SELECTED NITRO- AND NITRO- OXY-POLYCYCLIC AROMATIC … · 7.5.5.4 Bioassay-directed chemical analysis of airborne particulate matter using a human cell mutagenicity assay 334 7.5.5.5

This report contains the collective views of an international group of expertsand does not necessarily represent the decisions or the stated policy of theUnited Nations Environment Programme, the International LabourOrganization or the World Health Organization.

Environmental Health Criteria 229

SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATICHYDROCARBONS

First draft prepared by Drs J. Kielhorn, U. Wahnschaffe and I.Mangelsdorf, Fraunhofer Institute of Toxicology and AerosolResearch, Hanover, Germany

Please note that the pagination and layout ofthis web version are not identical to the printedEHC

Published under the joint sponsorship of the United NationsEnvironment Programme, the International Labour Organizationand the World Health Organization, and produced within theframework of the Inter-Organization Programme for the SoundManagement of Chemicals.

The International Programme on Chemical Safety (IPCS), established in 1980, is ajoint venture of the United Nations Environment Programme (UNEP), the International LabourOrganization (ILO) and the World Health Organization (WHO). The overall objectives of theIPCS are to establish the scientific basis for assessment of the risk to human health and theenvironment from exposure to chemicals, through international peer review processes, as aprerequisite for the promotion of chemical safety, and to provide technical assistance instrengthening national capacities for the sound management of chemicals.

The Inter-Organization Programme for the Sound Management of Chemicals(IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization ofthe United Nations, WHO, the United Nations Industrial Development Organization, theUnited Nations Institute for Training and Research and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations madeby the 1992 UN Conference on Environment and Development to strengthen cooperation andincrease coordination in the field of chemical safety. The purpose of the IOMC is to promotecoordination of the policies and activities pursued by the Participating Organizations, jointlyor separately, to achieve the sound management of chemicals in relation to human health andthe environment.

WHO Library Cataloguing-in-Publication Data

Selected nitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

(Environmental health criteria ; 229)

1.Polycyclic hydrocarbons, Aromatic - toxicity 2.Polycyclic hydrocarbons,Aromatic - adverse effects 3.Environmental exposure 4.Risk assessmentI.International Programme for Chemical Safety II.Series

ISBN 92 4 157229 9 (NLM classification: QD 341.H9)ISSN 0250-863X

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The mention of specific companies or of certain manufacturers’ products does not implythat they are endorsed or recommended by the World Health Organization in preference toothers of a similar nature that are not mentioned. Errors and omissions excepted, the namesof proprietary products are distinguished by initial capital letters.

The Federal Ministry of the Environment, Nature Conservation, and Nuclear Safety,Germany, provided financial support for, and undertook the printing of, this publication.

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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FORSELECTED NITRO- AND NITRO-OXY-POLYCYCLICAROMATIC HYDROCARBONS

PREAMBLE xiii

ACRONYMS AND ABBREVIATIONS xxiii

1. SUMMARY 1

1.1 Identity, physical and chemical properties, andanalytical methods 1

1.2 Sources of human and environmental exposure 21.3 Environmental transport, distribution and

transformation 41.3.1 Environmental transport and distribution 41.3.2 Biotransformation 41.3.3 Abiotic degradation 5

1.4 Environmental levels and human exposure 61.4.1 Indoor air 71.4.2 Food and beverages 71.4.3 Other products 81.4.4 Occupational exposure 8

1.5 Kinetics and metabolism in laboratory animalsand humans 9

1.6 Effects on laboratory mammals and in vitrotest systems 10

1.7 Effects on humans 141.8 Effects on other organisms in the laboratory and

field 14

2. IDENTITY, PHYSICAL AND CHEMICALPROPERTIES, AND ANALYTICAL METHODS 16

2.1 Identity 162.2 Physical and chemical properties 232.3 Conversion factors 232.4 Analytical methods 30

EHC 229: Selected Nitro- and Nitro-oxyPAHs

iv

2.4.1 Sampling 312.4.2 Extraction 382.4.3 Cleanup 382.4.4 Analytical separation and detection 39

2.4.4.1 Difficulties in analysis 422.4.4.2 Complex mixtures 432.4.4.3 Analysis of nitro-oxyPAHs 43

2.4.5 Use of bioassay (mutagenicity) fractionationand chemical analysis 43

3. SOURCES OF HUMAN AND ENVIRONMENTALEXPOSURE 47

3.1 Industrially produced nitroPAHs 473.1.1 Production levels and processes 473.1.2 Uses of commercially produced nitroPAHs 47

3.2 Other sources of nitroPAHs 483.2.1 Direct sources of nitroPAHs from

combustion processes 543.2.1.1 Diesel exhaust 543.2.1.2 Diesel compared with gasoline

exhaust 623.2.1.3 Aeroplane emissions 623.2.1.4 Emissions from combustion of

heating oils 633.2.1.5 Fumes from cooking oils 633.2.1.6 Other combustion sources 63

3.2.2 Atmospheric formation of nitroPAHs 643.2.2.1 Reactions of gas-phase PAHs (and

nitroPAHs) with the hydroxylradical (daytime reactions) 66

3.2.2.2 Reactions of gas-phase PAHs (andnitroPAHs) with the nitrate radical(nighttime reactions) 68

3.3 Oxygen-containing nitroPAHs 68

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTIONAND TRANSFORMATION 70

4.1 Transport and distribution between media 704.1.1 Distribution and transport in the atmosphere 70

v

4.1.1.1 Distribution of nitroPAHs betweenfine and coarse fractions ofinhalable atmospheric particulates 71

4.1.2 Distribution and transport in the hydrosphere 714.1.3 Adsorption onto soils and sediments 714.1.4 Bioaccumulation 724.1.5 Biomagnification 72

4.2 Transformation 724.2.1 Biotransformation 72

4.2.1.1 Bacteria 724.2.1.2 Fungi 754.2.1.3 Plants 754.2.1.4 Aquatic animals 76

4.2.2 Abiotic degradation 764.2.2.1 Direct photolysis 764.2.2.2 Other atmospheric

transformations 85

5. ENVIRONMENTAL LEVELS AND HUMANEXPOSURE 87

5.1 Environmental levels 875.1.1 Air 87

5.1.1.1 Ambient air 875.1.1.2 Indoor air 107

5.1.2 Water 1095.1.3 Soil, sewage sludge, sediment and

incinerator ash 1095.1.4 Food and beverages 110

5.1.4.1 Food 1105.1.4.2 Beverages 114

5.1.5 Other sources 1165.1.5.1 Carbon black and toners 1165.1.5.2 Cigarette smoke 116

5.2 General population exposure 1165.3 Occupational exposure 117

6. KINETICS AND METABOLISM IN LABORATORYANIMALS AND HUMANS 121

6.1 Overview of the metabolism of nitroPAHs 121


vi

6.2 1-Nitropyrene metabolism in mammals 1256.2.1 Absorption 1256.2.2 Distribution 1256.2.3 Metabolism 126

6.2.3.1 Introduction 1266.2.3.2 Identification of metabolites 1266.2.3.3 Cytochrome P450-mediated ring

C-oxidative pathway 1376.2.3.4 Nitroreduction pathway 1386.2.3.5 Human and rodent intestinal

microflora 1396.2.3.6 Suggested metabolic pathway 140

6.2.4 Elimination and excretion 1416.2.4.1 Elimination 1416.2.4.2 Excretion 1426.2.4.3 Biliary excretion and enterohepatic

circulation 1426.2.5 Reaction with body components 142

6.2.5.1 Protein binding 1436.2.5.2 DNA adducts 143

6.2.6 Biomonitoring studies 1516.3 Mononitropyrenes (1-, 2- and 4-nitropyrene) —

a comparison 1526.3.1 Faecal and urinary excretion 1526.3.2 Metabolism 1526.3.3 DNA adducts 154

6.4 2-Nitrofluorene 1556.4.1 Absorption, distribution and elimination 1556.4.2 Metabolism/mechanism of action 158

6.4.2.1 Metabolites 1586.4.2.2 DNA adducts 1606.4.2.3 Haemoglobin adducts 161

6.5 Dinitropyrenes (1,3-, 1,6- and 1,8-dinitropyrene) 1616.6 Mononitrobenzo[a]pyrenes (1-, 3- and

6-nitrobenzo[a]pyrene) 1646.7 The nitrofluoranthene family 1656.8 2- and 9-nitroanthracene 1676.9 6-Nitrochrysene 1686.10 K- and H-ras mutations in tumours produced by

nitroPAHs 170

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6.11 Human enzymes expected to be involved innitroPAH metabolism 171

7. EFFECTS ON LABORATORY MAMMALS ANDIN VITRO TEST SYSTEMS 172

7.1 Acute toxicity 1727.1.1 1-Nitronaphthalene 1727.1.2 2-Nitronaphthalene 1737.1.3 5-Nitroacenaphthene 1737.1.4 2-Nitrofluorene 1737.1.5 3,9-Dinitrofluoranthene 1737.1.6 1-Nitropyrene 173

7.2 Short-term and long-term exposure (non-neoplastic effects) 1747.2.1 1-Nitronaphthalene 1747.2.2 5-Nitroacenaphthene 1747.2.3 2-Nitrofluorene 2197.2.4 1-Nitropyrene 2197.2.5 1,3-Dinitropyrene 2207.2.6 1,6-Dinitropyrene 2207.2.7 1,8-Dinitropyrene 2207.2.8 6-Nitrochrysene 2207.2.9 1- and 3-nitrobenzo[a]pyrene 2207.2.10 1,6-Dinitrobenzo[a]pyrene 221

7.3 Skin and eye irritation and sensitization 2217.4 Reproductive toxicity, embryotoxicity and

teratogenicity 2217.5 Mutagenicity and related end-points 221

7.5.1 In vitro genotoxicity studies 2377.5.1.1 Salmonella typhimurium

microsome assay 2377.5.1.2 Comparison of the mutagenic

potency of nitroPAHs in theSalmonella microsome assay 293

7.5.1.3 Studies into the pathways ofmicrobial metabolism 294

7.5.1.4 Relationship between mutagenicpotency in S. typhimurium and thechemical structure of nitroPAHs 303


viii

7.5.1.5 Bacterial test systems other than theSalmonella microsome assay 305

7.5.1.6 Eukaryotic test systems 3067.5.1.7 High potency in the Salmonella

microsome assay in relation to genemutation results from other in vitroassays 307

7.5.1.8 Assessment of data on genotoxicityin vitro 309

7.5.2 In vivo genotoxicity studies 3107.5.2.1 Comparison with in vitro results 321

7.5.3 Genotoxicity of oxygen-containingnitroPAHs 3257.5.3.1 3-Nitrobenzanthrone 3257.5.3.2 Nitrodibenzopyranones 3267.5.3.3 Nitropyrene lactones 3287.5.3.4 Comparison of mutation frequency

at hprt versus tk locus in human B-lymphoblastoid cell lines 326

7.5.4 Summary of the genotoxicity of nitroPAHs 3297.5.5 Mutagenicity of complex mixtures 329

7.5.5.1 Difficulties encountered wheninterpreting the mutagenicity ofcomplex mixtures compared withindividual compounds 329

7.5.5.2 Mutagenicity of diesel engineexhaust 330

7.5.5.3 Mutagenic effect of urban airsamples 331

7.5.5.4 Bioassay-directed chemical analysisof airborne particulate matter usinga human cell mutagenicity assay 334

7.5.5.5 DNA adducts 3347.5.5.6 Mutagenic contribution of selected

nitroPAHs from their occurrence inair samples multiplied by themutagenicity in the Salmonellamutagenicity test 335

7.5.5.7 Municipal waste incineratoremissions 335

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7.5.5.8 Mutagenicity of soils exposed to automobile exhaust 336

7.5.6 Effects of antimutagenic compounds onnitroPAH mutagenicity 336

7.6 Carcinogenicity 3377.6.1 Route of administration 3417.6.2 Adequacy of data 3427.6.3 Type of induced tumours 3437.6.4 Ranking of carcinogenic potency in

comparative studies on nitroPAHs 3497.6.4.1 Comparison of the carcinogenicity

of nitroPAHs with parent PAHs 3527.6.4.2 Comparison of the dose 355

7.6.5 Carcinogenicity of oxygen-containingnitroPAHs 356

7.6.6 Carcinogenicity of the metabolites 3567.6.7 Carcinogenic potency of nitroPAHs in

diesel exhaust 3577.6.8 Genotoxicity in vivo and in vitro versus

carcinogenicity 3587.6.9 Potency equivalency factors for nitroPAHs 3597.6.10 Mechanisms of carcinogenesis 360

7.7 Special studies: Target organ effects 3607.7.1 1-Nitronaphthalene 3607.7.2 2-Nitronaphthalene 3617.7.3 1-Nitropyrene 362

8. EFFECTS ON HUMANS 363

8.1 General population exposure 3658.2 Occupational exposure 3668.3 Indicators of exposure to nitroPAHs in diesel

exhaust 3668.3.1 Biomonitoring of exposure/effect 366

8.3.1.1 DNA adducts 3668.3.1.2 Protein adducts 3678.3.1.3 1-Nitropyrene metabolites 3688.3.1.4 Immunochemical determination 369

8.3.2 Biomarkers of susceptibility 3698.3.2.1 Cytochrome P450 369


x

8.3.2.2 Influence of polymorphisms onbiomarkers 370

9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORYAND FIELD 372

9.1 Laboratory experiments 3729.1.1 Aquatic species 3729.1.2 Biotransformation studies in aquatic

species 3729.1.3 DNA damage in aquatic species 373

9.2 Field observations 375

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTSON THE ENVIRONMENT 376

10.1 Evaluation of human health risks 37610.1.1 Exposure levels 376

10.1.1.1 NitroPAHs 37610.1.1.2 Nitroketones 37810.1.1.3 Nitrolactones 378

10.1.2 Fate in the body 37910.1.2.1 NitroPAHs 37910.1.2.2 Nitroketones 38010.1.2.3 Nitrolactones 380

10.1.3 Toxic effects 38010.1.3.1 Non-neoplastic effects 38010.1.3.2 Genotoxicity 38110.1.3.3 Neoplastic effects 385

10.1.4 Evaluation of nitroPAHs,nitroketones and nitrolactones thatseem to be of importance in theenvironment 386

10.2 Evaluation of effects on the environment 38610.3 General considerations 39010.4 Overall evaluation 391

11. RECOMMENDATIONS FOR PROTECTION OFHUMAN HEALTH AND THE ENVIRONMENT 392

12. RECOMMENDATIONS FOR FURTHER RESEARCH 393

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13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES394

REFERENCES 395

RESUME 448

RESUMEN 465

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NOTE TO READERS OF THE CRITERIA MONOGRAPHS

Every effort has been made to present information in the criteriamonographs as accurately as possible without unduly delaying theirpublication. In the interest of all users of the Environmental HealthCriteria monographs, readers are requested to communicate any errorsthat may have occurred to the Director of the International Programmeon Chemical Safety, World Health Organization, Geneva, Switzerland,in order that they may be included in corrigenda.

* * *

The WHO Environmental Health Criteria Programme is financiallysupported by the US Environmental Protection Agency, EuropeanCommission, German Federal Ministry of the Environment, NatureConservation, and Nuclear Safety, and Japanese Ministry of Health,Labour and Welfare.

Environmental Health Criteria

Objectives

In 1973, the WHO Environmental Health Criteria Programme wasinitiated with the following objectives:

(i) to assess information on the relationship between exposure toenvironmental pollutants and human health, and to provideguidelines for setting exposure limits;

(ii) to identify new or potential pollutants;

(iii) to identify gaps in knowledge concerning the health effects ofpollutants;

(iv) to promote the harmonization of toxicological and epidemio-logical methods in order to have internationally comparableresults.

The first Environmental Health Criteria (EHC) monograph, onmercury, was published in 1976, and since that time an ever-increasingnumber of assessments of chemicals and of physical effects have beenproduced. In addition, many EHC monographs have been devoted toevaluating toxicological methodology, e.g. for genetic, neurotoxic,teratogenic and nephrotoxic effects. Other publications have beenconcerned with epidemiological guidelines, evaluation of short-termtests for carcinogens, biomarkers, effects on the elderly and so forth.

Since its inauguration, the EHC Programme has widened its scope,and the importance of environmental effects, in addition to healtheffects, has been increasingly emphasized in the total evaluation ofchemicals.

The original impetus for the Programme came from World HealthAssembly resolutions and the recommendations of the 1972 UNConference on the Human Environment. Subsequently the workbecame an integral part of the International Programme on Chemical

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Safety (IPCS), a cooperative programme of UNEP, ILO and WHO. Inthis manner, with the strong support of the new partners, theimportance of occupational health and environmental effects was fullyrecognized. The EHC monographs have become widely established,used and recognized throughout the world.

The recommendations of the 1992 UN Conference on Environmentand Development and the subsequent establishment of theIntergovernmental Forum on Chemical Safety with the priorities foraction in the six programme areas of Chapter 19, Agenda 21, all lendfurther weight to the need for EHC assessments of the risks ofchemicals.

Scope

The criteria monographs are intended to provide critical reviews onthe effects on human health and the environment of chemicals and ofcombinations of chemicals and physical and biological agents. Assuch, they include and review studies that are of direct relevance forthe evaluation. However, they do not describe every study carried out.Worldwide data are used and are quoted from original studies, not fromabstracts or reviews. Both published and unpublished reports areconsidered, and it is incumbent on the authors to assess all the articlescited in the references. Preference is always given to published data.Unpublished data are used only when relevant published data areabsent or when they are pivotal to the risk assessment. A detailedpolicy statement is available that describes the procedures used forunpublished proprietary data so that this information can be used inthe evaluation without compromising its confidential nature (WHO(1990) Revised Guidelines for the Preparation of Environmental HealthCriteria Monographs. PCS/90.69, Geneva, World Health Organization).

In the evaluation of human health risks, sound human data,whenever available, are preferred to animal data. Animal and in vitrostudies provide support and are used mainly to supply evidencemissing from human studies. It is mandatory that research on humansubjects is conducted in full accord with ethical principles, includingthe provisions of the Helsinki Declaration.

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The EHC monographs are intended to assist national andinternational authorities in making risk assessments and subsequentrisk management decisions. They represent a thorough evaluation ofrisks and are not, in any sense, recommendations for regulation orstandard setting. These latter are the exclusive purview of national andregional governments.

Content

The layout of EHC monographs for chemicals is outlined below.

• Summary — a review of the salient facts and the risk evaluation ofthe chemical

• Identity — physical and chemical properties, analytical methods• Sources of exposure• Environmental transport, distribution and transformation• Environmental levels and human exposure• Kinetics and metabolism in laboratory animals and humans• Effects on laboratory mammals and in vitro test systems• Effects on humans• Effects on other organisms in the laboratory and field• Evaluation of human health risks and effects on the environment• Conclusions and recommendations for protection of human health

and the environment• Further research• Previous evaluations by international bodies, e.g., IARC, JECFA,

JMPR

Selection of chemicals

Since the inception of the EHC Programme, the IPCS has organizedmeetings of scientists to establish lists of priority chemicals forsubsequent evaluation. Such meetings have been held in Ispra, Italy,1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and NorthCarolina, USA, 1995. The selection of chemicals has been based on thefollowing criteria: the existence of scientific evidence that thesubstance presents a hazard to human health and/or the environment;the possible use, persistence, accumulation or degradation of the

xvi

substance shows that there may be significant human or environmentalexposure; the size and nature of populations at risk (both human andother species) and risks for environment; international concern, i.e., thesubstance is of major interest to several countries; adequate data onthe hazards are available.

If an EHC monograph is proposed for a chemical not on the prioritylist, the IPCS Secretariat consults with the Cooperating Organizationsand all the Participating Institutions before embarking on thepreparation of the monograph.

Procedures

The order of procedures that result in the publication of an EHCmonograph is shown in the flow chart on p. xvii. A designated staffmember of IPCS, responsible for the scientific quality of the document,serves as Responsible Officer (RO). The IPCS Editor is responsible forlayout and language. The first draft, prepared by consultants or, moreusually, staff from an IPCS Participating Institution, is based onextensive literature searches from reference databases such as Medlineand Toxline.

The draft document, when received by the RO, may require aninitial review by a small panel of experts to determine its scientificquality and objectivity. Once the RO finds the document acceptable asa first draft, it is distributed, in its unedited form, to well over 150 EHCcontact points throughout the world who are asked to comment on itscompleteness and accuracy and, where necessary, provide additionalmaterial. The contact points, usually designated by govern ments, maybe Participating Institutions, IPCS Focal Points or individual scientistsknown for their particular expertise. Generally some four months areallowed before the comments are considered by the RO and author(s).A second draft incorporating comments received and approved by theDirector, IPCS, is then distributed to Task Group members, who carryout the peer review, at least six weeks before their meeting.

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Commitment to draft EHCCommitment to draft EHC

Document preparation initiated

Draft sent to IPCS Responsible Officer (RO)

EHC PREPARATION FLOW CHART

Revision asnecessary

Possible meeting of a few experts to resolve controversial issues

First DraftFirst Draft

Responsible Officer, Editor check for coherence of text and readability (not language editing)

Responsible Officer, Editor check for coherence of text and readability (not language editing)

International circulation to Contact Points (150+)

Comments to IPCS (RO)

Review of comments, reference cross-check;preparation of Task Group (TG) draft

Task Group meeting

Insertion of TG changes

Post-TG draft; detailed reference cross-check

EditingEditing

Word-processing

Camera-ready copy

Final editing

Approval by Director, IPCS

WHO Publication Office

Printer Proofs PublicationPublication

Graphics

Library forCIP Data

French/Spanish translations of Summary

Working group,if requiredEditor

routine procedure

optional procedure

xviii

The Task Group members serve as individual scientists, not asrepresentatives of any organization, government or industry. Theirfunction is to evaluate the accuracy, significance and relevance of theinformation in the document and to assess the health and environ-mental risks from exposure to the chemical. A summary and recommen-dations for further research and improved safety aspects are alsorequired. The composition of the Task Group is dictated by the rangeof expertise required for the subject of the meeting and by the need fora balanced geographical distribution.

The three cooperating organizations of the IPCS recognize theimportant role played by nongovernmental organizations. Repre-sentatives from relevant national and international associations may beinvited to join the Task Group as observers. Although observers mayprovide a valuable contribution to the process, they can speak only atthe invitation of the Chairperson. Observers do not participate in thefinal evaluation of the chemical; this is the sole responsibility of theTask Group members. When the Task Group considers it to beappropriate, it may meet in camera .

All individuals who as authors, consultants or advisers participatein the preparation of the EHC monograph must, in addition to servingin their personal capacity as scientists, inform the RO if at any time aconflict of interest, whether actual or potential, could be perceived intheir work. They are required to sign a conflict of interest statement.Such a procedure ensures the transparency and probity of the process.

When the Task Group has completed its review and the RO issatisfied as to the scientific correctness and completeness of thedocument, it then goes for language editing, reference checking andpreparation of camera-ready copy. After approval by the Director, IPCS,the monograph is submitted to the WHO Office of Publications forprinting. At this time, a copy of the final draft is sent to the Chairpersonand Rapporteur of the Task Group to check for any errors.

It is accepted that the following criteria should initiate the updatingof an EHC monograph: new data are available that would substantiallychange the evaluation; there is public concern for health or

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environmental effects of the agent because of greater exposure; anappreciable time period has elapsed since the last evaluation.

All Participating Institutions are informed, through the EHCprogress report, of the authors and institutions proposed for thedrafting of the documents. A comprehensive file of all commentsreceived on drafts of each EHC monograph is maintained and isavailable on request. The Chairpersons of Task Groups are briefedbefore each meeting on their role and responsibility in ensuring thatthese rules are followed.

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WHO TASK GROUP ON ENVIRONMENTAL HEALTHCRITERIA FOR SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

Members

Professor D. Anderson, Department of Biomedical Sciences,University of Bradford, Bradford, West Yorkshire, UnitedKingdom (Chairperson)

Professor J. Arey, Air Pollution Research Center, University ofCalifornia, Riverside, California, USA

Dr R.P. Bos, Department of Pharmacology & Toxicology, UMC St.Radboud, University of Nijmegen, Nijmegen, The Netherlands

Dr A. Cecinato, Istituto sull’Inquinamento Atmosferico-CNR, CP10Monterotondo Stazione, Rome, Italy

Dr K. El-Bayoumy, Division of Cancer Etiology & Prevention,American Health Foundation, Valhalla, New York, USA (Vice-Chairperson)

Dr P.C. Howard, Division of Biochemical Toxicology, National Centerfor Toxicological Research, Jefferson, Arkansas, USA (Co-Rapporteur)

Dr J. Kielhorn, Chemical Risk Assessment, Fraunhofer Institute ofToxicology and Aerosol Research, Hanover, Germany (Co-Rapporteur)

Professor M. Kirsch-Volders, Laboratory of Cell Genetics, FreeUniversity of Brussels, Brussels, Belgium

Dr I. Mangelsdorf, Chemical Risk Assessment, Fraunhofer Instituteof Toxicology and Aerosol Research, Hanover, Germany

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Dr S. Pavittranon, Toxicology and Environmental Laboratory,National Institute of Health, Department of Medical Sciences,Ministry of Public Health, Nontaburi, Thailand

Dr H. Tokiwa, Department of Environmental Health Science, KyushuWomen’s University, Kitakyushu, Japan

Dr U. Wahnschaffe, Consultant, Uetze, Germany

Professor Z. Yuxin, Institute of Occupational Medicine, ChineseAcademy of Preventive Medicine, Beijing, People’s Republic ofChina

Secretariat

Mr T. Ehara, International Programme on Chemical Safety, WorldHealth Organization, Geneva, Switzerland

Mrs P. Harlley, International Programme on Chemical Safety, WorldHealth Organization, Geneva, Switzerland

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ENVIRONMENTAL HEALTH CRITERIASELECTED NITRO- AND NITRO-OXY-POLYCYCLICAROMATIC HYDROCARBONS

The first and second drafts of this monograph were prepared bythe authors, Drs J. Kielhorn, U. Wahnschaffe and I. Mangelsdorf.

A WHO Task Group on Environmental Health Criteria for SelectedNitro- and Nitro-oxy-Polycyclic Aromatic Hydrocarbons met at theFraunhofer Institute of Toxicology and Aerosol Research, in Hanover,Germany, on 26–30 November 2001. The group reviewed the draft andthe peer review comments, revised the draft and made an evaluation ofthe risks for human health and environment from exposure to selectednitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

Dr P. Jenkins and Mr T. Ehara of the IPCS central unit wereresponsible for the scientific aspects of the monograph, and Ms. MarlaSheffer was responsible for the technical editing.

The efforts of all, especially the Fraunhofer Institute of Toxicologyand Aerosol Research, which helped in the preparation and finalizationof the monograph, are gratefully acknowledged.

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ACRONYMS AND ABBREVIATIONS

BaP benzo[a]pyrenebw body weightCAS Chemical Abstracts ServicecDNA complementary (or copy) DNACHO Chinese hamster ovaryCYP cytochrome P450D2 no. 2 diesel fueldA deoxyadenosinedA-C8-2-AP N-(deoxyadenosin-8-yl)-2-aminopyreneDCM dichloromethanedG deoxyguanosinedG-C8-AAF N-(deoxyguanosin-8-yl)-2-

acetylaminofluorenedG-C8-AF N-(deoxyguanosin-8-yl)-2-aminofluorenedG-C8-1-amino-6-NP N-(deoxyguanosin-8-yl)-1-amino-6-

nitropyrenedG-C8-1-amino-8-NP N-(deoxyguanosin-8-yl)-1-amino-8-

nitropyrenedG-C8-AP N-(deoxyguanosin-8-yl)-1-aminopyrenedG-C8-2-AP N-(deoxyguanosin-8-yl)-2-aminopyrenedG-C8-4-AP N-(deoxyguanosin-8-yl)-4-aminopyrenedG-N2-AAF C3-(deoxyguanosin-N2-yl)-2-

acetylaminofluorenedG-1-nitroBaP-DE 10-(deoxyguanosin-N2-yl)-7,8,9-

trihydroxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene

dG-3-nitroBaP-DE 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitro-benzo[a]pyrene

6-dG-N2-1-aminoBaP 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene

6-dG-N2-3-aminoBaP 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene

dI deoxyinosineDMSO dimethyl sulfoxideDNA deoxyribonucleic acidDNP dinitropyrene

xxiv

EC electron captureEC50 median effective concentrationECD electron capture detectorED50 median effective doseEHC Environmental Health Criteria monographEI electron impactELISA enzyme-linked immunosorbent assayEPA Environmental Protection Agency (USA)FAO Food and Agriculture Organization of the

United NationsFTP Federal Test Procedure (USA)GC gas chromatographyGPC semipreparative gel permeation

chromatographyGST glutathione S-transferaseHb haemoglobinHCFC-22 chlorodifluoromethaneHDD heavy-duty dieselHPLC high-performance liquid chromatographyIARC International Agency for Research on

CancerILO International Labour Organizationi.m. intramusculari.p. intraperitonealIPCS International Programme on Chemical

Safetyi.v. intravenousJECFA Joint FAO/WHO Expert Committee on

Food AdditivesJMPR Joint FAO/WHO Meeting on Pesticide

ResiduesKoc organic carbon/water partition coefficientKow n-octanol/water partition coefficientLC50 median lethal concentrationLC100 lethal concentration for 100% of test

organismsLD50 median lethal doseLOAEL lowest-observed-adverse-effect level

xxv

LOEL lowest-observed-effect levelLPG liquefied petroleum gasMA metabolic activationMN micronucleus inductionMS mass spectrometryMS/MS tandem mass spectrometryMTD maximum tolerated doseMW molecular weight (relative molecular

mass)NADH nicotinamide adenine dinucleotideNADPH reduced nicotinamide adenine

dinucleotideNAT N-acetyltransferaseNB nitrobenzanthronend not detectedNICI negative ion chemical ionizationNIST National Institute of Standards and

Technology (USA)nitroPAH nitro-polycyclic aromatic hydrocarbonNMR nuclear magnetic resonanceNOAEL no-observed-adverse-effect levelNPD nitrogen–phosphorus detectorNP-LC normal-phase high-performance liquid

chromatographyNPR NADPH-cytochrome P450 reductaseOCC oxidation catalytic converterOECD Organisation for Economic Co-operation

and DevelopmentOH-AAF N-acetyl-2-aminofluoren-x-olOH-2-NF 2-nitrofluorenolPAH polycyclic aromatic hydrocarbonPCB polychlorinated biphenylPEF potency equivalency factorPICI positive ion chemical ionizationPM2.5 particulate matter =2.5 µm in diameterPM10 particulate matter =10 µm in diameterp.o. per osppb part per billion

xxvi

ppm part per millionppt part per trillionPUF polyurethane foamRO Responsible OfficerRP-LC reversed-phase high-performance liquid

chromatographys.c. subcutaneousSCE sister chromatid exchangeSEOM solvent extractable organic matterSOF soluble organic fractionSPE solid-phase extractionSRM Standard Reference MaterialTA98AT+ N-hydroxyarylamine O-acetyltransferase-

overproducing S. typhimurium strainTA98NR+ nitroreductase-overproducing S.

typhimurium strainTA98AT– N-hydroxyarylamine O-acetyltransferase-

deficient S. typhimurium strainTA98NR– nitroreductase-deficient S. typhimurium

strainTEA thermal energy analyserTID thermionic detectorTLC thin-layer chromatographyUDS unscheduled DNA synthesisUN United NationsUNEP United Nations Environment ProgrammeUV ultravioletWHO World Health OrganizationXOC extractable organic component

1

1. SUMMARY

1.1 Identity, physical and chemical properties, andanalytical methods

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are deriva-tives of polycyclic aromatic hydrocarbons (PAHs), which contain twoor more fused aromatic rings made of carbon and hydrogen atoms.NitroPAHs occur in the environment as a mixture together with parentPAHs and hundreds of other organic compounds. NitroPAHs areusually present in much smaller quantities than PAHs.

NitroPAHs in the environment occur either in the vapour phase oradsorbed to particulate matter. NitroPAHs are insoluble or sparinglysoluble in water but mostly soluble in organic solvents.

The sampling of nitroPAHs is similar to that of PAHs. Ambient airis sampled by collecting particulate matter on special filters by meansof high-volume samplers. Vapour-phase nitroPAHs are commonlycollected on solid sorbents such as polyurethane foam.

Solvent extraction is followed by cleanup using liquid chroma-tography with silica gel or alumina, high-performance liquid chroma-tography (HPLC) or solid-phase extraction. The nitroPAH fraction mustbe separated from the PAH fraction and oxygenated PAH fraction byHPLC on silica. Methods used for the separation and detection ofnitroPAHs include gas chromatography with a variety of detectors,HPLC with fluorescence, chemiluminescence or electrochemical detec-tor, and mass spectrometric techniques. Analysis is dependent on thestandards available.

Another approach to analysis of complex mixtures is bioassay-directed chemical analysis, where mutagenically active fractions arebioassayed and characterized until the major class or specific com-pounds potentially responsible for the mutagenicity are identified. Theuse of bacterial tester strains selectively sensitive to nitroarenes has


2

led to the identification of nitroPAHs as potent mutagens in complexmixtures from diverse sources. Synthetic standards are required for thistype of analysis.

The nitroketone 3-nitrobenzanthrone and nitrolactones, such as 2-and 4-nitrodibenzopyranone, are nitro-oxy compounds, which havebeen detected together with nitroPAHs and are analysed by similarmethods.

1.2 Sources of human and environmental exposure

NitroPAHs originate primarily as direct or indirect products ofincomplete combustion. Only a few nitroPAHs are produced indus-trially; commercially produced nitronaphthalenes and 5-nitroacenaph-thene, for example, are used primarily as chemical intermediates.

NitroPAHs originate from PAHs (generally adsorbed on particu-late matter and themselves products of incomplete combustion) by atleast two distinct processes: (1) through nitration during combustionprocesses (e.g., in vehicle exhaust, particularly diesel, but also gasolineand aircraft emissions; industrial emissions; domestic residentialheating/cooking; wood burning) and (2) through atmospheric formationfrom PAHs by either gas-phase reactions — daytime hydroxyl radicaladdition to the PAH followed by reaction with nitrogen dioxide andloss of a water molecule and nighttime nitrate radical addition to thePAH followed by reaction with nitrogen dioxide and loss of nitric acid— or heterogeneous gas–particle interaction of parent PAHs adsorbedonto particles with nitrating agents.

The distribution of nitroPAH isomers in samples of ambient air hasbeen found to be significantly different from that in direct emissionsfrom combustion. 2-Nitrofluoranthene and 2-nitropyrene are ubiquitouscomponents of particulate matter, although they are not directly emittedfrom most combustion sources. The nitroPAH profile, or the relativequantities of certain “marker” PAHs, is a pointer to the source offormation of the nitroPAH. The most abundant nitro isomers of pyrene,fluorene and fluoranthene observed in diesel exhaust are 1-nitropyrene,2-nitrofluorene and 3-nitrofluoranthene, whereas the isomers formed

Summary

3

from the hydroxyl radical reactions of these PAHs are 2-nitropyrene, 3-nitrofluorene and 2-nitrofluoranthene.

The majority of ambient nitroPAHs are now thought to be formedin the atmosphere from the gas-phase reactions of PAHs with fourrings or less.

Many mono- and some di- and trinitroPAH isomers have beenidentified and quantified in various samples of diesel exhaust, 1-nitropyrene usually being the most abundant. 1-Nitropyrene is the“marker” nitroPAH for diesel exhaust, and its presence in ambient airsamples is a sign of pollution by diesel vehicle traffic. Diesel fuel,engine types and catalytic traps are continually being modified, so thevarious studies of nitroPAHs in diesel exhaust cannot be directlycompared. In general, the mass emission of particles, emissions ofparticle-bound PAHs and nitroPAHs, and mutagenic activity levelsgenerally decreased with the use of either particulate traps or catalyticconverters.

The concentration of 1-nitropyrene was much less in gasolineexhaust particles than in diesel exhaust particles, but the concen-trations of 1,3-, 1,6- and 1,8-dinitropyrenes were found to be almost thesame in gasoline and diesel exhaust particles.

There is evidence of the presence of nitroPAHs in jet aeroplaneexhaust.

NitroPAHs have been detected in the emissions of keroseneheaters, fuel gas and liquefied petroleum gas (LPG) burners, which areused in many countries for heating and cooking at home.

3-Nitrobenzanthrone has been detected in diesel exhaust particu-late and in urban air samples. 2-Nitrodibenzopyranone and 4-nitro-dibenzopyranone as well as nitropyrene lactones have been observedin ambient particulate matter.


4

1.3 Environmental transport, distribution andtransformation

1.3.1 Environmental transport and distribution

NitroPAHs can be transported in the vapour phase or adsorbedonto particulate matter. Those with liquid-phase vapour pressuresgreater than 10–4 Pa at ambient air temperature (i.e., two- to four-ringPAHs and two-ring nitroPAHs) will exist at least partially in the gasphase.

Owing to their low aqueous solubility or insolubility, nitroPAHsare not expected to be transported in water. Data available give highvalues for sorption coefficients (log Koc), suggesting that nitroPAHs,similar to PAHs, adsorb onto soil and sediments. Leaching into ground-water is thought to be negligible. Some nitroPAHs may be slowlybiodegradable under certain conditions.

The values for the n-octanol/water partition coefficient (log Kow)range from 2.5 for 1-nitronaphthalene to 6.3 for 3-nitroperylene,suggesting a potential for bioaccumulation. There were no data avail-able on biomagnification.

1.3.2 Biotransformation

Many anaerobic and aerobic bacteria reduce nitroPAHs to muta-genic aminoPAHs. Nitroreduction by intestinal microflora plays a majorrole in the metabolism of nitroPAHs in mammals. Although a widevariety of bacteria, fungi and algae have been shown to degrade theparent PAHs containing two to five rings, nitro-substituted PAHs areonly slowly degraded by indigenous microorganisms and may persistin soils and sediments. The recalcitrance of high molecular weightnitroPAHs is due in part to the strong adsorption to soil organic matter,low solubility, large molecular size and the polar character of the nitrogroup.

Summary

5

Time course studies in microcosms showed that 1-nitropyrene wasdegraded slowly under aerobic and anaerobic conditions in estuarinesediments.

Sphingomonas paucimobilis strain EPA 505 (a soil bacteriumcapable of utilizing fluoranthene as the sole source of carbon andenergy) biodegraded 1-nitropyrene to 48.6% after 6 h.

The filamentous fungus Cunninghamella elegans has beenshown to oxidatively metabolize, via a cytochrome P450monooxygenase, a number of nitroPAHs (1-nitropyrene, 2-nitrofluorene, 2- and 3-nitrofluoranthene, 6-nitrochrysene, 1-nitrobenzo[e]pyrene and 6-nitrobenzo[a]pyrene) to products that areless mutagenic than the nitroPAHs themselves.

A plant cell culture derived from alligator weed (Alternantheraphiloxeroides) detoxified 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitro-pyrene, all direct-acting mutagens, when incubated with them, asshown by mutagenicity response in the Salmonella typhimurium TA98assay.

1.3.3 Abiotic degradation

The photolysis of nitroPAHs has been studied under variedconditions of irradiation. The rate of photolysis depends not only onthe conditions of irradiation but also on whether the nitroPAH is in thegaseous phase (e.g., 1-and 2-nitronaphthalene), in solution (type ofsolvent) or bound to solids/particles. In the latter case, the type andage of the particle seem to influence the photochemistry of therespective nitroPAH. The rate of photodecomposition, identification ofphotolytic products and resulting loss or gain of metabolic activity asdetermined by the S. typhimurium assay have been the main end-points studied.

Calculated atmospheric lifetimes of nitroPAHs due to photolysisand gas-phase reactions with hydroxyl and nitrate radicals and withozone under atmospheric conditions show that the dominant lossprocess for nitroPAHs (e.g., 1- and 2-nitronaphthalene) is photolysis.


6

Particle oxidation of nitroPAHs by ozone may be the main loss processat night.

1.4 Environmental levels and human exposure

NitroPAHs that have been detected in ambient air include 1- and2-nitronaphthalene and methylnitronaphthalenes (predominantly in thevapour phase), 2-nitrofluorene, 9-nitroanthracene, 9-nitrophenanthrene,2-, 3- and 8-nitrofluoranthene, 1- and 2-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene.

At remote and forest sites, nitroPAHs were either not detected ordetected in the low picogram per cubic metre range (e.g., 17 pg/m3 for2-nitrofluoranthene; 4 pg/m3 for 1-nitropyrene). The concentration ofnitroPAHs in the atmosphere of urban regions depends on the season,the type of heating used and the number and regulation of trafficvehicles. Reported levels in air do not usually exceed 1 ng/m3, althoughmaxima of up to 13 ng/m3 have been reported.

Various studies have been performed monitoring certain isomericnitroPAHs. Investigators have concentrated on the nitroPAHs thatseem to be of quantitative/environmental (e.g., nitroPAHs of relativemolecular mass 247: 1-nitropyrene, 2-nitropyrene, 2-nitrofluoranthene)or carcinogenic (e.g., 1-nitropyrene, 2-nitrofluorene, dinitropyrenes)importance.

Studies of daytime/nighttime concentrations of specific isomericnitroPAHs in certain regions (in particular California, USA) and parallelenvironmental chamber studies have led to an understanding of theatmospheric formation of certain nitroPAHs (2-nitrofluoranthene and2-nitropyrene). Concurrent studies of certain nitroPAHs (1-nitropyrene,dinitropyrenes) and traffic volume have confirmed that traffic emissionis a source of nitroPAHs.

Most seasonal studies show higher winter/spring concentrationsof marker nitroPAHs, which parallels the use of domestic heating,although this is not always the case.

Summary

7

1.4.1 Indoor air

As nitroPAHs have been detected in the emissions of keroseneheaters, fuel gas and LPG burners used for heating and cooking athome, as well as in the fumes of cooking oils, there is therefore a poten-tial indoor exposure to nitroPAHs in poorly ventilated conditions.

Concentrations of polyaromatic compounds, including nitroPAHs,were measured in a study of indoor and outdoor air levels associatedwith 33 homes located in two US cities: Columbus, Ohio, and Azusa,California. The overall levels were much higher in homes occupied bysmokers, but the use of natural gas heating and cooking appliancesalso appeared to increase the nitroPAH levels slightly.

1-Nitropyrene (4.2–25 600 ng/litre) was detected in 36 of 55 sam-ples of wastewater from oil–water separating tanks of gasoline stationsand in used crankcase oil.

1- and 2-nitronaphthalene and 1,3- and 1,5-dinitronaphthalene weredetected in river water in Japan at concentrations of 1.3, 11.7, 1.7 and3.2 ng/litre, respectively. In another water sample, 1-nitropyrene wasidentified.

There are only limited data on the presence of nitroPAHs insamples of soil, sewage sludge, sediment and incinerator ash (e.g., for1-nitropyrene, 0.03–0.8 µg/kg dry weight in soil, 0.68 µg/kg in sewagesludge, 25.2 µg/kg in sediment and


8

Another survey of nitroPAH levels in various foods in Austriashowed mostly detectable levels of 2-nitrofluorene, 1-nitropyrene and2-nitronaphthalene. The highest concentrations were found in spices,smoked foods and teas, in particular Mate tea, which is roasted. Nitro-PAHs were also detected in vegetables and fruits, probably due toatmospheric pollution.

1-Nitropyrene was detected in grilled corn, mackerel and (inconsiderable amounts) pork and yakitori (grilled chicken) grilled withsauce (up to 43 ng/g).

1.4.3 Other products

In 1980, studies showed that extracts of selected xerographictoners and paper photocopies were mutagenic. The fraction of thecarbon black B responsible for 80% of the mutagenicity contained 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 1,3,6-trinitropyrene and1,3,6,8-tetranitropyrene. As a result of this finding, the manufacturersmodified the production of carbon black B, substantially reducing thelevels of nitropyrenes.

1.4.4 Occupational exposure

Occupational exposure to nitroPAHs has been demonstrated inworkplaces associated with the use of diesel engines. For example,concentrations of 1-nitropyrene in air were measured in various work-places associated with the use of diesel engines. The highest levels(42 ng/m3) reported were determined in the breathing zones of theunderground workers (drivers of diesel-powered excavators) at an oilshale mine in Estonia.

Summary

9

1.5 Kinetics and metabolism in laboratory animals andhumans

1-Nitropyrene and 2-nitrofluorene administered by various routesare rapidly absorbed, and the resulting metabolites are conjugated andexcreted. Radiolabelled 1-nitropyrene was found to be widely distrib-uted in the body of rats and mice following administration by all routes.Other nitroPAHs have not been as well studied.

The metabolism of nitroPAHs is complex. It seems that there are atleast five metabolic activation pathways through which mutations canbe induced by nitroPAHs in bacterial and mammalian systems and/orthrough which DNA binding occurs. These are 1) nitroreduction; 2)nitroreduction followed by esterification (in particular acetylation); 3)ring oxidation; 4) ring oxidation and nitroreduction; and 5) ringoxidation and nitroreduction followed by esterification. In bacteria,nitroreduction seems to be the major metabolic pathway, whereas thefungus Cunninghamella elegans is an example of a species in whichnitroPAHs are metabolized by ring oxidation.

Nitroreduction of nitroPAHs in vivo probably occurs mainly bybacteria in the intestinal tract. In oxidative metabolism, the first step istransformation to phase I primary metabolites such as epoxides,phenols and dihydrodiols, and then to secondary metabolites, such asdiol epoxides, tetrahydrotetrols and phenol epoxides. In mammaliansystems, the phase I metabolites are then conjugated with glutathione,sulfate or glucuronic acid to form phase II metabolites, which are morepolar and water-soluble than the parent hydrocarbons. On reaching theintestine, the conjugated metabolites can be deconjugated by theintestinal microflora and absorbed, entering enterohepatic circulation.Nitroreduction and N-acetylation can occur, resulting in the excretionin urine and faeces of metabolites such as acetylaminopyrenols after 1-nitropyrene administration.

Different cytochrome P450 enzymes may be involved in themetabolism of a specific nitroPAH, and these may differ in the relatedisomers, resulting in possibly different kinetics and pathways. Cyto-chrome P450 enzymes responsible for the metabolism of nitroPAHs may


10

vary between species and in different target organs and in different celltypes within target organs.

All nitroPAHs do not follow the same activation pathways. Someare mutagenic when reduced to an arylhydroxylamine (e.g., 1-nitro-pyrene is metabolized mainly by hydroxylation of the aromatic moiety,followed by nitroreduction and N-acetylation); others (e.g., 1,8- and 1,6-dinitropyrene) are reduced to the arylhydroxylamine and then requirefurther O-esterification (in particular O-acetylation) to an acyloxy esterfor mutagenicity. Some may be mutagenic only after activation byoxidation to reactive epoxides or dihydrodiol epoxides (as possibly in6-nitrobenzo[a]pyrene, similar to benzo[a]pyrene, or BaP). The mainDNA adducts detected with nitroPAHs in vivo and in vitro are C8-substituted deoxyguanosine adducts; however, N2-substituteddeoxyguanosine and C8-substituted deoxyadenosine derivatives havealso been detected and may predominate in nitroPAHs with greaterhydrocarbon character (e.g., 3-nitrobenzo[a]pyrene and 6-nitrochrysene). DNA adducts of dinitropyrenes are formed only vianitroreduction, presumably owing to the high electron deficiency in thearomatic rings caused by the presence of two nitro groups. The DNAadducts resulting from the nitroreduction of nitroPAHs are bettercharacterized than those arising from oxidative metabolism, althoughthe latter may be of more importance in mammalian metabolism.

1.6 Effects on laboratory mammals and in vitro testsystems

Only six nitroPAHs have been tested for acute toxicity. In rats, anLD50 of 86 mg/kg of body weight (kg bw) after intraperitoneal (i.p.)application was reported for 1-nitronaphthalene; in mice, an oral LD50of 1300 mg/kg bw was reported for 2-nitronaphthalene. In furtherstudies on both substances, systemic effects on the target organs lungand liver were observed after single high doses; however, 2-nitro-naphthalene seemed to be less toxic than 1-nitronaphthalene. 5-Nitro-acenaphthene at an i.p. dose of 1700 mg/kg bw was lethal to all treatedrats. For 2-nitrofluorene, an oral LD50 of 1600 mg/kg bw in mice wasreported, whereas gavaging with up to 5000 mg 1-nitropyrene/kg bwresulted in no observable toxic effects. Local inflammation and

Summary

11

ulceration were seen in rats after subcutaneous (s.c.) injection of 8 mg3-nitrofluoranthene/kg bw.

Data on systemic or local non-neoplastic effects caused by short-term or long-term treatment with nitroPAHs are limited, as the end-pointof most studies has been carcinogenicity. In most cases, non-neoplastic toxic effects were observed at doses at which carcinogenicresponses are also manifested. Systemic non-neoplastic toxic effects,such as reduced body weight or increased mortality, appeared presum-ably independently of carcinogenic effects in feeding studies with 5-nitroacenaphthene at a dose level of 500 mg/kg bw per day (rat) or40 mg/kg bw per day (mice) and with 2-nitrofluorene at a dose of25 mg/kg bw per day (rat). Medium-term exposure via inhalation to 1-nitropyrene resulted in metaplasia of the upper respiratory tract atconcentrations of ≥0.5 mg/m3.

No data are available on skin and eye irritation, sensitization orreproductive toxicity.

Data on genotoxicity in vitro are available on 95 nitroPAHs; for 74nitroPAHs, however, only one or two end-points, mainly in bacterialtest systems, were investigated. A sufficient database, includingeukaryotic test systems, has been found only with 21 nitroPAHs. Mostof these substances (67 out of 95) showed positive results, but theresults were derived from a small database. Clearly positive results wereobtained for 19 nitroPAHs, and questionable results for 8 nitroPAHs.With none of the nitroPAHs were clearly negative results obtained.

For 86 nitroPAHs, data on the S. typhimurium microsome test areavailable. In contrast to the parent PAHs, most nitroPAHs were clearlymore effective in the Salmonella microsome test without metabolicactivation. There are five nitroPAHs that showed exceptionally highmutagenic potency (≥100 000 revertants/nmol) in this test system: 3,7-and 3,9-dinitrofluoranthene, 1,6- and 1,8-dinitropyrene, and 3,6-dinitrobenzo[a]pyrene.


12

Bacterial nitroreductase and acetyltransferase are involved in themetabolic activation of nitroPAHs, but not all nitroPAHs follow thesame metabolic activation pathways. Furthermore, there is no uniformmutagenic effect of the different nitroPAHs, as they produce bothframeshift and base pair substitutions in the S. typhimurium microsometest. There is evidence that nitroPAHs with nitro groups perpendicularto the aromatic ring are not as mutagenic as isomers having parallelnitro orientation.

Data on the in vivo genotoxicity of nitroPAHs are available for 15nitroPAHs. All nitroPAHs that gave positive results in vivo were alsopositive in vitro . Four nitroPAHs that were positive in in vitro geno-toxicity tests revealed inconsistent or inconclusive genotoxicity (2-nitronaphthalene, 5-nitroacenaphthene and 3-nitrofluoranthene) ornegative genotoxicity (2,7-dinitrofluorene; limited validity) results invivo.

3-Nitrobenzanthrone, like 1,6- and 1,8-dinitropyrene, is highlymutagenic in bacteria through nitroreduction and O-esterification. 3-Nitrobenzanthrone is also an effective gene mutagen and causes micro-nuclei formation in human cells in vitro and in mice in vivo.

2-Nitrodibenzopyranone was reported to be highly mutagenic inthe S. typhimurium microsome test in strain TA98 (–S9), being moremutagenic than 2-nitrofluorene and 1-nitropyrene. 1- and 3-nitro pyrenelactones have been found to be highly mutagenic in the S. typhimuriummicrosome test.

Studies on the in vitro genotoxicity of 2-nitrodibenzopyranone inforward mutation assays using two human B-lymphoblastoid cell linesare conflicting. Nitropyrene lactones were found to induce mutationsat the tk and hprt loci in both cell lines. Further, they induced kineto-chore-positive and -negative micronuclei in the CREST modifiedmicronucleus assay, which detects chromosomal loss and breakageevents.

Data on carcinogenic effects are available for 28 nitroPAHs.Although inhalation is the main exposure route in humans, no long-

Summary

13

term inhalation study on any nitroPAH is available. Most studiesexamined the carcinogenic effects of nitroPAHs by oral administration,topical application, pulmonary implantation or intratrachealadministration.

Owing to the limitations in experimental design, none of thenegative studies confirmed the absence of carcinogenic effects inanimals. However, results showed carcinogenic effects in experimentalanimals for 5-nitroacenaphthene, 2-nitrofluorene, 3-nitrofluoranthene,3,7- and 3,9-dinitrofluoranthene, 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene. Some carcinogenic effects inexperimental animals were observed for 2-nitropyrene, 7-nitrobenz[a]-anthracene, 2- and 6-nitrobenzo[a]pyrene, 3,6-dinitrobenzo[a]pyrene,7-nitrodibenz[a,h]anthracene and 3-nitroperylene. For the remaining 10nitroPAHs tested, not enough data were available with which toevaluate their carcinogenicity in experimental animals.

Besides local effects at the site of injection, nitroPAHs inducedmainly systemic tumours in mammary tissue, lung, liver and thehaematopoietic system. 6-Nitrochrysene appears to be the most car-cinogenic of the nitroPAHs considered here. With systemic effectsafter s.c. or i.p. injection, 1-nitropyrene was more carcinogenic than thedinitropyrenes. The carcinogenicity of 1-nitropyrene and dinitro-pyrenes varies, depending on the route of administration.

Nitrated benzo[a]pyrenes are generally less potent carcinogensthan the parent compound BaP. However, the mono- or dinitratedpyrenes are more carcinogenic than pyrene. Similar results werepresented for 3-nitroperylene compared with perylene and for 6-nitrochrysene compared with chrysene; with local effects after dermalexposure, however, 6-nitrochrysene was less active than chrysene.

Data were available on carcinogenic effects of some metabolites of2-nitrofluorene, 1-nitropyrene and 6-nitrochrysene. Comparing 2-nitrofluorene with its metabolites in rats, the highest carcinogenicpotency was shown by 2-acetylaminofluorene. 1-Nitropyrene wassignificantly more carcinogenic after oral application in rats than either1-nitrosopyrene or 1-aminopyrene. In contrast, 1-nitrosopyrene


14

induced a higher incidence of liver tumours in mice after i.p. applicationthan 1-nitropyrene; no effects were observed with ring hydroxylatedmetabolites. 6-Nitrosochrysene and 6-aminochrysene were inactive, incontrast to the ring hydroxylated metabolites, which showedcarcinogenic activity in the liver similar to that of the parent compound6-nitrochrysene; this indicates that the metabolic activation of 6-nitrochrysene occurs by ring oxidation and/or a combination of ringoxidation and nitroreduction.

1.7 Effects on humans

There are no reports on the effects of individual nitroPAHs onhumans. As would be expected, since nitroPAHs occur in complexmixtures in the atmosphere and exhaust, the exact contribution ofnitroPAHs to the adverse health consequences of exposure to pollutedatmospheres and to exhaust cannot be elucidated.

At present, investigations on the effects of nitroPAHs on humanhealth are being carried out using biomarkers of exposure. Severalreports have described the development of methods for and provideddata on the evaluation of 1-nitropyrene as a biomarker for occupationalexposure to diesel exhaust. Urinary metabolites of PAHs andnitroPAHs were determined in the urine of diesel mechanics using theenzyme-linked immunosorbent assay (ELISA). In another study,metabolites of 1-nitropyrene (namely, N-acetyl-1-aminopyren-6-ol andN-acetyl-1-aminopyren-8-ol) were measured in the urine of workers ina shipping department. Several studies have focused on measuring thehaemoglobin and plasma adducts of metabolites of 1-nitropyrene andother nitroPAHs and may provide appropriate biomarkers in futuremolecular epidemiological investigations.

1.8 Effects on other organisms in the laboratory andfield

Data on the acute toxicity of nitroPAHs to aquatic organisms areavailable only for 1-nitronaphthalene. An LC50 (96 h) of 9.0 mg/litre wasreported for the fathead minnow (Pimephales promelas). Furthermore,

Summary

15

this nitroPAH inhibited the growth of the ciliate Tetrahymenapyriformis, with an EC50 (60 h) of 17.3 mg/litre.

Some studies have been concerned with the effect of nitroPAHson the metabolism of some aquatic species — for example, the sub-cellular and tissue distribution of two- and one-electron NAD(P)H-dependent nitroreductase activity in marine invertebrates from threephyla: mussel (Mytilus edulis), crab (Carcinus maenas) and starfish(Asteria rubens). NADPH-dependent two-electron nitroreductase activ-ity, occurring only under anaerobic conditions, was detected in themicrosomal and cytosolic fractions of the major digestive tissues ofmussel (digestive gland) and crab, but not in the gills of either species.1-Aminopyrene was the only metabolite identified. No activity wasdetectable in the pyloric caeca or stomach region of the starfish.NAD(P)H-dependent one-electron nitroreduction was present in allsubcellular fractions of the major digestive tissues of the three species.

In the presence of calf thymus DNA, adducts derived from 1-nitropyrene were detected in vitro using hepatic S9 fractions preparedfrom fish. The ability of 1-nitropyrene to form DNA adducts was alsoestablished in vivo using brown trout (Salmo trutta) and turbot(Scophthalmus maximus). These DNA adducts were comparable tothose obtained in Wistar rats treated with 1-nitropyrene.

16

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES,AND ANALYTICAL METHODS

2.1 Identity

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are deriva-tives of polycyclic aromatic hydrocarbons (PAHs), which contain twoor more fused aromatic rings made of carbon and hydrogen atoms,formed as a result of incomplete combustion (see IPCS, 1998). Nitro-PAHs occur in the environment as a mixture together with parent PAHsand hundreds of other organic compounds (see chapter 3). NitroPAHsare usually present in smaller quantities (by 2 orders of magnitude) thanPAHs.

Interest was focused on nitroPAHs in the early 1980s as correla-tions were found between the presence of nitroPAHs in diesel exhaustand environmental extracts and mutagenic activity. A large number ofgroups of nitro, oxy and mixed nitro-oxy compounds eluted together inthe mutagenic fractions. Analytical methods were developed to sep-arate and identify these compounds and to specify their isometric com-position, as the biological action of these compounds also depends ontheir stereospecificity (see chapters 6 and 7). As it would be impossibleto evaluate all these compounds in one document, a decision was madeto include mono- and dinitroPAHs (2–5 rings) but, in general, notmethylated or hydroxylated nitroPAHs. Some nitro-oxyPAHs are alsoincluded: nitroketones (3-nitrobenzanthrone) and selected nitrolactones(e.g., the nitrophenanthrene lactones: 2- and 4-nitrodibenzopyranone[2- and 4-nitro-6H-dibenzo[b,d]pyran-6-one] and nitropyrene lactones),which have recently been shown to be present in the extracts of thepolar fractions of diesel exhaust and airborne particulates.

The nomenclature, molecular formula, relative molecular mass andChemical Abstracts Service (CAS) number of selected nitroPAHs andnitro-oxyPAHs are given in Table 1. The structural formulas of someselected nitroPAHs and nitro-oxyPAHs are shown in Figure 1.

17

Table 1. Nomenclature, molecular formulas, relative molecular mass and CAS numbers of selected nitroPAHs and their oxygen-containing derivatives

Parent PAHs Nitro derivative Molecular formula Relative molecular mass CAS number

Two-ring PAHsNaphthalene 1-Nitronaphthalene C10H7NO2 173.17 86-57-7

2-Nitronaphthalene " " 581-89-51,3-Dinitronaphthalene C10H6N2O4 218.17 606-37-11,5-Dinitronaphthalene " " 605-71-01,8-Dinitronaphthalene " " 602-38-02,7-Dinitronaphthalene " " 24824-27-92,3,5-Trinitronaphthalene C10H5N3O6 263.17 87185-24-81,3,6,8-Tetranitronaphthalene C10H4N4O8 308.16 28995-89-3

Three-ring PAHsAcenaphthene 3-Nitroacenaphthene C12H9NO2 199.21 3807-77-0

5-Nitroacenaphthene " " 602-87-9Fluorene 1-Nitrofluorene C13H9NO2 211.22 22250-99-3

2-Nitrofluorene " " 607-57-83-Nitrofluorene " " 5397-37-54-Nitrofluorene " " 24237-68-12,7-Dinitrofluorene C13H8N2O4 256.22 5405-53-8

Anthracene 2-Nitroanthracene C14H9NO2 223.23 3586-69-49-Nitroanthracene " " 602-60-8

18

Table 1 (Contd).Parent PAHs Nitro derivative Molecular formula Relative molecular mass CAS numberAnthracene (contd) 9,10-Dinitroanthracene C14H8N2O4 268.23 33685-60-8Phenanthrene 2-Nitrophenanthrene C14H9NO2 223.23 17024-18-9

9-Nitrophenanthrene " " 954-46-12,6-Dinitrophenanthrene C14H8N2O4 268.23

Four-ring PAHsFluoranthene 1-Nitrofluoranthene C16H9NO2 247.25 13177-28-1

2-Nitrofluoranthene " " 13177-29-23-Nitrofluoranthene " " 892-21-77-Nitrofluoranthene " " 13177-31-68-Nitrofluoranthene " " 13177-32-71,2-Dinitrofluoranthene C16H8N2O4 292.25 33611-88-02,3-Dinitrofluoranthene " " 105735-66-82,4-Dinitrofluoranthene " " 102493-19-62,5-Dinitrofluoranthene " " 102493-21-03,4-Dinitrofluoranthene " "3,7-Dinitrofluoranthene " " 105735-71-53,9-Dinitrofluoranthene " " 22506-53-21,2,4-Trinitrofluoranthene C16H7N3O6 337.25 102493-20-91,2,5-Trinitrofluoranthene " " 102493-22-12,3,5-Trinitrofluoranthene " " 116331-54-5

19

Table 1 (Contd).Parent PAHs Nitro derivative Molecular formula Relative molecular mass CAS numberPyrene 1-Nitropyrene C16H9NO2 247.25 5522-43-0

2-Nitropyrene " " 789-07-14-Nitropyrene " " 57835-92-41,3-Dinitropyrene C16H8N2O4 292.25 75321-20-91,6-Dinitropyrene " " 42397-64-81,8-Dinitropyrene " " 42397-65-91,3,6-Trinitropyrene C16H7N3O6 337.25 75321-19-61,3,6,8-Tetranitropyrene C16H6N4O8 382.24 28767-61-5

Benz[a]anthracene 7-Nitrobenz[a]anthracene C18H11NO2 273.29 20268-51-3Chrysene 2-Nitrochrysene C18H11NO2 273.29 3989-90-0

5-Nitrochrysene " " 89455-17-46-Nitrochrysene " " 7496-02-8

Five-ring PAHsBenzo[e]fluoranthene 3-Nitrobenzo[e]fluoranthene C20H11NO2 297.31Benzo[a]pyrene 1-Nitrobenzo[a]pyrene C20H11NO2 297.31 70021-997

3-Nitrobenzo[a]pyrene " " 70021-98-66-Nitrobenzo[a]pyrene " " 63041-90-73,6-Dinitrobenzo[a]pyrene C20H10N2O4 342.31 128714-76-1

Benzo[e]pyrene 1-Nitrobenzo[e]pyrene C20H11NO2 297.31 91259-16-43-Nitrobenzo[e]pyrene " " 81340-58-1

20

Table 1 (Contd).Parent PAHs Nitro derivative Molecular formula Relative molecular mass CAS numberPerylene 3-Nitroperylene C20H11NO2 297.31 20589-63-3Dibenz[a,h]anthracene 7-Nitrodibenz[a,h]anthracene C22 H13 NO2 323.4

Six-ring PAHsBenzo[ghi]perylene 4-Nitrobenzo[ghi]perylene C22H11NO2 321.34

7-Nitrobenzo[ghi]perylene " "Coronene 1-Nitrocoronene C24H11NO2 345.36

Nitro-oxyPAHs3-Nitrobenzanthrone C17H9NO3 275.26 1711-34-92-Nitrobenzopyranone C13H7NO4 241.20 6623-66-13-Nitrobenzopyranone C13H7NO4 241.20 6638-64-84-Nitrobenzopyranone C13H7NO4 241.20 51640-90-5Nitropyrene lactones C15H7NO4 265.22

21

NO 2

NO 2

NO 2

1

3

456

7

8

9

10

11

12

4-Nitrobenzo[ghi]perylene1-Nitrocoronene

1

2

3

4

5

67

8

9

10

2

31

4

5

67

8

12

1 3

7-Nitrodibenz[a,h]anthracene

9

1

12

11

1 1

14

Fig. 1. Structural formulas of some nitroPAHs and some nitro-oxyPAHs.

NO2NO

2

O2N

NO2

NO2NO2

O2N NO

2

NO2

NO2

NO2

NO2

NO2NO2

N O2

NO2

NO2

1-Nitronaphthalene 2-Nitronaphthalene 9-Nitrophenanthrene

5-Nitroacenaphthene 9-Nitroanthracene 2-Nitrofluorene

2,7-Dinitrofluorene 2-Nitrofluoranthene 1-Nitropyrene

2-Nitropyrene 1,6-Dinitropyrene 6-Nitrochrysene

6-Nitrobenzo[a]pyrene7-Nitrobenz[a]anthracene 3-Nitroperylene

Fig. 1 Structural formulae of some nitro-PAHs

12

3

45

1

2

3456

7

8

9

6

7

8

4

3

21

1

1

3

45

6

8 9

3

456

78 9

2

3

4

56

7

8

9

1 0

5

4

3

2

1

67

8

910

11

12

2

56

7

8

1

2

3

45

6

7

8

1

7 2

1

2

3

4

567

8

9

101

2

3

4

56

7

8

9

10

2

3

4

5

68

9

10

11 12

1

1

2

3

4

567

8

9

10

11

12

12

4

5

6

78

9

10

1 1

1 2

12

3

4

5

67

8

9

10

1

2

3

4105

6

7

8 9

22

Fig. 1 (Contd).

NO2

O

3-Nitrobenzanthrone

O

O

NO21

2

3

67

8

910

nitropyrene lactone

O

NO2

O

O

O

NO2

4-Nitrodibenzopyranone2-Nitrodibenzopyranone

Identity, Physical and Chemical Properties, Analytical Methods

23

2.2 Physical and chemical properties

At ambient temperatures, nitroPAHs are yellowish to orange solidsthat tend to sublime (White, 1985). NitroPAHs in the environment occurin the vapour phase or are absorbed and/or adsorbed to particulatematter, depending upon their vapour pressure and the ambientconditions. Two- to four-ring nitroPAHs are present partially in thevapour phase under certain conditions; for example, 1-nitronaphthalene(in certain climates) occurs mainly in the vapour phase (Arey et al.,1987), whereas 2-nitrofluorene occurs equally in both vapour andparticulate phases, and 1-nitropyrene occurs in the particulate phase(Schuetzle & Frazier, 1986).

NitroPAHs are insoluble or sparingly soluble in water but aremostly soluble in organic solvents such as acetone, benzene, dimethylsulfoxide (DMSO) and methylene chloride.

Table 2 gives details of some environmentally relevant physicaland chemical properties of nitroPAHs together with those of the parentPAHs (see IPCS, 1998). Nitrodibenzopyranones have a lower vapourpressure than nitroPAHs and therefore are to be found predominantlyin the particulate phase.

2.3 Conversion factors

Atmospheric concentrations of nitroPAHs are usually expressedas micrograms, nanograms or picograms per cubic metre. At 25 °C and101.3 kPa, the conversion factors for a compound of given molecularmass are obtained as follows:

ppb = µg/m3 × 24.45/relative molecular massµg/m3 = ppb × relative molecular mass/24.45

where ppb is parts per billion, and one billion is 109.

For example, for 1-nitropyrene, 1 ppb = 10.1 µg/m3, and 1 µg/m3 =0.099 ppb.

24

Table 2. Physical and chemical properties of nitroPAHs and their parent PAHsa

Parent PAHs;nitro derivatives

Melting point(°C)

Boiling point (°C) at101.3 kPa

Vapour pressure(Pa at 25 °C)

Solubility in waterat 25 °C (mg/litre)

Henry’s lawconstant at 25 °C

(kPa·m3/mol)

Log Kowb Log Kocb

Two-ring PAHsNaphthalene 81 218 10.4 31.7 4.9 × 10–2 3.41-Nitro- 58–61.5

(56.5c)330; 314 sublimesd

312e0.0154 (20 °C)d

3.2 × 10–2 e34f

9.18e6.1 × 10–1 e 2.50c

3.19g,h3.022.98e

2-Nitro- 74–79 (76c) 304e 3.2 × 10–2 e 26f

9.24e6.1 × 10–1 e 2.78c

3.24g,h3.093.03e

1,3-Dinitro- 144–149 2.83g,h

1,5-Dinitro- 215–219 2.58g,h

1,8-Dinitro- 171–172 2.52g,h

2,7-Dinitro- 2341,3,6,8-Tetranitro- 195 2.29g

Three-ring PAHsAcenaphthene 95 279 2.9 × 10–1 3.93 1.5 × 10–2 3.923-Nitro- 1515-Nitro- 102

(101–102c)3.36c

3.85g,h

Fluorene 115 295 8 × 10–2 1.98 1.0 × 10–2 4.181-Nitro- 326 9.7 × 10–5 0.28 7.2 × 10–2 3.76e

25

Table 2 (Contd).Parent PAHs;nitro derivatives

Melting point(°C)





(kPa·m3/mol)

Log Kowb Log Kocb

2-Nitro- 154–158(158c)

326 9.7 × 10–5 0.216 9.5 × 10–2 4.08c

3.37g,h3.16e

3-Nitro- 105–106 326 9.7 × 10–5 0.28 7.2 × 10–2 3.76e

4-Nitro- 75–76 326 9.7 × 10–5 0.28 7.2 × 10–2 3.76e

2,7-Dinitro- 334 3.35g,h

Anthracene 216 342 8 × 10–4 0.073 7.3 × 10–2 4.52-Nitro- 172 4.23g

9-Nitro- 141–146(146c)

4.16c

4.50g

4.78 i

4.69 j

9,10-Dinitro- 263, 310 4.10c

Phenanthrene 100.5 340 1.6 × 10–2 1.292-Nitro- 119–120 4.23g

9-Nitro- 116–1172,7-Dinitro-

Four-ring PAHsFluoranthene 108.8 375 1.2 × 10–3 0.26 6.5 × 10–4 (20 °C) 5.221-Nitro- 4.69g

2-Nitro- 420e 9.9 × 10–7 e 0.019e 1.3 × 10–2 e 4.48e

26


Melting point(°C)





(kPa·m3/mol)

Log Kowb Log Kocb

3-Nitro- 156–162(166c)

5.154.69g

7-Nitro- 144–145 420e 9.9 × 10–7 e 0.017e 1.4 × 10–2 e 4.69g 4.48e

8-Nitro- 158–164 420e 9.9 × 10–7 e 0.017e 1.4 × 10–2 e 4.69g 4.48e

3,4-Dinitro- 279–280k

3,7-Dinitro- 203–204k

3,9-Dinitro- 275–276222–224k

Pyrene 150.4 393 6.0 × 10–4 0.135 1.1 × 10–3 5.181-Nitro- 151–152

(153c)472e 4.4 × 10–6 e 0.017e 6.4 × 10–2 e 5.29c

4.694.48e

2-Nitro- 197–199 472e 4.4 × 10–6 e 0.021e 6.4 × 10–2 e 3.53e

4-Nitro- 190–192 472e 4.4 × 10–6 e 0.017e 6.4 × 10–2 e 4.48e

1,3-Dinitro- 295–297 4.44g

1,6-Dinitro- 309–310 4.44g

1,8-Dinitro- 299–300 4.44g

1,3,6-Trinitro- 4.18g

1,3,6,8-Tetranitro- 335 3.92g

27


Melting point(°C)





(kPa·m3/mol)

Log Kowb Log Kocb

Benz[a]anthracene 160.7 400 2.8 × 10–5 0.014 5.617-Nitro- 161–162c 5.34c

Chrysene 253.8 448 8.4 × 10–5 0.002 5.912-Nitro-5-Nitro-6-Nitro- 208c 5.41c

5.41g

Five-ring PAHsBenzo[a]pyrene 178.1 496 7.3 × 10–7 0.004 3.4 × 10–5 (20 °C) 6.501-Nitro-3-Nitro-6-Nitro- 260c 567e 0.012e 6.13c

5.87e5.66e

3,6-Dinitro-Benzo[e]pyrene 178.7 493 7.4 × 10–7 0.005 6.441-Nitro- 5.87g

Perylene 277.5 503 0.0004 5.33-Nitro- 209c 6.34c

28


Melting point(°C)





(kPa·m3/mol)

Log Kowb Log Kocb

Six-ring PAHsCoronene 439 525 2 × 10–10 0.000 14 5.41-Nitro-Benzo[ghi]perylene 278.3 545 1.4 × 10–8 0.000 26 2.7 × 10–5 (20 °C) 7.104-Nitro-7-Nitro-

Nitro-oxyPAHs3-Nitrobenzanthrone 256–257l

2-Nitrodibenzo[b,d]-pyranone

368e 2.5 × 10–6 e 128e,f 2.9 × 10–6 e 2.04e

4-Nitrodibenzo[b,d]-pyranone

368e 2.5 × 10–6 e 128e,f 2.9 × 10–6 e 2.04e

a The data for parent PAHs are from IPCS (1998). The data for nitroPAHs are from White (1985), unless stated otherwise.b Log Kow = octanol/water partition coefficient; log Koc = sorption coefficient.c From Karcher et al. (1991).d From IUCLID dataset on 1-nitronaphthalene, 2000.e From Yaffe et al. (2001).

29

Table 2 (Contd).

f From Al-Bashir et al. (1994).g From Compadre et al. (1990).h Experimental values.i From Sinks et al. (1997).j From Nielsen et al. (1997).k From Nakagawa et al. (1987).l From Suzuki et al. (1997).


30

2.4 Analytical methods

A direct analysis of nitroPAHs from environmental sources is notpossible, as all environmental matrices are very complex. The samplesoften contain thousands of combustion products, including parentPAHs and other closely related derivatives (in particular oxygenatedPAHs such as aldehydes, ketones and carboxylic acids), which tend toco-elute with nitroPAHs under a variety of liquid and gas chromato-graphic conditions and are present at concentrations 1 or 2 orders ofmagnitude higher than those of the nitro-substituted compounds(Schuetzle, 1983; Vincenti et al., 1996; see Table 3). An extensive samplecleanup and prefractionation of the sample are tedious but necessaryprerequisites for trace analysis of nitroPAHs.

Isomer-specific identification is necessary, as the biological activ-ity depends on the position of the nitro substituent. The source of thenitroPAH (e.g., from combustion or from atmospheric reactions; seechapter 3) may determine the isomeric specificity of the nitroPAH.

Although 1- and 2-nitronaphthalene are expected to be found pre-dominantly in the gas phase, other semivolatile nitroPAHs will bedistributed between the particulate and gas phases, depending uponthe ambient temperature. Thus, many ambient measurements will under-estimate the total nitroPAHs present unless both gas- and particulate-associated species have been measured.

Analysis is hindered by a lack of adequate instrumental sensitivityor selectivity and limited availability of native and isotope-labelledstandards (Chiu & Miles, 1996).

Bioassay-directed fractionation (usually using the Ames test ormodifications of this with specific Salmonella typhimurium strains)and subsequent chemical characterization have been used for the iden-tification of nitroPAHs in a number of complex mixtures (see section2.4.5).

As most nitroPAHs are mutagenic, special precautions should betaken, even at ultratrace concentrations.


31

Table 3. Relative concentrations of various PAH compounds and PAHderivatives in the non-polar and moderately polar fractions of a diesel particle

extract, showing only the nitroPAH fraction in detaila

Compound Fraction concentration

Non-polar fractions 1000

Total PAHs, hydrocarbons, alkylbenzenes 1000

Moderately polar fractions 1000

PAH ketones 147PAH carboxyaldehydes 122.2PAH acid anhydrides 54.1HydroxyPAHs 113.1PAH quinones 71.3NitroPAHs 2.9

Nitrofluorenes 0.34Nitro(anthracenes andphenanthrenes)

0.71

Nitrofluoranthenes 0.05Nitropyrenes 1.5Methyl nitro(pyrenes andfluoranthenes)

0.25

Other oxygenated PAHs 83.4PAH carry-over phthalates, hydrocarboncontaminants

340

a Adapted from Schuetzle (1983).

It should be noted that many nitroPAHs, in particular 9-nitro-anthracene, are unstable in the presence of light; therefore, reducedlight conditions should be used (see chapter 4).

Details of methods used for the analysis of nitroPAHs in differentmatrices are given in Table 4. Reviews on the analysis of nitroPAHs aregiven by White (1985), Vincenti et al. (1996), CONCAWE (1998) andHayakawa (2000).

2.4.1 Sampling

Methods of collecting air particulates include a) Mega samplerwith 50% cut-off point of 20 µm and typically 6-h sampling periods

32

Table 4. Analysis of nitroPAHsa

Sample type Extraction Cleanup Analysis Detector Detection limitb References

AirAmbient air Soxhlet (DCM) NP-LC GC MS in SIM Arey et al. (1987)Reactionchamber study

Soxhlet (DCM) NP-LC GCRP-LC

MS in SIMUV

Atkinson et al. (1987a)

Ambient air Ultrasonication (DCM) Extensive, includingNP-LC

GC FPD; NPD; MS EI Fernández & Bayona(1992)

Air particulate Ultrasonication (DCM) Silica gel HPLC Electrochemical Galceran & Moyano(1993)

Air particulate Ultrasonication(benzene/ethanol)

NP-LC HPLC (with on-linereduction)

CL 0.3–5 fmol Murahashi &Hayakawa (1997)

Air particulate Ultrasonication (DCM) Silica/cyclohexane HPLC switchingtechniqueGCELISA

FL

HRMS

20 fmol(1-nitropyrene)

Zühlke et al. (1998)

Diesel exhaustDiesel Soxhlet NP-LC GC NPD (FL) Schuetzle & Perez

(1983)Dieselparticulate

Soxhlet (DCM) NP-LC GCGC

NPDMS

0.5 ppm5 ppm

Paputa-Peck et al.(1983)

Diesel or airparticulates

Soxhlet (DCM) Extensive; alsoNP-LC

GCGC

FIDTEA

Niles & Tan (1989)

33

Table 4 (Contd).Sample type Extraction Cleanup Analysis Detector Detection limitb ReferencesDiesel or airparticulates

Ultrasonication(acetone)

SPE and on-linereduction

GC MS EI 1 ng/g Scheepers et al.(1994a)

Air and dieselparticulates

Soxhlet (DCM) NP-LC LCOn-line reduction

ElectrochemicalFL

60 pg10 pg

MacCrehan et al.(1988)

Diesel or airparticulates andgaseous

DCM SPE HRGC HRMS low ng/g (dieselparticulate);pg/m3 range

Chiu & Miles (1996)

Vehicle or airparticulates

Ultrasonication(benzene/ethanol)

Precolumn reduction HPLC (columnswitching)

CL with on-linereduction

Hayakawa et al.(1999a)

WaterRiver water DCM; florisil column GC MS Takahashi et al. (1995)River water Concentration by blue

chitin column;methanol

GC MS Nagai et al. (1999)

Wastewater Fractionation intodiethyl ether-solubleneutral, acidic andbasic fractions

HPLC UV and FL Manabe et al. (1984)

Crankcase oil Extraction withmethanol, concentra-tion and dissolution inwater; then as above

HPLC UV and FL Manabe et al. (1984)

34

Table 4 (Contd).Sample type Extraction Cleanup Analysis Detector Detection limitb References

Soil, sewage sludge, sedimentUrban dust,residual ofincinerator, soil

Soxhlet (acetonitrile) SPE GC NPD Librando et al. (1993)

Sewage sludge Dimethyl formamide LSE or SPE GC FID; GC-MS Bodzek & Janoszka(1995)

River sediment Ether; sodiumhydroxide orbenzene methanol;chloroform

Silica gel column;reduced/acylated

GC ECD Sato et al. (1985)

Soil Methanol Silica gel column;hexane/benzene/chloroform; HPLCODS columns

Analytical ODScolumn for HPLC

Reduction toamino derivatives;fluorescence

Dinitropyrenes0.7–4 pg

Watanabe et al. (1999)

Soil Methanol/acetone SPE; HPLC GC MS EI or NICI 30 ng/kg dryweight

Niederer (1998)

35

Table 4 (Contd).Sample type Extraction Cleanup Analysis Detector Detection limitb References

Biological samplesRat tissue Homogenate treated

with acetonitrile;evaporated undernitrogen; residuedissolved in methanol;extraction with BlueRayon

HPLC; on-linereduction

Fluorescence van Bekkum et al.(1999)

Human lungspecimens

Ultrasonication (DCM) Bioassay-directedchemical analysis,HPLC, GC

MS Tokiwa et al. (1993a,1998a)

Food and beveragesVarious foodsamples

Homogenization;acetonitrile; hexane

Silica gel column;DCM

GC TEA 12 pg Dennis et al. (1984)

Various foodsamples

GCGC

MSNPD

5 pg Schlemitz &Pfannhauser (1996a)

Grilled andsmoked foodsamples


Fluorescence Schlemitz &Pfannhauser (1996b)

Fish, meatproducts andcheese


Fluorescence 50 ng/kg Dafflon et al. (2000)

36

Table 4 (Contd).

a Abbreviations:

CL chemiluminescenceDCM dichloromethaneECD electron capture detectorELISA enzyme-linked immunosorbent assayFID flame ionization detectorFL fluorescence detectionFPD flame photometric detectionGC gas chromatographyHPLC high-performance liquid chromatographyHRGC high-resolution gas chromatographyHRMS high-resolution mass spectrometryLC liquid chromatographyLSE adsorption column chromatography on silica gel

MS mass spectrometryMS EI mass spectrometer electron impact modeNICI negative-ion chemical ionizationNPD nitrogen–phosphorus detectionNP-LC normal-phase high-performance liquid chromatographyODS octadecylsilylppm parts per millionRP-LC reversed-phase high-performance liquid chromatographySIM selective ion monitoringSPE solid-phase microextractionTEA thermal energy analyserUV ultraviolet

b The percent recovery was not given in most cases.


37

(used in Claremont, California, USA); b) an electrostatic precipitatorwith an impact stage designed for a 15-µm cut-off (used in Aurskog,Norway); and c) specially designed filter baghouses collected over aperiod of a year (National Institute of Standards and Technology[NIST] Standard Reference Materials [SRMs]) (Ramdahl et al., 1986). A10-µm size-selective inlet (for particulate matter less than 10 µm indiameter, or PM10) sampler is also reported (Nishioka & Lewtas, 1992).

For investigations into the formation and photochemistry of nitro-PAHs, three different collection media for ambient air sampling havebeen used: Tenax-GC solid adsorbent, polyurethane foam (PUF) andfilters for high-volume sampling (Arey et al., 1991).

Three methods for the sampling of the semivolatile phase of dieselexhaust were compared: cryogenic sampling, adsorbent sampler withXAD-2 and PUF. The PUF technique gave the highest recovery ofPAHs and mutagenic activity. The three sampling techniques for thesemivolatile phase resulted in extracts with different chemical compo-sition, different mutagenic potency and different mutagenicity profiles(Westerholm et al., 1991).

In general, vapour-phase constituents are collected on solid sor-bents such as XAD and PUF, and particles are collected on Teflon-impregnated glass fibre filters (see Table 4).

For sampling of diesel exhaust particulates, a dilution tunnelmethod is mainly used (Hayakawa, 2000). The exhaust is diluted by thefiltered air in the tunnel to simulate the real road conditions, and analiquot is sampled on the filter. Gaseous substances are trapped in PUF.The sampling and analytical methods are reviewed by Levsen (1988).

Blue cotton bearing covalently linked copper phthalocyanine tri-sulfonates as a ligand adsorbs polyaromatic compounds and precon-centrates several nitroPAHs in water (Hayatsu, 1992).


38

2.4.2 Extraction

Extraction of filter and PUF samples can be carried out by dichloro-methane (DCM) in a Soxhlet apparatus for 16 h, the Soxhlet body beingloosely covered in aluminium foil to exclude light (Chiu & Miles, 1996).DCM was found to be the most efficient solvent for extraction ofmutagenic compounds from diesel particles (Montreuil et al., 1992).

Toluene as solvent has also been reported (Spitzer, 1993; Vincentiet al., 1996). Supercritical fluid extraction of nitroPAHs from dieselexhaust particulate matter using carbon dioxide–chlorodifluoromethane(HCFC-22) or carbon dioxide–toluene has been demonstrated (Paschkeet al., 1992).

Soil sample extraction has been described using the Soxhlet deviceusing 1:1 (v/v) toluene:methanol (Vincenti et al., 1996) or 5% ethanol intoluene (Spitzer, 1993).

2.4.3 Cleanup

Various procedures for fractionation of particulate extracts havebeen described:

• open-column liquid chromatography with either silica gel oralumina (Yu et al., 1984; Lindskog et al., 1987; Lewtas, 1988

SELECTED NITRO- AND NITRO- OXY-POLYCYCLIC AROMATIC … · 7.5.5.4 Bioassay-directed chemical analysis of airborne particulate matter using a human cell mutagenicity assay 334 7.5.5.5

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