Nisina é um peptídeo antimicrobiano natural produzido por Lactococcus lactis subsp. lactis ATCC 11454 durante a fase exponencial de crescimento. A bacteriocina é usada como conservante natural de alimentos, uma vez que mostra atividade antimicrobiana contra bactérias Gram-positivas e esporos. Tem potencial aplicação em inúmeros campos (farmacêutico, veterinário e cosméticos). O objetivo deste trabalho foi estudar a cinética de crescimento bacteriano e a produção de nisina em biorreator, utilizando leite desnatado diluído, como um meio de cultura a baixo custo. Também foram avaliados os consumos de açúcar e proteína, formação de ácido lático e adsorção de nisina nas células produtoras durante os processos de produção de nisina. Pré-cultivos com 107 UFC.mL-1 de Lactococcus lactis foram cultivados em biorreator de 2 L contendo 25% de leite desnatado diluído em água (1,5 L, pH 6,7). Os ensaios foram esenvolvidos a 30°C por 52 horas, variando a agitação e aeração: (i) 200 rpm (0,0, 0,5, 1,0 e 2,0 L.min-1) e (ii) 100 rpm (0,0 e 0,5 L.min-1). A atividade de nisina foi avaliada pelo método de difusão em ágar, utilizando Lactobacillus sakei ATCC 15521 como microrganismo sensível à ação de nisina. A melhor concentração de nisina (62,68 mg.L-1 ou 2511,89 AU.mL-1), foi obtida em 16 horas, 200 rpm e sem aeração (kLa = 5,29 x 10-3 h-1). A adsorção de nisina nas células produtoras foram baixas (6,8 - 15,1%), quando comparadas com a atividade do sobrenadante. Estes resultados mostraram que o meio de cultivo composto por leite desnatado diluído favoreceu o crescimento celular e produção associada ao crescimento da nisina. Foram realizados estudos preliminares de liofilização (bioconservação) e purificação por cromatografia da nisina produzida em biorreator. A liofilização apresentou perda da atividade de nisina (24,8%), enquanto a purificação por cromatografia de interação hidrofóbica com resina Butyl-Sepharose, recuperou 40% da atividade da biomolécula, mostrando que ambos os processos poderão ser aplicados à bacteriocina. / Nisin is a natural antimicrobial peptide produced by Lactococcus lactis subsp. lactis ATCC 11454 during its exponential growth phase. The bacteriocin is used as natural food preservative due to its antimicrobial activity against Gram-positive bacteria and outgrowth of spores. This property allows its application in numerous fields (pharmaceutical, veterinary and cosmetic). The aim of this work was to study the bacterial growth kinetics of L. lactis and respective nisin production in bioreactor, using diluted skimmed milk as an inexpensive medium. During the production, the consumption of sugar and protein, lactic acid formation and nisin adsorption on the producer strain cells were evaluated. Pre-cultivation with 107 UFC.mL-1 of L. lactis were expanded in a 2 L bioreactor containing 25% diluted skimmed milk in water (1.5 L, pH 6.7). The assays were performed at 30°C for 52 hours, varying agitation and airflow rate: (i) 200 rpm (0.0, 0.5, 1.0 and 2.0 L.min-1) and (ii) 100 rpm (0.0, 0.5 L.min-1). Nisin activity was evaluated through diffusion assays using Lactobacillus sakei ATCC 15521 as sensitive strain. The best nisin concentration (62.68 mg.L-1 or 2511.89 AU.mL-1), was achieved at 16 hours, 200 rpm and with no airflow rate (kLa = 5.29 x 10-3 h-1). The quantity of nisin adsorbed by the producer cells were low (6.8 -15.1%) when compared to the quantity released in the supernatant. These results showed that diluted skimmed milk supported cell growth and growth-associated nisin. Preliminary assays of lyophilization (biopreservation) and purification by chromatography of nisin produced in bioreactor were performed. Lyophilization presented a loss of nisin activity (24.8%) while purification by hydrophobic interaction chromatography with Butyl-Sepharose column recovered 40% of the activity, showing that both processes can be applied to the bacteriocin.

In this final thesis, an existing method for analysis of chlorophenols (CP) in bottom sediments has been updated and adjusted for analysis of chlorophenols in soil. The covalent bonds between the chlorophenols and the soil matrix were broken through basic hydrolysis and the chlorophenols were then separated from the water phase through addition of sulphuric acid followed by ether extraction. The chromatography was improved through extractive acetylation of the chlorophenols. The updated method was then applied on soil samples from a contaminated area (a former sawmill in Hyttsjö, Östergötland, Sweden). The analyse was preformed by GC/MS with respect to 2-MonoCP, 4-MonoCP, 2,4-DiCP, 2,6-DiCP, 2,4,6-TriCP, 2,3,4,6-TetraCP and pentachlorophenol (PCP). Contamination of chlorophenols in nature can be explained by the former use of wood preservative chemicals based on chlorophenols. In the 1960s and the 1970s these chemicals were used in Sweden, but due to their toxicity they were banned by the Swedish government in 1978. In Hyttsjö a pentachlorophenol-based product named Santobrite was used for several years. The concentration of PCP in the soil samples from Hyttsjö varied from 0.2->1.8 ng/mg dry substance. 2,3,4,6- Tetrachlorophenol was also detected in some of the soil samples.

<p>In this final thesis, an existing method for analysis of chlorophenols (CP) in bottom sediments has been updated and adjusted for analysis of chlorophenols in soil. The covalent bonds between the chlorophenols and the soil matrix were broken through basic hydrolysis and the chlorophenols were then separated from the water phase through addition of sulphuric acid followed by ether extraction. The chromatography was improved through extractive acetylation of the chlorophenols.</p><p>The updated method was then applied on soil samples from a contaminated area (a former sawmill in Hyttsjö, Östergötland, Sweden).</p><p>The analyse was preformed by GC/MS with respect to 2-MonoCP, 4-MonoCP, 2,4-DiCP, 2,6-DiCP, 2,4,6-TriCP, 2,3,4,6-TetraCP and pentachlorophenol (PCP).</p><p>Contamination of chlorophenols in nature can be explained by the former use of wood preservative chemicals based on chlorophenols. In the 1960s and the 1970s these chemicals were used in Sweden, but due to their toxicity they were banned by the Swedish government in 1978.</p><p>In Hyttsjö a pentachlorophenol-based product named Santobrite was used for several years. The concentration of PCP in the soil samples from Hyttsjö varied from 0.2->1.8 ng/mg dry substance. 2,3,4,6- Tetrachlorophenol was also detected in some of the soil samples.</p>

Submitted by Maria Suzana Diniz (msuzanad@hotmail.com) on 2015-11-09T12:32:47Z No. of bitstreams: 1 arquivototal.pdf: 1303304 bytes, checksum: 33ba8f9a576e8914a5915254673c5f79 (MD5) / Made available in DSpace on 2015-11-09T12:32:47Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1303304 bytes, checksum: 33ba8f9a576e8914a5915254673c5f79 (MD5) Previous issue date: 2014-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The study of vulnerability to sexually transmitted diseases seeks at understanding the different risks that any individual has of getting contaminated, due to individual and social characteristics that can be relevant to a greater exposure to the human immunodeficiency virus (HIV). Based on this premise, grounded on the Theory of Planned Behavior, the Theory of Control Locus, and the Vulnerability Model, two studies were conducted. Study 1 aimed at carrying out a survey about women’s beliefs about masculine preservative use by the partner. Study 2 aimed at investigating the behavior intention of women toward preservative use. Results from Study 1, based on a convenience sample of 35 women from low socioeconomic status in the city of João Pessoa, demonstrated behavioral beliefs regarding the advantages of using preservative, highlighting the knowledge of its contraceptive role and its function for preventing sexually transmitted diseases. Nevertheless, disadvantages were also mentioned, such as being uncomfortable and reducing pleasure when the partner uses preservative. As positive model references for preservative use, they mentioned family and doctors, and as negatives, friends and partner. Based on the results of the first study, an instrument was developed and answered by 244 women, comprising Study 2, which had as independent variables Indirect Attitudes, Direct Attitudes, Subjective Norm and Perceived Control, and as dependent variable, the Intention of using preservative. It was observed that the variable Perceived Control (β= .43) was the main responsible for the explained variance of the intention of preservative use, followed by the dimension Subjective Norm (β = .30). Direct Attitude (β= .15) had a good significance level, but small explanatory power. Moreover, it was verified that most women who declared using contraceptive pills declared safe sex is practiced “sometimes”. On the other hand, most of the women who do not make use of contraceptive pills declared that they “always” use preservative. Significant correlations between the four dimensions of locus of control and behavior intentions were not found. These results can be considered a reference for studies and interventions about the importance of preservative use in sexual relations. / O estudo da vulnerabilidade às doenças sexualmente transmissíveis busca entender as diferentes chances que todo e qualquer indivíduo tem de se contaminar, decorrente de características individuais e sociais que podem ser relevantes para uma maior exposição ao vírus da imunodeficiência humana (HIV). Com base nessa premissa, fundamentados na Teoria do Comportamento Planejado, na Teoria do Lócus de Controle e no Modelo da Vulnerabilidade, desenvolveu-se dois estudos. O Estudo 1 teve como principal objetivo realizar um levantamento das crenças de mulheres acerca do uso do preservativo masculino pelo parceiro. Já no Estudo 2, o objetivo principal foi investigar a intenção comportamental de mulheres quanto ao uso do preservativo. Os resultados do Estudo 1, a partir de amostra por conveniência formada por 35 mulheres, de classe social menos favorecida, da cidade de João Pessoa, apontaram crenças comportamentais acerca das vantagens do uso do preservativo evidenciando o conhecimento acerca do papel contraceptivo e a função de evitar doenças sexualmente transmissíveis. Não obstante, como desvantagem, ressaltaram incômodo e a diminuição do prazer quando o parceiro utiliza o preservativo. Como referentes modais positivos para o uso de preservativo, foram citados a família e os médicos, enquanto como referentes modais negativos, foram referidos os amigos e o parceiro. A partir dos resultados do primeiro estudo, foi construído um instrumento aplicado em 244 mulheres, constituindo o Estudo 2, que teve como variáveis independentes as Atitude Indireta, Atitude Direta, Normas Subjetivas e Controle Percebido e, como variável dependente, a Intenção do uso do preservativo. Observou-se que a variável Controle Percebido (β= 0,43, p < 0,01) foi o principal responsável pela explicação da variância da intenção de uso do preservativo, seguida da dimensão Norma Subjetiva (β = 0,30, p < 0,01). A Atitude Direta (β= 0,15, p < 0,01) obteve nível de significância bom, porém com baixo poder de explicação. Adicionalmente, verificou-se que a maioria das mulheres que declaram fazer uso da pílula anticoncepcional afirmam a prática do sexo seguro “às vezes”. Por outro lado, a maioria das mulheres que não usam pílula relatam que faz uso do preservativo “sempre”. Não foram observadas correlações consideráveis entre as quatro dimensões de lócus de controle e a intenção comportamental. Estes resultados podem ser considerados como mais uma referência para estudos e intervenções sobre a importância do uso de preservativo nas relações sexuais.

La qualité microbiologique d’un produit cosmétique se doit d’être conservée durant la totalité de sa durée de vie grâce à l’ajout notamment de conservateurs antimicrobiens. Néanmoins, les conservateurs organiques traditionnellement utilisés sont particulièrement décriés depuis quelques années car suspectés d’effets secondaires. Dans l’objectif de disposer d’alternatives à ces substances, les propriétés antimicrobiennes de l’oxyde de zinc (ZnO) ont été étudiées. L’efficacité antimicrobienne de ces particules inorganiques a ainsi été évaluée sur les cinq micro-organismes d’intérêt pour l’industrie cosmétique (E. coli, S. aureus, P. aeruginosa, C. albicans et A. brasiliensis). Pour cela des tests microbiologiques ont été mis en place en milieu gélosé et bouillon liquide pour évaluer la sensibilité de chaque souche microbienne au ZnO. L’efficacité de ces poudres a été évaluée au sein de diverses formules cosmétiques via la réalisation de Challenge Tests. Des études spécifiques visant à améliorer la compréhension des mécanismes antimicrobiens du ZnO ont été menées : les phénomènes de dissolution des particules générant des cations zinc, de production photochimique de radicaux libres ou encore de contact direct entre particules et cellules microbiennes ont été approfondis. Couplés à des études supplémentaires visant à affiner les relations structure/activité, ces travaux ont été menés dans l’objectif d’optimiser le potentiel antimicrobien de ces poudres pour la présente application. Ce projet a démontré que le ZnO permettait de conserver la qualité microbiologique de produits dermopharmaceutiques variés, allant des émulsions aux poudres. Dépendamment de sa concentration, les actions bactéricides, levuricide et fongistatique du ZnO confèrent aux produits la capacité de répondre aux exigences requises en termes de conservation. Les poudres minérales de ZnO apparaissent alors comme une alternative appropriée aux conservateurs organiques / The microbiological quality of a cosmetic product should be preserved during its whole shelf-life notably thanks to the addition of antimicrobial preservatives. Nevertheless, commonly used organic preservatives are particularly criticized since a few years because they are suspected of side effects. In order to propose alternatives to these substances, the antimicrobial properties of zinc oxide (ZnO) were studied. The antimicrobial efficacy of these inorganic particles was evaluated on the five microorganisms of interest for the cosmetic industry (E. coli, S. aureus, P. aeruginosa, C. albicans and A. brasiliensis). Microbiological tests were designed in agar medium and liquid broth to evaluate the sensitivity of each microbial strain to ZnO. The efficacy of these powders was evaluated in various dermopharmaceutical formulations via Challenge Tests. Some specific studies dedicated to improve the understanding of antimicrobial mechanisms of ZnO were carried out: (i) particles dissolution generating zinc cations, (ii) photochemical generation of free radicals (iii) direct contact between particles and microbial cells. Coupled with additional studies designed to refine structure/activity relationships, this work was performed in order to optimize the antimicrobial potential of these powders for the present application. All these studies demonstrated that ZnO enabled the preservation of the microbiological quality of various cosmetic products (emulsions and powders). The bactericidal, levuricidal and fongistatic activities of ZnO were dependent of its concentration and confer to the products the ability to comply with the demands in term of preservation. The inorganic powders of ZnO appear as suitable alternatives to organic preservatives

Orientador: Márjorie de Assis Golim / Resumo: O sistema de qualidade é de suma importância em laboratórios clínicos para avaliação de processos analíticos de maneira que os resultados liberados sejam verdadeiros. Para a metodologia de imunofenotipagem celular por citometria de fluxo as amostras devem ser frescas e os exames realizados preferencialmente dentro de 48 horas. É relevante utilizar amostras de controle de qualidade internos (CQI) padronizadas, de modo que possam ser repetidas rotineiramente, como referencial de qualidade. No Brasil, poucos serviços comercializam amostras preservadas para uso como CQI. Deste modo, a padronização in house com validação de processo para obtenção de amostras que possam ser utilizadas para esta finalidade é relevante. O objetivo deste trabalho foi desenvolver controle de qualidade interno para as rotinas de quantificação de células progenitoras hematopoéticas (CPH), utilizando solução preservante e avaliar a reprodutibilidade e estabilidade ao longo do tempo. Foram preparadas soluções preservantes contendo diferentes concentrações de anticoagulantes e fixadores, e destas, foi selecionada uma composição, originalmente padronizada neste estudo. Foram utilizados 5mL de sangue periférico, sendo este acrescido da solução a ser testada. Imediatamente, realizou-se a quantificação das populações de CPH em tubo Trucount®, usando anti-CD45, anti-CD34 e 7-AAD, conforme indicado pelo fabricante. A leitura foi realizada em citômetro de fluxo modelo FACSCalibur®-BD, para obtenção dos valores abs... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The quality system is of paramount importance in clinical laboratories for evaluating analytical processes in order to consider true the released results. The samples must be performed fresh preferably within 48 hours for the cell immunophenotyping methodology by flow cytometry. It is relevant to use standardized internal quality control (IQC) samples, thus they could be repeated routinely, as a quality benchmark. In Brazil, only a few services commercialize preserved samples for use as IQC. Therefore, it is relevant to use in-house standardization with process validation to obtain samples that can be used for this purpose. The objective of this work was to develop an IQC for a daily routine quantification of hematopoietic stem cells (HSCs) by using a preservative solution and to evaluate the reproducibility and stability over time. Preservative solutions containing different concentrations of anticoagulants and fixatives were prepared, and from these, a composition was selected, which was previously originally standardized in this study. 5mL of peripheral blood were used, which was added to the solution to be tested. The HSCs populations were immediately quantified in a Trucount® tube, using anti-CD45, anti-CD34 and 7-AAD, as indicated by the manufacturer. The reading was performed in a flow cytometer model FACSCalibur®-BD in order to obtain the absolute values of HSCs on day zero, 7, 21, 35 and 49. During this period, the samples were kept refrigerated (2 to 8ºC). The value... (Complete abstract click electronic access below) / Mestre

Cette thèse de doctorat a été réalisée dans le cadre du projet NATUBAVAL visant à découvrir de nouveaux conservateurs naturels pour l’industrie cosmétique, à partir de plantes issues d’un des hotspots mondiaux de biodiversité : le bassin méditerranéen. Dix-sept extraits ont ainsi été obtenus et évalués quant à leurs propriétés antimicrobiennes contre Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus brasiliensis et Candida albicans, ainsi que leur capacité antioxydante. Santolina chamaecyparissus a démontré des propriétés remarquables, et a été sélectionnée pour une étude plus approfondie. Une approche par fractionnement bioguidé a permis l’isolement du composé majoritaire de la fraction la plus active, également identifié comme actif : un spirokétal énol connu de la famille des polyacétylènes. Une optimisation de l’extrait brut d’intérêt a alors été entreprise dans le but de maximiser son activité et de faire face au challenge d’une transposition industrielle et de son incorporation dans un produit cosmétique. En définitive, cette étude introduit une stratégie de développement d’un ingrédient naturel pouvant potentiellement être utilisé comme une alternative aux conservateurs de synthèse dans les produits cosmétiques. / This PhD thesis is part of the NATUBAVAL project that aims at discovering new natural preservatives for the cosmetics industry, issued from one of the world’s biodiversity hotspots: the Mediterranean Basin. Seventeen plant extracts were obtained and screened for their antimicrobial properties against Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus brasiliensis and Candida albicans and their antioxidant capacity. Santolina chamaecyparissus extract was found to present superior properties and selected for further investigation. A bioguided fractionation permitted to isolate the major compound of the most active fraction, that was identified as the active compound, a known spiroketal enol from the polyacetylenes family. An optimization of the crude extract of interest was then performed in order to maximize its activity and to face the challenge of an industrial scale-up and its incorporation in a cosmetic formulation. Finally, this study introduces a natural ingredient development strategy that might potentially be used as an alternative to synthetic preservatives in cosmetics.

Biomaterials such as wood and bamboo are in high demand as a building material with the push for building with green technology. The wood product industry accounts for approximately 4% of the total U.S. manufacturing GDP (Gross Domestic Product), which is more than $100 billion. The industry supports over 752,000 full-time equivalent jobs, most of which are in rural areas where employment opportunities are limited. The estimated global market value of bamboo is estimated to be $60 billion annually. This research will explore the use of wood and bamboo in different end use products. The objectives of this research will 1) evaluate the behavior of two single bolt connections in the post-to-rail joint in a hardwood stairway system; 2) the potential of post-treating pre-fabricated cross-laminated timber (CLT) panels with two different copper based preservative treatments; and 3) estimated design values for a commercially sourced bolt laminated bamboo industrial mat. To accomplish these objectives, this dissertation is divided into five sections: 1) Introduction, 2) Structural performance of the post-to-rail connectors in a hardwood stairway handrail, 3) Development of preservative-treated cross-laminated timber: effects of panel layup and thickness on bonding performance and durability when treated with copper-azole (CA-C) and micronized copper-azole (MCA), 4) Strength and stiffness of 3-ply industrial bamboo matting, 5) Conclusion.

Whole cell biosensors have been extensively used for monitoring toxicity and contamination of compounds in environmental biology and microbial ecology. However, their application in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET) has been limited. According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to construct and develop whole cell microbial biosensors with Pseudomonas aeruginosa ATCC 9027. Constitutive promoters: PlysS, Pspc, Ptat, Plpp and PldcC and the lux-cassette were inserted into plasmid pME4510 and transformed into P. aeruginosa ATCC 9027 cells to produce bioluminescent strains. Plasmids were found to be maintained stably (~50 copies per cell) throughout the growth and death cycle. The novel bioluminescent strains were validated in accordance with the pharmacopoeia using bioluminescence detection and quantification followed by comparison with the traditional plate counting method. The bioluminescent method was found to be accurate, precise and equivalent at a range of 103 – 107 CFU/mL, as compared with plate counting. Recovery of bacterial cells was quantified using bioluminescence; this method proved to be accurate with percentage recoveries between 70-130% for all bioluminescent strains. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. Therefore, the bioluminescent constructs passed/exceeded pharmacopoeial specified criteria for range, limit of detection, accuracy, precision and equivalence. Physiology of the validated bioluminescent strains was studied by assessing the growth and death patterns using constitutive gene expression linked with bacterial replication. Promoter strengths were evaluated at various stages of the growth and death pattern and related to promoter sequences. PlysS, Ptat and Plpp were relatively strong promoters whilst PldcC and Pspc were relatively weak promoters. Relative promoter strength decreased in the order of Plpp>Ptat>PlysS>PldcC>Pspc during the exponential phase whilst Ptat was stronger than Plpp during the stationary phase of growth. Plpp had its highest level of expression during the exponential phase, while Ptat had relatively stable lux expression during the stationary phase. Correlations between relative bioluminescence and CFU at 24 hours were greater than 0.9 indicating a strong relationship for all bioluminescent strains. Reduction in correlation coefficients to approximately 0.6 between relative bioluminescence and CFU and between relative fluorescence and CFU beyond 24 hours indicated that a certain proportion of cells were viable but non-culturable. Tat-pME-lux showed steady bioluminescence compared to CFU count (R>0.9) throughout 28 days of growth. Equivalence analysis showed no significant difference between the bioluminescence and plate count method throughout 28 days of growth for all five bioluminescent strains. Applicability of these novel bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication and bioluminescence as a measure of constitutive gene expression. PET using benzalkonium chloride and benzyl alcohol showed no significant difference between the bioluminescent method and the plate count method. Good correlations between bioluminescence, CFU count and fluorescence were obtained for benzalkonium chloride (BKC) concentrations (R>0.9) between 0.0003% and 0.0025% against strains lysR25, lppR4 and tatH5. Similarly, good correlations (R>0.9) between the three parameters were obtained for benzyl alcohol (BA) concentrations between 0.125% and 2% against strains lysR25, lppR4 and tatH5. The bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003 - 0.02%) and BA (0.25 - 2%) during preservative efficacy tests. These bioluminescent constructs therefore are good candidates for selection for preservative efficacy testing. The bioluminescent method and traditional plate counting method were also found to be equivalent for construct tatH5 at a concentration of 0.125% BA. PET testing with BKC and BA showed that tatH5-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence. Together with the results from growth and death kinetics, where tatH5 showed the greatest constitutive expression, it can be concluded that P. aeruginosa ATCC 9027 tatH5-pMElux is the best construct for testing various antimicrobial agents. This study has shown that according to the pharmacopoeial requirements, the bioluminescent method is more accurate, precise and equivalent to the traditional plate counting method and therefore can be utilised instead of the traditional plate counting method for the purpose of preservative efficacy testing.

The effects of ACQ solution parameters such as copper to quat ratio, pH and copper to ligand ratio on distribution of copper complexes in solution and insoluble precipitates, and on fixation and leaching of copper in treated wood were evaluated. The distribution of ionic complexes, predicted by equilibrium speciation model (MINTEQA2), was related to laboratory fixation and leaching results at controlled ACQ solution parameters. A decrease in the relative proportion of copper in the ACQ formulation from a copper oxide (CuO) to didecyldimethylammonium carbonate (DDACb) ratio of 2:1 to 1:1 and 1:2 resulted in lower copper retention in the treated samples and substantially decreased the amount of copper leached per unit area. For monoethanolamine (Mea) based ACQ, solution parameters which favour a higher proportion of monovalent cationic complex, which consume one reactive site in wood, and the presence of insoluble carbonate precipitate of copper in wood during preservative treatment resulted in higher leach resistance compared to the neutral copper complex present at higher pH. Ammonia (NH3) based ACQ can fix more copper at high pH as there is no chelated neutral complex as in Mea based ACQ; however divalent copper-NH3 complexes may consume two sites to fix in wood. Addition of NH3 in Mea based ACQ at Cu:Mea:NH3 ratio of 1:4:6 at pH 10.6 significantly reduced copper leaching compared to 1:4:0 (without ammonia) at pH 9 due to increased divalent copper-ammonia complexes and decreased neutral copper amine complex at elevated pH. Ammonia addition with a lower proportion of Mea (1:2.5:4 at pH 10.5-10.7), significantly reduced copper leaching compared to 1:4:0 at pH 9; no reduction was observed for ammonia addition in ACQ with a higher proportion of Mea (1:4:4 at pH 10.45). The lower copper leaching from 1:2.5:4 resulted from the higher amount of divalent copper-NH3 complexes at higher pH without compromising the amount of copper precipitated at lower pH. The higher percent copper leached from tetramethylethylenediamine (Tmed) based ACQ compared to Mea and NH3 based ACQ suggested that highly stable complexes tend to stay in solution and do not result in leach resistant copper in the wood.