PROCEEDINGS OF THE
NORTH AMERICAN VETERINARY CONFERENCE
VOLUME 20
JANUARY 7-11, 2006
ORLANDO, FLORIDA
SMALL ANIMAL EDITION
Reprinted in the IVIS website (http://www.ivis.org) with the permission of the NAVC.
For more information on future NAVC events, visit the NAVC website at www.tnavc.org
Exotics — Aquatic Medicine
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ADVANCED WATER QUALITY TOPICS FOR
MARINE MAMMALS AND FISH
M. Andrew Stamper, DVM, Diplomate ACZM
Disney’s Animal Programs
The Living Seas, Epcot®, Walt Disney World® Resort
Lake Buena Vista, FL
Water quality is a complex process. Beyond the
fundamental practices such as mechanical and bacterial
filtration, there are many techniques used in modern
aquarium water systems which can ensure clean and
safe environments for aquatic life. Of the various marine
organisms, only marine mammals have specific
government enforced regulations. Animal and Plant
Health Inspection Service (APHIS) has water quality
standards for coliform count, pH, chemical additives, and
salinity. The first step in this process is to understand the
complexities of water chemistries as well as some of the
more advanced techniques to keep the parameters
within the acceptable biological limits.
WATER PARAMETERS AND TESTING
Salinity is very important for marine animals. Even
though most marine mammals can withstand freshwater
for very short periods of time, salts are needed for
periods of time beyond one or two days. Seals have
historically been housed in freshwater with oral salt
supplementation but this is not recommended by the
author. AHPIS now requires that primary enclosure
pools be salinized for marine mammals requiring
saltwater (everything excluding polar bears). The
difference between marine mammals and marine fish is
the type of salts needed within the salt mixture. Marine
mammals can live in saltwater composed of basic NaCl,
which is much more cost effective. Marine and estuarian
fish need not only NaCl but all the micronutrients upon
which natural sea water is based. There are
approximately 78 inorganic elements composing
seawater.1 The salinity concentration recommended for
marine mammal water is ≥22 ppt while others have
suggested keeping the salinity between 15 and 36 ppt.
Salinity of marine fish will depend on their native
environments with estuarian fish being more adaptive to
salinity changes. Marine system enclosures exposed to
the elements have evaporative and dilution effects (rain
and deck hosing) creating fluctuations which need to be
carefully monitored. The smaller the enclosure and the
larger the surface area will create greater variation.
Salinity can be measured indirectly with a salinometer or
refractometer, or by conductivity. Salt mixtures can be
bought premixed or formulas acquired from various
texts.
pH (p=puissance (power) and H=hydrogen) is
extremely important to marine fish and invertebrate
physiology and bacterial biological filtration. Most fish
have an optimal pH range in which they will thrive. Its
importance with marine mammals is mostly associated
with chemical reactions including sterilants that are often
unpredictable. pH decreases over time by addition of
organic acids from animal and bacterial waste products.
APHIS requires pH testing be performed daily for all
marine mammals and most facilities keep the pH
between 7.2 and 8.4.
Coliform count sampling is mandatory when housing
marine mammals but marine fish systems are not
monitored for one reason or another. Coliform counts
can be analyzed by the Multiple Tube Fermentation
test or the more accurate Membrane Filter Test which
pulls 100 ml of water through a filter membrane which is
placed on a culture to determine colony forming units.
Water samples should be taken in a consistent way to
ensure accuracy. This can be accomplished by adhering
to the following protocols: samples should be taken at
same place and same time of day, at least 2–3 feet
below surface, near middle of pool or drain, and prior to
emptying the pool rather than just after filling. Samples
must be processed within 30 hours of collection and
refrigerated if not tested within one hour. For marine
mammals, tests must be performed weekly and counts
must not exceed 1,000 MPN (most probable number)
per 100 ml of water. If the count exceeds the 1,000
mark, then two subsequent counts must be taken at
48-hour intervals; all three counts are averaged to
determine the accuracy of the first test. If the coliform
count is still high then the conditions are unacceptable
and the situation needs to be corrected immediately by
either changing or sterilizing the water. The water must
be retested after either of these options is exercised.
Total Organic Carbon (TOC) = Particulate Organic
Matter (POC) plus Dissolved Organic Matter (DOC).
DOC is defined as the fraction of the TOC that passes
through a filter of a stipulated pore size of 0.45 µm.
Two major components of DOC are humic and fulvic
acids which often color the water. High TOC increases
oxidant demand, reduces efficacy of sterilization, and
creates toxic byproducts. POC is usually efficiently
removed through mechanical filtration. Organic
compounds enter the ambient water from various
sources including influent water, animal waste products,
uneaten food, as well as drift from the air (ie, pollen,
dust). Biological filtration bacteria reduce both DOC and
POC by incorporating the carbon when growing and
reproducing but, even with this, TOC often overwhelms a
system. The easiest way to reduce TOC is to change the
water but this can be expensive or impractical so other
steps have been developed to condition the water.
Water treatments can be as simple as mechanical
filtration to much more advanced techniques with
oxidative sterilization. Basic mechanical and biological
filtration is the first line of purification but has been
discussed in previous abstracts within this series.
Therefore, further discussions will focus on more
advanced techniques.
Flocculation is a technique used to precipitate TOC,
in particular a fraction of the DOC, which becomes
trapped in the mechanical filter and is flushed out with
the other particulate matter. Common flocculates used in
freshwater include aluminum sulfate (alum) and natural
and synthetic polymers (cationic polyelectrolytes).
These act by reducing surface charges on dissolved or
suspended particles allowing them to collide and
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The North American Veterinary Conference — 2006
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coagulate. They reach their peak effectiveness at pH
<7.5. These are not notably toxic but lack of true
understanding of these chemicals does not justify their
use when other techniques are available. A safer
alternative is foam fractionation, where particles are
brought together mechanically by causing flow
conditions promoting particle collisions.
Mucopolysaccharides produced by bacteria and algae stick
together along with other substances as they collide.
Foam fractionation can be accomplished by slowly
pushing water (salinity ≥15 ppt) down a column. As this
is happening, air is injected at the bottom of the column
creating a steady upward stream of very small bubbles.
Organic molecules (including the mucopolysaccharides)
often have hydrophilic and hydrophobic parts causing
them to collect at air-water interfaces. As they come in
contact with the air bubbles, these molecules will collect
and form an organic film on surface of bubbles. When
the bubble reaches the surface the film stays intact for a
period of time which creates foam. The resulting foam is
then skimmed and flushed to a drain, thus removing the
organic waste from the system.
STERILIZATION
Once gross organic material is removed as well as
ammonia and nitrite, other materials such as basic
proteins as well as infectious agents (bacteria, viruses,
and fungi) need to be addressed through sterilizing
techniques. Sterilization can be characterized in two
forms…point-contact sterilization where water is
diverted in a side stream that is in continuous contact
with a sterilizing agent, then recycled back to the system
(in-line UV radiation and ozone) and bulk-fluid
sterilization, which oxidizes organics and kills microorganisms throughout the water system (chlorine). Note:
ozone can create residual oxidative products which
travel out into the main system which can easily kill fish.
Point-contact sterilization is subject to engineering
constraints (side-stream configuration, flow rate).
To improve water quality, rate of side stream treatment
must exceed rate of system contamination (bacterial
growth within the region holding the animals).
UV Sterilization uses UV radiation in the 254 nm
range to irradiate pathogens and proteinaceous material.
The advantage is there is no residual effect but, because
it is localized, microbial density in the body of water
nearest the animal may not be diminished. This
technique is best used in flow-through systems where
the incoming water is irradiated and has limited use in
closed and semi-closed systems.
Ozone is O3, an allotrope of oxygen. Ozone can
generate a high oxidation potential and is recommended
to be greater than 700 millivolts to sterilize water.
If ozone is used in fish systems, Oxidation Reduction
Potentials (ORPs) should not enter the ambient water
above 350 millivolts. Therefore, the higher ORPs levels
need to be in a contact area outside the fish holding
area. Most facilities regulate their water to be free of
residual dissolved ozone as this could be detrimental to
skin, eyes and the respiratory system. There are four
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processes for ozone to work safely and efficiently:
ozone gas generation, gas-to-liquid absorption, contact
time for reaction, and ozone residual removal. Ozone is
generated by passing high AC voltage across a
discharge gap in the presence of O2. O3 reacts directly
with oxidizable compounds which are any chemical
which will accept oxygen (proteins, fatty acids in cell
walls, etc.). This is a very short-lived process since O3 is
extremely unstable (within seconds). The oxidation
process is affected by TOC, pH, bicarbonate, and
temperature. Products of these oxidation events include
free radicals, hydroperoxide species, and unstable
ozonide inter-mediates, which in turn are weaker
oxidizers, but last longer in the water column. One such
product is bromine. O3 reacts with bromide to yield
hypobromous acid (HOBr) and hypobromite (OBr-).
Ozonation is less efficient in presence of bromide; Brcan be regenerated from OBr- causing catalytic
destruction of ozone and increasing ozone demand.
OBr- is a weak persistent oxidant. OBr- reacts with
organics to produce carcinogenic and mutagenic
compounds (bromoform, formaldehyde) but these are in
small concentrations and are not harmful if regulated
appropriately. To accomplish this, HOBr is measured
and used as an indicator to ensure ozone oxidation is
not excessive (ozone oxidation is kept at a level where
excess HOBr is not produced). Remembering ozone is
short-lived, many of its residuals can be limited by
increased contact time in the sidestream. Water is then
passed through a biofilter and/or activated carbon, UV
light or intense heat to remove residuals. “Packed
Column Aeration” is another way to remove ozone
residuals from the water. Post- ozonated water is trickled
down a tower filled with inert material as a fan blows air
upward into tower. Most residuals are volatile
compounds and are released at this point into the air.
The advantages to ozone are that it has extremely high
antimicrobial properties compared to chlorine (~3x more
effective). It destroys POC which normally decolorizes
the water. The biggest advantage to ozonation is it can
be used in exhibits with fish or in conjunction with
biofiltration. The big disadvantages of ozone are that its
use results in higher equipment and operational costs.
Ozone is unstable and must be generated on-site. There
are considerable human health and fire hazards if not
handled correctly, so in enclosed areas the ambient air
ozone concentration must be monitored.
Chemical oxidizing substances include chlorine or
bromine-based oxidants and ozone as well as other
techniques are not discussed here. Because of their long
lasting effects, these chemicals need to be carefully
monitored to ensure they stay within safe parameters
and can only be used in marine mammal systems which
do not house fish. They are regulated by APHIS, which
requires daily water tests to ensure any chemical added
does not cause harm or discomfort (higher levels of each
oxidant can cause corneal, skin or respiratory damage).
For the sake of brevit,y only chlorine will be discussed
but bromine has similar reactions and is found in high
concentrations in saltwater. Chlorine is probably the
Exotics — Aquatic Medicine
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most cost effective and well-known sterilant. Efficacy is
determined by chlorine concentration, contact time,
temperature, pH, number and types of micro-organisms,
and amount of organic matter. Chlorine is applied as a
gas or as salts of hypochlorous acid (sodium or calcium
hypochlorite). Chlorine dioxide (ClO2) is an effective
sterilizer but is not frequently used since it is sensitive to
temperature, pressure, and light, and can be explosive
even at low temperatures. Chlorine reacts with water to
form hypochlorous acid (HOCl) and hypochlorite (OCl-)
which are considered “Free Chlorine.” HOCl is the
superior sterilizing agent (150–300 times more effective
than OCl-). Efficacy of sterilization (ratio of HOCl to OCl) is dependent on pH. HOCl reacts with free ammonia to
form chloramines: mono- (NH2Cl); di- (NHCl2), and
trichloramines (NCl3), which are persistent oxidants;
however, oxidation potential is much less than free
chlorine and chloramines are more irritating. Thus
organic matter increases chlorine demand and
decreases efficacy. “Combined Chlorine” = sum of
NH2Cl, NHCl2, and NCl3 in the water sample.. “Total
Chlorine” = sum of free and combined chlorine. A testing
guideline for marine mammal pool water is to test the
water twice daily for concentration of chlorine and/or
other oxidizing agents. Total free and combined chlorine
should not exceed 1.8 parts per million (ppm). Others
have recommended that “Total Chlorine” to be less than
1 ppm and “Free Chlorine” to be at least 50% of the
total. Chlorine-based oxidants react with organic
compounds to produce trihalomethanes (THM), such as
chloroform, which are considered mutagens and
carcinogens. These can be removed through aeration
and do not result in concentrations that are considered
harmful to marine mammals or humans. Chlorination of
seawater is more difficult due to the high concentrations
of magnesium, iron, and manganese which interfere with
proper chlorination. Ozone can be used in combination
with chlorine or bromine which has a synergistic effect
and can lower the use of both chemicals.
References
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3.
4.
5.
Bidwell JP, Spotte S. Artificial Seawaters Formula
and Methods. Boston: Jones and Bartlett Publishers,
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Arkush KD: Water quality. In Dierauf LA, Gulland
FMD (eds). CRC Handbook of Marine Mammal
Medicine, 2nd ed. Boca Raton, FL: CRC Press,
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Reidarson TH. Cetacea (Whales, Dolphins,
Propoises). In Fowler ME, Miller RE. Zoo and Wild
Animal Medicine, 5th ed. St. Louis: WB Saunders,
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Spotte S. Sterilization of Marine Mammal Pool
Waters. Theoretical and Health Considerations.
United States Departments of Agriculture. Animal
and Plant Health Inspection Service. Technical
Bulletin No. 1797, 1991.
Spotte S. Captive Seawater Fishes. Science and
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