The authors thank Miss Pi-Fen Lin, Mr. Wei-Lung Tseng, Miss Pei-Hsuan Lin, and Dr. Jia-Lu Chuan for their assistance during the field sampling and lab analysis for the practical development and application of &#34;CATNET.&#34;&#160;The assistance of crew and technician on board research vessel Ocean Research-I and Ocean Research-II during the sampling expeditions is greatly appreciated. This work was supported partly by Taiwan Ministry of Science and Technology of grants 91-2611-M-002-007, 95-2611-M-002 -009, 96-2611-M-002-004, 97-3114-M-002-006, 104-2611-M-002-019. This manuscript is written in memory of Miss Wen-Huei Lee for her immense dedication and contribution to marine researches in Taiwan.

The following protocol involves working with harmful chemicals. Please read the Safety Data Sheets (SDS) carefully, and follow institutional chemical safety guidelines.
1. Multi-layer Gravity Filtration Sampler Preparation
<ol>
	<li>Sampler cleaning
	<ol>
		<li>Fill the tubing and filtration unit with 1% (w/v) of anionic protease enzyme detergent solution and soak it for 24 h. Flush the multi-layer gravity filtration sampler with reverse osmosis double distilled water (RO-DDW) thoroughly, then fill it with 0.1% (v/v) hydrochloric acid (HCl, Reagent Grade) and soak for 72 h.</li>
		<li>Thoroughly flush the multi-layer gravity filtration sampler with reverse osmosis double distilled deionized water (RO-DD-DIW) three to five times, at least 20 liters each time, and store the assemblage in plastic bags.</li>
	</ol>
	</li>
	<li>Particle sample container cleaning/preparation
	<ol>
		<li>Use low density polyethylene (LDPE, 125 mL) or fluorinated ethylene propylene (FEP, 125 mL) bottles as containers for particles. Clean the bottles by soaking them first in alkaline detergent (Micro, 1%), then in 50% (v/v) nitric acid (HNO3, Reagent Grade), then 10% (v/v) HCl solutions for at least 24, 48, and 24 h, respectively. Rinse the bottles with de-ionized water (RO-DD-DIW) between the two soaking steps.</li>
		<li>After a final HCl soaking, rinse the bottles thoroughly with de-ionized water (RO-DD-DIW), and dry the bottles in a clean room or class-100 clean bench. 
		Attach the cleaned bottle to the multilayer gravity filtration sampler, or seal cleaned bottles in PE zippered bags and double-bag them for transport.</li>
	</ol>
	</li>
	<li>Assemblage of multi-layer gravity filtration sampler
	<ol>
		<li>Connect six 4 m long chemically resistant thermoplastic elastomer tubes (outside diameter of 0.635 cm) to the six directional inlets on top of the sampler.</li>
		<li>Assemble the three different mesh nylon filters with low-density polyethylene sample containers (125 mL LDPE) in sequence in a clean room (bench) after they are cleaned (see below), with the 10 &#181;m mesh filter positioned on the outside, the 63 &#181;m mesh filter in the middle, and the 153 &#181;m mesh filter on the inside. For transportation, store the multi-layer gravity filtration sampler in two layers of polyethylene (PE) bags, then place it in the polypropylene (PP) shipping container.</li>
	</ol>
	</li>
</ol>
2. Sampling
<ol>
	<li>Sample collection
	<ol>
		<li>Upon arrival at the sampling site, have one person remove the multi-layer gravity filtration sampler from a shipping container on the deck of the research vessel and open the bag with the sampler. Then, have them put on the PE gloves, connect the six 4 m thermoplastic elastomer tubes to the water spigots of six 20 L PTFE-coated sampling bottles on the elevated multi-bottle sampling array, and guide the seawater into this filtration unit. The seawater will flow through the directional inlets, and the particles/plankton will be gently separated/fractionated through the nets and settle into the 125 mL LDPE bottles that are secured at the base of the nets.</li>
		<li>After the seawater has flowed through (usually 120 L for coastal sea water and 240 L for open ocean water), remove each net in sequence (firstly, the 153 &#181;m, then the 63 &#181;m, and finally the 10 &#181;m) in a class-100 clean bench, then spray the net with trace-metal-clean 0.4 &#181;m filtered seawater to rinse out any plankton stuck on the inner surface of the nets. Collect the seawater with concentrated particles/plankton in 125 mL polyethylene bottles.</li>
		<li>Unscrew these bottles from the nets, and filter the solutions with concentrated particles/plankton again through an acid-washed vacuum filtration apparatus with pre-weighed, acid-washed 47 mm, 10 &#181;m pore-size polycarbonate filters under low-vacuum conditions (&#60;5 kPa).</li>
		<li>To collect particles/plankton smaller than 10 &#181;m, wait for at least 20 L of seawater to flow through the sampler, then after that, collect two to five L of water in the 5 L PE container, and filter these sample waters by an acid-washed vacuum filtration apparatus with pre-weighed, acid-washed, 47-mm, 0.4-&#181;m pore size polycarbonate filters.</li>
		<li>After vacuum filtration, rinse the sample filters immediately with high purity DDW water to remove the residue of seawater, minimizing the influence of sea salts on determining the dry weights of particles/plankton. Keep the rinsing volume to only a couple of milliliters to prevent damaging the fragile plankton.</li>
		<li>Then, after this rinsing step, carefully remove the filter from the vacuum filtration unit, store the sample filters in acid-washed, pre-weighed acrylic plastic petri dishes, and seal in resealable plastic bags. Keep the bags in a -20 &#730;C freezer onboard until returning back to a land-based laboratory for further sample pretreatment and chemical analysis.</li>
	</ol>
	</li>
</ol>
3. Sample Treatment
<ol>
	<li>Freeze drying and digestion of particles
	<ol>
		<li>Place the filters with particle samples in the collector chamber of the freeze-drying machine, and turn on the machine. As the machine temperature reaches -40 &#176;C, turn on the vacuum pump of the machine and start the freeze drying processes. 
		NOTE: The vacuum level should be maintained steadily below 0.12 mBar. Please read the user manual carefully and follow the manufacturer&#39;s guidelines for each step.</li>
		<li>After 72 h, turn off the freeze-drying machine, remove the dried filters and weigh them. Then, place dried sample filters into pre-weighed perfluoroalkoxy alkane (PFA) vessels (60 mL capacity), and add 3 mL of concentrated ultrapure nitric acid into the vessels2,3,6,7.</li>
		<li>Tighten the vessels with a torque wrench to a constant torque of 2.5 kg-m, and place the vessels in a conventional oven at 130 &#176;C for 12 h for the first digestion sequence. After cooling, remove the vessels from the oven, open the vessels, and add 2 mL of ultrapure hydrofluoric acid into the vessels2,3,6,7.</li>
		<li>Tighten the vessels with a torque of 2.5 kg-m, and place the vessels in a conventional oven at 130 &#176;C for 12 h, which is the second digestion sequence. After cooling, open the vessels and add 16 mL of 4.5% ultra-pure boric acid solution into the vessels2,3,6,7.</li>
		<li>Tighten the vessels to a constant torque of 2.5 kg-meters, and digest the samples in oven at 130 &#176;C for 12 h for the final digestion sequence. After cooling, weigh each vessel and determine the final mass and specific mass of each digested solution to yield a final digestant volume. 
		NOTE: Specific mass is determined by measuring the weight of exactly 1.00 mL of digestant.</li>
		<li>Carefully pour the digestant into 30 mL acid cleaned PE bottle for further trace metal analysis.</li>
	</ol>
	</li>
	<li>Trace Metal Analysis
	<ol>
		<li>Determine trace metal concentrations (Cd, Cu, Fe, Mn, Ni, and Al) in digested solutions of particles using a graphite furnace atomic absorption spectrometer (GF-AAS)6.</li>
		<li>As an accuracy test, use certified reference material (CRM), such as marine sediment reference materials from the National Research Council of Canada, estuarine sediment standard reference material from the National Institute of Standards and Technology of the USA, and plankton reference material from the European Commission&#39;s science and knowledge service. The process gives 95% to 107% recovery of the certified value for the trace metals provided in the CRM.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jeandel, C., 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=What+did+we+learn+about+ocean+particle+dynamics+in+the+GEOSECS-JGOFS+era.">What did we learn about ocean particle dynamics in the GEOSECS-JGOFS era.</a> Progr. Oceanogr. 133, 6-16 (2015).</li><li>Lam, P., 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=Methods+for+analyzing+the+concentration+and+speciation+of+major+and+trace+elements+in+marine+particles.">Methods for analyzing the concentration and speciation of major and trace elements in marine particles.</a> Progr. Oceanogr. 133, 32-42 (2015).</li><li>Collier, R., Edmond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trace+element+geochemistry+of+marine+biogenic+particulate+matter.">The trace element geochemistry of marine biogenic particulate matter.</a> Progr. Oceanogr. 13, 113-199 (1984).</li><li>Donat, J. R., Bruland, K. W., Steinnes, E., Salbu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+elements+in+the+oceans.">Trace elements in the oceans.</a> Trace Elements in Natural Waters. , 247-280 (1995).</li><li>Wen, L. -. S., Santschi, P., Tang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+radioactively+labeled+colloids+and+natural+particles:+evidence+for+colloidal+pumping.">Interaction between radioactively labeled colloids and natural particles: evidence for colloidal pumping.</a> Geochim. Cosmochim. Ac. 61, 2867-2878 (1997).</li><li>Wen, L. -. S., Warnken, K., Santschi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organic+carbon,+iron,+and+aluminium+oxyhydroxides+as+trace+metal+carriers:+Comparison+between+the+Trinity+River+and+the+Trinity+River+Estuary+(Galveston+Bay,+Texas).">The role of organic carbon, iron, and aluminium oxyhydroxides as trace metal carriers: Comparison between the Trinity River and the Trinity River Estuary (Galveston Bay, Texas).</a> Mar. Chem. 112, 20-37 (2008).</li><li>Hurd, D., Spencer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+particles:+analysis+and+characterization.">Marine particles: analysis and characterization.</a> American Geophysical Union. , (1991).</li><li>Martin, J. H., Knauer, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elemental+composition+of+plankton.">The elemental composition of plankton.</a> Geochim. Cosmochim. Ac. 37, 1639-1653 (1973).</li><li>Morel, F., Price, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biogeochemical+cycles+of+trace+metals+in+the+oceans.">The biogeochemical cycles of trace metals in the oceans.</a> Science. 300, 944-947 (2003).</li><li>Ho, T. -. Y., 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=The+elemental+composition+of+some+marine+phytoplankton.">The elemental composition of some marine phytoplankton.</a> J. Phycol. 39, 1145-1159 (2003).</li><li>McDonnell, A., 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=The+oceanographic+toolbox+for+the+collection+of+sinking+and+suspended+marine+particles.">The oceanographic toolbox for the collection of sinking and suspended marine particles.</a> Prog. Oceanogr. 133, 17-31 (2015).</li><li>Wen, L. -. S., Li, W. -. H., Zhuang, G. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Multiple layer filtering and collecting device. , (2005).</li><li>Ho, T. -. Y., Wen, L. -. S., You, C. -. F., Lee, D. -. 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+trace-metal+composition+of+size+fractionated+plankton+in+the+South+China+Sea:+biotic+versus+abiotic+sources.">The trace-metal composition of size fractionated plankton in the South China Sea: biotic versus abiotic sources.</a> Limnol. Oceanogr. 52, 1776-1788 (2007).</li><li>Hsu, R., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+estimations+of+the+density+and+porosity+of+flocs+of+varying+sizes+in+a+submarine+canyon.">In-situ estimations of the density and porosity of flocs of varying sizes in a submarine canyon.</a> Mar. Geol. 276, 105-109 (2010).</li><li>Liao, W. -. H., Yang, S. -. C., Ho, T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+metal+composition+of+size-fractionated+plankton+in+the+Western+Philippine+Sea:+the+impact+of+anthropogenic+aerosol+deposition.">Trace metal composition of size-fractionated plankton in the Western Philippine Sea: the impact of anthropogenic aerosol deposition.</a> Limnol Oceanogr. , (2017).</li><li>Grasshoff, K., Kremling, K., Ehrhardt, 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> Methods of seawater analysis. , (2007).</li></ol>

The co-author, Mr. Alan Chuang is the patent co-owner and the general manager of the company (Sino Instruments Co., Ltd.) which manufactured this collection apparatus for interested users. The patent ended in May 9th, 201512.

Obtaining reliable trace metal concentrations on plankton and suspended particles in natural waters, which are generally present at very low concentrations, requires great care during sample collection, processing, pretreatments, and analysis, with the aim of reducing contamination. Therefore, the procedures to design and prepare sampling gear, sample containers, and materials used to collect and process samples are all critical steps toward obtaining high-quality data for trace metals in marine environments. With the ad...

Particles in the ocean play an important role in marine biogeochemical cycles1. Most of the properties of particles, such as size, mineralogy, and composition, can change profoundly from one geological or hydrographical setting to another2. In addition, the distributions of elements in the ocean are also associated with the life cycle of marine phytoplankton: growth, death, sinking, and re-mineralization3,4. Marine particles span at least 4 orders of magnitude in size, ranging from submicron particles to large aggregates (&#62;5 mm). Most particles are biologically derived, from processes such as viral lysis, exudation, secretion, fecal pellet production, etc. Other particles are formed from physical coagulation of cells, cellular debris, or lithogenic materials1. Various chemical and biological characteristics of particles control both the geochemical cycles and biological processes occurring on and within the particles4,5,6. These particles are important habitats as well as food sources for some organisms, such as zooplankton or saprotrophs. Accordingly, the fate of particles is often related to their size, which can be modified by biological processes on and around particles.
Sampling marine particles usually requires filtration, but this approach introduces a certain ambiguity in identifying the properties of particles, since marine particles are not homogenous in composition and size. Suspended particles, mainly composed of small and low density particles that are almost permanently in suspension, are mixed with varying amounts of larger and denser particles in suspension only for a short period of time, depending on hydrodynamic conditions7. The first reports of the trace metal composition of plankton samples were collected by plankton tows or suspending plankton nets on a research vessel8. The authors often found metal particles and paint chips in samples, suggesting a severe problem of contamination during marine particle sampling for chemical analysis. Other efforts include net towing by rubber rafts or using a polyvinyl chloride (PVC)-hand winch3. The difficulty of reliable sampling of particles makes progress in our understanding of the chemical composition of marine particles more difficult, especially for trace elements. As such, most crucial information on the concentration of trace elements in phytoplankton has come from culture studies9,10. This recognition has motivated marine scientists to create new methods for studying particles in the sea over the past thirty years11.
Oceanographers have used various sampling techniques, including shipboard filtration, in situ filtration, and sediment traps11. The processing of large volumes of sea-water to collect non-contaminated samples can be challenging, especially for open ocean and deep waters in which the particle concentrations are very low (0.001 - 0.1 mg/L). It is also necessary to filter large volumes of sea-water to obtain an adequate quantity of particles to measure trace metal concentrations. Some researchers have used the size-fractionation method to separate suspended particles from sinking particles. However, particle size, porosity, density, and shape can all influence particle sinking velocities. Sediment traps are not practical tools to collect suspended particles, since those are designed for sinking particles. Therefore, it is important to develop sampling and treatment methods that can collect sufficient quantities of suspended particles with minimal contamination. Hence, size-fractionation by in situ filtration is still a promising tool in the oceanographer&#39;s sampling toolbox, since it can reveal critical information on marine particle dynamics. Here, we describe a successfully tested trace-metal-clean, multi-layer gravity filtration sampling apparatus, which can treat large volumes (120 - 240 L) of seawater on board in one pass from polytetrafluoroethylene (PTFE) coated water sampling bottles in a multi-bottle sampling array. This sampling apparatus uses acid-washed synthetic nylon nets in sequence, and the nets are enclosed within a polycarbonate container to gently collect size-fractionated suspended matter and phytoplankton12,13,14,15 (Figure 1). The aim of this work is to provide a better tool for studying the metal-particle associations and their reaction dynamics in marine environments, and improve our understanding of the fate of a wide variety of planktons, particles, and trace metals in these environments.

<table>
	<tbody>
		<tr>
			<td>thermoplastic elastomer (C-Flex) Tubings</td>
			<td>Cole Palmer</td>
			<td>EW-06424-67</td>
			<td>O.D. 0.635 cm, Opaque White 1/8&#34;ID x 1/4&#34;OD, 25 ft/pack</td>
		</tr>
		<tr>
			<td>LDPE Bottle (Nalgene)</td>
			<td>ThermoFisher Scientific</td>
			<td>2103-0004</td>
			<td>125 mL, Nalgene Wide-Mouth LDPE Bottles with Closure</td>
		</tr>
		<tr>
			<td>anionic protease enzyme detergent detergent (Tergazyme)</td>
			<td>Alconox</td>
			<td>1104-1</td>
			<td>1&#215;4 lb box (1.8 kg)</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Sigma-Aldrich</td>
			<td>695025</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>alkaline detergnet (Micro)</td>
			<td>Cole Palmer</td>
			<td>EW-99999-14</td>
			<td>Micro-90 Cleaning Solution</td>
		</tr>
		<tr>
			<td>polycarbonate filter, 47 mm, 0.4 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>WHA111107</td>
			<td>Whatman Nuclepore Track-Etched Membranes, diam. 47 mm, pore size 0.4 &#956;m, polycarbonate</td>
		</tr>
		<tr>
			<td>polycarbonate filter, 47 mm, 10 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>WHA111115</td>
			<td>Whatman Nuclepore Track-Etched Membranes, diam. 47 mm, pore size 10 &#956;m, polycarbonate</td>
		</tr>
		<tr>
			<td>PFA vessel, 60 ml capacity</td>
			<td>Savillex</td>
			<td>300-060-03</td>
			<td>60 mL Digestion Vessel, Flat Interior, Flat Exterior, Buttress Threaded Top</td>
		</tr>
		<tr>
			<td>Nitric acid, ultrapure</td>
			<td>Seastar Chemicals</td>
			<td>N/A</td>
			<td>BASELINE Nitric Acid</td>
		</tr>
		<tr>
			<td>HF, ultrapure</td>
			<td>Seastar Chemicals</td>
			<td>N/A</td>
			<td>BASELINE Hydrofluoric Acid</td>
		</tr>
		<tr>
			<td>Boric acid, ultrapure</td>
			<td>Seastar Chemicals</td>
			<td>N/A</td>
			<td>BASELINE Hydrobromic Acid</td>
		</tr>
		<tr>
			<td>polyethylene (PE) gloves</td>
			<td>Safty Zone</td>
			<td>GDPL-MD-5</td>
			<td>Clear Powder Free Polyethylene Gloves</td>
		</tr>
		<tr>
			<td>Multiple layer filtering and collecting device</td>
			<td>Sino Instrumnets Co. Ltd</td>
			<td>not available</td>
			<td>Multiple layer filtering and collecting device, CATNET</td>
		</tr>
		<tr>
			<td>10 um Nylon filters, Nitex</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX 10</td>
			<td>Nitex - Standard Widths (40 - 44 inches)</td>
		</tr>
		<tr>
			<td>60 um Nylon filters, Nitex</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX 60</td>
			<td>Nitex - Standard Widths (40 - 44 inches)</td>
		</tr>
		<tr>
			<td>150 um Nylon filters, Nitex</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX 150</td>
			<td>Nitex - Standard Widths (40 - 44 inches)</td>
		</tr>
		<tr>
			<td>torque wrench</td>
			<td>Halfords</td>
			<td>200238</td>
			<td>Halfords Professional Torque Wrench 8-60Nm</td>
		</tr>
		<tr>
			<td>multi-bottle sampling array, Rosette</td>
			<td>General Oceanics</td>
			<td>Model 1018</td>
			<td>Rosette Sampler</td>
		</tr>
		<tr>
			<td>PTFE-coated sampling bottles, GO-Flo</td>
			<td>General Oceanics</td>
			<td>108020T</td>
			<td>GO-Flo water sampler teflon coated</td>
		</tr>
		<tr>
			<td>Marine sediment reference materials</td>
			<td>National Research Council Canada</td>
			<td>MESS-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Estuarine sediment standard reference material</td>
			<td>National Institute of Standards and Technology</td>
			<td>1646a</td>
			<td></td>
		</tr>
		<tr>
			<td>Plankton reference material</td>
			<td>The European Commission&#39;s science and knowledge service</td>
			<td>CRM414</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

With the development of modern oceanography, it is now a common practice to use &#34;clean techniques&#34; to obtain accurate trace metal concentrations in marine particles or plankton. Since most particles in natural waters are in the low mg/L to &#181;g/L range, the treatment of large volumes of seawater is necessary to investigate geochemical and biological effects of trace metals on various particles in ambient environments. With the use of clean, multi-layer gravity filtration (&#34;...

Watch this Scientific Journal Video about An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles at JoVE.com

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

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 ...

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8.7K Views.  National Taiwan University. 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.

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

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

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 ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...