EPA-600/3-76-033
May 1976
            Ecological Research Series
     ACUTE TOXICITY OF
CERTAIN  PESTICIDES TO
  ACARTIA  IQNSA  DANA
        Environmental Research Laboratory
        Office of Research and Development
       U.S. Environmental Protection Agency

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                 RESEARCH REPORTING SERIES

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

     1.    Environmental Health Effects Research
     2.    Environmental Protection Technology
     3.    Ecological Research
     4.    Environmental Monitoring
     5.    Socioeconomic Environmental Studies

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

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                                    EPA-600/3-76-033
                                    May 1976
ACUTE TOXICITY OF CERTAIN PESTICIDES TO
          ACARTIA TONSA DANA
                  by

           Fadhil H. Khattat
             Susan Farley

         Hazleton Laboratories
        Vienna, Virginia  22180
        Contract No. 68-01-0151
            Project Officer

            John H. Gentile
   Environmental Research Laboratory
   Narragansett, Rhode Island  02882
 U.S. ENVIRONMENTAL PROTECTION AGENCY
  OFFICE OF RESEARCH AND DEVELOPMENT
   ENVIRONMENTAL RESEARCH LABORATORY
   NARRAGANSETT, RHODE ISLAND  02882

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                             DISCLAIMER
     This report has been reviewed by the Environmental Research
Laboratory, Narragansett, U.S. Environmental Protection Agency,  and
approved for publication.  Approval does not signify that the
contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor endorsement or recommendation
for use.
                                ii

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                             ABSTRACT
The acute toxicity to the marine copepod Acartla tonsa Dana of four
technical grade insecticides was determined by bioassay using
standardized procedures, homogeneous populations and constant
laboratory conditions.  At a water temperature of 17 + 1°C, the
96-hour median lethal concentrations or tolerance limits for methyl
parathion, Azodrin, diazinon and toxaphene were computed as 0.89
milligrams per liter, 0.24 milligrams per liter, 2.57 micrograms per
liter and 7.2 nanograms per liter, respectively.  Residue analysis
for diazinon at zero and 96-hour exposure time revealed that the
amounts of diazinon uptake by three algal organisms is greater than
amounts concentrated by the copepod.  The toxicity of higher con-
centrations above 2.0 ppm (2 milligrams per liter) has offset copepod
uptake, while at lower concentrations, quantities concentrated by
Acartia are negligible.

Concurrently, the world literature was surveyed for supporting toxicity
data of these chemicals to closely related species.

This report was submitted in fulfillment of Project Number 68-01-0151
and Contract 68-01-0151 by Hazleton Laboratories under the sponsorship
of the Environmental Protection Agency.  Work was completed as of May 1972.
                                   iii

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                            CONTENTS
Section
Page
   I.      Conclusions




  II.      Recommendations




 III.      Introduction




  IV.      Culture Development




   V.      Bioassay Procedures




  VI.      Results




 VII.      Supporting Data




VIII.      References




  IX.      Appendix
  1




  2




  3




  4




 10




 13




 22




 24




 27

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                            FIGURES
1.     Growth Curves of Algal Clones                       7
                     i

2.     Schematic Plan of the Procedure of the             12


        Acute 96-Hour Test With Pesticides
                            vi

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1.    Average growth rates of R. baltica (H-), I. galbana      6
     (H2),  and C. nana (C,-) cultured at Hazleton Labora-
     tories, Inc., 1971-1972

2.    Results of acute 96-hour exposure of adult Acartia      14
     tonsa to technical methyl parathion

3.    Results of acute 96-hour exposure of adult Acartia      15
     tonsa to technical Azodrin

4.    Results of acute 96-hour exposure of adult Acartia      16
     tonsa to technical diazinon

5.    Results of a repeat acute 96-hour exposure of adult     17
     Acartia tonsa to technical diazinon

6.    Results of acute 96-hour exposure of adult Acartia      18
     tonsa to technical toxaphene
7.   Summary of acute 96-hour test and adjusted percentage   19
     mortalities and LC   values for Acartia tonsa exposed
     to methyl parathion, Azodrin, diazinon and toxaphene
8.   Computed LC1Q and LC5Q values with 95% confidence       20
     limits for 96-hour exposure of technical methyl
     parathion, Azodrin, diazinon and toxaphene to
     Acartia tonsa Dana

9.   Amounts of diazinon uptake by copepods and algae        21
     96 hours post-exposure in repeated acute toxicity
     test
                              vii

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                         ACKNOWLEDGMENTS
The support of the following individuals outside the company is
gratefully acknowledged:

Dr. Donald Heinle - Chesapeake Biological Laboratory, University of
Maryland, Solomons, Maryland - for initial copepod starting culture.

Mr. Donald Wilson and Miss K. Parish - U.S. Naval Research Laboratory,
Washington, D.C. - for initial algal starting cultures.

Mr. Michael Castagna - Virginia Institute of Marine Sciences,
Wachapreague, Virginia - for the supply of natural sea water for
the project.

Mr. David Clarke - State Inspection Service, University of Maryland,
College Park, Maryland - for verifying the chemical analysis of
water samples.

At Hazleton Laboratories, the extensive vigilance over water and
laboratory temperature and installation of equipment by Mr. Joseph. Trammell
and his assistants is hereby acknowledged with thanks.  Chemical analysis
of water samples, likewise, were performed by Dr. S.I. Shahied of the
Biochemistry Department.

The support of the project by the Environmental Research Laboratory at
Narragansett, U.S. Environmental Protection Agency, and the help
provided by Dr. John H. Gentile, the Project Officer and Mr. John Cardin
is acknowledged with sincere thanks.
                                  viii

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                             SECTION I

                            CONCLUSIONS

From experience in this study and from the work of others the following
conclusions can be made:

1.  It is possible to culture estuarine copepods for several generations
when due attention is made to curb contamination of water and culture
media from bacterial growth and other incidental contaminants.

2.  Relative toxicities of pesticides can be obtained only with homo-
geneous populations (reasonable uniformity in age, sex, size and
nutritional status of the animals exposed).

3.  Stability of extrinsic factors such as temperature, photoperiod
and population density are also important for interpretation of
bioassay results.

4.  Methods of dispensing the algal media, the water from one vessel
to another, the stability of salinity levels, and knowledge of the
mode of action and physical properties of the pesticides are
important parameters for conducting bioassay.

5.  Acute toxicities of toxaphene in the parts per trillion range,
diazinon in the parts per billion range, and methyl parathion and
Azodrin in the parts per million, range to the copepod Acartia
tonsa suggest possible pesticidal specificity.

These data augment the results obtained from pesticide residue analysis
in water samples performed by the National Monitoring Program at various
locations of the United States.

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                            SECTION II

                          RECOMMENDATIONS

1.  Reference organisms obtained from recognized reference labora-
tories should be used in starting cultures of Acartia, the
flagellates and diatoms.

2.  Acute 96-hour tests are too restrictive for sensitive species.
The measurement of susceptibility or tolerance should be based on
longer exposure duration, during which the peak effect of slow-
acting chemicals can be detected.

3.  Natural conditions should be simulated as far as possible.
Natural sea water of suitable quality with the appropriate
adjustment of salinity should be used as test and culture media.

4.  A short term continuous flow test is preferred over a static
one.

5.  Exposure techniques should be standardized so that workers in
various laboratories can communicate and verify their results in
comparable terms.

6.  Pesticide batches for bioassay should be selected from recently
manufactured samples and, as far as possible, standard reference
themicals of known composition should be utilized for controls.

7.  Five replicates and 20 organisms per replicate per concentration
should be used to reduce individual variations in response.

8.  Test mortalities of sensitive species must be given the oppor-
tunity of recovery within a reasonable period after terminating
observations at any one concentration, by transferring treated
individuals into food-containing test medium without the pesticide.
Recovered individuals should not be counted as dead.

9.  More than one closely related species, or preferably two,
biologically distinct species, should be tested at the same time
under similar conditions.  This will allow detecting species
specificity of the chemical tested.  If a chemical shows irratic
behavior in the dose-response curve due to some detoxification
mechanisms this can be cross-checked with the other species.

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                          SECTION III

                         INTRODUCTION


The scope of this contract was three-fold:

(1)  to produce levels of copepod populations sufficient for toxicity
bioassay (requires production of sufficient levels of three algal
species as food organisms);

(2)  to determine the acute and chronic toxicity of four selected
pesticides and define their median lethal concentration level.  In
the chronic phase, to define the effect of repeated exposure to the
pesticides on the growth and development of two generations of the
copepod; and

(3)  to review the literature for work of related scope.

Considerable effort was directed to raising several generations of
the copepod, to insure the development of healthy vigorous laboratory
stock, and to standardize the rearing protocol so that variations in
individual response to low concentration levels were minimized.

Acute or short term toxicity tests were carried out in accordance
with rigid procedures to safeguard against the influence of contamination
and experimental error.  Pesticide concentrations were prepared and
stored in a separate building and only on dosing were they brought to
the Bioassay Laboratory.  Conditions of temperature, light and salinity
were kept constant and uniform throughout.  Data were analyzed by a
computer program based on the Litchfield and Wilcoxon method of statis-
tical analysis.  Review of the literature was accomplished by consulting
a wide range of abstracting journals and information retrieval systems
available to private foundations, universities and government agencies.

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                               SECTION IV

                          CULTURE DEVELOPMENT

SEA WATER

As provided in the contract (Article II of the schedule), artificial
sea water based on a formula proposed by Kester et al (1967) was pre-
pared.  The object was to reduce travel time to the nearest ocean
source, provided that artificial sea water at 20% salinity level could
be produced in the laboratory that would sustain various stages of the
copepod's life cycle through at least two generations.  A pilot experi-
ment was conducted to ascertain this point.  Results revealed that
copepods originating from natural estuarine water with a salinity level
not exceeding 12% did not survive as adults on gradual adaptation up to
20% in artificial sea water.  Likewise, adult copepods reared in
Kester's artificial sea water at 32% at the Naval Research Laboratory,
Washington, D.C., did not survive at lower salinities of natural sea
water, when a few copepods were brought to our laboratory.  Conse-
quently, the artificial sea water was judged unsuitable for copepod
culture development.

With similar experiments, natural sea water yielded favorable
results at the conditions prevailing at our laboratory.  Estuarine
copepods survived and laid eggs; the eggs developed at graded levels
of salinity ranging from 29% natural sea water to 11.6% (or 40%
sea water).  It was therefore concluded that in order to reduce
rearing and sea water batch variations and insure high adult survival
and reproductive rates, copepods originally obtained from the
Chesapeake Bay at a salinity level of 12% could very well be adapted
to higher salinities of natural sea water.  In all experiments, cope-
pods were exposed to a salinity decrease or increase of 4% at a time
for a period of 24 hours (Lance 1963).  Subsequent transfers of cope-
pods into natural sea water at a salinity level of 20% gave encouraging
results, thus this concentration was finally chosen for all sea water
as culture and test media.  Natural sea water was diluted to this
salinity level by the addition of glass-redistilled water.  All water
batches were stored at 4°C prior to use.  Diluted sea water was left
overnight in an open vessel during which time 80% levels or higher
of oxygen saturation concentration and temperature equilibrium were
attained.

ALGAL CULTURES

Three species of marine algae, Isochrysis galbana, Rhodomonas baltica,
and Cyclotella nana  were grown axenically in enriched sea water
medium at a salinity level of 20%.  The algal medium used was the "f"
medium of Guillard and Ryther (1962), and its composition is outlined
in the appendix.  Before autoclaving, the medium was buffered with 50 ml
per liter of a 1% solution of tris (hydroxymethyl aminomethane) in
glass-redistilled water, and its pH was adjusted to 7.5.  All stock
solutions for the algal medium were prepared with glass-redistilled water,

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Final algal medium was autoclaved in quantities of 25 ml in 50 ml
screw-cap test tubes fitted with Teflon-lined caps, and 100 ml quanti-
ties in Erlenmeyer flasks at a temperature of 121°C and a pressure of
15 p.s.i. for no longer than 20 minutes.  Thereafter, two days were
allowed for pH equilibration before inoculation of new cultures.
Prior to subculture and when contamination of any culture was suspected,
sterility of the cultures was checked.  Sterility tests were performed
by inoculating the sterility test medium with 1.0 ml of the test sample.
Caps were tightened, and the inoculated tubes were stored in a dark
area for one to three days  (maximum of two weeks).  The appearance of
growth or turbulence in the test broth indicated presence of contami-
nation in the cultures tested.  Composition and preparation of the
sterility test medium is described in the appendix.

Marine algal cultures were  grown in sterile enriched sea water medium
under constant laboratory conditions.  Room temperature was 16 + 1°C
throughout, and culture tubes were placed in slightly inclined trays
under continuous illumination of 200 foot-candles originating from a
fluorescent, cool white light source.

Pure clones of Rhodomonas baltica  (H,.), Isochrysis galbana (lU) and
Cyclotella nana (C,-) obtained from the Naval Research Laboratory formed
the starting inocula.  Culture tubes containing 25 ml of sterile enriched
medium were inoculated weekly with approximately 1.0 ml of mature algal
cultures.  Larger quantities of algae were grown in Erlenmeyer flasks
and in a special fill and draw-off system.  Algal densities were
determined by direct measurement with a Model F Coulter counter.  Mature
algal cultures developed 11 to 14 days post-inoculation.  Average cell
densities attained at the optimum stage of development for providing
new inoculations and for feeding copepod cultures were approximately
2.3X10  cells per ml for Rhodomonas baltica, 5.7X10  cells per ml for
Isochrysis galbana and 3.8X10° cells per ml for Cyclotella nana (see
Table 1 and Figure 1).

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TABLE I:  AVERAGE GROWTH RATES OF R. BALTICA  (H-), I. GALBANA  (H,),-
AND C. NANA (C5) CULTURED AT HAZLETON LABORATORIES, INC.,  1971-1972
Average
Average initial
density at
inoculation
Day post-inoculation
6
13
20
27
Algal Densities
R. baltica
8.7 x 104
1.5 x 106
2.3 x 106
2.2 x 106
1.8 x 106
(cells/ml)
I. galbana
2.3 x 105
4.6 x 106
5.7 x 106
6.3 x 106
6.6 x 106

C . nana
2.2 x 105
2.9 x 106
3.8 x 106
3.9 x 106
3.6 x 106

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z
LU
O
_1
_l
LU
u
7x106
    6x1O6 -
5x106
4x10*
3x106
2x106
IxlO6
                    HU = Isochrysis go I ban a

                    C  = Cyclotella nana

                         Rhodotnonas baltica
                             Figure 1   Growth curves of algal clones

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Stock copepod cultures were fed the standard daily ration suggested by
Wilson and Parrish (1971) which is a combination of the three algal
species produced.  Cell densities for the feeding regimen were as
follows:

R.
I..
C.
Species
baltica
galbana
nana
Feeding Density
3.
5.
7.
OOX106
70X106
15X106
algal
algal
algal
cells
cells
cells
per
per
per
liter
liter
liter
of
of
of
copepod
copepod
copepod
culture
culture
culture
per day
per day
per day
The above densities were found to promote optimum copepod growth and
development.  Algal cultures showing any signs of contamination were imme-
diately discarded.

GOPEPODS

Acartia tonsa (Dana) was originally obtained from tows in the Chesapeake
Bay, by the Chesapeake Biological Laboratory, Solomons, Maryland.  Cope-
pods were cultured in natural sea water medium at an adjusted salinity
level of 20%.  Stable laboratory conditions were maintained throughout
the rearing period.  Room temperature was 17° + 1°C and cool white
fluorescent illumination was supplied at a photoperiod of 14 hours day
and 10 hours night cycle.  The rearing laboratory was kept as free from
contamination as possible.

All glassware was cleaned thoroughly by rinsing five times with acetone,
washing in hot soapy water, rinsing with distilled water, soaking in 3N
hydrochloric acid, and rinsing five times with acetone.  After air
drying and prior to use, dishes or vessels for copepod culture, bioassay
tests or chemical samples were autoclaved.  There procedures insured
freedom from contamination with bacteria and adsorption of trace levels
of detergents.

Copepods were cultured in 2.3 liter pyrex crystallizing dishes (190
mm x 100 mm) covered with a flat piece of glass.  Approximately 200
adult copepods were cultured in one liter of sea water, and each liter
of stock culture was fed a total algal cell density of 1.6X10  per day
comprising the three species.

Once a week, stock copepod cultures were transferred into fresh sea water
medium to reduce the possibilities of accumulation of toxic metabolites
and bacterial contamination.  For transfers, nylon-mesh Nitex netting
was used.  Separation of older copepodites and adults from immatures was
made with Nitex #63-T.  Transfers of the entire culture or of juvenile
and egg stages were made with Nitex #20-T.

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Before the transfer of organisms, heavy accumulations of algae and debris
were removed from the old culture dish with a dropping pipette.  Used
sea water medium was slowly drained off with the aid of a piece of
Nitex netting, sized according  to mode of  transfer or separation of
older stages from remaining populations and fitted snugly   to the rim
of the dish by a holding ring.  The net holding copepods was quickly
inverted and dipped into a clean sterile dish containing fresh sea
water,  Copepods were then immediately fed three times their normal
daily algal ration  (Wilson and  Parrish, 1971).

Generation cages were adapted for production and age standardization
of populations.  Twenty to 30 adults were  placed in each generation
cage made of a plexiglass cylinder  (125 mm X 90 mm) fitted with Nitex
#63-T screen netting approximately one inch from the base.  This
procedure allowed eggs laid by  females to  drop through the netting
and hatch without the possibility of cannibalism by adult copepods
 (Heinle, 1970).  In age standardization of stock populations, adult
cages were removed^after 24 hours.  Developing adults were  then set
aside for testing when gravid females appeared.  At our laboratory
conditions*, the average length of each developmental stage of
Acartia tonsa was as follows:

	Stage	       Length in Days	

    Egg  (newly oviposited)                    1
    Nauplius  (6 instars)                      7
    Copepodite  (6 instars)                    6
    Adult  (until gravid)                      3

    Total life cycle                         17

*During the course  of  technique development, the following references
were  especially useful on the biology and  behavior of Acartia tonsa:
Conover  (1956, 1959),  Lance  (1965) and Heinle (1966).  In the
culture of  copepods, emphasis was placed on some extrinsic  culture
requirements based  on  the experiences of Clarke (1959), Neunes and
Pongolini  (1965), Zillioux and  Wilson  (1966), Corkett (1967, 1968)
and Heinle  (1969).

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                            SECTION V

                       BIOASSAY PROCEDURES

TOXICANTS TESTED

The following materials were tested; CD 80% solution of technical grade
methyl parathion; (2) 80% solution of technical grade Azodrin; (3) 97.6%
solution of technical grade diazinon; and 100% technical grade toxaphene
(see Appendix for further details on the toxicants),
The following procedures were followed in Biological assay:

Pre-exposure

Ten adult copepods (of both sexes), of gravid age, were individually
transferred by means of a capture pipette into a small beaker containing
20 ml of sea water (20% salinity level).  Four such beakers were
prepared for each concentration per insecticide.  Prior to dose-ranging,
3 blind doses, using 10 copepods in 100 ml sea water per container at
an exposure of 24 hours, were made to gain an impression of the highest
dose to be finally used.  Insecticide doses from which a high dose and
subsequent four to six lower concentrations on a logarithmic regression
scale were selected.  In some instances, dose-ranging was based on
half the higher dose.  Higher concentrations were prepared in appropriate
solvent (double-distilled water for Azodrin and acetone for other
pesticides), using a precision Hamilton microliter syringe.  Lower
concentrations were obtained from the parent high concentration by
serial dilutions.  All insecticide solutions were tightly capped,
sealed with Parafilm and refrigerated.  Thirty minutes prior to use,
they were taken out and adjusted to room temperature before dosing.

Dosing

A number of 400-ml beakers formed the test vessels for the acute test.
Eighty milliliters of sea water were dispensed into each of 24 to 32
beakers.  0.1 ml of each parent concentration was added to each test
beaker to produce the desired concentration level before the addition
of a 20-ml sea water volume containing test organisms.  4.0X10  cells
per ml of test media of a ratio of 2:3.5:4.5 mixture of Rhodomonas,
Isochrysis and Cyclotella algal species respectively were added prior
to dosing.  Beakers were covered with a glass plate.   Comparable amounts
of the solvent carrier were added to the solvent control beakers.

A parallel dish for each concentration without food organisms was
similarly dosed and sent to the chemist for chemical analysis for
residue determination at the conclusion of the acute test.

Post-Dosing Observation

Organisms were observed 24, 48, 72 and 96 hours post-exposure (times
of exposure for each beaker were noted on the beaker, together with
concentration level and replicate number).  This included the number
                                   10

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of living and dead organisms in each replicate.  Attempts made
earlier in the tests to differentiate living from dead copepods by
use of vital stains yielded inconsistent results.  This method was
abandoned in favor of probing with a fine needle on the thorax of
dead adults to induce a response.  Those exhibiting slow diving
reaction, disoriented movement, tremor and discoloration were con-
sidered moribund and counted as dead individuals.  Water quality
measurement was made again on conclusion of the test.

LCcQ values with confidence limits were computed, using a computer
program based on the Litchfield and Wilcoxon (1949) method of
statistical analysis.  Figure 2 summarizes bioassay procedures.
                                 11

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           Figure  2    Schematic  plan  of the  procedure
                            acute 96-hour  test  with pesticides
PRB-EXPOSURE
      • Autoclave test vessels.

      • Dilute NSW to 20%o.

      • Oxygenate sea water
        overnight.

      • Measure and adjust water
        quality to provide optimum pH,
        temp., D.O.  and salinity.

      • Dispense water to
        test vessels:
        7 cone. x 4 - 28
        (80 ml. per vessel).

      • Prepare standard  age
        Acartia tonsa.
EXPOSURE
 Transfer copepods into small
 beakers corresponding with
 test vessels:
   10 copepods per vessel
   of 20ml. NSW.

 Feed test media with algae.

 Dose each cone, direct into
 test vessels.

 Dip copepods in beakers into
 test vessels.
                                    POST-EXPOSURE
•  Observe for mortality
   at 24, 48, 72, 96 hrs.
   post-treatment.

•  Observe controls for
   possible mortality,

•  Measure water quality
   at conclusion of test.

•  Calculate adjusted
   mortality rates.

•  Construct dose/mortality
   curve.

•  Estimate or compute
   LC10 ' LC50
   and LC9()for 96 hr.
   exposure.

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                            SECTION VI

                              RESULTS

As shown in Table 8, toxaphene is the most potent chemical of the four
tested to Acartia tonsa.  The LC50 value if  7.2 parts per trillion
(nanograms/liter).  Methyl parathion was the  least toxic with a value
of 0.89 ppm.  Tables 2 through 7 give raw data, adjusted mortalities,
and LCjo values of  the four pesticides  tested.

The order of toxicity from highest to lowest  of the four pesticides is
as follows:  toxaphene, diazinon, Azodrin and methyl parathion.  Azodrin
seems to be about four times as toxic as methyl parathion, while diazinon
is about 100-fold as toxic as Azodrin.

Unlike other cyclodiene insecticides, toxaphene is distributed through-
out the animal body by the hemolymph and has  no effect on the nervous
system.  Dehydrochlorination of this chemical under alkaline conditions
of sea water may enhance  its toxicity (Metcalf, 1955).  Azodrin, on the
other hand, is one  of a few water soluble pesticides, but this solubility
may enhance uptake  and metabolism of sub-acute levels or may promote
absorption throughout the organism and  result in quicker toxic action.
Chlorinated compounds, as a rule, are fat soluble, thus can be stored
in fat intact at concentrations greater than  the amounts ingested.

Organophosphorous compounds, such as diazinon, have different physical
properties and modes of action.  They hydrolyze in alkaline water media
and inhibit estrases of invertebrates (Metcalf, 1955).  Toxic phosphorous
compounds may produce different physiological manifestations because of
differences in stability, solubility and ability to inhibit the various
cholinesterases and aliesterases in the animal body.

The uptake of these pesticides by both  algae  and copepods is an indication
of their physiological tolerance and contributes to the rates affecting the
process of detoxification whereby conversion  to non-toxic metabolite generally
occurs at sub-lethal levels  (Metcalf, 1955).  To calculate the amounts
metabolized, either the remaining pesticide  residue in the aqueous phase
of the various dose levels or the residue in  the organisms must be
determined.  Minute quantities of some  pesticides in the parts per
billion range cannot be readily determined with available analytical
techniques from small quantities of test material.  This problem must
be resolved before  data derived from chemical analysis can be meaningful.
In the diazinon repeat test, the amounts concentrated by both algae and
copepods are summarized in Table 9.  Greater  amounts were taken up by
algae than by the copepods.  The lethality of higher concentrations above
2.0 ppm (2 mg./l) has apparently offset copepod uptake, while at lower
concentrations, quantities concentrated by Acartia are negligible.
                                      13

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-. Alive

: Dead
      T = Total
Table 2:   RESULTS  OF ACUTE 96-HOUR EXPOSURE OF ADULT ACARTIA  TOHSA TO
            TECHNICAL METHYL PARATHION  (10  COPEPODS EXPOSED PER REPLICATE)
                                              Acute Toxieity Tests

dose levels
1
115.0
8 p. p.m.

I 2 5
• 2
g * p. p.m.
8
I -1.0
g "p. p.m.

j .0.1
g P • P • m .
1 eO.Ol
§ Dp.p.m.
o 4) ^0.001
C P o
o t> r>.p.m.
o H - ^
x~^*
V
•P H fl
fl 0 0
a; fc .p
>• •£ 0)
H G o
O o d
01 O — -

1 I
5 §
1 °

replicates
1
2
3
4
T
I
2
3
4
T
I
2
3
4
T
1
2
3
4
T
1
2
3
4
T
1
2
3
4
~
1
2
3
4
T
1
2
3
4
T
Raw data from individual replicates at post-exposur
2k hrs.
A
1
it
1
1
T'
5
T
6
6
2U
8
9
8
9
31*
Y
10
10
9
36
10
8
10
10
38
9
10
10
30
8
10
10
10
38
10
10
10
10
1*0
0
y
6
9
9
33
5
3
It
it
16
2
1
2
1
6
3
0
0
1
1+
0
2
0
0
2
1
0
0
1
2
2
0
0
0
2
u
u
u
u
o
U8 hrs
A
0
0
0
0
0
it
6
5
it
19
7
9
7
7
30
7
8
8
8
3,1
10
9
9
9
37
y
10
9
10
~w
8
10
10
10
38
8
V
V
10
,te
D
10
10
10
10
ItO
6
It
5
6
21
3
1
3
3
10
3
2
2
2
9
0
1
1
1
3
1
0
1
0
2
2
0
0
0
2
'
-------
A = Alive
D= Dead
Total
         Table  3:    RESULTS OF ACUTE 96-HOUR EXPOSURE OF ADULT ACARTIA
                     TO TECHNICAL AZODRIN  (10 COPEPODS EXPOSED PER REPLICATE2
                                                    Acute Toxicily Tejh

dose levels

s
-. - 0.0001
1 p. p.m.
« n nm
!J Zp.p.m.
8
» n 01
u **p.p.m.
o
o
1 0.1
J *p.p.m.
8

a
• in
- 5 1-0
g P. P.m.
u
1 | Dis-
> c .
° 8 tilled
Water*
1 i
S | None
1 °

1
1
I
7
3
4
T
1
2
3
4
T
I
2
3
4
T
1
2
3
4
T
i
2
3
4
T
i
2
3
4
T
I
2
3
4

Raw data from individual replicates at post-exposur
2k hrs.
A
P
10
9
8
36
9
9
8
9
35
8
g^
8
8
33
8
T


T2
b
1
8
7
?H
10
10
10
10
40





0
1
n
i
2
it
1
1
2
1
5
2
1
2
2
7
2
3
1
2
8
k
3
2
3
12
0
0
0
0
u





1*8 hrs
A
Q
10
9
8
36
8
9
8
9
TU
8
9
8
8
33
'/
7
7
7
28
(S
6
7
?
2U
10
10
10
f°
Ho





D
1
n
i
2
t
2
1
2
1
6
2
1
2
2
7
3
3
3
3
12
k
k
3
"?
16
0
U
0
o
u





72 hrs.
A
0
q
9
8
35
8
7
8
8
31
7
7
6
7
27
b
5
6
7
2U
•7
it
6
5
20
10
y
10
10
^y





D
1
1
1
2
5
2
3
2
2
9
3
3
it
3
13
it
5
It
3
16
5
6
It
5
20
0
1
0
0
1





96 hrs.
A
Q
9
9
8
35
7
5
6
7
?8
6
6
6
7
25
i?
5
6
6
22
it
It
It
it
16
9
y
10
10
38





D
1
1
1
2
5
3
it
k
3
IS
It
it
it
a
15
1?
5
it
it
18
6
6
6
6
2it
1
1
0
0
2






A



































D




































A



































D




































A



































D



































j Percentage actual test mortalities from raw data
!lt hrs.
A




i




%




%




i




1-




%





D




10




12




17




20




30




u





1*8 hrs.
A



































D




10




15




17




30




iff)




0





72 hrs
A



































D




12




22




32




itO




50




=!.!>





96 hrs
A



































D




12




30




37




^5




60




? 0





_
A



































D











-
























A



































_
D




































A



































•»
D



































       *Azodrin Concentrations were  prepared in double-distilled water as  a  solvent.

-------
A= Alive

D= D«od
 TABLE 4:  RESULTS OF ACUTE 96-HOUR EXPOSURE OF ADULT ACARTIA TONSA  TO

             TECHNICAL DIAZINON  (10 COPEPODS EXPOSED PER REPLICATE)
F = Total                                        Acute Toxicity Tests

dose levels

I 8.0
a 1 PPB
5
u
o o
. J.
1 I
2 §
§ "

8
'o
o
~o_
u
1
2
3
4
T
I
2
3
4
T
i
2
3
4
T
1
2
3
4
T
1
2
3
4
T
i
2
3
4
T
1
2
3
4
T
i
2
3
4
T
1
2
3
4
T
Raw Data From Individual Replicates Post-Exposure
24 Hrs
A
7
9
7
9
3?
/
8
6
9
30




32*




32*
q
9
8
fi
34
9
8
10
9
36
in
10
10
9
39
9
10
9
9
3/
iU
10
10
10
4U
D
3
1
3
1
8
3
2
4
1
10




8*




8"
1
1
2
2
f>
1
?
0
1
4
o
0
0
1
1
J.
0
1
;
3
0
U
0
n
u
48 Hrs
A




29*




28*




30*




31*
q
7
7
7
30
9
8
8
9
34
10
10
10
9
3.9
y
9
9
9
36
10
10
10
in
w
o




11*




12*




10*




9*
1
3
3
3
10
1
2
2
1
6
0
0
0
1
1
1
1
1
1
4
0
0
0
n
u
72 Hrs
A




20*




24*
6
6
7
5
24
6
9
•}
6
26
q
7
6
fj
28
9
7
7
8
31
8
10
9
9
36
V
9
9
9
Jb
9
U
H
9
3.4
D




20*




16*
4
4
3
5
16
4
1
•i
4
14
1
3
4
4
1,2
1
3
3
?,
9
2
0
1
1
4
1
1
1
1
4
i
2
'/
1
b
96 Hrs
A
3
6
4
4
17
b
4
S
6
20
6
3
6
5
20
5
7
4
4
20
7
5
6
6
24
8
7
7
7
29
8
9
9
7
33
y
8
9
9
3i
B
8
H
q
M.
0
7
4
6
6
23
b
6
•>
4
20
4
7
4
5
20
5
2
6
6
19
3
5
4
4
16
2
3
3
3
11
2
1
1
3
7
i
2
1
1
i
2
2
?
—L-
1

A













































D














































A













































D














































A













































D













































Percentage Actual Test Mortalities From Raw Data
24 Hrs.
A




1




%




°r




%




of




fl




1-\




t




r,
D




?00




25.0




20D




20D




150




IQjO




2.5




7.1}




>,V
48 Hrs.
A














:




•

























D




?TH*




30. 9




25.0




22. 5«




i5.0




L5.0




2.5




in.r




y.v
72 Hrs
A













































D




™




40.0




400




35




300




225




10.0




inn




10.0
96 Hrs
A









t



































D




•i?,1?




50.0




50.0




48.7




4.0.J0




27.5




17. S




I?,1?




,7.'

A













































D














































A














































D















































A















































D















































-------
TABLE 5:
RESULTS OF A REPEAT ACUTE 96-HOUR EXPOSURE OF ADULT ACARTIA TONSA
A> Alive
D= Dead

dose levels
j 8.0
« 1 PPb
8
•> Q O
n J-t
g * PPb

1 - 1-6
g 3 ppb
1 0.8
,; 4 ppb
8
1 0.4
j 5 ppb

S - 02
J » ppb
8
» — 0 04
g ' PPb
o
*; -T
1 1
2 3
T3
II
§ u

1
f
1
2
3
4
J
1
2
3
4
T
i
2
3
4
J
1
2
3
4
T
i
2
3
4
T
i
2
3
4
1
1
2
3
4
T
i
2
3
4
T
i
2
3
4
T


Acute Toxicity Tests
Raw Data from Individual Replicates Post-Exposure
24 Hrs
A
8
R
8
in

8
4
4
fi
22
9
q
9
R

10
10
9
8
37
8
9
9
in
36
8
8
10
in
36
lu
9
7
7
33
in
10
in
9
39
in
9
10
10
39
D
2
2
2
0

2
6
6
4
18
1
1

2
5
o
0
1
2
3
2
•
1
n
4
2
2
0
0
4
0
1
3

7
0
0
0


0
1
0
n
I
48 Hrs.
A
fi
7

10

fi
2
2
4
I/
9
7
8
7

8
9
8
7
32
8
8
9
in
35
y
8
1C
9
3b
y
8
6
7
3
4

•>
3
8
2
2
1
0
5
2
2
0
1
b
1
2
4

10
2
1


)


4
0
'

\
0
i!
72 Hrs
A
4
6
5
9

2
2
1
4
9
8
7
8
fi

7
8
8
6
29
1
1
9
8
31
8
8
8
9
33
8
b
5
7
26
7
9
9
9
34
in
8
9
10
3/
D
fi
4
5
1
Ifi
8
8
9
fi
31
J>
•^
2
5
IP
3
2
2
4
11
3
3
1
2
9
2
2
2
1
7
2
4
b
•?
14
3
1
1
1
b
n

i
0
3
96 Hrs.
A
2
3
4
?

'




0
2
4
5
4
6
i

PO
6
6
6
6
2i
i
(
]


'

1
24
<
t
>
J
8
7
29
1
b
b
7
2b
7
8
9
9
33
in
8
9
9
3b
D
8
7

6
P
pc;

I
9
9
in
8
36
5
fi
4
5
PO
i
i
t
i




16
b
4
3
3
Ib
'



2
3
1!
3
4
b
3
Ib
3
2
1
1
7
0
2
1
1
4

A













































D














































A













































D














































A













































D













































Percentage Actual Test Mortalities from Raw Data
24 Hrs
A




%




%














%














%



—

D




t}t(




K,(




2.i




.5




3tC




o.t




7 F




2.5




'•r1'
48 Hrs.
A 1 D




$5.0
T



^5.0




22. 5




fo.c
i


j
12.5
I



12.5



I
25.0
1



10.0




|5.0
72 Hrs
A













































D




40.




77,




30.




?7i




'?if




7,5




$•(




b.f




.b
96 Hrs
A




I









|









4




t




^










0




'2.5




90.




50.




KM




flif




7,5




7,5




7 5




O.t

A 0









)

















































































•A













































D














































A













































D












,

































-------
         A= Alive
         D= Dead
T = Total
          TABLE  6:        RESULTS OF ACUTE 96-HOUR EXPOSURE OF ADULT ACARTIA TONSA
                         TO TECHNICAL TOXAPHENE (10 COPEPODS EXPOSED PER REPLICATE)
                                                         Acute Toxicifry Tests

dose levels

> 300.0
j 1 PPT*
8
1 30.0
~. O PPT
S *"
8

5 3.0
\ 3PPT
] „ 0-3
j 4 PPT
8
S n .U
72 Hrs,
A



































D




n*




1J.5




•?,*\




23.C




3I?iO









5-(
96 Hrs.
A



































D




77-




37.5




35-C




2T-1?




30^^









5.C

A



































D




































A



































D



































Data

A



































D



































00
         * Parts  per  trillion (nanograms/liter).

-------
                                 TABLE  7  -  SUMMARY  OF  ACUTE  96-HOUR TEST  AND  ADJUSTED
                                 PERCENTAGE MORTALITIES3  AND LCcg VALUES  FOR  ACARTIA
                                 TONSA  EXPOSED  TO METHYL  PARATHION,  AZODRIN,  DIAZINON
                                                    AND  TOXAPHENE

methyl parathion
Concentration Test M Adj . M LC50
Tested % %
(ppm)
5.0 100.0
2.5 62.5 57.1
1.0 47.5 40.0 0.89
0.1 25.0 14.3
0.01 15.0 2.9
(ppb)
8.0
3.2
1.6
1.0 12.5 0.0
0.8
0.4
0.3
0.2
0.1
0.04
0.03
3.0
0.3
0.03
Solvent Control 12.5
Untreated Control 10.0
diazinon
Azodrin (Average of Two Tests) toxaphene
Test M Adj . M LC5Q Test M Adj . M LC5Q Test M Ad j . M , LC5Q



60.0 57.9 0.24
45.0 42.1
37.0 33.7

65.0 58.8
70.0 64.7
50.0 41.2
30.0 26.3
44.4 34.6
40.0 29.4
2.57 77.5 76.3
27.5 14.7
12.0 7.4
27.5 14.7
67.5 65.8
35.0 31.6
27.5 23.7 7.20
30.0 26.3
5.0 15.0
5.0 5.0
aAbbott,  W.S.,  J.  Econ.  Ent.  1£,  265-267 (1925)
 Parts per trillion

-------
                             TABLE 8
            COMPUTED LCio AND LCso VALUES WITH 95% CONFIDENCE
            LIMITS FOR 96-HOUR EXPOSURE OF TECHNICAL METHYL
            PARATHION, AZODRIN, DIAZINON AND TOXAPHENE TO
            ACARTIA TONSA DANA (40 ANIMALS EXPOSED PER
            CENTRATION EXCEPT FOR DIAZINONa) .
Pesticide	        iP.10	             LC50
methyl parathion            0.07 ppm                   0.89 ppm
   (80% Technical)      (0.0418 - 0.1145 ppm).      (0.685 - 1.163 ppm)

Azodrin                     0.05 ppb                   0.24 ppm
   (80% Technical)      (0.0098 - 0.269 ppb)       (0.08536 - 0.66170 ppm)

diazinon                    0.04 ppb                   2.57 ppb
   (97.6% Technical)    (0.0164 - 0.0777 ppb)      (1.7259 - 3.8247 ppb)

toxaphene                   0.0035 ppt                 7.2 ppt
   (100% Technical)     )0.0006 - 0.0187 ppt)      (3.540 - 14.636 ppt)
    a Computation of a mean of two tests
          (80 animals exposed per concentration).
                                   20

-------
  TABLE 9.  AMOUNTS OF DIAZINON   UPTAKE BY COPEPODS AND ALGAE
     96 HOURS POST-EXPOSURE IN REPEATED ACUTE TOXICITY TEST
                              (ppb)
Amount of Residue   Amount of Residue in
in H90 (0 Hours)    in HJO (96 Hours)
Amount of Uptake
   (96 Hours)
Dose Level
at Exposure Copepods & Algae
0.2 0.16
0.4 0.34
0.8 0.73
1.6 1.3
3.2 3.0
8.0 7.7
a LC5Q =2.57 ppb
^C^ = 0.04 ppb
Copepods Copepods
Copepods Only & Algae & Algae Copepods Only Algae Only
0.17 0.06 0.10 0.0 0.11
0.35 0.24 0.10 0.0 0.11
0.68 0.54 0.19 0.05 0.14
1.1 1.1 0.2 0.2 0.0
2.0 2.6 0.4 ? ?
6.8 5.6 2.1 0.9 1.2



-------
                            SECTION VII

                          SUPPORTING DATA

To understand the extent and importance of pesticide pollution,
laboratory studies on acute and chronic toxicities are important.
From a columinous literature search, it seems that estuarine organisms
have received the least attention.  For the purpose of this contract,
we have retrieved 120 articles from the world literature dating back to
1950.  We found no reference to copepod toxicity studies relevant to
the pesticides being investigated.  Therefore, we are including, as
supporting data, some of the work relevant either to other marine
invertebrates or to fresh water organisms of related scope.  This review
is, therefore, a selective one aimed to include only valid data that
can be correlated with the results obtained in this study.

The subject of biological concentration of pesticides in aquatic
organisms has been reviewed by the excellent work of Dustman and
Stickel (1969), Macek (1969) and Pimentel (1971).

METHYL PARATHION

Methyl parathion is registered for use against cotton and small grain
insect pests where resistance to other insecticides had developed.

The LC5Q for blue gills was 8,500 ppb and for rainbow trout was
7,000 ppb (USDA, 1968).  Early toxicity studies with Anopheles quad-
rimaculatus larvae gave an LCgy of 0.0025 ppm for 48-hour exposure
(Negherbon, 1959).  A recent finding (Shim & Self, 1972) indicated
that the LC5Q to Culex tritaeniorhynchus larvae was 0.54 ppb.  For
Daphnia magna the LCjQ was 4.8 ppb (Frear and Boyd, 1967).  Macek and
McAllister (1970) gave data on the relative susceptibility of 12 fish
species exposed for 96 hours to methyl parathion which were in the
order of TL5Q values of 1.1 to 3.3 ppm.  The only toxicity tests with
marine species were those of Eisler (1969) in which the 96-hour LC$Q
value using the sand shrimp Crangon septemspinosa was 2 ppb and with
the grass shrimp Palaemonetes vulgaris the corresponding LC5Q was 3 ppb.

AZODRIN

Azodrin, being a new organophosphorous chemical used in the control of
cotton insects resistant to organochlorine pesticides, has not been
implicated in the environment as much as other agricultural chemicals.
Data on its toxicity to aquatic organisms is minimal.  F.W.P.C.A. (1968)
reported 48-hour chronic toxicity for rainbow trout as 7-0 ppm.  Eichel-
berger and Lichtenberg (1971) stressed that Azodrin, among other organo-
phosphorous compounds, was the most stable in raw river water, and per-
sisted over a two-month period.  This pesticide deserves further toxicity
study with aquatic organisms.
                                   22

-------
DIAZINON

This is a popularly used chemical both  in  agriculture  and  in animal and
human health.  Its toxicity  for  fish  is widely documented.  The LC$Q
for blue gills (48-hour exposure) was 86 ppb  and  170 ppb for rainbow
trout  (Cope, 1966).  Aquatic arthropods, on the other  hand, respond
very quickly to  trace  levels of  diazinon.  For example, Aedes aegypti
LC5Q for 24-hour exposure  was 3.3 ppb (WHO, 1970),  and the water flea
Daphnia pulex responded to 0.9 ppb  (LC5Q for  48-hour exposure),
Sanders and Cope,  1966).   Gammarus  lacustris, a fresh  water amphipod,
responded to 200 ppm  (LC5Q for 96 hours)  (Sanders,  1969).

TOXAPHENE

This insecticide is also used in agricultural pest  control.  Because of
its high mammalian toxicity, it  is  restricted to  a  low residue tolerance
level  on food commodities.  Its  LCjQ  for 24-hour  exposure  for the rain-
bow trout was 7.6 ppb  and  7.2 ppb for blue gills  (USDA, 1968).  This
chemical has been widely tested  against fresh water invertebrates and
some selected data are as  follows:
 Species
Exposure Time      LC50
   (Hours)	(ppm)
       Reference
 Gammarus lacustris
 Pteronarcys californica (naiad)
 Daphnia magna
 Daphnia pulex
 Simocephalus serrulatus
     96
     96
     26
     48
     48
26     Sanders,  1969
 2.3   Sanders & Cope,  1968
94     Frear & Boyd, 1967
15     Sanders & Cope,  1966
19     Sanders & Cope,  1966
 No references were found with effects shown in the parts  per  trillion dose
 range.

 Apart from toxicity to aquatic animals,  chemicals tend to accumulate in
 other forms of life, such as bacteria, diatoms, algae, and non-aquatic
 species.   Studies on the interaction between pesticides and aquatic plants
 and animals have been increasing within the last few years.   However,
 the pesticides under reference in this contract have not  as yet  been
 adequately studied in this regard.
                                        23

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                           SECTION VIII

                            REFERENCES

Clarke, G.L. 1959-  Culture methods for pelagic marine copepods.  In:
Culture Methods for Invertebrate Animals.  Dover Publications, Inc., New
York. pp. 221-224.

Conover, R.J. 1956.  Oceanography of Long Island Sound, 1952-1954.  IV
Biology of Acartia clausi and Acartia tonsa..  Bull. Bingham Oceanogr.
Coll. 15: 156-233.

	.  1959.  Regional and seasonal variation in the respiratory
rate of marine copepods.  Limnol Oceanogr, 4:259-268.

Cope, O.B. 1966.  Contamination of the freshwater ecosystem by pesticides.
Journal of Applied Ecology, 3:33-44.

Corkett, C.J. 1967.  Technique for rearing marine calanoid copepods in
laboratory conditions.  Nature, 216:58-59.

	.  1968.  La reproduction en laboratoire des copepods marine
Acartia clausi Ciesbrecht et Idya furcata (Baird).  Pelagos - Bull. Inst.
Oceanogr. Alger, 10:77-90.

Dustman, E.H. and L.F. Stickel. 1969.  The occurence and significance of
pesticide residues in wild animals.  Ann. N.Y. Acad. Sci. 160:162-172.

Eichelberger, J.W. and J.J. Lichtenberg. 1971.  Persistence of pesticides
in river water.  Environmental Science and Technology, 5(6):541-544.

Eisler,, R. 1969. Acute toxicities of insecticides to marine decopod crusta-
ceans.  Crustaceana. 16(3):302-310.

Federal Water Pollution Control Administration. 1968. Report of the
Committee on Water Quality Criteria. Government Printing Office, Washing-
ton, D.C. 62 pp.

Frear, D.E.H. and Boyd, J.E. 1967.  Use of Daphnia magna for the microbio-
assay of pesticides. I. Development of standardized techniques for rearing
Daphnia and preparation of dosage mortality curves for pesticides.
Journal of Economic Entomology. 60:1228-1239.

Geigy Agricultural Chemicals. 1972. Technical Data on Diazinon, Personal
Communication.

Guillard, R.R.L. and J.H. Ryther. 1962. Studies of marine planktonic diatoms.
Canadian Journal of Microbiology, 8:229-239.

Heinle, D.R.  1966. Production of a calanoid copepod, Acartia tonsa, in the
Patuxent River Estuary. Chesapeake Science, 7(2):59-74.
                                      24

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                       1969. Culture of calanoid copepods in synthetic sea
water. Journal Fisheries Research Board of Canada, 26(1):150-153.

_    	. 1970. Population dynamics of exploited cultures of
Calanoid Copepods.  Helgolander wiss.  Meeresunters, 20:360-372.

Hercules, Inc. 1970. Agricultural Chemicals. Technical Data Bull., No. AP-
103-A. 3 pp.

Kester, D.R., I.W. Duedall, D.N. Connors, and R.M. Pytkowicz.  1967.
Preparation of artificial  seawater. LimnoL. Oceanogr. 12:176-178.

Lance, J. 1963.  The salinity  tolerance of some estuarine planktonic
copepods. Limnol. Oceanogr. 8:440-449.

Lance, J. 1965-  Respiration and osmotic behavior of  the Copepod Acartia
 tonsa in dilute  sea water. Comp. Biochem. Physiol. 14:155-165.

Litchfield, J.T., Jr. and  F.  Wilcoxon. 1949.  Simplified method of evaluating
 dose-effect experiments. J. Pharmacol. Exptl. Therap. 96:99-113.

Macek, K.J. 1969- Biological  magnification of pesticide residues in
 food  chains.  In; The  Biological Impact of Pesticides in  the Environment
 Environmental Health  Series No. 1,  publisher, city,  pp. 17-21.

Macek, K.J. and  W.A.  McAllister. 1970.  Insecticide  susceptibility of some
 common fish family representatives.   Transactions of the American Fisheries
 Society. 99(1):20-27.

Menzel, D.W., et al.  1970. Marine phytoplankton vary in  their response to
 chlorinated hydrocarbons.  Science.  16751724-1726.

Monsanto Corp.  1971.  Parathion, Methyl Parathion and Stabilized Methyl
 Parathion, Technical  Bull. No. AG-lb. Monsanto	, St. Louis, Mo.
 pp.  12-13.

Metcalf, R.L. 1955. Organic  Insecticides: Their Chemistry and Mode of
Action. Interscience  Publisher, Inc., New York. 392-pp.

 Negherbon, W.O.  1957. Handbook of Toxicology  III. Publisher, city. 101 pp.

 Neunes, H.W.  and G.F. Pongolini. 1965. Breeding a pelagic copepod,
 Euterpina acutifrons  (Dana),  in the laboratory. Nature 208:571-573.

 Pimentel, D.  1971. Ecological Effects of Pesticides  on Non-Target Species.
 Executive Office of  the President,  Office of  Science and Technology,
 219  pp.

 Sanders, H.O. and O.B.  Cope.  1966.  Toxicities of several pesticides  to
 two  species of  cladocerans. Transactions of  the American Fisheries Society.
 95(2):165-169.
                                       25

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	. 1968.  The relative toxicities of several
pesticides to Naiads of three species of stoneflies. Limnol. Oceanogr.
13(1):112-117.

Sanders, H.O. 1969. Toxicity of pesticides to the crustacean Gammarus
lacustris.  Technical Paper No. 25, Bureau of Sport Fisheries and
Wildlife, Fish and Wildlife Service. 18 pp.

Shell Chemical Company. 1969. Technical Data Bulletin, Agricultural
Division, No. ACD65-151. 4 pp.

Shim, J.C. and L.S. Self. 1972. Toxicity of Agricultural Chemicals to
Larvivorous Fish in Korean Rice Fields. Unpublished World Health
Organization Document, No. WHO/VBC772.342. 6 pp.

U.S. Department of Agriculture. 1968. Suggested guide for the use of
insecticides to control insects affecting crops, livestock, households,
stored products, forests, and forest products. Agriculture Handbook,
No. 331, p xii.

Wilson, D.J. and K.K. Parrish.   1971. Remating in a planktonic marine
calanoid copepod. Marine Biology, 9:202-204.

World Health Organization. 1970. Insecticide resistance and vector control.
Techn. Rep. Ser. 443:77.

Zillioux, E.J. and D.F. Wilson. 1966. Culture of a planktonic calanoid
copepod through multiple generations. Science 151:996-998.
                                    26

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                             SECTION IK
                             APPENDIX

 1.  "F" ALGAL MEDIUM - COMPOSITION
 Sodium Salts
     Na N03                  150 mg
     Na H2P04.H20             10 mg
     Na2 Si03.9H20            30 mg
     Fe Sequestrene           10 mg
 Trace Metals
     CUS04.5H20                0.0196 mg
     Zn S04.7H20               0.044 mg
     CoCl2.6H20                0.022 mg
     MnCl2.4H20                0.36 mg
     Na2Mo04.2H20              0.0126 mg
 Vitamins
     Thiamine. HC1             0.2 mg
     Biotin                    1.0 yg
     B12                       1-0 yg
 Sea Water                     To 1 liter
 2.  STERILITY TEST MEDIUM-COMPOSITION
     1 gm       Yeast extract
     1 gm       Trypticase
     1 ml       FeCl3.6H20 stock solution
                (5 mg/ml glass redistilled water)
     0.5 ml     K-HPO, (anhydrous) stock solution
                (1 gm/100 ml glass redistilled water)
   850 ml       natural sea water
    50 ml       1 percent stock solution of tris
                (hydroxymethyl aminomethane) pH 7.5
ca. 100 ml      glass redistilled water
                                27

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Preparation

Combine substances listed above and mix thoroughly.  When a uniform
suspension has been obtained, heat with frequent agitation until
the mixture boils for one minute.  Dispense 10 ml amounts per 25 ml
screw-cap tube.  Autoclave at 120°C, 15 psi, for 20 minutes.  When
sterility test medium has cooled, tighten caps and store at room
temperature.

3.  PESTICIDE SPECIFICATIONS

Methyl parathion (Monsanto)

   Chemical name:  0,0-dimethyl 0-_p_-nitrophenyl phosphorothioate
   Formulation:  Technical, 80% in solution
   Solubility:  Insoluble in water; soluble in acetone and other
                organic solvents

Azodrin  (Shell)

   Chemical name:  Dimethyl phosphate of 3-hydroxy-N-methyl-CIS-
                   crotonamide

   Formulation: Technical, 80% in solution

   Solubility: Soluble in water, acetone and other organic solvents

Diazinon  (Geigy)

   Chemical name: 0,0-diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl)
                  Phosphorothioate

   Formulation:  Technical, 97.6% in solution

   Solubility:  Very slightly (40 ppra) soluble in water; soluble in
                acetone and other organic solvents

Toxaphene (Hercules, Inc.)

   Chemical name:  Chlorinated Camphene containing 67-69% chlorine

   Formulation:  100% Technical with a chlorine content of 67-69%

   Solubility:  Slightly soluble in water (3 ppm) and soluble in
                aromatic hydrocarbons.
                                28

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                                  TECHNICAL REPORT DATA
                           (Please read Instructions on the reverse before completing)
 FUPORT NO.
 EPA-600/3-76-033
            3. RECIPIENT'S ACCESSION-NO.
 TITLE AND SUBTITLE
                                                           5. REPORT DATE
 Acute  Toxicity of Certain Pesticides  to Acartia
 Tonsa  Dana
            6. PERFORMING ORGANIZATION CODE
                                                             May  1976 (Issuing Date)
 AUTHOR(S)

 Fadhil  H.  Khattat and  Susan Farley
            8. PERFORMING ORGANIZATION REPORT NO.
 PERFORMING ORG '\NIZATION NAME AND ADDRESS
 Hazleton Laboratories
 9200  Leesburg Turnpike
 Vienna,  Virginia  22180
             10. PROGRAM ELEMENT NO.

               1BA608
             11. CONTRACT/GRANT NO.

               68-01-0151
12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental Research Laboratory
  Office of Research  and Development
  U.S.  Environmental  Protection Agency
  Narragansett, Rhode Island  02882
                                                           13. TYPE OF REPORT AND PERIOD COVERED
             14. SPONSORING AGENCY CODE
               EPA-ORD
16. SUPPLEMENTARY NOTES
16. ABSTRACT
  The acute toxicity  to  the marine copepod Acartia tonsa Dana of  four  technical grade
  insecticides was  determined by bioassay- using standardized procedures,  homogeneous
  populations and constant laboratory  conditions.   At a water temperature of 17 + 1°C,
  the 96-hour median  lethal concentrations or tolerance limits for methyl parathion,
  Azodrin, diazinon and  toxaphene were  computed as 0.89 milligrams per .liter, Q.24
  milligrams per liter,  2.57 micrograms per  liter  and 7.2 nanograms per  liter,
  respectively.  Residue analysis for diazinon at zero and 96-hour exposure time
  revealed that the amounts of diazinon uptake by  three algal organisms  is greater
  than amounts concentrated by the copepod.   The toxicity of higher concentrations
  above 2.0 ppm (2  milligrams per liter)  has offset copepod uptake, while at lower
  concentrations, quantities concentrated by Acartia are negligible.

  Concurrently, the world literature was  surveyed  for supporting  toxicity data of
  these chemicals to  closely related species.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
  Bioassay
  Pesticides
  Toxicity
  Marine Biology
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                           c. COSATI Field/Group
 Calanoid copepod
 Toxicity review-
 Residue  Analysis
                                                                                6F
13. DISTRIBUTION STATEMENT
 Release Unlimited
19. SECURITY CLASS (This Report)
 UNCLASSIFIED
21. NO. OF PAGES
       37
                                              20. SECURITY CLASS (Thispage)
                                                UNCLASSIFIED
                                                                         22. PRICE
EPA Form 2220-1 J9-73)
                                             29
                                                 •ft U. S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5M1 Region No. 5-11

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