PB8l-2'm36
Influence of Diet and Starvation on Toxicity of
Endrin to Fathead Minnows (Pimephales Promelas)
Goteborg Univ. (Sweden)
Prepared for
Environmental Research Lab.
Duluth, KN
Aug 81
&& fopstwsS cj ©saswee
Ksfeatl Tectoxal iRtamation Service
mm
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TECHNICAL REPORT DATA
iPlease trad Instructions on tht reverse te/ori egmpktine!
t. BEPOnT NO. 2.
EPA-600/3-81-048 0RD Report
3. REC f'P F 1-2*4'l 16
4. TITLE AND SUBTITLE
Influence of Diet and Starvation on Toxicity of Endrin
to Fathead Minnows (Piaephales pronelas)
5. REPORT SATE
Aunust 1981
8. PERFORMING ORGANIZATION CODE
7, AUTMORISI
Goran Dave
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADORESS
Department of Zoophyaiology
University of Goteborg - Fack.
400 33 Goteborg, Sweden
10. PROGRAM ELEMENT NO.
1BA820
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon boulevard
Duluth, Minnesota 55804
>3. TYPE OP REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
IS, SUPPLEMENTARY NOTES
IS, ABSTRACT
Euitrin toxicity Co fathead alnnows, Plncjihsles prune Ian. was detora'inud In flow-throunh toxicity
teata ut 2b*C tor per lodl of up ca 10 days.
Three months prior to endrln exposure six groups of fish wars fed artificial diets contalnlnc an
Increasing percentage of (at, and two groups were fed either a high or low ration of rroien
brine shrlap. Klsh fed froirtn brine shrlrap allowed better growth, survival anil fewer detomltles
(hesorrhs«lc kidney lesions and lmdosls) than tlsh fed artificial diets. Based on 96-hr LC50
values, fish fed diets low in dleca'y lac were 2.A times aora susceptible Co endrln than fish
fed frozen brine shrlap. On cl» >a*e basis (96-hr LC50) fUh fed a low ration level of Urine
slirlnp ware 2.0 dees sore susceptible to endrln tlmn fish fed an unrestricted ration level of
brintf shrlap.
In another series of experiments, adult fathead minnows starved for 30 days were 2.0 tinea u,re
susceptible to endrln than recently feH fish. Fathead ulnnowa captured In a saall pond ,m April
30 uere also about 2.0 tines tsoro susceptible thfn recently fed fish. There was no detectlkla
relationship between body weight and susceptlbllUy of fathead alnnows to acuta uudrln
toxicity.
The reduced tolerances of endrln caused by a low percentage of dietary fat, red-iced ration level
and starvation were correlated to an Increased total body water content, Tim underlying reason
Is assuaed to be a coneoalcantly decreased total body lipid concent.
17, KEY WORDS AND DOCUMENT ANALYSIS
J. DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS
C. COSATI I'lcldfGttlUp
Effect of diet on acute toxicity
Toxicity tests
Fathead minnows
Diet
Nutrition
Mortality
Growth
Bioassay
0611
06F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS iThisHtpo't)
UNCLASSIFIED
21. NO. OF PAGES
95
20. SECURITY CLASS iThu pa ft 1
UNCLASSIFIED
22. PRICE
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EPA-600/3-81-048
August 1981
INFLUENCE OF DIET AND STARVATION ON TOXICITY OF EHDRIN TO FATHEAD MINNOWS
(PIMEFHALES PROMELAS)
by
Goran Dave
Department of ZoophysioLogy
University of Gotaborg
Fack
400 33 Goteoorg
Sweden
Grant Number
CR 806860-01
Project Officer
Jasses M. JlcKia
U.S. Environmental Protection Agency
Environmental Research Laooratory-Duluth
Duluth, Minnesota 55804
This study was conducted in cooperation with the
National Swedish Environmental Protection Board
Stockholm, Sweden
and the
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55304
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
6201 CONCDON BOULEVARD
DULUTH, MINNESOTA 55S04
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DISCLAIMER
Thia report haa been reviewed by the Environmental Research laboratory-
DuiuCh, U.S. Environmental Protection Agency, and approved for publication.
Approval does not aignify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade nanes or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
Man and his environment muse be projected from Che adverse effects of
pesticides, radiation, noise, and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay betveen the components of our physical,
environment—air, water, and land. The U.S. Environmental Protection
Agency, Environmental Research Laboratory-Dulath contributes to this
raultidisciplinary focus thvough progress engaged in
—studies on the effects of environmental contaminants on the
biosphere, and
—a search for ways to prevent contamination and to recycle
valuable resources.
iii
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ABSTRACT
Indrin toxicity to Cathead rainnowo, Fiaaphaleg promelaa, was determined
in flow-through toxicity tests at 25*C for periods up co 30 day*.
Three months prior co endrin exposure 6 groups of fish were fed
artificial diets containing an increasing percentage of fat, and 2 groups
were fed either a high or a low ration of frozen brine nhrirap. Fiah fed
frozen brine flhrimp showed better growth, survival, and fewer deformities
(hemorrhagic kidneys and lordosis) than fish fed artifical diets. Baaed on
95-hr LCSO-valuea, fiah fed dieta low in dietary fat were 2.4 times more
susceptible to endrin than fiah fed frozen brine ahrimp. On the sane basis
(96-hr LC5G) fiah fed a low ration level of frozen brine shrimp were 2.0
times more susceptible than fish fed an unrestricted ration level of brine
ahrimp.
In another series of experiments, adult fathead minnows stir"ed for SO
days were 2,0 timea more suaceptible to endrin than recently fed fiah.
Fathead minnows captured in a pond on April 30 were also about 2.0 times
more susceptible than recently fed fiah. There was no detectable
relationship between body weight and suceptibility of fathead minnows to
acute endrin toxicity.
The reduced tolerances to endrin caused by a low percentage of dietary
fat, reduced ration level and starvation were correlated to an increased
total body water content. The underlying reason is assumed to be a
concomitantly decreased total body lipid content.
iv
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CONTENTS
Foreword iii
Abstract . . iv
Figure# vii
Table* viii
Abbreviations and Symbols ..... ix
Acknowledgment ........... x
1. Introduce ion 1
2. Conclusions ....... . 3
3. Recoanendat ions 4
4. Materials and Methods ...... ..... 5
Sources of experimental fish . , , . 5
Experimental diets . 8
Aquaria system for rearing of fish 8
Aquaria and toxicant delivery syatea far toxicity tests . 10
Water charac ter iit ics 10
Treatment of effluent ....... .... 11
5. Experimental Procedures 12
Ceneral procedures ....... 12
Feeding routines ............ 13
Toxicity testing routines ... 14
Statistical treatment of data 14
Median survival times 14
6. Results 16
Dietary effects on the fathead minnow 15
Mortality 16
Growth and fooit consumption 18
Other «;f feces of diet . 18
Starvation and its effects on the fathead minnow .... 22
Dietary influence on er.drin toxicity . 22
Influence of starvation on endrin toxicity 32
Correlation between water content and susceptibility to
endrin . » , 36
Effects of endrin on total body water content . 40
7. Discussion ...... .... 46
References 49
Appendice¦
A. Measured flow rates and temperatures during toxicity tests
with endrin on fathead minnows 54
B, Alkalinity, hardness and pH of the dilution water . 56
V
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C, Measured concentrat ions of endrin (ug/1) in the water during
toxicity testa with fathead tsinnowe _ SB
B. Food consumption and nortality during rearing period prior
to toxicity tests ..... ... 59
1. Body weight, water content and survival tine for fathead
minnows subjected to starvation prior to tonicity teats
with endrin . . . ...... . • 67
F. Position of experimental groups of fish in screened
compartments during toxicity tests ..... 80
C. SuBssry on previous findings on endriii toxicity to fish ... 83
vi
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FIGURES
Number Page
1 Growth of fathead minnows fed 7 different diets . 20
2 Mortality of adult fathead minnows starved at 25 .O'C 23
3 Mortality of juvenile fathead ainnows starved at 23.0-24.5'C . 24
4 Toxicity of endrin to fathead minnows fed either a high or a
low ration of frozen brine shrimp prior to exposure 29
5 . Toxicity of endrin to fathead minnows fed either a low-fat diet
or frozen brine shrinp prior to exposure 31
6 Endrin toxicity to fathead minnowa atarved for i to 97 daya prior
to exposure 34
7 Effect of starvation on total body water in fathead minnows . , 35
1
8 Correlation between median survival time (MST), in 3 concen-
trations of enorin and mean total body water content for
fathead minnows 38
(
j 9 Correlation between survival time in 3 concentrations of endrin
j and total body water content for individual fish 39
10 Total body water content of intoxicated and surviving laboratory
brood stock fish starved for l-"7 days before exposure to endrin 42
11 Total body water content of intoxicated and surviving pond fish
held in the laboratory for 7 months before exposure to endrin 43 ;
12 Total body water content of intoxicated and surviving pond fish
, trapped betwaen 2 and 21 days before exposure to endrin ... 44
vir*
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TABLES
Number Page
Smaaary of Experimental Exposure* Co Endrin with Fathead Minnows
Fed Different Diets Prior to Exposure 6
Sunmary of Experimental Exposures to Endrin with Fathead Minnows
Starved for Various Periuds Prior to Exposure 7
Cooposition of Artificial diets (I-VI) . . 9
Cusmulacive Mortality of Fathead Minnows fed 7 Different Diets
for 100 Days 17
Mean Body Weights and Mean Daily Body Weight Cain for Fathead
Minnows fed 7 Different Diets Ad lib .19
Recovery of Abnormal Fathead Minnows when fed Frozen Brine Shrimp
for 3 Weeks .21
Water Content (Z w.w./w.w.) of Surviving and Starvation-Killed
Fathead Minnows Starved for Various Periods at 25*C ..... .25
8 Body Weights of Fathead Minnows Pre-fed with 8 Diets and Used in
Toxicity tests with Endrin 26
9 Toxicity of Endrin to Fathead Minnows fed a Low Ration of Brine
Shrimp Prior to Toxicity Tests, When Tented at Various Periods
and iti Different Exposure Chambers . 28
10 Toxicity of Endrin to Fathead Minnows fed 8 Diets for 80-100 Days
Prior to Toxicant. Exposure 30
11 Toxicity of Endrin to Adult Fathead Minnows Starve! for 1-97 Days
Ptior to Toxicant Exposure . .33
12 Total Body Water Content in Fatheau Minnows fed 8 Diets for 3
Months 37
13 Effects of Endrin on Total Body Weight .41
viii
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
B.W. — body weight
D.V. — dry weight:
LC50 — lethal concentration 501
HST — Medim Survival Time
w.w. — wet weight
SYMBOLS
C1CO3 — calcium carbonate
HjO — water, used for total body water content in Table E3
is
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ACKNOWLEDGMENTS
This vork waa performed at the Environmental Research Laboratory-
Duluth, Duluch, Minn. The cooperation of its entire staff is greatfully
acknowledged, a aa "srCicularly indebted to Dr. James McXim, Chief of the
Physiological Effects Section, and all the members of this section.
Cheoical analysis were carried out by Mrs. Diane Olson and Mr. Steve
Peterson.
Dr. David Weininger and Kr. Paul Telega provided help on the
statistical treatnent of the data.
Financial support wss provided by the Sweden-America Foundation, The
National Swedish Environment Protection Board, (Project Number 7-338-78),
and the United States Environmental Protection Agency (Grant N'umbe OR
806860-01). The help of the American Scandinavian Foundation is also
greatfully acknowledged.
/
x
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SECTION 1
INTRODUCTION
The miCricional aspects of Eoxi.ci.Ey studies with aquatic organisms have
only recently started to receive the attention of aquatic toxicologists, and
the importance of nutritional factors like dietary ingredients, percent
composition and ration level on the toxicity of selected toxics has become
apparent.
Cairns £t^ al. (1975) found that short-term starvation (0 to 72 hrs) in
Orsanco strain goldfish Carassius auratua (Orsanco, 1977) had no significant
influence on the acute toxicity of zinc indicating that short-term effects
of starvation on acute toxicity (<24 hrs) may be of minor importance.
However, the importance of dietary composition on response to toxicants has
been demonstrated in several previous studies, hehrle et al. (1977) found a
six-fold difference in the 96-hr LC50's (8.2 to 47 Ug/1) of rainbow trout
(Salmo gairdneri) when they were fed six different diets for 42 days prior
to exposure to chlordane. The underlying reason for these differences in
acute toxicity was not elucidated. A four-fold difference in the chronic
toxicity of copper to the water flea (Daphnia magna) was found between
daphnids fed an algal versus a trout pellet-based diet (Winner et al¦,
1974). In another stud/ on interactions between dietary levels of vitamin C
and exposure to toxaphene in channel catfish (Ictalurus punctatus) (Mayer jit
al., 1978), it was shown that increased levels of dietary vitamin C counter-
acted the effects of toxaphene on body weight, collagen content of the
vertebrae, incidence of spinal deformities, and histopathological effects on
the skin. Furthermore, increased dietary levels of vitamin C increased the
activity of livir aromatic hydrocarbon hydroxylase, an enzyme involved in
detoxification, and caused a reduction in toraphene residues. Phillips and
Buhler (1979) found that rainbow trout fed two different diets (Oregon Moist
Pellets or tubificid worms) at three ration levels for 61 days responded
differently in terms of fat elaboration and dieldrin accumulation.
Based on the existing knowledge of the effects of nutrition on the
response of aquatic organisms to toxicants, it can be concluded that
nutritional factors have definite effects on both acute and chronic
toxicity. Therefore, since one of the main objectives of toxicity tests
with aquatic organisms is to estimate maximum tolerable concentrations of
chemicals, it is important to understand to what extent results of
laboratory toxicity tests are influenced by nutritional factors, especially
when food supply (amount as well as composition) in nature is often quite
different from that in the laboratory.
The purpose of this study was to examine the influence of dietary
composition and ration level (including starvation) on the toxicity of a
1
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lipophilic (fat soluble) chemical to the fathead minnow (Pimephalea)
promelas). The chemical used for this purpose was endrin (1,2,3,4,10,10-
hexachloro-6,7,epoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4-endo-end o-5,8-
d j"ienh»ncnaphthalene), which is extremely toxic to fish, [96-hr LC50 from
0.26 Pg/1 for fathead minnows (Pimephalea promelaa) to 8.25 ug/1 for
bluegills (Lepomis macrochirus) (Solon ei£ ., 1969; Katz and Chadwick,
1961)]. Fathead minnows were selected as the fish species for this study
baa"j on availability and general importance in anuatic toxicology.
Since a lipophilic toxicant was studied, special emphasis was focused
on lipid metabolism. Pretreatment of fish prior to toxicity tests included
feeding for three months with diets containing various amounts of lipids as
well as specific types of lipids. In order to deplete the lipid depots,
various groups of fish were also subjected to starvation for up to three
months prior to toxicity tests. Zn the starvation experiments three sources
of fathead minnows were used in order to reveal a possible influence of
pretreatment prior to the starvation period. The hypothesis that the total
lipid content of a fish is important in its short-term survival when exposed
to a lipophilic toxicant like endrin was evaluated. This was measured by a
determination of survival time, total body weight, and total body moisture
content following endrin exposure. Body moisture? content in fish was shown
to be inversely proportional to lipid content (Love, 1970).
2
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SECTION 2
CONCLUSIONS
Fathead minnows (Pimcphales proaelaa) fed frozen brine shrimp showed
better growth and survival and fewer deformities (hemorrhagic kidneys and
lordosis) than those fed recommended reference research diets. In those
fish fed only the reference research diets supplemented with either 10% corn
oil, salmon oil, or a 1:1 mixture of these oils no differences were observed
in survival, growth or number of deformities.
Acute toxicity of endrin was increased 2.4 times by a lowered
percentage of dietary fat, 2.0 times by a restricted ration level, and
approximately 2 times following starvation for 80 days prior to exposure.
Fathead minnows trapped in a pond in the spring (April 30) were approxi-
mately 2 times more susceptible to endrin than the laboratory stock. The
higher susceptibility in wild minnows reflected their poor nutritional
status in the spring.
An increased susceptibility to endrin in fathead minnows was positively
correlated to an increased total body water content, reflecting a lowered
level of total body fat. Re-localizacion of endrin to non-target tissues
like adipose tissue seems to be an important mechanism for resistance to
endrin. A dose-dependenc reduction in total body water content of fathead
minnows exposed to endrin was also observed.
There uas no detectable relationship between body weight and
susceptibility of fathead minnows to acute endrin toxicity.
3
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SECTION 3
RECOMMENDATIONS
Reference research diets should not be used for the culture of fathead
minnows (Pimephales pronelas) since they result in on increased mortality, a
greater incidence of deformities (hemorrhagic kidneys and lordosis) and
decreased growth compared to fish fed frozen brine shrimp (Artenia galina)
A lowered level of dietary fat, a lowered ration level and long-term
starvation prior to toxicity testa with a lipophilic chemical such as endrin
can produce significantly lower LC50 values. This should be considered in
standardized toxicity tests with this type of chemical.
No influence of acclimation to test temperature from 2 to 23 days was
evident on the acute toxicity of endr\n in wild fathead minnows. On the
other hand, feeding for I week prior to exposure made the wild minnows more
resistant to endrin. Therefore, a more detailed study on differences in
susceptibility to lipophilic chemicals between laboratory fish and
field-collected fish on a seasonal bttsiB should ba made. Such a study
should preferably be complemented by measurements of condition factor, water
content and lipid content for different groups of fish, in order to achieve
a better understanding of the variations in susceptibility of different lots
of fish.
Considering the findings in the present studv, acclimation to test
conditions for 1 week without feeding in both laboratory stock fish and wild
fish seems to b«i a sound basis for the comparison of 96-hr LC50 values.
4
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SECTION 4
MATERIALS AM) METHODS
SOURCES OF EXPERIMENTAL FISH
Two sources of fathead minnows ware used in this experiment:, One wis
the laboratory brood atoek of the Environmental Research Laboratory-Duluth,
Minnesota, and the other was a private pond owned by one of the laboratory
staff members. This artificial pond (surface area; 18 acres; maximum depth;
1.6 a), supplif.d with water from a saall spring fed stream was, after its
construction in 1972, stocked with far iead minnows from the Duluth
laboratory brood stock in 1973. Since then, this population of fathead
rainnovo has gone through approximately 5 generations. Therefore, these fist
have a natural (wild) background in terms of diet (pond organisms), seasonal
variation, and natural reproduction.
For the diet experiments, fathead minnow larvae hatched December 8-22,
1978 were used. After hatching, these larvae ware fed live newly hatched
brine shrimp (Living World, San Francisco Bay Brand), and later supplemented
with frozen brine shrimp (San Francisco Bay Brand, Newark, California
94560; Stock #65006). On January 2? *.nd 23, 1979 one-month-old fish were
randomly divided into seven groups of 220 fish each. After determination ol
total body weight (ten fish at a tins) in a tarred aluminum foil cup
containing approximately 20 ml water, they were transferred to pretreatment
tanks. Experimental groups of fish in thi? study were designated by numbers
I through VIII (Table 1).
For the starvation experiments adult (more than five months old)
fathead minnows from tt-e laboratory stock were divided into 4 groups of 50
fish each on January 25, March 2, March 23, and April 9, 1979. Individual
body weights of fish were obtained by weighing each fish in a glass beaker
containing approximately 300 ml af water and designated numbers IX through
XII, A fifth group of >50 fish from this source (not weighed before the
toxicity tent) were netted on April 30 and fasted 24 hrs prior to the
toxicity test (group XIII, Table 2).
In addition, two <"-
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TABLE 1. SUMMARY OF EXPERIMENTAL FXPOS'JflES TO EJCRIN WITH FATHE/O MINNOBS
FED DIFFERENT DIETS PRIOR TO EXPOSURE
Group of Exparlmant Expetura to Enflrln
~ lah no#* Olatary protraatmant Cone. no. Period of wposura
ARTIFICIAL 01ETS
'1
I
"5!-* i for 78 days
1,2,1.4.5
- 29 days
»2
2
01 at 1 foi 92 daya
1,2,3
- 15 days
'3a
3
01 at 1 for 99 days
1,2
- 7 days
'3b
3
D'et 1 for 92 days plus
0lot VIII for 7 daya
1,2
- 7 days
"l
1
Olet 11 for 78 days
1.2.3,4,3
- 29 days
Il2
2
01 at II for 92 days
1.2,3
* 15 days
III)
1
01at III for 78 days
1.2.3.4,9
- 29 days
"'2
2
01 at III for 92 days
1,2,3
- 15 days
IV,
1
Olat IV for 78 days
1.2,3,4,3
- 29 days
IV2
2
01at IV for 92 days
1.2.3
- 15 days
V1
I
Diet V for 78 days
1.2,3,4,5
- 29 days
V2
2
Olat V for 92 days
1,2,3
- 15 days
VI,
1
Olat VI fop 78 days
1.2,3,4,5
" & days
viz
2
Diet VI for 92 days
BRINE SHRIMP DIETS
t .2,3
- 15 days
VII,
i
Olet VI* for 78 oays
thigh ration)
1,2,3,4,5
- 29 da/s
V112
2
Olat VII for 92 days
(high ration)
1.2,3
- 15 days
Villi
1
Olat VII1 for 78 days
£ low ration)
1,2,3,4,9
- 29 days
vhi2
2
Olat Vil1 for 92 days
(low ration)
1,2,3
- 15 days
villj
3
Olat VIII for 100 days
(low ration)
1.2
- 7 days
*in4
4
Olet VIII for 66 daya
(low ration)
1
- 3 days
0 Exparlnaits I, 2, 3 and 4 wara started April 30, Hay 14, May 22, and May 8,
respectively.
b Concentration No. 1 was tfca tilyh*t ona followed by 1:1 dllutlons producl ng
noalnal concentrations of 1/2, 1/4, 1/3 and 1/16 of this concentration.
Ccxicentralion No. 6 (not presented above! »as tna control.
6
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TABLE 2. SUMMARY OF EXPERIMENTAL EXPOSURES TO DOR IN Ml TO FATHEAD MINNOWS
STARVED FCR VARIOUS PER IKS PRIW TO EXPOSURE
Group e# ixfterlMMit StarvatIon prior Exposure to inflrIn
lllh no.* Source pretr#af*ient Cone, no.® Period of wpoaura
IX
1
Laboratory
fish
93 days
1,2,3,4,3
-
29 days
X
1
Laboratory
fish
39 days
1,2,3,4,5
-
29 days
XI
t
Laboratory
fish
38 day*
1,2.3,4.5
-
29 days
XII
»
Laboratory
fish
21 days
1,2,3,4,3
-
29 days
XIII
i
Laboratory
fish
24 hrs
1,2,3,4,5
-
IB day s
XIV
i
Pond fish
fad In the
laboratory
during »Inter
33 days
1,2,3,4,3
25 day*
XV,
se
Pond flsft
fed In the
laboratory
<);.rtng * Inter
24 hrs
1.2,3,4,3
29 days
XVII
ic
Pond fish
c pturad just
prior to twit
24 hrs
1,2,3,4,3
m
29 days
X*2
2
Pcao fish fad
lii ttia laboratory
during winter
14 days
1,2
w
4 days
XVI2
2
Pond f f i.fi
captured end
aixl 1 mated for
14 days
14 days
1,2
2 days
xv3
3
Group XV2 fad
brliM sltrlnp
for 7 days
14 days
and feeding
for 7 days
1.2
4 days
XV,So
3
Group XVIj fad
brine sftrTap
for 7 days
14 days
plus feeding
tor 7 days
1,2
3 days
*V,3b
3
Group XVI2
starved for
another 7 day*
21 days
1,2
1 day
a Experiment 1,2 and 3 >«ri start ad on April 30, May 14, and May 23,
respectively.
6 Concentration Mo. 1 was ttie highest mm fol loned by 1:1 dilutions producing
nominal concentrations of 1/2, 1/4, 1/8 end 1/16 of this concentration.
Concentration No, 6 (not presented above) was ttim control ¦
c Started on Hay I.
7
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In order to reveal Che ecological significance of toxicity data for
these groupi of fish another group of approximately 200 fathead minnows
(group XVI) were trapped on April 23 and 29 in the pr.nd mentioned above and
brought to the laboratory on April 30. After chore-term temperature
acclimation (25 *_ 1*C - 7 hrs) they were put into one of the pretreatment
tanka for 24 hrs prior to exposure to endrin.
Fish from group# I through XVI ware taken at two or more occasions («s
indicated by subscripts, Table* I and 2) for toxicity testa with endrin, A
susnary of the pretreatment# far the various groups of fish used in toxic ity
tests are presented in Tables I and 2.
EXPERIMENTAL DIETS
In order to study the influence of lipid content and composition, six
group* of fish (I-VI) were fad diets with increasing amounts of lipids. For
this purpose six diets were prepared based ?>n the recommendations given for
reference research diets (National Research Council, 1973, 1977; 3raun and
Schoettger, 1975). All of the experimental diets contained the same
percentage (dry weight basis) of proteins (lactic casein, and gelatin),
digestible carbohydrates (dextrin), vitamins (vitamin mixture 23-997), and
minerals (mineral mixture 28-998). Th»*. only ingredients and percentages
that were varied were the lipid source (corn oil and/or salmon oil), which
was compensated for by the nondigestible binder (o-cellulose). All
ingredients, except for the salmon oil (relatively free from pesticide
residues), which was kindly provided as a gift from Dow Chemical Co., were
prepared by Zeigler Brothers, Gardners, Pennsylvania. A description of the
artificial diets prepared, I through VI, is presented in Table 3.
The artificial diets, I through VI, were prepared by mixing appropriate
amounts o£ all dry ingredients including the lipid sources (core and/or
saloon oil (Table 3). After careful mixing, the dry powdered diets were
stored in sealed plastic boxes at 12*0. Each week (Monday) the daily
rations for the whole week, 6x7 rations consisting of 10.0 g dry diet
mixed with 20 ml water in an aluminum weighing cup, were prepared and stored
in 12*C.
In addition, two groups of fish, VII and VIII, were fed frozen brine
shrimp (San Francisco lay Brand, Newark, California 94560; Stock #65006).
Group VII was fed essentially ad lib, while group VIII, was fed a much lower
ration level (see Section 6).
AQUARIA SYSTEM FOR REARING OF FISH
During the pretreatment period, including feeding with diets I through
VIII and starvation for periods ranging from 1 to 95 days, all groups of
fish were held in a flow through test system previously described by BenoiS
et al. (1976). Briefly described, it consisted of six duplicate upper glass
tanka (55 x 23 x 15 cm; L x U x H) with a water volume of 20 1 and six
duplicate lower stainless steel tanks (91 x 33 x 30 cm; L x W x H) with a
8
-------
TABLE 3. COMPOSITION OF ARTIFICIAL DIETS Cl-Vl)
Amount of ingredients (g) in respective diet
Ingredient I II III IV V VI
Casein
280
280
280
280
280
280
Gelatin
120
120
120
12C
120
120
Dextrin
280
280
280
280
280
280
Vitamin mix.
10
10
10
10
10
10
Mineral nix.
40
AO
40
40
40
40
Sum of above
730
730
730
730
730
730
Com oil
-
100
-
50
75
100
Salmon oil
-
-
100
50
75
100
Sua of above
730
830
830
830
880
920
a cellulose
270
170
170
170
120
70
Sum of above
1000
1000
1000
1030
1000
1000
Added water
2000
2000
2000
2000
2000
2000
Moisture (X)
66.7
66.7
66.7
66.7
66.7
66
9
-------
water volume of 90 I. The water was supplied by a 2 1 proportional diluter
(Mount and Brungs, 1967) and flow-splitting cells (Benoit and Puglisi, 1973)
which provided a total water flow of 1 1/ain to each set of duplicate upper
and lower tanks. Water replacement times (°0X) for each tank were
approxinately 4.5 hrs (Bencit et al., 1976), Water temperatures in the
upper tanks were approximately 25 ~ l*c, while the lower tanks were 26 +
rc.
AQUARIA AND TOXICAIO.' DELIVERY SYSTEM FOR TOXICITY TESTS
A system identical to that used for rearing the fish was utilized
during the toxicity tests (Holcombe et al., 1976). Data for flow rates and
test temperatures are included in Appendix A. A schematic figure of the
exposure system is presented in Appendix P.
The toxicant, endrin, was delivered into the exposure system by a
safturator (Veith and Comstock, 1975) modified slightly by Jarvinen and Tyo
(1978), The column of the saturator was made up by coating 5.0 g of endrin
(Analytical Reference Standard; assay: 96.1%, std. no. RS: E-71172, Velsicol
Chemical Corp.) dissolved in acetone (P.A.) on 100 g of acetone-washed sand.
Tha delivery of toxicant from the saturator holding tank containing 14 1 was
achieved by a "dipping bird" (described by Lemke et ai., 1979). Thirty-five
milliliters of water saturated with endrin was mixed in Che diluter on each
cycle. Endrin was introduced into the exposure system 20 days prior to
exposing the animals in order to equilibrate the system. The concentration
of endrin in the saturator was monitored daily by gas chromatography (APHA
«tt al., 1975), Monday through Friday. Accuracy for total endrin in water
was checked by the method of known additions by adding endrin to control
water and determining the percentage recovery. The raaan recovery and 95%
confidence interval for the entire exposure period was 89 _+ 52. Precision
was determined by analyzing duplicate sanples from a different exposure tank
with each awapling period. This was repeated 8 timet* (over period May 1-30)
and the results were within 6Z of each other. A complete listing of all
measured endrin concentrations (from May 1-30) are presented in Appendix C,
while endrin concentrations for specific experimental groups are presented
in Tables 9, 10, and 11.
WATER CHARACTERISTICS
All fish used in the. present study were raised and tested in Lake
Superior water. The only exception was the minnows which were trapped in
the pond prior to experimental use. Water quality in this pond is, however,
considered to be close to Lake Superior water as the pond is located close
to the lake and supplied with water fron the same drainage area.
Before entering the test system all Lake Superior water was sand
filtered, sterilized by ultraviolet light and brought to the desired
temperature (25 _+ 1*C) in the head-box reservoirs (one for each experimental
system).
10
-------
Weekly water quality data on alkalinity, -.ardnesa and pH for the study
period are presented in Appendix ft. Ranges for pli, alkalinity and hardness
were 7.2 to 7.5, 39.5 to 44 tag/1 ao CsCOg, and 43.5 to 49.5 mg/1 aa
CaCOj, reBpectively. On one occasion, May 17 and 18, all of the upper
exposure tanks were aanpled for measurements of dissolved oxygen, ptt,
alkalinity, acidity and total hardness (data presented in Appendix B).
TREATMENT OF EFFLUENT
All test water leaving the exposure system was put through an activated
carbon filter designed to remove >90% of the endrin prior to going to the
city S£wer syscem. During the experioental period the percent removal
ranged £ro« 82-94%.
II
-------
SECTION 5
EXPERIMENTAL PROCEDURES
GENERAL PROCEDURES
To enable cuanti.ficati.on of minor differences in toxicity to the
experimental groups of fish, uniform conditions were created during
pretreatment of the test fish prior to testing as well as during the
toxicity tests. This was achieved in the following way.
a, Pretreatment of fish
1. Use of an exposure system (without addition of toxicant) which was
previously designed to produce uniform conditions in terms of water
quality flow, and temperature as well as the physical environment
(uniform tanks) and light (diffuse light at 16 hrs/day).
2. Establishment of uniform and realistic feeding routines and exposure
system maintenance - siphoning of tanks (daily), cleaning tank sides
and bottoms (weekly).
b. Toxicity Testing
1. In addition to the items mentioned above for pretreatment the
uniformity of the exposure conditions were preconditioned by careful
ad justments of dlluter function, especially flow rates to duplicate
tanks, and adjustment of temperature in the headbox.
Z. Uniformity in exposure conditions was monitored and controlled by
measurements of variations between and within duplicate canks.
3. By separating the groups of fish to be compared by stainless steel
screens, as all diet groups were exposed in the same, continuously
renewed solution of toxicant (five concentrations plus one
control).
4. Possible timewise differences in water quality, temperature and
toxicant concentrations as well as timewise differences in
pretreatment were eliminated by starting experimental exposures of
groups to be compared on the same day (Experiments 1-4). In the
present study the two main timewise differences were the adjustment
of fltjw-ratea in the upper tanks on May 7 and variations in the
concentrations of endrin in the saturator. For further information
on these matters see Appendix A and C.
12
-------
FEEDING ROUTINES
Aa mentioned previously portions of the food (Diet I-VI) were prepared
weekly by weighing out 10.0 g premade powdered dry food and mixing with 2D
ml of water. For diets VII and VIII consisting of frozen brine shrimp
(Artemia salina) sufficient amounts for 1 week were thawed once weekly,
weighed into the same kind of aluminum pans aa the other diets. For this
purpose, 40.0 g (wet weight, which corresponds to 4.2 g dry weight) w»s
weighed into each pan and stored in a freezer until used that week.
.is the fathead minnow does not have a differentiated stomach for the
storage of food, a sufficient time for faeding each day must be allowed in
order to to obtain optimal growth. It was shown at this laboratory
(unpublished observations) as well aa by other investigators (Smith et al.,
1978) that the growth rate ia positively correlated to the number (1-4
tines) of feedings per day. In order to create a feeding situation aa close
to ad lib feeding as possible, the following routine waa followed during the
entire pretrectaent period (February 9 through May 1, 14, and 22, Exp. 1-3).
Food previously frozen in aluminum weighing pans, was placed in the same
corner of the exposure tank each day (Monday through Friday) between 3 and 4
p.m. On Saturday and Sunday food was placed in the tanks between 9 and 10
a.m. Food waa removed between 8 and 8:30 a.m. '•he following day (all days),
taking care to siphon up all spilled food on the bottom. This waa usually
easy to do, except f<~<. the frozen brine shrimp (group VII) in which case
residual brine shiittp were siphoned up in about 2 I of water in a plastic
bucket. After settling for 15 mm the food was concentrated into less than
200 mi and put into new aluminum weighing pans. The residual uneaten food
was taken to dryness overnight in an oven at 105'C. By weighing dryed
samples of the prepared diets (dry weights 9.2, 9.2, 9.3, 9.3, 9.3, and 9.4
g for I-VI) and rhe residual uneaten diets, the food consumption for each
group of fish was determined as the difference (dry weight basis).
After removal of food from the tanks, tank bottoms were siphoned free
of fecal material and the number of dead fish were recorded. Once a week
(Mrnday) all tanks werfc cleaned by scrubbing and additional siphoning. No
visual differences in turbidity of the water between the various tanks was
observed.
On February 22 all groups (I-VII) of fish in the upper pretreatment
tanks were counted and total body weights determined, after which they were
transferred to corresponding lower tanks in the pretreatment system. On May
1, before the start of Experiment 1 (first toxicity test), all groupB of
fish (I-VII) were counted and total body weights determined.
During the toxicity tests and one day prior to starting tests no food
waa given, lor the starvation groups, no food was added after transfer to
the pretreatment system.
During endrin exposure, all tanks were inspected twice daily Monday
through Friday and once on Saturday and Sunday. Dead fish were removed and
wet weights and body water content (drying at 105'C overnight) were
determined on the starvation groups (Appendix E). Data recording on food
13
-------
consumption and mortality for fish groups I through VII are presented in
Appendix D.
TOXICITY TESTING ROUTINES
Randomization or experimental fish for toxicity tests was performed in
the following three steps.
1. After transfer of all fish in a group to a 10 1 bucket (aerated when
necessary), appropriate numbers of fish (8 to 10) for all groups, except
group IX where 4 to 5 fish per concentration only were available due to
previous starvation aortality, were randomized into 6 (Exp. 1), 3 ("xp.
2), or 2 (Exp. 3) plastic containers (approximately 3 1 wat&r).
7. Transfer of fish in each plastic container to various concentrations was
randouized.
3. Position of screened compartments was prerandomized (positions presented
in Appendix F).
Transfer to exposure compartments was performed with a small dip net. The
start of the exposure was recorded for each group of fish (1-XVI). The time
required to place fish into the toxicant solutions was less than 3 hrs for
groups I through VIII and less than 1 hr for groups IX through XVI in
Experiment 1 (started April 30). After a 24 hr acclimation to 25*C, the
exposure of groups XV and XVI were initiated on Hay 1.
Mortality rate and body weight for all groups of fish were recorded in
order to reveal possible relationships between body weight and sensitivity
to endrin. These measurements were per Termed more frequently during the
first 24 hrs (8 a.m.. 1 p.m., 4 p.m., and 11 p.m.) and twice daily
thereafter. Dry weights and body water content were also determined on all
dead fish as well as survivors at termination in those fish starved prior to
exposure (IX-XVI). Both live and dead fish from groups I-VIII were frozem
for iuture residue analysis. Fish (groups I-VIII) from concentrations 4, 5,
and 6 (controls) were also sampled (4 to 5 from each group) after .4 and 29
days of exposure to allow the determination of a bioconcent rat ior. factor.
Results of these residue analyses will be presented in a separate report.
STATISTICAL TREATMENT OF DATA
Hedian Survival Times
Median survival tioa (MST) was selected as the endpoint in these endrin
toxicity studies. Frost these data LCSO's can be estimated for any period of
time within the range wh^re 50% mortality occurred, and, although more
uncertain, to periods of time outside this range. For calculation of MST's
and 95a confidence limits a computerized procedure (Hamilton _al_., 1977)
for the Trimmed Spearman-Karber Method of calculating LC50's was used by
substituting dose (concentrations) for survival time. The survival time for
14
-------
each fish was considered to be closest to Che time between when mortality
was observed and the preceding observation, i.e., (tine when the fish was
removed and last time observed alive) divided by 2.
15
-------
SECTION 6
RESULTS
The results of this study are divided into the following subject
areas:
1. Dietary effects on the fathead minncw. '
2. Starvation and its effects on the fathead minnow.
3. Dietary influence on endrin toxicity
4. Influence of starvation on endrin toxicity
5. Correlatians between body water content and susceptibility to endrin
DIETARY EFFECTS ON THE FATHEAD MINNOW (prior to endrin exposure)
During the period lasting from February i to May 20 (100 days)
mortality and food consumption were measured daily for diet groups I through
VII.
In addition, total body weight aad total number of fish were determined
on January 22 and 23 (during randomization of fish groups for diet study),
on February 21 (during transfer from upper to lower tanks) and on April 30
(during start of Exp. 1). Total numbers of fish in each tank (I-VI) were
also determined on Hay 24 (after initiation of the last exposure to endrin,
Exp. 3) and on June 12 (after feeding these groups of fish with frozen brine
shrimp in order to look for possible recovery from visual abnormalities like
lordosis and hemorrhagic kidney regions).
Mortality
Daily recordings of mortalities in the tanks (I-VII) are presented in
Appendix D. Cumulative mortality for 100 days (February 9 through May 20)
is presented in Table 4. It was Sound that the brine shrimp diet produced
the least mortality.
After 100 days, the cumulative mortalities were similar asong the
groups fed the artificial reference research diets (I-VI). No influence of
either total lipid content (0 to 20%) or lipid type (corn oil in Diet II and
salmon oil in Diet III) was observed. The overall tendency, as seen for the
sum of the groups (I-VII), was that mortalities occurred at a very constant
rate throughout the entire pretreataent period. Although not actually
16
-------
TABLE 4. CUMULATIVE MORTALITY OF FATHEAD MINNOWS FED SEVEN
DIFFERENT DIETS FOE 100 DAYS
Days of
Diet no.
feeding
I
II
III
IV V
VI
VII
(I-VII)
0
0*(0)
0 (0)
0 (0)
0 (0) 0 (0)
0 (0)
0 (0)
0 (0)
50
10.5 (23)
8.6 (19)
10.0 (22)
7.3 (16) 13.2 (29)
3.6 (8)
0.5 (1)
4.3 (118)
100
23.6 (52)
28.2 (62)
29.1 (64)
21.8 (48) 24.5 (54)
20.5 (45)
0.9 (2)
21.2 (32?)
* Cugxnulat ive mortality expressed m preccntage of initial number, which was 220 Cish for each
dietary treatment. Number in parenthesis is cumulative mortality.
-------
quantified, size and body weight of dead compared Co live fish seemed to
follow Che same pattern, i.e., dead fish were neither smaller nor larger
thau live fish. Infectious disease as a course of deatn was suspected on a
few occasions, hot no general trends were observed. Therefore, the reasons
for the higher mortalities aooog fathead minnows fed the artificial diets
(I-VI) compared to the brine shrimp diet (VII) is so far not understood;
although, one would expect dietary deficiencies to play a major role. One
might speculate that the reason for this difference does not originate in
the lipid soluble cotapmenCs, aa no real difference in mortality was
observed between group 7, with no added lipid source, and the other groups
(II-VI) fed diets containing corn oil, high in 06 fatty acids, salmon oil,
high in n3 fatty acids, or a 1:1 mixture of then in increasing amounts.
Srowth and Food Consumption
The effectr of diet on growth (Table 5 and Fig, 15 were in agreement
with those on mortality, showing that brine shrimp produce better growth and
survival than the artifical diets.
Experimental data on food consumption (Appendir, D) have not been fully
evaluated. An interpretation of these data oust consider actual number of
fish as well as their body weight. This can be estimated from the data on
total body weight, mortality aad total number of fish in groups I-VII. Data
for caloric content, which varies among the diets, and digestibility are
also of importance in such an evaluation. However, the need for a more
detailed interpretation is not obvious because of the poor growth and
survival among fish fed the artifical diets.
Other Effects of Diet
In addition to the increased mortality and reduced body weight gain,
observed in fathead minnows fed artifical diets (group l-VII), live fish
shoved visual symptoms of dietary deficiencies. These were not observed in
brine shrimp-fed fish (group VII and VIII). Symptoms of deficiency which
were observed in some fish fed artifical diets included swollen bellies,
lordosis, hemorrhages (especially of the kidney region) and erratic
swimming. In severe caBes, cheae fish eventually died. Some fish were,
however, able to survive with these symptoms for .teveral weeks.
After Che last exposures to etidrin were started, the total number of
remaining unexposed fiah in group I-VI and the number of visually abnormal
fish with lordosis and/or hemorrhagic kidney regions were counted. These
fish were then fed frosen brine shrimp once daily, 5 days a week, for 3
weeks, after which they were differentially counted again. The results of
this alteration in diet is shown in Table 6. The good recovery of fish in
group IV (50%) compared to that for the other groups (0-152), and probably
the recoveries in group III (152) as well, was due to a high incidence of
hemorrhagic kidney lesions observed in these groups. The hemorrhagic kidney
lesions were not observed prior to the last "1 weeks of Che feeding period,
when the number of fish affected in this way seemed to increase. At that
time fish with hemorrhagic kidniy lesions were also observed in group III
and V. Only one dead fish with this symptom was observed.
18
-------
TABLE 5. MEAN BODY WEIGHTS AMD MEAN DAILY BODY HEIGHT GAIN FOR FATHEAD MINNOWS FED
SEVEN DIFFERENT DIETS AD LIB
Diet Group
Date
I
II
III
IV
V
VI
VII
01-23-79
.091 (220)a
.098 (220)
.093 (220)
.095 (220)
.091 (220)
.095 (220)
.092 (220)
Feeding with frozen 1
brine shrimp
02-C V79
Feeding with experimental
diets initiated
02-21-79
.239 (218)
.251 (216)
.255 (208)
.269 (217)
.210 (218)
.230 (222)
.340 (213)
Mean daily
.00510
.00528
.00559
.00600
.00410
.00466
.00855
body weight
gainb
04-30-79
.514 (147)
.688 (170)
.708 (160)
.751 (185)
.538 (154)
.536 (168)
1.175 (211)
Mean daily
.00404
.00643
.00666
.00709
.00482
.00450
.01228
body weight
gainc
a Mean body weight (g) and number of live fish in parenthesis.
k Mean body weight (g) at 02-21-79 minus that at 01-23-73 through number of days in between (29).
c Mean body weight (g) at 04-30-79 minus that at 02-21-79 through number of days in between (68).
-------
I-
0.8
f
5
0.6
a. 0.4
0.2
Bftnt KXrliKS c4 lib
S%C.CU 9* 3.0,
rs%c.o ~ ra* &o
tonco ~ to* s,o.
No tol
(fin* at
rulrtelM rairan
0 40 80
Period of feeding, days
Figure 1. Growth in fathead minnows fed 7 different diets. Percentages
shown are for the dry diets. C.O. (Corn Oil) and S.O. (Salmon
Oil). Composition of experimental diets la presented in Table
3.
20
-------
TABLE 6. RECOVERY OF ABNORMAL FaTHEAD MINNOWS WHEN FED FROZEN BRINE
SHRIMP FOR THREE WEEKS
Diet group no.
I
II
III
iv4
V
VI
Initial total no. of fish
5
48
36
66
43
50
Initial no. of abnormal3 fish
1
13
28
13
to
Mortalities''
0
0
1
3
2
2
Final total no. of fish
5
41}
35
63
41
46
Discrepancy
0
0
0
0
0
-2
Final no. of abnormal fish
1
2
10
10
10
6
Recovery
0/1
0/2
2/13
15/28
1/43
0/10
Recovery (X)
0
0
15
50
2
0C
a Visually abnormal fish. This number includes fish with lordosis
and/or hemtaorrages.
k Occurred vithin Che first week of feeding with brine shrimp.
c It is assumed that two of the abnormal fish were eaten by the
remaining fish.
21
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The h**aorrhagic kidney las ions could have been caused by some produce
produced during storage of the dry diets, as no dose-dependent relationship
with any of the ingredients existed. The only possible connection between a
dietary ingredient and the hemorrhagic kidney lesions is the salmon oil,
One possible explanation is, that some of its ingredients, e.g. the
polyunsaturated fatty acida, had deteriorated and thus produced nephrotoxic
components.
In contrast to the recovery of the hemorrhagic kidney lesions, no
recovery from lordosis was observed. Lordosis occurred in all groups fed
the artifical diets (I-VI), but not in group VII and only to a minor extent
in group VIII, which were both fed frozen brine shrimp.
STARVATION AND ITS EFFECTS ON THE FATHEAD MINNOW (prior to endrin exposure)
When starved at 25*C adult fathead minnows survived for about 40 days
with less Chan 1QZ mortality (Fig. 2). After 40 days, mortality due to
starvation increased gradually and 50% mortality wes reached at about 100
days. The survival time for adult fathead minnows, when starved at 25*C,
ranged from 12 days (1 fish in group X) to >126 days (1 surviving control in
group IX). For juvenile fish (33 days post-hatch; average body weight 0.095
g) starvation resulted in 50Z mortality after 17.5 days (23,0-24.5'C) ao
seen in Fig. 3.
Independent of survival time, fish killed by starvation had a body
water content (post-mortem determination) of 86.8+2.2% (Mean ~_ S.D. for fish
in group IX which were killed by starvation over a 32-95 day period) (see
Appendix E). This can be compared with the water content of surviving
control fish from group IX-XVI presented in Table 7. Surviving fish were
killed by i lasers ion in ice-water for 1-2 min prior to determination of total
body weight and water content. From the data presented in Table 7 it ia
evident that fish killed by starvation had a higher water content than
surviving fish. Furthermore, busc:d on the total body water content of
surviving fish starved for periods between 30 and 126 days and that of the
pond-fish trapped just prior to testing on April 30 (Table 7), it can be
concluded that the pond-fish after 30 days starvation at. 25*C, had a
nutritional status similar to laboratory reared fish starved for about 67 to
98 days (80 days for simplicity). A subtraction of the 30 days of
starvation, when these fish served as controls, then suggest that these fish
(group XVI) had a nutritional status similar to fish starved for 50 days (80
minus 30) at 25*C.
DIETARY INFLUENCE ON ENDRIN TOXICITY
As mentioned above diet per ie was shown to affect the fathead minnow
in terms of growth, survival and gross pathology. Only healt'.iy looking fish
were taken for bioassays vith endrin. As shown in Table 8 their mean body
weights (determined post-ourtern) differed slightly among the various groups.
However, there was no detectable relationship between body weight of fathead
minnows and susceptibility to acute endrin toxicity. Furthermore, it was
22
-------
I20-
t3
1
0 10-
1
h
20 40 60
Period of starvation, days
-p"
@0
100
Figure 2. Mortality of adult fathead mtmiows starved at 25.0 C.
syobola in the figure are for group U, (• •)i X (
mi XL (A A).
23
-------
MST, 175 days
100
50
10 20
Period of starvation, days
Figure 3. Mortality of juvenile fathead minnows starved at 23.0-24.5 C.
*Tha expariaent was terminated after 23 days. Age and weight at
day 0 was 33 days and .095 g, respectively.
24
-------
TABLE 7. WATER CONTENT (Z, v.w/w.w.) OF SURVIVING AND STARVATION-
KILLED FATHEAD MINNOWS STARVED FOR VARIOUS PERIODS AT 25*C
Group of fish® IX X XI XII XIII XIV XV XVI
Period of starvation 126 98 67 50 30 92 30 30
(days)
Hater content of 82.6 (I) 82.9 (5) 81.9 (7) 79.6 (7) 74.0 C10) 83.4 (7) 78.9 (10) 82.1 (7)
surviving fish + 2.8 ~ 3.3 + 4.7 + 3.1 + 2.7 _~ 3.1 + 1.9
CZ)b ~ _ _
Water content of 88.8 (2) 87.0 CD - (0) - CO) - (0) - (0) - CO) 87.6 (2)
dead fish + 0.8 ~ 2.5
C%)b
a Group 1X-XIII originated from '"he laboratory stock, and were fed frozen brine shrimp at
25*C prior to starvation. Groi.r XIV and XV originated from a pond and were fed trout
starter at 5-iO*C in the laboratory during the winter for 5 ana 7 months respectively
prior to starvation. Croup XVI were fathead minnows trapped in the same pond just prior
to starvation.
b Results are expressed in (Z) (= g water/100 g on a wet weight basis) and presented as
Hean + S.D. with number of fish Cn) in parenthesis.
-------
TABLE i. BOD* WEIGHTS* OF FATHEAD MINNOWS FRE-FED WITH EIGHT DIETS AND
USED IS TOXICITY TESTS WITH EKDRIN
Group of fish
I
II III
IV
V
VI
VII
VIII
Experiment 1.
(April 30-Mny 7)
Concentration
1
.676 (10)
~ ,321
.787 (9) .739 (10)
*_ .244 + .404
.764 (10)
+ .274
.558 (10)
t -244
.694 (10)
* .287
1.502 (10)
1 -137
.623 (10)
~ .2r*»
Concentrat ion
2
.570 (10)
1 «140
.795 (10) ,610 (10)
+ .311 ~_ .135
.587 (10)
± -2Z»2
.471 (10)
1 -147
.428 (10)
1 .103
.928 (9)
<_ .282
.334 U0)
1 -322
Concentration
3
.588 (10)
+ .137
,556 (10) .759 (10)
^ .159 _+ . 146
.700 (10)
+ ,183
.596 (10)
1 -263
.466 CIO)
~_ .130
1.273 (10)
1 *374
.520 (10)
~ -152
Experiment 2.
(May 14-May 30)
Concentration
I
.540 (10)
± *222
.816 (10) .869 (10)
£ .314 ~_ .425
.982 (10)
+ .383
.887 (10)
+ .245
.544 (10)
1 179
1.929 (10)
+ .672
.315 (10)
1 -122
Concentration
2
.614 (10)
+ .281
.840 (10) .764 (10)
+ .141 +_ .427
1.187 (10)
+ .340
.697 (9)
1 *236
.616 (10)
1 *209
1.607 (10)
1 "AS5
.330 (10)
1 *181
Concentration
3
.550 (9)
1 *328
.746 (8) .720 (U)
+ .284 + .218
.859 (9)
I .175
.539 (11)
1 *18B
.465 (8)
1 .156
1.670 (9)
1 -681
.350 (9)
± .223
* Body weights (g) expressed as Mean +_ S.D. with number of fish (n) in parenthsis. Body weights
determined on fish killed either by endrin during tests or by immersion in ic3-water at termination.
-------
not possible to visually decermine Che dietary treatment of individual fish,
sb variations in size and shape was as great within the groups a* between
them. Consequently, the observed differences in susceptibility to endrin
among the dietary groups originated from invisible, internal, biochemical
and physiological differences. These differences were in turn created by
the diets fed to the fish.
Before examining the differences or similarities in susceptibility to
endrin among the groups of fish (I-VIII), it is important to know how exact
and reproducible the method for determination of toxicity is. This can be
done bv examining the results obtained for group VIII, the group fed a
restricted ration of frozen brine shrimp.
From the results presented in Table 9 and Fig. 4, it can be seen that
the relatively small discrepancies obtained can be attributed to the slight
differences in exposure starting times. When different groups of fish from
the same period (May 8-11) are exposed to the same -ndrin concentration, but
in different exposure chambers, then the differences in MST are small «2.2£
maximal difference for the mean values).
From the daM presented in Table 10, it is evident that fish fed the
diet with no ado lipids (group I) was most susceptible, and that fish fed
frozen brine shrimp ad lib (group VII) was most resistant. Looking at
Figure 5 and comparing the 96-hr LCSGs of .£85 and 1.15 vg/1 interpolated
from it, the maximal variation in endrin toxicity du-s to diet becomes 2.4
times (1.15/.4B5), Within the periods of exposure examined (<384 hrs or 16
days), no incipient lethal threshold was established.
Comparing the susceptibility to endrin for fish fed various percentages ,
or composition of lipid in their diet, the only consistent difference wa3
found between fish fed no added lipid (group X) and the ether groups
(II-VI). Consequently, a variation in dietary lipid percentage between 10%
(groups II, III and IV) and 20Z ou a dry weight basis (group VI) seems to be
of minor importance in terms of susceptibility to endrin. Furthermore,
there was no difference in susceptibility to endrin among fish fed a diet
containing corn cil, which is high in 06 fatty acids (group II), and fish
fed a diet containing salmon oil, vrtiich is high in 03 fatty acids (group
III).
From the data presented in Table 10 and Figure 4, the influence of
feeding a restricted ration of brine shrimp (group VIII) and a high ration
level, essentially ad lib (group VII) can be examined. On April 30, after
80 days of feeding, group VIII had 733 fish with a total body weight of
204,5 g (average body weight .279 g). They were fed a daily ration of 40.0
g frozen brine shrimp (4.8 g dry weight) for the entire period (80 dp.ya)
prior to April 30. Based on an average initial body weight of .095 g/fish
(determined from groups I-VIl, Table 4) and an assumed initial number of 745
(based on the final number, 733, and mortality data for group VII, Table 4),
an average total body weight during the feeding period for group VIII can be
calculated as (204.5-745 x ,095)/2 « 66.36 g. The average daily ration fad
to group VIII then becomes 40.0 x 100/66.86 » 59.8 g/100 g body weight on a
wet weight/wet weight basis or 7.18 g/100 g body weight on a dry weight/wet
27
-------
TABU 9, TOXICITY OF EMKIN TO FATHEAD MINNOWS FED A LOW RATION OF BRINE
SHRIW5 PRIOR TO TOXICITY TESTS, WHEN TESTED AT VARIOUS PERIODS AM) IN
DIFFERENT EXPOSURE CHAMBERS
Period of
Cone. 1
MST
Cone. 2
KST
Cone. 3
KST
oxposuro
tug/1)
(hrsl
(ug/l)
(hrsl
(yg/l)
-------
K3Q0
500
iOO
50-
10-
OJ
0.5
5
EmJrln, jig/I
Figure 4, Toxicity of endrln Co fathead minnows fed either a high or a low
ration of frozen brine shrimp prior to exposure. Results are
shown as mean values and 951 confidence limits. Symbols are as
follows;
Experiment no.: 1 2 3 4
Diet group VII; A A
Diet group VIII: O a 0 a
29
-------
TABLE 10. TOXICITY OF EJfflRIN TO FATHEAD MINNOWS FED EIGHT DIETS FDR 80-100
DAYS PRIOR TO TOXICANT EXPOSURE
DIM-
Co we. I
tm
Cone. 2
m
&a*»c. *
HIT
(TO®
Cug/t)
(hr»)
ibr%)
(Hr»J
«»*
1.30s
t«0ft-t.5t
23.0*
17.1-30.9
-63
.49-,77
76*8
40,3-146,0
-
h
I.2S
1.22-1*33
».2
16.3-Z2.4
,67
.37-.76
33,4
46.4~66.2
.39
.29-.4«
138.9
112.4-171.7
>Sm
1.H
U39-I.76
no
4.7-10.4
.43
'3,3
6.3-16.3
-
•
i.fift
I.XM.7S
6,3
JO
•73-«06
46,8
3&. 0-36*6
-
"l
1*26
•97-f.M
92.2
7**3-100*2
*63
.44-.T?
>170
.
-
1.2a
1.2>I*3*
35,1
26.0-41,4
.74
•34-.93
122*2
101.3*147.3
.33-.M
s%
in,
1.39
1.09-1,31
54,7
43*4-49*0
.63
#46*,77
ISA
-
-
itu
1.23
1.2>i,34
43.6
37.0-51J
.71
.32-.33
61.6
(9.H9.3
.39
.,W.<6
24A.3
201.3-301.9
¦f,
1*30
1.SS-U3I
SS.9
43.0-68*3
.63
*44-*77
*170
-
•
!.4J
t.OJ-l.it
28.9
29.3-40.7
.74
J3-.0
131.5
f 10«CMf?*3
.39
.J1-.4#
jB4.fi
327-3-431.7
«i
1.36
1.32*!.«
33.3
43.2-46.2
.63
*4S-»77
2*3
i
I
H
1.34
33,6
.74
ia&,3
*39
291.3
1.43-1.53
49.6-39*3
.33**93
52*9-122*2
«Jil-«47
169.6-339.6
U3Q
74»S
*63
_
-
.
1.09-1.)1
67.6-S2.2
.44-.T7
M7Q
*
-
*i2
1*33
43,3
.73
150.6
.39
309.2
l«O»-l,0T
37.4*34*9
,54-,«
110.5-134,4
-31-.47
220,3-433.9
t.as
137.0
¦63
>170
-
*
t8T-l,|S
113.2-163.6
.4ft-. 77
•
-
-
1.34
76.4
.74
145L7
.39
m
1*16-1.33
S6.0-#£*2
*3*-.?3
130*4-134,1
*31—*47
-
VIII,
1*37
34,5
.63
9i*6
.
-
1.34-1.39
30.3-39.1
.44-.T7
94*9-109,6
-
•
mij
1.29
23.6
«7i
7?.6
•35
(7B
103-i.JS
19.4-29,2
•32-. 90
60.7-64.5
.30~.46
-
fiiij
1,41
21.6
.79
*1.1
.
-
17.4-26.6
.7S-.63
33.6-30*0
-
-
* Sy*»i» Ksiainaa In urn at trttmmiwmit aau msamtrm in T«si« I.
" DawilT* «muM ct ma ana fit ceattOMe. it.it.
c Ma eoatlmaea ll.IT, M 13 mrnilur IJOfl.
30
-------
1000-
500-
«•
100'
50-
10-
0J
OS
5
10
Endrlrt, pq/1
figure 5. Toxicity of eodrln to fathead minnows fed either s loir-fat diet
or frozen brine shricp prior to exposure. Results are shown as
mean values and 95% confidence lisits, Symbols are as follows;
Experiment no.: 1 2
Diet group I: O •
Diet group VII: A A
31
-------
weight: basis. Group VII, vhich was fed essentially a.d lib, constituted
initially 220 fish with a total body weight of 20.24 g (average body weight
0.092 g). On April 30 there was 211 fish left with a total body weight of
248.0 g (average body weight 1.175 g). The average total body weight for
group VII then becomes (248.0-20.24)/2 ¦ 113.9 g. From the daily recordings
on food consumption, a mean daily ration of 18.57 g dry weight or 71.45 g
wet weight can be determined (for further details see Appendix D). The
average daily ration fed to group VII then becomes 62.7 g/100 g body weight
on a wet weight/wet weight basis or 7.52 g/100 g fish on a dry weight/wet
weight basis. What this means is not that the fish were fed a similar
ration (59.8 and 62.7 g/100 g body weight respectively) but just that the
food conversion efficiency was sinilar in both groups. When the daily
ration is instead expressed in grams of food per fish independent of body
weight, chen it can be calculated that fish in group VII were given a 6.15
times higher daily ration than fish in group VIII (71.45/214.5 : 40.0/739 ¦
6.15). Tnis difference caused apparent differences in susceptibility to
endrin as seen in Figure 4, from which 96-hr LC50a of 1.15 and .58 can be
interpolated. The difference in susceptibility is 1.98 times. Thus, a low
ration of food can create the same order of increase in susceptibility to
endrin as a low-fat diet compared to a brine shrimp diet.
INFLUENCE OF STARVATION ON ENDRIN TOXICITY
From the results presented in Table 11 and Figure 6 it is quiie clear
that prolonged starvation made the fish more susceptible to endrin.
Furthermore, it was quite clear that the fish trapped in the pond just prior
to toxicity tests (April 30; group XVI), were anong the most susceptible
groups In fact they were as susceptible to endrin as the fish starved for
95 days prior to toxicity tests (group IX).
From the linear regression line for MST as a function of period of
starvation prior to testing (Fig. 6), an equivalent period of starvation
(25*C) for the pond-minnows, which had livsd in their natural environment
during the winter, was determined based on their susceptibility to endrin.
These fish behaved as if they were starved at 25*C for 75 days
(concentration 1), 95 days (concentration 2) or 96 days (concentration 3).
This estimated starvation period was compared to the one independently
estimated from total body water content (50 days). The discrepancy between
these estimates (50-96 days) becomes easier to understand by looking at
Figure 2 and Figure 7. During periods of starvation exceeding 50 days,
mortality due to starvation complicates the correlation between water
content and period of starvation. Because of this, the increase in water
content of surviving fish becomes less as the upper limit is approached.
Therefore, an estimate of the prehistory of feeding for fathead minnows with
a total body water content greater than about 81Z was less precise than
below 81Z. This might in part explain the discrepancy between the estimate
of 50 days based on water content and the estimate of 75-96 days based on
suscc -c'.bility to endrin. These results showed that fathead minnows at this
time c. the year (April 30) are starved and that they behaved like starved
fish i:. their response to endrin.
32
-------
TABLE II. TOXICITY OF ENGRIN TO AOULT FATHEAD MINNOWS STARVED TOR 1-97 DAYS
AT ?3*C PftlCR TO TOXICANT EXPOSURE
Srom <#
tec, i
»«r
Cane. 2
«T
Coac* 3
»ST
Ca*c. 4
mr
~ IM5
(BffU
H»1»
luO/ll
Cirti
(U&/M
i*i 1
<*•
i.»- i.a
.¦.t"
0*19.0
,U
9.1
ft.»-!«•?
,35
91,9
.19
• 14-.23
51.4
4«.>-l73LI
I
I.J7
l.M-I.Ji
t.4
J.I-I4.I
•m
.S4-.7S
a.»
22.2-47.A
*33
,2fc-,4*
&2.1
40.H7.4
.19
.it-.29
379.7
3i.l-4642.ft
XI
1.5J
15.4
I3.7-2U
¦61
.J4-.7J
*9.5
•3§
.I**.!?
176.3
lSI.^Uft.9
.19
.I4-.2S
409,1
222.3-4^*7.*
III
I.J?
I.JS-IJI
U.2
II.I-2J.*
.M
,49-.«
17.0
4f.d~i44.4
#33
«25*.4l
223.Q
I34.fr»3i2.7
.19
.14-.23
*720
XIII
I.J7
3.7
2B.J-J5.J
.49-.*1
at
il«fr4?4
.34
.2fr-.4i
229.J
rit*»»27«,l
.19
•14-.J3
501
XI*
1.37
1.J6-I.A
10.1
J.9-14.7
.41
Z7.S
ZS.*-37.a
.33
.2*-.44
iOf.I
13.3- 142.4
,19
.I4»«53
307
»l
t.JT
1.»I.J7
T.I
<.»ZU)
.M
.9KI)
6?.i
J7
197.3
.20
.I4-.2J
4«2
i.a
I.2J-I.1J
3.3
49
.W-.70
41.4
4C.o-ua
i
.
•
-
>.«!
i.is-i.m
t*.2
IJ.»-Z3U
•J»
.74-.U
*7.1
39.3-54.1
•
-
.
-
MI,
t.J?
1.JJ-1.JT
1.2
J.3-IJJ
M
IS.9
to. w uo
*32
«.0
33*2-Mu3
.20
¦!4»*2S
*720
(.a
1.22-1.iS
1.2
i.o-mu
**l-.§7
13*9
13.4-1*^
-
.
•
*
"'3.
I.*l
1.3V-1.U
13.*
»I0
.7S-.I3
Ji.i
23.7-M.I
-
-
-
K¥(j»
l.sj
io.;
$.1-14.4
.W
•77-.4J
13.0
5.&-I7.5
-
* HMIM tmlxl TIM IHTSJ far Cane. J M* m* ogmKei wm »720 me 111.i l«J,S-ioa.2l, cmotetlymit In
>M IX.
* ftMult* mermmm m *mm ma »Jf ca»H»«t» Haiti,
s for iMora«?tca m u/a at llu *M parte* of inrMflM, w* Tmi* t.
33
-------
400
300-
conc. 4
w
S
200-
100-
cone.
0
50
100
150
Period of starvation prior to exposure, days
Figure 6. Endrln toxicity to fathead minnows starved for X to 97 days prior
to exposure.
Data for linear regression analysis (MST's for group IX-XIV) are
presented in Table 11, Endrin concentrations (ug/1) are ranges
for corresponding nean values. Correlation coefficient (r),
slope (aO t y-intercept (a0), standard deviation (s) and number
of data points were as falloys.
Cmc, a#.
u| ladrla/1
I
1.3J
z
o.m-o.«s
J
9JM.U
J
o.iJ-o.ia
r
-0.9M
-a.ss*
-O.SH
-0.1U
-O.JiO
-J.OM
-MM
*a
. U.J
14.4
la.i
)*3.4
*
2.A
14,*
10, J
tO.4
a
«
«
i
i
34
-------
85
*
j. 80
€>
75
o
S
70
65
T
T
r
0 50 100 150
Period of starvation, days
Figure 7, Effect of starvation on total body water in fathead minnows.
Each point is a mean of the nusfaer of animals listed by each
point and ther vertical line through each point ia the standard
deviation,
35
-------
When tt»e recently trapped pond-fish (group XVI]i) were starved in the
laboratory at 25 *C for 14 to 23 day* prior to exposure (group XVI2 and
XVIjk respectively) their ousceptibility to endrin was not altered ouch
(Table 11), Thus, it seemed that the response to endrin after acclimation
from a teaperature of about 5*C in the pond to the test temperature of 25"C
was at least not influenced much by periods of accliaation between 2 and 23
dayi prior to exposure to endrin. When fish acclimated and starved for 14
days at 25* C (group X?^) were either starved (group XVIjl) or fed
with frozen brine shrimp (group XVl3fl) essentially ad lib (7,23 g/100 g
fioh on a dry weight/wet weight basis or 98.2 g/100 g fish on a wet
weight/wet weight basis per day during one week) and tested simultaneously,
an increased resistance in fed compared to starved pond fish was observed
(Table 11). A similar positive response to feeding with frozen brine shritsp
for one week prior to testing was also observed in fish previously fed a low
fat diet (group and in Table 10).
CORRELATION BETWEEN WATER CONTENT AND SUSCEPTIBILITY TO ENDRIN
In several studies it was shown that the tissue water content of fish
was inversely proportional to the fat or lipid content (Love, 1970).
Although such a fat-water relationship was not actually shown for the
fathead minnow, there is no reason to believe that this relationship should
not exist. In the present study the total body water content, expressed as
percent on a wet weight basis, was used as a simplified, indirect measure-
sent of the relative total body lipid content. Thus, if a negative
correlation between survival tine (resistance) and percent water was found,
then a positive correlation between survival time and percent lipid was
highly probable. Such a correlation would mean that a fat fish can
withstand longer periods of exposure or higher coneantrations of a
lipophilic chemical than a lean fish.
Total body water content for the diet-treated groups (I-VIII) are
presented in Table 12. Data for total body water in preacarved groups (IX-
XVI) were also determined (Appendix E). Mean value; for intoxicated fish in
the three highest concentrations of endrin were calculated. In order to see
if there was a correlation between total body inter and susceptibility to
endrin, mean values for total body water content were plotted against median
survival times for group I to XVI in the three highest toxicant concentra-
tions, and linear regression analysis was applied to these three sets of
data. As seen in Figure 8, a negative correlation existed for all three
concentrations. Considering the fat-water relationship (negative
correlation) this should support the hypothesis of a protective effect
against endrin toxicity by an increased lipid content.
If this relationship is true, then survival time for individual fishes
in a certain concentration of endrin should be positively correlated r.o
their respective lipid content. Again considering the fat-water relation-
ship, survival time should then be negatively correlated to water content.
The latter wis the case (Fig. 9) and represented a negative correlation
between total body water content and survival time for individual fishes
(group IX-XIII) exposed to the three highest concentrations of endrin.
36
-------
TABLE 12. TOTAL BODY WATER CONTENT IN FATHEAD MINNOWS
FED EIGHT DIETS FOR THRES MONTHS
Diet no.
I II
III IV V
VI
VII
VIII
78,8* 73,3
~ 2.8 2.1
73.5 70.1 73.1
~ 1.5 + 2.3 + 2.6
71.6
* 2«6
68.4
1 2-9
73.3
~ 2.5
* Mean +_ S.D. for eoeai body water £2 on w.w. basis). Body water determined
on a random sample of S fish for each group.
37
-------
300-
200
CQfiC, 2
100-
70
75 80
Maori total body water, %
85
Figure 8, Correlation between Median Survival Tisa, MST, ia 3 concentrations
of endrin and mean total body water concent for fathead minnows.
Data for linear regression analysis were obtained from group I.-
VIII. and I2-VIII_ (Table 10) and group IX-XVI, 50^ and XV ^
(Table 11). Endrin concentrations (yg/1) are ranges for
corresponding oean values. Correlation coefficient (r), slcpe
(a-), y-lntereept (a ), standard deviation (s) and number of
data points were as Sallows,
Cone. no.
pg Endrin/1
1
1.26-1.43
2
0.63-0.75
3
0.32-0.39
r
-0.763
-0.769
-0.892
al
-5.10
-8.94
-17.38
ao
424
774
1560
a
20.0
34.J
48.6
n
24
20
16
38
-------
500
400
- 300
2
»
=5
200
o 100
>
>
cone
COflC.
Total body water, %
Figure 9. Correlation between survival time in 3 concentrations of endrin
and total body water content for individual fish.
Data for linear regression analysis were obtained from group
IX-XIII and for conc. 3 also from group XIV-XVX. Endrin concen-
trations (jig/1) are ranges for corresponding mean values (Table
11), Correlation coefficient (r), slope (a^), y-Intercept (a0),
standard deviation (s) and number of data points were as follows.
Coae, no. 12 3 3—•—
il Eadrla/X 1,37 0.65-0,68 0.33-0.36 0,32-0.37
r -0,340 -0.758 -0.740 -0.332
^ -3.05 -14.M -2ft.69 -24.
•0 270 U&3 2384 22U
< 42.3 S3.9 109.3
a 37 34 34 29
Group* of lUh 3-1XXI 3-XHI Dt-mi X£V-m
39
-------
EFFECTS OF ENDRIN ON TOTAL BODY WATER CONTENT
The ur of total body water content, when determined post-mortem on
intoxicated fishes, in support of the hypothesis that the fat protected the
fish from acute intoxication with endrin, assumed that endrin had no or only
limited physiological effects on the total body water content. There were,
however, suae indications in the results presented above were against the
validity of such an assumption.
Examining the data in Figure 8 more carefully, it seemed the higher the
concentration of endrin was, the lower the mean total body water was. At
time aero (i.e., before start of exposure to endrin) the total body water
content should be the same independent of toxicant concentration. Total
body water content at time zero, estimated from the data in Figure 8, was
83.0, 86.5, and 89,71 at concentrations 1, 2, and 3, respectively. This
problem deserves further attention, especially since these data indicated a
dose-response relationship.
It was shown in a separate experiment that endrin actually affected the
total body water content of fathead minnows. In this experiment the body
weights of seven fish were determined before, during and after lethal
exposure to two acutely lethal concentrations of endrin (concentration 1 and
2). From the results of this small-scale experiment, presented in Table 13,
endrin was shown to affect the body weight of fathead minnows. Variations
in body wight during exposure to endrin must be caused mainly by the
passage of water between the fish and the environment. Due to the small
number of fiah that were used and to the fact that only the two highest
concentrations were tested, a more thorough interpretation of these results
was not possible. However, a dose-dependent effect of endrin on total body
water was indicated, in which the lower of the two concentrations (concen-
tration 2) produced a greater increase in body water content than the higher
one (concentration 1). Support in favor of this idea was that when the fish
were carefully blotted dry after exposure (i.e., according to the procedure
which was used fur post-mortem determination of body weight in the present
study), then the increase in body weight was significantly higher in fish
exposed to the lower concentration (concentration 2) compared to the higher
one (concentration 1). Further support for the idea that endrin produced a
dose-dependent effect on total body water in the fathead minnow is presented
in Figures 10, 11 and 12. These data on total body water content also
showed that endrin caused a concentration-dependent decrease in total bod"
water content. Thus, in Figure 8 the discrepancy in total body water
content at time zero between fish exposed to three different concentrations
of endrin, can be explained as a physiological effect of endrin on the
osmoregulatory process.
Such an effect of endrin does, however, not rule out the support for
the hypothesis of a protective effect of the body lipida on intoxication by
endrin. On the contrary the results suggested that in spite of the lowering
effect of endrin on total body water a high initial water content, which was
indicative of a low lipid content, made the fish more susceptible to
endrin.
40
-------
TABLE 13. EFFECTS OF ENDRIN ON TOTAL BODY WEIGHT
Period in endrin solution (hrs)
Fish no.
0 24 48 48
B,W.a(g> B.W. (g) &XZ) B.W. (g) ACX) B.W. (g) A(2)
48
b2o (%)
lb
3.59
4.21
+ 17.3
4,13
+15.0
3.89
+8.3
84.3
2
1.37
2.24
+ 19.8
2.14
+14.4
1.95
+4.3
84.6
3
1.17
1.40
+19.7
1.33
~13.7
1.18
+0.9
83.9
Haan
S.D.
18.9
t
14.4
+ 0.7
4.5
+ 3.7
84.3
+ 0.4
4
3.84
3.60
- 6.3
4.58
+19.8
4.46
+16.1
82.1
5
2.57
2.46
- 4.3
2.95
+14.8
2.92
+13.6
81.2
6
3.67
3,53
- 3.8
4.22
+15.0
4.17
+13,6
81.3
7
1.30
1.32
~ 1.5
1.48
+13.8
1.44
+ 10.5
81.3
Mean +_
S.D.
15.9
+ 2.7
+13.5
+ 2.2
81.5
+0.4
a Body weight measured after blotting the fiah dry on paper towels.
k Fiah No. 1-3 were exposed to 1.7 ug endrin/1 and Fish No. 4-7 to 0,9 ug
endrin/l.
41
-------
TOTAL BOGY MTEfl. %
SO-
05
80
75
ENWM COKC NO' S 5 4 3 2 1 65432
GROUP OF FiSM. an
PERWO OF STARVATION.day* I
zu
21
I x 5
3
JL
6 5 4 3 2
z
69
6 5 4 3 2 1
s
97
Figure 10, Total body racer content of intoxicated and surviving laboratory
brood stock fish starved for 1-97 days before exposure to endrin.
Open staples denote intoxicated fish. Shaded staples denote
surviving fish after 30 days. Bars denote standard deviation
with number of fish (n) above. Average endjin concentrations
(vig/1) during exposure were as follows: conc. 1(1.27), 2(0.66),
3(0.35), 4(0.19), 5(0.11), 6(0, control). More detailed
information is presented in Table 11.
42
-------
TOTAL BODY WATER. %
90-
85
80
75-
SOWi C3NC. NQ; 65432! 654321
GROUP OF FISH: IE X2,
WJ330 OF STARVATION,
-------
TOTAL BODY WATER, %
SO-
BS
80
75
ENDRIN CONC NO 6 5
GROUP OF FISH
to
10
2 I
2 I
2B4
of
1
2 I
221,
figure 12. Total body water content of Intoxicated and surviving pond fish
trapped between 2 and 21 days before exposure to endria.
Symbols as in Fig, 10,
Endrin concentrations (ug/1) are presented in Table 11.
Concentration no, 1 was highest concentration and no. 6 the
control.
44
-------
The lowering effect at endrin on total body water in fish has not been
reported previously. This effect should be considered during interpretation
of experimental data dealing with alterations m blood as well as tissue
constituents (when expressed on a wet weight basis) caused by endrin.
45
-------
SECTION 7
DISCUSSION
The aim of the present study was to study interactions between
nutritional factors and the toxicity of a lipophilic chemical to fioh.
Endrin was selected as a model toxicant because its toxicity and fate in
aquatic organises were studied quite extensively, providing a great deal of
basic knowledge oti various toxicological aspects (reviews by Grant, 1976 and
by Schiunel et^ _al_. , 1974).
The results of the present study are discussed from the point of
interactions between nutritional factors and the toxicity of endrin.
Special attention is focused on the role of lipid metabolism, as I believe
that alterations in lipid metabolism and lipid depots are of great
importance in the interpretation of results. 1 have also tried to interpret
the ecological implications of the results, and to relate the findings to
results from previous studies on interactions between ambient conditions,
biological factors, and endrin toxicity to fish.
From the results reviewed in Table G1 (Appendix G) it can be seen that
among various ambient conditions, (1) temperature (Katz and Chadwick, 1961;
Macek t at. 1969), (2) loading density (Katz and Chadwick, 1961), and (3)
the presence of activated charcoal (Brungs ££ al^., 1966) produced
significant effects on endrin toxicity to fish. Based on mean values (no
confidence limits reported), interactions producing a 2 times or greater
variation in acute toxicity (LC50) have also been found for (4) chemical
formulation of endrin (Henderson £it al. 1959), 2nd for (5) static compared
to dynamic test conditions (Lincer e£ al., 1970.
Among those ambient conditions, which were previously shown to affect
endrin toxicity to fish, only those related to renewal of toxicant solution,
(2) and (5) above, need to be considered during an interpretation of the
results obtained in the present study. Such factors were et least partly
responsible for the slightly lower survival times, which vere obtained after
increasing the flow rate between the first and second series of fathead
endrin exposures (Fig. 4 and 5, Table 10). Minor variations in water
temperature (Appendix A) and water characteristics (Appendix B) were not
considered an important variable in the present study.
Among the ambient conditions studied previously (Table G1, Appendix G),
water temperature is probably the one of greatest ecological importance for
endrin toxicity to fish. Based on the laboratory toxicity data reported by
Katz and Chadwick (1961), a 25-fold increase in acute toxicity (96-hr LC50
for bluegill) was found with an increase in water temperature from 3 to
25*C.
46
-------
Besides the influence of ambient conditions on endrin toxicity to fish,
the influence of certain biological factors have been shown. Among these
biological factors, species, population and size as well as developmental
stage have been shown to influence endrin toxicity to fish. To examine
these resulcs in more detail is beyond the scope of the present report.
However, it can not be excluded that lipid content may be an underlying
reason for significant variations in endrin toxicity due to species,
population, size and developmental stage.
In addition to ambient conditions and biological factors, the period of
exposure is certainly of utmost importance for the toxicity of a bioaccua-
ulating chemical like endrin. Reported bioconcentration factors for endrin
in fioh range between 1,640 (Argyle £t _al., 1973) and 15,000 (Heroanutz,
1978). Inspite of the high bioconcentrat ion of endrin in fish, the
variations between acute toxicity (96-hr LC50) and chronic toxicity (KATC)
are surprisingly small, as seen from reported application factor values of
0.46 for fathead minnows (Jarvinen and Tyo, 1978) and 0.30 for flagfish
(Hermanutz, 1976).
In the present study three nutritional factors (1) dietary composition,
(2) dietary ration level, and (3) starvation, were each shewn to influence
the acutfe toxicity of endrin by about 2 times. It is hard to imagine, in a
more natural or even in a laboratory situation, how these three nutritional
factors could have a simultaneous influence, ana thus, produce a combined
effect of 8 times (2x2x2) higher susceptibility. A more accurate way to
interpret these nutritional interactions is probably to regard them as three
separate ways to influence one cotanon factor important for resistance; to
endrin. This common factor was indicated to be the total body lipid
content. Concomitant with a decreased lipid content, there was generally an
increased water content in fish (Love, 1970). In the present study an
increased total body water content was shown to decrease the resistance to
endrin (Fig. 9). This correlation between total body water content was
found independent of pre-treatment (Fig. 8).
One ecological implication of the present findings was that laboratory
acute toxicity data, achieved in tests on well-fed fish, mi^ht underestimate
the toxicity of lipophilic chemicals to natural populations of less well-
fed fish. The magnitude of this underestimation might be at least 2-fold,
when based on 96-hr LC50 values. For a more unbiased and correct
interpretation of this finding, parallel toxicity tests with laboratory
stock fish and field-collected specimens should be undertaken. Moreover,
the influence of temperature must also be considered as temperature
interacts strongly with the acute toxicity of lipophilic chemicals like
endrin.
Three main factors determine the dose of a lipophilic chemical
dissolved in water to fish. These three main factors are (1) concentration,
(2) time of exposure, and (3) temperature. The toxic effect of a dooe is to
a great extent determined by its distribution within the body on an organ
as well as a cellular and a subcellular basis. As unionized lipophilic
chemicals are transferred over biological membranes by passive processes,
their distribution within the body at steady state is determined by the
47
-------
distribution of Che lipids. Considerations of lipid Metabolism and lipid
distribution Chen become very important for the understanding of the
bioconcentration, bj.omagnif icatica and effects of Lipophilic chemicals in
fish as well ao in other living organisms.
4S
-------
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49
-------
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iller, I#. L. 1971, Histopathologic lesions in cutthroat trout, Salao
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Karber method for estimating oedian lethal concentrations in toxicity
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417 (1978).
50
-------
Henderson, C., Q. U. Pickerlag, and C. M. Tarzvell. 1959. Relative
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(Salvelinua fontiualie). J. Fish. Res. Board Can. 33: 1731-1741.
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tissues. Bull. Environ. Contain. Toxicol. 16(5): 505-507.
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endrin in food and water. Arch. Environ. Contam. Toxicol. 7: 409-421.
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260-263.
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51
-------
Mayer, F. L., P. H. Mehrle, and P. L. Crutcher. 1978. Interactions of
toxaphene and vitamin C in channel catfish. Trans. Am. Fish. Soc.
107(2): 326-333.
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Orsanco. 1977. 0RSANC0 24-hour bioassay. Cincinnati, Ohio. Ohio River
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J. Fish. Res. Board Can. 36: 77 -80.
Schimsel, S. C., P. R. Psrrish, D. J. Hansen, J. M. Patrick Jr., and J.
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52
-------
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Environ. Contao. To*icol. 6(2): 171-176.
53
-------
APPENDIX A. MEASURED FlOW HATES AND TEMPERATURES DURING TOXICITY TESTS WITH ENORIN ON FATHEAD MINNOWS
Flow Ratos {mf/cyclo tins of I oln 43 soc)
Oat*
U
1
I
2
u
L
U
i
L
U
4
L
U
s
L
u
6
I
Consent
April 9
143
10B0b
136
980
131
980
126
930
148
tooo
108
730
v side0
134
990
134
M0
146
1000
132
1030
142
1100
1)2
630
w sldoc
May 7
67Qb
sso
050
960
910
410
v side
1060
800
900
WO
790
770
w si da
4
4
030
v slda
750
vv slda
d
May 6
SIS
800
780
860
870
370
940
740
930
840
80S
765
8 Tank no. 1-6 In dacreaslng ordor for toxicant oancenTratIons. Proportional dilution for l-S was lit.
Tank no. 6 Mas control (no toxicant).
II » upper tanks, L • luwar tanks
b Flows In lower tanks meosurod with flow to upper onas shut-off.
c V sldQ and w side relates to duplicate sides
d Adjustment In tha flow splitting calls porfortaod botwoen measurements. Flow rotes In upper tanks, which
ware drained into the lower anas were Increased by moans of the flow splitting cells.
-------
APPEWJIX A. (Continued — Koter Temperatures
Tank
no.
Data
U
1
L
U
2
L
U
3
L
U
4
L
U
5
t
U
6
L
Commont
Apr11 23
24.9
26.6
24.8
26. B
24.9
27.0
24.7
27,1
25.2
26.9
23,9
26.8
* s 1 do
24. 8
26,7
24,8
26.9
25,1
27,0
24.8
27.1
24,9
27.0
24.0
26.7
vv slits
Hay 1
24.6
26.6
24.7
26.6
24.8
27.1
24.9
27.0
24.9
26.8
24.6
26.5
v Side
24.7
26.6
24.5
26.8
25.1
27.1
24.7
27,1
24.8
27.1
24.4
26.6
w side
Hay 4
24.5
26,7
24.5
26.8
24,8
27,0
24.9
27.0
24.8
26.8
24,4
26.5
v si do
24.4
26.6
24.2
26.a
25,0
26.9
24.fi
27.0
24.8
27.0
24.4
26.8
vv side
Hay 7
24.0
23.7
24.2
24.1
24.4
24.6
24.4"
24.1
24.4
24.2
24.2
24.0
May 17
24.7
24.9
25.2
25,2
25,6
25.6
25.5
25.3
25.3
25.1
25.1
2S.0
24.9
28.1
25.1
24.8
25.4
25.6
25.4
25.3
25.4
23.4
25.0
24.9
* Temperatures always aeasured cioso to the drain. Temperature or Incoming wafer (flow sp! Ittlny ciiambar for
uppor tank dl vldor) was 25.7"C on this occasion ccmparod to 24.4*C for tho outlet. This means that tho
tomporaturo control lor the uppor tanks In alt experiments was approximately within _+ 0.7'C with a moan
tMsperafuro close to 25.1 *C. for tho lowor tanks tha Incroaso of flow rot# to tho upper tanks, which was
compensated for by adjusting tho tonporaturo control In the headbox (water supply), created a drop In
temperature by almost 2*C. Moan and ranoo of toraporatur* for tho lower tanks Is considered to be 26.0 +
1*0. -
-------
OTINQiX B. ALKALINITY, HARDNESS WD pH OF THE DILUTION WATER
TAB'.£ Bl. ALKALINITY, HARDNESS AM> PH OF THE WATER SUPPLY, UNTREATED
LAKE SUPERIOR HATER
Data
Alksi1nlty
(mg/l as CaC03)
Hardness
(mg/l as CaCO-j)
pH
Experiment
January 5
41
45
7.5
Feeding end starvation
January 13
41
45.5
7.3
January 22
42,5
46.5
7.3
January 30
42
46
7.3
February 8
42.5
46.3
7.4
February 15
42.5
46
7.3
February 22
42
47
7.3
March 1
42.5
46.5
7.3
March 8
44
46.5
7.2
March 18
43.5
49.5
7.4
March 23
44
46.5
7,2
April 1
42.5
45.5
7.3
April 7
42
44.5
7.3
April 11
41.5
44
7.2
April 21
39.5
44.5
7.3
April 28
40
45.5
7.3
May 7
40
45.5
7.3
Toxicity test
May 14
41
45
7.4
May 21
39.5
44
7.5
June 1
40
43.5
7.5
June 7
41.5
44
7.6
Termination
56
-------
i TABLE B2. KMER CHARACTERISTICS IN DIFFERENT EXPOSURE CHAMBERS
DUR1N3 BIOASSAY
Chambur No.
1
2
3
4
5
6
V
vv
V
vv
V
vv
V
VV
V
w
V
vv
•
Acidity
1.943
1.6665
1.300
1.943
1.665
1.665
2.220
1.368
1.665
1.665
1.943
1.665
AlKol 1 iilty,
42.4
42.4
42.4
42.4
42.4
42.4
42.4
42.4
42.4
42.4
41.4
41.4
mij/l as CeCOj
Dissolved
8.8
e.4
8.6
8.8
8.4
8.6
8.4
8.4
8.3
8.4
8.4
8.2
oxygon, mg/l
Hardness,
44.9
43.5
43.0
44.0
44.5
44.5
43.0
43.5
43.5
42.5
44.0
44.5
ma/I as CiCOj
PH
7.73
7.64
7.07
7.30
7.54
7.62
7.73
7.74
7.71
7.70
7.67
7.75
All somplas taken fran tha alddlo cf uppor tr.nka on Hay IS, except fcr dissolved oxygon, ahlch was dutvmlnod on
Hay 17.
-------
WPENDIX C.
TABLE CI. MEASURED CQNCENTRATI OfS OF E>ORIN ivq/li IN THE WATER DURING
TOXICI1Y TESTS WITH FATHEAD MINNOWS
LKposura concentration No.
Day No.
Stock solution
1
2
3
' 4
5
0
213
1.5
.74
.41
.19
.12
1
212
.66
.43
.24
.16
.07
2
212
1.1
.51
.26
. 17
.10
3
209
.79
.48
.34
.16
.083
4
236
¦ 66
.36
.19
.11
5
6
7
153
.42
.20
.11
.063
8
193
9
10
11
270
12
13
14
202
1.5
.76
.37
.18
.12
\5
195
1.3
.33
.092
16
203
1.6
.72
.35
.18
.090
17
195
1.4
.62
.34
.17
.113
IS
19
20
21
274
2.1
.44
.131
22
23
255
.94
.48
.24
.166
24
25
245
1.7
.90
.43
.23
.128
26
27
28
29
Z33
1.8
.89
.44
.24
.14
58
-------
,c,
.1
,2
.5
,9
.9
,3
,5
,5
,4
2
4
APPENDIX D. fOOO CONSUMPTION AND HSRTAUTf IXJRIfC REARING PERIOD PRIOR
TO TOXICITY TtSTS
TABLE 01. DAILY FOOO CONSUMPTION, F.C. (g/d&y/group of flshj AKG NUWJER OF
DEAD FtSH, N.D. FCK jEVLN GWXJFS OF FATHEAD MIWOKS FED DIFFERENT DIETS
(i-Vtl). EACH CROUP STARTED WITH 220 J-MONTH-OLD FlSH
Grow of f l&h
II III IV V VI VII
N.D, F.C. N.D. F.C. N.D. F.C. N.D. F.C N.D. F.C. N.D. F.C. N.D, CcnnenM
fi.B 7.2 0.4 6.8 7.9 3.6* Fab. 9, 1979
5.1 4.9 6.0 7,5 6.7 3.6*
3.9 4.1 5.6 5.1 4.1 3.6*
1.4 1.0 1.4 I.I 1.4 1.4 Food otfarad
2 hrs
5.9 5.5 7.1 4.3 1 7.6 3.6 I
4.8 4.5 7.2 3,3 7.2 48
5.1 3.6 6.6 2.3 5.3 8.6
7.0 4.4 7.2 3.B 4.9 B.9
6.0 6.3 5.6 4.3 4.4 9.6*
5.3 5.3 5.6 3.9 3.1 4.J
No feeding
3.6 4.0 6.2 4.5 3.5 9.6
-------
TABLE Dl. (Continual)
Group of fIsh
CT>
©
I It Hj. IV V VI VI!
Oay No. F.C. N.D. F.C. N.O. F.C. N.O. F.C. N.D. F.C N.D, F.C. N.O. F.C. N.D. Conoanft
12 6.4 6.0 6.4 6.3 S.I 5.1 9.4
13 7.4 6.8 6.0 6.6 5.7 6.5 9.6*
14 6.7 6.4 6.4 6.3 5.5 I 6.2 9.6"
15 5.S 6.9 4.8 8.6 4.2 5.4 9.6*
16 4.4 4.6 3.2 7.1 2.5 3.5 9.5
17 6.5 6.3 4.6 5.4 4.0 2 3.7 11.9
18 5.8 7.1 5.7 6.9 1 4.5 1 4.9 12.7
19 5.2 1 5.9 5.1 6.2 3.3 5.8 14.2
20 5.3 5.6 3.7 5.8 3.8 5.3 14.2
21 6.0 1 7.0 6.9 7.6 4.3 5.7 14.4"
22 6.0 7.9 7.8 7.4 5.3 5.3 14.4*
23 3.8 6.3 6.4 5.4 3.9 4.3 13.0
24 6.6 7.1 6.2 1 7.3 3.9 4.2 14.2
25 6.5 8.6 7.2 7.6 5.5 7.9 13.3
(contlnuod)
-------
TAtilE D1, (ContlnuMl)
Group of f)sh
I II ill IV V VI VII
Day No, F.C. N.D. F.C. M.O. f.C. N.D. F.C. N.O. F.C N.D. F.C. N.O. F.C. N.O. Carawnts
26 3.0 2 6.7 1 6.0 6.8 4.7 1 4.2 12.8
27 6.6 3 7,1 2 5.1 I 6,7 2 3.7 I 3.8 13.1
28 6.8 2 7.6 6.3 6.8 5.1 1 6.1 9.6
29 5.4 7.0 7.1 5.7 5.6 5.7 9,6
30 4.1 5.1 6.0 6.0 2.7 3.5 8.9
31 6.6 8.0 3 7.6 1 6.5 1 4.1 3 5.2 9.4
32 8.0 4 8.6 2 7.4 6.4 5,5 3 5.8 9.6
33 7.2 1 8,4 6.4 1 8.1 5,4 0.4 14,2
34 7.3 2 9,1 7.5 7.9 I 4,9 1 7.7 9.6
35 5.7 I 6.7 6,0 2 7.3 4.4 8. i 9.5
36 6.8 6.1 6.3 9.0 3.6 4.0 9.6
37 5.7 7.3 2 6.1 I 5.2 4 4.0 5.7 1 9.5
30 a.3 1 6.0 1 6.6 1 6.9 3 3.9 2 5.9 2 N.O
39 8.2 I 9.0 2 7.3 I 7.8 I 6.0 1 7.6 1 13.7
(continued)
-------
TABLE PI. (Continued)
Group ot Ush
I II III IV V VI VII
01
NJ
Day No. F.C. N.O. F.C. N.O. F.C. N.O. F.C. N.D. F.C N.O. F.C. N.O. F.C. N.D. CauaontJ
40 a.2 I 6.5 I 7.6 7.9 5.7 7.9 13.6
41 6.0 2 a.S 6.0 2 7.7 4.3 2 6.3 t 13.1
42 7.9 I 6.1 7.3 3 6.4 I 3.9 6.0 12.7
43 7.2 6.9 1 3.2 3.3 2.5 I 4.6 I 12.4
44 7.2 6.3 7.4 7.4 S.6 6.B 12.5
45 6.1 8.6 I 7.5 8.7 I 3.7 5.7 13.3
46 8.1 8.6 I 7.6 I 6.3 4.7 I 7.0 13.1
47 6.6 6.1 6.6 1 8.1 I 6.2 3 7.2 12.3
48 6.6 7.0 t 5.2 4 8.0 3.3 6.0 2 11.6
43 7.2 7.1 I 7.1 1 0.2 5.6 I 7.B 11.2
50 7.3 7.3 7.1 I 7.6 5.2 2 7.5 6.3
51 7.1 7.0 1 7.2 1 7.8 I 4.9 7.4 I 9.7
32 6.0 1 0.0 1 7.2 3 8.1 1 4.6 I 7.3 I 14.2'
53 B, I 1 8.6 6.3 B.5 5.5 1 7.6 I 12.0*
(continued)
-------
TABLE 01. (Continued}
Group o? fish
m
U
I II 111 IV V V) Vlt
Day No. F.C. H.O. F.C. N.D. F.C. N.O. F.C. H.D. F.C N.D. F.C. N.O. F.C. H.D. Ccnmonts
54 7.8 I B.2 6.6 8.1 1 4,3 7.7 1 0.1
55 6.9 1 7.9 6.8 6.1 I 3.8 7.3 2 U.O
56 7.1 B.I 5.7 7.5 3.7 6.7 8.7
57 6.1 6.6 6.5 8.4 3.9 1 7.3 9.5
58 6.7 2 7.7 1 6.2 6.4 1 4.4 6.9 9.5
59 7.5 6.2 I 7.5 2 0,6 5.9 8.3 2 10.4
60 7.7 8.5 7.8 6.5 6.4 8.1 8.9
61 6.0 6.4 1 6.6 6.5 1 4.4 2 6.5 4.8
62 8.1 7.1 2 4.9 I 7.5 2 5.1 7.0 1 9.4
63 7.7 2 8.3 1 6.6 1 8.0 1 5.0 3 8.0 6.5
64 7.3 6.2 6.3 I 9.0 4.5 7.3 2 9.5
65 8.1 7.0 4.8 8.9 4.8 1 7.5 10.0
66 #.3 V.2 2 7.3 I 9.2 5.6 I 9.3 3 6.4
67 7.5 7.6 1 6.5 8.7 I 5.2 3.8 8.3
68 6.8 I 7.9 2 8.5 1 7.6 5.4 2 8.7 I 6.2
(continued)
-------
TAUIE Dl. tCooTlnudd)
Group of tIsh
i n in iv ¥ ¥i »n
Day Ho. F.C. N.D. F.C. N.D. F.C. N,D. F.C, M.U. F.C N.D. F.C. N.D. F.C. N.D. Caaaaiits
69 7.9 8.2 7.5 S.4 5.0 8.6 2 8.6
TO 8.6 1 7.2 7.3 1 8.2 1 5.4 2 B.2 1 8.9
7) 8.2 7.5 I 6.3 I 7.6 6.9 8.1 I 9.3 1
72 11)2 No food off.
73 II No food off.
74 7.3 4 9.0 4 9.0 9.2 I 8.2 7.9 9.3
75 6.7 2 8.3 7.5 8.8 1 6.3 7.S 9.1
76 6.7 I 7.5 1 7.2 6.7 I 6.B I 7.S 8.5
77 7.2 5.7 6.8 2 8.2 1 6.3 7.9 1 7.0
78 B,Q I 6.0 2 7.6 3 6.9 7.2 8.2 1 8.9
79 2 1 to food off.
80 4.8 I 4.5 3 4.5 6.8 3 2.J 5.9 7.3
81 5.6 4 7,6 5 5.4 1 5.0 3.5 2 7.4 5 4.2
82 6.7 6.3 5.2 I 4.8 3.4 I 6.5 4.2
(continued)
-------
TABLE 01. (Continued)
Grouo of fish
1 II III IV V VI VII
Day No. F.C. M.D. F.C. N.D. F.C. N.O. F.C. N.D. F.C N.D. F.C. N.O. F.C. N.D. Ccraxunts
S3 4.7 I 6.7 t 6.4 5.0 3.1 6.3 4.6*
84 4.5 1 7.0 J 5.0 t 4.4 3.7 6.2 2 6.6
85 I 2 I No food off.
86 5.7 8.1 8.4 7.7 6.1 1 7.5 1 5.7
67 4.2 3 5.5 6.3 I 4.9 3.4 6.8 I 4.7
86 5.9 6.6 2 7.2 2 5.6 4.3 7.6 7.2
89 5.4 6.9 2 6.8 3 4.2 I J.5 I 7.0 I 4.8
90 5.5 4.8 6.0 3.6 3.1 3.9 4.7
91 4.6 5.2 I 5.7 2 4.5 I 3.6 I 6.8 1 4.7
92 5.2 6.4 > 5.4 2 4.7 1 4.2 6.2 8.6
93 2 3 1 No food off.
94 1.9 5.4 5.0 2 4.1 4 2.6 1 6.3 4.7
95 1.1 2.8 2.5 I 2.3 1.7 2.0 6.5
(continued)
-------
TABLE Dl. (Continued)
Group of fish
I II III IV V VI VII
Dfiy Mo* F.C. NaD* F.C. N*Di F.C. N«D» F«C> N.D. F.C N.D. F«C< !»,0» F.C. N«D* Ccnunonts
96 I.I 3.1 1 2.5 3.0 2.2 2 3.0 7.9
97 1.0 2.7 2.6 I 2.6 I 1.6 2.4 4.0
9fl 1.3 2.9 I 3.1 2 2.8 I 1.9 2.7 4.4
99 1.7 3.8 3.3 2.2 2.3 3.1 3.7
100 1.5 3.3 3.3 2.0 2 2.6 3.7 4.0
+ + * + ~ + +
1-100 52 62 64 48 54 45 2
* AlI food consumed
-------
APPENDIX E
BODY WEIGHT, WATER CONTENT AND SURVIVAL TIME FOR FATHEAD MINNOWS SUBJECTED
TO STARVATION PRIOR TO TOXICITY TESTS WITH ENDRIN
TABLE El. INITIAL BODY WEIGHT (g) OF FATHEAD MINNOWS SUBJECTED TO STARVATION*
Croup No.
IX
X
XI
XII
XIV
Mean 4.00 4.75 2.56 2.32 2.37
S.D. 1,80 1.97 0.93 0.98 1.07
No. of Fish (N) 50 50 50 50 60
Range 0.70-7.10 1.80-11.90 1.43-6,60 1.12-5,76 1,07-4.68
~Initial body weights were not determined for groups XIII, XV and XVI, which
were subjected to toxicity tests within 24-48 hours of starvation and
acclimation.
67
-------
TABLE 12, BODY WEIGHT (g) AND BODY WATER CONTENT (1) OP
FATHEAD MINNOWS KILLED BY STARVATION PRIOR TO TOXICITY
TESTS
Group Mo.
IX
X
XI
Body Weight
Mean
2.01
1.86
2.40
S.D.
0.63
0.39
0.57
Range
0.55-3.71
1.37-2.18
1.99-2.80
Water Content
Mean
86.8
86.0
83.7
S.D.
2.2
1.7
1.7
Range
81.8-90,0
84.4-88.4
82.5-84.9
No. of Fish (n)
24
4
2
68
-------
TABLE £3. BODY WEIGHT (BM), BODY HATER OCSfTENT (H-jO) AND SURVIVAL TIME OF FATHEAD MINNOWS
EXPOSED TO 5 CONCENTRATIONS OF EH3RIN AND CONTROLS
Group IX - FIsN starved for 95 days prior to exposure
Concentration No.
Survival time B.W. 3.W. B.W. HjO B.N. KjO B.W. HjO B.H. HgO
(hrsl {%) (g) l%i i%) Cg> (S)
6.6 3.37 04.0
14.7 4.24 82.; 2.22 85.6 3.10 85.6
4.69 86.6 2.01 85.1
0.64 85.?
22.6 1.95 §6,2
40.7 2,47 85,8
64.3 2,71 85,6
96,1 1.57 85.4 0.45 86.7 1.97 89.3
128.5 1.88 88.3
152,7 3.79 66.5
191.6 1.64 79.9
419,1 1.21 78.5
>696 0.59 79,7 2.57 82.1 1.55 82.6
1.06 82.1
Continued
69
-------
TABLE E3. (Continued)
Group X - Fish starved fo- 59 days prior to exposure
Concentration No.
1 2 3 4 5 6
Survival timtt S.W. H^O B.W. 1^0 B.W. H^O B.W. 1^0 B.U. HjO B.W. HjO
(hrs) (g) (J) (9) (I) 656
2.14 S5.0
3.33 86.3
4.93 95.4
3.53 85.6
3.44 81.7 2.12 85.8 2.12 88.7
3.30 84.5 2.76 87.0
86.1 5.55 87.2
3.58 86.6
2.08 80.5 4.17
2.09 81.3
3.01 78.1
3.02 88.7
3.82 85.6
4.38 81.5
6.04 82.1
2.29 80.3 4.28 86.4
3.00 81.3 5.42 65.1
3.45 77.4
7.34 83.4
3.II 78.8
1.76 76.3 2.34 85.5
4.99 82.6
2.39 87.9
2.76
72.5
2.56
78.9
l.ifl
82.8
1.62
77.8
2.23
70.6
2.31
36.6
1.78
77.5
2.27
84.1
2.75
84.7
1.89
75.9
1.78
79.8
1.87
77.5
2.34
SO. 8
1.39
83.5
Continued
70
-------
TABLE O. (Co-iT Inuad)
Group XI - Fltn starv«a for Ms aays prior to *posur«
^weMlrif iem
*!«•! tla*
Ihtt I
+•>*
til
(c« <**
tgj
ih
«ai
A.*.
igl
"2*
ih
Ll.
(g!
*•3
15
22-4
30J
3fW#
*1.1
M
72
507
1ZI.C
2I5J
£51
25?
34|
4H
5ift
tmTf »na
u«i
ns»
c-a
•?«i
§*,§
t.« rto
2JH
US
7«*2
it.*
1*30 >4.7
l.tf tf-2
U4t 73.3
tj» M.4
W»
IJJ
».n
uift
l*4f
90*3
13.3
71.*
•0*3
n*s
.m ?w
.3a ?aa
tj.}
3.N <3*3
i.u tun
:«M *V3
t.JF m.3
2.3a 10.1
t-f3 T?«J
1.2* ^.9
i.ta ss*6
M.2
U60
73.#
1.18
14.1
1.32
fU*
tpjs
1.47
7?,a
'.i3
S3.6
t*£2
**.3
urn
7«L2
S.JA
fl.ft
2»#l
?*_»
I..S
73*4
l.«1
52.3
fl*»3
u.a
t-13
79.J
la 13
IU*
1.33
77.4
;.3o
M.i
2a*7im+:
71
-------
TAPLE a. (Continued)
Group XII -Fish starved tor 21 d«y» pr lor ft) ntpcsur*
riM a.a. >o a,t. *+* i.v. pO a.** &,«« *jj
Ur«l t*J th (gl tU <3J ill <9) CM {91 IS) rgj (f)
64 3*03 U.ft
;J 1, to ei.*
3^4 M.t
22-7 1,97 t(.l
J»J4 7).«
30.* 1.54 78-J
104
W.i 5JI *2.1
K1 1.61 TS.2
¦3.5 LM fif.l
2J4 7«.«
na 1.73 cg»q
m i-T2 rr.i a-it 97.7
73L* 1.21 77.0
2)M 5-34 77.*
237.1 «.»« T4.2
230 1-47 U.«
2*37 79.0
273 2-*6 30.3
2.14 77.3
2.21
aa.7
t.M
57.7
1-63
tirt
i.n
74,9
1.93
tt.l
2*33
A3.3
i.»
M.4
1.34
73.9
I.ZZ
71,7
U4Q
T7.I
i.3t
71.2
1.92
73,3
1.20
72.3
1.23
T3-2
o.ao
76.J
1.22
74.6
3. IS
U*3
1.37
83.0
t.Ot
G.4
72
-------
TABLE £3. (uontlnuKj)
Group XIIJ - Fish itarvad tor 24 nr* prior to exposure
nwcttrwitu —.
I 2 > » > a
iKMIMI fin LI. S.«. !.<• P.«. «-> I.I. tfi
tw-tl (>• (11 19> (» l]l 111 «jU it) Igj sil tcJ (II
1.1 *.7* M.7
U <.81 M.C
22.* i.a rt.t
i.it n.a a.*
i.a ii.7
t.» »*,«
i*.$ l.M >4.1
».) l.M JT.» l.M M.I
l.M M.i
«*.» 4.M M.i
5.J Z.<2 '«.« I.J7 #SJ
U.« 2.71 M.1
13.7 2.SS 11.3
0j i»» nj
us a.*» «.?
2.72 T7.fi
IT* 3.23 7*.*
».t» JS.»
an i.a# tt.j
237 U'3 7t.2
2» I.a KM
1.17 M.J
2.0* «.>
its s.1? n, f
l.M Jfc*
ICZ 2.J7 M.I
4*5 *.* K.t
«n t.n m.i 2. as **.7
tu
70.2
13.92
71,?
l.2»
77.5
!«§»
n.a
1,46
7:. a
49.9
*~«*
13.2
l.Sf
7«W
1.!*
71 .j
U72
T«.3
l.M
72,7
1.#
75-6
n.9
U4V
22*3
l.fl
Tf.i
fl.37
S0»5
0,97
19.4
i,?2
13,3
1.23
63*2
2-l«
71.7
1*3
72-4
1*25
1.24
75.0
71.1
73
-------
TABLE E3. (Continued)
Group XIV - Pbnd-fIsh starved lor 53 days prior to exposure
Np.
tim S.tf. *2? *•** *2^ 6*>* **2^ *2^ *2^
lhr»l (gl U* (gl (SI (9! (I)
A.4 2.0 fiU
£.69 M.I
*3 Z.09 lt.i 0,44 19.1
Ul 91*6 1-17 U.)
1.49 <2*9
2.5T BL)
1.4f 13*1
1.62 B0w2
22.* 1^1 M.O 0,97 V4
JO. I l.:a 79.7 1.73 90.3 2,92 11.2
1,13 o*z
39*3 l.3f M*9
1J31 94,2
47 Ul 79.6
34.2 1,15 11*7 2*79 fl.3
71.7 !.»7 79.7
1.47 77.6
WJ 1,9 U.3
132 1.93 tZ.4
1.21 01.3
1.04 U.3
133 3.02 99-1
0.94 94.0
11V 1.4 13.9
2tJ 3.19 99.1
3M 0,49 87.5
472 1.23 9&.0
334
!,U
93.4
(.*1
93.9
0,50
62 .0
0.74
S2.4
0.90
93.0
1.00
83.0
1,30
90.7
1.4*
B3.a
O.M
M-fl
0.93
03.9
0.93
M.2
92.6
2.J6
96.4
t.H
91.1
a.&s
2.M
50.1
2.72
BO, 3
l.lt
79.3
74
-------
TABLE D. (Continued)
Oroup XV, - Pond-fish starved lor 24 hn prior to sxpesur.
Csac«»TrctH3w m*
SirvlMl ria* tt.». MjO «.*, MjO &,«, «j0 ».#, s.«,
Ikril 13) (II tgl (11 (gl (II
-------
TABLE ES. (Continued)
Groyp XVI| - Pond-fish rreppod 2 days prlcr to exposora
CaocitrrtlM to.
Survival Tla* l.>. HjS 3,«. HjO D.«. HjO t.<> KjO I.a. kl.
llnl tg! (SI ((I (I) (it (II lj< (!) Igl Ml t|J ill
j.j i.;i ».« zj» m.* ».i8 iy
2.M W.7
0.M ai.i
11.J t.» ».i
1.08 a.o
m.4 ?.*> ii.a 3,11 u.o
1.t7 M.7 I.IT 11.«
i.a ii.a i.is u.»
uid eo.o i M ai.a
zs.i «.a it.*
It 2.91 n. I 1.3) it.»
1.32 M.I
so i.m ai.e
i.it ea.i
I.4J II.*
77 1.09 eo.«
108 Ml M.3
1.SI 90.1
I» 1.78 19.1
m o.m s.j
373
J0I
0.M
1.00
1-55
flS.fl
QuW
9A*7
0.90
WO
0.47
03,1
1.01
ata
0.7*
03.9
1.02
§3*1
1*85
84.1
a.59
U.J
SQ.fi
o.n
78,1
I.51
82.i
0.67
a3.fi
O^f
U.I
9.3]
E5.3
U43
60.7
8»45
W.i
I.C3
94.0
31.2
0-34
33.2
I.S7
3**8
O.oO
M.a
0.43
3A.0
2.33
M.I
U33
Gearlnurt
76
-------
TABLE £3, (Continued)
Group xv2 - Group xv, starved 14
day* prior to axposura
Concentration No.
Survival tin# B,*» B.w. HjO
th.-ii tg) (S» (g> (SI
18.5
24.2
27.0
30.7
48.2
49.0
65.5
81
98
1.66 SI.5 1.36 86.3
1.25 61.6
1.02 78.4
1.30 78.7
1.93 77.7
2.16 79.2
1.29 76.1
1.62 80,2
2.02 78.7
1.31 7S.3 2.85 82.1
3.08 81.2
3.58 86.0
1.45 79.3
2.02 79.2
3.33 80.2
1.84 79.3
2.16 76.4
Group XVj - Croup XVj after 7 days
tending prior to axpeiura
Concentration No.
Survival time
(hrsJ
13.4
23.0
25.5
29.8
40.3
43.6
61.3
81.7
8.W. H^0 B.w, HjO
(9) (S3 (g> (I)
1,66 77,7
1.91 78,0
4.37 84,2
3,11 77.2
2.93 79,5
1.81 75,7
3.66 78.2
3,81 78.2
2.03 79.8
3,36 78.3
1.8? 77.1 4.74 81.9
2.20 78.6 1,99 78,4
3.61 83,1
2.99 77.3
2,64 77,3
2.0! 81.1
Continued
77
-------
TABLE E3. (Continued)
Group XVIj - Group XVIt starved 14
days prior to exposure
Group XVIj - Group XVI starved 21 days
prior to exposure
Concentration Mo.
Concontrntlon No,
2
1
2
Survival time
B.W.
"2°
3.M.
"2°
Survival tlm«
B.W.
"2°
B.M.
"2°
(hrs3
<>.'>
{«
(hrs>
c»
tg)
CS)
9,8
1.46
04.9
0.T9
86.1
12.5
1.40
68.6
2.96
81.3
1.78
84.3
1.29
84.5
3.35
83.6
3.94
82.7
1.19
66.6
3.65
79.7
2.80
86.1
1,34
63,6
2.31
tt.f
1.67
05.6
1.96
82.7
3.03
86.0
1.96
82.7
1.23
02.9
2.78
84.2
1.94
87. T
1.16
85.3
1.70
85.3
0.92
84.8
2.38
85.7
1.75
83.4
13.5
2.50
82.3
1.10
86.4
17.8
1.71
81.9
22
2.63
80.2
5.15
86.1
0.96
84.4
29
3.53
81.9
1.2B
84,4
0.71
83.1
23.5
1.74
86.2
27
0.93
87. \
41.2
2.30 81.3
Continued
78
-------
TABLE E3. (Continued)
Group XVI4 - Group XVIj ted for 7
daya prior Co exposure
Concentration Mo.
I 2
Survival tide B.W. H20 B.W. HjO
(hrs) (g) (Z) (g) (%>
12.8
23.9
30
38. a
48.5
59
3.38
2.96
3.21
1.61
1.59
82.0
83.1
84.1
84.5
81.1
2.54
1.82
2.16
2.51
1.11
2.13
2.42
2.79
3.16
3.87
83.5
85-8
84.3
80.5
82.0
82.6
80.2
81.0
82.9
81.4
2.34 80.3
1.84 83.2
1.76 84.7
3.59 80.8
79
-------
APPENDIX F.
POSITION OF EXPERIMENTAL GROUPS OF FISH IN SCREENED COMPARTMENTS DURING
TOXICITY TESTS.
A system for toxicant exposure previously used for chronic 1ife-cycle
testa (Holcocbe et al¦ 1976) was nodified slightly to fit the experiments.
Each tank was divided by screens according to Fig. F-l. Exposure chambers
for experimental groups of fis»h (I-XVI) were randoaized. The positions for
various groups in experiment i and 2 are presented in Figure F-2.
80
-------
Figure y-1." Arrangement of testing tanks used for toxicity tests with
16 pre-treated groups of fish.
UPPER TANK, from above
b c
i—
e
od
o6
100 mm
LOWER TANK, from above
a. Water fed fron flow splitting chamber of the diluter.
b. Flow-splitting chamber.
c. Notches for flow-splitting.
d. Drain to lower tank. Height of standpipe 130 nsn.
e. Safety drain. Height of standpipe 140 na.
f. Water fed from upper tank drain (d).
g. Drain to holding tank and carbon filter. Height of standpipe
300 am.
Material: ( ——- ) glass
( mmmmmm ) stainless steel
( ) screen, stainless steel, mesh 40, wire 0.40 nun
(¦mMwaa} screen, stainless steel, meah 4, wire 0.88 ran.
Volumes: Total upper tank: 20 1 Total lower tank: 81 1
Exposure chamber: 3 1 Exposure chambers 14 1.
81
-------
Figure F-2. fositions of experimental groups of fish during exposure to
endrin,
vvSIDE
3w
4vv
5vv
fivrv
A.
..a..
VI
V
IV
VI
IV
1
V
It
b mm
mmm
VI
!l
VIH
VII
til
Vl«
11
III
o
a
c
a
6v
3v
X
XV
XV
XI
XIV
XVI
••••'a—
•™*o™
XI
X
V VI
—
_ mm
1
via
Vtl
II
III
Vtl
1
III
o
0
0
0
4 it
9*
XVI
X
XV
XI
IX
XV
o
XI
IX
2v
lv
XII
XVI
XI
XII
XIII
mi
o--
o
XVI
IX
yvSIDE
vSlOE
vSlCE
EFFLUENT
TANK
CARBON
FILTER
lv to 6v and Iw to 6w refer to toxicant concentrations from the diluter.
1 was the highest concentration and concentrations 2 to 5 were achieved
by progressive 1:1 dilation in the dilucer. 6 was the control,
Roman numerals I-VIII indicate the diet-treated groups of fish which
were exposed to the upper tanks, and IS-XVI indicate the groups of adult
starved fish exposed in the lower tanks.
82
-------
APPENDIX G.
SUMMARY ON PREVIOUS FINDINGS ON ENDRIN TOXICITY TO FISH
In the lace 1950's tha extreme toxicity of endrin to fish was reported
by lyatooi eX_ aK (1958) and Henderson £t al. (1959). During the I960'a the
implications of this extreme toxicity to fish was realized in studies
related to the Mississippi River fish kills (Mount and Putnicki, 1966).
Lethal blood concentrations of endrin in two species of fish were
established (Mount et al., 1966; Brungs and Mount, 1967). Studies were
undertakes on bioconcentration, distribution and/or depuration (Mount, 1962;
Bennett and Day, 1970; Argyle ejt _al., 1973; Jackson, 1976) and more recently
on biotaagnification (Jarvinen and Tyo, 1978). In addition the physiological
mechanism of toxic action was studied (Mount, 1962; Colvin and Phillips,
196d; Cutcomp et^ a_l., 1971; Cller, 1971; Davis et al., 1972; Grant and
Mehrle, 1970, 1973; Cutcomp, 1974).
The toxicity of endrin to fish became extraordinarily complicated, when
fish populations in heavily sprayed areas were found to be much more
resistant to endrin and other pesticides than previously unexposed fish
(Boyd and Ferguson, 1964). Since then various aspects of this resistance
phenomenon to endrin in fish has been studied quite extensively (Ferguson
and Bingham, 1966; Ferguson e_t j_l., 1966; Ludtke et^ al^ , 1968; Kynard, 1974;
Fabacher and Chambers, 1971, 1976; Wells and Yarbrough, 1972; Yarbrough and
Coons, 1974; Yarbrough and Wells, 1971.
Interactions between endrin toxicity to fish and ambient conditions
like temperature, hardness, salinity and suspended solids, as well as with
biological factors like species, size and developmental stage were also the
subject of several studies. The results of these studies are summarized in
Table Gl.
83
-------
TAM.E CI. SJMM1 Or nljWIfcB INTERACTIONS11 BETWEEN AMBIENT FACTORS #WU EttKIN TOXICITY TO FISH
S(«CiN
Fvspofts* vftrlabltt
MaxIm
y mwt (nt«r«cf Ion
#n«
pa Uiau! l*nMutl]rt
*lM 0O13ft,
Alfctllnfff, mi
m i»iwh«a»ou»iv!
StutpwiUvd Aol Id*
AcH»at»d am bum
%wvmtMturm
f «t auUilun
mjQ »p4 r-i/l mm CaUBj
V3 .xt 10 »j/l it taCUj
1.4 i'>4 1*4
l« am) iQQ *^'l U CoUDj
n «nd J)l «^/l » U>Oj
5.9 **4 7.6
5-27 °/««
3-2> °/uo
U-W.4 *y/l
0 and IM k^/|
0 >nd 11.7 i^/i
HJ'E
12.7*25,S-C
tf.t-2J.VC
1.6-i2.7*C
l*fi-!2,7*C
ptwdtr, fachnlal In
tettarta. «r wMflalbl* oncMF
irtfflcM
¦ II* (1:1)
partthloo
vfIhouf sulbyl
pIivkw
f«1Ntfkf •titnua
Marin* }hr«MplM tfidiltbick
liflni ttrMtplw *fjcklabtck
fafhaod *lnra*
fatfiMl PttftfHMr
fathead •tnnw
SlypflJ1
BluagtH
8!u*gllt
fttlntur traul
Kalnbm trr^t
faflMtad mlmm
96-hr LCSO
4Q»hr LC50
96-hr U3C
9ft-fcr LOO
Vb-t* I.C50
96»hr LC3T
36-hr LC5Q
21-hr ICM
96-hr LC50
24-br LC5Q
96-hr t«0
ffr-l.r U50
Mltfi 11*1* or Mlt./t
pM-«lftien
U tfKcuth 24-fr UX
Urgoaatifft !**• 96-hr LOO
I.J/l.Q - 1.1
0.47/O.O » t,B
2.33/1.74 » 1.3
I.20/1,65 - 0.7
0.52AJ.J? - 1,4
H**%lw*on «t «)», I9JV
Orunys •; at», !VM
Mix Md U,*d •><:>, 1961
lUli and LhaJaltk, 1V6I
tifwnyi *1 at., 1W6
li.2/0.43 ** 40*4 (tela) andrln) Urwny» if ii», 196a
C,66/0.45 * 1,3 (<3U»o«r*d »hdrlf») Urunyt »f ii., il&n
«,25/0,13 ¦ 23.0
2.6/0 0 * j.W
0.6I/D.J7 - 1.64
15/2,3 * 5.33
if.3/i.I - I.it
1.9/0.36 • 3,4
0.48/0,4? - 1.0
£.37/0.27 * 1.4
Mtt and Chedalck, 1*61
NkA «f •!., 1969
Mki* *1 ¦!.. 1969
Mac** ol al., 1V69
Macah »T al., IVftV
Hotter»un a» •!., IV5¥
fttodiv, 1976
fai*ct*«-, 5976
-------
TAbLt G1. (Contlnuud)
Miitnt laclcr
Kan^a
5p*cfaa
fUaponM vv 1 aQla
Pacta** i
ma tntaractltfif
Katamnon
(J«r«rU*>nt (LA$)
0 mid 1,0 ny/l
UthNd alniM
4fl-hr LC50
o.jvo.29 -
1.9
iolon of »1„ 1967
0 and 1.0 ay/1
Fa«hfa»4 alnftOK
96-hr LC50
0.2VU.24 -
1.0
Solon at •!., ltd
Im4Ii^| dMtlty ol
tUh In atallc f^fa
0,6-4.19 g/t
BIungiH
72-hr LCM
2,10/Q.n -
l.t
K«t« wS Cftatfalch, 1961
loading 4»n«lty of
liitt In static tast«
0,6-0.»9 g/l
BIucqIH
94-ttf ICJ0
uia/o.6o *
1.0
tot! vd Cfiodalck, 1961
Static v». dynaalc
tMt
10 1 (atatlcl er 12.S
¦l/alft (dynaalc)
Fafhaad Blnnoo
*a-hr LC50
0.77/0,51 •
1.4
Linear nl nt.( IV?0
Static vt» dynamic
l«t
13 1 Utnllc) cr 12.3
al/ata titynnnl c)
Fat feud nlnnov
H-lr IC?0
0.77/D.W -
7.0
linear at a!., 1970
¦ Koil of fhata Inter actlmi balm*** andrln tonicity and «afrj«nt tnctora v«r« CEMldertd » InlfiftMlcenl by live Invaatlgator*. Sl^nIrlc«nt Intaracflonn «nr* only
•vldnir fix t«wf#raW» (Mrit and Oitf»lck, i9^l and Hecafe at al. ~ I969}« actt*eT*J carbon* vfi« baled m total onflrla (Brungi alii*• !H6>( and load!ng 4»ntlty
In atatlc ta«U (tall and Chadvtck, I961),
b Ha«lH0 nan Infraction • ICW ln*n) at tha lavol of tnlaracflng liwblant) factor jrctfuclrq taa»t To«lclty IMuhoat LC2Q) dldted bp I£Vi at im lav* I of
IntrKlIng toctcr producing hlghait tonicity (iwait ICM), tipoiun onrnVatlon lug mdrlo/l vttvl ««r* not wUiid bf chmlcal oAatytU In nil tlvdlm
cited. . A dlrnct o*p*rIton batvoau IM r«»ultt obtained althln, but ml bir*Mn, iipvifi ihidla I* postldln (fan to «p«rlaanfal daafgn, th«
I actor a iia«!nad im to to tl« only or Mln vvlaftlM*
------- |