Lichen biodiversity and biomonitoring of atmospheric pollution

Esta tese debruça-se sobre a biodiversidade de líquenes epífitos de pinhais dunares portugueses e sobre uso de líquenes como biomonitores de poluição atmosférica nesse habitat. A Mata Nacional das Dunas de Quiaios (Figueira da Foz) foi o ponto de partida dos estudos de biodiversidade efetuados nesta tese, mas alguns deles estenderam-se à maior parte da costa portuguesa. Como resultado, encontrou-se uma espécie nova para a ciência, Lecanora sorediomarginata Rodrigues, Terrón & Elix, epifítica sobre Pinus pinaster Aiton e P. pinea L, que se encontra distribuída na maior parte da costa. Esta espécie caracteriza-se morfologicamente por um talo crustáceo, de cor esbranquiçada a acinzentada ou esverdeada e que desenvolve sorálios a partir de pequenas verrugas marginais. Quimicamente caracteriza-se pela presença dos ácidos 3,5-dicloro-2'-O-metilnorestenospórico [maior], 3,5-dicloro-2 -O-metilanziaico [menor], 3,5-dicloro-2 -O-metilnordivaricático [menor], 5-cloro-2'-Ometilanziaico [traço] e úsnico [traço]; atranorina [menor] e cloroatranorina [menor]. É quimicamente semelhante a L. lividocinerea Bagl., com a qual apresenta afinidades filogenéticas com base na análise da sequência ITS do rDNA, e a L. sulphurella Hepp. Adicionalmente, espécies Chrysothrix flavovirens Tønsberg e Ochrolechia arborea (Kreyer) Almb, também se encontraram epifíticas sobre P. pinaster e P. pinea em vários pinhais ao longo da costa, representando novos registos para a flora liquénica portuguesa, bem como a espécie Lepraria elobata Tønsberg encontrada epifítica sobre P. pinaster apenas nas Dunas de Quiaios. Além disso, as espécies Hypotrachyna lividescens (Kurok.) Hale e H. pseudosinuosa (Asahina) Hale encontraram-se epifíticas sobre P. pinaster e outros forófitos nas Dunas de Quiaios, constituindo novos registos para a flora liquénica da Península Ibérica. Estes resultados põe em evidência a importância dos pinhais dunares como habitat para líquenes epífitos. Num estudo conduzido entre janeiro e julho de 2008 num pinhal dunar (Mata do Urso, Figueira da Foz), em cuja bordadura existe uma fábrica de celulose de papel, usaram-se transplantes de líquenes da espécie Flavoparmelia caperata (L.) Hale para avaliar a acumulação de trinta e três elementos putativamente emitidos por fábricas de papel e pasta de papel. A cinética da fluorescência da clorofila a foi estudada nos líquenes transplantados, através da análise dos parâmetros Fv/Fm, F0, Fm, qP, NPQ, PSII, e Exc, de forma a avaliar os efeitos decorrentes da acumulação de elementos na vitalidade dos líquenes. Pretendeu-se avaliar se a acumulação de elementos e a cinética da fluorescência da clorofila a variavam significativamente com o local e o tempo de exposição, tendo em consideração os resultados obtidos de transplantes colocados num local de referência (Dunas de Quiaios) durante o mesmo período de tempo. (Continua no verso) resumo A maior parte dos elementos — Al, B, Ba, Ca, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Na, Ni, P, S, Sb, Sc, Sr, Ti e V — ocorreu em concentrações significativamente mais elevadas nos transplantes expostos a 500 m da fábrica. Cerca de metade dos elementos estudados — B, Ba, Cr, Fe, Hg, Mg, Mn, Mo, Na, P, Pb, S, Sb e V — encontraram-se em concentrações significativamente mais elevadas nos transplantes expostos durante 180 dias. O solo foi identificado como uma fonte parcial da maior parte dos elementos. Os parâmetros Fv/Fm, Fm, PSII e Exc variaram significativamente com o local e/ou com o tempo de exposição. Observou-se um decréscimo significativo nos parâmetros Fv/Fm e Fm nos transplantes expostos a 500 e 1000 da fábrica, e também naqueles expostos durante 135 e 180 dias. Observou-se também um decréscimo significativo nos parâmetros PSII e Exc expostos durante 180 dias. Estes parâmetros correlacionaram-se de forma negativa e significativa com a acumulação de elementos: Fv/Fm: B, Ba, Co, Fe, Hg, Mg, Mn, Mo, N, P, S, Sb e Zn; Fm: Ba, Co, Hg, Mn, Mo, N, P, S, Sb e Zn; PSII: N e P; Exc: Mn, N, P e S. Estudos acerca da diversidade liquénica efetuados nos mesmos locais onde os transplantes foram colocados no local impactado, revelaram um menor valor de diversidade liquénica a 500 m da fábrica, que foi também o único local onde se encontraram espécies nitrófilas, o que se poderá dever à deposição de amónia e/ou poeiras. À semelhança de outros estudos, este trabalho confirma que os líquenes podem ser usados com sucesso em estudos de biomonitorização, mesmo em locais florestados. Além disso, traz também informações adicionais sobre como a acumulação de elementos pode influenciar a cinética da fluorescência da clorofila a em líquenes.

Environmental assessment and biomonitoring of the Twelve Mile Creek watershed, Niagara Peninsula, Ontario

In light of the heavy reliance of the people of the Niagara Peninsula on the T\\'elve Mile Creek (TMC) watershed for recreational activities and for municipal and industrial uses ( e.g., drinking water, shipping and discharge of effluents), it was deemed prudent to assess the envirol1tnental health of the system by analysing the sediments total and exchangeable metal, and TPH contents. The MOEE has set guidelines with limits for the protection and management of aquatic sediments, and the sediments from the headwaters of the TMC have total metal and TPH (subset of O&G) contents well below the lower provincial limits. Areas of environmental concern where total metal contents in sediments, either individually or collectively, exceed the guideline, are the south side of Lake Gibson, the Old WeIland Canal, a segment of TMC just south of the QEW and Martindale Pond. The total metal content of sediments does not in all instances identify areas of biological concern. Instead, it has been found that the exchangeable metal fraction of sediments is a better indicator of metal availability and thus potential accumulation in organisms. In some instances, the exchangeable metal fraction agrees with the total metal fraction defining areas of environmental concern, but it does vary from site to site reflecting the natural variability of the ambient environment. Overall, the exchangeable metal fraction of sediments appears to be a better indicator of anthropogenic pollution and ecosystem impact. A histochemical study of Anodon.ta sp., Elliptio sp. and zebra mussels (Dreissena polyn'101pha) was done in conjunction with passive biomonitoring of zebra and quagga mussels (Dreissena bugensis) from the Twelve Mile Creek watershed and Lake 51. Clair (Jeanette's Creek, Chatham, Ontario). The highest concentrations of divalent metals such as Cu, Ni, Cd, and Zn, and trivalent Al appear to accumulate in gill and kidney tissues. Metal contents of organ tissues in Anodonta sp. vary with size class. Organ metal content varies among size classes, thus requiring consideration of size in biomonitoring studies. Shucked zebra and quagga mussel tissues, exhibited similar size class to Al content trends. In addition they reflected the Al content trends of top (approximately 10 cm) most sediments in the Twelve Mile Creek watershed. Quagga mussels appear to have higher Al concentrations than zebra mussels, thus suggesting that quagga mussels may be better passive biomonitors of AI. Cd content in zebra mussel tissues, seemed to increase with size class trends. This was not demonstrated in the quagga mussel tissues. This suggests that Cd may be regulated by quagga mussels and not by zebra mussels, and that zebra mussels may be better passivebiomonitors of Cd than are quagga mussels. Zebra mussel, quagga mussel, Anodonta sp., and Elliptio sp. were used in a two part, active (translocated) biomonitoring study of the Twelve Mile Creek watershed. There was no statistical difference in death rates between zebra and quagga mussels after 65 days of biomonitoring. However there does appear to be a difference of death rates between sites. Unfortunately the data base did not permit us to differentiate between sites. Relative to Port Colborne Harbour (Port Colborne, Ontario), the Twelve Mile Creek watershed appears to be elevated in bioavailable AI. An area near the terminus of the Twelve Mile Creek appears to be an area of environmental concern since mussels seemed to have accumulated relatively large concentrations of Cd, Zn, and Pb. In addition to possible metal loading from a nearby outfalls, or possible upstream outfalls, road salt runoff from storm sewers may have contributed to metal accumulation through cation exchanges processes. Similar trends in cumulative quagga mussel metal concentrations during the two time periods (65 and 159 days), suggest that quagga mussels may reach equilibrium within 65 days of translocation. Differences in bioaccumulated metal concentrations of the two dreissenid species demonstrate that active biomonitoring studies must use a variety of organisms to adequately assess the environmental situation of specific waterways and/or bodies.

Polycyclic aromatic hydrocarbons at fire stations: firefighters’ exposure monitoring and biomonitoring, and assessment of the contribution to total internal dose

This work characterizes levels of eighteen polycyclic aromatic hydrocarbons (PAHs) in the breathing air zone of firefighters during their regular work shift at eight Portuguese fire stations, and the firefighters' total internal dose by six urinary monohydroxyl metabolites (OH-PAHs). Total PAHs (ΣPAHs) concentrations varied widely (46.4-428ng/m(3)), mainly due to site specificity (urban/rural) and characteristics (age and layout) of buildings. Airborne PAHs with 2-3 rings were the most abundant (63.9-95.7% ΣPAHs). Similarly, urinary 1-hydroxynaphthalene and 1-hydroxyacenaphthene were the predominant metabolites (66-96% ΣOH-PAHs). Naphthalene contributed the most to carcinogenic ΣPAHs (39.4-78.1%) in majority of firehouses; benzo[a]pyrene, the marker of carcinogenic PAHs, accounted with 1.5-10%. Statistically positive significant correlations (r≥0.733, p≤0.025) were observed between ΣPAHs and urinary ΣOH-PAHs for firefighters of four fire stations suggesting that, at these sites, indoor air was their major exposure source of PAHs. Firefighter's personal exposure to PAHs at Portuguese fire stations were well below the existent occupational exposure limits. Also, the quantified concentrations of post-shift urinary 1-hydroxypyrene in all firefighters were clearly lower than the benchmark level (0.5μmol/mol) recommended by the American Conference of Governmental Industrial Hygienists.

木本植物对大气污染指示与监测作用研究

本文研究了木本植物的不同部位即叶片、枝条和树皮以及植物的生理指标如气孔阻力对大气S02、TSP和重金属污染的指示和监测作用，并用树木年轮指示大气污染的历史和程度。结果认为： 承德市大气污染自1703年城市化以来开始出现，但达到严重污染水平则出现在本世纪50年代以来尤其是最近10-20年城市化与工业化的加剧，主要污染物以S02为主，从避暑山庄修建前的<0.1μg m-3达到目前的30μg m-3，重金属污染Fe自1927-45大庙铁矿开采后出现，Mn、Ni、Pb等出现在工业化以来的最近40-50年中，上述污染物含量在木质部年轮中明显升高，如S增加了10倍以上，Pb增加了560% (P<0.00l)。 不同城市功能区树皮pH和气孔阻力不同，主要与大气中的S02和TSP有关，据此可监测大气S02和TSP污染。前者以榆树、加拿大杨、垂柳和国槐最佳，相关系数分别可达-0.8384 (P<0.0l)，-0.7447、-0.6904和-0.6552 (P<0.05);后者则以白腊和旱柳下表皮最好，相关系数达0.9968和0.9951 (P<0.00l)。在扫描电镜下发现气孔受大气TSP影响出现不同程度的堵塞现象，主要有2种途径，小型颗粒物(<5μm)进入气孔腔，大型颗粒物(>30μm)可将气孔封盖。 植物不同器官部位污染物含量以树皮为最高，其次是枝条或叶，因而适宜的指示或监测部位是叶或枝条。主分量分析认为：承德市大气污染物以S为主，重金属Fe、Zn、Mn也有一定的贡献，Pb仅出现在繁忙道路区。不同季节污染物含量变化以休眠期最高，生长初期次之，生长旺盛期最低，如S和Pb分别从0.75 mg g-1和0.7 mg g-1上升到1.5 mg g-1和2.0 mg g-1(P<0.001)。植物不同季节污染物含量的变化反应了大气污染物季节变化特点，因而可以指示或监测大气污染尤其是S02污染。其中刺槐多部位复相关模型监测效果最佳，复相关系数可达0.987;某些植物单一部位的监测作用也较好，叶以珍珠梅最佳，相关系数为0.8695 (P<0.001)，枝以油松、珍珠梅、垂柳为好(r≥0.8，P <0.001)，树皮以刺槐为佳，r=0.8615 (P<0.0l)。植物不同部位的污染物含量还可用来评价大气环境质量，其中复合污染指数可以 评价总的大气环境质量，S污染指数和重金属污染指数可以评价S02、重金属和TSP污染，与直接利用污染物浓度法基本一致。油松不同部位对于大气S02的指示作用可表现为年轮对大气污染历史的指示或监测，针叶对现状S02污染的预测，并利用针叶对于S02的监测结果，绘制了大气S02污染分布图。 总之，本文利用古松年轮和现状城市植物的枝条、叶和树皮中的污染物含量以及树皮酸度等不同方面的指标，对承德市大气污染的历史和现状进行了指示与监测，即承德市大气污染从过去到现在均以S02为主，植物不同部位可以非常有效地进行大气S02污染的监测与评价，其中多部位的复相关模型预测效果极佳。另外，由植物监测而绘制的大气S02分布图，较准确地揭示了承德市大气S02现状分布规律。

Historical intake and elimination of polychlorinated biphenyls and organochlorine pesticides by the Australian population reconstructed from biomonitoring data

Quantifying the competing rates of intake and elimination of persistent organic pollutants (POPs) in the human body is necessary to understand the levels and trends of POPs at a population level. In this paper we reconstruct the historical intake and elimination of ten polychlorinated biphenyls (PCBs) and five organochlorine pesticides (OCPs) from Australian biomonitoring data by fitting a population-level pharmacokinetic (PK) model. Our analysis exploits two sets of cross-sectional biomonitoring data for PCBs and OCPs in pooled blood serum samples from the Australian population that were collected in 2003 and 2009. The modeled adult reference intakes in 1975 for PCB congeners ranged from 0.89 to 24.5 ng/kg bw/day, lower than the daily intakes of OCPs ranging from 73 to 970 ng/kg bw/day. Modeled intake rates are declining with half-times from 1.1 to 1.3 years for PCB congeners and 0.83 to 0.97 years for OCPs. The shortest modeled intrinsic human elimination half-life among the compounds studied here is 6.4 years for hexachlorobenzene, and the longest is 30 years for PCB-74. Our results indicate that it is feasible to reconstruct intakes and to estimate intrinsic human elimination half-lives using the population-level PK model and biomonitoring data only. Our modeled intrinsic human elimination half-lives are in good agreement with values from a similar study carried out for the population of the United Kingdom, and are generally longer than reported values from other industrialized countries in the Northern Hemisphere.

Detection and diversity of pathogenic Vibrio from Fiji

Here we investigate the diversity of pathogenic Vibrio species in marine environments close to Suva, Fiji. We use four distinct yet complementary analyses – biochemical testing, phylogenetic analyses, metagenomic analyses and molecular typing – to provide some preliminary insights into the diversity of vibrios in this region. Taken together our analyses confirmed the presence of nine Vibrio species, including three of the most important disease-causing vibrios (i.e. V. cholerae, V. parahaemolyticus and V. vulnificus), in Fijian marine environments. Furthermore, since toxigenic V. parahaemolyticus are present on fish for consumption we suggest these bacteria represent a potential public health risk. Our results from Illumina short read sequencing are encouraging in the context of microbial profiling and biomonitoring. They suggest this approach may offer an efficient and costeffective method for studying the dynamics of microbial diversity in marine environments over time.

Bioaccumulation of chlorinated pesticides and PCBs in the tropical freshwater fish Hoplias malabaricus: Histopathological, physiological, and immunological findings

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Extraction, partial characterization and susceptibility to Hg2+ of acid phosphatase from the microalgae Pseudokirchneriella subcapitata.

Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 μM Hg2+ .

Use of the multispecies freshwater biomonitor to assess behavioral changes of Corophium volutator (Pallas, 1766) (Crustacea, Amphipoda) in response to toxicant exposure in sediment

Automated sediment toxicity testing and biomonitoring has grown rapidly. This study tested the suitability of the marine amphipod Corophium volutator (Pallas, 1766) for sediment biomonitoring using the Multispecies Freshwater Biomonitor (MFB). Two experiments were undertaken to (1) characterize individual behaviors of C. volutator using the MFB and (2) examine behavioral changes in response to sediment spiked with the pesticide Bioban. Four behaviors were visually identified (walking, swimming, grooming and falling) and characterized in the MFB as different patterns of locomotor activity (0-2 Hz range). Ventilation was not visually observed but was detected by the MFB (2-8 Hz). No clear diel activity patterns were detected. The MFB detected an overall increase in C. volutator locomotor activity after Bioban addition to the sediments (56, 100, 121 mg kg(-1)). C. volutator was more active (both locomotion and ventilation) in the water column than the spiked sediment. C. volutator appears a sensitive and appropriate species for behavioral sediment toxicity assessment and biomonitoring. (c) 2005 Elsevier Inc. All rights reserved.

A Metacommunity Framework for Enhancing the Effectiveness of Biological Monitoring Strategies

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Methodological approaches to estimate human population exposure to chemical substances

The public awareness that chemical substances are present ubiquitously in the environment, can be assumed through the diet and can exhibit various health effects, is very high in Europe and Italy. National and international institutions are called to provide figures on the magnitude, frequency, and duration of the population exposure to chemicals, including both natural or anthropogenic substances, voluntarily added to consumers’ good or accidentally entering the production chains. This thesis focuses broadly on how human population exposure to chemicals can be estimated, with particular attention to the methodological approaches and specific focus on dietary exposure assessment and biomonitoring. From the results obtained in the different studies collected in this thesis, it has been pointed out that when selecting the approach to use for the estimate of the exposure to chemicals, several different aspects must be taken into account: the nature of the chemical substance, the population of interest, clarify if the objective is to assess chronic or acute exposure, and finally, take into account the quality and quantity of data available in order to specify and quantify the uncertainty of the estimate.

Macroinvertebrados aquáticos como bioindicadores no processo de licenciamento ambiental no Brasil

Resumo: Licenciamento ambiental no Brasil é um procedimento com vários níveis e etapas, concebido como ferramenta preventiva aos potenciais danos ambientais causados pela implantação de empreendimentos. Embora este procedimento seja obrigatório desde meados da década de 1980, ainda é limitado no que diz respeito ao uso de informações biológicas para a avaliação e o monitoramento de ambientes aquáticos. Neste processo, o órgão licenciador (federal, estadual ou municipal) define as variáveis a serem medidas, tendo como referência o tipo e a magnitude do empreendimento e as características específicas do local proposto para sua instalação. Respostas biológicas devem ser usadas para medir os impactos sobre ecossistemas aquáticos e os macroinvertebrados constituem um grupo que apresenta vantagens como bioindicadores, sendo os mais utilizados para este fim. Em 2011, o Grupo de Trabalho Intersetorial em Biomonitoramento foi criado para discutir o uso de macroinvertebrados em programas de monitoramento. Este trabalho apresenta as reflexões e propostas deste grupo e fornece subsídios para a inclusão destes organismos nos termos de referência a serem aplicados nos processos de licenciamento ambiental no Brasil. Abstract: In Brazil, the environmental licensing process has many stages, and it was designed as a tool to identify the potential and to prevent environmental damages caused by enterprises. Although this procedure is required since the mid-1980s, it is still limited regarding the use of bioassessment and biomonitoring of aquatic ecosystems. In this process, the agency responsible for the licensing (federal, state or local) is the responsible for defining the parameters to be measured. Those parameters are required based on the type and magnitude of the project, and the specific environment and location proposed for its installation. We recommend that biological responses should be used to assess and monitor stressors on aquatic ecosystems. Macroinvertebrates have been used worldwide as bioindicators because they have some advantages over other biological groups for this purpose. In 2011, the ?Intersectoral Working Group on Biomonitoring? was created to discuss the use of macroinvertebrates in biomonitoring programs. This paper presents proposals and recommendations aiming to stimulate and provide guidance for the use of this group in the context of the environmental licensing process in Brazil. Resumen: En Brasil, el proceso de licenciamiento ambiental tiene varios niveles y etapas. Fue diseñado como una herramienta de prevención de posibles daños ambientales causados por la ejecución de proyectos. Aunque este procedimiento exista desde la década de 1980, siegue con limitaciones en relación al uso de la información biológica para la evaluación y control de los ecosistemas acuáticos. En este proceso, la agencia de licenciamiento (federal, estadual o local) es la que define las variables a mensurar en función del tipo y la magnitud del proyecto y las características específicas del lugar propuesto para su instalación. Las respuestas biológicas deben ser utilizadas para evaluar los impactos a los ambientes acuáticos, y el grupo de los macroinvertebrados tienen ventajas sobre otros grupos biológicos y son los más utilizados para este fin. En 2011, el ?Grupo de Trabajo Intersectorial de Biomonitoreo? fue creado para discutir el uso de los macro-invertebrados en los programas de monitoreo ambiental. Este artículo presenta reflexiones y propuestas de este grupo y ofrece subsidios para la inclusión de estos organismos en los términos de referencia que serán aplicados en el proceso de licenciamiento ambiental en Brasil.

Pooled biological specimens for human biomonitoring of environmental chemicals : opportunities and limitations

Biomonitoring has become the ‘gold standard’ in assessing chemical exposures, and plays an important role in risk assessment. The pooling of biological specimens – combining multiple individual specimens into a single sample – can be used in biomonitoring studies to monitor levels of exposure and identify exposure trends, or to identify susceptible populations in a cost-effective manner. Pooled samples provide an estimate of central tendency, and may also reveal information about variation within the population. The development of a pooling strategy requires careful consideration of the type and number of samples collected, the number of pools required, and the number of specimens to combine per pool in order to maximize the type and robustness of the data. Creative pooling strategies can be used to explore exposure-outcome associations, and extrapolation from other larger studies can be useful in identifying elevated exposures in specific individuals. The use of pooled specimens is advantageous as it saves significantly on analytical costs, may reduce the time and resources required for recruitment, and in certain circumstances, allows quantification of samples approaching the limit of detection. In addition, use of pooled samples can provide population estimates while avoiding ethical difficulties that may be associated with reporting individual results.

Biomonitoring of polycyclic aromatic hydrocarbons exposure in small groups of residents in Brisbane, Australia and Hanoi, Vietnam, and those travelling between the two cities

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with adverse health outcomes. Concentrations of urinary PAH metabolites (OH-PAHs) provide an integrated measure of human exposure to PAHs but measurement of urinary OH-PAHs has not been done in Australia and rarely in Vietnam, where air pollution is of concern. In this study, we assessed exposure to PAHs in 16 participants living in Brisbane, Australia and Hanoi, Vietnam, with 4 participants travelling between the two cities during the monitoring period. A total of 312 first morning urine samples were collected over 10 weeks and were analysed for nine OH-PAHs. Concentrations of the urinary OH-PAHs were 2–10 times higher in participants from Hanoi than those from Brisbane. For example, the median concentrations of 1-hydroxypyrene were 292 pg/mL in Hanoi, compared to 64 pg/mL in Brisbane. For participants travelling from Brisbane to Hanoi and back, differences in exposure to PAHs in these two cities resulted in corresponding changes of urinary OH-PAH concentrations, demonstrating that the more polluted environment in Hanoi was likely the source for higher PAH exposure there.