The authors thank Drs. Bobby J. Presley, Robert Tayloy, Paul Boothe, Mr. Bryan Brattin, and Mr. Mike Metcalf for their assistance during the laborious field sampling and lab work for the practical development and application of "clean techniques".

1. Sampler Preparation
<ol>
	<li>Sampler
	<ol>
		<li>Assemblage of sampler
		<ol>
			<li>Connect a 4 m long fluorinated ethylene propylene (FEP) tubing (I.D. 0.635 cm, O.D. 0.95 cm or similar) to a 1.5-m chemically resistant silicone pumping tube (O.D. 0.635 cm).</li>
			<li>Insert a polypropylene Y-connector into the pumping tube, and connect a 50-cm pumping tube to one outlet, and a 0.45 &#956;m capsule filter (by a 20-cm pumping tube) to the other.</li>
			<li>Assemble the tubings in a clean room (bench) after they are cleaned (see below), and store the assemblage in two layers of polyethylene bags.</li>
		</ol>
		</li>
		<li>Sampler cleaning
		<ol>
			<li>Fill (by attaching the 1.5-m pumping tube onto a peristaltic pump) the tubing set with lab detergent solution and soak it for 24 hr. Flush the tubing set with de-ionized water, then fill it with 10% (v/v) HCl (Reagent Grade) and soak for 48 hr.</li>
			<li>Thoroughly flush the tubing set with de-ionized water several times, and store the assemblage in plastic bags. Clean the Y-connectors and short pumping tubes by soaking them in a 50% (v/v) HNO3 (Reagent Grade) solution for 24 hr.</li>
		</ol>
		</li>
		<li>Capsule filters
		<ol>
			<li>Clean the capsule filters by first flushing them with de-ionized water, then 10% (v/v) HCl solution for a 48-hr soaking step.</li>
			<li>After acid soaking, flush the filters with de-ionized water and add 1 ml of 21% (weight) NH4OH (sub-boiled) solution into each filter to neutralize the acid.</li>
			<li>Seal individual filter with a loop of 30-cm cleaned-pumping tube connecting the inlet and outlet, and store in polyethylene zippered bags.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sample containers
	<ol>
		<li>Use polyethylene (PE, 1,000-ml) and FEP (500 ml and 1,000 ml) bottles for containers for trace metal determination.
		<ol>
			<li>Clean the bottles by soaking them first in detergent (1%), then in 50% (v/v) HNO3 (Reagent Grade) and 10% (v/v) HCl (Reagent Grade) solutions for 24, 48 and 24 hr, respectively, and rinse the bottles with de-ionized water between the two soaking steps.</li>
			<li>After a final HCl soaking, rinse the bottles thoroughly with de-ionized water (DIW) and dry the bottles (with cap sealed) in a clean room or class-100 clean bench.</li>
			<li>Seal cleaned bottles in polyethylene zippered bags and double-bag them in polyethylene bags for transport.</li>
		</ol>
		</li>
		<li>Clean glass bottles for dissolved organic carbon (DOC) determination
		<ol>
			<li>Soak amber borosilicate glass bottles (40-ml), for analysis of dissolved organic carbon (DOC), in 10% HCl for 48 hr. Rinse cleaned glass bottles with de-ionized water, and combust them at 480 &#176;C for 2 hr before use. Seal the bottles individually in zippered polyethylene bags for transport.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Sampling
<ol>
	<li>Water sample collection
	<ol>
		<li>Upon arrival at the sampling site, mark sample identification number on the outer bag, and keep the bottles in their original bags.</li>
		<li>At the river bank or on a boat, have one person open the bag with the sampler and attach the 4-m FEP tubing to a 3-m polypropylene pole (cleaned). Extend the pole as far from the bank as possible and keep the inlet of the FEP tubing approximately 30 cm below the surface of running river water before the pump is turned on.</li>
		<li>Have another person attach the pumping tube to the pump head of a peristaltic pump (sampling pump with internal batteries). Start the pump and drain the water (downstream side) at least 3 times of the total sampler volume. Put on powder-free gloves and open the bag of bottles and caps to start filling the sample bottles.
		<ol>
			<li>Alternatively, if another person is available, have the third person be in charge of opening the inner bags and sample caps, and holding the sampling tube that drains the sample into the bottle.</li>
		</ol>
		</li>
		<li>Collect an unfiltered sample in a 125-ml plastic bottle for measurements of conductivity, temperature and pH in the field.</li>
		<li>Collect unfiltered samples (500-ml or 1,000 ml for particulate sample collection) first (through the outlet without a filter) and then close the outlet with a plastic clamp to force water to go through the capsule filter to collect a filtered water sample (1-L for dissolved trace metal determination).</li>
		<li>Collect filtered samples (through the outlet with the filter) in 40-ml amber glass bottles for DOC and EDTA measurements.</li>
		<li>Collect water samples at a flow rate of approximate 500 ml/min to 1,000 ml/min. Replace capsule filters when the pressure starts to build up (according to the filters&#39; specification). For each type of sample, collect extra samples, as well as field blanks, at selected locations to serve as quality control aliquots.</li>
		<li>Place the 40-ml glass bottles on dry ice and stored in an ice chest, and polyethylene bottles in ice chests.</li>
	</ol>
	</li>
	<li>Collection of suspended particulate matter (SPM)
	<ol>
		<li>Collect SPM on 0.4 &#956;m polycarbonate (PC) membrane filters (acid washed and pre-weighed) by vacuum filtration, using a plastic filtration funnel and vessel.</li>
		<li>Freeze dry membrane filters in the lab to yield SPM concentrations and provide particle samples for particulate trace metal determination.</li>
	</ol>
	</li>
</ol>
3. Sample Pretreatment (Dissolved Trace Metals)
<ol>
	<li>For dissolved trace metal determination, add 2-ml concentrated sub-boiled HNO3 (per 1-L sample) into sample bottles. Transfer dissolved trace metal samples (acidified) into FEP bottles. Alternately, samples can be collected directly into FEP bottles. UV-irradiate the samples in FEP bottles for 24 hr (8 15-watt UV lamps).</li>
</ol>
4. Preconcentration and Treatments for Trace Metal Analysis
<ol>
	<li>Preconcentration resin
	<ol>
		<li>Weigh 2 g of cation exchange resin into a small (10-30 ml capacity) plastic cup and add a small amount of 2 N HNO3 solution into the cup. Pour the resin into a 10-ml capacity chromatography column. Clean the resin by washing the column with 5-ml 2 N HNO3 (sub-boiled) twice, and rinse with high purity water (HPW) 3 times.</li>
		<li>Add 10 ml of 1 M NH4OH (sub-boiled) into the column to convert the resin to NH4+-form.</li>
	</ol>
	</li>
	<li>Buffer solution (1 M ammonium acetate)
	<ol>
		<li>Add 57 ml of glacial acetic acid (sub-boiled) in approximately 800 ml of HPW. Add ~60 ml of ammonium hydroxide (21%, sub-boiled) and mix with acetic acid. Adjust the final pH to 5.5 and the final volume to 1,000 ml.</li>
	</ol>
	</li>
	<li>Preconcentration procedure (dissolved trace metals)
	<ol>
		<li>Adjust the pH of the acidified, UV-irradiated samples to 5.5 &#177; 0.3 by adding 30 ml of 1 M ammonium acetate buffer solution and some (~2.8 ml) NH4OH (sub-boiled) into the samples.</li>
		<li>Place the sample bottle ~30 cm above the column packed with cationic exchange resin (Section 4.1) and connect the sample bottle and preconcentration column by a ~60 cm FEP tube and chromatography cap and connector (female Luer).</li>
		<li>Control the flow rate at 3-5 ml/min using a 2-way stopcock, connected above the column. Allow the samples to pass through the preconcentration columns. After the samples have passed the column, disconnect the tubes and caps from the columns.</li>
		<li>Treat the columns with 2 x 5-ml HPW and 4 x 5-ml of 1 M ammonium acetate (pH 5.5), and 2 x 5-ml of HPW to separate major cations from other trace metals.</li>
		<li>Place a 30-ml acid-washed PE bottle just below the column and wash the column with 7 x 1-ml 2 N HNO3 (sub-boiled) into the PE bottle.</li>
		<li>Determine the volume of 2 N HNO3 effluent (~8.0 ml) by obtaining the weight and specific gravity of the 2 N HNO3 effluent.</li>
	</ol>
	</li>
	<li>Digestion of suspended particulate matter
	<ol>
		<li>Freeze dry PC filters with SPM samples and weigh them after drying. Place SPM samples, with filters, into pre-weighed perfluoroalkoxy alkanes (PFA) vessels (60-ml capacity), and add 3 ml of concentrated HNO3 into the vessels.</li>
		<li>Tighten the vessels to a constant torque of 2.5 kg-meters, and digested in a conventional oven at 130 &#176;C for 12 hr. After cooling, open the vessels and add 2 ml of HF into the vessels.</li>
		<li>Tighten the vessels to a constant torque of 2.5 kg-meters, and digested in a conventional oven at 130 &#176;C for 12 hr. After cooling, open the vessels and add 16 ml of 4.5% boric acid solution into the vessels.</li>
		<li>Tighten the vessels to a constant torque of 2.5 kg-meters, and digest in a conventional oven at 130 &#176;C for 12 hr. Weigh each vessel and determine specific gravity of each digest solution to yield final digest volume.</li>
		<li>Calculate a dilution factor for each digest from the final volume and weight of SPM on filter (final digest volume divided by sample weight on filter).</li>
	</ol>
	</li>
</ol>
5. Sample Analysis
<ol>
	<li>Trace metals
	<ol>
		<li>Determine trace metals (Cd, Co, Cu, Fe, Mn, Ni, Pb, and Zn) concentrations in pre-concentrated dissolved sample and solution from particulates using flame atomic absorption spectrometry, graphite furnace atomic absorption spectrometry, and/or inductively coupled plasma mass spectrometer.</li>
		<li>Determine major ions concentrations and some trace metals, in sub-samples drawn prior to preconcentration and digests of suspended particulate matter, by an inductively coupled plasma-atomic emission spectrometry.</li>
	</ol>
	</li>
	<li>Ancillary parameters
	<ol>
		<li>Determine sample temperature, pH, salinity and conductivity in the field, using portable devices.</li>
		<li>Determine dissolved organic carbon (DOC) concentrations by a Total Organic Carbon Analyzer, based on wet oxidation with carbon dioxide detection by infrared spectrometry 21. Determine total EDTA concentration by a high performance liquid chromatography (with a SPD-M10AV Diodearray detector) following the established procedures 22,23.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Taylor, H. E., Shiller, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mississippi+River+Methods+Comparison+Study:+Implications+for+water+quality+monitoring+of+dissolved+trace+elements.">Mississippi River Methods Comparison Study: Implications for water quality monitoring of dissolved trace elements.</a> Environmental Science and Technology. 29, 1313-1317 (1995).</li><li>Windom, H. L., Byrd, J. T., Smith, R. G., Huan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inadequacy+of+NASQAN+data+for+assessing+metal+trends+in+the+nation's+rivers.">Inadequacy of NASQAN data for assessing metal trends in the nation's rivers.</a> Environmental Science and Technology. 25 (6), 1137-1142 (1991).</li><li>Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trace Metals in Aquatic Systems. , (2013).</li><li>Wen, L. -. S., Santschi, P., Gill, G., Paternostro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estuarine+trace+metal+distributions+in+Galveston+Bay:+importance+of+colloidal+forms+in+the+speciation+of+the+dissolved+phase.">Estuarine trace metal distributions in Galveston Bay: importance of colloidal forms in the speciation of the dissolved phase.</a> Marine Chemistry. 63, 185-212 (1999).</li><li>Wen, L. -. S., Stordal, M. C., Tang, D., Gill, G. A., Santschi, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultraclean+cross-flow+ultrafiltration+technique+for+the+study+of+trace+metal+phase+speciation+in+seawater.">An ultraclean cross-flow ultrafiltration technique for the study of trace metal phase speciation in seawater.</a> Marine Chemistry. 55, 129-152 (1996).</li><li>Benoit, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clean+technique+measurement+of+Pb,+Ag,+and+Cd+in+freshwater:+A+redefinition+of+metal+pollution.">Clean technique measurement of Pb, Ag, and Cd in freshwater: A redefinition of metal pollution.</a> Environmental Science and Technology. 28, 1987-1991 (1994).</li><li>Benoit, G., Hunter, K. S., Rozan, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+trace+metal+contamination+artifacts+during+collection,+handling,+and+analysis+of+freshwater.">Sources of trace metal contamination artifacts during collection, handling, and analysis of freshwater.</a> Analytical Chemistry. 69 (6), 1006-1011 (1997).</li><li>Jiann, K. -. T., Presley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+determination+of+trace+metal+partitioning+in+river+water+by+a+two-column+ion+exchange+method.">Preservation and determination of trace metal partitioning in river water by a two-column ion exchange method.</a> Analytical Chemistry. 74 (18), 4716-4724 (2002).</li><li>Fardy, J. J., Alfassi, Z. B., Wai, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Preconcentration Techniques for Trace Elements. , 181-210 (1992).</li><li>Pai, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-concentration+efficiency+of+Chelex-100+resin+for+heavy+metals+in+seawater.+Part+2.+Distribution+of+heavy+metals+on+a+Chelex-100+column+and+optimization+of+the+column+efficiency+by+a+plate+simulation+method.">Pre-concentration efficiency of Chelex-100 resin for heavy metals in seawater. Part 2. Distribution of heavy metals on a Chelex-100 column and optimization of the column efficiency by a plate simulation method.</a> Analytica Chimica Acta. 211, 271-280 (1988).</li><li>Pai, S. -. C., Fang, T. -. H., Chen, C. -. T. A., Jeng, K. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+contamination+Chelex-100+technique+for+shipboard+pre-concentration+of+heavy+metals+in+seawater.">A low contamination Chelex-100 technique for shipboard pre-concentration of heavy metals in seawater.</a> Marine Chemistry. 29, 295-306 (1990).</li><li>Pai, S. -. C., Whung, P. -. Y., Lai, R. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-concentration+efficiency+of+Chelex-100+resin+for+heavy+metals+in+seawater.+Part+1.+Effects+of+pH+and+salts+on+the+distribution+ratios+of+heavy+metals.">Pre-concentration efficiency of Chelex-100 resin for heavy metals in seawater. Part 1. Effects of pH and salts on the distribution ratios of heavy metals.</a> Analytica Chimica Acta. 211, 257-270 (1988).</li><li>Salbu, B., Oughton, D. H., Salbu, B., Steinnes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trace Elements in Natural Waters. , 41-69 (1995).</li><li>. U.S. Environmental Protection Agency. Method 1669. Sampling ambient water for trace metals at EPA Water Quality criteria levels Available from: <a target="_blank" href="https://www3.epa.gov/caddis/pdf/Metals_Sampling_EPA_method_1669.pdf">https://www3.epa.gov/caddis/pdf/Metals_Sampling_EPA_method_1669.pdf</a> (1996)</li><li>Horowitz, A. J., 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=Problems+associated+with+using+filtration+to+define+dissolved+trace+metal+concentrations+in+natural+water+samples.">Problems associated with using filtration to define dissolved trace metal concentrations in natural water samples.</a> Environmental Science and Technology. 30, 954-963 (1996).</li><li>Cortecci, 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=Geochemistry+of+trace+elements+in+surface+waters+of+the+Arno+River+Basin,+northern+Tuscany,+Italy.">Geochemistry of trace elements in surface waters of the Arno River Basin, northern Tuscany, Italy.</a> Applied Geochemistry. 24 (5), 1005-1022 (2009).</li><li>Markich, S. J., Brown, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+importance+of+natural+and+anthropogenic+influences+on+the+fresh+surface+water+chemistry+of+the+Hawkesbury-Nepean+River,+south-eastern+Australia.">Relative importance of natural and anthropogenic influences on the fresh surface water chemistry of the Hawkesbury-Nepean River, south-eastern Australia.</a> The Science of the Total Environment. 217, 201-230 (1998).</li><li>Shafer, M. M., Overdier, J. T., Hurley, J. P., Armstrong, D., Webb, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+dissolved+organic+carbon,+suspended+particles,+and+hydrology+on+the+concentration,+partitioning+and+variability+of+trace+metals+in+two+contrasting+Wisconsin+watersheds+(U.S.A.).">The influence of dissolved organic carbon, suspended particles, and hydrology on the concentration, partitioning and variability of trace metals in two contrasting Wisconsin watersheds (U.S.A.).</a> Chemical Geology. 136, 71-97 (1997).</li><li>Warren, L. A., Haack, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+controls+on+metal+behaviour+in+freshwater+environments.">Biogeochemical controls on metal behaviour in freshwater environments.</a> Earth-Science Reviews. 54, 261-320 (2001).</li><li>Jiann, K. -. T., Santschi, P. H., Presley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+geochemical+parameters+(pH,+DOC,+SPM,+EDTA+Concentrations)+and+trace+metal+(Cd,+Co,+Cu,+Fe,+Mn,+Ni,+Pb,+Zn)+concentrations+in+river+waters+of+Texas+(USA).">Relationships between geochemical parameters (pH, DOC, SPM, EDTA Concentrations) and trace metal (Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn) concentrations in river waters of Texas (USA).</a> Aquatic Geochemistry. 19 (2), 173-193 (2013).</li><li>Peltzer, E. T., 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+comparison+of+methods+for+the+measurement+of+dissolved+organic+carbon+in+natural+waters.">A comparison of methods for the measurement of dissolved organic carbon in natural waters.</a> Marine Chemistry. 54, 85-96 (1996).</li><li>Nowack, B., Kari, F., Hilger, S. U., Sigg, L. <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+dissolved+and+adsorbed+EDTA+species+in+water+and+sediments+by+HPLC.">Determination of dissolved and adsorbed EDTA species in water and sediments by HPLC.</a> Analytical Chemistry. 68 (3), 561-566 (1996).</li><li>Bergers, P. J. M., de Groot, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+EDTA+in+water+by+HPLC.">The analysis of EDTA in water by HPLC.</a> Water Research. 28 (3), 639-642 (1994).</li></ol>

The authors have no conflict of interests to disclose.

Obtaining reliable trace metal data in natural waters requires great care as emphasized during sample collection, processing, pretreatments, and analysis that aim to reduce contamination. Trace metal concentrations in natural waters obtained using &#34;clean techniques&#34; in the last two decades found that the concentrations can be orders of magnitude lower than previously reported. Water quality criteria for trace metals in waters are now more readily assessed when trace metal levels are accurately measured resulting ...

It has been commonly recognized that some trace metal results obtained for natural waters may be inaccurate owing to artifacts arising from inadequate techniques applied during sample collection, treatments and determination1,2. The true concentrations (in sub-nM to nM range in surface waters 3) of dissolved trace metals are now up to two orders of magnitude lower than previously published values. The same situation has been found in marine chemistry where the accepted dissolved trace metal concentrations in oceanic waters have decreased by orders of magnitude over the last 40 years or so as improved sampling and analytical methods have been introduced. Efforts have been made to improve data quality with the developments of &#34;clean techniques&#34; aiming at the reduction or elimination of trace metal contamination throughout all phases of trace metal analysis 4-8. For the determination of trace metal concentrations at ambient levels, preconcentration is often required. Ion-exchange techniques 8-12 have been commonly applied for efficient preconcentration.
Contamination can arise from the walls of containers, the cleaning of the containers, the sampler, sample handling and storage, and sample preservation and analysis 7,13. All studies using clean methods conducted more recently indicate that trace metal concentrations in natural waters are typically well below the detection limits of routine methods 7. Since the recognition of suspect trace metal data in the early 1990s, clean methods have been incorporated into US EPA (Environmental Protection Agency) Guidelines for trace metal determination 14 and the US Geological Survey has adopted clean methods for their water quality monitoring projects 15. Clean methods for trace metal studies need to be employed in all projects in order to create a firm and accurate data base.
In principle, water samples used for trace metal determination should be collected with appropriate sampling gears of a particular material and composition, stored and treated properly using appropriate containers and apparatus, before proceeding with instrumental analysis. Since suspended particulate matter (SPM) can undergo changes during the sample storage period and alter water composition, rapid separation of SPM from water samples is a common practice for trace metal studies in aquatic environments. For the determination of dissolved trace metal concentrations in natural waters, filtration is necessary and in-line filtration techniques are suitable and efficient.
Distribution and behavior of trace metals in aquatic environments such as surface and ground waters can be affected by natural (e.g., weathering) and anthropogenic (e.g., wastewater effluents) factors, as well as other environmental conditions, such as regional geology, morphology, land use and vegetation, and climate 16-19. This can then lead to differences in physicochemical parameters such as concentrations of suspended particulate matter (SPM), dissolved organic carbon (DOC), anthropogenic ligands (e.g., ethylenediaminetetraacetic acid, EDTA), salt, redox potential and pH 17-20. Therefore, accurate and relevant trace metal studies require appropriate collection of samples for trace metal analysis as well as for the determination of related factors and parameters.

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Nitric Acid</td>
			<td style="width:216px;">Seastar Chemicals</td>
			<td style="width:129px;"></td>
			<td style="width:188px;">Baseline grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Ammonium hydroxide</td>
			<td style="width:216px;">Seastar Chemicals</td>
			<td style="width:129px;"></td>
			<td>Baseline grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Acetic Acid</td>
			<td style="width:216px;">Seastar Chemicals</td>
			<td style="width:129px;"></td>
			<td>Baseline grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Nitric Acid</td>
			<td style="width:216px;">J. T. Baker</td>
			<td style="width:129px;">9601-05</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Hydrochloric acid</td>
			<td style="width:216px;">J. T. Baker</td>
			<td style="width:129px;">9530-33</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Chromatographic columns</td>
			<td style="width:216px;">Bio-Rad</td>
			<td>7311550&#160;</td>
			<td>Poly-Prep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Column stack caps</td>
			<td style="width:216px;">Bio-Rad</td>
			<td>7311555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Cap connectors (female luers)</td>
			<td style="width:216px;">Bio-Rad</td>
			<td style="width:129px;">7318223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">2-way stopcocks</td>
			<td style="width:216px;">Bio-Rad</td>
			<td>7328102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Cation exchange resin</td>
			<td style="width:216px;">Bio-Rad</td>
			<td>1422832&#160;</td>
			<td>Chelex-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Portable sampler (sampling pump)</td>
			<td style="width:216px;">Cole Palmer</td>
			<td>EW-07571-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">FEP tube</td>
			<td style="width:216px;">Cole Palmer</td>
			<td>EW-06450-07</td>
			<td>6.4 mm I.D., 9.5 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pumping tube</td>
			<td style="width:216px;">Cole Palmer</td>
			<td>EW-06424-24</td>
			<td>6.4 mm I.D. C-Flex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Capsule filter (0.4 mm)</td>
			<td style="width:216px;">Fisher Scientific</td>
			<td>WP4HY410F0</td>
			<td>polypropylene casing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">1 L low density polyethylene bottle</td>
			<td>NALGE NUNC INTERNATIONAL</td>
			<td>312088-0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">1 L (or 500 ml) FEP bottle</td>
			<td>NALGE NUNC INTERNATIONAL</td>
			<td>381600-0032</td>
			<td></td>
		</tr>
	</tbody>
</table>

clean sampling and analysis of river and estuarine waters for trace metal studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

With the development and use of &#34;clean techniques&#34;, it is now well recognized that in order to obtain accurate trace metal concentrations in ambient waters, preconcentration of trace metals in waters samples is a common practice. While most water quality criteria for trace metals in natural waters are in the low &#956;g/L range, lower detection limits are needed to investigate geochemical and biological effects on trace metals at ambient concentrations in aquatic environments....

Watch this Scientific Journal Video about Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies at JoVE.com

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

clean-sampling-analysis-river-estuarine-waters-for-trace-metal

Research

JoVE Journal

Environment

11.3K Views.  National Sun Yat-sen University. Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Video: Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

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

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

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

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

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

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 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 ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...