Interesting Publication!
Ozonation of a
recirculating rainbow trout culture system
I. Effects on bacterial gill disease and heterotrophic bacteria
Graham L. Bullock E, Steven T. Summerfelt, Alicia C,
Noble,
Amy L. Weber, Martin D. Durant, Joseph A. Hankins
Abstract
Ozone was added to water in a recirculating rainbow trout (Oncorhynchus
mykiss) culture system just before it entered the culture ranks in an
attempt to reduce the numbers of heterotrophic bacteria in system water and
on trout gills, and to prevent bacterial gill disease (BGD) in newly stocked
fingerlings. During four 8-week trials. ozone was added to the system at a
rate of 0.025 or 0.036-0.039 kg ozone/kg feed fed. In the control, where no
ozone was added, and in previously published research, BGD outbreaks
occurred within two weeks of stocking, and these outbreaks generally
required three to four chemotherapeutant treatments to prevent high
mortality. In three of four trials where ozone was added to the system, BGD
outbreaks were prevented without chemical treatments, but the causative
bacterium, Flavobacterium branchiophilum, still colonized gill tissue. The
one ozone test where BGD outbreaks required two chemical treatments
coincided with a malfunction of the ozone generator. Although ozonation did
reduce BGD mortality, it failed in all trials to produce more than a one log
reduction in numbers of heterotrophic bacteria in the system water or on
gill tissue. Failure of the ozone to lower numbers of heterotrophic bacteria
or to prevent the causative BGD bacterium from occurring on gills was
attributed to the short exposure time to ozone residual (35 sec contact
chamber) and rapid loss of oxidation caused by levels of total suspended
solids. Rationale for ozone's success at preventing BGD mortalities are not
fully understood but may in part be due to improved water quality. Use of
the lower ozone dosing rate (0.025 kg ozone / kg feed) appeared to provide
the same benefits as the higher dosing rate (0.036-0.039 kg ozone / kg feed
fed); however, the lower ozone dosing rate was less likely to produce a
toxic ozone residual in the culture tank and would also reduce ozone
equipment capital and operating costs.
Discussion
Prior to ozonation, BGD was a constant problem among newly stocked fish.
During an 11-month period previous to ozonation, five groups of rainbow
trout were stocked, and up to 30% of each group died because of BGD or a
secondary amoebic infection (Bullock et al., 1994) despite regular
chemotherapeutic treatments In the ozonation study, BGD associated
mortalities also occurred on a regular basis when ozone was not added or
insufficient ozone was added. Adding ozone appeared to lower total mortality
and the number of clumps of BGD bacteria on gill tissue in tests one, three
and four, compared to that in the control and test two, when the ozone
generator failed. A total of 14 treatments were required to reduce BGD
mortality in the two tanks in the control and test two, while no treatments
were needed in the other trials. After ozone addition, only 1.7-4.1% of
stocked fish died because of BGD, and chemical treatments were rarely
required.
The benefits of adding ozone to our system were an
overall improvement in water quality entering the culture tanks (Summerfelt
et al., 1997) and, more importantly, a reduction of mortality due to BGD and
a reduction in the need for chemotherapeutic treatments. The improvement in
water quality from ozonation may, at least indirectly, affect mortality from
BGD. MacPhee et al. (1995) found that feeding played an important role in
BGD mortality; fish fed after being challenged with F. branchiophilum
developed clinical signs of BGD and had high levels of mortality, while
those that were not fed after the challenge developed only moderate clinical
signs and were generally normal 72 h post challenge. They proposed that
feeding promotes active excretion of urea and ammonia which accumulates in
the mucus and static water layer surrounding the gills, and this provides a
nutrient-rich environment that allows colonization and growth of BGD
bacteria on gill tissue. They also proposed that acidification of the mucous
boundary layer of the gill, which can be produced from increased carbon
dioxide excretion as a result of feeding. may play an important role in F.
branchiophilum attachment and colonization of the gills. Because MacPhee at
al. (1995) used a single-pass system, it is unlikely that deterioration of
water quality or environmental stresses favored the development of BGD.
Within our recirculating system, however, it is more likely that the
nitrogenous and organic substrates in the water affected the growth of F.
branchiophilum. Better water quality (Summerfelt et al., 1997) and reduced
BGD mortalities both appeared to result from system ozonation; but the
connection between the two was not shown. Although limiting nutrients to F.
branchiophilum may be a reason for reduced BGD mortality, other factors are
probably involved.
Several factors contributed to the failure of ozone to
eliminate F. branchiophilum and the general failure to reduce numbers of
heterotrophic bacteria in our recirculating system by even one log10.
Bacterial reduction can be predicted from the product of the dissolved
oxidant concentration and the exposure times, as described by the
Chick-Watson model (Watson, 1908). Within our system, ozone was
co-transferred with oxygen in LHO® units and short (35 s) contact times were
provided for ozone reaction after transfer to the flow before entering the
culture tank. Even the roughly 55 daily exposures of recirculated water to
ozone within the LHO units did not offset the short contact time each pass.
The other factor that limited bacterial reductions was
the low ozone residuals (means ranged from 0.02 to 0.180 mg/I) at the end of
the ozone contact tank (Table 2). Within our recirculating system, ozone
demand produced by suspended solids, nitrite, and color (dissolved organic
molecules) reduced ozone's half-life to levels that were generally too short
to measure. The longest half-lives measured were only IS s In contrast, the
half-life of ozone in a solution of pure water is about 165 min at 20deg C
(Rice at al., 1981). The ozone demand of the water in the recirculating
system consumed the ozone's oxidative power and thus shielded the bacteria
from direct oxidation. The shortened half-life reduced the effective
concentration and the time of ozone contact within solution and thus reduced
the predictor of ozone disinfection power, the product of residual
concentration and contact time.
The product of the contact time and range of ozone
concentrations in these trials were less than those reported by others. In
the studies by Owsley (1991), the water supply was treated with 0.2 mg/I
ozone for 10 mm to kill infectious hematopoietic necrosis virus (IHN); after
treatment, water was degassed in packed columns to reduce ozone to a safe
level for the fish. Liltved et al. (1995) reported 99.99% inactivation (4
log reductions in viable count) of four bacteria (Aerornonas salmonicida
subsp. salmonicida, Vibrio anguillarum, Vibrio salmonicida, and Yerkinia
ruckeri) and the infectious pancreatic necrosis virus (IPNV) within 180 s at
residual ozone concentrations of 0.15 to 0.20 mg/I within distilled water in
bench-top studies. Tipping (1988) reported that a contact time and ozone
concentration product of 1 mg/I * min was necessary to kill the protozoan
Ceratomyxa shasta from the water entering a trout hatchery. And, Colberg and
Lingg (1978) reported 99% kill of four bacterial fish pathogens (A.
salmonicida subsp. Salmonicida, A. liquefaciens, Pseudornonas fluorescens.
and Y ruckeri) when exposed to 0.1 and 1.0 mg/I ozone for 60 s in simulated
recirculating system water.
Greater redactions in bacteria within our recirculating
system, with its high oxidation demand, would have required ozone loading
rates greater than those used here (i.e., >0.039 kg ozone/kg feed), which
would be difficult to achieve without: (1) wasting excess oxygen to carry
more ozone to the LHO® unit, and/or (2) replacing the ozone generator with a
larger unit that could produce a higher ozone concentration in the oxygen
feed gas (6-l0% instead of 4-5%), and/or (3) installing an ozone removal
unit (air stripper, UV light, or large hydraulic retention chamber) to
prevent the increased ozone residual from reaching toxic levels in the
culture tank.
One of the main reasons that ozone is not widely used in
aquaculture is its toxicity and a manager's unwillingness to risk losing
fish to an accidental overdose. Residual ozone is highly toxic to fish at
low levels. Ozone destroys epithelium covering the gill lamella which
results in a rapid drop in serum osmolality (Paller and Heidinger, 1979;
Wedemeyer et al., 1979) and, if mortality does not occur immediately, can
leave the fish highly susceptible to microbial infections (Paller and
Heidinger, 1979). Wedemeyer et al. (1979) reported that an ozone residual of
0.002 mg/I would be a safe level or ozone when culturing rainbow trout.
Based on the literature, the exact level of ozone that damages gills or
kills rainbow trout is between 0.008-0.06 mg/I (Roselund, 1975; Wedemeyer et
a!., 1979). In our research. ozone concentration rose to lethal levels on
five occasions when we attempted to maximize ozone dosages in trials three
and four. The high ozone concentrations were caused by variable ozone demand
in the water and the short hydraulic retention time provided before each
fish culture tank. Ozone levels as high as 0.08 mg/I were measured during
fish mortalities; however, higher ozone levels probably occurred but were
not measured because staff would first attempt to restore ozone-free water
flow to protect the fish; measuring ozone residual was less important. Ozone
mortalities were not observed in tests one and two, probably because the
ozone dosing rate per unit feed fed was lower than those in tests three and
four. Additionally, we observed that when fish stopped feeding from the
demand feeders after being stressed (for example, just after selective
harvest of the fish greater than about 0.34 kg) ozone accumulated more
readily within the region that was harvested. This indicated that the
production of organic compounds during and after feeding affected the race
hat ozone reacted, which decreased ozone concentrations
Occurrence of ozone produced mortalities illustrates a
serious liability of ozone Technology - the lack of instrumentation to
continually detect ozone at levels <0.1 mg/I and the lack of chemical tests
to readily measure ozone in water grab samples at concentrations <0.01 mg/I.
At present. there is no fail-safe system to directly measure and control
ozone in solution. An indirect measure of residual ozone is the water's
oxidation reduction potential (ORP), which is a measure of a water's
potential to oxidize and is thus a measure of the waters potential to
disinfect or to kill fish. ORP can be monitored and used to control ozone
addition to ensure that the desired treatment objective has been achieved
and to ensure tar ozone residual is not in the fish culture tank. A safe ORP
for freshwater appears to be between 300-350 mV, depending upon pH. Our
attempts to indirectly measure ozone residuals by ORP control strategies
were only partially successful. An ORP control system was identified that
could prevent ozone residual from accumulating in the culture tank within
the region of the ORP probe - However, because our recirculating system
contained two culture tanks, each partitioned into two areas to isolate
fingerlings from larger fish, a single ORP controller, no matter how
accurate, could not prevent mortalities from occurring within a given region
of a culture tank unless a probe was in that region. In a single completely
mixed freshwater environment, a good automatic ORP controller could probably
help to obtain maximum oxidative treatment with minimum toxicity to fish.
These results may indicate that adding ozone at a lower
rate (0.025 kg ozone/kg feed) could provide about the same benefits as a
higher dosing rate (0.036-0-039 kg ozone/kg feed fed): e.g., reduced BGD
associated mortalities and no required use of non-approved chemical
treatments to control BGD epizootics. Yet, the lower ozone dosage rate
apparently did not kill fish from ozone toxicity because ozone had such a
short half-life and its residual quickly reacted away. Accordingly, the
lower ozone addition rate could allow use of a shorter ozone contact time
before the completely mixed culture tanks and also avoid the use of ozone
residual removal units and the dependence upon expensive and sometimes
unreliable ORP control technologies. Hence, use of the lower dose could
provide all of the benefits but also reduce capital and operating costs
associated with the higher ozone dosing rate.
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WHY USE OZONE IN AQUACULTURE?
PRACTICAL SUGGESTIONS
Ozone can be used in water treatment for the following purposes:
Fine and colloidal solids consist of particles 1-30 microns
(m m) and 0.001-1 m m respectively. The small size of the particles enables
the solids to remain in suspension and avoid most mechanical methods of
separation. The accumulation of fine and colloidal solids can impair
biofilter nitrification efficiencies and stress fish stocks.
Ozone removes fine and colloidal solids by causing clumping
of the solids (microflocculation), which facilitates removal by
foam-fractionation, filtration and sedimentation.
Dissolved organic compounds (DOC’s) or refractory organics,
give the water a characteristic tea-coloured stain. DOC’s are
non-biodegradable and accumulate according to feed input, water exchange
rate and the rate of solids removal. High levels of DOC’s can stress fish
and reduce nitrification efficiencies of the biofilter.
Ozone removes dissolved organics by:
-
oxidation into products that are more readily nitrified in the
biofilter;
-
including precipitation, which enables removal of waste particles by
conventional filtration or sedimentation.
Nitrite can accumulate as production intensifies and organic
loadings on the biofilter increase. Bacteria that process ammonia into
nitrite (Nitrosomonas spp) operates more efficiently under high
organic loadings than bacteria that process nitrite to nitrate (Nitrobacter)
and levels of nitrite rise accordingly.
High levels of nitrite can be toxic to fish. Data available
for silver perch, Bidyanus bidyanus indicates levels of nitrite as
low as 2.8 parts per million (ppm) can reduce growth of fingerlings by 5%.
Ozone removes nitrite by:
-
direct oxidation to nitrate;
-
reducing organic loading, which improves biofiltration efficiency and
nitrification.
The high stocking densities,
associated fish stress and increased nutrient loads found in RAS create an
ideal environment for fish pathogens. An important step in reducing the risk
of disease outbreaks in RAS is the use of standard quarantine procedures for
any fish introduced. Facilities using surface waters, including RAS and
flow-through hatchery systems, are also interested in reducing the pathogen
load introduced via the source water. The disinfection of effluent waters
before introduction to the environment is also crucial to prevent the
translocation of exotic diseases.
Ozone can effectively inactivate a
range of bacterial, viral, fungal and protozoan fish pathogens. The
effectiveness of ozone treatment depends on ozone concentration, length of
ozone exposure (contact time), pathogen loads and levels of organic matter.
If high levels of organic matter are present, the demand created by
oxidising the organic matter can make it difficult to maintain enough
residual ozone for effective disinfection.
Most commercially available ozone
generators use either corona discharge to produce ozone. In corona
discharge generation, a high-energy electric field is established between
two metallic plates and dried air or oxygen gas is fed through the plates.
The electrical energy excites a proportion of the oxygen molecules creating
atoms of oxygen (O). The oxygen atoms then bond to oxygen molecules in the
feed gas to create ozone.
UV light of a certain wavelength
(140-190nm) can be used to excite and break the oxygen molecules to generate
ozone in a similar way. UV generators are less expensive to purchase than
corona discharge generators, but at present is a much less energy efficient
way of producing ozone.
Use of oxygen as the feed gas
increases the yield of ozone from both UV and corona discharge generation
substantially when compared with dried air, but has an associated cost.
The design of the ozone reactor or
contact vessel is very important for safe, successful ozonation. There is a
range of reactors available using various designs to transfer ozone to the
water. Designs include fine bubble diffusers, turbine contactors, injectors,
deep u-tube reactors, packed columns, static mixers and spray contact
chambers. Some designs are also used for oxygen transfer or aeration. Each
design has advantages and disadvantages not discussed here.
Important considerations when
choosing a reactor include:
-
ozone transfer efficiency;
-
leak-free design and
construction;
-
construction with ozone
resistant materials.
Materials used in an ozone
treatment system must be highly resistant or inert to ozone. Use of improper
materials can lead to erosion of the unit and cause dangerous and costly
leakages. Such systems are not suitable for the long-term application of
ozone and require on-going, high replacement costs. The generation of ozone
in systems with substandard materials is also less efficient as ozone is
lost as the materials of the reactor are oxidised. The use of some plastics,
such as polyvinyl chloride (PVC) and polycarbonate is not recommended for
long-term applications for this reason. Galvanised steel is also not
recommended.
Stainless steel contact chambers
and piping are recommended for use with ozone. Valves should be made of
stainless steel, with gaskets and membranes of Teflon® or similar.
Ozone can be applied continuously,
as a series of treatments per day or as a single batch treatment per day.
Application in most situations can be linked to the feeding strategy
employed in the culture system. Three to four hours after feeding fish, the
concentrations of ammonia, dissolved organics and other wastes products
reach a maximum. If fish are fed several times during the day, a series of
ozone treatments can be introduced after each feed to target the associated
rise in waste levels. If feed is introduced 24 hours per day, water quality
degrades continuously and so ozone application should be continuous. A
single batch ozone treatment can be used to target rises in waste levels in
the system associated with a moderate feed event or to treat batches of
exchange or inlet water from the supply source.
Continuous ozonation is beneficial
when compared to batch and serial treatments because water quality remains
relatively stable. However, the lower costs of serial and batch ozonation
make these treatments regimes viable management options.
The required amount of ozone for
treatment in an RAS
(Recirculating Aquaculture
Systems) is usually
calculated according to the daily feed rate. Rates of 10-15 g of ozone per
kilogram of feed are generally recommended to reduce accumulated organics.
Any background organic loadings of the source water used for the RAS should
also be taken into account.
If disinfection is the primary
goal of ozonation, the amount of ozone necessary is largely dependent on the
background organic loading of the water to be treated. In pure water,
residual concentrations of 0.01-0.1 ppm ozone for periods as short as 15
seconds can be effective in reducing bacterial loads. However, in water with
organic loadings the residual ozone concentration and/or contact time of
ozone must be increased to produce significant disinfection. Natural waters
(seawater, brackish and freshwaters) generally require residual
concentrations of between 0.1-0.2 ppm ozone and contact times of 1-5 minutes
for disinfection. Aquaculture effluent generally requires between 0.2-0.4
ppm residual ozone for 1-5 minutes for significant disinfection to occur
after oxidation of organics.
The optimum rate of ozone for
disinfection is highly variable and represents the sum of ozone demands from
dissolved organics, colloidal solids, nitrate and disinfection. In many
situations in RAS, the cost of production of sufficient residual ozone for
complete disinfection after all other ozone demands are met is prohibitive.
However, some reduction in pathogen loads can be achieved using moderate
levels of ozone, and water quality improvements are considerable.
Disinfection of exchange and
effluent water is more cost effective than treating the entire system due to
the relatively small volumes treated. Disinfection of source water with
ozone, in combination with quarantine procedures for incoming stock, reduces
the risk of disease outbreak within the system.
Ozone is reported to be toxic to a
wide range of fresh and salt-water organisms at residual concentrations
between 0.01 ppm and 0.1 ppm. When deciding where to introduce ozone the
effect of residual concentrations from the reactor on either the biofilter
or fish stocks should be carefully considered. There are several locations
in a RAS where ozone may be added depending on the desired outcome.
-
Oxygen feed gas
- A common method of
introduction uses existing oxygen transfer systems to add ozone with the
oxygen feed gas. This generally occurs after the biofilter and just prior
to the culture tank. Due to the proximity to the culture tank this method
carries a moderate risk of exposing fish to residual concentrations of
ozone. By retaining water in a contact chamber (de-ozonation unit) for
several minutes before it passes to the culture tanks, this risk can be
reduced. Advantages of this option include reduction of pathogen loads and
nitrite levels immediately prior to contact with fish stocks.
-
Pre-biofilter - Addition
of ozone before the biofilter is also popular. This method carries a lower
risk of exposing fish stocks to residual ozone. Any residuals present must
first pass through the biofilter and are used in the oxidation of biofilms.
In this way the biofilter effectively buffers the fish stocks from toxic
effects of ozone. However, if levels of residual are too high performance
of the biofilter may be affected, leading to decreased nitrification. An
advantage of applying ozone before the biofilter is that the oxygen
produced as a reaction end product of ozone increases dissolved oxygen
levels in the biofilter. This is particularly beneficial in submerged
biofilters. However, for trickling biofilters any by-product oxygen is
effectively lost to the atmosphere.
-
Incoming water supply -
Ozone can be introduced into the incoming supply water as it enters the
building to achieve disinfection. This prevents entry of pathogen loads to
the system and enables isolation of healthy stock. For RAS relying on
surface water supplies disinfection at this point is very important. It
should be noted that ozonation of incoming water alone would not address
the build-up of organics within the system.
-
Effluent - Conversely,
ozone can be used to disinfect effluent exchange water before it leaves
the building to prevent exotic disease loads from entering the
environment. This can be done most effectively on bath loads of effluent
prior to release to irrigation or discharge systems.
Ozonation of water prior to coarse
solids removal is not recommended. Treatment of coarse solids by ozonation
is prohibitively expensive as levels of residual ozone must be increased to
address the additional ozone demand.
Direct treatment of the culture
tank is not recommended. This method carries a high risk of exposing fish
stocks to residual ozone concentrations.
Ozonation of brackish or seawater
results in the production of different by-product oxidants to freshwater.
Ozone reacts with bromide and chloride ions in saltwater to produce
relatively stable oxidants that are toxic to aquatic organisms. Use of ozone
in saltwater systems is usually restricted to batch treatment of water
separate to the main recirculating flow. Activated carbon filtration can be
used to remove residual ozone and other oxidants from ozonated saltwater.
The direct measurement of ozone in
a water sample is generally achieved using colorimetric test kits and
spectrophotometry. However, these methods can be too coarse to detect the
low residual levels lethal to some fish species and are unsuitable for
continuous in-flow monitoring. A common way of providing some level of
continuous in-flow monitoring for ozone is the use of oxidation-reduction
potential (ORP) probes. Rather than measure ozone directly, an ORP probe
measures the total capacity, in millivolts (mV), or various oxidants in a
solution to oxidise an electrode. By keeping ORP measurements within a
certain range, the levels of total oxidants can be controlled, which gives
indirect control over ozone. A safe ORP level of freshwater fish culture is
generally considered to be 300 mV.
Many systems automate ozonation by
linking ORP measurement and the ozone generator, so that the generator
switches off once the required ORP is reached and cuts back in when ORP
drops again. Factors such as pH, temperature and species cultured will
determine the exact targeted ORP level. However, due to the lack of direct
measurement of ozone and because ORP probes can take several minutes to
register a charge in ORP, any use of ORP to measure and control ozone
application is approximate. For this reason it is recommended that ozone
control using ORP measurements allows for some error and limits are set
conservatively. Other water quality parameters, particularly nitrite, should
also be monitored in close association to ORP and used to gauge the effect
of ozonation.
Ozone is a very effective
oxidising agent for use in water treatment and reduction of pathogen loads
in RAS. However, use of any chemical of this nature is accompanied by
considerable risks. RAS viability may be threatened in several ways.
-
The reduction of nitrite levels
by ozone carries a risk. The biofilter receives less nitrite and the
population of bacteria responsible for processing nitrite to nitrate
diminishes. If any disruption to ozonation occurs, dangerous spikes in
nitrite concentration can subsequently develop.
-
High residual ozone
concentrations are a risk to cultured fish stocks causing gross tissue
damage and stock mortalities.
-
High residual ozone
concentrations are a risk to bacterial films on the biofilter. Disruption
to biofilter performance can cause large fluctuations in ammonia and
nitrite levels. This can have a lethal effect on fish stocks or at the
very least reduce stock health and growth performance.
It is recommended that a de-ozonation
unit be installed directly after ozone application in a RAS to prevent toxic
residual levels. This should be done regardless of the location of ozone
application in the system. A simple do-ozonation unit consists of a contact
chamber to increase the retention time of water, allowing ozone to degrade.
Alternatively, an in-line activated carbon filter or biofilter can also
function as a de-ozonation unit. Degassing of residual ozone also occurs in
packed column aerators and trickle filters. Any residual ozone gas should be
vented from the RAS building and destroyed before release.
Ozone is extremely toxic and any
exposure to humans constitutes a serious health hazard. Decrease in lung
function, aggravation of asthma, throat irritation and cough, chest pain,
shortness of breath and the inflammation of lung tissue are typical symptoms
of ozone exposure. In cases of prolonged or severe exposure, chronic
respiratory illnesses such as emphysema, chronic bronchitis and premature
aging of the lungs may occur.
Exposure standards for residual
ozone of various International occupational health and safety
administrations range between 0.05 and 0.1 ppm for an 8 hour work period and
a maximum single dosage of 0.3 ppm for less than 10 minutes.
It is therefore important to
repeat the requirements of a leak-free ozone reactor made of suitable ozone
resistant materials. Venting of sheds or areas of a RAS where ozone is used
is also highly recommended. Humans can detect low levels of residual ozone
as a sharp, pungent odour, but continued exposure can quickly dull the
senses. For this reason perceived odour should not be used as an indicator
of ozone presence.
Test-kits for the detection of
air-borne ozone are commercially available and are a useful tool in helping
to ensure the safety of RAS operators.
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