The authors thank Masayuki Kubo of the US Environmental Protection Agency for his help with the TSP/PM2.5 sampling. This work was supported in part by JSPS KAKENHI Grant Number JP16K15373 and JP18H03039.

1. Collection of Total Suspended Particles, PM2.5, or PM7, and the Hydrolysis Method for PM Proteins
<ol>
	<li>Collect the PM from a prefilter or backup filter.
	<ol>
		<li>Prior to the collection of total suspended particles (TSP), PM2.5, and PM7, weigh the quartz filter. 
		Note: Given that contamination by PM proteins and peptides should not affect the detection of tyrosine nitration (as they lack such a nitro group), we did not treat the quartz filter at a high temperature before the measurement. Additionally, 3-NT content in the quartz filter was under the detection limit, as determined by HPLC-ECD.</li>
		<li>Set the quartz filter on a high-volume air sampler with a particle size selector to collect PM7 at a flow rate of 1,000 L/min continuously for 7 d (Figure 1B). TSP can be collected on a quartz filter without a size selector (Figure 1A).</li>
		<li>Collect PM2.5 using a low-volume air sampler with a size classification unit at a flow rate of 116 L/min continuously for 7 d. Using this unit, particles can be classified as PM4.5-2.5, PM7.3-4.5, PM15-7.3, and TSP-PM15 and collected onto quartz filters for classification. PM2.5 can be collected onto a backup filter (Figure 1C).</li>
		<li>Record the total flow volume at the time of the filter collection. Measure the weight of the TSP, PM2.5, and PM7 on the filters by subtracting the precollection weight from the postcollection weight. Seal the filter in a plastic bag and store it at -30 &#176;C until the next step (see section 2).</li>
	</ol>
	</li>
	<li>Proteolyze the samples.
	<ol>
		<li>Dissolve nonspecific protease cocktail with a 0.1 M acetate buffer (pH 7.4). Put the solution into a dialysis membrane (molecular weight cutoff = 5,000 Da) and immerse it in 1 L of 0.1 M acetate buffer with stirring at 4 &#176;C. Replace the buffer 2x every 12 h, and then perform dialysis overnight in order to remove endogenous 3-NT and nitrite (NO2-). After the dialysis, collect the solution and measure the protein concentration by bicinchoninic acid (BCA) protein assay.</li>
		<li>Punch round circles of 6 mm out of the prefilter or the backup filter and put them into microtubes. Up to five pieces of 6 mm fractions can be used for the analysis (Figure 2).</li>
		<li>Add 300 &#181;L of 0.1 M acetate buffer containing 50 &#181;g of protein of the nonspecific protease cocktail (prepared in step 1.2.1) to the tube to hydrolyze the PM proteins at 50 &#176;C for 16 h with rotation. 
		Note: To subtract endogenous 3-NT from the nonspecific protease cocktail solution, it is necessary to prepare and digest a nonspecific protease cocktail-only sample.</li>
		<li>Rinse the ultrafiltration membrane along with its supplied tube by adding 0.1 M acetate buffer and centrifuging (10,000 x g for 30 sec; the exact speed depends on the membrane), due to contaminating chemicals similar to 3-NT in the membrane. Repeat the rinse 1x.</li>
		<li>Post-proteolysis, centrifuge the samples at 2,500 x g for 10 min. Load the supernatant into the ultrafiltration membrane and centrifuge it at 10,000 x g and 4 &#176;C for ultrafiltration (MWCO = 10,000), until sufficient volume of elution is collected. Store the eluates at 4 &#176;C in the dark. 
		CAUTION: Due to the detection of a similar peak to 3-NT in the spin column, it is necessary to wash it well with clean water or with a buffer such as HPLC water and a 0.1 M acetate buffer prior to use.</li>
	</ol>
	</li>
</ol>
2. Determination of 3-NT Content in TSP, PM2.5, and PM7 via a High-performance Liquid Chromatography and Electrochemical Detection (HPLC-ECD) System
Note: See Figure 3.
<ol>
	<li>Prepare the mobile phase.
	<ol>
		<li>Prepare 0.2 M phosphate buffer (pH 2.5) containing 50 mg/L EDTA. Measure off 98 volumes of the buffer and two volumes of acetonitrile (HPLC grade), pour them into a clean glass bottle, and mix vigorously with a cap.</li>
		<li>Settle the mobile phase at room temperature for several hours until the dissolved air goes flat. 
		Note: If the system is started earlier, the mobile phase can be sonicated and degassed under a vacuum. However, over-degassing will change the ratio between the water and organic phase via evaporation.</li>
	</ol>
	</li>
	<li>The HPLC-ECD system consists of a pump, an HPLC column, a degasser, an ECD, and an injector. Equilibrate the mobile phase and stabilize the system to reduce the background and noise.
	<ol>
		<li>Install the mobile phase and the HPLC column in the HPLC system. Turn on the HPLC pump and stabilize the column with the mobile phase at a flow rate of 500 &#181;L/min at 25 &#176;C column temperature from the day before the start day.</li>
		<li>On the next day, ignite the ECD and set the parameters (the reduction voltage at -900 mV and the excitation voltage at 400 mV). Stabilize it for at least 3 h.</li>
	</ol>
	</li>
	<li>Measure the 3-NT concentration in the PM7.
	<ol>
		<li>Prepare various concentrations of 3-NT standards (5 - 500 nM) and a blank (without the 3-NT standard and nonspecific protease cocktail, to make sure neither the vial nor the buffer is contaminated) in a 0.1 M acetate buffer.</li>
		<li>Run the HPLC data processor.</li>
		<li>Put sample vials in the auto-injector and set the injection volume to 25 &#181;L. Operate the same conditions of mobile phase and flow rate as mentioned in step 2.2.1. 
		Note: If the sample preparation is complete, there should be samples, a standard (5 - 500 nM), a nonspecific protease cocktail-only sample, and a blank vial.</li>
		<li>Make sure that the baseline in the chromatogram is flat and start the sample injection. 
		Note: Generally, 3-NT is detected in 20 - 21 min; however, approximately 30 min is recommended as the running time per sample injection. Otherwise, contaminant peaks will be included in the next run.</li>
	</ol>
	</li>
</ol>
3. Analysis of Chromatograms and Quantification of 3-NT Content in PM7
<ol>
	<li>Check the 3-NT peak in the chromatograms and calculate the content.
	<ol>
		<li>After the data collection, open the chromatogram data file with analysis software.</li>
		<li>Draw a line across the 3-NT peak from the flat baseline and read the peak height from the drawn line. 
		Note: The quantitative limit is approximately 5 nM (125 fmol in a 25 &#181;L injection). 
		CAUTION: The electrode in the ECD becomes depleted slowly, and the limit will be high due to the S/N ratio.</li>
		<li>Prepare a regression line from the 3-NT standard peaks, calculate the slope, and intercept.</li>
		<li>Assign these constants into the following equation and calculate the 3-NT content in the samples as follows. 
		Sample 3 &#8211; NT (nM) = [sample peak &#8211; intercept] / slope&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Eq. (1) 
		Note: The standard curve should be well represented as a straight line between 5 nM and 500 nM.</li>
	</ol>
	</li>
	<li>Calculate the 3-NT in the air environment and the PM7 from the sample vial.
	<ol>
		<li>Multiply the 3-NT concentration by the reaction volume (and the dilution factor, if any), and evaluate the absolute 3-NT content in PM7 from the circle.</li>
		<li>Measure the collected filter area and calculate the 3-NT content in the total PM7 filter by the following equation. 
		Total PM7 = [3-NT content in the PM7 of the circle] x [ratio of the PM7 filter area to the circle area] x 226.19 (in the case of converting moles to grams)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;Eq. (2)</li>
		<li>Evaluate the 3-NT content in the air environment or PM7 by dividing the total PM7 by the total air flow volume or PM7 weight (recorded in step 1.1.3).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Castillo, J. A., Staton, S. J. R., Taylor, T. J., Herckes, P., Hayes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+feasibility+of+bioaerosol+analysis+as+a+novel+fingerprinting+technique.">Exploring the feasibility of bioaerosol analysis as a novel fingerprinting technique.</a> Analytical and Bioanalytical Chemistry. 403 (1), 15-26 (2012).</li><li>Franze, T., Weller, M. G., Niessner, R., P&#246;schl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+nitration+by+polluted+air.">Protein nitration by polluted air.</a> Environmental Science &amp; Technology. 39 (6), 1673-1678 (2005).</li><li>Abe, R. Y., Akutsu, Y., Kagemoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+amino+acids+as+markers+for+biological+sources+in+urban+aerosols.">Protein amino acids as markers for biological sources in urban aerosols.</a> Environmental Chemistry Letters. 14 (1), 155-161 (2016).</li><li>D'Amato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergenic+pollen+and+pollen+allergy+in+Europe.">Allergenic pollen and pollen allergy in Europe.</a> Allergy: European Journal of Allergy and Clinical Immunology. 62 (9), 976-990 (2007).</li><li>Bolzacchini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+reaction+of+phenol+with+NO3.">Gas-phase reaction of phenol with NO3.</a> Environmental Science &amp; Technology. 35 (9), 1791-1797 (2001).</li><li>Shiraiwa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphase+chemical+kinetics+of+the+nitration+of+aerosolized+protein+by+ozone+and+nitrogen+dioxide.">Multiphase chemical kinetics of the nitration of aerosolized protein by ozone and nitrogen dioxide.</a> Environmental Science &amp; Technology. 46 (12), 6672-6680 (2012).</li><li>Gruijthuijsen, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitration+enhances+the+allergenic+potential+of+proteins.">Nitration enhances the allergenic potential of proteins.</a> International Archives of Allergy and Immunology. 141 (3), 265-275 (2006).</li><li>Kubo, M., Ogino, K., Tsukahara, H., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Procedures+for+Nitrative/Nitrosative+Stress.">Analytical Procedures for Nitrative/Nitrosative Stress.</a> Studies on Pediatric Disorders. , 149-158 (2014).</li><li>Thomson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-nitrotyrosine+modified+proteins+in+atherosclerosis.">3-nitrotyrosine modified proteins in atherosclerosis.</a> Disease Markers. 2015, 708282 (2015).</li><li>Kuhn, D. M., Sakowski, S. A., Sadidi, M., Geddes, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrotyrosine+as+a+marker+for+peroxynitrite-induced+neurotoxicity:+the+beginning+or+the+end+of+the+end+of+dopamine+neurons?.">Nitrotyrosine as a marker for peroxynitrite-induced neurotoxicity: the beginning or the end of the end of dopamine neurons?.</a> Journal of Neurochemistry. 89 (3), 529-536 (2004).</li><li>Teixeira, D., Fernandes, R., Prud&#234;ncio, C., Vieira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Nitrotyrosine+quantification+methods:+Current+concepts+and+future+challenges.">3-Nitrotyrosine quantification methods: Current concepts and future challenges.</a> Biochimie. 125, 1-11 (2016).</li><li>Hitomi, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposition+of+protein-bound+3-nitrotyrosine+in+rat+plasma+analysed+by+a+novel+protocol+for+HPLC-ECD.">Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD.</a> Journal of Biochemistry. 141 (4), 495-502 (2007).</li><li>Ogino, K., Wang, D. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+oxidative/nitrosative+stress:+an+approach+to+disease+prevention.">Biomarkers of oxidative/nitrosative stress: an approach to disease prevention.</a> Acta Medica Okayama. 61 (4), 181-189 (2007).</li><li>Tsikas, D., Mitschke, A., Suchy, M. -. T., Gutzki, F. -. M., Stichtenoth, D. 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B, Analytical Technologies in the Biomedical and Life Sciences. 827 (1), 146-156 (2005).</li><li>Ito, T., Ogino, K., Nagaoka, K., Takemoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+particulate+matter+and+ozone+with+3-nitrotyrosine+in+the+atmosphere.">Relationship of particulate matter and ozone with 3-nitrotyrosine in the atmosphere.</a> Environmental Pollution. 236, 948-952 (2018).</li></ol>

The authors have nothing to disclose.

This article describes a quantification method to evaluate 3-NT in airborne PM collected on quartz filters using highly sensitive HPLC-ECD techniques.
In general, 3-NT measurement methods have been developed as biomarkers of oxidative stress in human diseases. Antibody-based methods (e.g., the enzyme-linked immunosorbent assay) are considered as semi-quantitative because there is no strict assay validation and it is difficult to assess the test&#39;s reliability. HPLC with electrochemical detection (ECD) and mass spectrometry-based assays have adequate sensitivity for the quantification of 3-NT13. Although they are sensitive, mass spectrometry-based methods like GC-MS or GC-MS/MS require the derivatization of amino acids, and the process of derivatization often results in the formation of artifacts14.
Compared to both the GC and LC techniques, HPLC-ECD is relatively less expensive and has a sufficient sensitivity to measure 3-NT. Additionally, the derivatization step in GC-MS and LC-MS often requires additional time.&#160;Although the method presented here requires 16 hours for the protein digestion step, it can, nevertheless, be carried out overnight (as described above, about 30 minutes of hands-on time is required per sample).Automatic repeat measurement by using an autosampler may provide a high-throughput measurement system.Previous studies have reported immunological methods to measure 3-NT in PM2,7; however, such methods could not detect 3-NT in the winter season due to the low levels of 3-NT in the PM15.
The HPLC-ECD method has additional advantages over other standard methods; for example, (i) an extraction process is not required in this method, (ii) it is completely&#160;detergent-free, (iii) it has minimal sample requirement, and, importantly, (iv) it has high sensitivity. Particles that are collected onto filters are frequently separated by sonication for further investigations, including the quantification of various components. Detergents are also used to isolate PM-bound proteins from filters. However, these additional steps increase the risk of contamination, sample loss, and underestimation due to extraction efficiency, and furthermore, most detergents are incompatible with LC/MS. In the present HPLC-ECD method, HPLC samples can easily be separated from the filter by using a simple hollow punch and without the requirement of an additional sample preparation process. The area of the round-shaped sample is 28.3 mm2, which is very small compared to the size of the original quartz filter (8 x 10 inch = 203.2 x 254 mm = 51,600 mm2).
The HPLC-ECD condition has been reviewed carefully to avoid interference to the 3-NT signal, as described previously12.&#160;A strong acidic pH of mobile phase and an adequate concentration of acetonitrile is important for detection.Using these conditions, other compounds, including nitro base, can be separated from nitrotyrosine.The present condition is suitable for the detection of 3-NT from samples with a different physical property (e.g., particulate matter and plasma).
To evaluate 3-NT in the air, the measurement of the filter weight and the elimination of the background are critical steps. In general, over approximately 100 mg of PM7 are collected over a period of four to seven days; however, several factors affect the filter weight before and after the PM7 collection, including humidity and static electrical charge. To avoid these effects, the stabilization of the filter weight for long periods is necessary under subequal temperature and humidity. The location where the electronic balance is installed should be maintained in a stable environment. For sample preparation, it is important to correct the background effects from the nonspecific protease cocktail and the ultrafiltration membrane, which often show a similar peak to 3-NT.
This method can be applied to other particles, including those in indoor environments. The nitration of proteins may increase their allergenic potential2. Therefore, this method may help to evaluate environmental cleanliness and prevent nitrosative stress.
In this study, we report a highly sensitive measurement method for atmospheric 3-NT of air sampler filters with easy-to-handle and inexpensive apparatus. The generation of 3-NT in the atmosphere is associated with environmental pollutants such as O3, NO2, PM proteins, and meteorological elements which affect human allergenicity. In conclusion, the method developed in the present investigation may help to assess the atmospheric reaction of O3, various pollutants, and PM proteins under various meteorological conditions. With these endeavors of developing HPLC-ECD for an estimation of 3-nitrotyrosine in the atmosphere, we anticipate better environmental cleanliness, improving human health.

Aerosol or airborne PM contains proteins from various biological origins, including viruses, prokaryotes, fungi, plants (pollen), and animals (insects, human)1.&#160;The ratio of protein content in PM is estimated to be nearly 5%, which has been implicated to play a major role as an airborne allergen2. Recent reports suggest that urban ambient aerosol of various sizes contain amino acids with ratios ranging between 0.5% and 2%3. In addition, chemical modifications of PM proteins have been suggested to be generated by the reactions of proteins with various pollutants, such as O3, nitrogen dioxide (NO2), and sulfur dioxide (SO2)4.
3-NT&#8212;a protein modification&#8212;is generated by the nitration of tyrosine residues.&#160;A higher concentration of O3 and NO2 has been shown to promote the nitration of protein molecules in pseudo-atmospheric space5,6.&#160;The nitration of tyrosine residues in the polypeptide chain enhances the allergenic potential of pollen proteins7.Thus, the quantification and assessment of airborne 3-NT is an important aspect in addressing the concerns of environmental health.
3-NT is also known as a biomarker of oxidative and nitrosative stress8.&#160;An emerging body of evidence has shown a significant association of 3-NT content with several human diseases9,10.&#160;Owing to its detrimental effects, the detection and quantitative estimation of 3-NT in a biological sample hold great relevance in determining the condition of an individual&#39;s health. In recent years, diverse methods have been introduced to estimate the 3-NT content, including enzyme-linked immunosorbent assays, HPLC, and LC/MS11. In previous studies, we have reported a detection method using the HPLC-ECD technique bestowed with several advantages compared to previous methods. For example, the HPLC-ECD does not require an extraction or derivatization procedure which makes it a relatively simple assay system12.We have confirmed that this method is applicable to detect 3-NT from biological samples (plasma from human and rat).
Although many investigations have emphasized the detection of 3-NT in biological samples, its detection in nonbiological samples remains elusive, and hence the present study has been pursued. In fact, to assess the harmful effect of airborne 3-NT, a quantitative method that is useful to detect 3-NT directly from airborne particles is required; therefore, we applied the existing HPLC-ECD method to detect 3-NT from PM2.5 collected on a glass-fiber filter.&#160;By using the technique, 3-NT could be detected directly from small pieces (6 mm-diameter round holes) of the sample from a PM collection filter.We further evaluated the content of 3-NT for various PM sizes and measured the detection limit of 3-NT.The present article proposes a high-sensitive and high-throughput method to detect 3-NT directly from both nonbiological and biological samples.

<table border="0" cellpadding="0" cellspacing="0" style="width:940px;" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Quartz filter, backup quartz filter</td>
			<td style="width:240px;">PALL life sciences</td>
			<td style="width:168px;">7204</td>
			<td style="width:293px;">Tissuquatrz 2500QAT-UP, 8x10in</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High-volume air sampler</td>
			<td style="width:240px;">SIBATA SCIENTIFIC TECHNOLOGY</td>
			<td style="width:168px;">HV-1000F</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Particle size selector for SPM</td>
			<td style="width:240px;">SIBATA SCIENTIFIC TECHNOLOGY</td>
			<td style="width:168px;">080130-061</td>
			<td style="width:293px;">SPM (suspended particulate matters) is defined in Japan that particles with 10 um of 100% cut-off diameter.&#160; SPM is almost identical to PM7.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low-volume air sampler</td>
			<td style="width:240px;">SIBATA SCIENTIFIC TECHNOLOGY</td>
			<td style="width:168px;">AH-600F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Size classification unit for PM2.5</td>
			<td style="width:240px;">SIBATA SCIENTIFIC TECHNOLOGY</td>
			<td style="width:168px;">AH-600</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Quartz filters for classification (PM4.5&#8211;2.5, PM7.3&#8211;4.5, PM15&#8211;7.3, and TSP-PM15)</td>
			<td style="width:240px;">Tokyo Dylec</td>
			<td style="width:168px;">AHQ-630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Non-specific protease cocktail</td>
			<td style="width:240px;">Roche</td>
			<td style="width:168px;">11459643001</td>
			<td style="width:293px;">Pronase from Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">3-Nitrotyrosine</td>
			<td style="width:240px;">SIGMA</td>
			<td style="width:168px;">N7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ultrafiltration membranes</td>
			<td style="width:240px;">Millipore</td>
			<td style="width:168px;">UFC5010BK</td>
			<td style="width:293px;">AMICON ULTRA 0.5 mL - 10kDa cutoff</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">ECD</td>
			<td style="width:240px;">Eicom</td>
			<td style="width:168px;">HTEC-500, PEC-510</td>
			<td style="width:293px;">standalone HPLC-ECD unit (HTEC-510) and Pre-electrolysis cell (PEC-510), includes pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">HPLC column</td>
			<td style="width:240px;">Eicom</td>
			<td style="width:168px;">SC-5ODS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Data processor</td>
			<td style="width:240px;">Eicom</td>
			<td style="width:168px;">EPC-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Injector</td>
			<td style="width:240px;">Hitachi High-Tech Science&#160;</td>
			<td style="width:168px;">L-7200</td>
			<td style="width:293px;">Auto sampler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analyzing software</td>
			<td style="width:240px;">eDAQ Japan</td>
			<td style="width:168px;">ES280&#160;</td>
			<td style="width:293px;">PowerChrom software</td>
		</tr>
	</tbody>
</table>

detection of 3-nitrotyrosine in atmospheric environments via a high-performance liquid chromatography-electrochemical detector system

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

The HPLC-ECD principle is simple. Samples containing the target are separated by the HPLC column and mobile phase, and the separated target compound is reduced and/or oxidized in the electrochemical detector.
In the HPLC-ECD system, a 3-NT peak is detected in approximately 22 minutes. ECDs (PEC-510 and HTEC-500) are connected in tandem in the HPLC line. The first ECD reduces the hydroxyl groups in the compounds, and the next oxidizes the reduced chemicals. ECDs have a highly sensitive detection range of 10-15 M. The detection limit of the HPLC-ECD system was 1.13 pg/m3, which was calculated as the mean basal signal (mV) of 10 parts x 3.
Representative chromatograms are shown in Figure 4. By analyzing a pure 3-NT standard, a definitive peak with a stable baseline can be detected (Figure 4A). 3-NT in PM7 was also measured using 6 mm, round-shaped samples (Figure 4B).
Regarding the accuracy of 3-NT detection capability, when a 3-NT standard was injected in HPLC, the peak corresponding to 3-NT was detected at a retention time of 20 minutes. In PM samples, 3-NT was separated from other substances, and the independent peak was detected at the same retention time as the standard. A typical standard curve is shown in Figure 5, where linearity is observed between 5 and 40 nM. Linearity can be seen up to 500 nM (data not shown).
Table 1 shows the 3-NT contents in the atmosphere, calculated as pg/m3 air. The highest concentration of 3-NT in a PM2.5 prefilter was observed in #3 (PM7.3-4.5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58371/58371fig1.jpg" /> 
Figure 1: Collection scheme for TSP, PM7, and PM2.5. (A) The total suspended particles (TSP) are collected directly onto a collection filter (at a flow rate of 1,000 L/min). (B) When PM7 is collected, large particles are excluded by a size selector (at a flow rate of 1,000 L/min). (C) PM2.5 is collected through four prefilters (at a flow rate of 116 L/min). A quartz filter is commonly used as a backup filter for PM7 and PM2.5 or as a collection filter for TSP, as shown in this figure. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58371/58371fig1large.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/58371/58371fig2.jpg" /> 
Figure 2: Preparation of a 6 mm-round circle. The collection filter was cut using a hollow punch tool with a 6 mm diameter. Then, the sample was stored in a 1.5 mL microtube. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58371/58371fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58371/58371fig3.jpg" /> 
Figure 3: Schematic diagram of the HPLC-ECD system. 3-NT in an injected sample was separated in the HPLC column through which mobile phase flows. The 3-NT was reduced to aminotyrosine, oxidized to iminotyrosine, and detected electrochemically in the detector. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58371/58371fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58371/58371fig4.jpg" /> 
Figure 4: Typical chromatograms in the HPLC-ECD system. (A) Pure 3-NT analyzed by the HPLC-ECD system. (B) 6 mm circle sample from a PM7 filter used to detect 3-NT. A 3-NT peak is indicated by an arrow. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58371/58371fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58371/58371fig5.jpg" /> 
Figure 5: Standard curve of pure 3-NT samples. The results at a lower concentration are indicated in another graph. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58371/58371fig5large.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 colspan="2">Subject</td>
			<td>Days of 
			Collection</td>
			<td>Spot</td>
			<td>3-NT 
			(pg/m3 air)</td>
		</tr>
		<tr>
			<td>PM2.5</td>
			<td>Prefilter #1</td>
			<td>27</td>
			<td>4</td>
			<td>8.25 &#177; 0.37</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #2</td>
			<td>27</td>
			<td>4</td>
			<td>29.66 &#177; 1.94</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #3</td>
			<td>27</td>
			<td>4</td>
			<td>74.37 &#177; 4.09</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #4</td>
			<td>27</td>
			<td>4</td>
			<td>32.70 &#177; 0.75</td>
		</tr>
		<tr>
			<td></td>
			<td>Backfilter</td>
			<td>7</td>
			<td>5</td>
			<td>4.21 &#177; 0.91</td>
		</tr>
		<tr>
			<td>PM7</td>
			<td>Backfilter</td>
			<td>7</td>
			<td>1</td>
			<td>89.16 &#177; 6.36</td>
		</tr>
		<tr>
			<td>TSP</td>
			<td>Filter</td>
			<td>7</td>
			<td>1</td>
			<td>10.34 &#177; 2.09</td>
		</tr>
	</tbody>
</table>
Table 1: 3-NT in the atmosphere measured by the HPLC-ECD system. The 6 mm circular spots from each filter were measured using the HPLC-ECD system. Prefilters #1 - #4 were collected in the same period. Note that the concentrations of 3-NT in the air are influenced by various factors, such as the collected date, year, or location. The data (n = 3 - 10) are represented as the mean &#177; the standard error.

Watch this Scientific Journal Video about Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System at JoVE.com

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

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7.3K Views.  Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

Video: Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.

A protocol for the design and construction of a soil tank interfaced to a small climate controlled wind tunnel to study the effects of atmospheric forcings on evaporation is presented. Both the soil tank and wind tunnel are instrumented with sensor technologies for the continuous in situ measurement of environmental conditions.

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

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Chapters in this video

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea