Effects of different diets on the reproduction and naupliar development of the copepod Acartia bifilosa

The influence of diatoms on the reproduction and naupliar development of Acartia bifilosa was investigated under laboratory conditions, comparing initial in situ values and laboratory-food treatments. Egg production by A. bifilosa was significantly reduced by one diatom diet (Phaeodactylum tricornutum: Pt) and by two non-diatom diets (Platymonas subordiformis: Ps and Nannochloropsis oculata: No). It was less affected by the other diatom diet (Skeletonema costatum: Sc) or by two mixed-food treatments (D-mix and DG-mix), composed of two diatoms (Pt, Sc) and four species (Pt, Sc, Ps and No), respectively. The negative effect of Pt was eliminated when adult copepods were offered mixed-food diets. There were no significant differences between the hatching success values observed in filtered seawater and in algal exudates, indicating that diatoms did not produce active dissolved toxic substances under the different food concentrations tested. The mortality rate of nauplii was higher with Pt than the other diets, suggesting that this diatom species had a negative effect on egg production, hatching success and naupliar survival simultaneously. Compared to other diets, No and Pt were not beneficial food sources for reproduction and for female and larval survival. (c) 2007 Elsevier B.V. All rights reserved.

Concentrations of Cadmium and Zinc in Seawater of Bohai Bay and Their Effects on Biomarker Responses in the Bivalve Chlamys farreri

Both in-field chemical investigation and in the laboratory toxic tests were carried out to systematically understand the pollution status of cadmium (Cd) and zinc (Zn) in Bohai Bay. Samples collected from surface seawater were determined to describe the distributions of Cd and Zn in Bohai Bay. The average values in our study of Cd and Zn were 0.15 mu g/L and 19.68 mu g/L, respectively. Both of them were lower than the first class limit of seawater quality standard in China. In the laboratory, antioxidant enzymes [SOD (Cu/Zn-SOD, Mn-SOD), CAT], lipid peroxidation (MDA), phase I and phase II enzymes (CYP4501A and GST) were investigated in the bivalves Chlamys farreri exposed to Cd and Zn at the concentration levels of Bohai Bay seawater, which were obtained from our in-field investigation. The reduced SOD, CAT, and EROD (7-ethoxyresorufin-O-deethylase) activities (with the inhibitory rate of 16.8%, 31.5%, and 51.6%, respectively) in Cd treatment were observed and resulted in obvious lipid peroxidation damage. However, treatment of Zn showed elevations in SOD and GST by 13.3% and 29.9%, respectively, and with no influence on lipid peroxidation. In summary, seawater quality in Bohai Bay seawater was ranked as good in general, but it seemed that Cd might possess a potential environmental risk by effecting pro-oxidant/antioxidant balance and phase I detoxification in C. farreri.

Quantitative Cytochemical Effects Of 3 Metal-Ions On A Lysosomal Hydrolase Of A Hydroid

The quantitative effects of Cu8+, Cd2+ and Hg2+ on the cytochemical staining reaction for lysosomal N-acetyl-/?-D-glucosaminidase have been determined and related to the inhibitory effects of the metals on colonial growth rate in the experimentally cultured hydroid Campanularia flexuosa. Cytochemical threshold concentrations are comparable to known environmental levels and are about one order of magnitude lower than those obtained by measuring colony growth rates. Pretreatment of colonies with Cuz+ gave no indication of tolerance adaptation, although there is evidence of the cumulative toxicity of Cu2+ and the possible sequestration of this metal in endodermal cell lysosomes. There is also an indication that the Cu2+ may exert its toxic effect by decreasing the stability of the lysosomal membranes, thus increasing the level of free glucosaminidase activity.

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Do ectomycorrhizal fungi exhibit adaptive tolerance to potentially toxic metals in the environment?

The effects of potentially toxic metals on ectomycorrhizal (ECM) fungi and their higher plant hosts are examined in this review. Investigations at a species and community level have revealed wide inter- and intraspecific variation in sensitivity to metals. Adaptive and constitutive mechanisms of ECM tolerance are proposed and discussed in relation to proven tolerance mechanisms in bacteria, yeasts and plants. Problems with methodology and research priorities are highlighted. These include the need for a detailed understanding of the genetic basis of tolerance in the ECM symbiosis, and for studies of ECM community dynamics in polluted sites.

Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening

Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POP were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p'-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2h and 48h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced the ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC+Br mixture. No significant effects were detected in the Br+Cl, PFC+Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment.

Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts

BACKGROUND: Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS: Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS: Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS: Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Study of pesticide effects on non-target organisms

A utilização insustentável de pesticidas, especialmente em zonas com elevado valor ecológico constitui uma ameaça à integridade dos ecossistemas. Sendo um problema à escala mundial, e também no contexto nacional, o presente trabalho pretende ser um contributo para a avaliação dos efeitos de pesticidas em organismos não alvo terrestres e, principalmente, aquáticos, em contextos de progressiva relevância ecológica. Neste sentido, o estudo foi direccionado para áreas (A1 e A2) integradas numa zona agrícola extensa em Portugal, utilizada para a produção de milho e, principalmente, de arroz (Baixo Mondego), a qual sustenta uma elevada biodiversidade. O estudo teve início na área A1, onde a monitorização físico-química e os ensaios com amostras naturais (ensaios WET - whole effluent tests) provenientes desta área evidenciaram que, apesar da ausência de pesticidas, as amostras de água colhidas no canal que atravessava os arrozais foram as mais nocivas para o crescimento de Pseudokirchneriella subcapitata e Chlorella vulgaris. Uma vez que outras fontes de contaminação (produção de gado) actuavam em A1, o estudo prosseguiu apenas na área A2. Assim, em A2, começou-se por determinar a toxicidade individual e da mistura de dois herbicidas formulados aplicados nos campos de arroz (Viper®) e milho (Mikado®) em condições laboratoriais. Viper® foi o herbicida mais tóxico, tanto para o crescimento de P. subcapitata e C. vulgaris, como para a sobrevivência, reprodução e crescimento de Daphnia longispina e Daphnia magna. Adicionalmente, estimou-se que a mistura Viper®/Mikado® induz efeitos antagonistas no crescimento de P. subcapitata e efeitos sinérgicos no crescimento de C. vulgaris e na sobrevivência dos dafnídeos. A avaliação da toxicidade destes herbicidas formulados e seus ingredientes activos no comportamento de minhocas terrestres (Eisenia andrei), usando solos naturais, demonstrou que Viper® e penoxsulam causaram uma % de evitamento superior nos organismos expostos. Contudo, o risco para E. andrei será à partida reduzido se as taxas de aplicação dos herbicidas forem respeitadas. Ensaios WET foram novamente usados para testar amostras naturais da área A2. Verificou-se que a qualidade do sistema aquático e do arrozal diminuiu durante a estação agrícola, em paralelo com a presença de nutrientes e pesticidas. O crescimento algal foi inibido, apesar dos parâmetros de história de vida dos dafnídeos terem sido estimulados. O resultado desta avaliação subestimou, em certos casos, os impactos reais causados pela aplicação de pesticidas. A avaliação in situ simultânea à aplicação de herbicidas nos arrozais demonstrou que os efeitos registados foram de facto restritos aos pulsos de herbicidas. A inibição das taxas de alimentação de D. longispina e D. magna forneceram um sinal precoce de alterações no sistema, seguido pela diminuição da sua sobrevivência e do crescimento de P. subcapitata. Em suma, as diferentes fases da avaliação efectuada confirmaram a existência de condições desfavoráveis devido às práticas agrícolas, reforçando a necessidade de se conjugar ensaios laboratoriais com avaliações in situ de maior relevância ecológica, para reduzir o grau de incerteza aliado à determinação dos riscos.

From genes to population: effects of toxicants on Enchytraeus albidus

Nowadays, a systems biology approach is both a challenge as well as believed to be the ideal form of understanding the organisms’ mechanisms of response. Responses at different levels of biological organization should be integrated to better understand the mechanisms, and hence predict the effects of stress agents, usable in broader contexts. The main aim of this thesis was to evaluate the underlying mechanisms of Enchytraeus albidus responses to chemical stressors. Therefore, there was a large investment on the gene library enrichment for this species, as explained ahead. Overall, effects of chemicals from two different groups (metals and pesticides) were assessed at different levels of biological organization: from genes and biochemical biomarkers to population endpoints. Selected chemicals were: 1) the metals cadmium and zinc; 2) the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim. At the gene and sub-cellular level, the effects of time and dosage were also adressed. Traditional ecotoxicological tests - survival, reproduction and avoidance behavior - indicated that pesticides were more toxic than metals. Avoidance behaviour is extremely important from an ecological point of view, but not recommended to use for risk assessment purposes. The oxidative stress related experiment showed that metals induced significant effects on several antioxidant enzyme activities and substrate levels, as well as oxidative damage on the membrane cells. To increase the potential of our molecular tool to assess transcriptional responses, the existing cDNA library was enriched with metal and pesticide responding genes, using Suppression Subtractive Hybridization (SSH). With the sequencing information obtained, an improved Agilent custom oligonucleotide microarray was developed and an EST database, including all existing molecular data on E. albidus, was made publicly available as an interactive tool to access information. With this microarray tool, most interesting and novel information on the mechanisms of chemical toxicity was obtained, with the identification of common and specific key pathways affected by each compound. The obtained results allowed the identification of mechanisms of action for the tested compounds in E. albidus, some of which are in line with the ones known for mammals, suggesting across species conserved modes of action and underlining the usefulness of this soil invertebrate as a model species. In general, biochemical and molecular responses were influenced by time of exposure and chemical dosage and these allowed to see the evolution of events. Cellular energy allocation results confirmed the gene expression evidences of an increased energetic expenditure, which can partially explain the decrease on the reproductive output, verified at a later stage. Correlations found throughout this thesis between effects at the different levels of biological organization have further improved our knowledge on the toxicity of metals and pesticides in this species.

Exposure modelling to traffic-related air toxic pollutants

Atualmente, a poluição atmosférica constitui uma das principais causas ambientais de mortalidade. Cerca de 30% da população europeia residente em áreas urbanas encontra-se exposta a níveis de poluição atmosférica superiores aos valores- limite de qualidade do ar legislados para proteção da saúde humana, representando o tráfego rodoviário uma das principais fontes de poluição urbana. Além dos poluentes tradicionais avaliados em áreas urbanas, os poluentes classificados como perigosos para a saúde (Hazard Air Pollutants - HAPs) têm particular relevância devido aos seus conhecidos efeitos tóxicos e cancerígenos. Neste sentido, a avaliação da exposição tornase primordial para a determinação da relação entre a poluição atmosférica urbana e efeitos na saúde. O presente estudo tem como principal objetivo o desenvolvimento e implementação de uma metodologia para avaliação da exposição individual à poluição atmosférica urbana relacionada com o tráfego rodoviário. De modo a atingir este objetivo, foram identificados os parâmetros relevantes para a quantificação de exposição e analisados os atuais e futuros potenciais impactos na saúde associados com a exposição à poluição urbana. Neste âmbito, o modelo ExPOSITION (EXPOSure model to traffIc-relaTed aIr pOllutioN) foi desenvolvido baseado numa abordagem inovadora que envolve a análise da trajetória dos indivíduos recolhidas por telemóveis com tecnologia GPS e processadas através da abordagem de data mining e análise geoespacial. O modelo ExPOSITION considera também uma abordagem probabilística para caracterizar a variabilidade dos parâmetros microambientais e a sua contribuição para exposição individual. Adicionalmente, de forma a atingir os objetivos do estudo foi desenvolvido um novo módulo de cálculo de emissões de HAPs provenientes do transporte rodoviário. Neste estudo, um sistema de modelação, incluindo os modelos de transporteemissões- dispersão-exposição, foi aplicado na área urbana de Leiria para quantificação de exposição individual a PM2.5 e benzeno. Os resultados de modelação foram validados com base em medições obtidas por monitorização pessoal e monitorização biológica verificando-se uma boa concordância entre os resultados do modelo e dados de medições. A metodologia desenvolvida e implementada no âmbito deste trabalho permite analisar e estimar a magnitude, frequência e inter e intra-variabilidade dos níveis de exposição individual, bem como a contribuição de diferentes microambientes, considerando claramente a sequência de eventos de exposição e relação fonte-recetor, que é fundamental para avaliação dos efeitos na saúde e estudos epidemiológicos. O presente trabalho contribui para uma melhor compreensão da exposição individual em áreas urbanas, proporcionando novas perspetivas sobre a exposição individual, essenciais na seleção de estratégias de redução da exposição à poluição atmosférica urbana, e consequentes efeitos na saúde.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5ºC in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the “interactive” nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

Effects of copper in plasma, gills, liver and muscle of seabream (Sparus aurata, Linnaeus 1758) juveniles

Dissertação de Mestrado, Aquacultura e Pescas, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2006

Effects of nutrient and light enrichment on phytoplankton growth

Alterations of freshwater flow regimes and increasing eutrophication lead to alterations in light availability and nutrient loading into adjacent estuaries and coastal areas. Phytoplankton community respond to these changes in many ways. Harmful phytoplankton blooms, for instance, may be a consequence of changes in nutrient supply, as well as the replacement of some phytoplankton species (like diatoms, that contribute for the development of large fish and shellfish populations) by ohers (like cyanobacteria, that may be toxic and represent an undesirable food source for higher trophic levels). Nutrient and light enrichment experiments allow us to understand and predict the effects of eutrophication on the growth of phytoplankton. This is a fundamental tool in water management issues, since it enables the prediction of changes in the phytoplankton community that may be harmful to the whole ecosystem, and the design of mitigation strategies (Zalewski 2000).

Effects of Environmental Organochlorine Pesticides on Human Breast Cancer: Putative Involvement on Invasive Cell Ability

Human exposure to persistent organic pollutants (POPs) is a certainty, even to long banned pesticides like o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), and its metabolites p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE), and p,p′-dichlorodiphenyldichloroethane (p,p′-DDD). POPs are known to be particularly toxic and have been associated with endocrine-disrupting effects in several mammals, including humans even at very low doses. As environmental estrogens, they could play a critical role in carcinogenesis, such as in breast cancer. With the purpose of evaluating their effect on breast cancer biology, o,p′-DDT, p,p′-DDE, and p,p′-DDD (50–1000 nM) were tested on two human breast adenocarcinoma cell lines: MCF-7 expressing estrogen receptor (ER) α and MDA-MB-231 negative for ERα, regarding cell proliferation and viability in addition to their invasive potential. Cell proliferation and viability were not equally affected by these compounds. In MCF-7 cells, the compounds were able to decrease cell proliferation and viability. On the other hand, no evident response was observed in treated MDA-MB-231 cells. Concerning the invasive potential, the less invasive cell line, MCF-7, had its invasion potential significantly induced, while the more invasive cell line MDA-MB-231, had its invasion potential dramatically reduced in the presence of the tested compounds. Altogether, the results showed that these compounds were able to modulate several cancer-related processes, namely in breast cancer cell lines, and underline the relevance of POP exposure to the risk of cancer development and progression, unraveling distinct pathways of action of these compounds on tumor cell biology.