This research was supported by Basic Science Research program (Project number: 2016R1A2B4013467) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT &#38; Future Planning 2016 and also supported by Korea Basic Science Institute Research Program (Project number: C36707).

1. Synthesis of DMMTAV
<ol>
	<li>Chemical preparation and molar ratio mixing of DMAV, Na2S, and H2SO4 
	NOTE: DMAV:Na2S:H2SO4 = 1:1.6:1.6
	<ol>
		<li>Dissolve 5.24 g of DMAV in 40 mL deionized and N2-purged (purged for at least 30 min) water in a 50 mL centrifuge tube.</li>
		<li>Prepare Na2S reagent by dissolving 14.41 g of Na2S&#183;9H2O in 50 mL deionized and N2-purged water in a 250 mL flask.</li>
		<li>Prepare H2SO4 reagent by adding 3.3 mL of concentrated sulfuric acid (96%) to 40 mL of deionized and N2-purged water in a 50 mL centrifuge tube. 
		NOTE: Final molar ratio of DMAV:Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15.</li>
		<li>Add the prepared 40 mL of DMAV solution (step 1.1.1) to the 50 mL of Na2S solution contained in the 250 mL flask (step 1.1.2). Rinse the tube containing DMAV with 10 mL of purged water and add this to the 250 mL flask as well.</li>
		<li>Close the flask with a three-hole rubber stopper fitted with glass tubes. Use the glass tubes for N2 gas inflow, outflow, and H2SO4 solution inlet, respectively. Immediately after closing the flask, allow N2 gas flow into the flask. 
		NOTE: Gas pressure should be maintained to flow over the surface of the reaction solution without splashing.</li>
		<li>Connect acid resistant tubing to the glass tube of the H2SO4 solution inlet with a 50 mL syringe to add the prepared 40 mL of H2SO4 solution (step 1.1.3). Add 40 mL of H2SO4 solution, slowly and in a stepwise manner. 
		Caution: Upon adding sulfuric acid, white fumes will be generated; use a well-ventilated fume hood.</li>
		<li>Monitor color change of the reaction mixture in the flask while adding sulfuric acid at regular intervals. Maintain an interval of 4 - 5 mL drops of H2SO4; the mixture should be a white cloudy solution. 
		NOTE: Instantaneous yellow precipitation might appear due to rapid addition of concentrated sulfuric acid.</li>
		<li>Ensure the reaction solution has been standing for 1 h since the start of step 1.1.4.</li>
	</ol>
	</li>
	<li>DMMTAV extraction using liquid-liquid extraction method
	<ol>
		<li>After 1 h, pour the reaction solution into a separatory funnel containing around 200 mL of diethyl ether.</li>
		<li>Shake the funnel for 5 - 10 min, releasing gas several times by switching the stopcock. 
		NOTE: Synthesized DMMTAV will be transferred to the upper layer of diethyl ether (0.713 g&#183;mL-1). 
		Caution: Diethyl ether gas might be harmful; use a well-ventilated fume hood.</li>
		<li>Collect the reaction solution in a beaker, and separately collect the diethyl ether containing DMMTAV in a bottle. Place the reaction solution back in the same separatory funnel and add about 200 mL of fresh diethyl ether for reshaking. Repeat steps 1.2.2 - 1.2.3 three times.</li>
		<li>Pour the collected diethyl ether from step 1.2.3 into the same separatory funnel again, and add about 100 mL N2-purged deionized water. Shake for 5 - 10 min, and discard the N2-purged deionized water and a few mL of diethyl ether for purity purposes. Collect the remaining diethyl ether in a glass petri dish (minimum inner diameter of 160 mm and minimum height of 50 mm).</li>
		<li>Transfer the glass petri dish into a N2 atmosphere glove box to prevent species transformation. 
		Caution: Ensure the solvent is not pulled into the vacuum pump through the outlet of the pass box.</li>
		<li>Dry until a white precipitate of dimethylmonothioarsinate (crystalized DMMTAV) is formed on the glass petri dish. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Verification of synthesized DMMTAV and storage
	<ol>
		<li>Take the white precipitate of crystalized DMMTAV, and measure and record its total weight. 
		Caution: Use a well ventilated fume hood or glove box to prevent inhalation of hydrogen sulfide gas.</li>
		<li>Dissolve crystalized DMMTAV in 50 mL of N2-purged deionized water, and filter the yellow precipitate through a 0.2-&#181;m syringe filter.</li>
		<li>Assume the total As of used DMAV is converted into DMMTAV, i.e.&#8776;9,649 mg As&#183;L-1. Dilute DMMTAV stock solution to &#8776;1 mg&#183;L-1 and &#8776;40 &#181;g&#183;L-1 for verification analysis using Electrospray ionization Mass Spectromtery (ESI-MS) and HPLC-ICP-MS, respectively.</li>
		<li>Analyze m/z of DMMTAV using ESI-MS11,16 and fragments at m/z 155 in the positive-ion mode or at m/z 153 in the negative-ion mode (Table 1). 
		NOTE: See reference values of m/z (Table 1).</li>
		<li>Analyze chromatogram of DMMTAV in the stock solution using HPLC-ICP-MS11,16,17 with appropriate eluent conditions and confirm a major peak is found at the retention time described in the literature. 
		NOTE: Purity of synthesized DMMTAV should be calculated using the analysis results from step 1.3.5.</li>
		<li>Analyze the total arsenic concentration using ICP-MS after acid digestion11 and calculate the true DMMTAV concentration in synthesized DMMTAV stock solution using dilution factors and purity, as in the following equation: 
		Analyzed total As concentration (&#181;g&#183;L-1) &#183; Dilution factor &#183; Purity (%) = True DMMTAV concentration in DMMTAV stock solution (&#181;g&#183;L-1)</li>
		<li>Store DMMTAV stock solution at 4 &#176;C in the dark for further quantitative speciation analysis18.</li>
	</ol>
	</li>
</ol>
2. Synthesis of DMDTAV
<ol>
	<li>Chemical preparation and molar ratio mixing of DMAV , Na2S, and H2SO4 
	NOTE: DMAV:Na2S:H2SO4 = 1:7.5:7.5
	<ol>
		<li>Dissolve 1.38 g of DMAV in 40 mL deionized water in a 50 mL centrifuge tube.</li>
		<li>Prepare Na2S reagent by dissolving 18.01 g of Na2S&#183;9H2O in a 50 mL deionized water in a 250 mL flask.</li>
		<li>Prepare H2SO4 reagent by adding 4 mL of concentrated sulfuric acid (96%) to 40 mL of deionized water contained in a 50 mL centrifuge tube. 
		NOTE: Final molar ratio of DMAV:Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15.</li>
		<li>Add the prepared 40 mL of DMAV solution (step 2.1.1) to the 50 mL of Na2S solution contained in the 250 mL flask (step 2.1.2). Rinse the tube containing DMAV with 10 mL of deionized water and add this to the 250 mL flask as well.</li>
		<li>Add the prepared 40 mL of H2SO4 solution (step 2.1.3), slowly and in a stepwise manner, into the flask.</li>
		<li>Monitor the color change of the reaction mixture in the flask while adding sulfuric acid at regular intervals. Maintain an interval of 4 - 5 mL drops of H2SO4; the mixture should be a white/yellow cloudy solution. 
		NOTE: Instantaneous yellow precipitation might appear due to a rapid addition of concentrated sulfuric acid. 
		Caution: Upon adding sulfuric acid, white fumes will be generated; use a well ventilated fume hood.</li>
		<li>Maintain the reaction solution in the flask overnight without covering. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>DMDTAV extraction using solid-phase extraction (SPE) method
	<ol>
		<li>After the overnight standing reaction, filter the reaction solution using a C18 syringe type silica-based SPE in order to trap synthesized DMDTAV on the resin. 
		Caution: Use a well-ventilated fume hood.</li>
		<li>Prepare 10 mM ammonium acetate (pH 6.3) by dissolving 0.77 g ammonium acetate in 1 L of deionized water. Elute a sufficient volume of 10 mM ammonium acetate through the C18 syringe (step 2.2.1) to extract the adsorbed DMDTAV. Collect the filtered ammonium acetate in a glass Petri dish (minimum inner diameter of 160 mm and minimum height of 50 mm).</li>
		<li>Transfer the glass petri dish into a N2 atmosphere glove box to prevent species transformation. 
		Caution: Ensure solvent is not pulled into the vacuum pump through the outlet of the pass box.</li>
		<li>Dry until a white precipitate of dimethyldithioarsinate is formed (crystalized DMDTAV) on the glass petri dish. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Verification of synthesized DMDTAV and storage
	<ol>
		<li>Take the white precipitate of crystalized DMDTAV, and measure its total weight, recording the measurement. 
		Caution: Use a well-ventilated fume hood or glove box to prevent inhalation of hydrogen sulfide gas.</li>
		<li>Dissolve crystalized DMDTAV in 50 mL of N2-purged deionized water in a 50 mL centrifuge tube, and filter any precipitate through a 0.2-&#181;m syringe filter.</li>
		<li>Assume that the total As of used DMAV is converted into DMDTAV, i.e.&#8776;2,539 mg As&#183;L-1. Dilute DMDTAV stock solution to &#8776;1&#160;mg&#183;L-1 and &#8776;40 &#181;g&#183;L-1 for verification analysis using ESI-MS and HPLC-ICP-MS, respectively.</li>
		<li>Analyze m/z of DMDTAV using ESI-MS11,16 and fragments at m/z 171 in the positive-ion mode or at m/z 169 in the negative-ion mode (Table 1). 
		NOTE: See reference values of m/z (Table 1).</li>
		<li>Analyze the chromatogram of DMDTAV in the stock solution using HPLC-ICP-MS11,16,17 with appropriate eluent conditions and confirm that a peak is found at the retention time described in the literature. 
		NOTE: Purity of synthesized DMDTAV should be calculated using the analysis results from step 2.3.5.</li>
		<li>Analyze the total arsenic concentration using ICP-MS after acid digestion11 and calculate the true DMDTAV concentration in synthesized DMDTAV stock solution using dilution factors and purity as the following equation: 
		Analyzed total As concentration (&#181;g&#183;L-1) &#183; Dilution factor &#183; Purity (%) = True DMDTAV concentration of DMDTAV in stock solution (&#181;g&#183;L-1)</li>
		<li>Store DMDTAV stock solution at 4 &#176;C in the dark for further quantitative speciation analysis18.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The developed protocol has clarified critical steps that previous studies1,3,4,8,15 omitted or abbreviated, which may have led to difficulties with or failure during DMMTAV and DMDTAV synthesis. As DMMTAV is oxidation-sensitive1,5, chemical reagents for its synthesis were prepared using N2-purged deionized water (steps 1.1.1 - 1.1.3) to prevent the possible retardation of DMAV thiolation and the oxidation of DMMTAV. DMMTAV prepared using this protocol had a purity of 92%. The reference DMDTAV extraction method featured silica-based C18 column extraction with ammonium acetate or phosphate buffer solution as an eluent,3,4,7,8 with the purity of the thus prepared DMDTAV varying depending on the column compartments used, which is not described in references studies3,4,8. In contrast, the use of disposable SPE in the developed protocol (step 2.2.1) allowed the extraction of 88% pure DMDTAV. In addition, previous methods of crystallizing DMDTAV referenced only a freeze-drying procedure20, whereas in this protocol, simple drying of the extracted DMDTAV solution in an atmosphere of N2 inside the glove box (step 2.2.3) produced a white residue of crystalized dimethyldithioarsinate.
Identity verification of the synthesized DMMTAV and DMDTAV is a critical step for determining ideal standard chemical. In our previous work16 based on this protocol, major fragments at m/z 155 in the positive-ion mode and m/z 169 in the negative-ion mode were detected by ESI-MS analysis of as-synthesized DMMTAV and DMDTAV, respectively. The latter fragment was ascribed to [CH3]2AsS(=S)S]- (i.e., [M-H]-), in good agreement with previous results5,17,20,22, whereas the fragment at m/z 155 was assigned to [CH3]2As(=S)(OH) + H]+ (i.e., [M+H]+), again, in agreement with references13,14,17,19,21,24. Since ESI-MS analysis cannot be used as the only verification method, major peaks in the chromatograms of DMMTAV and DMDTAV stock solutions obtained by HPLC-ICP-MS were compared to those reported by Li et al.17 (Figure 1). Although DMAIII is known as an intermediate produced in the initial stage of DMMTAV formation1,7,8,25, it exhibits low stability, disappearing within 70 min19 and therefore is not detectable by this procedure (Figure 1).
Another goal of this study was to suggest storage conditions for DMMTAV and DMDTAV stock solutions with fewer impurities to prevent sulfide-to-oxide conversion and thus achieve greater stability18. The storage conditions used here (i.e., 4 &#730;C in the dark) allowed the preservation of the synthesized DMMTAV and DMDTAV as major species in stock solutions (Figure 2) for four weeks, with no drastic decomposition observed even after 13 weeks. Although the solvent pH (i.e., that of deionized water) may affect the transformation of species during storage due to the presence of native10 and excess sulfide species originating from chemical reagents such as HS- or H2S, the stock solutions maintained neutral pH without significant transformation of the species contained therein (Figure 2). Therefore, stock solutions could be stored at 4 &#730;C in the dark for 13 weeks prior to quantitative speciation analysis.
In this study, the reference molar ratio mixing method of synthesizing DMMTAV and DMDTAV 1,3,4,8,15 was modified to produce stable DMMTAV and DMDTAV stock solutions for HPLC-ICP-MS quantitative analysis. Due to a lack of critical step details, including not only chemical reagent preparation, extraction, and crystallization steps, but also the storage conditions of DMMTAV and DMDTAV, stock solutions had to be modified and optimized. Therefore, stock solutions prepared using this protocol are sufficiently applicable for quantitative analysis of DMMTAV and DMDTAV for environmental monitoring purposes.

Since dimethylarsinic acid (DMAV) has been demonstrated to exhibit both acute toxicity and genotoxicity due to undergoing methylation and thiolation upon ingestion1,2, the metabolic pathway of arsenic thiolation has been intensively studied both in vitro and in vivo3,4 as well as in environmental media (e.g., landfill leachate)5,6. Previous studies have found both reduced and thiolated analogs of DMAV in living cells, for example, dimethylarsinous acid (DMAIII), dimethylmonothioarsinic acid (DMMTAV), and dimethyldithioarsinic acid (DMDTAV)7,8,9, with dimethylated thioarsenicals such as DMMTAV exhibiting greater toxicity than other known inorganic or organic arsenicals10. The abundance of highly toxic thioarsenicals has serious environmental implications, since they may pose a risk to humans and the environment under highly sulfidic conditions11. However, the mechanisms of DMMTAV and DMDTAV (trans)formation and their fates in environmental media still require further study. Thus, the quantitative analysis of thioarsenicals is required to improve understanding of the environmental effects of DMMTAV and DMDTAV.
Although standard chemicals are the key requirement for quantitative analysis, the standards of DMMTAV and DMDTAV are difficult to obtain by replicating previous studies, owing to the high risk of species transformation into other species and unstandardized synthesis procedures12. Moreover, the methods referenced have limitations that may lead to practical difficulties in synthesizing the standard chemicals and performing quantitative analysis. DMMTAV and DMDTAV are commonly prepared by mixing DMAV, Na2S, and H2SO4 in a certain molar ratio1 or bubbling H2S gas through a solution of DMAV 13,14. The bubbling method features substitution of oxygen by sulfur using a direct supply of H2S gas, which, is highly toxic and difficult to control for an inexperienced user. Conversely, the above mixing method1, widely used for the qualitative analysis of DMMTAV and DMDTAV in environmental sudies5,6,12, features the thiolation of DMAV with H2S generated by mixing Na2S and H2SO4 and produces DMMTAV and DMDTAV, allowing easier stoichiometric control to produce target chemicals, as compared to the direct use of H2S gas.
The reference mixing method procedures1,3,4,8,15 mentioned in this study exhibit limitations in some of their critical experimental steps, which might lead to experimental failure. For example, the details of specific solvent (i.e., deionized water) preparation and the extraction and crystallization of the synthesized arsenicals are over-abbreviated or not described in sufficient detail. Such dispersed and limited information on procedural steps might lead to the inconsistent formation of thioarsenicals and unreliable quantification analysis. Therefore, the modified protocol developed herein describes the synthesis of DMMTAV and DMDTAV stock solutions with quantitative species separation analysis.

<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 such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Since DMMTAV has been mistakenly prepared by the DMAIII synthesis method19, verification of synthesized DMMTAV and DMDTAV is a critical step for synthesis and extraction and determining the ideal standard chemical materials. Synthesized chemicals can be verified by the peak of DMMTAV (MW 154 g&#183;mol-1) and DMDTAV (MW 170 g&#183;mol-1) mass-to-charge ratio (m/z) using either the positive or negative ion mode of electrospray ionization-mass spectrometer (ESI-MS) through real-time injection. The reference values of m/z are listed in Table 119. Additional verification of the successful synthesis of DMMTAV and DMDTAV was conducted by comparing the species separation analysis results of the retention time (RT) of major peaks to reference data using HPLC-ICP-MS. Figure 1 shows similar RT of major and minor peaks, DMAV and DMMTAV (Figure 1a) or DMDTAV (Figure 1b), using 1.0 mL&#183;min-1 of 5 mM formic acid as an eluent and a C18 Liquid Chromatography (LC) column as described in17. Note that the RT of the major peaks may vary depending on the instrumental and eluent conditions, and which LC column is used. Species included in the stock solutions of DMMTAV and DMDTAV should be examined prior to every analysis, although this protocol suggests storage conditions of 4 &#730;C in the dark, which maintains synthesized DMMTAV and DMDTAV with, respectively, 2.2% and 5.8% transformation during the 13 weeks of analysis (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56603/56603fig1.jpg" /> 
Figure 1: HPLC-ICP-MS chromatogram of the synthesized DMMTAV and DMDTAV. 1: DMAV, 2: DMDTAV, and 3: DMMTAV were measured as major peaks at 3.8 min, 5.9 min, and 8.0 min in each stock solutions of a: DMMTAV and b: DMDTAV, which were corresponded to those reported by Li et al. in 201017. Instrumental conditions of ICP-MS were 1550 V RF power and 50 &#181;L injection volume. A C18 column was used with 5 mM formic acid as an eluent17. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56603/56603fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56603/56603fig2.jpg" /> 
Figure 2: Stability of DMMTAV and DMDTAV at 4 &#730;C in the dark. Percentage changes of As species distribution in each of the stock solutions DMMTAV (a) and DMDTAV (b) for 13 weeks. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56603/56603fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Synthesized stock solution&#160;</td>
			<td>ion mode</td>
			<td>Fragments&#160;</td>
			<td>m/z</td>
			<td>References</td>
		</tr>
		<tr>
			<td rowspan="7">DMMTAV</td>
			<td rowspan="4">Positive</td>
			<td>[Me2As(SH)OH]+</td>
			<td>155</td>
			<td>13,14,19,17,21,24</td>
		</tr>
		<tr>
			<td>[Me2As(OH)2]+</td>
			<td>139</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td>[Me2AsS]+</td>
			<td>137</td>
			<td>13,19,17,24</td>
		</tr>
		<tr>
			<td>[Me2As(S)SAsMe2]+</td>
			<td>275</td>
			<td>13</td>
		</tr>
		<tr>
			<td rowspan="3">Negative</td>
			<td>[Me2AsOS]-</td>
			<td>153</td>
			<td>17,5,23</td>
		</tr>
		<tr>
			<td>[MeAsSO]-</td>
			<td>138</td>
			<td>17,5</td>
		</tr>
		<tr>
			<td>[AsSO]-</td>
			<td>123</td>
			<td>17,5</td>
		</tr>
		<tr>
			<td rowspan="6">DMDTAV</td>
			<td rowspan="3">Positive</td>
			<td>[Me2As(SH)2]+</td>
			<td>171</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td>[Me2As(SH)OH]+</td>
			<td>155</td>
			<td>19</td>
		</tr>
		<tr>
			<td>[Me2AsS]+</td>
			<td>137</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td rowspan="3">Negative</td>
			<td>[Me2AsS2]-</td>
			<td>169</td>
			<td>20,17,5,22</td>
		</tr>
		<tr>
			<td>[MeAsS2]-</td>
			<td>154</td>
			<td>20,17,5</td>
		</tr>
		<tr>
			<td>[AsS2]-</td>
			<td>139</td>
			<td>17,5,22</td>
		</tr>
	</tbody>
</table>
Table 1: Suggested structure of synthesized DMMTAV and DMDTAV, and minor fragment ions by positive and/or negative ion mode of ESI-MS. The list of DMMTAV, DMDTAV, and minor fragments m/z measured by ESI-MS was reproduced from literature5,13,14,17,19,20,21,22,23,24. Note that m/z peaks may vary with instrumental conditions and/or matrices of the stock solutions.

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

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

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

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

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Nanyang Technological University

Research

JoVE Journal

Environment

5.4K Views.  Korea Basic Science Institute, Seoul Center. This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in qua...

Video: 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 ...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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