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APPENDIX H Emerging Monitoring Technologies ADENOSINE TRIPHOSPHATE ANALYSIS Perhaps the most highly developed monitoring method for viable biological activity is the measurement of adenosine triphosphate (ATP). This analysis has been used by biologists for many years to determine the presence or absence of viable activity. The biochemical energy system, ATP, is only present when biological activity is occurring; ATP is absent from inactive biological material. Analysis for ATP is well defined and is performed with common laboratory equipment. There has been little change in ATP methods over the past five years. ATP must be extracted from cells to be stabilized before analysis. This procedure has been slightly simplified with the use of nitric acid as an extractant and new photometers have been developed for field use. These handheld photometers are relatively inexpensive ($400) and allow the presence of ATP to be determined after extraction. ATP levels decrease over a period of minutes or hours after cell death. Therefore, the technique is awkward to use for disinfection processes, as some ATP remains for a variable period of time after treatment. However, the technique can be calibrated for a given disinfection process to account for the time variability of measurements. The ATP content of cells varies widely with cell size; therefore, ratios of nucleic acids or protein to cell size are perhaps most representative of population viability. Such ratios would need to be generated through research efforts for standardized monitoring of ballast water microbial populations. Despite the aforementioned limitations, the ATP analysis technique does have universal application and has been shown to be effective for planktonic and benthic organisms, including both heterotrophic and autotrophic procaryotes and eucaryotes (Herbert, 1990).
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RNA/DNA ANALYSES A second group of promising monitoring techniques includes the determination of RNA/DNA ratios, enzyme analyses, and measurements of lipid content. These analyses of eucaryotic or procaryotic populations can be streamlined, but all currently require extractions. The analyses for each of the biological materials discussed above are simple to quantify and could be adapted for use on board ship. As with ATP monitoring, the analyses would need to be correlated with actual ballast water populations for predictive purposes. These analyses would probably require some preparation on the part of crew members, but the extractions and chemical additions could be simplified to be analogous to swimming pool analyses. Specifically, safe, premeasured portions of chemical additives and small amounts of glassware could be supplied to generate a visible residual. The residue could then be readily analyzed colorimetrically on board ship. Surrogate methods of analysis, such as ATP analysis and determination of RNA/DNA ratios, may be particularly useful if on board treatment procedures such as filtration or disinfection are used. In these cases, it would only be necessary to determine the absence or presence of biological materials such as ATP or DNA to indicate a high degree of treatment effectiveness. Such monitoring processes will probably be the least expensive to implement on board ship but will require some training to facilitate the analysis by crew members. FLOW CYTOMETRIC TECHNIQUES A promising technique that could eventually be applied to monitoring ballast water for unwanted biological organisms is flow cytometry. This technique uses a modification of a typical Coulter counter system and can be operated in a flow-through mode. The equipment allows for a small passage of water through an aperture of preset size (usually 2 to 20 µm). Organisms that pass through this aperture can be measured by the cytometer. The system not only quantifies the size and volume of organisms passing through the orifice, but can also detect the natural fluorescence associated with the presence of chlorophyll. Therefore, flow cytometry can be used to quantify all microorganisms without an outside excitation. This equipment is currently available commercially and has been installed and operated on research vessels where its portability, while requiring some improvement, has been proven. A flow cytometer could also be used to identify the presence of specific organisms if either dyes or DNA/RNA probes were added to the water prior to measurement. For instance, dyes added to a sample of water before passage through the flow cytometer affect the membrane potential of the cells. This potential can be detected by the flow cytometer, and it will be different depending on whether a cell is active or inactive. Because inactive cells have no induced membrane potential, the viability of organisms passing through the cytometer can be determined.
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A sample of ballast water incubated with specific DNA probes will hybridize with known sections of a genome of specific target organisms, such as unwanted dinoflagellates. These organisms can be detected by the cytometer. Therefore, specific taxonomic groups can be identified automatically using a flow cytometer with the addition of RNA or DNA probes. A great amount of research work is currently going on in the United States to develop specific DNA probes as a tool for ecological application (see, for example, Fell et al., 1992). Because of its specialized nature, the main drawback to the use of flow cytometry is expense, and unless a simplified version becomes available it is not clear that ships would voluntarily use such equipment on board. OTHER AUTOMATED TECHNIQUES Other possible procedures that may be adapted for automated shipboard use to determine the quality of ships ballast water are immunofluorescence, specific DNA probes, and extractable lipid phosphate analyses. These techniques are of interest because they allow identification of specific taxonomic groups and ships could determine if they had a specific organism within their ballast water that would not be accepted in a port of call. An analytical procedure with this capability would obviate the need for extensive research efforts to correlate surrogate analyses to the presence of specific unwanted species. Development of automated forms of these analyses would permit a specific identification that could be rapidly implemented to assist in controlling introductions of selected species. Using specific DNA probes in flow cytometry was discussed above. Research on lipid phosphates has not received much recent attention, but information is available from previous research. Lipids serve as a major form of energy storage in plant and animal tissues. They are the principal components of membranes and maintain the structural integrity of cells. Therefore, monitoring the presence or absence of lipids indicates the overall biological activity of a water sample. Lipids are routinely separated and analyzed using high-pressure liquid chromatography (Christie, 1987). Both DNA probes and lipid phosphate techniques would require more development before they could be used as a routine tool for monitoring ballast water. Perhaps the most promising of the group, however, is the immunofluorescence technique that uses antigen-antibody reactions to implant fluorescent molecules on viable biological material. This procedure is currently the subject of a substantial research effort by the U.S. Food and Drug Administration to identify unwanted toxic marine phytoplankton. Efforts are under way to identify antibody/antigen reactions specific to toxic phytoplankton such that fluorescent compounds can be selectively attached to these organisms. Once toxic phytoplankton have been ''tagged" in this way, they are easily identified using either flow cytometry or simpler photometry. If refined, this technique would provide a method to identify selected members of the biota within ballast water samples. However, the high level of specificity is accompanied by high equipment cost.
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REFERENCES Christie, W.W. 1987. High Pressure Liquid Chromatography and Lipids. Elmsford, New York: Pergamon Press. Fell, J.W., A.S. Tallman, and M. Lutz. 1992. Partial rRNA sequences in marine yeasts: A model for identification of marine eukaryotes. Molecular Marine Biology and Biotechnology 1(3): 175–186. Herbert, R.A. 1990. Methods for enumerating micro-organisms and determining biomass in natural environments. Pp. 1–29 in Methods in Microbiology, eds. R. Grigorova and J.R. Norris. New York: Academic Press.
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