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Marine Biological Laboratory, Woods Hole, Massachusetts 02543
1 Barnstable County AmeriCorps Cape Cod, PO Box 427, Barnstable, MA 02630.
2 Marine Biological Laboratory, Woods Hole, MA 02543.
The use of ozone to clean and disinfect fresh and seawater life-support systems has become increasingly popular. Many analyses have been performed on their initial setup and design, but as they age and disinfection technology advances, they may require adjustments to function at optimum levels (1). Finding and maintaining the ideal specifications for any given system can be difficult as there are no universally accepted standards (1) and relatively few studies have addressed recirculating seawater systems for fish.
The current efficacy of the ozone disinfection system installed in 1992 at the Marine Resources Center of the Marine Biological Laboratory is unknown. Over the past year, we have linked poor animal health to an increase in bacterial pathogens such as Vibrio spp., Edwardsiella sp., Pseudomonas sp. in the circulating seawater system (Smolowitz, unpublished data), which suggests that the ozone disinfection system is insufficient to reduce fish pathogens to a safe level. To examine the functioning of our current system, we monitored bacterial levels during normal and modified system setups.
One line of the recirculating seawater system was isolated and maintained at a temperature near 15 °C and an ozone dose of 0.52 mg/l ozone, over a period of several months. Three test conditions were set up for a minimum of 3 weeks each. Test 1 was a control period during which no fish were in the system. During Test 2, 30 toadfish (Opsanus tau) were maintained in a single tank. Finally in Test 3, a venturi injecting elbow (which increases the ozone gas-water contact) (Ozone Systems model 1584) was added to the disinfection unit at a point prior to the contact chamber. All other parameters were held constant. Feeding and cleaning schedules were controlled. Weekly microbial and chemical analyses were run on samples drawn from four locations in the system: immediately before and after the ozone contact chamber, and immediately before and after the tank. Appropriate dilutions of the water samples were filtered through a sterile 0.45-µm filter and plated on thiosulfate-citrate-bile salts (TCBS, specific for Vibrio spp.) and Levine eosin methylene blue (LEMB, specific for coliforms) agar (Difco). Colonies were counted after 48 ± 2 h incubation at 22–24 °C. To estimate the amount of ozone reaching the water, we monitored the oxidation reduction potential (ORP) using an ORP pinpoint monitor (American Marine).
Bacterial count data (Fig. 1) were analyzed using t tests to compare bacterial levels from different phases and locations. Bacterial levels remained fairly consistent throughout the system during the control phase without toadfish, averaging about 10,700 Vibrio and 500 coliform bacteria per 100 ml. Once toadfish were added to the tank, post-tank bacterial levels rose to 535,000 Vibrio/100 ml and 17,300 coliforms/100 ml, and average bacterial levels increased to 166,700 Vibrio/100 ml and 8,700 coliforms/100 ml.
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When compared to standard ozone disinfection, the post-ozone bacterial levels with the venturi were lower, but not significantly so (Vibrio P = 0.21, coliform P = 0.23). When compared to the control phase without toadfish, the post-ozone bacterial levels with a venturi injector were not significantly different (independent pooled-variances t test: Vibrio P = 0.83, coliform P = 0.88). The greatest decrease in bacterial levels occurred between the post-tank sample location and pre-ozone sample location. Coliform bacteria levels were consistently lower than those for Vibrio spp. and often more highly variable.
Measurements of the oxidation reduction potential (ORP) averaged 279 mV during Test 1, with a maximum of 288 mV in the post-ozone sample. These values averaged 322 mV during Test 2 (max 329 mV) and 325 mV during Test 3 (max. 349 mV). These ORP values are higher than those of the intake pipe before any treatment, which averaged 299 mV.
At current specifications, the ozone disinfection system does not kill significant numbers of bacteria in the recirculating seawater. The coliform bacteria counts without the venturi injector exceeded the standard set by the Animal and Plant Health Inspection Service for marine mammal aquariums of 1000 coliform bacteria/100 ml (2). Even with the new venturi injector added, bacterial counts were not significantly reduced.
Ozone has been shown to be an effective disinfectant in fresh and seawater systems, but its efficacy depends on system design and specifications (3,4,5, 6). In an analysis of multiple disinfection systems for a freshwater hatchery, Wilson (7) found that a venturi injector system using a commercially available source of ozone did not significantly reduce bacterial levels as compared with an air-stone system using onsite ozone production. The best reduction in bacterial counts was achieved by additional filtration followed by UV treatment (7). Preliminary studies at the Marine Resources Center comparing ozone disinfection with UV disinfection have shown similar results.
Literature Cited
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