Rozprawy doktorskie na temat „Biomonitoring”

Phthalates are a class of ubiquitous environmental contaminants that cause health problems including reproductive disorders, asthma, and obesity. Advocacy against phthalates has been taking place in the US since the mid-1990s, and eight in-depth interviews were conducted with advocates and scientists in order to construct a history of this advocacy. There have been a variety of campaigns and victories; those around medical devices, children’s products, and personal care products are examined in detail. Phthalate exposure data for a representative sample of the US population indicates that exposure to DEP, DEHP, DnBP, and BBzP went down between 1999 and 2010. As these were the phthalates that had the largest volume of advocacy during the period researched, this decrease suggests that advocacy around specific phthalates is effective in reducing exposure and that more advocacy around phthalates, and potentially other harmful chemicals, could result in further decreased exposure and improved health in the US population. Additional research using more finely graded biomonitoring data would help deepen understanding about correlations between advocacy and phthalate exposure. In reviewing the health effects of phthalates, it was found that a disproportionate amount of the research is on male reproductive health effects, which is partially responsible for the fact that a disproportionate amount of phthalate advocacy is on heath effects relating to men, particularly male babies. Both phthalate science and advocacy sometimes treat women instrumentally, objectifying them or regarding them as incubators. To combat this, scientists could do more research on the health effects of phthalates on women and advocates could take more care not to neglect or instrumentalize women in their efforts to reduce phthalate exposure for all people.

The introduction of instream biological monitoring to water resources management has been an increasing trend world-wide. This monitoring uses biological field assessments of instream biota such as macroinvertebrates, fish and riparian vegetation as an integrated and sensitive tool for diagnosing the condition of the ecosystems and assessing ecological impacts. Biomonitoring information has become an important component in the overall assessment of water resources and is used to drive and direct processes of decision-making and management of water resources. The River Health Programme (RHP) was initiated in South Africa to serve as a source of information regarding the ecological status of river systems, in order to support rational management of these natural resources. In this study, biomonitoring indices (SASS5 and FAII) were used to assess the present ecological status of two rivers located in contrasting catchments of the Eastern Cape. The first river is the Buffalo River located in an urban and industrialized catchment. The second river is the Inxu River draining a rural and afforested catchment. SASS5 was used successfully in both rivers and the results based on water quality and SASS5 indicated that most sites selected on the upper catchment of the Buffalo River have a fair water quality with most sites selected on the lower catchment having a poor water quality. The Inxu River sites (both upper and lower catchment) based on SASS5 and water quality results have a good to fair water quality. The majority of sites sampled on both rivers systems had very low FAII scores and fell within a critically modified water quality category. This result may be due to the fact that these rivers have low fish diversities (either low natural diversity or low diversity due to the presence of alien fish species), poor water quality or inadequate sampling methods. Observations from this study suggest that this index may not be suitable for rivers with low fish diversity. A fish index that is usable to all ecoregions of South Africa with minor adaptations to suit local conditions is still needed, as the present FAII index does not meet these requirements.

The economic, social and biological value of our forests makes their sustainability essential to our well-being. To ensure their long-term health, it is critical to regularly and effectively monitor their inhabitants, as well as to detect non-indigenous species early and accurately. These programs rely on the precise diagnosis of species, which can be complicated for terrestrial arthropods by sizeable trap samples, damaged specimens, immature life stages and incomplete taxonomy. The recent advent of DNA barcoding, a technique that differentiates species using sequence variation in a standard gene region, shows tremendous promise for circumventing these obstacles. This dissertation evaluates the integration of barcoding into forest arthropod biomonitoring and biosurveillance programs with several investigations of nocturnal moths (Lepidoptera) in British Columbia, Canada. Barcode reference libraries are constructed for looper moths (Geometridae) and Lymantria (Erebidae) tussock moths, and are determined to successfully discriminate species in over 93% and 97% of cases, respectively. The libraries demonstrate how barcoding might enhance biosurveillance programs by flagging two new records for geometrid moths, and by successfully diagnosing 32 intercepted tussock moth specimens. These two libraries, and a multi-gene phylogeny constructed for Geometridae, are used to conduct faunal inventories in modified forest systems, and investigate the influence of disturbance on three levels of moth diversity—species, genetic, and phylogenetic. A first level inventory of Stanley Park, Vancouver, produces a preliminary list of 190 species, the detection of four new exotic species, and the discovery of two potentially cryptic species. Surveys conducted across several harvest treatments at two silvicultural research forests display no evidence of increased diversity at intermediate disturbance levels, but do reveal a correlation between species and genetic diversity. And lastly, three levels of moth diversity are estimated in ponderosa pine systems that differ widely in attack by Dendroctonus bark beetles, and demonstrate a negative association between species diversity and tree mortality. In combination, all projects suggest that DNA barcoding provides several advantages over traditional biosurveillance and biomonitoring, including the ability to rapidly sort specimens, a reduction in specialist time, the detection of species at low density, and the ability to appraise multiple levels of diversity.

The validity of the use of sycamore tree rings for the reconstruction of atmospheric lead pollution histories was investigated. The lead concentration and ²⁰⁶Pb/²⁰⁷Pb profiles of several tree cores collected from the Loch Lomond region were compared with one another and with the established records at a Loch Lomond sediment core and as Scottish archival moss collection. Little similarity was observed among the lead concentration and isotopic histories of the tree cores, or between the records of the tree-cores and the sediment/moss records. Sycamore tree ring analysis was also used to investigate the temporal and spatial influence of lead pollution from two areas of former lead mining and smelting. Wanlockhead and Tyndrum, which produced lead with distinctive isotopic signatures. The investigation of lead concentrations and ²⁰⁶Pb/²⁰⁷Pb ratios revealed a waning influence of the mines with both distance and time since cessation of operations. Although general temporal trends of lead pollution in regions close to large point sources were identified in the tree-ring studies, accurate annual records were not preserved. The contemporary concentration and isotopic composition of atmospheric lead in Scotland was investigated by the analysis of tree bark. Some 85 samples of Scots pine tree bark were collected from throughout mainland Scotland and analysed for lead concentrations and isotopic ratios. The lead concentrations in bark allowed comparison of the relative atmospheric lead concentrations around the country, with higher lead concentration observed in areas of higher population and traffic density, and lower concentrations associated with more remote areas. The ²⁰⁶Pb/²⁰⁷Pb ratios allowed estimations of the source/s of the lead detected in the tree bark. Tree bark was seen to be less suitable for studies of contemporary atmospheric isotopic composition than moss.

Doutoramento Biologia
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.
This thesis focuses on the biodiversity of epiphytic lichens of Portuguese pine forests on sand dunes, and on the use of lichens as biomonitors of atmospheric pollution in this habitat. Mata Nacional das Dunas de Quiaios (Figueira da Foz) was the starting location of the biodiversity studies undertaken during this thesis, but some were extended to most of the Portuguese coast. As a result a new species to science, Lecanora sorediomarginata Rodrigues, Terrón & Elix was discovered epiphytic on Pinus pinaster Aiton and P. pinea L, in most of the coast. It is characterised morphologically by a crustose whitish-grey to greenish thallus developing soralia from small, marginal warts and chemically by the presence of 3,5-dichloro-2'-O-methylnorstenosporic acid [major], 3,5-dichloro- 2􀀂-O-methylanziaic acid [minor], 3,5-dichloro-2􀀂-O-methylnordivaricatic acid [minor], 5-chloro-2'-O-methylanziaic acid [trace], atranorin [minor], chloroatranorin [minor], and usnic acid [trace]. It is chemically similar to L. lividocinerea Bagl., to which it shows phylogenetic affinities based on ITS rDNA sequence analysis, and to L. sulphurella Hepp. Additionally, Chrysothrix flavovirens Tønsberg and Ochrolechia arborea (Kreyer) Almb, were also found epiphytic on P. pinaster and P. pinea in several pine forests along the coast, representing new records for Portuguese lichen flora, as well as that of Lepraria elobata, which was found epiphytic on P. pinaster only at Dunas de Quiaios. Furthermore, Hypotrachyna lividescens (Kurok.) Hale e H. pseudosinuosa (Asahina) Hale were found epiphytic on P. pinaster and other phorophytes at Dunas de Quiaios, and were new records for the lichen flora of the Iberian Peninsula. These results indicate the importance of pine forests on sand dunes has habitats for epiphytic lichens. In a study conducted in a pine forest on sand dunes (Mata do Urso, Figueira da Foz), impacted by a pulp mill at its border, between January and July 2008, lichen transplants of the species Flavoparmelia caperata (L.) Hale were used to evaluate the accumulation of thirty-three elements putatively emitted by paper and pulp mill industry. Chlorophyll a fluorescence kinetics studies were performed in the transplanted lichens, through the analysis of the parameters Fv/Fm, F0, Fm, qP, NPQ, 􀀁PSII, and 􀀁Exc in order to evaluate the effect of elemental accumulation on lichen vitality. It was intended to evaluate if elemental accumulation and chlorophyll a fluorescence kinetics varied significantly with site and period of exposure, taking into account the results from transplants performed in a reference location (Dunas de Quiaios) during the same period of time. Most elements — Al, B, Ba, Ca, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Na, Ni, P, S, Sb, Sc, Sr, Ti and V — were found in significantly higher concentrations in the transplants exposed at 500 m of distance from the point source. Nearly half of the elements — B, Ba, Cr, Fe, Hg, Mg, Mn, Mo, Na, P, Pb, S, Sb, and V — were also found in significantly higher concentrations in the transplants exposed during 180 days. (Continues on the verse) abstract Soil was identified as a partial source for most elements. The chlorophyll a fluorescence kinetics parameters Fv/Fm, Fm, 􀀁PSII, and 􀀁Exc varied significantly with site and/or period of exposure. Fv/Fm and Fm were significantly decreased in the transplants exposed at 500 and 1000 m from the pulp mill and in those exposed during 135 and 180 days. Both, 􀀁PSII and 􀀁Exc decreased significantly after 180 days of exposure. Significant negative correlations were identified between these parameters and the accumulation of elements: Fv/Fm: B, Ba, Co, Fe, Hg, Mg, Mn, Mo, N, P, S, Sb, and Zn; Fm: Ba, Co, Hg, Mn, Mo, N, P, S, Sb, and Zn; 􀀁PSII: N and P; 􀀁Exc: Mn, N, P, and S. Lichen diversity studies performed in the same locations where lichen transplants were placed at the impacted location revealed a lower lichen diversity value at the 500 m, which was also the only site were nitrophylous species were found, what could be due to the deposition of ammonia and/or dust. Similarly to other studies, this work confirms that lichens can be successfully used in biomonitoring studies, even in forested locations. Furthermore, it provides additional information on how chlorophyll a fluorescence kinetics of lichens can be influenced by elemental accumulation.

This study investigated trace essential metals (Cu, Zn and Cr) and non-essential metals (Pb, Cd and Hg) in water, sediment and tissues of water spinach, snake head fish and swamp eel from the Saigon River. Sampling was conducted from January to December 2004 at three sites. Three water and sediment samples, edible parts of twenty water spinach (Ipomea aquatica) samples (leaves and stems), twenty snake head fish (Channa striata) and twenty swamp eels (Monopterus albus) were collected at each site. Water, sediment and biota tissues were analysed for Cu, Zn, Cr, Pb, Cd and Hg following the method of USEPA, AOAC and NFTA. Results showed that Cu, Zn, Cr, Cd and Hg in water from the river was not higher than the maximum permitted level for protection of aquatic life by the national standards while the average Cu concentration in sediment was found to be close to the Effects range- low value and within the slightly polluted range (25- 50 ìg.g-1 dry weight). The results also showed that the river sediment was considered to be contaminated by Zn and Hg in comparison with contaminated sites in Australia and in Thailand. In water spinach, no Cu deficiency was found but Zn was accumulated. No accumulation of Cr, Cd and Hg was revealed but Pb was detectable. The results indicated that snake head and eel may have Cu deficiencies. Pb and Hg were found to be bioaccumulated in the muscle of snake head and eel at some sites on some occasions, but the levels were small. The results showed that metal in sediment liver of snake head muscle of snake head and eel water. The metals in water spinach decreased in the order Zn Cu Cr Pb Cd and Hg; in snake head the metal concentrations decreased in the order Zn Cu Cr PbHgCd and in eel as Zn Cu Cr PbCd and Hg. The ANOVA test showed no significant relationship between metals in the water and sediment and biota. Therefore it was concluded that the metals in the selected biota samples did not reflect the metals in the environment. The essential metals Cu, Zn and Cr were found to decrease at the end of the dry season and then increase at the end of the rainy season. The seasonal variations of metals in the river and biota maybe a result of the change of temperature in the area and the need for essential metals in the reproduction of fish. The results of this study clearly demonstrates that water spinach, snake head fish and swamp eel are not potential bio-indicators for metals in the Saigon River since they did not reflect concentrations of metals in their environment. It was also concluded based on consumption rates that water spinach, snake headfish and eel from the Saigon River do not present any potential health risk to consumers. However, attention should be paid to controlling the load of pollutants entering the river to avoid such risks and future problems.

A wide-ranging macroinvertebrate and physico-chemical survey of 230 3rd and 4th order streams throughout the Canterbury region was conducted between November 1999 and March 2000. Kick-net sampling, spot water sampling and habitat surveys were used. Invertebrate community composition appeared to be influenced by two overriding factors; the physical condition of the stream, and the amount of anthropogenic development within the catchment. Faunas dominated by Ephemeroptera and Trichoptera and with some Plecoptera present tended to occur in pristine high altitude streams with low conductivity, well vegetated riparian zones, heterogeneous streambed substrates and periphyton consisting primarily of diatom biofilms. Faunas dominated by Crustacea, Oligochaeta and Chironomidae occurred commonly at degraded lowland sites with high conductivity, little or no riparian vegetation, more homogeneous fine substrates and periphyton dominated by thick mats and filaments. Between these two extremes, gradual change in faunas was found, with Trichoptera dominating intermediately disturbed sites. A striking decrease in the relative abundance of Ephemeroptera along an ecological gradient appeared to be associated with increasing intensity of landuse. A comparative investigation of three biotic indices widely used in New Zealand for assessing stream health, indicated that the MCI, OMCI and SOMCI may not assess the health of all sites, consistently. The inconsistencies were probably brought about by two factors. Firstly, presence-absence data used in calculating the MCI may not detect subtle differences in community structure, whereas the quantitative data used by the OMCI and SOMCI may pick up small differences and therefore group sites into different degradation bands. Secondly, published degradation bands for the MCI, OMCI and SOMCI do not appear to be directly comparable in Canterbury. The utility of a quantitative MCI with low-level (order, class, phylum) identification was also investigated, and found to be a potentially viable alternative to the MCI and its derivatives when a low-cost, rapid assessment technique is needed, but expertise in identification is lacking. The health of streams in the Canterbury region as assessed by the MCI, was investigated. The MCI indicated that streams were generally more healthy if they were further inland, at higher altitudes, and were in forested or unmodified catchments. Stream health was poorest in lowland sites with pastoral and urban/city developed catchments, although 42 pastoral sites with MCI values> 100 and taxonomic richness >25 indicated that healthy streams were attainable in agriculturally developed land. Finally, a multimetric approach for assessing the health of Banks Peninsula streams using macroinvertebrates was developed. Five biological metrics (OMCI, % EPT, % Chironomidae, % Mollusca, No. Ephemeroptera) that best discriminated selected reference sites from sites impaired by habitat disturbance and organic pollution were combined into an index of biological integrity; the Banks Peninsula Macroinvertebrate Index (BPMI). Strong relationships between the BPMI and MCI and OMCI suggested that the extra effort required to produce a multimetric index did not result in improved assessment of stream condition. However, a multimetric index can provide additional information on the source of degradation to a stream and indicate where restoration or mitigation should be focussed.

This study investigated human exposure to inorganic arsenic (As), a risk factor for cancer and non-cancerous health effects, in Cornwall, UK - a region of elevated environmental As resulting from naturally occurring mineralisation and historical mining. Recent exposures to As from private water supplies (PWS) were detected by measuring As in drinking water samples (n=127) and urine samples (n=207). Exceedances of the WHO 10 As µg L-1 guidance value were measured in drinking waters from 5 % of households. The Spearman correlation calculated for drinking water versus unadjusted total urinary As concentrations was 0.36. Urinary As speciation was used to distinguish between environmental inorganic As exposure and non-toxic dietary sources. Seafood derived urinary arsenobetaine exclusion and osmolality hydration adjustment yielded an improved correlation of 0.62 between drinking water and urinary As concentrations. Urinary hydration adjustment methods were improved and comparatively assessed using data from the US National Health and Nutrition Examination Survey (NHANES). Correlations of urinary concentrations of As, iodine (I), lead (Pb) and cadmium (Cd) against urinary flow rate (UFR) (low correlations desired) and urinary Pb and Cd against respective blood concentrations (high correlations desired) were used as independent performance criteria. Osmolality adjustment and a modified UFR-based adjustment method using empirically derived coefficients (slopes of analyte concentrations as a function of UFR) generally performed better than creatinine, excretion rate and bodyweight-adjusted excretion rate methods. The findings demonstrated the analyte specific nature of adjustment methods, their misuse in the literature and suggested a pathway to a more robust adjustment framework. Prolonged exposure to As from PWS was identified by the stability of 127 drinking water As concentrations measured up to 31 months apart. Drinking water As concentrations were correlated with those measured in toenails (Pearson's r: 0.53; n=200) and hair (Pearson's r: 0.38; n=104). The successful elimination of external contamination of toenail samples was indicated by low As concentrations in final-stage rinse solutions (geometric mean contribution: 0.4 %). A positive association between seafood consumption and toenail As and a negative association between home-grown vegetable consumption and hair As was observed when As in drinking water was < 1 As μg/L. Elevated As concentrations measured in residential soil (12-992 mg kg-1; n=127) and household dust (3-1079 mg kg-1; n=99), particularly on mineralised geological domains and in the vicinity of former As mining sites, were indicative of additional As exposure routes. Bioaccessibility-adjusted assessment criteria of 190 (13 % bioaccessibility) and 129 (23 % bioaccessibility) As mg kg-1 were derived and 10 and 17 % of residential soils were in exceedance, respectively. The relative importance of different exposure routes in the study region, namely whether As intake from soil and dust is evident in the study population, will form the basis of further work. This will be addressed using multivariate analyses of drinking water, soil and dust in conjunction with urine, toenail and hair As concentrations.

Doutoramento em Biologia
Os ecossistemas de água doce – responsáveis por funções ambientais importantes e pelo fornecimento de bens e serviços insubstituíveis – têm vindo a ser severamente afectados por perturbações antropogénicas. A conversão de floresta em terreno agrícola afecta os sistemas aquáticos através de uma série de mecanismos: sedimentação; excesso de nutrientes; contaminação; alterações hidrológicas; e remoção de vegetação ripícola. As comunidades de macroinvertebrados de água doce – devido à sua diversidade, ubiquidade e sensibilidade às perturbações ambientais – revelam-se como particularmente adequadas para estudos de avaliação da integridade ecológica destes sistemas expostos simultaneamente a múltiplos factores de impacto. O uso sistemático de respostas biológicas para avaliação de mudanças ambientais – ou biomonitorização – pode ser levado a cabo através de diversas metodologias, que, de uma forma geral, não consideram aspectos funcionais das comunidades biológicas e têm aplicabilidade geograficamente restrita. A biomonitorização através de atributos biológicos (características que reflectem a adaptação das espécies ao seu meio ambiente) revela-se como uma ferramenta promissora na resolução dos problemas referidos, apresentando vantagens adicionais: relações causa-efeito directas; melhoria na diferenciação de impactos; e integração da variabilidade natural. O presente estudo apresenta uma revisão critica do estado-da-arte actual na área do uso de atributos biológicos em biomonitorização. Até à data de publicação, não estava disponível nenhum outro trabalho com a base conceptual do uso de atributos de macroinvertebrados enquanto descritores de comunidades e para efeitos de biomonitorização e gestão de sistemas de água doce. Descrevem-se as teorias ecológicas de suporte destas metodologias (conceitos de habitat-molde e de filtros paisagísticos) e os estudos que aplicaram estas teorias em cenários reais, tendo-se chamado a atenção para questões técnicas e possíveis soluções. As necessidades futuras nesta área englobam: o desenvolvimento de uma só ferramenta de biomonitorização de aplicação alargada; uma maior compreensão da variabilidade natural nas comunidades biológicas; diminuição dos efeitos de soluções de compromisso biológico e sindromas; realização de estudos autoecológicos adicionais; e detecção de impactos específicos em cenários de impacto complexos. Um dos objectivos deste estudo foi contribuir para a melhoria das técnicas de biomonitorização através de atributos, focalizando em comunidades de macroinvertebrados ribeirinhas em diferentes regiões biogeográficas (as bacias hidrográficas dos rios: Little e Salmon em New Brunswick, Canadá; Anllóns na Galiza, Espanha; Reventazón em Cartago, Costa Rica). Em cada região, foram estudados gradientes de uso agrícola de solo, incluindo desde bacias hidrográficas quase exclusivamente cobertas por floresta até bacias sob a influência maioritária de actividades agrícolas intensivas. Em cada gradiente de uso de solo, a caracterização da comunidade biológica (por amostragem de macroinvertebrados em troços de rápidos) foi acompanhada pela caracterização do habitat circundante (incluindo propriedades da bacia hidrográfica, análise química das águas e outras propriedades à escala local). A comunidade de macroinvertebrados foi caracterizada através de informação taxonómica, métricas estruturais, índices de diversidade, métricas de tolerância, índices bióticos e através da compilação de atributos biológicos e fisiológicos gerais, de história de vida e de resistência a perturbações. Análises estatísticas univariadas e multivariadas foram usadas para evidenciar os gradientes biológicos e físico-químicos, confirmar a sua co-variação, testar a significância da discriminação de níveis de impacto e estabelecer comparações inter-regionais. A estrutura de comunidades revelou os complexos gradientes de impacto, que por sua vez co-variaram significativamente com os gradientes de uso de solo. Os gradientes de impacto relacionaram-se sobretudo com entrada de nutrientes e sedimentação. Os gradientes biológicos definidos pelas medidas estruturais seleccionadas co-variaram com os gradientes de impacto estudados, muito embora apenas algumas variáveis estruturais tenham individualmente discriminado as categorias de uso de solo definidas a priori. Não foi detectada consistência nas respostas das medidas estruturais entre regiões biogeográficas, tendo-se confirmadado que as interpretações puramente taxonómicas de impactos são difíceis de extrapolar entre regiões. Os gradientes biológicos definidos através dos atributos seleccionados também co-variaram com os gradientes de perturbação, tendo sido possível obter uma melhor discriminação de categorias de uso de solo. Nas diferentes regiões, a discriminação de locais mais impactados foi feita com base num conjunto similar de atributos, que inclui tamanho, voltinismo, técnicas reproductivas, microhabitat, preferências de corrente e substrato, hábitos alimentares e formas de resistência. Este conjunto poderá vir a ser usado para avaliar de forma predictiva os efeitos das modificações severas de uso de solo impostas pela actividade agrícola. Quando analisadas simultaneamente através dos atributos, as comunidades das três regiões permitiram uma moderada mas significativa discriminação de níveis de impacto. Estas análises corroboram as evidências de que as mudanças nas comunidades de macroinvertebrados aquáticos em locais sob a influência de agricultura intensiva podem seguir uma trajectória convergente no espaço multidimensional, independentemente de factores geográficos. Foram fornecidas pistas para a identificação de parâmetros específicos que deverão ser tidos em conta no planeamento de novos programas de biomonitorização com comunidades de macroinvertebrados bentónicos, para aplicação numa gestão fluvial verdadeiramente ecológica, nestas e noutras regiões. Foram ainda sugeridas possíveis linhas futuras de investigação.
Freshwater ecosystems - engineers of environmental functions and important providers of irreplaceable goods and services - are being severely affected by anthropogenic perturbations. The conversion of forested land to agriculture is affecting these aquatic systems through a series of mechanisms: sedimentation; nutrient enrichment; contaminant input; hydrologic alteration; and riparian clearing. Freshwater macroinvertebrate communities, being diverse, widespread, and sensitive to environmental perturbation, are particularly interesting for the ecological integrity assessment of those aquatic systems affected by such a multitude of stressors. Biomonitoring - the systematic use of biological responses to evaluate environmental changes - can be undertaken through diverse methodologies. But these do not usually consider the functional intricacies of biological communities and are geographically constrained. Biomonitoring through sets of biological traits (characteristics that reflect species adaptation to their environment) appears as a promising tool to overcome these issues, offering a series of other advantages: direct causal relationships with stressors; better differentiation of impacts; and integration of natural fluctuations. In this study, the current state-of-the-art of the usage of biological traits in biomonitoring is presented in a critical review of the existing published literature. Until publication date, no such work was available to provide freshwater ecologists with the conceptual underpinning for the use of traits as community descriptors and for freshwater biomonitoring and management. The support from ecological theory (the habitat templet concept and the landscape filtering hypothesis) was reviewed and studies applying this knowledge under real scenarios were presented. Technical issues were addressed and solutions proposed. Specific future needs are: a broader unified trait biomonitoring tool; more accurate understanding of the natural variation of community patterns; approaches to deal with trait trade-offs and syndromes; additional life history and ecological requirement studies; and the detection of specific impacts under multiple stressor scenarios. The aim was to address the improvement of biomonitoring through traits, focusing on freshwater macroinvertebrate communities from streams of different biogeographic regions (in the Little and Salmon River, New Brunswick, Canada; Anllóns River, Galicia, Spain; Upper Reventazón River, Cartago, Costa Rica) spanning comparable gradients from low (watersheds with percentages of forest cover >75%) to high agricultural land use intensity. In each land use gradient, the characterization of the biological community (by macroinvertebrate kick sampling in riffle areas) was accompanied by the characterization of the surrounding habitat (watershed scale properties, water chemistry and other reach scale properties). The macroinvertebrate community was characterized through taxonomic information, structural metrics, diversity indices, tolerance metrics, biotic indices and through the attribution of traits reflecting general biological and physiological features, life history and resistance to disturbance. Univariate and multivariate statistical data analyses were used to highlight biological and physico-chemical gradients, confirm their co-variation, test the significance of impact level discrimination and establish interregional comparisons. Community structure was used to reveal complex impact gradients, that significantly covaried with watershed agricultural land use gradients. These 14 impact gradients were mostly related with nutrient input and sedimentation. The biological gradients defined by selected structural measures co-varied with the disturbance gradients, although few structural variables individually discriminated the a priori defined land use categories. No consistency in the responses of the structural measures across biogeographic regions was detected. It was therefore confirmed that pure taxonomic interpretations of potential impacts are difficult to extrapolate between regions. The biological gradients defined by the selected traits also co-varied with the disturbance gradients and an improved discrimination of land use categories was obtained. Across regions, a similar set of traits discriminated higher impact sites, including size, voltinism, reproductive techniques, microhabitat, current and substrate preferences, feeding habits and resistance forms. This set could be further studied and used to predictably assess the effects of severe land uses changes posed by agricultural scenarios. When analyzed simultaneously using traits, the communities of the three regions allowed a moderate but significant discrimination of impact levels. These analyses support the evidence that freshwater macroinvertebrate community shifts in sites impacted by intensive agriculture may follow convergent trajectories in multi-dimensional space, regardless of geography. Overall, clues were given to identify specific features that should be considered in the design of future freshwater biomonitoring programs using benthic macroinvertebrate communities for application in true ecologically oriented river management in these and other regions. Future research needs were also suggested.
FCT/FSE - SFRH/BD/18514/2004

Els bisfenols (BPs) són un grup de compostos orgànics que actuen com disruptors endocrins, causant efectes adversos per la salut com, malalties metabòliques, disrupció del sistema reproductor o càncer. Aquests compostos s’utilitzen en productes quotidians, especialment en menjar; per això, la seva presència és ubiqua al medi ambient i l’exposició humana és extensa. Degut a la regulació vers el bisfenol A (BPA) – l’anàleg més usat – la industria ha començat a substituir-lo per altres anàlegs. L’objectiu d’aquesta tesi doctoral és avaluar l’exposició dietètica a sis anàlegs de bisfenol a través d’un estudi de dieta duplicada. Es va dividir la cohort en dos grups, cadascun dels quals va seguir una dieta diferenciada: una consistia en menjar enllaunat i l’altra en menjar fresc. El BPA va ser l’anàleg més detectat en les mostres de menjar i d’orina. Els resultats mostren que seguir una dieta a base de menjar enllaunat està significativament relacionat amb una major exposició al BPA. No obstant, els participants que van seguir una dieta consistent en productes frescos també van presentar nivells de BPA en orina. Això és degut a que el menjar no enllaunat també contenia BPA, tot i que en una concentració menor que la trobada en el menjar enllaunat. A més, també es va avaluar l’exposició al BPA, i es va trobar que cap dels dos grups estaven exposat per sobre de la ingesta diària tolerable (TDI) de 4 µg/kg pc/dia. Per tant, la salut humana no es troba en risc tot i consumir una dieta alta en BPA. Els altres anàlegs es van detectar en mostres puntuals. Per tant, seria aconsellable que les organitzacions internacionals comencessin a regular els bisfenols ja que la seva estructura y els seus efectes potencials són similars. Finalment, és important tenir present els efecte derivats de la co-exposició a diversos compostos.
Los bisfenoles (BPs) son un grupo de compuestos orgánicos que actúan como disruptores endocrinos, causando efectos adversos para la salud como, enfermedades metabólicas, disrupción del sistema reproductor o cáncer. Estos compuestos se usan en productos cotidianos, especialmente en comida; por eso, su presencia es ubicua en el ambiente i la exposición humana es extensa. Debido a la regulación del bisfenol A (BPA) – el análogo más usado – la industria ha empezado a sustituirlo por otros análogos. El objetivo de esta tesis doctoral es evaluar la exposición dietética a seis bisfenoles a través de un estudio de dieta duplicada. La cohorte se dividió en dos grupos, que siguieron dietas diferenciadas: una consistía en comida enlatada y la otra en comida fresca. El BPA fue el análogo más detectado en muestras de comida y orina. Los otros sólo se detectaron en muestras puntuales. Los resultados muestran que seguir una dieta a base de comida enlatada está significativamente relacionado con una mayor exposición al BPA. Aun así, los participantes que siguieron una dieta de comida fresca también presentaban niveles de BPA en orina. Esto se debe a que la comida no enlatada también contiene BPA, aunque en niveles más bajos que la comida enlatada. Adicionalmente, se evaluó la exposición al BPA y se encontró que ninguno de los grupos estaba expuesto por encima de la ingesta diaria tolerable (TDI) de 4 µg/kg pc/día. Por lo tanto, la salud humana no está en riesgo aun consumiendo una dieta alta en BPA. Otros análogos se encontraron en muestras puntuales. Por tanto, las organizaciones internacionales deberían empezar a regularlos, ya que la estructura y los efectos potenciales son similares. Además, éstos se están empezando a detectar en muchas matrices. Finalmente, es importante tener en cuenta los efectos derivados de una co-exposición a diversos compuestos al mismo tiempo.
Bisphenols (BPs) are a group of organic compounds that act as endocrine disruptors causing adverse health effects such as, metabolic diseases, impairment of the reproductive system or cancer. They are used in everyday products, especially food products; hence, they presence is ubiquitous is the environment and the human exposure is extensive. Due to the strong regulation against bisphenol A (BPA) – the most used analogue – manufacture companies have started to replace it with other analogues. The aim of this doctoral thesis was to evaluate the dietary exposure to six bisphenol analogues performing a duplicate diet study. Cohort was divided in two groups and each of them followed a differentiated diet: one consisting of canned food and the other consisting of fresh food. BPA was the most detected analogue in food and urine samples. Results showed that following a canned food diet is significantly correlated with the exposure to BPA. Nonetheless, participants who followed a non-canned food diet also had detectable levels of urinary BPA. This is because non-canned food also presented levels of BPA, although they were lower than those found in canned food. In turn, exposure to BPA was also assessed, showing that none of the groups were exposed above the tolerable daily intake (TDI) of 4 µg/kg bw/day. Thus, human health is not at risk even when consuming a potential high-BPA diet. Besides, Other analogues were found only in punctual samples. Therefore, international organizations should start to regulate BPs compounds due to their similar structure and potential effects. Finally, it is important to keep in mind the health effects derived from the co-exposure to several compounds at the same time.

This study compared the use of biomarkers to assess sub-lethal effects of pollutants alongside a standard toxicity test and sediment analysis within a harbour environment predominantly affected by TBT and PAHs, in Aberdeen, NE Scotland. Analysis of sediment sampled twice yearly showed few clear trends for metals, TBT or PAHs over a 8 year period from 1997-2005. At one sample site, (29) PAHs were seen to increase during the study period. This could potentially be linked to the Denburn storm overflow, which can release untreated waste water during periods of prolonged heavy rain. Levels of contamination were highest at sample sites located within the inner harbour, sites where sediment would accumulate and tidal flushing would be less effective at redistributing contaminants. The Corophium bioassay was used to assess temporal and spatial variation in the toxicity of harbour sediment. Significant differences were seen between sample sites located in the inner harbour areas and control sediment. In most cases, mortality did not correlate to any one individual contaminant, however when mortality was relatively low, correlations were seen with heavy metals such as Pb, Cd and Cr. A seasonal fluctuation in toxicity was evident at sample site 29, which could relate to seasonal fluctuations in water quality due to the Denburn overflow, since metals such as zinc also followed a seasonal fluctuation. By 2005, no sample sites showed a significant increase in mortality when compared to controls. At 7 sample sites, a significant improvement in Corophium survival was seen during the study period, which strongly suggests that bioavailable levels of contaminants are decreasing. Intersex in L. littorea was assessed on an annual basis to monitor the effects of TBT within the harbour. Again, a significant difference was found between the inner harbour and a control site, whilst intersex levels in the outer harbour area were comparable to the control site. At the inner harbour site, levels of intersex were sufficient to reduce reproductive capability individual periwinkles, but would not affect reproduction rates at the population level. Intersex ISI values suggest that water within Aberdeen harbour does not yet meet the UK EOS of 2 ngl^-1. Again, a decrease in the degree of intersex was seen during the study period, this was not statistically significant, yet may indicate that the level of bioavailability pollutants is decreasing.

Philosophiae Doctor - PhD
The risk of increasing global pollution dictates the need to understand environmental processes and develop innovative ways to monitor pollution levels and address associated problems. In order to address this need, this study used a selection of plants leaves (Commelina benghalesis, Paspalum urvillei, Persicaria lapathifolia and Salix babylonica) as biomonitors to assess the state of the environment, more specifically the concentration of certain heavy metal pollutants (Cu, Zn, Fe, Ni, Pb and Cd) of river water and soils in the Eerste River catchment, Western Cape, South Africa.

A pollution survey was done in Milnerton industrial area along three transects, the coastal road, the R27 road, and the N7 road. In this project lichens were used as pollution monitors and their availability, abundance and percentage cover were used to estimate the level of pollution. Lichen specimens were collected from St. James, which is far from the pollution source and put in the vicinity of the Caltex oil refinery for four month after which they were analysed for their fluorescence and chlorophyll content. In all transects, lichen species richness and percentage cover increased with distance from the oil refinery, suggesting that indeed lichens are sensitive to pollution and are therefore good air pollution bio-monitors. The fruticose growth forms especially Teloschistes and Usnea were shown in this study to be the most sensitive to pollution because none of them was recorded close to the oil refinery. Photosynthetic pigment analyses revealed that Parmelia and Xanthoria are affected differently by pollution. Parmelia showed chlorophyll a and b injury under high pollution conditions while Xanthoria showed high carotenoid injury. The results of this study indicate that the atmosphere in the Milnerton industrial area is heavily polluted, with the Caltex oil refinery being the main pollution source.

The thesis presents the development of immunochemical methods for detection of trichlorophenols (TCP) in environmental and biological samples. An indirect enzyme linked immunosorbent assay (ELISA) for 2,4,5-TCP has been developed after a rational design of the immunizing hapten chemical structure, and the screening of 12 competitor haptens. The effect of the conjugation degree of the competitors and their homology with the target analyte, the physicochemical parameters (pH, ionic strength), the concentration of detergent, the time of incubation and the specificity were studied.

Two immunoassays (2,4,5- and 2,4,6-TCP ELISAs) were evaluated for the analysis of water, milk, serum and urine. Drinking water was analyzed directly after buffering the sample. The strong matrix effects in milk samples requires the sample clean up. Human serum can be analyzed after protein precipitation with absolute ethanol. The strong matrix effect of urine and its variability for samples from different individuals suggested the introduction of a purification step prior to ELISA. The C18- solid phase extraction (SPE) is an effective clean up method to remove an important part of the nonspecific interferences present in urine. The C18-SPE-ELISA method allows accurate quantification of TCPs in urine of occupationally exposed persons. SPE based on immunosorbents (immunoaffinity extraction, IAE) have been developed in single and 96-column formats. IAE is an effective clean up method to remove all nonspecific urinary interferences. The IAE step was optimized regarding sample volume, loading level, type of urine hydrolysis washing and elution conditions. The selectivity of the immunosorbents can be modulated by the washing conditions. The immunosorbents have sufficient capacity to effectively extract 2,4,6-TCP from urine samples of occupationally exposed persons and the general population. The HTS-IAE-ELISA method allows the processing of 100 samples/day with very good precision and accuracy. The method was validated with GC-MS and applied to the biomonitoring of three groups of population from Catalonia.

A quenching fluorescence immunoassay based on the laser-induced fluorescence detection in microdroplets (LIF-microdroplet-QFIA) for 2,4,6-TCP has been developed as a novel biodetection system. This approach offers significant improvement in method detectability compared to the microplate immunoassays and is the first application urine samples that can be directly analyzed after sample dilution.

The explosive growth of the biosensors and health-related wearable monitoring devices has accentuated the need for miniaturized, high-efficiency conformal bio-modules that can operate over a wide range of frequencies, while they can be integrated in wearable and lightweight configurations. One of the major issue for the implementation of Wireless Body Area Networks (WBAN) is the very limited range of commonly used metal antennas. Due to the high dielectric constant between the metal antenna material (as well as the metal-based circuitry) and the mostly "ionized-water" human body parts, the near-field gets significantly disturbed, while local reflections due to the dielectric mismatch further shorten the operation range. Even wearable bracelet-like sensing devices have a very low range due to this reason. Thus, there are two major aspects that are going to be addressed in this Thesis: enhanced-range wearable antennas for wireless biosensors and compact "rugged-polarization" wireless sensor readers.

DNA immobilization was demonstrated using the emulsification/internal gelation technique for calcium alginate bead production. The microspheres formed were recoverable magnetically, resistant to acidic environments, and strong enough to withstand gastrointestinal transit. Purification of DNA from alginate was accomplished sing a Sepharose CL-2B gel column, resulting in decreased alginate interference when quantifying DNA by absorbance. DNA microspheres were studied in vitro for the ability to trap ($ sp{14}$C) methyl iodide and in vivo with ($ sp{14}$C) benzo(a)pyrene. Assay of recovered DNA microspheres involved DNA purification by gel chromatography and liquid scintillation for the detection of bound radiolabel. Methylation resulted in a 2 fold increase in trapping of label within DNA microspheres compared to that of its control containing no DNA. The in vivo study showed a 5 fold increase in trapping with benzo(a)pyrene metabolites. Approximately 50% of the microspheres introduced by gavage to rats were recoverable magnetically, with a diameter reduction of 57.8% due to dewatering in the lower GI tract.

The application of molecular techniques for the identification of cellular damage in response to environmental exposure is presented in this thesis. Using the ³²P-postlabelling assay (³²PPL) buccal mucosa has been shown to represent an easily obtainable, alternative tissue for human biomonitoring. Relative adduct levels (RAL) in oral biopsies and buccal mucosa, taken from smokers and non-smokers, were analysed. Mean RAL for non-smokers were not statistically different in the two tissues: 2.1 X 10^-7 in buccal mucosa and 1.66 X 10^-7 in oral biopsies (p=0.72). Likewise no statistical difference between the two tissues was observed for smokers: 6.73 X 10^-7 and 6.16 X 10^-7 (p=0.75) respectively. Statistically higher levels of damage were seen in smokers compared to non-smokers. The p53 mutation spectra of oral tumours, obtained from smokers and non-smokers, was assessed using PCR amplification and Sanger sequencing. Four mutations were found: two in exon 7 at codon 247 (AAC to AC); one in exon 8 at codon 294 (GAG to GAAG) and one in codon 299 (CTG CCC to CTG AA CCC). No relationship was found between cigarette smoking and p53 mutation. A small study was carried out to assess persons living in an industrial area of South Wales. Using the butanol and nuclease P₁ enhanced ³²PPL methods to measure DNA adducts in WBC revealed the mean RAL to be statistically higher in the exposed group than the control group (p=<0.05). Analysis of buccal mucosa samples by butanol ³²PPL revealed no statistical difference between RAL in the exposed and control groups (p=>0.05). The p53 gene in exposed persons was analysed for mutations. WBC were analysed using the Restriction Site Mutation assay. No mutations were found.

With numerous bioassessment methodologies available, a regional protocol needs to be developed to ensure that results are comparable. A regional assessment protocol was developed that includes collecting five benthic macroinvertebrate samples, identifying organisms to genus, and calculating the following metrics: Number of Taxa, Total Number of Individuals, Simpson's Diversity Index, Shannon's Diversity Index, Percent Contribution of Dominant Taxa, Hilsenhoffs Biotic Index, and Percent Contribution of Dipterans. Once the protocol was developed, it was used to assess the Bayou Chico tributaries and watershed. All three tributaries had been significantly impacted by human activity as had the watershed as a whole. This study indicates that a regional protocol could be developed and is appropriate for biomonitoring at the watershed scale.

Freshwater ecosystems are currently amongst the most threatened habitats due to high levels of anthropogenic stress and increasing efforts are required to monitor their status and assess aquatic biodiversity. Biomonitoring, which is the systematic measurement of the responses of aquatic biota to environmental stressors, is used to evaluate ecosystem status. Macroinvertebrates are commonly used organisms for ecosystem assessment, due to their numerous biomonitoring qualities, which qualify them as ecological indicators. Traditional taxonomy-based monitoring is labour intensive, which limits the throughput, and is often inefficient in providing species level identification, which limits the accuracy of detections. The introduction of molecular based methods for biomonitoring, especially when coupled with High Throughput Sequencing (HTS) applications, offers a step change in ecosystem monitoring. Here I tested the utility of DNA based applications for increasing the efficiency of freshwater ecosystem biomonitoring, using benthic macroinvertebrates as a target group. For the first part of this work, I used DNA barcoding of the Cytochrome Oxidase Subunit I (COI), from individual specimens, to populate a barcode reference library for 94 species of Trichoptera, Gastropoda and Chironomidae from the UK. Then, I used High Throughput Sequencing (HTS) methods to characterise diversity from complex environmental samples. First, I used metabarcoding of aqueous environmental DNA (eDNA) and community invertebrate samples (Chironomidae pupal exuviae), collected on regular intervals throughout a year, to identify diversity levels and temporal patterns of community variation on ecosystem-wide and group specific scales. Finally, I used a structured design of mock macroinvertebrate communities, of known biomass content, to perform a comparison between PCR-based metabarcoding of the COI gene and PCR-free shotgun sequencing of mitochondrial genomes (mito-metagenomics), and evaluate their efficiency for accurate characterisation of biomass content of bulk samples. Overall, HTS has demonstrated great potential for advancing biomonitoring efforts, allowing ecosystem scale diversity detection from non-invasive types of samples, such as eDNA, whilst moving into mito-metagenomic work could improve the field even further by improving quantitative abundance results on the community composition level.

This PhD project has been focused on the improvement of the methods commonly used in biomonitoring surveys of persistent airborne pollutants based on native lichens. These protocols recommend to measure the content of pollutants in the outermost parts of foliose epiphytic thalli, which purportedly developed in the last year before sampling. This allows to associate the pollutant content to a known exposure period. Unfortunately, lichens are very sensitive to local environmental conditions, although the lack of data concerning most of the species used in biomonitoring surveys caused an underestimation of the problem. Therefore, in a first study, I investigated the influence of site-specific climatic factors on the inter-site variability of seasonal and long-term Radial Growth Rates (RaGR) of Xanthoria parietina, one of the most popular lichen biomonitor. For 17 months RaGR was monitored in 11 sites along an altitudinal transect by measuring the lobe length of 54 thalli, whereas site-specific climatic variables were monitored with thermo-hygrometric sensors. Results highlighted that site-specific climatic differences in terms of air water availability and wind frequency were strongly correlated to the observed inter-site variability of both RaGRs, with thalli of dry sites that had significantly lower seasonal RaGR with respect to those of moist ones. For this reason, it was concluded that biomonitoring surveys with native lichens should be limited to climatically homogeneous areas in order to ensure comparable exposure periods. In a second study, the correct reference exposure period revealed itself as an important yardstick if we want to correctly use lichen data in the validation of atmospheric dispersion models (ADMs). In this study, I used the element concentrations measured in lichen samples from the surroundings of a coal-fired power plant as the core dataset for the validation of two alternative ADMs. These ADMs simulated the dispersion of Total Suspended Particulate emitted by the plant during the year 2005, selected by the authorities as reference meteorological period (ADM1), and, the six months preceding the lichen sample collection (ADM2), because this was the age of the collected lichen samples, as estimated using the above-mentioned RaGR. After normalization for the element soil content, results showed that the Cr content of lichen samples were more correlated with the outcomes of ADM2 rather than with those of ADM1. These results were confirmed by a PM10 survey, carried out by ARPA FVG, which revealed that in two successive periods of activity and inactivity of the putative source, concentration of Cr was significantly higher in the former. Finally, a third study was aimed at investigating the influence of lichen melanins in trace element retention. Most of the species used as biomonitor of airborne trace elements belong to the Parmeliaceae, a highly differentiated family characterized by a highly melanised lower cortex, whose adaptive value is unknown. Here, I tested the hypothesis that melanins can affect the content of selected elements. Macro- (Ca, K and S) and micro- (Fe, Mn and Zn) nutrients in melanized and non-melanized pseudotissues of nine species was first evaluated by micro-XRF analysis on either the upper and lower cortex, and on the artificially exposed medulla. Afterwards, the total concentration of the same elements was measured by ICP-AES, and a sequential elution experiment was performed on one heavily melanised and one lightly melanised species. In this way, I could demonstrate that native lichens with heavily melanised surfaces are more Fe- and Zn- enriched than lightly or non-melanised lichens, possibly increasing the bioavailability of both elements in favour of the photobionts. Therefore, in order to exclude any bias related to the different degree of melanisation, the proposal is to compose analytical samples with lichen material chara
This PhD project has been focused on the improvement of the methods commonly used in biomonitoring surveys of persistent airborne pollutants based on native lichens. These protocols recommend to measure the content of pollutants in the outermost parts of foliose epiphytic thalli, which purportedly developed in the last year before sampling. This allows to associate the pollutant content to a known exposure period. Unfortunately, lichens are very sensitive to local environmental conditions, although the lack of data concerning most of the species used in biomonitoring surveys caused an underestimation of the problem. Therefore, in a first study, I investigated the influence of site-specific climatic factors on the inter-site variability of seasonal and long-term Radial Growth Rates (RaGR) of Xanthoria parietina, one of the most popular lichen biomonitor. For 17 months RaGR was monitored in 11 sites along an altitudinal transect by measuring the lobe length of 54 thalli, whereas site-specific climatic variables were monitored with thermo-hygrometric sensors. Results highlighted that site-specific climatic differences in terms of air water availability and wind frequency were strongly correlated to the observed inter-site variability of both RaGRs, with thalli of dry sites that had significantly lower seasonal RaGR with respect to those of moist ones. For this reason, it was concluded that biomonitoring surveys with native lichens should be limited to climatically homogeneous areas in order to ensure comparable exposure periods. In a second study, the correct reference exposure period revealed itself as an important yardstick if we want to correctly use lichen data in the validation of atmospheric dispersion models (ADMs). In this study, I used the element concentrations measured in lichen samples from the surroundings of a coal-fired power plant as the core dataset for the validation of two alternative ADMs. These ADMs simulated the dispersion of Total Suspended Particulate emitted by the plant during the year 2005, selected by the authorities as reference meteorological period (ADM1), and, the six months preceding the lichen sample collection (ADM2), because this was the age of the collected lichen samples, as estimated using the above-mentioned RaGR. After normalization for the element soil content, results showed that the Cr content of lichen samples were more correlated with the outcomes of ADM2 rather than with those of ADM1. These results were confirmed by a PM10 survey, carried out by ARPA FVG, which revealed that in two successive periods of activity and inactivity of the putative source, concentration of Cr was significantly higher in the former. Finally, a third study was aimed at investigating the influence of lichen melanins in trace element retention. Most of the species used as biomonitor of airborne trace elements belong to the Parmeliaceae, a highly differentiated family characterized by a highly melanised lower cortex, whose adaptive value is unknown. Here, I tested the hypothesis that melanins can affect the content of selected elements. Macro- (Ca, K and S) and micro- (Fe, Mn and Zn) nutrients in melanized and non-melanized pseudotissues of nine species was first evaluated by micro-XRF analysis on either the upper and lower cortex, and on the artificially exposed medulla. Afterwards, the total concentration of the same elements was measured by ICP-AES, and a sequential elution experiment was performed on one heavily melanised and one lightly melanised species. In this way, I could demonstrate that native lichens with heavily melanised surfaces are more Fe- and Zn- enriched than lightly or non-melanised lichens, possibly increasing the bioavailability of both elements in favour of the photobionts. Therefore, in order to exclude any bias related to the different degree of melanisation, the proposal is to compose analytical samples with lichen material characterized by a similar degree of melanisation.

The use of manure from animal feeding operations (AFOs) as fertilizer on agricultural land may introduce contaminants to aquatic environments that can negatively affect the health of aquatic organisms. This study utilized a landscape-scale regression-based design to assess the effects of AFOs on contaminant concentrations and resident populations of a pleurocerid snail, Leptoxis carinata, in streams within the Shenandoah River watershed (Virginia, USA). Individual characteristics of L. carinata were also evaluated to provide further understanding of observed population characteristics. In streambed sediment and mollusk tissue, concentrations of the trace element arsenic, used as an additive in poultry feed, were not directly related to watershed densities of AFOs. In-stream concentrations of dissolved nutrients and estrogenic compounds, measured as estrogenic activity, were directly related to watershed densities of AFOs. Population sex ratios of L. carinata varied across study sites, from balanced to female-biased, but were not related to concentrations of estrogenic compounds. However, the spatial variation in population sex ratios, coupled with little variation in site-specific sex ratios across seasons and generations, suggest an influence of site-specific environmental conditions. Individual-level studies of L. carinata revealed that there is an eight month lag between hatching and gametogenesis which could allow disruption of sexual differentiation by environmental contaminants, but further study of the effects of specific contaminants on sexual differentiation in this species is needed. Population densities of L. carinata were related to in-stream nutrient concentrations and landscape sources of nutrients, including AFOs, but none of these factors were directly related to the infection prevalence of digenetic trematodes in L. carinata populations. Although trematode infection rates in L. carinata populations do not appear to be viable indicators of the influence of eutrophic conditions on disease incidence in aquatic organisms, the identification of five types of trematodes in L. carinata populations highlights the utility of this snail species for further investigation of transmission dynamics of trematode parasites in lotic systems.
Ph. D.

Indoor microenvironments can have a relevant contribution towards the total exposure to pollutants. The biomarkers of exposure and effect were analysed in general population, to evaluate the impact of VOCs, PAHs and tobacco smoke exposure in everyday life. We designed a model for toxicological studies, as proof of concept for future studies. We analysed airborne personal exposure to air pollutants, the urinary and oxidative stress biomarkers of three exposure level groups in a non-smoking population from Birmingham, UK, a subset with high exposures to tobacco smoke, and a subset of schoolchildren in Saudi Arabia, living in high pollution areas. A significant correlation was observed between low concentration urinary cotinine and urinary VOCs. Urinary VOCs were found suitable for exposure assessment of general population, although the correlations with oxidative stress biomarkers were weak. No significant correlation between urinary PAHs and oxidative stress was observed in schoolchildren from Saudi Arabia, although certain areas showed a significant increase in urinary PAHs and oxidative stress biomarker. The cell exposure system was designed and tested so it could be applied in future toxicological studies. The observed ROS generation and DNA damage in the cells after exposure, proved the validity of the model for benzene exposure.

Research was conducted in three areas of water quality assessment. Long term ecological monitoring data from Shenandoah National Park (SNP) were analyzed and a protocol for data analysis was presented. Streams in SNP were found to be comparable to the best that can be found in the Blue Ridge ecoregion. Land use in SNP (mostly for recreational purposes) does not appear to be causing impairment to the macroinvertebrate assemblages. Streams in the SNP were found to recover quickly from disturbance. The Macroinvertebrate Aggregated Index for Streams (MAIS) was found to have an overall classification efficiency (CE) of 86% in the Ridge and Valley ecoregion, and an overall CE of 91% in the Central Appalachians ecoregion. Refinement of the MAIS for use in the Blue Ridge ecoregion resulted in an increase of the overall CE to 78%. The CE for reference sites in the Blue Ridge was 75%, and the CE for degraded sites was 87%. An intensive study of a stream (Peak Creek) with suspected heavy metal impairment showed that capping of an industrial waste site has resulted in improvements to the macroinvertebrate assemblages. The source of the impairment was not linked solely to heavy metals, but was found to be a mixture of pollution sources and environmental stress.
Ph. D.

A process for disposing of pesticide rinsewater generated from the rinsing of application equipment is being developed at Virginia Polytechnic Institute and State University. This process involves the sorption of pesticides onto an organic matrix followed by degradation in a composting environment. We are now evaluating the hazards that might be associated with land-applying composted pesticide waste. Diazinon was the first pesticide selected for evaluation, which consisted of two studies. The first used the earthworm species Eisenia foetida to evaluate the toxicity of soil amended with composted diazinon waste. The second study determined the bioavailability of delta-2-14C-diazinon and its degradation products to E. foetida in soil amended with composted delta-2-14C-diazinon. Results from the first study indicate that uncomposted diazinon sorbent and 30-day composted diazinon sorbent were toxic to E. foetida at sublethal and lethal levels. However, E. foetida exposed 60-day composted diazinon sorbent did not experience mortality or demostrate sublethal effects commonly associated with acetylcholinesterase inhibition. Earthworms exposed to diazinon that was uncomposted or composted for 30 days in the radiolabelled study experienced higher mortality than in the field study. After 30 and 60 days of composting 14C-diazinon became unextractably incorporated into organic matter and very little was mineralized. Earthworms were shown to accumulate radioactivity when exposed to soil amended with 60- day composted delta-2-14C-diazinon. The majority of this radioactivity was unextractably bound to earthworm tissue and that which was extractable contained only trace levels of delta-2-14C-diazinon. Based on the absence of toxicity in the field study and the low levels of 14C-diazinon present in earthworm tissues, 60 days of composting appears to greatly reduce the hazard that diazinon rinsate poses to E. foetida.
Master of Science

A biological monitoring tool to assess water quality using bivalve gape behavior was developed and demonstrated. The purpose of this work was to develop methodologies for screening water quality appropriate to the goals of the watershed paradigm. A model of bivalve gape behavior based on prediction of behavior using autoregressive techniques was the foundation of the bivalve biomonitoring system. Current technology was used in developing the system to provide bivalve gape state data in a continuous real-time manner. A laboratory version of the system, including data collection and analysis hardware and software, was developed for use as a toxicological assay for determination of effective concentrations of toxicant(s) or other types of stress on bivalve gape behavior. Corbicula fluminea was monitored and challenged with copper, zinc, and chlorpyrifos using the system. Effective concentrations of 176±23µg/L copper, 768±412µg/L zinc, and 68µg/L chlorpyrifos were observed using a natural water with high dissolved organic carbon concentrations. A rugged field version of the bivalve biomonitoring system was developed and deployed in two locations. The field systems were fitted with a photovoltaic array, a single board computer, and a CDPD telemetry modem for robust remote operation. Data were telemetered at a time relevant rate of once every ten minutes. One unit was deployed in Lake Lewisville, Denton County, TX in February 2000. Data were telemetered and archived at a 92% success rate. Bivalve gape data demonstrated significant behavioral deviations on average 5 times per month. A second unit was deployed in Pecan Creek, Denton, TX in June 2001. Data from this site were telemetered and archived at a 96% success rate. Over the months of June-August 2001, 16 significant behavioral deviations were observed, 63% of which were correlated with changes in physical/chemical parameters. This work demonstrated the relative sensitivity of bivalve gape as a toxicological endpoint and the feasibility of its use in a continuous, real-time, bivalve biomonitoring system. Technical aspects of collecting, telemetering, and analyzing this type of data in a time-relevant manner were developed resulting in a system appropriate for use as a means of data collection within the watershed paradigm.

Dans un contexte où les préoccupations environnement-santé sont croissantes à l’échelle globale. Améliorer la compréhension des mécanismes de biodisponibilité et d’(éco)toxicité des polluants métalliques persistants est un enjeu prioritaire, notamment en raison de leur omniprésence observée dans les écosystèmes en relation avec leur compartimentation et spéciation . Le ver de terre est utilisé pour évaluer la qualité des sols ; de plus cet organisme du sol de par ses activités de bioturbation a une influence sur les cycles biogéochimiques. Des tests d’écotoxicité en conditions contrôlées sur vers de terre ont donc été réalisés avec différents sols et espèces de vers. Puis une étude d’impact sur les communautés de vers a été effectuée sur un site pollué présentant un gradient de concentration. L’influence de la bioturbation du ver sur la phytodisponibilité des polluants a été étudiée grâce à des expériences en mésocosmes. Finalement, des mesures de bioaccessibilité des polluants ont été réalisées in vitro sur des sols et végétaux pollués dans différents contextes : friches industrielles, jardins potagers, terrain de sport. L’objectif étant d’étudier le lien entre biodisponibilité pour l’homme, caractéristiques des sols et contexte de pollution. Deux approches complémentaires ont été développées : recherche scientifique liés aux mécanismes et développement d’outils, de procédures pratiques utilisables par les gestionnaires et évaluateurs de risques. L’écotoxicité des métaux et métalloïdes (notés ETM) sur les vers de terre n’est pas simplement régie par leurs concentrations totales, mais dépend fortement des caractéristiques physico-chimiques des sols. Par ailleurs, l’analyse des communautés de ver de terre présentes sur un site contaminé permet d’évaluer la qualité des sols puisqu’on constate un impact sur les l’abondance, la diversité et le taux de juvénile des vers. De plus, la bioturbation du ver de terre augmente significativement la biodisponibilité des ETM pour les plantes potagères telles que la laitue. Finalement, la bioaccessibilité humaine des ETM est régi par de nombreux facteurs liés en particulier au contexte de pollution. Cette fraction bioaccessible des ETM est directement responsable de leur cytotoxicité sur les cellules intestinales. Ces différents résultats ont été complétés par des études mécanistiques (IR, EXAFS, XANES, µ-XRF)
In a context where environmental health concerns are globally increasing. Improve understanding of the mechanisms and bioavailability (eco) toxicity of persistent metals pollutants is a priority, especially because of their observed omnipresence in ecosystems in relation to their compartmentation and speciation. The earthworm is used to assess soil quality; moreover this soil organism through its bioturbation activities affects biogeochemical cycles. Ecotoxicity tests under controlled circumstances on earthworms have been conducted with different soils and worms’ species. Then an impact study on earthworms’ communities was conducted on a polluted site showing a concentration gradient. The influence of earthworms’ bioturbation on phytoavailability of pollutants was studied through experiments in mesocosms and Rhizotest. Finally, in vitro measures of pollutants bioavailability were performed on polluted soils and plants in different contexts: brownfields, gardens, sports field. The objective is to study the link between bioavailability for humans, soil characteristics, context of pollution and toxicity. Two complementary approaches have been developed: scientific research related to the mechanisms and development of tools, practical procedures which could be used by managers and risk assessors. Ecotoxicity of metals and metalloid (denoted ETM) on earthworms is not simply governed by their total concentrations, but strongly depends on the physico-chemical characteristics of soils. Furthermore, analysis of earthworm communities from a contaminated site can evaluate the quality of soil since seen an impact on the abundance, diversity and rate of juvenile worms. In addition, earthworms’ bioturbation significantly increases the bioavailability of ETM for vegetable plants such as lettuce. Finally, the human bioaccessibility of ETM is governed by many factors, in particular the context of pollution. The bioaccessible fraction of ETM is directly responsible for their cytotoxicity on intestinal cells. These results were complemented by mechanistic studies (IR, EXAFS, XANES, μ-XRF)

Thesis (MTech (Environmental Health))--Cape Peninsula University of Technology, 2008
The lower Diep River is a major freshwater ecosystem in the Western Cape. The river is surrounded by many possible sources of metal pollution such as an oil refinery, industries, a sewage treatment plant and a landfill site. However, metal contamination levels have not been monitored in this river. The aim of the study was therefore to monitor the degree of metal pollution in the lower Diep River, over a period of one year, and to investigate the use of the sedge Bolboschoenus maritimus, as biomonitor species. Three sampling sites were used. Site I was located in the vicinity of landfill sites and farm areas. Site 2 was located I km upstream from a wetland reserve, surrounded by heavy industrial activity and continuous residential developments. Site 3 was located downstream of the wetland reserve, 2 km from the river mouth. The following metals were investigated: aluminium, cadmium, chromium, cobalt, copper, iron, lead, manganese, nickel and zinc. Water and sediment samples were collected every two months for a period of one year. Plant specimens (roots, leaves and stems) were collected seasonally from site I and site 3. Samples were acid digested and metal analysis was done using an ICP - AES (Inductively Coupled Plasma- Atomic Emission Spectrophotometer). Statistical analyses were done to investigate possible differences between the sites, sampling occasions and various plant components.

This research focuses on the methodological standardization of bioaccumulation techniques by lichens. The leitmotif of this work is a biomonitor of airborne trace elements and other contaminants, the lichen Pseudevernia furfuracea (L.) Zopf, that was here repeatedly selected as test species. Firstly, the background element concentration values (BECs) for such species were assessed, since these are a prerequisite for the assessment of trace element pollution, by a large sampling effort over the Italian territory. In a first contribution, the occurrence of BEC differences of the two varieties of P. furfuracea (var. furfuracea and var. ceratea) was assessed using samples from remote areas. After having proved the absence of significant element content differences ascribable to taxonomic traits, in a second study, it was demonstrated that environmental descriptors were predictive of BECs, and these were provided for environmentally homogeneous Italian macro-regions. In this phase, the analytical outcomes of two acid mixtures for sample mineralization (i.e., a total digestion with hydrofluoric acid and a partial, aqua regia-based one) were also compared. The total digestion showed a better performance for the majority of elements. Two sets of digestion-specific BECs were provided, to be used as references in biomonitoring applications, depending on the selected mineralization procedure. Other approaches can be used to interpret bioaccumulation data. Case in point are the so-called “interpretative scales”. New scales for native lichens and transplants were respectively built up by analyzing the distribution of ratios between element concentration data and species-specific background concentration references (B ratio), contextually provided for two foliose lichens (Flavoparmelia caperata and Xanthoria parietina) and that of ratios between element concentration in exposed and unexposed samples (EU ratio) of two fruticose lichens (Evernia prunastri and Pseudevernia furfuracea). The new scales overcome critical points affecting previous ones. The second part of this research focuses on aspects related to the functioning of the lichen symbiosis, still using P. furfuracea as test species. Firstly, the effects of ozone (O3) on the physiology of previously field-stressed P. furfuracea samples were assessed. Samples were exposed for 6 weeks at sites with different levels of particulate and polycyclic aromatic hydrocarbon (PAH) emissions. Afterwards, samples were transferred to fumigation chambers, where they were O3-treated for 2 weeks. Physiological markers, elemental and PAH concentrations were measured in matched replicate sets at each experimental step. The O3-tolerance of the test species was proved, and interestingly, the content of some PAHs in samples with the highest enrichment levels significantly lowered after the ozonation, suggesting a role of O3 in degrading accumulated PAHs, with potential interpretative repercussions in transplant-based surveys aimed at evaluating PAH depositions in case of high O3 ground levels. A second contribution addresses a frequently investigated aspect for mosses, but not for lichens. Indeed, devitalization procedures are standardly carried out on moss samples, since these enhance the elemental capture by passive uptake processes. In this study, the accumulation performance of living and dead P. furfuracea samples is assessed through a high sample-sized transplant. Paired living-dead samples were exposed for 8 weeks at 40 sites in a study area of NE Italy. The two sample sets, consistently described low deposition levels over the study area, however, higher accumulation signals were revealed in dead lichens. When sites were classified according to the new bioaccumulation scale, some interpretational discrepancies arose, therefore the possibility of sample devitalization should be seriously considered, also to contribute to the methodological harmonization of biomonitoring techniques.

The aims of the work reported in this thesis were threefold; firstly, to investigate the effect of experimental exposure to polycyclic aromatic hydrocarbons (PAHs) on the immune response of dab (Limanda limanda), a sentinel species of flat fish for pollution studies, and rainbow trout (Oncorhynhus mykiss); secondly, to then go into the field to investigate the effects of exposure to PAHs on dab immunity through a major oil spill and thirdly, to increase current knowledge on the cellular immune functions of dab so as to provide better assays for biomonitoring purposes. Chapter 1 of this thesis is a general introduction to immunotoxicology, subspeciality of toxicology, which described brief accounts for both immunology and toxicology. Some brief accounts for the use of fish (lower vertebrates) in a pollution monitoring programme and the toxicology aspects of polycyclic aromatic hydrocarbons, were also given. In chapter 2, a laboratory exposure of animals to oil-contaminated sediment was established where four doses of diesel oil-based drilling mud used, i.e. 4, 8 (seen 500m from oil exploration platforms in the North Sea), 12 and 16% (w/w), with two exposure times employed, i.e. 2 and 4 weeks. Haematocrits and lymphocyte numbers tend to increase with low diesel oil doses, whereas high doses (particularly with longer exposure) gave significantly decreased values. Serum lysozyme levels were decreased, but serum bactericidal and anti-protease activities tended to increase following exposure. Kidney phagocyte respiratory burst and neutrophil migration activity also showed a trend to lower levels relative to control fish, whereas the number of antibody-secreting cells were increased with high drilling mud concentration. Lastly, there was a dose-dependent hepatomegaly. In chapter 3, rainbow trout were injected intra-peritoneally with an extract obtained from diesel oil-based drilling mud. Fish were exposed to different doses of extracts (made up to 2.4 mL/kg with olive oil), i.e. 0, 0.6, 1.2 and 2.4 mL/kg body weight (B.W.), for 6 weeks in the dose effect experiment.

Le but de ce travail de thèse est de démontrer l’intérêt d’un macrophyte aquatique submergé, Myriophyllum alterniflorum pour la détection de contaminants dans l’environnement. Des études in situ ont été réalisées pendant 28 jours sur cinq milieux aquatiques, affectés par différents types de pression anthropique, durant trois campagnes de terrain (printemps 2015, automne 2015 et printemps 2016), pour déterminer si l’accumulation des contaminants dans la plante et si des biomarqueurs du myriophylle peuvent être utilisés pour détecter la présence de contaminants dans les eaux. L’accumulation des contaminants au cours du temps était corrélée avec les teneurs des contaminants dans l’eau ; l’influence de la durée d’exposition a été étudiée. Au cours des trois campagnes, la réponse des biomarqueurs, teneur en MDA et activité de nitrate réductase était corrélée avec les teneurs en contaminants dans l’eau et dans la plante ; cependant l’influence de la durée d’exposition sur l’évolution de la réponse des biomarqueurs est moindre par rapport à celle de l’accumulation des contaminants. Ainsi, l’étude des biomarqueurs pourrait permettre une évaluation plus fiable de la qualité des masses d’eau que le suivi de l’accumulation dans la plante. Au cours de la campagne printemps 2015, un panel de biomarqueurs plus large a été étudié afin de définir les biomarqueurs les plus pertinents pour détecter spécifiquement les pressions liées aux activités urbaines ou industrielles. Une méthodologie d’évaluation de la qualité des eaux est proposée, en considérant un protocole d’analyse des biomarqueurs : un premier dosage de Chlb et MDA est simple et moins coûteux. Quand aucune preuve d'impact anthropique n'est montrée, aucune analyse complémentaire n'est requise. En revanche, lorsqu’une altération est mise en évidence, l’analyse d'autres biomarqueurs plus spécifiques tels que les activités G6PDH et γ-GCS sera nécessaire. La réponse de l'activité γ-GCS est spécifique du stress métallique, ainsi, la réponse de ce biomarqueur devrait amener à réaliser l’analyse des métaux/métalloïdes dans M. alterniflorum
The aim of this thesis is to demonstrate the interest of a submerged aquatic macrophyte, Myriophyllum alterniflorum for the detection of contaminants in the environment. In situ studies were conducted over 28 days in five aquatic environments, affected by different levels of anthropogenic pressure, during three field seasons (spring 2015, autumn 2015 and spring 2016), to determine whether the accumulation of contaminants in the plant and whether biomarkers can be used to detect the presence of contaminants in water. The accumulation of contaminants over time was correlated with the levels of contaminants in the water; the influence of the duration of exposure has been studied. In all three campaigns, biomarker response, MDA content, and nitrate reductase activity were correlated with contaminant levels in water and in the plant; however, the influence of the duration of exposure on the evolution of the biomarker response is lower compared to the accumulation of contaminants. Thus, the study of biomarkers could allow a more reliable assessment of the quality of water bodies than the monitoring of contaminant accumulation in the plant. During the spring 2015 campaign, a much broader biomarker panel was investigated to define the most relevant biomarkers for assessing water quality. A methodology for evaluating water quality is proposed, considering a biomarker analysis protocol: a first assay of Chlb and MDA is simple and less expensive. When no evidence of human impact is shown, no further analysis is required. On the other hand, when an alteration is highlighted, the analysis of other more specific biomarkers such as G6PDH and γ-GCS activities will be necessary. The response of γ-GCS activity is specific for metal stress, so the response of this biomarker should lead to the analysis of metals / metalloids in M. alterniflorum

Beluga teeth are evaluated as biomonitors of heavy metal accumulation in beluga soft tissues and contaminant exposure in people who consume beluga as part of a traditional diet. Selenium, which protects marine mammals from the toxic effects of mercury, was measured in beluga teeth for the first time using hydride-generation atomic fluorescence spectrometry. Tooth selenium concentrations are shown to be moderately strong predictors of liver and muscle selenium, validating the use of teeth as a selenium biomonitor. Dietary exposure to mercury from the consumption of beluga was compared between historic and modern Mackenzie Delta Inuit populations, based on measured mercury concentrations in archeological beluga teeth and modern beluga tissues. Despite higher mercury levels in modern beluga, estimated average mercury exposure from the consumption of beluga is higher for pre-industrial Inuit populations than for modern Inuit populations, due to the significantly decreased average consumption of beluga among the modern population.
Des dents de beluga sont évaluées pour prévoir l'accumulation des métaux lourds dans les tissus mous, et l'évaluation d'exposition diététique pour les personnes qui consomment le beluga en tant qu'élément d'un régime traditionnel. Le sélénium, qui protège les mammifères marins contre les effets toxiques du mercure, a été mesuré dans des dents de beluga pour la première fois. C'était trouvé que le sélénium dans les dents est un facteur prédictif modérément efficace de sélénium dans le foie et les muscles, validant l'utilisation des dents comme biomoniteurs de sélénium. L'exposition diététique au mercure venant de la consommation du beluga a été comparée entre les populations d'Inuit historiques et modernes, à l'aide des dents de beluga archéologiquement préservées et des tissus de beluga modernes. Malgré le plus haut nivelle de mercure dans les baleines modernes, l'évaluation de l'ingestion diététique du mercure venant de la consommation de baleine de beluga ont été plus hauts pour les populations historiques que pour les populations modernes, à cause d'une diminution de la consommation moyenne de beluga parmi la population moderne.

This study involved the development and evaluation of a number of biomonitoring techniques for use with members of the Ulvaceae, Enteromorpha intestinalis (L.) Link, and Viva iactuca (L.). The techniques included both previously established ones and those which required development. The two main procedures developed for these algae were the neutral red retention technique (making use of a vital stain) and the ion leakage Health Index (based upon electrolyte leakage). Following optimisation both were used to produce quantitative structure-activity relationships (QSARs) for a series of n-alcohols using log Kow as the physicochemical parameter. The resultant QSARs were statistically indistinguishable with line equations of -0.88 log Kow + 2.87 and -0.95 log Kow + 2.83 respectively, with r values of 0.98, suggesting that both reflect the same non-specific narcotic effect on membrane integrity. However the neutral red retention technique was less useful than the Health Index with low reproducibility and is therefore unsuitable for use as an environmental biomonitor. The effects of other compounds with specific modes of action were established using the ion leakage technique, the high toxicity of the antifouling compound tributyltin being reflected in the Health Index. Further development of the ion leakage technique involved inductively coupled - plasma mass spectrometry (ICP/MS) in the identification of ions lost during leakage. Synergistic toxicity was investigated exposing the algae to UV light and anthracene, simulating photo-induced toxicity. No photo-induced toxicity was observed although UV exposure did reduce the Health Index. An environmental assessment was carried out for algae from clean sites and organically and inorganically polluted sites, comparing the Health Index with other measures of health and bioaccumulation data. A long term study of a clean site showed the algal Health Index to be influenced by seasonally dependent physical parameters, but reflected reduced health in algae exposed environmentally to organic pollutants. Algae exposed to long term heavy metal pollution in the Fal Estuary had 'normal',. Health Indices, however chlorophyll fluorescence induction techniques did highlight a reduced photosynthetic efficiency in these algae. The ion leakage technique has potentially a wide application in field and laboratory based biomonitoring and direct toxicity studies. Chlorophyll fluorescence analysis was also shown to be useful although it requires further investigation and evaluation.