Malcolm S. Hill and April L. Hill

Background
Since receiving the ABLE Laboratory Initiative grant last summer, we have been working on a lab that deals with the use of freshwater sponges as indicators of water pollution. Specifically, the focus of this laboratory module is on the effects that chemicals of environmental concern (e.g., endocrine disrupters) have on sponge growth and development. Contamination of aquatic ecosystems is a serious issue in environmental science. Identifying which chemicals we should be concerned with, and determining the consequences of contamination by specific compounds, is a major area of current research. Undergraduates are constantly exposed to news of environmental deterioration, yet few have an idea of how bioassays can help set national drinking water standards or shape environmental guidelines/laws. Our laboratory activity represents an introduction to this area of biological exploration with an unusual animal model.

Biology of Freshwater Sponges
Freshwater sponges are common animals of most aquatic ecosystems. They utilize flagellated choanocytes to pump water through a series of canals. Incoming water enters through ostia, passes through choanocyte chambers, and exits through the osculum. Bacteria are filtered from incoming water, and large volumes of water can pass through a sponge in a 24-hour period. Due to their simple morphological construction, many cells come into direct contact with the surrounding water as the sponge pumps. Thus, a sponge's mode of feeding results in high levels of exposure to any compound present in an ecosystem. Watanabe and colleagues ("Life of the Freshwater Sponge", 1998, produced by Tokyo Cinema, Inc.) have produced a beautiful film using time-lapse videography to document the life cycles and water purification role of freshwater sponges. The running time of the film is 28 mins, and shows sponges in their natural environment. This film was produced by the BBC, and would make a nice introductory presentation to the topic.

A useful aspect of freshwater sponge biology (at least for the purposes of an undergraduate lab module) is the fact that they enter diapause as small gemmules. Gemmules are overwintering balls that are produced in the late summer/early fall by the adult sponge. Adult tissue disintegrates around the gemmule during the winter, and a new sponge emerges from the gemmules in the spring. The newly developing sponge exits the gemmule from a micropyle, and then quickly spreads around the gemmule. In a healthy sponge, a water vascular system is evident, and many sponges produce a long, striking osculum (Fig. 1). Gemmules may be stored for years at 4˚C and still remain viable.

Figure 1. In control or low concentration treatments, sponge growth is normal. Notice the well-defined choanotcytic chambers indicated by the circle (A) and the large osculum indicated by the large circle (B).

There are several other reasons why sponges are a model laboratory organism to explore the biological consequences of environmental pollution. For the purposes of ease of set-up, sponges represent a cost and time effective study organism. They are readily available at a low cost from the major teaching supply companies (e.g., Connecticut Valley Biological, Carolina Biological Supply Company). Finally, gemmules grow relatively quickly (within 3-5 days) and require very little equipment to grow (a few tissue culture plates, and a room of fairly constant temperature and photoperiod).

Laboratory Goals-Progress Report
This lab will introduce students to a simple bioassay that will allow them to explore the effects that a chemical's concentration has on the level of toxicity. By relying on morphological examination of sponges hatching from gemmules that are smaller than a millimeter in diameter, this module will help students develop their microscopy skills. Our major aim, however, is to have students strengthen their ability to design and test their own hypotheses. After students have familiarized themselves with the basics of the freshwater sponge life cycle, they would be expected to propose a hypothesis about the effects of pollutants on sponge growth and morphology. The students would be guided in the design of an experiment to examine 1) the effects of chemicals of their choice on sponge growth, and 2) the effects of pollutant concentration on sponge growth. The choice of chemicals would be at the discretion of the instructor, but our work has focused on endocrine disrupters (e.g., ethyl benzene, nonylphenol). As the experiments progress, students will be able to detect major growth abnormalities immediately (Fig 2), but should also look for developmental abnormalities such as the absence of a well-defined water vascular system (Fig 3). As mentioned earlier, normal sponge growth typically includes the production of a distinct water vascular system with a less dense cellular construction (Fig 1). We hope that results from this laboratory can be used for a discussion of the biological consequences of pollution and will provide students with an appreciation for how dilution influences a chemical's biological effect. {While we have focused specifically on the effect that chemical pollutants have on sponge growth, sponge gemmules provide a host of other hypothesis testing possibilities (e.g., effects of temperature, light, food concentration, water oxygen content, etc. on rates of gemmule hatching).}

Figure 2. Sponge growth in our concentrated methyl paraben (A), nonylphenol (B), and ethyl benzene (C) treatments was highly anomalous. Newly produced tissue exited the micropyle as a tight tube which was drastically different from a "normal" sponge.

Figure 3. Sponges grown in solutions with medium or low concentrations of chemicals often displayed unusual growth forms or failed to produce a water vascular system.

We have spent the past year identifying chemicals that cause gross morphological defects in sponge growth. As can be seen in the accompanying figure, the effects of a number of chemicals can have a significant effect of sponge morphology. This abnormality appears to be a common response by sponges to many organic compounds since other researchers have observed this response. The compounds we used in our trials produced dramatic effects within 3 d. An incomplete list of compounds that we have tested includes: nonylphenol, ethyl benzene, toluene, methyl paraben, benzo-a-pyrene, and bisphenol-a. We attempted to choose compounds that would be commonly encountered in a student's daily life (e.g., methyl paraben is used in sunscreens and in the color coatings of many candies). Countless other compounds would work equally well.

As we prepare our final report to the members of ABLE for the Chicago meeting, we would appreciate any feedback or suggestions that might improve this laboratory module (email Malcolm or April ).

 Malcolm S. Hill and April L. Hill
Biology Department
Fairfield University
Fairfield, CT 06430