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SCIENCE SERVING AGRICULTURE. FOOD AND THE ENVIRONMENT
( CONTRACT REPORT
STUDY NUMBER HT5601
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS):
AN ACUTE CONTACT TOXICITY STUDY V __________ WITH THE HONEY BEE
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CSL Study Number HT5601
TITLE PAGE
STUDY NUMBER: HT5601 PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): AN ACUTE CONTACT TOXICITY STUDY WITH THE HONEY BEE
REPORT U.S EPA SERIES 850-Ecological Effects Test Guidelines
OPPTS Number 850.3020 (draft)
EPPO GUIDELINE 170 OECD GUIDELINE 214
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 I of 6
Environmental Laboratory Project Number U2723 Page 1 of 25
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CSL Study Number HT5601
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Study number: HT5601 Report title: Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute contact 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
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CSL Study Number HT5601
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
Definitive test
18/09/00
Dosing bees
20/09/00
Assessments
Date reported 08/09/00
15/09/00 15/09/00
15/09/00
21/09/00 21/09/00
S ig n e d ^ - r A ^ .S ^ ...................................... .....Date.^3>|.S>?>.t..l.|...(dd/mm/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.
Signed. .
..... ................................... Date.
Mrs C V Walker QA Auditor Central Science Laboratory
(..O & i .\ ..(dd/mm/yy)
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SPONSOR: 3M Corporation
CSL Study Number HT5601 REPORT APPROVAL
TITLE:
Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute contact toxicity study with the honey bee
CSL STUDY NUMBER: HT5601
STUDY DIRECTOR:
DATE
( O S j 0 1 (dd/mm/vv)
Mr P Wilkins Environmental R&D Team, CSL
Approval for Report issue MANAGEMENT:
_____________________ ________________________________________
DATE________ ~1 Cr\ (dd/mm/vy) Mr M H Bew Head Environmental Biology Group, CSL
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CSL Study Number HT5601 TABLE OF CONTENTS
TITLE P A G E ............................................................................................................................................................................................. 1
GOOD LABORATORY PRACTICE COM PLIANCE S T A T E M E N T .................................................................................2
QUALITY A SSU RA N C E ST A T E M E N T S .................................................................................................................................... 3
REPORT A PPR O V A L........................................................................................................................................................................... 4
TABLE OF C O N T E N T S ............................................................... ...................................................................................................... 5
SUM M A R Y ................................................................................................................................................................................................ 7
IN TR O D U C TIO N .................................................................................................................................................................................... 8
O B J E C T IV E .............................................................................................................................................................................................. 8
EXPERIM ENTAL D E SIG N ................................................................................................................................................................. 8
M ATERIALS A N D M ETH O DS......................................................................................................................................................... 9
Test item ...................................................................................................................................................................................................... 9
T oxic reference........................................................................................................................................................................................ 10
Test b e e s.................................................................................................................................................................................................... 10
Conditions during the te sts..............................................................
10
D ose preparation..................................................................................
10
D ose administration............................................................................ ..................................................................................................11
O b s e r v a tio n s .............................................................
11
Analysis o f d ata....................................................................................................................................................................................... 11
RESULTS A N D D ISC U SSIO N ..................................................;.................................................................................................... 11
Range te s t..................................................................................................................................................................................................12
D efinitive t e s t .................................................................................................................................
12
C O N C L U SIO N S.....................................................................................................................................................................................12
Table 1. Contact LD50o f P F O S ..........................................................................................................................................................12
Table 2. Contact toxicity o f dimethoate toxic reference............................................................................................................ 12
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R EFER ENC ES............................................................................................................................................... PRINCIPAL W O R K E R S............................................................................................................................ DISTRIBUTIO N OF REPO RT................................................................................................................. APPENDIX 1 (TABLES A1-A8) DETAILED CONTACT TOXICITY TEST RESULTS A PPE N D IX 2 INTERIM CERTIFICATE OF A N A L Y SIS FO R T E ST IT E M .....................
13 13 14 15 23
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SPONSOR:
SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: CSL STUDY NUMBER: TEST ITEM: STUDY:
TEST CONCENTRATIONS: TEST DATES:
CSL Study Number HT5601
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
HT5601
Perfluorooctanesulfonate, Potassium Salt (PFOS)
Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute contact toxicity study with the honey bee
Negative control, Positive control, 45.0,20.5, 9.30,4.24 and 1.93 pg a.i./bee
Range test: 12-16 September 2000 Definitive test: 18-22 September 2000
LENGTH OF EXPOSURE: LENGTH OF TEST:
Single dose 96 hours
TEST ORGANISM: SOURCE OF TEST ORGANISMS:
LIFE STAGE OF ORGANISMS:
HONEY BEE (Apis mellifera L.)
National Bee Unit CSL Sand Hutton, York Y041 1LZ
Adult
CONTACT LD50 (96 hrs):
4.78 jig a.i./bee (95% CL 3,8 - 5.8 pg a.i./bee)
STATISTICALLY DETERMINED CONTACT
NO OBSERVED EFFECT LEVEL:
1.93 pg a.i./bee
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CSL Study Number HT5601
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 September to 22nd September 2000. The protocol, protocol amendment, 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 contact toxicity of Perfluorooctanesulfonate, Potassium Salt (PFOS) administered to the honey bee (Apis tnellifera).
EXPERIMENTAL DESIGN
Contact exposure evaluated cuticular absorption of the test item following the application of a droplet to the body surface. Three batches of bees in groups of 10 bees were topically dosed on the thorax with lpl drops containing PFOS in acetone.
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. At initiation of the test, each bee received a single topical dose of the test item applied to the thorax with a micro-applicator pipette. The bees were then observed for up to 96 hours for toxicological responses and an LD50 value and 95% confidence limits were calculated. All doses of test item are reported as jig active ingredient (a.i.)/bee. At the end of each test any bees remaining alive were killed by freezing and all bees used in the study were disposed of by incineration. All data are retained on the study file.
The doses administered are shown below. Mortality and sub-lethal effects were assessed at 4, 24, 48, 72 and 96 hours after dosing.
Dose group
1 2 3 4 Control 1
Control 2
PFOS (Mg a.iTbee)
103 10.3 1.03 0.103 Acetone
Undosed
Dimethoate (pg ai/bee)
0.30 0.15 0.075
-
Water containing lg/1 Triton X-100 N/A
jji
j =*
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Definitive contact test dose levels
Dose group
PFOS
(pg a.iTbee)
1 45.0
2 20.5
3 9.30
4 4.24
5 1.93
Control 1
Acetone
Control 2
Undosed
Dimethoate (pg ai/bee)
0.30 0.15 0.075
-
-
Water containing lg/1 Triton X-100 N/A
-^
Estimates of LD50values were calculated for the definitive contact test. The no observed effect levels were determined by a statistical evaluation of the mortality data.
Negative controls, solvent controls (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 and route of administration described in this report are based upon pro cedures specified in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.3020 Honey Bee Acute Contact Toxicity (draft); EPPO Guideline " * 170, Guideline on Test Methods fo r Evaluating the Side-Effects o f Plant Protection Products on Honey Bees', and the OECD Test Guideline 214, Honeybees, Acute Contact Toxicity 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 if: 1) control mortality is 10% or less and 2) the calculated dimethoate LD50 falls within the OECD standard, i.e. 0.10 - 0.30 pg ai/bee.
The droplet application method allows for a precise exposure to the test item that simulates a likely route of exposure to bees and other nontarget insects. Exposure is most likely to occur either through absorption or from grooming.
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
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I*
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 86.0 pg a.i./pt 1PFOS (the approximate concentration required to prepare the highest diluted dose in the test). The use of acetone negated the need to perform an application test as acetone solutions can be readily applied to B bees in contact dosing tests.
- B Toxic reference
r m 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-llC (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 LD50calculations are for corrected dimethoate concentration based on data supplied by BASF.
Test bees I
The honey bee (Apis mellifera) 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 in the range-finding and definitive test. Bees were examined prior to the start of the test and shown to be free of acarie, nosema and amoeba. The colony from which 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) until needed for the test.
Conditions during the tests
Test bees were housed in test chambers which were clean, well ventilated, inverted petri dishes, measuring approximately 9 cm in diameter. A small inverted petri dish (approximately 3 ml) containing the sucrose solution was affixed in each chamber. Each test chamber contained 10 worker bees and were 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.
Dose preparation
Doses of the test item were made by preparing a solution of 103 pg a.i./pl (range-finding test) and 90.4 pg a.i./pl (definitive test) PFOS and making a series of dilutions from these. All dose solutions were made in acetone. One set of control bees was treated with acetone and a second set of control bees was untreated.
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CSL Study Number HT5601
Stock solutions of the positive control, dimethoate, were prepared in deionised water containing
l g/l Triton X-100. Stock solutions of 2.8 pg ai/pl (range-finding test) and 3.0 pg ai/pl (definitive test) dimethoate in deionised water containing 1 g/l Triton X-100 were prepared. Control bees were treated with 1 g/l Triton X-100 in deionised water. The tests were carried out in parallel with those for PFOS.
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 bee was dosed on the thorax with a 1 pi drop of a given test item concentration or 1 pi acetone using an Anachem EDP Plus micropipette. The lid was then placed on the cage, the bees allowed to recover and kept in the incubator with a continuous supply of 50% w/v aqueous sucrose solution as food.
Observations
Mortality and any bees knocked down; i.e. alive but immobile, or stumbling were assessed at intervals of 4, 24,48,72 and 96 hours after dosing.
Analysis of data
Analysis of the data comprised plotting probit mortality recorded after 24,48, 72 and 96 hours against the logarithm of dose (CSL Probit 1 package). A least - squares regression (Finney 1971) was fitted to these. Toxicity was expressed as LDs0 in pg of active ingredient per bee (a.i.) 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 LD5ovalue 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-, 72- and 96-hour mortality results were analysed using the CSL probit program (Probit 1, version 4).
RESULTS AND DISCUSSION
The results of the tests are summarised in Tables 1 and 2 and detailed results are listed in Appendix 1. Further details are given below. The contact toxicity of the test item resulted in a 96 hr LD50value for the honeybee of 4.78 pg a.i./bee.
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CSL Study Number HT5601
Mortality significantly greater than seen in the controls was observed at all doses of 10.3 jig a.i./bee
and above with a steep dose response between a dose of 10.3 and 103 pg a.i./bee. No dose related sublethal effects were observed at any dose level.
i* Definitive test
i There was significant mortality at all doses above 1.93 pg a.i./bee with a steep dose response between 4.24 and 9.30 pg a.i./bee. The 96 hr LD50 was calculated as 4.78 pg a.i./bee, with 95% confidence I limits of 3.8 - 5.8 pg a.i./bee. The NOEL was 1.93 pg a.i./bee at 96 hours (Student's t-test p<0.05). j # No dose related sublethal effects were observed at any dose level.
The 24, 48, 72 and 96-hour contact LD50 values for dimethoate in the definitive test are shown in
m Table 2. Mortality of bees exposed to the toxic reference in the tests allowed calculation of a contact LD50of 0.19 pg ai dimethoate/bee at 24, 48 and 96-hours and 0.20 pg ai dimethoate/bee at 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.
CONCLUSIONS
Exposure of honeybees to PFOS by contact dosing resulted in a steep dose response curve with significant mortality above 1.93 pg a.i./bee and a 96-hour LD50 calculated as 4.78 pg a.i./bee. The NOEL based on statistical analysis of the data (Student's t test p<0.05) was 1.93 pg a.i./bee at 96 hrs.
These results demonstrate the toxicity of PFOS as 4.78 pg a.i./bee and according to the ICBB (1985) the test item is classified as moderately toxic by contact.
Table 1. Contact LDsn of PFOS
Time Hrs
LD50 (pg a.iJbee)
24 38.9
48 10.4.
72 6.00
96 4.78
95% Confidence Interval for LD50
28.2-71.2 8.2-13.0 4.7-7.6 3.8-5.8
Estimate of slope of response line 2.2 3.0 2.9 4.0
Table 2. Contact toxiciu of dimethoate toxic reference
Time Hrs
LD50 (pg a.i^bee)
95% Confidence Interval for LDS0
24 0.19 0 .1 6 -0 .2 4 48 0.19 0 .1 6 -0 .2 4 72 0.20 0.16 -0 .2 4 96 0.19 0.15-0.23
Estimate of slope of response line
4.2 4.7 4.8 5.1
=u
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CSL Study Number HT5601
REFERENCES
European and M editerranean Plant Protection Organization. 1992. Guideline on Test Methods for Evaluating the Side-Effects o f 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.
International Commission for Bee Botany (ICBB). 1985. Recommendations fo r harmonization o f methodsfor testing hazard o f 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 214 Honeybees, Acute Contact Toxicity Test.
U.S. Environmental Protection Agency. 1996. Honeybee Acute Contact Toxicity. Series 850Ecological Effects Test Guidelines (draft), OPPTS Number 850.3020.
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 Spinks Mr M J Kelly
QUALITY ASSURANCE
QA Manager QA Auditor
Mrs R M Brookes Mrs C V Walker
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CSL Study Number HT5601
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
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CSL Study Number HT5601
APPENDIX 1 (TABLES A1-A8) DETAILED CONTACT TOXICITY TEST RESULTS
Table A l. Results of range-finding contact dosing test with PFOS - M ortality
Actual dose (MS a.i./bee)
Cage No.
Cumulative number dead (n=10)
4 hrs
24 hrs
48 hrs
72 hrs
96 hrs
103 13 0 6 10 10 10 103 14 0 7 10 10 10 103 15 0 5 10 10 10
10.3 16 0 0 4 5 7 10.3 17 0 0 3 5 9 10.3 18 0 0 1 6 8
1.03 19 0 0 0 0 0 1.03 20 1 1 1 1 1 1.03 21 0 0 0 0 0
0.103
22
0
0
0
0
0
0.103
23
0
0
0
0
0
0.103
24
0
0
0
0
0
o1 25 0 0 0 0 1
o' 26 1 1 1 1 2 o' 27 0 0 0 0 0
o2 28 0 0 0 0 0 02 29 0 0 0 0 0 o2 30 0 0 0 0 0
1dosed with acetone 2 undosed
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CSL Study Number HT5601
Table A2. Results of range-finding contact dosing test with PFOS - Sub-lethal effects
Actual dose Cage
r (Mg a.iybee)
No.
Num ber knockdown (K) or stumbling (S)
i
t 4 hrs
24 hrs
48 hrs
72 hrs
96 hrs
Ii % 103 13 0 2K 0 0 0
r % 103 14 0 0 0 0 0
[ 103 15 0 2S 0 0 0
10.3 16 0 0 0 0 0 10.3 17 0 0 0 IK 0 10.3 18 0 0 0 0 0
1.03 19 0 0 0 0 0 1.03 20 0 0 0 0 0 1.03 21 0 0 0 0 0
0.103
22
0
0
0
0
0
0.103
23
0
0
0
0
0
0.103
24
0
0
0
0
0
O' 25 0 0 0 0 0
o 1 26 0 0 0 0 0 o' 27 0 0 0 0 0
o2 28 0 0 0 0 0 02 29 0 0 0 0 0 O2 30 0 0 0 0 0
1dosed with acetone 2 undosed
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CSL Study Number HT5601
Table A3. Results of range-finding contact dosing test with Dimethoate - Mortality
Actual dose Cage (jig a.iibee) No.
Cumulative number dead (n=10)
4 hrs 24 hrs
48 hrs 72 hrs 96 hrs
0.30 1 2 10 10 10 10 0.30 2 0 10 10 10 10 0.30 3 0 9 9 10 10
0.15 4 0 5 6 6 6 0.15 5 0 5 6 6 6 0.15 6 0 3 4 5 6
0.075
7
0
0
0
0
0
0.075
8
0
0
0
0
0
0.075
9
0
0
0
0
0
O1 10 0 0 0 0 0 o' 11 0 0 0 0 0 O1 12 0 0 0 0 0
1dosed with Triton X-100
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Table A4. Results of range-finding contact dosing test with Dimethoate - Sub-lethal effeP!
Actual dose Cage
* (Mg a.i^bee) No. Number knockdown (K) or stumbling (S)
f 4 hrs
24 hrs
48 hrs
72 hrs
96 hrs
l 0.30 1 2K 0 0 0 0 0.30 2 4K 0 0 0 0 0.30 3 3K 0 0 0 0
0.15 4 IK 0 0 0 0 0.15 5 0 0 0 0 0 0.15 6 0 0 0 IK 0
0.075
7
0
00
0
0
0.075
8
0
0
0
0
0
0.075
9
0
0
0
0
0
l 0 ` 10 0 0 0 0 0 O1 11 0 0 0 0 0 o' 12 0 0 0 0 0
1dosed with Triton X-100
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1
1 i* r* i* i*
t
f*
r f* T*
f* c 2 P* r 4. 4^ *
h
JL.
%
CSL Study Number HT5601
Table AS.
Results of definitive contact dosing test with PFOS - Mortality
Actual dose (pg a.i^bee)
Cage No.
Cumulative number dead (n=10)
4 hrs
24 hrs 48 hrs 72 hrs 96 hrs
45.0 213 0 45.0 214 0 45.0 215 0
7 9 10 10 3 9 10 10 5 10 10 10
20.5 216 0 20.5 217 0 20.5 218 0
4 10 10 10 3 8 9 10 5 10 10 10
9.30 219 0 1 3 5 9 9.30 220 0 1 5 9 9 9.30 221 0 0 4 5 9
4.24 222 0 0 2 4 4 4.24 223 0 0 2 4 4 4.24 224 0 0 0 3 3
1.93 225 0 0 0 0 0
1.93 226 0 1 1 1 1
1.93 227 0
113
3
o' 228 0 0 0 0 0 01 229 0 0 0 0 0 O1 230 1 1 1 1 1
02 231 0 0 0 0 0 02 232 0 0 1 1 1 02 233 0 0 0 0 0
dosed with acetone 1undosed
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i
r I*
i*
CSL Study Number HT5601
Table A6. Results of definitive contact dosing test with PFOS - Sub-lethal effects
Actual dose (jig a.iybee)
Cage No.
Number knockdown (K) or stumbling (S)
4hrs
24 hrs
48 hrs
72 hrs
96 hrs
45.0 213 0 45.0 214 0 45.0 215 0
IK IK
0
000
000
0 0 0
20.5 216 0 0 0 0 0 20.5 217 0 0 0 IK 0 20.5 218 0 IK 0 0 0
9.30 219 0 0 0 0 0 9.30 220 0 0 0 0 IK 9.30 221 0 0 0 0 0
4.24 222 0 0 0 0 0 4.24 223 0 0 0 0 0 4.24 224 0 0 0 0 0
1.93 225 0 0 0 0 0
1.93 226 0 0 0 0 0
1.93 227 IK
0
0
0
0
o1 228 0 0 0 0 0 o' 229 0 0 0 0 0 o' 230 0 0 0 0 0
o2 231 0 0 0 0 0 o2 232 0 0 0 0 0 o2 233 0 0 0 0 0
1dosed with acetone 2undosed
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Table A7. Results of definitive contact dosing test with Dimethoate - Mortality
Actual dose Cage (fig a.iybee) No.
Cumulative number dead (n=10)
4hrs
24 hrs 48 hrs 72 hrs 96 hrs
0.30 201 2 8 8 8 8 0.30 202 3 6 6 6 7 0.30 203 1 9 10 10 10
0.15 204 1 3 3 3 3 0.15 205 0 5 5 5 5 0.15 206 1 4 4 4 4
0.075
207
0
0
0
0
0
0.075
208
0
1
111
0.075
209
0
0
0
00
O1 210 0
12 22
o' 211 0 0 0 0 0
O1 212 0 0 0 1 1
t
1dosed with Triton X-100
t
Environmental Laboratory Project Number U2723 Page 21 of 25
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3D
rl
%
%
CSL Study Number HT5601
Table A8. Results of definitive contact dosing test with Dimethoate - Sub-lethal
Actual dose (fig a.iibee)
Cage No.
Number knockdown (K) or stumbling (S)
4hrs
24 hrs
48 hrs
72 hrs
96 hrs
0.30 201 0 0 0 0 0
0.30 202 0
IK 0
0
0
0.30 203 IK
0
0
0
0
0.15 204 0 0 0 0 0 0.15 205 0 0 0 0 0 0.15 206 0 0 0 0 0
0.075
207
0
0
0
0
0
0.075
208
0
0
0
0
0
0.075
209
0
0
0
0
0
O1 210 IK 0 0 0 0 o' 211 0 0 0 0 0 O1 212 0 0 0 0 0
1dosed with Triton X-100
Environmental Laboratory Project Number U2723 Page 22 of 25
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CSL Study Number HT5601
APPENDIX 2 INTERIM CERTIFICATE OF ANALYSIS FOR TEST ITEM
INTERIM CERTIFICATE OF ANALYSIS
Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A
3M Product: PFO S,Lot217
Reference U: SD-018
Teat Name Purity1
Purity: 86.9% Specifications
86.9%
Appearance
Identification NMR
Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese
Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC
Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate*
Organic A cids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA
Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine
White Crystalline Powder 5:ri :iiTf:*:{--ri-:i'S:X:: i-f I;1 ?:I {LL*-i::i'::i:: i j :H:vi i;i:
llllllllSliBilllllllSIWSi
^Miii.l':;:'i;iin-:?!i=:;H`^^ E
1VjH* ^ i!vij 5rr
ii:
1 2 J*
itIp illlllli
& >'
5&:.
it;i:ti:>3:-:i?-3r-:S?:
V:;:;:
1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% I 5. Theoretical Value = 60%
Conforms
Positive
1. 0.005 wtVwt.% 2. 0.001 wtVwt.% 3. 1.439 w t/w t.% 4. 6.849 wt./wt.% 5. <0.001 w t/wt.% 6. 0.005 wt./wt.% 7. <0.001 wt./wt.%
1.93 wtVwt.% 8.41 wtVwt.%
None Detected
0.33 wtVwt.% None Detected Not Applicable3
1. <0.015 wtVwt.% 2. 0.59 wt/wt.% 3. <0.040 wt/wt.% 4. <0.009 wt/wt.% 5. <0.006 wt/wt.% 6. <0.007 wt/wt.% 7. 8.76 wt/wt.%
1. <0.1 w t/w t.% 2. <0.1 w t/w t% 3. 0.10 wt/wt.% 4. 0.28 wt/w t.%
1. 12.48 w t/w t.% 2. 0.244 wt/wt.% 3. 1.74 w t/w t.% 4. 8.84 wt/wt.% 5. 54.1 w t/w t.%
COA023-OI8A
Page 1 o f3
3
Environmental Laboratory Project Number U2723 Page 23 of 25
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CSL Study Number HT5601
INTERIM CERTIFICATE OF ANALYSIS
Centre Analytical Laboratories COA Reference #: 023-018A D ate o f Last Analysis: 08/31/00
Expiration Date: 08/31/01
Storage Conditions: Frozen <-10C
Re-assessment Date: 08/31/01
`Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38% +POAA, 0.33% )
Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9%
'Potassium is expected in this salt form and is therefore not considered an impurity.
'Purity by DSC is generally not applicable to materials o f low purity. N o endotherm was observed for this sample.
4Sulfur in the sample appears to be converted to SO* and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO* is not considered an impurity.
5T F A HFBA NFPA PFPA
Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic add Pentafluoropropanoic acid
^Theoretical value calculations based on the empirical formula, CgFnSOjTC* (M W =538)
This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160).
(
COA023-018A
Environmental Laboratory Project Number U2723 Page 24 of 25
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[*>
r* f i* i*
CSL Study Number HT5601
INTERIM CERTIFICATE OF ANALYSIS
Centre Analytical Laboratories COA Reference #: 023-018A
LC/MS Purity Profile:
Impurity C4 C5 C6 C7
Total
wt./wt. % 1.22 1.33 4.72 1.14 8.41
Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 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.
Prepared By: _________________ _______________ ___________________ ;
David S. Bell
Date
Scientist, Centre Analytical Laboratories
Reviewed B y : _________________________________
____
John Flaherty
Date
Laboratory Manager, Centre Analytical Laboratories
COA023-018A
Environmental Laboratory Project Number U2723 Page 25 of 25
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CJSL Study Number HT5601
CENTRAL SCIENCE LABORATORY AMENDMENT TO PROTOCOL
Study title Test facility
Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS): An acute contact toxicity study with the honey bee
Environmental R&D Team, Environmental Biology Group Central Science Laboratory, Sand Hutton, York, Y 0 4 1 1LZ
Study number
HT5601
Amendment number: 1
Number o f pages: 2
Study Director Paul Wilkins ,
Signature
Date '4 / o f a (dd/nun/yy)
Management Representative Medwin Bew
USignature.
Date lu l- o~l (dd/mm/wl
Environmental Laboratory Project Number U2723 page 1 of 2
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ilUUiUVX U 1 J U U 1
CENTRAL SCIENCE LABORATORY PROTOCOL AMENDMENT
Amendment No 1 to Protocol HT5601
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:
Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS): An acute contact toxicity study with the honey bee
to
Perfluorooctanesulfonate, Potassium Salt (PFOS): An acute contact 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
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