Review of QSAR Models for Ready Biodegradation

Transcription

1 Review of QSAR Models for Ready Biodegradation Manuela Pavan and Andrew P. Worth 2006 EUR EN

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3 EUROPEAN COMMISSION DIRECTORATE GENERAL JOINT RESEARCH CENTRE Institute for Health and Consumer Protection Toxicology and Chemical Substances Unit European Chemicals Bureau I Ispra (VA) Italy Review of QSAR Models for Ready Biodegradation Manuela Pavan and Andrew P. Worth 2006 EUR EN

4 The mission of the IHCP is to provide scientific support to the development and implementation of EU policies related to health and consumer protection. European Commission Directorate General Joint Research Centre Institute for Health and Consumer Protection Contact information Address: E. Fermi, 1, Ispra (VA) Italy Tel.: Fax: ihcp.jrc.cec.eu.int LEGAL NOTICE Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server ( EUR EN ISSN European Communities, 2006 Reproduction is authorised provided the source is acknowledged Printed in Italy

5 ABSTRACT Many regulatory laws resulting from the enactment of the United Nations Stockholm Convention in May 2004, together with the new REACH legislation, have promoted significant new activity in the assessment of Persistent, Bioaccumulative and Toxic (PBT) substances. These are chemicals that have the potential to persist in the environment, accumulate within the tissues of living organisms and, in the case of chemicals categorised as PBTs, show adverse effects following long-term exposure. Under REACH, estimated data generated by (Q)SARs may be used both as a substitute for experimental data, and as a supplement to experimental data in weightof-evidence approaches. It is foreseen that (Q)SARs will be used for the three main regulatory goals of hazard assessment, risk assessment and PBT/vPvB assessment. In the Registration process under REACH, the registrant will be able to use (Q)SAR data in the registration dossier, provided that adequate documentation is given to argue for the validity of the model(s) used. The experimental determination of the persistence, bioconcentration and toxicity is generally expensive and demanding to perform. For this reason, measuring experimentally the potential PBT profiles of those chemicals that are of potential regulatory interest is considered not feasible. The limited empirical data, the high test costs together with the regulatory constraints and the international push for reduced animal testing motivates a greater reliance on QSAR models in PBT assessment. This report provides an overview of PBT regulations and criteria, and gives a detailed review of QSAR for estimating the biodegradation of chemicals. The role of biotransformation in the modelling of PBT substances is also described.

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7 CONTENTS 1. INTRODUCTION 1 2. PBT SUBSTANCES: DEFINITIONS Persistence Bioaccumulation / Bioconcentration Toxicity 5 3. REVIEW OF PBT REGULATIONS Stockholm Convention OSPAR Convention North American Regional Action Plans (NARAPs) EU Water Framework Directive (2000/60/EC) EU REACH programme US EPA PBT Profiler Canadian Domestic Substances List categorisation PBT Japanese chemical legislation OVERVIEW OF PBT AND vpvb CRITERIA REACH PBT criteria REACH vpvb criteria METHODS FOR PBT DATA GENERATION Persistence data generation Biodegradation data Bioaccumulation data generation Toxicity data generation BIODEGRADATION DATABASES BIODEG Database BIOLOG Database MITI Database ESIS Database University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD) California Department of Food and Agriculture Biodegradation Database QSARS FOR BIODEGRADATION Group contribution approaches Degner et al. OECD hierarchical model approach Multivariate Partial Least Squares (PLS) model Biodegradation Probability Program BIOWIN Linear and Non-Linear Biodegradation Model Ultimate and Primary Biodegradation Model Linear and Nonlinear MITI Biodegradation Model MultiCASE anaerobic program Biodegradation model based on diverse theoretical descriptors Expert system approaches Inductive machine learning method BESS 49

8 7.3.3 MultiCASE / META biodegradability CATABOL probabilistic assessment of biodegradability TOPKAT TGD models for persistence VALIDATION STUDIES ON BIODEGRADATION MODELS BIODEG/PLS/MultiCASE/ Machine learning method validation on MITI-I BIODEG validation PLS biodegradation model validation MultiCASE model validation Machine learning model validation BIODEG/PLS/MultiCASE validation on HPVC BIODEG/OECD/PLS/MultiCASE validation on 894 MITI-I test BIOWIN/PLS/MultiCASE/CATABOL validation performance comparison CATABOL validation on chemicals under the Japanese Chemical Substances Control Law CONCLUSIONS REFERENCES 72

9 LIST OF ABBREVIATIONS AQUIRE B BAF BCF CAS C&L CEPA CIS COMMPS CSCL CTV DSL EC EEV EFDB EINECS ELINCS ENEV EPA ESIS ESR EU EURAM GA HPVC Kow LPVC LRT MITI NACEC NARAP NLP OECD OPPT OSPAR PMN POP PBiT PBT QSAR QSPR Q2ext REACH Union) RTECS RMSE R2 AQUatic toxicity Information REtrieval system Bioaccumulation Bioaccumulation Factor Bioconcentration Factor Chemical Abstracts Service Classification and Labelling Canadian Environmental Protection Act Chemical Information System Combined Monitoring-based and Modelling-based Priority Setting Chemical Substances Control Law (Japan) Critical Toxicity Value Domestic Substances List (Canada) European Commission Estimated Exposure Value Environmental Fate Database (by SRC) European Inventory of Existing Commercial chemical Substances European List of Notified Chemical Substances Estimated No Effects Value Environmental Protection Agency European chemical Substances Information System Existing Substances Regulation (European Union) European Union EU Ranking Method Genetic Algorithm High Production Volume Chemical Octanol-water partition coefficient Low Production Volume Chemical Long-Range Transport Ministry of International Trade and Industry North American Commission for Environmental Co-operation North American Regional Action Plan No-Longer Polymers Organisation for Economic Cooperation and Development Office of Pollution Prevention and Toxics (U.S. EPA) Oslo-Paris Convention Premanufacture Notice (U.S. EPA) Persistent Organic Pollutant Persistent Bio-accumulating and inherently Toxic chemical Persistent Bio-accumulating Toxic chemical Quantitative Structure-Activity Relationship Quantitative Structure-Property Relationship Explained variance in prediction calculated by external validation Registration, Evaluation, Authorisation of Chemicals (European Registry of Toxic Effects of Chemical Substances Root Mean Squared Error Coefficient of determination

10 s SD SMOC SRC TC NES TGD TRI TSCA TSMP UM-BBD UNEP vpvb WFD Standard error of the estimate Standard Deviation Sound Management of Chemicals Syracuse Research Corporation Technical Committee on New and Existing Substances (European Union) Technical Guidance Document Toxic Chemical Release Inventory Toxic Substances Control Act (U.S. EPA) Toxic Substances Management Policy University of Minnesota Biocatalysis/Biodegradation Database United Nations Environment Programme very Persistent very Bioaccumulative Water Framework Directive

11 1. INTRODUCTION Persistent, bioaccumulative, and toxic chemicals (PBTs) are the subject of several national, and international effort to limit their production, and use. PBT chemicals exhibit low water solubility and high lipid solubility, leading to their high potential for bioaccumulation. In addition, multimedia releases and volatility lead to long range environmental transport both via water and the atmosphere, resulting in widespread environmental contamination of ecosystems and organisms, including humans. The possible effects of long term and cumulative exposure to such chemicals is not always addressed adequately in risk assessment methods that base the evaluation on acute toxicity and short term exposure. As a subgroup of PBT (Persistent, Bioaccumulating and Toxic) substances, Persistent Organic Pollutants (POP) are of global concern as these substances are not only extremely persistent and bioaccumulating but they can also be transported in the air or other environmental media far from their sources. POPs and PBTs have become the subject of growing attention and risk management measures all over the world. The UNEP (United Nations Environment Programme) global Stockholm Convention addressed POPs and aimed at elimination of the releases of the listed POP substances. Moreover, it provided a general obligation to take measures to prevent production and use of new substances that exhibit the characteristics of POPs and it established internationally agreed screening criteria for POPs. The Convention included a procedure for identifying new POPs to be put under global control. One of the criteria for persistence and long-range transport (LRT) is "scientific evidence", which can include model calculations. Quantitative structure-activity relationship (QSAR) models have been identified, both in scientific and policy communities, as a prominent tool for providing such evidence. The scientific and regulatory issues for PBTs require the identification of chemicals having these undesirable properties, and the assignment of priority to such groups. On 29 October 2003, the European Commission (EC) adopted a legislative proposal [1] for a new chemical management system called REACH (Registration, Evaluation and Authorisation of Chemicals), intended to harmonise the information 1

12 requirements applied to New and Existing Chemicals. The REACH Regulation, aims among other things at identifying, evaluating and regulating PBT substances effectively. To this end, it establishes clear criteria for the PBT properties of chemicals. Annex XI of the legislative proposal for REACH provides for the use of valid (Q)SARs for predicting the environmental and toxicological properties of chemicals, in the interests of time- and cost-effectiveness and animal welfare. An increased use of quantitative structure activity relationship (QSAR) models is thus foreseen for the hazard and risk assessment of chemicals in the European Union [2]. The purpose of this report is to review available QSAR models that could be used to estimate chemical biodegradability. This report also discusses how QSAR models can be used to provide reliable predictions of biodegradation in support of the identification and characterisation of PBTs, and highlights how these estimates can be used for regulatory and non-regulatory purposes. A concise summary of the main concepts and terminology used in the PBT field is provided together with a short section on a persistence testing strategy accepted in international and national programmes. 2

13 2. PBT SUBSTANCES: DEFINITIONS Persistent Organic Pollutants (POPs) and Persistent, Bioaccumulative and Toxic (PBT) substances are carbon-based chemicals that resist degradation in the environment and accumulate in the tissues of living organisms, where they can produce undesirable effects on human health or the environment at certain exposure levels. 2.1 Persistence The persistence of a substance is the length of time that a substance remains in a particular environment before it is physically transported to another compartment and/or chemically or biologically transformed [3]. The primary degradation of a substance refers to the process of producing organic derivatives. The resulting one or more products exhibit their own properties, reactivities, fates, and effects. The metabolites can be either less toxic (detoxification) or even more toxic (toxification). Mineralisation refers to the complete (ultimate) degradation of an organic chemical to stable inorganic forms of C, H, N, P, etc. Abiotic degradation is the transformation of organic substances by chemical reactions like oxidation, reduction, hydrolysis, and photodegradation. It does not usually result in a complete breakdown of the chemical (mineralisation). Biodegradation is the transformation by microrganisms of organic compounds by enzymatic reactions like oxidation, reduction, and hydrolysis. In soil and sediment, biodegradation is often the most important factor in the removal of the chemical from the environment. Depending on the ambient conditions, different modes and rates of biodegradation may predominate and may make a chemical readily biodegradable at one site, but not at another because of different degradative capacities. Microbial transformation is usually the only way by which a xenobiotic organic compound may be mineralised in the environment, while abiotic processes commonly yield other organic degradation products. 3

14 The bioavailability of a chemical depends on its chemical and physical reactivity with various environmental components and its ability to be absorbed through the gastrointestinal tract, respiratory tract and/or skin of susceptible species. It determines the fraction of compounds able to interact with the biosystem of organisms per unit time. 2.2 Bioaccumulation / Bioconcentration The terms bioaccumulation and bioconcentration refer to the uptake and build-up of chemicals that can occur in living organisms. Bioaccumulation is the process where the chemical concentration in an aquatic organism achieves a level that exceeds that in the water as a result of chemical uptake through all routes of chemical exposure (e.g. dietary absorption, transport across the respiratory surface, dermal absorption, inhalation). Bioaccumulation takes place under field conditions. The level of chemical bioaccumulation is usually expressed in terms of the bioaccumulation factor (BAF), defined as the ratio of the chemical concentrations in the organism (CB) and the water (Cw) [4]: BAF = CB/ CW Eq. 1 Bioconcentration is the process where the chemical concentration in an aquatic organism achieves a level that exceeds that in the water as a result of exposure of the organism to a chemical concentration in the water via npn-dietary routes. Bioconcentration refers to a condition, usually derived under laboratory conditions, where the chemical is absorbed from the water via the respiratory surface and/or the skin only. The extent of chemical bioconcentration is usually expressed in the form of the bioconcentration factor (BCF), which is the ratio of the chemical concentration in the organism (CB) and the water (Cw) [4]: BCF = CB / Cw Eq. 2 Several chemical properties limit the absorption and distribution of chemicals, thus reducing the uptake and distribution in such a way that the BCF can be considered of no or of limited concern. The EU PBT Working Group, established under the Technical Committee on New and Existing Substances (TC NES), identified some indicators (molecular weight, molecular length, a maximum cross-sectional diameter 4

15 and octanol solubility) that either alone or in combination indicate that chemicals may not bioconcentrate to a level of concern, recognising the uncertainties in the interpretation of experimental results. 2.3 Toxicity A toxic substance has the potential to generate adverse human health or environmental effects at specific exposure levels. The intrinsic toxicity of a substance can be identified by standard laboratory tests. For the environment, these properties include short-term (acute) or long-term (chronic) effects. For human health, the properties include toxicity through breathing or swallowing the substance, and effects such as cancer, mutagenicity, reproductive toxicity and neurological effects. 5

16 3. REVIEW OF PBT REGULATIONS In recent decades environmental pollution has been considered a problem of high concern which has motivated the idea of a required sustainable development as a comprehensive strategy to govern human activities and their relationship with the environment. The first announcement of pollution problems dates back to 1972 in the Stockholm United Nations Conference on the Human Environment. In this forum the need for countries to improve living standards was agreed and twenty six principles were stated to guarantee that development was sustainable [5]. The sustainability topic was addressed some years after at the Conference on Environment and Development held in Rio de Janeiro in The Rio Summit developed a major plan for sustainable development called Agenda 21 [6], which is a plan of actions to be taken globally, nationally and locally by organisations of the United Nations System, Governments, and major groups. The identification, banning and reduction of chemicals that are persistent, bio-accumulative and toxic were addressed as actions to be undertaken. Several programs and conferences were started during the 1990s related to the PBT policy. Several governments, as well as regional economic integration organisations, have established programs for identifying and assessing substances with PBT/POP properties. Similarly, regional and global regimes and organisations have adopted criteria or guidelines for identifying, assessing and managing such substances. The better known of these are described briefly in this chapter. 3.1 Stockholm Convention The UNEP Governing Council in May 1995 [7] agreed on an international action plan to protect human health and the environment by the reduction or elimination of POPs. The May 1995 decision targeted a short-list of twelve POPs: aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and toxaphene. In 1997 the UNEP Governing Council decided [8] to establish a negotiating committee to develop a global instrument to address POPs and to initiate a number 6

17 of immediate actions pertaining to exchanging information, identifying alternatives to POPs, identifying sources and managing and disposing of certain POP-containing materials and wastes. The main outcome of those negotiations was the Stockholm Convention on POPs, which was adopted by 127 countries in Stockholm in May 2001 [9]. The Stockholm Convention is the first global, legally binding instrument of its kind with scientifically based criteria for potential POPs and a process that ultimately may lead to elimination of a POP substance globally. The criteria for persistence in Annex D of the convention are expressed as single-media criteria as follows: Evidence that the half-life of the chemical in water is greater than two months, or that its half-life in soil is greater than six months, or that its halflife in sediment is greater than six months; or Evidence that the chemical is otherwise sufficiently persistent to justify its consideration within the scope of the Convention. In the Convention it was agreed that candidate substances to be considered under the Convention were initially screened against the criteria and further assessed based on additional information. Surrogate information might also be submitted for persistence and bioaccumulation, e.g. monitoring data indicating that the bioaccumulation potential was sufficient to warrant consideration of the substance. 3.2 OSPAR Convention The Oslo-Paris (OSPAR) Convention for the Protection of the Marine Environment of the North-East Atlantic adopted a Strategy with regard to Hazardous Substances at Sintra in 1998 which aimed to prevent pollution by continuously reducing discharges, emissions and losses of hazardous substances (identified by specific PBT criteria) by 2020 in order to reach close to zero concentrations in the marine environment [10]. Under the OSPAR Convention, a dynamic selection and prioritisation scheme for substances that may cause a risk to the marine environment (called DYNAMEC) was developed [11]. The scheme highlighted substances with PBT properties and was based on several steps: Step 1: Selection of candidates for priority setting. 7

18 Step 2: Elaboration of a priority list based on an exposure assessment using data from monitoring and effects assessment, and on scoring by applying a modified EURAM (European Union Risk Ranking Method) procedure. Step 3: Elaboration of a priority list based on predicted exposure data modelled from production volume, use pattern, distribution within the environmental compartments, persistence, and effects; and on scoring, again by applying the modified EURAM procedure. Step 4: Consolidation/validation of the higher-ranking substances through a comparison of the monitoring- and modelling-based lists, using expert judgment together with additional information. Step 5: Further detailed consideration, using expert judgment, of the substances ranking the highest in the risk-ranking exercise (step 4), establishing finally a priority list. Measured concentrations were used as input for the monitoring-based ranking. For the modelling-based ranking the scale of the model was at the European level, corresponding to the "continental scale" defined in the EU-Technical Guidance Document [12]. Emissions were supposed to be estimated from production volume, main use category and fractions of release, while distribution was evaluated by applying the Mackay Level 1 model. Degradation was evaluated by taking into account the results of biodegradability testing (e.g. ready biodegradability and inherent biodegradability). 3.3 North American Regional Action Plans (NARAPs) Within the North American Commission for Environmental Co-operation (NACEC) Sound Management of Chemicals (SMOC) initiative, substances with PBT properties were identified as a priority. A three-stage process was worked out for the nomination, evaluation and selection of substances for preparation of NARAPs. In stage I, substances were nominated by any of the Parties providing information in a complete and concise "Nomination Dossier" with key references, following an agreed format. Stage II was based on screening to collect all available information. Four basic information requirements were considered necessary: 8

19 valid monitoring or predicted data related to emissions, effluents or levels in environmental media or biota confirming that the substance may enter, is entering or has entered the North American ecosystem as a result of human activity; a comprehensive, scientifically based risk assessment document to characterise risks to the environment or human health; adequate measured or predicted data relating to the persistence, bioavailability and bioaccumulation of the substance; adequate indirect evidence of the potential for transboundary environmental transport such as persistence in biota/media and volatility. Stage III consisted of a detailed evaluation intended to provide valid reasons for supporting the selection of a substance as a candidate for regional action. The NACEC-SMOC process for developing NARAPs allowed predictive data. 3.4 EU Water Framework Directive (2000/60/EC) On 23 October 2000, the European Parliament and the Council adopted "Directive 2000/60/EC establishing a framework for the Community action in the field of water policy" (commonly referred to as the Water Framework Directive; WFD) The main purpose of the WFD was to protect the inland surface waters, transitional waters, coastal waters and groundwater. A distinction was clearly made between hazardous substances and priority substances and amongst those priority hazardous substances. Hazardous substances included PBT substances and other substances giving rise to an equivalent level of concern; Priority substances are substances identified through simplified risk assessment based on a hazard assessment focusing on aquatic toxicity and human toxicity via aquatic exposure routes and evidence of widespread environmental exposure; Priority hazardous substances were supposed to be identified by the Commission by taking into account the selection of substances of concern undertaken in the relevant Community legislation regarding hazardous substances or relevant international agreements. 9

20 Two different levels for emission controls of substances were defined, depending on whether these substances were classified as priority substances or priority hazardous substances. The Commission was then expected to submit proposals for emission controls and environmental quality standards within two years of the inclusion on the substance on the list of priority substances. The Directive aimed at the cessation or phasing out of discharges, emissions and losses of the substances concerned by an appropriate timetable for the implementation of these measures that should not exceed 20 years. Under the WFD the priority substances were selected on the basis of a comprehensive or, if not available in time, a simplified risk assessment. A procedure named COMMPS (Combined Monitoring-based and Modelling-based priority setting) was developed to prioritise chemical parameters, leading to a ranking of exposure based on both monitoring and model predicted data. Toxicity data were also ranked and the final product of these rankings was used in the final priority setting. This scheme provided a list containing a number of well-established PBTs as indicator compounds. In 2001, the European Commission adopted a proposal for the list of priority substances to include priority hazardous substances [13]. While many of the priority hazardous substances identified can be characterised as PBT compounds, no specific PBT cut-off criteria were developed. For the revision of the list of priority substances it was planned to identify priority hazardous substances on the basis of PBT criteria agreed in the European Community. 3.5 EU REACH programme On 29 October 2003, the European Commission adopted a proposal for a new EU regulatory framework for chemicals. The two most important aims of the new system called REACH [1] (Registration, Evaluation and Authorisation of CHemicals) are to enhance the competitiveness of the EU chemicals industry and to improve protection of human health and the environment from the risks of chemicals. REACH will create a single system for both existing and new chemicals. Under REACH, enterprises that manufacture or import more than one tonne of a chemical 10

21 substance per year are required to register information of the chemical in a central database. The Registration procedure will require manufacturers and importers of chemicals to obtain relevant information on their substances and to use that data to manage them safely. To reduce testing on vertebrate animals, data sharing is required for studies on such animals. Better information on hazards and risks and how to manage them will be passed down and up the supply chain. To prevent unnecessary testing, authorities will evaluate the proposals for testing made by industry and will check compliance with the registration requirements, on the basis of which they may ask industry for further information. The Evaluation procedure enables authorities to investigate chemicals with potential risks by asking industry for further information. Substances with properties of very high concern will be subject to the Authorisation procedure. Applicants will have to demonstrate that risks associated with uses of these substances are adequately controlled. In this case the Commission will grant an authorisation. Otherwise an authorisation may be granted for uses of these substances if the socio-economic benefits outweigh the risks and there are no suitable alternative substitute substances or technologies. The Restrictions procedure will provide a means of regulating the manufacture, placing on the market or use of certain dangerous substances, which will either be subject to conditions or prohibited. Thus, restrictions will act as a safety net to manage Community wide risks that are otherwise not adequately controlled. Article 56 of the REACH proposal outlines that substances which are persistent, bioaccumulative and toxic in accordance with the criteria set out in Annex XIII and substances which are very persistent and very bioaccumulative in accordance with the criteria set out in Annex XIII are considered of very high concern. These substances may be included in Annex XIV, and subsequently are considered subject to authorisation. The objective of the PBT and vpvb assessment will be to determine if the substance fulfils the criteria for the identification of PBT and vpvb substances given in Annex XIII and if so, to characterise the potential emissions of the substance. A hazard assessment addressing all the long-term effects and the estimation of the long-term 11

22 exposure of humans and the environment, cannot be carried out with sufficient reliability for substances satisfying the PBT and vpvb criteria, which necessitates the need for a separate PBT and vpvb assessment. The PBT and vpvb assessment should be based on all the information submitted as part of the technical dossier. If the technical dossier contains for one or more endpoints only information as required in Annexes VII and VIII, the registrant should consider whether further information needs to be generated to fulfil the objective of the PBT and vpvb assessment. The PBT and vpvb assessment will comprise the following two steps: Step 1. Comparison with the Annex XIII criteria to establish whether the substance fulfils or does not fulfil the criteria. If the available data are not sufficient to decide whether the substance fulfils the criteria, then other evidence giving rise to an equivalent level of concern should be considered on a case-by-case basis. Step 2. Emission Characterisation, if the substance fulfils the criteria. In particular, this should contain an estimation of the amounts of the substance released to the different environmental compartments during all activities carried out by the manufacturer or importer and all identified uses, and an identification of the likely routes by which humans and the environment are exposed to the substance. Non-test information may also under REACH be used in helping making best scientific interpretation of all available test data. Even though this type of assessment is relatively new, quite specific screening criteria, some of which include use of molecular structure considerations and QSARs, have already been developed, tested and used by the PBT Working Group under the TCNES (Technical Committee on New and Existing Substances). This experience together with use of available test data and expert judgment, should create the best scientific basis for deciding that the identification of PBT candidates and further testing needs are both rational and consistent. The general experience of the working group indicates that practical further development and acceptance of 12

23 non-testing approaches may best take place in a continuous process taking new scientific developments into account and by involvement of the stakeholders (i.e. governmental experts, industry and NGOs). Based on such work further guidance on use of non-test information for screening for PBTs should be extended and provided as guidance under REACH. It would also be relevant under REACH to periodically update such non-testing based PBT screening criteria and guidance in light of the further scientific development of non-testing modelling tools and approaches. The PBT and vpvb assessment requires information on three intrinsic properties of chemicals, i.e. persistence, bioaccumulation and toxicity, which are evaluated independently, but tested sequentially. Substances recognised as persistent, bioaccumulative and toxic (PBT) substances under Article 56 and very persistent and very bioaccumulative (vpvb) require the production of an Annex XV dossier to propose that a substance should be identified as a PBT or a vpvb substance. If agreed, the substance is then added to the pool of substances to be prioritised for inclusion in Annex XV and after inclusion it will be subject to authorisation. The overall process leading to the Annex XV dossier will normally be started by a Member State, or the Agency on behalf of the Commission, when they consider that a substance may be a PBT, vpvb or substance of equivalent concern. The next steps will be to obtain the relevant available information and review it. If the available data are considered to be sufficient then the Annex XV dossier can be prepared. In cases where the data are not considered sufficient in one or more areas, a substance evaluation should be performed in order to generate the required information. The information gained through the evaluation will also be reviewed in the same way. This may be a multi-step process with several iterations. The basic process is set out in the flow chart of Figure 1. 13

24 Figure 1 Process leading to the production of an Annex XV dossier. 3.6 US EPA PBT Profiler Syracuse Research Corporation (SRC), on behalf of the US EPA, has developed the PBT Profiler [14]. This is an internet-accessible program, designed to assess the hazard characteristics of a chemical against US EPA criteria. The PBT Profiler was developed jointly by EPA, The American Chemistry Council, the Chlorine Chemistry Council, the Synthetic Organic Chemical Manufacturers Association and Environmental Defence. The PBT Profiler is a subset of methods included in the P2 Framework, which is an approach to risk screening that incorporates pollution prevention principles in the design and development of chemicals. The objective of the P2 Framework is to inform decision-making at early stages of development and to promote the selection and application of safer chemicals and processes. This approach is implemented by means of a subset of estimation methods included in OPPT's P2 Framework [15]. 14

25 The tool includes methods for estimating environmental persistence (P), bioconcentration potential (B), and aquatic toxicity (T) built upon SRC s EPISUITE software that estimates physico-chemical properties, environmental fate and effects of molecules using models that are either fragment or Kow based QSARs, or expert systems, or some combination of the three. For persistence, the PBT Profiler determines a substance s half-life in air, water, soil, and sediment based on the AOPWIN and BlOWIN 3 models and certain assumptions. The medium (or media) in which a chemical is most likely to be found is identified by using a Mackay Level III multi-media mass balance model (fugacity model). This medium is then selected and the model assigns a rank of high, medium, or low to the chemical by comparing against US EPA criteria. Bioaccumulation is estimated according to the BCFWIN model. Finally, toxicity is determined from the chronic value estimated by the QSARs in ECOSAR and, again, after criteria comparison, the same rankings are applied. In addition, the PBT Profiler compares results with the PBT criteria established for Premanufacture Notices (PMNs) submitted under section 5 of TSCA; and the final rule for reporting chemicals under the Toxic Chemical Release Inventory (TRI). Results are displayed in three levels of detail, with useful information for management of any potential risks associated with the chemical. It is emphasised by the EPA that it does not rely solely on results of screening level methods, such as the PBT Profiler, to regulate chemicals. The PBT Profiler is used as a screening level method that provides estimates of PBT characteristics, and is useful for establishing priorities for chemical evaluation when chemical-specific data are lacking. If the PBT Profiler identifies an issue of potential concern, additional data should be gathered and/or additional analyses conducted to come to an informed decision about the chemicals under review. 3.7 Canadian Domestic Substances List categorisation Criteria for persistence, bioaccumulation and inherent toxicity (PBiT) are used by Environment Canada to assess approximately 23,000 substances listed on the Domestic Substances List (DSL). Criteria for persistence and bioaccumulation are defined in the Regulations for Persistence and Bioaccumulation [16]. These criteria were developed from the Toxic Substances Management Policy [17], which provides 15

26 a common science-based management framework for toxic substances in all Canadian federal programmes and initiatives. The definition of inherently toxic to non-human organisms is under consideration by Environment Canada. Those substances found to be persistent or bioaccumulating and inherently toxic proceed to the second phase, a screening level risk assessment. Depending on the outcome of the screening level risk assessment, one of the following outcomes can take place: if the screening level risk assessment indicates that the substance does not cause a risk to the environment or human health, no further action is taken; the substance is added to the Priority Substances List to assess more comprehensively the possible risks associated with the release of the substance; it is recommended to add the substance to the list of Toxic Substances in Schedule I of CEPA (Canadian Environmental Protection Act), if the screening level risk assessment indicates clear concerns. Substances on Schedule 1 can be considered for regulatory controls, including, if the substance is not a naturally occurring substance, virtual elimination. Under this process, risk assessment principles are applied to priority materials. The screening assessment is a tiered process, with decreasingly conservative assumptions as one proceeds up the tiers. An Estimated Exposure Value (EEV) and a Critical Toxicity Value (CTV) are derived. In Tier I, the EEV will likely be the highest estimated or measured environmental concentration available. The CTV, similarly, will be based on toxicity to the most sensitive organism tested. The CTV is then divided by the necessary assessment factor(s) to derive the Estimated No Effects Value (ENEV). A Tier 1 quotient is calculated by dividing the EEV by the ENEV. If the result is less than 1, the substance is considered not to be toxic under CEPA for the assessment endpoint and no further assessment is needed. If it is greater than 1, then the substance is assessed further, using less conservative (more data intensive) assumptions (Tiers II or III). If a substance fails in Tier III (EEV/ENEV > 1), then it is considered to be CEPA toxic and put on Schedule 1. 16

27 3.8 PBT Japanese chemical legislation The Chemical Substances Control Law ratified in 1973 [18] aims at preventing damage to human health caused by environmental pollution from chemical substances. According to the latest amendment to the Chemical Substance Control Law of 1 st April 2004 new chemical substances undergo a volume-dependent ecotoxicological and toxicological testing scheme by the notifier before approval for manufacture/supply to the Japanese market. In addition, under the Existing Chemicals Programme sponsored by the Japanese Government, existing substances which are not covered by the legislation for new chemicals also undergo systematic testing. Hazard endpoints, such as persistence in combination with ecotoxicity or long-term toxicity or confirmed potential for damage by environmental pollution, can lead to specific classification and regulation of chemical substances. In addition, substances identified as exhibiting persistence and bioaccumulative properties can be placed under legal control by classification as Type I Monitoring Substances or ultimately as Class I Specified Chemical Substances. Currently 13 substances have been designated as Class I specified Chemical Substances. Regulatory measures for Type I Monitoring Substances comprise mandatory reporting of quantities of manufacture, import and use, risk reduction measures according to a preliminary toxicological evaluation by the authorities and the requirement for further investigation of longterm ecotoxicity/toxicity. Class I Specified Chemical Substances are banned from production and import unless they are specifically approved for use by the authorities. 17

28 4. OVERVIEW OF PBT AND vpvb CRITERIA National, regional and international bodies are developing ways to manage PBT and POP chemicals to better protect human health and the environment. At present there is little coordination or consistency between the approaches and the criteria defined by different authorities to select and manage PBT substances. The OSPAR Convention for the Protection of the Marine Environment of the North- East Atlantic on the Marine Environment aims to prevent pollution by continuously reducing discharges, emissions and losses of hazardous substances (identified by specific PBT criteria), with the ultimate aim of achieving concentrations in the marine environment near background values for naturally-occurring substances or close to zero for man-made substances. The European Union REACH regulation under discussion considers PBT chemicals as substances of particular concern due to the uncertainty of predicting exposures and concentrations that cause unwanted effects. As such, the EU is proposing the use of specific criteria to identify PBT substances, and very persistent and very bioaccumulating substances (vpvbs). For this second category, the EU says it is not necessary to demonstrate toxicity as long-term effects can be anticipated. The Environmental Protection Agency (USA) has proposed two sets of criteria for PBTs under the Toxic Substances Control Act. These define substances that will have to be controlled and others that will have to be banned. The Canadian Government is also developing PBT criteria in the context of its Toxic Substances Management Policy. The assessment of persistence and bioaccumulation properties for new substances notified in Canada relies on the criteria listed in the Persistence and Bioaccumulation Regulations [16]. The inherent toxicity (it) of a new substance is determined and used in the risk assessment. Currently, Environment Canada is examining policy to address new substances that are PBiT, separately from conclusions of the risk assessment. New substances that are assessed as P and B and found suspected of being Canadian Environmental Protection Act (CEPA)-toxic, that is, found to be of risk to the environment, are subject to the 18

29 virtual elimination policy described under the Toxic Substances Management Policy (TSMP) [17]. The OECD conducted a survey of approaches in the assessment of new chemicals in different countries, in preparation for an OECD Workshop on new chemicals notification and assessment in This survey showed that, as in the US, Austria had recently developed criteria for PBT substances that reflected levels of concern. New chemicals with PBT properties may be judged persistent and bioaccumulating or very Persistent and very Bioaccumulating (vpvb). As in Canada and the US the P and vp criteria are half-lives in the various environmental compartments. In some other nations, notably Japan and the United Kingdom, there was no formal recognition of PBT substances as a category; nevertheless new chemical notification dossiers were reviewed for the core PBT characteristics of persistence, bioaccumulation and toxicity. The main PBT criteria are illustrated in Table 1. 19

30 OSPAR PBT criteria Persistence Bio-accumulation Toxicity Not readily biodegradable or half-life in water > 50 days LogK ow > 4 Or BCF 500 Acute aquatic toxicity L(E)C 50 1 mg/l or long term NOEC 0.1 mg/l or mammalian toxicity: CMR 1 chronic toxicity EU PBT criteria Half-life > 60 days in marine water, or > 40 days in fresh- or estuarine water, or > 180 days in marine sediment, or > 120 days in fresh- or estuarine water sediment is higher, or > 120 days in soil BCF > 2000 Chronic NOEC< 0.01 mg/l for marine or freshwater organisms, or the substance is classified as carcinogenic (cat. 1 or 2), mutagenic (cat. 1 or 2), or toxic for reproduction (cat. 1, 2, or 3). EU vpvb criteria US EPA Control action 2 Half-life > 60 days in marine, fresh or estuarine water, or > 180 days in marine, fresh or estuarine water sediment, or > 180 days in soil Transformation half-life > 2 months BCF > 5000 BCF > 1000 Not applicable Toxicity data based on level on risk concern US EPA Ban Pending 3 Canada Toxic Substance Management Program (TSMP) 4 Transformation half-life > 6 months Half life in Air > 2 days Water > 2 months Sediment > 6 months Soil > 1 year BCF 5000 BAF or BCF > 5000 or LogK ow > 5 Toxicity data based on level on risk concern Inherently toxic Table 1 - PBT criteria. 1 CMR - carcinogenic, mutagenic or toxic to reproduction. 2 Testing and release control required. 3 Commercialisation denied except if testing 20

31 justifies removing chemical from high risk concern. 4 The Canadian Domestic Substances List uses different criteria (water>6 months, sediment>1year, soil>6 months) to define substances which will undergo full elimination (P and B and T and predominantly anthropogenic) and those which will undergo in-depth risk assessment (P or B and T and predominantly anthropogenic) REACH PBT criteria A substance that fulfils all three of the criteria below is a PBT substance. Persistence A substance fulfils the persistence (P-) criterion when: the half-life in marine water is higher than 60 days, or the half-life in fresh- or estuarine water is higher than 40 days, or the half-life in marine sediment is higher than 180 days, or the half-life in fresh- or estuarine water sediment is higher than 120 days, or the half-life in soil is higher than 120 days. The assessment of the persistency in the environment should be based on available half-life data collected under the adequate environmental conditions which should be described by the registrant. Bioaccumulation A substance fulfils the bioaccumulation (B-) criterion when: the bioconcentration factor (BCF) is higher than The assessment of bioaccumulation should be based on measured data on bioconcentration in aquatic species. Data from freshwater as well as marine water species can be used. Toxicity A substance fulfils the toxicity (T-) criterion when: the long-term no-observed effect concentration (NOEC) for marine or freshwater organisms is less than 0.01 mg/l, or the substance is classified as carcinogenic (category 1 or 2), mutagenic (category 1 or 2), or toxic for reproduction (category 1, 2, or 3), or 21

32 there is other evidence of chronic toxicity, as identified by the classifications: T, R48, or Xn, R48 according to Directive 67/548/EEC REACH vpvb criteria A substance that fulfils the criteria below is a vpvb substance. Persistence A substance fulfils the very persistence criterion (vp) when: the half-life in marine, fresh- or estuarine water is higher than 60 days, or the half-life in marine, fresh or estuarine water sediment is higher than 180 days, or the half-life in soil is higher than 180. Bioaccumulation A substance fulfils the very bioaccumulative criterion (vb) when: the bioconcentration factor is greater than In order for a substance to be designated a PBT or a vpvb substance, all of the relevant criteria have to be demonstrated to be fulfilled for the substance. 22

33 5. METHODS FOR PBT DATA GENERATION For most substances the available data do not enable to decide with certainty whether the substance should be considered under the PBT assessment or not. This motivates the need to use screening data that identify whether the substance has the potential to be a PBT/vPvB. In deciding which information is requested (on P, B or T) special care should be taken to avoid animal testing wherever possible. This implies that when for several properties further information is needed the assessment should be focused 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 the B criterion, eventually followed by toxicity testing to clarify the T criterion. However, it is recognised that it may sometimes be more convenient to start the PBT assessment by evaluating the B criterion. 5.1 Persistence data generation The persistence of a substance reflects the potential for long-term exposure of organisms but also the potential for the substance to reach the marine environment and to be transported to remote areas. The assessment of the potential for persistency in the marine environment should in principle be based on actual half-life data determined under marine environmental conditions. When these key data are not available other types of available information on the degradability of a substance can be used to decide if further testing is needed to assess the potential persistence. In this approach three different levels of information are defined according to their perceived relevance to the criteria: experimental data on persistence in the marine environment; other experimental data; data from biodegradation estimation models. This approach reflects existing knowledge on biodegradation and is considered a pragmatic approach to make optimal use of the available data and methods. Research is ongoing to better estimate the persistence in the marine environment from existing biodegradation tests. Moreover, other degradation mechanisms such as hydrolysis and photolysis should be taken into account where they can be shown to be relevant. 23

34 In principle the persistence in the marine environment should be assessed in simulation test systems that determine the half-life under relevant environmental conditions. The determination of the half-life should include assessment of metabolites with PBT characteristics. The half-life should be used as the first and main criterion in order to determine whether a substance should be regarded as persistent. Hence appropriate half-life data from valid simulation tests override data from the other levels of information. Tests performed under marine conditions should use media from marine areas not directly influenced by freshwater outlets or runoffs. It is not possible to establish specific criteria and each test must be evaluated case-by-case. However, the content of freshwater in the sample should be low (i.e. a large dilution as determined, for example, by salinity), the sample should be taken from the water column (and not the surface), the content of microorganisms should be low (compared to freshwater) and cross-contamination during handling, transport and testing should be avoided. In case no half-life data are available for marine water or sediment the decision whether a substance is potentially persistent needs to be based on other experimental data. If available, use can be made of the half-life values from simulation tests of degradation in freshwater. Extrapolation of the existing biodegradation information (either measured data from ready and inherent tests or results from QSAR modelling) to degradation rates in the marine environment is very difficult and care should be taken not to over-interpret the outcome of such tests. However, in order to use the available information to select potentially persistent substances, this information should be used. For new substances, priority existing substances and biocides, information from a ready biodegradability test is normally available and therefore an initial decision whether the substance is potentially persistent can be taken. However, for many other substances no data will be available or the available information is difficult to interpret. For these substances it can be helpful to apply models that estimate the potential for biodegradation in the environment. In a preliminary assessment whether a substance has a potential for persistence in the marine environment and hence for asking for actual test data the use of the BIOWIN program is proposed [19]. This program estimates aerobic biodegradability of organic chemicals using six different models (linear, non-linear model, ultimate and 24