Rotifers as Bio-Indicators: Using EDS of protective secretions to track metal pollutants in NE ponds.

Methods

Summary

Specimens will be collected primarily from three ponds: Mascuppic Lake Dam and Flint Pond, In Tyngsboro, MA, and Ayers Pond, in Hudson, NH. 

Sessile rotifers have specific vegetation preferences, including the bottom of lily pads and bladderwort (Utricularia spp.). Vegetation will be collected by hand and stored in large plastic buckets full of pond water, prior to being transported back to the lab at UMass Lowell. Once there, air stones will be added to the buckets, to ensure proper aeration. All plant material will be examined using a dissection microscope, while submerged in glass bowls full of pond water. Select specimens will be monitored for several days to and any larvae produced will be moved to synthetic pond water, fed on cultured algae, and trace amounts of metals will be added to their water, to determine if trace metals are passively or actively incorporated into their protective tubes. 

Both cultured and field specimens of sessile rotifers, especially Floscularia and Ptygura species, will be carefully removed, with their protective tubes intact, and will be transferred to small (~5mL) glass bowls full of filtered water.  All specimens will be anesthetized with 0.5% bupivacaine and lidocaine, prior to being mounted on glass coverslips and being dehydrated in a desiccator for 48h. The glass coverslips will then be mounted on carbon tape on SEM stubs and sputter-coated with Au. 

SEM images and EDS data will be collected with a JEOL JSM-7401F FE-SEM. EDS data will be graphed and quantified using EDAX Genesis software (v4.61, Ametek, Inc., Berwyn, PA, USA). Graphical spectra represent a combination of elements that make up the specimen secretion and in some cases the coverslip if the secretion is very thin.

Challenges

Bacteria are likely to be source of contamination in species of Limnias and Ptygura (and possibly others) as rod-like shapes were embedded in their secretions. The presence of Hg in the EDS spectrum of L. melicerta and P. crystallina may be due to the presence of sulfate- and/or iron-reducing bacteria that generate methyl mercury. 

Preliminary data collected from prior studies also shows the need for corrections of EDS data due to background discrepancies. It may be possible to eliminate such "background noise" by mounting specimens directly onto aluminum SEM stubs.