Esta pesquisa foi apoiada pelo programa de pesquisa de ciência básica (número do projecto: 2016R1A2B4013467) através do nacional pesquisa Fundação da Coreia (NRF) financiado pelo Ministério da ciência, TIC & futuro planejamento 2016 e também apoiada pela ciência básica de Coreia Instituir o programa de pesquisa (número do projecto: C36707).

1. síntese de DMMTAV
<ol><li>
Preparação química e razão molar mistura de DMAVat2S e H2SO4 Nota: DMAV: at2S:H2então4 = 1:1.6:1.6<ol>
<li>Dissolver 5,24 g de DMAV em 40 mL de água deionizada e N2-expurgados (descartados pelo menos 30 min) água num tubo de centrífuga de 50 mL.</li>
		<li>Preparar o reagente de2S at dissolvendo 14,41 g de at2S·9H2O em 50 mL de água deionizada e N2-expurgados de água em um balão de 250 mL.</li>
		<li>Preparar H2para4 reagente adicionando 3,3 mL de ácido sulfúrico concentrado (96%) para 40 mL de água deionizada e N2-expurgados de água em um tubo de centrífuga de 50 mL. Nota: A relação molar Final de DMAV: Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15.</li>
		<li>Adicione o preparado 40 mL da solução DMAV (etapa 1.1.1) para os 50 mL de at2S solução contida no frasco de 250 mL (passo 1.1.2). Enxaguar o tubo contendo DMAV com 10 mL de água purgada e adicionar esta para o balão de 250 mL também.</li>
		<li>Feche o frasco com uma rolha de borracha do três-furo equipada com tubos de vidro. Use o vidro tubos de influxo de gás N2 , vazão e H2entrada de solução SO4 , respectivamente. Imediatamente depois de fechar o frasco, permita o fluxo de gás de2 N para o balão. Nota: A pressão do gás deve ser mantida a fluir sobre a superfície da solução de reação sem salpicos.</li>
		<li>Conectar-se resistente aos ácidos de tubos para o tubo de vidro do H2entrada de solução4 SO com uma seringa de 50 mL, adicionar o preparado de 40 mL de H2então4 solução (etapa 1.1.3). Adicionar 40 mL de H2então4 solução, lentamente e de forma gradual. Atenção: Após a adição de ácido sulfúrico, fumos brancos serão gerados; Use uma coifa bem ventilada.</li>
		<li>Monitorar a mudança de cor da mistura no recipiente de reação, enquanto a adição de ácido sulfúrico em intervalos regulares. Manter um intervalo de 4-5 mL gotas de H2então4; a mistura deve ser uma solução turva branca. Nota: Precipitação instantânea de amarela pode aparecer devido a rápida adição de ácido sulfúrico concentrado.</li>
		<li>Certifique-se de que solução a reação tem sido permanente por 1h, desde o início da etapa 1.1.4.</li>
	</ol>
</li>
	<li>
Extração deV DMMTA usando o método de extração líquido-líquido
	<ol>
<li>Após 1 h, despeje a solução de reação em um funil de separação contendo cerca de 200 mL de éter dietílico.</li>
		<li>Agite o funil durante 5-10 min, liberando gás várias vezes, alternando a torneira. Nota: Sintetizado DMMTAV vai ser transferido para a camada superior de éter dietílico (0.713 g·mL-1). Atenção: Gás de éter dietílico podem ser prejudicial; Use uma coifa bem ventilada.</li>
		<li>Recolher a solução de reação num copo e recolher separadamente o éter dietílico contendo DMMTAV em uma garrafa. Coloque a solução de reação volta no mesmo funil de separação e adicionar cerca de 200 mL de fresco éter dietílico para reshaking. Repita os passos 1.2.2 - 1.2.3 três vezes.</li>
		<li>Despeje o éter dietílico coletado da etapa 1.2.3 no funil de separação mesmo novamente e adicionar cerca de 100 mL N2-expurgados de água desionizada. Agitar durante 5-10 min e descartar o N2-expurgados de água desionizada e alguns mL de éter dietílico para fins de pureza. Colete o restante éter etílico em um prato de petri de vidro (diâmetro interno mínimo de 160 mm) e altura mínima de 50 mm.</li>
		<li>Transferi o prato de petri de vidro em uma caixa de luva N2 atmosfera para impedir a transformação de espécies. Atenção: Certifique-se que o solvente não é puxado para a bomba de vácuo através da saída da caixa de passagem.</li>
		<li>Seco até um precipitado branco de dimethylmonothioarsinate (Vde DMMTA cristalizada) é formada sobre o prato de petri de vidro. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Verificação de armazenamento e sintetizado DMMTAV
	<ol>
<li>Leve o precipitado branco de cristalizada DMMTAVe medir e registrar seu peso total. Atenção: Use uma coifa bem ventilada ou luvas para evitar a inalação de gás sulfeto de hidrogênio.</li>
		<li>Dissolver cristalizado DMMTAV em 50 mL de N2-purga de água desionizada e filtrar o precipitado amarelo através de um filtro de seringa 0,2 µm.</li>
		<li>Assumir que o total a partir DMA usadosV é convertido em DMMTAV, ou seja,≈9, 649 mg As· L-1. Diluir a solução-mãe DMMTAV para ≈1 mg· L-1 e ≈40 µg· L-1 para análise de verificação utilizando ionização Electrospray Mass Spectromtery (ESI-MS) e HPLC-ICP-MS, respectivamente.</li>
		<li>Analise a m/z de DMMTAV utilizando ESI-MS11,16 e fragmentos, em m/z 155 no modo positivo-íon ou em m/z 153 no modo negativo-íon (tabela 1). Nota: Ver referência os valores de m/z (tabela 1).</li>
		<li>Cromatograma do DMMTAV na solução estoque usando HPLC-ICP-MS11,16,17 com eluente adequadas condições de analisar e confirmar um grande pico encontra-se no tempo de retenção, descrito no literatura. Nota: Pureza de sintetizado DMMTAV deve ser calculada utilizando os resultados da análise da etapa 1.3.5.</li>
		<li>Analisar a concentração de arsênio total usando ICP-MS após digestão com ácido11 e calcular a concentração real de DMMTAV em solução-mãe de DMMTAV sintetizada usando fatores de diluição e pureza, como na seguinte equação: Total analisado como concentração (µg· L-1) · · Fator de diluição Pureza (%) = concentração de verdade DMMTAV DMMTAV solução estoque (µg· L-1)</li>
		<li>Armazenar a solução estoque de DMMTAV a 4 ° C no escuro por mais análise quantitativa especiação18.</li>
	</ol>
</li>
</ol>2. síntese de DMDTAV
<ol><li>
Preparação química e razão molar mistura de DMAV at2S e H2SO4 Nota: DMAV: at2S:H2então4 = 1:7.5:7.5<ol>
<li>Dissolva 1,38 g de DMAV em 40 mL de água desionizada num tubo de centrífuga de 50 mL.</li>
		<li>Prepare o reagente de2S at dissolvendo 18,01 g de Na2S·9H2O em um 50 mL de água desionizada num balão de 250 mL.</li>
		<li>Prepare-se H2para que4 reagente adicionando 4 mL de ácido sulfúrico concentrado (96%) para 40 mL de água desionizada contido num tubo de centrífuga de 50 mL. Nota: A relação molar Final de DMAV: Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15.</li>
		<li>Adicione o preparado 40 mL da solução DMAV (etapa 2.1.1) para os 50 mL de at2S solução contida no frasco de 250 mL (passo 2.1.2). Enxaguar o tubo contendo DMAV com 10 mL de água desionizada e adicionar esta para o balão de 250 mL também.</li>
		<li>Adicionar o preparado 40 mL de H2então4 solução (etapa 2.1.3), lentamente e de forma gradual, para o balão.</li>
		<li>Monitore a mudança de cor da mistura no recipiente de reação, enquanto a adição de ácido sulfúrico em intervalos regulares. Manter um intervalo de 4-5 mL gotas de H2então4; a mistura deve ser uma solução turva branco/amarelo. Nota: Precipitação instantânea de amarela pode aparecer devido a uma rápida adição de ácido sulfúrico concentrado. Atenção: Após a adição de ácido sulfúrico, fumos brancos serão gerados; Use uma coifa bem ventilada.</li>
		<li>Manter a solução de reação no frasco durante a noite sem cobertura. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Extração deV DMDTA usando o método de extração de fase sólida (SPE)
	<ol>
<li>Após a reação de pé durante a noite, filtro da solução de reação utilizando uma seringa de C18 digite SPE baseada em sílica para aprisionar sintetizado DMDTAV sobre a resina. Atenção: Use uma coifa bem ventilada.</li>
		<li>Prepare o acetato de amônio 10 mM (pH 6.3) pela dissolução do acetato de amónio de 0,77 g em 1L de água desionizada. Eluir um volume suficiente de acetato de amónio de 10 mM através da seringa C18 (etapa 2.2.1) para extrair o adsorvida DMDTAV. Recolha o acetato de amónio filtrada em um copo de Petri (diâmetro interno mínimo de 160 mm) e altura mínima de 50 mm.</li>
		<li>Transferi o prato de petri de vidro em uma caixa de luva N2 atmosfera para impedir a transformação de espécies. Atenção: Certifique-se de solvente não é puxado para a bomba de vácuo através da saída da caixa de passagem.</li>
		<li>Secar até um precipitado branco de dimethyldithioarsinate é formado (cristalizada DMDTAV) sobre o prato de petri de vidro. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Verificação de armazenamento e sintetizado DMDTAV
	<ol>
<li>Pegue o precipitado branco de cristalizada DMDTAVe medir seu peso total, a medição de gravação. Atenção: Use uma coifa ventilada ou luvas para evitar a inalação de gás sulfeto de hidrogênio.</li>
		<li>Dissolver cristalizado DMDTAV em 50 mL de N2-água desionizada purgado em 50 mL de um tubo de centrifugação e filtrar qualquer precipitado através de um filtro de seringa 0,2 µm.</li>
		<li>Suponha que o total a partir DMA usadosV é convertido em DMDTAV, ou seja,≈2, 539 mg As· L-1. Diluir a solução-mãe DMDTAV para ≈1 mg· L-1 e ≈40 µg· L-1 para análise de verificação utilizando ESI-MS e HPLC-ICP-MS, respectivamente.</li>
		<li>Analise a m/z de DMDTAV utilizando ESI-MS11,16 e fragmentos, em m/z 171 no modo positivo-íon ou em m/z 169 no modo negativo-íon (tabela 1). Nota: Ver referência os valores de m/z (tabela 1).</li>
		<li>Analisar o cromatograma da DMDTAV na solução estoque usando HPLC-ICP-MS11,16,17 com condições adequadas de eluente e confirmar que um pico encontra-se no tempo de retenção, descrito em a literatura. Nota: Pureza de sintetizado DMDTAV deve ser calculada utilizando os resultados da análise da etapa 2.3.5.</li>
		<li>Analisar a concentração de arsênio total usando ICP-MS após digestão com ácido11 e calcular a concentração real de DMDTAV em solução-mãe de DMDTAV sintetizada usando fatores de diluição e pureza, como a seguinte equação: Total analisado como concentração (µg· L-1) · · Fator de diluição Pureza (%) = concentração de verdade DMDTAV DMDTAV em solução-mãe (µg· L-1)</li>
		<li>Armazenar a solução estoque de DMDTAV a 4 ° C no escuro por mais análise quantitativa especiação18.</li>
	</ol>
</li>
</ol>

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O protocolo desenvolvido clarificou passos críticos que estudos anteriores1,3,4,8,15 omitido ou abreviado, que pode ter levado a dificuldades com ou falha durante Síntese deV DMMTAV e DMDTA. Como DMMTAV é sensível à oxidação1,5, reagentes químicos para sua sínt...

Desde que o ácido dimethylarsinic (DMAV) demonstrou-se a expor tanto a toxicidade aguda e a genotoxicidade devido a sofrer metilação e thiolation após a ingestão de1,2, a via metabólica de arsênico thiolation tem foi intensamente estudado tanto in vitro e in vivo3,4 , bem como em meios ambientais (por exemplo, percolado de aterro)5,6. Estudos anteriores descobriram ambos reduziram e células thiolated análogos de DMAV na vida, por exemplo, dimethylarsinous ácido (DMAIII), ácido dimethylmonothioarsinic (DMMTAV) e dimethyldithioarsinic (ácido DMDTAV)7,8,9, com dimethylated thioarsenicals como DMMTAV apresentando toxicidade maior do que outro conhecido inorgânicos ou orgânicos arsenicals10. A abundância de thioarsenicals altamente tóxicos tem sérias implicações ambientais, uma vez que eles podem representar um risco para os seres humanos e o ambiente sob condições altamente sulfidic11. No entanto, os mecanismos de DMMTAV e DMDTAV (trans) formação e seus destinos em meios ambientais ainda exigem um estudo mais aprofundado. Assim, a análise quantitativa de thioarsenicals é necessário para melhorar a compreensão dos efeitos ambientais de DMMTAV e DMDTAV.
Embora produtos químicos padrão são o requisito-chave para análise quantitativa, os padrões de DMMTAV e DMDTAV são difíceis de obter, replicando a estudos anteriores, devido ao alto risco de transformação de espécies em outras espécies e procedimentos de síntese unstandardized12. Além disso, os métodos referenciados têm limitações que podem levar a dificuldades de ordem práticas em sintetizar os químicos padrão e realizar a análise quantitativa. DMMTAV DMDTAV comumente preparam e misturando DMAV, Na2S e H2para4 , em uma certa relação molar1 ou borbulhamento H2S de gás através de uma solução de DMAV 13,14. A substituição de recursos de método subida de oxigênio por enxofre usando um fornecimento directo de gás H2S, que é altamente tóxico e difícil de controlar para um usuário inexperiente. Por outro lado, o acima mistura método1, amplamente utilizado para a análise qualitativa de DMMTAV e DMDTAV em sudies ambiental5,6,12, apresenta o thiolation de DMAV com H2S gerados pela mistura Na2S e H2para que4 e produz DMMTAV e DMDTAV, permitindo mais fácil estequiométrico controle para produzir produtos químicos de alvo, como em comparação com o direto uso do gás de H2S.
A referência que mistura método procedimentos1,3,4,8,15 mencionado em limitações de exposição deste estudo em alguns dos seus passos críticos experimentais, que podem levar a falha experimental. Por exemplo, os detalhes da preparação de solvente específico (ou seja, água deionizada, água) e a extração e cristalização dos arsenicals sintetizados são excessivamente abreviados ou não descritas em detalhes suficientes. Tais dispersaram e informações limitadas sobre actos processuais podem levar à formação inconsistente de thioarsenicals e análise de quantificação confiável. Portanto, o protocolo modificado desenvolvido neste documento descreve a síntese de DMMTAV e DMDTAV soluções estoque com análise de separação quantitativa de espécies.

<table width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Cacodylic acid</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">20835-10G-F</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Sodium sulfide nonahydrate</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">S2006-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Sulfuric acid 96%</td>
			<td style="width:179px;">J.T.Baker</td>
			<td style="width:179px;">0000011478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Ammonium acetate</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">A7262-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Formic acid 98%</td>
			<td style="width:179px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:179px;">066-00461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Diethyl ether (Extra Pure)</td>
			<td style="width:179px;">Junsei Chemical</td>
			<td style="width:179px;">33475-0380</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Adapter cap for 60 mL Bond Elut catridges</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">12131004</td>
			<td>Syringe type of SPE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Bond Elut C18 cartridge</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">14256031</td>
			<td>Syringe type of SPE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">HyPURITY C-18</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:179px;">22105-254630</td>
			<td>5 um, 125 x 4.6 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Glovebox</td>
			<td style="width:179px;">Chungae-chun, Rep. of Korea</td>
			<td style="width:179px;"></td>
			<td>Customized&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Agilent 1260 Infinity Bio-inert LC</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">DEAB600252, DEACH00245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Agilent Technologies 7700 Series ICP-MS</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">JP12031510</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:179px;height:63px;">Finnigan LCQ Deca XP MAX Mass Spectrometer System</td>
			<td style="width:179px;">Thermo Electron Corporation</td>
			<td style="width:179px;">LDM10627</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimethylated thioarsenicals, tais como o ácido dimethylmonothioarsinic (DMMTAV) e ácido dimethyldithioarsinic (DMDTAV), que são produzidas pela via metabólica de dimethylarsinic ácido (DMAV) thiolation, tem sido recentemente encontrado o ambiente, bem como os órgãos humanos. DMMTAV e DMDTAV podem ser quantificado para determinar os efeitos ecológicos de dimethylated thioarsenicals e sua estabilidade em meios ambientais. O método de síntese para esses compostos é unstandardized, fazendo replicando desafiador de estudos anteriores. Além disso, há uma falta de informação sobre técnicas de armazenamento, incluindo armazenamento de compostos sem transformação de espécies. Além disso, porque apenas informações limitadas sobre métodos de síntese estão disponíveis, pode haver dificuldades experimentais em sintetizar produtos químicos padrão e realizar a análise quantitativa. O protocolo apresentado neste documento fornece um método de síntese praticamente modificado para o thioarsenicals dimethylated, DMMTAV e DMDTAVe ajudará na quantificação da análise de separação de espécies usando líquido de alto desempenho cromatografia em conjunto com a espectrometria de massa de plasma indutivo (HPLC-ICP-MS). Os passos deste procedimento experimentais foram modificados, centrando-se na preparação de reagentes químicos, métodos de filtração e armazenamento.

Desde que DMMTAV foi erroneamente preparado pelo método de síntese a DMAIII 19, verificação de sintetizado DMMTAV DMDTAV é um passo fundamental para a síntese e a extração e a determinação do padrão ideal materiais químicos. Produtos químicos sintetizados podem ser verificados pelo pico de DMMTAV (154 MW g·mol-1) e proporção de massa-de-carga DMDTAV (170 MW g·mol-1</s...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Preparação de DMMTAV DMDTAV usando DMAV para aplicações ambientais: síntese, purificação e confirmação

Este artigo apresenta protocolos experimentais modificados por ácido dimethylmonothioarsinic (DMMTAV) e a síntese de ácido (DMDTAV) dimethyldithioarsinic, induzindo dimethylarsinic ácido (DMAV) thiolation através da mistura de DMAV , At2S e H2então4. O protocolo modificado fornece uma orientação experimental, desse modo, superando limitações as etapas de síntese que poderia ter causado falhas experimentais em análise quantitativa.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

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Environment

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

5.5K visualizações.  Korea Basic Science Institute, Seoul Center. Este artigo apresenta protocolos experimentais modificados por ácido dimethylmonothioarsinic (DMMTAV) e a síntese de ácido (DMDTAV) dimethyldithioarsinic, induzindo dimethylarsinic ácido (DMAV) thiolation através da mistura de DMAV , At2S e H2então4. O protocolo modificado fornece uma orientação experimental, desse modo, superando limitações as etapas de síntese que poderia ter causado...

Vídeo: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

Ambiente

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Uma Abordagem Bioreporter celular todo avalie Transportes e biodisponibilidade de contaminantes orgânicos em água insaturadas Sistemas

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

Preparação e Teste de impedância baseado em Biochips fluídicas com células RTgill-W1 para avaliação rápida das amostras de água para a toxicidade

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

A Bolsa Benthic de O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; e nutrientes dissolvidos Usando pequeno núcleo incubações

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

O uso de um cartucho de filtro para Amostras de filtração de água e extração de DNA Ambiental

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Ressurreição de dormentes Daphnia magna: protocolo e aplicações

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Composição e análise da distribuição de Bioaerossóis sob diferentes condições ambientais

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Identificação por- e espécies químicas perfluorados com um fluxo de trabalho combinada alvo e não alvo-triagem de alta resolução de espectrometria de massa

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Usando Caenorhabditis elegans para estudar trans-e multi-Generational efeitos de toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

Modelagem do espectro de tamanho para macroinvertebrados e peixes em ecossistemas de fluxo

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

Preparação de DMMTA^V DMDTA^V usando DMA^V para aplicações ambientais: síntese, purificação e confirmação

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