Document 0grZkbVgV5nm1Jqj88e2je8Dn
Commercial-in-Confidence Chemex reference: ENV8010/020737
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The growth inhibition of the marine alga Skeletonema costatum by
CJ
Report for Notox B.V
Report issued by:
Chemex Environmental International Limited Unit J Broad Lane Industrial Estate Cottenham Cambridge CB24 8SW England
April 2007
Sponsor:
Notox B.V Hambakenwetering 7 P.O. Box 3476 5203 DL `s-Hertogenbosch The Netherlands
ENV8010/02073 7
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Contents
Page Compliance with Good Laboratory Practice standards.......................................................... 3
Quality Assurance audit statement.................................................................................... 4
Summary................................................................................................................................ 5
1. Introduction................................................................................................................ 6
2. Materials and methods.................................................................................................. 6
2.1 Test material............................................................................................................... 6 2.2 Test organism............................................................................................................. 7 2.3 Preparation of test solutions....................................................................................... 7 2.4 Test methods and conditions...................................................................................... 8
3. Results.........................................
9
3.1 Water quality data....................................................................................................... 9 3.2 Percent inhibition by biomass integral and growth rate...............................................9 3.3 EC50 values by biomass integral (EbC50value) and growth rate (ErC50value)............9
4. Discussion.................................................................................................................. 9
5. References................................................................................................................. 10
Graphs
1. Growth curves for control and test concentrations......................................................11
2. Estimation of 0 to 48hours EbC50value..................................................................... 12
3. Estimation of 0 to 72 hours EbC50value........................................................
13
4. Estimation of 0 to 48 hours ErC5ovalue..................................................................... 14
5. Estimation of 0 to 72 hours EfCso value..................................................................... 15
Appendices
1. Cell density measurements - test sample................................................................... 16 2. Reference material results......................................................................................... 17 3. Culture media............................................................................................................. 18
ENV8010/02073 7
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Compliance with Good Laboratory Practice standards
I, the undersigned, hereby declare that the study described in this report was performed under my. supervision, and that the final report fully and accurately reflects the raw data generated during the conduct of the study, in compliance with international codes of Good Laboratory Practice including:
Section II o f Annex 1 to the European Parliament and council Directive 2004/10/EC and Annex 1 to the European Parliament and council Directive 2004/9/EC (Official Journal No. L 50) and embodied within:
The UK Good Laboratory Practice Regulations 1999 (The United Kingdom GLP Regulations 1999, Statutory Instrument 3106) as amended by:
The UK Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004 (Statutory Instrument No 994)
These principles are in accordance with the OECD Principles of Good Laboratory Practice
revised 1997 (ENV/MC/CHEM(98)17).
'
Study Director
Date
ENV8010/020737
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Quality Assurance audit statement
Studies of this type are subject to random process-based inspections by the Chemex Quality Assurance Unit.
Date of relevant inspection:
13 & 16 February 2007
Date relevant inspection reported: 16 February 2007
Date of study plan inspection:
28 February 2007
This report has been audited by the Chemex Quality Assurance Unit. It is considered to be an accurate description of the procedures and practices employed during the course o f the study and an accurate presentation of the findings.
Date audit started:
20 April 2007
Date audit reported:
23 April 2007
Signed: Date:
David Stittle Quality Manager
Z 3 A p r 1\ 2-0 0 1
All reports are individually audited.
Reports are issued to Management on a monthly basis.
Raw data used to derive results included in this report, supplied by either the sponsor or sub contractor (where applicable), are not audited by Chemex Environmental International Limited.
ENV8010/020737
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Summary
This section summarises aquatic toxicity test results obtained by Chemex Environmental International Limited on a sample as detailed below:
Test commissioned by: Substance under test: Chemex reference:
Test species: Test type: Protocol:
Test period: Test concentrations: Test performed at:
Notox B.V
CJ
Sample: EC0020737 Study: ENV8010
Skeletonema costatum, strain 1077/5
Acute toxicity: 72 hour EC5o
Static test conditions according to SOP E209 based on ISO/DIS BS EN ISO 10253: 1998 "Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricomutum.
From 2 to 5 April 2007
.
0, 2.2, 4.5, 10, 22, 45 and 100mg/l
Chemex Environmental International Limited Unit J
Broad Lane Industrial Estate Cottenham Cambridge CB24 8SW England
Results:
Period of exposure (hours)
0 to 48
Oto 72
NOECb (biomass) NOECr (growth rate)
EbC50value mg/1 biomass
(95% confidence limits)
14mg/l (10 - 19mg/l)
Umg/l (1 0 - 17mg/l)
1 |
|
Period of exposure (hours)
0to48
0to72
ErC50value mg/1 growth rate
(95% confidence limits)
23mg/l (21-25mg/l)
24mg/l (21 - 27mg/l)
4.5mg/l (0-72 hours)
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1. Introduction
This report contains a description of the methods used and the results obtained during a study to investigate the marine algal growth inhibition o f j J to Skeletonema costatum. The objective of this study was to determine the 72 hour median effective concentration (EC50) which is defined as the concentration which inhibits the growth of the marine algae by 50% after a 72 hour exposure period at 20C.
2. Materials and methods
Unless otherwise specified all methods mentioned in this report are according to Chemex Environmental International Limited standard procedures. All records of measurements and observations made during this test will be collated and held in the Chemex Environmental International Limited archives at Unit J, Broad Lane Industrial Estate, Cottenham, Cambridge CB24 8SW, England.
2.1 Test material
Identification: Supplied by: Date of receipt: Chemex reference: Required storage conditions: Actual storage conditions: Quoted stability: Quoted appearance: Observed appearance: Quoted odour: Quoted solubility in water:
Notox B.V 27 February 2007 EC0020737 Store in original containers at room temperature Room temperature in dark Stable Amber viscous liquid/paste Amber viscous liquid/paste Not Stated Complete
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2.2 Test organism Species: Source: Culture conditions:
Culture media:
Skeletonema costatum strain: 1077/5
Culture Collection of Algae and Protozoa Dunstaffnage Marine Laboratory, Scotland
Temperature: 20 1C Illumination: 6000 - 10000 lux continuous white light Shaking: 200rpm
Natural seawater with added nutrients according to the ISO standard, with concentrations of iron and EDTA at the levels recommended by VKI (Danish Water Quality Institute) (see Appendix 2).
2.3 Preparation of test solutions
The behaviour oi(2
was examined in seawater according to the method described in the
Harmonised Offshore Chemical Notification Format (OSPAR 1995).
A nominal l,000mg/l solution was prepared in dilution water (see section 2.4), shaken vigorously and allowed to stand for one hour.
A faintly grey solution was obtained. Test concentrations were prepared by mixing specified quantities of the sample with culture medium to obtain the required concentrations.
A preliminary study had identified the 72 hour EC50 as being between 10 and 32mg/l and therefore definitive test concentrations were prepared as 0, 2.2, 4.5, 10,22, 45 and 100mg/l.
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2.4 Test methods and conditions
Static test conditions according to ISO draft method (1991) "WATER QUALITY Marine algal
growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum" (ISO/DIS 10253).
Chemex SOP reference:
E209 "Marine Algal Growth Inhibition test with Skeletonema costatum and Phaeodactylum tricomutum,,
Test period:
2 to 5 April 2007
Test volume:
200ml
Test vessel:
250ml conical flask
Number of replicates: Seawater:
6 control flasks, 3 replicates at each test concentration
Collected on 6 December 2006 from the CEFAS laboratories at Bumham-on-Crouch directly from the River Crouch estuary.
Algal test inoculum:
From a pre-culture growing in exponential phase under conditions described in 2.2 above. Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.
Test conditions:
Temperature: 20 1C Illumination: 6000 - 10000 lux continuous white light Shaking: 200 rpm
Water quality measurements:
The pH (to 0.1) and temperature (to 0.5C) were measured on the pooled replicates for each test and control solution immediately prior to initiating the test and at the end of the full 72 hour test period.
Observations/frequency:
The cell density was determined at 24, 48 and 72 hours on a small volume removed from each replicate flask. The cell counts were made using a haemocytometer and microscope.
Calculation and expression of results:
Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (area under the growth curve) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", copyright 1994-1996.
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3. Results
3.1 Water quality data
Nominal concentration (mg/1)
0 2.2 4.5 10 22 45 100
Mean pH values Start of test End of test
8.0 8.6 8.0 8.7 8.0 8.7 8.0 8.3 8.0 8.0 8.0 8.0 8.0 8.0
Temperature C Start of test End of test
20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0
3.2 Percent inhibition by biomass integral and growth rate
Nominal concentration (mg/1)
2.2 4.5 10 22 45 100
Percent inhibition
by biomass integral
48 hours
72 hours
-6 -8
02
39 45
68 73
100 100
100 100
Percent inhibition
by growth rate
48 hours
72 hours
2 -3
-1 1
13 17
32 35
100 100
97 100
Note: Negative inhibition values indicate an increase in growth compared with controls.
3.3 EC50 values by biomass integral (EbC5ovalue) and growth rate (ErC50value)
Period of exposure (hours)
0 to 48
0 to 72
NOECb (biomass) NOECr (growth rate)
EbC50value mg/1 biomass
(95% confidence limits)
14mg/l (10 - 19mg/l)
13mg/l (10 - 17mg/l)
Period of exposure (hours)
0 to 48
Oto 72
ErCS0value mg/1 growth rate
(95% confidence limits)
23mg/l (21 -25mg/l)
24mg/l (21 - 27mg/l)
4.5mg/l (0-72 hours)
4. Discussion
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The 72 hour EC50 o .C ZU io Skeletonema costatum was estimated as 13mg/l using the biomass integral and 24mg/l by growth rate calculation. The 48 hour EC50 was 14mg/l by biomass integral and 23mg/l by growth rate. The 72 hour NOEC was estimated as 4.5mg/l as determined by the Bonferroni T test.
The growth curves illustrated in Graph 1 show that growth inhibition increases with increasing concentration, demonstrating a clear dose response to the test material. The inhibition values given in 3.2 support these observations.
The test organism sensitivity was also monitored during this study using the recommended reference substance 3,5 DCP. The algae were exposed to a 1.5mg/l concentration of 3,5 DCP for 72 hours under test incubation conditions and therefore the 42% inhibition by growth rate represents an acceptable level of sensitivity (ie. 72 hour inhibition was within the range of 20 to 80%).
The water quality measurements of the test solutions are given in 3.1 and were within acceptable limits. The cell density of the control during the 72 hour test period increased by a factor of 67, which is satisfactory.
There was no chemical analytical confirmation of the actual dissolved concentrations. The dissolved concentrations were likely to be as given in this report as the `sample was soluble in water.
5. References
ISO/DIS BS EN ISO 10253: 1998 "Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum.
VKI - "Addendum to PARCOM Ring Toxicity Test with Marine Unicellular Algae" (1991) (Danish Water Quality Institute).
OSPAR - Oslo and Paris Conventions for the Prevention of Marine Pollution Programmes and Measures Committee (PRAM) Harmonised Offshore Chemical Notification Format 1995.
ToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software",copyright 1994-1996.
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Graph 1 Growth curves for control and test concentrations
Concentration in mg/1
-- 0 -*-2.2 -- 4.5 -- 10 -- 45 -- 100
-- 22
Statistical data
NOECb (biomass) = 4.5mg/l
NOECr (growth rate) = 4.5mg/l
LOEC
= 10mg/l
As determined by the Bonferroni T test, based on the 0-72 hour values.
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Graph 2 Estimation of 0 to 48 hours EbC5ovalue
Statistical data
Estimated EC5ovalue = 14mg/l (as determined by the Maximum Likelihood-Probit Method)
95% confidence limits = 10 to 19mg/l
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Graph 3 Estimation of 0 to 72 hours EbCso value
---------
Denotes 95% confidence limits
Statistical data
Estimated EC50 value = 13mg/l (as determined by the Maximum Likelihood-Probit Method)
95% confidence limits = 10 to 17mg/l
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Graph 4 Estimation of 0 to 48 hours ErCso value
Statistical data Estimated EC50 value
95% confidence limits
23mg/l (as determined by the Spearman-Karber Method)
21 to 25mg/l
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Graph 5 Estimation of 0 to 72 hour ErC5ovalue
Statistical data
Estimated EC5ovalue = 24mg/l (as determined by the Spearman-Karber Method)
95% confidence limits = 21to27mg/l
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Appendix 1 Cell density measurements - test sample
Concentration (mg/1)
0
2.2 4.5 10 22 45 100
Replicate
1 2 3 4 5 6 Mean 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean 1 2 3 Mean
Cell density measurements
(cells/ml x 104)
24 hours
48 hours
72 hours
2.67 22.67 69.33
6.33
21.67
62.33
6.67
27.67
72.00
4.00
24.00
56.67
4.33
23.00
67.67
8.33
22.67
73.33
5.39
23.61
66.89
10.00
22.00
87.33
5.33
24.67
71.00
6.00
19.67
68.33
7.11
22.11
75.55
4.67
29.33
66.67
3.67 23.33 60.00
6.67 20.33 64.33
5.00 24.33 63.67
4.00
18.00
35.00
2.00
13.00
32.67
4.00
15.67
30.67
3.33
15.56
32.78
0.00 5.33 14.33
2.33
14.67
14.67
3.00 8.00 16.33
1.78 9.33 15.11
0.00 0.00 0.00
1.33 0.00 0.00
0.67 0.00 0.00
0.67 0.00 0.00
0.00 0.00 0.00
0.00 0.00 0.00
0.00 1.33 0.00
0.00 0.44 0.00
Mean initial cell density: 1 x 104 cells/ml.
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Appendix 2 Reference material results
The test organism sensitivity was monitored during this study using the recommended reference substance 3,5 DCP. The algae were exposed to a 1.5mg/l concentration of 3,5 Dichlorophenol for 72 hours under test incubation conditions.
Percent inhibition by biomass integral and growth rate inhibition
Nominal concentration
3,5-DCP
1.5mg/l
Percent inhibition
by biomass integral
48 hours
72 hours
60 75
Percent inhibition
by growth rate
48 hours
72 hours
38 42
Cell density measurements
Concentration 3,5-DCP (mg/1)
0
1.5
Replicate
1 2 3 4 5 6 Mean 1 2 3 Mean
Cell density measurements
(cells/ml x 104 )
24 hours
48 hours
72 hours
2.67
22.67
69.33
6.33
21.67
62.33
6.67
27.67
72.00
4.00
24.00
56.67
4.33
23.00
67.67
8.33
22.67
73.33
5.39
23.61
66.89
6.00 9.67 17.33
3.67 7.67 13.00
2.33 5.00 7.00
4.00 7.45 12.44
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Appendix 3 Culture media
Culture medium:
Prepared in natural seawater according to the ISO/DIS 10253 standard except that concentrations of iron and EDTA were at the levels recommended by VKI (Danish Water Quality Institute).
Seawater:
Filtered, and sterilised by autoclaving at 120C for 30 minutes.
Method of preparation:
Sterile nutrient stock solutions were prepared and added to the seawater to obtain the culture medium.
Composition of culture medium:
Nutrient
FeCl3 6H20 MnCl2.4H20 ZnS04.7 H20 CuS 0 4.5 H20 CoC12.6 H20 H3BO3 Na2 EDTA, 2H20 Thiamin hydrochloride Biotin Bi2 (cyanocobalamin) k 3 p o 4h 2o NaN03 Na2S i 0 3.5H20
Final concentration in test solution 16.4pg/l (Fe) 605pg/l (Mn) 150pg/l (Zn) 0.6pg/l (Cu) 1.5pg/l (Co) 17.1mg/l 100pg/l
25pg/l 0.005pg/l 0.05pg/l 3.25mg/l 50.0mg/l 14.9mg/l
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This TSCA 8(e) substantiation applies to the following studies for materials that have never been commercialized in the U.S.: [test substance]
In addition to the chemical identities or formula of a proprietary mixture, 3M maintains its 3M proprietary ID #s and names of 3M personnel.
1. Yes, the CBI claim is on behalf o f 3M Company, including its wholly-owned subsidiaries and affiliates.
2. The period o f time for 3M 's confidentiality claim is indefinite. 3M 's claim is for the chemical identities of components of materials that have been commercialized in the U.S. 3M engaged in significant R&D efforts to develop this product. 3M has always claimed the components of the chemical identities in question as proprietary and continues to do so today. With this knowledge, competitors could do one or more of the following: (a) deduce (and potentially duplicate) 3M 's process technology, process chemistry and process know-how; (b) develop products with components and/or functionalities identical or similar to 3M 's current products; and (c) devise marketing strategies and tactics to promote their current products as compared to 3M 's current products. In these ways, disclosure o f 3M's proprietary information would deprive 3M o f its intellectual property and its competitive advantage and would bestow a competitive advantage that 3M 's competitors would not otherwise be able to achieve using their independent "know how". 3M would be deprived of research and development investment return as a result of such disclosure.
3. If a PMN was written, the PMN number is provided in the confidential copy o f the Index to Studies Submitted. Disclosure would have been made to the U.S. EPA. Otherwise, no other disclosure was made.
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6. No, the chemical identities only appear as a generic name on the MSDS. 7. No. 8. Refer to response to Question #2. 9. No. 10. a) Yes b) Approximately 3 years. C) The mixture is a raw material used at a very
small percentage in hard surface coating formulations. Competitors are not aware of the exact formulation of the mixture. 11. Determining the exact chemical identity o f the substance would require the use of very sophisticated analytical equipment and methods by highly skilled chemists and would require an extended period of time. 12. a.) Yes, refer to response to Question #2. b.) Yes, refer to response to Question #2. c.) Yes, refer to response to Question #2. 13. No CAS number exists for the polymer in the product. 14. No.
0
3M Company Contact name: Deanna Luebker, PhD, 3M TSCA 8(e) Coordinator 3M Toxicology Assessment and Compliance Assurance 3M Center 220-06-E-03 St. Paul, MN 55144 651-737-1374 (office) 651-733-1773 (fax) di luebker@mmm.com
