D.S. Page, Professor of Chemistry and Biochemistry

CURRENT RESEARCH INTERESTS 12/2000

For the past 20 years I have been engaged in research problems relating to the fate and effects of petroleum and other pollutants in the marine environment in collaboration with Dr. Ed Gilfillan of the Environmental Studies Program. Some of the things I will be interesting in studying over the next few years are described below.

Fate and Effect of Pollutants on Marine Animals:

Tri-n butyltin (TBT) and di-n-butyltin (DBT) Studies

A. Effects of TBT:

TBT and DBT are organometallic compounds with a central tin (IV) covalently bound to 2 or 3 butyl groups. TBT has a charge of +1 and exists as neutral species associated with any one of a number of anions. DBT is a divalent cation in association with anions. Neither TBT nor DBT are very water soluble and are readily absorbed by lipid structures of marine animals. TBT is very toxic to invertebrate marine life at very low dose levels and is thought to act primarily as an uncoupler. DBT is much less toxic and is considered to be more of an enzyme inhibitor. The toxicolgy of TBT and DBT in vertebrates is much more complex.
The absorption and depuration of TBT by mussels is not a simple phase partitioning process, but involves at least 2 compartments in the mussels with different affinities for the pollutant. While the ability of marine animals to absorb and bioconcentrate most hydrophobic pollutants (i.e. hydrocarbons, PCB’s, DDT, etc.) from water can be predicted from the water solubility of a pollutant, the ability of marine animals to bioconcentrate TBT from water is much greater than one would expect from its water solubility. While it has been proposed that this is due to protein binding by TBT in addition to the usual hydrophobic effects, the hypothesis has not been tested. A good way to estimate binding for enzymes is to measure the inhibition constant for a number of enzymes associated with metabolism. Therefore, as a starting point, one could study the inhibitory effect of TBT and DBT on various glycolytic and Krebs cycle enzymes. This project involves the spectrophotometric measurement of the rates of enzyme reactions under various conditions of substrate and TBT and DBT concentrations.

B. Effects of Thermal Stress and TBT on Anaerobic Energy Metabolism in Mytilus Edulis.

The hypothesis of this study is that TBT impairs anaerobic energy metabolism resulting in a lowered ability of Mytilus edulis to withstand emersion. The study design includes 2 levels of anaerobic effect (air and nitrogen) and elevated temperature to accentuate any metabolic stress effects in a control group of mussels and a group of mussels exposed to TBT. Using a HPLC-based method, mono- and dicarboxylic acid metabolites associated with anaerobic energy metabolism can be quantified before and after treatment for subsamples of all groups. Differences in the concentration of metabolites reflect differences in rates of formation an breakdown and thus can provide insights into the metabolic effects of TBT.

C. Bioavailability of TBT in Sediments:

mitochondrial processes Organic residues in sediments associated with shipyard activity can contain high concentrations of TBT and DBT. Over time, as sediment layers build up, significant quantities of various sediment-associated pollutants become trapped in the sediment. Significant quantities of TBT and DBT can be released into the water column when these sediments are disturbed, for example by dredging. Even though these TBT sources are generally of a limited area, dispersion of these sediments beyond the collection site through dredging or other physical disturbance could make TBT residues available to biota over a large area.
The question of bioavailability is very important because pollutants associated with sediment may be in a form that is not readily taken up by marine animals even though chemical measurements of the pollutant in sediments indicates high concentrations are present. In the case of TBT in marine sediments, the presence of TBT associated with paint particles from boat hull refinishing limits its bioavailability. There is currently no good way to determine the form in which TBT is present in sediments. Using sediments from a shipyard site in Maine, one can determine the bioavailability of TBT in the sediments by suspension of a sediment sample in seawater followed by analysis of the water for TBT. This can be done successively to determine whether continued release of TBT occurs. It may be possible to separate paint residues from sediments by some floatation process. The goal is to devise methods to determine the form in which TBT is present in specific sediment samples.

D. Monitoring for TBT:

Since 1988, the use of TBT in the US has been restricted to vessels over 25 m in length. There are a number of boatyards in Maine and New Hampshire that service larger boats. Analysis of TBT in mussels collected from areas in Maine including active shipyards would reveal whether ongoing inputs of TBT are occurring.

Applied Ecotoxicology

A. Mussels as Sentinel Organisms for Measuring Pollutant Effects:

The presence of measurable quantities of a pollutant in a marine sediment sample does not necessarily infer an adverse effect. The measurement of sediment levels of pollutants alone, then, does not tell the whole story. There are locations in the Maine where marine sediments are known to have elevated levels of various organic and inorganic pollutants such as polycyclic aromatic hydrocarbons (PAH), TBT and heavy metals. Normally, the analysis of sediments for specific hydrocarbon-like pollutants is a very expensive process. If one wants to ask the question whether there are substances in a sediment sample that can have a harmful effect on marine life, then a simple screening process involving testing the effects of material in sediments on marine animals is a better approach than simply analyzing sediments alone.

mussel collecting and mussel sampling

The goal of this project is to determine whether the bioluminescent bacteria Microtox™ bioassay is able to screen for a toxic effect, using the common marine mussel as a sentinel organism. The toxicity of chemical compounds bioaccumulated in the tissues of mussels collected from field sites is assessed through a scheme which has been termed "musseltox". The mussel tissue obtained from the field is mechanically homogenized, and the base/neutral organic compounds present in the organisms were extracted using a steam distillation method (Donkin and Evans, 1984). The degree of toxicity of the compounds present in the mussel residue is determined by the Microtox® bioassay, a test which uses light-producing bacteria. The toxicity of the sample is thus measured by the degree of light output inhibition by the bacteria. Application of this scheme in 1998/99 involved the collection of mussel samples from test sites in Portland before and after a dredging project in the Fore River as well as a site heavily impacted by the Julie N oil spill of 1996. Mussels were also obtained from control sites in the Casco Bay which were well removed from the dredging activity and unimpacted by the oil spill. Part of the 1998/99 project was to determine if dredging released bioavailable base/neutral toxicants.

ec50 values for mussel extractsThe results of the 1999 Microtox™ measurements on extracts of mussel tissues from the Portland, Maine area are given in the accompanying graph. The data obtained in the 1999 project indicate that the "mussel-tox" procedure can be used to measure relative toxicity. The results show that activities not related to the oil spill of 1996 produced more bioavailable toxicants taken up by the mussels than the spill site mussels. Work in this study shows that in certain parts of the Fore River, where industrial sites exist, the compounds present have a detectable toxic effect. The results of the Microtox® bioassay suggest that the dredging activity in Portland did not have an impact upon the toxicity of the waters which could be detected. In a previous study, mussels which were obtained in 1997 from Thompson's Point on the Fore River produced lower EC50 values relative to controls, indicating high toxicity (Watson, 1998). In the present study, the spill site mussel extracts did not differ significantly in toxicity from the control mussels, consistent with overall weathering and non-bioavailability of any remnants of the spill at the Thompson's Point site.

The advantage of the "musseltox" approach as a bioassay-based environmental monitoring tool is that mussels integrate exposure to toxicants over time and area. This avoids location-specific sampling of "hot-spots" that may not represent an area as a whole. While sediment geochemistry plays an important role in oil spill injury assessment, the effects of hydrophobic toxicants actually taken up by organisms is the fundamental measure of injury. We plan to develop this method further in 1999/2000 with additional field studies of other estuaries, including the Piscataqua River in Maine/New Hampshire.