Aquacultural Engineering 55 (2013) 46–58

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Aquacultural Engineering
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Impact of aeration and alkalinity on the water quality and product

quality of transported tilapia—A simulation study
John Colt ∗ , Eric Kroeger
National Marine Fisheries Service, Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle, WA 98112-2097, USA

a r t i c l e i n f o a b s t r a c t

Article history: The effects of aeration and alkalinity on water quality and product quality of Nile tilapia (Oreochrmis
Received 20 September 2012 niloticus) were determined for simulated commercial hauling conditions. Three types of aeration were
Accepted 13 March 2013 tested: pure oxygen aeration with a fine bubble diffuser (Oxygen), air aeration with medium bubble
diffusers (Air), and a combination of both pure oxygen aeration with a fine bubble diffuser and air aer-
Keywords: ation with a medium bubble diffuser (Mixed). Simulated transport hauls were conducted at two initial
Fish transport
alkalinities: 1.74 ± 0.11 meq/L (Low) and 8.84 ± 0.55 meq/L (High).
Aeration
The Air treatments resulted in the lowest carbon dioxide concentration, and the highest pH and
Water quality
un-ionized ammonia concentrations. At high alkalinities, the Air treatments were unable to maintain ade-
quate dissolved oxygen levels. The Mixed treatment resulted in reduced carbon dioxide and dissolved
oxygen concentrations. The Oxygen treatment resulted the highest dissolved oxygen, highest carbon
dioxide, and lowest pH and un-ionized ammonia. Un-ionized ammonia concentrations were higher with
the High Alkalinity treatments because of higher pH. Significant mortality was observed in the Air treat-
ments in both the Low- and High-Alkalinity treatments. Mortality in the Oxygen and Mixed treatments
for both low and high alkalinities were comparable to that observed in commercial tilapia transport using
fine bubble diffusers and pure oxygen.
These results indicate that mortality due directly to hauling water quality will not be increased at
high alkalinity, if pure oxygen aeration is used. The potential effects of water quality during haul-
ing on survival and product quality may be less than the impact from (a) physical damage from
loading and un-loading and (b) physiological problems resulting from pH and temperature shock
during the transfer from the hauling tanks to retail holding systems, especially for fish of reduced
fitness.
Published by Elsevier B.V.

1. Introduction ammonia concentration. The alkalinity of waters used for tilapia

A significant live-haul industry for tilapia has developed in
North America, and fish are commonly transported as much as
1000–3000 km (10–30 h) to market (Colt et al., 2011). At the end
of the haul, fish are transferred to holding systems at individ-
ual retail stores and sold over the following week. While direct
mortality during hauling is typically low, high mortalities may
occur during the post-haul holding period for reasons not fully
understood.
Key water quality parameters in transport include temperature,
dissolved oxygen, carbon dioxide, pH, and un-ionized ammonia.
Alkalinity and carbon dioxide concentrations have a significant
impact on pH through the carbonate chemistry reactions; changes
in these parameters can have a major impact on the un-ionized
∗ Corresponding author. Tel.: +1 206 860 3243; fax: +1 206 860 3467.
E-mail address: john.colt@noaa.gov (J. Colt).
rearing in Idaho (Colt et al., 2011) range from 1.6 to 9.3 meq/L.
The concentration of carbon dioxide depends on the metabolism
of the fish, water temperature, type of aerator used, and gas flow
rates. Typical tilapia haulers in the Pacific Northwest use pure
oxygen with fine bubble ceramic or carbon diffusers (Colt et al.,
2011). Detailed information on water quality during tilapia trans-
port and holding is lacking because of the sensitivity of analytical
equipment to transport, logistical constraints, restricted access to
hauling trucks and retail stores, and the overall complexity of the
industry (Colt et al., 2011). The objectives of this research are
to document impacts of aeration type and alkalinity on impor-
tant water quality parameters by using simulated hauling trials
with Nile tilapia (Oreochromis niloticus). Hauling protocols were
selected to minimize the impacts of physical damage and hauling
tank-retail store transitions on mortality and product quality. The
intent is that this information will be used to direct future research
and develop improved fish transport protocols for commercial
haulers.

0144-8609/$ – see front matter. Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.aquaeng.2013.03.002
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58
2. Background
2.1. Water quality requirements of tilapia
Basic information on the water quality requirements of Nile
tilapia is needed to interpret the results of this work. Because of
the lack of some specific water quality information for Nile tilapia,
it is necessary to consider published information for other tilapia
and non-tilapia species.
The optimal water temperature for tilapia growth is between
29 and 31 ◦ C (Popma and Masser, 1999). Tilapia generally stops
feeding when the water falls below 17 ◦ C and mortality occurs
below 11 ◦ C (Atwood et al., 2003; Popma and Masser, 1999). The
24-h LC50 of Nile tilapia for dissolved oxygen is 1.41 mg/L (Mahdi,
1973a). The critical oxygen level is 3.1 mg/L (Abdel Magid and
Babiker, 1975). Above this concentration, the oxygen consumption
of Nile tilapia is independent of DO concentration. Because of the
extremely wide water quality conditions that this group of fish
occupies, it is generally assumed that tilapia will tolerant of high
carbon dioxide concentrations, although specific information is
lacking. In rainbow trout (Oncorhynchus mykiss), exposure to 12
and 24 mg/L free carbon dioxide had no effect on growth over 275
days. Fish growth was reduced at 55 mg/L, but only after about
330 days (Smart et al., 1979). O. niloticus appear to have typical
sensitivity to dissolved gas supersaturation. Ps between 85 and
100 mm Hg, resulted in 25% mortality with 270 g fish (Saeed and
Al-Thobaiti, 1997). Following reduction in the gas supersaturation
level, a heavy infection of monogenetic trematodes resulted. No
mortalities were observed when O. niloticus were exposed to
0.43 mg/L un-ionized ammonia for 75 days (El-Shafai et al., 2004).
Increased mortality was observed after exposure for several weeks
to >1 mg/L un-ionized ammonia (Popma and Masser, 1999), while
transfer to water above 2.0 mg/L resulted in massive mortality.
The lower and upper 24-h LC50 values of pH for Nile tilapia are 3.4
and 11.0, respectively (Mahdi, 1973b). Mozambique tilapia (Ore-
ochromis mossambicus) can acclimate to pH as low as 4.0 when the
acclimation rate is slow (6 h) and additional stressors are avoided
(Van Ginneken et al., 1997). No significant changes in hemoglobin,
glucose, cortisol, sodium chloride or energy status in liver or white
muscle was observed. In contrast, when fish were abruptly exposed
to low pH, a variety of adverse physiological changes resulted
(Van Ginneken et al., 1997). Of the commercially important tilapia
species, Nile tilapia are the least tolerant to salinity, but grow well
in 15 g/kg salinity (Popma and Masser, 1999).
Optimal water quality criteria and “action levels” for 1-day
commercial tilapia hauls (300–600 g fish) are presented below. Val-
ues exceeding these action levels are expected to have a negative
impact on survival and product quality. These action levels are
somewhat subjective because published toxicity tests are largely
based on single factor experiments and small fish (Colt, 2006),
where under production conditions, fish are exposed to all the
factors simultaneously.
Parameter Optimal Action level
◦ bacteria and other microorganisms (kg/h).
Temperature ( C) 29–31 <12–13
Dissolved oxygen (mg/L) >5–6e <3.1
Carbon dioxide (mg/L) <20e >60e
P (mm Hg) <30–40e >85–100
Un-ionized ammonia (mg/L) <0.43 >1.0e
pH 4–9 <3.9 and >10.1
e
Estimated value.
2.2. Gas transfer in transport systems
2.2.1. Aeration in fish hauling systems
Based on a survey of government, tribal, and commercial
haulers, Carmichael and Tomasso (1988) found that 83% of the
47
respondents used pure oxygen aeration during hauling. Of those
using pure oxygen aeration, 70% also used agitators to circulate the
water in the hauling tanks. Only 15% and 2% of the respondents
used pumps and blowers, respectively. Some form of air vent or
scoop to allow air circulation through the head space (between the
water surface and tank lid) was reported by 47% of the respondents.
The breakdown of species transported by the respondents was
39% coldwater species, 21% coolwater species, and 40% warmwa-
ter species. The three most frequently transported species were
rainbow trout, channel catfish, and largemouth bass (Micropterus
salmoides) (Carmichael and Tomasso, 1988).
Adequate air movement through the headspace in a hauling
tank can reduce the buildup of carbon dioxide (Forsberg et al.,
1999; Haskell and Davies, 1958). In the transport of channel cat-
fish to processing plants, the use of blowers resulted in undesirable
increases in water temperature during summer months (Bosworth
et al., 2004). Commercial tilapia haulers use pure oxygen aeration
without agitators and in some cases without air vents or scoops
(Colt et al., 2011).
Some of the differences between commercial tilapia haulers
(Colt et al., 2011) and those reported by Carmichael and Tomasso
(1988) are due to species differences, longer distances, and higher
densities. For tilapia hauling, conservation of heat may be more
important than carbon dioxide removal during winter conditions
in the Pacific Northwest.
The use of pure oxygen allows higher dissolved oxygen con-
centrations to be maintained during transport (Colt and Watten,
1988) with higher reliability than mechanical agitation or blow-
ers. In commercial tilapia transport, it is not uncommon for the
dissolved oxygen to range between 10 and 15 mg/L during loading
and 30–35 mg/L at the end of haul (Colt et al., 2011). Because of
the stripping of nitrogen and argon gases by oxygen bubbles, total
gas pressures are below saturation even at these elevated dissolved
oxygen concentrations (Colt et al., 2011).
2.2.2. Gas transfer in hauling systems
The rate of oxygen transfer is generally assumed proportional
to the driving force (Lewis and Whitman, 1924):
dCO2
= KL a(CO∗ − CO2 ) (1)
dt 2
whereKL a = Volumetric transfer coefficient (1/h).CO∗ =
2
Saturation concentration of dissolved oxygen (mg/L).CO2 =
Concentration of dissolved oxygen (mg/L).
This equation is restricted to a simple batch system (no inflow
or outflow) and with no other sinks or sources of oxygen.
For hauling systems, the oxygen consumption of the fish and
bacteria must be included:
dCO2 R R
= KL a(CO∗ − CO2 ) − − bacteria (2)
dt 2 Vol Vol
whereR = Oxygen uptake of fish (kg/h).Rbacteria = Oxygen uptake of
At this time, there is no published information on Rbacteria in
hauling systems. Therefore, for the purposes of this discussion, it
is assumed that the R » Rbacteria and all of the oxygen consumption
during hauling is due to the fish. For hauling applications where
temperature, pressure, and salinity corrections can be ignored, the
steady-state dissolved oxygen concentration can be computed from
the following equation (ASCE, 2007; Stenstrom and Gilbert, 1981):
 
(ˇO2 CO∗ − CO2 )
R = ˛O2 SOTR 2
(3)
CO∗
2, standard
48 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58

or 10
 
R CO∗ , standard 9
C*
CO2 = ˇO2 CO∗ − 2
(4) 1 mg/L
2 ˛O2 SOTR
8 10 mg/L

pH
where˛O2 = KL a − hauling water/KL a − clean water.ˇO2 =
CO∗ − hauling water/CO∗ − clean water.SOTR = Standard 7 100 mg/L
2 2
oxygen transfer rate (kg/h).CO∗ = 6
2, standard
Standard oxygen saturation concentration (20 ◦ C,1 atm, moist air).
Assuming that ˇ ≈ 1.00 and SOTR = 0.50 kg/h (Boyd and Moore, 5
0 2000 4000 6000 8000 10000 12000
1993), Eq. (4) can be written as:
Alkalinity (ueq/L)
 
R
CO2 = CO∗ − 18.2 (5) Fig. 1. Impact of carbon dioxide and alkalinity on pH (pure carbonate system, tem-
2 ˛O2 perature = 25 ◦ C, salinity = 0.0 g/kg.).

The value of CO∗ depends on temperature, salinity, pressure, and

2
where
oxygen purity (Colt, 2012). Increases in R or decreases in ˛O2 result
in lower dissolved oxygen concentrations (CO2 ). More efficient aer- Alkminor = [NH3 ] + [HPO4 2− ] + 2[PO4 3− ] − [H2 PO4 − ] + [B(OH)4 ]
ators (larger values of SOTR) will increase the dissolved oxygen
concentration. − [HSO4 − ] − [HF] + [SiO(OH)3 ] + [HS− ] + 2[S2− ] (10)
Similar transfer equations can be written for carbon dioxide
(Colt et al., 2012a): The importance of the minor components of alkalinity will
  depend on the particular system under consideration in relation-
∗ R
CCO2 = CCO + 0.680 (6) ship to the total carbonate concentration (Eq. (8)). Significant
2 ˛CO2
concentrations of ammonia (NH3 ) and phosphates (PO4 3− , HPO4 2− ,
It is unclear at this time if ˛CO2 = ˛O2 (Eqs. (5 and 6)). and H2 PO4 − ) may be present in hauling systems. If hauling tanks
Carbon dioxide is much more soluble than oxygen, therefore are filled from a reuse system, humic and fulvic acids must also be
carbon dioxide removal depends on the gas flow rate or gas-to- included in Eq. (10).
liquid ratio (Colt et al., 2012b). Because of low gas flow rates used Detailed analysis for the carbonate components and analytical
in pure oxygen systems, pure oxygen aeration systems are ineffi- solution to Eq. (7) can be found in DOE (1994) and Dickson et al.
cient at removing carbon dioxide (Colt and Watten, 1988; Colt et al., (2007). One commonly used program (CO2SYS) for freshwater and
2012b). Foam production during hauling and the resulting reduced seawater carbonate chemistry applications was developed at Oak
air movement through hauling tank headspace may significantly Ridge National Laboratory (Lewis and Wallace, 1998).
reduce carbon dioxide removal.
2.3.1. Effect of alkalinity and carbon dioxide on pH (pure
2.3. Water chemistry in fish transport carbonate system)
For a pure carbonate system, the effect of carbon dioxide (C*
The major water quality parameters in fish transport are tem- to 100 mg/L) and alkalinity (100–10,000 ␮eq/L) on pH is presented
perature, dissolved oxygen, carbon dioxide, un-ionized ammonia, in Fig. 1 (Lewis and Wallace, 1998). For a fixed alkalinity, raising
and pH. In typical waters, it is generally assumed that pH is con- the carbon dioxide concentration depresses the pH. For a given
trolled by the carbonate system. concentration of carbon dioxide, the pH increases with higher alka-
The carbonate system consists of the following parameters: linity. The pH vs. alkalinity curves are logarithmic curves so pH vs.
log(alkalinity) is a straight line. High concentrations of free carbon
CO2 (gas) Carbon dioxide gas in the gas phase
dioxide are undesirable (Colt, 2006; Good et al., 2010), although
CO2 (aq) Carbon dioxide gas in the liquid phase
H2 CO3 Carbonic acid in the liquid phase the water quality criterion for carbon dioxide is not well defined,
HCO3 1− Bicarbonate ion in the liquid phase especially for hauling applications.
CO3 2− Carbonate ion in the liquid phase The pH for the carbon dioxide and alkalinity combinations, pre-
H+ Hydrogen ion in the liquid phase sented in Fig. 1, results in pH that ranges from 5 to 9, a change of
It is difficult to distinguish between dissolved carbon dioxide 10,000 × the hydrogen ion concentration. This pH change is well
gas (CO2 (aq)) and carbonic acid (H2 CO3 ) in solution (Stumm and within the tolerance of tilapia (see Section 2.1).
Morgan, 1981). Therefore, it is convenient to represent the sum of
dissolved carbon dioxide gas (CO2 (aq)) and carbonic acid (H2 CO3 ) 2.3.2. Effect of pH on un-ionized ammonia nitrogen (pure
by H2 CO3 * and the overall carbonate reaction can then be written carbonate system)
as The impact of pH on un-ionized ammonia nitrogen over a 24-h
haul is presented in Fig. 2 for representative alkalinity-carbon diox-
CO2 (gas) ⇔ H2 CO3 ∗ ⇔ H+ + HCO3 1− ⇔ H+ + CO3 2− (7) ide values in Fig. 1. It is assumed that the ammonia excretion rate
and carbon dioxide concentrations are constant with time. While
In carbonate chemistry work, it is also convenient to define
there may be significant variability in the carbon dioxide concen-
two additional chemical parameters (Dickson, 1981; Stumm and
trations at the start of the haul, these differences are not important
Morgan, 1981):
because the TAN and resulting un-ionized ammonia concentrations
Total carbonate or CT (mol/kg)=[H2 CO3 ∗ ]+[HCO3 − ]+[CO3 2− ] (8) are low during this period.
The concentration of un-ionized ammonia (at a constant tem-
and perature and conductivity) depends strongly on pH, and therefore,
on alkalinity and carbon dioxide levels. For an alkalinity of
Alkalinity (eq/kg)
1000 ␮eq/L (Fig. 2), an action level of 1000 ␮g/L un-ionized ammo-
= [HCO3 − ] + 2[CO3 2− ] + [OH− ] − [H+ ] + Alkminor (9) nia is reached in 11 and 19 h for C* (saturation) and 1 mg/L,
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58
2000
10,000 ueq/L, C*
Un-ionized Ammonia (ug/L)
10,000 ueq/L, 1 mg/L
1500
Action Level
1000
500
0
0 4 8 12 16
Time (hours)
Fig. 2. Variation of un-ionized ammonia nitrogen (␮g/L) with time as function of
carbon dioxide and alkalinity (pH values from Fig. 1, initial TAN = 0.2 mg/L, TAN
excretion rate = 1.03 mg/h, conductivity = 200 ␮S/cm).
respectively. At higher levels of carbon dioxide, the action lev-
els are not reached during the 24-h haul. This is in contrast with
10,000 ␮eq/L alkalinity where the action levels are reached for C*
and 1 mg/L in only 2–3 h. While high concentrations of carbon diox-
ide are undesirable because of their biological impacts (Good et al.,
2010; Smart et al., 1979), low values of carbon dioxide are even
more undesirable when TAN concentrations are high.
2.3.3. Effect of “minor” alkalinity components during Hauling
In Fig. 2, the impacts of excreted phosphates and ammonia have
been ignored. Ammonia is excreted across the fish’s gills as un-
ionized ammonia (NH3 ). The increase in alkalinity due to ammonia
excretion (over a given time period) is equal to:
 
1000 g/mg × TANexcreted
AlkalinityTAN =
14.007 g/eq
whereAlkalinity = Increase in alkalinity
(␮eq/L).TANexcreted = Excreted ammonia (mg TAN/L).
For commercial tilapia hauling (Colt et al., 2011), the increase in
alkalinity from excreted ammonia ranged from 40% to 77% of the
initial alkalinity at loading. For a pure carbonate system, an increase
in alkalinity at a fixed carbon dioxide concentration will raise the
pH (Fig. 1) and the un-ionized ammonia concentration (Fig. 2).
In fed rainbow trout, the P/N excretion ratio is only 0.0346 on
a molecular basis or 0.0765 on a weight basis (Clarke et al., 1985).
The P/N ratio is likely to be different for unfed fish. As a result of
this reduced excretion and the larger equivalent weight, the con-
tribution of phosphate to the change in alkalinity will be less than
that of ammonia.
For very low initial alkalinities and in longer hauls, pH may
depend on both the carbonate system (Eq. (7)) and the excreted
ammonia and phosphate reactions. Impacts of large amounts of
added ammonia or phosphate have not been considered in existing
carbonate models.
Table 1
Initial alkalinities and hauling densities.
Experiment Initial alkalinity (meq/L) Fish/Tank (#)
1 1.74 ± 0.11 36–37
2 8.84 ± 0.55 33–35
3 1.20 ± 0.01 31–32
Commercial
Commercial hauling information from Table 1, Colt et al. (2011).
49
3. Materials and methods
3.1. Fish
Nile tilapia were obtained from First Ascent Fish Farms, a com-
1,000 ueq/L, C* mercial farm in Central Idaho and delivered to Seattle on November
1,000 ueq/L, 1 mg/L
1,000 ueq/L, 10 mg/L
22, 2011. Fish were fed a 35% protein catfish feed (Silver Cup, Nelson
1,000 ueq/L, 100 mg/L and Sons, Murray Utah) at 0.8% of body weight, at approximately
8:00 am 5 days a week.
20 24
3.2. Holding system
The fish were held in a lightly loaded reuse system (cumula-
tive feed burden = 124 ± 10 mg/L; Colt et al., 2006) that consisted of
three 1800 L holding tanks, six 400 L rearing tanks, a sedimentation
basin, two 125 L bead filters, an aeration/degassing column, and
water heater/heat exchange system. The system pH was controlled
by the addition of saturated sodium bicarbonate solution by a dos-
ing pump controlled by a YSI-5200A (Yellow Springs Instruments,
Yellow Springs, Ohio).
3.3. Simulated hauling system
The hauling system consisted of six insulated coolers (120
Quart Ice Blue MaxCold, Igloo Product Corporation, Houston,
Texas). The average inside dimensions of the coolers was
86.0 cm × 35.7 cm × 33.7 cm. A single 8 cm hole was drilled in the lid
to allow air exchange and for aerator hose access. A 100 or 200 W
electrical heater was installed in each hauling tank. The coolers
were filled with 75.6 L of water, resulting in an initial water depth
of 23.5 cm. The hauling densities ranged from 176 to 188 g fish/L of
water (Table 1). These experimental densities are comparable those
(11) used by commercial tilapia hauler from Central Idaho to Vancou-
ver, B.C. (Colt et al., 2011). Un-iodized sodium chloride (5000 mg/L)
was added to all treatments 1 h prior to adding the fish.
3.4. Oxygen/aeration systems
Two types of diffusers were used: fine bubble ceramic plate dif-
fuser (MBD 100, Point Four Systems, Coquitlam, British Columbia)
or a medium pore diffuser (AS8L, Aquatic Ecosystems, Apopka,
Florida). Oxygen gas flow to each individual hauling tank was reg-
ulated by a Victor flow meter (Model MOF7 4H, Denton, Texas). A
single ceramic plate diffuser was used in all hauling tanks. A sin-
gle medium pore diffuser was used in the Mixed treatment and
three medium pore diffusers were used in the Air treatment. Each
treatment consisted of two replicates.
3.5. Environmental analysis
Dissolved oxygen and temperature were measured using a
ProODO optical sensor (YSI, Yellow Springs, Ohio). pH was mea-
sured using an Orion Ross Ultra Combination pH probe and
Orion 710A+ pH meter (Thermo Fisher Scientific, Waltham,
Mean weight (g) Hauling density
(g fish/liter water) (g fish/liter tank)
384 ± 86 182 ± 2 154 ± 1
430 ± 70 176 ± 2 150 ± 1
453 ± 53 188 ± 0 158 ± 0
158–290 136–225
50 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58
Massachusetts). Alkalinity (unfiltered) was determined by Hach
Digital Titrator and Hach cartridges (Hach Company, Loveland,
Colorado) using Gran function (Rounds, 2006). Total ammonia
nitrogen (APHA, 1998) was measured using an Orion 95-12 Ammo-
nia Electrode (Thermo Fisher Scientific, Waltham, Massachusetts).
Un-ionized ammonia nitrogen (UIAN) was computed using the pKa
developed by Emerson et al. (1975) and the spreadsheet presented
by Wedemeyer (2001). Carbon dioxide was measured using an Oxy-
Guard CO2 Analyser (OxyGuard International, Birkerod, Denmark).
Soluble reactive phosphorus was determined by U.S. EPA Method
365.2 (USEPA, 1983).
In Experiments 1 and 2, samples for TAN and bacterial oxy-
gen uptake were frozen (−4 ◦ C) until analysis (approximately 3–4
weeks). In Experiment 3, analysis for TAN, oxygen uptake, KL a was
done on fresh samples. Additional final samples in Experiment 3
were frozen to evaluate the impact of freezing on bacterial oxygen
uptake and KL a.
Samples used for pH and carbon dioxide were returned to the
individual haul tanks after analysis. To maintain initial hauling den-
sities, replacement water was added equal to the amount removed
for the total ammonia nitrogen and oxygen uptake samples.
3.6. Bacterial oxygen uptake determinations
The bacterial oxygen uptake of an aerated water sample was
determined in a 250 mL BOD bottle. The LDO oxygen probe (with
the probe guard removed) was inserted directly into the BOD bottle.
A 2.5 cm magnetic stir bar was used to gently mix the water. Dis-
solved oxygen values were logged on a 1-min interval for 20 min.
Slope of DO vs. time was determined by linear regression and the
results adjusted to a base temperature of 20 ◦ C using  = 1.047.
3.7. KL a determinations
In Experiment 3, the oxygen transfer coefficients (KL a) were
determined daily. To reduce the impact of biological oxygen uptake
on the oxygen transfer coefficient, 1 mL of 6% sodium hypochlorite
was added to each sample and allowed to react for 8 h. Next, 7 mL
of 10% sodium thiosulfate was added and allowed to react for 16 h
to remove residual chlorine. Then the samples were aerated for 4 h
to remove any residual sodium thiosulfate.
Approximately, 0.6 L of hauling water was added to a cylindrical
reactor and the water deoxygenated with nitrogen gas. KL a values
were determined by the ASCE method (ASCE, 2007) and corrected
to 20 ◦ C using  = 1.047. Duplicate final samples were frozen (−4 ◦ C)
and re-analyzed after 4, 11, 18, and 32 days.
3.8. Statistical analysis
Statistical analysis was performed using Prism 5.0 for Mac OS
X. Significant differences were determined by either analysis of
variance or analysis of variance for repeated measures followed
by Bonferroni’s multiple comparisons test (P = 0.05). Significant
differences for survival curves were determined by the log-rank
(Mantel–Cox) test.
3.9. Experimental protocols
Prior to each experiment, fish were acclimated for at least 1 week
at the specific alkalinity. Two days prior to hauling, feed was with-
held. At the start of a haul, the fish had not been fed for 2.25 days.
One day prior to transport, the fish were randomly transferred to
holding tanks (400 L). The fish were weighed prior to transfer to
the hauling tanks. At the end of the simulated haul, fish were trans-
ferred back to the holding tanks. Mortalities were recorded and
removed for the following 7 days (except in Experiment 3). The
fish were not fed during the post-haul period. At the end of post-
haul period, the fish were individually examined for scale loss and
physical damage. A fish was classified with “Minor Damage” if it
had less than four small (<0.5 cm in diameter) areas of scale loss
and ulceration. In the lighter colored fish, these areas were red and
very visible. Fish with extensive fin damage, eye damage, or more
than four areas of scale loss were classified with “Major Damage”.
The number of saleable fish was equal to:
#Saleable Fish = #Total Fish − #Mortalities − #Major Damage (12)
One hour prior to the start of the simulated haul, all tanks were
aerated with pure oxygen at 1.5 L/m. After the fish were added, the
oxygen flow was stopped in the Air treatments and airflow turned
on in the Air and Mixed treatments. The airflows for the Mixed
and Air treatments were 5.24 ± 0.43 and 138.93 ± 9.55 L/m, respec-
tively. Four hours after the start, the oxygen flow was reduced to
1.0 L/m.
The following simulated transport experiments were con-
ducted:
3.9.1. Experiment 1
This experiment consisted of Oxygen, Mixed, and Air treat-
ments, low alkalinity process water, 24-h simulated transport, and
a 7-day post-haul evaluation.
3.9.2. Experiment 2
Similar to Experiment 1, except a high alkalinity process water
was used.
3.9.3. Experiment 3
This experiment consisted of an Oxygen treatment, low alkalin-
ity process water, 72-h simulated transport, and a 1-day post-haul
evaluation. Sampling intensity was reduced to allow analysis of
fresh water quality and oxygen transfer samples.
3.9.4. Experiment 4
This experiment investigated the impact of aerator type on
water temperature over a 24-h simulated haul. No fish or heaters
were used.
3.9.5. Experiment 5
This experiment evaluated oxygen uptake and oxygen transfer
coefficient samples that were frozen from Experiments 1 and 2 and
fresh samples from Experiment 3.
4. Results
4.1. Experiment 1 – one day transport, low alkalinity
Water quality in the pre- and post-haul periods is presented
in Table 2. Compared to the pre-haul period, DO was higher and
carbon dioxide was lower for the post-haul period. The reduction
in carbon dioxide resulted in higher pH and un-ionized ammonia
in the post-haul period.
The initial and final alkalinity means were 1.74 ± 0.11 and
2.42 ± 0.32 meq/L, respectively (Table 3). Type of aeration did not
have a significant impact on the change in alkalinity (F = 5.968,
P < 0.090). The water quality during the simulated haul is presented
in Fig. 3A for dissolved oxygen, Fig. 3B for carbon dioxide, Fig. 3C
for pH, Fig. 3D for total ammonia nitrogen, Fig. 3E for un-ionized
ammonia nitrogen, and Table 4 for temperature. Dissolved oxygen,
carbon dioxide, and pH were relatively constant over the transport
period (Fig. 3A, B, and C). Aeration type had similar impacts on dis-
solved oxygen (Fig. 3A), carbon dioxide (Fig. 3B), and TAN (Fig. 3D):
highest for Oxygen, intermediate for Mixed, and lowest for Air.
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58 51

Table 2 mortality in the Air treatments was significantly higher than in

Water quality during pre- and post-hauling periods (mean ± SD).
the Oxygen treatment (Chi-squared = 26.66, df = 3, P < 0.0001) or the
Parameter Experiment 1 Experiment 2 Mixed treatment (Chi-squared = 23.74, df = 3, P < 0.0001). At the end
of the 7-day post-haul period, there were 11 and 13 mortalities in
Pre-haul Post-haul Pre-haul Post-haul
the Air treatments and only three other mortalities in the other
Temperature (◦ C) 24.7 ± 0.8 25.1 ± 0.4 25.6 ± 0.1 24.7 ± 0.3
four tanks. The impact of aeration on product quality is presented
DO (mg/L) 4.69 ± 0.61 6.98 ± 0.54 6.18 ± 0.22 7.50 ± 0.27
CO2 (mg/L) 4.7 ± 0.8 1.5 ± 1.0 2.0 ± 0.7 1.4 ± 0.5 in Table 5. The number of saleable fish was significantly reduced
pH 7.42 ± 0.21 7.77 ± 0.07 8.14 ± 0.19 8.53 ± 0.11 in the Air treatment due to increased mortality, but the number of
TAN (mg/L) 0.28 ± 0.13 0.25 ± 0.04 0.23 ± 0.16 0.20 ± 0.04 fish with minor damage (0–28% of fish) and major damage (0–17%
UIAN (␮g/L) 3.30 ± 2.04 7.28 ± 1.07 16.6 ± 10.5 28.8 ± 7.67 of fish) was not related to aeration type.
Alkalinity (meq/L) 1.08 ± 0.31 1.47 ± 0.35 6.63 ± 0.97 7.54 ± 1.09

4.2. Experiment 2 – one day transport, high alkalinity

The impact of aeration type was reversed for pH (Fig. 3C) and
UIAN (Fig. 3E): lowest for Oxygen, intermediate for Mixed, and
highest for Air. The variation of TAN (Fig. 3D) and UIAN (Fig. 3E)
was linear with time.
Aeration type had a significant impact on dissolved oxy-
gen (F = 542, P < 0.0001), carbon dioxide (F = 325, P < 0.001), pH
(F = 146, P < 0.0001), TAN (F = 10.28, P < 0.0025), and UIAN (F = 10.57,
P < 0.0023). All of the treatments were significantly different for
dissolved oxygen, carbon dioxide, and pH. For TAN, the Oxygen
and Air treatments were significantly different. For UIAN, the Oxy-
gen and Mixed treatments were significantly different from the Air
treatment.
The variation of mortality with time is presented in Fig. 4. At
the end of the haul, there was only one mortality, in the Air treat-
ment (Tank 6), during the transport period. In the post-haul period,
Water quality in the pre- and post-haul periods is presented in
Table 2. The changes in water quality between the pre- and post-
haul periods were similar to Experiment 1.
The initial and final mean alkalinity were 8.84 ± 0.55 and
9.63 ± 1.22 meq/L, respectively (Table 6). Aeration type had a sig-
nificant impact on the change in alkalinity (F = 18.70, P < 0.0202)
and the mean change for Air and Mixed were significantly different.
Water quality parameters during the simulated haul is presented in
Fig. 5A–E for dissolved oxygen, carbon dioxide, pH, total ammonia
nitrogen, un-ionized ammonia nitrogen, and Table 4 for tempera-
ture. The major impact of aeration on dissolved oxygen (Fig. 5A),
carbon dioxide (Fig. 5B), and TAN (Fig. 5D) was similar to Experi-
ment 1: highest for Oxygen, intermediate for Mixed, and lowest for
Air. The impact of type of aeration was also reversed for pH (Fig. 5C)

Table 3
Initial, final, and change in alkalinity in Experiment 1a .

Treatment Initial (meq/L) Final (meq/L) Change (meq/L) Mean change (meq/L)

Air 1.74 ± 0.01 2.83 ± 0.25 1.09 1.06 ± 0.03a

1.75 ± 0.06 2.79 ± 0.06 1.04

Mixed 1.60 ± 0.25 2.19 ± 0.01 0.59 0.50 ± 0.13a

1.83 ± 0.00 2.24 ± 0.02 0.41

Oxygen 1.76 ± 0.04 2.46 ± 0.04 0.70 0.48 ± 0.31a

1.78 ± 0.11 2.04 ± 0.13 0.27

Mean ± SD 1.74 ± 0.11 2.42 ± 0.32

a
Values within columns sharing a common superscript letter were not significantly different (Bonnferroni’s multiple comparisons test, P < 0.05).

Table 4
Temperature variation during simulated hauling experiments.

Experiment/conditions Oxygen Mixed Air All

1/1-day, low alkalinity 25.18 ± 0.11 25.06 ± 0.12 24.73 ± 0.18 24.73 ± 0.03 24.75 ± 0.19 24.78 ± 0.41 24.88 ± 0.27
2/1-day, high alkalinity 25.81 ± 0.18 25.60 ± 0.24 26.01 ± 0.23 25.70 ± 0.30 25.56 ± 0.31 25.48 ± 0.58 25.72 ± 0.38
3/3-day, oxygen 25.26 ± 0.45 25.12 ± 0.42 N/A N/A N/A N/A 25.19 ± 0.43

Table 5
Product quality at end of 7-day post-haul (Experiments 1 and 2).

Alkalinity Treatment Total fish Ending product qualitya

Mortalities Minor damage Major damage Saleable fish

Low (Exp. #1) Oxygen 37 1 4 1 35

37 0 0 2 35
Mixed 37 1 4 0 36
37 1 2 1 35
Air 36 13 10 6 17
36 11 4 0 25

High (Exp. #2) Oxygen 34 2 12 0 32

33 0 11 0 33
Mixed 35 3 20 2 30
35 2 8 1 33
Air 34 34 NA NA 0
33 27 4 2 4
a
See Section 3.9 for scoring criteria.
52 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58

Table 6
Initial, final, and change in alkalinity in Experiment 2a .

Treatment Initial (meq/L) Final (meq/L) Change (meq/L) Mean change (meq/L)

Air 8.42 ± 0.76 10.47 ± 1.19 2.06 2.54 ± 0.69b

8.23 ± 0.90 11.26 ± 0.36 3.03

Mixed 9.29 ± 0.14 8.51 ± 2.02 −0.78 −0.40 ± 0.54a

9.16 ± 0.08 9.14 ± 0.21 −0.02

Oxygen 8.91 ± 0.31 9.26 ± 0.33 0.36 0.24 ± 0.17a

9.02 ± 0.17 9.14 ± 0.17 0.12

Mean ± SD 8.84 ± 0.55 9.63 ± 1.22

a
Values within columns sharing a common superscript letter were not significantly different (Bonnferroni’s multiple comparisons test, P < 0.05).

45
3A. Dissolved Oxygen
and UIAN (Fig. 5E): lowest for Oxygen, intermediate for Mixed, and
40 Oxygen Mixed Air
35 highest for Air.
DO (mg/L)

30
25 The dissolved oxygen concentration in the Air treatment
20 showed a slow decrease over the hauling period; at the end of the
15
10 haul, the dissolved oxygen was 3.5 mg/L. The carbon dioxide con-
5 centration generally increased over most of the hauling period for
0
0 4 8 12 16 20 24 Oxygen and Mixed treatments (Fig. 5B), while the Air only increased
35
30 3B. Carbon Dioxide in the last 3 h. The impact of aeration type on pH was similar to
CO2 (mg/L)

25 Experiment 1 except that the average pH was 0.5–0.7 units higher

20 in Experiment 2. The variation of TAN with time was linear for Oxy-
15
10
gen and Mixed (Fig. 5D), but was non-linear Air. The variation of
5 UIAN was linear with time (Fig. 5E). The increase in conductivity
0 was significantly larger for Air compared to either Mixed or Oxygen
0 4 8 12 16 20 24
9.0 (Table 7).
3C. pH
8.5 Aeration type had a significant impact on dissolved oxygen
8.0 (F = 436, P < 0.0001), carbon dioxide (F = 310, P < 0.001), pH (F = 140,
pH

7.5 P < 0.0001), and UIAN (F = 10.57, P < 0.0023). All treatments were
7.0
6.5
6.0
0 4 8 12 16 20 24 45
40 40 Oxygen Mixed Air 5A. Dissolved Oxygen
UIAN (ug/L) TAN (mg/L)

3D. TAN 35
DO (mg/L)

30 30
20 25
20
10
15
0 10
0 4 8 12 16 20 24 5
2000
3E. UIAN 0
1500
35 0 4 8 12 16 20 24
1000 5B. Carbon Dioxide
30
500
CO2 (mg/L)

25
0 20
0 4 8 12 16 20 24
15
Time (hours) 10
5
Fig. 3. Water quality in Experiment 1, low alkalinity process water. (A. dissolved 0
oxygen; B. carbon dioxide; C. pH; D. total ammonia nitrogen; and E. un-ionized 0 4 8 12 16 20 24
9.0
ammonia.) 5C. pH
8.5
8.0
pH

7.5
7.0
6.5
100% 6.0
Air, Tank 3 Air, Tank 6
0 4 8 12 16 20 24
40
Cumulative Mortality (%)

TAN (mg/L)

80% Mixed, Tank 1 Mixed, Tank 5 5D. TAN

30
Oxygen, Tank 2 Oxygen, Tank 4 20
60% 10
0
40% 0 4 8 12 16 20 24
UIAN (ug/L)

2000
5E. UIAN
1500
20% 1000
500
0% 0
0 24 48 72 96 120 144 168 0 4 8 12 16 20 24
Time (hours) Time (hours)

Fig. 4. Mortality during post-haul holding; Experiment 1, low alkalinity process

Fig. 5. Water quality in Experiment 2, high alkalinity process water. (A. dissolved
water.
oxygen; B. carbon dioxide; C. pH; D. total ammonia nitrogen; and E. un-ionized
ammonia.)
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58 53

100% 40

O 2 or CO2 (mg/L)
7A. Dissolved Oxygen and Carbon Dioxide
Cumulative Mortality (%)

35
80%
30

60% 25 Dissolved Oxygen

Air, Tank 1 Air, Tank 4

Mixed, Tank 2 Mixed, Tank 6 20 Carbon Dioxide

40% Oxygen, Tank 3 Oxygen, Tank 5 15
0 12 24 36 48 60 72
20% 9.0
7B. pH
8.5
0% 8.0
0 24 48 72 96 120 144 168

pH
7.5
Time (hours) 7.0
6.5
Fig. 6. Mortality during post-haul holding; Experiment 2, high alkalinity process
6.0
water. 0 12 24 36 48 60 72
150
7C. TAN
significantly different for dissolved oxygen, carbon dioxide, and pH. 125

TAN (mg/L)
For UIAN, both Oxygen and Mixed were significantly different from 100
75
the Air treatment.
50
Post-haul mortality over time is presented in Fig. 6. At the end of
25
the haul, there were three mortalities in the Air treatment (Tank 1).
0
Mortality in the Air treatments was significantly higher during the 0 12 24 36 48 60 72
post-haul period than in the Oxygen treatment (Chi-squared = 116,

Conductivity (mS/cm)
11.0
7D. Conductivity
df = 3, P < 0.0001) or the Mixed treatment (Chi-squared = 117, df = 3,
10.5 Tank 1
P < 0.0001). At the end of the 7-day post-haul period, there were
27 and 34 mortalities in the Air treatments and only seven other 10.0
Tank 2
mortalities in the other four tanks. The impact of aeration on prod- 9.5
uct quality is presented in Table 5. The number of non-saleable fish
9.0
was significantly increased in the Air treatment due to increased 0 12 24 36 48 60 72
mortality and the level of fish with “minor damage” was greater in Time (hours)
Experiment 2 (27% of fish) compared to Experiment 1 (11% of fish).
Fig. 7. Water quality in Experiment 3, low alkalinity process water. (A. dissolved
oxygen and carbon dioxide; B. pH; C. total ammonia nitrogen; and D. conductivity.)
4.3. Experiment 3 – 3 day transport, pure oxygen

The initial and final mean alkalinity were 1.20 ± 0.01 and
6.19 ± 0.31 meq/L, respectively. The water quality during the sim- 26
ulated haul is presented in Fig. 7A–D for dissolved oxygen, carbon 24
dioxide, pH, total ammonia nitrogen, conductivity, and Table 4 for
22
temperature. The impact of aeration on dissolved oxygen and car-
Temperature (C)

20
bon dioxide was similar to Experiment 1. During the first 8 h, pH
18
decreased and then slowly rose over the rest of hauling period
(Fig. 7B). TAN (Fig. 7C) and conductivity (Fig. 7D) showed a linear 16

increase with time. Conductivity increased over the 3 days simu- 14

lated haul by 0.560 and 0.898 mS/cm for Tanks 1 and 2, respectively. 12 Oxygen Mixed Air

10
4.4. Experiment 4 – impact of aerator type on water temperature 0 200 400 600 800 1000 1200 1400
Time (Minutes)

Ambient water temperature for the three aeration types tested Fig. 8. Variation of ambient water temperature for the three aerators tested in
in Experiment 4 is presented in Fig. 8. Aeration type had a signifi- Experiment 4. The Oxygen and Mixed treatment data plot as a single curve. No
cant impact on water temperature (F = 2346, P < 0.0001). The mean fish were present.
decrease in water temperature was significantly larger for the Air
treatment (−14.13 ◦ C) compared to either the Mixed (−4.37 ◦ C) or
Oxygen (−4.28 ◦ C) treatments. During the experiment, ambient air
9.0
temperature was 12.3 ± 1.0 ◦ C.
Dissolved Oxygen (mg/L)

8.5
4.5. Experiment 5 – bacterial oxygen uptake and oxygen transfer
8.0
coefficient
7.5
For a given sample, the bacterial oxygen uptake of the hauling
7.0 Tank 4, Oxygen
water was linear with time (Fig. 9). For both Experiments 1 and 2,
Tank 5, Mixed
the oxygen uptake was highest for Air, intermediate for Mixed, and 6.5
Tank 6, Air
lowest for Oxygen. Type of aeration had no impact in Experiment
6.0
1 (F = 2.98, P < 0.1938) but had a significant impact in Experiment 0 5 10 15 20 25 30
2 (F = 47, P < 0.0054). In Experiment 2, the Air treatments were Time (Minutes)
significantly different from both the Oxygen and Mixed treatments.
In Experiment 3, the bacterial oxygen uptake rose up to 28 h, Fig. 9. Representative oxygen uptake curves for Experiment 1.
decreased from 28 h to approximately 48 h, and then rose again
54 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58

Table 7
Initial, final, and change in conductivity in Experiment 2a .

Treatment Initial (mS/cm) Final (mS/cm) Change (uS/cm) Mean change (uS/cm)

Air 10.86 11.26 400 475 ± 106b

10.10 10.65 550

Mixed 10.40 10.43 30 68 ± 54a

9.914 10.02 106

Oxygen 10.45 10.43 −20 27 ± 66a

9.817 9.890 73

Mean ± SD 10.26 ± 0.39 10.45 ± 0.49

a
Values within rows sharing a common superscript letter were not significantly different (Bonnferroni’s multiple comparisons test, P < 0.05).

Foam Production Table 8

5 Comparison of pH values for Experiments 1 and 2.
Oxygen Uptake (mg/hour)

Tank #1 Treatment Alkalinity (meq/L)

4
1.74 ± 0.11(Experiment #1) 8.84 ± 0.55(Experiment #2)
3 Oxygen 6.896 ± 0.383 7.578 ± 0.265
Final Sample - Frozen Mixed 7.290 ± 0.330 7.797 ± 0.234
2 Air 7.936 ± 0.155 8.476 ± 0.176
Tank #2
1
100%
0 Experiment 1
0 12 24 36 48 60 72 18 days 32 days Total Mortality (%) 80% Experiment 2
Time (hours)
60%
Fig. 10. Oxygen uptake in Experiment 3; values reported at a standard temperature r2 = 0.979
of 20 ◦ C (mg/h). 40%

20% r2 = 0.965
for the rest of the haul (Fig. 10). The oxygen uptake of the frozen
samples was comparable to, or larger than, the fresh sample at the
0%
end of the haul (72 h). Over the 72-h simulated haul, ˛O2 (Eq. (5)) 0 200 400 600 800 1000 1200 1400 1600 1800
decreased to approximately 73% of the clean water value (Fig. 11). Final Un-ionized Ammonia Nitrogen (ug/L)
Freezing did not appear to change the measured ˛O2 .
Fig. 12. Final mortality for Experiments 1 and 2 vs. final un-ionized ammonia.

5. Discussion
The only parameter that exceeded its action level (Section 2.1)
5.1. Impact of aerator type on water quality
was un-ionized ammonia, although dissolved oxygen was close for
some of the Air treatments. Aerator type had a significant impact on
The aerator treatments (Oxygen, Mixed, and Air) resulted in dis-
the final UIAN in both experiments. The final UIAN concentrations
tinct variations in dissolved oxygen and carbon dioxide levels. The
were significantly higher for Air compared to either Mixed or Oxy-
concentrations of these two gases were highest in the Oxygen treat-
gen. There was a highly significant linear relationship between the
ment, intermediate in the Mixed treatment and lowest in the Air
final mortality during the post-haul period and un-ionized ammo-
treatment (Figs. Fig. 33A,B and Fig. 55A,B). The Mixed treatment
nia concentration at the end of the haul (Fig. 12). Final mortality
maintained a relatively high dissolved oxygen concentration (21
was also positively correlated with oxygen uptake (Fig. 13). For
and 25 mg/L) while reducing the carbon dioxide concentration (9
transportation of Nile tilapia, the limiting water quality parameter
and 13 mg/L). The resulting pH was higher in the high alkalinity
appears to be un-ionized ammonia. No clear indication of problems
experiments compared to the low alkalinity treatments (Table 8).
with low pH or high carbon dioxide was found in these experi-
These changes are due to differences in SOTR between the two types
ments. Lower pH and higher carbon dioxide are routinely observed
of aerators used, oxygen content of the gases, and gas flowrates.
in commercial tilapia hauls (Colt et al., 2011).

110%
100%
100%
Tank #1 Final Sample - Frozen
80%
Mortality (%)

90%
Alpha

y = 0.136x - 0.051 r2 = 0.809

60%
80%
40%
70%
Tank #2 20%

60%
0 24 48 72 4 days 11 days 18 days 32 days 0%
Time (hours) 0 2 4 6 8
Oxygen Uptake (mg/hour)
Fig. 11. Oxygen transfer coefficient (KL a); values reported at a standard temperature
of 20 ◦ C (1/min). Fig. 13. Final mortality for Experiments 1 and 2 vs. oxygen uptake rate.
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58
Cod (Gadus morhua) are intolerant of pH below 7.0 (Treasurer,
2012) and buffer addition or aeration with air is needed to maintain
pH and prevent mortalities. Turbot appear to be more tolerant of
lower pH than cod and mortalities were recorded at un-ionized
ammonia concentrations above 400 ␮g/L (Grøttum et al., 1997).
The limiting water quality parameters may also depend strongly on
species, water temperature, hauling density, and hauling duration;
extrapolation to different conditions must be made with care.
Under the conditions used in these experiments, the Oxygen and
Mixed treatments produced very high survival. The Mixed treat-
ment may offer some advantages, if necessary, to reduce the carbon
dioxide concentration while maintaining moderately high DO con-
centrations. Significant mortality occurred in the Air treatment and
is related to high un-ionized ammonia concentration, excessive
scale loss, or lower dissolved oxygen concentrations. Under winter
conditions, heat losses could also be an important consideration.
Except for very short hauls or special conditions, aeration with air
should be avoided.
5.2. Impact of alkalinity on water quality
Concentrations of dissolved oxygen and carbon dioxide were
similar between the two alkalinities tested (Figs. Fig. 33A,B and
Fig. 55A,B). Changes in alkalinity had a significant impact on pH
(Figs. Fig. 33C and Fig. 55C), un-ionized ammonia (Figs. Fig. 33D
and Fig. 55D) and mortality (Table 5). These differences were more
pronounced in the Air treatments because of the resulting higher
pH. For higher densities or longer hauling durations, it may prudent
to limit initial alkalinities to less than 4 meq/L (or less) and carefully
adjust oxygen flow to control carbon dioxide stripping and pH.
5.3. Variation of oxygen and carbon dioxide during transportation
Comparison of dissolved oxygen and carbon dioxide with time
(Figs. Fig. 33A,B and Fig. 55A,B) indicate that a large change in gas
concentrations occurred during the first 2 h of hauling. Dissolved
oxygen concentrations were relatively constant for the remainder
of the haul, except with the Air treatments (Fig. 5A). This is in con-
trast with carbon dioxide, where there was a general rise in carbon
dioxide over time in all three experiments. This increase is espe-
cially prominent in the Air and Mixed treatments from Experiments
#2 (Fig. 5B) and #3 (Fig. 7A).
It is generally assumed that the oxygen consumption of fish (R),
is elevated following handling and decreases during the haul to
a basal rate. In the transport of adult Atlantic salmon, Farrell et al.
(2010) found that oxygen uptake was elevated from 1.4 to 2.9 × the
basal rate. Up to 6 h were needed for the oxygen uptake to return to
a basal rate. From Eqs. (5 and 6), a reduction in the respiration rate
should increase both dissolved oxygen and carbon dioxide assum-
ing that the ˛ values remain constant. The consistent decrease in
dissolved oxygen for the Air treatment in Experiment 2 (Fig. 5A)
may be due to the impact of mucus on ˛ (see following discussion
in Sections 5.4 and 5.5). More research is needed to evaluate the
impact of aeration type on oxygen uptake and the oxygen transfer
coefficient.
5.4. Variation in bacterial oxygen uptake
Aerator type also had significant impact on bacterial oxygen
uptake, conductivity, and color. In both Experiments 1 and 2, the
Air treatment had the highest bacterial oxygen uptake and Oxygen
treatment was the lowest (Table 9). There was significant linear
increase in conductivity over the simulated haul (Fig. 7C). The tint
of the hauling water was visibly darker in the Air treatments.
These changes may be related to production and loss
of mucus. Mucus is produced by goblet cells and consists
55
Table 9
Oxygen uptake at the end of haul for Experiments 1 and 2a . Uptake is expressed in
mg oxygen/hour adjusted to a base temperature of 20 ◦ C.
Treatment Exp. #1 Exp. #2
Air 2.45 ± 1.81a 6.39 ± 0.82b
Mixed 0.390 ± 0.197a 2.27 ± 0.25a
Oxygen 0.0934 ± 0.00266a 1.06 ± 0.51a
a
Values within columns sharing a common superscript letter were not signifi-
cantly different (Bonnferroni’s multiple comparisons test, P < 0.05).
primarily of glycoprotein but also contains lysozyme, immunoglob-
ulins, complement, carbonic anhydrase, lectins, crinotoxins,
calmodulin, C-reactive protein, pheromones, and proteolytic
enzymes (Shephard, 1994). Most glycoproteins are acidic and act as
ion exchangers. Adverse water quality often leads to the production
of mucus that is released to the water. Mucus has been thought to be
involved in osmotic and ionic regulation and it is hypothesized that
the buildup of mucus on body surfaces contributes to the ability of
freshwater fish to retain electrolytes (Shephard, 1994).
The very high flow rate in the Air treatment resulted in much
higher turbulence levels than in the other two treatments. This
increased turbulence may be responsible for the increased (a) loss
of mucus from the fish, (b) conductivity, (c) color, and (d) bacterial
oxygen uptake.
The decrease in bacterial oxygen uptake in Experiment 3
(Fig. 10) that occurred from 36 to 48 h may have been due to foam
production, which would have removed of surface-active com-
pounds and bacteria. In Experiments 1 and 2, foam production was
stronger in the Air treatments but would typically stop before the
end of the trial. The foam filled the head space in the tank and
spilled out the 8 cm access port and onto the floor. There was less
foam production in the Mixed and Oxygen treatments, it started
later than in the Air treatment. The oxygen uptake in the previ-
ously frozen samples appears to be slightly higher than the fresh
samples, so the reported values for Experiments 1 and 2 may be
slightly over-estimated.
5.5. Oxygen transfer coefficient and alpha
 
In Experiment 3, the mean alpha value ˛O2 decreased from
approximately 88% at 24 h to 72% at 72 h (Fig. 11). A decrease in ˛O2
will reduce dissolved oxygen (Eq. (5)) and an raise carbon dioxide if
˛CO2 = ˛O2 . Additional research is needed to document the changes
in ˛O2 and ˛CO2 during hauling and impact on dissolved oxygen and
carbon dioxide concentrations.
5.6. Comparison of simulated hauls with commercial hauling
conditions
The hauling protocols used in this work did not attempt to
simulate the sound, vibration, water movements, or temperature
changes that occur in commercial transport. In addition, it is likely
that the fish used in these simulated hauls were treated more gen-
tly than is typical of many commercial haulers (see Colt et al., 2011
for description of loading procedures). While these differences may
have a significant impact on product quality and long-term survival,
they should have little effect on water quality during transport.
The hauling water was also aerated with pure oxygen for 1 h prior
to loading to reduce the risk of hypoxia during loading. Not all of
the commercial haulers pre-aerate their hauling water. The mean
water quality parameters in Experiments 1 and 2 are compared
to the two commercial haulers reported by Colt et al. (2011) in
Table 10.
Dissolved oxygen and TAN values are comparable for the
two systems. The carbon dioxide concentrations in commercial
56 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58

Table 10 5.7. Impact of aeration on water temperature

Comparison of mean final water quality for Experiments 1 and 2 (Oxygen treatment
only) with commercial haulers (Table 3, Colt et al., 2011).
The use of large volumes of cold air significantly reduced the
Parameter Simulated hauling Commercial haulers water temperature in the simulated hauling tanks for the Air treat-
Exp. 1 Exp. 2 Hauler A Hauler B ment (Fig. 8). The resulting water temperature would be lethal to
tilapia over a 24-h haul. During more moderate air temperatures,
DO (mg/L) 32 32 31 25
CO2 (mg/L) 23 21 46 57 the use of Air could lower the water temperature enough to shock
TAN (mg/L) 25 22 17 26 fish when they are transferred to the retail holding facilities.
UIAN (␮g/L) 86 62 20 55 The water–air turnover rate (volume of water/gas flow rate) was
pH 7.9 8.6 6.5 6.8 equal to
O2 flow (kg fish/lpm O2 ) 13 14 82–136 91–136
Treatment Turnover (minute)

Oxygen 76
Mixed 11–14
Air 0.53–0.61

tanks were approximately double that found in the simulated
hauling system. This reduced the pH, which resulted in lower un-
ionized ammonia concentrations in the commercial tanks. On a fish
mass/oxygen flow basis, 6–10× more oxygen was used in this study
than used by commercial haulers (Table 11). While the commer-
cial haulers used similar fine bubble diffusers, greater water depths
(2–3 times the depth of simulated hauling system) resulted in much
higher SOTR rates and transfer efficiencies (Boyd and Moore, 1993).
The temperature reduction during simulated transport was
less than that which occurs during some seasons with com-
mercial haulers (Colt et al., 2011). Temperature control was
necessary in the simulated hauls because cooling in the Air
treatments would have lowered the water temperature below
the action levels. Larger hauling tanks with better insulation
may also reduce temperature changes in commercial tanks. The
largest potential temperature change in the commercial trans-
port occurs at the end of trip when fish are transferred to
retail holding tanks, rather than the temperature changes en
route.
The pH changes during the simulated hauling experiments
are compared with data from commercial farms and haulers
in Table 11. The final pH in the Oxygen-Low Alkalinity treat-
ment is comparable with the final pH from commercial transport.
The final pH Mixed-Low Alkalinity and Air-Low Alkalinity treat-
ments are higher than the final pH from commercial transport.
The final pH of the High Alkalinity treatments is much higher
than that found in commercial hauling. The large increases in
pH from pre-haul to post-haul are comparable for the two
systems. In contrast, commercial transport exhibits a greater down-
ward shift in pH because of the low pH in some retail holding
systems.
Lower gas flow rates are possible in commercial hauling tanks
because of the impact of water depth on SOTR (Boyd and Moore,
1993). The use of air aeration is unlikely to have much application
in this type of fish transport unless temperature control systems
are used or hauls are very short.
5.8. Impact of TAN and phosphate excretion on alkalinity and pH
estimates
Excreted TAN and phosphates significantly raise the computed
alkalinity during the simulated hauls (Table 12). The majority of the
theoretical increase in alkalinity was due to excreted TAN (94%). In
most cases, the computed final alkalinity was higher than the mea-
sured final alkalinity (Table 12, Fig. 14). Reported alkalinities were
based on unfiltered samples. In Experiment 1, filtration through
0.45 ␮m glass fiber filters raised the alkalinity by only 8 ± 4%. Based
on visual observations, filtration did not appear to remove much of
the mucus from these samples.
The increase in alkalinity from TAN and phosphates results in
a slow raise in the pH from Hour 12 to the end of the haul. This is
especially evident in Experiment 3 where the final measured alka-
linity was 416% of the initial alkalinity. In very low alkalinity waters,
it may be necessary to raise the alkalinity to limit this pH shift.
Methods for accurate measurement of alkalinity in the presence
of high TAN, phosphate, and mucus levels remain to be determined.
Accurate prediction of pH will require water quality models that
consider the reactions of carbonate compounds, ammonia, phos-
phates, and mucus. Thus carbonate-only models such as CO2SYS
(Lewis and Wallace, 1998) are unlikely to give accurate results.

Table 11
Variation in pH during the hauling process and comparison with commercial hauling conditions (Colt et al., 2011).

Experiment Treatment/Farm Pre-haul holding Simulated hauling Post-haul holding Maximum pH increase Maximum pH decrease

Start +1 h Final

1 Oxygen 7.42 8.23 6.92 6.82 7.77 +0.81 −0.50

Mixed ¨ 8.38 7.34 7.23 ¨ +0.94 −0.19
Air ¨ 8.34 7.84 7.95 ¨ +0.92 None

2 Oxygen 8.14 8.53 7.59 7.57 8.53 +0.39 −0.57

Mixed ¨ 8.54 7.88 7.63 ¨ +0.40 −0.51
Air ¨ 8.52 8.63 8.26 ¨ +0.49 None

3 Oxygen 7.65 6.58 6.53 7.01 7.49 None −1.12

Commercial Farm ID Farm Commercial hauling Retail holding Maximum pH increase Maximum pH decrease

Start +1 h Final

A 7.38–8.71 8.13–8.14 6.88–6.92 6.41–6.51 6.73–7.49 +0.75 −2.30

B 7.21–7.58 7.64–7.69 6.73–6.86 6.73–6.95 4.21–7.44 +0.48 −3.00
C 7.56–7.79 – – – – – –
D 7.89–8.12 – – – – – –
 J. Colt, E. Kroeger / Aquacultural Engineering 55 (2013) 46–58 57

Table 12
Contribution of ammonia and phosphate to alkalinity in Experiments 1–3.

Exp. # Treatment Tank Initial Alk (meq/L) Final TAN (meq/L) Final P (meq/L) Final Alk (measured) Final Alk (computed) Mean final Alk (computed)

1 Oxygen 2 1.76 2.09 N/A 2.46 3.85 3.74

4 1.78 1.84 N/A 2.04 3.62
Mixed 1 1.60 1.73 N/A 2.19 3.33 3.43
5 1.83 1.69 N/A 2.24 3.52
Air 3 1.74 1.69 N/A 2.83 3.43 3.29
6 1.75 1.41 N/A 2.79 3.16

2 Oxygen 3 8.91 1.77 N/A 9.26 10.68 10.58

5 9.02 1.46 N/A 9.14 10.48
Mixed 2 9.29 1.57 N/A 8.51 10.86 10.79
6 9.16 1.56 N/A 9.14 10.72
Air 1 8.42 1.93 N/A 10.47 10.35 10.13
4 8.23 1.68 N/A 11.26 9.91

3 Oxygen 1 1.21 9.15 0.55 6.46 10.90 10.77

2 1.19 8.90 0.55 5.92 10.63

12 product quality may be less than the impact from (a) physical dam-
age resulting from loading and un-loading and (b) physiological
problems resulting from pH and temperature shock during transfer
10 to retail holding systems, especially for fish of reduced fitness. The
hauling protocols documented in this article can be used as a base-
Computed Final Alkalinity (meq/L)

line to further improve survival and product quality of transported

tilapia.
8

Acknowledgments
6
This project was supported by Western Regional Aquaculture
Center Grant no. 2008-38500-19230 from the USDA Coopera-
4 tive State Research, Education, and Extension Service (now the
Experiment 1
National Institute for Food & Agriculture (NIFA)). We would like
Experiment 2 to thank Andrew Dickson, Michael Stenstrom, and Steward Rounds
Experiment 3
for advice on analytical protocols.
2

0
0 2 4 6 8 10
Measured Final Alkalinity (meq/L)
Fig. 14. Comparison of final measured alkalinity with computed alkalinity based on
initial alkalinity plus contribution of alkalinity from final TAN and P concentrations.
6. Conclusions
The Air treatments resulted in lowest carbon dioxide, high-
est pH, and highest un-ionized ammonia concentrations. At high
alkalinities, the Air treatment was unable to maintain adequate
dissolved oxygen levels. Compared to the Air Treatment, the Mixed
treatment resulted in increased carbon dioxide and dissolved oxy-
gen concentrations. The Oxygen treatment resulted the highest
dissolved oxygen, highest carbon dioxide, lowest pH, and lowest
un-ionized ammonia. There was more un-ionized ammonia in the
High Alkalinity treatments because of higher pH.
Significantly increased mortality was observed in the Air treat-
ments in both the Low- and High-Alkalinity treatments. The
mortality in the Oxygen and Mixed treatments was comparable to
that observed in commercial hauling of tilapia. These results indi-
cate that direct hauling mortalities will not be increased at high
alkalinities if pure oxygen aeration or mixed aeration is used.
These experiments were designed to document water quality
and survival under relatively good conditions: low capture and
loading stress, limited pH and temperature shock during trans-
fer, and using fish of exceptional physiological condition. The
potential effect of water quality during hauling on survival and
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