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

-------
                RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency,  have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination  of  traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.   Environmental Health Effects Research
      2.   Environmental Protection Technology
      3.   Ecological Research
      4.   Environmental Monitoring
      5.   Socioeconomic Environmental Studies
      6.   Scientific and Technical  Assessment Reports (STAR)
      7.   Interagency  Energy-Environment Research and Development
      8.   "Special" Reports
      9.   Miscellaneous Reports

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on  the effects of pollution on  humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. Th is work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial,  and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia  22161.

-------
                                             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

-------
                                 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

-------
                                  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

-------
                                   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

-------
                                  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

-------
                                   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

-------
                                   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

-------
                               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

-------
                                   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

-------
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.

-------
                                   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.

-------
                                   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.

-------
                                   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

-------
            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

-------
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.

-------
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.

-------
                                  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.

-------
   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.

-------
   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.

-------
                                   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

-------
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

-------
                                  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

-------
                                   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

-------
 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

-------
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

-------
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

-------
   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.

-------
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

-------
                     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.

-------
                     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.

-------
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.

-------
                 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.

-------
                                    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

-------