Etofogiwl Research Series
USE OF EXPOSURE UNITS FOR ESTIMATING
AQUATIC TOXICITY OF ORGANOPHOSPHATE
PESTICIDES
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Dulnth, Minnesota 55804
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EPA-600/3-77-077
July 1977
USE OF EXPOSURE UNITS FOR ESTIMATING
AQUATIC TOXICITY OF ORGANOPHOSPHATE PESTICIDES
by
Donald T. Allison
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
ii
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FOREWORD
Our nation's fresh waters are vital for all animals and plants, yet our
diverse uses of water for recreation, food, energy, transportation, and
industry physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The environmental Research Laboratory in Duluth, Minnesota, develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects
aquatic life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through
use of models
—to measure bioaccumulation of pollutants in aquatic
organisms that are consumed by other animals, including
man.
This report describes the results of a preliminary study of the
relationships of exposure concentration, duration, and periodicity to the
toxicity of an organophosphate pesticide. A hypothesis is proposed for
estimation of aquatic environmental impact over a range of exposure
conditions.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
lii
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ABSTRACT
This study investigated the relationship of exposure duration,
concentration, and periodicity with regard to the toxicity of one
organophosphate insecticide to a freshwater fish. Flagfish (Jordanella
floridae) were exposed to uniform levels of diazinon throughout one and a
half generations to establish baseline chronic toxicity values. Three
additional tests were conducted with concentrations increased and duration
decreased fourfold. These exposures were administered as a single pulse in
each test to newly hatched, pre-spawning, and spawning flagfish, respectively.
Subsequent effects on population success were observed until parents and
progeny corresponded in age to the one and a half generations of the baseline
test. To facilitate comparison, the toxicant challenges administered in each
test were expressed as the product of exposure concentration and exposur"
duration ("exposure units").
All four types of exposure had similar overall effects on population
growth and survival at equivalent exposure units. However, initiation of
exposure during spawning caused temporary but complete inhibition of
reproduction at concentrations which did not produce this effect in fish
exposed continuously since hatch. This could have a severe impact on other
species with a restricted reproductive period.
This study suggested the hypothesis that exposure units can be used in
conjunction with previously established toxicity data to assess the
environmental impact of fluctuating water concentrations of organophosphate
pesticides over a wide range of concentration, duration, and periodicity.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables vii
Acknowledgment viii
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Methods 5
Experimental design 5
Statistical analysis 5
5. Results 9
Terminal biomass in pulse and baseline chronic exposures .... 9
Survival of parental fish in pulse, chronic, and acute tests . . 9
6. Discussion 12
References 14
Appendix: Details of test techniques and results 15
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FIGURES
Number Page
1 Schematic profiles of the diazinon concentrations administered
in the larval-juvenile, juvenile-adult, and adult-spawning
pulse exposures 6
2 Schematic profiles of baseline-chronic and pulse exposure
regimens 7
Estimated terminal biomass after one and a half generations
expressed as percentage of control 10
Survival of parental fish to reproductive age expressed as
percentage of control 11
vi
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TABLES
Number Page
1 Chronic Effects on Flagfish (Jordanella floridae) of Continuous
Life-Cycle Exposure to Diazinon 19
2 Relative Effects on Flagfish Progeny of Continuous Diazinon
Exposure of Parents and Progeny from Paired Observations
of Subdivided Egg Samples 20
3 Chronic Effects on Flagfish (Jordanella floridae) of Exposure
During Larval-Juvenile Stage of Parents to 21-day Pulse
of Diazinon 21
4 Chronic Effects on Flagfish (Jordanella floridae) of Exposure
During Juvenile-Adult Stage of Parents to 21-day Pulse of
Diazinon 22
5 Chronic Effects on Flagfish (Jordanella floridae) of Exposure
During Adult-Spawning Stage of Parents to 21-day Pulse of
Diazinon 23
6 Comparison of the Survival of Brook Trout and Fathead Minnows
Versus Exposure Units of Diazinon 24
vii
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ACKNOWLEDGMENTS
The author wishes to thank Leonard H. Mueller and his assistants for
diazinon analyses; and John G. Eaton and other members of the Environmental
Research Laboratory-Duluth for advice, assistance, and critical review of the
manuscript.
viii
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SECTION 1
INTRODUCTION
During the last decade many studies have been made of the long-term
effects on aquatic organisms of continuous exposure to toxicants throughout
complete or partial life cycles. However, pesticide concentrations in
natural waters fluctuate widely because of use patterns (Bradshaw et al.,
1972) and variation in the physical, chemical, and biological characteristics
of the water itself (Gomaa e_t a.L. , 1969; Cowart et^ £LL., 1971; Sethunathan
and Pathak, 1972).
Periodicity of exposure seems to influence the toxicity of some
pollutants. Acute toxicity of fluctuating levels of ammonia or zinc equaled
that of constant-concentration exposure if the cycle times were short.
Extending the cycle period increased the toxicity of ammonia but not of
zinc (Brown et^ al., 1969). Allison et_ ad. (1963) reported accretion of
tissue residues and progressive chronic toxicity over a period of 20 months
in trout exposed for one-half hour every 4 weeks to near-lethal concentrations
of DDT. A similar study by Allison (unpublished data) with the organo-
phosphate pesticide malathion failed to produce conclusive chronic effects.
However, continuous exposure to low levels of malathion will produce chronic
effects (Mount and Stephan, 1967; Eaton, 1970).
The influence of duration and periodicity of exposure on chronic toxicity
became a pertinent consideration following toxicity tests with diazinon
(Allison and Hermanutz, unpublished data). Continuous exposure caused chronic
effects in fathead minnows and brook trout at concentrations less than 10 3
times their 96-hr LCSO's. This great disparity makes the setting of a maximum
permissible environmental level from the results of long-term continuous
exposures to some toxicants unreasonable. A standard so derived would ignore
the influence of exposure duration on the toxicity of a relatively non-persist-
ent pesticide when the allowed maximum concentration probably would persist for
only part of the life cycle of many aquatic species,
The transport, distribution, and persistence of organophosphate pesti-
cides are subject to a large number of variable and generally unquantified
factors. Estimates of exposure profiles under field conditions can be
expected to have wide confidence limits. In addition, it would be impossible
to simulate all probable field combinations of pesticide concentration and
exposure duration in tests. Therefore, the current study was designed to
compare in general the relative environmental impact of long-term, low-
concentration exposure to a "non-persistent pesticide" with that of shorter
term, higher concentration exposures intermediate between the currently
standard acute and chronic toxicity tests. The possibility that major
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differences might follow exposures limited to different life stages was
included in the experimental design. It was also hoped that insight might
be gained into any basic time-concentration relationship for the toxicity of
organophosphate pesticides. However, no attempt was made to develop an
experimental design capable of distinguishing and quantifying small
differences for specific responses (growth, survival, egg hatch, etc. per
se).
The purposes of this paper are: (1) To note some problems inherent
in assessments of the environmental hazards of "non-persistent" pesti-cides;
(2) to present the method used to investigate the time-concentration
relationship of organophosphate-pesticide toxicity; and (3) to present the
hypothesis suggested by the results of the study.
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SECTION 2
CONCLUSIONS
Similar overall response displayed at equivalent exposure units independent
of the life stages exposed and exposure concentration or duration per se, led
to the conclusion that use of exposure units might permit calculation of
organophosphate pesticide toxicity over a wide range .of water concentration,
duration, and periodicity. Such a hypothesis would have important application
to estimating the impact of organophosphate pesticides in practical field
situations but it requires further testing and evaluation to verify its utility.
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SECTION 3
RECOMMENDATIONS
The hypothesis that the exposure-unit concept can be used in estimating
the impact of pesticides on aquatic populations rests on a limited data base.
Therefore, the results of this study should be verified for other organophos-
phate pesticides, other species of fishes having longer egg-to-egg life spans,
and aquatic invertebrates.
If acute toxicity tests are used to formulate water-quality criteria for
short-term contamination by non-persistent pesticides, the possibility of
residual chronic effects on survivors should be considered in addition to
concurrent mortality.
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SECTION 4
METHODS
EXPERIMENTAL DESIGN
Baseline chronic effects of several concentrations of the organophosphate
Insecticide diazinon on the flagfish (Jordanella floridae) were determined by
continuous exposure throughout one and a half generations. The time factor
involved was included by conversion to "exposure units" (exposure concen-
tration x exposure duration) to permit comparison with the "challenge"
experienced in exposures of shorter duration.
Three tests in which only a part of the life cycle was exposed used
exposure units equivalent to the baseline chronic test (i.e., the same
amounts of diazinon used in each level of the baseline chronic were delivered
in a shorter period of time by increasing concentration accordingly). These
tests were designed so that the highest concentration experienced was about
two-thirds the level known to be acutely toxic. To avoid shock from sudden
exposure to near-acute concentrations and to approximate a profile of
environmental contamination, maximum test concentrations were built up over a
period of a few days and then decreased more slowly at termination of
exposure. The result was a regimen in which concentrations were increased
from zero to maximum in four equal daily increments. Maximum levels were
maintained for 12 days and then reduced to zero in four equal decrements at
2-day intervals. Therefore, exposures in each test consisted of single
pulses of diazinon delivered over a period of 21 days (Figure 1). These
pulse exposures were administered to the separate test populations at 1, 29,
and 66 days post hatch, respectively (Figure 2). The pulse tests were
identified by the approximate life stage exposed as larval—juvenile,
juvenile-adult, and adult-spawning. Post-exposure observations were made of
the impact on treated parents and their untreated progeny to an age
comparable to the baseline-chronic-test fish. In the adult-spawning test the
progeny reared during exposure as well as the post-exposure progeny of
treated parents were observed.
Details of test techniques are given in the appendix.
STATISTICAL ANALYSIS
The indices chosen to reflect population impact were parental survival
and growth, egg production and hatch, and progeny survival and growth. The
tests were run sequentially with different generations or spawnings from a
common stock. Much of the extraneous variance inherent in sequential testing
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1000° h
(1070,1120,1170)°
h- , 500
< (510, 450, 380)
o:
LU
o
O 250
(290, 250, 210)
125
(130, 120, 130)
67.5
(60. 60, 60)
0
0
16
21
DAYS
Figure 1. Schematic profiles of the diazinon concentrations administered
in the larval-juvenile, juvenile-adult, and adult-spawning
pulse exposures. aNominal concentration.
concentrations in parentheses.
Average measured
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CHTONIC EXPOSURE'
(tester/
- Juvenile- Adult r Spanning ,
Parental
Egg Hatch
LARVAL- JUVENILE
PULSE EXPOSURE0
(test #2)
Progeny Egg
Hatch and
Larval Development
JUVENILE-ADULT,
PULSE EXPOSURE0
(test #3)
ADULT-SPAWNING
PULSE EXPOSURE*
(test
O.. ..DAYS.
.60.
Figure 2. Schematic profiles of baseline-chronic and pulse exposure
regimens. aOnly one set of equivalent exposure units is
represented for each test.
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was eliminated by expressing results as a percentage of the respective
control. Comparison was further simplified by combining all data for specific
effects into a common index of overall impact on population success which has
been named terminal biomass. Details of the calculation of terminal biomass
are given in the appendix.
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SECTION 5
RESULTS
TERMINAL BIOMASS IN PULSE AND BASELINE-CHRONIC EXPOSURES
Detailed results used to calculate terminal biomass are presented in
appendix, Tables 1, 3, A, and 5.
Response regressions of terminal biomass for the baseline chronic and
pulse tests are shown in Figure 3. As explained in the appendix, a time
factor of 90 days was chosen to calculate exposure units in the baseline
chronic test. For Jordanella floridae this time period included the life
stages normally exposed in standard "life-cycle" tests with fishes. To show
that a precise time value is not critical for demonstrating similar response
between the pulse exposures and the life-cycle baseline exposure, the
computational effects of a range of arbitrary baseline time frames between
60 and 120 days have also been delineated in Figure 3. The four types of
exposure had similar impact on the test populations when treatment was
quantified by exposure units.
SURVIVAL OF PARENTAL FISH IN PULSE, CHRONIC, AND ACUTE TESTS
In this study death of parental stock was limited to the actual exposure
period in the pulse tests and to the pre-reproductive period in the baseline
chronic test. This loss of breeding stock was a major factor influencing
reproductive potential and estimates of terminal biomass. It seemed likely
that deaths from acutely toxic exposure before the reproductive period would
have a similar influence on population success. Calculations were therefore
carried out to determine if exposure-unit relationship would hold true for
exposures of short duration.
Four-day acute data were available for flagfish from a previous study
(Allison and Hermanutz, unpublished data). Baseline chronic exposure units
for comparison of parental deaths were based on 60 days of pre-reproductive
exposure as no deaths occurred beyond this age. The regressions of parental
survival for pulse, chronic, and acute exposures (Figure 4) indicate that
correlation of effects with exposure units may have some validity in expo-
sures as short as 4 days.
Calculations of parental survival in acute and chronic exposures of
fathead minnows and brook trout were based on unpublished data from Allison
and Hermanutz (appendix, Table 6). Although chronic data for survival below
75% were not available, there is some indication that the exposure-unit
relationship may hold true for these species also.
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100
gso
H
§60
1
o
GQ
40
20
5O
Baseline chronic-calc.
for 90 days of exposure
Baseline chronic range
for 60 to 120 days
Larval-juvenile pulse
Juvenile-adult pulse
Adult-spawning pulse
500 5000
EXPOSURE UNITS (jjg/l x days)
50,000
Figure 3. Estimated terminal biomass after one and a half generations expressed as
percentage of control.
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100
o: so
cr
O)
40
20
0^ r Baseline chronic
(60-day pre-spawning
exposure;
Larva I-juvenile pulse
Juvenile-adult pulse
Adult-spawning pulse
Acute (96-hr exposure)
50 500 5000 50,000
EXPOSURE UNITS (jjg/l x days)
Figure 4. Survival of parental fish to reproductive age expressed as percentage of
control.
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SECTION 6
DISCUSSION
This study was designed as a preliminary investigation of the possible
relationship of exposure concentration, duration, and periodicity to the
toxicity of organophosphate pesticides. If very different degrees of chronic
response follow exposures of distinct life stages or intermittent versus
continuous exposure, these factors should be considered when evaluating the
environmental impact of non-persistent pesticides. The single-pulse exposure
used here was intermediate to the acute and chronic exposures currently used
to derive water-quality criteria. The pulse tests also investigated the
impact on population success of exposure limited to three different periods
in the life cycle of a freshwater fish.
Many of the specific effects accepted as significant in laboratory
bioassays cannot be correlated with overall survival of a population. It was
not the intent of this study to demonstrate differences in degree of sensi-
tivity for specific observed effects. The objective was to determine if
different patterns of exposure resulted in variations in impact on population
success. This study did not indicate any extreme population sensitivity to any
profile or timing of exposure. Instead response was correlated rather closely
to the product of exposure concentration and exposure duration ("exposure
units") over a wide range of time factors. However, there are obvious time
and concentration constraints on the use of exposure units. Validity must at
some point diminish on approaching very high or low concentrations for very
short or long durations respectively.
Subjective evaluation would be required to determine the advisability of
initiating exposure during a breeding season. Spawning was temporarily
inhibited in the adult-spawning exposure (see appendix), but terminal biomass
of Jordanella was not greatly affected. However, if the physical and
biological conditions necessary for successful reproduction in another
species are of limited duration, an entire year class might be severely
damaged by inhibition of spawning. Allison and Hermanutz (unpublished data)
observed that brook trout, severely distressed for several weeks after initial
exposure to diazinon, recovered and were capable of spawning after 6 months of
continuous exposure. Although the continuous exposure did appear to cause
some attrition, subjection of the trout to the same concentrations without
acclimation just before their limited spawning period might have been more
damaging to productivity.
In summary, the exposure-unit technique may have value In estimating the
environmental impact of non-persistent pesticides over a range of exposure
conditions. Estimated exposure units of a potential field situation could be
summed and compared to exposure units of field or laboratory studies for which
12
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results were already available. The concept should be verified with other
species and toxicants and its limitations established and recognized before it
is put to general use. In spite of restrictions it seems possible that the
hypothesis may well be validated with sufficient confidence to warrant its
inclusion in management decisions based on mass—balance modeling of water
pollution.
13
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SECTION 7
REFERENCES
Allison, D., B. J. Kallman, and 0, B. Cope. 1963, Insecticides, effects on
cutthroat trout of repeated exposure to DDT. Science 142:958-961.
Bradshaw, J. S., E. L. Loveridge, K. P. Rippee, J. L. Peterson, D. A. White,
and D. K. Fuhriman. 1972. Seasonal variations in residues of chlorinated
hydrocarbon pesticides in waters of the Utah Lake drainage system, 1970-1971.
Pest. Monit. J. 6:166-170.
Brown, V. M., D. H. M. Jordan, and B. A. Tiller. 1969. The acute toxicity to
rainbow trout of fluctuating concentrations and mixtures of ammonia, phenol
and zinc. J. Fish Biol. 1:1-9.
Cowart, R. P., F. L. Bonner, and E. A. Epps. 1971. Rate of hydrolysis of
seven organophosphate pesticides. Bull. Environ. Contain. Toxicol. 6:231-234.
Eaton, J. G. 1970. Chronic malathion toxicity to bluegill (Lepomis macro-
chirus Rafinesque). Water Res. 4:673-684.
Gomaa, H. M., I. H. Suffet, and S. D. Faust. 1969. Kinetics of hydrolysis of
diazinon and diazoxon. Residue Rev. 29:171-190.
Mount, D. I., and C. E. Stephan. 1967. A method of establishing acceptable
toxicant limits for fish — malathion and the butoxyethanol ester of 2,4-D.
Trans. Am. Fish. Soc. 96:185-193.
Sethunathan, N., and M. D. Pathak. 1972. Increased biological hydrolysis of
diazinon after repeated application in rice paddies, J, Agr, Food Chem.
20:586-589.
14
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APPENDIX
DETAILS OF TEST TECHNIQUES AND RESULTS
METHODS
Baseline Chronic Exposure
Flagfish (Jordanella floridae) were used as the test species. Their
relatively short life cycle (about 2 months egg-to-egg) allowed this series of
tests to be conducted sequentially within a period of 2 years.
A modification of the proportional diluter equalized solvent concentra-
tions in all test tanks. Temperatures were maintained between 25.5 and 26.5
C. Diazinon concentrations were nominally doubled between levels. Five
concentrations (14-240 yg/1) and the control were duplicated. Exposure began
with thirty 1-day-old flagfish per duplicate. Survival and growth were
determined at 35 days and at termination. Each duplicate was thinned to two
mature males and five mature females just prior to spawning. Two yarn-covered
spawning substrates were placed in each duplicate chamber at 60 days, and
spawning commenced immediately. After 60 days of spawning parental fish were
killed and weighed.
Egg production was recorded daily. If numbers permitted, one sample of
50 eggs per duplicate was retained each day to measure hatch success. Twenty
newly hatched larvae were placed in each of two larval chambers per duplicate
on a space-available basis and were reared for 35 days to measure growth and
survival (eight larval groups per treatment level).
Pulse Exposures
The pulse exposures were run sequentially in the test system used for the
baseline chronic. The same basic test techniques were used. Fish initially
exposed at 29 and 66 days (juvenile-adult and adult-spawning) were raised
as pooled stock before testing. Observation of progeny growth and survival
was limited to four groups per treatment level in the larval-juvenile and
juvenile-adult tests as exposure had been discontinued before the repro-
ductive period. Progeny produced early in the adult-spawning exposure (four
groups per level) were maintained for 21 days before being terminated to make
space available for post-exposure progeny studies of 35 days of growth and
survival.
15
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Chemical Analysis
Water levels of diazinon were monitored weekly in the baseline chronic
and daily during the 21-day pulse exposures. Concentrations were determined
by gas chromatography and electron capture detector.
Statistical Analysis
This study was conceived as a preliminary investigation to determine if
exposures of different duration and seasonal occurrence might cause major
differences in population success. Evidence of wide discrepancy might then
warrant more sophisticated and costly experimental designs to quantify the
factors involved. The possibility of extraneous variables (due to genetic and
physical differences) as well as an inherent problem in the test design of
precisely defining the time frame for each effect precluded direct comparison
by standardized methods of statistical analysis and the use of mathematically
derived tests of significance.
Exposure in the baseline chronic test lasted about 120 days. However,
dl deaths of parental fish took place before reproduction at 60 days. In
addition, prolonged exposure of adults during reproduction apparently did not
cause any correlated increase in effects on progeny. Therefore, for the pur-
pose of comparison, 60 days was chosen as the exposure duration for parental
deaths, and 90 days (60 days of parental exposure plus 30 days of progeny
exposure) was chosen to represent a baseline chronic exposure for this
species. The baseline chronic exposure period also encompassed the relative
duration of a standard life-cycle exposure for fishes. The five levels of
challenge could then be approximately quantified as "exposure units" to be
duplicated in the pulse exposures. This same problem of defining the true
exposure duration causing specific effects existed in the pulse exposures.
Consequently less confidence can be placed on the relationship of exposure
units to specific effects than to overall impact on population from the entire
exposure. Direct comparison and overall effects between these pulse tests is
probably justified to the same extent as that usually made between different
acute or chronic exposures with similar experimental designs. However,
extrapolation of comparisons between baseline chronic, pulse, and acute
exposures requires a greater degree of subjective evaluation.
The specific responses recorded in this study were parental survival and
weight of survivors, egg production per spawning female and hatch success,
and progeny survival and weight of survivors. These indices were used to
derive an index of overall impact on populaiton success, named terminal bio-
mass. As a matter of convenience, terminal biomass for each treatment was
computed and compared per each theoretical mating pair (one male and one
female) present at the start of exposure. The average weights of parental
males and females at test termination were multiplied by percentage parental
survival, and the results were added to give an estimated index for parental
biomass excluding the influence of thinning before reproduction. Progeny
biomass was estimated by multiplying percentage parental survival to spawning
by eggs per female by percentage hatch by percentage progeny survival to
16
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termination by average weight of surviving progeny. The values for parental
and progeny biomass were added for each treatment and expressed as a percen-
tage of their respective test control. Extraneous variance due to sequential
rather than simultaneous testing of the baseline and pulse exposures was
thereby removed by equating all control results to 100%, and comparison could
be made for population response versus exposure units.
Since actual exposure units delivered (determined by water analyses)
differed somewhat between tests, comparison was made with calculated regres-
sions of response. The regressions for the pulse tests are based on four
data points, as the highest levels of exposure killed virtually all of the
parental stock. Response within the range of the semilog plots presented in
Figures 3 and 4 was fundamentally linear as represented.
Because true time factors in these tests could not be exactly defined,
exposure—unit values must be considered as best-estimate approximations. Any
tendency to assign unwarranted accuracy to the results should be avoided.
RESULTS
Specific results of the baseline chronic and 21-day pulse exposures are
summarized in Tables 1 through 5 of this section. The response of progeny in
the baseline test (Table 2) is of special interest. In previous tests with
fathead minnows and brook trout (Allison and Hermanutz, unpublished data) all
responses of the progeny at lower test concentrations could be attributed to
the pre-spawning exposure of the parents. However, effects on flagfish
progeny appear correlated to both parental and progeny exposure. In flagfish,
progeny exposure alone apparently caused greater effects than parental expo-
sure alone, but this evaluation is subjective because paired data could not be
used in this case and analysis of unpaired data did not demonstrate a
statistically significant difference. Diazinon also reduced the incubation
time of flagfish eggs, an effect not seen with fathead minnows or brook trout.
Four samples of eggs per treatment were collected at the beginning of the
adult-spawning test just before spawning was interrupted by the exposures.
These eggs did not show any distinct evidence of reduced or early hatch as
noted in the baseline chronic, even though the average concentrations
experienced before hatch were generally higher than those to which eggs were
continuously exposed in the baseline test. Graduated increase in concen-
tration may have reduced toxicity, but more probably the sample size was
simply too small to identify differences in response. During the remaining
exposure period of 15 - 16 days following hatch of these eggs, the larvae
displayed a range of response equivalent to that of fish of the same age
exposed during the larval-juvenile pulse test.
In the highest concentration of each pulse test the fish were subjected
to about two-thirds their 96-hr LC50 during the 12-day pulse peak. Sixty
fish per test were exposed at this level, and only one of the 180 survived.
Survival in lower exposures was adequate for comparison of growth, survival,
and reproductive success. The three pulse tests did differ in degree of
response for specific indices. For example, parental stock survived best in
the larval-Juvenile exposure and poorest in the juvenile-adult exposure.
17
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Subsequent egg production per female was higher in the juvenile-adult expo-
sure, but survival of progeny was poorer. These apparent differences may
simply reflect variance due to different stocks of fish or slightly different
physical conditions in sequential tests. In any event, the differences tended
to cancel out during calculation of terminal biomass.
One effect was noted that was unique for the life stage exposed.
Exposure in the adult-spawning pulse test was delayed until all groups
commenced spawning. Spawning ceased abruptly when the various concentration
profiles reached about 130 yg/1. Fish exposed to no more than 130 yg/1 for
12 days resumed spawning as soon as the concentration was reduced, but fish
exposed to higher concentrations did not resume spawning until 1-2 weeks
after concentrations were reduced below 130 pg/1. During the post-exposure
period egg production in the treated groups equaled or exceeded the control.
The temporary inhibition of spawning appears to be the result of initial
exposure during the reproductive period. Fish in the baseline chronic
exposed to about 240 yg/1 since hatch showed no evidence of spawning
inhibition.
The actual impact of spawning inhibition on a fish population would
depend on the length of the spawning season of a given species. In the flag-
fish the 21-day pulse exposure resulted in only a temporary interruption.
Terminal biomass was calculated from equally weighted intra-exposure plus
post-exposure reproductive data versus post-exposure data alone. Both methods
of calculation resulted in biomass regressions within the range of those
derived for exposure during the other two life stages.
18
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TABLE 1. CHRONIC EFFECTS ON FLAGFISH (JORDANELLA FLORIDAE) OF CONTINUOUS
LIFE-CYCLE EXPOSURE TO DIAZINON
Average measured concentration (ug/1)
Exposure unit range (pg/1 x days) for
exposure period of 60 - 120 days
240
14,400 5
28,800 10
88
,280
,560
54
3,240
6,480
26
1,560
3,120
14
840
1,680
0
0
0
Parental effects
Survival to 35 days post hatch
Average length at 35 days post hatch (mm)
Survival to spawning (61 days post hatch)
Average weight of males at termination (g)
Average weight of females at termination (g)
Egg production per female parent
Hatch success of eggs
Average start of hatch (days)
Average end of hatch (days)
Survival to 35 days post hatch
Average weight at 35 days post hatch (g)
507.
17.7
307.
2.6*
2.2
Embryo progeny
722 1,
40%*
3.0*
4.1*
Larval progeny
72%
0.18*
77%
17.7
55%
3.3*
2.3
effects
174
52%*
2.9*
4.2*
effects
73%
0.20
78%
19.5
62%
3.4
2.3
966
60%
3.2*
4.3*
89%
0.21
87%
19.6
73%
3.7
2.4
1,324 1
62%
3.4
4.5*
93%
0.18*
80%
19.5
70%
4.2
2.5
,034
60%
3.5
4.6*
96%
0.18*
85%
21.0
80%
4.3
2.8
1,348
66%
3.5
4.9
85%
0.25
*Significantly different from control (P=0.05) using one-way analysis of variance and Dunnett's procedure.
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TABLE 2. RELATIVE EFFECTS ON FLAGFISH PROGENY OF CONTINUOUS DIAZINON EXPOSURE
OF PARENTS AND PROGENY FROM PAIRED OBSERVATIONS
OF SUBDIVIDED EGG SAMPLES
Exposure of both Exposure of progeny only
parents and progeny versus
Concentration versus No exposure of parents
(yg/1) Exposure of parents only or progeny
Start of hatch (days)
240 2.1 vs 2.7* 2.5 vs 3.5*
88 2.2 vs 2.2 2.5 vs 3.8*
End of hatch (days)
240 3.6 vs 4.8* 4.0 vs 5.2*
88 4.5 vs 4.9* 4.4 vs 4.9*
Hatch success
240 29% vs 46% 38% vs 62%
88 41% vs 52% 44% vs 59%
Larval survival3 - hatch to 35 days
240 48% vs 92% 40% vs 75%
88 88% vs 92% 92% vs 78%
Larval weight (g)a - 35 days post hatch
240 0.20 vs 0.26 0.19 vs 0.30
88 0.26 vs 0.27 0.24 vs 0.28
aAverage of two subsamples per treatment.
*Pairs statistically different (P=0.05) on the basis of "t" test. At least
eight pairs were used for each comparison
20
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TABLE 3. CHRONIC EFFECTS ON FLAGFISH (JQRDANELLA FLORIDAE) OF EXPOSURE DURING
N3
LARVAL- JUVENILE STAGES
OF PARENTS TO 21-DAY
PULSE OF DIAZINON3
Exposure units (ug/1 x days)
Average 12-day peak concentration (yg/1)
Survival to 35 days post hatch
Average length at 35 days post hatch (mm)
Survival to spawning (58 days post hatch)
Average weight of males at termination (g)
Average weight of females at termination (g)
Egg production per female parent
Hatch success of eggs
Survival to 35 days post hatch
Average weight at 35 days post hatch (g)
18,000 8,600
1,070 510
Parental effects
27.* 577.
22.9*
27.* 57%
4.3
2.1
Embryo progeny effects
687*
47%
Larval progeny effects
83%
0.32
4,800
290
87%
24.1
87%
3.7
1.6*
925*
45%
96%
0.32
2,300
130
93%
24.4
90%
3.8
2.1
1,314
567.
96%
0.29
1,000
60
787.
25.4
78%
4.9
2.9*
1,510
60%
85%
0.33
0
0
87%
25.3
87S
3.8
2.2
1,577
58%
93%
0.28
aParental stock exposed between 1 and 22 days after hatch.
Spawning begun 36 days after termination of diazinon exposure.
*Significantly different from control (P-0.05) using one-way analysis of variance and Dunnett's procedure.
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TABLE 4. CHRONIC EFFECTS OF FLAGFISH (JORDANELLA FLORIDAE) OF EXPOSURE DURING
ro
to
JUVENILE-ADULT STAGE OF PARENTS TO 21-DAY PULSE OF DIAZINON3
Exposure units (ug/1 x days) 17,600 7,100.
Average 12-day peak concentration (ug/1) 1,120 450
Parental effects
Survival to spawning (57 days post hatch)b 0%* 10%*
Average length 53 days post hatch (mm) 34.2
Average weight of males at termination (g) 4.1
Average weight of females at termination (g) 2.6
Embryo progeny effects
Egg production per female parent 924
Hatch success of eggs 42%
Larval progeny effects
Survival to 35 days post hatch 61%*
Average weight at 35 days post hatch (g) 0.45
3,900 2,000 950 0
250 120 60 0
43%* 95% 100% 98%
33.0 33.3 34.0 36.8
3.0 3.8 3.5 3.7
2.1 2.1 2.2 2.3
884 768 1,026 1,036
42% 40% 362 43%
91% 93% 65%* 94%
0.36 0,37 0.40 0.33
Parental stock exposed between 29 and 50 days after hatch.
Spawning begun 7 days after termination of diazinon exposure.
*Significantly different from control (P-0.05) using one-way analysis of variance and Dunnett's procedure.
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K>
TABLE 5. CHRONIC EFFECTS ON FLAGFISH (JORDANELLA FLORIDAEj OF EXPOSURE DURING
ADULT-SPAWNING STAGE OF PARENTS TO 21-DAY PULSE OF DIAZINON3
Exposure units (ug/1 x days) 18,200 6,100 3,500 2,100 1,000 0
Average 12-day peak concentration (bg/1) 1,170 380 210 130 60 0
Parental effects
Survival15
Average weight of males at termination (g)
Average weight of females at termination (g)
Embryo progeny
Egg production per pre-exposure female parent
Hatch success of eggs
Larval progeny
Survival to 21 days post hatch
Average weight at 21 days post hatch
Post-exposure
Egg production per female parent0
Hatch success of eggs
Post-exposure
Survival to 35 days post hatch
Average weight at 35 days post hatch (g)
07.* 577.
4.3
2.1
effects during exposure
31* 109*
767. 727,
effects during exposure
67.* 827.
0.09 0.49
embryo progeny effects
759
56%
larval progeny effects
95%
0. 25
795;
4.4
2.5
238
557.
697.
0.64
851
402
92Z
0.26
93%
5.2
2.3
137*
602
90%
0.70
718
59%
78%
0.31
100X
5.3
2.3
284
70%
82%
0.89
630
52X
927.
0.28
100?
5.1
2.6
454
69%
992
0.83
636
63%
86 7.
0.27
Spawning begun 60 days after hatch. Exposure initiated at 66 days when all groups were spawning.
Deaths of parental stock were limited to the 21-day exposure period.
From 10 days of spawning immediately following exposure plus 7 days of spawning 1 month after exposure
was terminated.
*Significantly different from control (P=0.05) using one-way analysis of variance .and Dunnett's procedure.
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TABLE 6. SURVIVAL OF BROOK TROUT AND FATHEAD MINNOWS VERSUS
EXPOSURE UNITS OF DIAZINON*
4-day exposure
Exposure units'3 Survival
680 90%
1,520 85%
2,040 75%
2,700 60%
3,640 40%
4-day
Exposure units
880
1,480
1,880
1,120
2.520
Brook trout
91-day exposure 173-day exposure
Exposure units Survival Exposure units Survival
870 92% 620 96%
1,660 75%
Fathead minnow
exposure 70-day exposure0
Survival Exposure units Survival
957, 945 98%
90% 1,220 95%
80% 4,421 83%
75%
60%
aUnpublished data from Allison and Heraanutz.
bug/1 x days.
cSurvival rates between 97th and 167th day of exposure. High mortality from causes unrelated to exposure concentration
precluded use of data from first 97 days.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-600/3-77-077
3. RECIPIENT'S ACCESSION* NO.
4. TITLE AND SUBTITLE
USE OF EXPOSURE UNITS FOR ESTIMATING AQUATIC
TOXICITY OF ORGANOPHOSPHATE PESTICIDES
5. REPORT DATE
July 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Donald T. Allison
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, MN 55804
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
SAME
13. TYPE OF REPORT AND PERIOD COVERED
In-House
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Environmental water concentrations of organophosphate pesticides can be expected to
fluctuate widely due to use patterns and rapid hydrolysis. This study investigated
some relationships of exposure concentration, duration and periodicity to the
chronic toxicity of diazinon to flagfish (Jordanella floridae). Effects were
compared on the basis of "exposure units" (exposure concentration x exposure
duration). Treatments at equivalent exposure units caused similar overall effects
on the test populations regardless of the life stages exposed or exposure duration
per se. The hypothesis is proposed that exposure units could be used to estimate
the environmental impact of fluctuating water concentrations of organophosphate
pesticides over a wide range of concentration, duration, and periodicity.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Pesticides*
Organic phosphates*
Diazinon*
Toxicity*
Freshwater fishes
Exposure units
Chronic toxicity
Pulse exposure
Flagfish
6F
7C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
TTnp laacH f -for!
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
25
U.S. GOVERNMENT PRINTING OFFICE: 1977—757-066/6466
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