TABLE A9. MEAN MONTHLY TEMPERATURES, BROOK TROUT CHRONIC
Month Deg C+2 Month Deg C+2 Month Deg C+2
May 14.0 Sept 16.7 Jan 13.1
June 13.8 Oct 15.0 Feb 13.6
July 14.2 Nov 13.4 Mar 14.2
Aug 15.8 Dec 13.3
54
-------
TABLE A10. MEAN MONTHLY ANALYSIS OF PARATHION IN WATER,
BROOK TROUT CHRONIC TEST, ug/L
Tank
OA OB 1A IB 2A 2B 3A 3B 4A 4B 5A 5B
May 0.07 0.07 9.50 8.40 3.41 3.27 1.84 1.83 0.86 0.94 0.40 0.46
June 0.04 0.05 6.12 6.40 2.70 2.57 1.59 1.53 0.63 0.63 0.46 0.44
July 0.04 0.03 9.49 9.52 4.43 4.50 2.94 2.84 0.76 1.07 0.59 0.62
Aug 0.02 0.01 11.86 11.70 5.36 5.30 3.84 3.84 0.91 0.90 0.36 0.42
Sept 0.02 0.02 1.32 1.32 0.80 0.80 0.59 0.59 0.31 0.31 0.11 0.11
Oct 0.16 0.16 7.19 7.19 3.77 3.77 2.56 2.56 l.il 1.11 0.29 0.29
Nov 0.02 0.02 3.68 4.84 2.16 2.54 0.94 0.89 0.83 0.83 0.21 0.21
Dec 0.01 0.01 3.77 3.77 2.15 2.15 0.72 0.72 0.42 0.42 0,11 O.LL
Jan 0.01 0.01 6.62 6.62 3.06 3.06 2.13 2.13 1.12 1.12 0.13 0.13
Feb 0.05 0.07 9.30 9.73 4.96 4.53 3.25 3.26 1.49 1.47 0.60 0.65
Mar 0.01 0.01 12.34 12.34 6.03 6.02 4,43 4.43 1.92 1.92 1.07 1.07
Mean 0.05 0.05 7.12 7.17 3.38 3.35 2.12 2.10 0.88 0.92 0.34 0.36
N 45 45 45 45 45 45 45 45 45 45 45 45
SD 0.11 0.11 4.34 4.17 1.99 1.98 1.44 1.41 0.62 0.60 0.31 0.32
High 0.23 0.23 16.90 14.00 6.74 6.82 5.01 5.01 2.30 2.30 1.06 1.14
Low nil nil 0.28 0.28 0.62 0.62 0.06 0.06 0.19 0.19 0.02 0.01
55
-------
TABLE
All.
BROOK
TROUT
SURVIVAL IN
CHRONIC
EXPOSURE
Tank
OA
OB
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
May 1
14
14
14
14
14
14
14
14
14
14
14
14
Jun 1
14
6
10
7
14
13
0*
12
13
13
14
14
Jul 1
14
5
8
6
14
1
0
12
13
13
14
13
Jul 19
14
5
8
6
15**
9**
12**
12
13
13
14
13
Aug I
14
4
8
6
15
7
9
12
7
12
14
13
Sep 1
14
4
8
6
15
7
9
12
7
12
14
13
Oct 1
14
4
8
6
12
7
8
12
6
12
14
13
Oct25§
14
4
8
6
12
6
7
12
6
12
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
Feb 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 27.
**One remaining fish from 2B moved to 2A on July 19. Nine new fish added to
2B, twelve added to 3A.
§Thinning. All tanks reduced to 2 males, 4 females. All fish from 2B and
3A removed. Six fish transferred from each corresponding duplicate.
56
-------
TABLE
A12.
BROOK TROUT WEIGHTS,
CHRONIC
EXPOSURE
May
1
August 25
February
Mean
Mean
Mean
we i gh t,
weight,
weight
J
Tank
I®
No.
S.D. gm
No.
S.D.
gm
No
S.D.
OA
63.9
14
12.8 182.3
14
32.8
331.7
6
80.7
OB
62.8
14
11.1 201.9
4
64.8
439.7
6
78.3
1A
60.6
14
8.8 179.7
8
42.5
347.6
5
72.8
IB
62.6
14
13.6 204.2
6
25.2
436.2
5
78.6
2A
60,6
14
6.9 163.8
15
17.4
370.2
5
119.9
2B
67.6
14
9.5
» •
290.5
6
47.7
3A
60.0
14
8.4
• •
...
394.5
6
100.6
3B
68.2
14
8.7 198.2
12
19.2
320.8
4
17.3
4A
67.0
14
10.5 209.0
7
28.6
358.8
4
149.7
4B
59.3
14
8.1 184.9
12
21,5
337.8
5
63.7
5A
59.9
14
8.2 172.4
14
20.4
351.2
5
45.9
5B
63.0
14
10.0 183.3
13
31.9
416.2
6
74.6
TABLE
A13. BROOK TROUT GONADOSOMATIC INDEX,
SIX MONTH
EXPOSURE, PERCENT
Tank
OA
1A
2B 3A
4A
4B
5A
5B
Female
0.21
0.28
1.27 3.87
0.30
0.33
0.31
0.34
0.24
3.11
0.36
9.75
0.34
4.25
7.10
5.01
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
1.73
1.25
2,51
TABLE
A14, BROOK TROUT GONADOSOMATIC INDEX,
NINE MONTH
EXPOSURE, PERCENT
Tank
OA OB
1A
IB 2A 2B
3A
3B
4A
4B
5A
5B
Female
0.77 0.65
0.05
0.68 0.02 0.41
0.48 0.70
0
.39 0
.05 0
.51
0.78
0.51 0.48
0.58
0.62 0.04 nil
0,14 nil
0
.49
nil 0
.47
0.10
1.26 nil
1.49
nil 0.67 1.00
0.46
0
.53 0
.70 0
.33
0.50
0.50 0.28
0.35
nil 0.43 0.63
0.58
0
.57 0
.50
0.42
0.40 0.41
Male
0.26 3.39
1.68
0.45 0.26
1.06 0.38
0
.18 2
.36 0
.14
0.36
0.34 0.56
0.70
0.08
-------
TABLE A15. SUMMARY OF RESIDUE RESULTS FOR BLOOD AND MUSCLE OF TROUT
EXPOSED TO PARATHION FOR SIX AND NINE MONTHS
Res idue,
ppb
Cone
Tank Avg water
No. of
Months
ratio,
no. cone, ppb
fish
exposure
Blood (B)
Tissue(T)
B/T
21 7.1
2
6
1302
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
TABLE A16.
ROUTINE
WATER QUALITY,
FATHEAD MINNOW
CHRONIC TEST
N
Mean
Range
D.O.,mg/l
384
7.1
8.1
-6.3
?H
90
7.94
8.05'
-7.78
Alkalinity, mg/1
90
153
168
-135
Acidity, rag/1
90
2.5
9.1-
-10.5
Hardness, mg/1
90
238
261-
-194
TABLE A17. MEAN
MONTHLY
WATER TEMPERATURE, FATHEAD MINNOW CHRONIC TEST
Month Deg C
Month
Deg C
Month
Deg C
Apr 24.8
July
23.8
Oct
24.3
May 23.9
Aug
23.2
Nov
23.5
June 23.5
Sept
24.2
Dec
24.3
58
-------
TABLE A18. MEAN MONTHLY ANALYSIS OF PARATHION IN WATER,
FATHEAD MINNOW CHRONIC TEST, ug/1
Tank
OA OB 1A IB 2A 2B 3A 3B 4A 4B 5A 5B
Apr 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 11.07 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 18.31 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 2.81 2.24 1.13 1.60
Tank
TABLE A19.
GROWTH AND SURVIVAL
OF FATHEAD
MINNOWS IN PARATHION
30 days
60
days
Mean
growth, ram
Number
present
Mean
length, mm
Number
present
Mean
length, nun
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
3B
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
5B
26
14.8
22
23.8
9.0
Number at start: 35 per tank.
59
-------
TABLE A20. DAPHNIA MAGNA ACUTE TEST, FLOW-THROUGH, 18°C
a. Number surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3B
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
b
. Parathion,
Uj
l/l
.
0.11
0.10
1.56
1.55
0.85
0.86
0.45
0.44
0.23
0.2<
c.
LC50, u
g/1
24 hour
2.70
(5
.61 -
1.30)
48 hour
1.00
(1
.73 -
0.58)
96 hour
0.62
(0
.90 -
0.43)
60
-------
TABLE A21. DAPHNIA MAGNA CHRONIC TEST
a. Number surviving
Tank
OA
0B
1A
IB
2A 2B 3A
3B
4A
4B
5A
5B
Start
10
10
10
10
10 10 10
10
10
10
10
10
1 wk
10A
89Y
9A
34 Y
0
0
0 0 10
9
10
10
10
10
2 wk
8A
47 6Y
6A
589Y
0
0
0 0 8A
271Y
8A
233Y
10A
387Y
9A
219Y
10A
37 5Y
10A
416Y
3 wk
7A
205Y
3A
51Y
0
0
0 0 OA
10 3Y
OA
176Y
9A
268Y
8A
280Y
10A
114Y
9A
367Y
Total
young
770
674
... ... 374
409
655
499
489
783
A: adult, Y:
young
•
b. Parathion analysis,
ug/1
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
^.48^0.47y 0.23
0.23
0.10
0.10
0.08
0.07
/ iJ '
. >; 7
I : J
77 f
."J-V t>b .75
c. LC50, ug/1
, 1 ^
, f t/ 7
1 week
0.28 (0.29 -
¦ 0.26)
2 week
0.25 (0.22 -
¦ 0.28)
3 week
0.14 (0.16 -
• 0.13)
RI50: 0
MATC: 0
.24 (0.32 - 0.17)
.08 ug/1
61
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TABLE A22. GAMMARUS ACUTE TEST
a. Number surviving
Tank
OA
0B
1A
IB
2A 2B 3A
3B
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
72 hr 10
10
0
0
0 0 8
7
9
10
96 hr 10
10
0
0
0 0 5
5
8
10
24 hour
b.
LC50, ug/1
2.1 (3.8 - 1.2)*
48 hour
0.62
96 hour
0.43 (0.65 - 0.29)
*For
juveniles at 20°C.
TABLE A23
. GAMMARUS
SUBCHRONIC TEST, 20°C, NUMBER
SURVIVING.
Tank
OA
OB
1A
IB
2A 2B 3A
3B
4A
4B
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
14A
8Y
0
0
0 0 0
1
6A
5Y
7A
4A
43 days 13
14A
0
0
0 0 0
1
3A
7 A
Mean para-
thion cone,
ug/1 0.03
0.02
1.64
1
.65
0.24 0.24 0.15
0.15
0.04
0.05
A: adults, Y: young.
62
-------
TABLE A24. CHIRONOMUS TENTANS ACUTE TEST
a. Number surviving
Tank
OA
0B
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
Start
10
10
10
10
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
72 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
b. Analyzed parathion,
ug/1
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
c.
LC50,
ug/1
48 hour
135
(703 -
26)*
96 hour
31.0
(43.4 ¦
- 22.1)
*For 4th instar larvae at 21°C.
TABLE A25. CHIRONOMUS TENTANS SUBCHRONIC TEST
a. Number surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3B
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
b. Analyzed parathion, ug/1
0.3 0.3 52.9 52.9 23.9 23.6 11.0 11.0 5.0 5.1 3.1 3.1
c. LC50, ug/1
14 days 2.3 (3.4 - 1.6)
63
-------
APPENDIX B
BROWN TROUT ACUTE TEST
Young brown trout, Salmo trutta Linnaeus, 16 to 19 cm total length,
were acutely exposed to parathion in an LC50 test and also in a residue up-
take test. The acute LC50 test method was identical to that of the brook
trout. The LC50 results for 24 and 96 hours {Table Bl) show essential
agreement with those for brook trout. Symptoms of parathion poisoning were
also similar.
TABLE Bl. BROWN TROUT ACUTE TEST
a. Number surviving
Tank
OA
OB
1A
IB
2A
2B
3A
3B
4A
4B
5A
5B
Start
10
10
10
10
10
10
10
10
10
10
10
10
24 hr
9
10
6
0
10
10
10
10
10
10
10
10
48 hr
9
10
0
0
9
10
10
10
10
10
10
10
96 hr
9
10
0
0
5
1
10
10
10
10
10
10
144 hr
9
10
0
0
0
0
9
6
10
1
10
10
b.
Analyzed
parathion i
concentration,
ug/1
Pooled
daily
nd
nd 2
.40
2.40
1.63
1.63 1.04
1.04
0.68
0.68
0.47
0.4;
c. LC50,
ug/1
Hour
LC50
Upper CL
Lower
CL
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
64
-------
In a separate test, brown trout of the same stock were exposed to the
two highest parathion levels which had caused no acute mortalities, 0.68 and
0.47 rag/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 were
transferred to two duplicate tanks with uncontaminated flowing water. Again
three fish per tank were removed at time intervals until all fish were gone
at 79 hours. Muscle samples dissected from the dorsal fin region were pre-
served at -18°C and later analyzed for parathion residues. The residue val-
ues shown in Table B2 are each the mean of true duplicates. Precision for
the clean-up and chromatographic method was ^+5 percent.
A simple model was applied to the data from the 0.47 rag/1 exposure and
plotted (Figure Bl). Assuming first order kinetics, the change in fish res-
idue , C, can be expressed as a function of Cw, the toxicant concentration
in water:
dc/dt = kiCw - k2C
C = K!Cw/k2 + I(C0 - k1Cw)/k2]e"k2t
where k\ is the rate constant for uptake from water, k2 is the rate of
elimination, metabolism, or other removal, and CQ is the initial fish res-
idue. The washout data were fitted to a least-square line of log C vs. time,
giving K2 = 0.002/hr. Substituting for k2 and C at 64 hours, the value of
Kj_ is about l.Q/hr. The calculated stady state level of parathion in the
muscle is 250 tng/kg, reached in about 90 days, assuming no mortality or
change in mechanism. The concentration factor of 500 then agrees very well
with the concentration factors for chronically exposed brook trout muscle.
Although the data are not as precise as one would like, they show that a
simple model can be used to describe parathion uptake in fish. The rate
constants would, of course, vary with the temperature, fish species, and,
perhaps, order of magnitude of the dosage.
65
-------
TABLE B2, PASATHION RESIDUES IN BROWN TROUT MUSCLE
Parathion
in water, mg/1
0.47
0.68
Uptake residues, mg/kg 2 hr
4.10
5.48
4,53
10.61
4 hr
4.87
7.48
6.48
8 hr
3. U
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
46.49
34.80
57.99
Washout residues, mg/kg 2 hr
64.05
72.85
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%.
66
-------
100
-------
APPENDIX C
CHLORINATED HYDROCARBON PESTICIDES IN FISH FOOD
Samples of fish food used for chronic tests were analyzed for chlori-
nated hydrocarbon pesticides. Initial attempts to attain effective clean-up
of these food samples using standard Florisil chromatography failed. The
silicic acid chromatography procedure reported by Kadoum (46) was subse-
quently used. Food samples were extracted with hexane in the presence of
Na2S04« Concentrated extracts were applied to columns of silicic acid (Mall-
inckrodt 100 mesh, chromatography grade) and eluted with hexane and 2%, 10%,
40% and 70% benzene in hexane and benzene. Fractions were pooled, evaporat-
ed and rechromatographed a second time with collection of individual frac-
tions: hexane, 2% benzene, 10% benzene, 40% benzene, 70% benzene, and ben-
zene. These were analyzed by GC on 3% OV - 17 on Chromosorb W at 195°C us-
ing electron capture detection. Standard solutions of 13 chlorinated hydro-
carbon 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 frac-
tion. The presence at low level (ppb) or absence of these pesticides could
not be verified. The overall results are summarized in Table CI. In gener-
al it was concluded that chlorinated hydrocarbon pesticide levels were low
in each food source used and should not have had a significant affect on the
tes ts.
68
-------
TABLE CI. PESTICIDE RESIDUES IN FISH FOOD, ug/kg
DDE
DDD Heptachlor Dieldrin
Food sample
Aldrin
Heptachlor
DDT
Epoxide*
Lindane**
Endrini
Starter mash
<1
<0.1
<0.5
nil
<0.3
ND
No. 3 pellet
nil
<0.2
<0.5
nil
<0.3
ND
No. 6 pellet
nil
<0.1
<0.5
nil
<0.6
ND
No. 5 pellet
§§
<0.1
<0.5
<1
<1
ND
*No true match of retention time (RT) with standard; nearest GLC peak taken
as heptachlor epoxide for calculation of no. 5 sample.
**No true match of RT with standard; nearest peak taken as Lindane for calcu-
lation. RT was 256 sec, vs. 249 sec. for lindane standards. Peak may be a
phthalate ester,
§These pesticides could not be detected (ND) unless present at very high
levels because of broad peaks of relatively high level interferences pres-
ent in the 70 percent benzene fraction.
§§A GLC peak near the RT of aldrin, but probably not aldrin, gave a value of
10-15 ppb calculated on the basis of aldrin.
69
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
ACUTE AND CHRONIC PARATHION TOXICITY
TO FISH AND INVERTEBRATES
5. REPORT DATE
Date of Preparation April 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Anne Spacie, Algirdas G. Vilkas, Gerald F.
William J. Kuc and Gerald R. Iwan
Doebbler,
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Union Carbide Corporation Environmental Services
10. PROGRAM ELEMENT NO.
1131021
Tarrytown, New York 10591
11. CONTRACT/GRANT NO.
Contract 68-01-0155
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Acute and chronic aquatic bioassays were conducted with a variety of organisms
using parathion [0,0-diethyl 0-(p-nitrophenyl) phosphorothioate] as the challenge
compound. Acute LC50 values ranged from a low of 0.38 ug/1 in invertebrates to a
high of 2.0 mg/1 in freshwater fish. Non-lethal effects were documented in bluegill
and brook trout chronically exposed to parathion concentrations in excess of 0.17
Ug/1 and 32.0 ug/1 respectively. Chronic exposure of fathead minnows to concen-
trations of 4 ug/1 resulted in deformation and reproductive impairment. Biocon—
centration of parathion in freshwater fish tissue ranged from five to several
hundred times that of the exposure water. Chronic no effect concentration of
parathion for D. magna was 0.08 ug/l. for G- fasciatus less than 0.04 ug/1 and
for C. tentans less than 3.1 Ug/1.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.lOENTlFIERS/OPEN ENDED TERMS
c. cosati Field/Croup
Bioassay
Pesticides
Hazardous materials
Toxicity
Parathion
Freshwater fishes
Invertebrates
Aquatic biology
06 Biological
and medical
science/toxi-
cology
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF pages
79
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R«». 4-77) phevioui edition is obsolete
------- |