Calcium nitrate addition to control the internal load of phosphorus from sediments of a tropical eutrophic reservoir: Microcosm experiments

The main objective of this study was to perform laboratory experiments on calcium nitrate addition to sediments of a tropical eutrophic urban reservoir (Ibirite reservoir, SE Brazil) to immobilize the reactive soluble phosphorus (RSP) and to evaluate possible geochemical changes and toxic effects caused by this treatment. Reductions of 75 and 89% in the concentration of RSP were observed in the water column and interstitial water, respectively, after 145 days of nitrate addition. The nitrate application increased the rate of autotrophic denitrification, causing a consumption of 98% of the added nitrate and oxidation of 99% of the acid volatile sulfide. As a consequence, there were increases in the sulfate and iron (II) concentrations in the sediment interstitial water and water column, as well as changes in the copper speciation in the sediments. Toxicity tests initially indicated that the high concentrations of nitrate and nitrite in the sediment interstitial water (up to 2300 mg L-1 and 260 mg L-1, respectively) were the major cause of mortality of Ceriodaphnia silvestrii and Chironomus xanthus. However, at the end of the experiment, the sediment toxicity was completely removed and a reduction in the 48 h-EC50 of the water was also observed. Based on these results we can say that calcium nitrate treatment proved to be a valuable tool in remediation of eutrophic aquatic ecosystems leading to conditions that can support a great diversity of organisms after a restoration period. (C) 2012 Elsevier Ltd. All rights reserved.

In vitro cytotoxicity of white MTA, MTA Fillapex® and Portland cement on human periodontal ligament fibroblasts

The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5X3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and ¼ dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Effects of daunorubicin, mitomycin C, azathioprine and cyclosporin A on human retinal pigmented epithelial, corneal endothelial and conjunctival cell lines

BACKGROUND: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines. METHODS: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days. RESULTS: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml. CONCLUSION: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Effect of oral D-tagatose on liver volume and hepatic glycogen accumulation in healthy male volunteers

Standard toxicity tests with high levels of D-tagatose showed a reversible enlargement of the liver in Sprague-Dawley rats without increase of liver enzymes. The present study tests the hypotheses that partial substitution of dietary sucrose by D-tagatose for 28 days increases the volume of human liver and the concentration of liver glycogen. Twelve healthy, male volunteers were studied in a double-blind crossover study with ingestion of D-tagatose (3x15 g daily) and placebo (sucrose, 3x15 g daily) for periods of 28 days each. Liver volume and glycogen concentration have been determined by magnetic resonance (MR) imaging and spectroscopy, which were accompanied by routine medical examinations. MR examinations before and after the treatments revealed no effects (P>0.05) of treatment, period, or subject for changes in liver volume or glycogen concentration. A steady increase of liver volumes, independent of the D-tagatose or placebo intake, has been observed over the study in parallel with a slight increase in body weight. The treatment with D-tagatose was not associated with clinically relevant changes of the examined clinico-chemical and hematological parameters, including liver enzymes and uric acid.

In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter

BACKGROUND: Several epidemiological studies show that inhalation of particulate matter may cause increased pulmonary morbidity and mortality. Of particular interest are the ultrafine particles that are particularly toxic. In addition more and more nanoparticles are released into the environment; however, the potential health effects of these nanoparticles are yet unknown. OBJECTIVES: To avoid particle toxicity studies with animals many cell culture models have been developed during the past years. METHODS: This review focuses on the most commonly used in vitro epithelial airway and alveolar models to study particle-cell interactions and particle toxicity and highlights advantages and disadvantages of the different models. RESULTS/CONCLUSION: There are many lung cell culture models but none of these models seems to be perfect. However, they might be a great tool to perform basic research or toxicity tests. The focus here is on 3D and co-culture models, which seem to be more realistic than monocultures.

Predator-induced stress makes the pesticide carbaryl more deadly to gray treefrog tadpoles (Hyla versicolor)

Global declines in amphibians likely have multiple causes, including widespread pesticide use. Our knowledge of pesticide effects on amphibians is largely limited to short-term (4-d) toxicity tests conducted under highly artificial conditions to determine lethal concentrations (LC50). We found that if we used slightly longer exposure times (10–16 d), low concentrations of the pesticide carbaryl (3–4% of LC504-d) killed 10–60% of gray treefrog (Hyla versicolor) tadpoles. If predatory cues also were present, the pesticide became 2–4 times more lethal, killing 60–98% of tadpoles. Thus, under more realistic conditions of increased exposure times and predatory stress, current application rates for carbaryl can potentially devastate gray treefrog populations. Further, because predator-induced stress is ubiquitous in animals and carbaryl's mode of action is common to many pesticides, these negative impacts may be widespread in nature.

Efeitos dos agrotóxicos Kraft®36EC e Score®250EC (e seus princípios ativos) em ecossistemas aquáticos: análises comparativas e ecossistêmicas

Com o advento da agricultura ampliou-se a produção alimentar e os bens de consumo, no entanto, os riscos ambientais também foram maximizados em função da adoção de técnicas produtivas baseadas no uso intensivo de insumos agrícolas. Esta problemática é mundial, embora mais evidenciada nos países em desenvolvimento e que tem, na produção agrícola, a base de sua economia. O Brasil enquadra-se nesta situação e desde 2009 é considerado o maior consumidor de agrotóxicos do mundo, criando um cenário de risco ambiental e de saúde humana. Os efeitos ambientais, base deste estudo, estão relacionados não somente à perda de espécies não-alvo, uma vez que os agrotóxicos não são seletivos, mas também as alterações em nível ecossistêmico, a qual se relaciona com as perdas das funções e dos serviços gerados pelos sistemas naturais. Adiciona-se a esta complexidade, a forma de ação de cada agrotóxico, a distribuição dos mesmos nos diferentes compartimentos (ar, solo e água), o período de permanência de cada um, as relações sinérgicas decorrentes das interações entre diferentes produtos, a formação de subprodutos no processo de degradação, entre outros fatores, como as diferenças existentes entre o ingrediente ativo e a formulação comercial, na qual existem os chamados ingredientes inertes em sua composição, os quais podem ser muito mais tóxicos para espécies e ecossistemas. Considerando esta abordagem, a presente pesquisa foi desenvolvida com base na realidade de um local de referência, o município de Bom Repouso (MG/BR), no qual a intensificação da produção de morango e batata tem trazido uma série de riscos sociais e ambientais. Semelhante a outras regiões produtivas do país, o uso de agrotóxicos é recorrente, amplo e irrestrito, com destaque para as formulações comerciais Kraft®36EC e Score®250EC, as quais, juntamente com seus respectivos ingredientes ativos (abamectina e difenoconazol), foram avaliadas por meio de testes de toxicidade com espécies de diferentes níveis tróficos representativas de um ecossistema aquático, gerando informações que foram avaliadas em nível de espécie e de ecossistema, simulando o cenário de aplicação dos produtos no local de referência. Os resultados obtidos permitiram concluir sobre as diferenças de sensibilidade das espécies e quais seriam as mais indicadas para se avaliar os efeitos tóxicos de ambos os agrotóxicos; os efeitos diferenciados entre a formulação comercial e os ingredientes ativos; bem como as respostas em termos de espécies e de ecossistemas, demonstrando a necessidade de que ambas as análises sejam consideradas na avaliação de risco ecológico.

Comparação de métodos ecotoxicológicos na avaliação de sedimentos marinhos e estuarinos

A amônia não ionizada pode ser interferente nos testes de toxicidade com a água intersticial de sedimentos marinhos e estuarinos. Com o objetivo de se encontrar em uma alternativa que minimizasse o efeito da amônia avaliou-se, em paralelo à água intersticial, o método que utiliza a interface sedimento/água. Foram analisadas 25 amostras de sedimento através do teste de toxicidade crônica de curta duração com Lytechinus variegatus. Foi observada correlação significativa entre os efeitos tóxicos para os diversos estágios embrio-larvais e a concentração de amônia não ionizada para os dois métodos avaliados. Quando comparadas com a interface sedimento/água, foram observadas concentrações superiores de amônia não ionizada em todas as amostras de água intersticial, que isoladamente, poderiam causar os efeitos tóxicos observados. Conclui-se que o teste de toxicidade na interface sedimento/água é o método mais adequado para avaliação de sedimentos marinhos e estuarinos, pois possibilitou a utilização de um maior número de resultados de testes de toxicidade do que aquele com água intersticial, uma vez que minimizou a influência da amônia. Foi evidenciada correlação significativa entre os resultados das concentrações de HPAs determinadas nas amostras de sedimento bruto e a toxidade observada nos diversos estágios embrio-larvais, pelo método da água intersticial. Para os metais, obteve-se correlação significativa com os efeitos tóxicos nos referidos estágios determinados na interface sedimento/água. Não foi evidenciada correlação entre a granulometria e o efeito tóxico nas amostras. O registro dos efeitos nos diferentes estágios embrio-larvais mostrou-se bastante útil, pois além de oferecer informações a respeito da toxicidade da amostra (ausência ou presença) possibilitou a hierarquização, em termos da intensidade de efeito adverso, daquelas amostras tóxicas.

Aplicação de testes de toxicidade com organismos marinhos para a análise de efluentes industriais lançados em áreas estuarinas

Com o objetivo de aplicar e avaliar a viabilidade de uso dos métodos disponíveis com organismos marinhos, no controle da toxicidade de efluentes líquidos que são lançados em ambientes estuarinos, foram realizados testes de toxicidade aguda com os crustáceos Mysidopsis juniae, Artemia sp, Temora stylifera e Acartia IiIljeborgi e testes de toxicidade crônica de curta duração com o equinodermo Lytechinus variegatus, utilizando-se os efluentes industriais de uma indústria siderúrgica, COSIPA e uma fábrica de fertilizantes, ULTRAFÉRTIL/JARDIM SÃO MARCOS, ambos lançados no estuário do Rio Cubatão. Dentre os organismos-testes utilizados, para avaliação do efeito tóxico agudo, o misidáceo M. juniae foi o mais sensível para ambos os efluentes, sendo que Artemia sp foi o menos sensível. Testes de toxicidade crônica com L. variegatus também se mostraram bastante úteis para avaliação de efeitos subletais. Os efluentes analisados apresentaram grande variabilidade durante o período de estudo, o que foi evidenciado através do cálculo do coeficiente de variação para testes com M. juniae. Foi avaliado, também, o efeito da salinidade sobre a sensibilidade dos crustáceos M. juniae e Artemia sp a agentes químicos (zinco e DSS) e aos efluentes industriais. A salinidade não interferiu significativamente nos resultados observados, com exceção de um experimento realizado a 15x10-3 com Artemia sp, com o efluente da COSIPA. Verificou-se, ainda, o possível efeito da utilização de salmoura obtida através dos processos de congelamento e evaporação da água do mar, sendo que o primeiro processo foi indicado para salinização de efluentes.

Cost analysis methodology and protocol estimates : OECD minimum pre-market data test protocols /

"EPA-560/4-81-002."

Phototoxicity of photosystem II (PSII) herbicides to coral

Recent reports of contamination of the Great Barrier Reef Marine Park by herbicides used in antifouling paints and in agriculture have caused concern over the possible effects on corals in nearshore areas. Pulse-Amplitude Modulated (PAM) chlorophyll fluorescence techniques were used to examine changes in the maximum effective quantum yield (ΔF/Fm′) of symbiotic dinoflagellates within the host tissues (in hospite) of the coral Seriatopora hystrix exposed to a number of Photosystem II (PSII) inhibiting herbicides in short-term toxicity tests. The concentration of herbicide required to reduce ΔF/Fm′ by 50% (median effective concentration [EC50]) differed by over 2 orders of magnitude: Irgarol 1051 (0.7 μg l-1) > ametryn (1.7 μg l-1) > diuron (2.3 μg l-1) > hexazinone (8.8 μg l -1) > atrazine (45 μg l-1) > simazine (150 μg l-1) > tebuthiuron (175 μg l-1) > ionynil (> 1 mg l-1). Similar absolute and relative toxicities were observed with colonies of the coral Acropora formosa (Irgarol 1051 EC50: 1.3 μg l-1, diuron EC50: 2.8 μg l-1), Time-course experiments indicated that ΔF/Fm′ was rapidly reduced (i.e. within minutes) in S. hystrix exposed to Irgarol 1051 and diuron. On return to fresh running seawater, ΔF/Fm′ recovered quickly in diuron-exposed corals (i.e. in minutes to hours), but slowly in corals exposed to Irgarol 1051 (i.e. hours to days). Time-course experiments indicated that the effects of diuron (3 μg l-1) on S. hystrix were inversely related to temperature over the range 20 to 30 °C, although initially the effects were less at the lower temperatures. Repeated exposure to pulses of Irgarol 1051 (daily 2 h exposure to 30 μg l -1 over 4 d) resulted in a 30% decrease in the density of symbiotic dinoflagellates in the tissues of S. hystrix.

Valutazione del rischio ecologico e sviluppo di strategie innovative per il monitoraggio della qualita’ dei corpi idrici: applicazione di un nuovo approccio integrato in un bacino pilota.

Freshwater is extremely precious; but even more precious than freshwater is clean freshwater. From the time that 2/3 of our planet is covered in water, we have contaminated our globe with chemicals that have been used by industrial activities over the last century in a unprecedented way causing harm to humans and wildlife. We have to adopt a new scientific mindset in order to face this problem so to protect this important resource. The Water Framework Directive (European Parliament and the Council, 2000) is a milestone legislative document that transformed the way that water quality monitoring is undertaken across all Member States by introducing the Ecological and Chemical Status. A “good or higher” Ecological Status is expected to be achieved for all waterbodies in Europe by 2015. Yet, most of the European waterbodies, which are determined to be at risk, or of moderate to bad quality, further information will be required so that adequate remediation strategies can be implemented. To date, water quality evaluation is based on five biological components (phytoplankton, macrophytes and benthic algae, macroinvertebrates and fishes) and various hydromorphological and physicochemical elements. The evaluation of the chemical status is principally based on 33 priority substances and on 12 xenobiotics, considered as dangerous for the environment. This approach takes into account only a part of the numerous xenobiotics that can be present in surface waters and could not evidence all the possible causes of ecotoxicological stress that can act in a water section. The mixtures of toxic chemicals may constitute an ecological risk not predictable on the basis of the single component concentration. To improve water quality, sources of contamination and causes of ecological alterations need to be identified. On the other hand, the analysis of the community structure, which is the result of multiple processes, including hydrological constrains and physico-chemical stress, give back only a “photograph” of the actual status of a site without revealing causes and sources of the perturbation. A multidisciplinary approach, able to integrate the information obtained by different methods, such as community structure analysis and eco-genotoxicological studies, could help overcome some of the difficulties in properly identifying the different causes of stress in risk assessment. In synthesis, the river ecological status is the result of a combination of multiple pressures that, for management purposes and quality improvement, have to be disentangled from each other. To reduce actual uncertainty in risk assessment, methods that establish quantitative links between levels of contamination and community alterations are needed. The analysis of macrobenthic invertebrate community structure has been widely used to identify sites subjected to perturbation. Trait-based descriptors of community structure constitute a useful method in ecological risk assessment. The diagnostic capacity of freshwater biomonitoring could be improved by chronic sublethal toxicity testing of water and sediment samples. Requiring an exposure time that covers most of the species’ life cycle, chronic toxicity tests are able to reveal negative effects on life-history traits at contaminant concentrations well below the acute toxicity level. Furthermore, the responses of high-level endpoints (growth, fecundity, mortality) can be integrated in order to evaluate the impact on population’s dynamics, a highly relevant endpoint from the ecological point of view. To gain more accurate information about potential causes and consequences of environmental contamination, the evaluation of adverse effects at physiological, biochemical and genetic level is also needed. The use of different biomarkers and toxicity tests can give information about the sub-lethal and toxic load of environmental compartments. Biomarkers give essential information about the exposure to toxicants, such as endocrine disruptor compounds and genotoxic substances whose negative effects cannot be evidenced by using only high-level toxicological endpoints. The increasing presence of genotoxic pollutants in the environment has caused concern regarding the potential harmful effects of xenobiotics on human health, and interest on the development of new and more sensitive methods for the assessment of mutagenic and cancerogenic risk. Within the WFD, biomarkers and bioassays are regarded as important tools to gain lines of evidence for cause-effect relationship in ecological quality assessment. Despite the scientific community clearly addresses the advantages and necessity of an ecotoxicological approach within the ecological quality assessment, a recent review reports that, more than one decade after the publication of the WFD, only few studies have attempted to integrate ecological water status assessment and biological methods (namely biomarkers or bioassays). None of the fifteen reviewed studies included both biomarkers and bioassays. The integrated approach developed in this PhD Thesis comprises a set of laboratory bioassays (Daphnia magna acute and chronic toxicity tests, Comet Assay and FPG-Comet) newly-developed, modified tacking a cue from standardized existing protocols or applied for freshwater quality testing (ecotoxicological, genotoxicological and toxicogenomic assays), coupled with field investigations on macrobenthic community structures (SPEAR and EBI indexes). Together with the development of new bioassays with Daphnia magna, the feasibility of eco-genotoxicological testing of freshwater and sediment quality with Heterocypris incongruens was evaluated (Comet Assay and a protocol for chronic toxicity). However, the Comet Assay, although standardized, was not applied to freshwater samples due to the lack of sensitivity of this species observed after 24h of exposure to relatively high (and not environmentally relevant) concentrations of reference genotoxicants. Furthermore, this species demonstrated to be unsuitable also for chronic toxicity testing due to the difficult evaluation of fecundity as sub-lethal endpoint of exposure and complications due to its biology and behaviour. The study was applied to a pilot hydrographic sub-Basin, by selecting section subjected to different levels of anthropogenic pressure: this allowed us to establish the reference conditions, to select the most significant endpoints and to evaluate the coherence of the responses of the different lines of evidence (alteration of community structure, eco-genotoxicological responses, alteration of gene expression profiles) and, finally, the diagnostic capacity of the monitoring strategy. Significant correlations were found between the genotoxicological parameter Tail Intensity % (TI%) and macrobenthic community descriptors SPEAR (p<0.001) and EBI (p<0.05), between the genotoxicological parameter describing DNA oxidative stress (ΔTI%) and mean levels of nitrates (p<0.01) and between reproductive impairment (Failed Development % from D. magna chronic bioassays) and TI% (p<0.001) as well as EBI (p<0.001). While correlation among parameters demonstrates a general coherence in the response to increasing impacts, the concomitant ability of each single endpoint to be responsive to specific sources of stress is at the basis of the diagnostic capacity of the integrated approach as demonstrated by stations presenting a mismatch among the different lines of evidence. The chosen set of bioassays, as well as the selected endpoints, are not providing redundant indications on the water quality status but, on the contrary, are contributing with complementary pieces of information about the several stressors that insist simultaneously on a waterbody section providing this monitoring strategy with a solid diagnostic capacity. Our approach should provide opportunities for the integration of biological effects into monitoring programmes for surface water, especially in investigative monitoring. Moreover, it should provide a more realistic assessment of impact and exposure of aquatic organisms to contaminants. Finally this approach should provide an evaluation of drivers of change in biodiversity and its causalities on ecosystem function/services provision, that is the direct and indirect contributions to human well-being.

Synthesis and evaluation of novel polymer modifiers for biodegradable polymers

The effects of ester plasticizers and copolymers on the mechanical properties of the natural biodegradable polymers, poly(3-hydroxybutyrate) [PHB] and poly(lactic acid) [PLA] have been studied after subjecting to melt processing conditions. Ester plasticizers were synthesized from citric, tartaric and maleic acids using various alcohols. A variety of PLA copolymers have also been prepared from poly(ethylene glycol) derivatives using stannous octanoate catalysed ring opening polymerisations of DL-lactide. A novel PLA star copolymer was also prepared from an ethoxylated pentaerythritol. The structures of these copolymers were determined by NMR spectroscopy. The plasticizing effect of the synthesised additives at various concentrations was determined. While certain additives were capable of improving the mechanical properties of PLA, none were effective in PHB. Moreover, it was found that certain combinations of additives exhibited synergistic effects. Possible mechanisms are discussed. Biotic and abiotic degradation studies showed that the plasticizers (esters and copolymers) did not inhibit the biodegradability of PHB or PLA in compost at 60°C. Simple toxicity tests carried out on compost extract and its ability to support the growth of cress seeds was established. PLA was found to be susceptible to limited thermal degradation under melt processing conditions. Conventional phenolic antioxidants showed no significant effect on this process, suggesting that degradation was not predominantly a free radical process. PLA also underwent photo-oxidative degradation with UV light and the process could be accelerated in the presence of a photoactivator such as iron (III) diisononyl dithiocarbamate. The mechanisms for the above processes are discussed. Finally, selected compounds were prepared on a pilot plant scale. Extruded and blown films were prepared containing these additives with conventional polymer processing equipment. The mechanical properties were similar to those obtained with laboratory produced compression moulded films.

Ecological studies on:(a) the use of colonisation samplers in relation to biological surveillance of river water quality (b) the requirements of freshwater gastropoda

Some of the factors affecting colonisation of a colonisation sampler, the Standard Aufwuchs Unit (S. Auf. U.) were investigated, namely immersion period, whether anchored on the bottom or suspended, and the influence of riffles. It was concluded that a four-week immersion period was best. S. Auf. U. anchored on the bottom collected both more taxa and individuals than suspended ones. Fewer taxa but more individuals colonised S. Auf. U. in the potamon zone compared to the rhithron zone with a consequent reduction in the values of pollution indexes and diversity. It was concluded that a completely different scoring system was necessary for lowland rivers. Macroinvertebrates colonising S. Auf. U. in simulated streams, lowland rivers and the R. Churnet reflected water quality. A variety of pollution and diversity indexes were applied to results from lowland river sites. Instead of these, it was recommended that an abbreviated species - relative abundance list be used to summarise biological data for use in lowland river surveillance. An intensive study of gastropod populations was made in simulated streams. Lynnaea peregra increased in abundance whereas Potamopyrgas jenkinsi decreased with increasing sewage effluent concentration. No clear-cut differences in reproduction were observed. The presence/absence of eight gastropod taxa was compared with concentrations of various pollutants in lowland rivers. On the basis of all field work it appeared that ammonia, nitrite, copper and zinc were the toxicants most likely to be detrimental to gastropods and that P. jenkinsi and Theodoxus fluviatilis were the least tolerant taxa. 96h acute toxicity tests of P. jenkinsi using ammonia and copper were carried out in a flow-through system after a variety of static range finding tests. P. jenkinsi was intolerant to both toxicants compared to reports on other taxa and the results suggested that these toxicants would affect distribution of this species in the field.