Guidance on information requirements and chemical safety assessment : Chapter R.11: PBT and vPvB assessment.
Date: 2017-07-03
Institutions: European Chemicals Agency
Subjects: chemical industry, chemical product, environmental policy, environmental risk prevention, industrial hazard, pollutant, technical regulations, toxic substance, user guide

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

1 

G U I D A N C E    

Guidance on Information Requirements 
and Chemical Safety Assessment 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 

June 2017 

  
  
2 

Legal notice 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

This document aims to assist users in complying with their obligations under the REACH 
Regulation. However, users are reminded that the text of the REACH Regulation is the only 
authentic legal reference and that the information in this document does not constitute legal 
advice. Usage of the information remains under the sole responsibility of the user. The 
European Chemicals Agency does not accept any liability with regard to the use that may be 
made of the information contained in this document. 

Guidance on Information Requirements and Chemical Safety Assessment 
Chapter R.11: PBT/vPvB Assessment 

Reference: ECHA-17-G-12-EN 
Cat. Number: ED-01-17-294-EN-N 
ISBN: 978-92-9495-839-6 
DOI: 10.2823/128621 
Publication date: June 2017 
Language: EN 

© European Chemicals Agency, 2017 

If you have questions or comments in relation to this document please send them (indicating 
the document reference, issue date, chapter and/or page of the document to which your 
comment refers) using the Guidance feedback form. The feedback form can be accessed via 
the ECHA Guidance website or directly via the following link: 

https://comments.echa.europa.eu/comments_cms/FeedbackGuidance.aspx 

European Chemicals Agency 

Mailing address: P.O. Box 400, FI-00121 Helsinki, Finland 
Visiting address: Annankatu 18, Helsinki, Finland 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

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Preface 

This document describes the information requirements under the REACH Regulation with 
regard to substance properties, exposure, use and risk management measures, and the 
chemical safety assessment. It is part of a series of guidance documents that are aimed to 
help all stakeholders with their preparation for fulfilling their obligations under the REACH 
Regulation. These documents cover detailed guidance for a range of essential REACH 
processes as well as for some specific scientific and/or technical methods that industry or 
authorities need to make use of under the REACH Regulation. 

The original versions of the guidance documents were drafted and discussed within the REACH 
Implementation Projects (RIPs) led by the European Commission services, involving 
stakeholders from Member States, industry and non-governmental organisations. After 
acceptance by the Member States competent authorities the guidance documents had been 
handed over to ECHA for publication and further maintenance. Any updates of the guidance 
are drafted by ECHA and are then subject to a consultation procedure, involving stakeholders 
from Member States, industry and non-governmental organisations. For details of the 
consultation procedure, please see: 

http://echa.europa.eu/documents/10162/13559/mb_63_2013_consultation_procedure_for_gui
dance_revision_2_en.pdf  

The guidance documents can be obtained via the website of the European Chemicals Agency 
at: 

http://echa.europa.eu/web/guest/guidance-documents/guidance-on-reach      

Further guidance documents will be published on this website when they are finalised or 
updated. 

This document relates to the REACH Regulation (EC) No 1907/2006 of the European 
Parliament and of the Council of 18 December 20061.  

1 Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 
concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing 
a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) 
No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and 
Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (OJ L 396, 30.12.2006, p.1; 
corrected by OJ L 136, 29.5.2007, p.3). 

  
 
 
 
 
 
                                                 
 
4 

Document History 

Version 

Changes 

Version 1 

First edition 

Version 1.2 

Corrigendum: 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Date 

May 2008 

November 2012 

November 2014 

Version 2.0 

(i) replacing references to DSD/DPD by references 
to CLP; 

(ii) further minor editorial changes/corrections. 

Second edition.  Full revision of this document was 
necessary to take into account the amendment of 
Annex XIII to REACH (according to Commission 
Regulation (EU) No 253/2011 of 15 March 2011, OJ 
L 69 7 16.3.2011). Main changes in the guidance 
document include the following: 

  Chapter R.11 title has been changed to 

“PBT/vPvB assessment”; 

  Chapter R.11 has been re-structured to 

differentiate more clearly between the 
obligations of the registrant arising directly 
from the legal text (Section R.11.3) and the 
description of the scientific method to 
assess PBT/vPvB properties (Section 
R.11.4); 

  Description of the registrant’s obligations in 
Section R.11.3 has been expanded upon; 

 

 

The description of the scope of PBT/vPvB 
assessment regarding relevant 
constituents/impurities/additives and 
transformation/degradation products has 
been expanded upon and divided into two 
Sections: Section R.11.3.2.1 for legal 
aspects and Section R.11.4 for the aspects 
related to assessment; 

The different steps of the PBT/vPvB 
assessment process, in particular the first 
step of comparison with the PBT and vPvB 
criteria, and the subsequent conclusions 
and consequences for the registrant have 
been refined to take account of the case 
where the registrant concludes that further 
information is needed but he decides not to 
generate additional information by 
considering the substance “as if it is a 
PBT/vPvB”;  

 

The number of conclusions deriving from 
the first Step of the PBT/vPvB assessment 
process has been reduced from four to 
three in Section R.11.4.1.4 “Conclusions on 

 
 
Chapter R.11: PBT/vPvB assessment 

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PBT or vPvB properties”; 

  Consequences for the registrant of the 

conclusions deriving from the first Step of 
the PBT/vPvB assessment process are 
described in the new Section R.11.3.2. 

  Section R.11.3.2.2 is new and describes the 

situation of substances concluded as being 
PBT/vPvB by ECHA’s Member State 
Committee in relation to the inclusion in the 
Candidate List of Substances of Very High 
Concern; 

 

The basic approach to bioaccumulation 
assessment described in Section R.11.4.1.2 
has been slightly extended to reflect in 
particular the revised OECD test guideline 
305 and the possibility to take other 
bioaccumulation information into account. 
The molecular length screening threshold 
value has been removed; 

  As the screening threshold values for 
PBT/vPvB assessment are part of the 
scientific methodology and not part of legal 
text, they are now presented in relevant 
parts of Section R.11.4 only.  

 

The document has been re-formatted to 
ECHA new corporate identity. 

Version 3.0 

Revision of this document was necessary in sections 
related to the scientific assessment approach to 
take into account recent scientific and technical 
developments in the field, including recently 
adopted or revised OECD TGs. Main changes in the 
guidance document include the following: 

June 2017 

 

 Update of Section R.11.4.1.1 on 
“Persistence assessment” and of the 
corresponding Integrated Testing Strategy 
described in Section R.11.4.1.1.1 and 
Figure R.11-3; 

  Update of Section R.11.4.1.2 on 

“Bioaccumulation assessment” and of the 
corresponding Integrated Testing Strategy 
described in Section R.11.4.1.2.1 and 
Figure R.11-4; 

  Update of Section R.11.4.1.4 on 

“Conclusions on PBT or vPvB properties”; 

  Revision of Section R.11.4.2.2 on 

“Assessment of substances containing 
multiple constituents, impurities and/or 
additives”; 

  Update of Appendix R.11—3 on “PBT 

  
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Chapter R.11: PBT/vPvB assessment 

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assessment of UVCB petroleum 
substances”; 

  Update of cross references and links to the 
revised sections of Chapters R.7b and R.7c. 

 
 
Chapter R.11: PBT/vPvB assessment 

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Convention for citing the REACH Regulation 

7 

Where the REACH Regulation is cited literally, this is indicated by text in italics between 
quotes, or text in green boxes.  

Table of Terms and Abbreviations  

See Chapter R.20.   

Pathfinder 

The figure below indicates the location of Chapter R.11 within the Guidance Document: 

NoIterationYesStopNoYesInformation: available   -required/neededHazard Assessment (HA)Emission CharacterisationExposure Scenario BuildingRisk Characterisation (RC)Risk controlled?Document in CSRCommunicate  ES via SDSArticle 14(4) criteria?R11R11.3.4  
 
 
 
 
 
8 

 Table of Contents 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

R.11  PBT and vPvB Assessment ...................................................... 11 

R.11.1  Introduction ................................................................................................ 11 

R.11.2  Overview of Annex XIII to the REACH Regulation ....................................... 14 
Elements and terminology of Annex XIII to the REACH Regulation ................. 14 
PBT and vPvB criteria and information listed in Annex XIII to the REACH 

R.11.2.1 
R.11.2.2 
Regulation   ............................................................................................................. 17 

R.11.3  Duties of the registrant ............................................................................... 20 
R.11.3.1  Objective and overview of the PBT/vPvB assessment process ........................ 20 
Comparison with the criteria (Step 1) ........................................................ 23 
R.11.3.2 
  Scope of the PBT and vPvB assessment (relevant constituents, 

R.11.3.3 

transformation/degradation products) ..................................................................... 24 
  Specific cases: substances fulfilling the PBT/vPvB criteria according to ECHA’s 
Member State Committee in relation to the inclusion of substances in the Candidate List of 
Substances of Very High Concern ........................................................................... 25 
Consequences of Step 1 ........................................................................... 25 
  No consequences ................................................................................ 26 
  Conduct emission characterisation and risk characterisation ..................... 26 
  Generate relevant additional information (including, where relevant, 
submission of a testing proposal) ............................................................................ 26 
  Treat the substance “as if it is a PBT or vPvB” ........................................ 28 
Emission characterisation, risk characterisation and risk management measures . 
 ............................................................................................................. 28 
  Emission characterisation ..................................................................... 29 
  Risk characterisation and risk management measures for “PBT or vPvB 

R.11.3.4 

Substances”  ........................................................................................................ 30 
R.11.3.4.2.1  Options and measures to minimise emissions and exposure ............... 30 
R.11.3.4.2.2  Risk Characterisation for humans in cases of direct exposure to “PBT or 
vPvB substances” .............................................................................................. 32 
R.11.3.5  Documentation of the PBT/vPvB assessment ............................................... 32 
R.11.3.6  Documentation of the risk characterisation and communication of measures ... 34 

R.11.4.1 

R.11.4  Assessment of PBT/vPvB properties – the scientific method ...................... 35 
Standard approach .................................................................................. 35 
  Persistence assessment (P and vP) ........................................................ 40 
R.11.4.1.1.1 
Integrated assessment and testing strategy (ITS) for persistence 
 .................................................................................................. 40 
assessment 
Introduction to persistence assessment ........................................... 47 
R.11.4.1.1.2 
R.11.4.1.1.3  Test data on biodegradation ........................................................... 50 
R.11.4.1.1.4  Assessment based on estimation models (QSAR, SAR) ...................... 62 
R.11.4.1.1.5  Field studies for persistence ........................................................... 64 
R.11.4.1.1.6  Monitoring data ............................................................................ 65 
  Bioaccumulation assessment (B and vB) ................................................ 66 
R.11.4.1.2.1 
Integrated Assessment and Testing Strategy (ITS) ........................... 66 
R.11.4.1.2.2  Experimental aquatic bioconcentration factor (BCF) data ................... 69 

 
 
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R.11.4.1.2.3  Experimental dietary biomagnification in fish (experimental dietary BMF) 
 .................................................................................................. 71 

R.11.4.1.2.4  Experimental sediment bioaccumulation data (experimental 
Bioaccumulation Factors BAF and BSAF for sediment) ............................................ 73 
R.11.4.1.2.5  Experimental soil bioaccumulation data (experimental Bioaccumulation 
Factor BAF and BSAF for soil) .............................................................................. 75 
R.11.4.1.2.6  Field data and biomagnification ...................................................... 77 
R.11.4.1.2.7  Addressing uncertainty of field data in the assessment ...................... 78 
R.11.4.1.2.8  Use of a fugacity approach for bioaccumulation assessment ............... 79 
R.11.4.1.2.9  Other testing data ......................................................................... 80 
R.11.4.1.2.10  Further data ............................................................................... 81 
R.11.4.1.2.11  Conclusion on the endpoint .......................................................... 85 
  Toxicity assessment (T) ....................................................................... 87 
R.11.4.1.3.1 
Integrated testing and assessment strategy (ITS) for T-testing in support 
of PBT assessment for the aquatic environment .................................................... 87 
R.11.4.1.3.2  The toxicity criterion ..................................................................... 90 
R.11.4.1.3.3  Use of QSAR data ......................................................................... 92 
R.11.4.1.3.4  Screening information and screening threshold values ....................... 92 
R.11.4.1.3.5  Water accommodated fraction (WAF) .............................................. 94 
R.11.4.1.3.6  Use of non-testing data ................................................................. 94 
  Conclusions on PBT or vPvB properties .................................................. 96 

R.11.4.1.4.1  (i) The substance does not fulfil the PBT and vPvB criteria. The available 
information show that the properties of the substance do not meet the specific criteria 
provided in REACH Annex XIII Section 1, or if the information does not allow a direct 
comparison with all the criteria there is no indication of P or B properties based on 
screening information or other information. .......................................................... 96 
R.11.4.1.4.2  (ii) The substance fulfils the PBT and/or vPvB criteria. The available 
information show that the properties of the substance meet the specific criteria detailed 
in REACH Annex XIII Section 1 based on a Weight-of-Evidence determination using 
expert judgement comparing all relevant and available information listed in Section 3.2 
of Annex XIII to REACH with the criteria (for more specific terminology, also used in 
IUCLID, please, see subsection “Terminology”). .................................................... 97 
R.11.4.1.4.3  (iii) The available information does not allow to conclude (i) or (ii). The 
substance may have PBT or vPvB properties. Further information for the PBT/vPvB 
assessment is needed. ....................................................................................... 99 
Assessment of PBT/vPvB properties – consideration of specific substance 

R.11.4.2 
properties   ............................................................................................................ 101 

  Assessment of substances requiring special considerations with regard to 

testing 

additives 

 ....................................................................................................... 101 
R.11.4.2.1.1  Substances with very high sorptivity .............................................. 101 
R.11.4.2.1.2  Substances with low solubility in octanol and water .......................... 102 
  Assessment of substances containing multiple constituents, impurities and/or 
 ....................................................................................................... 106 
R.11.4.2.2.1 
Initial profiling of the substance composition ................................... 107 
R.11.4.2.2.2  Assessment approaches ................................................................ 108 
R.11.4.2.2.3  Specific aspects ........................................................................... 114 
R.11.4.2.2.4  Test items ................................................................................... 115 

R.11.5  References ................................................................................................ 117 

  
 
 
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Chapter R.11: PBT/vPvB assessment 

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Table of Figures 

Figure R.11—1: Overview of the conclusions from Step 1 (“Comparison with the criteria”) and 
their consequences. ................................................................................................... 21 
Figure R.11—2: Overview of the PBT/vPvB assessment process for the registrant. ............. 22 
Figure R.11—3: Integrated Assessment and Testing Strategy for persistence assessment – 
maximising data use and targeting testing.................................................................... 41 
Figure R.11—4: Integrated assessment and testing strategy for B-assessment. ................ 68 
Figure R.11—5: T testing in support of PBT assessment for the aquatic environment. ........ 88 
Figure R.11—6: Example of the first assessment tier of a UVCB substance for which fraction 
profiling has been applied ........................................................................................... 112 
Figure R.11—7: Log BCF v calculated Log Kow. .............................................................. 136 
Figure R.11—8: LogBCF v measured log Kow. ................................................................ 137 
Figure R.11—9: LogBCF derived from feeding studies versus calculated Log Kow. ............... 138 
Figure R.11—10: Relationship between lipid and organic carbon normalised BSAF values and 
Log Kow as indicator for the B and vB criterion. .............................................................. 156 

Tables 

Table R.11—1: PBT and vPvB criteria according to Section 1 of Annex XIII to the REACH 
Regulation. ............................................................................................................... 17 
Table R.11—2: Screening information as listed in Section 3.1 of Annex XIII to the REACH 
Regulation. ............................................................................................................... 18 
Table R.11—3: Assessment information according to Section 3.2 of Annex XIII to the REACH 
Regulation. ............................................................................................................... 19 
Table R.11—4: Screening information for P and vP. ....................................................... 49 
Table R.11—5: Persistence (P/vP) criteria according to Annex XIII to the REACH Regulation 
and related simulation tests. ....................................................................................... 50 
Table R.11—6: Screening threshold values for toxicity. .................................................. 92 
Table R.11—7: Solubility of some pigments and comparison of their Co/Cw values with 
estimated Kows .......................................................................................................... 104 
Table R.11—8: Tissue absorption potentials .................................................................. 128 
Table R.11—9: Summary of various ranges of CBB - lethality (mmol/kg ww). ................... 132 
Table R.11—10: List of antioxidants (from Ullmann, 1995). ............................................ 144 
Table R.11—11: Properties of the antioxidant. .............................................................. 145 
Table R.11—12: Properties of the antioxidant. .............................................................. 146 
Table R.11—13: Properties of the antioxidant. .............................................................. 148 
Table R.11—14: Octanol and water solubility of pigments, critical body burden for narcotic 
mode of action and Log Coctanol/Cwater (ETAD, 2006). ....................................................... 149 
Table R.11—15: Data for Pigment Yellow 12. ................................................................ 151 

Appendices with examples  

Appendix R.11—1: Indicators for limited bioconcentration for PBT assessment. ................. 124 
Appendix R.11—2: Assessment of substances requiring special consideration during testing.144 
Appendix R.11—3: PBT assessment of UVCB petroleum substances. ................................ 152 
Appendix R.11—4: Bioconcentration studies with benthic and terrestrial invertebrate species 
(BSAF). .................................................................................................................... 156 

 
 
 
 
 
 
 
 
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R.11  PBT and vPvB Assessment 

R.11.1  Introduction 

According to Section 4 of Annex I to the REACH Regulation the objective of the persistent, 
bioaccumulative and toxic (PBT) and very persistent and very bioaccumulative (vPvB) 
assessment is to determine if the substance assessed in Chemical Safety Assessment (CSA) 
fulfils the criteria set out in Annex XIII. It furthermore states that a conventional hazard 
assessment of the long-term effects and the estimation of the long-term exposure cannot be 
carried out with sufficient reliability for the purpose of assessing the safety of substances 
satisfying the PBT and vPvB criteria in Annex XIII. Therefore a PBT and vPvB assessment is 
required to be carried out for all substances for which CSA is carried out. 

This guidance document contains a description of scientific principles for the PBT and vPvB 
assessment in accordance with Section 4 of Annex I to the REACH Regulation, and a 
description of the obligations of the registrant in carrying out a PBT and vPvB assessment as 
part of CSA.  

PBT substances are substances that are persistent, bioaccumulative and toxic, while vPvB 
substances are characterised by a particular high persistence in combination with a high 
tendency to bioaccumulate, which may, based on experience from the past with such 
substances, lead to toxic effects and have an impact in a manner which is difficult to predict 
and prove by testing, regardless of whether there are specific effects already known or not. 
These properties are defined by the criteria laid down in Section 1 of Annex XIII to the REACH 
Regulation (CRITERIA FOR THE IDENTIFICATION OF PERSISTENT, BIOACCUMULATIVE AND 
TOXIC SUBSTANCES, AND VERY PERSISTENT AND VERY BIOACCUMULATIVE SUBSTANCES, 
henceforth “the PBT and vPvB criteria”). 

A PBT/vPvB assessment2 is required for all substances for which a CSA must be conducted and 
reported in the chemical safety report (CSR). These are, according to Article 14(1) of the 
REACH Regulation, in general all substances manufactured or imported in amounts of 10 or 
more tonnes per year that are not exempted from the registration requirement under the 
Regulation. However, some further exemptions apply as described in Article 14(2), e.g. for 
substances present in a mixture if the concentration is less than 0.1% weight by weight (w/w), 
for on-site or transported isolated intermediates, and for substances used for Product and 
Process Oriented Research and Development (for further information see the Guidance on 
Registration). Therefore, this guidance is mainly targeted at registrants manufacturing or 
importing a substance in amounts of 10 or more tonnes per year and to downstream users 
who have an obligation to conduct their own CSA. This guidance is also relevant for ECHA and 
for Member State competent authorities who carry out PBT/vPvB assessment related tasks 
under REACH. 

Experience  with  PBT/vPvB  substances  has  shown  that  they  can  give  rise  to  specific  concerns 
that may arise due to their potential to accumulate in parts of the environment and 
 
 

that the effects of such accumulation are unpredictable in the long-term; 
such  accumulation  is  in  practice  difficult  to  reverse  as  cessation  of  emission  will  not 
necessarily result in a reduction in substance concentration. 

Furthermore, PBT or vPvB substances may have the potential to contaminate remote areas 
that should be protected from further contamination by hazardous substances resulting from 
human activity because the intrinsic value of pristine environments should be protected. 

2 The term “PBT/vPvB assessment” is applied in this document to denote “PBT and vPvB assessment” and 
covers both “screening” and “assessment” as described in the following sections. 

  
                                                 
 
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Chapter R.11: PBT/vPvB assessment 

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These specific concerns occur particularly with substances that can be shown both to persist 
for long periods and to bioaccumulate in biota and which can give rise to toxic effects after a 
longer time and over a greater spatial scale than substances without these properties. These 
effects may be difficult to detect at an early stage because of long-term exposures at normally 
low concentration levels and long life-cycles of species at the top of the food chain. In the case 
of vPvB substances, there is concern that even if no toxicity is demonstrated in laboratory 
testing, long-term effects might be possible since high but unpredictable levels may be 
reached in man or the environment over extended time periods. 

The properties of the PBT/vPvB substances lead to an increased uncertainty in the estimation 
of risk to human health and the environment when applying quantitative risk assessment 
methodologies. For PBT and vPvB substances a “safe” concentration in the environment cannot 
be established using the methods currently available with sufficient reliability for an acceptable 
risk to be determined in a quantitative way3. Therefore, a separate PBT/vPvB assessment is 
required according to Article 14(3)(d) of the REACH Regulation in order to take these specific 
concerns into account. Registrants are required to perform this specific PBT/vPvB assessment in 
the context of their CSA. 

According to Section 4 of Annex I to the REACH Regulation, the objective of the PBT/vPvB 
assessment is to determine if the substance fulfils the criteria given in Annex XIII to the 
REACH Regulation (“Step 1: Comparison with the Criteria”), and if so, to characterise the 
potential emissions of the substance to the different environmental compartments during all 
activities carried out by the registrant and all identified uses (“Step 2: Emission 
characterisation”). In addition, in the latter step it is also necessary to identify the likely routes 
by which humans and the environment are exposed to the substance. According to Section 6.5 
of Annex I to the REACH Regulation the registrant then needs to use the information obtained 
during the emission characterisation step, when implementing on his site, and recommending 
to downstream users, risk management measures (RMMs) which minimise emissions and 
subsequent exposures of humans and the environment throughout the life-cycle of the 
substance that results from manufacture or identified uses. The authorities may further subject 
substances with PBT or vPvB properties to restrictions or the authorisation requirement, with 
substitution of the substance as objective in the latter case where economically and technically 
viable.  

The registrant’s process for assessing the substance and consequences to the registrant of the 
conclusions are outlined in detail in Section R.11.3. Guidance on scientific methods that can be 
used for carrying out Step 1 is given in Section R.11.4 of this Chapter. The sub-sections of 
Section R.11.4 on the assessment of the P, B and T properties of a substance provide guidance 
on how a registrant or an authority can make best use of the different types of information 
available in order to conclude with least efforts on the PBT/vPvB–properties of the substance. 
These sub-sections also contain guidance on specific assessment and testing strategies for 
substances that are difficult to test, including adaptation of tests, specific rules for 
interpretation of results, consideration of monitoring data and cut-off criteria.  

The guidance explains how all available evidence can be considered in order to decide with 
sufficient certainty whether the PBT/vPvB criteria are fulfilled or not without always requiring 
the generation of such types of data that numerically match with the Annex XIII criteria. 
Generating such data may for instance not be possible because the properties of the substance 
do not permit the respective tests to be conducted. In these cases a conclusion may need to 
be drawn on the basis of screening information and all further evidence available. In many 
cases further information may need to be generated before it can be judged whether the 

3 It should be noted that over the last years a number of methods have been proposed in the scientific 
literature that could eventually be used to reduce the uncertainty in the risk estimation (on either the 
exposure or effects side) of PBTs and vPvBs and hence may lead to a better understanding of the level of 
risk associated with these substances, in particular in a comparative sense. 

 
                                                 
 
Chapter R.11: PBT/vPvB assessment 

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13 

substance fulfils the Annex XIII criteria, and the guidance provides detailed testing strategies 
that the registrant should use for each endpoint in Section R.11.4. 

Substances are considered as PBT or vPvB substances when they fulfil the criteria for all three 
inherent properties P, B and T or both of the inherent properties vP and vB, respectively. It is 
the task of the registrant to assess if the information that is available and/or produced is 
sufficient to assess whether the substance is a PBT or a vPvB substance or not.  

It is to be noted that this guidance is not meant to guide authorities directly in identifying 
substances fulfilling the criteria of Article 57(f) of the REACH Regulation (substances of 
equivalent level of concern). However, this guidance may in such cases be used as one 
reference for understanding what indications may be needed to identify a substance to be of 
equivalent level of concern to PBT or vPvB substances.  

  
 
 
 
 
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Chapter R.11: PBT/vPvB assessment 

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R.11.2  Overview of Annex XIII to the REACH Regulation 

The purpose of this section is to introduce the content and terminology of Annex XIII to the 
REACH Regulation. The interpretation of the content is presented mainly from Section R.11.3 
onwards. Only some key clarifications of the legal text are included in this section. 

R.11.2.1 

Elements and terminology of Annex XIII to the REACH Regulation 

The introductory section of Annex XIII to the REACH Regulation defines the PBT/vPvB 
assessment scope regarding substance groups: 

Introductory Section of Annex XIII to REACH  

[…] This Annex shall apply to all organic substances, including organo-metals. 

Annex XIII to the REACH Regulation is generally applicable to any substance containing an 
organic moiety. Based on the common definition of an organic substance in chemistry, PBT and 
vPvB criteria are not applicable to inorganic substances.   

The PBT/vPvB criteria as set out in Annex XIII to the REACH Regulation are presented in 
Section R.11.2.2, Table R.11—1.  

Annex XIII defines two levels of assessment within the PBT/vPvB assessment (“screening” 
and “assessment”) and two sets of information (“screening information” and “assessment 
information”). The two sets of information are presented in Table R.11—2 and Table R.11—3, 
respectively. The differentiation of the two assessment levels within the PBT/vPvB assessment 
is mainly designed to help the registrant identify his obligations specifically with respect to the 
PBT/vPvB assessment. 

The combination of several passages of extracts of the text of Annex XIII, as cited below, 
stipulate that all relevant and available “assessment information” and “screening 
information” must be used in the PBT/vPvB assessment: 

Introductory Section of Annex XIII to REACH  
[…] For the identification of PBT substances and vPvB substances a weight-of-evidence determination 
using expert judgement shall be applied, by comparing all relevant and available information listed in 
Section 3.2 with the criteria set out in Section 1. […] 
Section 2.1 of Annex XIII to REACH  

For the identification of PBT and vPvB substances in the registration dossier, the registrant shall 
consider the information as described in Annex I and in Section 3 of this Annex. […] 
Section 2.2 of Annex XIII to REACH  

For dossiers for the purposes of identifying substances referred to in Article 57(d) and Article 57(e), 
relevant information from the registration dossiers and other available information as described in 
Section 3 shall be considered. […] 

Recital 5 of Commission Regulation (EU) No 253/2011 

Experience shows that, for the adequate identification of PBT and vPvB substances, all relevant 
information should be used in an integrated manner and applying a weight-of-evidence approach by 
comparing the information to the criteria set out in Section 1 of Annex XIII. 

The screening information can be understood as one subtype of assessment information, as 
Sections 3.2.1.(d), 3.2.2.(b) and 3.2.3(f) of Annex XIII to the REACH Regulation allow “other 
information” to be used as assessment information, provided that its suitability and reliability 
can be reasonably demonstrated. However, it should be noted that screening information 
cannot be directly (numerically) compared with the PBT/vPvB criteria, i.e. the screening 

 
 
 
 
 
 
 
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information does not contain degradation half-life values or BCF values, which could be directly 
compared with the criteria. Screening information involves simple data, typically information 
from Annexes VII and VIII endpoints, that must be used to assess whether further information 
is needed. 

A Weight-of-Evidence determination by expert judgment must be used in the PBT/vPvB 
assessment (see the green boxes above). It is defined as follows:  

Introductory Section of Annex XIII to REACH  

[…] 

A weight-of-evidence determination means that all available information bearing on the identification 
of a PBT or a vPvB substance is considered together, such as the results of monitoring and modelling, 
suitable in vitro tests, relevant animal data, information from the application of the category approach 
(grouping, read-across), (Q)SAR results, human experience such as occupational data and data from 
accident databases, epidemiological and clinical studies and well documented case reports and 
observations. The quality and consistency of the data shall be given appropriate weight. The available 
results regardless of their individual conclusions shall be assembled together in a single weight-of-
evidence determination. […] 

The Weight-of-Evidence determination by expert judgement enables the use of all (screening 
and assessment) information types listed in Section 3 of Annex XIII to the REACH Regulation 
in the PBT/vPvB assessment for comparing with the criteria, although not all of these 
information types can be directly (numerically) compared with the criteria. 

Examples and principles of Weight-of-Evidence determination for the PBT/vPvB assessment 
further applying the introductory section of Annex XIII to the REACH Regulation are provided 
in Section R.11.4. In addition, the Practical Guide on “How to use alternatives to animal testing 
to fulfil your information requirements for REACH registration” provides a general scheme for 
building a Weight-of-Evidence approach.  

As regards the registrants’ specific duties for the PBT/vPvB assessment, the following 
provision of Annex XIII to the REACH Regulation must be considered further to Annex I: 

Section 2.1 of Annex XIII to REACH  
 […] If the technical dossier contains for one or more endpoints only information as required in 
Annexes VII and VIII, the registrant shall consider information relevant for screening for P, B, or T 
properties in accordance with Section 3.1 of this Annex. If the result from the screening tests or other 
information indicate that the substance may have PBT or vPvB properties, the registrant shall 
generate relevant additional information as set out in Section 3.2 of this Annex. In case the 
generation of relevant additional information would require information listed in Annexes IX or X, the 
registrant shall submit a testing proposal. Where the process and use conditions of the substance 
meet the conditions as specified in Section 3.2(b) or (c) of Annex XI the additional information may be 
omitted, and subsequently the substance is considered as if it is a PBT or vPvB in the registration 
dossier. No additional information needs to be generated for the assessment of PBT/vPvB properties if 
there is no indication of P or B properties following the result from the screening test or other 
information.  

When fulfilling the data requirements of Annexes IX and X to the REACH Regulation, 
adaptations according to Column 2 and Annex XI should be applied wherever possible to 
minimise testing on animals, which must be only as a last resort under REACH (see REACH 
Articles 13(3) and 25(1)).  

In addition, the following principles must be applied while performing a PBT/vPvB 
assessment:  

  
 
 
 
 
 
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Chapter R.11: PBT/vPvB assessment 

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Introductory Section of Annex XIII to REACH  
[…] The information used for the purposes of assessment of the PBT/vPvB properties shall be based 
on data obtained under relevant conditions. […] 

By “relevant conditions”, relevant environmental conditions and relevant testing conditions are 
generally meant. These are further discussed in Section R.11.4.   

Introductory Section of Annex XIII to REACH  
[…] The identification shall also take account of the PBT/vPvB properties of relevant constituents of a 
substance and relevant transformation and/or degradation products. […] 

The term “constituent” refers to the main constituents, impurities and additives of substances 
of well-defined composition and constituents of UVCB substances as defined in the Guidance 
for identification and naming of substances under REACH and CLP. The implication in terms of 
PBT/vPvB assessment requirement for the registrant is described in Section R.11.3.2.1 and 
further guidance on what should be considered as relevant constituents is provided in 
Section R.11.4.1.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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R.11.2.2  PBT and vPvB criteria and information listed in Annex XIII to the 

REACH Regulation 

The following tables (Table R.11—1, Table R.11—2, and Table R.11—3) summarise the PBT 
and vPvB criteria given in accordance with Section 1 of Annex XIII to REACH and the relevant 
information to be used for the PBT/vPvB assessment as provided in Sections 3.1 and 3.2 of 
Annex XIII to the REACH Regulation.  

Table R.11—1: PBT and vPvB criteria according to Section 1 of Annex XIII to the 
REACH Regulation. 

Property 

PBT criteria  

vPvB criteria  

Persistence 

A substance fulfils the persistence 
criterion (P) in any of the following 
situations: 

A substance fulfils the “very persistent” 
criterion (vP) in any of the following 
situations: 

(a) the degradation half-life  in marine 
water is higher than 60 days; 

(b) the degradation half-life  in fresh or 
estuarine water is higher than 40 days; 

(c) the degradation half-life in marine 
sediment is higher than 180 days; 

(d) the degradation half-life in fresh or 
estuarine water sediment is higher than 
120 days; 

(e) the degradation half-life in soil is 
higher than 120 days. 

(a) the degradation half-life in marine, 
fresh or estuarine water is higher than 
60 days; 

(b) the degradation half-life in marine, 
fresh or estuarine water sediment is 
higher than 180 days; 

(c) the degradation half-life in soil is 
higher than 180 days. 

Bioaccumulation 

A substance fulfils the bioaccumulation 
criterion (B) when the bioconcentration 
factor in aquatic species is higher than 
2000. 

A substance fulfils the “very 
bioaccumulative” criterion (vB) when the 
bioconcentration factor in aquatic 
species is higher than 5000. 

Toxicity* 

- 

A substance fulfils the toxicity criterion 
(T) in any of the following situations: 

(a) the long-term no-observed effect 
concentration (NOEC) or EC10  for 
marine or freshwater organisms is less 
than  0.01 mg/L;   
(b) the substance meets the criteria for 
classification as carcinogenic (category 
1A or 1B), germ cell mutagenic 
(category 1A or 1B), or toxic for 
reproduction (category 1A, 1B or 2) 
according to Regulation EC No 
1272/2008; 

(c) there is other evidence of chronic 
toxicity, as identified by the substance 
meeting the criteria for classification: 
specific target organ toxicity after 
repeated exposure (STOT RE category 1 
or 2) according to Regulation EC No 
1272/2008. 

* EC10 preferred over NOEC (see further explanation in Section R.11.4.1.3). 

  
 
 
 
 
 
 
 
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Table R.11—2: Screening information as listed in Section 3.1 of Annex XIII to the 
REACH Regulation.  

Indication of P and vP properties 

(a)  Results from tests on ready biodegradation in accordance with 

Section 9.2.1.1 of Annex VII; 

(b)  Results from other screening tests (e.g. enhanced ready test, 

tests on inherent biodegradability); 

(c)  Results obtained from biodegradation (Q)SAR models in 

accordance with Section 1.3 of Annex XI; 

(d)  Other information provided that its suitability and reliability 

can be reasonable demonstrated. 

Indication of B and vB properties 

(a)  Octanol-water partitioning coefficient experimentally 

Indication of T properties* 

determined in accordance with Section 7.8 of Annex VII to 
REACH or estimated by (Q)SAR models in accordance with 
Section 1.3 of Annex XI;  

(b)  Other information provided that its suitability or reliability can 

be reasonably demonstrated. 

(a)  Short-term aquatic toxicity in accordance with Section 9.1 of 
Annex VII to REACH and Section 9.1.13 of Annex VIII;  

(b)  Other information provided that its suitability or reliability can 

be reasonably demonstrated. 

* Acute or short-term aquatic toxicity data are considered to be screening information (Annex XIII, 
Section 3.1) and may be used as an indication that the substance may fulfil the T criterion. However, 
when acute/short-term aquatic toxicity data show that the substance is very toxic (L(E)C50 < 0.01 
mg/L), a definitive conclusion can be drawn that the substance fulfils the T criterion and no further 
testing is necessary. Acute data cannot be used for concluding definitively “not T”. If long-term or chronic 
aquatic toxicity data are available, a definitive assessment can be made.  

 
 
 
 
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Table R.11—3: Assessment information according to Section 3.2 of Annex XIII to the 
REACH Regulation.   

Assessment of P or vP 
properties 

Assessment of B or vB 
properties* 

(a)  Results from simulation testing on degradation in surface water; 

(b)  Results from simulation testing on degradation in soil; 

(c)  Results from simulation testing on degradation in sediment; 

(d)  Other information, such as information from field studies or monitoring 
studies, provided that its suitability and reliability can be reasonably 
demonstrated. 

(a)  Results from a bioconcentration or bioaccumulation study in aquatic 

species; 

(b)  Other information on the bioaccumulation potential provided that its 
suitability and reliability can be reasonably demonstrated, such as:  

-  Results from a bioaccumulation study in terrestrial species;  

-  Data from scientific analysis of human body fluids or tissues, such 

as blood, milk, or fat;  

-  Detection of elevated levels in biota, in particular in endangered 
species or in vulnerable populations, compared to levels in their 
surrounding environment; 

-  Results from a chronic toxicity study on animals; 

- 

Assessment of the toxicokinetic behaviour of the substance; 

(c) Information on the ability of the substance to biomagnify in the food 

chain, where possible expressed by biomagnification factors or trophic 
magnification factors. 

Assessment of T 
properties 

(a)  Results from long-term toxicity testing on invertebrates as set out in 

Section 9.1.5 of Annex IX; 

(b)  Results from long-term toxicity testing on fish as set out in Section 

9.1.6 of Annex IX; 

(c)  Results from growth inhibition study on aquatic plants as set out in 

Section 9.1.2 of Annex VII; 

(d)  The substance meeting the criteria for classification as carcinogenic in 
Category 1A and 1B (assigned hazard phrases: H350 or H350i), germ 
cell mutagenic in Category 1A or 1B (assigned hazard phrase: H340), 
toxic for reproduction in Category 1A, 1B and/or 2 (assigned hazard 
phrases: H360,H360F, H360D, H360FD, H360Fd, H360 fD, H361, 
H361f, H361d or H361fd), specific target organ toxic after repeated 
dose in Category 1 or 2 (assigned hazard phrase: H372 or H373), 
according to Regulation EC No 1272/2008; 

(e)   Results from long-term or reproductive toxicity testing with birds as 

set out in Section 9.6.1 of Annex X; 

(f)  Other information provided that its suitability and reliability can be 

reasonably demonstrated. 

* At present, there is no guidance on how to apply in the PBT/vPvB assessment the information coming 
from:  
- data from scientific analysis of human body fluids or tissues, such as blood, milk, or fat;  or  
- the detection of elevated levels in biota, in particular in endangered species or in vulnerable 
populations, compared to levels in their surrounding environment. 
Such guidance needs to be developed in the future. 

  
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Chapter R.11: PBT/vPvB assessment 

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R.11.3  Duties of the registrant  

The purpose of this section is to delineate the obligations of the registrant within the PBT/vPvB 
assessment workflow. For further details, the registrant may refer to the recommendations 
provided in Section R.11.4. 

R.11.3.1  Objective and overview of the PBT/vPvB assessment process  

Section 4.0.1 of Annex I to the REACH Regulation defines the objective of the PBT/vPvB 
assessment:  

Annex I to REACH  

[…] 

4. PBT AND VPVB ASSESSMENT 

4.0. Introduction 

4.0.1. The objective of the PBT/vPvB assessment shall be to determine if the substance fulfils the 
criteria given in Annex XIII and if so, to characterise the potential emissions of the substance. […] 

It furthermore states that a hazard assessment and exposure assessment for CSA cannot be 
carried out with sufficient reliability for substances satisfying the PBT or vPvB criteria and that 
therefore a separate PBT/vPvB assessment is required.  

According to Section 4.0.2 of Annex I to the REACH Regulation, the process of the PBT/vPvB 
assessment consists of the following two steps: Step 1: “Comparison with the criteria” and 
Step 2: “Emission characterisation”. Section 6.5 of Annex I to the REACH Regulation 
requires the registrant to implement for PBT/vPvB substances risk management measures 
which minimise exposures and emission to humans and the environment, throughout the 
lifecycle of the substance that result from manufacture and identified uses. The obligations of 
the registrant for carrying out the PBT/vPvB assessment are defined more in detail in Section 
2.1 of Annex XIII to the REACH Regulation. In the following paragraphs the main assessment 
steps are described.  

Step 1 comprises a scientific PBT/vPvB assessment where the relevant available information 
must be compared with the PBT/vPvB criteria (for detailed guidance on this step, see Section 
R.11.4).  In Step 1 the registrant must come to one of the conclusions presented in Figure 
R.11—1. Each conclusion leads to specific consequences, which the registrant must comply 
with. The conclusions are described in more detail in Section R.11.4.1.4 and consequences in 
Section R.11.3.3. 

 
  
 
Chapter R.11: PBT/vPvB assessment 

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21 

Conclusion (i): The substance does not fulfil the PBT and vPvB criteria. 
For screening assessment: there is no indication of P or B properties. 



No consequences for the registrant. The PBT/vPvB assessment stops.  

Conclusion (ii): The substance fulfils the PBT or vPvB criteria.  



The registrant must carry out emission characterisation and ensure minimisation of 
exposures and emissions throughout the life-cycle of the substance that results from 
manufacture and identified uses. 

Conclusion (iii): The available information does not allow to conclude 
(i) or (ii). The substance may have PBT or vPvB properties.  
Further information for the PBT/vPvB assessment is needed. 





The registrant must generate relevant additional information (including, where necessary, 
submission of a testing proposal) and carry out Step 1 again, OR 
The registrant must treat the substance as if it is a PBT or vPvB. 

Figure R.11—1: Overview of the conclusions from Step 1 (“Comparison with the 
criteria”) and their consequences. 

The registrant is only allowed to finalise Step 1 of the assessment process if he is able to reach 
an unequivocal conclusion on the PBT or vPvB properties (conclusion (i) or conclusion (ii)4). 

Conclusion (iii) is an interim conclusion in Step 1. This conclusion triggers the requirement for 
the registrant to generate all necessary additional information and to continue in Step 1 until 
the available information allows a definitive conclusion. Section 2.1 of Annex XIII to the REACH 
Regulation requires information to be generated by the registrant irrespective of the standard 
information requirements of the registrant. This may require several iterative steps of 
acquisition of further information, testing and assessment. Alternatively, the registrant can 
decide after conclusion (iii) to apply an exemption from the requirement to generate additional 
data by considering the substance “as if it is a PBT or vPvB”. This is only allowed if the 
registrant applies specific exposure based adaptation conditions as specified in Section 3.2(b) 
or (c) of Annex XI to the REACH Regulation. 

The consequences of each conclusion for the registrant are described in more detail in Section 
R.11.3.3. Figure R.11—2 provides an overview of the PBT/vPvB assessment process of the 
registrant as a flowchart. 

4 Conclusion (i) and (ii) are either based on a) data directly comparable with the PBT/vPvB criteria or b) 
based on Weight-of-Evidence expert judgement of information which is not directly (numerically) 
comparable with the PBT/vPvB criteria or c) a combination of both situations a) and b). 

  
 
 
 
 
                                                 
 
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Chapter R.11: PBT/vPvB assessment 

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Step 1: Compare all 
relevant and available 
information with the 
PBT/vPvB criteria 

- beyond the standard 
information requirements, 
if necessary for the 
PBT/vPvB assessment 

Generate further 
relevant 
information 
(including, where 
relevant, 
submission of a 
testing proposal) 

Registrant must 
draw one of the 
following three 
conclusions  

(iii) Further 
information is 
needed 

Registrant must 
choose one of 
the following 
two options 

If specific exposure-based 
adaptation conditions are 
met1, the substance can be 
considered as if it is a 
PBT/vPvB  

(ii) PBT/vPvB 
criteria are 
fulfilled2 

(i) PBT/vPvB 
criteria are not 
fulfilled 

Step 2: Emission 
characterisation 

The PBT/vPvB 
assessment can be 
stopped 

Minimise exposures3 and 
emissions to humans and the 
environment 

Communicate the outcome of the 
PBT/vPvB assessment and risk 
management measures within 
the supply chain 

1 Please refer to the conditions as specified in Section 3.2(b) or (c) of Annex XI to the REACH Regulation. 
2 Normally not applicable if only screening information is available.  
3 For further information on exposure minimisation please refer to Section R.11.3.4.2. 

Figure R.11—2: Overview of the PBT/vPvB assessment process for the registrant.  

Relevant constituents, impurities, additives, degradation/transformation products must also be 
encompassed in this process.  

 
 
 
Chapter R.11: PBT/vPvB assessment 

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R.11.3.2 

Comparison with the criteria (Step 1) 

In the following Sections the formal obligations for Step 1 (“Comparison with the criteria”) of 
the PBT/vPvB assessment are described.  

In Step 1 of the PBT/vPvB assessment, the standard information requirements are first applied 
by the registrant as described in the Guidance on Information Requirements & Chemical Safety 
Assessment (IR&CSA). It should be noted that any data adaptations according to Column 2 of 
Annexes VII to X or Annex XI to the REACH Regulation should be justified according to the 
relevant ECHA documents (e.g. Practical Guides on “How to use and report (Q)SARs” and on 
“How to use alternatives to animal testing to fulfil your information requirements for REACH 
registration”, and Chapter 5 and Chapter 6 of the Guidance on IR&CSA,). The information 
included in the registration dossier as a result of adaptations of standard information 
requirements and their justifications are part of the available information for the PBT/vPvB 
assessment, where relevant. The PBT and vPvB assessment must initially be based on all the 
relevant information available which is as a minimum the information as listed in Annexes VII 
and VIII to the REACH Regulation. This information normally corresponds to PBT/vPvB 
screening information as listed in Section R.11.2.2.  

The registrant must conclude Step 1 by selecting one of the three conclusions presented in 
Figure R.11—1 and Figure R.11—2. If conclusion (iii) “The available data information does not 
allow to conclude (i) or (ii)” applies, Step 1 continues after the necessary new information has 
been generated (see more details in Section R.11.3.3). 

In cases where only screening information as listed in Section R.11.2.2 is available for one or 
more endpoints, Step 1 of the PBT/vPvB assessment implies first that the registrant is not able 
to compare the information directly (numerically) with the PBT/vPvB criteria. Although it might 
be theoretically possible to calculate degradation half-life values or BCF values from screening 
information, such values must not be directly compared with the criteria. At this stage, the 
registrant is required to analyse whether the information indicates that the substance may 
meet the PBT/vPvB criteria, in which case the registrant must draw conclusion (iii) “The 
available data information does not allow to conclude (i) or (ii)”, or whether the information 
shows that there is no indication on P or B properties, in which case the conclusion (ii) “The 
substance does not fulfil the PBT and vPvB criteria” applies. In Section R.11.4 several 
screening threshold values and conditions for applying them are described, which the 
registrant should consider while drawing a conclusion for screening. The screening threshold 
values are indicative and the registrant must use all relevant pieces of information on his 
substance to justify his conclusion. Also, where only screening information is available, the 
choice of the conclusion should be based on a Weight-of-Evidence consideration by expert 
judgement where all relevant and available data for all endpoints are considered in 
conjunction.  

If only screening information is available, it is normally not possible to conclude (ii) (“The 
substance fulfils the PBT or vPvB criteria”) due to the uncertainties related to screening 
information. However, if scientifically justified, it is in principle possible to draw conclusion (ii) 
based on screening information. In Section R.11.4 few such exceptional cases are described, 
where the registrant may make use of screening information for concluding (ii).  

The conclusion of Step 1 should be derived by the registrant taking into account also all 
aspects as described in Section R.11.4.1.4.  

The consequences of the individual conclusions to the registrant are described in more detail in 
Section R.11.3.3. 

  
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  Scope of the PBT and vPvB assessment (relevant constituents, 

transformation/degradation products) 

For the purpose of this Guidance it should be noted that the term “constituent” as mentioned 
in Annex XIII to the REACH Regulation refers to constituents and impurities of well-defined 
substances, constituents of UVCB substances, and additives to all substances.  

The PBT/vPvB assessment must, according to Annex XIII to the REACH Regulation, take 
account of the PBT/vPvB properties of relevant constituents and relevant transformation 
and/or degradation products of organic substances (including organo-metals).  

Generally, the PBT/vPvB assessment obligations as described in Sections R.11.3.1 and 
R.11.3.2  have to be applied for relevant constituents, impurities, additives and 
transformation/degradation products. The registrant cannot stop the PBT/vPvB assessment if 
there is not enough information available to take into account the PBT/vPvB properties of 
relevant constituents, impurities, additives and transformation/degradation products. This 
means that if there is not enough information available on the PBT/vPvB properties of relevant 
constituents, impurities, additives and transformation/degradation products to derive for the 
registrant’s substance either conclusion (i) (“The substance does not fulfil the PBT and vPvB 
criteria”) or conclusion (ii) (”The substance fulfils the PBT or vPvB criteria”), the registrant 
must generate the necessary further information on the PBT/vPvB properties of the relevant 
constituents, impurities, additives and transformation/degradation products until one of these 
two definitive conclusions can be achieved. The other option, as provided in Sections R.11.3.1 
and R.11.3.3 is to treat the substance “as if it is a PBT or vPvB”. 

If the registrant deems as a result of the PBT/vPvB assessment an uncharacterized 
constituent, impurity, additive or transformation/degradation product relevant for the 
PBT/vPvB assessment, the registrant must characterize its substance identity as required in 
the Guidance for identification and naming of substances under REACH and CLP.    

The interpretation of the term “relevant” constituent, impurity, additive, 
transformation/degradation product, is described in Section R.11.4.1. It is recommended that 
the registrant follows this interpretation in the PBT/vPvB assessment, in defining which 
constituents, impurities, additives, transformation or degradation products are relevant. 

The registrant must show in the PBT/vPvB assessment that he has taken into account the 
relevant constituents, impurities and additives. This is normally possible only if he includes in 
the PBT/vPvB assessment appropriate justifications for all constituents, impurities and 
additives or for all fractions/blocks of the substance composition on why these are considered 
to be relevant or judged to be not relevant for the PBT/vPvB assessment, regardless of 
whether the substance identity of these could be ultimately determined or not5. The registrant 
may derive such reasoning quantitatively or qualitatively, by using the PBT/vPvB assessment 
principles as described in Section R.11.4. This also applies to the transformation/degradation 
products. It should be noted that also Section 9.2.3 of Annex IX to the REACH Regulation 
requires identification of degradation products. 

5 The PBT/vPvB assessment of short-chain chlorinated paraffins (EC 287-476-5) used for the 
identification of the substance to the Candidate List is one of the examples where the constituents were 
not characterized ultimately. See related Member State Committee SVHC Support Document at 
http://echa.europa.eu/documents/10162/414fa327-56a1-4b0c-bb0f-a6c40e74ece2. 

 
                                                 
 
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  Specific cases: substances fulfilling the PBT/vPvB criteria according 
to ECHA’s Member State Committee in relation to the inclusion of 
substances in the Candidate List of Substances of Very High Concern 

According to REACH Article 59, ECHA’s Member State Committee (MSC) agrees on substances 
to be included to the Candidate List of Substances of Very High Concern (SVHC), i.a., if they 
fulfil the PBT and/or vPvB criteria. These agreements are published as ECHA decisions on 
ECHA’s website. If a registrant’s substance has been included in the Candidate List as a 
PBT/vPvB substance, the registrant must align his PBT/vPvB assessment and conclusion with 
the PBT/vPvB assessment which was the basis of the MSC agreement. This PBT/vPvB 
assessment is reported in a support document of the decision on inclusion of the substance in 
the Candidate List and is available on ECHA’s website. In such cases, it is appropriate to 
replace in the CSR the documentation of Step (1) of the PBT/vPvB assessment with a reference 
to the relevant ECHA decision. If the registrant has new information available which was not 
referred to in the support document of the relevant ECHA decision, the registrant must include 
the new information in the registration dossier and may reflect his opinion of the relevance of 
the new information to the conclusion in the CSR. Although the registrant would in this case 
present in the CSR the opinion that the new information would trigger another conclusion than 
the one drawn by the MSC, the registrant is further obliged to implement the conclusion of the 
MSC as the conclusion in force in his CSR. In case ECHA’s Committee for Risk Assessment 
provides an opinion recommending restriction of a substance because it meets PBT/vPvB 
criteria, it is highly recommended that the registrant(s) recognise and implement the PBT/vPvB 
status of the substance in their dossiers, minimise releases and exposures in their activities 
and inform their downsteam users about the PBT/vPVB status. 

If a registered substance contains a constituent, impurity or additive or transforms/degrades 
to a substance which is in the Candidate List because of meeting the PBT and/or vPvB criteria, 
the registrant must conclude his substance to meet the PBT or vPvB criteria accordingly. To 
help the registrant, Section R.11.4 provides definitions on what are relevant constituents, 
impurities, additives and relevant transformation and degradation products.  

There are several substances on the Candidate List which have been identified as fulfilling PBT 
or vPvB criteria because their constituents or transformation/degradation products fulfil PBT or 
vPvB criteria6. The support documents of ECHA decisions on the Candidate List inclusion 
identify in these cases the constituents or transformation/degradation products of concern and 
contain a PBT/vPvB assessment of them. If a registered substance contains one of these as 
constituent, impurity, additive, or transforms/degrades into one of these substances, the 
registrant should reflect the conclusion presented in such support documents in his own 
PBT/vPvB assessment. This applies by analogy also to any future cases where inclusion to the 
Candidate List was due to PBT/vPvB properties of impurities or additives. 

R.11.3.3 

Consequences of Step 1  

The three conclusions from Step 1: “Comparison with the criteria” trigger four different 
consequences for the registrant (see Figure R.11—1 and Figure R.11—2). These are: 

  No consequences: after conclusion (i) 

  Conduct emission characterisation and risk characterisation: after conclusion (ii) 

  Generate relevant additional information (including, where relevant, submission of 
testing proposal) and continue under Step 1: after conclusion (iii) or Treat the 
substance “as if it is a PBT or vPvB”: after conclusion (iii) 

6 Such substances are for example: Coal tar pitch, high temperature (EINECS No: 266-028-2) 
and Bis(pentabromophenyl) ether (EC 214-604-9). 

  
                                                 
 
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In the following sections the consequences are described more in detail. 

  No consequences 

If the registrant concludes (i): The substance does not fulfil the PBT and vPvB criteria, 
this is the end of the PBT/vPvB assessment process. In this case, the general obligation of 
REACH Article 22 to take into account relevant new information or relevant changes in the 
substance composition applies for triggering the need to revise the PBT/vPvB assessment. 

  Conduct emission characterisation and risk characterisation 

If the registrant concludes (ii): The substance fulfils the PBT or vPvB criteria, he must 
carry out an emission characterisation and implement and recommend such risk management 
measures which minimise emissions and subsequent exposures of humans and the 
environment from manufacture and identified uses (see Section R.11.3.4). 

Also substances concluded according to the principles described in Section R.11.4.1.4 as 
fulfilling PBT or vPvB criteria because their constituents, impurities, additives or 
degradation/transformation products fulfil the PBT or vPvB criteria must be subjected to 
emission characterisation and minimisation of releases for their whole life-cycle. 

It should be noted that if the registrant draws this conclusion within his CSA, it does not 
automatically lead to initiation of the REACH Article 59 process for inclusion of the substance in 
the Candidate List but the registrant has the primary responsibility to implement the necessary 
risk management measures for minimisation of the exposure and emissions.  

  Generate relevant additional information (including, where relevant, 

submission of a testing proposal) 

If the registrant concludes (iii): The available information does not allow to conclude (i) 
or (ii), the registrant must generate relevant additional information and continue the 
PBT/vPvB assessment Step 1 until the comparison with the criteria can be reliably done and a 
final conclusion (i) “The substance does not fulfil the PBT and vPvB criteria” or (ii) “The 
substance fulfils the PBT or vPvB criteria” can be unequivocally drawn (see flowchart in Section 
R.11.3.1). The obligation of the registrant to generate relevant additional information for the 
PBT/vPvB assessment concerns also relevant constituents, impurities, additives and 
transformation/degradation products. This means that if there is not enough information 
available on the PBT/vPvB properties of relevant constituents, impurities, additives and 
transformation/degradation products to derive for the registrant’s substance either conclusion 
(i) or conclusion (ii), the registrant must generate the necessary further information on the 
PBT/vPvB properties of the relevant constituents, impurities, additives and 
transformation/degradation products until one of these two definitive conclusions can be 
arrived at. 

This obligation to generate relevant additional information is valid regardless of whether the 
registrant’s dossier contains experimental information on the registered substance for all 
standard information requirements or whether he has made use of the data adaptation 
possibilities of Annex XI and Column 2 of Annexes VII to X to the REACH Regulation. In certain 
cases this may mean that the adaptation the registrant originally made (or planned to make) 
in the registration needs to be replaced by results from a study which needs to be carried out 
for the purpose of the PBT/vPvB assessment as required in Section 2.1 of Annex XIII to the 
REACH Regulation. Especially for such Column 2 waivers of Annexes VII to X to the REACH 
Regulation which are based on limited or unlikely exposure, it is important to note that the 
registrant, if not able to conclude (i) (“The substance does not fulfil the PBT or vPvB criteria”), 
may need to carry out the tests he originally wished to waive in order to be able to conclude 

 
 
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the PBT/vPvB assessment ultimately either by conclusion (i) or (ii), unless he decides to treat 
the substance “as if it is a PBT or vPvB” (see next Section). For example, a registrant may 
apply the Column 2 adaptation rule “The study need not be conducted if direct and indirect 
exposure of the aquatic compartment is unlikely” for the testing requirement (bioaccumulation 
in aquatic species) of Section 9.3.2 of Annex IX to the REACH Regulation. If he concludes the 
PBT/vPvB assessment with the conclusion (iii) (“The available data information does not allow 
to conclude (i) or (ii)”) because the substance fulfils the P or vP criteria and due to a Log Kow 
> 4.5 potentially fulfils the B/vB criteria, he must either carry out the bioaccumulation test he 
originally wished to waive or he must treat the substance “as if it is a PBT or vPvB” (see next 
Section). 

The additional relevant information needed to be generated by the registrant must be 
identified by the registrant in the technical dossier and CSR. This additional information can 
relate to one or several tests as listed in Annexes IX or X to the REACH Regulation. The 
additional relevant information can also be an “other type” of information, which the registrant 
considers to be optimal for the PBT/vPvB assessment, as Section 3.2 or Annex XIII to the 
REACH Regulation allows the use of such other information. The other type of information can 
be experimental information not falling under Annex IX or X, but it may also be a combination 
of experimental research information and monitoring research or solely research based on 
monitoring/measured field data. Section R.11.4 provides guidance to the registrant for 
deciding which information could be necessary in pursuing an unequivocal conclusion (i) or (ii). 
The additional information can be generated by the registrant in a tiered way by means of a 
testing strategy, if this is deemed necessary. Elements of such testing strategies include 
avoiding unnecessary animal or other testing and ensuring efficient use of resources while 
optimising the generation of data that can be used to reach definitive conclusion (i) or (ii). 

If the registrant, based on the PBT/vPvB assessment, identifies that information listed in Annex 
IX or X to the REACH Regulation is needed, he must submit appropriate testing proposal(s). 
Such testing proposals are subject to the normal testing proposal evaluation process of REACH.  

If the registrant is using his right to generate for the purpose of the PBT/vPvB assessment an 
“other type” of information as described above, testing proposals cannot be submitted. The 
registrant should, however, inform ECHA about his plans to generate any such other 
information by specifying in the CSR to the degree of detail possible an appropriate information 
gathering or testing strategy and an estimated time needed to update the PBT/vPvB 
assessment and the registration dossier. This is the only way the registrant can inform ECHA 
that he is using this possibility for complying with the data generation obligation in his 
PBT/vPvB assessment. 

The registrant should strive to plan generation of further relevant information in a way that 
leads to submission of a minimum number of updates of the PBT assessment and technical 
dossier. However, it is recognized that PBT assessment can be challenging and the information 
generated may sometimes provide results which indicate that further information not initially 
foreseen by the registrant needs to be generated to come to final conclusion (i) or (ii). In such 
cases the registrant is obliged to update the registration dossier (including the CSR) without 
delay each time new information becomes available. Hence, the registration dossier may in the 
most complex cases need to be updated several times before the PBT assessment Step 1 can 
be concluded. 

Section 0.5 of Annex I to the REACH Regulation, requires of the registrant that: “[…] While 
waiting for results of further testing, he shall record in his chemical safety report, and include 
in the exposure scenario developed, the interim risk management measures that he has put in 
place and those he recommends to downstream users intended to manage the risks being 
explored.” It is thus the duty of the registrant to identify appropriate interim risk management 
measures. 

Section 2.1 of Annex XIII to the REACH Regulation requires relevant further information to be 
generated regardless of the tonnage band for the substance of the registrant conducting the 
PBT/vPvB assessment. This obligation is illustrated by the following example: a registrant with 

  
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a tonnage band for a substance of 10-100 t/y identifies that more information is needed and 
that (a) degradation simulation test(s) would be the first test(s) needed, followed by a fish 
bioaccumulation test if the substance is deemed persistent after simulation testing. He must 
submit a testing strategy and testing proposals, even though the degradation simulation test 
and the fish bioaccumulation test are not listed as standard information requirements for 10-
100 t/y registrations. 

  Treat the substance “as if it is a PBT or vPvB” 

If the registrant arrives at the conclusion (iii): The available information does not allow to 
conclude (i) or (ii), he can also decide - based on REACH Annex XIII, Section 2.1 - not to 
generate further information, if he fulfils the conditions of exposure based adaptation of Annex 
XI, Section 3.2(b) and (c). Uniquely to the PBT assessment, the registrant must additionally 
consider the substance “as if it is a PBT or vPvB”, i.e. state that he wishes to regard the 
substance as a PBT/vPvB without having all necessary information for finalising the PBT/vPvB 
assessment. This option has exactly the same consequences for the registrant and his supply 
chain, as if the substance had been identified as PBT or vPvB based on a completed PBT/vPvB 
assessment. This includes the obligation that if a substance is considered “as if it is a PBT or 
vPvB”, the registrant must compile and provide recipients with a Safety Data Sheet (SDS) in 
accordance with REACH Article 31 even if the substance does not already meet the criteria in 
Article 31(1)(b) for supply of an SDS. It is important that the registrant clearly flags in the 
registration dossier and in the supply chain communication that the substance is considered 
“as if it is a PBT or vPvB”. 

R.11.3.4 

Emission characterisation, risk characterisation and risk 
management measures  

The registrant must develop for a “PBT or vPvB substance”7 exposure assessments including 
the generation of Exposure Scenario(s) (ES(s)) for manufacturing and all identified uses as for 
any other substance meeting the criteria for classification for any of the hazard classes or 
categories of Article 14(4) of the REACH Regulation8.  

Whereas for substances meeting the classification criteria for Article 14(4) hazard classes or 
categories the objective of an exposure assessment is to make qualitative or quantitative 
estimates of the dose/concentration of the substance to which humans and the environment 
are or may be exposed, the main objective of the emission characterisation for “a PBT or vPvB 
substance” is to estimate the amounts of the substance released to the different environmental 
compartments during all activities carried out by the registrant and during all identified uses.  

7 For the purpose of this section including the sub-sections, it is noted, that when reference to a “PBT or 
vPvB substance(s)” in italics is made, this covers both the case that the substance has been concluded to 
fulfil the PBT/vPvB criteria and the case that the registrant considers the substance “as if it is a 
PBT/vPvB” (for when these terms apply, see Section R.11.3.2.1). However, it is noted, that the registrant 
needs to clearly flag in the technical dossier, CSR and Safety Data Sheet which of the two cases applies 
to his substance. 

8 i.e.: 
 

 

 
 

hazard classes 2.1 to 2.4, 2.6 and 2.7, 2.8 types A and B, 2.9, 2.10, 2.12, 2.13 categories 1 and 
2, 2.14 categories 1 and 2, 2.15 types A to F 
hazard classes 3.1 to 3.6, 3.7 adverse effects on sexual function and fertility or on development, 
3.8 effects other than narcotic effects, 3.9 and 3.10 
hazard class 4.1 
hazard class 5.1 

 
                                                 
 
 
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Additionally, for a substance to be considered “as if it is a PBT/vPvB” (i.e., the substance is 
regarded as a PBT/vPvB without finalising the PBT/vPvB assessment), appropriate parts of the 
CSR and the technical dossier must clearly demonstrate that the registrant fulfils the 
conditions for exposure based adaptation. This is the prerequisite as defined by Section 2.1 of 
Annex XIII to to the REACH Regulation for avoiding the further information needed to finalise 
the PBT assessment Step 1. All use and exposure related information of the registration 
dossier must in this case be in line with the specific conditions for exposure based adaptation 
as stipulated in Section 3.2(b) and (c) of Annex XI to to the REACH Regulation. For a 
description of the required conditions please refer to the Guidance on intermediates and 
Chapter R.5: Adaptation of information requirements of the Guidance on IR&CSA.  

The subsequent risk characterisation for “PBT or vPvB substances” requires a registrant to use 
the information obtained in the emission characterisation step to implement on his site, or to 
recommend to his downstream users, Risk Management Measures (RMM) and Operational 
Conditions (OC) which minimise emissions and subsequent exposure of humans and the 
environment throughout the life-cycle of the substance that results from manufacture or 
identified uses (Section 6.5 of Annex I to the REACH Regulation). RMMs and OCs are 
documented in an ES(s). 

  Emission characterisation 

The objective of the emission characterisation is: 

 

 

to  identify  and  estimate  the  amount  of  releases  of  a  “PBT  or  vPvB-substance”  to  the 
environment; and  

to identify exposure routes by which humans and the environment are exposed to a “ PBT 
or vPvB-substance”. 

The principal tool to achieve this objective is exposure scenarios. Part D and Chapters R.12 to 
R.18 of the Guidance on IR&CSA provide guidance on how to develop exposure scenarios for 
substances in general. Parts of the exposure assessment guidance are relevant also for “PBT or 
vPvB substances” (i.e. emission estimation and assessment of chemical fate and pathways). 
However, since the objectives are not the same, the general scheme for exposure assessment 
needs to be adapted to the requirements of emission characterisation for “PBT or vPvB 
substances”. Guidance is given below on some issues where special considerations are needed 
for “PBT or vPvB substances”. 

Throughout the development of an ES for a particular use, the objective of the risk 
characterisation for “PBT or vPvB substances”, namely the minimisation of emissions and 
(subsequent) exposures of humans and the environment that results from that use, needs to 
be considered. Hence the need or a potential to (further) minimise emissions may be 
recognised at any point in the development of the ES. In this case, the appropriate RMMs or 
OCs must be included in the risk management framework and their effectiveness be assessed. 
In particular, for a substance to be considered “as if it is a PBT or vPvB”, the exposure 
scenarios must be in line with the fact that the adaptation criteria of REACH Annex XI Section 
3.2(b) and/or (c) are fulfilled. The final ES, or ES(s) in case of different uses, must be 
presented under the relevant heading of the chemical safety report, and included in an annex 
to the SDS. It must describe the required OCs and RMMs in a way that downstream users can 
check which measures they have to implement in order to minimise emissions or exposures of 
humans and the environment.  

It should be noted that a registrant has to take care of his own tonnage (manufactured and 
imported). In co-operation with his downstream users the registrant has to cover, where 
relevant, his own uses and all identified uses including all resulting life-cycle stages. However, 
it can be useful to consider on a voluntary basis exposure resulting from emissions of the same 
substance manufactured or imported by other registrants (i.e. the overall estimated market 
volume), c.f. Part A.2.1. 

  
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As “PBTs or vPvB substances” are substances of very high concern, the registrant must pay 
attention to the level of detail of his assessment as well as to whether its accuracy and 
reliability is sufficient for a “PBT or vPvB substance”. Where generic scenarios and assumptions 
may be sufficient for exposure assessment of non PBT/vPvB-substances, specific scenarios and 
data will be needed throughout an emission characterisation for “PBT or vPvB substances”. The 
emission characterisation must, in particular be specific in the use description and concerning 
RMMs, and must furthermore contain an estimation of the release rate (e.g. kg/year) to the 
different environmental compartments during all activities carried out during manufacture or 
identified uses. Emissions and losses may e.g. be addressed by performing mass balances. The 
total amount of a substance going to each identified use must be accounted for and the whole 
use-specific life-cycles be covered. This can, for instance, be done by performing a substance 
flow analysis covering manufacture, all identified uses, emissions, recovery, disposal, etc. of 
the substance. If the total amount of the substance cannot be accounted for, the identification 
of emission sources should be refined. All effort necessary should be made to acquire for 
manufacture and any identified use throughout the life-cycle, site- and product-specific 
information on emissions and likely routes by which humans and the environment are exposed 
to the substance. However, information on environmental concentrations is normally not 
needed because minimisation of emissions and exposure is required for “PBT or vPvB 
substances” (data on environmental concentrations, if available, may however be useful in the 
assessment and should be considered). Gathering of the mentioned information is not required 
for uses that are advised against as mentioned under heading 2.3 of the CSR and in Section 
1.2 of the SDS. 

  Risk characterisation and risk management measures for “PBT or 

vPvB Substances” 

According to REACH, the objective of a risk characterisation for PBTs or vPvBs is to minimise 
emissions and subsequent exposure to these substances. Section 6.5 of Annex I to to the 
REACH Regulation further requires that: “For substances satisfying the PBT and vPvB criteria 
the manufacturer or importer shall use the information as obtained in Section 5, Step 2 when 
implementing on its site, and recommending for downstream users, RMM which minimise 
exposures and emissions to humans and the environment, throughout the life-cycle of the 
substance that results from manufacture or identified uses.” 

Risk characterisation for PBT/vPvB substances includes, as for other hazardous substances, the 
consideration of different risks. These are: 

  Risks for the environment 

  Risks for different human populations (exposed as workers, consumers or indirectly via the 

environment and if relevant a combination thereof) 

  Risks due to the physico-chemical properties of a substance. 

For the assessment of the likelihood and severity of an event occurring due to the physico-
chemical properties of a PBT/vPvB substance, the same approach for risk characterisation 
applies as for any other substance (see Section R.7.1 of Chapter R.7a of the Guidance on 
IR&CSA). 

The estimation of emissions to the environment and exposure of humans performed in the 
emission characterisation provides the basis for risk characterisation and risk management of 
PBT/vPvB substances. 

R.11.3.4.2.1 

Options and measures to minimise emissions and exposure 

A registrant has to generate ES(s) which describe how emissions and exposures to PBT/vPvB 
substances are controlled. These ES(s) have to cover manufacturing, registrants own uses, all 
other identified uses and life-cycle stages resulting from manufacturing and identified uses. 
Life-cycle stages resulting from the manufacture and identified uses include, where relevant, 
service-life of articles and waste. The registrants are advised to consider at an early stage 

 
Chapter R.11: PBT/vPvB assessment 

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which uses they wish to cover in their CSR. Obviously, if the registrant substitutes a PBT/vPvB 
substance in his own uses or he decides to stop supplying for certain downstream uses, he 
does not need to cover these uses in his CSR. Supply chain communication is of high relevance 
for such cases.  

For the uses the registrant decides to include in his CSA and therefore develops ES(s) for, 
supply chain communication can be crucial for getting detailed enough information on 
conditions of use applied in practice. The registrant can conclude on the basis of the ES(s) he 
develops that he is not able to demonstrate that emissions can be minimised from a specific 
use. He must list any such uses as ‘uses advised against’ under heading 2.3 of the CSR. 
Furthermore, this information has also be documented under heading 3.7 of the technical 
dossier and communicated to the downstream users in Section 1.2 of the SDS. 

The registrant has to implement the risk management measures and operational conditions 
described in the final ES(s) for manufacture and his own uses. He has to communicate as an 
annex to the SDS the relevant ES(s) for his downstream users. The downstream users have to 
implement the recommended ES(s) or alternatively prepare a downstream user CSR. 

One possibility to develop ES(s) that minimise emissions and exposure is to use a similar 
approach as for isolated intermediates (outlined below, for further details see the Guidance on 
intermediates). 

Rigorous containment of the substance 

The “PBT or vPvB substance” must be rigorously contained by technical means during its whole 
life-cycle. This covers all steps in the manufacturing of the substance itself as well as all its 
identified uses. It further includes cleaning and maintenance, sampling, analysis, loading and 
unloading of equipment/vessels, waste disposal, packaging, storage and transport. This 
containment may only become unnecessary from a step in the life-cycle on for which it can be 
demonstrated that the substance is being transformed to (an)other substance(s) without 
PBT/vPvB properties or that the substance is included into a matrix from which it or any of its 
breakdown products with PBT/vPvB properties will not be released during the entire life-cycle 
of the matrix including the waste life stage. Note however that residues of the original “PBT or 
vPvB substance” in the matrix or impurities with PBT/vPvB properties resulting from side-
reactions must additionally be considered (see Section R.11.3.2.1). 

Application of procedural and control technologies 

Efficient procedural and/or control technologies must on the one hand be used to control and 
minimise emissions and resulting exposure when emissions have been identified. For example, 
in case of emissions to waste water (including during cleaning and maintenance processes), it 
will be considered that the substance is rigorously contained if the registrant can prove that 
techniques are used that give virtually no emissions The same applies to emissions to air or 
disposal of wastes where technologies are used to minimise potential exposure of humans and 
the environment. It is important to consider that RMM which protect humans, for instance from 
direct exposure at the workplace, can in some cases lead to emissions to the environment 
(e.g. ventilation without filtration of exhaust air). For a “PBT or vPvB substance”, such a 
measure is insufficient as exposure of both humans and the environment must be minimised 
(ventilation plus filtration of exhaust air may thus be an option in the case of the example). 

On the other hand, procedural and/or control technologies must also be implemented to 
guarantee safe use, i.e. to prevent accidents or to mitigate their consequences. Regarding this, 
the clarifications according to the Directive 2012/18/EU on the control of major-accident 
hazards involving dangerous substances and the Directive 2014/34/EU concerning equipment 
and protective systems intended for use in potentially explosive atmospheres might be 
consulted. 

Handling of the substance by trained personnel 

In order to minimise emissions and any resulting exposure, it is important that only trained 
personnel handle “PBT or vPvB substances” or mixtures. From this perspective any consumer 

  
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use of these substances on their own or in mixtures is probably inappropriate, because in 
these cases sufficient control of the emissions is in practice difficult to ensure. 

R.11.3.4.2.2 

Risk Characterisation for humans in cases of direct exposure to 
“PBT or vPvB substances” 

Although quantitative risk assessment methodologies can, due to the associated high 
uncertainties regarding the extent of long-term exposure and effects, generally not be used for 
estimating the risk posed by “PBT or vPvB substances” to the environment or to humans via 
the environment (indirect exposure of humans), it may be possible to use the quantitative 
approach for assessing the risk for workers caused by direct exposure to the substance at the 
workplace, because in this case exposure under the controlled conditions of the working 
environment is predictable. A quantitative approach can only be applied to characterise the 
risk for workers resulting from direct exposure.  

In case of assessing exposure at the workplace the quantitative approach (i.e. 
Exposure / DNEL) must be used, wherever possible, to demonstrate that workplace exposure 
does not result in health risks. If a DNEL cannot be derived (e.g. for substances for which effect 
thresholds cannot be established), the respective approach for assessing the health risk posed 
by non-threshold substances must be applied9. The overall risk for workers (resulting from all 
types and routes of exposure) can normally only be assessed in qualitative terms and in doing 
so the increased uncertainty in estimating the risk via indirect exposure through the 
environment must be taken into due consideration. As a consequence, the application of a 
higher margin of safety (i.e. a risk quotient Workplace Exposure / DNEL << 1) than usually 
applied to non-“PBT or vPvB substances” may be required to account for this increased 
uncertainty and to consider workplace exposure as safe. Guidance on risk assessment for 
human health is given in Chapter R.8 of the Guidance on IR&CSA. 

It should further be noted that even if a quantitative assessment of health risks at the 
workplace would indicate low risks, this does not imply that the RMM and the OC at the 
workplace can be considered sufficient where it is technically and practically possible to further 
minimise emissions and exposure at the workplace. 

R.11.3.5  Documentation of the PBT/vPvB assessment  

The documentation of the PBT/vPvB assessment in the registration dossier consists of several 
elements depending on the outcome. Section 8 of the CSR and Section 2.3 “PBT assessment” 
of the technical dossier generated in IUCLID 10 should be provided by all registrants who need 
to conduct a CSA. Furthermore, for substances with conclusion (iii) “The available data 
information does not allow to conclude (i) or (ii)”, the registrant must identify the additional 
information needed in the CSA and in the technical dossier. These elements are described 
further in the following. 

When the registrant conducts a CSA and submits a CSR he needs to conduct the PBT/vPvB 
assessment based on the relevant and available data (Step 1). This should be reported in 
detail in Section 8.1 “Assessment of PBT/vPvB properties” of the CSR. One of the three 
conclusion options described in Section R.11.4.1.4 must be recorded in this chapter as well. 
Furthermore, if the registrant as the result of conclusion (iii) “The available data information 
does not allow to conclude (i) or (ii)” considers his substance “as if it is a PBT or vPvB”, this 
must be recorded in Section 8.1 as well.  

9 Note that, apart from predictable exposure, a further prerequisite for quantitative assessment of risk is 
the possibility to derive  the no-effect level for humans with an appropriate level of certainty. 
10 The IUCLID software is downloadable from the IUCLID website at http://iuclid.eu for free by all parties, 
if used for non-commercial purposes. 

 
                                                 
 
 
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If the registrant concludes that the substance fulfils the PBT/vPvB criteria or considers the 
substance “as if it is a PBT or vPvB”, emission characterisation and risk characterisation shall 
be conducted and the CSR must contain also a section “Emission characterisation”, reported as 
Section 8.2 of the CSR. It is noted, that the CSR-plugin of IUCLID automatically creates these 
two section titles. It is recommended that the registrant lists in Section 8.2 all relevant 
sections of the CSR (Sections 9 and 10), including the details of the emission characterisation 
elements. 

All available relevant data must be recorded in the technical dossier in relevant endpoint study 
records and those relevant to the PBT/vPvB assessment must be reflected in the CSR, Section 
8.1. Furthermore, the conclusions of the PBT/vPvB assessment including brief justification 
should be recorded in IUCLID Section 2.3. Support on how to fill in the information in Section 
2.3 “PBT assessment” of IUCLID in practice is given in the IUCLID  End-User Manual. In this 
section, it is possible to create one endpoint summary and several endpoint records. Note that 
the objective of the PBT Section 2.3 in IUCLID is not to repeat information already provided in 
other IUCLID sections. A reference to other IUCLID sections can be made.  

If the conclusion (iii): “The available data information does not allow to conclude (i) or (ii)” is 
drawn in the PBT assessment Step 1 the registrant must as part of the technical dossier submit 
testing proposals, if the information needed is listed in Annex IX or X to the REACH Regulation. 
Instructions for recording the testing proposals in the technical dossier are provided in Data 
Submission Manual 5. If the additional information needed to finalise the PBT assessment Step 
1 is not listed in Annex IX or X, the registrant cannot submit a testing proposal as testing 
proposals on other items than those listed in Annex IX or X will be rejected by ECHA. If the 
additional information is not listed in Annex IX or X, the registrant should describe in his CSR, 
Section 8.1 what information is envisaged to be generated. In this case the CSR should also 
contain the estimated timeline.  

After relevant studies have been conducted, the PBT/vPvB assessment must be updated. The 
same applies to the CSR and the technical dossier including endpoint study records for newly 
generated information. The tasks of generation of further information and subsequent updating 
of the CSR and the technical dossier should ideally be carried out in one step. However, it is 
recognised that PBT/vPvB assessment sometimes may be a challenging task where several 
updates and cycles of generation of additional information may be needed until the PBT/vPvB 
assessment can be finalised by the registrant.  

Furthermore, the registrant must differentiate in the registration dossier, CSR and Safety Data 
Sheet between the status of a substance fulfilling the PBT/vPvB criteria and a substance 
considered “as if it is a PBT or vPvB”. This ensures that the downstream user receives enough 
information to be able to make use of his rights and obligations under Article 37 of REACH. 
Furthermore, this requirement is consistent with the purpose of the SDS, as stated in Section 
0.2.1 of Annex II to to the REACH Regulation: ‘The safety data sheet shall enable users to take 
the necessary measures relating to protection of human health and safety at the workplace, 
and protection of the environment (…) a safety data sheet must inform its audience of the 
hazards of a substance or a mixture and provide information on the safe storage, handling and 
disposal of the substance or mixture’. Correct information on the hazard is provided when 
there is a differentiation between substances which meet the PBT/vPvB criteria based on data 
and those which are treated "as if it is a PBT or vPvB". 

If a registrant’s substance is included in the Candidate List as a PBT or vPvB substance, please, 
see also Section R.11.3.2.2. 

  
 
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R.11.3.6  Documentation of the risk characterisation and communication of 

measures  

Given the potential risk exerted by “PBT or vPvB substances”11, the descriptions of the 
implemented or recommended, RMMs and OCs in an ES need to be sufficiently detailed to 
demonstrate rigorous control of the substance and to allow examination and assessment of 
their efficiency by authorities. The level of detail communicated in the ES attached to the 
Safety Data Sheet must further permit downstream users to check that their use(s) are 
covered by the ES developed by their supplier and that they have implemented the 
recommended RMMs and OCs correctly. 

The risk characterisation for all ESs developed for the identified uses of the “PBT or vPvB 
substance” have to be documented under heading 10 of the CSR. The registrant is obliged 
according to REACH Article 14 to keep his CSR available and up to date. It should be further 
noted that any update or amendment of the CSR will require an update of the registration by 
the registrant without undue delay.  

If the registrant concludes based on available information (ii) “The substance fulfils the PBT or 
vPvB criteria” or he considers the substance “as if it is a PBT or vPvB”, this triggers the 
obligation to generate a Safety Data Sheet according to REACH Article 31. For both cases, the 
general obligations of Article 31 apply. Furthermore, the registrant must differentiate in the 
Safety Data Sheet which of the two cases applies for his substance. This differentiation is 
necessary in order to provide the downstream users the possibility to take own action for 
assessing further the PBT/vPvB properties of the substance. 

11 “PBT or vPvB substance(s)” covers both the case that the substance has been concluded to fulfil the 
PBT/vPvB criteria and the case that the registrant considers the substance “as if it is a PBT/vPvB” (for 
when these terms apply, see Section R.11.3.2.1).  

 
 
                                                 
 
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R.11.4  Assessment of PBT/vPvB properties – the scientific method 

This section describes the method for comparison of the available information with the criteria, 
which for the registrant is Step 1 of the PBT/vPvB assessment process. It should be noted that 
this section is not meant to set obligations/requirements for the registrant, but the registrant 
should nonetheless use this part of the guidance for pursuing the overall requirement to clarify 
unequivocally whether a substance fulfils the PBT or vPvB criteria or not. The method is the 
same as used by authorities for PBT/vPvB assessments, e.g., for identifying a substance as 
“Substance of Very High Concern” for the ECHA Candidate List according to REACH Article 59. 
The method has been developed on a scientific basis and as such lays out the rules of 
convention.  

As in several areas of PBT/vPvB assessment scientific development activities are on-going, it is 
underlined that the assessor has the responsibility to critically scrutinize and apply in the 
PBT/vPvB assessment any relevant new scientific developments. 

Sections R.11.4.1.1, R.11.4.1.2 and R.11.4.1.3 contain an assessment and testing strategy at 
the beginning of those sections. It should be noted that there is a high number of different 
combinations of property–specific conclusions, which a registrant may reach after the 
assessment. Due to the high number of the possible outcomes, they are not presented in this 
section. However, Section R.11.4.1.4 (conclusion (iii)) provides an overview of the different 
situations that may arise for which further information is needed.  

Before starting the assessment at the level of individual properties, it is recommended to 
become familiarised with Section R.11.4.2.2. Any substance containing multiple constituents, 
impurities and/or additives should be assessed according to that section. 

R.11.4.1 

Standard approach 

The PBT/vPvB assessment must cover a consideration of each property persistence, 
bioaccumulation and toxicity against each respective criterion (P or vP, B or vB, and T) in order 
to arrive at an informed decision on the properties of a substance or of its relevant individual 
constituents, impurities, additives or transformation/degradation products. In principle, 
substances are considered as fulfilling the PBT or vPvB criteria when they are deemed to fulfil 
the criteria P, B and T or vP and vB, respectively. 

The assessment strategies set out in this section and Section R.11.4.2 should normally be 
followed and further information be searched for or generated, if necessary. In deciding which 
information is required on persistence, bioaccumulation or toxicity in order to arrive at an 
unequivocal conclusion, care must be taken to avoid vertebrate animal testing when possible. 
This implies that, when for several properties further information is needed, the assessment 
should normally focus on clarifying the potential for persistence first. When it is clear that the P 
criterion is fulfilled, a stepwise approach should be followed to elucidate whether the B 
criterion is fulfilled, eventually followed by toxicity testing to clarify the T criterion. 

It should be noted that for some elements of the PBT/vPvB assessment there may be, for the 
purpose of a particular PBT/vPvB assessment, a need to take the recent scientific 
developments into account although they have not yet been implemented in this guidance. In 
such a case the assessor should duly justify the reasons for deviation from, or extension of, 
the approach presented in this document.  

Weight-of-Evidence determination 

As described in Section R.11.2.1, a Weight-of-Evidence determination using expert judgement 
is to be applied in the PBT/vPvB assessment. This applies for all assessment situations 
employing screening and/or assessment information. In order to decide whether the substance 
must be considered as a potential PBT/vPvB substance based on screening information or as a 

  
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substance meeting the PBT or vPvB criteria, all relevant available information must be taken 
into account.  

The requirement to use a Weight-of-Evidence approach using expert judgement implies, 
according to the introductory section of Annex XIII to to the REACH Regulation, that “The 
available results regardless of their individual conclusions shall be assembled together in a 
single Weight-of-Evidence determination”. This normally means that the individual pieces of 
data available do not need to be compared individually to each of the P, B, T or vP, vB criteria 
but all information are assembled together for each of the properties, respectively, for the 
purpose of a single comparison with the respective criteria. This does not exclude the option to 
compare information directly with each of the P, B, T or vP, vB criteria to support the 
assessment, where appropriate. It should be noted that Weight-of-Evidence determination is 
not a mechanism to justify disregarding valid, standard test data. The quality and consistency 
of the data should be given appropriate weight.  

The use of quantitative Weight-of-Evidence approaches for the whole or a part of the available 
information is encouraged, although the derivation of a conclusion property by property needs 
expert judgement, especially when very different types of information are available and when 
the information cannot be directly (numerically) compared with the criteria12.  

The Practical Guide on “How to use alternatives to animal testing to fulfil your information 
requirements for REACH registration” provides a general scheme for building a Weight-of-
Evidence approach. It should be noted that further development of the Weigh-of-Evidence 
approach is on-going and further Guidance may become available in the near future.It is 
underlined that an essential prerequisite for applying a Weight-of-Evidence approach is that 
the reliability and suitability of experimental studies and non-experimental data are evaluated 
according to Chapters R.4, R.7b and R.7c of the Guidance on IR&CSA. The suitability and 
relevance of information to the PBT/vPvB assessment is further described in the following sub-
sections. This evaluation must be well documented in the assessment report.  

For particular cases, further described in Section R.11.4.1.4, the Weight-of-Evidence 
determination should consider all three properties (i.e. persistence, bioaccumulation and 
toxicity) in conjunction. In particular, if for one or more of these properties only screening 
information is available and screening threshold values as provided in the following sub-
sections are applied to draw a conclusion, all three properties must be considered in 
conjunction.  

Relevant constituents, impurities, additives and transformation/degradation 
products 

The PBT/vPvB assessment should be performed on each relevant constituent, impurity and 
additive. It is not possible to draw overall conclusion if, e.g., the assessment of persistence has 
been concluded for one constituent and the assessment of bioaccumulation or toxicity for 
another constituent.  

Constituents, impurities and additives should normally be considered relevant for the PBT/vPvB 
assessment when they are present in concentration of ≥ 0.1% (w/w). This limit of 0.1% (w/w) 
is set based on a well-established practice recognised in European Union legislation to use this 

12 In particular, it should be noted that although it might be theoretically possible to calculate 
degradation half-life values or BCF values from screening information, such values must not be directly 
compared with the criteria. 

 
 
                                                 
 
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limit as a generic limit13. Individual concentrations < 0.1% (w/w) normally need not be 
considered.  

In practice, this means that the registrant should carry out a comparison of the available data 
with the criteria for all constituents, impurities and additives present in concentration of ≥ 
0.1% (w/w). Alternatively, the registrant should provide a justification in the CSR for why he 
considers certain constituents, impurities or additives present in concentration of ≥ 0.1% 
(w/w) or certain constituent fractions/blocks14 as not relevant for the PBT/vPvB assessment. 

It may not always be possible or even necessary to fully characterize and identify for the 
purpose of the PBT/vPvB assessment UVCBs (substances of Unknown or Variable composition, 
Complex reaction products or Biological materials) or fractions of impurities based on the 
information given in Section 2 of Annex VI to the REACH Regulation for substance 
identification. This is because (i) the number of constituents/impurities may be relatively large 
and/or (ii) the composition may, to a significant part, be unknown, and/or (iii) the variability of 
composition may be relatively large or poorly predictable. Regardless of whether full 
substance identification is possible or not for the whole composition, the registrant 
should make efforts for carrying out a PBT/vPvB assessment for all constituents, 
impurities and additives present in concentrations above 0.1% (w/w). Section 
R.11.4.2.2 provides further insight into how to carry out PBT/vPvB assessment for 
fractions of the substance that cannot be fully identified by the registrant. For an 
example of application of this recommendation in a specific industry sector, please see the 
Environmental assessment guidance on essential oils15. 

In specific cases it may be considered, for the sake of proportionality of assessment efforts and 
the level of risk being considered, to elevate or reduce the threshold value above or below 
0.1% (w/w) for the PBT/vPvB assessment. Account could be taken of, e.g. the use pattern of 
the substance and the potential emissions of the constituents, impurities or additives having 
PBT or vPvB properties. Careful consideration should be given especially when uses are known 
or anticipated to cause significant emissions.  

An elevated threshold value should not exceed 10% (w/w) for the total amount of all 
constituents, impurities and additives with PBT/vPvB properties, and the total amount of these 
within the manufactured/imported substance should in no case exceed 1 tonne/year. A 
reduced threshold might be necessary to derive information relevant for PBT/vPvB assessment, 
e.g. for very toxic substances, and the information on the toxicity derived for the classification 

13 The limit of 0.1% (w/w) is indicated in the European Union legislation, where there is no specific 
reason (e.g., based on toxicity) to establish a concentration limit specific to the case. Examples of this 
generic concentration limit are, i.a., another category of substances of very high concern according to 
Article 57 of REACH, where the default concentration of Carcinogenic/Mutagenic (category 1A/1B) 
ingredients in a mixture requiring a Carcinogen/Mutagen (1A/1B) classification of the mixture under 
Regulation (EC) No 1272/2008 is 0.1% (w/w). Furthermore, Articles 14(2)(f), 31(3)(b) and 56(6)(a) of 
REACH apply a similar principle and the same concentration limit for PBT/vPvB substances in mixtures 
regarding some obligations under REACH. Additionally, the Judgments of the General Court (Seventh 
Chamber, extended composition) of 7 March 2013 in cases T-93/10, T-94/10, T-95/10 and T-96/10 (see 
in particular paragraphs 117 to 121) confirmed the validity of this approach for PBT/vPvB constituents of 
a substance.  
14 The terms “constituent fractions” refer to a situation where for a UVCB substance not all its 
constituents can be identified individually and the substance identity needs then to be based on its 
fractions/groups of constituents. “Block” is a term analogous to fraction/group and is used in the 
hydrocarbon block–approach (see Section R.11.4.2.2). 

15 http://echa.europa.eu/support/substance-identification/sector-specific-support-for-substance-
identification/essential-oils  

  
                                                 
 
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and labelling purposes could be used for defining such a lower concentration limit for PBT/vPvB 
assessment. 

Especially for very complex UVCBs it is possible that individual constituents are present in 
concentrations <0.1% (w/w) and that these have not been characterised by chemical analysis 
individually. For UVCBs even the whole substance may consist of individual constituents only 
present in such low concentrations. The fact that all individual constituents of a UVCB-
substance are present in concentration <0.1% (w/w) does not automatically exempt the 
registrant from the obligation to carry out the PBT/vPvB assessment. A close structural 
similarity of individual constituents within a fraction of a UVCB substance, i.e. constituents with 
the same carbon number, chain lengths, degree and/or site of branching or stereoisomers, 
triggers the need to sum up the concentrations of these constituents and to compare the total 
concentration with the limit of 0.1% (w/w) in order to determine whether these constituents 
need to be covered in the PBT/vPvB assessment. Criteria for grouping or read across, as 
mentioned in the Practical Guide on “How to use alternatives to animal testing to fulfil your 
information requirements for REACH registration” and the “Introductory note to the illustrative 
example of a grouping of substances and read-across approach” , should be applied to the 
determination and justification of such fraction and (an) appropriate approach(es) as provided 
in Section R.11.4.2.2 should be applied for the PBT/vPvB assessment. 

Similarly, a UVCB substance which contains constituents in concentrations well above 0.1% 
(w/w) each, but also (a) large fraction(s) where constituents are individually <0.1% (w/w), 
cannot be concluded as “not PBT/vPvB” unless it can be justified with sufficient reliability that 
none of the constituents and fractions of minor constituents would cause a concern. For 
example, a UVCB-substance may contains ten constituents, present in a total concentration of 
60% (w/w) and the remaining 40% of the composition consists of not fully identified 
constituents. All latter minor constituents are individually present in concentration of <0.1% 
(w/w) but are expected to be similar to each other structurally and hence expected to have 
similar degradation, bioaccumulation and toxicity-properties. Not only the ten constituents 
making the largest part of the substance, but also the remaining 40% of the composition 
would need to be assessed using the appropriate approach provided in Section R.11.4.2.2 and 
testing, where necessary.  

The same principles, as described in the two previous paragraphs above for UVCB-substances, 
apply also to the constituents of well-defined substances and their impurity fractions. 

It should be noted in this connection that in cases where large fractions of unidentified 
constituents are present at <0.1% w/w, the assessment efforts need to remain proportionate.  

A close structural similarity of individual constituents within a fraction, determined by criteria 
of grouping or read across as mentioned above, means that the concentrations of constituents 
with P, B and T (or vP and vB) properties should normally be summed up in order to compare 
with the threshold of 0.1% (w/w). Structural similarity of the constituents (justify assessing 
the constituents as if they were one substance in terms of their physico-chemical, degradation 
and bioaccumulative properties and effects. This recommendation relies on the assumption 
that the mode of action of similar constituents is the same and the fate properties are very 
similar, hence causing an exposure which triggers effects in humans and the environment as if 
the exposure were to one substance. This understanding of aggregated exposure (aggregated 
concentration) leading to corresponding aggregated effects draws from the same scientific 
basis as the concept of additivity (“joint action”, “dose additivity”, “concentration additivity”, 

 
Chapter R.11: PBT/vPvB assessment 

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“additivity of toxicity”), used in many regulatory activities, e.g. in the CLP-Regulation (EC, 
2012; ECB, 2003; Feron et al., 2002). However, it should be noted, that if the criteria for read 
across are not fulfilled for degradation, bioaccumulation and (eco)toxicity in PBT-assessment 
and for the first two properties in the vPvB-assessment, such summing up is not applicable 
and the normal 0.1% (w/w) threshold should be applied.     

Similar arguments apply to relevant transformation/degradation products. The PBT/vPvB 
assessment should normally be carried out for each relevant transformation or degradation 
product.  

It is not possible to draw an overall conclusion for the substance if the assessment of 
persistence has been concluded for one transformation/degradation product and the 
assessment of bioaccumulation or toxicity for another transformation/degradation product. 

The registrant should endeavour to carry out a comparison of the relevant available data with 
the PBT/vPvB criteria for each relevant transformation/degradation product (or in case those 
cannot be ultimately identified: for each group or block of transformation or degradation 
products), respectively. If the registrant considers degradation/transformation products that 
are formed (or groups/blocks of them) as not relevant for the PBT/vPvB assessment, he should 
also clearly explain in the PBT/vPvB assessment the reasons why they are not relevant.  

If the available and relevant screening and other information allows the registrant to conclude 
that the substance is not persistent using the screening threshold values as provided in Table 
R.11—2, then it may normally be assumed that the substance is mineralized quickly and is not 
likely to form transformation/degradation products relevant for the PBT/vPvB assessment. 
However, the available relevant screening or other information (including information from 
hydrolysis tests and field data) may indicate that transformation or degradation products 
relevant for the PBT/vPvB assessment are indeed formed. These indications should be 
addressed in the registrant’s PBT/vPvB assessment either qualitatively or quantitatively.   

Following the obligation of the registrant under Article 13(3) of REACH in the situation where 
new degradation simulation testing is necessary, the transformation and degradation products 
relevant for the registrant’s own PBT/vPvB assessment are those products, which must be 
identified in tests C.23, C.24 and C.25 carried out in accordance with Council Regulation No 
440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 
(REACH) (“Test Methods Regulation”). It should be mentioned in particular that guideline C.24 
requires that “…in general transformation products detected at ≥ 10% of the applied 
radioactivity in the total water-sediment system at any sampling time should be identified 
unless reasonably justified otherwise. Transformation products for which concentrations are 
continuously increasing during the study should also be considered for identification, even if 
their concentrations do not exceed the limits given above, as this may indicate persistence. 
The latter should be considered on a case by case basis....”  The latter case always applies 
when the registrant is in the situation of generating new degradation simulation data for the 
purpose of the PBT/vPvB assessment because he will have previously concluded that the 
substance may have PBT/vPvB properties,  

For the situation where information from tests comparable to the standard degradation 
simulation tests mentioned above are already available to the registrant or the registrant 
considers it more appropriate to generate new degradation information in accordance with 
Section 2.1 of Annex XIII to the REACH Regulation other than degradation simulation test data 
(see Section R.11.4.1.1 for the other possibilities), the principles of the standard test 
guidelines mentioned above for identifying relevant transformation and degradation products 
should be applied by analogy.  

It should be noted that authorities are not bound under the REACH Substance Evaluation and 
SVHC-identification processes to the stipulations of the Test Methods Regulation or other 
standards for defining what is a relevant transformation/degradation product but have the 
possibility to use other types of justified (concentration or formation rate) limits to define on a 

  
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case-by-case basis which transformation/degradation products are relevant for their PBT/vPvB 
assessment (e.g, see the Support Document of the Decision to identify Bis(pentabromophenyl) 
ether as Substance of Very High Concern16). Guidance is given in Section R.11.4.2 on the 
assessment and testing strategy for substances with specific substance properties such as 
UVCBs or multi-constituent substances with several constituents, in relation to 
transformation/degradation products, and for substances with low water solubility, high 
adsorption or volatility requiring deviations from the standard PBT/vPvB assessment.  

  Persistence assessment (P and vP) 

R.11.4.1.1.1 

Integrated assessment and testing strategy (ITS) for persistence 
assessment 

A strategy for degradation assessment and testing in the context of PBT/vPvB assessment is 
proposed in Figure R.11—3. A tiered approach to assessment and testing is necessary until a 
definitive conclusion on persistence can be drawn.  

Available data consisting solely of screening information can be employed to derive a 
conclusion mainly for “not P and not vP” or “may fulfil the P or vP criteria”. After the latter 
conclusion on screening, higher tier information generally needs to be made available. 
Appropriate data need to be available to conclude the P/vP-assessment with a conclusion “not 
P/vP” on all three compartments (or five, with marine compartments): water (marine water), 
sediment (marine sediment) and soil. Either the available data, including in normal case 
simulation test data from one or two compartments, can be interpreted so that a conclusion 
can be derived on the remaining compartment(s) for which no higher tier data are available, or 
data need to be available directly on all compartments, or there is another justification for why 
a conclusion does not need to be drawn for all three (five) compartments. In the opposite 
situation, if a conclusion “P” or “vP” is reached for one compartment, no further testing or 
assessment of persistence of other environmental compartments is normally necessary. In 
certain cases it may be possible to draw a conclusion “P” or “vP” based on screening 
information (e.g. tests on inherent biodegradation) combined with other useful information in a 
Weight-of-Evidence approach, as described later in this section and indicated in the ITS in 
Figure R.11—3.  

For substances containing multiple constituents, impurities and/or additives, the guidance 
provided below apply to that/those “part(s)” of the substance, which is/are the target(s) of the 
assessment and testing. The criteria for selecting an appropriate assessment approach is 
provided in Section R.11.4.2.2. 

16 https://echa.europa.eu/candidate-list-table/-/dislist/details/0b0236e1807dd2e6  

 
 
 
                                                 
 
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Figure R.11—3: Integrated Assessment and Testing Strategy for persistence 
assessment – maximising data use and targeting testing. 

1. Substance is readily biodegradable?Not P/vPOECD TG 309 technically feasible?Start with    OECD TG 309 (freshwater or marine water)6. Can the result(s) be used, together with other relevant available information, to conclude persistence in the remaining compartment(s)?Conclude P & vP assessment for the compartment74.2 Specify/justify the test compartment (OECD TGs 308, 307, or other) and test conditions- technical aspects- compartment of concern aspectsyes4. Potentially P/vP: Further information needed if substance also potentially B*. Develop a testing strategy for simulation testing7. Conclude P and/or vP assessment for all compartments7yesChoose the next compartment for testingno5. Further information needed for P-assessment for the chosen compartment (e.g. on specific degradation products)?nonovesno3. Other information useful in a weight-of-evidence approach: Negative enhanced ready biodegradation test?Specific inherent biodegradation test negativePositive enhanced ready biodegradation test and other data supporting?Specific inherent test positive with non-adapted inoculumNot P and not vPPotentially P and vP2. Screening information (Table R.11—4):Abiotic degradationApplicable QSARsMonitoringdataOther (testing and non-testing information)Simulation test resultsIn situ/field  degradation study resultsyesyes4.1 Is there compartment specific concern for soil or sediment?- water compartment is not at all relevant (based on fate and compartment of release),- persistence criteria are most likely to be exceeded in sediment or soil,- High hydrolysis rate etc...noyesnoyes* In the context of the Biocidal Product Regulation (BPR), it is worth noting that the P-criteria has to be assessed also when the T-criterion is (potentially) fulfilled.  
 
 
 
 
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Integrated assessment and testing of Persistence - Explanatory Notes to Figure 
R.11—3. 

1.  Evidence of ready biodegradation  

If the substance is readily biodegradable, or if the criteria for ready biodegradability are 
fulfilled with the exception of the 10-day window, there is no reason to perform further 
biodegradation tests for the PBT/vPvB assessment. The conclusion is that the substance is 
generally not regarded as fulfilling the criteria for Persistence (P or vP) (see Sections R.7.9.4 
and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA, and for multi-constituent substances 
see Section R.11.4.2.2). 

2.  Other sceening information (Table R.11—4) 

Following the ITS, and based on the screening information, the substance can be concluded as 
potentially P/vP or not P/vP according to the criteria and conditions described in Table R.11—4 
and Sections R.7.9.4 and R.7.9.5 of Chapter R.7b of the Guidance on IR&CSA. After 
consideration of the Explanatory Notes bulleted below, and before concluding that a substance 
is “not P" or "not vP”, it should be carefully examined if counter-evidence to that conclusion 
exists, e.g. from monitoring data or other available information (see Points 3-7 below for more 
information). When combined with all available information on persistence in a Weight of 
Evidence, the conclusion on persistence may cover one or multiple environmental 
compartments. 

If the substance is confirmed to degrade in other biodegradation screening tests than the tests 
for ready biodegradability, the results may be used to indicate that the substance will not 
persist in the environment. Specific enhancement conditions described in Sections R.7.9.4 and 
R.7.9.5 of Chapter R.7b of the Guidance on IR&CSA can be used for this purpose. For example, 
a result of more than 60% ultimate biodegradability (ThOD, CO2 evolution) or 70% ultimate 
biodegradability (DOC removal) obtained under the conditions specified in Chapter R.7b in an 
enhanced ready biodegradability test may be used to indicate that the criteria for P are not 
fulfilled (see Sections R.7.9.4 and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA). The 
enhancements may also be applied to standardised marine biodegradability tests (OECD TG 
306, Marine CO2 Evolution test, Marine BODIS test, and the Marine CO2 Headspace test).  

  Assessment of inherent biodegradation test data - Results of a Zahn-Wellens test 
(OECD TG 302B) or MITI II test (OECD TG 302C) only (not SCAS-test) may be used to 
confirm that the substance does not fulfil the criteria for P provided that certain additional 
conditions are fulfilled. In the Zahn-Wellens test, a level of 70% mineralisation (DOC 
removal) must be reached within 7 days, the log phase should be no longer than 3 days, 
and the percentage removal in the test before degradation occurs should be below 15% 
(pre-adaptation of the inoculum is not allowed). In the MITI II test, a level of 70% 
mineralization (O2 uptake) must be reached within 14 days, and the log phase should be no 
longer than 3 days (pre-adaptation of the inoculum is not allowed). A lack of degradation in 
an inherent biodegradation test (≤20%) can provide evidence that degradation in the 
environment would be slow (see further consideration under “Tests on inherent 
biodegradation in the main text). It should however be noted that the very low solubility of 
many PBT/vPvB substances may reduce their availability and hence their degradability in 
the test. The lack of degradation in an inherent test does not always imply that the 
substance is intrinsically persistent and in some cases further testing might be needed.  

  Enhanced screening tests – Positive results from enhanced screening tests may be used 
together with other supporting information to conclude that the substance is not P/vP. 
However, it is important that the following conditions are met: 1) the enhancements should 
only be about an extended test duration or an increased test vessel size, 2) the test should 
be performed with non-pre-adapted/non pre-exposed inocula, 3) the test duration should 
never been extended beyond 60 days, and 4) the test criteria set for ready biodegradability 

 
 
 
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tests should be applied, i.e. 60% or 70% degradation, depending on analyte, without the 
10-day window. If the results are negative, then it is generally not possible to definitively 
conclude on the persistence or absence of persistence of the substance and further testing 
will be needed. More information on enhanced screening tests can be found in Sections 
R.7.9.4 and R.7.9.5 of Chapter R.7b of the Guidance on IR&CSA. 

3.  Other information useful for a Weight-of-Evidence approach (not exhaustive) 

All available information on (bio)degradation, including testing, non-testing and monitoring 
data, should be considered. The overall evaluation could either show that the information 
available coherently provides proof of (non-)persistence and is sufficient to allow concluding 
the P/vP assessment, or indicate that further testing is needed. If further testing is needed a 
testing strategy should be developed following the ITS starting from step 4 below. 

  Use of (Q)SAR (both QSARs and SARs) estimates – Refer to Section R.11.4.1.1.4 

below on “Assessment based on estimation models (QSAR, SAR)”, which describes QSARs 
appropriate for specific P/vP screening.  

  Use of pure culture data – The data derived from studies with pure culture(s), single 

species or mixture of species, cannot be used on their own within persistence assessment 
but should be considered as part of a Weight-of-Evidence approach.  

  Use of information on anaerobic degradation – The data derived from anaerobic 

degradation studies cannot be used on their own within persistence assessment but should 
be considered as a part of a Weight-of-Evidence approach.  

  Use of information on any other degradation studies – The data derived from 

degradation studies other than those described above cannot be used on their own within 
persistence assessment but should be considered as a part of a Weight-of-Evidence 
approach (e.g. OECD TG 314).  

  Abiotic degradation – Concern for P/vP screening cannot be removed by significant and 
substantial loss of the parent substance by hydrolysis alone. Careful consideration of the 
hydrolysis test is required (for example mass balance is needed to address concerns for 
losses by volatilisation or absorption to glassware). Rapid hydrolysis also needs to be shown 
across all environmentally relevant pH. Additional evidence is also needed to examine 
whether the fate properties of the substance would cause attenuation of the hydrolysis rate 
in sediment or soil, or whether DOC would similarly affect the rate in aquatic media such as 
river or sea water. Additional studies, e.g. examining the influence of dissolved organic 
carbon / adsorption processes on hydrolysis rates, may be necessary for this. The 
degradation half-lives obtained in a hydrolysis test cannot be compared to the persistence 
criteria of Annex XIII. As abiotic degradation is primary degradation, careful consideration 
will need to be given to the potential formation of stable degradation products with 
PBT/vPvB properties. Hydrolysis products should be identified in accordance with the 
recommendations contained in the test guidelines (e.g. OECD TG 111). 

  Use of other abiotic data – Data derived from other abiotic studies (e.g. 

photodegradation, oxidation, reduction) cannot be used on their own within persistence 
assessment, but may be used as part of a Weight-of-Evidence approach. Due to the large 
variation in the light available in different environmental compartments, the use of 
photolysis data is not generally recognised for persistence assessment. This is discussed in 
more details in the Chapter R.7b of the Guidance on IR&CSA. 

  Field studies – Data derived from field studies (e.g. mesocosm) may be used as part of a 

Weight-of-Evidence approach. This is discussed in more detail in Section R.11.4.1.1.5 below 
named “Field studies for persistence”. 

  Monitoring data – If monitoring data, used as part of a Weight-of-Evidence analysis, show 
that a substance is present in remote areas (i.e. long distance from populated areas and 
known point sources, e.g. Arctic sea or sub-Arctic/Arctic lakes in Scandinavia), it may be 
possible to conclude a substance as P or vP. Monitoring data obtained in areas closer to the 

  
 
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sources may also be useful for P/vP assessment and can be used as one line of evidence for 
supporting the conclusions(in both directions: P/vP or not P/vP). Use of monitoring data in 
P/vP-assessment encompasses several uncertainties and conclusions should be drawn on 
the basis of monitoring data only when there is sufficient understanding of the substance 
distribution and transport behaviour and under the condition that the uncertainties in the 
monitoring data presented are adequately addressed. The lack of detection of a substance 
in monitoring data should be considered carefully as it does not necessarily mean that a 
substance is not persistent (e.g. shortcomings in analytical methods may affect monitoring 
of substances in the environment). If monitoring data show that the substance levels in 
environmental media or biota are rising, the reasons for such a time trend should be 
assessed very carefully against the information on the time trends of volumes, uses and 
releases. Where monitoring data clearly indicate that the substance fulfils the vP-criterion 
or, depending on the case, that the P criterion is fulfilled in addition to other supporting 
information (and without any conflicting data), it may not be necessary to generate 
simulation degradation data. In the latter case, conclusions on the fulfilment of the P/vP 
criteria may be drawn based on the monitoring data, the information on the substance 
distribution/transport behaviour, in addition to other supporting information used as part of 
a Weight-of-Evidence analysis.  

4.  Further information needed to conclude on P/vP – Testing strategy to be 

developed as described below  

If further degradation testing is needed based on steps 1 to 3 of the ITS, a testing strategy on 
persistence should be developed. The testing strategy should aim to conclude on persistence 
with the least possible efforts in testing and at the same time cover the assessment of 
persistence in all environmental compartments (marine water, fresh or estuarine water, 
marine sediment, fresh or estuarine sediment and soil).  

4.1. 

Identification of any specific environmental compartment(s) of concern  

This paragraph describes the part of the ITS where the need for further testing has been 
identified and there is a need to make a decision on the test compartment(s).  

In general, it is recommended to start testing with the OECD TG 309 if it is technically feasible. 
However, if there is evidence that the OECD TG 309 does not provide means to reflect the 
persistence of the substance in the environment, other environmental compartments may be 
considered as first test environment. For example, in case a P/vP criterion is expected to be 
exceeded in (a) compartment(s) other than water or if the substance hydrolyses fast in 
environmentally relevant conditions, this should be taken into account in the testing strategy. 
If, based on the fate and release(s) of the substance, it is considered that water compartment 
is not a relevant environmental compartment at all, this should also be taken into account in 
the testing strategy. This is not expected to be the case for most of the potential P/vP 
substances, as explained in the section below. If the OECD TG 309 is not technically feasible, 
selection of the most relevant environmental compartment to test first should be justified 
(Step 4.2).   

OECD TG 309 should be preferred for the following reasons:   

  Firstly the aquatic compartment is considered to be a relevant environmental compartment 
due to the large global volume of water: by default water compartment receives significant 
amount of emissions directly or indirectly, and transports/distributes the substance through 
e.g. deposition and run-off (unless based on the fate and release(s) of the substance, it is 
considered that the water compartment is not a relevant environmental compartment at 
all). Once entering water, a substance may stay there for very long time and be spread over 
long distances before it reaches other environmental compartments (via environmental 
transport, partitioning and distribution processes) such as sediments or (via air) the soil 
compartment;  

 
 
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45 

  Particularly for lower water solubility substances which tend to be adsorptive, the OECD TG 
309 (with a default concentration of suspended solids of 15 mgdw/L, see section below on 
OECD TG 309) minimizes potential NER formation. If NER is formed at significant levels in 
the OECD TGs 307 and 308 studies, this can be difficult to interpret and compare with  
degradation half-lives criteria of Annex XIII to the REACH Regulation; 

  OECD TG 309 is conducted under aerobic condition (there is no “anaerobic” option). This is 

considered as a relevant test condition as P assessment should first consider aerobic 
degradation. In general, a test using exclusively anaerobic conditions is not required as a 
first step. For further information, see Section R.11.4.1.1.3 below, under “aerobic and 
anaerobic conditions”. 

It should be noted that, at this step, considerations of complete absence of uses/releases, and 
thereby exclusion of the need to test a certain environmental compartment, is not discussed. 
Further information on exposure-based exclusion of testing may be found in this Guidance 
under Section R.11.3). 

Information on degradation and from environmental monitoring data, emissions estimated in 
the CSR, distribution modelling data (e.g. Mackay Level III) and physico-chemical information 
should be assessed to determine whether there is an environmental compartment (pelagic 
surface water, pelagic marine, sediment, marine sediment or soil) of specific concern for 
persistence. The driving factor for the assessment is that a conclusion needs to be derived for 
all three (five) environmental compartments with the least possible testing efforts. The specific 
concern for persistence is normally present for the environmental compartment for which the 
P/vP criteria are most likely to be exceeded or where the degradation half-life is the closest to 
the criteria (if the criteria are not exceeded). Consideration of the environmental 
compartment(s) of most relevant exposure may also play a role in the identification of the 
specific environmental compartment for testing. Absence of exposure in a specific 
environmental compartment may, in exceptional cases, be acceptable to exclude certain 
compartments from the P/vP assessment.  

The following pieces of evidences may help in the identification of the potential environmental 
compartment of specific concern: 

  Any available information suggests that (abiotic and bio-) degradation rates/half-lives are 

expected to meet the P/vP criteria for a specific environmental compartment;  

  Environmental monitoring data suggesting persistence is likely in a particular environmental 

compartment for a substance; 

  Direct discharge to an environmental compartment is expected to occur; 

  The life-cycle is well characterised and the environmental emission and exposure 

assessment (including environmental fate, modelling and/or monitoring data) show that a 
specific environmental compartment is exposed. 

If any environmental compartment other than surface water is chosen for simulation 
degradation testing, a justification should be provided (see step 4.2 below).   

4.2.  Specify/justify the test compartment  

As explained above (step 4.1) the OECD TG 309 is the preferred test. If another test is 
selected for further testing, this should be justified. Possible reasons are listed below: 

  OECD TG 309 is typically performed at concentrations between 1 and 100 μg/L and 

preferably in the range of <1-10 μg/L (to ensure that biodegradation follows first order 
kinetics). 

  Generally, when water solubility of a substance is very low (typically below 1 μg/L), testing 
on sediment (OECD TG 308) and/or soil (OECD TG 307) may be needed instead of a pelagic 
test (OECD TG 309). The detection limit(s) of analytical methods of quantification needs to 
be taken into account when designing the test setup.  

  
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  Aquatic testing is not technically feasible. Technically feasible means that it has been 

impossible, with allocation of reasonable efforts, to develop suitable analytical methods and 
other test procedures to accomplish testing in surface water so that reliable results can be 
generated. Appropriate analytical methods should have a suitable sensitivity and be able to 
detect relevant changes in concentration (including that of metabolites). 

  Indications from available data (e.g. literature) suggest that persistence is likely to occur in 

a different environmental compartment (i.e. in soil or sediment), including evidence of 
direct or indirect exposure.  

  The substance is a multi-constituent / UVCB which affects the test substance concentration 
at which the test can be performed (i.e. due to different multiple water solubilities of the 
individual constituents). 

Please see also further considerations on the simulation testing strategy in Section R.11.4.1.1 
below. 

5.  Is there further information needed to conclude on persistence for the tested 

environmental compartment? 

The information obtained from the performed tests should be assessed and the results 
compared with the REACH Annex XIII criteria for P/vP:  

  If the substance or its degradation products are concluded to be persistent or very 

persistent, there is no need for further testing for persistence assessment.  

  If the substance and its degradation products are concluded to be non-persistent in the 
tested environmental compartment it should be verified that there is no concern in 
remaining compartments (see step 6).    

6.  Remaining concern in untested environmental compartments 

It should be considered whether the available information is adequate to conclude persistence 
assessment for all or some of the remaining environmental compartments for which there are 
no testing data. If it can be concluded that the P and/or vP criteria are fulfilled in one 
environmental compartment, then no further information is needed for the other 
compartments (see above step 5). 

In general, results of a single simulation degradation study cannot be directly extrapolated to 
other environmental compartments. However, the results could be sufficient to conclude on 
persistence in other compartments, provided that the environmental media in environmentally 
realistic conditions have been selected for the study and the interpretation of the 
results/bridging is backed by proper justifications. Availability or generation of multiple 
simulation test data may allow more Weight-of-Evidence based conclusions to be drawn by 
expert judgement regarding environmental degradation half-lives for one or more 
environmental compartments. At this point of the flow chart, a decision on whether the data 
cover one, two or all five environmental compartments should be made on a case-by-case 
basis.  

It should be highlighted that the requirement is to draw a conclusion for all three (five) 
environmental compartments (see REACH Annex I, Section 3.0.2). If for the first tested 
compartment a conclusion “not P” could be derived, but the available data are not sufficient for 
drawing conclusions in (an)other compartment(s), further data generation is necessary to 
complete the assessment for the compartments for which a conclusion could not be drawn. 
Exclusion of (a) certain environmental compartment(s) from the P/vP assessment based on 
absence of exposure may be acceptable only in very exceptional cases and upon justification. 
A justification of absence of exposure in (a) certain environmental compartment(s) is different 
from a justification for the purpose of normal quantitative risk assessment, because for 
(potential) PBT/vPvB substances, and hence for the PBT/vPvB assessment, distribution over a 
very long timespan would need to be considered as well. 

 
 
 
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7.  Evaluation versus the P and vP criteria  

47 

The half-life(lives) obtained from the simulation data is evaluated against the criteria of Annex 
XIII to the REACH Regulation for the three (five) environmental compartments to determine 
whether the P or vP criteria are met or not. Before finally concluding  that a substance is “not 
P" or "not vP”, it should be carefully examined if there exists conflicting evidence from 
monitoring data, either from national monitoring programmes of Member States (e.g. Swedish 
national monitoring data collection17), from European monitoring programmes (e.g. NORMAN 
Network18) or internationally acknowledged organisations (such as OSPAR or the Danube 
Convention). For example, findings of significant concentrations of the substance under 
consideration in remote and pristine environments such as the Arctic sea or Alpine lakes need 
to be scrutinized carefully as there may be evidence of high persistence. Also, significant 
concentrations of the substance in higher levels of the food chain in unpolluted areas may 
indicate high persistence (beside a potential to bioaccumulate). If such evidence indicates that 
the substance may be persistent, further investigations are required. 

R.11.4.1.1.2 

Introduction to persistence assessment  

When assessing data concerning the persistence of a substance and, if necessary, determining 
the next steps of the assessment, there are a number of stages to go through. The first part of 
the assessment should address the extent to which available data enable an unequivocal 
assessment to be made. These data may comprise simple screening biodegradation tests (e.g. 
OECD TG 301C ready biodegradability MITI I test) or complex, high-tier simulation tests (e.g. 
OECD TG 308 aerobic and anaerobic transformation test in aquatic sediment systems). 
At this stage, it is only necessary to assess the strength of the data in one direction or 
another. Thus, for example, when an OECD TG 301 study indicates that the substance is 
readily biodegradable the decision that a substance is not P could be taken. Conversely, if a 
simulation test indicates for example a half-life of over 200 days, this might be sufficient to 
decide that the substance meets the P and vP criteria. However, as described in Section R.7.9 
of Chapter R.7b of the Guidance on IR&CSA, a negative result in a test for ready 
biodegradability does not necessarily mean that the substance will not be degraded under 
relevant environmental conditions and persist in the environment. Indeed, there are several 
references reporting that ready biodegradation tests underestimate the potential for 
degradation in real environmental conditions (Guhl and Steber, 2006). A failed ready 
biodegradability test may indicate the need for further testing under less stringent test 
conditions (e.g. enhanced biodegradation tests, simulation tests…). In addition, all relevant 
degradation pathways (biotic, abiotic, aerobic, anaerobic conditions) need to be considered 
with regard to the relevant route of exposure before concluding on persistence.  

Often, biodegradation data are not so clear-cut, and frequently they are different and/or 
contradictory. Therefore careful consideration is needed before a decision is taken in order to 
avoid a false negative or false positive conclusion. The strategy outlined in this section is a 
recommendation and is not intended to be an explicit prescriptive description of the sequence 
of steps to be taken. Ultimately the actual route taken will depend upon the data available and 
the physico-chemical properties of the substance being assessed. As a minimum, and where 
possible and technically feasible, information on vapour pressure, water solubility, 
octanol/water partition coefficient (Kow), other partition coefficients (such as the octanol-air 
partition coefficient (Koa) and organic carbon normalised adsorption coefficient (Koc)), basic 
dissociation behaviour (if relevant), surface active properties (if relevant) and Henry's law 
constant must be available. The impact of these data on the test design and data 
interpretation should be considered. 

17 http://dvsb.ivl.se/dvss/DataSelect.aspx  

18 http://www.norman-network.net/  

  
 
                                                 
 
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With regard to persistence, it is insufficient to consider removal alone where this may simply 
represent the transfer of a substance from one environmental compartment to another (e.g. 
from the water phase to the sediment). Degradation may be biotic and/or abiotic (e.g. 
hydrolysis) and result in complete mineralisation, or simply in the transformation of the parent 
substance (primary degradation). Where only primary degradation is observed, it is necessary 
to identify the degradation products and to assess whether they possess PBT/vPvB properties. 
In addition to the substance intrinsic properties, its transformation and/or degradation is 
dependent on the surrounding environment.  

The following sections give guidance on how to address data from biodegradation studies, 
abiotic degradation studies and information available from estimation models (QSARs/SARs). A 
subsequent section addresses information generation and particularly how to choose the 
correct compartment for further testing. As mentioned above, the sequence in which the 
subjects of these sections are addressed will depend upon the data available. Furthermore, 
most of the information reported in this guidance is further developed under the endpoint-
specific guidance on degradation, which should also be consulted (see Section R.7.9 in Chapter 
R.7b of the Guidance on IR&CSA). 

In case only screening information is available, screening threshold values listed in Table 
R.11—4 can be used to judge whether an ultimate conclusion on the persistence of a 
substance can be made or whether further information is needed. It should be noted that 
screening criteria can only be applied as provided. The triggers were originally derived for 
drawing only those conclusions indicated in Table R.11—4 and are not recommended to be 
used to draw other conclusions. (However, it should be noted that these criteria are indicative 
and the assessor should consider the relevance of any other indications before drawing a 
conclusion.)  

 
Chapter R.11: PBT/vPvB assessment 

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49 

Table R.11—4: Screening information for P and vP. 

Screening information 

Conclusion 

Persistence 

Biowin 2 (non-linear model 
prediction) and Biowin 3 
(ultimate biodegradation time) 

Does not biodegrade fast (probability < 0.5)* 
and ultimate biodegradation timeframe 
prediction: ≥ months (value < 2.25 (to 
2.75)**) 

Potentially P or vP 

or 

or 

Biowin 6 (MITI non-linear model 
prediction) and Biowin 3 
(ultimate biodegradation time) 

Does not biodegrade fast (probability < 0.5)* 
and ultimate biodegradation timeframe 
prediction: ≥ months (value < 2.25 (to 
2.75)**) 

or  

other models * 

or 

Model specific values 

Ready biodegradability test 
(including modifications allowed 
in the respective TGs) 

≥70% biodegradation measured as DOC 
removal (OECD TGs 301A, 301E and 306) or 
≥60% biodegradation measured as  ThCo2 
(OECD TG 301B) or ThOD (OECD TGs 301C, 
301D, 301F, 306 and 310)*** 

<70% biodegradation measured as DOC 
removal (OECD TGs 301A, 301E and 306) or 
<60% biodegradation measured as  ThCo2 
(OECD TG 301 B) or ThOD (OECD TGs 301C, 
301D, 301F,306 and 310) 

Potentially P or vP 

Potentially P or vP 

Not P and not vP 

Potentially P or vP 

Enhanced  screening tests**** 

biodegradable 

not biodegradable**** 

Not P and not vP 

Potentially P or vP 

Specified tests on inherent 
biodegradability: 

- Zahn-Wellens (OECD TG 302B)  ≥70 % mineralisation (DOC removal) within 7 

Not P and not vP 

- MITI II test (OECD TG 302C) 

d; log phase no longer than 3d; removal 
before degradation occurs below 15%; no 
pre-adapted inoculum 

Any other result***** 

≥70% mineralisation (O2 uptake) within 14 
days; log phase no longer than 3d; no pre-
adapted inoculum 

Potentially P or vP 

Not P and not vP 

Any other result***** 

Potentially P or vP 

* The probability is low that it biodegrades fast (see Section R.7.9.4.1 in Chapter R.7b of the Guidance on 
IR&CSA). Other models are described in Section R.7.9.3.1 of Chapter R.7b of the Guidance on IR&CSA 
and in this section below. 
** For substances fulfilling this but BIOWIN 3 indicates a value between 2.25 and 2.75 more degradation 
relevant information is generally warranted.   
*** These pass levels have to be reached within the 28-day period of the test. The conclusions on the P 
or vP properties can be based on these pass levels only (not necessarily achieved within the 10-d 
window) for monoconstituent substances. For multi-constituents substances and UVCBs these data have 
to be used with care as detailed in Section R.11.4.2.2 of this Guidance. 
**** see Sections R.7.9.4 and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA. Expert judgement 
and or use of Weight of Evidence also employing other information may be required to reach a conclusion 
(i.e. concerning « biodegradable/ not biodegradable »)  
***** See section below for concluding ultimately on persistence in particular cases (in particular “Tests 
on inherent biodegradation”). 

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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In the ITS for persistence assessment (Figure R.11—3), the types of simulation degradation 
tests that should be considered is indicated. The information in Table R.11—5 below presents 
the criteria for the assessment of persistence (P/vP) and identifies relevant test systems for 
determining environmental degradation half-lives. 

Table R.11—5: Persistence (P/vP) criteria according to Annex XIII to the REACH 
Regulation and related simulation tests. 

According to REACH, Annex 
XIII, a substance fulfils the P 
criterion when: 

According to REACH, Annex 
XIII, a substance fulfils the 
vP criterion when: 

Biodegradation simulation 
tests from which relevant 
data may be obtained include: 

The degradation half-life in 
marine water is higher than 60 
days, or 

The degradation half-life in 
marine, fresh- or estuarine water 
is higher than 60 days, or 

OECD TG 309: Simulation test – 
aerobic mineralisation in surface 
water 

The degradation half-life in fresh- 
or estuarine water is higher than 
40 days, or 

The degradation half-life in 
marine sediment is higher than 
180 days, or 

The degradation half-life in fresh- 
or estuarine water sediment is 
higher than 120 days, or 

The degradation half-life in 
marine, fresh- or estuarine 
sediment is higher than 180 
days, or 

OECD TG 308: Aerobic and 
anaerobic transformation in 
aquatic sediment systems 

The degradation half-life in soil is 
higher than 120 days 

The degradation half-life in soil is 
higher than 180 days 

OECD TG 307: Aerobic and 
anaerobic transformation in soil 

R.11.4.1.1.3 

Test data on biodegradation  

In principle, there are three types of tests that measure biological degradation: 

1.  Tests on ready biodegradation (e.g. OECD TG 301 series, OECD TG 306, OECD TG 310 

and enhanced ready test) 

2.  Tests on inherent biodegradation (OECD TG 302 series) 
3.  Tests on simulation degradation and transformation (surface water, sediment or soil)  

Tests on ready and inherent biodegradability contribute information at a screening level whilst 
simulation tests are adequate to assess degradation kinetics, degradation half-lives, 
information about mineralisation, non-extractable residues (NERs) and degradation products 
(metabolites, extracted residues).  

In order to select the appropriate test type, careful consideration of the physico-chemical 
properties and the environmental behaviour of a substance is required, which is discussed later 
on in this section.  

For further information on test descriptions refer to the degradation guidance (see Sections 
R.7.9.3 and R.7.9.4 in Chapter R.7b of the Guidance on IR&CSA). 

Tests on ready biodegradation 

Tests on ready biodegradation are described in OECD TG 301 A-F and OECD TG 310. 
Biodegradability in Seawater test (OECD TG 306) can also be used to describe the ready 
biodegradability in sea water. Degradation is followed by determination of parameters such as 
dissolved organic carbon (DOC), CO2 production or oxygen uptake. The parameter measures 
the mineralisation and the pass level is set to 60% (ThOD or ThCO2) or 70% for DOC removal 
assuming that the yield for growth of the microbial biomass is 30-40%. In the context of ready 
biodegradability,  test substance-specific analysis can also be used and primary degradation 
and formation of any metabolites can be assessed. Measurement of primary degradation is 
however a requirement only in the MITI I test (OECD TG 301C). 

 
 
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Due to the fact that the test methodology for the screening tests on ready biodegradability is 
stringent, a negative result does not necessarily mean that the substance will not be degraded 
relatively fast under environmental conditions. A lack of biodegradability may for example be 
caused by toxicity of the substance towards microorganisms due to the very high 
concentration employed in ready biodegradability tests compared with lower, environmentally 
relevant concentrations. Another reason for negative outcomes in ready biodegradability tests 
can be low water solubility of the test substance. A low solubility could constitute the rate 
limiting step for degradation at the environmentally unrealistic high test substance 
concentrations and not the intrinsic recalcitrance towards microbial transformation. ISO 
method 10634 and Annex III of OECD TG 301 also describe options to address poorly soluble 
substances. 

Given the time, costs and, in some cases, practical difficulties associated with conducting and 
interpreting a simulation degradation test, an enhanced ready biodegradation test design 
offers a cost-effective intermediate screening test in those cases where persistence in the 
environment is not expected although (a) standard ready biodegradation test(s) give(s) the 
result “not readily biodegradable”. If sufficient degradation is shown in an enhanced 
biodegradation screening test, i.e. the pass level as given in the test guidelines for ready 
biodegradation is reached, the substance can be considered as “not P”. It should be noted that, 
in this case, the 10-day window indicated in the corresponding test guideline does not need to 
be fulfilled. More information on modifications of ready biodegradability tests with respect to 
such enhanced screening tests is contained in Sections R.7.9.4 and R.7.9.5 of Chapter R.7b of 
the Guidance on IR&CSA. Please note that these tests are referred to as “enhanced 
biodegradation screening tests”. 

Tests on inherent biodegradation 

Tests on inherent biodegradability are useful to give an indication of biological degradability on 
a screening level. Inherent tests are similar to ready biodegradability tests as they usually 
measure sum parameters and are conducted with a high test substance concentration and an 
even higher microbial concentration. In general, they use more favourable, if not optimal, 
conditions than ready biodegradability tests (e.g. with increased biomass to test substance 
ratio and allowing pre-adaptation of the microbial inoculum), and are hence designed to show 
whether a potential for degradation exists. 

Due to the more favourable conditions of an inherent test, results need to meet specific criteria 
(specified in Table R.11—4 above and Section R.7.9.4.1 “Data on degradation/biodegradation” 
of Chapter R.7b of the Guidance on IR&CSA) in order for a substance to be considered as not 
P/vP. 

Lack of degradation (<20% degradation) in an inherent biodegradability test equivalent to the 
OECD TG 302 series may provide sufficient information to confirm that the P-criteria are 
fulfilled without the need for further simulation testing for the purpose of PBT/vPvB 
assessment. Additionally, in specific cases it may be possible to conclude that the vP-criteria 
are fulfilled with this result if there is additional specific information supporting it (e.g., specific 
stability of the chemical bonds). The tests provide optimum conditions to stimulate adaptation 
of the micro-organisms thus increasing the biodegradation potential, compared to natural 
environments. A lack of degradation therefore provides evidence that degradation in the 
environment would be slow. Care should be taken in the interpretation of such tests, however, 
since, for example, a very low water solubility of a test substance may reduce the availability 
of the substance in the test medium. These issues are discussed in more detail in Sections 
R.7.9.4 and R.7.9.5 of Chapter R.7b of the Guidance on IR&CSA. 

Tests on simulation of biodegradation 

In principle, degradation simulation studies performed in appropriate environmental media and 
at environmentally realistic conditions are the only tests that can provide a definitive 
degradation half-life that can be compared directly to the persistence criteria as defined in 

  
 
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REACH Annex XIII. Such tests allow both biotic and abiotic degradation processes to operate. 
The simulation tests as described in OECD TGs 307, 308 and 309 address the fate and 
behaviour of a substance as it may be expected in the environment including information 
about partitioning in the test system, primary or complete degradation, adsorption behaviour 
and route(s) of degradation (degradation products). The endpoints that need to be addressed 
are primary or ultimate degradation rate and degradation half-lives (DegT50) or dissipation 
half-lives (DT50) for the compartments included in the test system as well as the route of 
degradation, metabolites and non-extractable residues. In addition, a mass balance is included 
in these tests and therefore possible losses from the test system during the test period can 
also be quantified. A simulation study should be performed using a radio-labelled molecule, 
whenever feasible. 

In order to evaluate the outcome of a simulation test, the reporting of the results should follow 
the respective test guideline(s). 

Tests should report the degradation rate (or degradation half-life) in each medium determined 
through mineralisation, e.g. volatile 14C-CO2, and/or direct substance analysis. An option, if 
measuring mineralisation, is to measure the mineralisation rate for the whole system: if the 
mineralisation half-life for the whole system is below the respective half-life –value of P/vP 
criteria, it has been shown that the substance is not persistent in the tested environmental 
compartment (surface water, sediment or soil). However, investigation of degradation 
pathways/transformation products would be needed since it cannot be excluded that a second 
transformation route forms a persistent metabolite in concentrations relevant for the P 
assessment. When the mineralisation half-life for the whole system is not below the P criterion, 
a full mass balance of the substance and any degradation products/metabolites should be 
determined (or justification provided if this is not technically feasible), and a determination of 
the level of non-extractable residues should be included. In general, determination of non-
extractable residues is recommended in soil and water-sediment studies (OECD TG 307, OECD 
TG 308 and Kästner et al., 2014). Determination of non-extractable residues is also 
recommended in surface water simulation degradation studies. In all cases, the extraction 
method and the choice of extraction solvents should be justified. Where primary degradation is 
observed, the identity of possible relevant metabolites must also be determined and/or 
evaluated as regards their possible PBT/vPvB-properties. Where only degradation of the parent 
substance is monitored, this does not address all the concerns and further assessment of the 
degradation products may be required in order to complete the PBT/vPvB assessment (see 
Sections R.7.9.4 and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA). 

It should be noted that for direct comparison to the P/vP criteria only estimates of degradation 
half-life are appropriate. When the kinetics of transformation are first-order, single-first order 
(SFO) kinetic models can be used for predicting degradation half-lives. The predicted 
degradation half-lives should be used for comparison with the P/vP criteria. Use of bi-phasic 
kinetic models is recommended to be limited to cases where clear deviations from first-order 
kinetics occur. When the kinetics of transformation are bi-phasic, the best-fit model (FOMC, 
DFOP, HS) should be selected and used for predicting a DT50. The DT50 predicted from the 
best-fit bi-phasic model should be used for comparison with the P/vP criteria. When applicable 
(DFOP or HS), the DT50 predicted from the slow phase should be preferred and used for 
comparison with the P/vP criteria. In case other DT50 are used, a justification should be 
provided with adequate and reliable documentation of the applied method.  

Further information on degradation models can be found in the Generic Guidance Document 
for Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on 
Pesticides in EU Registration (FOCUS, 2014). It is recommended to consult that guidance 
document for in-depth analysis of simulation degradation test results.  

 
 
 
  
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Considerations for simulation testing strategy  

Annex IX to the REACH Regulation lists three simulation degradation tests as standard 
endpoints for the CSA (which, according to Annex I to the REACH Regulation, includes the 
quantitative risk assessment and the PBT/vPvB assessment).  

The P/vP assessment should cover all three (five) environmental compartments (water, marine 
water, sediment, marine sediment, soil). However, a substance can already be concluded as P 
or vP if the criteria are fulfilled for one compartment only. For the purpose of reducing efforts 
of testing, the test should be selected in such a way that it reflects the worst case of 
persistence potential (for which the expected degradation half-life is the closest to the 
corresponding criterion). This would also ideally be the environmental compartment with the 
best possibility to use the results for concluding the P/vP-assessment (as being “worst case”).    

The influence of the relevant environmental compartment(s) in terms of exposure potential 
based on fate properties, the identified uses and release patterns to the order of testing also 
need to be considered. In some cases, it may be necessary, and hence acceptable, to choose 
an environmental compartment for simulation degradation testing other than the one normally 
considered as the first preference (see discussion below).  

A flow diagram for considering the appropriate environmental compartment(s) for simulation 
degradation testing is illustrated in the ITS described in Figure R.11—3. 

The further elements to be considered when choosing the environmental compartment for 
testing are described in the context of the ITS (Figure R.11—3). 

Before testing, the simulation test(s) that is(are) the most appropriate for addressing 
degradation should be identified. This is further discussed below.  

Simulation studies on ultimate degradation in surface water are warranted unless the 
substance is highly insoluble in water. If a substance is highly insoluble in water it may not be 
technically possible to conduct a simulation study that provides reliable results, and at very low 
concentrations technical issues may make it very difficult to establish a reliable degradation 
curve in the study. Therefore, depending on the substance physico-chemical properties and the 
availability of good quality analytical methods for identification and quantification, it may not 
be possible to conduct this study if the water solubility of the substance is very low (typically 
<1 µg/L). The surface water transformation test (OECD TG 309) recommends using a test 
substance concentration for the kinetic part of the study in a range which is environmentally 
realistic, i.e. in a range of “less than 1 to 100 µg/L”. The pathways part of the study may be 
employed at a higher test substance concentration to ease the analytical identification and 
characterisation of the metabolites. Further considerations on the OECD TG 309 study are 
provided below. 

Testing in the aquatic compartment (OECD TG 309) is the preferred first step when there is a 
need for further information on persistence in the environment, considering the following 
reasons:  

  Firstly, the aquatic compartment is considered to be a relevant environmental 

compartment for persistence assessment because the criteria for B/vB and T are mainly 
based on tests performed in this compartment. In addition, by default, water compartment 
receives a significant amount of emissions, directly or indirectly, and transports/distributes 
the substance through e.g. deposition and run-off (unless evidence from substance 
emission data suggests otherwise). Once entering water, a substance may reside there for 
very long time and be spread over long distances before it reaches other environmental 
compartments (via environmental transport, partitioning and distribution processes) such 
as sediments or (via air) the soil compartment. 

  The OECD TG 309 minimises potential NER formation. If NER is formed at significant levels 
in OECD TGs 307 and 308 tests, this can be difficult to interpret and compare with the 
degradation half-life criteria of Annex XIII to the REACH Regulation. 

  
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Reasons to deviate from this general approach can be that: 

  The substance is a multi-constituent / UVCB substance, which affects the concentration at 

which the test can be performed (due to different multiple water solubilities of the 
individual components);  

 

Indications from available  data (e.g. literature) suggest that persistence is likely to occur 
in a different environmental compartment (i.e. in soil or sediment), including evidence of 
direct or indirect emission; 

  Aquatic testing is not technically feasible, i.e. it has been impossible, with allocation of 
reasonable efforts, to develop suitable analytical methods and other test procedures to 
accomplish testing in surface water so that reliable results can be generated. This may be 
the case in particular if the water solubility of the test substance is very low. Appropriate 
analytical methods should have suitable sensitivity to detect relevant changes in 
concentration (including metabolites). 

  OECD TG 309 should be performed at concentrations between 1 and 100 μg/L and 

preferably in the range of <1-10 μg/L (to ensure that biodegradation follows first order 
kinetics). Generally, when water solubility of a substance is very low (typically <1 μg/L), 
testing on sediment and/or soil will be preferred, if aquatic simulation degradation testing 
is not technically feasible due to analytical limitations and low solubility of the test 
substance. 

Soil/sediment simulation degradation testing may be warranted as a first test in the above-
listed cases. In addition, as described in the ITS (Figure R.11—3), the soil and sediment 
degradation simulation tests may be needed when results from simulation tests in water do 
not exceed the P/vP criteria but there are indications that the substance or its degradation 
products could persist in soil and sediment, meeting the respective P criteria.  

Before performing a soil or a sediment simulation degradation test, it is worth noting that for 
the purpose of quantitative risk assessment and for adsorptive substances, a simulation test in 
soil (OECD TG 307) could be more relevant than a simulation test in sediment (OECD TG 
308)19. Degradation rates/half-lives from simulation tests in soil can be used instead of generic 
values for the assessment of PECsoil. While degradation rates/half-lives from simulation tests in 
sediment can be taken into account for the calculation of the PECregional, in practice this would 
have only a negligible influence on risk assessment. 

Once the appropriate simulation test(s) have been identified and conducted, data need to be 
interpreted to determine environmental degradation half-lives. A prerequisite for data 
interpretation is that exhaustive extraction methods are used to ensure that suitable data are 
generated. Guidance on how to conduct the test and interpret data from a simulation test is 
available in the present Guidance document and in Section R.7.9.4 of Chapter R.7b of the 
Guidance on IR&CSA. 

OECD TG 309 

OECD TG 309 should be performed at concentrations between 1 and 100 µg/L and preferably 
in the range of <1-10 μg/L (to ensure that biodegradation follows first order kinetics). 
However, for low solubility substances, even if their water solubility is within this range, it is 
acknowledged that the feasibility of the test depends, inter alia, on the possibility to develop 

19 Removal of the substance during the WWTP process may be taken into account when 
considering the emissions through WWTP in relation to the relevance of the simulation test 
compartment (e.g. incineration of the sludge and removal or degradation during the water 
treatment process). 

 
                                                 
 
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with reasonable efforts appropriate analytical methods with suitable sensitivity to detect 
relevant changes in concentration (including metabolites). 

OECD TG 309 uses as a default one matrix sample, which is in contrast to the soil (4 soils) and 
sediment (2 sediments) simulation studies. Nothing prevents registrants from employing or 
authorities from requesting simulation degradation testing in more than one surface water. It 
is generally recommended to consider performing the test with more than one water source.  

In OECD TG 309, there are options to perform the test as a ‘pelagic test’ or as a ‘suspended 
sediment test’. In both cases, the coarse particles are removed from the water sample, for 
example by filtration through a filter with 100 µm mesh size or with a coarse paper filter, or by 
sedimentation. For the ’suspended sediment test’, surface sediment is added afterwards to 
obtain a suspension. 

For the PBT/vPvB assessment, the amount of suspended matter in the pelagic test should be 
representative of the level of suspended solids in EU surface water. For large rivers, the 
concentration of suspended matter (SPM) is reasonably constant and an EU default of 
15 mgdw/L has been proposed, e.g. for the implementation of the Water Framework Directive 
(European Communities, 2011) or in EUSES. For marine waters, a default SPM concentration 
of 3 mgdw/L has been proposed for the Water Framework Directive. Similarly an SPM 
concentration of 5 mgdw/L has been implemented in EUSES for marine waters. For REACH, 
using natural surface water containing between 10 and 20 mgdw/L SPM for simulation tests in 
freshwater and ca. 5 mgdw/L for simulation tests in marine water is considered acceptable. 
Further details are available in Section R.7.9.4.1 of Chapter R.7b of the Guidance on IR&CSA. 

Even if more than one water source is used in the assessment, it is recommended that the 
amount of suspended solids still reflects realistic concentrations for the EU surface waters (e.g. 
samples from different water bodies or to reflect seasonal variations in the concentration of the 
suspended solids). When the test concentration is well under the water solubility limit of the 
substance, one might consider testing several water sources instead of testing two 
concentrations of the test substance. In any case, a reference substance should be used to 
demonstrate the viability of the system.  

According to the OECD TG the ’suspended sediment test’ can be used to simulate surface 
water free of coarse particles or turbid surface water (which might exist near the water-
sediment interface). Ingerslev and Nyholm (2000) further indicate that conducting the tests 
with added suspended sediment significantly enhance the biodegradability of some of the test 
substances. However, this test design is generally not recommended for P testing purposes as 
such highly sediment particle loaded surface water systems are not the most prevailing ones. 
There is also a high probability that increasing the suspended solids concentration will increase 
the potential for NER formation and to avoid this the pelagic test without artificially added 
particular material/sediment particles is preferred. In specific cases where there is a need to 
address the influence of the suspended solids to the abiotic degradation rate in the surface 
waters, the addition of suspended solids may be justified. If suspended solids are added, it is 
recommended that a magnetic stirrer bar should not be used as it may grind the 
solids/sediment and result in increased levels of NER. Other methods are recommended 
instead, e.g. shaking of test vessels (Shrestha et al., 2016). 

In order to minimise the formation of NERs, it is recommended that the pelagic test be 
considered first before conducting any other simulation tests. However, it is worth noting that 
even for the ‘pelagic test’, the test water will contain suspended matter onto which the test 
substance and/or its metabolites can adsorb. Therefore, the formation of NERs may be 
significant in the ‘pelagic test’ too. It is thus necessary for this test as well to quantify the 
NERs and to explain and scientifically justify the extraction procedure and solvent used (see 
also Section R.7.9.4.1. in Chapter R.7b of the Guidance on IR&CSA). 

Unless there is a specific concern for the marine compartment, for the REACH PBT assessment, 
generally the OECD TG 309 would be performed using a freshwater rather than salt water 
media. However, the degradation in marine compartment should always be considered in PBT 

  
 
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assessment. It should therefore be assessed if the information on degradation in freshwater 
may be used to extrapolate the degradation rate in marine environment.  

The OECD TG 309 proposes quite a flexible framework for designing the test. The registrants 
should provide justifications in the robust study summaries and/or test plan proposals for the 
different options taken with regard to, for example the type and characteristics of the water 
used, whether suspended sediments were added, whether shaking or stirring of the test 
vessels was used, whether the test was performed in the dark or with diffuse light. 

Please note that scientific work is on-going to develop the understanding on NER and that the 
recommendation above is based on current knowledge and experience. Registrants are advised 
to follow-up the recent and future developments in the field, e.g. via the ECHA website. The 
role of NER in P/vP assessment is discussed further in the section on “non-extractable 
residues” below. 

OECD TG 308 & TG 307 

Testing on sediment (OECD TG 308) or soil (OECD TG 307) may be needed instead of a pelagic 
test (OECD TG 309) if the latter is not technically feasible; i.e. if the water solubility of the test 
substance is very low (typically below 1 µg/L) or if it is not possible with reasonable efforts to 
develop a suitable analytical method or other test procedures for conducting the test in water. 
Besides, in some situations it can be anticipated that the simulation test in water will not be a 
worst case and that the persistence criteria will possibly be exceeded in sediment and/or soil 
but not necessarily in water. This may be the case for example for hydrolysable substances, as 
hydrolysis may be hindered by adsorption onto sediment and soil. 

Testing on sediment and/or soil may also have to be conducted in addition to the test in water, 
to demonstrate that the substance is persistent in none of the compartment relevant for the 
PBT/vPvB assessment. Testing in the sediment and/or soil compartments should be considered 
in particular if there is a specific concern for this compartment, for example, if direct or indirect 
releases to these compartments are likely, or if the substance is predicted to accumulate in 
these compartments. Koc or Kd values can be used as an indicator of whether the substance is 
likely to be of concern for the sediment and soil compartments. As a rule of thumb, substances 
with log Koc > 4 are generally regarded as highly adsorptive and likely to distribute in sediment 
and soil.  

For the PBT/vPvB assessment, a half-life in sediment should be estimated. However, from 
OECD TG 308 simulation tests, the half-lives calculated for the sediment phase and the water 
phase separately are less reliable than the half-life calculated for the total water-sediment 
system. Still, because of the low volume and depth of water relative to the volume of sediment 
and the surface of the water-sediment interface used in OECD TG 308, even moderately 
adsorptive substances will tend to rapidly partition from the water phase to the sediment 
phase. Therefore, for adsorptive substances, the half-life in the sediment can reasonably be 
estimated from the half-life for the total water-sediment system. This approach avoids the 
need to determine specific half-lives for each phase separately (Honti and Fenner, 2015)20. 
However, the parent substance may degrade to more soluble and less adsorptive degradation 
products that can be released from the sediment to the water phase. This should be taken into 
account in the assessment. 

OECD TG 308 outcome can be affected by test vessel geometry and the associated water-
sediment interface size. There is no specification of the vessel size or geometry in the test 

20 Part of LRI ECO18 – “Improved strategy to assess chemical persistence at the water-sediment 
interface” http://cefic-lri.org/projects/lri-eco18-eawag-improved-strategy-to-assess-chemical-
persistence-at-the-water-sediment-interface/  

 
 
 
                                                 
 
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guideline and so it is recommended to record the dimensions of the test vessel, and include 
this in the IUCLID robust study summary. 

Based on the OECD 308 TG it is expected that “the sediment is disturbed as little as possible” 
when introducing the test substance. Sediment spiking instead of addition of the test 
substance via water may in some cases be necessary to ensure realistic exposure of sediment 
in the test. Currently, however, deviations from the test guideline may only be justified on a 
case-by-case basis as there are no generic criteria available when spiking would be 
appropriate. If sediment spiking is undertaken, the overall half-life from such a test should be 
assumed to be the sediment half-life (unless there is significant desorption, which seems 
unlikely in the case of most PBT substances). Sediment spiking methods have been developed 
so far for the purpose of sediment toxicity testing. These are mentioned in Section R.7.8.10.1 
“Laboratory data on toxicity to sediment organisms” in Chapter R.7b of the Guidance on 
IR&CSA. However, none can currently be directly recommended for the purpose of sediment 
simulation testing but further approach development would be necessary.  

Multiple simulation test results  

A substance can be concluded to be not-P only if it can be demonstrated that it is persistent in 
none of the environmental compartments relevant for the PBT/vPvB assessment, i.e. water, 
sediment and soil. 

Generally, for substances registered under REACH, the likelihood of having more than four 
different results from the same environmental compartment is deemed to be limited. For 
determining transformation rates, OECD TGs 307, 308 and 309 recommend respectively that 
at least four different soils and two different sediments and one type of water should be 
tested.  

For the same environmental compartment, when four or less results are available, the most 
stringent result should be used with respect to the PBT assessment. 

Where more than four results are available for the same compartment, the first step is to 
assess the validity of the data and whether the different tests are equivalent (for example 
temperature, pH, organic carbon content, microbial biomass, etc). Only test results 
corresponding to equivalent test conditions can be compared. In all cases, the approach should 
be well justified and documented and should be supported by the Weight of Evidence analysis. 
This should include a discussion of outlying results. In particular, the representativeness of the 
test conditions should be carefully assessed for each test result. Particular scrutiny should be 
given if results from the tests are close to P or vP threshold.  

Aerobic and anaerobic conditions 

The following options are available in the environmental simulation test guidelines: 

  OECD TG 307 – Aerobic and Anaerobic Transformation in Soil: The test is usually 

conducted under aerobic conditions. The test can be performed also under partial or 
strict anaerobic conditions.  

  OECD TG 308 – Aerobic and Anaerobic Transformation in Aquatic Sediment Systems: 
The normally employed test includes aerobic and anaerobic sub-compartments. The 
test can be performed also under strict anaerobic conditions. 

  OECD TG 309 – Aerobic Mineralisation in Surface Water – Simulation Biodegradation 

Test; There is no “anaerobic” option. 

In the anaerobic OECD TG 307 study, the anaerobic conditions can be achieved by covering 
the soil with water, i.e. mimicking a flooded field, in the absence of oxygen (the soil is purged 

  
 
 
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with nitrogen and oxygen excluded for the test duration). A further option is a flooded soil but 
without the specific exclusion of oxygen (paddy field simulation). Anaerobic degradation in soil 
may also have influence on the results in some study cases, for example, if water covered soil 
environments are studied in the field. However, for REACH PBT/vPvB assessments, neither of 
the solely anaerobic test conditions are considered to be especially relevant scenarios for the P 
assessment in the EU. Nevertheless, if anaerobic soil data are available, they may be used as 
part of a Weight-of-Evidence approach in the P assessment. 

The OECD TG 309 is an aerobic test. There is no anaerobic option in the test guideline - this 
would effectively be stagnant water. The main discussion here therefore focuses on OECD TG 
308.  

Sediment test: 

The “aerobic” OECD TG 308 is a mixture of aerobic and anaerobic sediment. The OECD TG 
states that the “aerobic test simulates an aerobic water column over an aerobic sediment layer 
that is underlain with an anaerobic gradient”. By comparison, the anaerobic test “simulates a 
completely anaerobic water-sediment system”. 

It is not recommended to judge whether a substance has an environmental half-life exceeding 
the P and/or vP thresholds using only anaerobic simulation data. Generally it would be 
expected that an anaerobic half-life would be greater than an aerobic half-life where the main 
route of degradation is aerobic, i.e. if there is no oxygen, degradation will be hindered21. Care 
should also be taken where the anaerobic data show rapid degradation of a substance. This is 
because there is generally no immediate discharge of a substance to anaerobic sediment or 
soil. Instead, the substance will usually need to cross an aerobic zone before reaching the 
anaerobic zone. This means it is important to understand the rate degradation across that 
aerobic zone to assess the persistence22.  

Where anaerobic data are already available, these might be useful as part of a Weight of 
Evidence of whether the P or vP thresholds are met. For example the presence of oxygen may 
be less relevant if the primary degradation step is hydrolysis. 

Sediment core data might provide some indication of anaerobic degradation capacity. However 
some caution should be exercised as the initial starting concentration is rarely known. 
Therefore any derived degradation kinetics estimating a half-life will have uncertainty due to 
the assumptions required. The history of any local emissions and contamination at the sample 
site also provides useful information to help interpret the data. It is more likely that core data 
can be used in an evidence base for anaerobic degradation, as part of a broader Weight of 
Evidence in the persistence assessment. 

When new sediment simulation testing is assessed to be required for P/vP characterisation, 
metabolism route prediction23 or prior knowledge24 should be used to judge whether additional 
information will be gained from performing the anaerobic-only test. Exploring an anaerobic 
route of degradation may be useful in specific cases where a metabolite may be of concern. 
However, in general a test using exclusively anaerobic conditions is not required. For the OECD 
TG 308 sediment simulation test the “aerobic”’ test will include anaerobic sediment. Therefore, 

21 For example, some widely degradable materials may take considerably longer to degrade under 
anaerobic conditions such as newspapers in landfill waste sites. 

22 New information on anaerobic degradation may be needed in specific cases to understand 
the degradation dynamics in the sediment compartment. 

23 E.g. with the EAWAG-BBD Pathway Prediction System (http://eawag-bbd.ethz.ch/aboutBBD.html).  

24 For example consider the application of substance – an anti-oxidant would be expected to be affected 
by oxygen and therefore aerobic degradation is likely to be more relevant. 

 
                                                 
 
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if a substance is expected to degrade only under anaerobic conditions, an OECD TG 308 may 
not be the most suitable test to study the persistence of the substance. Even in the aerobic 
version of the OECD TG 308 a large part of the sediment is anaerobic. The substances that 
degrade only anaerobically may degrade in an OECD TG 308 study but not in an OECD TG 309 
study. This has been shown for example with nitro-containing substances, like musk xylene. 
OECD TG 308 might therefore overestimate the degradation rate in the aerobic environment. If 
only an OECD TG 308 study is conducted, wrong conclusions on persistence may be drawn. In 
such cases, to exclude potential false negative results in relation to the P/vP assessment, 
strictly aerobic degradation should also be assessed if technically feasible, i.e. the surface 
water simulation degradation tests with its strictly aerobic conditions.   

Test temperature 

Guidance on test temperature for the simulation test(s) is provided in Section R.7.9.4.1 of 
Chapter R.7b of the Guidance on IR&CSA. The reference temperature for providing results on 
higher tier tests (and carrying out tests, where relevant) is 12°C for surface water 
environment and 9°C for marine environment. 

Non-extractable residue 

With regard to evaluation of soil or sediment simulation degradation test results, it is 
important to differentiate between actual degradation of a substance and formation of non-
extractable residues (NERs); especially in the soil or sediment but also in a surface water test 
which may lead to NERs formation depending on the SPM concentration (including its OC 
content). The formation of NERs should not be confused with the degradation phenomenon.   

The NER should ideally be differentiated in remobilisable and irreversibly bound fractions. 
While the irreversibly bound part (e.g. biogenically bound) can be assessed as a potential 
removal pathway, the remobilisable fraction (heavily sorbed, physical inclusion) pose a 
potential risk for the environment. There is, however, no simple relationship between 
extraction by the different individual extraction methods and the type of chemical binding to 
soil/sediment. This is discussed in Sections R.7.9.4 and R.7.9.5 of Chapter R.7b of the 
Guidance on IR&CSA.  

Another issue to address is whether the parent substance, or its degradation products, via 
their interaction with sediment or soil organic matter become bound to or entrapped in the 
organic matrix. The environmental significance of NERs is related precisely to the extent to 
which they become “indistinguishable” from existing soil, sediment or organic matter. 
However, the term “indistinguishable” cannot currently be defined because the relationship 
between extraction by the different individual extraction methods and the type of chemical 
binding to soil/sediment is not simple to understand or to describe. For example, NER 
formation might be an indication of degradation only if the NER level decreases concurrently 
with gradual increase in mineralisation or metabolite formation. In contrast, a lack of 
degradation of the parent compound may be assumed if fast NER formation (with extensive 
NER formation in several days without any degradation observed) is followed by a period of 
relative constant levels of NER. This might indicate the fact that the parent compound has 
become non-extractable, and thus is not readily available to degradation. Information obtained 
by comparing results from NER formation in sterile and non-sterile soils/sediments can 
sometimes provide insight into the mechanisms of the process. If NER is only formed at high 
levels in non-sterile soils/sediments, this may indicate degradation of the parent substance. In 
this case the formed NER in the non-sterile soil/sediment is unlikely to consist of the parent 
substance. 

There is currently no procedure to measure which part of the residue is not bound irreversibly 
(see Chapter R.7b of the Guidance on IR&CSA for more details). Neither is there a standard 
concept currently available to measure different fractions of the residue.  

  
 
 
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Therefore, the residues should be regarded, in the absence of systematic methodology, as 
non-degraded substance25, unless, on a case-by-case basis, it can reasonably be justified or 
analytically demonstrated that a certain part of the residues can be considered to be 
irreversibly bound. 

Please note that scientific work is on-going to develop the understanding on NER and that the 
recommendation above is based on current knowledge and experience. Registrants are advised 
to follow-up the recent and future developments in the field, e.g. via the ECHA website. 

Assessment of relevant degradation/transformation products (“relevant 
metabolites”) 

Where a substance is degraded by abiotic means or partly biodegraded, it may be necessary to 
consider whether there are any breakdown products or metabolites formed that could be 
potential PBTs/vPvBs. Where the original substance forms a breakdown product or metabolite 
that could be PBT/vPvB, there should be an assessment of the amount of this breakdown 
product or metabolite compared with the parent substance. In relation to degradation testing 
results, including those from simulation degradation tests which also include investigation of 
degradation pathways (OECD TGs 307, 308 and 309), there are often practical constraints to 
the analytical identification of transformation products. Biotransformation/ degradation 
pathways may be complex and many different degradation products may be formed and some 
only in small amounts (or rates). Practical constraints in relation to analytical methodologies 
for identification of degradation products may thus limit the possibility for identifying them 
chemically, when they occur in very small concentrations. In the simulation degradation test 
guidelines for soil, water-sediment and surface water, transformation products detected at 
≥10% of the applied concentration of the parent substance at any sampling time (principal 
metabolites) should at least be identified unless reasonably justified otherwise. The test 
guidelines furthermore stipulate that values even lower than 10% may be warranted 
depending on the specific case. However transformation products for which concentrations are 
continuously increasing or seem to be stable during the study should also be considered for 
identification, even if their concentrations do not exceed the general limit given above, as this 
may indicate persistence. The need for quantification and identification of transformation 
products should be considered on a case-by-case basis with justifications. See also the 
definition of relevant degradation/transformation products in Section R.11.4.1. 

It should be noted that neither a readily biodegradable substance (based on ultimate 
degradation) nor its metabolites will normally need to be assessed because any metabolites 
can be assumed to be minimal and transient. 

To assess whether the breakdown products or metabolites may be potential PBT or vPvB 
substances, the following approaches may be helpful: 

  Based on the structure of the parent molecule, predictions of the structures of the 
breakdown products/metabolites may be made. These can be based on QSAR 
models/expert systems e.g. the freely available EAWAG-BBD Pathway Prediction 
System (available at: http://eawag-bbd.ethz.ch/predict/index.html), KEGG 
biodegradation database/prediction tool, the OECD QSAR Tool Box (see microbial 
metabolism functionality) or the commercial CATABOL or Multicase modelling tools, and 
by use of expert judgement, supported by appropriate substance-relevant scientific 
documentation. 

25 Meaning non-degraded parent substance or as relevant metabolite(s) if such is or are 
formed. 

 
 
                                                 
 
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For further PBT/vPvB assessment of the relevant degradation/transformation products, the 
normal PBT/vPvB assessment approach and data generation principles apply, as described in 
this Guidance document. See also the definition of and discussion on relevant 
degradation/transformation products in Section R.11.4.1. 

Assessment of abiotic degradation data 

Abiotic degradation tests are not required in a P assessment for readily biodegradable 
substances, or for substances shown to be (ultimately) degraded in “enhanced” biodegradation 
tests and modified ready biodegradability tests, or for substances with a degradation half-life 
in a simulation test not fulfilling the P-criterion. If abiotic degradation tests are available, there 
is a need to assess the properties of abiotic degradation products against the screening P, B 
and T criteria (see Sections R.7.9.4. and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA). 

It should be noted that the abiotic degradation processes typically concern only primary 
degradation. Hence, when assessing such data for PBT/vPvB characterisation, the identification 
of the transformation product(s) should be performed.   

There are several abiotic degradation/transformation processes in the environment to be 
considered, including e.g. hydrolysis, direct and indirect photodegradation, 
oxidation/reduction, surface-controlled catalytic reactions, molecular internal conversions etc. 
The most important of these processes is usually hydrolysis, which is relatively independent 
from the mode of entry of the substance into the environment. Hydrolysis may proceed 
effectively in aquatic, sediment and soil compartments but it is however noted that there are 
substances reaching rapid hydrolysis rates which are well known to be persistent in soil and/or 
sediment, e.g. endosulfan. Therefore, rapid hydrolysis rates cannot alone lead to concluding 
that a substance is not persistent. Test results showing rapid hydrolysis rates always need to 
be evaluated carefully in context with other information on the substance, such as partitioning 
and ionogenic properties both of which may significantly influence the extent and strength of 
sorption to soil and sediment. Hydrolysis also needs to be consistently rapid across the range 
of environmentally relevant pH. To provide confidence in the hydrolysis results, analytical data 
identifying metabolites to provide a mass balance are also needed. These both demonstrate 
that primary degradation has occurred, and allow subsequent PBT assessment of the 
degradants.   

There is currently no cut off for hydrolysis rate, which could alone be used as justification to 
conclude that a substance is not persistent. Hydrolysis data always need to be considered in 
connection with the other properties, such as partitioning properties and the knowledge on the 
abiotic and biotic degradation pathways.  

Due to the number of factors that affect photodegradation rates, this process is not generally 
considered in the persistence assessment for substances registered under REACH. Further 
discussion on photodegradation is provided in Chapter R.7b of the Guidance on IR&CSA. 

According to Castro-Jiménez and de Meent (2011), light absorption in natural water is 
significantly slower than measured in laboratory water with photo degradation occurring 
around 30 times more slowly for typical fresh water, 400 times more slowly for typical coastal 
sea water, and 500 times more slowly for ocean water. These authors also conclude that the 
“contribution of photodegradation in water to overall degradation is significant only for 
substances that reside in water to a considerable extent”. They highlight that many substances 
reside in sediment and soil, rather than in water. They give as an example bromophenyl 
ethers, which are “photochemically labile in water” but only slowly photodegrade in the 
environment. The relative importance of direct photolysis versus the indirect process varies 
and is dependent both on the composition of the substance as the prevailing conditions of the 
media. Indirect photodegradation is stimulated in natural environmental waters by the 
presence of dissolved organic matter (which is not present in pure laboratory water). 

  
 
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The tests used and their interpretation are discussed in Sections R.7.9.4 and R.7.9.5 of 
Chapter R.7b of the Guidance on IR&CSA. 

R.11.4.1.1.4 

Assessment based on estimation models (QSAR, SAR) 

The use of QSAR and SAR predictions for identifying substances for persistence (P and vP) 
might be used at the screening level, as described below and in detail in Sections R.7.9.4 and 
R.7.9.5 of Chapter R.7b of the Guidance on IR&CSA. 

Biodegradation QSAR models – screening 

Generally, it is recommended to consider both the validation status of any QSAR model and 
whether the substance for which predictions are made may be regarded as being within the 
applicability domain of the model (see Section R.6.1 in Chapter R.6 of the Guidance on 
IR&CSA). 

(Q)SAR estimates may be used for a preliminary identification of substances with a potential 
for persistence. For this purpose, the combined use of results from three estimation models in 
the EPI suite (US EPA, 2000) is suggested, as described above in the Explanatory Note 2 to the 
ITS for persistence assessment (Figure R.11—3). 

Other QSAR approaches 

Pavan and Worth (2006) describe a number of models and approaches that specifically address 
the issue of identifying structures that meet or do not meet the P criteria.  

In the same way, Nendza et al. (2013) provide an inventory of in silico screening tools that 
could be used for the assessment of the degradation potential of substances under the REACH 
Regulation. Such estimates may be used for preliminary identification of substances with a 
potential for persistence (see also Section above). The combined results of the three freely 
available estimation models BIOWIN 2, 6 and 3 in the EPI suite (US EPA, 2000) may be used 
as follows: 

  Non-linear model prediction (BIOWIN 2): does not biodegrade fast (probability < 0.5) 
and ultimate biodegradation timeframe prediction (BIOWIN 3): ≥ months (value < 
2.25), or 

  MITI non-linear model prediction (BIOWIN 6): does not biodegrade fast (probability < 

0.5) and ultimate biodegradation timeframe prediction (BIOWIN 3): ≥ months (value < 
2.25) 

QSAR predictions can be used as part of a Weight-of-Evidence approach: predictions that the 
substance is not rapidly degradable would support the conclusion that the substance is 
potentially P/vP. In the contrary situation, predictions indicating that the substance could 
degrade rapidly would support the conclusion that the substance is not persistent. However, 
QSAR results alone are in most cases not sufficient to conclude on non-persistence but should 
be supported by additional information. In every case, it should be verified that the QSAR 
model and predictions are reliable and applicable to the substance. While the QSAR predictions 
using these models are reliable and the estimation results clearly indicate that the substance is 
not persistent, all other available information should still be taken into account together with 
QSAR estimation(s) in order to be able to consider the substance as not fulfilling the criteria for 
P. Borderline cases should be carefully examined, e.g. when the estimate of the ultimate 
degradation time predicted by BIOWIN 3 gives a result in the range of 2.25 to 2.75 (see 
Sections R.7.9.4 and R.7.9.5 in Chapter R.7b of the Guidance on IR&CSA). Note however that, 
in any case, all other existing and reliable QSAR predictions, read across and test data 
information should be considered for deriving a conclusion regarding the persistence status of 
the substance. 

 
 
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The use of QSAR model predictions are of particular relevance and interest when test data are 
lacking and when assessing multi-constituent substances for which it may often be difficult to 
find or even to generate test data on relevant individual constituents (including impurities) due 
to analytical, technical, practical and cost implications (see Section R.11.4.2.2). 

Abiotic degradation models 

There are very few software models available for predicting hydrolytic degradation, 
atmospheric and hydrolysis or aquatic photodegradation (e.g. AOPWIN and HYDROWIN 
programs are freely available in the Syracuse Research Corporation’s Estimation software 
(EPISuite)), and a few published models (Peijnenburg et al., 1992, Stegeman et al., 1993). 
These are reviewed in Section R.7.9.4 of Chapter R.7b of the Guidance on IR&CSA. 

Other modelling data 

Another useful source of information is programmes that predict metabolic pathways for the 
degradation of a substance. These can be useful for exploring likely routes of degradation as 
well as for helping identify potential metabolites (both for analysis and evaluation). One 
programme is the EAWAG-BBD Pathway Prediction System (formally from the University of 
Minnesota), which can be found at http://eawag-bbd.ethz.ch/predict/. 

Multi-media modelling 

Results from multi-media modelling (e.g. Mackay level III model as this is included in the 
EPIWIN QSAR package) could also be explored in order to evaluate the environmental 
exposure and compartment(s) of specific concern. Typically, the results used from such models 
are the relative (%) mass of the substance (in a steady state situation with continuous 
environmental release) in each environmental compartment, in a simple “Unit world” 
consisting of air, surface water, sediment and soil. Typically, the default situation is 
assumption of an environmental release pattern with equal release to air, surface water and 
soil (see the default settings in the Mackay level III part of the EPIWIN). It should be noted 
that the results of such models should be regarded as qualitative or at most semi-quantitative 
as they strongly depend on the relative size of the environmental compartments, the emission 
pattern (see below) and partitioning and transformation parameters employed in the 
modelling. Contrary to the result of Mackay fugacity level I modelling, Mackay level III 
modelling is also dependent on the release pattern (fraction of emission between air, water, 
soil) and thus also on the use of the substance.  

Therefore, if a more relevant /realistic release pattern than equal emission rate to air, water 
and soil can be assumed based on knowledge about use of the substance, the model should be 
run with an appropriately changed release pattern (for example, this can easily be done in the 
EPIWIN model package). Typically, but depending on the use profile of the substance, it is 
relevant to run such models assuming the default environmental risk assessment emission 
pattern, e.g. release to water only. Alternative and freely available models exist beside that 
included in EPIWIN, e.g. EQC (Mackay et al., 1996; see also 
http://www.trentu.ca/academic/aminss/envmodel/models/EQC.html), SIMPLEBOX (Schoorl et 
al., 2016; see also www.rivm.nl/en/Topics/S/Soil_and_water/SimpleBox). 

Another option is to consider comparing the results of the modelling with the normally 
employed environmental exposure assessment where emission normally takes place via 
emission to STP. This can easily be done by modelling the fate in a suitable STP model where 
the fractions at steady state are presented: volatilisation to air, adsorption to STP- sludge, 
STP-degradation and the emission fraction to surface water. Such models also typically employ 
the fugacity concept. The fraction adsorbed to STP sludge is normally assumed to be disposed 
of on soil and hence indirect exposure of the soil compartment has to be assumed. 

For some substances which have distinctive use patterns and pulsed releases into the 
environment, more specific models could be considered, e.g. the FOCUS models for 
agrochemicals. The FOCUS modelling framework relies on mechanistic process-based models 
to predict the exposure from substances, either directly applied in agricultural areas or driven 

  
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by weather-related compartmental transfer processes such as run-off and drainage. FOCUS 
models can thus be used to identify the relevant compartment(s) to which agrochemicals will 
partition, taking into account the specific use and release patterns of those substances. 

Finally, freely available multi-media models focussing on the potential for long range 
environmental (mainly air) transport also exist like the OECD Long Range Transport model 
(OECD, 2006). They could be employed for considering possible relevance of certain 
environmental compartments of concern for simulation degradation testing, in particular 
whether or not pristine environmental compartments (e.g. open sea) may be exposed to a 
significant extent.  

With respect to the results of the distribution modelling results, they should only be regarded 
as qualitative or semi-quantitative and a case-by-case evaluation26 of the results is needed. A 
robust study summary should be provided and give sufficient information on the modelling (i.e. 
default assumptions and input parameters of the model). 

R.11.4.1.1.5 

Field studies for persistence 

If field studies are available, they are an option to additionally assess the persistence of 
substances under realistic outdoor conditions. In contrast to laboratory studies that often 
include artificial elements such as drying and sieving of soils (e.g. OECD TG 307 study) it is 
possible to study the degradation of a substance under natural conditions in the undisturbed 
environment. One of the most important advantages of field studies over laboratory studies is 
the option to run them over long periods up to several years. There is no risk that the system 
gets exhausted as what happens with longer-lasting laboratory studies where the 
microbiological activity might significantly decrease if the study period needs to be extended to 
derive reliable half-lives. With field studies, it is also possible to study the accumulation 
potential of substances over several years. However, compared to laboratory studies, field 
studies are semi-controlled with a range of varying environmental factors. These factors and 
uncertainties derived therein should be taken into account in the assessment.   

Field studies do not represent higher-tier studies in the sense that their results would override 
other (lower-tier) results, but they can be used in a Weight-of-Evidence approach. PBT 
assessment is normally not bound to local conditions whereas field studies are particularly 
dependent on local conditions. Therefore, results from field studies are not directly comparable 
with one another, laboratory tests or P/vP criteria. .  

When including field studies in the Weight of Evidence, the varying temperature conditions 
should be taken into account (if available). Consideration should be given to whether 
temperature correction should be applied. Guidance on test temperature is provided in Section 
R.7.9.4.1 of Chapter R.7b of the Guidance on IR&CSA. 

In general, field studies can be carried out for the different compartments of interest. For the 
soil compartment several guidance documents exist on how to conduct terrestrial field 
dissipation studies. These guidance documents were mainly developed for PPP but can also be 
used for any other chemical substance. The NAFTA guidance (NAFTA, 2006) is based on the 
degradation behaviour of substances under realistic exposure conditions considering all 
possible dissipation and degradation pathways. The use of a conceptual model of the 

26 This should include consideration of the values of water solubility, octanol-water and organic-carbon 
partitioning coefficients, vapour pressure and half-life coefficients used in the modelling. This is because 
these values may be predicted by the model, even if measured values have been input to the 
programme. A robust study summary should be provided giving sufficient information on the modelling. 
(i.e. default assumptions and input parameters of the model). Finally consideration of how the substance 
is likely to be released to the environment should be made. This is important to understand which 
fugacity model may be most appropriate – for example 100% release to water, soil etc. A sense check 
should also be made to review whether the predictions seem reasonable. 

 
 
                                                 
 
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substance behaviour that would depend on results from laboratory studies should be supported 
and the results confirmed by different modules of the field study.  

EFSA developed a guidance (EFSA, 2014) focused on biodegradation in the soil matrix. It 
describes how surface processes such as volatilization and photolysis as well as dissipation by 
leaching to deeper soil layers are taken into account in order to get a DegT50 value that can 
be used in exposure modelling. In order to avoid surface processes, it is recommended for 
instance to mix the substance with the topsoil layer of the field or to cover the field after 
substance application with a sand layer. For mobile substances that can be leached down to 
deeper soil layers during the course of the study, the EFSA guidance requires sampling down 
to a depth were no substance can be found anymore to account for all residues.  

The OECD Guidance document 232 (OECD, 2016) considers aspects from both the NAFTA and 
the EFSA guidance and is the most recent guidance document. It should be used for the 
conduct of field degradation studies. 

Lysimeter studies, which are often carried out with radiolabeled substances (OECD, 2000a), 
can also provide useful information about the degradation behaviour of a substance to be used 
in the context of the P-assessment. Guidance for deriving DegT50 values from lysimeter 
studies is provided in FOCUS (2014). 

For studying the behaviour of a substance in water or sediment, less guidance is available. 
However, meso- or macrocosm studies, which are sometimes used in ecotoxicology, can in 
general be used to provide valuable information on the fate of the substance, e.g. on the 
partition behaviour of the substances. Guidance on how to derive DegT50 values from cosm 
studies is provided in Deneer et al. (2015). 

For further references, please see Section R.7.9.4.2 in Chapter R.7b of the Guidance on 
IR&CSA. 

R.11.4.1.1.6 

Monitoring data 

Monitoring data in themselves cannot demonstrate persistence because the presence of a 
substance in the environment is dependent on a range of factors other than degradation rates, 
namely emission and distribution rates. Potential sources, trends of volume, uses and releases 
should be considered when evaluating the suitability of monitoring data in the P/vP 
assessment. Nevertheless, if monitoring data as a part of a Weight-of-Evidence analysis show 
that a substance is present in remote areas (i.e. long distance from populated areas and 
known point sources, e.g. arctic sea or Alpine lakes), it may be possible to conclude a 
substance as P or vP. . Monitoring data obtained in areas closer to the sources may also be 
useful for P/vP assessment and can be used as one line of evidence for supporting the 
conclusions(in both directions: P/vP or not P/vP). Use of monitoring data in P/vP-assessment 
encompasses several uncertainties and conclusions should be drawn on the basis of monitoring 
data only when there is sufficient understanding of the substance distribution and transport 
behaviour and under the condition that the uncertainties in the monitoring data presented are 
adequately addressed. The lack of detection of a substance in monitoring data should be 
considered carefully as it does not necessarily mean that a substance is not persistent (e.g. 
shortcomings in analytical methods may affect monitoring of substances in the environment). 
If monitoring data show that the substance levels in environmental media or biota are rising, 
the reasons for such a time trend should be assessed very carefully against the information on 
the time trends of volumes, uses and releases. Where monitoring data clearly indicate that the 
substance fulfils the vP-criterion or, depending on the case, that the P criterion is fulfilled in 
addition to other supporting information (and without conflicting data), it may not be 
necessary to generate simulation degradation data. In the latter case, conclusions on the 
fulfilment of the P/vP criteria may be drawn based on the monitoring data, the information on 
the substance distribution/transport behaviour, in addition to other supporting information 
used as part of a Weight-of-Evidence analysis.  

  
 
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  Bioaccumulation assessment (B and vB) 

This section deals with assessment of bioaccumulation data accepted for use in the PBT and 
vPvB assessment and further provides guidance on how to evaluate whether a substance 
meets the B or the vB criteria. To this end, the section comprises a decision scheme on how to 
use data of different experimental tests as well as non-testing information. For a B and vB 
assessment all available relevant information should be taken into account. In accordance with 
Annex XIII all available information/evidence on bioaccumulation must be considered in a 
Weight-of-Evidence approach. This comprises results from bioaccumulation experiments, 
monitoring data from the field and toxicokinetic information from toxicity studies on 
accumulation as well as other testing and non-testing indications of bioaccumulation. The order 
of data types presented in the below ITS and in the following subsections are not meant to 
define the order of importance or weight of individual data types. The data types are presented 
so that the experimental data providing information on bioaccumulation are described first and 
other data relevant for the assessment as last.  

Guidance on the evaluation and validation of both testing data and non-testing information can 
be found in Section R.7.10 of Chapter R.7c of the Guidance on IR&CSA.   

For substances containing multiple constituents, impurities and/or additives, the guidance 
provided below applies to that/those “part(s)” of the substance, which is/are the target of 
assessment and testing. The criteria for selecting an appropriate assessment approach are 
provided in Section R.11.4.2.2. 

R.11.4.1.2.1 

Integrated Assessment and Testing Strategy (ITS)27 

If a substance is imported or produced in an amount of more than 100 t/y, information to fulfil 
REACH Annex IX, 9.3.2. standard information requirement is mandatory. The option of waiving 
the bioaccumulation test according to Column 2 of REACH Annex IX can only be taken if the 
information from the experimental test is not required for the conclusion on the PBT/vPvB-
properties (see also Section R.11.3.3). Similarly, the standard aquatic bioaccumulation test 
requirement cannot be adapted according to REACH Annex XI, if the PBT/vPvB assessment 
shows that a bioaccumulation test in aquatic species is necessary (and it is technically 
feasible). However, it is noted that the possibility to use information referred to in REACH 
Annex XI should be investigated in the frame of the PBT/vPvB assessment first before 
proposing a bioaccumulation test. In that case the evaluation of the B and vB criteria for the 
PBT and vPvB assessment should be performed simultaneously with the assessment of the BCF 
value. Detailed guidance regarding an ITS for BCF assessment is presented in Section R.7.10 
of Chapter R.7c of the Guidance on IR&CSA. Figure R.11—4 in this section should be seen as a 
detailed scheme of the B-assessment block within the ITS. 

If the tonnage produced or imported is below 100 t/y, normally a bioaccumulation test is not 
required and therefore a BCF value may not be available. In that case it should be first 
considered if the available testing and non-testing data are sufficient to conclude on the B-
properties for those substances produced or imported at <100 t/y or if bioaccumulation testing 
is needed and hence required to draw a reliable conclusion. 

If the Weight-of-Evidence approach described under "Conclusions on the Endpoint" is not 
sufficient to draw a conclusion, the performance of an experimental bioaccumulation test or 
generation of other appropriate bioaccumulation information is required. However, before such 
a study is conducted for assessing the B and vB criteria, the P criterion should be investigated 
in order to prevent unnecessary testing of animals. Further generation of information on 

27 The mitigating factors that are listed below only refer to the assessment of the B and vB criteria in the 
context of the PBT and vPvB assessment. If bioaccumulation appears to be a critical parameter in the risk 
assessment process, it could still be necessary to perform a bioaccumulation test, although this may not 
be needed from the perspective of the PBT and vPvB assessment. 

 
                                                 
 
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bioaccumulation is only necessary, if the P criterion has been confirmed to be fulfilled for the 
substance. 

If generation of further bioaccumulation data is necessary, there are several options for the 
most appropriate strategy. Additional data should always be generated in a tiered way 
revisiting the B-assessment after each time new data are made available. In normal case it 
may be possible to conclude on the B/vB properties after one study, but in specific cases 
several bioaccumulation studies may be needed.  

The available data define the choice of the study/test. Hereby, the understanding of in which 
type of species/compartment the bioaccumulation potential seems highest is crucial for the 
choice of the test. In very specific cases, the most relevant compartment(s) of exposure may 
also influence the choice of the study.   

  
 
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Figure R.11—4: Integrated assessment and testing strategy for B-assessment. 

Is there sufficient information to conclude the B-assessment taking all information into account in a weigh-of-evidence approach?ConcludeNot B Screening criteria for aquatic organisms:Log Kow > 4.5Screening criteria for air-breathing organisms:Log Kow > 2 and log Koa > 5Non-lipophilic bioaccumulation?NoConclude the B-assessment (B, vB or not B). If it can be concluded that the substance is not B, no further information for a definitive P assessment needs to be  generated.NoNoYesNo- aquatic bioconcentration  or bioaccumulation studies (fish BCF, fish dietary test results, mussels)- in vitro data on metabolism in combination with kinetics of uptake and depuration- read-across with structurally similar substance- terrestrial or benthic accumulation studies- field data concerning biomagnification and bioaccumulation- results of assessment of the toxicokinetic behaviour- detection of elevated levels in biota- physicochemical properties - toxicokinetics in aquatic organisms, birds and mammals- uptake and absorption efficiency- absence or presence of chronic toxicity- toxicokinetic information on laboratory mammals and humansThe substance is potentially B/vB.More data should be generated if the substance fulfils the P or vP criteria. What type of further data are needed, depends on the screening criteria and other available information.Consider whether other type of data, e.g. in vitro assays, provide sufficient information, before conducting an in vivo BCF test.Start B assessmentYesYesPhysicochemical indicators for hindered uptake:     due to molecular size Dmax aver > 17.4 Å1 OR     uptake and distribution in general Log KOW > 10 OR     for low potential mass storageOctanol solubility [mg/L] <0.002 [mM] x MW [g/mol]AND:Experimental indicators for hindrance of uptake No chronic toxicity for mammals and birds No uptake in mammalian toxicokinetic study Very low uptake after chronic exposureYesAny compartment/taxa of specific concern  based on available information? (highest likelihood of bioaccumulation in a specific compartment/taxa or non-lipid binding)OECD TG 305  preferred to be carried out. Flow-through -test preferred, if feasible.Generate the further information based on case-by-case assessment NoYes 
  
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R.11.4.1.2.2 

Experimental aquatic bioconcentration factor (BCF) data 

For the start, it should be noted that, in normal cases where experimental information on 
bioaccumulation is needed, a flow-through bioaccumulation test with fish according to OECD 
TG 305-I or OECD TG 305-II is preferred due to the best possibilities of reliably comparing the 
results from such test with the B/vB criteria.  

Only in specific cases, described in following subsections, other study/test types may be 
warranted as the option for generating further information. 

In line with Annex 1 of the OECD TG 305, the following definitions are used in this guidance: 

  The bioconcentration factor (BCF) at any time during the uptake phase of this 

accumulation test is the concentration of test substance in/on the fish or specified 
tissues thereof (Cf as mg/kg) divided by the concentration of the substance in the 
surrounding medium (Cw as mg/L). BCF is expressed in L·kg-1. Please note that 
corrections for growth and/or a standard lipid content are not accounted for in this 
definition of the BCF. 

  The steady-state bioconcentration factor (BCFSS) does not change significantly over a 

prolonged period of time, the concentration of the test substance in the surrounding 
medium being constant during this period. 

  The kinetic bioconcentration factor (BCFK) is the ratio of the uptake rate constant, k1, to 
the depuration rate constant, k2 (i.e. k1/k2 – see corresponding definitions in Annex 1 of 
the OECD TG 305). In principle the value should be comparable to the BCFSS (see 
definition above), but deviations may occur if steady-state was uncertain or if 
corrections for growth have been applied to the kinetic BCF. 

  The lipid normalised kinetic bioconcentration factor (BCFKL) is normalised to a fish with 

a 5% lipid content. 

  The lipid normalised, growth corrected kinetic bioconcentration factor (BCFKgL) is 

normalised to a fish with a 5% lipid content and corrected for growth during the study 
period as described in Annex 5 of the OECD TG 305. 

Bioconcentration data from controlled laboratory experiments can be used in assessing the 
bioaccumulation potential of a substance. For example, OECD TG 305-I: Aqueous Exposure 
Bioconcentration Fish Test (OECD, 2012) or an equivalent test protocol in fish is preferred for 
producing experimental bioconcentration data. Valid results from this test can be used directly 
for comparison with the B and vB criteria. Nevertheless, it is underlined, that in addition to BCF 
values, other relevant information should be considered. The REACH Annex XIII Introduction 
requires all other available bioaccumulation data to be taken into account in an integrated 
manner and applying a Weight-of-Evidence approach using expert judgement to derive the 
conclusion. If BCFs seem not coherent with other data or there are very different BCF-values 
available, it is important to address the reasons for inconsistency and discuss in which way this 
inconsistency impacts the overall conclusions on bioaccumulation potential. 

Also use of other taxonomic groups than fish (e.g. mussel bioconcentration test, ASTM, 2003) 
is possible for measuring bioconcentration in the aquatic environment and the valid BCFs 
determined in other taxonomic groups can be used in assessing whether or not the B/vB 
criteria are met. Furthermore, in case a Kow as screening information is considered likely to be 
reliable for estimating the bioaccumulation potential of a substance while still some 
experimental information is needed to refute or confirm this assumption, the OECD TG 305-II: 
Minimised Aqueous Exposure Fish Test may also be used to assess B or vB, provided that the 
final results will most likely not result in borderline cases of meeting either the B or vB 
criterion. This should be investigated before the test is initiated, e.g. by the use of QSARs, to 
avoid the results of the test being insufficient for the B assessment after the test has been 
completed. Conditions for selecting the minimised OECD TG 305-II instead of the OECD TG 
305-I are described in the OECD TG 305 and it should be noted that the OECD TG 305-II test 

  
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conducted within those conditions can be used for the bioaccumulation assessment in order to 
minimise animal use. Whether minimised tests should be carried out depends on a range of 
factors including the required level of precision of the determination of the BCF value for a 
particular substance. For instance, if it is estimated that the BCF-value may be close to the 
threshold values of either 2000 for 'B' or 5000 for 'vB', the BCF determination by OECD TG 
305-II is not warranted because the result may be associated with too much uncertainty. In 
such a case an OECD TG 305-I test would be appropriate.  

Bioconcentration can be tested experimentally for substances that are water soluble to an 
extent allowing that the exposure concentration(s) can be maintained constant throughout the 
uptake phase of the test, as demonstrated by regular analytical verification of the exposure 
concentrations. A proper analytical method should be available to measure the test substance 
concentration not only in the animal tissues but also in water at the used test concentrations 
that should always be below the water solubility limit of the substance. In bioconcentration 
tests accumulation via the water phase must be the only route of exposure and any 
accumulation via feed must be avoided. 

The aim of the bioconcentration testing is to produce a reliable estimate of how much 
substance could concentrate from the aquatic compartment (Cw) to fish (Cf) so that a 
bioconcentration factor (BCFSS) can be calculated by using ratio Cf/Cw at steady-state. 
However, a BCFk value is preferred, and it may also be calculated as the ratio of the uptake 
rate constant (k1) and the depuration rate constant (k2). This approach is especially useful in 
those cases in which steady-state is not reached during the uptake phase, as BCFk in these 
cases will generally provide a statistically more robust value. If uptake follows first order 
kinetics and the BCFSS was really based on steady state data, both methods should in principle 
lead to the same result. However, for bioaccumulative substances a real steady state is often 
not attained during the uptake phase, and the conclusion of steady-state from the 
concentrations in fish at three consecutive time points could be erroneous. If the BCFk based 
on first order kinetics is significantly different from the BCFSS, this is a clear indication that 
steady-state has not been attained in the uptake phase. 

Besides that, the BCFss cannot be corrected for the growth of fish as no agreed method is 
available to correct BCFSS for growth. The increase in fish mass during the test will result in a 
decrease of the test substance concentration in the growing fish (= growth dilution) and thus 
the BCF may be underestimated if no correction is made. Growth dilution may affect both 
BCFSS and BCFK and therefore the BCFK should be calculated and corrected for growth dilution, 
BCFkg, if fish growth is significant during the test (this is especially important for fast growing 
juvenile fish, such as juvenile rainbow trout, bluegill sunfish and carp). OECD TG 305 (Annex 
5) contains two different methods for growth dilution correction. For bioaccumulative 
substances the kinetics of bioaccumulation are slow and growth dilution may have a major 
impact on the BCF. In conclusion, BCFKg is preferred for PBT substances due to i) the slow 
kinetics possibly leading to non-equilibrium within the timeframe of the experimental 
bioaccumulation test, and especially ii) the correction for growth dilution, which is not included 
in the BCFSS. More emphasis on BCFKg is also given in OECD TG 305. 

For older fish bioaccumulation studies, information on growth may not be available. In this 
case, an assessment of the likely significance of growth on the results should be made to 
determine what weight should be given to the study in the Weight-of-Evidence assessment. As 
noted in the OECD TG 305 (paragraph 32), juvenile fish may be fast growing at the life-stage 
(and size) they are tested in the OECD TG 305. Small Rainbow Trout (O. mykiss) are an 
example of this. In contrast, fish such as Zebra fish (D. rerio) are usually adults and therefore 
significantly slower growing (for example see an analysis in Brooke and Crookes, 2012). In the 
absence of growth data, the uncertainty in a BCF value derived from a fast-growing fish will be 
greater than a slow growing fish, which is important for results near a regulatory threshold. 
Overall, any approach to using fish bioaccumulation data where growth data are not available 
needs to be considered on a case-by-case basis with justification for the conclusion drawn. 

 
 
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The preferred way to derive k1 and k2 is in most cases to fit both parameters simultaneously 
by non-linear regression to the combined data for both the uptake phase and the depuration 
phase (see Annex 5 of the OECD TG 305), because this procedure represents the best fit for 
both parameters to all available data and yields a consistent fit for the uptake and depuration 
phase. Another way to derive k1 and k2 is to use sequential fit procedure and find values of k1 
and k2 independently. This may sometimes lead to a gap in the fit between the uptake and 
depuration phase. However, a benefit of sequential fitting is that k2 is fitted first, and is 
therefore unaffected by the uptake fitting. K2, i.e. depuration, is the parameter of most 
interest in a bioaccumulation test given that the uncertainties in its derivation are understood 
and can be addressed. As recommended in Annex 5 of OECD TG 305, visual inspection of the 
modelled uptake and depuration curves when plotted against the measured sample data can 
be used to assess and compare the goodness of fit of both methods. This is a reporting 
requirement of OECD TG 305. 

The data could be transformed by taking the natural logarithms, if this transformation reduces 
the variation in the replicates and/or leads to a better fit of the data. However, care must be 
taken as such a transformation could give too much weight to very low concentrations 
observed at the end of the depuration phase, leading to a worse fit towards the end of the 
uptake phase and beginning of the depuration phase. If fish concentrations are lognormal-
transformed, a geometric mean for the water concentration should be used instead of an 
arithmetic mean.  

Normally, the concentration of the test substance in fish tissues should be lipid normalised. A 
5% lipid normalisation as recommended in OECD TG 305 should be performed unless it is 
evident that the substance does not primarily accumulate in lipid tissues; growth dilution 
should also be considered in the BCF estimation. The resulting BCF that is preferred for a 
comparison with the bioaccumulation criteria is the kinetic growth corrected and lipid 
normalised (to 5% lipids) BCF value (BCFKgL). A justification is needed in case no normalisation 
is carried out.  

It should be noted that the greatest weight under PBT assessment for REACH is placed on a 
valid BCF test due to the current understanding that BCF is in the most representative way of 
reflecting the bioaccumulation potential of a substance, where aquatic bioaccumulation is 
relevant. If BCF-values are incoherent with other data types, it is very important to address 
the reasons for such incoherence and discuss carefully about the plausibility of the BCF-values 
in this context. If a substance has a valid and plausible aquatic BCF > 2000 or 5000 (indicating 
a significant accumulation in the test organism), the substance is defined as B or vB regardless 
of whether biomagnification or trophic magnification occurs.  

R.11.4.1.2.3 

Experimental dietary biomagnification in fish (experimental 
dietary BMF) 

A dietary exposure test, preferably OECD TG 305-III: Dietary Exposure Bioaccumulation Fish 
Test, should be considered for substances for which it is not possible to maintain and measure 
aqueous concentrations reliably and/or potential bioaccumulation may be predominantly 
expected from uptake via feed (e.g. for substances with extremely low water solubility and 
high Koc, which will usually dissipate from water to organic matter). For strongly hydrophobic 
substances (log Kow > 5 and a water solubility below ~ 0.01-0.1 mg/L), testing via aqueous 
exposure may become increasingly difficult. However, an aqueous exposure test is preferred 
for substances that have a high log Kow but still appreciable water solubility with respect to the 
sensitivity of available analytical techniques, and for which the maintenance of the aqueous 
concentration as well as the analysis of these concentrations do not pose any constraints. 
Therefore, an improved analytical technique or the use of a radiolabelled substance should be 
considered first to improve the detection limit in the aqueous test before deciding on whether a 
dietary test is indeed the only feasible option. Also, if the expected fish concentration (body 
burden) via water exposures within 60 days is expected to be below the detection limit, the 
dietary test may provide an option to achieve body burdens that exceed the detection limits for 

  
 
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the substance. The endpoint for a dietary study is a dietary biomagnification factor (dietary 
BMF), which is the concentration of a substance in predator (i.e. fish) relative to the 
concentration in the prey (i.e. food) at steady state. The dietary test also provides valuable 
toxicokinetics data including the dietary chemical absorption efficiency and the whole body 
elimination rate constant (k2) and half-life for substances for which obtaining aquatic BCF data 
is technically not feasible. 

The following definitions are used in this guidance: 

  The biomagnification factor (BMF) is the concentration of a substance in a predator 

relative to the concentration in the predator’s prey (or food) at steady-state.  

  The dietary biomagnification factor (dietary BMF) is the term used in OECD TG 305 to 

describe the result of dietary exposure test, in which exposure via the aqueous phase is 
carefully avoided and thus the dietary BMF from this test method differs from a BMF 
value from a field study in which both water and dietary exposure may be combined. 
The laboratory dietary study is usually not performed using environmentally relevant 
concentrations, but uses high concentrations in food to dose the organism quickly to a 
level sufficient to assess the depuration. Another important difference that can occur 
between the lab BMF and the field BMF for substances with biomagnification potential is 
the variability of growth rates under laboratory and field conditions. However, it is 
possible to simulate field BMFs from lab BMFs to address these two differences using 
mass balance toxicokinetics (bioaccumulation) models. 

Annex 8 of the OECD TG 305 summarises some approaches currently available to estimate 
tentative BCFs from data collected in the dietary exposure study. This calculation is based on a 
model predicted uptake rate constant (k1) and the depuration rate constant (k2) determined 
from the dietary bioaccumulation study. For the PBT assessment, it is possible to translate the 
dietary experimental data to tentative BCFs for comparison against the BCF criteria outlined in 
Annex XIII. However, it should be noted that these calculated BCFs may be more uncertain 
than experimental BCFs due to the uncertainty in the k1 prediction. In particular, k1 is a 
function of chemical properties relating to the chemical transfer efficiency from water (e.g., 
membrane permeation or absorption efficiency), the physiology of the fish (body size, 
respiration rate) and the experimental conditions (e.g., dissolved oxygen concentrations, water 
temperature, gill water pH for ionic substances). Thus assuming k1 is accurately and 
appropriately predicted for the substance and the conditions of the experiment, the tentative 
BCF values can be determined.  

For poorly soluble non-polar organic substances first order uptake and depuration kinetics is 
assumed, and more complex kinetic models should be used only for substances that do not 
follow first order kinetics. Several models are available to estimate a k1 value needed to 
calculate an aqueous BCF from a dietary bioaccumulation study. Although there is some 
variation in the results of the k1 models and the models are restricted to predominantly neutral 
organic substances, the 13 presented models span a range of a factor 2.7 for some examples 
of a hydrophobic potential PBT substances (Crookes and Brooke, 2011). As noted by Crookes 
and Brooke (2011) “The uncertainty in the estimated uptake rate constant was relatively large, 
however, even for the best performing methods.” Therefore, the uncertainty of the k1 models 
and their applicability domains (e.g. mostly restricted to neutral organic substances but 
including some weakly acidic or basic substances as well, log Kow above 3.5 etc.) require 
consideration for the factors mentioned above. Accordingly, no one model can be 
recommended over the others and results must be used with caution, with reference to 
assumed applicability domains. If the method of deriving a BCF from a dietary BMF study is 
used, estimates of k1 should be derived according to all the models available to give a range of 
BCFs. 

Besides the calculation of a BCF from the depuration phase, the laboratory BMF derived from 
the test can be compared with laboratory BMF values for substances with known 
bioaccumulation potential in a benchmarking exercise. For example, such an approach has 

 
 
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been described for dietary bioaccumulation studies with carp (Inoue, Hashizume et al., 2012). 
Based on a regression between BCF and BMF for nine compounds tested in this set-up, it was 
shown that a BCF value of 5000 L/kg, normalized to a lipid content of 5%, corresponds to a 
lipid normalized BMF from the dietary test of 0.31 kg food/kg fish, and a BCF of 2000 L/kg 
corresponds to a BMF of 0.10 kg food/kg fish. Of the five substances that had a BCF value 
higher than 5000 L/kg, two of them had a BMF value in excess of 1. A different benchmarking 
could be obtained from aqueous and dietary bioaccumulation studies for perfluorinated 
compounds with rainbow trout (Martin et al., 2003a, b). A BCF value of 5000 L/kg 
corresponded to a BMF from the dietary test of 0.49 kg food/kg fish, and a BCF of 2000 L/kg 
corresponded to a BMF of 0.36kg food/kg fish. Of the three substances with a BCF > 2000, one 
had a BMF of 1.0, while the two others had substantially lower BMF values. These two different 
examples showed that there is no uniform relationship between BCF and BMF. Moreover, the 
studies emphasise the fact that even if a BMF from an OECD TG 305 dietary bioaccumulation 
study is found to be <1, it cannot be considered as a good discriminator for concluding 
substances not to be (very) bioaccumulative according to the BCF criteria of Annex XIII. 
Further examination of differences between BCF data (and criteria) and BMF data (and criteria) 
with mass balance models and with larger datasets may in future provide further insights into 
relationships between the two bioaccumulation metrics and their respective bioaccumulation 
criteria. If benchmarking is used for comparing dietary BMF values with BMF values for 
substances with a known bioaccumulation potential, it must be ensured that these BMF values 
were obtained under similar conditions. 

In conclusion, OECD TG 305 III: Dietary Exposure Bioaccumulation Fish Test provides a range 
of valuable information which should all be discussed in the bioaccumulation assessment. 
Paragraph 167 of the test guideline lists all the relevant measured and calculated data from 
the study which should be reported and considered for the bioaccumulation assessment, 
including the BMF values, substance assimilation efficiency and overall depuration rate 
constant. When interpreting the study results, the tentative calculated BCFs and a 
benchmarking exercise to compare the k2 and BMF derived from the test with other substances 
with known bioaccumulation potential also provide useful evidence for the bioaccumulation 
assessment and are recommended to be reported. The k2 (or half-life) value itself may be 
useful for the assessment of the bioaccumulation potential (see paragraph on “Overall 
depuration rate constants in fish” in Section R.11.4.1.2.9). Further guidance on the OECD TG 
305 is available (OECD, 2017). 

R.11.4.1.2.4 

Experimental sediment bioaccumulation data (experimental 
Bioaccumulation Factors BAF and BSAF for sediment)  

For the start, it should be noted that, in normal cases where experimental information on 
bioaccumulation is needed, a bioaccumulation test with fish (OECD TG 305) is preferred due to 
the better possibilities of comparing the results from such test with the B/vB criteria. However, 
there may be some very specific cases, where fish bioaccumulation test is not expected to 
reflect sufficiently the bioaccumulation potential but testing of bioaccumulation potential in soil 
or sediment might provide the necessary information for deriving conclusions on the B/vB-
assessment. Whether in such specific situation a sediment bioaccumulation test or soil 
bioaccumulation test is the first option, should be considered case by case. Targeting the 
testing to compartment where bioaccumulation potential is expected to be the highest should 
be the main consideration. Additionally, relevance of exposure may also be considered for the 
choice between sediment and soil invertebrates bioaccumulation testing. The choices should 
always be well justified. 

In line with Annex 1 of the OECD TG 315, the following definitions are used in this guidance: 

  The non-normalised biota-sediment accumulation factor (BAF) at any time during the 
uptake phase of this bioaccumulation test is the concentration of test substance in/on 
the test organism (Ca in g·kg-1 wet or dry weight) divided by the concentration of the 

  
 
 
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substance in the surrounding medium (Cs as g·kg-1 of wet or dry weight of sediment). 
In order to refer to the units of Ca and Cs, the BAF has the units of kgsediment·kg-1

worm. 

  The steady state biota-sediment bioaccumulation factor (BAFss) is the BAF at steady 

state and does not change significantly over a prolonged period of time, the 
concentration of the test substance in the surrounding medium (Cs as g·kg-1 of wet or 
dry weight of sediment) being constant during this period of time. 

  Biota-sediment accumulation factors calculated directly from the ratio of the sediment 
uptake rate constant divided by the elimination constant kinetic rate constants (ks and 
ke, respectively - see Annex 1 of the OECD TG 315) are termed kinetic biota-sediment 
accumulation factor (BAFK). 

  The biota-sediment accumulation factor (BSAF) is determined by normalising the BAFK 

(or BAFss) for the worm lipid content and the sediment total organic carbon content.  
Ca is then expressed as g·kg-1 lipid content of the organism, and Cs as g·kg-1 organic 
content of the sediment. BSAF is expressed in kgsediment OC·kg-1

worm lipid content. 

The units of the concentration values used for the calculations must all be related either to dry 
weight or to wet weight. The unit used should be reported. Optimally, calculations based on 
both the wet and the dry weights are presented. 

Bioaccumulation studies on sediment dwelling organisms can be used both for the screening 
and as part of the Weigh-of-Evidence assessment of bioaccumulation properties. It should be 
considered that (soil or sediment) invertebrate species may have a lower metabolic capacity 
than fish species, e.g. as is the case for polycyclic aromatic hydrocarbons (Bleeker and 
Verbruggen, 2009). Bioaccumulation in these invertebrates may therefore be higher than in 
fish under the same exposure conditions and this situation should be considered in a Weight-
of-Evidence approach. 

The OECD TG 315 Bioaccumulation in Sediment-dwelling Benthic Oligochaetes is the preferred 
method for generating additional information. The recommended oligochaeta species are 
Tubifex tubifex (Tubificidae) and Lumbriculus variegatus (Lumbriculidae). The species 
Branchiura sowerbyi (Tubificidae) is also indicated but it should be noted that it has not been 
validated in ring tests at the time of writing. The biota-sediment accumulation factor 
(expressed in kg wet (or dry) sediment·kg-1 wet (or dry) worm) is the main relevant outcome 
and can be reported as a steady state biota-sediment accumulation factor BAFss or as the 
kinetic biota-sediment accumulation factor (BAFK). In both cases the sediment uptake rate 
constant ks (expressed in kg wet (or dry) sediment·kg-1 of wet (or dry) worm d-1), and 
elimination rate constant ke (expressed in d-1) should be reported as well. The normalised 
biota-sediment accumulation factor (BSAF) is the lipid-normalised steady state factor 
determined by normalising the BAFK and should be additionally reported for highly lipophilic 
substances.  

OECD TG 315 recommends the use of artificial sediment. If natural sediments are used, the 
sediment characteristics should be specifically reported as described in the test guideline. For 
lipophilic substances, BSAFs often vary with the organic carbon content of the sediment. 
Typically a substance will have greater availability to the organism when the sediment OC is 
low, compared to a higher OC. It should be considered to test at least two natural sediments 
with different organic matter content, and the characteristics of the organic matter, in 
particular the content of black carbon, should be reported. To ensure comparability of results 
between different sediments, a normalised BSAF is derived from a non-normalised BSAF by 
converting the results to a sediment OC content. This allows tests on the same substance and 
tests on different substances to be comparable. The load rate should be as low as possible and 
well below the expected toxicity, however it should be sufficient for ensuring that the 
concentrations in the sediment and in the organisms are above the detection limit throughout 
the test. 

 
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The relevance of bioavailability of the substance for the test organism should also be 
considered and if relevant and possible, bioaccumulation could be expressed as a BCF between 
organism and dissolved pore water concentrations.    

It should be noted that it is not possible to give any threshold values for using sediment BSAF 
values in PBT assessment. A case-by-case assessment based on expert judgement of the 
reliability and relevance of the available information is required in order to be able to give 
BSAF values an appropriate weight in the B and vB assessment.  

Other indications of a high bioaccumulation potential, such as a bioaccumulation process not 
reaching the steady state at the end of the exposure period of OECD TG 315 test or a low 
depuration rate, both representing slow kinetics, are relevant parts of a Weight-of-Evidence 
approach when considering whether B or vB criteria are fulfilled. Especially substances having 
background sediment concentrations and potentially adaptable uptake mechanisms require 
careful consideration, as the sediment-dwelling organisms may have adapted to such 
substances which potentially affects the bioaccumulation process. 

R.11.4.1.2.5 

Experimental soil bioaccumulation data (experimental 
Bioaccumulation Factor BAF and BSAF for soil)  

For the start, it should be noted that, in normal cases where experimental information on 
bioaccumulation is needed, a bioaccumulation test with fish (OECD TG 305) is preferred due to 
the better possibilities of comparing the results from such test with the B/vB criteria. However, 
there may be some very specific cases, where fish bioaccumulation test is not expected to 
reflect sufficiently the bioaccumulation potential but testing of bioaccumulation potential in soil 
or sediment might provide the necessary information for deriving conclusions on the B/vB-
assessment.  Whether in such specific situation a soil bioaccumulation test or sediment 
bioaccumulation test is the first choice, should be considered case by case. Targeting the 
testing to compartment where bioaccumulation potential is expected to be the highest should 
be the main consideration. Additionally, relevance of exposure may also be considered for the 
choice between sediment and soil invertebrates bioaccumulation testing. The choices should 
always be well justified. 

In line with Annex 1 of the OECD TG 317, the following definitions are used in this guidance: 

  The non-normalised biota-soil accumulation factor (BAF) at any time during the uptake 
phase of this bioaccumulation test is the concentration of test substance in/on the test 
organism (Ca in g·kg-1 dry weight of worm) divided by the concentration of the 
substance in the surrounding medium (Cs as g·kg-1 of dry weight of soil); the BSAF has 
the units of kg wet (or dry) soil·kg-1 wet (or dry) worm. 

  The steady state biota-soil accumulation factor (BAFss) is the BAF at steady state and 
does not change significantly over a prolonged period of time, the concentration of the 
test substance in the surrounding medium (Cs as g·kg-1 of dry weight of soil) being 
constant during this period of time. 

  Biota-soil accumulation factors calculated directly from the ratio of the soil uptake rate 
constant and the elimination rate constant (ks and ke,) are termed kinetic biota-soil 
accumulation factor (BAFK). 

  The biota-soil accumulation factor (BSAF) is determined by normalising the BAFK (or 

BAFss) for the worm lipid content and the sediment total organic carbon content.  Ca is 
then expressed as g·kg-1 lipid content of the organism, and Cs as g·kg-1 organic content 
of the soil; the BSAF has the units of kgOC·kg-1

lipid. 

The units of the concentration values used for the calculations must be all related either to dry 
weight or to wet weight. The unit used should be reported. Optimally, calculations based on 
both the wet and the dry weights are presented. 

  
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Bioaccumulation studies with terrestrial organisms, especially those obtained from established 
experimental protocols, such as the OECD TG 317 Bioaccumulation in Terrestrial Oligochaetes 
can be used as part of the Weight-of-Evidence assessment of B and vB properties.  

It should be considered that (soil or sediment) invertebrate species may have a lower 
metabolic capacity than fish species. Bioaccumulation in these invertebrates may therefore be 
higher than in fish under the same exposure conditions and this situation should be considered 
in a Weight-of-Evidence approach.  

Earthworms and enchytraeids are the recommended taxonomic groups to be tested. In case of 
lipophilic substances the steady state biota-soil accumulation factor (BSAFss) and the kinetic 
biota-soil accumulation factor (BSAFK) are preferably presented as the normalised biota-soil 
accumulation factor (BSAF), which is the lipid and soil organic carbon -normalised BSAF.  The 
dependence of these values on the concentrations of the substance in soil, and when relevant, 
the soil characteristics should be specifically reported.  

The bioaccumulation often varies with the organic carbon content of the soil. Typically a 
substance will have greater availability to the organism when the soil organic carbon content is 
low, compared to a higher OC. To ensure comparability of results between different soils, a 
BSAF should be derived by normalising the results both to the soil organic carbon content and 
the lipid content of the organisms employed. The load rate should be as low as possible and 
well below the expected toxicity, however it should be sufficient for ensuring that the 
concentrations in the soil and in the organisms are above the detection limit throughout the 
test. 

The relevance of bioavailability of the substances potentially containing irreversibly binding 
fractions should also be considered and, if relevant and possible, the BSAF should be corrected 
for the bioavailable fraction.    

It should be noted that it is not possible to give any threshold values for BSAF in soil. A case-
by-case assessment based on expert judgement of the reliability and relevance of the available 
information is required in order to be able to give BSAF values an appropriate weight in the B 
and vB assessment. 

Other indications of a high bioaccumulation potential such as a bioaccumulation process not 
reaching the steady state at the end of the exposure period of an OECD TG 317 study or a low 
depuration rate, both representing slow kinetics, are relevant parts of a Weight-of-Evidence 
approach when considering whether the B or vB criteria are fulfilled. It should be noted that 
organo-metals and other substances with background soil concentrations and potentially 
adaptable uptake mechanisms require particularly careful consideration, as the soil-dwelling 
organisms may have adapted to such substances which potentially affects the bioaccumulation 
process. 

Some additional parameters relevant to bioaccumulation that can potentially be used for 
screening or in a Weight-of-Evidence approach, may be derived from other invertebrate 
studies. For the OECD TG 222 earthworm reproduction test, in which earthworms are exposed 
for 28 days to a test substance spiked into soil, it has been demonstrated that at test end 
(provided that the relevant analytical procedures are available) the concentration of the test 
substance in the adult worms can give an indication of uptake into the organism (Kinney et al., 
2012). Care must be taken that the bioaccumulation assessment is performed at a non-toxic 
test concentration (i.e. at which less than 10% mortality and no significant loss of body weight 
compared to control occurs over the 28d test period). It must also be noted that only uptake is 
measured at test termination and that elimination of the substance is not considered. As such, 
the results of this test should be interpreted with caution, but it can provide valuable screening 
information on substance accumulation that can help as preliminary information for considering 
whether more specific testing for bioaccumulation according to OECD TG 317 is needed. The 
same approach could potentially be useful for other guideline studies on invertebrate species 
as well, such as the 21 day larval survival test on dung beetles (OECD GD 122), the 
developmental test with dipteran flies (OECD TG 228) or the collembolan reproduction test 

 
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(OECD TG 232), depending on the expected route of exposure. However, measuring tissue 
residues in these studies could be hampered by the small size of the test organisms (Hoke et 
al., 2015). 

R.11.4.1.2.6 

Field data and biomagnification 

Bioaccumulation factors (BAF calculated from monitoring data, field measurements or 
measurements in mesocosms) or specific accumulation in food chains/webs expressed as 
biomagnification factors (BMFs) or trophic magnification factors (TMFs) can provide 
supplementary information indicating that the substance does or does not have 
bioaccumulation potential. Furthermore, the same information may be used to support the 
assessment of persistence, in particular for possible long range transport, if significant 
concentrations are found in biota in remote areas. If field data indicate that a substance is 
effectively transferred in the food chain, this is a strong indication that it is taken up from food 
in an efficient way and that the substance is not easily eliminated (e.g. excreted and/or 
metabolized) by the organism (this principle is also used in the fish feeding test for 
bioaccumulation). A relevant BMF or TMF value significantly higher than 1 (see also Section 
R.7.10.1.1 in Chapter R.7c of the Guidance on IR&CSA) can also be considered as an indication 
of very high bioaccumulation. For aquatic organisms, this value indicates an enhanced 
accumulation due to additional uptake of a substance from food next to direct accumulation 
from water. However, as dietary and trophic biomagnification represent different processes 
than bioconcentration in aquatic organisms, BMF and/or TMF values <1 cannot be directly used 
to disregard a valid assessment based on reliable BCF data indicating that a substance meets 
the numerical B/vB criteria in Annex XIII to the REACH Regulation, but in this kind of cases all 
available data need to be considered together in a Weight-of-Evidence approach. 

To be able to compare BMF values in a direct and objective manner, they should, as far as 
possible, be lipid normalized for the assessment of substances that partition into lipids in order 
to account for differences in lipid content between prey and predator. It should however be 
noted that non-lipophilic substances may bioaccumulate by other mechanisms than 
partitioning/binding to lipids. In such a case, another reference parameter than lipid content 
may be considered for normalisation, e.g. dry weight or protein content. 

In principle, BMF values are not directly related to the BCF values, and in fact BMFs and BCFs 
represent complementary bioaccumulation pathways. Food chain transfer and secondary 
poisoning are basic concerns in relation to PBT and vPvB substances, and therefore an 
indication of a biomagnification potential (BMF and/or TMF > 1) can on its own be considered 
as a basis to conclude that a substance meets the B or vB criteria. However, absence of such a 
biomagnification potential cannot be used to conclude that these criteria are not fulfilled. This 
is because a field BMF only represents the degree of biomagnification in the predatory/prey 
relationship for which it was measured. Biomagnification will vary between predatory/prey 
relationships, so a low BMF in one does not mean that it will be low in other predatory/prey 
relationship. Conversely, evidence of high biomagnification in one predatory/prey relationship 
is cause for significant concern and it is then in accordance with a cautious approach to 
assume that biomagnification may also occur in other (unmeasured) predatory/prey 
relationships. The same applies for bioaccumulation factors (BAF) calculated from field data 
(i.e. by relating concentrations in field sampled aquatic organisms to the concentration in their 
habitat). If such BAF values are above the criteria for B or vB it should be considered whether 
this information is sufficient to conclude that the substance meets the B or vB criteria. Care 
should be taken that the exposures from all relevant routes and compartments are considered 
when BAF values are evaluated. 

The quality of field data needs to be assessed and interpreted correctly. Difficulties in 
interpretation arise especially for trophic magnification factors (TMFs), which describe the 
accumulation throughout the whole food chain. The TMF for a food chain is calculated as the 
exponent of the slope of the natural logarithm transformed concentrations for organisms in the 
food chain as a function of the trophic level of these organisms. Currently, there is no standard 
procedure for studying TMFs. Hence, the conductance and sampling may vary considerably 

  
 
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between different studies. As such, TMF represents the average biomagnification per trophic 
level within that food chain. The validity of the TMF is strongly dependent on the spatial and 
time scales over which the samples were retrieved. The most reliable TMFs are derived from 
data for non-migratory species originating from a confined area and sampled in the same 
period, or from food chains for which low variability in time and space can be assumed (e.g. 
for vast remote areas). See also publications from Borgå et al. (2012) and ECETOC (2014) for 
discussion on uncertainties.  

The way data, on the basis of which the TMF values are calculated, are treated has a great 
impact on the outcome of the TMF value. Not only the magnitude of the TMF value can be 
impacted, but also whether biomagnification or biodilution occurs. In addition, the setup of the 
field study could have an influence on the resulting TMF values as well. These aspects cover 
both spatial and temporal variability in sampling, but also the selection of species belonging to 
the ecosystem. Spatial variability can lead to different organisms being exposed to different 
environmental concentrations. Temporal differences could have a strong impact on trophic 
magnification as well. Such temporal variability further complicates the interpretation of the 
observed TMF values. Further, it appears that TMF values could be strongly dependent on the 
inclusion or exclusion of certain species and on which part of the food chain is considered, for 
example pelagic species only or the benthopelagic food chain. Apart from that, even from 
similar food chains widely varying results can be obtained for the TMF (Houde et al., 2008).  

R.11.4.1.2.7 

Addressing uncertainty of field data in the assessment 

The uncertainties related to field data apply to all field metrics described above. If field data 
are available, these should be considered in the assessment. In particular, if the number of 
field studies is not very high, covering all different study conditions and/or species) the data 
presented should be accompanied with a comprehensive discussion on the uncertainties. The 
following elements are essential to be discussed for each study (where relevant) and when 
compiling the information from the studies together to draw an overall conclusion from the 
field studies: 

  Thorough elucidation of the food-web structure (feeding ecology; determination of the 

trophic level). The position in the food web is quantified using relative abundances of 
naturally occurring stable isotopes of N (15N/14N, referred to as δ15N). However the 
relative abundance of these isotopes and thus the determination of the trophic level and 
TMF is influenced by the physiology of the organism and its life trait history. Rapid 
growth with a higher protein demand for new tissue leads to lower enrichment factors 
than those with slower growth rates. Insufficient food supply and fasting and starvation 
leads to catabolism of body proteins and an increase of 15N in organisms relative to 
those organisms with adequate food supply; 

  Evidence to demonstrate that the steady-state has been achieved in the food web 

considered. Opportunistic feeders vary their diet over seasons or with life stage and 
point sources may influence observed TMFs. Additionally, apart from the diet there is 
always the possibility of a direct uptake of the substance under scrutiny and the relative 
importance of food versus e.g. water exposure can influence the magnitude of the TMF; 

 

Influence of sampling location(s) and timing(s), concentration gradients/migration 
behaviour;  

  Difference between poikilotherms and homeotherms (cold and warm blooded). An 

investigation of an Arctic food web revealed the unequal magnification behaviour of 
POPs within both thermal groups (Hop, 2002). These results may be explained by a 
higher food intake, caused by a higher energy demand, and a longer life span of birds 
and mammals. Intrinsic differences in gastrointestinal absorption mechanisms have also 
been suggested as an explanation for these differences between homeotherms and 
aquatic poikilotherms (Drouillard, 2000). Therefore, when the trophic magnification 

 
 
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potential of a substance is determined via a single regression for the overall food web, 
the magnification in poikilotherms may be overestimated and the magnification in 
homeotherms, in particular apex predators, may be underestimated (Fisk, 2001). 

Influence of species physiological characteristics (e.g. typical lipid content, whether air-
inhaler or water inhaler); 

Influence of digestion rate/diet energy content, size and growth, ability to biotransform, 
sex, age;  

 

 

  The number of organisms sampled at each point of the food web; 

  Sample  type.  Sample  collection  is  often  restricted  to  tissue  or  serum  samples  in  large 
predators  due  to  ethical  reasons  and  due  to  the  challenging  logistics  with  respect  to 
sampling  and laboratory  constraints.  Tissue-to-whole  body  extrapolations  of  measured 
concentrations,  where  this  cannot  be  avoided,  introduce  additional  uncertainties  which 
need to be addressed; 

  Analytical information such as detection and quantification limits; 

  Quality assurance throughout the sampling, sample treatment, storage and analysis 

(including such as blanks and spiked samples); 

  etc… 

Also where a high number of field studies are available, the discussion on uncertainties 
mentioned above may support the assessment. It should also be noted that field studies often 
sample vertebrate species. Therefore, as Annex XI to the REACH Regulation requires 
vertebrate testing to be the last resort, the need for additional field studies requires careful 
consideration for whether alternative sources (e.g., already existing stored samples from 
specimen banks) could provide the same information, particularly in the light of uncertainties 
stated above. 

Further considerations on field evaluation of bioaccumulation (with particular focus on 
terrestrial bioaccumulation) can be found in Van den Brink et al. (2016). 

R.11.4.1.2.8 

Use of a fugacity approach for bioaccumulation assessment 

The use of fugacity ratios (Burkhard et al., 2012; Mackay et al., 2013) has been proposed as a 
technique for bioaccumulation assessment. This method converts laboratory and field 
bioaccumulation metrics into a common fugacity ratio scale. However, there is a lack of 
agreement on how to interpret fugacity ratios and the method has not yet been validated 
sufficiently, for example with existing POP and PBT substances.  

The calculation of a fugacity ratio is an approximation based on certain assumptions. One of 
the assumptions made is that the partitioning to lipids is equal to the octanol-water 
partitioning and this may not always be the case. Therefore, use of a fugacity approach in 
bioaccumulation assessment under REACH cannot be recommended at this stage.  

Apart from these considerations, it must be realised that the use of fugacity ratios is only 
justified in cases of thermodynamic equilibrium between the different compartments that an 
organism is exposed to. When applied to field studies, this is seldom the case. If for example a 
ratio between biota and sediment is used as basis for the fugacity ratio the assessment might 
be strongly hampered by strong sorption to the sediment and consequently very slow 
depuration of the substance from the sediment into (pore-)water. In such cases, which for 
example could be expected for many well-known PBT substances, the fugacity ratio between 
biota and sediment will be low, while the fugacity ratio between biota and the depleted pore-
water could be high. However, also in laboratory studies, thermodynamic equilibrium between 
different exposure media (water and food) is even prevented. In both the aqueous and dietary 

  
 
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OECD TG 305 studies, fish are exposed to only one exposure route, either water or diet. The 
consequence is that the remaining medium to which fish are exposed simultaneously have 
arbitrarily a very low fugacity compared to fish and the exposure medium. 

The fugacity ratio only considers a substance of concern for bioaccumulation if there is an 
increase in fugacity, i.e. biomagnification occurs. Indeed if biomagnifications is confirmed this 
is a clear indication of bioaccumulative properties of a substance (Gobas et al., 2009). 
Nevertheless, the bioaccumulative properties of substances that do not biomagnify could be 
considered of concern as well. Polycyclic aromatic hydrocarbons (PAHs) could be considered as 
an example of this concern. These substances are very efficiently taken up in invertebrates 
with very high bioaccumulation factors. However, they are not biomagnified in higher trophic 
levels, such as fish. Still, the additional uptake due to the consumption of high concentrations 
in invertebrates can lead to significantly higher bioaccumulation factors in the field (e.g. Khairy 
et al., 2014) than would be predicted based on laboratory bioconcentration data. This example 
illustrates that high bioaccumulation in a part of the food chain may have unpredictable effects 
throughout other parts of the food chain as well. 

Even though the fugacity approach in bioaccumulation assessment under REACH cannot be 
recommended at this stage, it is noted that the approach allows various lines of evidence to be 
put into a consistent framework to apply a quantitative Weight-of-Evidence determination as to 
whether or not a substance biomagnifies.  

R.11.4.1.2.9 

Other testing data 

In the following section other testing information which may be relevant for the 
bioaccumulation assessment is discussed. It should be noted from the outset that this other 
information does not override valid information on aquatic bioaccumulation of the substance if 
the aquatic data indicate high bioaccumulation potential.  

Overall depuration rate constants in fish 

Upon prolonged exposure and after internal redistribution of a compound, the rate of 
elimination is independent of the uptake route: aqueous exposure, dietary exposure or both 
routes simultaneous as in the field. Besides that, uptake rates in fish are rather similar for 
neutral organic compounds and dependent on e.g. ventilation rates of gills for aqueous 
exposure and feeding rate for dietary exposure. So, the elimination rate is a discriminating 
factor in the bioaccumulation potential of such compounds. For this reason the half-life has 
been suggested as a useful metric for the bioaccumulation assessment (Goss, Brown et al., 
2013). 

The kinetic processes of especially bioconcentration from water, which are the uptake and 
elimination rate constants, are dependent on the size of a fish (e.g. Barber 2008, Brooke and 
Crookes, 2012). This implies that setting one value for the depuration rate constant for 
different organisms is not sufficient. If aqueous bioconcentration is considered, an uptake rate 
constant of 520 L/kg/d could be estimated for fish with a weight of 1 g (see Section R.7.10.4.1 
in Chapter R.7c of the Guidance on IR&CSA). The depuration rate constants that lead to 
bioconcentration factors of 2000 and 5000 could thus be estimated to be 0.26 d-1 and 0.10 d-1. 
For fish weighing ten grams these values would be approximately half of these values (0.12 d-1 
and 0.05 d-1). A similar limit of 0.085 d-1 for the depuration rate corresponding with a BCF of 
5000 was reported resulting from a comparison of lipid normalized BCF values with their 
corresponding depuration rate constants (Brooke and Crookes, 2012). These ranges could be 
used in interpreting and comparing data obtained from different studies (laboratory aqueous 
and dietary exposure, field exposure) in a Weight-of-Evidence approach for the assessment of 
bioaccumulation. 

 
 
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Chronic toxicity studies with mammals 

81 

If chronic toxicity studies with mammals are available, the complete absence of effects in the 
long-term is an indication that the compound is either chronically non-toxic and/or that it is 
not taken up to a significant extent. Although this is only indirect information on the uptake of 
a substance, it may be used together with other indicators, e.g. referring to non-testing 
information, to conclude in a Weight-of-Evidence approach that a substance is likely to be not 
B or vB. 

Toxicokinetic studies with mammals 

More direct information on the potential of a substance to bioaccumulate can be obtained from 
toxicokinetic studies with mammals, if available. Information on absorption, distribution, 
biotransformation and excretion of a substance in mammals may be used in a Weight-of-
Evidence approach. Information on absorption and systemic bioavailability indicate if a 
substance is taken up after the exposure and, depending on other substance properties 
influencing toxicokinetics, whether there is a possibility for bioaccumulation. Distribution 
information may indicate possible location(s) of bioaccumulation. Some substances go through 
a biotransformation (i.e. metabolism). Also transformation products may accumulate and that 
possibility needs to be scrutinised in the PBT/vPvB assessment. The elimination process of a 
substance includes metabolism and excretion. Different elimination parameters may provide 
information on the bioaccumulation potential. 

Elimination rates and half-lives are acknowledged as useful metrics indicative of the 
bioaccumulation potential (Arnot, Brown and Wania, 2014; Gobas et al., 2009; Goss, Brown 
and Endo, 2013; Gottardo, Hartmann and Sokull-Gluttgen, 2014; ECETOC, 2014; ECHA 
Member State Committee, 2015).  

There is no universal elimination process–related threshold in B-assessment available which 
would cover all (aquatic/terrestrial - water breathing/air breathing) organisms because the 
elimination rate depends on several factors (e.g. species). Nor can any more specific cut off 
criteria be recommended to compare elimination data with the B/vB criteria. Nevertheless, 
prolonged elimination half-lives may indicate the potential of a substance to bioaccumulate.  

Particular attention should be drawn to the toxicokinetic studies considered to be included in 
the PBT/vPvB-assessment. For further information, see Sections R.7.10.14 and R.7.12 in 
Chapter R.7c of the Guidance on IR&CSA.  

Please note that the use of toxicokinetic data in B-assessment is under scientific development 
and that the qualitative recommendations above are based on current knowledge and 
experience. Registrants are advised to follow-up recent the recent and future developments in 
the field, e.g. via the ECHA website. 

R.11.4.1.2.10 

Further data 

In this section several types of non-animal data are discussed that can be used in a Weight-of-
Evidence approach for the B and vB assessment. The way in which the information on 
molecular size (average maximum diameter and maximum molecular length), molecular 
weight, Log Kow, and octanol solubility should be used is briefly addressed in the following 
(background information on these parameters can be found in Appendix R.11—1). It should be 
noted from the outset that this information does not override valid information on aquatic 
bioaccumulation on the substance if the aquatic data indicate high bioaccumulation potential. 

If average molecular size, log Kow, and octanol solubility are above or below certain values (as 
described below), they can be considered as indicators for a limited bioaccumulation potential 
due to the lack of uptake. However, these parameters should never be used on its own to 
conclude that a substance is not bioaccumulative. The information from these parameters 
should be accompanied by other information confirming the low uptake of the substance in 

  
 
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living organisms, e.g. by read-across with similar substances, absence of toxicity or lack of 
uptake in toxicokinetic studies with mammals. 

Other methods such as in vitro methods or biomimetic extraction procedures may also be 
useful and are mentioned briefly at the end of the section. 

(Q)SAR models 

BCF-QSARs and other computer models may be used, provided that the model is appropriate 
for the chemical class (see Section R.7.10.3.2 in Chapter R.7c of the Guidance on IR&CSA and 
Annex 1 to Appendix R.11—1 of this guidance document). 

Read-across with other substances 

If a valid and reliable BCF value for a structurally closely-related substance is available, read-
across can be applied. In addition to the normal criteria for application of read across, when 
applying read-across data in bioaccumulation assessment, two generally important aspects 
have to be considered, which are the hydrophobicity and the centre of metabolic action for 
both substances. An important parameter for PBT and vPvB assessment is the molecular size 
of the substance since it has an influence on the bioaccumulation behaviour (see Appendix 
R.11—1). 

Molecular size 

Information on molecular size can be an indicator to strengthen the evidence for a limited 
bioaccumulation potential of a substance. One parameter for molecular size is the maximum 
molecular length of a substance. From a certain minimum length upwards  it may be assumed 
that the substance disturbs the entire interior structure of the lipid bilayer of cell membranes 
and therefore does not accumulate to a significant amount, i.e. has a BCF value lower than 
2000. Folding of long linear structures may alter the effective length of the molecule of the 
substance, which renders it more easily transferable across cell membranes. Therefore, the 
criterion for molecular length should only be used in a Weight-of-Evidence approach together 
with other information as described under "conclusion on the endpoint". In conclusion, an 
assessor may justify that, in certain cases when information on the effective length and other 
information indicating a low bioaccumulation potential is available, the criterion for B and 
hence also for vB as not being met. It is noted, that there is no agreed cut-off criterion for 
molecular length available at the moment and therefore the use of molecular length as one 
indicator of low bioaccumulation potential needs to be well justified.  

Another parameter that directly reflects the molecular size of a substance is the average 
maximum diameter (Dmaxaver). Very bulky molecules will less easily pass through the cell 
membranes. This results in a reduced BCF of the substance. From one study of a diverse set of 
substances it appeared that for compounds with a Dmaxaver larger than 1.7 nm28 the BCF value 
will be less than 2000. However, the applicability of a numeric cut-off should be considered on 
a case-by-case basis. Also, it should be noted that the estimate of molecular size depends on 
conformation of the substance as well as the method used. 

Log Kow 

For the PBT and vPvB assessment a screening threshold value has been established, which is 
log Kow greater than 4.5. The assumption behind this is that the uptake of an organic 
substance in aquatic organisms is driven by its hydrophobicity. For organic substances with a 
log Kow value below 4.5 it is assumed that the B criterion, i.e. a BCF value of 2000 (based on 
wet weight of the organism, which refers to fish in most cases), is not exceeded. 

28 Please note that the indicator value of 1.7 nm for the average maximum diameter was derived using 
the descriptor Dmax from OASIS. However, it appears from the Environment Agency (2009) that the use 
of different software tools could lead to variable results for the same substance. 

 
                                                 
 
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Care must be taken in case a substance is known to bioaccumulate by a mechanism other than 
passive diffusion driven by hydrophobicity. E.g. specific binding to proteins instead of lipids 
might result in an erroneously low BCF value if this value is estimated from log Kow. 
Perfluorinated compounds (PFCs) are examples of such partitioning behaviour, of which 
perfluorooctanoic acid (PFOA) and perfluorooctane sulphonic acid (PFOS) are well-known 
examples. 

For some groups of substances, such as organometals, ionisable substances and surface active 
substances, log Kow is not a valid descriptor for assessing the bioaccumulation potential. 
Information on bioaccumulation of such substances should therefore take account of other 
descriptors or mechanisms than hydrophobicity. 

At log Kow values between 4 and 5, Log BCF increases linearly with log Kow, if the substance is 
absorbed at the same rate and if it is not biotransformed. This linear relationship is the basis 
for the B screening threshold value of log Kow > 4.5. However, at very high log Kow (>6), a 
decreasing relationship between the two parameters is observed. Apart from experimental 
errors in the determination of BCF values for these very hydrophobic substances, reduced 
uptake due to the increasing molecular size may play a role as well. Moreover, the 
experimental determination of log Kow for very hydrophobic substances is normally also very 
uncertain due to experimental difficulties. The reliability of measured and modelled log Kow 
values > about 8 is often lower than the reliability of measured and modelled log Kow values < 
about 8. Ideally the results of several model predictions for log Kow should be considered. The 
aquatic BCF of a substance is probably lower than 2000 if the calculated log Kow is higher than 
10. Given that none of the models have experimental information in this range, more than one 
model should be used to estimate the log Kow value and the results evaluated by expert 
judgement. If a log Kow value indicates that the substance screens as B/vB, but a registrant 
concludes it is not B/vB based on other data, there should be specific reference to the REACH 
guidance indicating how such a conclusion was drawn. It should be noted that neither a high 
Koc value nor low water solubility value can be used to argue that a substance lacks significant 
bioaccumulation potential. Instead these properties may influence the form of PBT testing 
required. 

Log Koa 

For the PBT and vPvB assessment other than bioconcentration factors in aquatic organisms 
have to be considered as well. For bioaccumulation in aquatic organisms a screening threshold 
value has been established, which is log Kow greater than 4.5. Equivalent to log Kow for aquatic 
organisms, log Koa (octanol-air partition coefficient) has been recognised as a parameter 
indicating that bioaccumulation can occur in air-breathing (terrestrial) organisms.   

Available information on the combination of log Koa and log Kow as provided in the ITS, may 
indicate that the substance is potentially bioaccumulative in air-breathing organisms. In case 
such a substance is already confirmed as P or vP, it should be carefully considered whether 
aquatic bioaccumulation testing already is expected to reflect the “worst case” in terms of 
concluding on the B/vB -properties or whether it is instead more efficient to directly generate 
information on accumulation in air-breathing species.  

In case a substance screens to be potentially bioaccumulative in air-breathing organisms and 
aquatic bioaccumulation testing indicates no bioaccumulation, further information and 
potentially further assessment on bioaccumulation in air-breathing organisms may be 
necessary. This could include monitoring data, mammalian toxicokinetics data (see Section 
“Toxicokinetic studies with mammals” above) and other information for air-breathing 
organisms as described above.  

Reporting of log Koa is not required under REACH but it can be calculated based on the 
information available in the registration dossier: Kow and Henry’s Law Constant (H). In case H 
is also unavailable, it can be estimated based on water solubility (WS), vapour pressure (VP), 
and molecular weight (MW). An efficiently absorbed, non-biotransformed neutral organic 
substance with a log Koa ≥ 5 in combination with a log Kow ≥ 2 has the potential to biomagnify 

  
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in terrestrial food chains and air-breathing marine wildlife as well as in humans, while the 
substances with log Kow < 2 are being quickly eliminated by the urinary excretion, and 
therefore do not biomagnify even though their Koa is high (Armitage and Gobas, 2007; Kelly et 
al., 2007; Gobas et al., 2009; McLachlan et al., 2011; Goss et al., 2013). 

The precise values for the Kow and Koa values indicated in the ITS are a function of the 
modelled organisms, food webs and environments used to obtain these values (e.g., Kelly et 
al., 2007; Armitage and Gobas, 2007). Furthermore, all of the studies used to develop these 
partition coefficient combinations have emphasized that these partitioning property 
combinations relate to biomagnification potential only when predicated by the assumptions of 
high chemical absorption efficiency from the diet and no biotransformation after absorption 
and negligible active transport (in or out). In particular, considerations for absorption efficiency 
and biotransformation rates are thus also necessary for bioaccumulation assessment. Whole 
body half-lives (see e.g. Goss et al., 2013) and biotransformation rates (see e.g., Armitage 
and Gobas, 2007) have been proposed that would counteract biomagnification potential. 
However, these toxicokinetic values to mitigate biomagnification are a function of the defined 
conditions in which they were derived. 

For example, for the soil-earthworm-shrew food-chain a model illustrates that substances with 
a log Koa > 5.25 and with a log Kow between 1.75 and 12 have a biomagnification potential 
unless they are metabolized at a sufficiently rapid rate, e.g., in excess of 0.3 d-1 or a half-life 
time of 2.5 d for shrews (Armitage and Gobas, 2007). Evaluative, representative 
biomagnification models for adult humans (e.g., Goss et al., 2013; Arnot et al., 2014) have 
indicated that biotransformation half-lives of about 70 days or faster may be sufficient to 
mitigate biomagnification potential. The differences between the half-lives required to mitigate 
biomagnification potential in the two systems (shrews and humans) relate primarily to 
differences in maximum gastrointestinal biomagnification and bioenergetics (Kelly et al., 2004; 
de Bruyn and Gobas, 2006) and body size (ca. 0.01 kg for shrews vs. ca. 70 kg for humans), 
i.e. allometry in physiological and metabolic processes (e.g. Hendriks et al., 2001), 
emphasizing the requirement for context-specific data. However, it should be noted that the 
above mentioned cut-off values for elimination rates/half-lives are not currently recommended 
to be used in the B-assessment. Development of an approach to better understand 
toxicokinetic information is necessary and on-going (see also subsection “Toxicokinetic studies 
with mammals” above).  

If sufficiently reliable and condition-specific data for chemical absorption efficiency and 
biotransformation rates are available from in vivo, in vitro or in silico methods, such data can 
be used to parameterize the models for terrestrial bioaccumulation assessment. As necessary, 
in vitro data and in vitro to in vivo extrapolation models can be used for evaluating substances 
that have Kow values lower than the BCF screening threshold values (i.e., log Kow < 4.5 and > 
2), but with log Koa values greater than about 5.5. In vitro methods for mammals are 
reasonably well-established as a result of decades of pharmaceutical testing and development 
(see below) and technical challenges relating to the solubility of such substances are expected 
to be minimal, i.e. substances with log Kow <4.5 are generally amenable to in vitro testing. 
Additionally, in silico models for hepatic (Pirovano et al., 2016) and whole body clearance 
(Arnot et al., 2014; Berellini et al., 2012) may also provide valuable insights for 
bioaccumulation assessment of substances that fall into the aforementioned chemical 
partitioning range (2 < log Kow < 4.5 and log Koa > 5.5). The absorption efficiency is another 
critical parameter that can mitigate the biomagnification potential indicated by the proposed 
Kow and Koa values. In general, and when deemed necessary, combining the relevant 
information in the form of a mass balance bioaccumulation or toxicokinetic model is 
recommended.  

Octanol solubility 

Octanol is often used as a surrogate for fish lipids. With a low solubility in octanol, the Log Kow 
and hence the BCF can be either high or low, depending on the water solubility of the 
substance. Therefore, the solubility in n-octanol is not a parameter that is directly related to 
the BCF value. However, if the solubility of a substance in octanol is so low that the maximum 

 
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concentration levels that can be attained in organisms do not reach levels sufficient to elicit 
any toxic effects, it can be reasoned that such accumulation would not be of concern. The 
concentration of a substance at which the occurrence of toxic effects normally can be excluded 
is 0.002 mmol/L in n-octanol. Furthermore, octanol solubility is only an indicator for 
substances accumulating in fatty tissues and certain substances may bind to proteins instead 
of partition into lipids. Finally, information on octanol solubility should in particular be 
accompanied and complemented by information on mammalian toxicity or toxicokinetics to 
confirm the absence of uptake and/or chronic toxicity. 

In vitro data on biotransformation in fish  

In vitro methods such as fish liver S9 and primary hepatocyte assays provide information on 
metabolism and hence biotransformation in the organism. Because biotransformation is 
considered to be the dominant mechanism of elimination of hydrophobic substances, such in 
vitro tests have potential to support the assessment of bioaccumulation and may contribute to 
a reduction in (or refinement of) animal testing. For further details, see Section R.7.10.3.1 In 
vitro data on aquatic bioaccumulation in Chapter R.7c of the Guidance on IR&CSA.  

In evaluating the test results of an in vitro test care must be taken that the dissipation of the 
substance indeed relates to biotransformation. As the current procedures for in vitro 
metabolism tests are not suitable for constructing a mass balance, it cannot be excluded that 
the dissipation may be due to other processes. Especially for potential PBT substances that 
have generally a very low water solubility, the dissipation might be caused by processes such 
as adsorption and volatilisation. 

To estimate a BCF the in vitro metabolism rate constant is extrapolated to an overall in vivo 
metabolism rate constant. This overall rate constant is used to calculate a kinetic BCF from the 
kinetic rate constants k1 (gill uptake rate constant), k2 (gill elimination rate constant), kD 
(dietary uptake rate constant), kE (faecal egestion rate constant), kM (metabolic rate constant), 
kG (growth rate constant), which are defined for the whole fish. The more hydrophobic a 
substance is, the slower the internal redistribution kinetics between lipid compartments and 
blood will become, which will likely reduce the overall metabolism rate. The in vitro to in vivo 
extrapolation to estimate the overall metabolism rate constant seems to be insufficiently 
validated at present for highly hydrophobic substances.  

Biomimetic extraction procedures 

Biomimetic extraction procedures with semi-permeable membrane devices (SPMD) and solid 
phase micro extraction (SPME) are used to mimic the way organisms extract substances from 
water. These types of methods are at the moment only well described for hydrophobic 
substances. For more detailed information, see Section R.7.10.3.1 in Chapter R.7c of the 
Guidance on IR&CSA. 

R.11.4.1.2.11  Conclusion on the endpoint 

A substance meets the B or vB criterion if it is considered bioaccumulative or very 
bioaccumulative in one or more of the relevant food chains or receptors, e.g. the aquatic 
environment, the terrestrial environment or wildlife or humans. To determine these 
classifications, all reliable and relevant information on the bioaccumulation potential of a 
substance has to be gathered by the registrant and considered in the CSA, including the 
PBT/vPvB assessment. If available, such information might be sufficient to conclude whether 
the substance is vB, B, or not B. 

 

If the substance has a log Kow lower than 4.5 and no specific mechanism of uptake apart 
from hydrophobic partitioning is known and the possibility for accumulation in other food 
chains than the aquatic food chain can be ruled out, then the substance can be considered 
as not B and not vB. In such a case further evaluation of the B and vB criteria is not 
necessary. A partitioning process other than lipophilic partitioning could for example be the 

  
 
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binding to proteins. The possibility of a substance to accumulate in air-breathing 
organisms instead of aquatic organisms is indicated by the combination of a log Koa > 5 
with a log Kow >2. A high metabolism rate for the substance could mitigate such a 
potential for bioaccumulation in air-breathing organisms. 

 

If the substance has very limited potential to be taken up by biota, this might be indicated 
by several factors based on substance properties listed below. These indicators should be 
confirmed by other information to exclude the possibility of a high bioaccumulation 
potential. If such a lack of significant uptake is proven, the substance can be considered as 
not B and not vB. In such a case, further evaluation of the B and vB criteria is not 
necessary. It should be noted that the only conclusion drawn based on this information is 
that the substance is not (very) bioaccumulative, and not that the substance can’t be 
taken up at all. A substance is unlikely to meet the B criterion (i.e. unlikely to have a BCF 
> 2,000) if some or all of the following indicators are met: 

1.  an average maximum diameter (Dmax aver) of greater than 1.7 nm28  

2.  octanol-water partition coefficient as Log10 (Log Kow) > 10 (calculated 
value, preferably by several estimation programs, for substances for 
which Log Kow can be calculated and the model is reliable) 

3.  a measured octanol solubility (mg/L) < 0.002 mmol/L × MW (g/mol) 
(without observed toxicity or other indicators of bioaccumulation) 

Indicator 1. recommended here as non-testing information influences uptake and 
distribution of substances. The log Kow (2.) is a general indicator for uptake, distribution 
and excretion whereas the octanol solubility (3.) reflects the potential for mass storage, 
which might further prevent uptake in significant amounts in the organism.  

The supplementary information to confirm this limited uptake may comprise data from a 
chronic toxicity study with mammals (≥ 90 days, showing no toxicity), a toxicokinetic 
study with mammals or birds, a bioconcentration study with invertebrates, or reliable 
read-across from a structurally similar compound (all showing no uptake). These types of 
information should be examined in a Weight-of-Evidence approach together with the non-
testing information on the substance to conclude whether the B or vB criteria are met. 
Evidence of significant uptake of a substance in vertebrates after prolonged exposure is a 
contra-indication to using the above indicators. This approach is based on the report 
provided in Appendix R.11—1. 

 

If there is a reliable aqueous bioaccumulation study available, such as an aqueous 
exposure OECD TG 305 study, the result from this test can be directly related to the 
criteria for B and vB. If the BCF is higher than 2000 or 5000 the substance can be 
assigned to be B or vB. If a reliable BCF is lower than the B criterion (BCF < 2000), this is 
an indication of reduced uptake or metabolism for hydrophobic substances with a Log Kow 
> 4.5. Rapid metabolisation of a substance may lead to a lower BCF value. Methods such 
as fish liver S9 and fish hepatocyte assays may be useful as supporting information, but in 
vitro data alone should not be used to conclude on lack of bioaccumulation potential at the 
present point in time. However, further research in future may increase the predictive 
capacities of in vitro methods. Reduced uptake and metabolism will most likely also 
mitigate the bioaccumulation potential in general. If there are no other indications for 
accumulation outside the pelagic food chain, such as elevated concentrations in terrestrial 
and air-breathing organisms, the substance can be considered as not B and not vB. Such a 
conclusion could also be drawn for substances having log Kow < 4.5. However, in that case 
additional consideration should be given to the possibility of accumulation in food chains 
containing air-breathing organisms or humans. 

 

The results of a dietary bioaccumulation study with fish, such as an OECD TG 305 dietary 
exposure study, can be used in a similar way to that described above to conclude on the B 
criterion. However, because there are no direct criteria to compare the outcome of the 
dietary exposure test with B criterion, such a conclusion can only be drawn if the 

 
Chapter R.11: PBT/vPvB assessment 

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87 

substance clearly fulfils the B criterion or clearly does not (i.e. both the benchmarking 
approach in which BMF are compared to BMFs for known POPs and PBTs obtained under 
the same conditions and the method to derive a BCF calculated from the depuration rate 
from the dietary study in combination with an estimated uptake rate constant warrant a 
conclusion not B, B, or vB).  

 

In some cases, a conclusion can be drawn from additional information only. This could be 
information from field studies showing clear accumulation in a food chain, or long half-lives 
from monitoring studies in humans or wildlife. Often, this type of information yields 
variable results, which renders it insufficient to draw a conclusion on the bioaccumulation 
potential. Instead, this type of information will merely be used in a Weight-of-Evidence 
approach to support results from other studies.  

In any other case, no conclusion on the bioaccumulation potential can be drawn and the B and 
vB properties should be evaluated in more detail. Based on the above described information, 
this refers to the following cases: 

 

 

no direct information on bioaccumulation (e.g. BCF, BAF or BMF data) are available and 
the substance has a Log Kow higher than 4.5, or the partitioning process into aquatic 
organisms is not driven by lipophilicity. 

information on bioaccumulation is available for aquatic compartment indicating that 
substance is not B, but the screening information indicates potential to bioaccumulation in 
air-breathing organisms and no conclusion could be derived for them based on available 
data. In this case new information may need to be generated on bioaccumulation potential 
in air-breathing organisms (mammals), e.g. by appropriate testing or by generating 
suitable biomonitoring data, based on a case-by-case assessment of the needs.  

 

direct data on bioaccumulation are available but these data are not reliable and/or 
consistent to a degree sufficient to conclude whether the B or vB criteria are met. 

  Toxicity assessment (T) 

R.11.4.1.3.1 

Integrated testing and assessment strategy (ITS) for T-testing in 
support of PBT assessment for the aquatic environment 

In this section guidance on the recommended testing and assessment strategy is provided as 
an annotated flow chart (Figure R.11—5). The strategy is based on the T criteria (Table R.11—
1), which state that the T criterion is fulfilled if at least one of the data types listed in the 
criteria is fulfilled. If P and B criteria are fulfilled, information would need to be generated until 
for each (eco)toxicity data type it is clear whether the criterion is fulfilled or not. 

  
 
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Step 1 

yes 

T 

Start 

Classified H350, 
H340, H372, H373, 
H350i, H360, H361 or 
Acute data E(L)C50 < 
0.01 mg/L 

no 

Step 2 

Step 5 

yes 

No further 
assessment 
necessary 

Acute data 
E(L)C50 
 < 0.1 mg/L ? 

yes 

Potentially T 

no 

Carry out screening 
assessment for P 
and B: Log Kow < 
4.5 or readily 
biodegradable ? 

no 

Step 6 

yes 

Not T 

Other convincing 
evidence chronic 
T > 0.01 mg/L ? 

Step 3 

no 

P and B 
confirmed ? 

no 

No further 
assessment 
necessary 

Step 4 

yes 

Chronic T studies (order and 
selection of test organisms, 
see main text) 

Not T 

no 

NOEC or EC10 
< 0.01 mg/L ? 

yes 

PBT 

Figure R.11—5: T testing in support of PBT assessment for the aquatic environment. 

 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

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89 

According to Article 14 of REACH, PBT assessment starts at levels ≥ 10 t/y (it is assumed that 
at least acute algae, daphnia and fish data are available): 

Step 1: Assessment of mammalian toxicity data and acute aquatic toxicity data; 

 

 

IF classified or likely to be classified as carcinogenic (cat. 1A or 1B), germ cell 
mutagenic (cat. 1 or 1B) or toxic to reproduction (class 1A, 1B or 2) or STOT RE 1, 
STOT RE 2 or any EC50 or LC50 < 0.01 mg/L, THEN define the substance as T and stop 
assessment  
IF not classified or likely to be classified as carcinogenic (cat. 1A or 1B), germ cell 
mutagenic (cat. 1A or 1B) or toxic to reproduction (cat. 1A, 1B or 2) or STOT RE 1, or 
STOT RE 2 or any EC50 or LC50 ≥ 0.01 mg/L, THEN move to step 2. 

Step 2: Assessment of acute aquatic toxicity data; 

 

 

IF any EC50 or LC50 < 0.1 mg/L, THEN the substance is a Potential T candidate. Move to 
step 3. 
IF all EC50 or LC50 ≥ 0.1 mg/L, THEN it needs to be confirmed that this is not a false 
negative (i.e. a substance with possibly a high chronic toxicity). Move to step 5. 

Step 3: Consider outcome of P and B assessment* (Note.: it is considered good practice to 

assess P, B and T in that order) 

 
 

IF P and B confirmed, THEN proceed to Step 4 (chronic T testing) ** 
IF confirmed not P or not B, THEN STOP 

Step 4: Chronic T testing (on fish, daphnids, algae). The approach here is that chronic aquatic 

toxicity testing should be firstly carried out on non-vertebrate species, unless there 
are indications that fish is the most sensitive group (NB: it is not defined in this ITS 
how to rank the sensitivities) 

 
 

IF NOEC or EC10 < 0.01 mg/L, THEN PBT confirmed 
IF NOEC or EC10 ≥ 0.01 mg/L, THEN not T, and STOP 

Step 5: Screening of the substance for P and B * 

 

 
 

IF Log Kow ≤ 4.5*** or other B-cut-off criteria met, and no other indications are 
available that the substance might bioaccumulate in other ways than by absorption to 
lipids, then not B and STOP. 
IF substance is readily biodegradable, then not P and STOP 
IF Log KOW > 4.5 AND not readily biodegradable, THEN move to step 6 

Step 6: Other long term T-evidence (e.g. by means of read across and Weight-of-Evidence or 
group approach) 

 
 

IF chronic toxicity cannot be excluded, THEN move to step 3 (P & B confirmation) 
IF strong evidence for non-T properties, THEN STOP. 

*  For specific guidance on the identification of P & B substances, please refer to Section R.11.4.1.1 for 

persistence and Section R.11.4.1.2 for bioaccumulation 

** If B is likely but vB is not and a reliable BCF is not available, consider conducting tests on 

invertebrates to check the T status for these organisms before considering tests on fish (either for 
chronic toxicity or for obtaining a BCF). 

*** Care must be taken in case a substance is known to bioaccumulate by a mechanism other than 

passive diffusion   driven by hydrophobicity; e.g. specific binding to proteins instead of lipids might 
result in an erroneously low bioaccumulation potential if it is estimated from Log Kow.  

Care must also be taken for substances classified as polar non-volatiles (with low Log Kow and high 
Log Koa). This group of substances has a low bioaccumulation potential in aquatic organisms but a high 
bioaccumulation potential in air-breathing organisms (unless they are rapidly metabolised). 

  
 
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The toxicity criterion 

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According to Section 1.1.3 of Annex XIII to REACH, a substance is considered to fulfil the 
toxicity criterion (T) when: 

 

 

 

the long-term no-observed effect concentration (NOEC) or EC10 for marine or freshwater 
organisms is less than 0.01 mg/L; or 
the substance meets the criteria for classification as carcinogenic (category 1A or 1B), 
germ cell mutagenic (category 1A or 1B), or toxic for reproduction (category 1A, 1B or 2) 
according to the CLP Regulation; or 
there is other evidence of chronic toxicity, as identified by the substance meeting the 
criteria for classification: STOT RE 1, or STOT RE 2 according to the CLP Regulation. 

For the assessment of aquatic toxicity, EC10 values are preferred compared to NOEC values for 
deriving long-term toxicity to marine or freshwater organisms29.  

The evidence of CMR and chronic toxicity specified above does not only refer to substances 
that are already classified accordingly (i.e. DSD R-phrases: R45, R46, R48, R49, R60 – R63 or 
CLP hazard statements H350, H340, H372, H373, H350i, H360 and H36130)31 but also implies 
an obligation to check whether the criteria for assigning the respective classifications are 
fulfilled in accordance with the provisions of Annex I to REACH (Section 1.3 Step 3: 
Classification and Labelling)32. If any classification criterion leading to the assignment of the 
mentioned classifications is met, the substance fulfils the T criterion and there is no need to 
perform any further aquatic studies for T assessment. If data are available for birds these 
cannot be directly (numerically) compared with the T criterion (see Section 1.1.3 to Annex 
XIII). However, reprotoxicity studies or other chronic data on birds, if they exist, should be 
used in conjunction with other evidence of toxicity as part of a Weight-of-Evidence 
determination to conclude on the substance toxicity (a NOEC of  30 mg/kg food in a long term 
bird study should in this context be considered as strong indicator for fulfilling the T criterion). 

The rest of this document is limited to testing of the T criterion on the basis of evidence from 
aquatic tests. 

Due to animal welfare concerns, the general scheme of testing is sequentially first P, B and 
then T if there are no specific reasons for deviation from that sequence. Furthermore, 
vertebrate animal testing should be generally minimised by first testing non-vertebrate species 
if data from invertebrates are equivalent to vertebrate data in the context of the PBT/vPvB-
assessment. This is the case for aquatic toxicity testing but not for the B testing. For 
determination of whether a substance fulfils the criteria for aquatic toxicity, and in the absence 
of any long-term ecotoxicity data on aquatic species, a 21-d Daphnia reproduction test (OECD 
TG 211) would normally be the preferred test to perform with the few exceptions described 
later in this section where the results from short-term tests can already lead to concluding that 
the criteria are fulfilled. Under most circumstances, the T criterion of 0.01 mg/L (NOEC or 
EC10) can be compared to results from tests listed in REACH annexes VII to X. Existing data 

29 An OECD workshop (OECD, 1998) recommended that the NOEC should be phased out from 
international standard. Indeed, concerns were expressed about deciding to abandon the NOEC since it 
may not be sufficiently protective because of the danger of false negatives. According to the Report of 
the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (OECD, 1998), NOECs are leading to 
misunderstandings, misinterpretations and NOECs are statistically unfounded. 

30 H360 and H361 here include also all the possible combinations (e.g H360F, H360FD, etc). 

31 See Annex VII to CLP – (translation table from classification under DSD to classification under CLP) 

32 The criteria for classification of substances and mixtures in hazard classes and in their differentiations 
is provided in Annex I to the CLP Regulation, Mixtures must be classified and labelled according to the 
CLP Regulation  from 1 June 2015 but may be classified according to Directive 1999/45/EC until then.  

 
                                                 
 
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91 

from other equivalent test methods must be assessed on a case by case basis based on the 
recommendations described in the effects assessment methodology. 

As the aquatic T criterion is based on a NOEC or EC10 for pelagic organisms, the standardised 
chronic tests on fish, daphnids and algae are preferred to assess the NOEC or EC10. However, 
for poorly water-soluble substances, the feasibility of performing a test via the water phase 
needs to be considered carefully. Such a study may be technically difficult to perform as the 
substance will partition out of solution, especially if it is known to partition strongly to 
sediment and suspended solids. In such cases, it may be both impractical and uninformative to 
test pelagic species via the water phase. Tests with sediment dwelling species may provide 
more useful information on the toxicity of the substance in the compartment in which it will be 
mainly found. However, the T criteria do not include a chronic value for sediment as only NOEC 
or EC10 values related to pelagic toxicity are accounted for in Annex XIII. A possible way to 
determine whether a substance has equivalent toxicity in sediment to that in the water column 
could be to extrapolate the sediment toxicity value (e.g. NOEC) to a pelagic toxicity value by 
assuming that sediment toxicity occurs mainly through the pore water and using the 
equilibrium partitioning (EqP) theory. The EqP theory is normally used to calculate a 
PNECsediment from a pelagic PNECwater (see Section R.7.8 in Chapter R.7b of the Guidance on 
IR&CSA).  

However, the EqP theory may also be used to back-calculate a NOEC or EC10 value of an 
existing sediment test to a corresponding pelagic NOEC or EC10. The pelagic NOEC or EC10 
derived can then be compared with the T criterion of 0.01 mg/L given in Annex XIII. The 
sediment concentration equivalent to a pelagic NOEC or EC10 value of 0.01 mg/L increases 
linearly with the suspended matter-water partitioning coefficient (see Section R.7.8 in Chapter 
R.7b of the Guidance on IR&CSA). 

To check whether the T criterion of 0.01 mg/L is fulfilled, the equation for the equilibrium 
partitioning method used in order to calculate the PNECsediment is slightly revised: 

Equation 11-1 

NOEC(EC10)water (mg.L-1) 
Kpsusp (L.kg-1
NOEC(EC10)sed dw (mg.kgdw

 dw) 

-1) 

Kpsusp (L.kg-1
Focsusp is the mass fraction of organic carbon in dry suspended matter. 

 dw) can be estimated from the Koc of the substance as Kpsusp= Focsusp.Koc where 

It should be noted that NOECsed derived from experimental studies are given in dry weight (as 
mg/kg dw).  

As the equilibrium between sediment and water is influenced by the suspended solid-water 
partition coefficient (Kpsusp), it is necessary to calculate the T criterion for each substance, 
using its own partitioning coefficient. 

For substances with water solubility below 0.01 mg/L, a chronic limit test (Csed,lim) can be 
performed at the spiked sediment concentration that is calculated to be at equilibrium with the 
water solubility limit of the test substance. 

Equation 11-2 

Cwatersol (mg.L-1) 
Kpsusp (L.kg-1
 dw) 
Csed,lim (mg.kg-1 dw) 

suspdw ,sed,watersolKpECNOECECNOEC)10()10(suspwatersollimsed,KpCC.  
 
 
 
  
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If no chronic effects are found from this limit test, the result can be regarded as experimental 
evidence that the substance does not meet the pelagic T criterion for invertebrates provided 
that the equilibrium partitioning theory holds in the particular case (for guidance on the 
limitations of the equilibrium partitioning method, see Section R.7.8.10.1 in Chapter R.7b of 
the Guidance on IR&CSA). However no final conclusion on pelagic toxicity can be drawn if no 
further reliable toxicity data on fish and algae are available. If chronic effects are found then 
this is an indicator that T could be met in a pelagic test and consideration should be given to 
further testing (although care has to be taken at high spiking concentrations that the test 
substance does not cause indirect effects, e.g. by oxygen depletion as a result of 
biodegradation). 

R.11.4.1.3.3 

Use of QSAR data 

Only a few QSAR models predicting chronic aquatic toxicity are available but further research 
on the QSAR prediction of chronic toxicity may increase their predictive capacities. Therefore at 
the current state of the art, QSAR models generally seem not to be applicable for an 
unequivocal assessment of the T criterion. However, it should be noted that the registrant is, 
within the frame of Annex XI to REACH, allowed to make use of QSARs when they are 
applicable. 

R.11.4.1.3.4 

Screening information and screening threshold values 

If only screening information is available for the PBT/vPvB assessment, screening criteria listed 
in Table R.11—6 can be used for screening. It should be noted that these criteria are indicative 
and further description on the application of these criteria is provided below. 

Table R.11—6: Screening threshold values for toxicity. 

Screening information*** 

Conclusion 

Toxicity 

Short-term aquatic toxicity  

EC50 or LC50 < 0.01 mg/L**** 

T, criterion considered to be  

(algae, daphnia, fish)* 

definitely fulfilled 

Short-term aquatic toxicity  

EC50 or LC50 < 0.1 mg/L**** 

Potentially T 

(algae, daphnia, fish)** 

* From acute tests.  
** From acute tests or valid/applicable QSARs. 
*** The screening assignments should always be considered together for P, B and T to decide 
if the substance may be a potential PBT/ vPvB candidate. 
**** These threshold values only apply for the aquatic compartment. 

A substance is considered to potentially meet the criteria for T when an acute E(L)C50 value 
from a standard E(L)C50 toxicity test (REACH Annexes VII to X) is less than 0.1 mg/L. In 
addition to data from standard toxicity tests, data from reliable non-standard tests and non-
testing methods may also be used if available. These data should be particularly assessed for 
their reliability, adequacy, relevance and completeness (see Chapter R.4 of the Guidance on 
IR&CSA). 

The toxicity criterion (T) for PBT assessment cannot be decided upon the basis of acute studies 
alone. If the screening threshold value is met, the substance is referred to T testing and 
chronic studies are needed unless E(L)C50 < 0.01 mg/L. Normally, the testing order for 

 
 
 
 
 
 
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conclusion on T based on chronic data is Daphnia and then fish33. If the T-criterion is fulfilled 
by the chronic algae or Daphnia data, a chronic fish test is not necessary and should therefore 
not be carried out as it would be an unnecessary vertebrate animal test. 

For certain lipophilic substances (with a Log Kow > 4) acute toxicity may not occur at the limit 
of the water solubility of the substance tested (or the highest concentration tested). In such 
situations, chronic toxicity with a NOEC/EC10 < 0.01 mg/L cannot be excluded. Therefore, it 
may not be possible to draw a screening conclusion for T (see decision tree for aquatic 
endpoints, steps 2, 5 and 6, and Figure R.11—5). 

In the absence of conclusive information on T, for substances with very high lipophilicity, a 
Weight-of-Evidence or grouping approach for long-term toxicity may be used to predict 
whether long-term effects are likely to occur. If convincing evidence is available that aquatic 
toxicity is not expected to occur at < 0.01 mg/L, chronic testing may not be required. Such 
evidence should be based on expert judgement and Weight-of-Evidence of data including 
reliable QSAR predictions/read-across/grouping approaches indicating a narcotic mode of 
action together with measured low chronic fish toxicity from a related substance. Supporting 
information could be chronic data on aquatic species such as, e.g., daphnids, algae or 
sediment dwelling species and/or low acute or chronic mammalian and avian toxicity. 

If data from this approach provide insufficient evidence that toxicity will not occur in a chronic 
test a conclusion on the P and B properties should be drawn before further T-testing is 
considered. If the substance is found to be both P and B, a chronic study is required (testing 
order see above).  

In choosing the appropriate test organism, the data from the available base set of toxicity 
tests for algae (acute / chronic), Daphnia (acute) and fish (acute) should be evaluated under 
consideration of the possible hydrophobic properties of the test substance, and hence the 
expected time to steady-state. Any specific mode of action of the test substance also needs to 
be considered.  

If it can be concluded that one taxonomic group is significantly more sensitive than the others, 
e.g. because there is evidence for a specific mode of action, this sensitive group should be 
chosen for chronic testing and conclusion on the T-properties34. If no conclusive evidence for 
significant differences in sensitivity between the groups can be found the testing order as 
mentioned above applies. 

If the relevant test species is selected in accordance with the suggested approach in the 
paragraph above, lack of toxicity at or below the T criterion for the tested species is evidence 
that further studies on T are not necessary. If however a long-term test on Daphnia or algae 
provides a NOEC close to but above 0.01 mg/L, a long-term fish study is likely to be needed to 
confirm “not T” unless, taking into consideration the above-mentioned approach, convincing 
evidence exists that the fish NOEC will be higher than 0.01 mg/L. Supporting evidence in such 
considerations could be an acute fish value that is a factor of 10 or more greater than that of 
the other two trophic levels under the provision that the acute daphnid test showed toxicity at 
least one order of magnitude lower than the limit of solubility. 

Certain chemical characteristics (such as high adsorption or extremely low solubility) are likely 
to make any toxicity testing extremely laborious if not technically impossible. Guidance has 
been developed by OECD on toxicity testing of difficult substances (OECD, 2000b)35. Some 

33 Algae are not mentioned here because chronic algae data (i.e. 72h NOEC) normally will be available, 
as it can be easily obtained from the same 72h standard test from which the acute endpoint (72h EC50) 
is derived. 
34 This could mean that no further testing is necessary if it is concluded that algae are significantly more 
sensitive than daphnids or fish and the available chronic algae data are well above a NOEC of 0.01 mg/L. 

35 As of December 2016, the OECD "guidance document on aquatic toxicity testing of difficult test 
chemicals" is under revision. The revised version will introduce additional recommendations for poorly 

  
                                                 
 
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examples together with recommendations to overcome the technical difficulties are provided in 
the chapter on assessment of problematic substances (see Chapter R.7b of the Guidance on 
IR&CSA).  

R.11.4.1.3.5  Water accommodated fraction (WAF) 

For any substance with very low water solubility, all efforts should first be made to produce a 
reliable and stable test concentration. Only if this is not feasible due to the properties of the 
substance or due to disproportionate efforts, can the water accommodated fraction (WAF) be 
considered as last resort to generate exposure in a test (OECD, 2000b; Girling et al., 1992, 
see also Appendix R.7.8-1 in Chapter R.7b of the Guidance on IR&CSA). Test results are 
expressed as a lethal or effective loading that causes a given adverse effect after a specified 
exposure period. For complex multi-constituent substances, the principal advantage of this test 
procedure is that the observed aquatic toxicity reflects the multi-component dissolution 
behaviour of the constituents at a given substance to water loading. Expressing aquatic toxicity 
in terms of lethal loading enables multi-constituent substances comprised primarily of 
constituents that are not toxic to aquatic organisms at their water solubility limits to be 
distinguished from substances that are more soluble and which may elicit aquatic toxicity. As a 
consequence, this test procedure provides a consistent basis for assessing the relative toxicity 
of poorly water soluble substances. Effects concentrations in tests based upon WAFs can be 
calculated from (1) the loading rates and are identified as either LL50 or EL50 values and/or (2) 
the measured mass of test substance in the WAF and are identified as either LC50 or EC50 
values. LL50 or EL50 values are comparable to LC50 or EC50 values determined for pure (i.e. 
mono-constituent) substances tested within their solubility range. Similarly the NOEC (No 
Observable Effect Concentration) becomes the NOELR (No Observable Effect Loading Rate). 
The statistical methods used to determine LL50, EL50 and NOELR values are the same as those 
used to determine LC50, EC50 and NOEC values. The WAF procedure has been adopted for use 
in environmental hazard classification (for acute and long-term hazard classification) (OECD, 
2000b; UNECE, 2003). Poorly soluble substances that exhibit no observed chronic toxicity at a 
substance loading of 1 mg/L indicate that the respective constituents do not pose long term 
hazards to the aquatic environment and, accordingly, do not require hazard classification 
(CONCAWE, 2001; UNECE 2003). By its nature the WAF-method is testing several 
constituents. Where toxicity is exhibited, this can be problematic when a test substance 
containing several constituents is used. In such a case, the toxicity cannot be allocated to 
specific constituents directly, but the interpretation of the results (given that use of WAF is the 
last resort) should be supported by use of other data, such as QSAR –values or read-across 
values from a structurally similar substance. The loading cannot be compared to the toxicity 
criterion. Only in the case of analytical verification of the water-soluble fraction this type of 
tests might be used in the T assessment. 

R.11.4.1.3.6 

Use of non-testing data 

At preliminary stages in the assessment, in cases where no acute or chronic toxicity data are 
available, the assessment of the T criterion at a screening level can be performed using data 
obtained from quantitative structure activity relationships (QSARs) for acute aquatic toxicity as 
described in Table R.11—6. In order to be suitable, the QSAR prediction should comply with 
the general principles described in Chapter R.6.1 of the Guidance on IR&CSA. Long-term 
testing is required if QSAR estimations indicate that the substance fulfils the screening 
threshold values for T (EC50 or LC50 < 0.1 mg/L). It may, on a case by-case-basis, be decided 
whether confirmatory chronic testing on fish is necessary if valid QSAR prediction indicates 
that the acute E(L)C50 is < 0.01 mg/L. Alternatively either first an acute fish toxicity limit test 
could be performed to check whether the acute toxicity is below 0.1 mg/L or the QSAR-
prediction could be accepted as providing sufficient evidence of the T criterion being fulfilled.  

water-soluble chemicals, and in particular with regard to the use of liquid/liquid saturator units and of 
passive dosing. 

 
 
                                                                                                                                                                  
 
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If the substance is confirmed to fulfil the P and B criteria, testing on long-term toxicity should 
be performed to determine whether the substance meets the criteria for T. Alternatively, 
QSARs for chronic toxicity, if applicable, may be used by the registrant to conclude that the 
substance fulfils the T criterion, but normally, due to the uncertainties of the present QSAR-
models, not for concluding “not T”. 

When considering the use of non-testing data, it is important for substances containing 
multiple constituents, impurities and/or additives, to consider first the appropriate assessment 
approach provided in Section R.11.4.2.2. 

  
 
 
 
 
 
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  Conclusions on PBT or vPvB properties 

A detailed analysis of the Persistence, Bioaccumulation and Toxicity should be brought 
together into a clear overall conclusion.  Three conclusions for the comparison of the relevant 
available information on the PBT properties with the criteria listed in REACH Annex XIII Section 
1 are possible. 

(i) 

(ii) 

The substance does not fulfil the PBT and vPvB criteria. The available 
information show that the properties of the substance do not meet the specific 
criteria provided in REACH Annex XIII Section 1, or if the information does not allow 
a direct comparison with all the criteria there is no indication of P or B properties 
based on screening information or other information. 

The substance fulfils the PBT or vPvB criteria. The available information show 
that the properties of the substance meet the specific criteria detailed in REACH 
Annex XIII Section 1 based on a Weight-of-Evidence determination using expert 
judgement comparing all relevant and available information listed in Section 3.2 of 
Annex XIII to REACH with the criteria. 

(iii) 

The available data information does not allow to conclude (i) or (ii). The 
substance may have PBT or vPvB properties. Further information for the PBT/vPvB 
assessment is needed. 

The sub-chapters below provide more details on the circumstances that would lead to each of 
these conclusions. The consequences of each conclusion for the registrants are described in 
Section R.11.3.  

The prerequisite for drawing a correct overall conclusion is that the endpoint –assessments 
described in Sections R.11.4.1.1, R.11.4.1.2 and R.11.4.1.3 are carried out and concluded 
correctly. Additionally, the assessment described in Section R.11.4.2.2 for substances 
containing multiple constituents, impurities and/or additives needs to be carried out in such 
manner that the principles for choosing an approach are fulfilled (see Section R.11.4.2.2 for 
details). A very high number (tens) of combinations of end-point conclusions is possible. . If a 
substance contains multiple relevant constituents, impurities and/or additives, the overall 
picture may be highly complex. In such cases the overall conclusion(s) can be best presented 
by providing conclusion tables for all relevant constituents, impurities and/or additives (or 
fractions, where relevant). 

R.11.4.1.4.1 

(i) The substance does not fulfil the PBT and vPvB criteria. The 
available information show that the properties of the substance 
do not meet the specific criteria provided in REACH Annex XIII 
Section 1, or if the information does not allow a direct 
comparison with all the criteria there is no indication of P or B 
properties based on screening information or other information.  

This would be the case if, as a result of an analysis of existing data, or of data generated after 
conclusion (iii) any one of the parameters, i.e. environmental degradation half-life in an 
appropriate environmental compartment, the BCF for aquatic species or, in the case of a 
decision on PBT, long-term aquatic toxicity and the appropriate human health hazard 
classification do not meet the criteria in Annex XIII. 

In many cases, the information available, while not allowing a direct comparison with the 
criteria in Annex XIII, can be considered sufficient for a decision to be made, by applying 
Weight-of-Evidence based expert judgement, that the substance is not PBT/vPvB. Such would 
for instance be the case if the screening threshold values as provided in Section R.11.4 were 
not met for any particular endpoint based on screening information. Furthermore, when the 

 
 
 
 
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screening threshold values for persistence or bioaccumulation as defined in the following sub-
sections are not fulfilled, further PBT/vPvB assessment can stop when there is a well justified 
lack of counter evidence which would raise concern for the substance to have PBT or vPvB 
properties. In this case, the registrant can also draw the conclusion (i).  

It has to be kept in mind that the fact that a substance does not meet the T criterion is not a 
sufficient basis on which to stop the evaluation of the remaining endpoints in the PBT/vPvB 
screening step.  

Where supplementary information is available, such as sufficient evidence based on  
monitoring data, that indicates that a particular property, such as persistence or high 
bioaccumulation may in fact be present, a cautious approach should be followed and 
conclusion (iii) may need to be drawn (see below). 

When drawing conclusion (i), the registrant should show in the PBT/vPvB assessment that 
there is no indication that the relevant constituents, impurities, additives or 
transformation/degradation products do not have PBT or vPvB properties.  

It should be noted that where toxicity is a critical parameter for PBT assessment, i.e. the 
substance is persistent and bioaccumulative but there are insufficient (only acute valid) toxicity 
data, it will be necessary to conduct further testing (unless the registrant decides to treat the 
substance “as if it is a PBT or vPvB”). In such cases, the assessor must choose conclusion (iii) 
instead of conclusion (i). 

R.11.4.1.4.2 

(ii) The substance fulfils the PBT and/or vPvB criteria. The 
available information show that the properties of the substance 
meet the specific criteria detailed in REACH Annex XIII Section 1 
based on a Weight-of-Evidence determination using expert 
judgement comparing all relevant and available information 
listed in Section 3.2 of Annex XIII to REACH with the criteria (for 
more specific terminology, also used in IUCLID, please, see 
subsection “Terminology”).  

In principle, substances are only considered as PBT or vPvB when they are deemed to fulfil the 
PBT or vPvB criteria for all inherent properties. This would be the case if, as a result of an 
analysis of existing data, or of data generated after concluding that further information is 
needed (conclusion iii), the environmental degradation half-life in an appropriate 
environmental compartment, the BCF for aquatic species or a comparable metric and, in the 
case of a decision on PBT, long-term aquatic toxicity or an appropriate human health hazard 
classification show the criteria to be met. The data must show that all three criteria are met in 
the case of PBT, or both vP and vB criteria in the case of vPvB. In this context it is important to 
note that even where one criterion is marginally not fulfilled but the others are exceeded 
considerably, the assessor may, based on a justification relying on the available evidence and 
considering weigh-of-evidence, conclude in specific cases that the substance fulfils the Annex 
XIII criteria.  

If a constituent, impurity or additive of a substance fulfils the PBT/vPvB properties (based on 
the assessment of the registrant or of ECHA), a ≥0.1 % (w/w) threshold applies for concluding 
the substance as fulfilling the same PBT or vPvB criteria. For substances containing PBT/vPvB 
constituents, impurities or additives in individual amounts <0.1 % (w/w) of the substance, the 
same conclusion need not normally be drawn. This is in line with the threshold used for 
considering PBT and vPvB substances in mixtures (Article 14(2)(f) of REACH).  

Furthermore, where a substance contains a high number of constituents, impurities or 
additives <0.1% (w/w) which are structurally similar and therefore can be considered together 
as a fraction, the concentration limit is considered to apply for the fraction. This in particular 
applies to highly complex substances where all or most individual constituents are present in 
concentration <0.1 % (w/w) but also to other substances containing blocks of similar 
constituents whereby the assessment efforts should remain proportionate (for further details, 

  
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please, see Section R.11.4.1 on “Relevant constituents, impurities, additives and 
transformation/degradation products” and Section R.11.4.2.2).  

Additionally, there may be other particular cases for which specification of percentages below 
0.1% is required. This requirement is then driven by the toxicological profile of the constituent, 
impurity or additive (e.g. high potency carcinogenic, mutagenic or reprotoxic (CMR) and the 
provisions for classification and labelling and not by the fact that the respective constituent is 
concomitantly a PBT/vPvB. If a substance (its constituents, impurities or additives) degrades 
or is transformed into transformation/degradation products which fulfil the PBT or vPvB criteria 
(based on the assessment of the registrant or of ECHA) and if these are formed in relevant 
amounts, the substance is concluded to fulfil the PBT or vPvB criteria. The definition of 
“relevant” transformation/degradation product for the registrant’s substance is provided in 
Section R.11.4.1. Authorities should justify case by case what they consider as relevant 
transformation/degradation in their PBT/vPvB assessments. Terminology provided at the end 
of this section must be applied in the registration dossier to the substance subject to PBT/vPvB 
assessment to distinguish which of the cases above the substance represents.  

Overview of case types of conclusion (ii) 

The following differentiation is used for substances which have to be concluded to fulfil the PBT 
and/or vPvB criteria: 

  The substance is PBT/vPvB. This conclusion is drawn because this is a mono-constituent 
substance and it has a main constituent present at a concentration of 80% or more with 
PBT and/or vPvB properties; 

 

 The substance is PBT/vPvB. This conclusion is drawn because this is a mono-constituent 
substance, well-defined multi-constituent substance or UVCB substance. and it contains 
one or more relevant36 (group(s) of) constituent(s)37 which fulfil the PBT and/or vPvB 
criteria38;  

  The substance is PBT/vPvB. This conclusion is drawn because one or more (group(s) of) 

constituent(s), impurity or additive of the substance degrade(s) or is/are transformed into 
substance(s) which fulfil the PBT and/or vPvB criteria and these transformation or 
degradation products are formed in “relevant”36 amounts.  

  Combination of two or all of the above types. 

It should be noted that there is no difference in risk management between the different types. 
The consequences of conclusion (ii) for the registrant are described in Section R.11.3.  

36 “Relevant” is defined in section R.11.4.1. 

37 “Constituent” as referred to in Annex XIII of REACH means “constituent”, “impurity” or “additive” as 
described in the Guidance for identification and naming of substances under REACH and CLP.  

38 The terminology corresponds with IUCLID 6 section 2.3 terminology. The constituent(s) or constituent 
group(s) fulfilling the PBT/vPvB criteria should be specified in specific endpoint study records in section 
2.3 of IUCLID. 

 
 
 
 
                                                 
 
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99 

R.11.4.1.4.3 

(iii) The available information does not allow to conclude (i) or 
(ii). The substance may have PBT or vPvB properties. Further 
information for the PBT/vPvB assessment is needed. 

The consequences of this conclusion for the registrant are described in Section R.11.3.3.  

This conclusion is derived when one or more of the following combinations of endpoint–specific 
conclusions apply: 

Potential P/vP + Potential B/vB + any T -conclusion 
Potential P/vP + B but not vB + Potential Teco 
Potential P/vP + B but not vB + Potential Thh 
Potential P/vP + B but not vB + Teco 
Potential P/vP + B but not vB + Thh 
Potential P/vP + vB + any T -conclusion 
Potential P/vP + B/potential vB + any T -conclusion 

P/potential vP + Potential B/vB + any T -conclusion 
P/potential vP + B but not vB + Potential Teco 
P/potential vP + B but not vB + Potential Thh 
P/potential vP + vB + any T -conclusion 
P/potential vP + B/potential vB + any T -conclusion 

P but not vP + Potential B/vB + Potential Teco 
P but not vP + Potential B/vB + Potential Thh 
P but not vP + Potential B/vB + Teco 
P but not vP + Potential B/vB + Thh  

P but not vP + B/vB + Potential Teco 
P but not vP + B/vB + Potential Thh 

vP + Potential B/vB + Any T-conclusion 

vP + B + Potential Teco 
vP + B + Potential Thh 

Where the data on the PBT properties of a substance do not allow a direct (numerical) 
comparison with the criteria specified in Annex XIII, but there are nevertheless indications 
from other data such as screening data, that the substance may be PBT/vPvB, then it is 
necessary to consider which information is needed to draw a final conclusion. 

Where it is concluded that further information is needed, consideration should first be given to 
clarifying the persistence of the substance since persistence is a critical property in 
determining PBT/vPvB properties and since degradation testing does not involve the use of 
vertebrate animals39.  

Once the new information is available, comparison with the criteria in Annex XIII should be 
carried out according to the principles described above and a decision be taken on whether the 
substance falls under conclusion (i) (is not a PBT/vPvB) or (ii) (i.e. is a PBT/vPvB). In certain 
cases the revised assessment may again lead to the conclusion that further information still 

39 Depending on the substance properties it may, however, be appropriate to consider bioaccumulation 
testing first. Guidance on the general approach to P, B and T testing is given in Section R.11.4. 

  
 
 
 
 
 
 
 
 
 
                                                 
 
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needs to be generated. If for one of the relevant constituents, impurities, additives or 
transformation/degradation products there is indication that it may have P and B properties, 
the registrant should draw conclusion (iii) and generate the necessary additional information 
until the available information allows to draw one of the two ultimate conclusions in relation to 
the whole composition (see Section R.11.4.1 for description of “relevant” and Section 
R.11.4.2.2 for the relevant assessment approaches). 

There may be cases where a clear decision on the properties of a substance cannot be made, 
but there are indications from available information that the substance may fulfil the PBT or 
vPvB criteria. In these cases conclusion (iii) applies. For instance, where there is a reason to 
expect that a substance may contain a known PBT constituent ,  impurity or additive (or 
fractions thereof) but it is not possible to characterise a substance identity to an extent that 
will allow the registrant to state with enough confidence that his substance does not contain 
PBT/vPvB constituents/impurities/additives or that it does not generate 
degradation/transformation products with PBT/vPvB properties above the relevant threshold 
levels as specified in Section R.11.4.1.  

Finally, there may be cases where it is simply technically not possible to conduct testing, either 
at screening or at confirmatory level and therefore not possible to derive conclusion (i) or (ii). 
If there are no indications or justification which would exclude the possibility that the 
substance could potentially fulfil the criteria, conclusion (iii) should be drawn. 

 
  
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R.11.4.2  Assessment of PBT/vPvB properties – consideration of specific 

substance properties 

  Assessment of substances requiring special considerations with 

regard to testing 

For substances that have exceptional properties (e.g. very high sorptivity, very low water 
solubility, or high volatility), or which consist of multiple constituents, test guidelines used to 
determine persistence, bioaccumulation and toxicity in the PBT/vPvB assessment may not be 
directly applicable. Instead specific testing and assessment strategies may be warranted. 

R.11.4.2.1.1 

Substances with very high sorptivity 

The assessment strategy should be applicable to strongly sorbing substances in general. For 
illustrative purposes certain antioxidants are used as examples (see List of Antioxidants,    
Appendix R.11—2). 

General considerations 

In Appendix R.11—1 indicators for limited bioaccumulation are described. For substances with 
very high calculated Log Kow, e.g. > 10, reduced bioaccumulation is expected. Log Kow values > 
8 cannot be measured reliably due to technical issues and need therefore to be calculated by 
property estimation methods based on the concept of Linear Free Energy Relationship (LFER). 
Before using a specific LFER method the extent to which the structural elements of the 
substance under consideration are covered by the applicability domain of the LFER needs to be 
checked. For example, organometallic substances like tin organics may not be covered 
whereas the corresponding carbon analogue of the substance is. 

It is very important to realise that the calculated Log Kow values > 10 are used simply to 
indicate a degree of hydrophobicity that is extreme. Such values should not be used in a 
quantitative manner. 

Assessment steps 

STEP 1 

Calculated / measured Log Kow 

Check/generate the calculated / measured Log Kow of the substance of interest. 

STEP 2 

Assessment type to be applied 

If the Log Kow is < 10 an assessment of P, B and T should follow the standard approach as 
described in Section R.11.4.1. 

If the Log Kow is > 10 it should be checked if available ecotoxicity and / or mammalian data 
do not meet the T criteria. If the T criteria are not met, a specific vPvB assessment might be 
applicable as described below. 

If for a substance with Log Kow > 10 data are available demonstrating toxicity in accordance 
with the T criteria for PBT substances, then a standard PBT assessment as described in Section 
R.11.4.1 is warranted. 

STEP 3 

vPvB Assessment for substances with Log Kow > 10 

Step 3a  Persistence check 

Substances with transformation potential 

If the substance can be transformed abiotically or biotically (e.g. when it has structural 
moieties like ester groups, phosphites or phosphonites see Appendix R.11—2,  

  
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Table R.11—10, Antioxidants No. 2, 4, 6-17 as examples) it should be checked if a specific 
biodegradation test at low concentrations and specific analysis or a specific hydrolysis test (see 
Section R.7.9.4 in Chapter R.7b of the Guidance on IR&CSA) could be carried out to 
demonstrate transformation with a primary half-life of < 40 d. In such circumstances, the 
transformation products will need to be checked to ensure they do not have PBT or vPvB 
properties. If the substance is transformed into substances not having PBT or vPvB properties 
it can be considered not to fulfil the vPvB criteria. In this case Step 3b can be omitted. 

Substances with limited transformation potential 

If a substance may not be easily transformed based on the structure (e.g. it has no ester 
functions or the transformation rate is limited by very low (bio)availability) it is nevertheless 
recommended to estimate the metabolic pattern, using e.g. Catabol (Mekenyan, 2006). For all 
relevant metabolites it must be checked that they do not fulfil the criteria for PBT or vPvB 
substances. For these substances Step 3b is mandatory. 

Step 3b  Bioaccumulation check for substances with limited transformation potential 

The low bioaccumulation potential indicated by the Log Kow > 10 should be supported by 
additional information (see Appendix R.11—1 “Indicators for limited bioaccumulation”). This 
information may comprise results from an animal study (mammalian or fish) confirming no or 
low bioaccumulation. 

Log Kow >10 and at least one additional indicator for limited bioaccumulation 

If for a substance with Log Kow > 10 at least one additional criterion (1. or 2.) mentioned 
above is fulfilled the substance should not be considered as vPvB, provided that potential 
metabolites are themselves not PBT or vPvB. 

Log Kow >10 and no additional indicator for limited bioaccumulation 

If none of the additional criteria (1. or 2.) mentioned under Step 3b is met, then an 
appropriate test as described in Section R.11.4.1.2 is warranted. 

STEP 4 

Overall conclusions 

Log Kow >10 and ready biodegradability in a specific biodegradation confirmed 

No further investigation necessary, if metabolites are neither PBT nor vPvB. In this case the 
(parent) substance is not vPvB. 

Log Kow >10 and no ready biodegradability confirmed 

If at least one additional indicator for limited bioaccumulation is fulfilled and potential 
metabolites are not PBT or vPvB, then the substance is not vPvB. 

If no additional indicator for limited bioaccumulation is fulfilled a standard vPvB assessment as 
described in Section R.11.4.1 is warranted. 

Examples for the above assessment strategy are presented in Appendix R.11—2 “Assessment 
of substances requiring special consideration during testing”. 

R.11.4.2.1.2 

Substances with low solubility in octanol and water 

The assessment strategy should be applicable to substances with low solubility in octanol and 
water and for which lipid is the target compartment for accumulation in organisms. For 
illustrative purposes certain organic pigments are used as examples (see List of Pigments, 
Table R.11—12, in Appendix R.11—2).  

 
 
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103 

It should be noted that these examples are presented under the assumption that the named 
pigments would not have specific nanoform -related properties. Whether the assumption is 
correct or not is not relevant for the purpose of the examples. 

General considerations 

1) Critical body burden (CBB) concept and octanol solubility 

In Appendix R.11—1 “Indicators for limited bioaccumulation” it is described how octanol 
solubility could be used in the B assessment (Critical Body Burden approach) as well as the 
limits of the approach. 

As octanol is a reasonable surrogate for fish lipid, a low substance concentration in octanol 
may indicate reduced bioconcentration / bioaccumulation potential. The concept is based on 
available measurements for substances using a safety factor of 10 for the uncertainty of the 
available CBB measurements. It is proposed that where a substance shows no specific mode of 
action and has a 

Coctanol [mg/L] < 0.002 [mMol/L] x Mol weight (g/Mol) 

Equation 11-3 

it can be assumed that the compound has only a limited potential to establish high body 
burdens and to bioaccumulate. If it does bioaccumulate, it would be unlikely to rise to levels in 
biota that would cause significant effects. 

2) Octanol water partitioning 

For substances with very low solubility specific methods exist to derive a Kow, e.g. OECD TG 
123 slow stirring method. However, this method is not always applicable due to experimental 
constraints caused e.g. by the low solubility and the available analytical methods. 

Kow values derived from fragment based LFER methods like KOWWin (US EPA, 2000) often 
overestimate the actual Kow of such substances e.g. organic pigments (Table R.11—7). In order 
to overcome the difficulties in measuring the Kow, the solubility in octanol (Co) and water (Cw) 
may be determined separately. With these solubilities the quotient Log Co/Cw can be 
calculated. This quotient is not exactly identical to Log Kow, as the latter is related to the 
partitioning of the substance in water-saturated octanol and octanol-saturated water. For 
Pigment Yellow 12, Log Co/Cw as well as Log Kow (from solubility measurements using water-
saturated octanol and octanol-saturated water) have been determined as 2.1 and 1.8, and 
hence being in the same order of magnitude (see Table R.11—7). This single comparison 
between Log Co/Cw and Log Kow needs further verification but the figures available for Pigment 
Yellow 12 can be interpreted as follows: as water saturation in octanol diminishes the octanol 
solubility of the substance and octanol saturation in water enhances the water solubility, the 
Log Kow of the substance should normally be smaller than Log Co/Cw (see values for Pigment 
Yellow 12, Appendix R.11—2, Table R.11—15). A measured Log Co/Cw = 4.5 would mean that 
the measured Log Kow should be < 4.5. 

In Table R.11—7 solubility data are given for some other organic pigments as well. The 
comparison of the measured quotient Log Co/Cw with estimated Log Kow using KOWWIN (US 
EPA, 2000) shows that the estimated Kow exceeds Co/Cw by between 1 and 8 orders of 
magnitude (more data see Appendix R.11—2). 

  
 
 
 
 
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Table R.11—7: Solubility of some pigments and comparison of their Co/Cw values 
with estimated Kows 

(US EPA, 2000) 

Colour Index 
Name 

Mol weight 
(g/Mol) 

Co (µg/L)    
at ambient 
temperature 

Cw (µg/L)   
at ambient 
temperature 

Log Co/Cw 

Log Kow 
(KOWWin) 

Pigment Yellow 12 

630 

Pigment Red 122 

340 

Pigment Red 168 

464 

Pigment Red 176 

573 

Pigment Violet 23 

589 

48* 

50 

600 

124 

15 

330 

0.8 

0.4 

19,6 

10,8 

1,9 

25 

1.8* 

2.1 

1,5 

1,1 

0,9 

1,1 

7,1 

2,5 

7,1 

7,3 

9,4 

* values relating to saturated solvents = water saturated octanol, octanol saturated water, this Log 
Co/Cw corresponds to Log Kow. 

3) Additional Indicators to be used for the ‘B’ Assessment 

As described in Appendix R.11—1 “Indicators for limited bioaccumulation”, additional indicators 
for low bioaccumulation potential, such as results from an animal study (mammalian or fish) 
confirming no or low uptake into the organism, might also be applicable for substances with 
low solubility in octanol and water. 

Assessment steps 

STEP 1 Solubility measurements for Substances with low Octanol & Water Solubility 

For the determination of the water solubility the column elution method and the flask method 
exist (OECD TG 105) but it needs to be checked which one is the most appropriate (Section 
R.7.1.7 in Chapter R.7a of the Guidance on IR&CSA). No OECD Guideline exists for the 
measurement of the octanol solubility but in principle the OECD TG 105 methods may be used 
in adapted form. 

STEP 2 B and T Assessment 

The octanol solubility of the substance is compared with the critical body burden (CBB) 
according to equation (1) given above using the Mol weight of the substance. 

Result 2A: Co < CBB 

If the octanol solubility is below the CBB, the maximum uptake of the substance can be 
expected to be below the CBB and toxicity is not likely. 

Animal studies should, in addition, be checked to confirm reduced uptake and low toxicity. In 
this case the substance has low bioaccumulation potential and low toxicity. 

Result 2B: Co > CBB and Log Co/Cw ≤ 4.5 

If the octanol solubility is above the CBB a build-up to a critical concentration of the substance 
in lipid cannot be excluded and additional information on adsorption is required. If the quotient 
Log Co/Cw of measured solubilities is ≤ 4.5 (if measurable / available) a reduced uptake is 
expected as well. Animal studies should, in addition, be assessed to confirm reduced uptake 

 
 
 
 
 
 
 
 
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and low toxicity. In this case the substance can be considered to have low bioaccumulation 
potential. 

Result 2C: Co > CBB and Log Co/Cw > 4.5 

For this substance a standard approach of P, B and T assessment as described in Section 
R.11.4.1 must be applied. No conclusion on B and T can be drawn. 

In addition indicators like molecular weight and average size of the molecule and reduced 
uptake in mammalian studies should be checked for further evidence, if necessary, and be 
used in a Weight-of-Evidence approach. 

STEP 3 Weight-of-Evidence approach for Results 2A & 2B 

Based on the results of Step 2 (2A and 2B) a Weight-of-Evidence approach with the elements 
Co, CBB, Log Co/Cw, possibly molecular weight and Dmax (size) as well as ecotoxicity and 
uptake behaviour in animal studies, is warranted to demonstrate that the substance is not a 
vPvB or PBT substance. An example for this type of assessment and conclusion is presented in 
Appendix R.11—2 under “2. Example for an assessment strategy for substances with low 
octanol and water solubility”. 

  
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  Assessment of substances containing multiple constituents, 

impurities and/or additives 

Annex XIII to the REACH Regulation requires that relevant constituents are taken into account 
in the PBT/vPvB assessment. Section R.11.3.2.1 describes registrants’ obligations in this 
matter and Section R.11.4.1 (under “Relevant constituents, impurities, additives and 
transformation/degradation products”) provides ECHA’s interpretation of the term “relevant”. 

This section gives recommendations on how to assess a substance containing several/many 
constituents, impurities and/or additives. In the following the term “constituent” is used to 
cover all these, in line with the legal text. A particular emphasis is given to UVCB substances, 
but the guidance should be applied by analogy for those well-defined substances40 which 
contain several/many relevant constituents.  

The assessment stages, listed briefly below, are the same as for assessing pure (i.e. mono-
constituent) substances but contain some additional features due to the complexity of 
assessment. The additional features are highlighted in bold and discussed in the 
corresponding subsections. The purpose of these additional features is to enhance the 
assessment efficiency by showing ways to use the limited information normally available on 
different constituents and to help in building an effective strategy for generating further 
information, where needed. Ultimately this helps to avoid the elaborate option of taking into 
account – i.e. assessing – all relevant constituents individually. 

  Gathering of available information: similar requirements as for any substance under REACH 
apply. However, for substances containing multiple constituents specific attention needs to 
be paid that all relevant information on identity and properties of the constituents and on 
the whole substance is gathered. In addition, specific attention needs to be paid that all 
relevant information on the test item identity/composition is gathered in order to be able to 
assess to which extent the gathered data actually represents the registered substance. 

  Assessment: 

o  Initial profiling of the substance composition for the purpose of the PBT/vPvB 
assessment, including profiling of the unidentified constituents/constituent fractions 
using available information on substance identity 

o  Assessment using one or more of the assessment approaches described below. If the 

approaches and principles defined in this section are correctly applied, guidance in 
sections R.11.4.1.1, R.11.4.1.2 and R.11.4.1.4 can be applied to the target “entities” of 
assessment and testing but additionally also taking into account specific aspects of 
assessing substances containing multiple constituents.   

 

If necessary, generation of further information: For the purpose of further 
specification of identity of specific constituents or fractions of constituents. It should 
be noted that the PBT/vPvB assessment may eventually require characterisation of 
constituents or fractions of constituents to a level beyond what is normally 
sufficient and necessary to identify constituents of the registered substance 
according to section 2 of Annex VI to the REACH Regulation. However, the level of 
detail to be pursued is also dependent on the feasibility and proportionality of 
efforts and is therefore case dependent. 

  Testing selected constituent(s)/fractions of constituents (or in well justified cases 
the whole substance) for necessary properties. For substances containing various 
constituents the choice of appropriate test items is essential. Furthermore, the 

40 For definition of UVCBs, well-defined multi-constituent and mono-constituent substances, please see 
the Guidance for identification and naming of substances under REACH and CLP.  

 
                                                 
 
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order in the normal tiered testing strategy (P first, then B, then T) may in some 
cases be changed, depending upon the ease and cost of generating such data and 
animal welfare considerations. Testing process may, e.g. start after a P and B–
screening assessment with B–testing of the most relevant fractions with appropriate 
analytical characterisation of all constituents. Based on these results the specific 
fractions tested in degradation and ecotoxicity tests could be narrowed further. Due 
to animal welfare considerations such reverse order of testing should, however, 
only be carried out when it is likely that B-testing will anyway be needed and that 
the reverse order does in no case lead to more vertebrate testing than what would 
be the case when starting with degradation testing. 

o  Next tier of the assessment will include change/modification of the assessment 

approach, where needed, and repetition of the previous steps, if needed. 

o  Conclusion (see Section R.11.4.1.4). 

R.11.4.2.2.1 

Initial profiling of the substance composition  

The complexity of the composition differs greatly between substances. Even for some UVCBs, 
the composition may be fully known. For other UVCBs as well as for large fractions of 
impurities of well-defined substances knowledge of the exact composition may be limited. 

The Guidance for identification and naming of substances under REACH and CLP prescribes 
that unknown constituents are reported as far as possible by a generic description of their 
chemical nature for the identification of a substance. This description must be fit-for-purpose 
in light of determining the properties of the substance. For the PBT/vPvB -assessment, the 
description of these unknown constituents needs to be provided to the level of detail making 
screening PBT/vPvB -assessment possible and feasible. Type and expected variation of 
constituents (in terms of chemical groups or classes) will determine the level of detail. For 
example, for petroleum substances it would be hydrocarbon class, like mono-aromatics, n-
alkanes, etc... For UVCB substances of botanical origin (e.g. essential oils) it could be 
terpenoid blocks, such as "monoterpene" and "sesquiterpene", subdivided by the appropriate 
functional descriptors "hydrocarbon", "alcohol", "ketone", etc and/or carbon skeletons 
"acyclic", "monocyclic", "bicyclic", etc… 41 The limitations of the analytical methods and 
proportionality of efforts to make other related information available may define the achievable 
level of detail and are case dependent. Therefore, the level of detail to be used to describe the 
constituents will vary from substance to substance and is case dependent. However, the level 
of available detail should allow defining chemical classes/functions present or modelling of the 
individual structures present.  

Descriptors such as identity of the chemical functionalities present, molecular weight range, 
carbon number range, etc. may be useful as specifications. In some cases, these constituents 
may be best reported as a group (e.g. ‘alkanes, C10-13, chloro’ or “sesquiterpene 
hydrocarbons, C15H24”). Raw material(s) and manufacturing process details may help in 
generating the necessary information on substance composition. Profiling of the composition 
with new methods, e.g, as reviewed by Dimitrov et al. (2015) is recommended for the purpose 
of filling the data gaps at screening level.  

41 For further guidance provided by the fragrance industry, please, see: 
http://echa.europa.eu/support/substance-identification/sector-specific-support-for-substance-
identification/essential-oils 

  
 
 
                                                 
 
 
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An example of an initial profiling strategy of a fraction of unidentified constituents is given 
below:  

1.  Assess the available data that is used to characterise/describe the substance. 

Information derived by chemical identity characterisation is of highest value, but if such 
cannot be derived for technical feasibility reasons, other information sources can also 
be used. For example boiling point range is typically one of the main descriptors of 
petroleum substances and, if used combined with other more specific manufacturing 
information, it can be used to generate a list of structures that could reasonably be 
predicted to be present in the substance. For example with petroleum substances this 
would probably be hydrocarbon classes within specified chain lengths, degree of 
branching,  and content of (iso)alkane, cyclic and aromatic constituents. For other 
classes of similar substances that are also UVCB (e.g. many surfactants, essential oils, 
halogenated mineral oil derived UVCBs) the composition could potentially be described 
as the distribution of non-polar and polar functional groups, as a function of molecular 
weight or chain length. Halogenated UVCBs could be described based on the nature of 
halogenation, chain length, degree of branching, saturation, cyclic and aromatic 
constituents and degree and nature of halogenation. Whatever approach is used to 
characterise the composition of the UVCB substance, a scientific and technical 
justification should be provided. 

2.  Determine the structures that are to be used as representative structures of each 
fraction for which full analytical identification is not available, detailing why these 
structures are regarded as representative and, if possible, give the approximate 
concentrations of the fraction for which they are considered representative. 

3. 

In general it would not be necessary to generate representative structures if it were 
possible to demonstrate that the fraction for any representative structure were present 
at less than 0.1%. In practice this may be difficult to achieve. 

R.11.4.2.2.2 

Assessment approaches 

Below the approaches which are recommended to be applied are described. These approaches 
are based on the idea that different “parts” (i.e. constituents or constituent fractions) of the 
substances are assessed separately (see the concept of “Assessment entity” 42), unless the 
whole manufactured/imported substance is consisting of such similar constituents, that read 
across criteria can be applied amongst them for the purpose of the PBT/vPvB assessment. 
Whichever approach is considered suitable for a particular substance, the assessment 
document should contain a clear justification for the choice. Issues related to feasibility and/or 
proportionality of efforts may play a role in the choice of the assessment approach in addition 
to the technical elements listed under each approach. These should also be duly described in 
the assessment document, where appropriate.  

The approaches described below do not necessarily cover all possible cases exhaustively, 
hence there may be situations where a different approach, not described below, could be 
justified.  

42 Presentation by Magaud H et al. at SETAC Europe 25th Annual Meeting (3-7 May 2015 - Barcelona, 
Spain): Abstract 311 available at: 
https://c.ymcdn.com/sites/www.setac.org/resource/resmgr/Abstract_Books/SETAC-Barcelona-
abstracts.pdf).  

 
 
 
 
 
                                                 
 
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“Known constituents” –approach 

109 

This can be applied when a substance is “a priori” known to contain specific constituents at 
relevant concentrations, these constituents are suspected based on available information to 
represent the worst case of the (v)P, (v)B and T properties of all constituents of the substance, 
and these specific constituents can be isolated or separately manufactured or otherwise 
acquired for the purpose of testing.  

In this approach, the known constituents of the substance are first subjected to screening 
assessment individually. Hereby assessment approaches applied to pure (i.e. mono-
constituent) substances can be applied (e.g. using experimental data, read across, QSARs). 
Specific constituents that are considered to be (the most) suspected ones with regard to the 
PBT/vPvB properties are targeted in the further steps. Testing, if necessary, is done by using 
individual constituents (or their surrogates) as test items. Each selected constituent is 
assessed for its P, B and T status, on its own, using available data on that constituent (or on 
read across–substances, if justified). The fact that a constituent can be more easily isolated or 
manufactured than another constituent may play a role in the choice of the constituent for 
assessment and testing but that should not be taken as the main criterion to test this specific 
constituent. The need to test a constituent should be driven by its relevance and 
representativeness for the overall PBT assessment of the substance (or fraction addressed).  

In this approach known constituents present at ≥0.1 % w/w concentration in the substance 
should normally be considered as relevant (see section R.11.4.1 for further discussion on the 
concentration limit). The substance can be deemed as “not PBT/vPvB” if none of the relevant 
constituents individually is PBT or vPvB. This does not mean that all known constituents need 
to be tested but step-wise assessment and testing is crucial for focussing on the known 
constituents which represent the worst case in relation to the PBT/vPvB properties among all 
constituents of the substance.  

In the opposite situation, if at least one of the relevant constituents meets the combination of 
P, B and T or vP and vB screening criteria, the assessment needs to progress to testing of 
those individual constituents following the normal P-, B- and then T-testing strategy. If one or 
more of the constituents are proven to fulfil either the vPvB or PBT criteria, the entire 
(registered) substance must be concluded as “The substance fulfils the PBT and/or vPvB 
criteria” and the (group(s) of) constituent(s) causing this conclusion must be specified in the 
dossier. 

This approach has been applied, e.g., in the SVHC identification of substances originating from 
coal tar distillation (e.g., coal tar pitch, high temperature; anthracene oil). It was also applied 
e.g. for phenol, styrenated (EC 262-975-0) under Substance evaluation.  

Advantages of the known constituents-approach are, i.a.: 

  Actual tests are performed on a pure (i.e. mono-constituent) discrete organic 

substance, and are easy to perform and interpret; 

 

 

In addition to being the preferred option, this approach may be the most efficient option 
in cases where substances contain constituents with diverse properties; 

It may in some cases require less effort to characterise the composition of the 
substance than the fraction profiling approach described below; 

  The specific constituents may in some cases already be known for their properties and 

hence assessment effort can be reduced. 

Disadvantages of the known constituents -approach are, i.a.: 

 

In many situations requires greater analytical ability to characterise the composition of 
the substance at the start of the PBT/vPvB assessment than the “whole substance -
approach” described below; 

  May require synthesis or other type of generation of specific constituent(s) for testing, if 

  
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not otherwise available (e.g., from commercial providers of laboratory grade 
standards);  

  May require more than one test for each P, B, T –endpoint. This might raise testing 

costs and needs for vertebrate testing ; 

  Requires justification that any representative constituent chosen for testing is a 

reasonable worst case. 

“Fraction profiling” (or “block profiling”) approach 

This approach is applied when, due to the complexity of the substance, it is not feasible to fully 
identify, assess or isolate single constituents but the substance can be divided into 
fractions/blocks, in which the constituents are structurally similar or in which the constituents 
are to such extent similar that their degradation, bioaccumulation and toxicity properties can 
be predicted to follow a regular predictable pattern(e.g., C14 chlorinated n-alkane with a 
chlorine content of 50-52 % by weight43). A prerequisite for application of this approach is that 
the PBT/vPvB-properties are assumed to be the same in the fraction (in this case the fraction 
should behave with regard to the PBT/vPvB-concern as if it were a single constituent or in a 
predictable manner relative to the single constituents) or to follow a regular – predictable - 
pattern. The assessment report should justify why the constituents in the blocks can be 
considered to be sufficiently similar for the purpose of the PBT/vPvB assessment. For the 
purpose of testing, an actual physical fractionation or separate manufacturing of a fraction of 
the substance may be carried out to derive appropriate test substance(s) (for more details, see 
the subsection R.11.4.2.2.4 below). 

A useful way to approach and document the assessment of the different fractions is via a 
matrix of the different blocks vs. P, B and T properties.  

Two possible variations of this approach are described below: 

i.  The substance is conceptually divided into fractions containing similar constituents 
based on structural fragments and/ or other relevant molecular descriptors. The 
fraction itself is the main target of the testing and assessment, not individual (or 
surrogate) constituents therein, as is the case in the method described below in (ii).  

This approach can be applied in particular to complex UVCBs, however, application to 
other UVCBs or large impurity fractions of well-defined substances may also in some 
cases be appropriate. This approach has been used in the PBT assessment of, e.g. EC 
no 293-728-5 under the previous legislation and is applied in several ongoing 
PBT/vPvB assessments of the MSCAs (e.g., “tetrabutane”, EC 292-461-1; medium 
chain chlorinated paraffins, EC 287-477-0).  

One example of this approach is where the substance is conceptually divided into 
fractions containing constituents having the same degradation behaviour (e.g. based 
on ready biodegradation tests). For these fractions the P assessment is clarified. The 
fractions identified as potentially P/vP  may  then  b e   divided  f u r t h e r   into  fractions 
containing similar constituents and assessed and tested in the same way as above.  

ii.  The so-called block method: this method is applied when a substance can be divided 

conceptually into fractions containing constituents which are very similar with regard 
to the properties to be assessed. Within a fraction read-across criteria can be applied 
among the constituents. For each of the fractions one or more representative 
constituent(s) is/are chosen for which testing and assessment is carried out. The 

43 See for example this decision on substance evaluation: 
https://echa.europa.eu/documents/10162/d489cc70-7b49-46d8-b208-56e5b738a35e   

 
 
                                                 
 
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111 

constituent can be selected based on several considerations, e.g. that it can be easily 
retrieved for testing, there are already data on that constituent available or that it 
represents the worst case PBT-properties of the fraction (in case the constituents in 
the fraction are expected to exhibit a pattern of P, B, and/or T -properties within the 
boundaries of read across). 

In all these variations of the “fraction profiling approach” fractions present at ≥0.1% w/w 
concentration in the UVCB are normally considered as relevant.  

Advantages of the “fraction profiling approach” are, i.a.: 

  More targeted and refined assessment compared to the “whole substance approach”  

  Assessment of a complex substance fraction-wise allows efficient targeting of testing; 

  May be the only practical option for some very complex UVCBs; 

  Provides a refinement option if the “known constituents approach” is not feasible. 

Disadvantages of the “fraction profiling approach” are, i.a.: 

  May require in some cases greater analytical effort to characterise the substance 
composition at start of PBT assessment than the “whole substance approach”; 

  May requires synthesis or other type of generation of specific substance/test item for 
testing, if not otherwise available (e.g. raw material may in some situations be used 
as representative of a fraction which consists of unreacted raw material); 

  May require more than one test for each P, B, T endpoint. This might increase needs 

for vertebrate testing. 
Requires demonstration that any test item chosen for testing is a reasonable worst 
case. 

Figure R.11—6 below shows an anonymised example of the first assessment tier of a UVCB 
substance for which fraction profiling has been applied. 

  
 
 
 
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Figure R.11—6: Example of the first assessment tier of a UVCB substance for which fraction 
profiling has been applied 

Whole substance approach 

The substance is considered to be one chemical substance for the purpose of the assessment 
and testing. This is possible, if all the constituents therein can be justified to be very similar 
with regard to the PBT-properties relevant for the assessment based on information on, e.g. 
manufacturing method, raw materials and/or chemical composition/analyses.  

Due to the disadvantages and limitations, the application of the “whole substance” approach 
may only be possible in certain limited cases for the complete PBT/vPvB assessment of a 
substance. If one of the above mentioned approaches is feasible, these should be used instead 
of the ‘whole substance’ -approach as they are generally more transparent and regarded as 
providing information of higher certainty. For certain tests and for certain endpoint-specific 
assessments it may be possible to address the substance as a whole despite some slight 
differences in the properties of the constituents. For example, if it is known or can be 
reasonably assumed (e.g. based on the known chemical composition and/or relevant 
description of raw materials and production process but in addition also relative to the known 
or likely chemical identity of constituents) that (all) the constituents are structurally similar 
and therefore can be expected to have a reasonably similar PBT-properties, using the whole 
substance as test item may be considered – especially if such an analysis can be supported by 
non-testing or experimental data.  

In cases where “not PBT/vPvB” is concluded based on results from tests with the whole 
substance, there should be a clear case made in the assessment for why all constituents are 
structurally sufficiently similar and hence also similar with regard to the PBT properties to 
justify such a conclusion. For such similarity criteria, please refer to Chapter R.6 of the 
Guidance on IR&CSA. 

The “whole substance approach” is often applied by the registrants. It has been observed that 
the use of this approach should be better justified in the CSRs. 

Advantages of the “whole substance approach” are, i.a.: 

 
 
 
 
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113 

  The registered substance itself is used for testing and thus there is no need for 

generation of new material; 

 

 

It may be the only option if it is technically not feasible within reasonable efforts to 
establish the exact identity of the constituents in the registered substance to the level 
needed;  

In some cases the analytical requirements for whole substance identification may be 
simpler than for identification of individual constituents. 

Some disadvantages and considerations of situations where the “whole substance approach” 
should not be applied are described below: 

  Conclusion provides a single profile for the whole substance. This may be too 

inaccurate in some cases. Test results may not be representative of all constituents: 
Possible risk of miss-screening, for instance using a single log Kow value to represent 
a range of constituents or assuming ready biodegradability for a UVCB, where 
constituents are not sufficiently similar in reality.  

  Some tests using the whole substance as test item may not produce reliable results 

(e.g. if physico-chemical properties of the constituents vary significantly, the exposure 
concentrations cannot in some cases be maintained in such way that the test would be 
considered valid according to the test guideline); 

  Available whole substance test data may not be relevant and/or may be unreliable 

and/or be difficult to interpret (either due to differences of physico-chemical properties 
between constituents or because the composition may be partly unknown/uncertain 
/vary, and hence data may not be shown to be representative enough for the 
registered substance); 

  May trigger the need for the water accommodated fraction (WAF) approach for 

ecotoxicity testing (see discussion in Section R.11.4.1.3). 

 

Isolation or synthesis of relevant constituent(s) may not be technically feasible.  

Combination of more/several approaches described above 

It may be most efficient with regard to resources and time needed to combine several 
approaches in the assessment of one substance. E.g., for a complex UVCB it may be necessary 
to carry out an assessment of certain known constituents always present in the substance, but 
also to carry out a profiling fraction-wise for the remaining parts of the composition of the 
substance, if the remaining parts are anticipated to be so different from the known 
constituents that they may make a difference for the assessment conclusion. 

CONCAWE has used an approach which combines information from tests where the whole 
substance has been tested and information from tests utilising the block approach. This 
approach is presented in Appendix R.11—3.  

Different approaches may also be applied at different stages of the assessment, e.g. if 
information and knowledge on the substance increases during the assessment. 

A particular example is that for bioaccumulation, simultaneous testing at low concentration of 
several constituents each below its water solubility and sampling and analysis of their 
concentration in water and in the organism (fish), if technically feasible, may be a cost efficient 
testing option. The approach may also be applied in the dietary bioaccumulation study. It may 
be employed on separate fractions or blocks – or in some cases even on the whole substance. 
A prerequisite for obtaining reliable results is that the co-occurrence of each constituent does 
not interfere with the bioaccumulation behaviour of other constituents also being tested (e.g. 
through enzyme induction, etc.) 

  
 
 
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Choice of the assessment approach 

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Finally, the choice of the assessment approach may be dependent on the data already 
available. In any case, results from relevant studies carried out by using the whole (registered) 
substance as test item should always be included into the dataset, where these are already 
available, regardless of the assessment approach chosen. Such results may in some cases 
support profiling of the substance, even in such cases where the “whole substance approach” 
will not be chosen as the main assessment approach for the case. Additionally, readily 
available test results on individual constituents need to be taken into account in the 
assessment even if the “whole substance approach” is applied. In such cases the results on 
individual constituents need to support the choice of the “whole substance approach”. If they 
do not support the use of the “whole substance approach”, another approach would need to be 
considered. 

R.11.4.2.2.3 

Specific aspects 

When assessing P, B and T it is important to understand that there is a difference in testing 
and interpretation of the data, that relates to the concentration of the test substance and that 
this has consequences for the assessment of substances containing various constituents. For 
degradation (hence persistence) and bioaccumulation, the concentration of the substance in 
the test vessel is not included within the measure of the endpoint (Mackay et al., 2001). This is 
not the case for toxicity which is expressed in terms of concentration. The impact this has 
when assessing P, B and T is discussed under each of the endpoints below. 

When evaluating P, B and T -related studies it is important to pay attention to the available 
physico-chemical data and its representativeness. For example, a water solubility or Kow –test 
carried out with the whole substance where whole substance–related analytics has been 
followed does not give information on the specific water solubility or Kow of individual 
constituents, in case these genuinely have different properties (due to structural differences). 
Therefore, the basic physico-chemical data may also need to be generated for the constituents 
or constituent fractions depending on the assessment approach chosen, before other results 
can be evaluated or further testing decided. 

QSARs-profiling, where applicable, is often crucial for the assessment to screen the potential 
properties of expected constituents and hence for the search for the worst case 
fractions/constituents which can be targeted for further assessment and testing. QSAR results 
of P, B, T and relevant physico-chemical properties of the expected constituents or 
representatives of fractions often have important role in justifying selected assessment 
approach and test items. It should be remembered, that individual QSAR-model predictions are 
not normally able to accommodate the multi-constituent nature of a substance but they 
represent the results for a particular chemical structure (i.e, for one selected constituent at a 
time). Otherwise, for the use of QSARs in the assessment of constituents the same principles 
apply as for the use of QSARs in the assessment of pure (i.e. mono-constituent) chemical 
substances.   

The following specific considerations on data interpretation take as prerequisite that there is 
differentiation between the test item and the registered substance (of course, in the whole 
substance-approach these are the one and same).  

Where new data are generated for a fraction profiling or known constituent-approach, it should 
be kept in mind that the most persistent constituent may not be the most bioaccumulative or 
toxic – and vice versa. 

 
 
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(i) Persistence 

115 

One cannot easily assess the persistence of complex substances that contain many 
constituents using biodegradation testing methods that measure parameters (e.g. CO2 
evolution), since these tests measure the properties of the whole substance but do not provide 
information on the individual constituents.  

If the selected test item consists of sufficiently similar structures and is shown to meet the 
stringent ultimate ready biodegradation test criterion (>60% in 28 days), it can be concluded 
that the underlying constituents comprising the complex substances are not expected to be 
persistent (OECD, 2001).  

If the test item composition does not consist of similar structures or is not well characterised, 
it may still contain a certain amount of constituents that are persistent although the amount of 
easily degradable constituents is high enough to lead to an overall degradation percentage 
sufficient to meet the criteria for ready biodegradation.  

(ii) Potential for Bioaccumulation 

Similar difficulties apply to bioaccumulation assessment.  

Estimates for the individual constituents based on Kow, QSARs or other methods may be used. 
Also multi-component measuring techniques such as SPME or HPLC could be useful to give an 
initial estimate of bioaccumulation potential. For example, if all the peaks in the HPLC 
chromatogram have a log Kow <4.5, it may be assumed that all constituents of the substance 
have log Kow < 4.5. For interpretation of such results and estimates, please see Section 
R.11.4.1.2. 

(iii) Toxicity 

Toxicity is defined via a concentration response and is dependent on the bioavailability. If the 
tested substance contains many constituents having differences in the response and 
bioavailability, this makes the interpretation very difficult. For example, the physical form may 
prevent the dissolution of the individual constituents of such a substance to any significant 
extent where the whole substance is applied directly, as required in normal ecotoxicity test 
guidelines, to the test medium. The apparent exposure concentration(s) in the test system 
may lead to incorrect interpretation on toxicity of individual constituents. Therefore, care 
should be taken to interpret the observed (lack of) effect(s) in relation to actual exposure 
concentrations of individual constituents.  

R.11.4.2.2.4 

Test items  

If new testing is considered necessary, the set of tests, test sequence and test item(s) should 
be determined so that the results serve in the most efficient way the assessment with the 
chosen approach.   

The test items are allowed to deviate from the registered UVCB substance, if that is justified by 
the selected assessment approach. It should be noted, that the test item(s) may 
itself/themselves be UVCB(s), well-defined multi-constituent substance(s) or mono-constituent 
substance(s), depending on the case and purpose.  

The choice of the test item(s) is always dependent on the type of the substance but also on 
the case-specific understanding of which testing strategy is most efficient to conclude on the 
PBT/vPvB properties. Furthermore, feasibility and proportionality of efforts may also play a role 
in selecting the test item. It may in some cases be necessary to run a test on a particular 
property, e.g., simulation degradation test, for several test items, where one or more test 
items per fraction are used in parallel or in sequential tests.  

  
 
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In the “known constituent–approach” the test item consists of a single chemical structure. It 
can be extracted from the substance itself or be a separately synthesised as surrogate for a 
constituent (a similar chemical substance to the constituent). In block method the test item(s) 
per block targeted for testing and assessment may consist of one or more substances which 
are present as constituent(s) in the block or surrogate substances. Test item of a block may 
also be the whole block or similar multi-constituent substance. In the other fraction profiling 
approaches, the test item is either the whole fraction itself or a fraction of the fraction hence 
always consisting of multiple constituents. In that case also, the test item can be extracted 
from the substance or be separately synthesised. Similarly, also in fraction profiling, the test 
item may be a representative multi-constituent substance/mixture, if no extraction or 
synthesis of the target fraction of the registered substance is feasible. 

Justification of test item selection should also be documented in the CSR or authority’s 
assessment report.  

The choice of the assessment approach and the test item may in some cases also affect the 
selection of the test method. For instance an aqueous BCF study can only in practice be 
performed with a substance where exposure concentration of constituents can be verified by 
measurements. Any uncertainty due differences in constituent properties of a test item (e.g., 
such as increased leaching of test substance from food pellets due to variation in physchem 
properties) need to be considered when interpreting the results. For this purpose a GC-
characterisation of the test substance in the test system and/or in different test system 
matrixes before, during and after the test has been conducted might be useful. 

. 

 
 
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117 

R.11.5  References 

Anon. (2004a) Fish, Dietary Bioaccumulation Study – Basic Protocol, 20 Jan. 2004. Document 
071121_Chapter_R.11_Final.doc submitted to TC-NES by the PBT working group.  

Anon. (2004b) Fish, Dietary Bioaccumulation Study – Background document to the fish dietary 
study protocol, 20 Jan. 2004. Document 071121_Chapter_R.11_Final.doc submitted to TC-NES 
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Armitage JM and Gobas FAPC (2007) A terrestrial food-chain bioaccumulation model for POPs. 
Environ Sci Technol 41:4019-25. 

Arnot J, Gouin T and Mackay D (2005) Practical Methods for Estimating Environmental 
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Arnot JA, Brown TN and Wania F (2014) Estimating screening-level organic chemical half-lives 
in humans. Environ Sci Technol 48:723-30. 

ASTM (2003) E1022-94. Standard Guide for Conducting Bioconcentration Tests with Fishes and 
Saltwater Bivalve Mollusks. ASTM International, West Conshohocken, PA, USA. 

Banerjee S, Howard PH, Rosenberg AM, Dombrowski AE, Sikka H and Tullis DL (1984) 
Development of a general kinetic model for biodegradation and its application to chlorophenols 
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Barraclough D, Kearney T, and Croxford A (2005) Bound residues: environmental solution or 
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Berellini G, Waters NJ, Lombardo F (2012) In silico Prediction of Total Human Plasma 
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Bleeker EAJ and Verbruggen EMJ (2009) Bioaccumulation of polycyclic aromatic hydrocarbons 
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the Environment, Bilthoven, The Netherlands. 

Borga K, Kidd KA, Muir DCG, Berglund O, Conder JM, Gobas FAPC, Kucklick J, Malm O and 
Powell DE (2012) Trophic magnification factors: Considerations of ecology, ecosystems, and 
study design. Integr Environ Assess Manag 8:64–84. 

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Appendices 

Appendix R.11—1:   Indicators for limited bioconcentration for PBT assessment 

Appendix R.11—2:   Assessment of substances requiring special consideration during testing 

Appendix R.11—3:   PBT assessment of UVCB petroleum substances 

Appendix R.11—4:   Bioconcentration studies with benthic and terrestrial invertebrate species 

(BSAF) 

  
 
 
 
 
 
 
 
 
 
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Appendix R.11—1: Indicators for limited bioconcentration for PBT assessment. 

Summary 

This document was originally drafted as part of an ECETOC report on the use of alternatives in 
assessing the environmental safety of substances (ECETOC, 2005). Subsequently, the TC NES 
(Technical Committee for New and Existing Substances) subgroup addressing persistent, 
bioaccumulative and toxic (PBT) and very persistent/very bioaccumulative (vP/vB) substances 
(PBT working group) considered the recommendations and agreed to use them as part of the 
strategy of determining whether a substance should be placed on a screening PBT/vPvB list 
and/or should be tested to determine whether it is B/vB. The document has been altered as a 
result of discussions in the PBT WG, and the following is the last version of the text being 
discussed by the TC-NES WG on PBTs44. 

The indicators below should not be considered as definitive, but should be considered with 
other information, e.g. data derived from toxicokinetic and/or chronic mammalian studies. 
Such data indicating extremely low or no uptake and/or no chronic systemic toxicity will 
increase confidence in the use of the guiding indicators below. The TC-NES WG on PBTs, 
therefore will consider the following provisional indicators case by case by employing expert 
judgement in assessing substances (note each term, their definition and derivation as well as 
the recommended values are further discussed later). 

Used within a Weight-of-Evidence approach and with expert judgment a substance may be 
considered as not B (i.e. unlikely to have a BCF > 2,000) using the following types of 
evidence: 

1.  An average maximum diameter (Dmax aver) of greater than 1.7 nm45 plus 

a molecular weight of greater than 1100 

2.  a maximum molecular length (MML) of greater than 4.3 nm46 

3.  Octanol-water partition coefficient as Log10 (Log Kow) > 10 

4. 

 measured octanol solubility (mg/L) < 0.002 mmol/L × MW (g/mol) 
(without observed toxicity or other indicators of bioaccumulation) 

In addition to indicators 2, 3 and 4 above, and again within a Weight-of-Evidence approach 
and with expert judgment, an indicator for considering a substance as possibly not being a vB 
(i.e. unlikely to have a BCF > 5,000) is if it has: 

  a Dmax aver of greater than 1.7 nm45 plus a molecular weight of greater than 700 

In using the indicators above it should be noted that 1 and 2 are generally considered as 
potential barriers to uptake, 3 is considered a general indicator of uptake, distribution and 
availability (i.e. bioaccumulation in lipid containing parts of the organism) and the fourth 
parameter an indicator of potential mass storage in lipid tissues. 

44 Please note that only editorial changes to the text of the TC-NES PBT WG were made during the first 
revision of this Guidance. 

45 Please note that the indicator value of 1.7 nm for the average maximum diameter was derived using 
the descriptor Dmax from OASIS. However, it appears from the Environment Agency (2009) that the use 
of different software tools could lead to variable results for the same substance. 

46 Please note that this indicator value was based on a small dataset and cannot be recommended in this 
Guidance as agreed by the Partner Expert Group consulted during the first revision of this Guidance (v2.0 
– Nov 2014). 

 
 
                                                 
 
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Evidence of high biotransformation/metabolisation rate in fish may be used in support for the 
above mentioned indicators. Similar evidence in mammalian species may also be considered, 
though the possibility that mammalian species may transform substances at a higher rate than 
fish should be considered. 

Evidence of significant uptake in fish or mammals after longer time exposure would imply that 
the indicators 1-3 above should not be used. 

Discussion 

Assessing the potential of substances to bioconcentrate - indications for reduced or 
hindered uptake 

The magnitude of bioconcentration (i.e. the BCF) or bioaccumulation (i.e. the BAF) of a 
substance in an (aquatic) organism is estimated by a ratio of the concentration of the 
substance in the body of the animal to that of the environment or food. The BCF or BAF is the 
result of four processes, which occur when a substance is taken up from an animal’s 
surrounding environment or food. The BCF refers to the process where uptake is only via 
aqueous exposure, the BAF takes into account multiple uptake routes. The four processes are: 

 

Absorption - after the introduction of a substance through food, water, air, sediment, or 
soil, its transport across a biological membrane into systemic circulation e.g. across fish 
gills, intestine, skin (Hodgeson and Levi, 1994). 

  Distribution - after absorption, a substance may bind to plasma proteins for circulation 

throughout the body, as well as to tissue components like fat or bone. The substance may 
be distributed to a tissue and elicit a toxic response; other tissues may serve as 
permanent sinks, or as temporary depots allowing for slow release into circulation 
(Hodgeson and Levi, 1994). 

  Metabolism - after reaching a tissue, enzymes may biotransform the substance. During 

Phase I, a polar group is normally introduced into the molecule, which increases its water 
solubility and renders it a suitable substrate for Phase II reactions. In Phase II, the altered 
molecule combines with an endogenous substrate and is normally readily excreted. 
Metabolism is often a detoxification mechanism, but in some cases, metabolism may 
activate the parent compound and intermediates or final products may cause toxicity 
(Hodgeson and Levi, 1994). 

 

Excretion - a substance with similar characteristics, primarily water solubility, to 
endogenous waste is eliminated by the same mechanisms. Substances with nutritional 
benefit may be broken down and ultimately exhaled as CO2; volatile substances may also 
be exhaled directly through the lungs, Polar molecules that are freely soluble in plasma are 
removed through renal filtration and passed into urine. Fat soluble substances may be 
conjugated and excreted in bile (faeces) (Hodgeson and Levi, 1994). 

In addition to excretion, growth of the organism may also be relevant in reducing the 
substance concentration in the organism when the rates of other elimination processes are of 
the same order of magnitude as the dilution due to growth rate. Elimination through the 
transfer of substance to the offspring through gestation or lactation may also be important. 

This section describes several chemical properties that limit the absorption and distribution of a 
substance, which would sufficiently hamper the uptake, distribution or the body burden of a 
substance so that the BCF can be assumed to be of no or limited concern. Metabolism, 
excretion processes and growth also lead to a reduction of BCF/BAF but are not discussed in 
this paper. 

  
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Regulatory context 

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Version 3.0 – June 2017 

This text should be seen in the context of the European PBT and vPvB assessment of 
substances with a focus on the B or vB-assessment. Currently, if a substance has a calculated 
or measured BCF > 2,000 it fulfils the criterion for B. If it has a calculated or measured BCF > 
5,000 it fulfils the criterion for vB. Based on a screening threshold value, a substance could be 
either B or vB when its (estimated) Log Kow is > 4.5. In this case, if a substance meets the 
screening criterion for B or vB and it is also shown to be or likely to be (very) persistent, 
further consideration of its bioaccumulation potential is warranted. This may include critical 
review of its bioaccumulation potential according to (Q)SARs and bioaccumulation models 
taking into account its potential for uptake and metabolism (EC, 2003). The result of such an 
assessment may be so uncertain that further bioconcentration or bioaccumulation testing may 
have to be undertaken to determine whether the substance is B or vB. 

Experimental testing to determine the BCF 

The standard test to study the BCF in fish is the OECD TG 305 (bioconcentration test 
guideline). In this guideline, BCF is experimentally estimated using a flow through exposure 
regime with an initial uptake phase of up to 28 days followed by a depuration phase in clean 
water. The BCF can be estimated from the ratio Cf/Cw (Cf: concentration of test substance in 
fish at steady state; Cw: concentration of test substance in the exposure phase (water) or 
Ku/Kd (Ku: rate constant for uptake and Kd: rate constant for depuration; provided that first 
order – one compartment kinetics apply). In cases where substances meet the screening 
threshold value for B or vB, it is probable that these substances are very hydrophobic and 
have a very low aqueous solubility. Due to these properties it can be very difficult to test them 
in aqueous exposure systems such as an OECD TG 305 study. Alternatively, a recently 
developed dietary test (Anonymous, 2004) could be used to determine bioaccumulation 
potential through food or to derive data to estimate a BCF. However, many studies to 
determine the BCF of hydrophobic substances have been performed following aqueous 
exposure. The interpretation of such studies must be done with care. Many such studies were 
conducted following earlier versions of the OECD TG 305, and may include the following 
possible artefacts or shortcomings: 

  Difficulties in measuring the ‘true’ aqueous concentration due to sorption of the substances 

to particulate and dissolved (organic) matter; 

  Unstable concentration of the test substance in water and thus highly fluctuating exposure 

conditions 

  Adsorption of the test substance to glass walls or other materials; 

  Volatilisation. 

  Testing at concentrations clearly above the water solubility of the test substance, normally 
via the inclusion of dispersants or vehicles which would lead to an underestimation of the 
BCF 

  Determination of a BCF as the ratio between the concentration in fish and in water but 

under non steady state conditions 

It is important to realise that in many of the studies that have investigated relationships 
between molecular dimensions and reduced uptake, i.e. based on ‘lower’ BCFs than expected, 
it was not always possible to exclude occurrence of some of the above mentioned 
shortcomings or artefacts and truly reduced uptake. Thus rules relating to molecular 
dimensions or mass proposed in the past and claiming reduced uptake should be critically 
reviewed. 

Some studies have proposed a reduced uptake based on experimental bioconcentration 
studies. The reduced uptake then usually refers to reduced uptake via the fish gills. This does 
not imply that there will be reduced or no uptake possible via the gut uptake, i.e. from food, 

 
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where other uptake mechanisms may play a role. The extent to which those additional uptake 
mechanisms play a role in bioaccumulation, however, is inadequately quantified for fish and 
aquatic invertebrates. There is evidence, however, for certain highly persistent and 
hydrophobic substances that significantly accumulate via the food, even for gill breathing 
organisms, but particularly for predatory fish higher in the food chain. 

Mechanisms of absorption 

The route a substance follows from the point of initial exposure to the site of action or storage 
involves passage through a number of tissues and every step involves the translocation of the 
substance across multiple membranous barriers (e.g. mucosa, capillary wall, cell membrane), 
each containing distinct lipid types and proteins. Four primary mechanisms operate to absorb a 
compound into the body from the environment (Hodgeson and Levi, 1994): 

Passive transport - molecules diffuse across cell membranes into a cell, and they can pass 
between cells. 

Active transport - like passive transport, works in both directions to absorb and exsorb a wide 
range of substances. This special protein, or carrier-mediated, transport is important for 
gastrointestinal absorption of essential nutrients. In rare instances, toxicants can be actively 
transported into the cell. Efflux proteins, such a P-glycoprotein, shunt molecules out of the cell. 
Because of the specificity of this mechanism, it cannot be generally modelled. 

Filtration - small molecules can fit through channels, but molecules with molecular weights 
(MWs) greater than 100 g/Mol are excluded. Most compounds have limited access through 
these pores; filtration is considered more important for elimination than absorption. 

Endocytosis - the cell membrane flows around the toxicant to engulf it and transfer it across 
the membrane. This mechanism is rare except in isolated instances for toxicants, such as for 
carrageenans with MW around 40,000 g/mol. 

This appendix focuses on passive transport as the significant mechanism of absorption for 
most toxicants. This mechanism is the only one that can be modelled due to recent work to 
determine the physico-chemical parameters affecting simple diffusion across a membrane. 

Molecular properties 

Lipinski et al. (1997) first identified five physico-chemical characteristics that influence 
solubility and absorption across the intestinal lumen using more than 2,200 drug development 
tests. These characteristics have been rigorously reviewed (Wenlock et al., 2003; Proudfoot, 
2005), used to develop commercial models to estimate absorption in mammals, and are 
commonly used by the human and veterinary pharmaceutical industry. Although less research 
in absorption, distribution, metabolism and excretion (ADME) processes have been conducted 
in fish, data indicate significant similarity among all vertebrates, as described below. 

‘Lipinksi’s Rule of 5’ allows the prediction of poor solubility, and poor absorption or permeation 
from chemical structure. A substance is not likely to cross a biological membrane in quantities 
sufficient to exert a pharmacological or toxic response when it has more than 5 Hydrogen (H)-
bond donors, 10 H-bond acceptors, molecular weight > 500, and has a Log Kow value > 5 
(Lipinksi et al., 1997). Wenlock et al. (2003) studied about 600 additional substances and 
found that 90% of the absorbed compounds had < 4 Hydrogen (H)-bond donors, < 7 H-bond 
acceptors, molecular weight < 473, and had a Log D value < 4.3. More recent work by Vieth et 
al. (2004) and Proudfoot (2005) supports the lower numbers. Molecular charge and the 
number of rotational bonds will also affect absorption by passive diffusion across a membrane 
or diffusion between cells. 

Although these studies on almost 6,000 substances focussed on absorption, generally of per 
orally dosed drugs across the intestinal wall, the similarity in tissue structures of mammals and 

  
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fish imply the equations and concepts can be reapplied to estimate absorption in fish. The 
‘leakiness’ of a tissue, or its ability to allow a substance to passively diffuse through it, can be 
measured using trans-epithelial electrical resistance (TEER) and can be used to compare tissue 
capabilities. A low TEER value indicates the tissue has greater absorption potential. Data 
indicate that fish and mammalian intestines are equally ‘leaky’ and that fish gills are more 
restrictive, similar to the mammalian blood brain barrier (Table R.11—8). The table also shows 
whether P-glycoprotein has been detected and could be a functional efflux protein active in the 
tissue. 

Table R.11—8: Tissue absorption potentials 

Tissue 

P-glycoprotein 
efflux? 

TEER ohm cm2  References 

Fish intestine 

Yes 

25-50 

Trischitta et al. (1999) 

Mammal intestine 

Yes 

20-100 

Okada et al. (1977); Sinko et al. (1999) 

Blood-brain barrier 

Yes 

400-2000 

Borchardt et al. (1996) 

Fish gill 

Human skin 

Yes 

No 

3500 

Wood and Pärt (1997) 

20,000 

Potts and Guy (1997)  

Octanol-water partition coefficient (Log Kow) 

Following an assessment of the database used by Dimitrov et al. (2002), a cut-off for the Log 
Kow of 10 has been suggested, which used within a Weight-of-Evidence scheme supports the 
observation that a substance may not be B/vB (see Appendix R.11—1 Annex 1). 

It should be noted that there are very few reliable measured values of Log Kow above 8 and 
that measurements in this region are very difficult (see Section R.7.1.8 in Chapter R.7a of the 
Guidance on IR&CSA). Consequently, measured values above 8 must be carefully assessed for 
their reliability. It is a consequence of this lack of data that most models predicting Log Kow are 
not validated above a Log Kow value of 8. Such predictions should therefore be considered in 
qualitative terms. As described in Appendix R.11—1 Annex 1, based on the current limited 
knowledge (both with respect to measured Log Kow and BCFs), a calculated Log Kow of 10 or 
above is taken as an indicator for showing reduced bioconcentration. 

Molecular size 

Molecular size may be considered as a more refined approach, taking into account molecular 
shape and flexibility explicitly rather than molecular weight alone. However, in the following 
section, certain definitions are needed; 

  Maximum molecular length (MML) – the diameter of the smallest sphere into which the 

molecule would reside, as written, i.e. not accounting for conformers 

  Maximum diameter, Dmax – the diameter of the smallest sphere into which the molecule 

may be placed. Often this will be the same as the MML, especially for rigid molecules. 
However, when flexible molecules are assessed, energetically reasonable conformers could 
be present for which this is very different. In the document the average value for this Dmax 
for “energetically stable” conformers is used, i.e. Dmax ave. 

 

(Maximum) Cross-sectional diameter – the diameter of the smallest cylinder into which the 
molecule may be placed. Again different conformers will have different cross-sectional 
diameters. 

 
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These definitions are shown graphically in Annex 2 to this Appendix, together with examples of 
software that may be used for their calculations. 

In the discussions although various values are referred to, the PBT WG recognise that firstly 
these values will probably alter as experience and the available data increase, and that 
secondly the actual value for a molecule’s Dmax, will depend on the conformer used and to a 
degree the software used. In interpreting the data these uncertainties need to be borne in 
mind. 

Opperhuizen et al. (1985) found a limiting molecular size for gill membrane permeation of 0.95 
nm, following aqueous exposure. In their study on polychlorinated naphthalenes (PCNs), 
bioconcentration increased with increasing hydrophobicity, i.e. the degree of chlorination, with 
uptake and elimination rate constants comparable to those of chlorinated benzenes and 
biphenyls. For the PCN-congeners studied, BCFs increased with increasing hydrophobicity up to 
higher Log Kow values (>105). No further increase was observed at higher Kow values. For the 
hepta- and the octachloronaphthalenes no detectable concentrations were found in fish. It was 
suggested that the absence of increasing bioconcentration was due to the inability of the 
hepta- and octachloronaphthalenes to permeate the gill lipid membrane, due to the molecular 
size of these compounds, brought about by the steric hindrance of the additional chlorine 
atoms. A cut-off of 0.95 nm was proposed as the cross-sectional diameter which limited the 
ability of a molecule to cross the biological (lipid) membrane. 

Anliker and Moser (1987) studied the limits of bioconcentration of azo pigments in fish and 
their relation to the partition coefficient and the solubility in water and octanol. A 
tetrachloroisoindolinone type and a phenyl azo-2-hydroxy-naphthoicacid type, both had low 
solubility in octanol, < 1 and < 0.1 mg/L, respectively. Their cross-sectional diameters were 
0.97 nm and 1.68 nm, respectively. Despite the high Log Kow calculated for these substances, 
the experimentally determined Log BCFs were 0.48 and 0.70, respectively. The explanation for 
this apparent inconsistency of high Log Kow and low BCF is the very limited absorption and fat 
(lipid) storage potential of these pigments, indicated by their low solubility in n-octanol (see 
next sub-chapter) and their large molecular size. 

Anliker et al. (1988) assessed 23 disperse dyestuffs, two organic pigments and a fluorescent 
whitening agent, for which the experimental BCFs in fish were known. Sixteen halogenated 
aromatic hydrocarbons were included for comparison. Two characteristics were chosen to 
parameterise the size of the molecules: the molecular weight and the second largest van der 
Waals diameter of the molecules, measured on conformations optimised by force field 
calculations (Opperhuizen et al., 1985). None of the disperse dyestuffs, even the highly 
lipophilic ones with Log Kow > 3, accumulated significantly in fish. Their large molecular size 
was suggested to prevent their effective permeation through biological membranes and thus 
limit their uptake during the time of exposure. Anliker et al. (1988) proposed that a second 
largest cross section of over 1.05 nm with molecular weight of greater than 450 would suggest 
a lack of bioconcentration for organic colorants. While some doubts have been raised 
concerning the true value of the BCFs in these papers, as experiments were conducted at 
exposure concentrations in excess of the aqueous solubility, the data support the underlying 
hypothesis for reduced uptake for larger molecules. 

Other studies addressing molecular dimensions have included Opperhuizen et al. (1987) who 
proposed that a substance greater than 4.3 nm would not pass membranes at all, either in the 
gills or in the gut based on a series of bioaccumulation and bioconcentration studies with linear 
and cyclic polydimethylsiloxanes (PDMS or “silicones”) varying in chain length. To allow such 
large substances to pass is very unlikely since it would mean that the entire interior of the lipid 
membrane would be disturbed. Molecular weight did not explain reduced uptake, since one of 
the substances with a molecular weight of 1,050 was found in fish. The cross-sectional 
diameter of these substances could in itself also not explain the reduced uptake since those 
were smaller or equal to those of PCBs that did bioaccumulate strongly. 

Opperhuizen et al. (1987) also referred to a study by Hardy et al. (1974) where uptake of long 
chain alkanes was disturbed for alkanes longer than C27H56 in codling. This chain length 

  
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corresponds to a molecular dimension, i.e. molecular length, of 4.3 nm, equal to the length of 
the PDMS congener where reduced uptake was observed. 

Loonen et al. (1994) studied the bioconcentration of polychlorinated dibenzo-p-dioxins and 
polychlorinated dibenzofurans and found that the laterally substituted (2,3,7,8 substituted) 
were bioconcentrated while the non-laterally substituted were not. The main reason for this 
was attributed to metabolism (previously reported by Opperhuizen and Sijm, 1990, and Sijm 
et al., 1993b), however, lower lipid solubility and lower membrane permeability were also 
considered to have played a role in the reduced BCFs observed. The non-accumulating 
structures would all have exceeded the effective cross-sectional diameter of 0.95 nm. 

Although the lack of bioconcentration of some substances with a cross section of > 0.95 nm 
has been explained by limited membrane permeability, a number of other studies have 
demonstrated the uptake of pollutants with large cross sections (e.g. some relevant dioxin and 
PBDE congeners) by fish and other species. Therefore a simple parameter may not be 
sufficient to explain when reduced BCF/BAF occurs. Dimitrov et al. (2002, 2003, 2005) have 
tried to develop a more mechanistic approach to address this concept, using molecular weight, 
size, and flexibility in their BCF estimates. 

In a review made by Dimitrov et al. (2002) it is suggested that for compounds with a Log Kow 
> 5.0, a threshold value of 1.5 nm for the maximum diameter, Dmax ave, could discriminate 
substances with Log BCF > 3.3 from those with Log BCF < 3.3. This critical value was stated to 
be comparable with the architecture of the cell membrane, i.e. half the thickness of the lipid 
bilayer of a cell membrane. This is consistent with a possible switch in uptake mechanism from 
passive diffusion through the bilayer to facilitated diffusion or active transport. In a later 
review paper, Dimitrov et al. (2003) used this parameter to assess experimental data on a 
wide range of substances. Their conclusion was that a substance with Dmax ave larger than 1.5 
nm would not have a BCF > 5,000, i.e. would not meet the EU PBT criteria for vB substances. 
More recently, Dimitrov et al., 2005, have revised this figure to 1.7 ± 0.02 nm following 
further assessment of the data set published. It is likely that the absolute value for this Dmax 
may alter with further assessment and generation of database containing high quality BCF 
values. 

Currently a value of 1.7 nm is recommended, however, with more experience and data this 
value may alter. Indeed it is recommended that the BCF data used in the various papers cited 
(Dimitrov et al., 2002, 2003 and 2005), and in particular the data for the larger molecules, for 
which the testing is undoubtedly difficult, undergo critical quality and reliability review. Further 
assessment of these cut-offs should also be conducted following publication of the CEFIC LRI 
database containing high quality BCF data. 

Conclusion: Again there would appear to be no clear cut-off. While recognising the 
uncertainties in the interpretation of experimental results, it is recommended that: 

  Possibly not B : a Dmax ave of > 1.7 nm plus a molecular weight greater than 1100 

  Possibly not vB : a Dmax ave of > 1.7 nm plus a molecular weight greater than 700 

  Possibly not B and possibly not vB: A maximum molecular length of 4.3 nm may suggest 

significantly reduced or no uptake. This criterion appears, to be based on older studies and 
a limited number of chemical classes and should be treated with caution until further case 
studies are generated; 

Solubility in octanol 

The concept of having a value relating a substance’s solubility in octanol to reduced BCF/BAF is 
derived from two considerations: firstly, that octanol is a reasonable surrogate for fish lipids, 
and secondly, that, if a substance has a reduced solubility in octanol (and therefore by 
extrapolation in lipid) this may result in a reduced BCF/BAF. The former is reasonably well 
understood and indeed forms the basis of the majority of models for predicting BCF using Log 
Kow. Further, octanol solubility (or better, the ratio of n-octanol/water solubilities) can 

 
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131 

characterise the transport of some small molecular sized, neutral compounds through 
biological membranes (Józan and Takács-Novák, 1997). 

When a substance has a low solubility in octanol (Soct) as well as a low solubility in water (Sw), 
the resulting ratio Soct/Sw could range from very low to very high, with no clear idea on how 
this would affect the magnitude of the BCF/BAF. Still, it could be argued that a very low 
solubility in octanol could be used as an indication that only low body burdens can be built up 
in an aquatic organism (however, this may not apply to other mechanisms of uptake, and 
when the bioaccumulation may not be related to the lipophilicity of the substance, e.g. when 
there is binding to proteins. 

Chessells et al. (1992) looked at the influence of lipid solubility on the bioconcentration of 
hydrophobic compounds and demonstrated a decrease in lipid solubility with increasing Kow 
values for super-hydrophobic compounds (Log Kow > 6). It was suggested that this led to 
reduced BCFs. Banerjee and Baughman (1991) demonstrated that by introducing a term for 
lowered octanol/lipid solubility into the Log Kow BCF relationship, they could significantly 
improve the prediction of bioconcentration for highly hydrophobic substances. 

Body burdens 

The meaningful implication of bioaccumulation that needs to be addressed for PBT substances, 
e.g. as in the EU TGD (ECB, 2003), is to identify the maximum concentration(s) in organisms 
that would give rise to concern. The concept of critical body burdens (CBB) for acute effects is 
reasonably well established (McCarty and Mackay, 1993; McCarty, 1986) especially for 
substances that act via a narcosis mode of action. Recently there have been a number of 
reviews of this concept, Barron et al. (1997, 2002), Sijm and Hermens (2000) and Thompson 
and Stewart (2003). These reviews are summarised as follows: 

  There are very few data available, especially for specifically acting substances and for 

chronic effects, upon which to make decisions relating to generic CBBs; 

  The experimental data for CBBs show considerable variation both within specific modes of 

action and for those substances with a specific mode of toxic action. The variation appears 
to be around one order of magnitude for the least toxic type of substances (narcotic 
substances) but extends over several orders of magnitude for substances within the same 
types of specific toxic action. Much of the variability in CBBs can probably be explained by 
differences in species sensitivities, biotransformation, lipid content, whether the 
measurements relate to organ , whole body or lipid and whether the substance was 
correctly assigned to a mode of action category; 

  Some of the data in these reviews need to be checked for quality and need clear 

interpretation, particularly, those 
  Studies based on total radiolabel, and 
  Studies that quote no effect data which were derived from tests without establishing 

either a statistical NOEC (EC10) and/or a dose response curve. 

Notwithstanding this, it may with some caution be possible to group ranges of CBB values for 
specific modes of toxic action. This is easier for narcosis type mode of actions, and becomes 
increasingly prone to error moving towards more specifically acting substances. 

Table R.11—9 summarises three sources of information: 

1.  Sijm (2004) - an expert judgement view to arrive at an approximate 

single value based on three references, McCarty and Mackay (1993), Van 
Wezel and Opperhuizen (1995) and Sijm and Hermens (2000). 

2.  Thompson and Stewart (2003) - based on a literature review, the data 
range beyond the narcosis mode of actions has been drawn from their 
report. 

3.  Barron et al. (2002) - based on Figure 10 of Barron et al. (2002). 

  
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When comparing the expert judgement of Sijm to the ranges indicated and to the figures in 
the respective publications, it is clear that the values chosen are in the approximate mid-point 
of the ranges/data. However, there is clearly a lot of variability and therefore uncertainty in 
deciding on the actual CBB value to use. Choosing the value of 0.001 mmol/kg ww (mid-point 
for respiratory inhibitors) allows for approximate protection for all the modes of action with the 
exception of the most toxic substances. The rationale for this choice would be that substances 
that act by the most specific mode of toxic action would probably be toxic (T) and hence 
sufficiently bioaccumulative to be of immediate concern. 

Table R.11—9: Summary of various ranges of CBB - lethality (mmol/kg ww). 

Mode of action and source 

Narcosis 

AChE inhibitors  Respiratory inhibitors 

Sijm (2004) 

2 

0.01 

0.001 

Thompson and Stewart (2003) 

2-8 

0.000001 – 10 

0.000001 – 10 

Barron et al. (2002) 

0.03 – 450 

0.00004 – 29 

0.00002 - 1.1 (CNS seizure agents) 

McCarty and Mackay (1993) 

1.7 – 8 

0.05 - 2.7 

0.00005 - 0.02 (CNS seizure agents) 

Lipid normalising the chosen CBB of 0.001 mmol/kg ww, and assuming a lipid content of 5%, 
gives a lipid normalised CBB of 0.02 mmol/kg lipid or 0.02 × molecular weight mg/L lipid. 
However, given the uncertainty involved in deciding on the CBB that should be used, it is 
suggested that an application factor of 10, to account for species differences and organ versus 
body differences be applied to this solubility in lipid/octanol, giving an octanol solubility (mg/L 
lipid) of 0.002 × molecular weight. This would mean octanol solubilities of 1 and 2 mg/L n-
octanol (or lipid), respectively, for substances with molecular weights of 500 and 1,000. 

Conclusion: it is proposed that where a substance has a solubility of less than (0.002 × 
molecular weight) mg/L in octanol it should be assumed that the compound has only a limited 
potential to establish high body burdens and to bioaccumulate. If it does bioaccumulate, it 
would be unlikely to give rise to levels in biota that would cause significant effects. 

When there are fish or mammalian toxicity or toxicokinetic studies available, all showing no 
chronic toxicity or poor absorption efficiency, and a substance has, in addition, a low solubility 
in octanol, no further bioaccumulation testing would be needed, and the substance can be 
assigned as no B, no vB. In theory, such a substance could elicit toxic effects after prolonged 
times in aquatic organisms. However, the chance such a thing would occur would be very low. 

When there are no other studies available, and a substance has a low solubility in octanol, it is 
probable that other types of information (persistence, molecular size) would need be taken 
into account in deciding on bioaccumulation testing. It would also be helpful if testing, of the 
nature discussed above, were needed for other regulations, that might be useful in this 
evaluation, then the need for bioconcentration testing could be assessed when the new data 
became available. 

Other indicators for further consideration 

The two indicators, molecular size and lipid solubility, are the most frequently cited physical 
limitations for low bioconcentration. However, there are other indicators that could also be 
used for indicating whether the bioconcentration of a substance is limited or reduced despite 
having a Log Kow > 4.5. These include: 

 
 
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  Biotransformation - discussed in the TF report, ECETOC, 2005, (de Wolf et al., 1992, 1993; 
Dyer et al., 2003) and clearly needing development to improve how such information may 
be used; 

  Other indicators for low uptake, these could for example include 

 

lack of observed skin permeability (this alone not without substantiating that it is 
significant less than uptake in fish), 

  very low uptake in long term mammalian studies, and/or 
 

low chronic systemic toxicity in long term mammalian and/ or ecotoxicity (fish) studies. 

Both these approaches would benefit from further research and investigation for their potential 
to indicate limited or reduced bioconcentration. While it is not recommended, based on the 
current level of information, to use such indicators alone to predict low bioconcentration, they 
can act as supporting information to other indicators in arriving at this conclusion. 

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Anliker R, Moser P and Poppinger D (1988) Bioaccumulation of dyestuffs and organic pigments 
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Anonymous (2004) Fish, Dietary Bioaccumulation Study Protocol, based on a version adapted 
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Barron MG, Anderson MJ, Lipton J and Dixon DG (1997) Evaluation of critical body residue 
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de Wolf, W, Seinen W, Hermens JLM (1993) Biotransformation and toxicokinetics of 
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135 

Sijm DTHM and Hermens JLM (2000) Internal effect concentrations: Link between 
bioaccumulation and ecotoxicity for organic chemicals, In: The Handbook of Environmental 
Chemistry, Beek B (Ed) Volume 2-J. Bioaccumulation. New Aspects and Developments, pp. 
167-99. 

Sijm DTHM, Wever H and Opperhuizen A (1993) Congener specific biotransformation and 
bioaccumulation of PCDDs and PCDFs from fly ash in fish. Environ Toxicol Chem 12:1895-907. 

Sijm DTHM (2004) Personal communication to some members of the TCNES sub-group on 
PBTs. 

TGD (2003) Technical Guidance Document on Risk Assesment, Part II, EUR 20418 EN/2, 
European Chemicals Bureau. 

Thompson RS and Stewart KM (2003) Critical Body Burdens: A review of the literature and 
identification of experimental data requirements, BL7549/B, CEFIC-LRI. 

US EPA (1999) Category for persistent, bioaccumulative and toxic new chemical substances. 
Feb Reg 64:60194-60204. 

Van Wezel AP and Opperhuizen A (1995) Narcosis due to environmental pollutants in aquatic 
organisms: residue-based toxicity mechanisms and membrane burdens. Crit Rev Toxicol 
25:255-79. 

Vieth M, Siegel MG, Higgs RE, Watson IA, Robertson DH, Savin KA, Durst GL and Hipskind PA 
(2004) Characteristic physical properties and structural fragments of marketed oral drugs. J 
Med Chem 47:224–32. 

Wenlock MC, Austin RP, Barton P, Davis AM and Leeson PD (2003) A Comparison of 
Physiochemical Property Profiles of Development and Marketed Oral Drugs. J Med Chem 
46:1250-6. 

  
136 

Appendix R.11—1 Annex 1 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

DEVELOPMENT OF A LOG KOW CUT-OFF VALUE FOR THE B-CRITERION IN THE PBT-
ASSESSMENT 

The following assessment was based on the same data set used for development of the Dmax ave 
indicators (Dimitrov et al., 2005, see main paper). Since publication the data set has been 
extended by Dimitrov. This was the dataset used for this exercise. With respect to the 
database used for the development of the cut-off value it is important to realize that the 
database comprises two data sets obtained from ExxonMobil and MITI. A quality assessment 
was made of the MITI data (as described in Dimitrov et al.) and consequently the assessed 
data does not contain all the MITI data and may contain values that may not be considered as 
reliable by the TC-NES PBT WG. The experimental data from ExxonMobil are generated from 
fish-feeding studies, but only cover substances with Log Kow values of < 7. For these reasons, 
it is recommended that this indicator (and those in the main paper) be re-evaluated when the 
CEFIC LRI Gold Standard database on BCF is available. 

The fitted lines in Figure R.11—7, Figure R.11—8 and Figure R.11—9 are based on subsets of 
the BCF-dataset and are use to illustrate a limited bioconcentration potential for substances 
with high Kow-values. However, they are not to be used as a QSAR to estimate BCF from Log 
Kow (see Section R.7.10 in Chapter R.7c of the Guidance on IR&CSA). 

For substances with a Log Kow higher than 9.3 (based on CLogP) it was estimated that the 
maximum BCF value is equal to 2000. The 95% confidence interval for this exercise is 9.5 
(Figure R.11—7). 

Figure R.11—7: Log BCF v calculated Log Kow. 

-5.0-2.50.02.55.07.510.012.515.0-1.5-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.5logKow (ClogP)log BCF 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

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137 

Figure R.11—8 plots the available BCF data against measured Log Kow values. No experimental 
were available above Log Kow of 8.5 apart from estimates by HPLC. This supports the belief 
that this is the limit of current state-of-the-art techniques for the determination of Log Kow (i.e. 
slow-stirring and column elution). 

Figure R.11—8: LogBCF v measured log Kow. 

The relevance and experimental difficulties of conducting aqueous exposure on substances 
with very high Log Kow must be questioned. Therefore it was decided to repeat the calculation 
with the BCFs from feeding experiments only (Figure R.11—9). The data for very hydrophobic 
compounds are limited and there were 15 values for substances with calculated Log Kow values 
above 7. None of these 15 reached the same level of BCF as the highest BCFs between Log Kow 
values of 6.5 and 7.0 when compared to the parabolic relationship in Figure R.11—8. Of these 
15, three substances had calculated Log Kow values above 8, one is a vB substance and one is 
a B substance (very close to vB). 

-3-2-1012345678910-1.5-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.5logKow (experimental)log BCF  
 
 
 
 
 
 
 
 
 
 
 
 
 
138 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Figure R.11—9: LogBCF derived from feeding studies versus calculated Log Kow. 

Summarized, the results of Figure R.11—7 to Figure R.11—9 suggest that the B-criterion is 
unlikely to be triggered for substances with a Log Kow higher than 10. As with the other 
indicators described in the main paper, a Log Kow-value higher than 10 should be used in a 
Weight-of-Evidence approach in combination with the other indicators. 

1234567890.00.51.01.52.02.53.03.54.04.55.05.56.06.5logKow (ClogP)log BCF (feeding test only) 
 
 
 
Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Appendix R.11—1 Annex 2 

139 

GRAPHIC DEFINITIONS FOR THE MOLECULAR DIMENSIONS USED IN THE MAIN 
PAPER 

  Maximum molecular length (MML) – the diameter of the smallest sphere into which the 

molecule would reside, as written, i.e. not accounting for conformers 

  Maximum diameter, Dmax – the diameter of the smallest sphere into which the molecule 

may be placed. Often this will be the same as the MML, especially for rigid molecules. 
However, when flexible molecules are assessed, energetically reasonable conformers could 
be present for which this is very different. The average value of Dmax for “energetically 
stable” conformers is used, i.e. Dmax ave. 

 

(Maximum) Cross-sectional diameter – the diameter of the smallest cylinder into which the 
molecule may be placed. Again different conformers will have different cross-sectional 
diameters. 

Conformer 1 (Ho = -84.5 kcal/mol), Dmax = 21.4; Deff = 4.99; Dmin = 4.92 

Conformer 2 (Ho = -71.8 kcal/mol), Dmax = 19.8; Deff = 6.63; Dmin = 5.12 

  
 
 
 
 
 
 
 
 
140 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Conformer 3 (Ho = -68.5 kcal/mol), Dmax = 14.0; Deff = 11.5; Dmin = 5.52 

Example Softwares 

OASIS 

To calculate Dmax ave conformational analysis of the molecule needs to be conducted. This is 
done by estimating Dmax of each conformers and then the average Dmax values across the 
conformers. An OASIS software module is used to generate the energetically stable conformers 
representing conformational space of the molecules. The method is based on genetic algorithm 
(GA) generating a final number of structurally diverse conformers to best represent 
conformational space of the molecules (Mekenyan et al., 1999 and 2005). For this purpose the 
algorithm minimizes 3D similarity among the generated conformers. The application of GA 
makes the problem computationally feasible even for large, flexible molecules, at the cost of 
non-deterministic character of the algorithm. In contrast to traditional GA, the fitness of a 
conformer is not quantified individually, but only in conjunction with the population it belongs 
to. The approach handles the following stereochemical and conformational degrees of freedom: 

 

 

 

 

rotation around acyclic single and double bonds, 

inversion of stereocenters, 

flip of free corners in saturated rings, 

reflection of pyramids on the junction of two or three saturated rings. 

The latter two were introduced to encompass structural diversity of polycyclic structures. When 
strained conformers are obtained by any of the algorithms the possible violations of imposed 
geometric constraints are corrected with a strain-relief procedure (pseudo molecular 
mechanics; PMM) based on a truncated force field energy-like function, where the electrostatic 
terms are omitted (Ivanov et al., 1994). Geometry optimization is further completed by 
quantum-chemical methods. MOPAC 93 (Stewart, 1990 and 1993) is employed by making use 
of the AM1 Hamiltonian. Next, the conformers are screened to eliminate those, whose heat of 
formation, DHfo, is greater from the DHfo associated with the conformer with absolute energy 
minimum by user defined threshold - to be within the range of 20 kcal/Mol (or 15 kcal/mol) 
threshold from the low(est) energy conformers (Wiese and Brooks, 1994). Subsequently, 
conformational degeneracy, due to molecular symmetry and geometry convergence is detected 
within a user defined torsion angle resolution. 

Calculation of the 3D Dimension of a Molecule 

A molecular modelling program, e.g. Molecular Modelling Pro, uses a 2D molecular structure as 
a starting point for the calculation. In the 1st step the program calculates the least strained 3D 

 
 
 
Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

141 

conformer using e.g. MOLY Minimizer as built in the Molecular Modelling Pro. Normally this 
minimizing of strain requires multiple steps. If the strain energy is minimized the program 
calculates the 2nd step the 3D molecular dimensions (x length, y width, z depth) e.g. in 
Angstrom. Based on these x,y,z dimensions Molecular Modelling Pro is able to calculate a 
global maximum and minimum which can be used a Dmax. 

OECD QSAR Toolbox 

The development of this resource, which is currently in development, will include a database of 
chemical structures and associated information, CAS numbers etc. Currently, it is understood 
that included in the associated information will be a calculated Dmax, derived by OASIS and 
based on a 2D structure. A value of this type should be used with extreme caution and as an 
indicator as to the possible utility of the approach. It is not recommended at this stage to use 
this value in the same way as a derived Dmax ave as described in the full paper. 

References 

Ivanov JM, Karabunarliev SH, Mekenyan OG (1994) 3DGEN: A system For an Exhaustive 3D 
Molecular Design. J Chem Inf Comput Sci 34:234-43. 

Mekenyan OG, Pavlov T, Grancharov V, Todorov M, Schmieder P and Veith G (2005) 2D-3D 
Migration of Large Chemical Inventories with Conformational Multiplication. Application of the 
Genetic Algorithm. J Chem Inf Model 45:283 -92. 

Mekenyan OG, Dimitrov D, Nikolova N and Karabunarliev S (1999) Conformational Coverage 
by a Genetic Algorithm. Chem Inf Comput Sci 39:997-1016. 

Stewart JJP (1993) MOPAC 93. Fujitsu Limited, 9-3, Nakase 1-Chome, Mihama-ku, Chiba-city, 
Chiba 261, Japan, and Stewart Computational Chemistry. 15210 Paddington Circle, Colorado 
Springs, Colorado 80921, USA. 

Stewart JJP (1990) MOPAC: A semi empirical molecular orbital program. J Comput-Aided Mol 
Des 4:1-105. 

Wiese T and Brooks SC (1994) Molecular modelling of steroidal estrogens: novel conformations 
and their role in biological activity. J Steroid Biochem Molec Biol 50:61-72. 

  
142 

Appendix R.11—1 Annex 3 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

EXAMPLES - USE OF THE INDICATORS FOR LIMITED BIOACCUMULATION 

Example R.11-1 

Indicator : n-Octanol solubility 

Name 

Pigment Red 168 

CAS No. 

4378-61-4 

Mol weight (g/Mol) 

464 

Co (µg/L) 

CBB (µg/L) 

Co < CBB 

Log Co/Cw 

Remark: 

124 

928 

YES 

1.1 

The n-octanol solubility Co of Pigment Red 168 is well below the Critical Body Burden (CBB) 
which is an indicator of low bioaccumulation potential. In addition the Log Co/Cw 
(octanol/water) is 1.1 which means low uptake through biological membrane. 

Example R.11-2 

Indicator : Kow > 10 

Name 

ODBPA 

CAS No. 

2082-79-3 

Mol weight (g/Mol) 

531 

Log Kow 

13.4 

Remark: 

ODBPA has a reduced potential for bioaccumulation. 
In a Biodegradation test at low substance concentration and specific substance analysis ready 
biodegradability could be achieved. The transformation products formed are neither PBT nor 
vPvB. 

OOBrBrOHOOCH38 
 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

143 

Example R.11-3 

Indicator : Average Size > 17A and MW > 1100 g/Mol PLUS Log Kow > 10 

Name 

PETP 

CAS No. 

6683-19-8 

Mol weight (g/Mol) 

1178 

Average size (A) 

17.9 

log Kow 

19.6 

Remark: 

The indicators average size > 17A and MW > 1100 g/Mol are fulfilled (substance is considered 
not B). In addition Log Kow is > 10 which means that the bioaccumulation potential is low. For 
more information see Appendix R.11—2, Example R.11-6. 

Example R.11-4 

Indicator :  Average Size > 17A and MW > 700 g/Mol PLUS Octanol solubility 

Name 

Pigment Red 83 

CAS No. 

5567-15-7 

Mol weight (g/Mol) 

818 

Average size (A) 

Co (µg/L) 

CBB (µg/L) 

Co < CBB 

Remark: 

20 

9 

1636 

YES 

The indicator average size > 17 A & MW > 700 g/Mol are fulfilled (substance is considered not 
vB). In addition the octanol solubility is very well below the Critical Body Burden (CBB) which 
means that the bioaccumulation potential is low. 

OOOOOOOOOHOHOHOHClClNNNNNOCH3ONOCH3OOOCH3OCH3OClCH3ClCH3  
 
 
 
 
144 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Appendix R.11—2: Assessment of substances requiring special consideration during 
testing. 

Table R.11—10: List of antioxidants (from Ullmann, 1995). 

Antioxidant type 

Hindered Phenols 

CAS No.  

MW  
(g/Mol) 

calc. Kow 
(KOWWin) 

1  Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl- (BHT) 

128-37-0 

220 

5.1 

2 

3 

4 

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-
hydroxy-, octadecyl ester 

Phenol, 4,4',4"-[(2,4,6-Trimethyl-1,3,5-
benzentriyl)tris(methylene)] 

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-
hydroxy-, 2,2-bis[[3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]-1-oxopropoxy]methyl]-1,3-propanediyl ester 

2082-79-3 

531 

1709-70-2 

775 

13.4 

17.2 

6683-19-8 

1178 

19.6 

Amines 

5  1,4-Benzenediamine, N-(1-methylethyl)-N'-phenyl- 

101-72-4 

226 

3.3 

Phosphites & Phosphonites 

6 

7 

8 

9 

2,4,8,10-Tetraoxa-3,9-diphosphaspiro 5.5 undecane, 3,9-bis 
2,4-bis(1,1-dimethylethyl)phenoxy - 

26741-53-7 

605 

12H-Dibenzo[d,g][1,3,2]dioxaphosphocin, 2,4,8,10-
tetrakis(1,1-dimethylethyl)-6-fluoro-12-methyl- (9CI) 

12H-Dibenzo[d,g][1,3,2]dioxaphosphocin, 2,4,8,10-
tetrakis(1,1-dimethylethyl)-6-[(2-ethylhexyl)oxy]-  

118337-09-0  487 

126050-54-2  583 

2,4,8,10-Tetraoxa-3,9-diphosphaspiro 5.5 undecane, 3,9-
bis(octadecyloxy)- 

3806-34-6 

733 

10  Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) 

31570-04-4 

647 

11  Phenol, nonyl-, phosphite (3:1) (TNPP) 

26523-78-4 

689 

10.9 

12.8 

14.9 

15.1 

18.1 

20.1 

12 

Phosphonous acid, [1,1 -biphenyl]-4,4 -diylbis-, tetrakis[2,4-
bis(1,1-dimethylethyl)phenyl] ester 

38613-77-3 

1035 

27.2 

Organosulfur compounds 

13  Propanoic acid, 3,3'-thiobis-, didodecyl ester 

123-28-4 

515 

14  Propanoic acid, 3,3 -thiobis-, ditetradecyl ester 

16545-54-3 

571 

15  Propanoic acid, 3,3'-thiobis-, dioctadecyl ester 

693-36-7 

683 

16  Disulfide, dioctadecyl 

2500-88-1 

571 

11.8 

13.8 

17.7 

18.6 

17 

Propanoic acid, 3-(dodecylthio)-, 2,2-bis[[3-(dodecylthio)-1-
oxopropoxy]methyl]-1,3-propanediyl ester 

29598-76-3 

1162 

24.8 

Oxamides 

18 

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-
hydroxy-, 2-[3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]-1-oxopropyl]hydrazide 

32687-78-8 

553 

7.8 

 
 
Chapter R.11: PBT/vPvB assessment 

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145 

1. Examples for Assessment of Substances with high Log Kow 

Example R.11-5 

Propanioic acid, 3,3’-thiobis-, dioctadecyl ester, CAS No. 693-36-7 

Table R.11—11: Properties of the antioxidant. 

Parameter 

Molecular weight (g/Mol) 

Water solubility (mg/L) 

Log Kow (calculated) 

Ready biodegradable (OECD TG 301B) 

T Criteria fulfilled 

Structure  

Value 

683 

<< 1 

17.7 

No 

No 

STEP 1 

Calculated / measured Log Kow 

Log Kow calculated is 17.7 

STEP 2 

Assessment type to be applied 

Log Kow is > 10 and the T criteria is not fulfilled, this means a vPvB Assessment 
according Step 3 

STEP 3 

vPvB Assessment 

STEP 3a 

Persistence check 

The substance has two ester bonds. Cleaving the ester would lead to 2 Mol of 1-
Octadecanol (1) and 1 Mol of 3,3’-Dithiobispropionic acid (2). Both substances 
(1) and (2) are readily biodegradable and are therefore no PBT or vPvB 
substances. The antioxidant itself is not readily biodegradable in a classical OECD 
TG 301B Sturm test at the usual high substance concentrations although the 
esters could be cleaved. The reason is the very low bioavailability of the 
substance. The biodegradation rate is therefore controlled by the dissolution 
rate. When the ready test (OECD TG 301D Closed Bottle Test) is carried out at 
low concentrations with stirring ready biodegradation can be achieved. In this 
case the assessment is finished with step 3a. 

Conclusion  The antioxidant can be transformed in a ready test to metabolites which 
are itself readily biodegradable. Therefore the substance Propanoic acid, 
3,3’-thiobis-, dioctadecyl ester, CAS No. 693-36-7 is not a vPvB 
Substance. 

SOOOO88  
 
 
 
 
 
 
 
 
 
146 

Example R.11-6 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2,2-bis[[3-[3,5-
bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1-oxopropoxy]methyl]-1,3-propanediyl 
ester, CAS No. 6683-19-8 

Table R.11—12: Properties of the antioxidant. 

Parameter 

Mol weight (g/Mol) 

Water solubility (µg/L) 

Log Kow (calculated) 

Ready biodegradable (OECD TG 301B) 

T criteria fulfilled 

Structure 

Value 

1178 

<< 1 

19.6 

No 

No 

STEP 1 

Calculated / measured Log Kow 

Log Kow calculated is 19.6 

STEP 2 

Assessment type to be applied 

Log Kow is > 10 and T criteria is not fulfilled means vPvB Assessment according 
Step 3 

STEP 3 

vPvB Assessment 

STEP 3a 

Persistence check 

The substance has 4 ester bonds. Cleaving the ester would lead to 4 Mol of 3,5-
bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid (1) and Pentaerythrol 
(2). The acid (1) is not readily biodegradable but in an assessment it was 
demonstrated that (1) is not a PBT substance. Pentaerythrol (2) is readily 
biodegradable and is therefore not a PBT or vPvB substance. The antioxidant 
itself is not readily biodegradable in a classical OECD TG 301B Sturm test at high 
substance concentrations although the esters could be cleaved. The reason is the 
very low bioavailable of the substance. The biodegradation rate is therefore 
controlled by the dissolution rate. Due to the extremely low water solubility of 
the antioxidant a ready test at lower substance concentration will not result in 

OOOOOOOOOHOHOHOH 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

147 

ready biodegradation. In this case the assessment needs to proceed with step 
3b. 

STEP 3b 

Bioaccumulation check 

Supporting information 

Results from Animal studies 

a) OECD TG 305 BCF Study 

The Study is regarded as invalid as the substance was tested above water 
solubility but indicate low bioaccumulation 

b) Animal ADE Studies 

Adsorption, Distribution and Eliminations (ADE) Studies carried out with 
radiolabelled material show low adsorption of the substance. Adsorbed 
radioactivity is most likely starting material 

MW and size criteria 

Dmax > 1.7 nm and MW > 700 g/Mol is fulfilled, substance has a Dmax of 1.79 nm 
and a MW of 1178 g/Mol 

Conclusion  Although the antioxidant has ester bonds which could be cleaved ready 

biodegradation cannot be achieved due to the very low (bio)availability of the 
substance. But there are several information available which support the low 
bioaccumulation potential based on the Log Kow > 10. There are animal studies 
available (fish and rat) demonstrating low adsorption of the substance. In 
addition the MW and size criteria for low bioaccumulation potential are fulfilled as 
well (see Annex 1 ‘Indicators for limited Bioaccumulation’). 

Based on the available information with respect to the bioaccumulation 
potential and the likely metabolites it can be concluded in a Weight-of-
Evidence approach that the antioxidant is not a vPvB substance. 

  
 
 
 
 
 
 
 
 
 
 
 
 
148 

Example R.11-7 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Tris(2,4-di-tert-butylphenyl)phosphite, CAS No. 31570-04-0 

Table R.11—13: Properties of the antioxidant. 

Parameter 

Mol weight (g/Mol) 

Water solubility (mg/L) 

Log Kow (calculated) 

Ready biodegradable (OECD TG 301B) 

T Criteria fulfilled 

Structure  

Value 

632 

<< 1 

18.1 

No 

No 

STEP 1 

Calculated / measured Log Kow 

Log Kow calculated is 18.1 

STEP 2 

Assessment type to be applied 

Log Kow is > 10 and the T criteria is not fulfilled, this means a vPvB Assessment 
according Step 3 

STEP 3 

vPvB Assessment 

STEP 3a 

Persistence check 

The substance has three ester bonds. Cleaving the ester would lead to 3 Mol of 
2,4-Ditert.butylphenol (1) and 1 Mol of phosphite (2). (1) is not a PBT or vPvB 
Substance (EU, 2005) and (2) is an inorganic salt and no PBT or vPvB substance. 
The antioxidant itself is not readily biodegradable in a classical OECD TG 301B 
Sturm test. For metabolic reasons ready biodegradation may not be achieved 
even at lower concentration. But hydrolysis at low concentration using 
radiolabelled material may result in abiotic transformation. 

STEP 3b 

Bioaccumulation check 

Log Kow is > 10 but no further indication for limited bioaccumulation is fulfilled. 

STEP 4 

Overall conclusion 

In this case the indicator Log Kow > 10 is of limited value as the substances does 
not readily biodegrade even at low concentrations and no additional indicators 
for limited bioaccumulation are available. 

In this case further degradation tests are warranted.  

OPOO 
 
 
 
 
 
 
 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

149 

Table R.11—14: Octanol and water solubility of pigments, critical body burden for 
narcotic mode of action and Log Coctanol/Cwater (ETAD, 2006). 

Pigment class 

Colour  

MW 

index 

(g/Mol) 

Octanol  
solubility  
Co (µg/L)   

Critical  
Body  

Burden 
(CBB) 
(µg/L) 

Co<CBB 

Water  
solubility  
Cw (µg/L) 

Log Co/Cw 

Anthanthrone  

P.R. 168  

464 

   124 

   928 

Anthraquinone  

P.R. 177  

444 

   70 

   888 

YES 

YES 

   10.8 

   230 

Benzimidazolone  

P.R. 176  

573 

   15 

   1146 

YES 

   1.9 

Benzimidazolone  

P.R. 208  

524 

   83 

   1048 

YES 

   3.2 

Benzimidazolone  

P.Y. 151  

381 

   210 

   762 

YES 

   17.8 

b-Naphthol  

P.O. 5 

338 

   1760 

   676 

445 

   1250 

   890 

NO 

NO 

   7 

   1250 

b-Naphthol  

BONA *  

BONA  

P.R. 53:1 
(salt)  

P.R. 48:2 
(salt)  

P.R. 57:1 
(salt)  

461 

   170 

   922 

YES 

   650 

426 

   850 

   852 

YES 

   1800 

Diarylide Yellow*  

P.Y. 12  

630 

   48 

   1260 

YES 

   0.8 

Diarylide Yellow  

P.Y. 12  

630 

   50 

   1260 

YES 

   0,4 

Diarylide Yellow  

P.Y. 13  

686 

   22 

   1372 

YES 

   0.8 

Diarylide Yellow  

P.Y. 14  

658 

   3 

   1316 

YES 

Diarylide Yellow  

P.Y. 83  

818 

   9 

   1636 

YES 

Diketopyrrolopyrrole 
Pigment (DPP)  

P.R. 254 

357 

   30 

   714 

YES 

analytical 
problems  

analytical 
problems  

analytical 
problems  

Dioxazin  

P.V. 23  

589 

   330 

   1178 

YES 

   25 

Disazo 
Condensation  

P.Y. 93  

937 

   200 

   1874 

YES 

   110 

BONA = beta Oxynapthoic acid 

* octanol is saturated with water, water is saturated with octanol 

1.1 

-0.5 

0.9 

1,4 

1.1 

2.4 

0.0 

-0.6 

-0.3 

1.8 

2.1 

1.4 

1.1 

0.3 

  
 
 
  
  
  
  
  
  
150 

Chapter R.11: PBT/vPvB assessment 

Version 3.0 – June 2017 

Table R.11—14 (continued) Octanol and water solubility of pigments, critical body 
burden for narcotic mode of action and Log Coctanol/Cwater (ETAD, 2006). 

Pigment class 

Colour  

MW 

index 

(g/Mol) 

Octanol  
solubility  
Co (µg/L)   

Critical  
Body  

Burden 
(CBB) 
(µg/L) 

Co<CBB 

Water  
solubility  
Cw (µg/L) 

Log Co/Cw 

Disazopyrazolone   P.O. 13   624 

  51 

  1248 

YES 

  1.4 

Isoindolinone  

P.Y. 110   642 

  315 

  1284 

YES 

  230 

Monoazo Yellow  

P.Y. 74  

386 

  740 

  772 

YES 

  7.6 

Naphthol AS  

P.R. 112   485 

  3310 

  970 

NO 

  9.8 

Naphthol AS  

P.R. 170   454 

225 

908 

Perinone  

P.O. 43   412 

  13 

  824 

YES 

YES 

  11.9 

  7.2 

Perylene  

P.R. 149   599 

<  12 

>  1198 

YES 

Perylene  

P.Black31  599 

  96 

  1198 

YES 

analytical 
problems  

analytical 
problems  

Perylene  

P.R. 179   576 

<  10 

>  1152 

YES 

<  8 

Perylene  

P.R. 224   392 

<  100 

>  784 

YES 

<  5 

Phthaloblue, 
metalfree  

P.Blue16   515 

<  10,1 

>  1030 

YES 

<  10 

Phthalocyanine  

P.G. 7  

1127 

<  10 

>  2254 

YES 

<  10 

Phthalocyanine  

P.B.15  

576 

<  7 

>  1152 

YES 

<  7 

Quinacridone  

P.R. 122   340 

  600 

  680 

YES 

  19.6 

Quinacridone  

P.V. 19  

312 

  1360 

  624 

NO 

  10.3 

Quinophthalone  

P.Y. 138   694 

  225 

   1388 

YES 

   10 

1.6 

0.1 

2.0 

2.5 

1.3 

0.3 

0.1 

1.3 

0.0 

0.0 

0.0 

1.5 

2.1 

1.4 

 
 
 
 
 
 
 
 
 
 
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151 

2. Example for an assessment strategy for substances with low octanol and water 
solubility 

Example 

Pigment Yellow 12, CAS No. 6358-85-6 

Table R.11—15: Data for Pigment Yellow 12. 

Parameter 

Mol weight (g/Mol) 

Water solubility (µg/L) 

Octanol solubility (µg/L) 

CBB (µg/L) 

Co << CBB 

Log Co/Cw 

Log Co/Cw << 4.5 

Aquatic ecotoxicity L(E)C50 (mg/L) 

14-C Pharmacokinetic male rat 

Value 

630 

0.4 

50 

1260 

YES 

2.1 

YES 

>> 0.1 

No uptake 

Complete excretion through faeces 

STEP 1 

Solubility measurement of Octanol and Water 

Octanol solubility is 50 µg/L and Water solubility 0.4 µg/L, Log Co/CW = 2.1 

STEP 2 

B and T Assessment 

Co < CBB and Log Co/CW < 4.5  

Neither exceedance of CBB nor uptake via membrane is likely. Rat 14C 
Pharmacokinetic study confirms reduced uptake. 

STEP 3 

Weight-of-Evidence approach 

In a Weight-of-Evidence approach based on Co, Log Co/CW as well as on 
pharmacokinetic data it can be concluded that Pigment Yellow 12 is not a vPvB 
Substance and no further test is warranted. 

References 

ETAD (2006) Measurements of Octanol and Water solubility of Pigments, carried out by ETAD 
Member companies, Data ownership is with ETAD. 

Ullmann (1995) Encyclopaedia of Industrial Chemistry, Section Antioxidants. 

  
 
 
 
 
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Appendix R.11—3: PBT assessment of UVCB petroleum substances. 

UVCB petroleum substances are assessed using the same principles as other UVCBs, as 
introduced in Section R.11.4.2.2. However, at the time of developing PBT assessment 
principles for UVCBs the available knowledge on the composition and behaviour of petroleum 
substances was broader than the knowledge available on other types of UVCBs, thereby 
warranting the development of a specific methodology to assess petroleum substances. The 
following subsections introduce how such knowledge can be used. The specific assessment 
path presented is called the hydrocarbon block method, developed by CONCAWE. An 
analogous assessment path may be used for other UVCB categories, if appropriate. 

Step 1: Characterisation of the petroleum substance 

Due to their derivation from natural crude oils and the refining processes used in their 
production, petroleum substances are complex mixtures of hydrocarbons, often of variable 
composition. Many petroleum substances are produced in very high tonnages to a range of 
technical specifications, with the precise chemical composition of particular substances, rarely 
if ever characterized. Since these substances are typically separated on the basis of distillation, 
the technical specifications usually include a boiling range. These boiling ranges correlate with 
carbon number ranges, while the nature of the original crude oil and subsequence refinery 
processing influence the types of hydrocarbon structures present. The CAS name definitions 
established for the various petroleum substance streams generally reflect this, including final 
refinery process; boiling range; carbon number range and predominant hydrocarbon types 
present. 

For most petroleum substances, the complexity of the chemical composition is such that it is 
beyond the capability of routine analytical methodology to obtain complete characterisation. 
Typical substances may consist of predominantly mixtures of straight and branched chain 
alkanes, single and multiple naphthenic ring structures (often with alkyl side chains), single 
and multiple aromatic ring structures (often with alkyl side chains). As the molecular weights 
of the constituent hydrocarbons increase, the number and complexity of possible structures 
(isomeric forms) increases exponentially. 

For the purposes of a PBT assessment of petroleum substances, when required, it is suggested 
that an analytical approach using GCxGC is used when feasible. This method offers a high 
resolution that may also be helpful in being more precise as to the exact type of structures 
present, (Forbes et al., 2006), in contrast to more generic methods based on Total Petroleum 
Hydrocarbon (e.g. TNRCC Method 1005). Still other methods could be used to characterize the 
composition of petroleum substances as the GCxGC method has the caveat that it can only be 
used for carbon numbers up to around C30. 

The outcome of this step should be a matrix of hydrocarbon blocks, containing the % 
contribution of the block to the petroleum substance. With GCxGC this characterisation will be 
extended to include broad descriptions of structures including alkanes, isoalkanes, 
naphthenics, aromatics, etc. 

Step 2: Assessment  

The next step is to collate the available information on persistence, bioaccumulation and 
toxicity of the petroleum substance(s) being assessed. Where this is done as part of a 
category, there will be need for a good justification, which could also include analytical 
characterisation of a category. The assessment of the data will follow similar lines as for any 
data examination, including the extent to which the petroleum substances were characterised 
or described, the type of protocol followed and the quality of the information obtained for the 
respective endpoints. 

 
Chapter R.11: PBT/vPvB assessment 

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Persistence (P)  

153 

The first part of the P assessment would be to examine the available data, and in particular 
attempt to identify whether the data on the petroleum substance(s) under investigation can be 
considered representative for the whole composition. The principles as provided for applying 
the “whole substance” approach as specified in Section R.11.4.2.2 and elements as discussed 
in Section R.11.4.1.1 (Persistence) need to be considered. Where there is convincing evidence 
of ready biodegradation of the whole substance under these principles, it can be reasonably 
assumed that the individual components are unlikely to be persistent. 

If there is insufficient evidence for ready biodegradation or the substance composition is not 
sufficiently homogenous (i.e. the known or assumed constituents are structurally too 
dissimilar) to interpret data on the whole substance, then the assessment should proceed to 
the next stage. This involves generating typical structures either from the chemical analysis 
conducted or from other sources of information relevant to the petroleum substances being 
assessed. For example, Redman et al. (2012, 2014) describe how a set of over 1500 
structures are available for assessing hydrocarbon blocks of petroleum substances. The 
structures cover a wide range of hydrocarbon types including isoparaffinic, normal paraffinic, 
mono-naphthenic (1-ring cycloalkanes), di-naphthenic (2-ring cycloalkanes) and poly-
naphthenic, mono-aromatic, di-aromatic and aromatic (3 to 6-ring cycloalkanes) classes and 
mixed aromatic/napthenic hydrocarbons. By correlating the predicted boiling point of these 
structures to the available analytical information, a series of blocks can be generated in which 
these structures are representative of the types potentially present in the petroleum 
substance. 

The assessment can then proceed by evaluating available degradation half-life information on 
any known individual constituents, e.g. benzene, hexane, pristane etc. This information will in 
every case be insufficient for the assessment of petroleum UVCB substances due to the wide 
range of potential structures and the relatively limited information currently available on most 
of the individual structures that have normally not been tested, as they are rarely isolated or 
manufactured. Consequently, the information will need to be supplemented with data from 
predictive models. 

For hydrocarbons, there are two QSAR models that could be considered for assessing 
environmental degradation half-lives and a third that could be used for assessing potential 
metabolites: 

  Howard et al. (2005) describe a model that predicts the degradation half-life of a 

hydrocarbon in the environment. The model is well described, including information on 
the test/training sets. In using the model it would be advisable to assess the training 
and tests sets to ensure suitable coverage of the structures being assessed. This model 
is freely available in EPISUITE as BIOHCWIN. 

  Dimitrov et al. (2007) also describe a new model that combines CATABOL (Jaworska et 
al., 2002) with assumptions of first order catabolic transformations. The training and 
test sets include information of petroleum substances as well as observed catabolic 
pathways compiled from various sources including public web sites such as EAWAG BBD 
(http://eawag-bbd.ethz.ch). 

  Finally, for demonstrating that there are no concerns, caused by potential degradation 
metabolites (the previous assessments are all addressing primary biodegradation), it is 
recommended that available information is collected and predictions made of relevant 
PBT properties of potential degradation metabolites. CATABOL is an example of 
integration of such an approach in a commercial modelling system (Jawoska et al., 
2002). 

If these assessments indicate that there are structures or blocks that are of concern, the 
assessment can either proceed to the generation of new information as described in the main 

  
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report, or conclude that the assessed blocks can be considered persistent and proceed to the 
bioaccumulation assessment. 

Bioaccumulation (B)  

The B assessment essentially follows the same process as that described for the P assessment 
except that it is highly unlikely that there will be good quality experimental data on petroleum 
UVCB substances. Instead the B assessment is more likely to address the individual structures 
for their potential to bioaccumulate. This, as with the P assessment, will start with addressing 
where there is available experimental evidence to be able to draw a conclusion on the B 
properties of blocks or individual constituents. 

Where there are insufficient experimental data to be able to make a judgement there are 
several QSAR models available for continuing the process. These are discussed in Section 
R.7.10.3.2 of Chapter R.7c of the Guidance on IR&CSA and Annex 1 to Appendix R.11—1 of 
this Guidance document. An assessment of the predictions from these QSAR models, with 
available experimental information should lead to the identification of those blocks where there 
are concerns for their potential (or realised, if specific structures are assessed) ability to 
bioconcentrate. The use of experimental fish bioaccumulation data is preferred over that from 
other sources, including invertebrates, because fish bioaccumulation data are generally more 
reliable as standard test methods/guidelines are used to determine them. Fish bioaccumulation 
data include the effect of biotransformation in fish which can be substantial for some 
hydrocarbons. Such data also provide indications of whether the potential for food-chain 
magnification at higher trophic levels exists. This type of data, with further information on 
trophic level biomagnification or dilution, can be used in a Weight-of-Evidence approach to 
demonstrate whether the longer term uncertainties associated with bioaccumulation of 
constituents may exist. 

Toxicity (T) 

Assessment of the toxicity of all individual constituents within a petroleum substance would in 
many cases be extremely difficult or practically impossible. While the whole substance 
assessment using the Water Accommodated Fraction (WAF) methodology has been accepted 
for classification purposes (OECD, 2001), the use of this information for the T assessment is 
problematic. 

For petroleum substances, a model, PETROTOX, has been developed (Redman et al., 2012), 
based on previous work assuming a non-polar narcosis mode of action (i.e. baseline toxicity, 
McGrath et al., 2004, 2005). The equations underlying the hazard portion of this model, which 
was developed to predict the acute and chronic ecotoxicity of petroleum substances and 
hydrocarbon blocks, may be used to address the predicted baseline toxicity of individual 
structures when no experimental data are available.  

It should be noted that for the ultimate conclusion on the T property, long-term toxicity test 
results are generally necessary as, at present, no appropriate prediction tools for long-term 
ecotoxicity are available. The prediction tools may, however, be used as supporting tools for 
designing tests and for the interpretation of experimental results. Before initiating 
experimental fish toxicity tests it should be considered whether data exist allowing a robust 
conclusion to be drawn on whether the substance fulfils the Tmammalian criteria (see Section 
R.11.4.1.3). 

How to proceed further 

Where there are constituents or blocks that show a concern for both P and B properties, there 
is a need to generate further higher tier information on these properties. Exceptions to this 

 
 
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155 

conclusion might be in case there are sufficient ecotoxicological data on specific constituents or 
representative structures in the blocks that demonstrate no concern for the T criterion and 
where the P and B properties are concluded not to indicate vPvB-properties. 

References 

Dimitrov S, Pavlov T, Nedelcheva D, Reuschenbach P, Silvani M, Bias R, Comber M, Low L, Lee 
C, Parkerton T and Mekenyan O (2007) A Kinetic Model for Predicting Biodegradation. SAR 
QSAR Environ Res 18:443-57. 

Dimitrov S, Dimitrova N, Parkerton T, Comber M, Bonnell M, Mekenyan O (2005). Base-line 
model for identifying the bioaccumulation potential of chemicals. SAR QSAR Environ Res 
16(6):531-54. 

Ellis LBM, Roe D and Wackett LP (2006) The University of Minnesota 
Biocatalysis/Biodegradation Database: The First Decade. Nucleic Acids Res 34:D517-D521. 

Forbes S, Eadsforth C, Dmytrasz B, Comber M and King D (2006) Application of comprehensive 
two-dimensional gas chromatography (GCxGC) for the detailed compositional analysis of gas-
oils and kerosines – SETAC Den Haag. 

Howard P, Meylan W, Aronson D, Stiteler W, Tunkel J, Comber M, Parkerton T (2005) A new 
biodegradation prediction model specific to petroleum hydrocarbons, Environ Toxicol Chem 
24:1847-60. 

Jaworska J, Dimitrov S, Nikolova N and Mekenyan O (2002) Probabilistic assessment of 
biodegradability based on metabolic pathways: catabol system. SAR QSAR Environ Res 
13:307-23. 

McGrath JA, Parkerton TF and Di Toro DM (2004) Application of the narcosis target lipid model 
to algal toxicity and deriving predicted no effect concentrations. Environ Toxicol Chem 
23:2503–17. 

McGrath JA, Parkerton TF, Hellweger FL and Di Toro DM (2005) Validation of the narcosis 
target lipid model for petroleum products: gasoline as a case study. Environ Toxicol Chem 
24:2382–94. 

OECD (2001) OECD Series on Testing and Assessment, Number 27, Guidance Document on 
the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for 
the Aquatic Environment. ENV/JM/MONO(2001)8 (available at: 
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2001)8
&doclanguage=en).  

Redman AD, Parkerton TF, McGrath JA and Di Toro DM (2012) PETROTOX: An aquatic toxicity 
model for petroleum substances. Environ Toxicol Chem 31:2498–2506. 

Redman AD, Parkerton TF, Comber MH, Paumen ML, Eadsforth CV, Dmytrasz B, King D, 
Warren CS, den Haan K, Djemel N (2014) PETRORISK: a risk assessment framework for 
petroleum substances. Integr Environ Assess Manag. 10(3):437-48. 

TNRCC (2001) TNRCC Method 1005, TOTAL PETROLEUM HYDROCARBONS, Texas Natural 
Resource Conservation Commission, Revision 03, June 1, 2001. 

US EPA (2012) Estimation Programs Interface Suite™ for Microsoft® Windows, v 4.11. United 
States Environmental Protection Agency, Washington, DC, USA. 

  
 
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Appendix R.11—4: Bioconcentration studies with benthic and terrestrial invertebrate 
species (BSAF). 

In case data are available from bioconcentration studies on benthic and terrestrial invertebrate 
species they may be used as indicator for a high bioaccumulation potential. Results of these 
studies are expressed as biota-to soil/sediment accumulation factor (BSAF). In order to 
compare BSAF with BCF values care must be taken if a species with a very low lipid content 
was used because BCF values are normaly reported on a wet weight basis. Lipid normalization 
(to 5% lipid content) should therefore always be performed, whenever possible for substance 
that are lipid binding.  

The relationship between BSAF and BCF is epressed in the following equation, in which BCF 
could be replaced by the criterion for B or vB. 

A terrestrial or benthic (lipid and organic carbon normalized) BSAF value for a substance with a 
Log Kow of 4.5 that exceeds the value of 2 is an indication of a BCF of 2000 and higher, based 
on pore water concentration. Similar for a substance with a Log Kow of 4.5 a BSAF value higher 
than 5 is an indication that the BCF exceeds the value of 5000, based on pore water 
concentration.  

Figure R.11—10: Relationship between lipid and organic carbon normalised BSAF 
values and Log Kow as indicator for the B and vB criterion.  

The solid line is calculated with a BCF value (5% lipids) from pore water of 2000, the dotted 
line is calculated with a BCF value of 5000. The Log Koc has been calculated according to the 
equation Log Koc = Log Kow - 0.21 by Karickhoff et al. (1979). 

Due to increasing sorption with Log Kow, the BSAF values for calculated BCF values of 2000 and 
5000 rapidly decrease. Therefore, for a substance exceeding Log Kow of 5.5, a BSAF value in 
the order of 0.5 and above indicates high bioaccumulation potential.  

However, lower BSAF values are difficult to interpret in the context of the B and vB assessment 
due to several confounding factors. Sorption and bioconcentration increase with 

vBofindicationforKorBofindicationforKKlipidBCFBSAFocococ05.0/500005.0/200001234544.555.566.57log KowBSAF 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chapter R.11: PBT/vPvB assessment 

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157 

hydrophobicity, and as it is not necessarily in the same manner, sorption is an important 
parameter dependent on soil and substance properties. Bioconcentration might be reduced 
compared to what is expected from Log Kow value but even low BSAF values of 0.1 and lower 
do not necessarily mean that the BCF value based on pore water concentration do not exceed 
5000, because of the strongly increased sorption for highly hydrophobic substances. Moreover, 
sorption might be higher than what is expected from Log Kow because sorption to carbonaceous 
materials may play an important role. Besides that, for these low BSAF values it is often 
difficult to distinguish between real uptake and adsorption to the organisms or interference of 
gut content in the determination of the BSAF values. 

In conclusion, lipid and organic carbon normalized BSAF values of 0.5 and higher are an 
indication of high bioaccumulation. In some cases these values might be considered to be 
enough evidence in itself to assess the substance as B and vB, especially if reliable 
experimental data on pore water concentrations are available and the system is in equilibrium. 
However, lower BSAF values should not be used to the contrary, because low uptake from 
sediment or soil does not imply a low aquatic BCF value. 

  
 
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