Slow-Stirring Method for Highly Hydrophobic …

1 validation study Slow-Stirring Method for POW-determination 1 Ring TestProposal of the Netherlands for a new OECD guidelinePartition Coefficient n-Octanol/Water (POW) Slow-Stirring Method for Highly Hydrophobic ChemicalsValidation ReportRIVM contract-Nrs602730M/602700/01 Johannes TollsIRAS Environmental Toxicology and ChemistryUtrecht UniversityValidation study Slow-Stirring Method for POW-determination 2 Table of contentsThe validation 3 Annex 1 The validation management 9 Parties and 9 Persons 10 Annex 2 Original guideline proposal - Draft submitted to 11 Annex 3 - The validation study 25 Annex 4 Test compound selection and establishing reference 27 Number of test 27 Test compound 28 Derivation of log POW,ref 29 Annex 5 - Guideline for participants in the validation 31I. General:.. :.. 31 Annex 6 - Results of the validation 37 Corollary 38 Overview of the 39 Repeatability and reproducibility of the Slow-Stirring 46 Conclusions and 48 Annex 7 Error analysis / Method 49 Error 49 Optimization of Technical Guideline 51 Recommendations for validation study of optimized Technical Guideline 51 Derivation of log POW,ref for 2,2 ,3,3 5,5 ,6.

2 6 52 Annex 8 Results of the validation study with the optimized 54 Data 54 Overview of the log POW 55 Statistical 57 Discussion and 57 Annex 9 - Synopsis of the results of the validation 59 Overview of the 60 Repeatability and reproducibility of the Slow-Stirring 61 Accuracy of the Slow-Stirring 63 validation study Slow-Stirring Method for POW-determination 3 The validation studyThis document describes the validation procedure in the process of seeking approval for theproposal of a Test Guideline of the Slow-Stirring Method for determination of log POW. Thevalidation procedure followed the scheme laid down in Draft Guidance Document on theDevelopment, validation and Regulatory Acceptance of New and Updated InternationallyAcceptable Test methods in Hazard Assessment [1].Pre- validation phaseIn the prevalidation phase the need for an OECD approved direct experimental Method fordetermination of POW of Highly Hydrophobic organic compounds was recognized.

3 The Slow-Stirring Method for determination of POW is employed by academic scientists for the preciseand accurate determination of log POW for Highly Hydrophobic compounds (log POW > 5) [2-7].This was first demonstrated through experimental work by Brooke et al. [2] and de Bruijn andcoworkers [3] and is acknowledged by the rating scheme for the evaluation of the reliability ofPOW determination as cited by Pontolillo and Eganhouse [8] which allows the acceptableratings for log POW > 5 for data determined by the Slow-Stirring Method , more than 15 years of application of this technique adequate protocols have been developedfor POW determination using the Slow-Stirring Method . Therefore, the Netherlands NationalCoordinator of the OECD Test Guidelines Programme started a validation procedure in orderto evaluate the performance of the Slow-Stirring phase1. The Netherlands National Coordinator of the OECD Test Guideline Programme involvedthe Institute of Risk Assessment Sciences (formerly RITOX, Utrecht University) with theperformance of an inter-laboratory comparison (ring test) as a study for the validation study ofthe Slow-Stirring IRAS developed an initial draft proposal for the respective OECD test guideline and adesign of the The Netherlands National Coordinator of the OECD Test Guideline Programme and IRAS established a validation management group.

4 It consists of the leading laboratory (IRAS), andNetherlands National Coordinator of the OECD Test Guideline Programme, and a review taskgroup of scientists. The management group is presented in Annex The initial draft proposal for the respective OECD guideline and the ring test design(including the selection of the test compounds) were reviewed by the review task group. Thereviewed draft proposal (Annex 2) served as the basis for the validation Volunteer participants for the ring test were recruited by alerting candidate laboratories viathe national OECD Coordinators of the OECD Test Guideline Programme and also byemploying the network of the RITOX contacts. Annex 3 specifies the participants for the ringtest. The participating laboratories work according to Good Laboratory Practice (GLP)schemes except for the university laboratories. Their ring test activities were performed in linewith study Slow-Stirring Method for POW-determination 46.

5 Four test compounds were selected and reference log POW data were derived (Annex IV).The criteria and considerations employed in test compound selection are presented there A laboratory manual for the ring test was drafted involving the expertise at the RITOX aswell as at the Dutch National Institute of Health and the Environment (RIVM) (Annex V).8. Test compounds were distributed among the participating laboratories, along with thelaboratory manual and diskettes for data reporting. The concentrations of the test compoundsare specified in Annex The tests were performed by the participating The results were reported to The experimental data were evaluated by RITOX according to the procedure described inthe draft proposal for the OECD test guideline (Annex 1). The raw data are included in The results and discussion were documented in the report (Annex VII). The test results arereproducible and accurate for 1,2,3,4-tetrachlorobenzene and hexachlorobenzene.

6 They arereproducible but inaccurate for the two most Hydrophobic chemicals. Thus the reliabilitycriterion was not An analysis of possible sources of error was performed and proposals for optimization ofthe Method are made (Annex 8). The inaccuracy noted for the two most Hydrophobic testcompounds was found to be due to the solubility of these compounds being exceeded in theoctanol solution employed in the The Method was optimized on the basis of this finding. Optimization was achieved byusing test compound concentrations that do not exceed the solubility in The optimized Method underwent a new validation Two test compounds, decachlorobiphenyl and 2,2 ,3,3 ,5,5 ,6,6 -octachlorobiphenyl wereselected. The latter replaces p,p -DDT. This was necessary due to the low number of reliablelog POW data available for derivation of a reference The tests were performed by the participating The results were reported to The experimental data were evaluated by RITOX according to the procedure described inthe draft proposal for the OECD test guideline (Annex I).

7 20. The results were documented in a report pertaining to the test of the optimized guideline(Annex VIII). The test results are reproducible and in good agreement with the referencevalues of log A synopsis of the results of the first and second validation study was prepared (Annex 9).The conclusions of the synopsis are that the optimized Method is reliable since the combinedtest results for 1,2,3,4-tetrachlorobenzene and hexachlorobenzene of the first and fordecachlorobiphenyl and 2,2 ,3,3 ,5,5 ,6,6 -octachlorobiphenyl in the second validation studyare precise and stepsFormal approval of the Netherlands proposal for the enclosed OECD test guideline will haveto take place in the OECD National Coordinators meeting after the proposal has beencirculated within the OECD member countries for results of the validation exercise are to be published in a peer-reviewed journal.

8 Thecorresponding manuscript is presently in preparation [9]. validation study Slow-Stirring Method for POW-determination 5 References[1]OECD. 2001. Draft Guidance Document on the Development, validation and RegulatoryAcceptance of New and Updated Internationally Acceptable Test methods in HazardAssessment - OECD Environment, Health and Safety Publications - Series on Testingand Assessment No. 34, OECD, Paris.[2]Brooke DN, Dobbs AJ, Williams N. 1986. Octanol:Water partition coefficients (P):Measurement, estimation, and interpretation, particularly for chemicals with P > and Environmental Safety 11: 251-260.[3]de Bruijn JHM, Busser F, Seinen W, Hermens J. 1989. Determination of octanol/waterpartition coefficients with the Slow-Stirring Method . Environ. Toxicol. Chem. 8: 499-512.[4]de Maagd PGJ, ten Hulscher DTEM, H. van den Heuvel, Opperhuizen A, Sijm Physicochemical properties of polycyclic aromatic hydrocarbons: aqueoussolubilities, n-octanol/water partition coefficients, and henry s law constants.

9 Chem. 17: 251-257.[5]Fisk AT, Rosenberg B, Cymbalisty CD, Stern GA, Muir DCG. 1999. Octanol/waterpartition coefficients of toxaphene congeners determined by the Slow-Stirring 39: 2549-2562.[6]Sijm DTHM, Sinnige TL. 1995. Experimental octanol/water partition coefficients ofchlorinated paraffins. Chemosphere 31: 4427-4435.[7]Tolls J, van Dijk J, Verbruggen EMJ, Hermens JLM. in prep. Aqueous solubility and 1-octanol-water partition coefficients of selected long chain (C8 to C19) saturatedhydrocarbons.[8]Pontolillo J, Eganhouse RP. 2001. The search for reliable aqueous solubility (SW) andoctanol-water partition coefficient data for Hydrophobic organic compounds: DDT andDDE as a case study, Water-Resources Investigations Report 01-4201, US GeologicalSurvey, Reston, VA.[9]Tolls J, Bodo K, De Felip E, Dujardin R, Schneider J, Moeller-Jensen L, Mullee D,Nakajima A, Pawliczek J-B, Tadeo J-L, Tognucci AC, Webb J, Zwijzen AC.

10 Inpreparation. The slow stirring Method for Determination of the n-Octanol/WaterPartition Coefficient (POW) for Highly Hydrophobic Chemicals - PerformanceEvaluation in a Ring study Slow-Stirring Method for POW-determination 6 validation study Slow-Stirring Method for POW-determination 7 validation study slow stirring Method for POW-determination AnnexesValidation study Slow-Stirring Method for POW-determination 8 Annex 1 The validation management group9 Annex 1 The validation management groupParties and tasksThe validation management group was assembled by the Netherlands National Coordinator ofthe OECD Test Guideline Programme and the leading laboratory in order to plan, perform,and supervise the validation study and to forward the validation report to the OECD in orderto seek formal approval as official test Method . The validation management group consists ofthe Netherlands National Coordinator of the OECD Test Guideline Programme, a statistician,a scientific review committee, and the leading (s)