Document remvGr5qnXVnBjzrzG2X76deG
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ACUTE ORAL TOXICITY TO TERRESTRIAL INVERTEBRATES (HONEY BEE)
TEST SUBSTANCE
Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3)
Remarks field: The 3M production lot number was 217. The test substance is a white powder. Sample was stored AT 16-20C prior to testing. Purity determined to be 86.9% by LC/MS, ' H-HMR, 19F-NMR and elemental analyses techniques.
METHOD
Method: OECD Guideline 213, EPPO Guideline 170 Test type: Acute Oral GLP: Yes Year Completed: 2001 Species: Apis mellifera L. Analytical monitoring: None - nominal concentrations Test honey bee source: Obtained from colony number 32 belonging to the Central Science Laboratory (CSL), Sand Hutton, York, UK, National Bee Unit. Test honey bee age at study initiation: Young adult Test honey bee type: Worker honey bees, free of acarie, nosema and amoeba. Varroacide treatment: None within the 4 weeks prior to test initiation.
Test conditions Humidity: 65% + 5% Temperature: 25 + 2C Lighting: Conducted in darkness
Stock and test solutions preparation: Test substance: Initial stock solution prepared in analytical grade acetone to a final concentration of 47.8 pg PFOS/pL (nominal concentration). Final test concentrations prepared from dilutions of this solution with 50% w/v sucrose. Resulting acetone concentration was 5%. Reference toxicant: Primary stock solution of dimethoate was prepared in deionized water containing 1 g/L. Triton X-100 to a final concentration of 3.0 pg/pL. Secondary stock solutions were made
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by diluting the primary stock solution in deionized water containing 1 g/L Triton X-100. Final test concentrations prepared from dilutions of these solutions with 50% w/v sucrose. Stability of the test chemical solution: A dispersion test was carried out on an 86 pg PFOS/pL acetone solution before the toxicity study was performed. The homogeneity of the mixture was assessed after 2 hours. The test item formed a clear solution on mixing; after 2 hours at room temperature, slight sediment was noted. For the toxicity test, all solutions were re-mixed prior to use. The contract laboratory considered the solutions of the test doses to be homogenous for the purpose of administration. Exposure vessels: Clean, well-ventilated, inverted petri dishes, measuring approximately 9 cm in diameter. Feeding: During the first four hours of the test, bees provided with 50% w/v aqueous sucrose solutions containing the appropriate PFOS dose. After 4-hours, dosed sucrose removed, and bees provided with 50% w/v aqueous sucrose solutions, continuously available through the end of the exposure period. Number of replicates: Three Number of bees per replicate: Ten Negative control: 50% w/v sucrose Solvent control: 50% w/v sucrose plus 5% acetone Reference substance: Dimethoate Reference substance control: Triton X-100 Number of concentrations: five plus a negative and a solvent control Dose administration: The bees were anaesthetized with carbon dioxide immediately before dosing and gently tipped out onto filter paper and counted into the petri dish cage (drones were discarded). Each group of 10 bees was offered 0.2 mL of a given test concentration or control solution. The dose was measured into a small, pre-weighed, glass feeder within the cage using a variable volume pipette. This volume of solution is equivalent to 20 pL per bee. Dose frequency: Once, for 4 hours of exposure Dose calculation: Feeders were weighed after removal from the cages to determine the dose consumed per bee. Element basis: Mortality
RESULTS_______________________________________________
Nominal concentrations: Negative control (sucrose only), acetone + sucrose control, 0.205, 0.450, 0.991, 2.17, 4.78 pg/bee
Element value and 95% confidence interval: 24-hour LDso = 0.72 (0.60 - 0.85) pg/bee 48-hour LDso = 0.46 (0.32 - 0.55) pg/bee
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72-hour LD50 = 0.40 (0.33 - 0.48) pg/bee
72-hour NOEL = 0.21 pg/bee
All element values based on nominal concentrations
Statistical Evaluation: Probit mortality plotted against the logarithm of dose using the contract laboratory Probit 1 package. A least-squares regression (Finney 1971 ) was fitted to these. The NOELs were estimated using Student's t-test (p<0.05)
Biological observations: There was significant mortality at all doses
above a mean intake of 0.21 pg/bee with a steep dose response between mean intakes of 0.45 and 2.2 pg/bee.
Cumulative percent mortality:
N o m in a l T e s t C o n e ., p g /b e e
4 -h o u rs
2 4 -h o u rs
4 8 -h o u rs
7 2 -h o u rs
N e g a tiv e C o n tro l S o lv e n t C o n tro l
0 .2 0 5 0 .4 5 0 0 .9 9 1 2 .1 7 4 .7 8
0 0 0 0 0 6 .7 30
0 3 .3
0 20 70 100 100
0 3 .3 6 .7 50 93 100 100
0 3 .3 10 60 97 100 100
Sub-lethal Effects - Percent Knockdown (K) or Stumbling (S):
Nominal Test Cone.,
p g /b e e
4 -h o u rs
2 4 -h o u rs
4 8 -h o u rs
7 2 -h o u rs
N e g a tiv e C o n tro l
0
0
0
0
S o lv e n t C o n tro l
0
0
0
0
0 .2 0 5
00
0
0
0 .4 5 0
0
3 .3 (S )
3 .3 (K )
3 .3 (K )
0 .9 9 1
00
0
0
2 .1 7
00
0
0
4 .7 8
1 0 (K )
0
Control response: satisfactory
0
0
Reference toxicant response: satisfactory - dimethoate 72-hour LD50= 0.11 pg/bee
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CONCLUSIONS___________________________________ The potassium perfluorooctanesulfonate 72-hour oral LD50for the honey bee was determined to be 0.40 pg/bee with a 95% confidence interval of 0.3 3-0 .4 8. The 72-hour no observed effect level was 0.21 pg/bee. The dose response was steep between a mean uptake of 0.45 and 2.2 pg/bee. Subm itter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133
DATA QUALITY R eliability: Klimisch ranking 1
REFERENCES This study was conducted at Central Science Laboratory, Sand Hutton, York, UK, under contract by Wildlife International, Ltd, Easton, MD at the request of the 3M Company, Lab Request Number U2723, 2001. OTHER___________________________________ __ _____________ Last changed: 5/1/01
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CENTRAL SCIENCE LABORATORY
SCIENCE SERVING AGRICULTURE, FOOD AND THE ENVIRONMENT
( CONTRACT REPORT STUDY NUMBER HT5602
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS):
AN ACUTE ORAL TOXICITY STUDY WITH THE HONEY BEE
r)
MAFF
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CSL Study Number Mi DOUi
TITLE PAGE
<B L
STUDY NUMBER: HT5602 PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): AN ACUTE ORAL TOXICITY STUDY WITH THE HONEY BEE
REPORT AMENDMENT NO 1
EPPO GUIDELINE 170 OECD GUIDELINE 213
AUTHOR: -fi Paul Wilkins
STUDY INITIATION DATE: 11 September 2000 STUDY REPORT DATE: 23 March 2001
SUBMITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St Paul, Minnesota 55106
USA
Performing Laboratory: EnEvnivriornomnmenetnatlaBl Rio&loDgyTGearomup Central Science Laboratory
Sand Hutton York Y 0 4 1 1LZ, UK
Environmental Laboratory Project Number U2723 Page 1 of 5
CSL
Study
Numbei
l
i
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CENTRAL SCIENCE LABORATORY
Study title
Test facility Study number Number of pages:
REPORT AMENDMENT 1 Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee Environmental R&D Team, CSL, Sand Hutton, York, UK HT5602 ,,5
Signature. Study Director P Wilkins Environmental R&D Team, CSL
Dat(dd/mm/yy)
Authorisation of release of report amendment Signature _____l U t k ^ ____ MMaHnaBgeewment Representative Head Environmental Biology Group, CSL
Date f'U--crx (dd/mm/yy)
This amendment has been noted by Quality Assurance as correction to the report
Signature
L 3JL Icw
Mrs C V Walker QA Auditor Central Science Laboratory
Date CS 1ck|olidd/mm/vv)
Environmental Laboratory Project Number U2723 Page 2 of 5
CSL Study Numbe] BACK TO MAIN Reason for amendment: There was a typographical error on pages 7 and 8 of the final report where the Test Item had been mis-spelt as Perfiiorooctanesulfonate, Potassium Salt (PFOS). This has been corrected to Perfluorooctanesulfonate, Potassium Salt (PFOS). A formatting error had occurred on page 7, which had obscured the length of exposure. This has been corrected.
Distribution
Sponsor's Representative Sponsor's Monitor Study Director CSL Management Representative QA Manager
Ms R Robideau Mr D Palmer Mr P Wilkins MMrrsMRHMBBerwookes
2 copies 1 copy 1 copy 1 copy 1 copy
Environmental Laboratory Project Number U2723 Page 3 of 5
SPONSOR:
SUMMARY
<lLCSL Study Numbei ***BACK TO MAIN
3M Corporation
Environmental Laboratory
935 Bush Avenue
St Paul, Minnesota 55106
USA
SPONSOR'S REPRESENTATIVE:
Ms Rochelle Robideau
LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT:
Central Science Laboratory Sand Hutton, York, Y041 1LZ UK
CSL STUDY NUMBER:
HT5602
TEST ITEM:
Perfluorooctanesulfonate, Potassium Salt (PFOS)
STUDY:
Perfluorooctanesulfonate, Potassium Salt (PFOS):,An acute oral toxicity study with the honey bee
NOMINAL TEST CONCENTRATIONS: Negative control, Positive control, 4.78, 2.17, 0.991, 0.450 and 0.205 pg a.i./bee
TEST DATES:
Range test: 12-15 September 2000 Definitive test: 21-24 September 2000
LENGTH OF TEST: LENGTH OF EXPOSURE: TEST ORGANISM: SOURCE OF TEST ORGANISMS:
LIFE STAGE OF ORGANISMS:
72 hours 4 hours
HONEY BEE (Apis mellifera L.) National Bee Unit CSL Sand Hutton, York Y041 1LZ Adult
ORAL LD50 (72 hrs): STATISTICALLY DETERMINED ORAL NO OBSERVED EFFECT LEVEL (BASED ON MEAN INTAKE):
0.40 pg a.i./bee (95% CL 0.33 - 0.48 pg a.i./bee) 0.21 pga.i./bee
Environmental Laboratory Project Number U2723 Page 4 of 5
INTRODUCTION
lCSL Study Number n iBoAouCzK TO MAIN
This study was carried out by the Environmental R&D Team, Central Science Laboratory c(ConSdLu)c, tfeodrf3roMm C1o2rthpSoerapttieomnbaetrtthoe2C4S*LSefpatceimlitbyerin20S0a0n.dTHhuetptorno,toYcoolr,kp, rUotKoc. oTl haemteensdtsmwenetrse, raw data, all notes to file and the final report associated with this study will be retained in the CSL GLP archives for a minimum of 20 years after submission of the final report. The test item will be disposed of by CSL within two months after issue of the final report.
OBJECTIVE
TPohteaossbijuemctiSvaelto(fPtFhiOs Sst)uaddymwinaisstteoreedvatlouathtee thhoenaecyubteeeor(aAlptiosxmiceitllyifoerfaP)e. rfluorooctanesulfonate,
EXPERIMENTAL DESIGN
The oral ingestion method with administration of the test item in aqueous sucrose solution allows for precise exposure over the 4-hour dosing period. This reflects one main potential route of exposure for bees and other non-target insects: oral intake of contaminated food (pollen, nectar etc.). At the initiation of the test three batches of bees in groups of 10 bees at each dose level received a single dose of the test item dissolved in sucrose. This was removed after 4 hours and replaced with untreated sucrose.
To determine an approximate toxicity a range-finding test was performed. Four doses of the test item separated by a factor of ten were administered to groups of 30 young adult worker honey bees. Five geometrically spaced doses of the test item were then administered to groups of 30 young adult worker honey bees to define the LD50. The toxicity of the test item could not be accurately determined in that trial due to high levels of mortality. Therefore another test (second main test) was carried out with using a lower dose of test item (issued as arepferrorteodcotol aams ethnedmdeefnint)i.tiOvenltyoxthiceitryestuesltts) oarfethreeproarntgede theesrtea. nAdllsedcaotandarme areintatiensetd(hoenrethaeftesrtudy file. At initiation of the test, each group of 10 bees was offered 200 pi of the test item in 50% w/v sucrose. After 4 hrs the test feed was removed and the dose taken by each group of bees calculated. Mortality and sub-lethal effects were assessed at 4 hrs then a further 24 and 48 hrs amftaedreraetmaofvuarlthoefrfe7e2dherrss.aIfntetrhreermaonvgaeltoesfttahnedtedsetffieneidtievres.teAstlal ndoasdedsitoiofnteasltaistseemssamreenrtepwoarsted as pg a.i. (active ingredient)/bee and have been adjusted for analysed content. At the end of the tests any remaining live bees were killed by freezing and incinerated.
* *r"v
Environmental Laboratory Project Number U2723 Page 5 of 5
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CSL Study Number HT5602 TITLE PAGE STUDY NUMBER: HT5602 PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): AN ACUTE ORAL TOXICITY STUDY WITH THE HONEY BEE
REPORT EPPO GUIDELINE 170 OECD GUIDELINE 213
AUTHOR: Paul Wilkins STUDY INITIATION DATE: 11 September 2000 STUDY REPORT DATE: 23 March 2001 SUBMITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St Paul, Minnesota 55106
USA
Performing Laboratory: Environmental R&D Team Environmental Biology Group Central Science Laboratory
Sand Hutton York Y041 1LZ, UK
Report No 1 of 6
Environmental Laboratory Project Number U2723 Page 1 of 25
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CSL Study Number HT5602
3 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Study number: HT5602 Report title: Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee
I, the undersigned, declare that the objectives laid down in the protocol were achieved and that the data generated are valid. As stated in the protocol no samples of dosing solutions were collected for chemical analysis. The report fully and accurately reflects the procedures used and the raw data generated in the above study. This study was conducted in accordance with the UK GLP Regulations SI 1999 (No. 3016). These regulations are in accordance with the Organisation for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice (1997 ENV/MC/CHEM (98) 17). It is my understanding that the UK and OECD regulations meet the following standard: Japan Ministry of Agriculture, Forestry and Fisheries, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, (Tokyo, 1984). The test item was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The Sponsor has stated that the test item is being recharacterized in accordance with GLP. No samples of the dosing solutions were collected for chemical analysis.
Mr P Wilkins Study Director Environmental R&D Team, CSL
Date:.
_(dd/mm/yy)
Environmental Laboratory Project Number U2723 Page 2 of 25
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CSL Study Number HT5602 QUALITY ASSURANCE STATEMENTS Quality assurance inspections of this study were made on the following dates:
Date Activity
08/09/00 Verification of study plan
Range test 11/09/00 12/09/00 14/09/00
Dispensing (test item and toxic reference) Preparation of dilutions Dosing bees Assessments Assessments
Main test 18/09/00 Dosing bees
20/09/00 Assessments
Definitive test 20/09/00 Dispensing (test item and toxic reference) 21/09/00 Preparation of dilutions
Dosing bees Assessments Weighing feeders 22/09/00 Assessments
Date reported 08/09/00 15/09/00 15/09/00 15/09/00 21/09/00 21/09/00 25/09/00 25/09/00
25/09/00
Signed.^lsA Ck.hA.
...............Date.
..(dd/mm/yy)
MQArs ACuVditWoralker Central Science Laboratory
This report has been audited by the Quality Assurance Unit of the Central Science Laboratory.
S ig n e d .. Mrs C V Walker QA Auditor Central Science Laboratory
.............D a t e . | . j .Cl.1... (dd/mm/yy)
Environmental Laboratory Project Number U2723 Page 3 of 25
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I CSL Study Number HT5602 REPORT APPROVAL
SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the
honey bee CSL STUDY NUMBER: HT5602
STUDY DIRECTOR:
Mr P Wilkins Environmental R&D Team, CSL Approval for Report issue MANAGEMENT:
Mr
DATE_______ 7-2>< 3>- 0~1______(dd/mm/yy) Mr M H Bew Head Environmental Biology Group, CSL
Environmental Laboratory Project Number U2723
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CSL Study Number HT5602 TABLE OF CONTENTS
TITLE PAGE.............................................................................................................................................................1
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT........................................................2
QUALITY ASSURANCE STATEMENTS........................................................................................................3
REPORT APPROVAL.............................................................................................................................................4
TABLE OF CONTENTS........................................................................................................................................ 5
S U M M A R Y ...............................................................................................................................................................7
INTRODUCTION.....................................................................................................................................................8
OBJECTIVE......................................
8
EXPERIMENTAL DESIGN..................................................................................................
8
MATERIALS AND METHODS......................................................................................
9
Test item....................................................................................................................................................................10
Toxic reference.........................................................................................................................................................10
Conditions during the tests.................................................................................................................................... 10
Dose preparation..................................................................................................................................................... 11
Dose administration............................................................................................................................................... 11
Observations.............................................................................................................................................................11
Analysis of data....................................................................................................................................................... 12
RESULTS AND DISCUSSION...........................................................................................................................12
Range test................................................................................................................................................................. 12
Definitive test..........................................................................................................
12
CO NCLUSIO NS..................................................................................................................................................... 13 Table 1. Oral LDsoof PFOS from definitive test data......................................................................................13 Table 2. Oral toxicity of dimethoate toxic reference from definitive test data...........................................13 Environmental Laboratory Project Number U2723
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CSL Study Number HT5602
REFERENCES..........................................................................................................................................................13
PRINCIPAL WORKERS....................................................................................................................................... 14
DISTRIBUTION OF REPORT.............................................................................................................................14
APPENDIX 1 (TABLES A 1-A 8)........................................................................................................................ 15
DETAILED ORAL TOXICITY TEST RESULTS..........................................................................................15
APPENDIX 2 ............
23
INTERIM CERTIFICATE OF ANALYSIS FOR TEST SUBSTANCE....................................................23
Environmental Laboratory Project Number U2723
SPONSOR:
SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT:
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CSL Study Number HT5602 SUMMARY
3M Corporation Environmental Laboratory 935 Bush Avenue St Paul, Minnesota 55106 USA Ms Rochelle Robideau Central Science Laboratory Sand Hutton, York, Y041 1LZ UK
CSL STUDY NUMBER:
HT5602
TEST ITEM:
Perfuorooctanesulfonate, Potassium Salt (PFOS)
STUDY:
Perfuorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee
NOMINAL TEST CONCENTRATIONS: Negative control, Positive control, 4.78, 2.17, 0.991, 0.450 and 0.205 pg a.i./bee
TEST DATES:
Range test: 12-15 September 2000 Definitive test: 21-24 September 2000
LENGTH OF TEST:
r riT m nT /ynm m /^fL T m ir
TEST ORGANISM: SOURCE OF TEST ORGANISMS:
LIFE STAGE OF ORGANISMS:
72 hours
_ 41
HONEY BEE (Apis mellifera L.) National Bee Unit CSL Sand Hutton, York Y041 1LZ Adult
ORAL LD50 (72 hrs): STATISTICALLY DETERMINED ORAL NO OBSERVED EFFECT LEVEL (BASED ON MEAN INTAKE):
0.40 pg a.i./bee (95% CL 0.33 - 0.48 pg a.i./bee) 0.21 pga.i./bee
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CSL Study Number HT5602
INTRODUCTION
This study was carried out by the Environmental R&D Team, Central Science Laboratory (CSL), for
3M Corporation at the CSL facility in Sand Hutton, York, UK. The tests were conducted from 12th
I
September to 24thSeptember 2000. The protocol, protocol amendments, raw data, all notes to file and the final report associated with this study will be retained in the CSL GLP archives for a minimum of
20 years after submission of the final report. The test item will be disposed of by CSL within two
months after issue of the final report.
OBJECTIVE
zap
The objective of this Potassium Salt (PFOS)
asdtumdiyniswtearsedtotoetvhaeluhaotneeythbeeea(cAutpeis omraelllitfoexraic)i.ty
of
Perfuorooctanesulfonate,
EXPERIMENTAL DESIGN
The oral ingestion method with administration of the test item in aqueous sucrose solution allows for precise exposure over the 4-hour dosing period. This reflects one main potential route of exposure for bees and other non-target insects: oral intake of contaminated food (pollen, nectar etc.). At the initiation of the test three batches of bees in groups of 10 bees at each dose level received a single dose of the test item dissolved in sucrose. This was removed after 4 hours and replaced with untreated sucrose.
To determine an approximate toxicity a range-finding test was performed. Four doses of the test item separated by a factor of ten were administered to groups of 30 young adult worker honey bees. Five geometrically spaced doses of the test item were then administered to groups of 30 young adult worker honey bees to define the LD50. The toxicity of the test item could not be accurately determined in that trial due to high levels of mortality. Therefore another test (second main test) was carried out with using a lower dose of test item (issued as a protocol amendment). Only the results of the range test and second main test (hereafter referred to as the definitive toxicity test) are reported here. All data are retained on the study file. At initiation of the test, each group of 10 bees was offered 200 pi of the test item in 50% w/v sucrose. After 4 hrs the test feed was removed and the dose taken bfuyrtehaecrh24groaunpd o4f8 bheress acfatelcrurlaemtedov. alMoofrtfaeleidtyeras.ndInsuthbe-lertahnagleetfefesct tasnwd edreefiansisteivsesedtesatt a4n hardsdtihtieonnaal assessment was made at a further 72 hrs after removal of the test feeders. All doses of test item are reported as |ig a.i. (active ingredient)/bee and have been adjusted for analysed content. At the end of the tests any remaining live bees were killed by freezing and incinerated.
Environmental Laboratory Project Number U2723
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The nominal doses administered are shown below.
Range finding oral test dose levels
Dose group
PFOS
Dimethoate
(jug a.iTbee)
(tig ai/bee)
1 103 0.60
2 10.3 0.30
3 1.03 0.15
4 0.103 0.075
Control 1
50% w/v sucrose
50% w/v sucrose
r-m
containing 5% acetone
containing Triton X-100
Control 2
50% w/v sucrose
N/A
Definitive test dose levels
Dose group
PFOS
(tig a.i^bee)
1 4.78
2 2.17
3 0.991
4 0.450
5 0.205
Control I
50% w/v sucrose
containing 5% acetone
Control 2
50% w/v sucrose
Dimethoate (pg ai/bee)
0.60 0.30 0.15 0.075
-
50% w/v sucrose containing Triton X-100 N/A
Estimates of LD50 values were calculated for the main oral test. The no observed effect levels were determined by a statistical evaluation of the mortality data.
Negative controls, solvent controls (5% acetone) and positive control groups (dimethoate as BASF 40 formulation nominally containing 400 g/1) were maintained concurrently to check that the bees were reacting normally to the test item during the studies.
MATERIALS AND METHODS
The methods, species used cedures specified in EPPO
and route Guideline
o1f70a,dmGuiniidsetrlianteioonndTesecsrtiMbeedthinodtshfisorprEovtoacluoal tainreg
bthaseeSdiduep-oEnffpercots
of Plant Protection Oral Toxicity Test.
Products on Honey Bees', and the In order to control bias, bees were
OECD Test Guideline impartially distributed
213, Honeybees, Acute to treatment and control
groups. No other potential sources of bias are expected to affect the results of the study. The study
was considered valid with: a) control mortality of 10% or less; and b) the calculated dimethoate LD50
fell within the OECD standard, i.e. 0.10 - 0.35 ftg ai/bee.
Environmental Laboratory Project Number U2723 Page 9 of 25
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CSL Study Number HT5602
Test item The PFOS (IUPAC Name 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-potassium salt, CAS Number 2795-39-3, white powder, lot 217) used in this study was supplied by Wildlife International Ltd. on behalf of 3M Corporation. Each sample of the material tested in this study was uniquely labelled with the Test Item number NBU105. The test item was stored, as recommended by the sponsor, at 16-20 C prior to use. PFOS was dispersed in acetone (analytical grade) before use due to a low solubility in water (approximately 500 mg/1). A dispersion test was carried out, before the toxicity tests were performed, in which PFOS was dispersed in acetone at the approximate concentration required to deliver the highest doses. The homogeneity of the mixture was assessed after 2 hours. In this report "solution" is used to include material which may be suspended or dispersed. Homogeneity of such solutions of the PFOS were checked visually during dispersion tests before the day of the test and immediately before use. All solutions were re-mixed prior to use. Solutions of the test doses were homogenous for the purpose of administration. The test item formed a clear solution on mixing at the highest dose, after two hours there was a slight sediment at room temperature at a concentration of 86 |ig a.i./pl PFOS (the approximate concentration required to prepare the highest diluted dose in the test). Toxic reference Dimethoate toxic standard (NBU90), BASF 40 lot 37M 9020106 (a blue liquid EC formulation, 37.4% w/w ai, 400 g/1 nominal concentration), was purchased from UAP York on 14/06/00 and was stored at 0-11C (on one occasion the maximum temperature recorded was 14C - this was after the door to the refrigerator had been opened) suitable to maintain stability for 2 years according to information supplied by the manufacturer. Dose and LDS0calculations are for corrected dimethoate concentration based on data supplied by BASF. The honey bee (Apis melliferd) is useful in evaluating the potential hazards of agricultural chemicals to nontarget insects since it is an important pollinator of various agricultural crops. There is also a substantial data base on the effect of agrochemicals upon bees with which to categorize potential hazards. Worker honey bees (Apis mellifera) were obtained from colonies belonging to the CSL National Bee Unit. Bees from the colony 32 were used throughout the tests. Bees were examined prior to the start of the test and shown to be free of acarie, nosema and amoeba. The colony from which the bees were taken had not been treated with a varroacide within the last 4 weeks. Worker bees were collected from the hive by using a small amount of smoke, gently shaking them from the combs and transferring them (40-50 per cage) into cylindrical mesh cages. In the laboratory the mesh cages were placed into the incubator (25 + 2C, 65 + 5% relative humidity) to starve the bees for 1.5 to 2 hrs before the test. Conditions during the tests Test bees were housed in test chambers which are clean, well ventilated, inverted petri dishes, measuring approximately 9 cm in diameter. A small inverted petri dish (approximately 3 ml) to contain the sucrose solution was affixed in each chamber. Each test chamber contained 10 worker bees and was identified by study number, dosage group and replicate. After dosing, test cages were kept in darkness at 25 2C and 65% 5% humidity, except during observations. Temperature and relative humidity within the environmental chamber were recorded continuously during the test.
Environmental Laboratory Project Number U2723 Page 10 of 25
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CSL Study Number HT5602 Dose preparation Doses of the test item were made by preparing a solution of 103 pg a.i./pl (range-finding test) and 47.8 pg a.i./pl (definitive test) PFOS and making a series of dilutions from this. Doses were prepared in acetone and then further diluted in 50% w/v sucrose for dosing. In the range test and definitive test 0.5 ml of each acetone solution was then diluted to 10 ml with 50% w/v sucrose giving a final acetone concentration of 5%. Although this concentration of acetone was greater than recommended in the OECD guidelines (1%) it was required to ensure the highest concentrations remained in solution for the oral test doses. One set of control bees was treated with the same level of acetone in 50% w/v sucrose (0.5 ml acetone in 10 ml sucrose) and a second set of control bees was treated with 50% w/v sucrose. Stock solutions of the positive control, dimethoate, were prepared in deionised water containing 1 g/1 Triton X-100. Stock solutions of 2.80 pg ai/pl (range-finding test) and 3.00 pg ai/pl (definitive test) dimethoate in deionised water containing 1 g/1 Triton X-100 were prepared and a series of dilutions made in 1 g/1 Triton X-100. This series of dilutions (0.5 ml) were then further diluted to 10 ml with 50% w/v sucrose. Control bees were dosed with 0.5 ml 1 g/1 Triton X-100 in deionised water diluted to 10 ml with 50% w/v sucrose. The tests were carried out in parallel with those for PFOS. Samples of the sucrose solution used in the study were analysed by Wildlife International Ltd. and no PFOS background levels were found. Dose administration The bees were anaesthetised with carbon dioxide immediately before dosing and gently tipped out onto filter paper and counted into the petri dish cage (drones were discarded). Each group of 10 bees was offered 0.2 ml of a given concentration (or controls as above), the dose being measured into a small, pre-weighed, glass feeder within the cage using a variable volume pipette. This is equivalent to 20 pl/bee. At each dose for the test item and dimethoate and for each control there were 3 replicate cages of 10 bees. Doses were administered within 2 hours of preparation. After 4 hours the glass feeders were removed and weighed and the sucrose feeders filled with approximately 3 ml 50% w/v aqueous sucrose so that bees had continuous access to sucrose for the remainder of the study. The dose consumed was determined by comparison of the weight of the dose remaining in the glass feeders with the weight of a known volume of the test solutions. Observations Mortality and any bees knocked down, i.e. alive but immobile, or stumbling were assessed when the feeders were removed and 24, 48 and 72 hours after removal of the test feeders (i.e. 4, 28, 52 and 76 hours after the start of the test).
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CSL Study Number HT5602
Analysis of data
Analysis of the data comprised plotting probit mortality recorded after 24,48 and 72 hours against the logarithm of dose (CSL Probit 1 package). A least - squares regression (Finney 1971) was fitted to these. Toxicity was expressed as LD50 in pg a.i. per bee with the 95% confidence limits, NOEL and slope of the response curve. The NOELs (no observed effect levels) were estimated using Student's t-test (p<0.05).
The LD50 was used to classify the toxicity of the test item according to the ICBB (1985). The categories used were:
Highly toxic: Moderately toxic: Slightly toxic: Virtually non-toxic:
less than lpg ai/bee 1-10 pg ai/bee 10-100 pg ai/bee greater than 100 pg ai/bee
Data for the dimethoate 24-, 48- and 72-hour mortality results were analysed using the CSL probit program (Probit 1, version 4).
RESULTS AND DISCUSSION
The results of the oral tests are summarised in Tables 1 and 2 and detailed results are listed in Appendix 1. Further details are given below. The oral toxicity of the test item resulted in a 72 hr LD50 value for the honeybee of 0.40 pg a.i./bee.
Range test
Mortality was observed at all doses from mean intake 1.0 pg a.i./bee with a steep dose response between a mean intake of 1.0 and 10 pg a.i./bee. Sublethal effects were observed as knockdown and stumbling at 4 hrs at the two highest dose levels with these individuals dying by 24 hrs.
Definitive test
There was significant mortality at all doses above a mean intake of 0.21 pg a.i./bee with a steep dose response between a mean intake of 0.45 and 2.2 pg a.i./bee. The 72 hr LD50 was calculated as 0.40 pg a.i./bee (95% confidence limits 0.33 - 0.48 pg a.i./bee). The NOEL was 0. 21 pg a.i./bee at 72 hours based on mean intake data (Student's t-test p <0.05). Sublethal effects were seen observed as knockdown at the highest dose level at 4 hrs with these individuals dying after 24 hours.
The 24, Table 2.
48 and 72 -hour Mortality of bees
contact exposed
LtDo5t0hevatoluxeics
for dimethoate reference in the
in the definitive test are tests allowed calculation
shown in of an oral
LDso of 0.12 pg ai dimethoate/bee at 24-hours and 0.11 pg ai dimethoate/bee at 48 and 72-hours.
These results show a toxicity level within the ranges reported by the OECD guidelines showing the
bees were reacting normally in this test.
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CSL Study Number HT5602
CONCLUSIONS
Exposure of honeybees to PFOS by oral dosing resulted in a steep dose response curve with significant mortality above a mean intake of 0.21 pg a.i./bee and a 72 hr LD50 calculated as 0.40 pg a.i./bee. The NOEL based on statistical analysis of the data (Student's t test p<0.05) was 0.21 pg a.i./bee at 72 hrs.
These results demonstrate the oral toxicity of PFOS as 0.40 pg a.i./bee and according to the ICBB (1985) is classified as highly toxic by ingestion.
Table 1. Oral LDsnof PFOS from definitive test data
Time Hrs
LD50 (pg a.i^bee)
95% Confidence Interval for LDS0
24 0.72 0.60 - 0.85
48 0.46 0.32 - 0.55
72 0.40 0.33-0.48
Estimate of slope of response line 5.0 4.7 4.8
Table 2. Oral toxicity of dimethoate toxic reference from definitive test data
Time Hrs
LD50 (pg a.i./bee)
95% Confidence Estimate of slope of
Interval for LD50
response line
24
0.12 0.10-0.14
6.4
48 0.11 0.096-0.13 7.3
72
0.11 0.096 -0 .1 3
8.1
REFERENCES
European and Mediterranean Plant Protection Organization. 1992. Guideline on Test Methods for Evaluating the Side-Effects of Plant Protection Products on Honey Bees. EPPO Bulletin,
22,203-215.
Finney, D. J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
I
International Commission for Bee Botany (ICBB). 1985. Recommendationsfor harmonization of methodsfor testing hazard ofpesticides to honeybees. Third Symposium on harmonization of
methods for testing hazard of pesticides to bees, England.
Organization for Economic Cooperation and Development. 1997. Guideline 213 Honeybees, Acute Oral Toxicity Test.
Environmental Laboratory Project Number U2723
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CSL Study Number HT5602
PRINCIPAL WORKERS
STUDY MANAGEMENT
Study Director CSL Management Representative
Mr P Wilkins Mr M H Bew
OPERATIONAL SUPERVISORS
Principal Scientist Scientist & Test Item Controller Scientist & Deputy Test Item Controller Scientist Scientist Scientist
Dr H M Thompson M iss U B Pietsch Miss S C Green Mr A H Battersby Dr R J Spinks Mr M J Kelly
QUALITY ASSURANCE
QA Manager QA Auditor
Mrs R M Brookes Mrs C V Walker
DISTRIBUTION OF REPORT
Sponsor's Representative Sponsor's Monitor Study Director CSL Management Representative QA Manager
Ms R Robideau Mr D Palmer Mr P Wilkins Mr M H Bew Mrs R M Brookes
2 copies 1 copy 1 copy 11 ccooppyy
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CSL Study Number HT5602
APPENDIX 1 (TABLES A1-A8) DETAILED ORAL TOXICITY TEST RESULTS Table A l. Results of range-finding oral dosing tests with PFOS - Mortality Nominal Actual dose2 Cage dose1 (jig a.iTbee) No. (pg a.i^bee)
Cumulative number dead (n=10) 4 hrs 24 hrs 48 hrs 72 hrs 103 96 46 5 10 10 10 103 94 47 5 10 10 10 103 101 48 7 10 10 10
10.3 10 49 0 10 10 10 10.3 10 50 0 10 10 10 10.3 10 51 0 10 10 10
1.03 1.0 52 0 0 2 3
1.03 1.03
1.0 53 0 1 1 2 1.0 54 0 0 0 4
0.103 0.10 55 0 0 0 0 0.103 0.10 56 0 0 0 0 0.103 0.10 57 0 0 0 0
O3 0 58 0 0 0 0
O3 0 59 0 0 0 0
O3
0
60 0
0
00
04 0 61 0 0
04
0
62 0
0
o4
0
63 0
0
1based on nominal consumption of test solution of 20pl/bee 2based on amount of test solution consumed (average of 10 bees) 3 acetone dosed 4undosed
0 0 0
0 0 0
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CSL Study Number HT5602
Table A2. Results of range-finding oral dosing tests with PFOS - Sub-lethal effects
Nominal Actual dose2 Cage dose1 (pg a.i./bee) No.
(pg a.i./bee)
Number knockdown (K) or stumbling (S)
4 hrs 24 hrs 48 hrs 72 hrs
103
96
46 4K
0
0
0
103 94 47 1S,4K 0 0 0
103 101 48 1S.2K 0 0 0
10.3 10 49 2K 0 0 0 10.3 10 50 1S,3K 0 0 0 10.3 10 51 2K 0 0 0
1.03 1.0 52 0 0 0 IK 1.03 1.0 53 0 0 0 0 1.03 1.0 54 0 0 IK 0
0.103 0.10 55 0 0 0 0 0.103 0.10 56 0 0 0 0 0.103 0.10 57 0 0 0 0
O3 0 58 0 0 0 0 O3 0 59 0 0 0 0 O3 0 60 0 0 0 0
04 0 61 0 0 o4 0 62 0 0 o4 0 63 0 0 1based on nominal consumption of test solution of 20pl/bee 2 based on amount of test solution consumed (average of 10 bees) 3acetone dosed 4undosed
0 0 0
0 0 0
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CSL Study Number HT5602
Table A3. Results of range-finding oral dosing tests with dimethoate - Mortality
Nominal Actual dose2 dose1 (pg a.iVbee)
(pg a.i7bee)
Cage No.
Cumulative number dead (n=10)
4 hrs 24 hrs 48 hrs 72 hrs
0.60 0.60 0.60
0.56 0.54 0.60
31 3 10 10 10
32 33
0 0
10 10
10 10
10 10
0.30 0.30
0.25 0.30
34 2 10 10 10 35 0 10 10 10
0.30 0.30 36 1 10 10 10
0.15 0.15 0.15
0.15 37 0 8 0.15 38 0 4 0.15 39 0 5
8 5 7
8 5 7
0.075 0.075 40 0 0 0 0
0.075 0.075
0.074 0.074
41 42
0 0
0 1
0 1
0 2
0 0 0
0 0 0
43 44 45
0 0 0
0 0 0
0 0 0
0 0 0
1based on nominal consumption of test solution of 20|il/bee based on amount of test solution consumed (average of 10 bees)
Environmental Laboratory Project Number U2723 Page 17 of 25
Table A4.
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ICSL Study Number HT5602
Results of range-finding oral dosing tests with dimethoate - Sub-lethal effect^
Nominal Actual dose12 Cage dose1 (pg a.iybee) No.
(jjg a.iybee)
Number knockdown (K) or stumbling (S)
4 hrs 24 hrs 48 hrs 72 hrs
0.60 0.56 31 2K 0 0 0 0.60 0.54 32 2K 0 0 0 0.60 0.60 33 7K 0 0 0
0.30 0.25 34 IK 0 0 0 0.30 0.30 35 0 0 0 0 0.30 0.30 36 0 0 0 0
0.15 0.15 37 0 0 0 0 0.15 0.15 38 0 0 0 0 0.15 0.15 39 0 0 0 0
0.075 0.075 40 0 0 0 0 0.075 0.074 41 0 0 0 0 0.075 0.074 42 0 0 0 0
0 0 43 0 0 0 0 0 0 44 0 0 0 0 0 0 45 0 0 0 0
1based on nominal consumption of test solution of 20pl/bee 2 based on amount of test solution consumed (average of 10 bees)
t 4 Environmental Laboratory Project Number U2723
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CSL Study Number HT5602
Table A5. Results of definitive oral dosing tests with PFOS - Mortality
Nominal Actual dose12 Cage dose1 (ng a.iJbee) No.
(pg a.iJbee)
Cumulative number dead (n=10)
4 hrs 24 hrs 48 hrs 72 hrs
4.78 4.8 316 3 10 10 10
4.78 4.78
4.8 317 3 10 10 10 4.8 318 3 10 10 10
2.17 2.2 3 19 1 10 10 10 2.17 2.2 320 0 10 10 10 2.17 2.2 321 1 10 10 10
0.991 0.96 322 0 7 10 10
0.991 0.991
0.96 323 0 0.96 324 0
8 6
9 10 99
0.450 0.450
0.45 325 0 0.45 326 0
1 3
4 7
5 7
0.450 0.45 327 0 2 4 6
0.205 0.21 328 0 0 0 1 0.205 0.21 329 0 0 0 0 0.205 0.21 330 0 0 2 2
03 0 331 0 1 1 1
O3 03
0 332 0 0 0 0 0 333 0 0 0 0
04 04 04
0 334 0 0 0 0 0 335 0 0 0 0 0 336 0 0 0 0
1based on nominal consumption of test solution of 20jil/bee based on amount of test solution consumed (average of 10 bees) acetone dosed 4undosed
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CSL Study Number HT5602
Table A6. Results of definitive oral dosing tests with PFOS - Sub-lethal effects
Nominal Actual dose12 Cage dose1 (fig a.i^bee) No.
(fig a.iybee)
Number knockdown (K) or stumbling (S)
4 hrs 24 hrs 48 hrs 72 hrs
:13
4.78 4.78
4.8 316 IK 0 4.8 317 IK 0
00 00
4.78 4.8 318 IK 0 0 0
2.17 2.2 319 0 0 0 0
r! *
2.17 2.17
2.2 320 0 2.2 321 0
0 0
00 00
_r
\ l
0.991 0.991 0.991
0.96 0.96
322 323
0 0
0.96 324 0
0 0 0
0 0 0
0 0 0
0.450 0.45 325 0 IS 0 0
I
0.450 0.45 326 0 0.450 0.45 327 0
0 0
00 IK IK
0.205 0.21 328 0 0 0 0
0.205 0.205
0.21 329 0 0.21 330 0
0 0
0 0
0 0
0J O3
0 0
331 0 332 0
0 0
0 0
0 0
1 O3 0 333 0 0 0 0
! ^3^
04 0 334 0 0 0 0
O4 O4
0 0
335 0 336 0
0 0
0 0
0 0
1based on nominal consumption of test solution of 20ftl/bee based on amount of test solution consumed (average of 10 bees) 3acetone dosed 4 undosed
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CSL Study Number HT5602
Table A7. Results of definitive oral dosing tests with dimethoate - Mortality
Nominal Actual dose2 Cage dose1 (jig a.iTbee) No.
(pg a.iJbee)
Cumulative number dead (n=10)
4 hrs 24 hrs 48 hrs 72 hrs
0.60 0.60 0.60
0.57 301 8 0.58 302 2 0.60 303 5
10 10 10 10 10 10 10 10 10
0.30 0.30 0.30
0.30 304 4 0.30 305 2 0.30 306 5
10 10 10 10 10 10 10 10 10
0.15 0.15 0.15
0.15 307 1
0.15 0.15
308 309
0 2
9 7 6
10 10 99 67
0.075 0.074 310 0 0.075 0.075 311 0 0.075 0.074 312 0
2 1 1
2 1 1
2 1 1
0 0 0
0 0
313 0 314 0
0 0
0 0
1 0
0 315 0 1 1 1
1based on nominal consumption of test solution of 20pl/bee based on amount of test solution consumed (average of 10 bees)
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CSL Study Number HT5602
Table A8. Results of definitive oral dosing tests with dimethoate - Sub-lethal effects
Nominal Actual dose2 Cage dose1 (jig a.iTbee) No.
(fig a.iTbee)
Number knockdown (K) or stumbling (S)
4 hrs 24 hrs 48 hrs 72 hrs
0.60 0.57 301 2K 0 0 0
0.60 0.60
0.58 302 6K 0 0.60 303 5K 0
0 0
0 0
0.30 0.30 304 3K 0 0 0 0.30 0.30 305 3K 0 0 0 0.30 0.30 306 2K 0 0 0
0.15 0.15 307 0 0 0 0 0.15 0.15 308 0 0 0 0 0.15 0.15 309 0 0 IK 0
0.075 0.074 310 IK 0 0 0 0.075 0.075 311 0 0 0 0 0.075 0.074 312 0 0 0 0
0 0
0 0
313 0 0 0 314 0 0 0
0 0
0 0 315 0 0 0 0
1based on nominal consumption of test solution of 20pl/bee based on amount of test solution consumed (average of 10 bees)
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CSL Study Number HT5602
APPENDIX 2 INTERIM CERTIFICATE OF ANALYSIS FOR TEST SUBSTANCE
IN TER IM CERTIFICATE OF ANALYSIS
CentreAnalytica3lMLaRPbRreooferedvaruistecointor:cnieePs#1:(FC9O/OS7DS/A0,-0L0R)1oe8tf2er1e7nce#: 023-018A
Test Name Purity1 Appearance IdentificNatiMonR Meta234561l......s (MPCNSIIrCooaoiacPdtlngacki/snuieMuselmimsSui)mum2 Tota7l.%MImapnugarintyes(eNMR) TT((LGooCCttaa//llMM%%SS)I)Immppuurriittyy RPOelAatAed Compounds RPuersiitdyubalySDoSlvCents (TGA) Inor4g235671....a... niNCNFBPcSlhuhriAiuottollrrofonmsiakrrtiptiteoiiedheddn4eaeeste(IC) Orga2341n....icTNPHAFFFFcPABPiAdAAs5(IC) Elem2341e....ntaNHCSluiayAtlrdrfbonruoogargnelynesnis6:
5. Fluorine
Specifications White Crystalline Powder
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COA023-0I8A
Page I of3
Environmental Laboratory Project Number U2723 Page 23 of 25
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CSL Study Number HT5602
CeInNtrTe AEnRalIyMticaCl LEabRorTaItForIiCesACOTAEROefFereAncNeAfkL0Y23S-0IS18A Date ofLastAnalysis: 08/31/00
ExpirationDate: 08/31/01
StorageConditions: Frozen<-10C Re-assessmentDate: 08/31/01
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01.0509%%-+(NsuMmRoimfmpuetraitlieims,p1u.9ri3t%ies+, o1r.4g5a%nic+aLcCid/MimSpuimriptiuesr,it0ie.3s,8%8.4+1P%O+AInAo,rganic ToPtuarliitmyp=ur1it0y0%from- 1a3l.l0t9e%sts==8163.9.0%9%
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This workwas conducted under EPA Good LaboratoryPractice Standards (40 CFR 160). /
COA023-018A
Environmental Laboratory Project Number U2723 Page 24 of 25
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CSL Study Number HT5602
CeInNtrTeAEnRalIyMticaCl LEabRorTaItForIiCesACOTAEROefeFreAncNeA#:L0Y23S-0IS18A LC/MS Purity Profile:
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COA023-018A
Environmental Laboratory Project Number U2723
CSL Study Numbt CENTRAL SCIENCE LABORATORY
AMENDMENT TO PROTOCOL
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< te>
Study title Test facility
Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee
Environmental R&D Team, Environmental Biology Group Central Science Laboratory, Sand Hutton, York, Y 041 1LZ
Study number
HT5602
Amendment number: 2
Number of pages: 2
Study Director Paul W ilkins Signature
Date (dd/mm/wl
Management Representative
Medwin Bew Signature
Date I>4- t-M (dd/mm/wl
Environmental Laboratory Project Number U2723 page 1 of 2
CSL Study Numbei
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CENTRAL SCIENCE LABORATORY PROTOCOL AMENDMENT
Amendment No 2 to Protocol HT5602
After clarification by the Sponsor, it was requested that the Test Item name be changed from Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) to Perfluorooctanesulfonate, Potassium Salt (PFOS) and consequently the study title changed from:
Phoernfelyuobreoeoctane Sulfonic Acid, Potassium Salt (PFOS): An acute oral toxicity study with the to
Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee.
This has no impact on the Study
Circulation list
Sponsor's Representative Sponsor's Monitor Study Director CSL Management Representative CSL, QAUnit
Ms R Robideau, 3M Mr D Palmer, W ildlife International Mr P Wilkins, CSL Environmental R&D M rM Bew, CSL Mrs R Brookes, CSL
Environmental Laboratory Project Number U2723 page 2 of 2
V
International, Ltd.
ECOTOXICOLOGY & ANALYTICAL TESTING SERVICES
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ANALYSIS FORPFOS INAQUEOUS AND SUCROSE SOLUTIONS USEDBY CENTRAL SCIENCE LABORATORIES FOR CONDUCTING HONEY BEE TESTING
Enclosed please find a data summary for our screen for PFOS in aqueous and sucrose solutions employed by Central Science Laboratory in the honeybee studies (Sample IDs HT5601/02 Water and HT5601/02 Sucrose). Included are the results table, calibration curve, and standard/sample ion chromatograms for the screens performed on ourtriple quadrupole LC/MS/MS system. As can be seenin the data, the samples were less than the limit of quantitation, 0.0500 mg a.i./L (0.000500 mg a.i./L low standard x sample dilution factor (100)). The screen was conducted on October 26, 2000. These results applyto solutions representative ofthose used in thefollowing studies.
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): An Acute Contact Toxicity Study with the Honey Bee (CSL Study Number: HT5601)
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): An Acute Oral Toxicity Study with the Honey Bee (CSL Study Number: HT5602)
RayijKjnd Scientist
-t/lW ol DATE
MANAGEMENT:
*
WDirilelcatrodr,BA. nNailyxtoicna,lPChhe.ml /istry
DATE '
8598 C om m erce Drive Easton, M aryland 21601 Tel. 410-822-8600 Fax 410-822-0632 E-mail: ecotox@ w lldllfeintem ational.com
European Office: B ergkam pweg 1 7231 CL W amsveld The Netherlands Tel. 31 (0) 575-573848 Fax 31 (0)575-574813 E-mail: aleopold@wortdonline.nl
MManoMQutaTn.hVu,MOioafll 21.t, 2000 IM I
C M o n FI Cal PfOS 02100MO Padi: Madntnh H&APOOOO-PfOJECT 0ATA:4S4C-10l:4#4C101_102a00MO CommanU; PFOS. 4MC-101: MaOisd Dauatopmanl Semnlng M i flaka. Malar, auORM-Q.CMMM44RM)
ms
No Intim ai Standard 499.0->99.1 W ilghtad (1/x) Intarcapc - 323.17 Slop* - B193J2 Corralatkm Coalf. > 0.99949 Uw Ana
filename PFOS_1
PFOS_2 PFOS.3 PFOS.4 PFOS_5
PF06J8 PF08.7 PFOSJ PFOSJ9 PTOS_10
PFOS.11 PFOS_12
PFOS.13 PFOS.14 PFOS_15
Fllotvoa Standard Standard Standard Standard Standard Bank Blank Blank Blank Blank Standard Standard Standard Standard Standard
Sample Nairn STD 0.500 ug a JA STD 1.00 ug a .IA STD 2.50 ug a .lA STD 3.60 ug a JA STD 5.00 ug a JA NEOH OIL SOLVENT RSHFLAKE WATER SUCROSE
STD 0.500 ug a-IA STD 1.00 ug a .lA STD 2.50 ug a J A STD 3.50 ug a J A STD 5.00 ug a .IA
Cone.
0.6000 1.0000 2.5000 3.5000 6.0000
0 .0 0 .0 0 .0 0 .0 0 .0 0.5000 1.0000 2.5000 3.5000 5.0000
Dll. Factor 1.0 1.0 1.0 1.0 1.0 1.0 1.0
100.0 100.0 100.0
1 .0 1 .0 1.0 1.0 1.0
BACK TO MAIN
Cab. Cene. Un
0.4770 ug a .lA 0.9884 ug a.L/L
2.5378 ug a .lA 3.8246 ug a .lA 9.0132 ug a J A
n / a ug a JA n / a ug a JA n / a ug ax/g n / a ug a lA 0.8041 ug a ,IA 0.5087 ug a JA 1.0267 ug a .lA 2.4725 ug a-IA 3.S0S8 ug a .lA 4.8413 ug a .lA
Aceuracv 95.4 98.8
101.5 103.8 100.3 n/ a n/ a n/ a n/ a n/ a 101.7 102.7 98.9 100.3 96.8
A fa
4231.52 8421.33 21116.08 30020.62
41397.58 n/ a n/ a n/ a n/ a
389.06 4491.33 8735.45 20581.27 29079.89 39989.71
Hatght 3 9 3 .9 0 748.57 1925.75 2739.86 3819.74
n/ a n/ a n/ a n/ a 41.96
3 8 7 .0 9 8 2 6 .5 7 1836.54 2538.52 3515.79
Saniote Dasc. 4675A-011D-6 4875A-011D-7 4675A-011D-8 46 75A .01 1D -9 4 6 7 5 A -0 1 1 0 -1 0 IUGCH DASOLVENT 454C -10 1454C -10 1454C -10 14 6 7 5 A -0 1 1 0 -8 4675A-011D-7 457SA-011D-8 4675A-011D-9 46 7 5 A -0 110-10
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Page 2 of 4
M a c Q u a n , v e rs io n 1 .6
P rin te d : T h u , O c t 2 6 , 2 0 0 0 1 2 :5 8 C a lib ra tio n R ie : C a l P F O S 1 0 2 6 0 0 M D
P a th : M a c in to s h H D :A P I3 0 0 0 :P R O J E C T D A T A :4 5 4 C -1 0 1 :4 5 4 C 1 0 1 _ 1 0 2 6 0 0 M D :
C o m m e n ts : P F O S : 4 5 4 C -1 0 1 : M e th o d D e v e lo p m e n t - S c re e n in g fis h fla k e , w a te r, s u c ro s e -(L C /M S /M S -M R M )
BACK TO MAIN
9
P R O S 4 9 9 .0 - > 9 9 .1 N o In te rn a l S ta n d a rd W e ig h te d (1 /x )
In te r c e p t = 3 2 3 .1 7 S lo p e = 8 1 9 3 .3 2 C o rre la tio n C o e ff. = 0 .9 9 9 4 9
A re a
^CERTIFIED COPY OF THE ORIGINAL
^ or>
4 -r c .
M a c Q u a n , v e rs io n 1 .6
Page 3 of 4
P rin te d : T h u , O c t 2 6 , 2 0 0 0 1 2 :5 9
C a lib ra tio n R ie : C a l P F O S 1 0 2 6 0 0 M D P a th : M a c in to s h H D :A P I3 0 0 0 :P R O J E C T D A T A :4 5 4 C -1 0 1 :4 5 4 C 1 0 1 _ 1 0 2 6 0 0 M D :
C o m m e n ts : P F O S : 4 5 4 C -1 01 : M e th o d D e v e lo p m e n t - S c re e n in g fis h fla k e , w a te r, s u c ro s e -(L C /M S /M S -M R M )
BACK TO MAIN
PFO S_1
S T D 0 .5 0 0 u g a .IV L
4 6 7 5 A -0 1 1 D -6
4 .9 8
in 1 p e rio d
PFOS
N o In te rn a l S ta n d a rd
U s e A re a
1: 4 .9 7 M R M , 2 9 8 s can s
4 9 9 .0 -> 9 9 .1
N o is e T h re s .
2 .0
Q u a n t T h re s .
1 .0
M in . W id th
12
M u lt. W id th
10
B a s e . W id th
40
R T W in . (s e c s )
io
S m o o th
2
E x p e c te d R T
3 .7 0
A re a
4 2 3 1 .5 2
H e ig h t
3 9 3 .9 0
S ta rt T im e E n d T im e
In te g ra tio n W id th
R e te n tio n T im e
In te g ra tio n T y p e
3 .5 0 4 .1 7 0 .6 7 3 .7 0 A - BB
T h u , O c t 2 6 , 2 0 0 0 1 0 :3 4
in te n s ity : 4 0 0 0 c p s
PFO S_2
S T D 1 .0 0 u g a .IV L
4 6 7 5 A -0 1 1 D -7
4 .9 8 PFOS
in 1 p e rio d
N o In te rn a l S ta n d a rd
U s e A re a
1: 4 .9 7 M R M , 2 9 8 scan s
4 9 9 .0 -> 9 9 .1 N o is e T h re s . Q u a n t T h re s . M in . W id th M u lt W id th
2 .0 1 .0 12 10
B a s e . W id th R T W in . (s e c s ) S m o o th
40 10 2
E x p e c te d R T
3 .7 0
A re a H e ig h t
8 4 2 1 .3 3 7 4 6 .5 7
S ta rt T im e E n d T im e In te g ra tio n W id th
3 .4 7
4.14
0 .6 7
R e te n tio n T im e In te g ra tio n T y p e
3 .6 9 A - BB
T h u , O c t 2 6 , 2 0 0 0 1 0 :4 0
in te n s ity : 4 0 0 0 c p s
CERTIFIED COPY OF THE ORIGINAL
0; '\v.v*VUD; ** V "-~
M a c Q u a n , v e rs io n 1 .6
ae
0
P rin te d : T h u , O c t 2 6 , 2 0 0 0 1 2 :5 9 C a lib ra tio n P ile : C a i P P O S 1 0 2 6 0 0 M D
13000P a t h : M a c i n t o s h H D : A P
:P R O J E C T D A T A :4 5 4 C -1 0 1 :4 5 4 C 1 01 ,, 1 0 2 6 0 0 M D :
C o m m e n ts : P F O S : 4 5 4 C -1 0 1 : M e th o d D e v e lo p m e n t - S c re e n in g fis h (la k e , w a te r, s u c ro s e -(L C /M S /M S -M R M )
BACK TO MAIN
PFO S_9 4 5 4 C -1 0 1 -
W ATER
T h u , O c t 2 6 , 2 0 0 0 1 1 :2 2
4 .9 8
in 1 p e rio d
PFOS
N o In te rn a l S ta n d a rd
U s e A rea
1: 4 .9 7 M R M , 2 9 8 scan s 4 9 9 .0 -> 9 9 .1
N o is e T h re s . Q u a n t T h res. M in . W id th M u lt. W id th B a s e . W id th R T W in . (s e c s ) S m o o th E x p e c te d R T
2 .0 1 .0 12 10 40 10 2 3 .7 0
A re a
0 .0
H e ig h t
o .O
S ta rt T im e
0 .0 0
E n d T im e In te g ra tio n W id th
0 .0 0 0 .0 0
R e te n tio n T im e In te g ra tio n T y p e
0 .0 0
in te n s ity : 4 0 0 0 c p s
PFO S_10 4 5 4 C -1 0 1 -
SUCROSE
4 .9 8
in 1 p e r io d
PFOS
N o In te rn a l S ta n d a rd
U s e A re a
T h u , O c t 2 6 , 2 0 0 0 1 1 :2 8
1: 4 .9 7 M R M , 2 9 8 s c a n s
4 9 9 .0 -> 9 9 .1
N o is e T h re s .
2 .0
Q u a n t T h re s .
1 .5
M in . W id th
12
M u lt W id th
10
B a s e . W id th
Sm oothR T W i n . ( s e c s )
40
21 0
E x p e c te d R T
3 .7 0
A re a
3 8 9 .0 6
H e ig h t
4 1 .9 6
S ta rt T im e
E n d T im e
In te g ra tio n W id th
R e te n tio n T im e
In te g ra tio n T y p e
3 .5 0 3 .7 7 0 .2 7 3 .5 9 A * - BB
In te n s ity : 4 0 0 0 c p s
CERTIFIED COPY OF THE ORIGINAL
HALS
^ JOT RECEIVED QA AUDIT
