EPA-600/4-81-076
September 1981
INITIAL TOXICOLOGICAL ASSESSMENT OF
AMBUSH, BOLERO, BUX, DURSBAN, FENTRIFANIL,
LARVIN, AND PYDRIN: STATIC ACUTE TOXICITY
TESTS WITH SELECTED ESTUARINE ALGAE, INVERTEBRATES,
AND FISH . ..
by
'Patrick W. Borthwick and Gerald E. Walsh
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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..INTRODUCTION
'.$ , The sensitivity of estuarine organisms to seven pesticides granted
experimental use permits or awaiting registration was evaluated in response
to a request of the Office of Pesticide Programs, Ecological Effects Branch,
Hazard Evaluation Division, U.S. Environmental Protection Agency (EPA).
Acute static toxicity tests were conducted with six insecticides (Ambush ,
Bux , Dursban , Fentrifanil , Larvin , and PydrinK), and one
herbicide (Bolero ) by the EPA Environmental Research Laboratory,
Experimental Environments Branch, at Gulf Breeze, Florida (ERL.GB). Sources,
.purities, target pests, applications, and registration status are summarized
in Appendix A. v ; -. >.
This report is the first of a series and includes the biological effects
"screening" data generated in 96-h EC50 (concentration of pesticide_that
would inhibit growth by 50%, as compared to control growth in 96 h) bioassays
-with marine algae, 48-h EC50 (concentration of pesticide that would cause -50%
of the exposed larvae to develop abnormally in 48 h) tests with estuarine
. 'mollusk larvae, and static acute 96-h LC50 (concentration of pesticide that '.
is lethal to 50% of the test organisms during a 96-h exposure) toxicity tests
with estuarine crustaceans and fish. ' .''"- -- c . - -f :" *
.....: . : : MATERIALS-AND METHODS .. . -
^-^Algal Bioassays -;; :ป"",;;: ..:;.':";/',.: , - - .'< -?.-: .-V '':: '::''-:<..: \,
Algal Species Tested and Procedures' .;- " . ; :',: . .-
- One to four species of algae were used in tests with Ambush, Bolero,
Dursban, Larvin, and Pydrin. Tests with all five pesticides were conducted
with Skeletonema costatum. In additional tests, Isochrysis ga'lbana and
Thalassiosira pseudonana were exposed to Dursban and Pydrin. Nitzschia
angularum was also tested with Pydrin. .All were obtained from the Culture
Collection of Algae, University of Texas, Austin, TX, except S. costatum
which was obtained from the University of Rhode Island, Kingston, RI.
Media for stock algal cultures and for tests pesticides were prepared by
dissolving a commercial artificial sea salt formulation (Rila Products,
Teaneck, NJ*) in deionized water to the concentration of 30 parts per
thousand. Before the solution was taken to final volume, trace, metals,
vitamins, and minor salts were added as nutrients (Apprendix B). Medium was
prepared on the day before a test was to begin to allow the pH to stablize at
8.1.
Algal stocks were maintained in 150 ma of culture medium with-EDTA^in
500 ma Eclenmeyer flasks under the following conditions: light intensity,
150 uE/m /sec(PAR, 400-700 nm waveband) from overhead "cool .white"
B
Registered trade marks, generic names and manufacturers are listed in
Appendix A. Mention of commercial products or trade names does not
constitute endorsement by the U.S. Environmental Protection Agency.
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*?.<:
., 'if^f -, , 'r j
.^';~J *'ฃ' '.
- : -c' '"";
, - # <-;,
"' f>;'- t" -
.- ^T,'ij. .-.
fluorescent tubes; lighting cycle, 14 h light followed by 10 h darkness;
temperature, 20.0 _+ 0.5ฐC; transfer to new stock medium every 10 days. Stock
cultures were not shaken. . . .
Pyrex (Corning Glass Works, Corning, NY) glassware was used and was
prepared as follows: (1) soaked in detergent overnight, (2) brushed lightly,
(3) rinsed thoroughly in tapwater, (4) rinsed three times in pesticide-grade
acetone, (5) soaked overnight in 1% HC1, (6) rinsed thoroughly with
tapwater, (7) rinsed five times with deionized water, (8) dried in an oven,
and (9) stored in a relatively dust-free container or individually sealed
with aluminium foil. All glassware and media were sterilized by autoclaving
at 121ฐC and 1.4 kg/cnr for 20 min.
Bioassay Procedure :-: ''*>- ''' '' - ; - < .-':.".
". '-''* One week prior to the tests approximately 5 ma, of an algal culture in
logarithmic growth phase was added to 150 m of culture medium that contained
. ethylenediaminetetracetic acid (EDTA), thus ensuring healthy culture in the
logarithmic growth phase. "On the day of the test, cultures were diluted with
growth media to yield 2000 eel 1 s/mm for SVcostatum and .1000 cells/mm ป)
for all other species; 1 mฃ of this cell'suspension was added to-each algal
culture tube.--. ; .... -r. -J..? .-, -. -.._,.,,.:... .
''.- -In tests with pesticides, 25 ma of culture medium without EDTA were
pipetted into optically matched test tubes (25X200mm) which then were capped
with stainless steel microbiological closures and autoclaved. On the second
day, a known quality of pesticide in 0.1 mx, of acetone was added to exposure
tubes, and 0.1 ml of nanograde acetone was added to control tubes. Then 1 mi
of algal suspension was added aseptically to each tube.' Treated cultures as
well as controls were run in triplicate. . .: ,
Cultures were incubated while shaking "at approximately 60 excursions/min
on Model G2 Gyratory shakers fitted with No. AG2-Ta25 platforms (New ; . .
Brunswick Scientific Cor, New Brunswick, CN) under 150 pE/nr/sec PAR
illumination from .".cool-white" fluorescent tubes; temperature was 20.0 +_
0.5ฐC; the "1 ighting cycle was 14 h-of light followed by 10 h of darkness;
test duration was 96 h. " -: --.r-
After 96 h incubation, absorbances of cultures and optical blanks were
measured at 525 nm on a Fisher Electrophotometer II (.Fisher Scientific
Co., Pittsburg, PA).
'. '-'^Concentrattons^that inhibited .growth' by 50% (EC50)1 and 95% confidence
. intervaTs-were calcu:Ta'ted by. moving .averages :on' a Digital Equipment
Corporation.-POP-.11/7:0 computer. . "... - '
INVERTEBRATE AND FISH TOXICITY TESTS
Animal Species
Invertebrates and fish used in static acute toxicity tests were spawned
or cultured at ERL, GB. The parent Eastern oyster (Crassostrea virginica)
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stock was purchased from oystermen, then conditioned and induced to spawn at
ERL, GB. Gametes were mixed to produce <2-h-old embryos for 48 h EC50 tests.
Stone crab (Menippe mercenaria) zoea larvae were hatched from a gravid
female collected from Santa Rosa Sound at Sabine Island. Mysid shrimp
(Mysidopsis bahia) <24-h-old were hatched from brooding females from
laboratory mysid cultures isolated overnight in a brood chamber.
Sheepshead minnow (Cyprinodon variegatus) brood stock was captured from
embayments on Santa Rosa Island, then conditioned and induced to spawn in the
laboratory. Fry for toxicity tests were reared to 28 days of age.
Animal Food , ...
Oyster parent stock were fed on natural plankton assemblages in
unfiltered seawater, while oyster larvae were not fed. Stone crab adult
females were fed raw fish muscle. Mysid shrimp and stone crab larvae were
fed living brine shrimp (Artemia sp.) nauplii during culture. The sheepshead
minnow brood stock diet included fresh-frozen adult Artemia and a commercial
dried flake food. Fry were fed Artemia nauplii. *'"/'-- ;...,-,. ~ ., ,,, .
Chemical analysis .of all foods given to brood stock or test animals
indicated that non detectable concentrations of chlorinated hydrocarbons less
than 0.1 (yg/g) or parts per million (ppm) wet weight were present. -,.-.
With the exception of adult oysters, which received raw unfiltered
seawater, all animals were spawned or cultured in intermittent-flow seawater
filtered through coarse sand and 10 to 20 micrometer (ym) polypropylene-core
filters at ambient salinity and 25ฐC. Static toxicity tests were conducted in
seawater filtered through 1.0 pm polypropylene-core filters and adjusted to 20
ฐ/oo salinity by addition of Rila Salt Mix K (Rila Products, Teaneck, NJ)
or deionized water. Temperature was maintained at_25 +_ 1ฐC in all cultures
and toxicity tests.
Test Pesticides . . .
Pesticide samples used in this study are described in Appendix A.
Seawater concentrations and median effect concentrations,.of each pesticide
are reported here as mi.crograms (yg) of pestircide!.-pe.r,rfli.tler (a) of seawater,
or parts per billion. All concentrations are nominal based on weight of
sample per volume and are uncorrected for purity or specific gravity.
Static Acute Toxicity Tests
All toxicity tests were conducted in accordance with Draft Toxicity Test
Standards of the American Society of Testing and Materials; 48-h EC50s for
larval oysters in accordance with "Standard Practice for Conducting Static
Acute Toxicity Tests with Larvae of Four Species of Bivalve Molluscs" (ASTM,
1978a), and 96-h LCBOs for stone crab larvae, mysids, and fish in accordance
with the proposed "New Standard Practice for Conducting Basic Acute Toxicity
Tests with Fishes, .Macroinvertebrates, and Amphitxians" (ASTM, 1978b).
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During the 96-h test period, stone crab larvae and mysid shrimp were fed
48-h-old Artemia hauplii each day to minimize cannibalism and starvation.
Both..48-h EC50s (and 95% confidence intervals) for Eastern oyster larvae
and 96-h LC50s (and 95% confidence intervals) for crustaceans and fish were
calculated by either the probit, binomial, or moving averages.
. ;",... RESULTS AND DISCUSSION . .".
Results of algal bioassays (Table 1) and static acute toxicity tests
with invertebrates and fish (Table 2) provide an initial foundation for the
toxicological assessment of Ambush, Bolero, Bux, Dursban, Fentrifanil, j.
Larvin, and Pydrin on estuarine organisms. The 48-h EC50 for Eastern oyster-
larvae was not determined since this species was determined to be
insensitive to four pesticides in a sbort-terjn acute exposure. The estimate
of toxicity (expressed as 48-h EC50 >1,000 mg'/i) provides a confirmatory
result. Test concentrations >1,000 mg/ฃ cause cross-contamination of
-equipment and glassware and increase the exposure hazard to workers. .When an
. estuarine test species -is insensitive at--1,000 mg/ฃ,^further acute testing
jshould be continued until toxicity to several phyla has been determined. :
. '.the synthetic pyrethroids, Ambush and :Pydrin, were .the most toxic of the
- -seven pesticides tested and were extremely-.toxic to crustaceans. '/Toxicity,._
f,-in .decreasing order,-to algal population-growth (Table 1) was Ambush, ;;-'
Dursban, Bolero, Larvin, Pydrin, and oyster larvae (Table 2), Ambush, Pydrin,
/ Dursban, Bolero. : Acute lethality, in decreasing order, to crustaceans was
'Pydrin, "Ambush;; Dursban, Fentrifanil, Bux, Larvin,'Bolero and to fish,
" Fentrifanil, Bux, Ambush, Pydrin, Dursban, Larvin, Bolero. ';-,-,- . <-
Bolero, the only herbicide'tested, was less toxic than the six
insecticides. Phylo'geneticany,'the descending order of-sensitivity was:
.v -crustaceans, fish, algae, and oyster .ilarvae. .!,./v^^Vc. ,:ซ;&.-.j, .:;';?; :u ,..;
-""' " ' " ' ' * ' ' ' '.-.'.,.'
-The synthetic pyrethroids'were extremely insoluble in seawater. In
..tests with algae, oyster larvae, and fish, median effects were higher than
the solubility in rseawater. ".Consequently? actual- exposure concentrations of
Ambush and Pydrin were probably much Tower than the 'dosed nominal
concentrations ;due to their-hydrophobia nature. 'Results of several
pyrethroid toxicity'"tests with filsh varied, indicating that exposure
concentrations {above- so-1 ubiTity) were fricd'nsistent among tests. A
subsequent reportrfro"rn^ERL jGB (SchimmeT.et-ฐal. ', "1981) confirms that
pyrethroid toxi'crties' tri< fTowihg^acute::tests;/w-ith sheepshead minnows were an
order of ma'gnitude'Towe^'-than in Vta'tnc ac-Ute tests reported here. -Since
problems with .pyrethroid exposure cohcentTati^ons4 occurred-with fish,"" static
acute'values-'reported here";'should"not be considered final'. ' Crustaceans were
extremely sensitive to "Ambu-sh "and Pydrin at concentrations below the limit of
solubility in seawater. When compared to flowing acute-test results, static
tests with mysid shrimp were characteristically 2 and 3 X less sensitive.
. ,,,:,. , .. 4 -.; . ; i
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REFERENCES
American Society .for Testing and Materials, -.Committee ฃ-35 on
Pesticide's,' '1978a. Proposed -Standard iRiractice.^d^^ondiuc.t-ing ,S.tat-ic
Basic Acute ''Toxi city Trests jWittKLarvae o.firlto.Ur.fSp.eGii>es
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Table 1. Results of algal bioassays using pesticides and unicellular algae.
'Values are the 96-h EC50s (95% confidence interval), or the
calculated concentration that would inhibit growth by 50%, as
- - compared to control growth, expressed in units of parts per
: 'billion (micrograms per liter). .
.ป ป *J> .-' * -
Pesticide
ป ' Species :: . ; - .
'Tested" .'-'"'."'
'''-^ Test
No.
96-h EC50 (95%CI) ^
' ug/ฃ
Ambush . , .:.Skeletonema costatum ,;.;.,".' . 1..
. '. ;.'.'..,. /,; -,v-..-;,-... .. -. ,.-... -vi ''.- ^
Bolero ; ji: - -Skeletonema costatum ;,,]; ^;':>^:1 '[
'. ' ^i;'.'' ->..',"', .-'.'' ,-.fr'; - >-. ป: ,;.:..{..- ". ",,.- ,-jt: 4 .
"Dursban v Skeletonema costatum ; .; ""''*'' !-'t
. .., .. . , ::,;'.""'"2 "
':"."'* ,. :. ' "3-
:'" " .';' *' : . " .'-"'4 :
.124 (104-154)
92 ( 71-115)
459 (327-692)
:.410 (368-461).
327 (290-368)
377 (326-439)
:389 (338-452)
,\"i'(" "' *','',;' V ''''
:255 (228-286)
289 (257-334)
326 (283-402)
328 (290-391)
297 (268-335)
':'''-' ' '*-':
Larvin
Pydrin
Isochrysis'-galbaria ':' -ป
-,ซ-... --' Thai assi osi ra' pseudonana
Skeletonema costatum
.
Skeletonema costatum
Isochrysis qalbana
_ Thai assi osi ra pseudonana
Nitzschia angularum
i-l'Sr: -
1 ' ' '
.1
2
3
4
1
138 "(112-169) .
148 (121-182)
457 (386-547)
462 (414-518)
404 (366-449)
454 (410-509)
M ,000*
>1 ,000*
>1 ,000*
>1,000*.
-ซ.
* No effect on growth
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Table 2. Acute static toxicity of pesticides to several,estaurine animals. Values are 96-h LC50
(95% confidence interval) or *48-h EC50 (based on abnormal development) expressed in
units of parts per billion (micrograms/liter).
Pesticide
*Crassostrea
virginica
<2-h larvae
Mysidopsis
bahia
1-d juveniles
Menippe
mercenaria
zoea larvae
Cyprinodon
variegatus
28-d fry
Ambush >1,000
Bolero 1,0001,000
0.046(0.032-0.056)
370(317-433)
1.0 by graphical
interpolation
0.056(0.032-0.100)
0.01: - - >1,000
- . 46(41-51)
270(235-309)
9(8-10).
>1,000
121(96-149):
Note: Greater than (>) values-, are the result of confirmatory tests.
Ranges (
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:, .r'r )'-'''""' .-"-I : -.r;."". i'-"'"^!''--"''!"""Vv' 'X''''-'.'" >:
Appendix A. Pesticide Sources, Purities, Major Pests, Applications, and'Registration Status >'-/ ..;?''
CO
REGISTERED TRADENAMES , v
Parameter
Conmon Name
Technical
Material Source
Lot
Purity, %
Manufacturer
In-house
Date Received
Use
Major Target
Pests
Application
-, .A
Status
Ambush3 .'i !
Permethrlri ' i
ICI
America's, 'Inc.
47/82- :-\ -.
Si ;: .'.]!'.!
" '.'',' ''
93
93
5/4/79
Insecticide
budworm
bollworm ..
bollweevil ;
Cotton '"
Registered
(uses pending)
Bolero15
Thlobencarb
Chevron !
- Chemical Co\
1039T" ' '- '
95.5Sr>
90
5/15/79 '
Herbicide-
preemergent and
early postemer-
gent grasses and
broadleafs
Rice. -"
Section 18
Emergency
Exemption
Buxc
Bufencarb
Chevron
Chemical Co.
Analytical
Standard
from RTP"
* '.-r
'.'.
":.
Insecticide
corn rootworm
rice water
weevil
Corn
R1ce '-' :
>-
Registered
Dursband
Chlorpyrlfos '\ .-
Dow ." .:-, -'''"
Chemical Co. ,' '..'
1-5503-2 :."'
* . ' -f -
- ''.-"'
97.7 v.uv^'i
92 . ' :.;. ,-:l<
4/24/79 . .". .
Insecticide ' ;
fire ants ''. '. -.
household pests-
worms ;
scale ; !
borers ,
soil '
turf .
foliage _; :.. '
cotton
corn
cattle
Registered
(uses pending) _.;
. i ";:. '"','
Fentr1fan1le
PP 199
Fentrlfanil .
ICI
United States,
Inc.
Analytical
Standard ';
from RTP
- - ' . "
,; . .-- ' .'
... '_ .
Insectlcde
mites
aphids
. scale Insects
apples
citrus
!''
Experimental
- ' Use Permit
/ '- ".' ' '
Larv1nf
Union ,
Carbide
511-01-2828
99 '
6/7/79
Insecticide
lepldopterous-
pests
I
.;'.
cotton !
' soybeans ;'
j
Experimental
Use Permit
Pydrln9
fenvalerate
Shell ,
Development
Co.
80115
.
100 '
4/20/79
Insecticide
budworms
bollworm
bollweevil
cotton
vegetables
fruit
. Registered
(uses pending)
a 3-(phenoxyphenyl) methyl (ฑ)-c1s, trans-3-(2,2-dichloroethenyl)-2,2-d1methyl cyclopropanecarboxylate
(approx. 60% trans, 40X c1s Isomers)
h^ '.': .-;-',..,'
b S-(4-chlorophenyl) methyl dlefehylcarbamothloate ' ' > . ,'..';:
c mixture of m-(ethylpropyl) phenyl methylcarbamate and m-(1-methylbuty1) phenyl irethylcarbamate 1n
approx. ratio of 1:3 I ' '
d 0,0-diethyl 0-(3,5,6-tr1chloro-2-pyr1dyl)-phosphoroth1oate
e N-[2-chloro-5-(trifluoromethyl) phenyl]-2,4-din1tro-6-(tr1fluoromethyl) benzenamlne ':
f ethanimidotMoic add, N.N1 -Cth1ob1s[(methyl-1m1no)carbonyloxy]]b1s-dimethylester " . '
Jano (3-phenoxyphenyl) methyl 4-chloro-alpha (1-methylethyl) ben^pacetate
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Appendix -B
Composition of mixes added to algal growth media 2.
\ - -
Nutrient Mixes Amount per-liter
Metal Mix3
FeCl3-6H20 0.048g
MnCl2'4H20 O'.144g
, ZnSO,'7H~0 . -. ' 0.045g
2
CuSO/,'5H90- ' 0.157mg
2
CoCl2'6H20 - 0.404mg
H3B03 : . , - . 1.140g
Na2EDTAd . Ig
Glass-distilled or deionized water lฃ
Vitamin mix
Thiamin hydrochloride 50mg
. . Biotin . ... ". . O.Olmg
B12 O.lOmg
Glass-distilled or deionized water 100mฃ
Minor Salt Mixc
K3P04 3.0g ;
NaN03 50.Og
Na2Si03'9H20 20.Og
Glass-distilled or deionized water U
3Add 15.0 nu/ฃ of test solution.
"Add 0.5 nu/ฃ of test solution.
cAdd 1.0 mi/a of test solution.
ฐEDTA added only to stock medium.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO. 2.
EPA-600/4-81-076
4. TITLE AND SUBTITLE
Initial Toxicological Assessment of Ambush, Bolero, Bux,
Dursban, Fentrifanil, Larvin and Pydrin: Static Acute
Toxicity Tests with Selected Estuarine Aglae, Invertebra"
7. AUTHOR(S)
Borthwick, Patrick W. and Gerald E. Walsh
9. PERFORMING ORGANIZATION NAME AND ADDRESS
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory Pi
Office of Research and Development
Environmental Protection Agency
Gulf Breeze, Florida 32561
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE 1
;es, and Fish
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
ACBE1A
11. CONTRACT/GRANT NO.
In-house
13. TYPE OF REPORT AND PERIOD COVERED
oblem Oriented Rebort5/79-8/81
14. SPONSORING AGENCY CODE
ORD-600
15. SUPPLEMENTARY NOTES
16. ABSTRACT
; Selected static toxicity tests were conducted -with /
Dursp'anฎ, Fentrifanilฎ, Larvinฎ,. and Pydrinฎ to determine
species representing four major phyla. Algal bioassays v
algae to determine the concentration of pesticide that we
growth by 50% in 96 h." "Static toxicity tests with molluj
concentration of pesticide that would cause 50% of the e>
abnormally in 48 h. Static acute lethality tests with cป
determined the concentration of: pesticide that is- lethal
organisms during a 96-h exposure.
\mbushฎ, Boleroฎ, Buxฎ, -. '
2 the sensitivity of
vere conducted with marin'e"
Duld inhibit population
;k larvae estimated the
';('.' ' ' '; ' ' L. " -" 0 " -' "- f. /1 '; ..I. '
17.. .: ^,. ... , .., - .- ,.\. .,"-," KEY WORDS AND DOCUMENT ANALYSIS
a. - -. .DESCRI.PTp.RS b.lDENTIFIERS/OPEN^ ENDED TERMS
.,,.,,..
.<.".' '"hC '-. '-.-' ' ~ '.'".'': : ' . L 5P'* '.'.'"',.. C .V: '".'?"'.' ". ' ('',- . ;. . '-. ; .' '
18. DISTRIBUTION-STATEMENT, :. ฃ p .- Crl . '. .i ' ' L'T !.. ' 19. SECURITY CLASS fT/iu /{tporf/ .-.
-', ' . "- " 'c ^v. -..,-.., v":-v. -",. -Unclassified -.
Rel ease to ' pub! i C ' ..' 20 SECURITY CLA
'" ' . ' - J. ;- '": " ' '' . Unclassifiec
SS./Tllis pagei .. . ~*
I -
- -
c. COS AT I Ejeld/Group
21. NO'. OF PAGES
9
22. PRICE
EPA.Form'2220-1 '(Rev. 4V*77)". ;' P.kevibys E'piT.iON.f.
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