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 overall goal of this procedure is to filter large volume of seawater under low stress and contamination free condition for the collection of suspended particles and planktons. This method can help answer key question in the oceanography field such as stoichiometry and trace metals composition of suspended particles and marine planktons. This method can also be used to study traffic transfer, biotransformation, and biomagnification of marine planktons.

The main advantage of this technique is flexible, super clean, and easy to use. First, clean the sampler, fill the tubing and fill filtration unit with 1%anionic protease enzyme detergent, and let it soak for 24 hours. Next thoroughly flush the multi-layer, gravity filtration sampler with reverse osmosis, double distilled water.

Then fill it with 0.1%hydrochloric acid and let it soak for three days. After 72 hours of soaking in dilute acid, thoroughly flush the sampler with reverse osmosis, double distilled deionized water three to five times. Using a minimum of 20 liters per flush.

When handling the cleaned sampler, always wear polyethylene gloves. To assemble the sampler, connect six four meter long, chemically resistant, thermoplastic elastomer tubes to the six directional inlets on the top of the sampler. Then attach three mesh nylon filters to three 125 milliliter low density polyethylene sample containers.

Set up the containers with the 10 micron mesh filter positioned on the outside of the assembly. The 63 micron mesh filter in the middle, and the 153 micron mesh filter on the inside. For transportation, double bag the assembled sampler in polyethylene bags.

And then package it in a polypropylene shipping container. To use the sampler, connect the six, four meter thermoplastic elastomer tubes to the water spigots of six different 20 liter PTFE coated sampling bottles on the elevated multi-bottle sampling array. Next guide the seawater into the filtration unit via the directional inlets.

Now particles get gently separated by the filtration nets and settle into the 125 milliliter LDPE bottles that are secured at the base of the device. After flowing through at least 20 liters, transfer two to five liters of the flow through water into a five liter polyethylene container. To have a sample with particulates smaller than 10 microns.

After seawater has filled the sampling bottles, take the filtration unit to a class 100 clean bench. There remove each filter in sequence. Starting with the 153 micron filter and finishing with the 10 micron filter.

Now rinse off the plankton stuck to the filters using trace metal clean, 0.4 micron filtered seawater. Collect the seawater in the appropriate 125 milliliter collection bottle. Then detach the bottles from the assembly.

Then filter the collections one at a time through an acid washed vacuum filtration apparatus. With pre-weighed, acid washed, 47 millimeter, 10 micron polycarbonate filters. Use less that five kilopascals of pressure for this filtration.

Next filter the 10 micron water sample using an acid washed vacuum filtration apparatus with under five kilopascals of pressure and using pre-weighed, acid washed, 47 millimeter, 0.4 micron polycarbonate filters. Immediately after each filtration gently rinse the filter using a couple of milliliters of highly pure, double distilled water to remove the sea salts without disturbing the trapped particles and plankton. Then carefully remove the filter and store it in a acid washed, pre-weighed acrylic plastic petri dish.

Seal the dish in a plastic bag and store it at negative 20 degrees Celsius until they can be analyzed at the laboratory. First load the filters into the freeze drying machine. Once the temperature drops to negative 40 degrees celsius turn on the vacuum pump to start the freeze drying process.

Three days later, remove the dried filters and weigh them. Then place the filters into pre-weighed 60 milliliter PFA vessels and add three milliliters of concentrated, ultra pure nitric acid to each vessel. Tighten the vessels using a torque wrench to 2.5 kilogram meters and place them in a conventional over at 130 degrees Celsius for 12 hours for the first digestion sequence.

The next day, open the vessels once they have cooled and add two milliliters of ultra pure, hydrofluoric acid to each. Then incubate them again for the second digestion. The next day, after the vessels have cooled, open them and add 16 milliliters of 4.5%ultra-pure boric acid to each.

Then return them to the oven for the final digestion. Now after cooling, weigh each vessel. Determine the final mass of each digested solution and determine the specific mass of exactly one milliliter of digestant taken from each vessel.

Use these figures to calculate the final digestant volume. Then carefully pour each digestant into an acid cleaned 30 milliliter polyethylene bottle for further trace metal analysis. When data collected using the described multi-layer gravity filtration sampling technique, CATNET, was compared with conventional pressurized dead end filtration data, there was good agreement in particle concentrations.

More than 90%of those particles were between 0.4 and 10 microns. CATNET filtered seawater has very low to no analytes and no noticeable contamination when compared with ambient unfiltered seawater. Particles were collected at the depth of the chlorophyll a maximum in the South China Sea in March of 2002.

While over 80%were in the smaller range, larger particles corresponding to zoa plankton were also collected and showed diurnal vertical migration patterns. Analysis of surface water taken from the Taiwan Straight showed marine particle distribution and composition changed dramatically. Generally most particles were enriched in iron and aluminum and concentrations decreased with increasing size.

However, cadmium concentrations increased with increasing particle size possibly due to a bioconcentration process. After watching this video, you should have a good understanding of how to collect uncontaminated particles of various size and plankton from seawater collections. Once mastered, the lab protocol can be completed within hour.

While attempting this procedure it is important to remember to carry out trace metal clean sampling technique. Though this method can provide insight into ocean biogeochemistry, it can also be applied to fresh water systems. I first had the idea for this method when I tried to collect some live planktons to feed my aquarium fishes during my senior year in National Taiwan Ocean University back in 1980s.

Now since it's development, this technique paved the way for researchers in the field of aquatic science to explore particle concentration effect biomagnifications and traffic transfer in element biogeochemical cycles and eco systems.