EPA-600/3-76-061b
July 1976
Ecological Research Series
                STANDARD TEST  FISH  DEVELOPMENT
                                                      Parti
                                          Environmental Research Laboratory
                                         Office of Research and Development
                                        U.S. Environmental Protection Agency
                                               Duluth, Minnesota  55804

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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 assessed 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-600/3-76-06lb
                                         July 1976
         STANDARD TEST FISH DEVELOPMENT

                     PART II

Chronic Toxicity of Guthion to the Fathead Minnow

        (Pimephales promelas Refinesque)
                       by

                 Ira R. Adelman
                       and
               Lloyd L. Smith, Jr.
Department of Entomology, Fisheries, and Wildlife
             University of Minnesota
           St. Paul, Minnesota  55108
                Grant No. R800940
                 Project Officer

                Robert W. Andrew
        Environmental Research Laboratory
            Duluth, Minnesota  55804
      U.S. ENVIRONMENTAL PROTECTION AGENCY
       OFFICE OF RESEARCH AND DEVELOPMENT
        ENVIRONMENTAL RESEARCH LABORATORY
            DULUTH, MINNESOTA  55804

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                              DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory -
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, rior does mention
of trade names or commercial products constitute endorsement or recommen-
dation for use.
                                      ii

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                               ABSTRACT

                                  (£>
Three chronic bioassays of Guthioir^were conducted with fathead minnows.
All tests were begun with eggs and the longest lasted 20 days after
termination of spawning, a total of 250 days.  Parameters measured were
survival, growth, fecundity, and growth and survival of second genera-
tion fry and eggs.  The most sensitive criteria for effect of the
toxicant was fecundity.  The maximum acceptable toxicant concentration
                                           (£>
was between 0.33 and 0.51 pg/liter Guthion,  and the application factor
was between .0017 and .0027 based on a soft water acute  and a hard
water chronic bioassay.
                                  iii

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                       CONTENTS

                                                    Page

Abstract                                            iii
List of Tables                                       vi
Acknowledgements                                    V11
Sections
   I  Conclusions                                     1
  II  Recommendations                                 2
 III  Introduction                                    3
  IV  Materials and Methods                           4
        Number of Experiments                         4
        Apparatus                                     4
        Fish                                          5
        Chemical Analysis                             9
   V  Results                                        11
                         (
        Egg and Fry Survival                         11
        Growth                                       13
        Reproduction                                 13
        Second Generation                            16
  VI  Discussion                                     17
 VII  References                                     18
VIII  Publications                                   20
  IX  Glossary                                       21

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                              TABLES

No.                                                              Page

 1    Analysis of Well Water                                       6

 2    Means and Standard Deviations  (in Parentheses) of
      Conditions in All Test Chambers                              7

 3    Percentage Survival of Fathead Minnow Eggs and Young
      Exposed to Various Concentrations of Guthion^              12

 4    Mean Weight of Fathead Minnows at Various Time
      Intervals After Spawning  (Number of Fish Weighed in
      Parentheses)                                                14

 5    Fecundity of Fathead Minnows Exposed to Various
      Concentrations of Guthion^                                 15
  6     Effect of Guthiori^on Survival  and  Growth of  Second
       Generation Eggs  and Fry                                    16
                                  vi

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                          ACKNOWLEDGEMENTS

The authors wish to thank Richard W. Frenzel and particularly
Gary D. Siesennop for assistance in conducting the experiments.
                             vii

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                              SECTION I
                             CONCLUSIONS

The results described in the following report permit certain conclusions
concerning the toxicity of Guthiorr to fathead minnows.

1.  The maximum acceptable toxicant concentration for fathead minnows is
    between 0.33 and 0.51 yg/liter Guthion.

2.  The application factor comparing an acute bioassay in soft water and
    a chronic bioassay in hard water is between .00017 and .00027.

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                             SECTION II


                           RECOMMENDATIONS
       (iv)
Guthion^ concentrations in the environment should not exceed


0.33 yg/liter.

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                             SECTION III
                             INTRODUCTION

GuthiorP (azinphosmethyl) is an important and widely used insecticide
on deciduous fruits and cotton as well as many other fruits and vege-
tables.  Although acute toxicity data have been reported by many workers
for a variety of fish species (Katz, 1961; Pickering, Henderson, and
Lemke, 1962; Meyer, 1965; and Macek and McAllister, 1970), no informa-
tion has been reported on the effect of exposures over a complete life
cycle.  Since data on chronic toxicity are important for establishment
of water quality standards, fathead minnows were exposed to various
                         ®
concentrations of Guthion  over a complete life cycle, from egg through
fry in the second generation, in order to determine a maximum acceptable
toxicant concentration (MATC) (Mount and Stephan, 1967).

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                             SECTION IV
                        MATERIALS AND METHODS

NUMBER OF EXPERIMENTS
Three experiments were conducted, subsequently referred to as tests I,
II, and III.  Tests I and II were preliminary and were terminated after
26 and 68 days, respectively, when it became apparent that the lowest
concentration was causing significantly greater mortality than the
control.  Furthermore chemical analysis of small Guthion  concentrations
had not been perfected, and residues on glassware and water sampling
equipment made analyzed concentrations inaccurate. This problem was
particularly true for test II where desired concentrations were low
enough to be influenced.  Therefore, information from the first two
tests is only roughly quantitative but does indicate an area of possible
effect.  For test III all glassware was soaked in a strong sodium hy-
droxide solution (pH = 12"^ for at least two days prior to use and no
residues were found in blank analyses.

APPARATUS
A proportional diluter  (Mount and Brungs, 1967) that dispensed seven
toxicant concentrations with a 60% dilution factor and one control was
used for administration of the toxicant in a flow-through system.
Technical grade Guthioir^ (93%) was added directly to a single mixing
chamber by  the injector described by DeFoe  (1975).  An air dispersion
stone and baffles thoroughly mixed the Guthion^with incoming water
before it was siphoned to the toxicant cells in the diluter.  The test
chambers for first generation young and adult fish were constructed of

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glass and G.E. Silicone Seal, measured 50 x 25 x 20 cm, and contained
20 liters.  With a flow rate of 250 ml/min, 90% replacement of water
occurred in approximately 3-1/4 hr.  Temperature was maintained at a
constant 25 C by a hot water exchange system in the head tank where
aeration also occurred.  All test chambers in test III contained an air
dispersion stone in order to maintain oxygen concentrations.  A dense
bacterial growth occurred in the two highest concentrations apparently
using the Guthion^ as a nutrient and the aeration was necessary to
replace oxygen consumed by the bacterial respiration.  Chambers were
siphoned daily except when fry were present to keep this bacterial
growth to a minimum.  A complete analysis of hard water from a deep
well is reported in Table 1.  Those water quality characteristics which
varied in individual test chambers are listed in Table 2.

Fry in the second generation were reared in glass chambers measuring
20 x 20 x 21 cm and containing 6.4 liters.  For each concentration
water flowed from the outlet of the adult test chamber into the first
fry chamber, then from the outlet of that to a second fry chamber so
that two groups of offspring from the same adults could be reared simul-
taneously.  Eggs were hatched in a small jar with a nylon screen bottom.
One jar in each adult test chamber was oscillated so that water flowed
past the eggs without raising them from the screen.

All adult chambers were exposed to the direct illumination of a 40-watt
cool white fluorescent tube, suspended 50 cm above the water surface.
Second generation fry chambers received only indirect illumination from
laboratory light.  Tests were begun with a photoperiod equivalent to
January 1 at Evansville, Indiana and adjustments were made every 15 or
16 days.

FISH
Fathead minnows originally obtained from the Environmental Research
Laboratory at Duluth, Minnesota were reared in a laboratory culture
system.  All tests were started with eggs from five spawning pairs which

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         Table 1.  ANALYSIS OF WELL WATER
                 (milligrams/liter)
         Item
Concentration
Total hardness as CaCO,
Calcium as CaCO_
Iron
Chloride
Sulfate
Sulfide
Fluoride
Total phosphates
Sodium
Potassium
Copper
Manganese
Zinc
Cobalt* nickel
Cadmium, mercury
Ammonia, nitrogen
Organic nitrogen
   220
   140
     0.02
    <1
    <5
     0.0
     0.22
     0.03
     6
     2
     0.0004
     0.0287
     0.0044
    <0.0005
    <0.0001
     0.20
     0.20
       taken from well head before aeration and heating;
 pH 7.5.

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   Table 2.  MEANS AND STANDARD DEVIATIONS  (IN PARENTHESES)  OF CONDITIONS IN ALL TEST CHAMBERS

Item
Test chamber
12345678
Guthiori^Xug/l)    0        7.6(1.3)
Temp. (C)      24.8(.6)   25.0(.6)
pH (reading)   7.73(.13)  7.53(.11)
DO (mg/1)      6.60(.66)  5.95(.24)

Guthion®(yg/l)    0        1.8(.9)
Temp. (C)      24.6(1.7)  24.6(1.8)
pH (reading)   7.75(.09)  7.58(.11)
DO (mg/1)      6.28(.77)  5.54(.45)
Guthion®(yg/l)   .04(.OS)-''  .10(.04)
Temp. (C)
pH (reading)
DO (mg/1)
24.1(1.1)  24.4(1.2)
6.96(.34)  6.52(.57)
          Test I
11.7(1.9)  20.5(1.9)  34.5(3.2)
24.9(.6)   25.0(.6)   25.0(.6)
7.52(.10)  7.49(.09)  7.51(.ll)
5.55(.56)  5.03(.05)  5.20(.74)
          Test II
 1.9(1.0)   2.8(1.1)   3.4(1.2)
24.6(1.9)  24.4(1.9)  24.6(1.9)
7.57(.10)  7.60(.09)  7.60(.09)
5.55(.45)  5.58(.31)  5.36(.38)
         Test III
      Adult chambers
 .16(.05)   .24(.06)   .33(.08)
24.4(1.2)  24.4(1.2)  24.6(1.4)
8.07(.ll)  8.06(.ll)  8.05(.ll)
6.49(.34)  6.27(.64)  5.79(.59)
                                                       61.4(2.4)  96.7(6.6)  165.9(13.1)
                                                       24.9(.6)   25.1(.6)    25.1(.6)
                                                       7.52(.16)  7.50(.09)   7.56(.17)
                                                       4.48(1.28) 4.46(.61)   5.13(.51)

                                                        4.8(.8)     6.5(.8)    15.0(2.2)
                                                       25.4(.6)   25.3(.6)    25.4(.6)
                                                       7.58(.08)  7.60(.09)   7.63(.10)
                                                       5.22(.57)  5.17(.61)   4.90(1.07)
     Ml)
24.5(1.4)
8.05(.ll)
5.77(.90)
 .72(.15)
24.4(1.2)
8.06(.12)
     :.86)

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               Table 2  (continued).   MEANS AND STANDARD DEVIATIONS (IN PARENTHESES) OF CONDITIONS


                                               IN ALL TEST CHAMBERS
00

Test chamber
Item
1
2
3
4

5

6

7
8
Test III
Fry chamber 1
Guthion^yg/l)-^ . 04 ( . 03)^ . 10 ( . 04)
Temp . (C)
pH (reading)
DO (mg/1)

Temp. (C)
pH (reading)
DO (mg/1)
23.6(.8)
8.22(.08)
7.30(.41)

23.0(.6)
8.23(.08)
7.46(.32)
23.8(.5)
8.17(.04)
6.86(.22)

23.0(.6)
8.16(.05y
6.96(.32)
.16(.05)
23.8(.4)
8.15(.05)
6.81(.30)
Fry
22.9(.4)
8.16(.05)
7.09(.29)
.24(.06)
23. 8(.
8.16(.
6.65(.
chamber
23. 0(.
8.15(.
6.99(.
5)
04)
33)
2
6)
05)
34)
.33(
23. 9(
8.12(
6.17(

23. 1(
8.13(
6.45(
.08)
.4)
.02)
• 41)

.6)
.07)
.51)
.51(.
23. 6(.
8.15(.
6.00(.

23.2(1
8.18(.
6.49(.
11)
6)
10)
76)

.0)
08)
84)
.72(.
23. 6(.
8.14(.
5.87(.

22.4(1
8.15(.
6.06(.
15)
4)
08)
60)

.4)
08)
83)
       a/
       j-,Concentration may be high by as much as 50% for the four^lowest concentrations.

       —/Unknown source of contamination resulted in some Guthion^ in control.

       — Concentrations measured  in adult chambers.

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were distributed in a random stratified manner to egg chambers in each
toxicant concentration and control.  Initial numbers of eggs were 90,
70, and 100 in tests I, II, and III, respectively.  Eggs were in the
middle to late blastula stage when first exposed to the toxicant and
more than 99% were fertile.  Mortality of eggs and fry was  recorded at
24-hr intervals in tests I and II.  In test III eggs were checked at
24-hr intervals but mortality of young was recorded only after 22 days
to avoid increased stress from handling.  At that time two runts or
malformed fish were removed from each chamber and fish were randomly
thinned to 15 fish.

Ten-cm sections of transite pipe were split lengthwise to form a
spawning substrate.  After 90 days six of these sections were placed in
each aquarium, three on each end with an open area in the center.  As
secondary sexual characteristics developed,  males were removed at bi-
weekly intervals to keep no more than five per chamber.   Spawning sub-
strates were checked at 1000 hours each day and if spawning occurred,
eggs were counted and a random sample of 50 was placed in the egg basket
for determination of survival through hatching unless the basket was
already occupied.  Two groups of fry from each concentration were reared
for 50 days, otherwise all hatched fry were discarded.

Fry were fed ground Glencoe trout pellets and live brine shrimp twice  a
day for the first 30 days.  Older fish were fed Oregon Moist frozen
pellets twice a day and live brine shrimp once a day.

CHEMICAL ANALYSIS
Concentrations of Guthion6^ in each adult test chamber were usually
analyzed twice per week for the first 5 months of test III and usually
once per week thereafter.  Samples from either or both fry chambers were
analyzed periodically.  Since the percentage difference between fry
chambers and adult chambers analyzed at the same time ranged from -24%
to +21% with a mean percentage difference of iO%, the analyses from
adult chambers were used.

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A 1-liter sample of water from each test chamber was extracted with 10
ml benzene  (pesticide grade) and the benzene was then evaporated to 0,5
to 2 ml, depending on the concentration.  With each series of samples
                                                       (§)
1 liter of  deionized water was spiked with 1 ml Guthion^ in acetone and
this sample was treated  the same as all others to determine a recovery
efficiency  which averaged 99.2 - 6.5%.  Samples were analyzed on a Beck-
man GC 72-5 gas chromatograph with a 122-cm glass column packed with 50%
DC 200 on 80/100 mesh Gas Chrom-Q and a nonradioactive electron capture
detector.   Standards were prepared from 93% technical grade Guthion^ and
were injected  alternately with unknowns.  Final Guthion*^concentrations
were corrected for the recovery efficiency and the technical grade pro-
duct.

The pH was  measured with a meter twice per week in adult chambers and
once per week  in fry  chambers when they were  occupied.   Temperature was
measured with  a thermister  three times per week in all chambers and dis-
solved oxygen  was analyzed weekly by the azide modification of the
Winkler analysis.
                                    10

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                               SECTION V
                               RESULTS

EGG AND FRY SURVIVAL
Survival of eggs was probably not directly affected by any concentration
          /g\
of Guthion** tested.  In Test I percentage survival through hatching
                                         ®
decreased from 90 to 29 to 17% as Guthion  concentration increased
from 34.5 to 61.4 to 96.9 yg/liter but then survival increased to 76%
          ®
as Guthion  concentration increased to 165.9 yg/liter (Table 3).  This
decreased survival was caused by eggs in these concentrations being
                                                            /&,
enmeshed in a bacterial network that apparently used Guthioir* as an
energy source and grew in  proportion to the concentration.  Dead eggs
in chambers with reduced survival were partly hatched as the fry seemed
to become enmeshed in the bacteria and could not break free.  Since the
chorion of these eggs softened, fry may also have been weakened.  Sur-
vival in the highest concentration was apparently greater because most
eggs hatched 1 day sooner, when the bacteria were less dense and before
the chorion had softened.

At 22 days there was decreased survival of fry in comparison to controls
at 7.6 yg/liter in Test I, and at approximately 6.5 yg/liter in Test II
(Table 3).  By 57 days survival decreased at approximately 1.8 yg/liter
       (R)
Guthion^ in Test II but did not decrease at 0.51 yg/liter in Test III.
In Test III Guthion  caused no decrease in survival at any life history
stage with the possible exception of the highest concentration where
fry were lost wheh the chamber overflowed.  The MATC in terms of sur-
vival was between 0.51 yg/liter in Test III and a maximum of 1.8 yg/liter
                                    11

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Table 3.  PERCENTAGE SURVIVAL OF FATHEAD MINNOW EGGS AND YOUNG EXPOSED


              TO VARIOUS CONCENTRATIONS OF GUTHIOt

Test chamber
Item

Guthion^Cug/l)
% hatched
% survival
22 days


Guthion (yg/1)
% hatched
% survival
22 days
57 days
98 days
/**>
Guthion^Xyg/l)
% hatched
% survival

22 days
from 22-
60 days
1

0
99

54


0
91

57
54
52

2

7.6
84

43


1.8
92

48
42
35

.O^.IO
86


61
100

84


80
100

3
Test
11.7
97

29

Test
1.9
90

57
34
21
Test
.16
82


74
93

4
I
20.5
95

16
a/
II-
2.8
90

43
5
0
III
.24
85


82
100

5

34.5
90

1


3.4
87

48
3
0

.33
85


76
100

6

61.4
29

0 -


4.8
93

48
0
0

.51
86


67
93

7 8

96.9 165.9
17 76

0 0


6.5 15.0
63 84

37 35
0 0
0 0

.72
83

c/
	 v/ _
88

 a/
 — Concentration may be high by as much as 50% for the four  lowest  con-

 centrations .

 — Unknown source of contamination resulted in some Guthion  in the

 control.
 c7
 — Tank overflowed and fry were lost.
                                   12

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where increased mortality occurred in Test II.

GROWTH
In test II fish were ewighed twice en masse during the experiment and
at termination.  Each time the control fish weighed more than those  in
the lowest concentration, 1.8 yg/liter (Table 4).  In test III the first
two weighings were of individual fish that were randomly thinned from
the experiment.  The third weighing was of fish that had remained in the
experiment.  Individual t-test comparisons indicated no significant  dif-
ference between the control and any of the treatments (p>.05) at 22  days
(Table 4).  At 60 days the 0.10 and 0.24 yg/liter treatment levels
weighed significantly more than the controls (p<.02) but since the
0.16 yg/liter treatment level was not different from the control,
Guthion  was probably not the cause of the above differences.  After
90 days there was no significant difference (p>.05) between the control
and any treatment.  Although dissolved oxygen concentrations were lower
with increasing Guthioir^ concentrations, these oxygen levels did not
seem to affect growth.

By 120 days males in some treatments started to defend territories and
fish were not weighed to avoid any influence of handling on spawning
behavior.  At termination of the experiment after 250 days there was no
consistent pattern of growth related to Guthioir concentration.   There-
fore, Guthion5^ affected growth of fathead minnows through 90 to 100 days
at essentially the same concentrations that caused mortality, or between
0.72 and 1.8 yg/liter.

REPRODUCTION
Interpretation of fecundity data was complicated by the variation in sex
ratios in test chambers.  Although an attempt was made to maintain five
males and seven females in each chamber,  in some chambers that many
males were not produced, in some immature males could not be identified,
and in some individuals were lost to accidental death or escapement.
                                   13

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         Table  4.  MEAN WEIGHT OF FATHEAD MINNOWS AT VARIOUS  TIME INTERVALS AFTER SPAWNING
                              (NUMBER OF FISH WEIGHED  IN PARENTHESES)

Item
Test chamber
12 3 4 5 6 7 8
Guthion® (ug/1)
Weight (mg)
  57 days
  85 days
  98 days
Guthion® (yg/1)
Weight (mg)
           1.8
             Test II
          1.9       2.8
                    3.4
          4.8
162(33)   111(29)    55(21)    72(3)     20(2)
320(33)   250(24)   230(14)
380(33)   350(24)   300(13)
 0.4
0.10
   Test III
0.16      0.24
0.33
0.51
          6.5
0.72
a/
j-.Fish randomly thinned from experiment.
—.Fish remaining in experiment.
— Termination.
         15.0
22
60
90
250

days^
days-
days—
days— - male
female
24(8)
179(15)
580(15)
2834(3)
1434(9)
24(27)
242(15)
580(15)
3047(5)
1594(7)
28(25)
216(13)
640(15)
3455(5)
1458(5)
27(19)
231(15)
660(14)
3051(5)
1353(5)
23(21)
260(15)
600(15)
2628(5)
1558(5)
21(11)
165(13)
600(13)
2965(6)
1360(6)
_
_
720(15)
3136(5)
912(1)

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 Spawning  occurred  after  121  days when  the  day  length  reached  12  hr and
 45 min.   Spawning  ceased after  230 days when the day  length was  reduced
 to 12 hr.  Intensive  spawning occurred first at 0.33  yg/liter GuthionY
 followed  12  to 14  days later by spawning at 0.24, 0.16 and 0.10  yg/liter.
 Intensive spawning by the control fish occurred 29 days later and  56
 days later in 0.51 and 0.72  yg/liter Guthiort-'  although at 0.51 yg/liter
 spawning  occurred  infrequently  during the  test.  The  number of spawnings
 and eggs  were greatest in the control chamber  (Table  5).  This result
 may have  been caused  by  the  highest female to  male sex ratio  throughout
 the test. The more significant  measurements in terms  of GuthiorHeffect
 were the  number of eggs  per  spawning and per female since these  nor-
 malize the dissimilar sex ratios.  On this basis Guthioff-'affected
 fecundity between  0.33 yg/liter which was  similar to  controls  and 0.51
 yg/liter  where there  was about  a 50% reduction in eggs per spawning and
 per female (Table  5).  The decreased oxygen concentrations with  increas-
 ing Guthion-'was apparently  not responsible for the effect on fecundity
 since the oxygen concentration  at 0.33 and 0.51 yg/liter GuthioA-^was
 similar.
 Table 5.  FECUNDITY OF FATHEAD MINNOWS EXPOSED TO VARIOUS CONCENTRATIONS
                              OF GUTHION5^

GuthiorP
yg/l
0.04
0.10
0.16
0.24
0.33
0.51
0.72
Number of
spawnings
88
54
57
49
52
21
27
Number
of eggs
16401
t
8541
9567
7091
8590
1791
2408
Number of
eggs per
spawning
186
158
168
145
165
85
89
Number of
eggs per
female^/
1691
1220
1611
1239
1718
256
782
a/
— Computed by intervals between thinnings.
                                   15

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SECOND GENERATION
Random samples of 50 eggs were taken from 12 spawnings from each test
chamber except the second highest where samples from seven spawnings
were taken.  These eggs were hatched in the oscillating egg baskets.  As
with the first generation eggs, there was no effect of Guthiott^ at the
tested concentrations  (Table 6).
Table 6.  EFFECT OF
                             ON  SURVIVAL AND GROWTH OF SECOND GENERATION
                               EGGS AND FRY

GuthionS/ concentration, yg/1
Item
% survival - eggs
% survival - fry-
Is t group
2nd group
Mean weight (mg)-
1st group
2nd group
0.04
86.2

79
69
94
143
0.10
84.2

97
89
130
102
0.16
90.9

100
88
94
135
0.24
86.2

97
89
126
85
0.33
82.3

89
100
115
144
0.51 0.72
86.4 90.2

- -1 78
100
78 128
222 80
 a/
 — After 50 days from hatching.
 — Some fry lost due to overflow.

 Survival or growth of fry was not consistently affected by the tested
 Guthion*^ concentrations (Table 6).  Survival was poorest among controls.
 Considerable differences in growth occurred between test chambers but
 these differences were not related to Guthiori-' concentrations (Table 6).
                                     16

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                              SECTION VI
                              DISCUSSION
The lowest concentration of Guthion^ that had an adverse effect  on  fat-
head minnows was 0.51 yg/liter where fecundity was much reduced  (Table
5).  An estimation of the maximum acceptable toxicant concentration
based on this parameter is between 0.33 and 0.51 yg/liter Guthionr
Effects on survival and growth during early life history stages  occurred
at slightly higher concentrations (Tables 3 and 4). Although no  effect
occurred in test III at the highest concentration (0.72 yg/liter) growth
and survival were reduced at the lowest concentration (1.8 yg/liter) in
test II.

The 96-hr LC50 for 11-week-old fathead minnows tested in a relatively
soft water was 1900 yg/liter (Part I of this report).  An application
factor based on this acutely toxic concentration and the MATC in the
present study is between .00017 and .00027.  Although this application
factor is extremely small compared to factors found for many other
toxicants (U.S. Environmental Protection Agency, 1973), it would prob-
ably be even smaller if the acute tests were conducted in hard water.
Henderson, Pickering and Tarzwell (1960) found that the 96-hr LC50 of
Guthioir-^to fathead minnows was approximately 25 to 40% less in soft
water than in hard (20 ing/liter vs.  400 mg/liter total hardness).  This
differential in hardness was more extreme than the acute vs. chronic
test in the present study, but if the application factor was based on an
acute test conducted in hard water,  the implication is that the factor
would be even smaller.
                                   17

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                              SECTION VII
                              REFERENCES

DeFoe, D.L.  1975.  Multichannel Toxicant Injection System for Flow-
through Bioassays.  J. Fish. Res. Board Can.  32;544-546.

Henderson, C., Q.H. Pickering, and C.M. Tarzwell.  1960.  Toxicity of
Organic Phosphorus and Chlorinated Hydrocarbon Insecticides to Fish.
In:  Biological Problems in Water Pollution.  Trans. 1959 Seminar.
Cincinnati, R.A. Taft Sanit. Eng. Center,  p. 76-92.

Katz, M.  1961.  Acute Toxicity of Some Organic Insecticides to Three
Species of Salmonids and to the Three-spine Stickleback.  Trans. Amer.
Fish. Soc.  90(3):264-268.

Macek, K.J., and W.A. McAllister.  1970.  Insecticide Susceptibility of
Some Common Fish Family Representatives.  Trans. Amer. Fish. Soc. 99:
20-27.

Meyer, F.P.  1965.  The Experimental Use of Guthion as a Selective Fish
Eradicator.  Trans. Amer. Fish. Soc.  94(3);203-209.

Mount, D.I., and W.A. Brungs.  1967.  A Simplified Dosing Apparatus for
Fish Toxicology Studies.  Water Res.  1(1):21-29.

Mount, D.I., and C.E. Stephan.  1967.  A Method for Establishing Accept-
able Toxicant Limits for Fish - Malathion and the Butoxyethanol Ester of
2,4-D.  Trans. Amer. Fish. Soc.  96:185-193.
                                    18

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Pickering, Q.H., C. Henderson, and A.F. Lemke.  1962.  The Toxicity of
Organic Phosphorus Insecticides to Different Species of Warm Water
Fishes.  Trans. Amer. Fish. Soc.  91(2);175-184.

U.S. Environmental Protection Agency.  1973.  Water Quality Criteria
1972.  Ecol. Res. Ser. EPA-R3-73-033.  594 p.
                                  19

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                             SECTION VIII
                             PUBLICATIONS

Adelman, I.R., L.L. Smith, Jr., and G.D. Siesennop.  Chronic Toxicity
of Guthion to the Fathead Minnow  (Pimephales promelas Rafinesque).
Bulletin of Environmental Contamination and Toxicology  (submitted).
                                  20

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                             SECTION IX
                              GLOSSARY

Adult - A fish older than 4 months.

Application Factor - The ratio of the MATC to the 96-hr LC50.

Bioassay - A toxicity test; the estimation of the strength of a poison
by its effect on a living organism.

Bioassay, acute - A toxicity test of short duration, usually less than
3 weeks.

Bioassay, chronic - A toxicity test of long duration, usually 1 month
or longer.

Bioassay, flow-through - A toxicity test where the toxicant and diluent
water are continuously replaced by fresh material.

Biastula - Early embryonic stage where cellular division has resulted
in cells arranged as a hollow spherical body.

Chorion - The outer membraneous covering of a fish egg.

Fry - A fish younger than about 50 days.

LC50 - The concentration of poison that will kill 50% of the test
organisms at a specified time.
                                  21

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Maximum Acceptable Toxicant Concentration (MATC) - A concentration of
toxicant determined from a chronic bioassay and considered an estimation
of the level at which no harm will occur in the natural environment.
It is bracketed by the highest level of no effect and the lowest level
of effect in the bioassay.
                                    22

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.
  EPA-600/3-76-061b
                                                            3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE

    STANDARD TEST FISH DEVELOPMENT, PART  II
              5. REPORT DATE
               July 1976 (Issuing  Date)
                                                            6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

 Ira R.  Adelman and Lloyd L. Smith, Jr.
                                                            8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Department of Entomology,  Fisheries,  and Wildlife
 University of Minnesota
 St.  Paul, Minnesota 55108
              10. PROGRAM ELEMENT NO.

                1BA608
              11. CONTRACT/GRANT NO.

                 Grant   R800940
 12. SPONSORING AGENCY NAME AND ADDRESS
 U.S.  Environmental Protection Agency
 Office of Research and  Development
 Environmental Research  Laboratory
 Duluth,  Minnesota 55804	
              13. TYPE OF REPORT AND PERIOD COVERED

               T7TNAL  	
              14. SPONSORING AGENCY CODE

               EPA-ORD
 15. SUPPLEMENTARY NOTES

  See Part I, EPA-600/3-76-061a
 16. ABSTRACT
        Three chronic bioassays of GuthiorTwere conducted with  fathead minnows.
  All  tests were begun with eggs, and the longest lasted 20 days  after termination
  of spawning, a total of  250 days.  Parameters measured were survival, growth,
  fecundity, and growth and survival of second  generation fry and eggs.  The most
  sensitive criteria for effect of the toxicant was fecundity.  The maximum   ^
  acceptable toxicant concentration was between 0.33 and 0.51 yg/liter Guthion^
  and  the  application factor between .00017 and .00027.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS  C.  COS AT I Field/Group
    Bioassay
    Criteria
    Fecundity
    Growth
    Minnows
    Water quality
    Survival
 Guthion
 Chronic toxicity
 Bioassay survival
 Application factor
 Maximum acceptable
   toxicant concentration
06C
06F
06S
18. DISTRIBUTION STATEMENT
    RELEASE TO PUBLIC
                                              19. SECURITY CLASS (ThisReport)
                                                 UNCLASSIFIED
                           21. NO. OF PAGES

                               31
20. SECURITY CLASS (Thispage)
  UNCLASSIFIED
                                                                          22. PRICE
EPA Form 2220-1 (9-73)
                                             23
                 •OUSGPO: 1976 — 657-695/5461 Region 5-11

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