The environmental agencies has been monitoring water reservoirs such as the Billings dam with the purpose of protection and conservation of water resources based on periodical water and sediment analyses, but it does not use fish as bioindicators routinely. The study aimed to characterize water quality of the Billings dam based on integrated analyses of water and sediment parameters in addition to pathological assessment of tilapias (n=103), acaras (Geophagus brasiliensis, n=246), and trairas (Hoplias malabaricus, n=78). Samplings of water, sediment and fish were executed in six sites of the dam in summer and spring of 2014 and 2015. Analytical methods for water and sediment parameters were carried out according to standard methods, while the pathological evaluation of fish involved the Fulton index, gross lesions evaluations, the macroscopic index and histopathological diagnosis of gills, liver, kidney, and spleen lesions, and their histopathological indices. Water and sediment analyses characterized the water as a eutrophic environment and polluted by metals. Water parameters showed improvement in 2015, but physicochemical, biological, chronic toxicity results, and metals criteria were in part above historic average and legal limits required. The pathological evaluation revealed that the three fish species responded to environmental conditions in different ways and presented worse indices in 2015. Gills were the main organ affected in all species studied and the principal tissue response was regressive and reversible pattern. The indices of trairas were the highest and this species was considered the best for biomonitoring the Billings dam. Comprehensive approach of water, sediment and fishes parameters allowed the evaluation of water quality of the Billings dam and it may represent one of the ways to evaluate its environmental conditions as well as the quality of the fish consumed in the region. / Os reservatórios de água, como a represa Billings, são monitorados com o propósito de proteger e conservar os recursos hídricos baseando-se na análise periódica de água e do sedimento, porém não são utilizados de rotina peixes como bioindicadores. O objetivo do estudo foi caracterizar a qualidade de água da represa Billings, considerando a análise integrada dos parâmetros de água e sedimentos somados à avaliação anatomopatológica de tilápias (n=103), acarás (Geophagus brasiliensis, n=246), e traíras (Hoplias malabaricus, n=78). Amostras de água, sedimento e peixes foram coletadas em seis pontos da represa durante o verão e a primavera de 2014 e 2015. Métodos analíticos padronizados foram utilizados para avaliação da água e do sedimento, e a avaliação anatomopatológica dos peixes, incluiu o índice Fulton, lesões macroscópicas e histopatológicas de brânquias, fígado, rins e baço. As análises de água e sedimentos caracterizaram a água como ambiente eutrofizado e poluído por metais. Os parâmetros da água mostraram melhora no ano de 2015, porém os resultados físicoquímicos, biológicos, de toxicidade crônica e de metais estiveram acima da média histórica e dos limites permitidos pela legislação. A avaliação anatomopatológica revelou que as três espécies de peixes responderam às condições ambientais de formas diferentes, apresentando índices mais altos de lesão em 2015. A brânquia foi o principal órgão afetado em todas as espécies de peixes. O principal padrão de resposta tecidual foi o regressivo e reversível. Os índices das trairas foram os maiores e esta espécie foi considerada a melhor espécie para o biomonitoramento da represa Billings. A análise integrada de parâmetros de água, sedimento e dos peixes permitiu a avaliação da qualidade da água da represa Billings, e pode representar uma das formas de avaliar as condições ambientais bem como a qualidade dos peixes consumidos na região.

Orientador: Mario Henrique Gonzalez / Coorientador: Ana Rita de A. Nogueira / Banca: Márcia Cristina Bisinoti / Banca: Edilene Cristina Ferreira / Resumo: O mercado de pescados no Brasil tem se expandido muito nos últimos anos e com previsão de um crescimento ainda maior. A busca crescente por essa fonte de alimento no país leva à uma preocupação associada a qualidade do pescado, principalmente se tratando de metais tóxicos presentes nas águas e sedimentos dos rios. Os peixes, assim como outros frutos do mar, têm a capacidade de bioacumular estes metais em seu organismo, seja por contato direto ou indireto. O arsênio é um destes contaminantes e está presente naturalmente em águas e sedimentos, e também advindo de atividades antropogênicas como queima de combustíveis fósseis e agrotóxicos. A concentração de arsênio total nos principais tecidos da tilápia foram determinados e uma estimativa dos riscos associados ao consumo deste peixe na região Noroeste de São Paulo foi realizada, levando em conta as diretrizes estabelecidas pela FAO/OMS. As análises foram realizadas por espectrometria de massa com plasma indutivamente acoplado (ICP-MS) no modo de discriminação de energia cinética (KED). Materiais de referência certificados foram analisados com recuperações de 101, 110 e 80% de NIST 1640a, NIST 1566b e DORM-3, respectivamente. Na parte consumível (o tecido muscular dos peixes), a concentração média encontrada foi de 0,030 ± 0,008 μg g-1, que é inferior ao valor máximo permitido de arsênio estabelecido pelas agências reguladoras nacionais e internacionais. A concentração média encontrada nas vísceras (estômago, brânquias e fígado... / Abstract: Seafood market in Brazil has expanded greatly in recent years and is expected to grow even more. The search for this food source in the country leads to a concern associated with the quality of the fish, mainly by the treatment of toxic metals in the waters and sediments of the rivers. Fish, like other seafood, have the ability to bioaccumulate these metals in your body by direct or indirect contact. Arsenic is one of these contaminants and is naturally present in waters and sediments, as well as from anthropogenic activities such as the burning of fossil fuels and agrochemicals. The concentration of total arsenic in the main tissues of tilapia were evaluated and an estimation of the risks associated with the consumption of this fish was done, taking into account the guidelines established by FAO/WHO. The analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) in kinetic energy discrimination mode (KED). Certified reference materials were analyzed with recoveries of 101, 110, and 80% from NIST 1640a, NIST 1566b, and DORM-3 respectively. In the consumable portion (the muscle fish tissue), the average concentration found was 0,030 ± 0,008 µg g-1 which it is below the arsenic maximum level established by international regulatory agencies. The mean concentration found in the viscera (stomach, gills and liver) was in the range of 0,016 - 1,170 μg g-1, demonstrating a higher bioaccumulation capacity of arsenic. Bioaccumulation tests were performed with As(III) (most toxic arsenic species) with laboratory grown tilapia. The assay was divided into 7 days of exposure and 7 days of clearance to the contaminant. The results demonstrated that the stomach and liver play an important role in the absorption and accumulation of this semimetal present in contaminated water. The estimation of the toxicokinetic parameters (absorption, depuration and Bioconcentration ... / Mestre

A hidroquinona (HQ) é um composto fenólico encontrado em grandes quantidades no cigarro, em medicamentos e alimentos, além de ser um dos mais importantes metabólitos tóxicos do benzeno (BZ). Temos mostrado que a exposição sistêmica à HQ compromete a resposta inflamatória in vivo. Complementando estas investigações, o objetivo do presente projeto foi investigar o efeito da exposição ambiental à HQ sobre o recrutamento leucocitário para o pulmão induzido pelo lipopolisacarídeo de E.coli (LPS) e sobre os eventos celulares envolvidos neste processo, bem como sobre a formação de adutos de DNA no tecido pulmonar. Para tanto, 12,5, 25 ou 50 ppm de HQ ou veículo (solução salina 5% de etanol) foram nebulizadas em caixa de exposição (60 mL/1h; 5 dias) onde 5 camundongos Swiss machos estavam alocados. Uma hora após as últimas exposições, a inflamação pulmonar foi induzida pela inalação de LPS (0,1 mg/mL; 10 min). Os animais expostos à HQ e na vigência ou ausência de inflamação foram empregados para: 1) coleta do lavado broncoalveolar (LBA) três horas após a inalação de LPS para quantificação do número de leucócitos (câmara de Neubauer e esfregaços corados por Panótico®); 2) coleta de sangue para quantificação do número de leucócitos circulantes antes e 3 horas após a inalação de LPS (câmara de Neubauer e esfregaços corados por Panótico®); para a quantificação da expressão de moléculas de adesão em leucócitos de animais não inflamados induzida ou não pelo formil-metionil-leucil-fenilalanina (fMLP) in vitro (citometria de fluxo); para obtenção de plasma para quantificação de malonaldeído (HPLC) e de neutrófilos para quantificação de espécies reativas de oxigênio intracelular (EROs; citometria de fluxo) de animais não inflamados pelo LPS; 3) coleta de tecido pulmonar para quantificação da expressão de moléculas de adesão em células endoteliais e para quantificação da atividade da enzima mieloperoxidase (MPO) 3 horas após a inalação de LPS e para quantificação de adutos de DNA em tecido de animais não inflamados. Adicionalmente, a concentração de HQ na caixa de exposição foi quantificada por HPLC. Os resultados obtidos mostraram que a exposição à HQ não afetou os números de leucócitos circulantes, mas reduziu o número de leucócitos polimorfonucleares (PMN) e mononucleares (MN) no LBA; aumentou a atividade de MPO no tecido pulmonar; reduziu a expressão de L-selectina em PMN estimulados in vitro pelo fMLP; aumentou a expressão de β2 integrinas em PMN na vigência ou ausência (basal) de estimulação pelo fMLP; aumentou a expressão de β3 integrinas e PECAM-1 em condições basais; aumentou a formação de EROs por PMN; não alterou a expressão das moléculas de adesão endoteliais PECAM-1, VCAM-1, ICAM-1, JAM-C, P e Eselectinas e VE-Caderina; não aumentou significantemente a formação de adutos 8-oxo-7,8-dihidro- 2\'-desoxiguanosina e 1,N2-propano-2\'-deoxiguanosina no tecido pulmonar; aumentou a concentração de malonaldeído plasmático. A saturação da concentração de HQ na caixa de exposição foi 10 vezes menor que as preconizadas para a exposição ocupacional pelas agências regulamentadoras, indicando que mesmo a baixas concentrações de exposição, a HQ prejudica a resposta do hospedeiro a um agente infeccioso. O mecanismo tóxico pode estar relacionado à ativação das células na circulação, dependente da produção de espécies reativas de oxigênio, e que a toxicidade pode não ser detectada na ausência de resposta do organismo ao trauma. / Hydroquinone (HQ) is a phenolic compound found in large quantities in cigarettes, medicines and food, besides it is one of the most important toxic metabolites of benzene (BZ). We have shown that systemic exposure impairs in vivo inflammatory response. Following these investigations, this work aimed to study the effects of environmental exposure to HQ on leukocyte recruitment to the inflamed lung induced by lipolissacarideo of E. coli (LPS), the cell events involved in this process, and adducts formation on DNA of pulmonary tissue cells . For that 12.5, 25, or 50 ppm of HQ or vehicle (saline solution with 5% of ethanol) we aerosolized into an exposure box (60 mL/1h; 5 days) containing 5 male Swiss mice. One hour after the last exposures, the pulmonary inflammation was induced by LPS inhalation (0.1 mg/mL; 10 min). Animals exposed to HQ in presence or absence of inflammation were used for: 1) BAL collection three hours after LPS inhalation to quantify the leukocytes in BAL (Neubauer chamber and Panótico® stained smears); 2) blood collection to quantify the circulating leukocytes before and three hours after LPS inhalation (Neubauer chamber and Panótico® stained smears); to quantify the expression of adhesion moleculas in leukocytes from non-inflamed animals induced or not for formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro (flow citometry); to obtain blood plasma and quantify the formation of malondialdehyde (MDA; HPLC) and neutrophils to quantify the intracellular generation of reactive oxygen spices (ROS; flow citometry) from non-inflamed animals; 3) pulmonary tissue collection to quantify the expression oh adhesion molecules on endothelial cells and quantify the mieloperoxydase (MPO) activity, three hours after LPS inhalation and to quantify the DNA adducts on pulmonary tissue obtained from non-inflamed animals. Additionally, the concentration of HQ in the exposure box was quantified by HPLC. The results showed that exposure to HQ did not affect the numbers of circulating leukocytes, but reduced the number of polymorphonuclear leukocytes (PMN) and mononuclear (MN) in BAL, increased the activity of MPO in lung tissue; reduced the expression of L-selectin in PMN stimulated by fMLP in vitro, increased the expression of β2 integrins in PMN in the presence or absence (basal) stimulation by fMLP, increased the expression of β3 integrin and PECAM-1 in basal conditions; increased ROS formation by PMN, did not alter the expression of endothelial adhesion molecules PECAM-1, VCAM- 1, ICAM-1, JAM-C, P and E-selectin and VE-Cadherin; not significantly increased the formation of 8-oxo-7,8-dihydro-2\'-desoxyguanosine e 1,N2-propano-2\'-deoxyguanosine adducts in lung tissue; increased the concentration of plasma malondialdehyde. The concentration of HQ into the exposure box was 10 times lower than those recommended for occupational exposure from regulatory agencies, indicating that even at low exposure concentrations, HQ affect the host response to an infectious agent. The toxic mechanism may be related to activation of circulating cells, dependent on the generation of reactive oxygen species, and toxicity can not be detected in the absence of body response to trauma.

O crescimento desordenado das cidades tem trazido à tona problemas de saneamento e degradação dos recursos naturais, entre eles a água. O despejo de efluentes domésticos e industriais têm ocasionado a eutrofização dos mananciais, culminando com a proliferação dos fitoplânctons. Esta proliferação tem chegado ao ponto, em certos momentos, de acarretar episódios de floração de algas. Entre os organismos fitoplanctônicos que se desenvolvem no ambiente, estão as cianobactérias, com vários gêneros capazes de produzir diversas cianotoxinas, tais como as microcistinas, anatoxinas, cilindrospermopsinas (CY), saxitoxinas (PSPs), entre outras. Com o aumento da freqüência dos episódios de floração de algas, a probabilidade da ocorrência destas toxinas também aumenta. Como conseqüência disto e devido às exigências legais, os corpos dágua devem ser monitorados para garantir a qualidade da água para consumo humano. Com vistas ao monitoramento da presença das cianotoxinas CY e PSPs, foram realizadas investigações em alguns corpos dágua do Estado de São Paulo. Das investigações realizadas, a neosaxitoxina foi identificada pela primeira vez no Reservatório Billings e os congêneres, saxitoxina, goniautoxina 2, goniautoxina 3, foram identificados de forma inédita em amostras de água deste reservatório. Com relação à CY, foi desenvolvido um método analítico, parcialmente validado. Entretanto, esta cianotoxina não foi localizada nas amostras de água ou cianobactérias das águas superficiais dos corpos dágua estudados. Este estudo mostra a importância do monitoramento da qualidade das águas dos mananciais quanto à presença de cianotoxinas, especialmente daqueles corpos dágua com a finalidade do consumo humano. / Cities growth usually occur in an unorganized manner. This tendence can generate a variety of sanitary problems, including the degradation of natural resources, such as water bodies. As a consequence, domestic and industrial efluents cause eutrofication of water reservoir, increasing the natural level of phytoplancton, what may form algal bloom. Among the phytoplanktonic organisms that grow in this modified environment it is found the cyanobacteria. Some of them can produce different types of cyanotoxins such as microcystin, anatoxin, cylindrospermopsin (CY) and saxitoxin (PSPs). The probability of production of these cyanotoxins increase according to frequent occurrence of algal blooms episodes. Consequently, water bodies monitoring becomes important to assure water quality. The aim of this project was to develop a specific method to identify the presence of cyanotoxins CY and to investigate PSPs in water bodies in São Paulo State. The results confirmed the presence of neosaxitoxin (NEO), a toxin of PSPs family. It was the first time that Neo was indentified in Billings Reservoir along with other PSPs types: saxitoxin, gonyautoxin 2, gonyautoxin 3. Although the study also included CY monitoring, CY was not identified in the tested samples. The present study confirmed the importance of continuous searching and monitoring of water bodies to grant quality to water used for human consumption.

A hidroquinona (HQ) é um composto fenólico encontrado em grandes quantidades no cigarro, em medicamentos e alimentos, além de ser um dos mais importantes metabólitos tóxicos do benzeno (BZ). Temos mostrado que a exposição sistêmica à HQ compromete a resposta inflamatória in vivo. Complementando estas investigações, o objetivo do presente projeto foi investigar o efeito da exposição ambiental à HQ sobre o recrutamento leucocitário para o pulmão induzido pelo lipopolisacarídeo de E.coli (LPS) e sobre os eventos celulares envolvidos neste processo, bem como sobre a formação de adutos de DNA no tecido pulmonar. Para tanto, 12,5, 25 ou 50 ppm de HQ ou veículo (solução salina 5% de etanol) foram nebulizadas em caixa de exposição (60 mL/1h; 5 dias) onde 5 camundongos Swiss machos estavam alocados. Uma hora após as últimas exposições, a inflamação pulmonar foi induzida pela inalação de LPS (0,1 mg/mL; 10 min). Os animais expostos à HQ e na vigência ou ausência de inflamação foram empregados para: 1) coleta do lavado broncoalveolar (LBA) três horas após a inalação de LPS para quantificação do número de leucócitos (câmara de Neubauer e esfregaços corados por Panótico®); 2) coleta de sangue para quantificação do número de leucócitos circulantes antes e 3 horas após a inalação de LPS (câmara de Neubauer e esfregaços corados por Panótico®); para a quantificação da expressão de moléculas de adesão em leucócitos de animais não inflamados induzida ou não pelo formil-metionil-leucil-fenilalanina (fMLP) in vitro (citometria de fluxo); para obtenção de plasma para quantificação de malonaldeído (HPLC) e de neutrófilos para quantificação de espécies reativas de oxigênio intracelular (EROs; citometria de fluxo) de animais não inflamados pelo LPS; 3) coleta de tecido pulmonar para quantificação da expressão de moléculas de adesão em células endoteliais e para quantificação da atividade da enzima mieloperoxidase (MPO) 3 horas após a inalação de LPS e para quantificação de adutos de DNA em tecido de animais não inflamados. Adicionalmente, a concentração de HQ na caixa de exposição foi quantificada por HPLC. Os resultados obtidos mostraram que a exposição à HQ não afetou os números de leucócitos circulantes, mas reduziu o número de leucócitos polimorfonucleares (PMN) e mononucleares (MN) no LBA; aumentou a atividade de MPO no tecido pulmonar; reduziu a expressão de L-selectina em PMN estimulados in vitro pelo fMLP; aumentou a expressão de β2 integrinas em PMN na vigência ou ausência (basal) de estimulação pelo fMLP; aumentou a expressão de β3 integrinas e PECAM-1 em condições basais; aumentou a formação de EROs por PMN; não alterou a expressão das moléculas de adesão endoteliais PECAM-1, VCAM-1, ICAM-1, JAM-C, P e Eselectinas e VE-Caderina; não aumentou significantemente a formação de adutos 8-oxo-7,8-dihidro- 2\'-desoxiguanosina e 1,N2-propano-2\'-deoxiguanosina no tecido pulmonar; aumentou a concentração de malonaldeído plasmático. A saturação da concentração de HQ na caixa de exposição foi 10 vezes menor que as preconizadas para a exposição ocupacional pelas agências regulamentadoras, indicando que mesmo a baixas concentrações de exposição, a HQ prejudica a resposta do hospedeiro a um agente infeccioso. O mecanismo tóxico pode estar relacionado à ativação das células na circulação, dependente da produção de espécies reativas de oxigênio, e que a toxicidade pode não ser detectada na ausência de resposta do organismo ao trauma. / Hydroquinone (HQ) is a phenolic compound found in large quantities in cigarettes, medicines and food, besides it is one of the most important toxic metabolites of benzene (BZ). We have shown that systemic exposure impairs in vivo inflammatory response. Following these investigations, this work aimed to study the effects of environmental exposure to HQ on leukocyte recruitment to the inflamed lung induced by lipolissacarideo of E. coli (LPS), the cell events involved in this process, and adducts formation on DNA of pulmonary tissue cells . For that 12.5, 25, or 50 ppm of HQ or vehicle (saline solution with 5% of ethanol) we aerosolized into an exposure box (60 mL/1h; 5 days) containing 5 male Swiss mice. One hour after the last exposures, the pulmonary inflammation was induced by LPS inhalation (0.1 mg/mL; 10 min). Animals exposed to HQ in presence or absence of inflammation were used for: 1) BAL collection three hours after LPS inhalation to quantify the leukocytes in BAL (Neubauer chamber and Panótico® stained smears); 2) blood collection to quantify the circulating leukocytes before and three hours after LPS inhalation (Neubauer chamber and Panótico® stained smears); to quantify the expression of adhesion moleculas in leukocytes from non-inflamed animals induced or not for formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro (flow citometry); to obtain blood plasma and quantify the formation of malondialdehyde (MDA; HPLC) and neutrophils to quantify the intracellular generation of reactive oxygen spices (ROS; flow citometry) from non-inflamed animals; 3) pulmonary tissue collection to quantify the expression oh adhesion molecules on endothelial cells and quantify the mieloperoxydase (MPO) activity, three hours after LPS inhalation and to quantify the DNA adducts on pulmonary tissue obtained from non-inflamed animals. Additionally, the concentration of HQ in the exposure box was quantified by HPLC. The results showed that exposure to HQ did not affect the numbers of circulating leukocytes, but reduced the number of polymorphonuclear leukocytes (PMN) and mononuclear (MN) in BAL, increased the activity of MPO in lung tissue; reduced the expression of L-selectin in PMN stimulated by fMLP in vitro, increased the expression of β2 integrins in PMN in the presence or absence (basal) stimulation by fMLP, increased the expression of β3 integrin and PECAM-1 in basal conditions; increased ROS formation by PMN, did not alter the expression of endothelial adhesion molecules PECAM-1, VCAM- 1, ICAM-1, JAM-C, P and E-selectin and VE-Cadherin; not significantly increased the formation of 8-oxo-7,8-dihydro-2\'-desoxyguanosine e 1,N2-propano-2\'-deoxyguanosine adducts in lung tissue; increased the concentration of plasma malondialdehyde. The concentration of HQ into the exposure box was 10 times lower than those recommended for occupational exposure from regulatory agencies, indicating that even at low exposure concentrations, HQ affect the host response to an infectious agent. The toxic mechanism may be related to activation of circulating cells, dependent on the generation of reactive oxygen species, and toxicity can not be detected in the absence of body response to trauma.

<p>This thesis reports the results of biomarker measurements in three environmental monitoring projects. In the first project, which was part of the Swedish national environmental monitoring, biomarkers were measured annually in female perch (<i>Perca fluviatilis</i>) in two background areas in the Baltic Sea during 1988–2000, resulting in a unique 13-year series of measurements. The most important results were a strong decreasing temporal trend in the gonadosomatic index (GSI) and a strong increasing temporal trend in the hepatic ethoxyresorufin O-deethylase (EROD) activity in the Baltic Proper. In the second project, biomarkers and concentrations of classic pollutants were measured in female perch in the Stockholm recipient 1999–2001. This was the first time a large city was investigated as a point source of pollution, and the gradient was longer and included more stations than customary. Severe pollution conditions in central Stockholm were indicated by the poor health status of the perch: retarded growth, decreased frequency of sexually mature females, low GSI, disturbed visceral fat metabolism, increased hepatic EROD activity, decreased muscle acetylcholinesterase activity, increased frequency of hepatic DNA adducts, and a high concentration of biliary 1-pyrenol. Muscle ΣDDT and ΣPCB were measured as pollution indicators and were 10–28 respectively 12–35 times higher than the background levels in perch from the Baltic Proper. In the Stockholm archipelago two superimposed gradients were found. Whereas the response of several biomarkers consistently decreased with increasing distance from central Stockholm, the response of others first decreased from Stockholm to the middle archipelago and then increased to the open Baltic Sea. The latter biomarkers included the frequency of sexually mature females, GSI, hepatic EROD activity, and hepatic DNA adducts. In the third project, potential toxicity from munitions on the seafloor, at a dumpsite in the Stockholm archipelago, was analysed by the nanoinjection of sediment extracts into newly fertilised rainbow trout (<i>Oncorhynchus mykiss</i>) eggs, followed by the measurement of biomarkers in the developing larvae. No biological effects of the dumped munitions were found. The same stations in the Stockholm archipelago as in the second project were investigated as a positive control. The results of the three projects agreed well, which demonstrated the continuous pollution of the Baltic Sea and the severe pollution conditions and adverse biological effects in central Stockholm. Further investigations are urgently needed to identify which pollutants or other factors are causing the observed biological effects, both in the background areas in the Baltic Sea and in the Stockholm recipient.</p>

<p></p><p>The antifouling paint Fabi 3959 is painted on the hulls of vessels to avoid fouling caused by marine organisms attached to surfaces. The paint is registered for use on pleasure boats and other vessels weighing over 200 kg which are mainly running on the Swedish west coast (www.kemi.se).</p><p>Fabi 3959 contains copper as its active component, which is highly toxic to marine organisms and thus classified as a biocide.</p><p>Fabi antifouling paint was tested under laboratory conditions on the red macro alga <em>Ceramium tenuicorne</em>, in natural brackish water taken from the Baltic Sea. The <em>Ceramium</em> growth inhibition-test was performed using cloned algae exposed to leakage water with and without sediment. The samples containing only water held concentrations in the range of 0.11% of volume-18% of volume per liter, while the samples using sediment held doses measuring between 0.11% of volume-36% of volume leakage water per liter.</p><p>The study showed a growth inhibiting effect on the <em>Ceramium</em> in both water and sediment samples down to the lowest concentration used in the test. There was a difference between the water series and the sediment series in the EC₅₀ values of the leakage water. The mean EC₅₀value was almost 10 times lower within the sediment series compared to the water series (0.114±0.10 and 1.024±0.75, respectively). This indicates that the sediment series are more toxic to <em>Ceramium</em> than the water series. However, if the mean values of EC₅₀ are expressed as copper-concentration, there is no clear difference between the two series (0.59 ± 0.13µg/l for the sediment series and 0.62 ± 0.12 µg/l for the water series). Apparently, the test did not indicate that the sediment was absorbing the copper. Instead it cannot be excluded that another substance involved could have a growth inhibiting impact on <em>Ceramium</em>.</p><p> </p> / <p> </p><p>Båtbottenfärgen Fabi 3959 målas på fartygsskrov för att undvika påväxt av marina organismer. Färgen är registrerad att användas på fritidsbåtar och andra fartyg med en egenvikt på över 200 kg och med huvudsaklig fart på Västkusten (www.kemi.se). Den aktiva komponenten i Fabi 3959 är koppar, vilket är mycket giftigt för marina organismer och därför klassificeras den som en biocid.</p><p>Fabi båtbottenfärg testades i laborativ miljö, på den röda makroalgen <em>Ceramium tenuicorne</em> i naturligt brackvatten från Östersjön. Ett tillväxthämningstest utfördes på <em>Ceramium</em>-kloner vilka exponerades för lakvatten i bägare med och utan sediment. Proverna endast innehållande vatten bestod av koncentrationer i intervallen 0,11-18 volym% per liter medan proverna med sedimentvatten hade koncentrationer på 0,11-36 volym% per liter.</p><p>Studien visade på en signifikant tillväxthämningseffekt på <em>Ceramium</em> i både vatten och sediment, ner till den lägsta använda koncentrationen. Det förelåg en tydlig skillnad mellan vattenserierna och sedimentserierna med avseende på resultaten av EC₅₀-värdena på lakvattnet; EC₅₀-värdena av den toxiska nivån för lakvattnet visade sig ligga tio gånger lägre i sedimentserierna än i vattenserierna (0,114 ± 0,10 i sediment och 1,024 ± 0,75 i vatten). Medelvärdena av EC₅₀för koppar visar inte någon påtaglig skillnad mellan serierna (0,59 ± 0,13µg/l för sediment 0,62 ± 0,12 µg/l för vatten). Testet indikerade därmed inte att sedimentet absorberade koppar. Istället föreligger en möjlighet att en annan substans orsakade tillväxthämning på<em> Ceramium.</em></p><p> </p>

This thesis reports the results of biomarker measurements in three environmental monitoring projects. In the first project, which was part of the Swedish national environmental monitoring, biomarkers were measured annually in female perch (Perca fluviatilis) in two background areas in the Baltic Sea during 1988–2000, resulting in a unique 13-year series of measurements. The most important results were a strong decreasing temporal trend in the gonadosomatic index (GSI) and a strong increasing temporal trend in the hepatic ethoxyresorufin O-deethylase (EROD) activity in the Baltic Proper. In the second project, biomarkers and concentrations of classic pollutants were measured in female perch in the Stockholm recipient 1999–2001. This was the first time a large city was investigated as a point source of pollution, and the gradient was longer and included more stations than customary. Severe pollution conditions in central Stockholm were indicated by the poor health status of the perch: retarded growth, decreased frequency of sexually mature females, low GSI, disturbed visceral fat metabolism, increased hepatic EROD activity, decreased muscle acetylcholinesterase activity, increased frequency of hepatic DNA adducts, and a high concentration of biliary 1-pyrenol. Muscle ΣDDT and ΣPCB were measured as pollution indicators and were 10–28 respectively 12–35 times higher than the background levels in perch from the Baltic Proper. In the Stockholm archipelago two superimposed gradients were found. Whereas the response of several biomarkers consistently decreased with increasing distance from central Stockholm, the response of others first decreased from Stockholm to the middle archipelago and then increased to the open Baltic Sea. The latter biomarkers included the frequency of sexually mature females, GSI, hepatic EROD activity, and hepatic DNA adducts. In the third project, potential toxicity from munitions on the seafloor, at a dumpsite in the Stockholm archipelago, was analysed by the nanoinjection of sediment extracts into newly fertilised rainbow trout (Oncorhynchus mykiss) eggs, followed by the measurement of biomarkers in the developing larvae. No biological effects of the dumped munitions were found. The same stations in the Stockholm archipelago as in the second project were investigated as a positive control. The results of the three projects agreed well, which demonstrated the continuous pollution of the Baltic Sea and the severe pollution conditions and adverse biological effects in central Stockholm. Further investigations are urgently needed to identify which pollutants or other factors are causing the observed biological effects, both in the background areas in the Baltic Sea and in the Stockholm recipient.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

In this Masters Thesis emissions to water from diesel engines in pleasure boats when driven with alternative fuels have been investigated. Two alternative fuels, Fischer-Tropsch-diesel (FT-diesel) and a blend of FT-diesel and rapeseed methyl ester (RME) have been compared with diesel of Swedish environmental class 1 (diesel EC1). The alternative fuels have been compared using two older marine diesel engines, and water samples have been taken from the water in the exhaust system before the exhaust compounds reaches the recipient. The water samples have been analysed with gas chromatography (GC) and mass spectrometry (GC/MS), to analyse volatile and semi volatile compounds including aldehydes and ketons. To investigate the acute toxicity of the exhaust water a 24 h ecotoxicological test was preformed with the crustacean Artemia fransiscana. The results showed considerable differences in emissions of poly aromatic hydrocarbons (PAHs) and alkylated benzenes. From diesel EC1, the amounts of PAHs and alkylated benzenes were significantly higher than from pure FT-diesel and FT-diesel containing 20 % of RME. Pure FT-diesel generated the lowest amounts of PAH and alkylated benzenes. The FT-diesel containing 20 % RME generated higher amounts of benzene than diesel EC1. FT-diesel and FT-diesel with a 20 % blend of RME generated the same amount of acetone, which in turn was higher than the concentration of acetone in the exhaust water, using diesel EC1. Based on the analysis, FT-diesel is the best alternative from an exhaust emission point-of-view, concerning both the amounts and the difference in chemical composition of the combustion products released into the water. The ecotoxicological test showed no effect on the test organism using either of the samples. / Fritidsbåtsintresset är stort i Sverige och båtlivet betyder mycket för många svenskar. Tyvärr är användningen av fritidsbåtar förknippad med en rad miljöproblem. Avgasutsläpp till vatten och luft från fritidsbåtsmotorer påverkar den lokala miljön avsevärt. Gammal teknik med dålig förbränning används fortfarande i stor utsträckning. På kort sikt, innan den gamla tekniken är utbytt, är det viktigt att den äldre motorparken använder miljöanpassade bränslen för att utsläppen till sjöar och hav ska bli så skonsamma som möjligt. Det är dock viktigt att utvärdera miljöpåverkan av dessa alternativa bränslen, så att det verkligen är ett bättre alternativ ur miljösynpunkt. I detta examensarbete har utsläpp till vatten från dieselinombordsmotorer vid drift med olika bränslen undersökts. Två miljöanpassade dieselbränslen; FT-diesel (Fischer-Tropsch) och en procentinblandning av rapsmetylester (RME) i FT-diesel, har jämförts med diesel av svensk miljöklass 1 (MK1). Bränslena har testkörts i två äldre, marina dieselmotorer och prover har tagits på det avgasblandade kylvattnet innan det når recipienten. Det avgasblandade kylvattnet har analyserats med avseende på delvis flyktiga och flyktiga föreningar, aldehyder samt ketoner. Analyserna har skett med gaskromatografi och masspektormetri (GC/MS-screening) på externt laboratorium. För att testa avgasvattnets akuta giftighet har även ett akut (24h) ekotoxikologiskt test utförts på kräftdjuret Artemia fransiscana. Analysresultatet visade skillnad i utsläpp av polyaromatiska kolväten (PAH) och alkylbensener. Vid körning med diesel MK1 genererades betydligt högre halter av PAH:er och alkylbensener, än vid körning med FT-diesel och FT-diesel med 20 % inblandning av RME. Ren FT-diesel gav de lägsta halterna av PAH:er och alkylbensener. Utsläppshalterna av bensen uppvisade inte lika stor skillnad mellan de olika bränslena. Blandningen FT-diesel med 20 % RME gav lite högre halter bensen än vad diesel MK1 gjorde. Ren FT-diesel uppvisade lägst halt bensen. Utsläpp av aceton visade sig vara lika för FT-diesel och FT-diesel med 20 % RME och gav högre halter än vad diesel MK1 gjorde. Baserat på analysresultaten är FT-diesel det bästa bränslet ur emissionssynpunkt med avseende på vilka ämnen som hamnar i vattenfasen. Det ekotoxikologiska testet visade ingen skillnad mellan de olika bränslena. Avgasvatten från bränslena hade inte någon akut toxisk effekt på testdjuret.