Introduction

Perspectives from space have accentuated public awareness that almost three quarters of our globe is covered with water. What is not generally appreciated is that more than eighty percent of the world ocean is below a mile in depth - and over half the planet is beneath two miles of sea. For the greater part of mankind this vast area has been both out of sight and out of mind - and scientific literature is replete with examples of strange life forms recovered once, dead or dying, and never seen again. Descents in modern submersibles have discovered entire ecosystems divorced from the energy of the sun, and scientists have now observed abundant numbers of great primitive sharks hunting the floor of the sea a half mile down.

Sealed beneath a door of immense pressure and cold, life in the deep sea has evolved in forms both strange and remarkable. Only a privileged few have crossed the threshold of the earth's abyss and returned with accounts of the journey - and each time science and public have attended in fascination. The high pressure aquarium, life support system, and deep retrieval devices we have developed will help to bring that world, alive, to the surface.

Maintenance of the abyssal animals under natural conditions has been a major dilemma for institutions interested in developing the display and research potential of the deep marine fauna. The main difficulty in maintaining demersal animals involves the high hydrostatic pressures to which such organisms are anatomically and physiologically adapted. Reproduction of this aspect of deep environments requires support systems that can produce the necessary pressure conditions without compromising accessibility to the animals in terms of feeding, water circulation, and viewing of occupants. In addition, successful capture of demersal animals, particularly fish having swim bladders, often is impossible without trapping devices that can preserve ambient pressures during ascent to the surface.

In this paper we describe a deep water animal maintenance technology that resolves many pressure-related problems connected with the capture and care of demersal animals. Equipment such as this is imperative if zoological institutions, public aquariums, and universities are to advance the display and research capabilities involving this important component of the marine realm.

Design

The design of our high pressure animal maintenance system is diagrammed in Figure 1. The sketch illustrates the apparatus as it is presently configured and assembled for operation. It is a prototype of a larger more powerful machine that we are currently constructing.

In essence, our concept involves the linking of two aquariums - one at low (i.e. atmospheric) pressure, and one at high pressure. The low pressure side of the system consists of a 50 gallon aquarium (Figure 1g) connected by spillway and return pump to an external filter/chilling unit (Figure 1h). Filtration is accomplished by pumping water through a gravel-bacterial medium covering the base of the unit. Water circulates continuously between the low pressure aquarium and satellite filter/chiller unit. This set-up provides an uninterrupted supply of cooled, filtered, and oxygenated water to the high pressure side of the apparatus.

Figure 1 Schematic diagram of high pressure animal maintenance apparatus. a - high pressure animal maintenance chamber containing Nautilus specimen; b - back pressure regulator; c - pressure pump; d - DC motor; e - pulse dampener; f - pressure gauge; g - low pressure satellite aquarium; h - low pressure filter/chiller unit; j - main viewing window; k - optical port.

The high pressure part of the apparatus consists of several components linked together into a single functional unit by stainless steel pressure tubing. Water is drawn from the low pressure aquarium through a wire mesh filter into the high pressure line by a multiple piston/positive displacement pump (Figure 1c), manufactured by Cat Pumps, Inc., Minneapolis, MN. This pump pressurizes the system and circulates water through the other high pressure components. The pump is powered by a 3/4 horsepower, 12 volt, variable speed motor (Dayton Motors, Inc., Dayton, OH; model # 2Z846A) (Figure 1d), which delivers its output to the pump via a 16:1 reduction drive.

Pressurized water leaves the pump through either of two exit ports. One of these ports leads to a pulse dampener (Figure le), the function of which is to suppress small, transient pressure fluctuations deriving from the reciprocating movements of the pump's pistons. The pulse dampener consists of a reinforced steel container 1 meter long and 30 cm in diameter which is filled partly with water and partly with air compressed to the pressure in the system. The free air/water interface rises and falls in response to instantaneous pressure fluctuations thus damping them out. Tests of the effectiveness of the pulse dampener show that even at pressures in excess of 100 atm (1500 psi), short term pressure fluctuations are in the range of 1/3 atm (5 psi) or less. Also attached to the pulse dampener is a pressure relief valve (not shown in Figure 1), which protects the entire system by automatically releasing the pressure if it inadvertently rises above a pre-set limit.

The second pump outlet leads to a high pressure aquarium (Figure la) in which specimens are maintained. This animal maintenance chamber is a machined, spin cast stainless steel cylinder 50 cm long and 30 cm in diameter and enclosing a water volume of about 8 liters (2.1 gallons). It rests on two axisymmetric pivots so that it can be rotated from the horizontal position shown in Figure la, to one more nearly vertical. As diagrammed in Figure 1, it is fitted with a variety of ports which give access to the animals inside.

Viewing and optical access is provided through a combination of conical disc and spherical sector acrylic windows. The main viewing aperture is an 8 inch diameter 150 degree spherical sector (Figure 1j) based on designs developed by the Ocean Technology Department of the Naval Undersea Center in San Diego, California, (J.D. Stachiw, Acrylic Plastic Viewports for Ocean Engineering Applications; NUC TP 562, Feb. 1977). Fiber optic illumination is introduced through one or more of the three conical windows spaced equidistantly around the pressure chambers circumference, (Figure 1k). Primary pressure sealing relies on Buna-N O'Rings configured to specifications provided by the Parker Seal Group in Lexington, Kentucky. Operating the aquarium at pressures equivalent to one mile in depth produces head and window loads exceeding 100 tons. Potential energy stored in both the elasticity of seawater and the pulse dampener's compressed gasses require that the aquarium be designed to ASME PVHO, (Pressure Vessels for Human Occupancy), standards. These safety and engineering criteria and the additional consideration of a metals/saltwater interface dictate the use of robust, seawater compatible, high pressure control and fluid flow components. Standardization is achieved through the use of highly reliable single ferrule fittings, connectors, flexible tubing, and selfseating high pressure valves, (Parker Hannifin Corporation, Cleaveland, OH.; CPI connectors, Parflex hose, stainless steel needle and ball valves).

Physical access is accomplished by removal of the main viewing port or through a captive door which also houses the upper concave window. Additionally, there are several direct access ports used for sampling purposes. Three such ports provide attachment for instruments monitoring chamber environment, particularly pressure and temperature. We used a temperature compensated, Bourdon tube pressure gauge (Figure If), manufactured by Heise Gauges, Inc., Norwalk, CN, to measure pressure inside the chamber. In this regard, the apparatus is rated for a maximum operating pressure of 160 atm (2350 psi) which is equivalent to the pressure acting at a depth of about 1610 m (1 mile). Other ports (not shown in Figure 1) provide attachment sites for temperature probes and other electronic monitoring gear, and allow the operator to remove samples of chamber water for chemical or other analysis without releasing pressure. The animal chamber is also provided with its own cooling system which is activated when temperatures beyond the capacity of the low pressure chiller are desired. This secondary unit consists of an evaporative alcohol chiller which circulates coolant through coils inside the animal chamber. The use of two chillers operating in tandem allows us to achieve water temperatures inside the chamber equivalent to the near freezing conditions of the abyssal plain floor.

Water exits the animal maintenance chamber through a high pressure line leading to a wire mesh filter and then to a Grove saltwater back pressure regulator assembly (Figure 1b). The back pressure regulator is used to set the pressure in the high pressure side of the system. Upon exiting the regulator, the water flow is again at atmospheric pressure, and is returned to the low pressure filter (Figure 1h) by plastic tubing.

Operation

As diagrammed in Figure 1, our high pressure system consists of two water circulation loops - one at atmospheric pressure, and one at high pressure. The low pressure loop runs continuously. It is the operation of the high pressure loop that we describe here. We discuss our procedures in three steps: 1) placing a specimen in the animal pressure; 2) pressurizing the system; and 3) long-term maintenance at pressure.

To introduce an animal into the chamber, we begin with the low pressure system actively circulating water between the satellite aquarium and filter box. The high pressure side of the system is inactive, however. The high pressure pump is shut off, and the high pressure aquarium is empty of water and has the acrylic viewing window removed so that the chamber is open at one end. The high pressure aquarium is oriented on its pivots so that the open end is directed upward. Water from the low pressure aquarium is poured directly into the open chamber, and the specimen placed inside when it contains sufficient water. The acrylic viewing window is then bolted into position, and the high pressure aquarium swung into its normal horizontal operating position. The system is now ready for pressurization.

The high pressure loop is brought into service by switching on the DC motor and setting its speed to produce the flow rate desired. The system, is operated at atmospheric pressure until any air remaining in the animal chamber is drawn off in the chamber discharge. When all trapped air has been evacuated, pressure is increased by stopping down the turncock on the back pressure regulator. The rate at which pressure increases in the system can be controlled by adjusting the motion of the turncock. When the system has attained the desired operating pressure, no further adjustments of the regulator are needed.

Long-term maintenance of specimens under pressure is enhanced by daily monitoring of chamber environment. Our procedure is to sample about 100 ml of chamber water on a daily basis in order to measure pH and the concentration of 02, N03, NH3, and dissolved metals. We found that setting the pump to produce a flow rate of about 48 1/hr, which is sufficient to flush the chamber once every 10 minutes, will adequately maintain water quality in most instances. At such flow rates 02 remained at saturation concentration, and we could detect no abnormal levels of dissolved constituents or pH.

Feeding is done using a pressure lock apparatus (not shown in Figure 1) incorporated into the pressure line entering the chamber from the pump. The advantage of our pressure lock set-up is that it allows food to be passed into the chamber without releasing pressure. The lock actually consists of a bypass line leading to a port through which food objects up to 1 cm in diameter may be introduced into the system. To present food to an animal in the chamber, the bypass line is isolated from the main high pressure line by closing valves at either end on the feeding bypass. The feeding port is then opened, releasing pressure in the bypass line only. The food is inserted into the bypass, and the port closed again. The bypass valves are then opened. This restores pressure to the bypass line, and the resulting flow of water through it forces the food into the animal chamber.

Dissolved waste (NH3, NO3, etc.) is removed in the chamber effluent and subjected to bacterial action in the low pressure filter. Solid waste (feces, uneaten food) removal is accomplished using a port at the base of the chamber. In the prototype described here, the waste port is not provided with a pressure lock. However, by judicious adjustment of the flow rate, we can open this port sufficiently to force out waste without causing a significant drop in pressure. A pressure locked waste port, similar to the feeding port pressure lock described above is planned for the next generation of high pressure aquariums presently under construction, and will improve this phase of operation.

Performance

We tested the effectiveness of the prototype machine in supporting both vertebrate and invertebrate animals. Species studied include killifish (Fundulus heteroclitus); goldfish (Carassius auratus); octopus (octopus bimaculatus) and the pearly nautilus (Nautilus pompilius). In all cases, our procedure was essentially the same. One or more conspecifics were introduced into the chamber, and pressure was then raised to at least 10 atm - 30 atm for periods of varying length. Feeding and waste removal were done as necessary.

The most extensive experimentation involved Nautilus. Tests with this species focused primarily on the length of time over which specimens can be safely kept in the machine. We found that juvenile specimens of about 100 gm total weight could be kept successfully for at least 8 weeks in the chamber. During this time no serious diminution in water quality within the chamber was observed, although we did find that waste particles too large to be easily siphoned off through the waste removal port built up to form a sediment on the floor of the chamber. This build-up would presumably ultimately limit longevity of a trial, but in the present case with Nautilus, termination of the experiment resulted from our need to use the equipment for other purposes, not from any serious environmental threat to its occupants. With second-generation, pressure locked machines as described above, waste accumulation should not longer pose a potential problem. As long as a rational approach to specimen size and numbers is exercised, and reasonable flow conditions and feeding regimens are invoked, occupancy periods of indefinite lengths should be practicable.

Applications

The technology we describe here opens up a host of possibilities of interest to public aquariums, universities, and research institutes. In our view, the principal value of our high pressure system lies in three areas: capture, research, and display of demersal organisms. The importance of such machines should become even more evident as improvements in design and system capacity are incorporated in the future.

The difficulty of capturing live demersal animals has been a major impediment to display and research applications involving such creatures. The grotesque fate of deep water teleosts with swim bladders when raised to the surface in nets or trawls is well known. Use of pressurized traps similar in design to our animal maintenance chamber should obviate many problems relating to pressure loss during capture.

Research involving demersal organisms is at a rudimentary level when compared to the state of investigations on shallow water forms. A main reason for this is the overwhelming problems associated with maintaining demersal life forms at ambient habitat pressures. Our methods represent a major step forward in resolving these problems. In fact, our investigation of the sensitivity of Nautilus to pressure change (Jordan et. al. in press), which is the first paper in the cephalopod literature to deal with pressure-related phenomena, could not have been conducted without such equipment.

With few exceptions, public aquariums and zoological parks have focused their efforts on displaying animals inhabiting neuritic, epipelagic, and other shallow water ecosystems. There are essentially three reasons for this: 1) such animals, of all marine forms, are the most readily caught; 2) the most easily maintained; and 3) the most familiar to the ticket purchasing public. In developing displays, planners must be cognizant of these facts; yet, much good can accrue to those institutions willing to devote at least some energy to unusual and exotic organisms. In this regard, displays of demersal animals under natural conditions, which our technology makes possible, seem especially promising for at least two prime reasons. First, virtually everyone has an interest in the unusual and unique; and second, the technological aspect of the maintenance system, if incorporated as a component of the display, is certain to intrigue most members of our technological society. The great potential of such displays is evidenced by the enthusiasm generated by the public showing of Nautilus in the high pressure prototype which the New York Aquarium and members of the Atlantic Foundation set up at the 1986 meeting of the American Association for the Advancement of science in Philadelphia. This success underscores our view, and emphasizes the wisdom of this approach.

Acknowledgements

We are grateful to the Griffis Foundation, Nixon Griffis Director, for its support in developing this technology. We also thank the late G.D. Ruggieri, S.J., Director of the New York Aquarium, and members of the Aquarium staff for their help in carrying this work forward.

Reference

1.  Jordan, M., Chamberlain, J.A., Jr., & Chamberlain, R.B.1988. Response of Nautilus to variation in ambient pressure. Jour. Exp. Biol. in press.