&EPA
            United States
            Environmental Protection
            Agency
            Environmental Research
            Laboratory
            Duluth MN 55804
EPA-600/3-80-005
January 1980
            Research and Development
Acute Toxicity of
Toxaphene to
Fathead  Minnows,
Channel  Catfish, and
Bluegills

-------
                 RESEARCH  REPORTING SERIES

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

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

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the  effects of pollution on hurnans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations  include formation, transport, and  pathway studies to deter-
mine the fate of pollutants and their effects 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.

-------
                                             EPA-600/3-80-005
                                             January 1980
ACUTE TOXICITY OF TOXAPHENE TO FATHEAD MINNOWS,
       CHANNEL CATFISH, AND BLUEGILLS
                      by
               W. Waynon  Johnson
               Arnold M.  Julin
         U.S.  Fish  and Wildlife  Service
      Fish-Pesticide  Research Laboratory
           Columbia,  Missouri  65201
       Contract No. EPA-IAG-141(D)
               Project Officer

             Leonard H. Mueller
      Environmental Research Laboratory
          Duluth, Minnesota  55804
      ENVIRONMENTAL RESEARCH LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U. S. ENVIRONMENTAL PROTECTION AGENCY
          DULUTH, MINNESOTA  55804

-------
                                 DISCLAIMER

     This report has been reviewed by the Environmental Research Laboratory -
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorcement or recommendation
for use.
                                     ii

-------
                                  FOREWORD

     This report describes the toxicity of toxaphene, a widely used
insecticide, to three species of fish, fathead minnows, channel catfish,
and bluegills.  Research compared the acute toxicity of static tests with
continuous flow exposure.  Also studied was the importance of various life
stages of aquatic organisms when exposed to a chemical pollutant.  Environ-
mental variables, pH, water hardness, and temperature were studied under
laboratory test conditions to determine their effects on toxaphene toxicity.
                                      J. David Yount, Ph.D.
                                      Deputy Director
                                      Environmental Research Laboratory-Duluth

-------
                                   ABSTRACT

      Toxaphene  was  very  toxic  to  fathead  minnows  (Pimephales  promelas),  chan-
 nel  catfish (Ictalurus punctatus),  and  bluegills  (Lepomis macrochirus)  in
 static  tests; 96-h  LCSO's  ranged  from 2.6 to  20 ug/1  at  20~.  Fathead  min-
 nows were  the least susceptible and bluegill  and  channel catfish were about
 equal in susceptibility.   Prolonged exposures  of  12 to 34 days in  flow-through
 tests produced  time-independent LC50  values of 0.6 to 1.9 ug/1, significantly
 lower than the  96-h values.

      The toxicity of toxaphene was  not  influenced by  variations in pH or water
 hardness in static  tests.  Temperature  seemed  to  influence  rate of onset
 rather  than degree  of response.  Toxicities were  similar at 20 and 25 C  in
 flow-through tests,  however, a decrease in the time required  for mortality to
 become asymptotic with time was observed.  Time-independence was reached af-
 ter  7 to 16 days of  exposure at 25  C while 24  to  34 days were required at
 20 C.

      The life stage  of channel catfish most sensitive to toxaphene poisoning
was  the swim-up fry with a 96-h LC50 of 0.8 ug/1.  Early yolk sac  fry were
extremely resistant with a 24-h LC50 of 4.7 mg/1 however, within 96 hours the
yolk had absorbed and the LC50 had  declined to 8.0 ug/1.

     This report was submitted in partial fulfillment of Contract  No. EPA-IAG-
141(D) by the Fish-Pesticide Research Laboratory under the  sponsorship of the
U.S.  Environmental Protection Agency.   This work was completed as  of April,
1976.
                                     iv

-------
                           CONTENTS
Foreword	iii
Abstract	iv
Tables	vi
Figures	vi
Acknowledgments	vii

     1.  Introduction	1
     2.  Conclusions	2
     3.  Recommendations	3
     4.  Methods and Materials	4
     5.  Results	6
     6.  Discussion	14

References	17

-------
                                  TABLES

Number
       Acute toxicity of toxaphene to three fishes at two
          temperatures in static toxicity tests 	  7

       Acute toxicity of toxaphene to three fishes at two
          temperatures in flow-through toxicity tests 	  8

       Influence of temperature, pH,  and water hardness on the
          acute toxicity of toxaphene to fingerling channel catfish
          in static tests 	  9

       The acute toxicity of toxaphene to three life stages of
          channel catfish in static tests at 25 C	10
                                     vi

-------
                                 FIGURES

Number                                                                Page

  1    Response curve for fathead minnows exposed to toxaphene in
         flow-through toxicity tests 	 11

  2    Response curve for channel catfish exposed to toxaphene in
         flow-through toxicity tests 	 12

  3    Response curve for bluegills exposed to toxaphene in flow-
         through toxicity tee«s  	 13
                                    vii

-------
                               ACKNOWLEDGMENT

     We thank Donnie W. Short for assisting us in performing the static
toxicity tests and Michael J. Nevins for performing the flow-through toxicity
tests.  Special appreciation is expressed to Mr. Nevins for performing all
of the LC50 calculations.
                                    viii

-------
                                   SECTION  1

                                INTRODUCTION
     Within recent years a general concern has developed among scientists
about the use of persistent pesticides  that are becoming more abundant and
widespread in the environment.  This concern has induced an increased aware-
ness and closer scrutiny of the effects these chemicals may impose on organ-
isms in the aquatic environment.  As more chemicals  face either registration
cancellation or increased restrictions  on their use, pesticide users are
forced to depend more and more on fewer alternative  chemicals.  As a conse-
quence, these chemicals are receiving greater attention from researchers and
many are being found to produce detrimental environmental effects.

     One compound, toxaphene, has replaced many of the agricultural applica-
tions of DDT, for which registration has been canceled.  This use has result-
ed in an increased environmental distribution of toxaphene, and it has been
identified as a residue in water and aquatic organisms in various studies
(1-4).  Toxaphene, like many of the organochlorine chemicals, is persistent
(5-9) and highly toxic to fish and other aquatic organisms (10-16).  Because
of its toxicity to fish, toxaphene was proposed and  used as a fish eradicant
(17-23).  However, the treated waters often remained highly toxic throughout
a detoxification time of a few weeks to several years (7, 8, 24-26).  This
slow detoxification rate led to the eventual elimination of toxaphene as a
piscicide, but only after a number of lakes were contaminated with its
residues.

     Recent data from the National Pesticide Monitoring Program indicate that
toxaphene is becoming a more common pollutant in the aquatic environment
(D. Walsh, personal communication), thereby increasing the likelihood of its
occurrence in diverse habitats and during different  stages of a fish's life
cycle.  The literature contains few data on the effects of environmental
variables on toxaphene toxicity or on the susceptibility of different life
stages(10).   We therefore undertook the present study with the following
objectives:   (1) to compare the susceptibilities of bluegills, channel cat-
fish, and fathead minnows to toxaphene; (2)  to compare the toxicity of
toxaphene to these species under static and flow-through test conditions; (3)
to determine the effects of temperature, pH, and water hardness on the
toxicity of toxaphene to channel catfish;  and (4) to compare the susceptibil-
ities of different life stages of channel catfish to toxaphene.

-------
                                 SECTION 2

                                CONCLUSIONS
     Toxaphene is highly toxic to fingerling bluegills, channel catfish, and
fathead minnows in static toxicity tests,'as evidenced by 96-h LCSO's ranging
from 2.6 to 20 ug/1 at 20 C and from 2.4 to 23 ug/1 at 25 C.

     Time-independent LC50 values (indicating the concentrations where mortal-
ity becomes asymptotic with time) for all three species were in the range of
0.6 to 1.9 ug/1.

     Water hardness and pH did not appreciably alter toxaphene toxicity.

     Temperature appeared to hasten the rate of onset but not the degree of
response.  For an equivalent response fish required a longer time at 20 C
than at 25 C.

     In channel catfish, early swim-up fry were the most susceptible life
stage (96-h LC50, 0.8 ug/1); yolk-sac fry, before yolk absorption, were the
least susceptible (24-h LC50, 4.7 mg/1).

-------
                                   SECTION 3

                               RECOMMENDATIONS
     The 96-h LC50 should be used in preference to the lethal threshold con-
centration when calculating application factors or maximum acceptable toxicant
concentrations.

     Thorough studies of the acute toxicity of toxicants to different life
stages of fish species and the influence on toxicity by environmental variables
such as temperature, pH, and hardness should be performed when "safe" levels
of pollutants are being estimated from acute toxicity data.

-------
                                   SECTION 4

                             METHODS AND MATERIALS
      Channel catfish (Ictalurus punctatus),  and bluegllls (Lepomis macrochirus)
 used in the toxicity tests  were obtained from National fish hatcheries as
 fingerlings (weight range,  0.5 to 1.5 g).   Fathead minnows (Pimephales
 promelas)  were reared in ponds from stock obtained from the Environmental
 Research Laboratory,  Duluth,  Minnesota.   Although the minnows were in the
 same size  range,  they were  immature adults  rather than fingerlings.   Early
 life stages of channel catfish were reared  from eggs  produced at the Fish-
 Pesticide  Research  Laboratory.   Fish used in any single test were of the same
 lot  and were held in laboratory facilities  similar to those described by
 Brauhn  and Schoettger (27)  for at least  14  days before testing.   Holding
 facilities were supplied with 17 C well  water with a  pH of 7.4 and alkalinity
 and  total  hardness  of 237 and 272 mg/i (as  CaCO.,),  respectively.   Channel cat-
 fish eggs  were hatched and  the fry reared at 25 C.

      Toxicity tests were conducted according to the procedures outlined in
 American Public Health Association (28)  and  Committee for Methods for Toxicity
 Tests with Aquatic  Organisms  (29).   Basic static tests were conducted in re-
 constituted water with a pH of  7.4 and alkalinity  and total hardness of 35 and
 40 mg/1 (CaCO ),  respectively.   The influence of water quality was investigat-
 ed by measuring toxicity at various pH's and water  hardnesses.   Appropriate
 buffers and salt  mixtures were  used to attain the  desired values  while main-
 taining other variables  constant (29,  30).   Water  quality values  were measured
 prior to each test  to assure  that they were  within  the recommended range of
 values.  Tests  were conducted at temperatures of 15,  20,  and 25  C and main-
 tained  (+  1 C)  by constant  temperature water baths.   Flow-through tests were
 conducted  in the  well water described  above  using a proportional  diluter
 system  modeled  after  Mount  and  Brungs  (31) and modified as recommended by
 McAllister et al. (32).   The  dilution  ratio  between test concentrations was
 0.75.   Well water used in the flow-through tests was  first passed through an
 ultraviolet  light sterilizer.   Ten  fish  per  concentration were used  in static
 tests and  30  per  concentration  in  flow-through tests;  at least 8  concen-
 trations were used  in each  test.

     Technical  toxaphene used in this  study was  supplied by Hercules,  Incorpo-
rated, Wilmington, Delaware.  It  is described  as being chlorinated camphene
containing 67 to  69%  chlorine and is considered  to be  100% active  ingredient
 (33).  Stock solutions were prepared by  dissolving  the required amount  of
chemical in acetone.   Test  concentrations were accomplished by the addition of
an appropriate  amount of the stock solution  to a test vessel.  Amounts  of
acetone to water were  1 ml/1 or  less.  All concentrations were based on the

-------
total amount of chemical used and all data are expressed as concentrations of
total material.  In flow-through tests, concentrations delivered to each test
vessel were determined by dye calibration of the diluter system.  All concen-
trations are expressed as nominal rather than measured values.

     Toxicity values are expressed as LC50's (estimated concentration lethal
to 50% of the test population) and were calculated according to the log-
probit method of Litchfield and Wilcoxon (34).  Values were considered
significantly different when the 95% confidence limits about the LCSO's did
not overlap (28).  If less than 50% of the organisms died in the highest con-
centration, the LC50 for that test was stated as being greater than that con-
centration.  Time-independent lethal concentrations (TILCSO-concentration
estimate at which 50% of the test population can survive for an indefinite
period of exposure) were calculated by the method of Green (35).  For these
determinations, except for the bluegill test at 25 C, exposures were con-
tinued for 30 days or until total mortality over a 4 day period was 5% or less
of the test population and the daily mortality in any single concentration
did not exceed 10% of the initial number of animals per concentration.

-------
                                   SECTION 5

                                    RESULTS
      Static toxiclty tests showed that fathead minnows were the least sensi-
 tive to toxaphene and bluegills and channel catfish were about equal in
 susceptibility (Table 1).   The 96-h LC50 of 20 ug/1 for fathead minnows at
 20 C was 7.7 times the value for bluegills and 4.8 times that for channel cat-
 fish.  The greater resistance of the fathead minnows, however, may have been
 a consequence of the difference in maturity between them and the bluegills
 or channel catfish.   An increase in test temperature from 20 to 25 C did not
 appear to alter the toxicity for any of the three species.

      After continuous exposure in flow-through tests at 20 C, the 96-h LC50
 of 7.0 ug/1 observed for fathead minnows was about one-third that observed
 in static tests (Table 2).  Such a large within species difference was not
 apparent between the two test methods in bluegills or channel catfish.  When
 exposures were continued until response became asymptotic with time,  LC50
 values decreased to  one-half or less of the 96-h values;  the estimated
 TILCSO's were 0.6 ug/1 for bluegills,  1.0 ug/1 for channel catfish,  and 1.8
 ug/1 for fathead minnows.

      Although differences  in response were observed,  the  changes in  LCSO's
 that occurred in flow-through tests when temperatures were increased from 20
 to 25 C were irregular and relatively small.   It was  noted,  however  (except
 in the test  with bluegills where system failure forced early termination of
 the test),  that the  tests  at 25 C reached time-independence  in response at an
 earlier date (7 to 16  days)  than those at 20  C (24 to 34  days)  (Figs.  1-3).
 Even so,  the TILCSO's  for  all species  and temperatures tested were similar.

      The  lethality of  toxaphene to  channel catfish did not appear  to be al-
 tered by  pH, water hardness,  or temperature in static tests  (Table 3).   The
 96-h LCSO's were  not materially changed when  temperatures  varied from  15 to
 25  C,  pH  from  6.5 to 8.3,  or  total  hardness from 10 to 320 ug/1  (CaCO_).   An
 exception was  the 24-h LC50  of  12.5  ug/1  at 15  C,  which was  considerably
higher  than  the values at  20  and  25  C.

     A  substantial difference in  susceptibility was observed  between early
life  stages of  channel catfish.  Newly hatched  fry, before absorption  of  the
yolk, were extremely resistant  to toxaphene poisoning  as indicated by  the
24-h LC50 of 4700 ug/1 (Table 4).  However, the toxicity sharply increased as
 the yolk was observed.  After 96 hours, when all yolk  had  been absorbed,  the
LC50 decreased  to 8.0 ug/1.  This increase  in susceptibility  continued  to  the
swim-up fry stage where LCSO's were 4.0 ug/1 at 24 hours and  0.8 at 96 hours.
Thereafter, resistance increased; the swim-up fry were  the most  sensitive  life
stage.
                                       6

-------
TABLE 1.  ACUTE TOXICITY OF TOXAPHENE TO THREE FISHES AT TWO TEMPERATURES IN
          STATIC TOXICITY TESTS.
                                     (ug/1)

*a
Species
Fathead minnow

Channel catfish

Bluegill

20 C
24 h
24
(20-28)
7.8
(6.3-9.6)
6.8
(6.2-7.4)

96 h
20
(15-27)
4.2
(2.9-6.0)
2.6
(2.2-3.0)
25 C
24 h
23
(18-29)
6.4
(4.9-8.4)
6.6
(5.5-7.4)

96 h
23
(18-29)
3.7
(2.3-6.0)
2.4
(2.1-2.7)

      size ranged from 0.5 to 1.5 g.

-------
oo
     TABLE 2.  ACUTE TOXICITY OF TOXAPHENE  TO  THREE FISHES AT TWO TEMPERATURES IN FLOW-THROUGH TOXICITY
               TESTS.
                                                  (ug/1)

LC50 values (and 95% confidence limits)
Species
Fathead
minnow

Channel
catfish

Bluegill

Temp 4
(OG) days
20 7.0
(6.3-7.
25 5.0
(3.4-7.
20 5.5
(4.9-6.
25 7.5
(6.0-9.
20 4.7
(4.4-5.
25 3.4
(3.0-3.
8
days
4.3
7) (3. 8-4.
2.1
4) (1.5-3.
4.4
2) (4. 0-4.
3.7
4) (3. 0-4.
2.1
0) (1.9-2.
8)
12
days
3.6
9X3.3-3
1.8
0) (1.3-2
3.2
9) (2. 9-3
3.7
5) (3. 0-4
1.4
3) (1.3-1

16
days
2.9
,9)(2. 7-3.1)
1.5
.3) (1.0-2.1)
2.9
.5X2.6-3.2)
.5)
1.2b
.5)

20 24
days days
2.7 2.6
(2.5-2.9X2.5-2.7)

2.5 2.1
(2. 2-2. 8) (1.9-2. 3)

0.9b 0.8
(0.6-0.8)

Terminal
Day
24
(2
16
(1
29
(1
12
(3
34
7
(1
value
LC50
2.6
.5-2.7)
1.5
.0-2.1)
1.9
.7-2.1)
3.7
.0-4.5)
0.7
1.4
.2-1.7)
TILC50
1.8
1.0
1.0
1.9
0.6

           size ranged  from 0.5  to  1.5  g except for channel catfish which were 4 g in the 20 C test and
      O.lSgin the 25 C  test.

      Data inadequate to  calculate  confidence limits.

-------
TABLE 3.  INFLUENCE OF TEMPERATURE, pH, AND WATER HARDNESS ON THE
          ACUTE TOXICITY OF TOXAPHENE TO FINGERLING CHANNEL CATFISH3
          IN STATIC TESTS.

                             (ug/1)

Test Conditions
Temp
(°C) PH
15 7.4

20 7.4

25 7.4

20 6.5

20 7.5

20 8.3

20 8.2

20 8.2

20 8.2

20 8.2

Alkalinity
(mg/1)
35

35

35

35

35

35

220

220

220

220

Hardness
(mg/1)
40

40

40

40

40

40

10

40

160

320

LC50 values (and
95% confidence limits)
24 h
12.5
(9.7-16.1)
7.8
(6.3-9.6)
6.4
(4.9-8.4)
6.2
(3.9-10.0)
7.9
(5.9-10.5)
7.5
(6.3-9.0)
5.6
(4.2-7.5)
6.7
(5.2-8.6)
6.1
(4.6-8.0)
7.3
(5.8-9.3)
96 h
4.7
(3.2-6.8)
4.2
(2.9-6.0)
3.7
(2.3-6.0)
2.7
(1.9-3.8)
3.4
(2.5-4.6)
3.0
(2.1-4.3)
3.9
(3.1-4.8)
3.2
(2.3-4.4)
3.9
(2.0-5.1)
4.7
(3.7-5.9)
SFlsh size ranged from 0.5 to 1.5 g.

-------
 TABLE  4.   THE  ACUTE  TOXICITY OF TOXAPHENE TO THREE LIFE STAGES OF
           CHANNEL  CATFISH IN STATIC TESTS AT 25 C.

                                 (ug/1)
          a
                                LC50  values  (and
                                95% confidence  limits)
Life stage                24 h                     96  h
Yolk-sac fry              4700                     8.0
                       (3040-7260)               (6.0-10.6)

Swim-up Fry                4.0                     0.8
                         (3.2-5.0)               (0.5-1.2)

Fingerling                 7.0                     2.8
                         (5.4-9.1)               (2.0-4.0)
^oik-sac fry were 1 to 4 days old, swim-up fry were 5  to  8 days  old,
 and fingerlings size ranged from 0.5 to 1.5 g.
                                  10

-------
      10-
LC50
       4-
       2-
                                            = 20 C
                                            = 25 C
                                                 o—o
                        r
                       8
12
—T-
 16
—i—
 20
24
                                 Days
Figure 1.  Response curve for fathead minnows exposed  to  toxaphene in
           flow-through toxicity tests.

-------
ro
iu-
8-

LC50 fi
(MO/D
4-
2-
o = 20C
• =25C
0\
\ •
\\
O 
-------
LC50
      8-
      6-
      4-
                                   o=20C
                                   .= 25 C
                                          -o—o.
                                                 o - o
                                                            0 - 0.
                      8     12     16     20     24     28     32

                                       Days
36
    Figure 3.  Response curve for bluegills exposed to toxaphene in
               flow-through toxicity tests.

-------
                                   SECTION 6

                                  DISCUSSION
      Toxaphene,  one of the more toxic of the organochlorine insecticides  to
 fish  (12),  has a lethal range  of concentrations  reported  to be between 5  and
 100 ug/1 for most freshwater species  (36).   Data on similar species from  the
 Fish-Pesticide Research Laboratory (37)  show a range of 96-h LCSO's from
 static toxicity  tests  of 2 to  23 ug/1,  the  upper limit  for lethality being
 lower than  previously  reported.   Such differences in LC50 values,  however,
 could be due to  variations in  test methods  and precision  rather than to
 toxicity limits.   Data from the present  study are in general agreement with
 ranges reported  for bluegills,  channel  catfish,  and fathead minnows (38).

      In comparison with the static tests, exposure of bluegills and channel
 catfish to  toxaphene in flow-through  test systems did not produce  an ap-
 preciable increase in  toxicity.   In contrast,  fathead minnows were notably
 more  susceptible  to toxaphene  poisoning  in  the flow-through system,  thereby
 eliminating the  differences in susceptibility observed  in static tests; in
 effect,  LCSO's were similar for  all three species.   The similarity in response
 was maintained throughout  the  prolonged  exposures and the terminal values
 were  all within  the range  of 0.7 to 3.7  ug/1.

      Time-independent  LCSO's were similar for all three species, although  the
 rate  at  which response became  asymptotic with time  varied considerably.
 TILC50 values are  of interest because they  represent the  theoretical concen-
 tration  at  which 50% of the test population can  survive toxicant exposure
 for a prolonged period (39).  Presumably, the surviving animals are  suffi-
 ciently  able  to handle these concentrations of chemical,  through detoxifica-
 tion,  metabolism,  or other means,  to  the extent  that the  imposition  of mortal-
 ity on the  test population due  to the toxicant exposure is  no longer signif-
 icant.   In  this context, the toxicant concentration should  represent the
 range  where the response is  no longer acute and  becomes chronic in nature.

      It has been suggested by Eaton (40) that the lethal  threshold concen-
 tration  (LTC) is the more  appropriate value to use  in the calculation of ap-
plication factors  as defined by  Mount (41).   He  stated  that it provides bet-
 ter acute values to  use with experimentally determined  application factors
when  computing "maximum acceptable  toxicant concentrations" (MATC)  for spe-
 cies  for which only acute  toxicity  data  are available.  He  identified the LTC
value  as  the point  in  an acute toxicity  test  at  which the mortality  rate is
 10% or less of the  original number  of fish  in  any concentration during the
preceeding  24 hours.   Using  this  criterion,  LTC  values  determined  for our
 (20 C) tests were  4.3,  4.5, and  2.0 ug/1 for  fathead  minnows,  channel catfish,


                                      14

-------
and bluegills, respectively.  When compared with the 4 day LCSO's (Table 2),
the values are not greatly different for these species and would produce re-
latively small differences in calculated MATC values.  If the definition of
an application factor presented by the National Academy of Sciences (38) is
used (the numerical value for the ratio of the safe concentration to the
acutely lethal concentration), it would seem that the 96-h LC50 would be as
acceptable as the LTC.  Since, as a general rule, 96-h LCSO's would be less
expensive and time consuming to generate and there are broadly accepted,
standardized procedures for conducting 96-h toxicity tests (29), we recom-
mend the continued use of the 96-h LC50 for calculating MATC values when
experimentally determined values are not available.  This would maintain
continuity with the National Academy of Sciences (38) since it uses the 96-h
LC50 in generating calculated MATC values.

     If the arbitrary value of 0.01 is used as the application factor for
toxaphene (38), then the calculated values of the MATC's for bluegills,
channel catfish, and fathead minnows are 47, 55, and 70 ng/1, respectively.
From a chronic toxicity study with brook trout (Salvelinus fontinalis), Mayer
et al. (42) reports that the MATC is below 39 ng/1 and recommended that the
water concentration of toxaphene be below this value to protect this species.
Our data, however, indicate that this concentration may be adequate for the
protection of these three species.

     Of the environmental variables studied, only temperature effected the
acute toxicity of toxaphene.  Considering the chemical stability of toxaphene,
it would be expected that pH and water hardness, under laboratory test con-
ditions, would have little effects on toxicity.  Although irregular in na-
ture, it appeared that higher temperatures increased the rate at which the
response occurred and a decrease in the time required for mortality to be-
come asymptotic with time was observed in the flow-through tests conducted
at 25 C.  Such an influence on response time has previously been reported by
Hussein et al. (43) and Kopp (13).  An explanation of these results is not
immediately apparent.  Macek et al. (44) have suggested that rates of uptake
and enzyme activity may offer at least partial explanations of these data.

     Variations in the susceptibility of different life stages appeared to be
a factor of major importance.  Although the early yolk-sac fry were highly
resistant to toxaphene poisoning, susceptibility sharply increased after the
yolk was absorbed.  We believe that the resistance of the yolk-sac fry may be
correlated with the lipid-solubility of toxaphene and that the yolk is a
reservoir in which toxaphene is sequestered.  Data reported by Mayer et al.
(42), which indicated that brook trout fry accumulated toxaphene up to 76,000
times the water concentration within 15 days after hatching, appear to sup-
port this view.  As yolk absorption advanced, toxaphene residues would be
mobilized and mortality would increase.

     It was not surprising that swim-up fry were the most sensitive stage
studied.  They were in the phase where feeding and movement were being in-
itiated and embryonic differentiation was being completed.  It would be ex-
pected that an organism undergoing the stress of such physiological change
would be highly susceptible to externally superimposed stress.  Significantly,
our data show that the 96-h LC50 for swim-up fry is equivalent to the as-

                                      15

-------
ymptotic LC50 for fingerlings.  Such data point out the importance of
studying various life stages of aquatic organisms as well as various species
when "safe" concentrations of pollutants are being determined.
                                     16

-------
                                 REFERENCES

 1.   Bartel, W.F., J.C. Hawthorne,  J.H.  Ford,  G.C.  Bolton,  L.L.  McDowell,  E.H.
     Grissinger,  and P.A. Parsons.   Pesticides in Water - Pesticide Residues  in
     Sediments of the Lower Mississippi  River  and its  Tributaries.   Pestic.
     Monit. J. .3(1)58-66.  1959.

 2.   Ginn, T.M.,  and P.M. Fisher, Jr.  Studies on the  Distribution  and  Flux of
     Pesticides  in Waterways Associated  with a Ricefield-Marsh-Land Ecosystem.
     Pestic. Monit. J. 8/1):23-32.   1974.

 3.   Reinold, R.J., and C.J. Durant.  Toxaphene Content of Estuarine Fauna  and
     Flora Before, During, and After Dredging  Toxaphene-Contaminated Sediments.
     Pestic. Monit. J. j?(l): 44-49.   1974.

 4.   Schafer, M.L., J.T. Peeler, W.S.  Gardner, and  J.E. Campbell.   Pesticides
     in Drinking Water - Waters  from the Mississippi and Missouri Rivers.
     Environ. Sci. Technol. .3(12):1261-1269.   1969.

 5.   Hughes, R.A. Persistence of Toxaphene in Natural Waters.   M.S. Thesis.
     Water Chemistry Department, University of Wisconsin, Madison,  Wisconsin.
     1968.  135  pp.

 6.   Hughes, R.A. Studies in the Persistence  of Toxaphene  in Treated Lakes.
     Ph.D. Thesis.  Water Chemistry Department, University  of Wisconsin,
     Madison, Wisconsin.  1970.   270 pp.

 7.   Johnson, W.D., G.F. Lee, and D. Spyridakls. Persistence of Toxaphene in
     Treated Lakes.  Air Water Pollut. Int. J. 1.0:55-60.  1966.

 8.   Terriere, L.C., U. Kiigemagi,  A.R.  Gerlock, and R.L.  Borovicka.  The
     Persistence of Toxaphene in Lake  Water and Its Uptake  by Aquatic Plants
     and Animals. J. Agric. Food  Chem.  14(1):66-69.  1966.

 9.   Veith, G.D., and G.F. Lee.   Water Chemistry of Toxaphene -  Role of Lake
     Sediments.   Environ.  Sci.  Technol.  5.(3):230-234.   1971.

10.   Courtenay,  W.R.,  Jr.,  and M.H. Roberts.   Environmental Effects on Toxa-
     phene Toxicity to Selected Fishes and Crustaceans.  Environmental
     Protection Agency, Washington, D.C.  Ecological Research Series No.
     EPA-R-3-73-035.   1973.   73 pp.

11.   Duodoroff,  P., M.  Katz,  and C.M.  Tarzwell.  Toxicity of Some Organic
     Insecticides to  Fish.   Sewage Ind.  Wastes 25_(7): 840-844.  1953.
                                       17

-------
 12.  Henderson,  C.,  Q.H.  Pickering,  and  C.M.  Tarzwell.   Relative Toxicities  of
     Ten  Chlorinated Hydrocarbons  to Four Species  of  Fish.   Trans.  Am.  Fish.
     Soc.  88(1):23-32.  1959.                                	

 13.  Koppe, R.   Bin  Beitrag  zur  Toxikologie des  Toxaphens gegenuber Fischer
     und  deren Nahrtieren owie die Wirkung bereits durch Toxaphen begifteten
     Nahrtiere und Fische.   (The Toxicology and  Toxicity of  Toxaphene with
     Respect  to  Fish and  Aquatic Food Animals).  Z. Fisch. Hilfswiss.
     2:771-794.  1961.

 14.  Needham, R.G.   Effects  of Toxaphene  on Plankton  and Aquatic Invertebrates
     in North Dakota Lakes.  U.S.  Bureau  Sport Fisheries and Wildlife,
     Washington, B.C.  Resource  Publ. No.  8.  1966.   16  pp.

 15.  Surber,  E.W.  Chemical  Control  Agents and Their  Effects on  Fish.   Prog.
     Fish-Cult.  10(8):125-131.   1948.                                   	

 16.  Workman, G.W., and J.M.  Neuhold.  Lethal  Concentrations  of Toxaphene on
     Goldfish, Mosquitofish, and Rainbow  trout with Notes on Detoxification.
     Prog. Fish-Cult. 25_(1) :23-30.   1963.

 17.  Fiyoshi, G.F.,  and F.F. Hooper.  Toxaphene  (Chlorinated Camphene)  as a
     Selective Fish  Toxin.   Prog.  Fish Cult.  2(h 189-190.  1958.

 18.  Hemphill, J.E.   Toxaphene as  a  Fish  Toxin.  Prog. Fish - Cult.  16(1);
     41-42.   1954.

 19.  Henegar, D.L.  Minimum  Lethal Levels of  Toxaphene as a  Piscicide in
     North Dakota Lakes.  U.S. Bureau Sport Fisheries and Wildlife, Washington,
     D.C.  Resource Publ. No. 7.   1966.   16 pp.

 20.  Huish, M.T.  Toxaphene  as a Fish Eradicant  in Florida.  Proc.  Fifteenth
     Annu. Conf. of Southeast. Assoc. Game Fish  Commi.   Atlanta, Georgia.
     1961.  536 pp.

 21.  Rose, E.T.  Further Notes on Toxaphene in Fish Population Control.  iQ.
     Rev.  Biol. Res.  10:5-7.   1958.                                      ~

22.  Stringer, G.E.,  and R.G. McMynn.  Three Years Use of Toxaphene as  a
     Fish Toxicant in British Columbia.   Can. Fish Cult. ^8_:37-44.  1960.

23.  Tanner,  H.A.,  and M.L. Hayes.   Evaluation of Toxaphene  as a Fish Poison.
     Colorado Coop.   Fish. Res.  Unit, Quart. Rep. 324:31-39.  1955.

24.  Johnson,  W.C.   Toxaphene Treatment of Big Bear Lake, California.   Calif.
     Fish Game 42(3);173-179.  1966.

25.  Kallman,  B.J.,  O.B.  Cope, and R.J. Navarre.  Distribution and Detoxifica-
     tion of Toxaphene in Clayton Lake, New Mexico.  Trans. Am. Fish. Soc.
     91(l):14-22.  1962.
                                     18

-------
26.  Mayhew, J.  The Use of Toxaphene as a Fish Poison in Strip Mine Ponds
     with Varying Physical and Chemical Characteristics.  Proc. Iowa Acad.
     Sci. 6.6:513-517.  1959.

27.  Brauhn, J.L., and R.A. Schoettger.  Acquisition and Culture of Research
     Fish:  Rainbow Trout, Fathead Minnows, Channel Catfish and Bluegills.
     Environmental Protection Agency, Duluth, Minnesota, Ecological Research
     Series No. EPA-660/3-75-011.  1975.  45 pp.

28.  American Public Health Association.  Standard Methods for the Examination
     of Water and Wastewater.  14th Edition.  New York, American Public Health
     Association, 1975.  1193 pp.

29.  Committee on Methods for Toxicity Tests with Aquatic Organisms.  Methods
     for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians.
     Environmental Protection Agency, Duluth, Minnesota, Ecological Research
     Series No. EPA-660/3-75-009.  1975.  61 pp.

30.  Marking, L.L.  Toxicological Protocol for the Development of Piscicides.
     In:  Rehabilitation of Fish Populations with Toxicants:  A Symposium.
     P.H. Eschmeyer, ed. Washington, D.C., American Fisheries Society, North
     Central Division.  Spec. Publ. No. 4.  1975.  pp. 26-31.

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

32.  McAllister, W.A., Jr., W.L. Mauck, and F.L. Mayer, Jr.  A Simplified
     Device for Metering Chemicals in Intermittent-flow Bioassays.  Trans.
     Am. Fish. Soc. 101:555-557.  1972.

33.  Hercules, Incorporated.  Physical and Chemical Properties of Hercules
     Toxaphene.  Hercules Incorporated, Wilmington, Delaware.  Bulletin AP-
     103A.  1974.  4 pp.

34.  Litchfield, J.T., Jr., and F. Wilcoxon.  A Simplified Method of Evaluat-
     ing Dose-Effect Experiments.  J. Pharmacol. Exp. Therap.  96:99-113.
     1949.

35.  Green, R.H.  Estimation of Tolerance Over an Indefinite Period.  Ecology
     46(6):887.  1965.

36.  McKee, J.E., and H.W. Wolf.  Water Quality Criteria.  2nd Ed. The
     Resources Agency of California, Sacramento, California Publ No. 3-A.
     1971.  548 p.

37.  Fish-Pesticide Research Laboratory.  Unpublished Toxicity Data.  Fish-
     Pesticide Research Laboratory, Columbia, Missouri  1974.

38.  National Academy of Sciences.  Water Quality Criteria- 1972.  Environ-
     mental Protection Agency, Washington, D.C.  Ecological Research Series
     No. EPA-R3-73-033.  1973.  594 pp.
                                      19

-------
39.  Brown, V.M.  Concepts and Outlook in Testing the Toxicity of Substances
     in Fish.  In:  Bioassay Techniques and Environmental Chemistry.  G.E.
     Glass, ed. Ann Arbor Science Publishers, Ann Arbor, Michigan, 1973.
     pp.  73-95.

40.  Eaton, J.G.  Chronic Malathion Toxicity to the Bluegill  (Lepomis
     macrochirus Rafinesque).  Water Res., 4^673-684.  1970.

41.  Mount, D.I.  Chronic Toxicity of Copper to Fathead Minnows (Pimephales
     promelas, Raf inesque) .  Water Res. 2^:215-223.  1968.

42.  Mayer, F.L., Jr., P.M. Mehrle, and W.P. Dwyer.  Toxaphene Effects on
     Reproduction, Growth, and Mortality of Brook Trout.  Environmental
     Protection Agency, Duluth, Minnesota  Ecological Research Series No. EPA-
     600/3-75-013.  1975.  43 pp.

43.  Hussein, M.F., N. Badir, and R. Boulos.  Studies on the Effect of Insect-
     icides as an Ecologically Induced Limiting Factors to the Life of Some
     Fresh Water Fishes.   Ill.  Effect of Temperature on Toxaphene Toxicity to
     Gambusia sp. and Tilapia zilli.  Zool. Soc. Egypt Bull.  21:22-28.  1967.

44.  Macek, K.J., C. Hutchinson, and O.B. Cope.  The Effects of Temperature on
     the Susceptibility of Bluegills and Rainbow Trout to Selected Pesticides.
     Bull. Environ. Contain. Toxicol. 4(3) :174-183.  1969.
                                     20

-------
                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
  EPA-6QO/3-80-005
             3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
 Acute Toxicity of Toxaphene to Fathead Minnows,
 Channel Catfish, and Bluegills
                                                           5. REPORT DATE
                                                             January  1980 issuing date
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

 W.  Waynon Johnson and Arnold M. Julin
             8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 U.S.  Fish and Wildlife  Service
 Fish-Pesticide Research Laboratory
 Columbia, Missouri   65201
             10. PROGRAM ELEMENT NO.

               1BA021
             11. CONTRACT/GRANT NO.
                EPA-IAG-141(D)
12. SPONSORING AGENCY NAME AND ADDRESS
 Environmental Research Laboratory - Duluth, MN
 Office of Research  and Development
 U.S. Environmental  Protection Agency
 Duluth, Minnesota   55804
             13. TYPE OF REPORT AND PERIOD COVERED
             14. SPONSORING AGENCY CODE
                 EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT

      Toxaphene was  very toxic to fathead minnows (Pimephales promelas),  channel
 catfish (Ictalurus  punctatus), and bluegills  (Lepomis macrochirus)  in static tests;
 96-h LCSO's ranged  from 2.6 to 20 ug/1 at  20  C.   Fathead minnows were the least
 susceptible and bluegill and channel catfish  were about equal in susceptibility.
 Prolonged exposures of 12 to 34 days in flow-through tests produced time-independent
 LC50 values of 0.6  to 1.9 ug/1, significantly lower than the 96-h values.

      The toxicity of toxaphene was not influenced by variations in  pH or water hardness
 in static tests.  Temperature seemed to influence rate of onset rather than degree of
 response.  Toxicities were similar at 20 and  25  C in flow-through tests, however, a
 decrease in the time required for mortality to become asymptotic with time was
 observed.  Time-independence was reached after 7 to 16 days of exposure  at 25 C while
 24 to 34 days were  required at 20 C.
      The life stage of channel catfish most sensitive to toxaphene  poisoning was the
 swim-up fry with a  96-h LC50 of 0.8 ug/1.   Early yolk sac fry were  extremely
 resistant with a 24-h LC50 of 4.7 mg/1, however, within 96 hours the yolk had adsorbed
 and the LC50 had declined to 8.0 ug/1.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
a.
                  DESCRIPTORS
                                               b.lDENTIFIERS/OPEN ENDED TERMS
                             COS AT I Field/Group
 Pesticides
 Bioassay
 Fishes
 Mortality
 Toxaphene
 Fathead minnows
 Channel catfish
 Bluegills
 Acute  toxicity
06 T,F
18. DISTRIBUTION STATEMENT
 Release to Public
                                               19. SECURITY CLASS (This Report}
                                                 Unclassified
                           21. NO. OF PAGES
                                29
20. SECURITY CLASS (Thispage)
  Unclassified
                                                                          22. PRICE
 EPA Form 2220-1 (R«v. 4-77)   PREVIOUS EDITION is OBSOLETE 01
                                                                     JUS GOVERNMENT MINTING OFFICE: I9M -657-146/5541

-------