Toxic Potency and Effects of Diffuse Air Pollution
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Toxic potency and effects of diffuse air pollution Timo Hamers Promotor: Prof Dr JH Koeman Hoogleraar in de Toxicologie Wageningen Universiteit Co-promotor: Dr AJ Murk Universitair Hoofddocent, Leerstoelgroep Toxicologie Wageningen Universtiteit Promotiecommissie: Dr TCM Brock Alterra Green World Research, Wageningen Prof Dr NM van Straalen Vrije Universiteit Amsterdam Prof Dr HA Udo de Haes Rijksuniversiteit Leiden Prof Dr M Scheffer Wageningen Universiteit Toxic potency and effects of diffuse air pollution Timotheus HM Hamers Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof Dr Ir L Speelman, in het openbaar te verdedigen op vrijdag 4 oktober 2002 des namiddags te half twee in de aula Title Toxic potency and effects of diffuse air pollution Author THM Hamers Thesis Wageningen University – with ref. – with summary in Dutch ISBN 90-5808-709-3 The air is precious to the red man, for all things share the same breath – the beast, the tree, the man – they all share the same breath. Chief Seattle, 1854 Voor mijn vader die zo trots was op zijn kinderen Contents Chapter 1 General introduction 9 Chapter 2 The application of reporter gene assays for the determination of the 25 toxic potency of airborne particulate matter Chapter 3 A small-volume bio-assay for quantification of the esterase 49 inhibiting potency of mixtures of organophosphate and carbamate insecticides in rainwater: development and optimization Chapter 4 Biological and chemical analysis of the toxic potency of pesticides 67 in rainwater Chapter 5 Estrogenic and esterase-inhibiting potency in rainwater in relation to 93 pesticide concentrations, sampling season and location Chapter 6 Lack of a distinct gradient in biomarker responses in small 121 mammals collected at different distances from a highway Chapter 7 Summary and concluding remarks 143 References 155 Samenvatting 163 Curriculum vitae 170 Publications 171 Main abbreviations 173 Dankwoord 174 Chapter 1 General introduction The umu-assay is a bio-assay in which genetically modified bacteria specifically to respond DNA damaging compounds (genotoxicants). It is performed in small volumes (270 µl) in a so-called microtiter 96-wells plate. In the example plate shown above, the genotoxicity of two model compounds is demonstrated. The color intensity is a measure for the genotoxicity added to each well. For each column 1 to 6, rows A to F are filled with replicate concentrations of the first genotoxicant. Declining concentrations in columns 1 to 6 are reflected by a decreased color intensity from left to right. Similarly, columns 7 to 12 contain declining concentrations of a second genotoxicant. Rows G and H contain control (no genotoxicants added) and background (no bacteria added) wells, respectively. 9 Chapter 1 Diffuse air pollution – the problem In terms of tons per year, most wastes from our industrialized society are emitted to the air compartment. For instance, the Dutch emission registration estimated that 285 kton of organic compounds (excluding CH4) were emitted to the air compartment in The Netherlands in 1999 compared to 35 and <1 kton to the water and soil compartment, respectively (CCDM, 2000). Most of the organics emitted to the air originate from dispersed sources including traffic (42%), industries (22%), households (11%), and the energy sector (7%). Another important source of diffuse air pollution is agriculture, which contributes significantly to emission of pesticides, NH3, CH4, and N2O. Generally, only emissions of known compounds are registered or analyzed in monitoring programs. For instance within the yearly emission registration, assessments are made of the emissions of the acidifying, eutrophicating, and greenhouse gasses NH3, NOx, SOx, CH4,N2O, and CO2. Additionally, emissions are assessed for toxic compounds including heavy metals, pesticides, and halogenated and non- halogenated aliphatic and aromatic hydrocarbons (e.g. CCDM, 2000). However, the mixture of air pollutants consists of many unknown compounds, such as degradation products and reaction products resulting from combustion or atmospheric photomodification processes. Knowledge on the chemical identity and toxicity of the constituents of the mixtures is still rather limited. Due to atmospheric transport, air pollution can reach relatively remote areas, which are not in the close vicinity of a notorious site of emission and which are therefore supposed to be clean. For instance, studies from our own laboratory demonstrated mutagenic activity and tumor promoting effects of airborne particulate matter collected in rural and background locations in The Netherlands (Alink et al., 1983; Van Houdt et al., 1987; Heussen, 1991). Applying trajectory analyses, Van Houdt et al. (1987) further demonstrated that relatively undiluted pockets of air with high mutagenic activity originated from the industrial Ruhr area in Germany and the Antwerp harbor area in Belgium. De Raat and De Meijere (1988) confirmed that a substantial part of the mutagenic airborne activity in an urban area in The Netherlands probably originated from long-range transport. Similarly, atmospheric presence and deposition of pesticides has been demonstrated in remote areas far away from their site of application. In samples of air and wet deposition collected in the Sierra Nevada mountains, organophosphate insecticides were found that probably had been applied ±200 km further in the fruit orchards of Central Valley, CA (Zabik and Seiber, 1993). Persistent organic chlorine 10 General introduction pesticides appear in arctic air, probably after long-range atmospheric transport by polluted aerosols and by global distillation (Biddleman et al., 1993; Allsopp et al. 1999). In summary, diffuse air pollution can be characterized as a very complex mixture that is omnipresent as a “gray veil”. It is emitted by numerous national and international sources, which can impossibly be traced individually. It has a heterogeneous composition in time and space and contains a substantial fraction with an unknown chemical identity and toxicity. Toxicity of air pollution Episodes of increased air pollution that coincided with increased mortality have focused attention on the potency of air pollution to affect human health (see overviews by Goldsmith and Friberg, 1977 and ACE, 2000). Such disastrous incidents often occurred in river valleys under extraordinary atmospheric conditions of temperature inversion in combination with foggy weather. Emissions to the air compartment were trapped in the fog, and could not be diluted into a larger volume of air due to the stagnant atmospheric conditions preventing air circulation to higher atmospheric layers. The first smog (smoky fog) episodes were reported th in London at the end of the 19 century. As a result of increased coal burning in winter, citizens were exposed to yellow/black clouds of smoke particles and SO2 that covered the city for days. In December 1952, the most severe London smog period resulted in 3500 to 4000 excess death numbers ascribed to bronchitis, broncho-pneumonia and heart diseases (Goldsmith and Friberg, 1977). Especially the Los Angeles basin is known for a different type of smog consisting of ozone and other reactive oxidants including peroxyacyl nitrates (PAN) that cause respiratory and eye irritation. This so-called photochemical smog arises when hydrocarbons and NOX emitted by industry and traffic emissions are trapped in fog and react together under the influence of sunlight. In The Netherlands, Biersteker (1966) performed the first studies on the effects of air pollution on human health. The author demonstrated that urban air pollution was a very minor cause of mortality and morbidity in Rotterdam, even during the smog episode of 1962. Much focus has further been given to traffic emissions. For instance, Brunekreef et al. (1983) demonstrated that blood levels of lead in inner city children were higher than in suburban children, probably as a consequence of elevated exposure to lead emitted by traffic. Leaded gasoline was banned in The Netherlands in 1996, but traffic emissions still contribute significantly to air pollution. In a later paper, Brunekreef et al. (1997) demonstrated that 11 Chapter 1 especially diesel exhaust particles might cause reduced lung functions in children living near motorways. With respect to ecological effects of airborne pollution, most studies have focused on lichens and plants. Already in 1859, declining lichen flora in England was attributed to air pollution. Based on the presence and absence of lichens on bark trees, a scale was developed in the 1970s by which sites could be allocated to one of 11 zones, indicating mean winter airborne concentrations of SO2 (Hawksworth and Rose, 1970; 1976). Plant exposure to ethylene has been known to cause growth abnormalities, senescence, and reduced growth th since the beginning of the 19 century (reviewed by Abeles et al., 1992). Later studies on air pollution have focused on the phytotoxicity of acidifying gasses as SO2, HF and HCl (reviewed by Guderian, 1977) and photochemical oxidants as ozone and PAN (reviewed by Guderian, 1985) causing chlorosis, necrosis and reduced growth. Airborne herbicides such as 2,4-D can also negatively affect plants, especially after long-term exposure (Tonneijck and Van Dijk; 1993). Risk characterization of complex mixtures Traditionally, a toxicological risk characterization of mixtures is based on an evaluation of the individual constituents of the