Trying to find juveniles was too much of a risk for a summer project. We needed something concise and publishable as a short report at the end of the summer. Chuck’s new project, and by default and responsibility, my project as well, was to analyze the nitrogen content of horseshoe crabs, their prey, and their environment through a process called nitrogen stable isotope analysis.
Without getting too deeply into the chemistry, this means that there are two naturally occurring forms of nitrogen, 14N and 15N. The basic concept of nitrogen stable isotope analysis is a comparison of these two isotopes to obtain a ratio. They are stable isotopes because they don’t degrade radioactively, as a radioisotope of, say, uranium would. Differences in ratios between organisms indicate their position in the food chain. Differences in ratios among organisms of the same species indicate that they are feeding in places that have different base levels of the two isotopes. More 15N indicates an area that has more wastewater entering the system, since our bodies build with 14N preferentially and we excrete the 15N.
Through this analysis we were hoping to show, among other things, that horseshoe crabs living in an area with more houses (more septic systems, more wastewater) would have a higher signature (more 15N). Barnstable Harbor is relatively pristine and has a ten-foot tidal range so it is flushed regularly, while Stage Harbor has a lot of activity on its shores and has only a two-foot tidal range.
We needed to collect tissue samples from horseshoe crabs, prey items (worms and clams), sediment, and particulate organic matter suspended in the water column, called seston. These would represent the top, middle, and base signatures of the food chain that interested us. Sites were chosen that overlapped with my survey sites, and we went out with labeled Ziploc bags and liter bottles to collect what we needed.
Collecting the water and sediment was easy. We simply used a large syringe with the end cut off to take sediment cores and filled up the bottles. Collecting worms and clams was more difficult. We had sieves, which we would fill with mud or sand and rinse until hopefully worms revealed themselves. We were looking for polychaetes, which are segmented worms with small paddles. The most common is the clam worm, Nereis, which is iridescent green with large black jaws. There were also bloodworms, Glycera, which are transparent so you can see the blood moving inside them. They have an eversible proboscis tipped with four tiny jaws that lunges out and presumably could nip you.
The clams we found by digging, of course, the way most people looking for clams must do, professional or otherwise. We started out using rakes but had the shellfish constable show up to ask us what we were doing clamming in a closed area. I’m convinced someone called in to report us. We stopped using the rakes so we wouldn’t attract as much attention. We were looking for quahogs, hard-shelled clams that serve a variety of dietary purposes for humans depending on size. Horseshoe crabs eat them also, crushing and grinding them with a densely spiked “mouth.” We were fortunate that the crabs are mostly indiscriminate with regard to the clam’s size, because it is much easier to search for clams without worrying about legal sizes.
Collecting tissue samples from horseshoe crabs required cutting off one of their legs. Chuck and Alison usually had me do the dirty work. Some crabs ooze, their thick, cloudy, light blue blood congealing almost immediately. Others hardly bleed at all, just a pale meniscus at the edge of the cut. One very memorable crab bled in a thin steady stream that squirted everywhere. As soon as the crabs were returned to the water the bleeding would stop.
When we returned to the lab, the samples had to be rinsed with deionized water, dried out in an oven, and ground into a fine powder. Tiny scoopfuls of each powdered sample then had to be packed for shipment to the lab in California where the actual analysis would occur. If you imagine a small aluminum foil cup, about 7mm tall x 5 mm in diameter, that’s the packing tin. We have to put approximately 2–3 mg of powdered material into this cup. The edge is crimped closed with sterilized tweezers, and then the cup is flattened, folded in thirds, folded in half, and shaped into a cube. It is a tedious process that requires me to swear off caffeine on the day I’m going to pack. We sent our miniscule gems of tissue west and waited. A couple weeks later, if all went well, we would get our results back and hopefully have something worthwhile to say.