Document DM8Qbz2B6BNVXKgpz03mDXrM
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ACUTE ORAL TOXICITY TO TERRESTRIAL INVERTEBRATES (HONEY BEE)
TEST SUBSTANCE
Identity: Perfluorooctanesulfonate;may also be referredto as PFOS or FC-95. (1-0ctanesulfonic 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 productionlot number was 217. The test
substance is a white powder. Sample was stored AT 16-20C prior to testing. Punty determined to be 86.9% by LC/MS, `H-HMR, "F-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: Obtainedfrom colony number 32 belongingto 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 acarine, nosema and amoeba. Varroacide treatment: None within the 4 weeks prior to test initiation.
Test conditions Humidity: 65% $- 5% Temperature: 25 5 2OC 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 concentrationspreparedfrom dilutions of this solution with 50% w/v sucrose. Resulting acetone
concentration was 5%.
Referencetoxicant: Primary stock solution of dimethoatewas prepared in deionizedwater containing 1g/L. Triton X-lo0 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 Ig/L Triton X-100. Final test concentrationspreparedfrom 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-I 00 Number of concentrations: five plus a negative and a solvent control Dose administration: The bees were anaesthetizedwith 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 yL 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 LDW= 0.72 (0.60 - 0.85) pg/bee
&hour LDw = 0.46 (0.32 0.55) pg/bee
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72-hour LD- = 0.40 (0.33 - 0.48) pg/bee
72-hour NOEL = 0.21 vg/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 NOELSwere 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 pghee with a steep dose response between mean intakes of 0.45 and 2.2 pg/bee. Cumulative Dercent mortalitv:
2.1 7
6.7
100
100
100
4.78
30
I00
100
100
Nwninal Test Conc.,
@bee
&hours
24-hours
&hours
72-hOUtS
I
4.78
0 I
0
I
Control response: satisfactory
Reference toxicant response: satisfactory - dimethoate 72-hour LDm =
0.1 1 pg/bee
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CONCLUSIONS The potassium perfluorooctanesulfonate72-hour oral LDwfor the honey
- bee was determined to be 0.40 pg/bee with a 95% confidence intervalof
0.33 0.48. The 72-hour no observed effect level was 0.21 yg/bee. The dose response was steep between a mean uptake of 0.45 and 2.2 pg/bee.
Submitter: 3M Company, EnvironmentalLaboratory, P.O.Box 33331, St.
Paul, Minnesota, 55133
DATA QUALITY Reliability: 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 ElT5602
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS):
AN ACUTE ORAL TOXICITY STUDY WITH THE HONEY BEE
~~
~
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CSL Study Number HT5602
TITLE PAGE
STUDY NUMBER: HT5602 PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS):
AN ACUTE ORAL TOXICITY STUDY WlTH THE HONEY BEE
REPORT AMENDMENT NO 1
EPPO GUIDELINE 170 OECD GUIDELINE 213
AUTHOR.
: '4 Paul Wilkins
STUDY INlTIATION 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 YO41 lLZ, UK
Environmental Laboratory Project Number U2723 Page 1 of 5
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CSL Studv Number HT5602
CENTRAL SCIENCE LABORATORY
REPORT AMENDMENT 1
Study title
Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute oral toxicity study with the honey bee
Test facility
Environmental R&D Team, CSL, Sand Hutton, York, UK
Study number
HT5602
Numberofpages: . 5
Signature P k q
Study Director P Wilkins Environmental R&D Team, CSL
Authorisation of release of report amendment
Signature-
Management Representative M H Bew Head Environmental Biology Group, CSL
Date s *L c - d (dd/mm/yy)
This amendment has been noted by Quality Assurance as correction to the report
Mrs C V Walker QA Auditor Central Science Laboratory
Environmental Laboratory Project Number U2723 Page 2 of 5
CSL Study Number HTB5A60C2K 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 Perfuorooctanesulfonate, 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
Mr M H Bew
Mrs R M Brookes
2 copies
1 COPY 1COPY 1 COPY 1 copy
Environmental Laboratory Project Number U2723 Page 3 of 5
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SPONSOR:
SPONSOR' S REPRESENTATIVE:
LOCATION OF STUDY,RAW DATA
AND A COPY OF THE FINAL REPORT:
3M Corporation Environmental Laboratory 935 Bush Avenue St Paul, Minnesota 55106 USA
Ms Rochelle Robideau
Central Science Laboratory Sand Hutton, York, YO41 1LZ UK
SL STUDY NUMBER:
HT5602
T ITEM:
Perfluorooctanesulfonate, Potassium Salt (PFW
STUDY:
Perfluorooctanesulfonate,Potassium Salt (PFOS):,An acute oral toxicity study with the honey bee
OMINAL TEST CONCENTRATIONS:
rST DATES: ENGTH OF TEST:
Negative control, Positive control, 4.78,2.17, 0.991,0.450 and 0.205 pg a.i./bee Range test: 12-15 September 2000 Definitive test: 21-24 September 2000
72 hours
ENGTH OF EXPOSURE:
4 hours
ST ORGANISM:
I OURCE OF TEST ORGANISMS:
IFESTAGE OF ORGANISMS:
iORAL LDso (72 hrs):
HONEY BEE (Apis melliferu L.) National Bee Unit
CSL
Sand Hutton, York YO41 1LZ Adult
0.40 pg a.i./bee
(95% CL 0.33 - 0.48 pg a.i./bee)
STATISTICALLYDETERMINED ORAL NO OBSERVED EFFECT LEVEL (BASED ON MEAN INTAKE):
0.21 pg a.i./bee
I
I
Environmental Laboratory Project Number U2723 Page 4 of 5
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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 12'hSeptember to 24* September 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
The objective of this study was to evaluate the acute oral toxicity of Perfluorooctanesulfonate, Potassium Salt (PFOS) administered to the honey bee (Apis rnelliferu).
EXPERIMENTAL DESIGN
<
-4
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
: '1
(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 LD,,. 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 pl 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 after removal of feeders. In the range test and definitive test an additional assessment was
made at a further 72 hrs after removal of the test feeders. All doses of test item are reported
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.
Environmental Laboratory Project Number U2723 Page 5 of 5
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TITLE PAGE CSL Study Number HT5602
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
SUBMITI'ED 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 YO41 lLZ, UK
Report No 1 of 6
Environmental Laboratory Project Number U2723 Page 1 of 25
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CSL Study Number HT5602 E!
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
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 1 1/09/00 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 2 1/09/00
22/09/00
Dispensing (test item and toxic reference) Preparation of dilutions Dosing bees Assessments Weighing feeders Assessments
Date reported 08/09/00
15/09/00 15/09/00
15/09/00
2 1/09/00 2 1/09/00
25/09/00 25/09/00
25/09/00
fi
S i g n e d . m & . ! A ......b . 4.............. Date.2%.lQ.%.].d..(dd/rnm/yy)
Mrs C V Walker QA Auditor Central Science Laboratory
This report has been audited by the Quality Assurance Unit of the Central Science Laboratory.
Mrs C V Walker QA Auditor Central Science Laboratory
Date..?3.1.@,%l.Q.l...(dd/rnm/yy)
Environmental Laboratory Project Number U2723 Page 3 of 25
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SPONSOR: 3M Corporation
CSL Study Number HT5602
REPORT APPROVAL
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:
DATE
23r3 - bt ( d d d y y )
Mr M H Bew
Head EnvironmentalBiology Group, CSL
Environmental Laboratory Project Number U2723
Dnrra A , F 3 C
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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 SUMMARY ............................................................................................................................................ 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
CONCLUSION.S................................................ ........................................................................ .........13
Table 1. Oral LDmof PFOS from definitive test data........................................................................... 13 Table 2. Oral toxicity of dimethoate toxic reference from definitive test data..................................... 13
Environmental Laboratory Project Number U2723 Page 5 of 25
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CSL Study Number HT5602
REFERENCES ...................................................................................................................................... 13 PRINCIPAL WORKERS ...................................................................................................................... 14 DISTRIBUTION OF REPORT............................................................................................................. 14 APPENDIX 1 (TABLES Al-A8) ......................................................................................................... 15 DETAILED ORAL TOXICITY TEST RESULTS............................................................................... 15 APPENDIX 2 ........................................................................................................................................ 23 INTERIM CERTIFICATEOF ANALYSISFOR TEST SUBSTANCE.............................................. 23
Environmental Laboratory Project Number U2723
Pnop f i nf 3 5
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~PONSOR:
PONSOR'S REPRESENTATIVE: OF STUDY, RAW DATA
A COPY OF THE FINAL
PORT:
CSL Study Number HT5602
SUMMARY 3M Corporation Environmental Laboratory 935 Bush Avenue St Paul, Minnesota 55 106 USA
Ms Rochelle Robideau
Central Science Laboratory Sand Hutton, York, YO41 1LZ UK
ZSL STUDY NUMBER:
HT5602
ST ITEM:
Perfuorooctanesulfonate, Potassium Salt (PFOS)
TUDY:
Perfuorooctanesulfonate,Potassium Salt (PFOS): An acute oral toxicity study with the honey bee
VOMINAL 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
PmOFTEST:
72 hours
ST ORGANISM:
________
HONEY BEE (Apis mellifera L.)
OURCE OF TEST ORGANISMS:
National Bee Unit CSL Sand Hutton, York YO41 1LZ
IFE STAGE OF ORGANISMS:
Adult
IRAL LDm (72 hrs):
;TATISTICALLY DETERMINED ORAL 40 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 pg a.i./bee
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CSL Study Number HT5602
INTRODUCTION This study was camed 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 12'h
September to 241hSeptember 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
The objective of this study was to evaluate the acute oral toxicity of Perfuorooctanesulfonate, Potassium Salt (PFOS) administered to the honey bee (Apis rneflifera).
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 LDso. 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 10bees was offered
200 p1 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 after removal of feeders. In the range test and definitive test an additional assessment was made at a further 72 hrs after removal of the test feeders. All doses of test item are reported 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.
Environmental Laboratory Project Number U2723
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CSL Study Number HT5602
The nominal doses administered are shown below.
1 I I Range finding oral test dose levels
Dose group
PFOS
(pg a.iJbee)
1
103
2
10.3
3
1.03
4
0.103
Control 1
50% wlv sucrose
containing 5% acetone
Control 2
50% wlv sucrose
Dimethoate I
(pg ailbee) 0.60
0.30 \ 0.15
0.075
50% wlv sucrose containing
Triton X-100
N/A
Dose group 1
PFOS (pg a.iJbee)
4.78
Dimethoate
(pg ailbee)
0.60
5 Control 1
Control 2
0.205 50% w/v sucrose containing 5% acetone
50%wlv sucrose
-
50% wlv sucrose containing
Triton X-100
N/A
.Estimatesof LDsovalues 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/l) 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 and route of administration described in this protocol are based upon procedures specified in EPPO Guideline 170, Guideline on Test Methods for Evaluating the Side-Efiects
of Plant Protection Products on Honey Bees; and the OECD Test Guideline 213, Honeybees, Acute
Oral Toxiciry Test. In order to control bias, bees were impartially distributed 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 LDm
fell within the OECD standard, Le. 0.10 0.35 pg ai/bee.
Environmental Laboratory Project Number U2723 Pane 9 of 25
Test item
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CSL Study Number HT5602 E!
The PFOS (IUPAC Name l-oCtanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluo~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 NBUIOS. The test item was stored, as recommended by
the sponsor, at 16-20"Cprior to use.
PFOS was dispersed in acetone (analytical grade) before use due to a low solubility in water (approximately 500 mg/l). 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 pg 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/l 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 14OC - 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 LDso calculations are for corrected dimethoate concentration based on data supplied by BASF.
The honey bee (Apisrnellifera) 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 (Apisrnelliferu) 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 acarine, 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& 2"C, 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 2 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
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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 ( O S 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 I g/l Triton X-100.Stock solutions of 2.80 pg aVp1 (range-finding test) and 3.00pg aVp1 (definitive test) dimethoate in deionised water containing 1 g/l Triton X-100were prepared and a series of dilutions made in 1 g/l 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/l Triton X-100in 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 @/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
Analvsis 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 LDm 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 ( ~ ~ 0 . 0 5 ) .
The LD5o 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 1pg aihee 1-10 pg ai/bee 10-100 pg aihee greater than 100 pg aihee
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 LDso 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 0.2.1 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 . 0 5 )S. ublethal effects were seen observed as knockdown at the highest dose level at 4 hrs with these individuals dying after 24 hours.
The 24, 48 and 72 -hour contact LDm values for dimethoate in the definitive test are shown in Table 2. Mortality of bees exposed to the toxic reference in the tests allowed calculation of an oral LD50 of 0.12 pg ai dimethoatehee at 24-hours and 0.11 pg ai dimethoatehee 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.
Environmental Laboratory Project Number U2723 Page 12 of 25
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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 LDso calculated as 0.40 pg a.i./bee. The NOEL based on statistical analysis of the data (Student's t test ~ ~ 0 . 0 5w)as 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.
Time Hrs 24 48 72
LDSO (pg a.iJbee)
0.72 0.46 0.40
95 % Confidence
Interval for LDso
0.60 - 0.85
0.32 - 0.55 0.33 - 0.48
Estimate of slope of response line 5 .O 4.7 4.8
Time Hrs 24 48 72
LDSO (pg a.iJbee)
0.12 0.1 1 0.1 1
95% Confidence
Interval for LD50
0.10 - 0.14
0.096 - 0.13
0.096 - 0.13
Estimate of slope of response line 6.4 7.3 8.1
REFERENCES
European and Mediterranean Plant Protection Organization. 1992. Guideline on Test Methods for Evaluating the Side-Effectsof Plant Protection Products on Honey Bees. EPPO Bulletin, 22,203-2 15.
Finney, D. J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
International Commission for Bee Botany (ICBB). 1985. Recommendationsf o r harmonization of methodsfor testing hazard of pesticides to honeybees. Third Symposium on harmonization of methods for testing hazard of pesticides to bees, England.
Organization for Economic Cooperation and Development. 1997. Guideline 2 13 Honeybees, Acute Oral Toxicity Test.
Environmental Laboratory Project Number U2723 Page 13 of 25
BACK TO MAIN 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 Miss 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 1 COPY
1 COPY
Environmental Laboratory Project Number U2723 Page 14 of 25
Table Al.
aBACK TO MAIN
CSL Study Number HT5602
APPENDIX 1 (TABLES Al-A8) DETAILED ORAL TOXICITY TEST RESULTS
Results of range-finding oral dosing tests with PFOS - Mortality
Nominal dose'
(pg a.iJbee)
Actual dose' (pg a.iJbee)
Cage No.
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.o
52
0
0
2
3
1.03
1.o
53
0
1
1
2
1.03
1.o
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
0
0
o4
0
61
0
0
0
0
o4
0
62
0
0
0
0
o4
0
63
0
0
0
0
1 based on nominal consumption of test solution of 20pLVbee 2 based on amount of test solution consumed (average of 10 bees)
acetone dosed 4 undosed
Environmental Laboratory Project Number U2723 Page 15 of 25
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CSL Study Number HT5602
Table A2. Results of range-finding oral dosing tests with PFOS - Sub-lethal effects
Nominal
dose' (pg a.iJbee)
Actual dose' (pg a.iJbee)
Cage No.
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 .o
52
0
0
0
1K
1.03
1 .o
53
0
0
0
0
1.03
1 .o
54
0
0
1K
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
o4
0
61
0
0
0
0
o4
0
62
0
0
0
0
o4
0
63
0
0
0
0
I based on nominal consumption of test solution of 20plhee 2 based on amount of test solution consumed (average of 10 bees) 3 acetone dosed 4 undosed
Environmental Laboratory Project Number U2723 Page 16 of 25
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Table A3.
CSL Study Number HT5602
Resultsof range-findingoral dosing tests with dimethoate - Mortality
Nominal dose'
(pg a.iJbee)
Actual dose2 (pg a.iJbee)
Cage No.
Cumulative number dead (n=10)
4hrs
24 hrs 48 hrs
72 hrs
0.60
0.56
31
3
10
10
10
0.60
0.54
32
0
10
10
10
0.60
0.60
33
0
10
10
10
0.30
0.25
34
2
10
10
10
0.30
0.30
35
0
10
10
10
0.30
0.30
36
1
10
10
10
0.15
0.15
37
0
8
8
8
0.15
0.15
38
0
4
5
5
0.15
0.15
39
0
5
7
7
0.075
0.075
40
0
0
0
0
0.075
0.074
41
0
0
0
0
0.075
0.074
42
0
1
1
2
'0
0
43
0
0
0
0
0
0
44
0
0
0
0
0
0
45
0
0
0
0
I based on nominal consumption of test solution of 20pVbee 2 based on amount of test solution consumed (average of 10 bees)
Environmental Laboratory Project Number U2723
*
Page 17of 25
BACK TO MAIN
Table A4.
CSL Study Number HT5602
Results of range-finding oral dosing tests with dimethoate - Sub-lethal effec
Nominal
dose' (pg a.iJbee)
Actual dose' (pg a.iJbee)
Cage No.
Number knockdown (K) or stumbling (S)
4hrs
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
1K
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 '. 0
0
43
0
0
0
0
0
44
0
0
0
0
0
45
0
0
0
0
1 based on nominal consumption of test solution of 20pVbee 2 based on amount of test solution consumed (average of 10 bees)
Environmental Laboratory Project Number U2723 Page 18 of 25
BACK TO MAIN
Table AS.
CSL Study Number HT5602
Results of definitive oral dosing tests with PFOS - Mortality
Nominal dose'
(pg a.iJbee)
Actual doseZ (pg a.iJbee)
Cage No.
Cumulative number dead (n=10)
4hrs
24 hrs 48 hrs 72 hrs
4.78
4.8
316
3
10
10
10
4.78
4.8
317
3
10
10
10
4.78
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
32 1
1
10
10
10
0.99 1
0.96
322
0
7
10
10
0.99 1
0.96
323
0
8
9
10
0.99 1
0.96
324
0
6
9
9
0.450
0.45
325
0
1
4
5
0.450
0.45
326
0
3
7
7
0.450
0.45
327
0
2
4
6
0.205
0.21
328
0
0
0
1
0.205
0.2 1
329
0
0
0
0
0.205
0.2 1
330
0
0
2
2
03
0
33 1
0
1
1
1
03
0
332
0
0
0
0
o3
0
333
0
0
0
0
o4
0
334
0
0
0
0
o4
0
335
0
0
0
0
o4
0
336
0
0
0
0
I based on nominal consumption of test solution of 20pYbee
2 based on amount of test solution consumed (average of 10bees)
3 acetone dosed 4 undosed
Environmental Laboratory Project Number U2723 Page 19 of 25
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CSL Study Number HT5602
Table A6. Results of definitive oral dosing tests with PFOS - Sub-lethal effects
Nominal dose'
(pg a.iJbee)
Actual dose' (pg a.iJbee)
Cage No.
Number knockdown (K) or stumbling (S)
4hrs
24hr5 48 hrs 72 hrs
4.78
4.8
316
1K
0
0
0
4.78
4.8
317
1K
0
0
0
4.78
4.8
318
1K
0
0
0
2.17
2.2
319
0
0
0
0
2.17
2.2
320
0
0
0
0
2.17
2.2
32 1
0
0
0
0
0.99 1
0.96
322
0
0
0
0
0.99 1
0.96
323
0
0
0
0
0.99 1
0.96
324
0
0
0
0
0.450
0.45
325
0
15
0
0
0.450
0.45
326
0
0
0
0
0.450
0.45
327
0
0
1K
1K
0.205
0.2 1
328
0
0
0
0
0.205
0.2 1
329
0
0
0
0
. 0.205
0.2 1
330
0
0
0
0
o3
0
33 1
0
0
0
0
o3
0
332
0
0
0
0
o3
0
333
0
0
0
0
o4
0
334
0
0
0
0
o4
0
335
0
0
0
0
o4
0
336
0
0
0
0
I based on nominal consumption of test solution of 20pVbee 2 based on amount of test solution consumed (average of 10 bees)
acetone dosed 4 undosed
Environmental Laboratory Project Number U2723 Page 20 of 25
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Table A7.
Nominal dose'
(pg a.iJbee)
CSL Study Number HT5602
Results of definitive oral dosing tests with dimethoate - Mortality
Actual dose' (pg a.iJbee)
Cage No.
Cumulative number dead (n=10)
4hrs
24 hrs 48 hrs 72 hrs
0.60
0.57
30 1
8
10
10
10
0.60
0.58
302
2
10
10
10
0.60
0.60
303
5
10
10
10
0.30
0.30
304
4
10
10
10
0.30
0.30
305
2
10
10
10
0.30
0.30
306
5
10
10
10
0.15
0.15
307
1
9
10
10
0.15
0.15
308
0
7
9
9
0.15
0.15
309
2
6
6
7
0.075
0.074
310
0
2
2
2
0.075
0.075
311
0
1
1
1
0.075
0.074
312
0
1
1
1
0
0
313
0
0
0
1
0
0
3 14
0
0
0
0
0
0
315
0
1
1
1
I based on nominal consumption of test solution of 20pVbee 2 based on amount of test solution consumed (average of 10 bees)
Environmental Laboratory Project Number U2723 Page 21 of 25
~~ _ _ _ ~ ~
BACK TO MAIN
Table AS.
CSL Study Number HT5602
Results of definitive oral dosing tests with dimethoate - Sub-lethal effects
Nominal dose'
(pg a.iJbee)
Actual dose' (pg a.iJbee)
Cage No.
Number knockdown (K) or stumbling (S)
4hrs 24hrs 48hrs 72 hrs
0.60
0.57
301
2K
0
0
0
0.60
0.58
302
6K.
0
0
0
0.60
0.60
303
5K
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
1K
0
0.075
0.074
310
1K
0
0
0
0.075
0.075
31 1
0
0
0
0
0.075
0.074
312
0
0
0
0
0
0
313
0
0
0
0
0
0
314
0
0
0
0
0
0
315
0
0
0
0
1 based on nominal consumption of test solution of 20pVbee 2 based on amount of test solution consumed (average of 10bees)
Environmental Laboratory Project Number U2723
1
Page 22 of 25
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CSL Study Number HT5602
APPENDIX 2
INTERIM CERTIFICATEOF ANALYSIS FOR TEST SUBSTANCE
INTERIM CERTIFICATE OF ANALYSIS
Revision I (9mOO) Centre Analytical Laboratories COA Reference #: 023-018A
3M Product: PFOS,Lot 217 Reference #: SD-018
1. carbon 2. Hydrogen 3. Nitrogea 4. Sulfur 5. Fluorine
1. TheoreticalValue = 17.8%
2. ?hcorctical Value = 0% 3. 'Ihborctical Value = 0% 4. Thcontical Value = 5.95%
5. TheareticalValue = 60%
1. 12.48wt&.% 2. 0.244 wt.lwt.% 3. 1.74 wt.hvt.% 4. 8.84WLlwt.%
5. 54.1 wt./wt.%
Environmental Laboratory Project Number U2723 Page 23 of 25
P
T)
I
-i=B
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CSL Study Number HT5602
INTERIM CERTIFICATE OF ANALYSIS
Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00
Expiration Date: 08131/01
Storage Conditions: Frozen ClO'C
Re-assessmentDate: 08/31/01
'Purity 100%- (sum of metal impurities, 1.45% +LC/IvfS impurities, 8.41%+Inorganic
Fluoride, 0.59??+NMRimpurities, 1.93%+organicacid impurities, 0.38%+POAA 0.33%)
- Total impurityfiom all tests = 13.09%' Purity = 100% 13.09% = 86.9%
'Potassium is expected in this salt form andis thereforenot consideredan impurity.
%punty by DSC is generally not applicableto materials of low purity. No endotherm was observed for this sample.
'Sulfur in the sampleappears to be convertedto SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determinationin the elemental analysis, lendingconfidenceto this interpretation Based on the results, the SO4is not consideredan bnpurity.
%A
HFBA "PA PFPA
Trifluoroaceticacid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid
6Theoreticalvalue dculations based on the empiricalformula, CsF1,SO3(' (Mw=538)
This work was conducted under FiPA Good LabonttoryPractice Standards(40 CFX 160).
/
COA023418A
Environmental Laboratory Project Number U2723 Page 24 of 25
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CSL Study Number HT5602 E!
INTERIM CERTIFICATE OF ANALYSIS
Centre Analytical Laboratories COA Reference #: 023-01SA
LUMS Purity Profile:
Impurity
c4
cs
C6
c7
Total
wtfwt. Yo 1.22 1.33 4.72 1.14 8.41
Note: The C4 and C6 values were calculatedusing the C4 and C6 standard calibration
curves, respectively. The CS value was calculated using the average response factors
from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves.
- AI -a -.
Prepared By:
David S. Bell
Date
Scientist, Centre AnalyticalLaboratories
Reviewed By:
-
I
John Flaherty
Date
Laboratory k g e r , Centre ~nalyticaLl aboratories
COA023418A
EnvironmentalLaboratoryProject Number U2723 Page 25 of 25
CSL Study Number HTB5A6C02K TO MAIN
CENTRAL SCIENCE LABORATORY AMENDMENT TO PROTOCOL
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, YO41 1LZ
Study number
HT5602
Amendment number: 2
Number of pages: 2
Study Director
PaulWilkins n
Signature I
Date N1/4Jo/ (ddmmlyy)
Management Representative
Medwin Bew
Environmental Laboratory Project Number U2723
page i of2
CSL Study Number HT5B6A0C2K TO MAIN
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 PeffluorooctaneSulfonic Acid, Potassium Salt (PFOS)to Perfluorooctanesulfonate, Potassium Salt (PFOS)and consequentlythe study title
changed from:
Perfluorooctane SulfonicAcid, PotassiumSalt (PFOS):An acute oral toxicity study with the honey bee
to
Peffluorooctanesulfonate,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, QA Unit
Ms R Robideau, 3M
Mr D Palmer, Wildlife International Mr P Wilkins, CSL Environmental R&D Mr M Bew, CSL Mrs R Brookes, CSL
Environmental Laboratory Project Number U2723 page 2 of 2
wildlife International, Ltd.
:... ...i ...........................<.........,....;....;....:.....:,........;.: ECOTOXICOLOGY & ANALYTICAL TESTING SERVICES
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ANALYSIS FOR PFOS IN AQUEOUS AND SUCROSE SOLUTIONSUSED BY CENTRAL SCIENCE LABORATORIES FOR CONDUCTINGHONEY 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 standardhample ion chromatogramsfor the screens performed on our triple quadrupole LC/MS/MS system. As can be seen in 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 apply to solutions representative of those used in the following studies.
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): An Acute Contact Toxicity Study with the Honey Bec (CSL Study Number: HT5601) PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): An Acute Oral Toxicity Study with the Honey Bee (CSL Study Number: HT5602)
R a w d d Van Hoven, Ph.D.
Scientist
4/r SI61
DATE
MANAGEMENT:
Willard B. Nixon, Ph.D' Director, Analytical Chemistry
DATE I .
8598 Commerce Drive Easton, Maryland 21601 Tel. 410-822-8600 Fax 410-822-0632 E-mail: ecotox@wildlifeinternational.com
EuropeanOffice: Bergkarnpweg 1 7231 CL Wamrveld The Netherlands Tel. 31(0)575473848 F a x 31(01575-574813 E-mail: aleopoldOworldonline.nl
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sanmuam STD 0.500 ug 8.m
STD 1.00 ug 8.IA
STD 250 ug aid
STD 3.60 ug 8.IA STD 5.00 ug 8.iA
m
ULsaVpIT
FISHRAKE WAlw
saax
STD 0.600 ug 8.M. STD 1.00 ug 8.lA
STDZWugUA STD3.Wugal.A
STD 5.00 ug am
koc.
0.5000 1 .0000 2.5000 3.5000 5.0000
0.0 0.0 0.0 0.0 0.0 0.5000 1.0000 2.5000 3.5000 5.0000
Qluaw 1 .o 1 .o 1 .o 1 .o 1 .o 1 .o 1 .o
100.0
100.0
100.0
- 1.o 1 .o 1 .o 1 .o 1 .o
Cat.CawzllPltl 0.4770 ug alA 0.0884 ug r.IA
2.5378 ug ..Id 3.6246 ug alA 5.0132 ug 1M
n l r ug 8.W 1118 ugalA n l a ug alJg n l r ug alA 0.6041 ug 8.IA 0.6087 ug alA 1.0287 ug 8.IA 2.4725 ug alA 3.5008 ug aiA 4.0413 ug 8.111
ACeUnCY
05.4 98.8
101.5 103.8
100.3 nla nlr n18 1118 1118 101.7
102.7 08.0 100.3 98.8
ALM 4231.52 84213 3 21 116.03 30020.82 41307.58
1118 nla nla n/8 389.06 44013 3 8735.45 20581.27 20070.80 39980.71
HBmaunQkbL
393.90 4675A-011D-6 746.57 4875A-011D-7 1925.75 4675A-011D-8 2739.86 4675A-011D-0 3810.74 4875A-0110.10
n l r hW2i n l r DLsoLvpir n I 8 454C-101n I 8 454C-10141.08 454C-101-
387.00 4675A-01lD-8 826.57 4675A-OllD-7 1836.54 4875A-011D-8 2536.52 4875A-011D-0 3515.70 4875A-011D-10
.-.. ..: t .,..,.I
-?
Page 2 of 4
MacQuan. version 1.6 Printed: Thu, Oct 26, 2000 12:s
- mlbrabion File: cal PFOS 102600 MD Path: MacintoshHD:AP13000:PROJECf DAT~454C-l01:454C101~1026oMOD:
Comments: PFOS: 4SC-101: Method Development Scmnlng fish fiake, water, sucrose -(LCIMS/MSMRM)
ffC6 499.0-~99.1No Internal Standard Weighted (l/x)
Intercept = 323.17 Slope = 8193.32 Correlation Coeff. I0.99949
Area
360 142000-1
3000
2400
BACK TO MAIN
1200 600
Conc. ug a.iA
I -7 ,..>. -:..=.-----.CERTfFiED COPY OF:THE ORlGlNAL
Mad;lum,version 1.6
Page 3 of 4
Printed: Thu, oct 26, 2000 1259
- Calibration File: Cal PFOS 102600 MD Path: Macintosh HD:AP13000:PROJECT DATtk454G-101:454C101-102600 MD:
Comments: PFOS: 454C-101: Method Development Screening fish flake, water, sucrose -(LCIMSNS-MRM)
BACK TO MAIN
/.*m)C'=--
PFOS-1
STD 0.500 ug a.IJL
4675A-011D-6
4.98 in 1 period
FITS No Internal Standard
U s e Area
1: 4.97 MRM, 298 scans
499.0-299.1
Ndse Thnw.
2.0
QuanlThres.
1.0
Min. Width
12
Mull. Width
10
Base. w m
40
RTWin.(secs) 1 0
Smooth
2
RT
3.70
h a 4231.52
Height 393.90
start rime
3.50
Endm
4.17
lntegratlon Width
0.67
Retention Time Integration Type
-3.70
A BB
Thu. Oct 26, 2000 10%
roc7
intensity: 4000 cps
7 6 5 4 3 2 1
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
PW-2
STD 1.00 ug a . i R
4675A-0110-7
4.98 in 1 period
FUX
No internal Standard U s e Area
1: 4.97 MRM, 298 scans
499.0->99.l
Ndse Thres.
2.0
QuantThres.
1.0
Min. Width
12
Muftwidul
10
Base. w m
40
RTWin.(secs) 1 0
Smth
2
I~QIORG T M 3.70
h a 8421.33
Height 746.57
start Time
3.47
EndTim
4.14
Integration Width
0.67
Retention Tlme Integration Type
-3.69
A EB
Thu, Ocl 26, 2000 10:40
intensity: 4000 cps
j 4
3
2d
2?0
14' 3 8 75 116 156 4'1 ' 8`1 ` 151 ' 161
0.69 1.36 203 2.70 3.37 4.04 4.71 Time
MacQuan, venion 1.6
Page 4 of 4
Printed: Thu, OcI26, 2000 1259
- Calibralion file: Cat PFos 102600 MD Path: Madntosh H D W w : P R O J E C T DAT&454C-101:454C101~102600M D
Comments: PFOS: 454C-101: Method Development screening fish flake, water, sucrose -(LCIMSIMS-MRM)
BACK TO MAIN
PFOS-9 454C-101-
WATER
Thu, Oct 26, 2000 1122
4.98 in 1 period Ros No Internal Standard
Use Area
intensity: 4000 cps
1: 4.97 MRM, 298 scans
499.0->99.1
Ndse Thres.
2.0
QuantThres.
1.0
Min. Width
12
Mult. Width
10
Base. Width
40
RTWln.(secs) 1 0
Smooth
2
w e d RT
3.70
Area 0.0
Height 0.0
Start Time
0.00
EndTim
0.00
Integration Width
0.00
Retention Time
0.00
Integration Type
0 38 66 105 138 166 189 217 266 ' 4'1 ' 81 121 I d 1 201 241 281 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
- PFOS-10
454C-101
sucRosE
4.98 in 1 peiiod
Ros No Internal Standard
Use Area
1: 4.97 MRM, 298 Scans
499.0->99.1
Noise Thres.
2.0
QuantThres.
1.5
Min. Width
12
Mult Width
10
Base. width
40
RTWin.(secs) 1 0
Smooth
2
EXiXUedRT 3.70
h a 388.06
Height 41.96
Start Time
3.50
End Time
3.77
Integralion Width
0.27
Retention Time Integration Type
-3.59
A* EB
Thu, Oct 26, 2000 11:28
"8 3
*I 1 10 40 0 ' 4'1 0.89
intensity: 4000 cps
9 0 1 1 1 152 177 214 244265
81 121 161 ' 2 6 7 2 4 1 ' 281 scan
1.36 2.03 270 3.37 4.04 4.71 Tim
