necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.
Concise International Chemical Assessment Document 20
MONONITROPHENOLS
First draft prepared by Dr A. Boehncke, Dr G. Koennecker, Dr I. Mangelsdorf, and Dr A. Wibbertmann, Fraunhofer Institute for Toxicology and Aerosol Research, Hanover, Germany
Please note that the layout andpagination of this pdf file are not identicalto those of theprinted CICAD
Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.
World Health Organization
Geneva, 2000
of the United Nations Environment Programme (UNEP), the International Labour Organisation (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.
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WHO Library Cataloguing-in-Publication Data Mononitrophenols.
(Concise international chemical assessment document ; 20)
1.Nitrophenols - toxicity 2.Risk assessment 3.Environmental exposure I.International Programme on Chemical Safety II.Series
ISBN 92 4 153020 0 (NLM classification: QD 341.P5) ISSN 1020-6167
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iii
FOREWORD . . . 1
1. EXECUTIVE SUMMARY . . . 4
2. IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES . . . 5
3. ANALYTICAL METHODS . . . 6
4. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE . . . 6
5. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION . . . 8
6. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE . . . 10
6.1 Environmental levels . . . 10
6.2 Human exposure . . . 10
7. COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS . . . 11
7.1 2-Nitrophenol . . . 11
7.2 4-Nitrophenol . . . 11
8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS . . . 11
8.1 Single exposure . . . 11
8.2 Irritation and sensitization . . . 12
8.3 Short-term exposure . . . 12
8.3.1 Oral exposure . . . 12
8.3.2 Inhalation exposure . . . 13
8.3.2.1 2-Nitrophenol . . . 13
8.3.2.2 4-Nitrophenol . . . 13
8.3.3 Dermal exposure . . . 13
8.4 Long-term exposure . . . 13
8.4.1 Subchronic exposure . . . 13
8.4.2 Chronic exposure and carcinogenicity . . . 14
8.5 Genotoxicity and related end-points . . . 14
8.6 Reproductive and developmental toxicity . . . 14
8.6.1 Reproductive toxicity . . . 14
8.6.2 Developmental toxicity . . . 18
8.6.2.1 2-Nitrophenol . . . 18
8.6.2.2 4-Nitrophenol . . . 18
8.7 Immunological and neurological effects . . . 18
8.8 Methaemoglobin formation . . . 18
9. EFFECTS ON HUMANS . . . 18
10. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD . . . 19
10.1 Aquatic environment . . . 19
10.2 Terrestrial environment . . . 20
iv
11. EFFECTS EVALUATION . . . 21
11.1 Evaluation of health effects . . . 21
11.1.1 Hazard identification and dose–response assessment . . . 21
11.1.2 Criteria for setting guidance values for 2- and 4-nitrophenol . . . 22
11.1.3 Sample risk characterization . . . 22
11.2 Evaluation of environmental effects . . . 22
12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES . . . 23
13. HUMAN HEALTH PROTECTION AND EMERGENCY ACTION . . . 23
14. CURRENT REGULATIONS, GUIDELINES, AND STANDARDS . . . 23
INTERNATIONAL CHEMICAL SAFETY CARD . . . 24
REFERENCES . . . 26
APPENDIX 1 — 3-NITROPHENOL . . . 31
APPENDIX 2 — SOURCE DOCUMENTS . . . 33
APPENDIX 3 — CICAD PEER REVIEW . . . 33
APPENDIX 4 — CICAD FINAL REVIEW BOARD . . . 34
RÉSUMÉ D’ORIENTATION . . . 35
RESUMEN DE ORIENTACIÓN . . . 38
FOREWORD
Concise International Chemical Assessment Documents (CICADs) are the latest in a family of publications from the International Programme on Chemical Safety (IPCS) — a cooperative programme of the World Health Organization (WHO), the International Labour Organisation (ILO), and the United Nations Environment Programme (UNEP). CICADs join the Environmental Health Criteria documents (EHCs) as authoritative documents on the risk assessment of chemicals.
CICADs are concise documents that provide summaries of the relevant scientific information concerning the potential effects of chemicals upon human health and/or the environment. They are based on selected national or regional evaluation documents or on existing EHCs. Before acceptance for publication as CICADs by IPCS, these documents undergo extensive peer review by internationally selected experts to ensure their completeness, accuracy in the way in which the original data are represented, and the validity of the conclusions drawn.
The primary objective of CICADs is characterization of hazard and dose–response from exposure to a chemical. CICADs are not a summary of all available data on a particular chemical; rather, they include only that information considered critical for characterization of the risk posed by the chemical. The critical studies are, however, presented in sufficient detail to support the conclusions drawn. For additional information, the reader should consult the identified source documents upon which the CICAD has been based.
Risks to human health and the environment will vary considerably depending upon the type and extent of exposure. Responsible authorities are strongly encouraged to characterize risk on the basis of locally measured or predicted exposure scenarios. To assist the reader, examples of exposure estimation and risk characterization are provided in CICADs, whenever possible. These examples cannot be considered as representing all possible exposure situations, but are provided as guidance only. The reader is referred to EHC 1701 for advice on the derivation of health-based guidance values.
While every effort is made to ensure that CICADs represent the current status of knowledge, new
information is being developed constantly. Unless otherwise stated, CICADs are based on a search of the scientific literature to the date shown in the executive summary. In the event that a reader becomes aware of new information that would change the conclusions drawn in a CICAD, the reader is requested to contact IPCS to inform it of the new information.
Procedures
The flow chart shows the procedures followed to produce a CICAD. These procedures are designed to take advantage of the expertise that exists around the world — expertise that is required to produce the high- quality evaluations of toxicological, exposure, and other data that are necessary for assessing risks to human health and/or the environment.
The first draft is based on an existing national, regional, or international review. Authors of the first draft are usually, but not necessarily, from the institution that developed the original review. A standard outline has been developed to encourage consistency in form.
The first draft undergoes primary review by IPCS to ensure that it meets the specified criteria for CICADs.
The second stage involves international peer review by scientists known for their particular expertise and by scientists selected from an international roster compiled by IPCS through recommendations from IPCS national Contact Points and from IPCS Participating Institutions. Adequate time is allowed for the selected experts to undertake a thorough review. Authors are required to take reviewers’ comments into account and revise their draft, if necessary. The resulting second draft is submitted to a Final Review Board together with the reviewers’ comments.
The CICAD Final Review Board has several important functions:
– to ensure that each CICAD has been subjected to an appropriate and thorough peer review;
– to verify that the peer reviewers’ comments have been addressed appropriately;
– to provide guidance to those responsible for the preparation of CICADs on how to resolve any remaining issues if, in the opinion of the Board, the author has not adequately addressed all comments of the reviewers; and
– to approve CICADs as international assessments.
Board members serve in their personal capacity, not as representatives of any organization, government, or
1 International Programme on Chemical Safety (1994) Assessing human health risks of chemicals: derivation of guidance values for health-based exposure limits.
Geneva, World Health Organization (Environmental Health Criteria 170).
S E L E C T I O N O F H I G H Q U A L I T Y N A T I O N A L / R E G I O N A L A S S E S S M E N T D O C U M E N T ( S )
CICAD PREPARATION FLOW CHART
F I R S T D R A F T P R E P A R E D
R E V I E W B Y I P C S C O N T A C T P O I N T S / S P E C I A L I Z E D E X P E R T S
F I N A L R E V I E W B O A R D 2
F I N A L D R A F T 3
E D I T I N G
A P P R O V A L B Y D I R E C T O R , I P C S
P U B L I C A T I O N S E L E C T I O N O F P R I O R I T Y C H E M I C A L
1 Taking into account the comments from reviewers.
2 The second draft of documents is submitted to the Final Review Board together with the reviewers’ comments.
3 Includes any revisions requested by the Final Review Board.
R E V I E W O F C O M M E N T S (P R O D U C E R / R E S P O N S I B L E O F F I C E R), P R E P A R A T I O N
O F S E C O N D D R A F T 1 P R I M A R Y R E V I E W B Y I P C S (REVISIONS AS NECESSARY)
industry. They are selected because of their expertise in human and environmental toxicology or because of their experience in the regulation of chemicals. Boards are chosen according to the range of expertise required for a meeting and the need for balanced geographic
representation.
Board members, authors, reviewers, consultants, and advisers who participate in the preparation of a CICAD are required to declare any real or potential conflict of interest in relation to the subjects under discussion at any stage of the process. Representatives of nongovernmental organizations may be invited to observe the proceedings of the Final Review Board.
Observers may participate in Board discussions only at the invitation of the Chairperson, and they may not participate in the final decision-making process.
1. EXECUTIVE SUMMARY
This CICAD on the isomers 2-, 3-, and 4-nitro- phenol was prepared by the Fraunhofer Institute for Toxicology and Aerosol Research, Hanover, Germany. It was based on reviews compiled by the German Advisory Committee on Existing Chemicals of Environmental Relevance (BUA, 1992) and the US Agency for Toxic Substances and Disease Registry (ATSDR, 1992) to assess the potential effects of 2- and 4-nitrophenol on the environment and on human health. Data identified up to 1992 were considered in these reviews. A compre- hensive literature search of several databases was con- ducted in 1998 to identify any relevant references on 2- and 4-nitrophenol published subsequent to those in the source documents and to identify all references contain- ing relevant data on the isomer 3-nitrophenol. Informa- tion found on 3-nitrophenol was very scarce, precluding a meaningful assessment. As a result, data on this isomer are summarized in Appendix 1. Information on the nature of the peer review and the availability of the source documents is presented in Appendix 2. Informa- tion on the peer review of this CICAD is presented in Appendix 3. This CICAD was approved as an interna- tional assessment at a meeting of the Final Review Board, held in Washington, DC, USA, on 8–11 December 1998. Participants at the Final Review Board meeting are listed in Appendix 4. The International Chemical Safety Card (ICSC 1342) for mononitrophenols, produced by the International Programme on Chemical Safety (IPCS, 1998), has also been reproduced in this document.
The nitrophenol isomers are water-soluble solids that are moderately acidic in water as a result of dissoci- ation. 2-Nitrophenol and 4-nitrophenol are used as inter- mediates in the synthesis of a number of organophos- phorus pesticides and some medical products. Releases into the environment are primarily emissions into air, water, and soil from diffuse sources, such as vehicle traffic and hydrolytic and photolytic degradation of the respective pesticides. Further releases into the hydro- sphere and the geosphere are caused by the dry and wet deposition of airborne nitrophenols from the atmos- phere. The photo-oxidative formation of 2- and 4-nitro- phenol in the atmosphere is still under discussion.
From the available data, only a slow rate of volati- lization of 2-nitrophenol and no significant volatilization of 4-nitrophenol from water to air are to be expected.
2-Nitrophenol is enriched in the liquid phase of clouds, whereas more 4-nitrophenol than expected from physico- chemical data can be found in the gas phase owing to extensive binding to particles. In view of the water solubilities and the expected occurrence in the vapour
phase, wet deposition of nitrophenols from air to surface waters and soil is to be expected. The major
transformation pathway for 2-nitrophenol emitted to the troposphere should be rapid nitration to 2,4-dinitro- phenol, whereas the major portion of airborne 4-nitro- phenol is expected to be particle bound and therefore only to a minor extent available for photochemical reactions. Most of the 4-nitrophenol should be washed out from air by wet and dry deposition. Nitrophenols are not considered to contribute directly to the depletion of the stratospheric ozone layer or to global warming.
Measured half-lives for the photochemical decomposi- tion of 4-nitrophenol in water ranged from 2.8 to 13.7 days. Numerous studies on the biodegradation of 2- and 4-nitrophenol indicate the isomers to be inher- ently biodegradable in water under aerobic conditions.
Mineralization of nitrophenols under anaerobic condi- tions requires extended adaptation of microbial commu- nities.
Measured coefficients of soil sorption (Koc) in the range of 44–530 indicate a low to moderate potential for soil sorption. Nitrophenols released to soil should be biodecomposed under aerobic conditions. Infiltration into groundwater is expected only under conditions unfavourable to biodegradation. For 2- and 4-nitrophe- nol, measured bioconcentration factors ranging from 11 to 76 indicate a low potential for bioaccumulation.
There is only limited information concerning the toxicological profiles of 2- and 4-nitrophenol. In experi- mental animals given 4-nitrophenol orally, intravenously, or intraperitoneally, most of the applied dose was excreted via the urine within 24–48 h as glucuronide and sulfate conjugates, while only very small amounts were excreted via faeces or as unchanged 4-nitrophenol. The percentages of glucuronide and sulfate conjugates were shown to be species and dose dependent. After oral dosing in rabbits, 4-nitrophenol undergoes reduction to p-aminophenol as well as glucuronidation and sulfation.
The available data from in vivo and in vitro studies give an indication for dermal uptake of 4-nitrophenol. The data for 2-nitrophenol are very limited. However, based on the available data, a comparable metabolic transfor- mation is assumed. Bioaccumulation of 2- and 4-nitro- phenol in organisms is not to be expected owing to their rapid metabolism and excretion.
In acute studies, 4-nitrophenol is harmful after oral uptake and was found to be more toxic than 2-nitrophe- nol. A dose-dependent increase in the formation of methaemoglobin was seen in cats after oral exposure to 2-nitrophenol and in rats after exposure by inhalation to 4-nitrophenol. After repeated exposure to 4-nitrophenol, the formation of methaemoglobin was shown to be the
most critical end-point for exposure by inhalation and is assumed to be relevant for oral exposure too. Other noted effects included decreases in body weight gain, differences in organ weights, focal fatty degeneration of the liver, and haematological changes. For these effects, it was not possible to identify a clear dose–response or reliable no-observed-(adverse-)effect levels (NO(A)ELs).
2-Nitrophenol is slightly irritating to the skin but non-irritating to the eye. The substance proved to have no sensitizing effects in a Buehler test. Based on valid studies with experimental animals, irritating effects on skin and eye are assumed for 4-nitrophenol. In a guinea- pig maximization test, 4-nitrophenol was considered as slightly sensitizing. In humans, a possible sensitization after contact with 4-nitrophenol cannot be excluded, especially as skin sensitization has been found in patch tests on factory workers who may have been exposed to 4-nitrophenol.
Neither of the two isomers of nitrophenol has been fully tested for genotoxicity. Insufficient data are avail- able on 2-nitrophenol to allow any conclusions to be made about its possible mutagenicity. More mutageni- city studies are available for 4-nitrophenol, although some were inadequately reported. There is evidence to suggest that 4-nitrophenol can cause chromosomal aberrations in vitro. In the absence of any in vivo mutagenicity studies in mammals, it is not possible to conclude whether or not the mutagenic potential of 4- nitrophenol is expressed in vivo.
In mice, the dermal application of 4-nitrophenol for 78 weeks gave no indication of carcinogenic effects. In another study with mice, which has several limitations, no skin tumours were noted after dermal application of 2- or 4-nitrophenol over 12 weeks. Carcinogenicity studies using the oral or inhalation routes were not available for either of the isomers.
For 4-nitrophenol, the available data gave no evidence of specific or statistically significant reproduc- tive or developmental toxicity effects after dermal or oral application to rats and mice. In an oral study with rats, 2- nitrophenol induced developmental effects in the offspring only at doses that also produced maternal toxicity. However, in these studies, the fetuses were not examined for internal malformations.
The database for 2-nitrophenol is extremely limited, and the database for 4-nitrophenol is insufficient for deriving reliable NO(A)EL values. Therefore, at present, no tolerable daily intakes (TDIs) or tolerable con- centrations (TCs) can be derived for either 2- or 4-nitro- phenol.
From valid test results available on the toxicity of 2- and 4-nitrophenol to various aquatic organisms, nitrophenols can be classified as substances exhibiting moderate to high toxicity in the aquatic compartment.
The lowest effect concentrations found in chronic studies with freshwater organisms (Scenedesmus subspicatus, 96-h EC50: 0.39 mg 2-nitrophenol/litre;
Entosiphon sulcatum, 72-h minimum inhibitory concen- tration, or MIC: 0.83 mg 4-nitrophenol/litre) were 40–
50 times higher than maximum levels determined in a densely populated and highly industrialized Asian river basin (0.0072 mg 2-nitrophenol/litre and 0.019 mg 4- nitrophenol/litre). Therefore, despite biotic and photo- chemical decomposition, nitrophenols emitted to water could pose some risk to sensitive aquatic organisms, particularly under surface water conditions not favour- ing both elimination pathways. Because of their use patterns and release scenarios, it is likely that nitrophe- nols pose only a minor risk to aquatic organisms.
The available data indicate only a moderate toxicity potential of nitrophenols in the terrestrial environment.
From calculations of the toxicity exposure ratio (TER) of nitrophenols from the degradation of pesticides, only a minor risk for organisms in this compartment is to be expected, even under a worst-case scenario.
2. IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES
2-Nitrophenol (CAS No. 88-75-5; 2-hydroxy-1- nitrobenzene, o-nitrophenol) and 4-nitrophenol (CAS No. 100-02-7; 4-hydroxy-1-nitrobenzene, p-nitrophenol) share the empirical formula C6H5NO3. Their structural formulas are shown below.
OH
NO
2OH
NO
22-nitrophenol 4-nitrophenol
Technical-grade 2- and 4-nitrophenol from the German producer have a typical purity of >99%. Named impurities are the corresponding isomer for each product (0.3%) and traces of 3-nitrochlorobenzene (<0.05%).
Polychlorinated dibenzo-p-dioxin/dibenzofuran (PCDD/
PCDF) and tetrachlorodibenzo-p-dioxin/dibenzofuran (TCDD/TCDF) isomers were not detected at detection limits between 0.1 and 0.4 :g/kg product (BUA, 1992).
The pure nitrophenol isomers form pale yellow to yellow crystals at room temperature. The substances are characterized by the physicochemical properties given in Table 1 (Sax & Lewis, 1987).
Table 1: Physicochemical properties of 2- and 4-nitrophenol.
Parameter 2-Nitrophenol 4-Nitrophenol Molecular mass
(g/mol)
139.11 139.11
Melting point (°C) 44–45 (1)(2)(3) 113–114 (1)(2)(3) Boiling point (°C) 214–217 (1) 279
(decomposition) (3) Vapour pressure
(kPa)
6.8 × 10–3 (19.8 °C) (4)
3.2 × 10–6
(20 °C) (5)
Water solubility (g/litre)
1.26
(20 °C) (4) 12.4
(20 °C) (6)
n-Octanol/water partition coefficient (log Kow)
1.77–1.89 (7) 1.85–2.04 (7)
Dissociation constant (pKa)
7.23 (21.5 °C) (8)
7.08
(21.5 °C) (8) Ultraviolet spectrum 8max (water):
230; 276 nm;
log ,max: 3.57;
3.80 (9)
8max (methanol):
no absorption maxima #290 nm(9)
Conversion factors 1 mg/m3 = 0.173 ppmv 1 ppmv = 5.78 mg/m3 References: (1) Budavari et al. (1996); (2) Booth (1991);
(3) Verschueren (1983); (4) Koerdel et al. (1981);
(5) Sewekow (1983); (6) Andrae et al. (1981); (7) BUA (1992);
(8) Schwarzenbach et al. (1988); (9) Weast (1979)
Additional physicochemical properties for mononitrophenols are presented in the International Chemical Safety Card (ICSC 1342) reproduced in this document.
3. ANALYTICAL METHODS
The nitrophenol isomers are usually determined by gas chromatography combined with mass spectrometric detection, flame ionization detection, electron capture detection, or nitrogen-sensitive detection, which are generally applied after derivatization (BUA, 1992; Nick &
Schoeler, 1992; Geissler & Schoeler, 1994; Harrison et al.,
1994; Luettke & Levsen, 1994; Mussmann et al., 1994).
For liquid samples (water, urine, blood), high-
performance liquid chromatography in combination with concentration-gradient elution (acetonitrile/methanol or ammonium acetate, acetic acid with potassium chloride/
methanol) and ultraviolet or electrochemical detection, which can be carried out without derivatization, is also used (BUA, 1992; Nasseredine-Sebaei et al., 1993; Ruana et al., 1993; Paterson et al., 1996; Pocurull et al., 1996;
Thompson et al., 1996). The separation of the different isomers is carried out either by steam distillation (BUA, 1992) or by the formation and subsequent extraction of different ion pairs (León-González et al., 1992).
The following enrichment techniques are used (BUA, 1992; see also review by Puig & Barcelo, 1996):
# solid-phase adsorption with thermal or liquid extraction for air and water samples (Luettke &
Levsen, 1994; Mussmann et al., 1994)
# liquid/liquid extraction after derivatization for water samples (initial purification by acid/base
fractionation of highly polluted samples, increased recovery rates with continuous extraction methods) (León-González et al., 1992; Nick &
Schoeler, 1992; Geissler & Schoeler, 1994; Harrison et al., 1994)
# liquid extraction with acid/base fractionation or solid-phase enrichment and subsequent desorption following aqueous extraction for soil samples (Vozñáková et al., 1996)
# acid hydrolysis of the glucuronide with subsequent derivatization for blood and urine samples or denaturation (Nasseredine-Sebaei et al., 1993; Thompson et al., 1996).
The detection limits are <10 ng/m3 for air, 0.03–
10 :g/litre for water, and 200–1600 :g/kg for soil. A detection limit for the determination of the nitrophenol isomers in biological materials was given only for rat liver perfusate (0.5–1 mg/litre; Thompson et al., 1996).
4. SOURCES OF HUMAN AND
There are no known natural sources of the nitro- phenol isomers.
Within the European Union, 2- and 4-nitrophenol are produced mainly by three companies. Six other large manufacturers are known in the USA and Japan (as of 1989). In 1983, the production volume for Western Europe was estimated at about 6400 t 2-nitrophenol and about 20 500 t 4-nitrophenol. In 1988–89, the German production volumes originating from one manufacturer were approximately 500 t 2-nitrophenol and about 2000 t 4-nitrophenol, with about 20 t of each being exported.
Both 2- and 4-nitrophenol are intermediates in the synthesis of azo dyes and a number of pesticides, mainly insecticides (2-nitrophenol: carbofuran, phosalon; 4- nitrophenol: parathion, parathion-methyl, fluorodifen) and, to a lesser extent, herbicides (4-nitrophenol:
nitrofen, bifenox). The corresponding aminophenols that are gained by reduction are used as a photographic developer (2-aminophenol) and as an intermediate in the synthesis of the tuberculostatic 4-aminosalicylic acid and the analgesic 4-acetaminophenol (paracetamol) (4- aminophenol) (see also Booth, 1991). In the 1980s, the production volumes for 2- and 4-nitrophenol showed a decreasing tendency in Germany as a result of changes in and termination of the production of some organo- phosphorus pesticides.
The releases of 2- and 4-nitrophenol during pro- duction and processing at the only German manufacturer appear to be of minor importance. In 1988–89, about 2.5 kg 2-nitrophenol and 10 kg 4-nitrophenol were emitted to air, and #93 kg 2-nitrophenol and <64 kg 4- nitrophenol were emitted to surface water.
For 1996, the following releases of 2- and 4-nitro- phenol to the environment were reported by manufac- turers in the USA (TRI, 1998):
# 2-nitrophenol: from three manufacturers (one production site each) with production volumes between 450 and 45 000 kg/year, total releases of 15 kg to air and 23 kg into water were reported.
# 4-nitrophenol: from three manufacturers (six production sites) with production volumes of 45–450 kg/year up to 45 000–450 000 kg/year, a total release of 420 kg to air was reported. Data on releases into water were not given.
2-Nitrophenol and 4-nitrophenol have been detected in the exhaust gases of light-duty gasoline and diesel vehicles. Depending on the motor load, the exhaust concentrations of the isomers were <50 :g/m3 exhaust gas (idle) and about 1000 :g 4-nitrophenol/m3 and 2000 :g 2-nitrophenol/m3 (driving at constant velocity) (Nojima et al., 1983; Tremp et al., 1993). A regulated three-way catalytic converter reduced the nitrophenol emissions to about 8% at high motor load
and to about 2% at normal motor load (Tremp et al., 1993). A rough estimation combining the above- mentioned exhaust gas concentrations with estimations of the total exhaust gas volumes from vehicle traffic for Germany resulted in an airborne nitrophenol load of at least several tonnes per year from this source (BUA, 1992). Data concerning nitrophenol releases from other combustion processes (heating, burning of refuse) were not identified.
From laboratory experiments, there is some evidence that 2- and 4-nitrophenol are generated in the atmosphere during the photochemical degradation of aromatic compounds such as benzene and toluene in the presence of nitric oxide or hydroxyl radicals and nitrous dioxide. These results were at least partly obtained in model experiments with unrealistically high nitric oxide concentrations, and there are competing reactions with- out nitrophenol formation for which the rate constant is not known (BUA, 1992). However, smog chamber experiments confirmed the formation of nitrophenol isomers during irradiation (Leone & Seinfeld, 1985;
Leone et al., 1985). Recent cloud water model experi- ments showed that 2- and 4-nitrophenol are also formed from the reaction of phenol with nitrogen pentoxide or monochloronitrogen dioxide, especially under alkaline conditions (Scheer et al., 1996). Estimations of the contribution of photochemically formed nitrophenols to total emissions into the atmosphere are not possible with the available data.
Significant releases of 4-nitrophenol into the hydrosphere may occur from the hydrolytic degradation of the insecticides parathion and parathion-methyl and
— although to a lesser extent — from the photolytic degradation of the herbicides nitrofen and bifenox.
Quantification of releases is not possible with the avail- able data. Furthermore, a considerable portion of air- borne nitrophenols, especially 4-nitrophenol, can be released to the hydrosphere and the geosphere by wet and dry deposition (see section 5) (Herterich & Herr- mann, 1990; Luettke et al., 1997). Numerous studies concerning the concentrations of 2- and 4-nitrophenol in wet deposition samples are available (see section 6.1).
From precipitation data (average 746 mm rain per year for land masses, according to Baumgartner & Liebscher, 1990) and the measured concentrations of 2- and 4- nitrophenol in rainwater, the release of nitrophenols via rain can be estimated to be at least in the order of several thousand tonnes per year on a global basis.
The application of the herbicides nitrofen and bifenox, which are photolytically degraded to 4-nitro- phenol in aqueous solutions, may especially lead to emissions into the geosphere and the biosphere. Further, nitrophenol-contaminated rain, snow, and other wet and
dry deposition may contribute to nitrophenol levels in soils. Data concerning the release of nitrophenols into the biosphere are not available.
5. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION
Environmental releases of nitrophenols are mostly to ambient air, surface waters, and — to a smaller extent
— soil. Using a non-steady-state equilibrium model, the following distribution of 4-nitrophenol in different envi- ronmental compartments was predicted: air, 0.0006%;
water, 94.6%; sediment, 4.44%; soil, 0.95%; biota, 0.000 09% (Yoshida et al., 1983). The distribution pat- terns of 2- and 4-nitrophenol sprayed on a natural soil in a standardized terrestrial ecosystem were determined via radiotracer technique (14C). Of the applied radioactivity (2-nitrophenol/4-nitrophenol), 49.45%/20.01% was recovered in air, 27.38%/40.21% in soil (including animals), 12.73%/7.57% in plants, and 0.05%/0.02% in leachate (Figge et al., 1985). Distribution of 4-nitrophenol in a terrestrial microcosm chamber with artificial soil largely corresponded to this result (Gile & Gillett, 1981).
Owing to the expected decomposition within the incuba- tion periods of 30 and 28 days, respectively, it can be assumed that most of the recovered radioactivity referred to breakdown products of the applied nitrophenols.
In volatility experiments conducted according to Organisation for Economic Co-operation and Develop- ment (OECD) guidelines, half-lives of 2-nitrophenol in water ranged from 14.5 to 27.3 days, indicating a slow rate of volatilization (Koerdel et al., 1981; Rippen et al., 1984; Scheunert, 1984; Schoene & Steinhanses, 1984).
Measurements concerning the partitioning between the gas and liquid phases of clouds during different rain events showed that 2-nitrophenol is enriched in the liquid phase to a larger extent than would be predicted from its water solubility and vapour pressure. On the other hand, 4-nitrophenol is extensively adsorbed to particles. Therefore, elevated levels of this isomer are detected in the gaseous phase of clouds (Luettke et al., 1997). From the available data, a significant volatilization of 4-nitrophenol from water to air is not expected. Since nitrophenols dissociate in aqueous solution, volatiliza- tion may further decrease with increasing pH in surface waters. This leads to the conclusion that dry and wet deposition of nitrophenols from air to surface waters and soil are to be expected. The occurrence of this partition mechanism is supported by the detection of 2- and 4- nitrophenol in rainwater and wet deposition samples (see section 6.1).
From experimental results on direct photodegrada- tion (Koerdel et al., 1981) and the atmospheric photo- oxidation by hydroxyl radicals (Zetzsch et al., 1984), both pathways were found to be of minor importance for the removal of 2-nitrophenol emitted to the troposphere.
Thus, the major degradation pathway for airborne 2- nitrophenol should be rapid nitration to 2,4-dinitrophe- nol (Herterich & Herrmann, 1990; Luettke et al., 1997).
The major portion of airborne 4-nitrophenol is expected to be particle bound and therefore only to a minor extent available for photochemical reactions. Thus, most of the 4-nitrophenol can be washed out from air by wet and dry deposition. Measured half-lives for the photochemical decomposition of 4-nitrophenol in water exposed to sunlight ranged from 2.8 to 13.7 days (Hustert et al., 1981; Mansour, 1996), being longer with increasing pH (Hustert et al., 1981). Traces of 4-aminophenol were found as a photoproduct in river water (Mansour, 1996).
In experiments conducted according to OECD guide- lines, Andrae et al. (1981) and Koerdel et al. (1981) found no hydrolysis of 2- or 4-nitrophenol under environmental conditions.
Numerous studies on the biodegradation of 2- and 4-nitrophenol have been conducted. Standardized tests on ready or inherent biodegradability provide data of large variability, indicating 2- and 4-nitrophenol to be inherently biodegradable under aerobic conditions (depending on origin and density of inoculum and the applied test method) (see Table 2). Results from different tests point to a possible bacteriotoxic effect of 4- nitrophenol at concentrations above 300 mg/litre (Gerike
& Fischer, 1979; Nyholm et al., 1984; Kayser et al., 1994).
Non-standardized experiments with different inocula (e.g., natural water, soil, sediment) showed that microbial decomposition of nitrophenols can occur in different environmental compartments after adaptation of the microflora (Rubin et al., 1982; Subba-Rao et al., 1982;
Van Veld & Spain, 1983; Spain et al., 1984; Ou, 1985;
Hoover et al., 1986; Aelion et al., 1987; Wiggins et al., 1987). Time for acclimation and degree of removal depended mostly on substance concentration, microbial population, climate, and additional substrates.
Biotic degradation of nitrophenols under anaerobic conditions requires extended acclimatization of microbial communities. In tests with sewage sludge and sludge from the primary anaerobic stage of a municipal sewage treatment plant, respectively, initial 2- and 4-nitrophenol concentrations in the range of 96.5–579 mg/litre were not degraded at all within 7–60 days (Wagner & Braeutigam, 1981; Battersby & Wilson, 1989). Boyd et al. (1983) found complete anaerobic removal of 50 mg/litre for all nitrophenol isomers within 1 week, but complete
Table 2: Biotic degradation of nitrophenols under aerobic conditions.
Test Substance
Concentration (mg/litre)
Additional carbon source
Test duration (days)
Removal
(%) Reference
Tests on ready biodegradability
AFNOR test 2-NP 40 OC no 14 16 Gerike & Fischer (1979)
Sturm test 2-NP 10 no 28 32 Gerike & Fischer (1979)
MITI I 2-NP 100
50
no no
14 14
0 7
Urano & Kato (1986) Gerike & Fischer (1979)
Closed bottle test 4-NP 2 no 28 55 Rott et al. (1982)
Modified OECD screening test
4-NP 20 DOC no 28 1 Rott et al. (1982)
Shake flask test 4-NP 20 OC no 21 50 Means & Anderson
(1981)
AFNOR test 4-NP 40 OC no 14 97 Gerike & Fischer (1979)
Sturm test 4-NP 10 no 28 90 Gerike & Fischer (1979)
MITI I 4-NP 50
100 100
no no no
14 14 14
1 0 4.3
Gerike & Fischer (1979) Urano & Kato (1986) CITI (1992) Tests on inherent biodegradability
Zahn-Wellens test 2-NP 400 no 14 80 Gerike & Fischer (1979)
SCAS test 2-NP 20 TOC
13.3 TOC
yes yes
24 24
107 110
Broecker et al. (1984)
Bunch & Chambers 2-NP 5–10 yes 28 100 Tabak et al. (1981)
Coupled units test 2-NP 12 OC yes 7 61 Gerike & Fischer (1979)
Batch test, aerated 2-NP 200 COD no 5 97 Pitter (1976)
Zahn-Wellens test 4-NP 300
100 DOC
no no
14 28
8 100
Andrae et al. (1981) Pagga et al. (1982) Activated sludge test 4-NP 50
100
no no
19 19
100 90
Means & Anderson (1981)
SCAS test 4-NP 20 TOC yes 33
27 25/39 12–15
>90
>97 100 100
Marquart et al. (1984) Scheubel (1984) Ballhorn et al. (1984) Koerdel et al. (1984)
Coupled units test 4-NP 12 OC yes 7 100 Gerike & Fischer (1979)
Batch test, aerated 4-NP 200 COD no 5 95 Pitter (1976)
Abbreviations used: 2-NP = 2-nitrophenol; 4-NP = 4-nitrophenol; OC = organic carbon; DOC = dissolved organic carbon; TOC = total organic carbon; COD = chemical oxygen demand.
mineralization was demonstrated only if the incubation period was extended to 10 weeks. Anaerobic degradation even of high initial nitrophenol concentrations was found by Tseng & Lin (1994), who observed >90%
removal of 2- and 4-nitrophenol (350–650 mg/litre) in a biological fluidized bed reactor with three different kinds of wastewater. From the available results, a slow degradation of nitrophenols under anaerobic conditions by adapted microorganisms can be expected.
Soil sorption coefficients (Koc) were found to increase with increasing organic carbon content. Mea- sured Koc values ranged from 44 to 230 (2-nitrophenol)
and from 56 to 530 (4-nitrophenol) (Boyd, 1982; Broecker et al., 1984; Koerdel et al., 1984; Løkke, 1984; Marquart et al., 1984). Nitrophenols emitted to soil are expected to be biodecomposed under aerobic conditions. Infiltration into groundwater is expected only under conditions unfavourable for biodegradation (e.g., anaerobic conditions). From the available experimental results, nitrophenols have to be classified as substances with a low to moderate potential for soil sorption.
A low potential for bioaccumulation is to be expected from the available valid test results for 2- and 4- nitrophenol. Bioconcentration factors ranging from 14.6
to 24.4 were determined for 2-nitrophenol in a semistatic test system with zebra fish (Brachydanio rerio) (Koerdel et al., 1984); in a flow-through experiment,
bioconcentration factors ranged from 30 to 76 for common carp (Cyprinus carpio), including possible conjugates (Broecker et al., 1984). In static tests, accumulation factors for 4-nitrophenol of 11 for the green alga Chlorella fusca after 1 day (Geyer et al., 1981) and 57 for the freshwater golden orfe (Leuciscus idus melanotus) after 3 days of exposure were determined (Freitag et al., 1982). Zebra fish exposed in tap and river water nearly completely eliminated the accumulated 14C- 4-nitrophenol within 48 h (Ensenbach & Nagel, 1991).
Star fish (Pisaster ochraceus) and sea urchin
(Strongylocentrotus purpuratus) eliminated 89% and 36%, respectively, of injected 14C-4-nitrophenol (3.48 and 3.70 mg/kg body weight, respectively) within 8 h (Landrum & Crosby, 1981).
6. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
6.1 Environmental levels
From the concentrations in rainwater, the total atmospheric nitrophenol pollution in Switzerland is estimated at about 1 :g/m3 (Leuenberger et al., 1988).
Recent measurements in the air of remote areas in Europe (German Alps, Fichtelgebirge, Germany; Mount Brocken, Germany; Great Dun Fell summit, United Kingdom) gave 2-nitrophenol concentrations between 0.8 and 25 ng/m3 and 4-nitrophenol levels between 1.2 and 360 ng/m3 (Herterich & Herrmann, 1990; Luettke et al., 1997). The higher atmospheric 4-nitrophenol levels are apparently due to the higher photochemical stability of this isomer (see section 5). 2-Nitrophenol was found in 22 out of 27 samples of air (range 1–140 ng/m3;detection limit 1 ng/m3) in Japan in 1994, and 4-nitrophenol was detected in 27 out of 27 air samples (range 1–71 ng/m3;detection limit 1 ng/m3) (Japan Environment Agency, 1995). In street dust samples from a Japanese city, up to 3.9 mg 2- nitrophenol/kg and up to 42 mg 4-nitrophenol/kg were detected (Nojima et al., 1983).
Numerous studies deal with the distribution, depo- sition, and degradation behaviour of airborne 2- and 4- nitrophenol in clouds and rainwater. 2-Nitrophenol levels in rainwater and snow between 0.03 and 5.7 :g/litre and 4-nitrophenol concentrations from <0.5 to 19 :g/litre are given in reports mainly from Germany and the USA (BUA, 1992). The recent measurements in rainwater, cloud water, and “fog” (water vapour; not further char- acterized) from rural and urban areas in Europe confirm
these concentration ranges (Herterich & Herrmann, 1990;
Levsen et al., 1990; Richartz et al., 1990; Capel et al., 1991;
Geissler & Schoeler, 1993; Levsen et al., 1993; Luettke et al., 1997). The 2-nitrophenol levels are mostly below or slightly above the detection limit (i.e., <0.1 :g/litre), whereas mean 4-nitrophenol concentrations of about 5 :g/litre rainwater and cloud water and 20 :g/litre fog water were detected. The nitrophenol concentrations in fog are significantly higher than those in rainwater or cloud water owing to the higher droplet surface and longer residence times of the droplets in air compared with rain. The lower concentrations of 2-nitrophenol in the deposition samples compared with 4-nitrophenol are presumably due to the lower photochemical stability of this compound (see section 5).
In the 1970s and early 1980s, the 2- and 4-nitro- phenol concentrations in the German and Dutch parts of the river Rhine and some of its tributaries were between 0.1 and 1 :g/litre (BUA, 1992). 2-Nitrophenol and 4- nitrophenol were not detected in 177 samples of Japa- nese surface waters (detection limits 0.04–10 :g/litre) or in 177 sediment samples (detection limits between 0.002 and 0.8 :g/kg) in 1978, 1979, and 1994 (Japan Environ- ment Agency, 1979, 1980, 1995). Whereas 4-nitrophenol was not detected in 129 fish samples (detection limits 0.005–0.2 :g/kg) in Japan in 1979 and 1994, 2-nitro- phenol was detected in 1 out of 129 saltwater fish samples (detection limits 0.005–0.3 :g/kg) in 1994 (Japan Environment Agency, 1980, 1995). 2-Nitrophenol concentrations between <0.15 :g/litre (detection limit) and 7.2 :g/litre and 4-nitrophenol levels between <0.1 and 18.8 :g/litre were reported for the densely populated and highly industrialized Malaysian Klang river basin in 1990 and 1991 (Tan & Chong, 1993).
6.2 Human exposure
Workers may be exposed to 2- and 4-nitrophenol via inhalation and skin contact during production and processing (mainly in the manufacturing of pesticides).
However, data on nitrophenol concentrations at the workplace were not identified.
Based on the measured concentrations given in section 6.1, an exposure of the general population to nitrophenols via the environment — predominantly through ambient air and drinking-water — cannot be excluded.
4-Nitrophenol accumulates in fog, whereas 2- nitrophenol is rapidly photochemically transformed (see sections 5 and 6.1). The mean measured level of 4- nitrophenol in fog water is about 20 :g/litre.
In Dutch drinking-water samples, maximum con- centrations of 1 :g 2-nitrophenol/litre and <0.1 :g 4-nitrophenol/litre were reported in 1988 (BUA, 1992).
Further data are not available.
7. COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS
AND HUMANS
Studies providing quantitative information on the absorption, metabolism, or elimination of 2- or 4-nitro- phenol in humans were not identified.
7.1 2-Nitrophenol
There is only very limited information available for 2-nitrophenol. In rabbits given a single dose of
200–330 mg/kg body weight via gavage, most of the applied dose (o80%) was excreted via the urine within 24 h. About 71% was conjugated with glucuronic acid and about 11% with sulfate, whereas about 3% was reduced to aminophenols (Robinson et al., 1951).
Skin permeation for 2-nitrophenol was shown in several in vitro experiments (Huq et al., 1986; Jetzer et al., 1986; Ohkura et al., 1990).
Although the information is limited, bioaccumula- tion of 2-nitrophenol in organisms is not to be expected owing to its rapid metabolism and excretion.
7.2 4-Nitrophenol
After oral, dermal, intravenous, or intraperitoneal application of 4-nitrophenol to several test species (rats, mice, dogs, or rabbits), most of the applied dose (up to 95%) was excreted as glucuronide and sulfate conjugates of 4-nitrophenol via the urine within 24–48 h. Only small amounts were excreted via faeces (about 1%) or as unchanged 4-nitrophenol (about 2–7%). The
percentages of glucuronide and sulfate conjugates were shown to be species, sex, and dose dependent.
Although sulfate conjugation dominates at lower 4- nitrophenol concentrations, the percentage of glucuronide conjugates increases at higher dosages (Robinson et al., 1951; Gessner & Hamada, 1970;
Machida et al., 1982; Rush et al., 1983; Snodgrass, 1983;
Tremaine et al., 1984; Meerman et al., 1987). As shown in rabbits after oral dosing, 4-nitrophenol undergoes
reduction to 4-aminophenol as well as glucuronidation and sulfation. Up to 14% of the administered dose was detected as amino compounds in the urine (Robinson et al., 1951). After intraperitoneal administration in mice, 4-
nitrophenyl glucoside was identified as a minor metabolite of 4-nitrophenol (about 1–2% of the administered dose) (Gessner & Hamada, 1970).
For 4-nitrophenol, the pretreatment of laboratory animals with ethanol (induction of cytochrome P-450) resulted in a marked increase in hepatic microsomal hydroxylation. The 4-nitrocatechol then formed com- peted with 4-nitrophenol for the glucuronidation and sulfation pathways (Reinke & Moyer, 1985; Koop, 1986;
McCoy & Koop, 1988; Koop & Laethem, 1992).
Specific investigations on dermal resorption under non-occlusive conditions showed dermal uptake of about 35% and 11% of the applied dose of 14C-4-nitro- phenol within 7 days in rabbits and dogs, respectively.
Skin permeation for 4-nitrophenol was also shown in several in vitro experiments (Huq et al., 1986; Jetzer et al., 1986; Ohkura et al., 1990).
Owing to its rapid metabolism and excretion, bioaccumulation of 4-nitrophenol in organisms is not to be expected.
8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
8.1 Single exposure
For 2-nitrophenol, the oral LD50 is in the range of 2830–5376 mg/kg body weight in rats (BASF AG, 1970;
Vasilenko et al., 1976; Vernot et al., 1977; Koerdel et al., 1981) and 1300–2080 mg/kg body weight in mice (Vasilenko et al., 1976; Vernot et al., 1977). Clinical signs following oral exposure were unspecific and included dyspnoea, staggering, trembling, somnolence, apathy, and cramps. The macroscopic examination performed in some studies revealed congestion in liver and kidneys and ulcers of the stomach in high-dose rats. The inhalation exposure of rats to an atmosphere saturated with the test substance at 20 °C for 8 h (no further information available) resulted in no mortality and no signs of toxicity (BASF AG, 1970). In a limit test, the dermal LD50 for the rat was >5000 mg/kg body weight (Koerdel et al., 1981). In cats (two animals per dose group), the oral application of 2-nitrophenol (50, 100, or 250 mg/kg body weight; no controls) resulted in a dose- dependent increase in methaemoglobin (6, 44, and 57%, respectively).1 One animal dosed with 250 mg/kg body
1 Methaemoglobin formation is discussed in greater detail in section 8.8.
weight died. No formation of methaemoglobin was detected after dermal application of a 50% solution of 2- nitrophenol in water to rabbits (dose not specified, exposure time 1 min to 20 h on the back or 20 h on the ear) (BASF AG, 1970).
The oral LD50 of 4-nitrophenol is in the range of 220–620 mg/kg body weight in rats (BASF AG, 1969;
Vasilenko et al., 1976; Hoechst AG, 1977a; Vernot et al., 1977; Andrae et al., 1981) and 380–470 mg/kg body weight in mice (Vasilenko et al., 1976; Vernot et al., 1977).
Clinical signs following oral exposure of rats were unspecific and included tachypnoea and cramps, and the macroscopic examination performed in some studies revealed a greyish discoloration with dark red patches of the lungs. No mortality was observed in rats after single exposure (head only) to 4700 mg/m3 (application as dust [sodium salt]; particle size not given) for 4 h. In four of six rats, a corneal opacity was observed at the end of exposure, which persisted through the 14-day observation period. In two extra rats exposed to 1510 mg/m3, the methaemoglobin concentrations were not altered compared with controls. A determination of methaemoglobin concentrations after exposure to 4700 mg/m3 was not performed (Smith et al., 1988). In another inhalation study with rats (exposure to an atmosphere saturated with the test substance at 20 °C for 8 h; no further information available), no mortality and no signs of toxicity were seen (BASF AG, 1969). The dermal LD50 for rats and guinea-pigs is $1000 mg/kg body weight (Hoechst AG, 1977b; Eastman Kodak Co., 1980; Andrae et al., 1981). In contrast to 2-nitrophenol, no formation of methaemoglobin was noted in cats (two animals per dose group) after oral dosing with 100, 200, or 500 mg 4-nitrophenol/kg body weight. The mortality rate was 0/2, 1/2, and 2/2, respectively (BASF AG, 1969).
8.2 Irritation and sensitization
From studies comparable to OECD Guidelines 404 and 405, it can be concluded that 2-nitrophenol is slightly irritating to the skin but not to the eye (scores not given). In a Buehler test with guinea-pigs compa- rable to OECD Guideline 406, the substance showed no skin-sensitizing effects (Koerdel et al., 1981).
In a study performed according to US Food and Drug Administration (FDA) guidelines, non-dissolved 4- nitrophenol was slightly irritating to the skin (score 2 of 8) (Hoechst AG, 1977c); in another study comparable to OECD Guideline 404, however, the non-dissolved substance showed no skin-irritating effects (score 0 of 4) (Andrae et al., 1981). 4-Nitrophenol applied as a 10%
solution to the eyes was slightly irritating in a test conducted according to FDA guidelines (scores not
given; Hoechst AG, 1977c). Results with the non- dissolved substance were either strongly irritating in a test conducted according to FDA guidelines (scores not given; Hoechst AG, 1977c) or slightly irritating in a test comparable to OECD Guideline 405 (score 1–2 of 4;
Andrae et al., 1981).
In a guinea-pig maximization test comparable to OECD Guideline 406, skin sensitization was shown in 5 of 20 animals (Andrae et al., 1981).
Data on respiratory tract sensitization for 2- and 4- nitrophenol were not identified in the literature.
8.3 Short-term exposure 8.3.1 Oral exposure
The effect of 2-nitrophenol in rats was studied in a 28-day study to evaluate OECD Guideline 407 (five animals per sex per dose group; daily oral doses of 0, 22, 67, or 200 mg/kg body weight via gavage). Food intake decreased in high-dose males and in mid- and high-dose females, and final body weight decreased non-signifi- cantly in all dosed animals. The absolute liver and kid- ney weights were decreased in mid-dose animals, and the relative testes weight increased in low- and mid-dose males and decreased in high-dose males. In all dosed animals, the relative and absolute weights of the adrenal glands increased. The haematological examination, clinical chemistry, and histopathological examination of the major organs and tissues did not give any indication of a substance-related toxic effect in comparison with controls (Koerdel et al., 1981). Owing to insufficient documentation and the fact that there were minor effects (weight of adrenal glands) shown by all exposed animals, a reliable NO(A)EL cannot be deduced.
In a 28-day study that was also conducted to eval- uate OECD Guideline 407, Sprague-Dawley rats (10 per sex per dose group) received daily oral doses of 0, 70, 210, or 630 mg 4-nitrophenol/kg body weight via gavage.
After dosing, locomotor inhibition, which lasted for about 2 h, was seen in mid- and high-dose animals. In mid-dose animals, 1/10 males died; in high-dose males and females, the mortality rate was 4/10 and 6/10, respec- tively (specific signs of intoxication were not given). In the lowest dose group, the macroscopic examination revealed seven cases of pale liver, and the histo- pathological examination showed 14 cases of finely dispersed fatty degeneration. A focal fatty degeneration of the liver was also observed in 13/20 rats of the mid- dose group, but not in high-dose animals. However, it must be noted that finely dispersed fatty degeneration was also seen in 6/20 control animals. In 4/10 high-dose
males but not females, a hydropic liver cell swelling was noted, and all high-dose rats that died before the end of the study showed vascular congestion of the liver. A slight increase in the leukocyte count was seen at 210 and 630 mg/kg body weight in males and females; the increase was significant in high-dose females. In high- dose males, the alanine aminotransferase (ALAT) activity was significantly increased. Other substance- related effects in high-dose animals included increased nephrosis (two males and five females), testicular atrophy and inhibition of spermatogenesis (one and two males, respectively), and follicular atresia in the ovaries (four females) (Andrae et al., 1981). Because of unclear effects in the liver, a NO(A)EL cannot be deduced.
8.3.2 Inhalation exposure 8.3.2.1 2-Nitrophenol
In Sprague-Dawley rats (15 per sex per group), no mortality was observed after exposure to 0, 5, 30, or 60 mg 2-nitrophenol vapour/m3 (“whole body” exposure;
to generate the vapour, melted 2-nitrophenol was used) for 6 h/day, 5 days/week, over a period of 4 weeks.
Except for squamous metaplasia of the epithelium lining the maxilloturbinates and nasoturbinates in all high-dose animals, the clinical and histopathological examinations gave no consistent exposure-related effects. The met- haemoglobin values determined after the 11th exposure were significantly increased only in low-dose animals (males: 1.0, 2.3, 1.8, and 1.6%; females: 2.0, 4.1, 2.1, and 1.1%), but were within control values at the end of the study (Hazleton Lab., 1984).
8.3.2.2 4-Nitrophenol
No mortality was observed in male albino Crl:CDR rats (10 per group) after exposure to 0, 340, or 2470 mg 4- nitrophenol dust/m3 (application as sodium salt; “head only” exposure; mass median aerodynamic diameter [MMAD] 4.6–7.5 :m) for 6 h/day, 5 days/week, over a period of 2 weeks. Both exposure concentrations resulted in signs of irritation (not further specified). After exposure to 340 and 2470 mg/m3, darker urine,
proteinuria, elevated aspartate aminotransferase (ASAT) values, and a dose-dependent increase in methaemo- globin values were observed. These effects were still evident after a 14-day recovery period; however, the methaemoglobin value was then still elevated in only 2/5 high-dose animals. The methaemoglobin values were 0.2, 0.87, and 1.53% after 10 exposures and 0.2, 0.13, and 0.7% after 14 days’ recovery. The erythrocyte, haemoglobin, and haematocrit values decreased during exposure but were elevated after the 14-day recovery period. In treated rats, the urine volume decreased in a dose-dependent manner during exposure and during the 14-day recovery period. In high-dose animals, the
absolute spleen weight was significantly lower than that of controls after 10 exposures, and the absolute/relative spleen and lung weights were significantly lower in comparison with controls at the end of the recovery period. According to the authors, the biological signifi- cance of the changes in organ weights is unknown owing to the absence of corroborating pathological effects (Smith et al., 1988).
In a second trial (exposure to 0, 30, or 130 mg/m3; MMAD 4.0–4.8 :m), both exposure concentrations again resulted in signs of irritation (not further speci- fied). Methaemoglobinaemia, an effect that was rever- sible within a 14-day recovery period, was seen only at 130 mg/m3. The methaemoglobin values were 0.5, 0.3, and 1.5% after 10 exposures and 0.4, 0.5, and 0.2% after 14 days’ recovery. The gross and histopathological examination revealed no adverse effects in any dose group. From these results, the authors of the study decided upon a NO(A)EL of 30 mg/m3 (Smith et al., 1988).
Groups of Sprague-Dawley rats (15 per sex) were exposed to 0, 1, 5, or 30 mg 4-nitrophenol dust/m3 (“whole body” exposure; MMAD 5.2–6.7 :m) for 6 h/day, 5 days/week, over a period of 4 weeks. The exposure resulted in no deaths, and no exposure-related effects were noted in terms of haematology or clinical chemistry values, gross examination, histopathology, and body or organ weights. In high-dose animals, unilateral and bilateral diffuse anterior capsular cataracts were observed. The methaemoglobin values determined after 2 weeks of exposure showed great variability and appeared to be unusually high (>3 %) in some unex- posed controls. However, the group total methaemo- globin value was increased at a concentration of 5 mg/m3, which was significant in males and not significant in females (males: 0.8, 0.5, 2.2, and 1.1%;
females: 1.3, 1.1, 2.0, and 1.0%) (Hazleton Lab., 1983).
Therefore, a NO(A)EL of 5 mg/m3 can be derived for local effects (cataracts), whereas the NO(A)EL for systemic effects (formation of methaemoglobin) may be lower.
8.3.3 Dermal exposure
Data concerning short-term dermal exposure were not identified in the literature.
8.4 Long-term exposure
In the literature, subchronic and chronic studies are available only for 4-nitrophenol.
8.4.1 Subchronic exposure
In a 13-week gavage study with Sprague-Dawley rats (20 per sex per dose group) given 0, 25, 70, or 140 mg 4-nitrophenol/kg body weight in water 5 days/week,
premature deaths were seen in animals dosed with 70 and 140 mg/kg body weight (1 male/1 female at 70 mg/kg body weight and 15 males/6 females at 140 mg/kg body weight); these were usually preceded by clinical signs, including pale appearance, languid behav- iour, prostration, wheezing, and dyspnoea, shortly after dosing. The histopathological examination of these animals revealed minimal to moderately severe conges- tion in the lung, liver, kidney, adrenal cortex, and pitui- tary; in surviving animals, no treatment-related changes compared with controls were reported. A statement concerning altered methaemoglobin values cannot be given owing to a non-reliable analytical method (about 13% in controls at week 7) (Hazleton Lab., 1989).
Therefore, only a provisional NO(A)EL (changes in liver, kidneys, and lungs) of 25 mg/kg body weight can be derived from this study. The NO(A)EL based on the formation of methaemoglobin may be lower.
The dermal application of 4-nitrophenol to Swiss- Webster mice (10 per sex and dose group; given 0, 22, 44, 88, 175, or 350 mg/kg body weight in acetone, 3 times per week over 13 weeks) resulted in dose-dependent mortality as well as skin irritation/inflammation and necrosis at $175 mg/kg body weight.1
8.4.2 Chronic exposure and carcinogenicity In a long-term study with Swiss-Webster mice (50 per sex per dose group), 4-nitrophenol in acetone was applied to the interscapular skin at doses of 0, 40, 80, or 160 mg/kg body weight, 3 days/week for 78 weeks. At termination of the study, the survival rates were 29/60, 17/60, 26/60, and 24/60 for males and 35/60, 26/60, 33/60, and 27/60 for females. The increased mortality after 60 weeks was due to a generalized amyloidosis (the severity of the amyloidosis was similar among dosed and control animals) and secondary kidney failure. The final mean body weights of the dosed animals were similar to those of the controls. NTP (1993) stated that there were no substance-related neoplastic or non-neoplastic effects associated with the dermal administration of 4-nitro- phenol and that there was no evidence of a carcinogenic activity of the substance in male or female mice.
In another study, which had several procedural deficiencies (only the skin was examined; only 12 weeks of exposure), no skin tumours were observed in 31 female Sutter mice after dermal application of a 20%
solution (25 :l of solution applied twice weekly) of 2- or 4-nitrophenol in dioxane (Boutwell & Bosch, 1959).
8.5 Genotoxicity and related end-points The available in vitro and in vivo genotoxicity studies on 2- and 4-nitrophenol are summarized in Table 3.
2-Nitrophenol showed no mutagenicity in several limited bacterial assays. From the available data, it is not possible to draw any conclusions regarding its mutagen- icity.
For 4-nitrophenol, positive results were obtained in in vitro tests for chromosomal aberrations in mammalian cells. However, apart from one well-documented study published by NTP (1993), the other available assays were inadequately reported. 4-Nitrophenol was shown to be mutagenic in some but not all of the bacterial assays, whereas other studies (i.e., host-mediated bacterial assay, mouse lymphoma assay, unscheduled DNA synthesis assay [apparently in vitro], sister chromatid exchange assay, sex-linked recessive lethal [SLRL] assay in Drosophila) gave negative results. In the absence of any in vivo mutagenicity studies in mammals, it is not possible to conclude whether or not the mutagenic potential of 4-nitrophenol is expressed in vivo.
8.6 Reproductive and developmental toxicity
8.6.1 Reproductive toxicity
In a valid two-generation study with groups of 24 female and 12 male Sprague-Dawley rats carried out by Angerhofer (1985), 4-nitrophenol dissolved in ethanol was applied dermally at doses of 0, 50, 100, or 250 mg/kg body weight per day, 5 days/week. The F0 generation was exposed over a period of 140 days before mating.
Dosing of the F0 females continued throughout breeding, gestation, and lactation. Groups of 26 females and 13 males of the F1 generation were then exposed for 168 days in the same manner as had been the F0 rats; the females were again exposed throughout breeding, gesta- tion, and lactation. Apart from dose-related signs of skin irritation (erythema, scaling, scabbing, and cracking) in dosed animals, the gross and histopathological examina- tions provided no indication of significant adverse effects. The calculated indices concerning fertility, gesta- tion, viability, and lactation were not different from those of controls. The testis to body weight ratios in the F0 generation were not affected, and histological lesions were not observed in the testes. In a 28-day study in rats (see section 8.3.1), testicular atrophy and inhibition of spermatogenesis were observed in some animals after oral dosing at a level of 630 mg/kg body weight, but not at 210 mg/kg body weight.
1 Gulf South Research Institute, not dated; no further information available; results cited from NTP (1993).
