Document xdrRZOdq3qZJrmMaEQX36Gmzb
ACUTE TOXICITY TO AQUATIC INVERTABRATES (DAPHNIA MAGNA)
TEST SUBSTANCE
Identity:Perfluorooctanoiaccid,ammonium salt;may also be referredto as PFOA ammonium saltA,mmonium perfluorooctanoateP,FO, FC-1 16, FC-126, FC-1 69, FC-143, oras a major component of FX-1003. (Octanoicacid,pentadecafluoro-a,mmonium salt, CAS # 3825-26-1)
Remarks: The 3M productionlotnumber was 2327. The testsample is FX-1003. It'psudtywas notsufficientclhyaracterizedt,hough current informationindicatesitis-asolutionof<45% ammonium perfluorooctanoat5e0,% water,<3% inertpernuorinatedcompound and I -2% C5 and C7 perfluoroanalogue compounds.
The followingsummary appliesto the testsample as a mixture of the testsubstance in water solutlonwith incompletely characterized concentrationsof impurities.Data may not accuratelyrelatetoxicity of the testsample with thatof the testsubstance.
METHOD:
Method: OECD 202 Type: Staticacute GLP: Yes Year completed: 1990 Species: Daphnia magna Supplier:EnviroSystems,Inc.in-houseculture Analyticalmonitoring:Temperature,pH, conductivitayn,d dissolved
oxygen Exposure period:48-hours Statisticamlethods: Non-linearinterpolationm,oving average, or probit
analysis Test organism age: <24 hours oldand produced from daphnids thatwere maintainedunder testconditionsforatleast14 days. Pretreatment: None.
Test conditions:
Dilutionwater: Hampton, New Hampshire wellwater
Dilutionwater chemistry:
pH:
8.2
Conductivity: 900 pmhos/cm
Hardness:
180 mg/L (as CaC03)
LightinCgo:ol-whiftleuorescbeunltbswithanintensiotf4y5 uE!M--2l A dailyphotoperiodof16 hours lighatnd 8 hoursdarkwas maintained throughoutthetestingperiod.
Test conditionsremarks: Aerationwas notrequiredto maintain dissolvedoxygen concentrationasbove acceptablelevels.
Daphnids were fedyeast,troutchow, and thefreshwateralga Selenastrumcapricomutum once dailyduringculturingb,utwere notfed duringthetest.
Stock and testsolutionpreparation:Test solutionwsere createdby directweightsaddition. Concentrationsdo-singrate:Once Stabilitoyf thetestchemical solutions:Not noted Exposure vessels: 250 mL glassbeakers containin2g00 mL test .soluti(o-n7 cm depth). Number ofreplicatesf:our Number ofdaphnids per replicate5: Number ofconcentrationsf:iveplusa blankcontrol Water chemistryduringthe study:
Conductivityrange:(0-48hours) 800 - 900 pmhos/cm (controelxposure) 800 - 900 pmhos/cm (1,000mg/L exposure)
pH range (0-48hours): 8.1- 8.2(controelxposure 8.3- 8.4(1,000mg/L exposure)
Temperature range (0-48hours): 19.5- 20.1*C (controelxposure) 19.5- 20.1 OC (1,000mg/L exposure)
Dissolvedoxygen range (0-48hours): 9.0- 9.5mg/L (controelxposure) 9.1- 9.5mg/L (1,000mg/L exposure)
RESULTS
Nominal concentrations: Bk control1,50,250, 400, 600, and 1,000 mg/L Element value: 48-hour EC50 = 584 (400 - 1,000)mg/L
Element valuebased on nominalconcentrations.
Remarks: Testingwas conductedon themixtureofthetestsubstanceas describedinthetestsubstanceremarksfieldT.he valuesreportedapply tothatmixtureand notthetestsubstance.
CONCLUSIONS
The testsample 48-hour EC50 forDaphnia magna was determined to be 584 mg/L witha 95% Confidence Intervaolf400 to 1,000 mg/L.
Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133
DATA QUALITY
ReliabilitKyl:imischranking2. Testingmeets the criterifaorquality testing.However, sample puritywas not properlycharacterizedand it lacksanalyticaclonfirmationoftestsubstance concentrations.
REFERENCES
Test was conducted by EnviroSystems,Inc.ofHampton, NH atthe requestofthe 3M Company, 1990. The EnviroSystems study number was 9013-3
OTHER
Last changed: 5/25/00
Static Acut* Toxicity of FX-1003 to the Daphoid, Dephnis magas
Authors Timothy J. Ward Robert L. boeri
Study Completed April 9, 1990
P*rf ormina Labora r Znviro3yatess, vivi les*urce'Analyst
Box'778 One' La f4YOtte Roa
or 9013-@
1. GOOD LABORATORYPITACTICISTATZKUT
This study was conducted according to OZCD Good Laboratory Practice Regulations. Neither the Study Director nor the sponsor are aware of any circumstances that would affect the integrityof this study. No deviationo &xisted that significantlyaffected the validity of the study.
Tirth
v
Au boryani Study Director
R6bert L. 87oeri Coauthor
fto
anne P. liag!@% Aquatic Toxicol st
PPvttoer-rL,L. Kowalski Aquatic,Toxicologist@
@@@Zoviroiystems.Study x w&bor 9013-3
CZRTIFICATion or OOD LABORATORY PRACTICES
Submitted by:
. EaviroSystons Division Resource An lYStS, Incorporated P.O. Box 77a8
-H0:aaptoLna,fayNeotwteRR&oRad
Pshire 03842
Certification: Data presented in this report were dh and with materials Identified in arived by methods a section of the report entitled .*Xethodsand Xaterials." The test was
performed in accordance with EnviroSysteas Protocol
9013-3 and the Product RegistrationAquatic Toxicology
Ltaobxoircaittoyry Standard operating Procedures Xanual. The Kowalski, t;st was performed by Jeanne Xagazu, Peter
obert Boeri, and Timothy Ward. The original
rcaewrtidfaitead and final reportwere sent to the sponsor. A
copy of the raw data and final report will be
archived years.
at
Resource Analysts, Inc.
for at least 10
All data transcribed from the raw data to the report
were chocked ,for accuracy,and all data were verified
by Quality Assurance Auditors. Quality assurance audits
w19e9r0e..performed,on,January 30, _rebruary 13, and Harch 9,
Audits ,.irirereported to the Study Director on
January 31,.:February,13, were reported to
and
Narch 9, 1990.
Audits
1990.
management on Xarch 19 and April 18,
panette@ Te Glen]burg Quality Assurance Auditor
i,4
e_
Dore Newhouse Quality Assurance Of ficar
-virosystons
7@
4'
irucy,Nunber.9
Ill. TABLZ Of conws
SZCTION:
PACE
Good Laboratory Practice Statement
2
Certification of Good Laboratory Practices
3
111. Table of Contents
4
IV. Index of Tables and Figures
5
V.
Summary
6
Vi. Introduction
7
V'Il. Metbods and Materials
7
viii.,Results
10
IX. Ref or onces
13
Appendii
ater Quality Data from Toxicity'T
4f
@kA
tongstudy-mus ber,9013-@i@ 16
inzi OF TABLZS AND FIGUES
Table 1.
Chemical characterizationof a representative saws of natural wall water used as dilution water-for toxicity test
Table 2. Survival data from toxicitytest
Table 3. Median effective -concentrations(ZCSOS) from toxicity test
Table A.l. Conductivity, ps, temperature, and dissolved
Oxygen concentration measured during toxicity test
PAGZ 8
11 12 is
r-,orsy"sats-*@'Study numb* 16"
zs
W4.
d
sumnity
The &cut& tox
described in this f,,4'1clrteypoOrft.YX-1003 to kbe daphD14,paphei,
R4e8sohuoricres duringF*bruary12 to 1T4bv test wax conductedfor 3H ComAptaengyilfeoi,rs
jinalystsI,nc. in Haupt1 1990' at the EaviroSyst*nsDivision
J*""O
on' New Hampshire. it was
of
the protn0acgo4t-z-ud,ePv**tloorpKefodowralEsnkvii,rRooSbyesrtteDn,oeri,and
7,1OthY Vard Accocndourcted 9
by
Tle
Study Numbe1r1 013-3.
ding to
concentrationst*sot1f188 Prforned
temperature
tsst substan
under tatic conditionswith fiv
of 19.90C. The collected fr
cO and a dilutionwatercontrolat a 0eare
to maintain0d8iwseslollsvaetd Hampdtiolnu#tNieownHwaamtpesrhwiarsef.i)ltetorDneawatarustrenadlotwglwlater
Nominal concentrations Oxygen concentrations abov 250 gg/L,
OPloyed
400 ag/L, 600 Of YX-1003 war,: o gll,e(&cDonAtcrcoelp)t,abl: level
used for all calculationmsg./L, and 1,000 mg/L.
iso
.
Nominal concentrationswar;
Dephaids used
Of the test.
main
in tb@ testwerelessthan24 hoursold At the start
They were producedfrom au in-houseCulturethatwas
t4ined under test conditionsfor at least14 days. After 48 hours of
exposure the control daphaidshad an averagewet weight (blotteddry) of 0.0007 g, resulting1. a loadingrate Of 0-0189/L
gteosotd sucbosntdaintcieorneastutlhteedbienginningof the study. Expo* All daphaids were in a 48 hour sc5o of 584 mg/L3uFrXe_1o0f0d3a.phnids to the
T
uv I os 0o
t*1M6E
i@tudY-Nuabee
9 13@;3
Vi. iirmoDUCTION
This study was sponsored by )K Company, St. Paul, Minnesota. The' objective of the study was to determinethe &cut* toxicityof Irz-1003to the d&Pbuid,a freshwateirnvertebrate.The reportcontainssectionsthat describe the methods and materialsemployed in the study,and the results of the investigation.The reportalso containsan appendixthat presents the water qualitydata collectedduring the test.
TEST SUBSTANCE:
VII. METHODS AND MATERIALS
TI-1003 (EaviroSystessSample Number 2308E) was delivered to
EnviroSystesson January 29, 1990. it was containedin a 500 aL plastic
bottle that was labelledwith the followinginformation:G2882-1. rx-1003 (a yellow liquid) was suppliedby 3H Company,035 Bush Avenue, St. Paul, Minnesota. Prior to use the test material was stared in the dark at room temperature. A reserve sample (approximately 1 gram) will be archived at
EnviroSystess for a minimum of 10 years.
DILUTION WATER:
Water used for acclimationof testorganismsand for all toxicity testingr was collected from wells at EnviroSystemsin Hampton, New Hampshire. Water was adjusted to a hardness of 180 mg/L as C&COZ and stored in 500-gallonpolyethylenetanks,where it was aerated.Resultsof
chemical analysis of a representative sampleof water are presentedin Table 1.
TEST ORGA]TISM:
.4
Juvenile dapbuids employed as test organismswere from a single
source and were identifiedusing an appropriatetaxonomickey.
used in the test were less than 24 hours old at the startof the test.
They .were producedfrom an in-houseculturethat was maintainedunier,tast
conditions for at least 14 days. Control daybuids were weighed at the
conclusion of the toxicity test. Prior to- testing, daphnids1,we
B&iutain*d in 100% dilutionwaterunderstaticconditionsIn 4 liter lass-,;,.
During -acclimationdaphnids were not treated for disease an 0
were' free of apparent sickness, injuries, and abnoraalities-'a
@:"7
beginning-@of the test. During the acclimationperiod 24 hours r
the test initiatiotnhe temperature was 19.BOC. Dapbuids were fe as @i trout chow, and the freshwater alga, Solonestrus capricorj3Uust.., C .",,dailbyefore the test.
UviroSystans Study Number 9013-3 -,Page.@7of 16
ROD" N-Vft w- amfty of
Table 1:
ChGaical characterization of a representative sample f natural well water used as dilution water for toxicity tgsto
Parameter
Conductivity Bardness Organochlorice pesticides
unit of N*asurement
Reporting Limit
pw units
uahos/ca
ug/L as CaCO2
ug/L
0.5
Measured Value 8.2 900 ISO ND-
Opregsatniocpihdoesspborus uo/L ND&
POIYChlorinated biphenyis
ug/L
0.5
ND&
anot detected above the reporting limit.
;rE-nv: rosyst, as Study N usbar..-9013@@3
o f 16
7;,4
4t"4
TOXICITY TESTING:
A screening test with the test substancewas conducted during -February 6 -- 8, 1990. Nominal concentrations of test substance were 0.1 mg/L, 1 mg/L@ 10 no/L. 100 mg/L, and 1,000 mg/L. After 48 hours of exposure there was 60% survival at 1,000 mg/L and 100% survival at all other tested concentrations.
The definitive toxicity test was performed during February 12 14, 1990, according to EnviroSystess Test Protocol 9013-3 (Static,
Acute Toxicity of TX-1003 To The Daphnid Dapbais magna), which wa0s, signed by the Study Director on February 6, 2989. It is based
procedures of the OECD (1984).
The test was conducted at a target temperature of 20 t IOC with five concentrations of test substance and a dilution water control.
No stock solution was prepared as test material was added directly to
dilution water contained in the test vessels without the use of-&
Solvent.
Nominal concentrations of the test material were: 0 mg/L
(control), 150 mg/L, 250 mg/L, 400 mg/L, 600 no/L, and 1,000 mg/L.
Twenty dapbnids were randomly and equally distributed among four
replicates of each treatment. The test was performed in 250 al glass
beakers that contained 200 al of test solution (water depth was
approximately 7 ca). Test vessels were randomly arranged in an
incubator during the 49 hour test (a random numbers table was used to select the location of each vessel). A 16 hour light and 8 hour dark
photoperlod was automatically maintained with cool-whit* fluorescent
lights that provided a light intensity of 45 uZs
Aerat on
was not required to maintain dissolved oxygen concentrations above
acceptable levels. Daphnids were not fed during the test.
The number of surviving organisms and @the occurreac e' of'
immobilization (inability to swim within 15 seconds after gentle agitation) or other sublethal effects (lossof equilibrium, errati@'
loss
behavior) were and 43 hours.
of reflex, excitability, discoloration, or ebango'irii,.,, determined visually and recorded initially and after 24,
Dead test organisms were removed when first obso-----
Dissolved -oxygen (YSI Model 57 aet*r; instrument number PRL-3), pa@
(Beckman model pRi 12 eater; instrument number PRL-4), conducti wity,
(Labcoup SCT motor, instrument number PRL-9), and temperature (ASTK
"rcury .'thermometer,
tb*rnom*t*r number 2040) were measured and
recorded daily in *ach test chamber that contained live animal s.,:@
STATISTICAL )=RODS:
Results o f the toxicity';tost were nterproted by xiiid rd miiiti'itic4l.,t,e,chniques :(Stophan,:1983). The probit, moving avo 'a
lnear' lnt*e@rpolationm,ethod was"used by the author to-calculat bmr ZCSO (based.,o:n".imwbi lization) using nominal concentra test substance
48 8 of
vi o Optess
Study'Number, @,;.,'.'t--,
1-13..-.1...
5
I of 26
Vill. RTSULTS
Jkll test vessels containing FX-1003 maintained a clear
appearance throughout the test.
Biological and water quality data
generated by the acute toxicity test are presentedIn Tabl* 2
and
Appendix A, respectively. control .*xpaturo.
on& hundred percent survival occurred in the
Control dapbnids had an average vot weight (blotted
-dry) of 0.0007 9 at the end of the test. Loadingrate duringthe
toxicity test was approximately 0.018 9/L. Exposure of dapholds to the reforence..toxicantsodium dodecyl sulfate resulted in a 43 hour LC50 of
33 mg/L, and a 48 hour EC50 of 16 mg/L.
The 24 and 48 hour EC509 for dapbuids exposed to FX-1003 ar presented in Tabl* 3. The 48 hour ECSO is 584 mg/L FX-1003.
ZoviroSystess Study'Ru'm"i Fags 10 01-16
eel,
kb"W
Table 2. Survival data from toxicity test
Nominal Concentration
(mg/L)
-rep.
Number Alive 0 hr 24 hr 48 hr
Number Affected
Ohr
24br 48br
0 (control) 1 2 3 4
150
1
2
3
4
250
1
2
3
4
400
1
2
3
4
600
2 3 4
11000
2 3 4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
4
5
5
5
5
5
3
5
5
.4
5
5
5
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0-
0
.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
2
0
-0
2
0
0
2
0
0
3
0
0
1
.............................. AsIeKc*oCntdesd aofrtgearnigsemnstlewearegiitmamtoiboinl)ized (unable to,swin witbin 15
ZOilrONYstftsStudy Xunb*r.9013-@3
page 11 of..is 7@
Ariai^kr-&"dkxy of
Table 3. "Odisu Off*ctiv* concentrations (ZCSOS)from toxicity t-*Bt'@
Zxposuro period
95 percent
ZC50
confidence limits
Calculation method
24 hours )1,000 mg/L
48 hours
584 mg/L 400 1,000 mg/L
nonlinear interpolation
. .. .. 4A
DMUJPOK
IX. RZMZNCIS
7
OZCD.
1981. Decision of the Council Concerning the mutual Acceptance of
Data in the Assessment of Chemicals. Annex 2. OZCD Principles of Good
LaboratoryPractice. -C (81)30 (lPinal).
ozcb.
1984. -Guidelines for Testing of chemicals. Section 2: Zffects on
Biotic systems. -M*thod 202, Daphaid sp., Acute Imobilisation Test and
Reproduction Test. Adopted April 4, 1984.
Stephan, C.Z. 1983. Computer program for calculation of LC50 values. Personal communication.
490,13 of
-St'"d
z
APPOndiXA: WATU OUALITY DATA "ON TOXICITY TSST
d, ZmiviroSyst*ass'tiay Nuab*r go
6f"l6 my;
Table X.J. Conductivity,pg, tesperaturt and dissolvedoxygen concentration measured during toxicity test
Nominal Conductivity (usho/ce) PH
concentration
0
24
48
(ag/L)
Rep.
hr
hr
hr
0
24
48
hr
hr
hr
(control) 150 250 400 600 1 006
1
900
Soo
2
900
too
3
900
Soo
4
900
800
1
900
Soo
2
900
Soo
3
900
too
4
900
goo
1
900
Soo
2
900
goo
3
900
Soo
4
900
goo
1
900
goo
2
900
$00
3
900
Soo
4
900
Soo
900
2 -.4 -,900"-,
3
900-
4." @900
@i7., 1900
2
900
3
00
4
-'900
1
$00 800
800
800 800 Soo
$001@ r,
900
8.2
8.1
8.2
900
8.2
8.2
8.2
900
8.2
8.2
8.2
900
9.2
8.2
8.2
900
8.2
8.2
8.2
900
8.2
9.2
8.2
900
9.2
8.2
8.2
900
8.2
8.2
8.2
900
8.3
8.3
8.2
900
8.3
8.3
1.2
900
8.3
8.3
8.2
900
8.3
8.3@
8.2
900.
8.3
8.3
8.2
900
3.3
8.3
8.2
900
8.3
8.3
8.2
@goo
4.3
8.3
8.2
900 900 900 900 @'r
83
8. 3'*
'-.-8.3 @@'.,8.3
S.3,
8.3
'@'8 3@... 8 3
8.3
S. 3
8.3 l@ .@.,-8.3
9
9o o i, 900. 900
a4
8 '3
8.4.
8.3
',4.8.4-t@Q@"-I 'S 3
8.4
..8.3
83 93 83 -ZF8 3
'rableii.l. Continuld.
Youlual concentration
(mg/L) xep.-
7toPerature (oC)
0
24
48
br
hr.
br
0 (control) 1 2 3 4
150
1
2
3
4
250
1
2
.3
4
400
1
2 3
4
600
1
2
3
4
l,ooo
2 3 4
20.1 20.1 .20.1 20.1
20.1 20.1 20.1 20.1
20.1 - 20.1
20.1 20.1
20.0 20.o 20.0 20.o
20.o 20.0 20.o 20.0
20.0 ' 20 20:00 20-0
19.5 19.5 19.5 19.5
19.5 19.5 19.5 19.5
19.5 19.5 19.5 19.5
20.1 20.1 20.1 20.1
20.1
20.0 20-0 20 o 20:0
20.0
19.5 19.5
19-5 '19.5
19
20.1 20.1 , :
20 o *
'.
@-'I19
20. 0
19 5
220 "-@2 0 0" 9
-2 U JL
0
20 1
DissolY&d Oxygen (no/L)
0
24
da
ur
hr
hr
9.5 9.5 9.5 9.5
9.5 9.5 9.5 9.5
9.5 9.5 9.5 9.5
9.3 9.3 9.3 9.3
9.5 9.5 9.5 9.5
9.3 9.3 9.3 9.3
93
9.3 93
9.3 9.3 9.3 9.3
9.3 9.3 9.3 9.3
9.3 @9.3 9.3 ''9.3
P.3 9.3 93 9.3
9.0 9.0 9.0 9.0
9.1 9.1 9.1 9.1
9.1 9.1 9.1 9.1
9.1 9.1 9.1 9.1
9.1 9.1 9.1 9.1
91 9 1. 91
i4
Fcaivgilo r1o6sysOtf"s16 Stu dy
Invirosyxtems StudY liumber901
Eff*ct'v* Datf: January, 1990
ITESOURCI ANALYSTS, INCORPORJTTZD ENVIROSYSTEMs Dry,SIOH
AQ?UJTAOTDIUCCT TOIXtIECGOILSTO]GtYATsrToUmDLyABOPIITtOATTOOCiOtLy
StaticAcut* ToxicitYTest i
And
The
Daphaid,
V th FX-1003
DINPh,7i& Magna
3N compa., PO Box 33428 St. Paul, Minnesota 55133
. SPOusor APP'rov&I Of Pr to 1
Dte
t
v roSYstems, Inc
Lafa ampton
Ye'tWtHs
)toad
at
ati
on Date,. j arluary, 1990
S est,conpIe,ti6nDit@ March,, tudy Direiti
mothy@'J ard. study Dir*ctpr..APproial..-@i......
Date
0f 6 -
1-HISISAC F-FMFIED TRUE Copy,
RE4ZW_DAT@of
Envirosystomsstudy iiu@-ba9r013-3
1.0 'TITLF.
Static Acute me"&
To)xicitY Test With FX-1003 And The Daphnid, Daphnis
2.0 PURPOSE..
To determine the 24 hour and 48 hour median lethal concentrations (LC50s) and median effective concentrations (EC50s)of the test substance to the daphaid, when exposed for 48 hours under static conditions.
3.0 TEST MATERIAL
@3.1 The test substance and related stability and purity data will be supplied by the spomror. Test material will be stored at 2-4*C in the original shipping container unless alternate storage conditions are requested by the sponsor. Handling of the test material will be in accordance with information contained in the sponsor-suppliedMaterial Safety Data Shoot and RAI Standard Operating Procedure 01-02-1106. A subsample of test substance (approximately1 gram) will be removed from the original container and transferred to a glass vial for archiving. All unused test substance will be returned to the sponsor.
3.2 Calculations are based an nominal concentrationsof the test substance. Test substance stock solutions are prepared In deionized or dilution water without the use of a solv(!nt(carrier)if possible.If a solvent is required, dinethylformamide, triethylene glycol, or acetone will be used. The concentration of solvent will be limited to 100 mg/L.
4.0 TEST SPECIES
4.1 The selection of test species is determinedby the sponsor. Healthy
juveniles (free from known diseases) from a single source will be used to
initiate the test. They will be obtained from an in-houseculture with a
known history and will be less than 24 hours old at the initiationof the
test. The the test.
wet weight of
control organisms will be
determined at
the
end
of
4.2 Identification of the appropriate taxonomic keys.
t'est animals shall be verified using
PRETEST OBSERVATIONS AND PROCEDURES
5 1 Pretest receipt
observation
the source, handling procedures
-@'date'"disease*@@treatment (ifany) 'health,feeding, and mortalit
of test aninals,will be recorded and reportd.
Pago,2 of 6'
R,,Ot$M
IL
En7iro:ystems
study ;I&izb-or9-oii-3
5.2 Daphnids will be maintained
Prior to test in!tis
Under static or replacement condition
lteeamspteraOtnurcee dtahialty wwitIht'obnein utsheed safmeorwattsesrtianngd.at ths same PwhOitlOlpbeeriofdedaandt2
freshwater algae. Daphni&dmciuxlttuuyrera*sots,oftroutTchheowa,nl&aaadlslcu*trioopnhyl and/or-'
test conditions
will be maintained in di
water &6@:
days prior to test(itnegm.perature, hardness,and pbotoperio)d for.at least l@
6.0 EXPOSURE CONDITIONS
6will The test will be conductedunder I " added only at the beginningof thestteastti.cconditionsa,nd testmedia
6me.a2su'r-aDbille uticoonnwcaetnttrra*w-iilolns be natural ground water. It will be free of
specified. in U.S@EPA- (1986,of pesticides, and me*t the requirements
micron
filter and
chemically
1987). Wat*r will betpwaiscseeyd characterized (at least
through
a
(20
at RAI) prior to us*.
early analysis.
6.3 Vattr temperature will be 20 1 10C.
s6a.t4urDaitsisoonl.vAeedratoixoyngewnill ncootncbeneturtaitiloinzed.will be maintained above 70% 6.5 Photoperiod will be light and 8 hours dark. automatically controlled and adjusted to 16 hours
6.6 The test vessels will be 250 al glass beakers
0
test media. Loading rate will be adjusted to assurethaantadcoenqtuaaitne 200 al f
oxygen concentration.
dissolved
7.0 STUDY CONDUCT
7.1 The results of data will be used
a
range
finding investigation and/or
previous
to select the concentration range for the testing-'@,
test. If a range findingtest is
definitive
series of concentrations of,.testconducted animals will be exposed t6'a
control (if required).
substance plus.a.control and a solvi@t@',
7.2 During the definitive test the animals
to at least five,concentrationsof.test substawniclleplbeu@se-xiptocso@netd'rfool@rt4d$irlhu6t6Iroin''@..@. .
water without test static conditions.
substance)
and
a
solvent control (if necessary)" 'd
,
anticipated LCSO and 'TECe5s0t concentrations--will@ ba '@.bcosen,:'to.brackuinttehri-* r
7.3 At least 20 -'animal;@@wlll"'bi
will betiexposed in gro
exposePder'to'each"@t'reatment
a' n "as 3
Ir
of solu on. The ups of 5 animals
v'isiel each containini!20 al
animals,@ will be equally and randomly assigned
replicate test vessels (a random numbers table will be used to choosteo,-!hi
test vessel for each sp ocim*n), and they.will not,be f ed.
pkgo
7.4 Animals will be added to test*mediaafter'test
added and aeration will not be emplo ed.'@
substance
y@,
3 of 6
has"b on
17
d
Env%roCyste=s Study flum@er901)-) 7.5 The highest tested concentration will be I 0/@.
7s.e6aleVdocloantlial0inetress.t substances Vill be tested in completely filled and
7.7 A toxicity test should also be conducted with a reference toxicant and the batch of daphuids used for the test.
7.8 Dissolved oxygen concentration, pH, temperature, and conductivity
will be measured and recorded at the beginning of the test and daily
thereafter in all
those vessels.
test vessels, as long as living animals are present in
7.9 lfortality and sublethal effects (imobility) will be recorded at 24
hour into-rvals during the test. Dead animals will be removed when first observed.
7.10 Data Analysis and Statistical Methods
7.10.1
The LCSO and ECSO values and their 95% confidence intervals
will be calculat*d by the non-linear interpolation, moving average and/or probit methods.
7.10.2
If
toxicity occurs the slope of the dose-response curve
will be provided.
8.0 QUALITY ASSURANCE AND QUALITY CONTROL
8.1 The test will be repeated if any of the following conditions occur:
8.1.1
Control survival at the end of the test is less than 90%,
or more than 10% of the control daphnids are stressed or immobilized,
or any control animals are trapped at the surface.
8.1.2
Temperature in any test vessel is outside of the range of
19 - 21*C, or dissolved oxygen falls below 70% saturation.
8.2 The 'test will be conducted according to OECD Good Laboratory Practice Standards.
w8i.l3l Abellaucdritietdicbayl tphheasQeusilitoyf tAhsesurtaensctecUonnidutc.ted at Resource Analysts Inc
8.4 The Study Director will be responsible for reviewing all
recording any ,changes
data' and fo@r to procedures outlined in this..-
protocol amendment.
protocol in
Protocol amendments will be approved by the sponsor.
31.05yeOarrigsoinrail t dwaitlalwbeilltransafrercrheidvteoadtthReesspoonusrocrAefnoarlayrsctbIiinvci.ngf.or at east
Page 4 of 6
'd
EnviroSystgat Stu4y I:umber901)-3 9.0
9.1 Three original reports ill be prepared
after review of a draft
report by the sponsor. ThWe final report will be signed by the Study
Director and staff that conducted the study and prepared the report, and
by the Quality Assurance Unit Representative.
9.2 The final report will consist of at least the following information:
9.2.1
Title page, including the study title,data requires 0 author, study completion dat*, testing facility,-,:',and sponsor.
9.2.2
Sponsor-supplied confidentiality claims.'
9.2.3 9.2.4 9.2.5
Good Uboratory Practice Statement. Certification of Good Laboratory Practices. Table of Contents.
9.2.6 9.2.7
Summary of test procedures and results. Introduction.
9.2.8
Methods and Materials, including a description of test methods, organisms, dilution water, and statistical'@'-.-. techniques.
9.2.9 9.2.10
Results, including all data from range-finding and.@' definitivetests, LC50 values and EC50 values. References.
9.2.11 10.0 REMEN CES
Appendices, including'-all @ater qualitydata and protocol with amendments (if any).-
e@' as
OECD. 2984. OECD.' Guidelines for Testing of Chemicals' section 2: E
t
@on Biotic Systems..'.Method 202, Daphnis sp., Acute'Tmmobilisa tic
and Reproduction Test. Adopted April 1984.
EPA.
4 0."v.CFR Pi'rt'@791 . Toxi6 Substaiciii.controAlc
Guidelines linal'R@Iii." Federal @"iegister,Monday,-January 6,-1986%@
Section.797@l 300.
'i,
v S" t?Jk G
s-@', 40-;-C@FR
Section 797.1300.,
o -,4tii Pirt'-@i'9*i -i@iic Subata"n-c@es Cootioi ct--'@aTt,
ednesdaye-May 20f 1987"'-@
Page 5 of 6
En,.,ireSycl-2nSsttidyflti-ibl9-0r13-3
SUMMARY OF TEST COIIDITIOIIS
Acceptable Species: Test Type: Test Duration: Flow Rate:. Dilution Vater: Ag*/&120 of Test Organisms: -AccliigitionPeriod-Temperature: Photop*riod: Light Intensity: Number of Concentrations: Dilution Factor: Number of Replicates: Number of Organisms/Replicate: Test Vessel size: Test Vessel Volume: Solvent:
Loading Rate: Feeding: Aeration: Effects Measured: Test Concentration Analysis: Vater Quality Measurements:
Acceptability Criteria:
Daphnis magna Static 48 Hours Not Applicable Natural Ground Vater Juveniles; Less Than 24 Hours Old Continuous 20 1 JOC
16 Hours hight/O Hours Dark Ambient laboratory Lighting 5 or More
!91.6 4 or More 5 250 al or larger 200 al or larger Dinethylformaxide, Acetone, or Triethylene Glycol. Less than or equal to 100 mg/L (0.5 g/L None During Exposure None Death; Immobilization None DO, pH, Temperature and Conductivity at 0, 24, and 48 Hours S10% Control Mortality, Stress,"-or.-.Immobilization Acceptable Temperature Range
Page 6 of 6 d.