The authors thank Dr. F. Cordeiro of the JRC for the useful discussions about statistical treatment of data. Expert laboratories in the analysis of iAs in biological matrices that provided results to be used as assigned value in PTs and laboratories that took part in the studied PTs are acknowledged.

NOTE: All the material used needs to be decontaminated with 10% (m/v) HNO3 and rinsed at least twice with deionized water.
1. Hydrolysis
<ol>
	<li>Weigh accurately ca. 0.5 to 1 g of lyophilized sample (or the equivalent quantity of freshly homogenized sample e.g. 1 to 4 g) in a 50 mL polypropylene centrifuge tube with screw cap.</li>
	<li>Add 4.1 mL of deionized water.</li>
	<li>Agitate with a mechanical shaker for about 5 min until the sample is completely wet.</li>
	<li>Add 18.4 mL of concentrated hydrochloric acid (HCl), not less than 37% m/v.</li>
	<li>Agitate with a mechanical shaker for 15 min.</li>
	<li>Let rest for 12-15 h (for instance overnight).</li>
</ol>
2. Extraction
<ol>
	<li>Add 2 mL of hydrogen bromide (HBr) not less than 48% m/v and 1 mL of hydrazine sulfate (N2H6SO4) solution (15 mg/mL) to the hydrolyzed sample.</li>
	<li>Shake for 30 s with a mechanical shaker.</li>
	<li>Add 10 mL of chloroform (CHCl3).</li>
	<li>Shake for 5 min with a mechanical shaker.</li>
	<li>Centrifuge for 5 min at 800 x g.</li>
	<li>Pipette the chloroform phase (lower phase) into another 50 mL polypropylene centrifuge tube.</li>
	<li>Add again 10 mL chloroform to the remaining acid phase and repeat the extraction. At the end around 20 mL of chloroform should have been collected. Take care to avoid cross-contamination from the acid phase.</li>
</ol>
3. Clean-up of the Chloroform Phase
<ol>
	<li>Centrifuge the pooled chloroform phases for 5 min at 800 x g. The centrifugation time or speed may be increased if needed to achieve a clear separation of the two phases.</li>
	<li>Remove all acid phase residues remaining on the chloroform with a 1 mL pipette. This step is crucial. Any acid phase residues remaining in the chloroform phase will lead to overestimated iAs results because all other arsenic species in the sample are present in the acid phase.</li>
	<li>Filter through a hydrophobic PTFE membrane (25 mm diameter) to remove the remaining solid or acid phase residues present in the chloroform phase and collect the chloroform phase in a 50 mL polypropylene centrifuge tube.</li>
</ol>
4. Back-extraction
<ol>
	<li>Add 10 mL of 1 M HCl to back extract iAs from the chloroform phase collected after the filtration step.</li>
	<li>Shake for 5 min with a mechanical shaker.</li>
	<li>Centrifuge for 5 min at 800 x g.</li>
	<li>Pipette the acid phase (upper phase) and pour it into a 250 mL glass beaker (e.g. Pyrex) for mineralization.</li>
	<li>Repeat the back-extraction and combine the collected HCl phases.</li>
</ol>
5. Sample Mineralization
Note: This step allows elimination of interferences and pre-concentration in samples in which the iAs mass fraction is close to or below the quantification limit, and it is frequently omitted by laboratories which use this protocol with ICP-MS for final determination instead of HG-AAS .
<ol>
	<li>Suspend 20 g of magnesium nitrate hexahydrate [Mg(NO3)26H2O] and 2 g of magnesium oxide (MgO) in 100 mL of deionized water. Add 2.5 mL of this suspension to the glass beaker. Shake the suspension while adding it to avoid precipitation.</li>
	<li>Add 10 mL of concentrated HNO3 of at least 65% m/v and evaporate to dryness in a sand bath (or a thermal plate), avoiding any projections. To verify that the samples are totally dry, place a watch glass on top of the glass beaker and check that no condensation is formed.</li>
	<li>Cover the beakers with watch glasses and place them in a muffle furnace at an initial temperature not exceeding 150 &#186;C and progressively increase the temperature to 425 &#177; 25 &#176;C at a rate of 50 &#176;C/h. Maintain at 425 &#176;C for 12 h. This step is critical. To avoid any projections the rate of increase in temperature must be strictly implemented.</li>
	<li>Allow the ashes to cool down to room temperature.</li>
	<li>Add 0.5 mL of deionized water to wet the ash and add then 5 mL of 6 M HCL. Take care to recover all the ash from the walls of the glass beaker. Dissolve the ash completely, shaking if necessary.</li>
	<li>Add 5 mL of pre-reducing agent, prepared by dissolving 5 g of potassium iodide (KI), and 5 g of ascorbic acid in 100 mL of deionized water, and wait 30 min to achieve a quantitative reduction of iAs to As(III).</li>
	<li>Filter the solution through a Whatman number 1 paper or equivalent and collect it in a 50 mL polypropylene centrifuge tube. Rinse the glass beaker twice with 6 M HCl. Collect the rinsing liquids in a 25 mL tube and make it up to a final volume with 6 M HCl 
	NOTE: When the iAs concentration in a sample is expected to be close or below the quantification limit of the method (0.010 mg/kg), or on the contrary, high, the mineralization steps 5.5-5.7 should be modified using the volumes given in Table 1, which would provide a lower quantification limit. Re-dissolved and pre-reduced samples are stable for 24 h at 4 &#176;C. At least two reagent blanks should be used for the whole analytical process.</li>
</ol>
6. Calibration
NOTE: For quantification purposes use an external calibration curve of As(III) in the range 0.5 - 10 &#181;g/L. Use a 1000 mg/L As(V) commercially available certified standard solution to construct the calibration curve applying subsequent dilutions.
<ol>
	<li>Prepare a 10 mg/L As(V) standard solution by pipetting 1 mL of the 1,000 mg/L standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare a 0.1 mg/L As(V) standard solution by pipetting 1 mL of the 10 mg/L As(V) standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare a 25 &#181;g/L As(V) standard solution pipetting 25 mL of the 0.1 mg/L As(V) standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare the calibration curve of As(III) as follows: pipette from the 25 &#181;g/L As(V) standard solution the volumes given in Table 2 into 50 mL volumetric flask, add 10 mL of the pre-reducing solution in each volumetric flask, wait 30 min, then fill to the mark with 6 M HCl. Other volumes are suitable provided that they maintain the proportions described above.</li>
	<li>Prepare a calibration blank as follows: pipette 10 mL 6 M HCl and 10 mL pre-reducing solution in a 50 mL volumetric flask. Wait 30 min and fill then to the mark with 6 M.</li>
	<li>Use the standards marked as QC1 and QC2 in Table 2 as quality control: QC1 ensures that the quantification at low concentration level is correct and QC2, ensures that the response is stable at high concentrations, with no significant drift in time.</li>
</ol>
7. Determination
<ol>
	<li>Use an atomic absorption spectrometer equipped with an auto-sampler, a flow injection-hydride generation system, and an electro-thermally heated quartz cell for detection and quantification purposes, following the instrumental conditions for quantification of iAs by FI-HG-AAS as listed in Table 3.</li>
</ol>
8. Quantification
<ol>
	<li>Calculate the iAs mass fraction in the samples analyzed (expressed in mg/kg), using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/55953/55953eq1.jpg" /> 
	Where: 
	Cx: Concentration in the extract (&#181;g/L), calculated from the calibration curve 
	CBI: Concentration in the reagent blank sample (&#181;g/L), extrapolated from the calibration curve 
	V: Final volume of the sample mineralization step (5.7), usually V= 25 mL 
	w: Weight of sample (in grams)</li>
</ol>

<ol>
	<li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Commission Regulation (EC) 1881/2006 setting maximum levels for certain contaminants in foodstuffs. , (2006).</li><li>EFSA Panel on Contaminants in the Food Chain (CONTAM). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+Opinion+on+Arsenic+in+Food.">Scientific Opinion on Arsenic in Food.</a> EFSA J. 7 (10), 1351 (2009).</li><li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+exposure+to+inorganic+arsenic+in+the+European+population.">Dietary exposure to inorganic arsenic in the European population.</a> EFSA J. 12 (3), 3597 (2014).</li><li>de la Calle, M. B., 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=Does+the+determination+of+inorganic+arsenic+in+rice+depend+on+the+method?.">Does the determination of inorganic arsenic in rice depend on the method?.</a> TrAC. 30 (4), 641-651 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GB/T5009.11-2003.">GB/T5009.11-2003.</a> Determination of total arsenic and abio-arsenic in foods. , (2003).</li><li>European Committee for Standardisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EN 15517:2008 "Determination of trace elements-Determination of inorganic As in seaweed by hydride generation atomic absorption spectrometry (HG-AAS) after digestion". , (2008).</li><li>de la Calle, M. B., 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=Is+it+possible+to+agree+on+a+value+for+inorganic+arsenic+in+food?+The+outcome+of+IMEP-112.">Is it possible to agree on a value for inorganic arsenic in food? The outcome of IMEP-112.</a> Anal Bioanal Chem. 404 (8), 2475-2488 (2012).</li><li>Schmeisser, E., Goessler, W., Kienzl, N., Francesconi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+analytes+formed+from+arsenosugars:+determination+by+HPLC-HG-ICPMS+and+implications+for+arsenic+speciation+analyses.">Volatile analytes formed from arsenosugars: determination by HPLC-HG-ICPMS and implications for arsenic speciation analyses.</a> Anal. Chem. 76 (2), 418-423 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EN 16802:2016. Foodstuffs. Determination elements and their chemical species. Determination of inorganic arsenic in foodstuffs of marine and plant origin by anion-exchange HPLC-ICP-MS. , (2016).</li><li>European Committee for Standardisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal feeding stuffs &#8211; Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE). , (2012).</li><li>Rasmussen, R. R., Qian, Y., Sloth, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPE+HG-AAS+method+for+the+determination+of+inorganic+arsenic+in+rice.+Results+from+method+validation+studies+an+a+survey+on+rice+products.">SPE HG-AAS method for the determination of inorganic arsenic in rice. Results from method validation studies an a survey on rice products.</a> Anal Bioanal Chem. 405 (24), 7851-7857 (2013).</li><li>Rasmussen, R. R., Hedegaard, R. V., Larsen, E. H., Sloth, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+method+for+the+determination+of+inorganic+arsenic+in+rice.+Results+from+method+validation+studies+and+a+survey+on+rice+products.">Development and validation of a method for the determination of inorganic arsenic in rice. Results from method validation studies and a survey on rice products.</a> Anal Bioanal Chem. 403, 2825-2834 (2012).</li><li>Fiamegkos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+a+method+based+on+atomic+absorption+spectrometry+to+determine+inorganic+arsenic+in+food:+Outcome+of+the+collaborative+trial+IMEP-41.">Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.</a> Food Chem. 213, 169-179 (2016).</li><li>Llorente-Mirandes, T., Barbero, M., Rubio, R., L&#243;pez-S&#225;nchez, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+inorganic+arsenic+in+edible+Shiitake+(Lentinula+edodes)+Products.">Occurrence of inorganic arsenic in edible Shiitake (Lentinula edodes) Products.</a> Food Chem. 158, 207-215 (2014).</li><li>de la Calle, M. B., Linsinger, T., Emteborg, H., Charoud-Got, J., Verbist, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+the+seventh+interlaboratory+comparison+organised+by+the+European+Union-Reference+Laboratory+for+Heavy+Metals+in+Feed+and+Food.">Report of the seventh interlaboratory comparison organised by the European Union-Reference Laboratory for Heavy Metals in Feed and Food.</a> IMEP-107: Total and inorganic As in rice. , (2010).</li><li>Cordeiro, F., Cizek-Stroh, A., de la Calle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+and+inorganic+arsenic+in+rice.">Determination of total and inorganic arsenic in rice.</a> IRMM-PT-43 Proficiency Test Report. , (2016).</li><li>de la Calle, M. B., 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=.">.</a> IMEP-112: Total and inorganic arsenic in wheat, vegetable food and algae. , (2011).</li><li>Fiamegkos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+As,+Cd,+Pb,+Hg+and+inorganic+arsenic+in+chocolate.">Determination of total As, Cd, Pb, Hg and inorganic arsenic in chocolate.</a> EURL-HM-20 Proficiency test Report. , (2015).</li><li>ISO-Geneva (CH), International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 17043: Conformity assessment - General requirements for proficiency testing. , (2010).</li></ol>

The authors have nothing to disclose.

A critical step in the described protocol is the clean-up of the chloroform phase (Step 3.2) because any acid phase residues remaining in the chloroform phase will lead to overestimated iAs results since all other arsenic species in the sample are present in the acid phase. This is of particular relevance when analyzing marine samples due to the presence of a plethora of organic species, which could account for most of the arsenic mass fraction present in the sample. The use of a hydrophobic PTFE (3.3) membrane is of paramount importance. If an emulsion is formed during the extraction of iAs into chloroform, the speed of centrifugation (3.1) can be increased. Other traditional approaches to eliminate emulsions can also be applied. Another critical step is the mineralization (Step 5.3). The rate of increase in temperature must be strictly implemented to avoid any projections which would reduce the iAs recovery leading to an uncontrolled negative bias and could be dangerous for the analyst.
As mentioned above some laboratories have used the evaluated method using ICP-MS instead of FI-HG-AAS. In such a case the dry ashing step (Step 5 in the protocol) is not needed and the 1 M HCl phase can be introduced into the ICP-MS. In the case of HG-AAS, due to its higher detection limit, a pre-concentration step which also eliminates possible interferences, is needed.
The percentage of satisfactory results obtained with the method described in this paper, both with and without the results reported for algae, is comparable to that of HPLC-ICP-MS and higher than that of HG-AAS. The latter technique (HG-AAS) is widely available but prone to interferences from organic arsenic species, especially in food commodities with a complex arsenic species distribution pattern. The lowest percentage of satisfactory results characterizes those obtained with &#34;Other methods&#34; but it must be kept in mind that it covers several analytical approaches, each one of them represented by a small amount of results, Figure 1. The method presented in this paper is an alternative to the more sophisticated/expensive HPLC-ICP-MS, still being characterized by a similar performance even in complex matrices. Frequently the use of hyphenated techniques, such as HPLC-ICP-MS, requires highly qualified operators and expensive infra-structures. The method presented in this paper can be implemented by any analyst trained in basic analytical chemistry.
There are some main drawbacks associated to the method. It is time-consuming since several steps must be followed to separate iAs from other arsenic species and to pre-concentrate iAs down to even sub-ppm levels. It implies the use of chloroform. There is a tendency to avoid the use of chlorinated compounds in the laboratories, due to the negative health effects that they could have. Nevertheless, if good laboratory practices are kept and samples are handled in fume hoods, those negative effects could be avoided. MMA will interfere in the determination of iAs. This must be kept in mind when analyzing samples in which MMA could be present, such as algae, fish and other seafood. However, MMA is normally present in small amounts which would be covered by the uncertainty associated to the results obtained for iAs.

Since January 2016 maximum levels for inorganic arsenic (iAs) in several rice commodities have been included in Commission Regulation (EC) 1881/2006 setting maximum levels for certain contaminants in foodstuffs1 with 0.10 &#181;g/L for rice destined for the production of food for infants and young children, 0.20 &#181;g/L for non-parboiled milled rice (polished or white rice), 0.25 &#181;g/L for parboiled rice and husked rice and 0.30 &#181;g/L for rice waffles, rice wafers, rice crackers and rice cakes. This update of the European legislation for contaminants in food followed the Scientific Opinion on Arsenic in Food of the European Food Safety Authority (EFSA)2 in which it is estimated that the exposure through diet to iAs for average and high consumers in Europe is such that can pose a risk to some consumers, keeping in mind that chronic exposure to iAs causes cancer of the lung, skin and bladder, and skin lesions. In the scientific report of EFSA on Dietary exposure to inorganic arsenic in the European population3, published in 2014, it is concluded that the main contributors to iAs in the diet for consumers of all ages are processed products made of cereals other than rice and that also rice, milk, dairy products and drinking water contribute significantly to iAs intake, with milk and dairy products being the main contributors for toddlers and infants.
In 2010 the European Union Reference Laboratory for Heavy Metals in Feed and food, EURL-HM, ran a proficiency test, IMEP-107, for the determination of iAs in rice, demonstrating that it was possible to determine iAs in rice with sufficient accuracy, regardless of the analytical method used4.
Several analytical methods have been validated for the determination of iAs in foodstuffs. China was the first country to introduce in its legislation a maximum level for iAs in rice. To make the implementation of legislation possible, a standard method was published in 2003 for the determination of what in the standard is called &#34;abio-arsenic&#34;5. The European Committee for Standardisation (CEN), published in 2008 a standardized method, EN 15517:2008, for the determination of iAs in seaweed6. The two methods are based on the use of optimized conditions to generate arsine only from iAs. In that way separation of iAs from other arsenic species that can also generate arsenic hydride is not needed. The final determination is done by atomic fluorescence5 or by hydride generation atomic absorption spectrometry, HG-AAS6. However, it is difficult to set the exact conditions to generate arsenic hydride without suffering from interference of other arsenic compounds and all the iAs mass fractions in algae reported in IMEP-112 (PT organized by the EURL-HM) obtained with those two methods, were scored as unsatisfactory7. Organoarsenic species, such as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenosugars present in algae samples, can generate volatile hydrides too and could interfere in the determination of iAs leading to a positive bias in the results8.
Recently, CEN published a new standard method, EN 16802:2016, for the determination of iAs in foodstuffs of marine and plant origin using HPLC-ICP-MS9. Not all laboratories are equipped with that type of instrumentation and non-expensive, straight-forward methods are needed, in particular in countries with less developed laboratory infrastructures.
In 2012 CEN standardized a method for the determination of iAs in animal feeding stuffs by HG-AAS after microwave extraction and off-line separation of iAs by solid phase extraction (SPE), EN 16278:201210. This method which proved to be fit for analyzing iAs in feed could lack the sensitivity required to determine iAs in food of non-marine origin, which according to EFSA seems to be the main dietary contributors in Europe3. However, the same group that developed and validated EN 16278:2012 tested and successfully applied and validated the method to determine iAs in seafood and rice in a collaborative trial11,12.
An alternative method for the determination of iAs in food matrices after selective extraction of iAs into chloroform and further quantification by HG-AAS, was recently validated by the Joint Research Centre (JRC) in a collaborative trial13. The selectivity of the method is better than that of direct HG-AAS and is easy to implement not requiring the use of sophisticated instrumentation such as HPLC-ICP-MS. In this manuscript, the feasibility of using this method to determine iAs in a wide range of food matrices: vegetables, grains, mushrooms and food of marine origin, has been evaluated. Furthermore, the performance of laboratories that used the method in proficiency tests organized by the EURL-HM and the JRC covering several matrices is described.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deionised water</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">18.2 M&#937; cm</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Concentrated hydrochloric acid (HCl).&#160;</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Not less than 37 % m/v, 
			c(HCl) = 12 mol/L, with a 
			density of 
			approx. &#961; (HCl) 1.15 g/L</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Concentrated nitric acid (HNO3)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Not less that 65 % m/v, 
			c(HNO3) = 14 mol/L, with a 
			densitiy of approx. 
			&#961; 1.38 g/L</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Chloroform&#160;</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Harmful by inhalation and if swallowed. Irritating to skin. 
			Wear suitable protective clothing and gloves.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen bromide (HBr)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td>Not less than 48 % m/v</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrazine sulphate (N2H6SO4)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Harmful if swallowed. 
			Causes burns. 
			May cause cancer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnesium nitrate hexahydrate [Mg(NO3)6H2O]</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnesium oxide (MgO)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium iodide (KI)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ascorbic acid (C6H8O6)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium hydroxide (NaOH)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium borohydride (NaBH4)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Arsenic (V) standard solution</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1000 mg/L 
			Use certified standard 
			solutions commercially available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mechanical shaker</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sand bath</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Muffle furnace</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polypropylene centrifuge (PC) tubes</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td>50 mL with screw cap</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe filters with hydrophobic PTFE membrane</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td>25 mm diameter</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pyrex glass beaker&#160;</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Tall form 250 mL, 
			capable of withstanding 500 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watch glasses</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Volumetric flasks</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">10, 25, 100 or 200, 
			Class A.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic funnels</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Whatman n&#176; 1 paper or equivalent</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic absorption spectrometer equipped with a flow injection system (FI-AAS)</td>
			<td style="width:71px;">Any available</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of inorganic arsenic in a wide range of food matrices using hydride generation - atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The method was applied to determine the iAs mass fraction in several food commodities purchased from various Spanish markets. The results obtained with this method for a series of different matrices are classified in Table 4 following the categories used by EFSA3 in a report in which dietary exposure to inorganic arsenic in the European population is evaluated on the basis of data reported by Official Control Laboratories (OCL). The results in Table 4 represent the mean of three replicates &#177; the reproducibility standard deviation (SR) for the different food categories, calculated during the collaborative trial in which the present method was validated13. The results shown in Table 4 are in good agreement with other previously published in similar matrices11,12,14.
Of particular relevance are the results obtained for iAs in different types of rice because maximum limits are included for them in the European legislation for contaminants in food1. The highest values being obtained for brown rice and the lowest for white rice, in agreement with the findings of the OCLs3. The highest levels were found for the sea weed Hizikia fusiforme, whose consumption has been discouraged by several authorities as indicated in the report by EFSA.
The performance of laboratories that participated in PTs organized by the EURL-HM and the JRC and that used this method for the determination of iAs, has been compared to the performance of laboratories using other methods. Most of the other methods are based on HPLC-ICP-MS (around 50% of the evaluated results) and on HG-AAS without previous separation of iAs from other arsenic species (25% of the total), Figure 1. Other approaches used (around 15% of the evaluated results), were based on electrothermal atomization (ETAAS), fluorescence detection and ICP coupled to atomic emission spectroscopy (ICP-AES), with and without hydride generation, and are evaluated together under the name &#34;Other methods&#34; because the individual numbers would be too few to be of any statistical significance.
Some of the laboratories that have used the evaluated method introduced some variations to the original protocol and used ICP-MS instead of FI-HG-AAS. Frequently those laboratories did not apply the dry ashing step (Step 5 in the protocol) and just introduced the 1 M HCl phase into the ICP-MS. The PTs evaluated covered various matrices: rice15,16, wheat, spinach, algae17 and chocolate18.
The performance of laboratories was expressed as z-score: 
<img alt="Equation 2" src="/files/ftp_upload/55953/55953eq2.jpg" /> 
Where: 
xlab &#160;&#160;&#160;&#160;is the measurement result reported by a participant in a PT 
Xref &#160;&#160;&#160;&#160;is the assigned value (used to benchmark laboratories). In all PTs dealt within this paper the assigned value was established by a group of expert laboratories in the field of iAs analysis using different analytical methods. 
&#963; &#160;&#160;&#160;&#160;&#160;is the standard deviation for proficiency assessment, fixed by the PT provider taking into consideration the state of the art in a certain area of analysis. In the PTs considered in this paper &#963; was 15% of the assigned value for rice and wheat, 22% in algae and 25% for spinach and chocolate.
The interpretation of the z score is done according to ISO 17043:201019: 
|score| &#8804; 2&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;satisfactory (S) performance 
2 &#60; |score| &#60; 3&#160;&#160;&#160;&#160;questionable (Q) performance 
|score| &#8805; 3&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;unsatisfactory (U) performance
Seventy-five per cent of the results obtained with the method described above, got a satisfactory z-score. The determination of the iAs mass fraction in algae turned out to be challenging as expected, taking into consideration the complex distribution of arsenic species in matrices of marine origin. Two out of the three values reported in IMEP-112 for iAs in algae, using this method, got an unsatisfactory z-score. The same difficulty was observed among the results obtained with other methods. Excluding the results reported for iAs in algae, 85% of the results obtained with the evaluated method were satisfactory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55953/55953fig1.jpg" /> 
Figure 1: Comparison of Performances (expressed as z-scores) of Laboratories Taking Part in PTs (IMEP-107, IMEP-112, EURL-HM-20 and IRMM-PT-43) with the Method Described in this Paper and with Other Commonly Applied Methods. S: satisfactory, Q: questionable and U: unsatisfactory. <a href="http://cloudflare.jove.com/files/ftp_upload/55953/55953fig1large.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></td>
			<td>Expected iAs mass fraction 
			lower than 0.010 mg/kg</td>
			<td>Expected iAs mass fraction 
			higher than what is covered 
			by the calibration curve</td>
		</tr>
		<tr>
			<td>6 mol L-1 HCL volume used to re-dissolve the ashes (mL)</td>
			<td>2</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Pre-reducing agent volume (mL)</td>
			<td>2</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Final volume (mL)</td>
			<td>10</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 1: Modifications of the Protocol when Analyzing Samples in which Very Low or Very High iAs Concentrations are Expected.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Concentration in the 
			calibration curve (&#181;g/L)</td>
			<td>Aliquot (mL)</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2 (QC1)</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>10 (QC2)</td>
		</tr>
		<tr>
			<td>7.5</td>
			<td>15</td>
		</tr>
		<tr>
			<td>10</td>
			<td>20</td>
		</tr>
		<tr>
			<td colspan="2">All As(III) calibration standard solutions shall be prepared freshly before each calibration.</td>
		</tr>
	</tbody>
</table>
Table 2: Aliquots to be taken from the 25 &#181;g/L As(V) standard solution to construct the As(III) calibration curve in a 50 mL final volume.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="4">Flow injection 
			Hydride generation</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Loop sample: 0.5 mL (To be adapted when the reconstitution volume of the final pre-reducing solution is different from 25 mL).</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Reducing agent: 0.2 % (w/v) NaBH4 in 0.05 % (w/v) NaOH; 5 mL/min flow rate.</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; HCl solution 10 % (v/v), 10 mL/min flow rate.</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Carrier gas: Argon, 100 mL/min flow rate.</td>
		</tr>
		<tr>
			<td rowspan="5">Atomic absorption 
			spectrometer</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Wavelength: 193.7 nm</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Spectral band-pass: 0.7 nm</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Electrodeless discharge lamp system 2</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Lamp current setting: 400 mA</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Cell temperature: 900 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 3: Instrumental Conditions used for iAs Quantification by HG-AAS.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Food</td>
			<td>i-As (&#181;g/kg fresh weight)</td>
		</tr>
		<tr>
			<td>Grain and grain-based products&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rice</td>
			<td>White</td>
			<td>113 &#177; 18</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>73 &#177; 12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>56 &#177; 9</td>
		</tr>
		<tr>
			<td></td>
			<td>Brown</td>
			<td>197 &#177; 32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>125 &#177; 20</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>275 &#177; 44</td>
		</tr>
		<tr>
			<td></td>
			<td>Parboiled</td>
			<td>134 &#177; 21</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>159 &#177; 25</td>
		</tr>
		<tr>
			<td></td>
			<td>Wafers</td>
			<td>162 &#177; 26</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>127 &#177; 20</td>
		</tr>
		<tr>
			<td>Vegetable and vegetable products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dehydrated mushroom</td>
			<td>Boletus edulis</td>
			<td>174 &#177; 10</td>
		</tr>
		<tr>
			<td></td>
			<td>Galocybe gambosa</td>
			<td>74 &#177; 4</td>
		</tr>
		<tr>
			<td></td>
			<td>Marasmius oreades</td>
			<td>104 &#177; 6</td>
		</tr>
		<tr>
			<td></td>
			<td>Cantharellus lutescens</td>
			<td>16 &#177; 1</td>
		</tr>
		<tr>
			<td></td>
			<td>Lentinula edodes</td>
			<td>96 &#177; 6</td>
		</tr>
		<tr>
			<td>Sea weed</td>
			<td>Hizikia fusiforme</td>
			<td>97000 &#177; 14550</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>44943 &#177; 6742</td>
		</tr>
		<tr>
			<td></td>
			<td>Fucus vesiculosus</td>
			<td>288 &#177; 43</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>433 &#177; 65</td>
		</tr>
		<tr>
			<td>Fish and other seafood</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fish meat</td>
			<td>Flathead grey mullet</td>
			<td>53 &#177; 12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>21 &#177; 5</td>
		</tr>
		<tr>
			<td></td>
			<td>European eel</td>
			<td>72 &#177; 16</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>42 &#177; 9</td>
		</tr>
		<tr>
			<td></td>
			<td>Crayfish</td>
			<td>33 &#177; 7</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>20 &#177; 4</td>
		</tr>
		<tr>
			<td></td>
			<td>Tuna</td>
			<td>11 &#177; 2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>5 &#177; 1</td>
		</tr>
		<tr>
			<td>Molluscs</td>
			<td>Clam</td>
			<td>243 &#177; 54</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>133 &#177; 29</td>
		</tr>
		<tr>
			<td></td>
			<td>Mussel</td>
			<td>32 &#177; 32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>139 &#177; 31</td>
		</tr>
	</tbody>
</table>
Table 4: Results Obtained for a Range of Different Matrices Applying the Described Method.

Watch this Scientific Journal Video about Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry. at JoVE.com

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

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14.5K Views.  Joint Research Centre. The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

Video: Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

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 ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often used on a limited number of stolons or rhizomes. For this reason, these traits are limited in their use for morphological characterization of plants. The use of digital image analysis software technology may overcome measurement errors due to human mistakes, which tend to increase as the number and size of samples also increase. The protocol can be used for any kind of crop but is particularly suitable for forage or grasses, where plants are small and numerous. Turf samples consist of aboveground biomass and an upper soil layer to the depth of maximum rhizome development, depending on the species of interest. In studies, samples are washed from the soil, and stolons/rhizomes are cleaned by hand before analysis by digital image analysis software. The samples are further dried in a laboratory heating oven to measure dry weight; therefore, for each sample, the resultant data are total length, total dry weight, and average diameter. Scanned images can be corrected before analysis by excluding visible extraneous parts, such as remaining roots or leaves not removed with the cleaning process. Indeed, these fragments normally have much smaller diameters than stolons or rhizomes, so they can be easily excluded from analysis by fixing the minimum diameter below which objects are not considered. Stolon or rhizome density per unit area can then be calculated based on sample size. The advantage of this method is quick and efficient measurement of the length and average diameter of large sample numbers of stolons or rhizomes.

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Removal of Arsenic Using a Cationic Polymer Gel Impregnated with Iron Hydroxide

In this work, we prepared an adsorbent composed of a cationic polymer gel containing iron hydroxide in its structure designed to adsorb arsenic from groundwater. The gel we selected was the N,N-dimethylamino propylacrylamide methyl chloride quaternary (DMAPAAQ) gel. The objective of our preparation method was to ensure the maximum content of iron hydroxide in the structure of the gel. This design approach enabled simultaneous adsorption by both the polymer structure of the gel and the iron hydroxide component, thus, enhancing the adsorption capacity of the material. To examine the performance of the gel, we measured reaction kinetics, carried out pH sensitivity and selectivity analyses, monitored arsenic adsorption performance, and conducted regeneration experiments. We determined that the gel undergoes a chemisorption process and reaches equilibrium at 10 h. Moreover, the gel adsorbed arsenic effectively at neutral pH levels and selectively in complex ion environments, achieving a maximum adsorption volume of 1.63 mM/g. The gel could be regenerated with 87.6% efficiency and NaCl could be used for desorption instead of harmful NaOH. Taken together, the presented gel-based design method is an effective approach for constructing high-performance arsenic adsorbents.

In this work, we prepared an adsorbent composed of the cationic N,N-dimethylamino propylacrylamide methyl chloride quaternary (DMAPAAQ) polymer gel and iron hydroxide for adsorbing arsenic from groundwater. The gel was prepared via a novel method designed to ensure the maximum content of iron particles in its structure.

In this work, we prepared an adsorbent composed of a cationic polymer gel containing iron hydroxide in its structure designed to adsorb arsenic from ...

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...