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

The overall goal of this method is to clean the collector of water inhaled and exhaled by Benthic suspension feeders under natural conditions so that the particulate and dissolved compounds they removed and excreted can be quantified. When preparing collection vessels for dissolved organics and ammonia analysis, use new pre-cleaned EPA vials. For sampling other nutrients, use EPA glass vials.

Use HDPE vials for silicate. Rinse the vials and caps with high quality double distilled water. Then replace punctured silicon septum with new ones and soak the vials and caps in 10%hydrochloric acid for at least three days followed by three rinses with high quality double distilled water.

To further prepare the glass vials for nutrients, combust them at 450 degrees Celsius for four hours and allow them to cool in the furnace. Then attach the caps and wrap the vials in aluminum foil. Next mix DDW with 0.2 micron filtered surfactant to prepare the five percent solution.

Now place a combusted binder free glass fiber filter inside the cleaned inline stainless steel swinnex filter holder. Then use an acid-cleaned syringe to run 100 milliliters of the filtered surfactant solution through the entire assembly. Rinse the assembly clean using 100 milliliters of high quality double distilled water.

If the filter assembly is for sampling silica, then prepare it with a polycarbonate filter and run 30 milliliters of five percent hydrochloric acid through it followed by 30 milliliters of high quality double distilled water. Assemble the system for underwater work using PEEK tubing. Use a sharp knife or PEEK cutter to prepare the required lengths.

Fit each tube with a male luer connector attached to a syringe needle. Make sure that the flat side of the blue flangeless ferrule is aligned with the end of the tube, then tighten the green nut by hand. Then, using insulating tape, secure the PEEK tubing to the tripod arms for a custom built manipulator.

Next, attach capped disposable syringe needles to the male luer connectors. Clearly label the sampling gear and color code the inhale and exhale sampling components such as with blue tape for in and yellow for out. Similarly, color code the sampling vessels to better identify pairs of vessels that are sequentially numbered.

Now evacuate the sampling vessels before the dive, ideally with a good vacuum pump. A standard Lyophilizer provides a high vacuum and works as well. Finally line up all the sampling vessels as needed for the dive.

Plan all sampling steps out in advance and pack accordingly. First confirm whether the specimen is pumping by releasing filtered fluorescing dye next to the inhalant orifice and confirm that the dye is emerging through the exhale end orifice. Next install the VacuSIP device and make sure it is stable.

Then place the inhalant sampling tube within the inhalant orifice or very close to it, making certain that the inhalant tube is not in the proximity of any other exhalant orifices. Next carefully direct the exhalant sampling tube towards the exhalant syphon and very gently insert it one to five millimeters into the syphon. Take great care not to make contact with or otherwise disturb the sampled organism.

Then secure the sampling assembly in close working proximity. If you use the customized manipulator, take the same care in positioning the inhalant and exhalant tube. Ultra-plankton sampling requires no filter.

To sample ultra-plankton use the needle to pierce the inhaled and exhaled evacuated tube's septum. Check that the water is dripping and collect two to six milliliters of water from each tube. For silicates, install the inline stainless polycarbonate filter holders with the polycarbonate membranes between the needle and the luer male connector at the distill end of the tube.

Then pierce the septum cap of the prepared HDPE vials to start sampling. To sample dissolved organics and inorganics, have three pairs of vials ready. One for nitrates and phosphates, one for ammonia, and a third pair for dissolved organics.

To sample dissolved organics or nutrients, replace the polycarbonate filter assembly with a pre-cleaned inline stainless steel filter holder that contains a pre-combusted glass filter. Then pierce the septum cap of an EPA glass vial to start sampling. Dissolved inorganics were collected in new EPA glass vials cleaned by the manufacturer or re-used EPA vials cleaned according to the described protocol.

The recycled vials reported higher ammonia concentrations but other values were similar. Stainless steel filter holders and polycarbonate inline Swinnex filter holders were compared by filtering pure water and testing for dissolved organic and inorganic nutrients. Polycarbonate membrane and pre-combusted glass fiber filter were tested with each holder.

The combination of stainless steel filter holder and combusted glass fiber filter worked best. Cell counts were made with the flow cytometer to test the sample quality. When in-ex sample pairs are properly collected, most cells were removed from the exhaled water.

In contrast, improper collection did not garner the same result. When the VacuSIP is properly employed, patterns of removal or excretion were evident for most analysed compounds. The measurements seen here were taken from a mediterranean sponge.