Co2 in Aquaculture

8
CO2 IN AQUACULTURE
PETER VILHELM SKOV1
Technical University of Denmark, Hirtshals, Denmark
1
Corresponding author: pvsk@aqua.dtu.dk
1. Introduction
2. CO2 Sources in Aquaculture
2.1. Fish Metabolism
2.2. Bacterial Metabolism
3. Safe Levels and Welfare Guidelines
4. Free CO2, pH and Alkalinity
4.1. Current Removal and Control Practices
5. Dynamics of CO2 in Aquaculture
6. Effects of Dissolved CO2 on Growth
6.1. Feed Intake and Appetite
6.2. Digestive Function
7. Seawater Transfer
8. Pathological Effects of CO2
8.1. Nephrocalcinosis
8.2. Swim Bladder Inflation
8.3. Cataract Formation
9. Confounding Water Quality Effects
9.1. Hypercapnia and Hypoxia
9.2. Hypercapnia and Metal Toxicity
9.3. Carbon Dioxide and Aluminum
10. Conclusions and Perspectives
References
Aquaculture of fishes is increasingly using recirculation technology to con-

serve water and reduce the environmental footprint from production. Rec-
irculating aquaculture systems (RAS) require an intensification of
production, which gives rise to a number of water quality issues that must
be considered, including the accumulation of carbon dioxide (CO2). This
chapter provides an initial overview on the excretion of CO2 from fish and
287
Carbon Dioxide, Volume 37 Copyright # 2019 Elsevier Inc. All rights reserved
FISH PHYSIOLOGY DOI: https://doi.org/10.1016/bs.fp.2019.07.004
288 PETER VILHELM SKOV
bacterial metabolism, before discussing what is currently being considered as

safe levels, or levels of no-effect, of dissolved CO2 for fish, highlighting some
differences between aquaculture and ecological studies. The correlation
between alkalinity and pH, and the chemical form of CO2 is briefly covered,
before providing an overview of current removal practices and technologies.
The chapter provides an overview of the physiological effects of CO2 in rela-
tion to aquaculture production, specifically feed intake, appetite, digestive
function and growth, before considering the effects of CO2 exposure on the
ability of fish to migrate from fresh to seawater, in light of structural and func-
tional changes in the gills. Finally, some pathologies associated with CO2
exposure, such as nephrocalcinosis and eye cataracts are highlighted, before
closing with an overview of interacting effects between CO2 and other water
quality variables, such as hypoxia or dissolved metals.
1. INTRODUCTION
Aquaculture production of fish, molluscs, and crustaceans has increased

fivefold in volume over the past 3 decades, and has now reached 80 million
tons. Of this, approximately 10 million tons represents finfish farmed in a
marine environment, while 50 million tons is fish farmed in inland production
systems (FAO, 2018). The exact contribution from different production sys-
tems is unknown, but it is fair to assume that inland production is dominated
by pond culture in many parts of the world. In European and North American
aquaculture, the implementation of recirculating aquaculture systems (RAS)
has facilitated part of the growth in aquaculture production in these areas.
Although net pen production has contributed significantly in the growth of
the aquaculture industry, as has been seen in Norway, fish produced in the
marine environment also spend a part of their life on land in RAS facilities
until ready for transfer to sea.
Past and ongoing development of recirculation technology stems from a
multitude of requirements, including eliminating the risk of escapees that
mix with wild populations, reducing nutrient discharge to the environment,
reducing water consumption, and preventing the transmission of disease (from
wild to farmed, and vice versa) (Martins et al., 2010). In addition to this, RAS
presents a possibility to provide fish with a stable environment, in which
physical and chemical water quality parameters can be controlled.
The availability of dissolved oxygen is generally considered the first limit-
ing factor that determines the biomass carrying capacity of an aquaculture
production system (Wurts, 2000). However, oxygen as a limiting factor can
be overcome in land-based aquaculture with the application of pure oxygen
8. CO2 IN AQUACULTURE 289
from either on-site production systems or liquid oxygen (Colt and Watten,
1988), as well as a variety of other aeration technologies. Logically, other
water quality parameters then become limiting in production; which is deter-
mined by the design of the water treatment components in a facility, along
with cultured species, rearing density and temperature. The water quality
parameters of interest to RAS managers, apart from oxygen, include CO2,
pH, dissolved nitrogen (total ammonia nitrogen and nitrite) (Dalsgaard
et al., 2013), and more recently hydrogen sulfide in saltwater systems. Control
of pH can be achieved by adding buffering capacity in the form of alkalinity,
or directly by the addition of base, while a properly dimensioned nitrification
system (biofilter) will efficiently convert ammonia and nitrite to the less toxic
nitrate. Arguably, CO2 can also be removed with the use of degassing technol-
ogy, but such devices are not always installed, and may not be sufficiently
efficient to fully remove CO2.
2. CO2 SOURCES IN AQUACULTURE
2.1. Fish Metabolism

In natural waters, the increase in dissolved levels of CO2 is considered to
originate largely from the absorption from atmospheric air as well as increased
productivity (and subsequent bacterial decomposition) of algae, due to fertilizer
run-off (Sunda and Cai, 2012). In aquaculture production systems, dissolved
CO2 originates from the metabolism of organisms in the system, predominantly
fish, but also from bacterial populations.
Although standard metabolic rates (SMR), the metabolic rate of an inac-
tive individual in an unfed state, have been determined for a wide range of spe-
cies, it is difficult to generalize about the oxygen consumption rates (Ṁ O2) to
fish in aquaculture systems, due to contributions from routine activity (routine
metabolic rate, RMR) and feeding (specific dynamic action, SDA). However,
tank based measurements from a number of species are available in the litera-
ture. For tilapia (Oreochromis niloticus) reared at 28°C, routine metabolic rates
(RMR) range from 5.3 to 7.0 mmol O2 kg1 h1, while peak O2 requirements
during feeding range from 9.9 to 15.4 mmol O2 kg1 h1 (Skov et al., 2017). For
rainbow trout (Oncorhynchus mykiss) reared at 15 °C, RMR may vary between
4.9 and 6.4 mmol O2 kg1 h1, with peak O2 requirements between 6.8 and
10.0 mmol O2 kg1 h1 (Skov et al., 2015). For Atlantic salmon (Salmo salar)
reared at 12 °C, RMRs are in the range of 5.0–7.5 mmol O2 kg1 h1 (Atkins
and Benfey, 2008). These values are likely to represent the range of ṀO2 for
cultured fish, and can thus be used to estimate CO2 excretion rates. Since fish
do not exclusively consume oxygen to satisfy their SMR and feeding metabo-
lism, but also for swimming and other routine activity, the true oxygen
requirements for fish in aquaculture may be higher. For a more detailed insight
into O2 consumption rates and correlation with culture conditions, Forsberg
(1994) provided a detailed model taking into account growth trajectories, tem-
perature, swimming speed and feeding rates. Assuming a mixed substrate utili-
zation with equal contribution from fat and carbohydrate, fish would have a
respiratory quotient of 0.85, meaning that fish excrete 0.85mol CO2 for every
mole of O2 consumed. As such, the corresponding CO2 excretion for the oxygen
consumption rates outlined above would be in the range between 4.2 and
13.1 mmol CO2 kg1 fish h1. As rearing densities in intensive aquaculture pro-
duction may exceed 50 kg of fish per m3 of water, this means that the CO2 excre-
tion of fish may add up to 0.7 mmol CO2 h1 to every liter of water.
2.2. Bacterial Metabolism

Feeding and activity of fish in aquaculture leads to the excretion of large
amounts of nitrogenous waste to the water, predominantly in the form of
ammonia, with smaller contributions from urea and other nitrogenous com-
pounds (Wood, 2001). In RAS, unlike net pens, this necessitates nitrification
of ammonia into nitrite and then nitrate, a process which is achieved by bio-
filters, in which media with a high specific surface area are used to culture bio-
film, ideally comprised of nitrifying bacteria. As a rule of thumb, a biofilter
should have a specific surface area of 100 m2 per kg feed used. Stoichiometry
dictates that for every mole of ammonia nitrogen nitrified, 2 mols of oxygen
are consumed. Actual O2 consumption rates may be higher, and we have
shown that biofilters that are not substrate or oxygen limited may consume
0.85 mmol O2 m2 h1 (P.V. Skov et al., unpublished observation). At equi-
molar CO2 excretion rates, feeding fish a 2% ration under the conditions
described for fish above, this would contribute an additional 0.085 mmol
CO2 L1 h1.
In addition to the oxygen consumption of fish and biofilter, comes a
smaller contribution from the biological O2 demand (BOD) of bacteria in
the water. This rarely exceeds 0.3 mmol O2 L1 day1 (Dalsgaard and
Pedersen, 2011), and thus makes only a moderate contribution to dissolved
CO2, although in some production systems, the breakdown of particulate
organic matter in the system may contribute on a more significant level.
3. SAFE LEVELS AND WELFARE GUIDELINES
Sensitivity to dissolved CO2 is likely to be highly species specific, and esta-

blishing generally applicable safety levels is not possible. As such, there are no
unequivocally accepted guidelines for safe levels of dissolved CO2 in
aquaculture systems. The majority of research on safe levels of CO2 in aqua-

culture systems has been conducted on salmonids, and even within this species
group, it has been difficult to find consensus. Colt (1991) proposed a division
between cold- and warm water fishes, with CO2 thresholds of 10–20 mg L1
and 20–40 mg L1, respectively, with no apparent distinction between species
or life stage. Currently, there are few countries with regulations or guidelines
on this issue. Norway has set a threshold for dissolved CO2 at 15 mg L1
(FOR, 2004) while the UK has set a more conservative threshold at 10 mg L1.
The latter is in good agreement with no effect concentrations of dissolved CO2
found for Atlantic salmon in both sea- and freshwater (Fivelstad et al., 1998;
Khan et al., 2018; Noble et al., 2012).
The tolerance levels of salmonids to dissolved CO2 are largely based on
data relating to production variables, such as specific growth rates, feed con-
version efficiencies, and condition factors. The levels at which animal welfare
may become compromised has received little attention, although it is known
that already at PCO2 levels below 0.15 kPa a range of fishes show changes in
their sensory ability, cognition, behavior, and learning abilities (Heuer and
Grosell, 2014, and references therein, but see also Chapter 5, Vol 37: Heuer
et al., 2019; Chapter 9, Vol 37: Munday et al., 2019).
As highlighted by Ellis et al. (2017), tolerance studies in aquaculture are typ-
ically characterized by the use of control treatment groups that experience CO2
concentrations well above saturation levels, and this may represent a bias in data
interpretation. In the absence of operational welfare indicators for fish, such
studies are limited to investigating conventional stress response parameters, such
as changes in plasma cortisol and glucose concentrations (Petochi et al., 2011),
which are poor indicators of chronic stress in fishes (Aerts et al., 2015).
4. FREE CO2, pH AND ALKALINITY
In aquaculture production systems, it is most common to refer to the

amount of CO2 present in free form, as dissolved CO2 L1, rather than
expressing CO2 in partial pressures. Measurement of CO2 in water can be done
using a CO2 electrode, which is an infrared gas analyzer situated behind a gas
permeable membrane, or by coulometric titration. The former is a more prac-
tical approach for fish farmers, allowing for a direct assessment of the levels of
CO2 in a system, without having to measure temperature, alkalinity and pH of
the water. In this chapter, the originally reported CO2 concentrations or par-
tial pressures are used throughout the text, but to facilitate comparison, Fig. 1
shows a correlation between partial pressures and dissolved CO2 levels for
freshwater and saltwater at three different temperatures.
Fig. 1. The correlation between partial pressure of CO2 and dissolved CO2 concentrations, and the
effects of temperature (black 5 °C, red 15 °C, green 25 °C). Solid lines represent fresh water, while
dotted lines represent seawater.
As CO2 is introduced into the water from the metabolism of fish and bac-
teria, it becomes hydrated to form carbonic acid, H2CO3. In all practical
senses, this is still considered part of the free CO2 pool, as it can rapidly be
dehydrated and revert back to free CO2 gas. Once dissolved in water,
H2CO3 can further enter into the carbonic acid-base equilibrium, where it
may donate one or two protons, to become either bicarbonate (HCO 3 ) or car-
bonate (CO2 3 ). The concentration of dissolved inorganic carbon (DIC) in
water constitutes the sum of carbonic acid, bicarbonate, and carbonate,

½DIC ¼ ½H2 CO3 + ½HCO3 + CO3 2 :
Ultimately, the chemical form of inorganic carbon dissolved in water, is
determined by the pH of the water. Alkalinity, the stoichiometric sum of bases
in a solution, is the main determinant of the pH buffering capacity of water, of
which carbonate alkalinity makes up the main part. Carbonate alkalinity is
consumed by nitrifying bacteria, while, at the same time, protons are liberated
from the conversion of ammonia to nitrite. Therefore, it is necessary to add
alkalinity, typically achieved by the use of various hydroxide, carbonate or
lime complexes. Due to the pH buffering capacity of alkalinity, this also means
that in waters with different alkalinities, the absolute amount of dissolved
inorganic carbon present as free CO2 at a given pH value will increase with
increasing alkalinity. For most water sources, the amount of DIC present
as free CO2 at pH 7–8, ranges from 1 to 10%, but below pH 7, CO2 quickly
becomes the dominant carbon species, and at pH 5, nearly all inorganic car-
bon is present as CO2.
4.1. Current Removal and Control Practices

The necessity of a dedicated CO2 stripper or degassing unit depends largely
on the oxygenation method in a given aquaculture facility. In facilities that use
air for the oxygenation of water, CO2 removal is achieved as a by-product, due
to the large volumes of air delivered to the system. On the other hand, in
facilities that use pure oxygen injection systems, there will be a build-up of
CO2 that requires removal. In intensive systems, the concentration of CO2
in the water is much higher than in the ambient air. The air that is brought
into contact with water during degassing quickly becomes laden with CO2.
An example of this is given by Summerfelt et al. (2000), showing that the
CO2 concentration in the outlet air from degassing may reach 6–8000 ppm.
This rapid increase in CO2 reduces the transfer gradient and impairs removal
efficiency. With the use of airlift pumps, Moran (2010b) showed that with an
incoming CO2 concentration between 10 and 40 mg L1, the maximum
obtained removal efficiency did not exceed 40%, even at considerable flow
rates. Efficient removal of CO2 from water therefore requires that degassers
operate at gas to liquid (G:L) ratios as high as 10:1 in order to function
efficiently (Summerfelt et al., 2000). Such high G:L ratios make it difficult
to perform subsurface aeration, and instead, degassing facilities frequently
use surface aeration, where water is brought into contact with air, rather than
vice versa. This is achieved by the use of cascade columns or packed columns,
where water is pumped to height and allowed to trickle down through perfo-
rated plates or a packed media, while air is delivered via the bottom to achieve
a counter current flow. Using this approach enables removal efficiencies as
high as 75–85% even at moderate CO2 concentrations (Moran, 2010b;
Summerfelt et al., 2000). The efficiency of these degassing installations are
influenced by the height of the degassing unit (contact time) and design of
the packing media, and have been reviewed in a number of publications
(Colt et al., 2012a,b; Moran, 2010a,b; Summerfelt et al., 2000).
Reportedly, CO2 control is occasionally also achieved by chemical means
by increasing alkalinity of the water. This has multiple effects, one being that a
higher pH of the water drives a larger fraction of the inorganic carbon into
bicarbonate or carbonate. The other is that this allows for an overall increase
in the total amount of dissolved inorganic carbon that can accumulate in the
system. This means that for any given decrease in water pH, the amount of
dissolved CO2 increases proportionally with alkalinity (Loyless and Malone,

2004), and furthermore introduces the risk that in the face of quite modest
decreases in pH considerably more free CO2 can be mobilized.
In the aquaculture of European eel (Anguilla anguilla) efficient CO2
removal is achieved by maintaining the majority of dissolved inorganic carbon
in the form of CO2. RAS facilities for eel control water pH in a range between
5.8 and 6.5. This ensures that close to all DIC is present as free CO2 and
facilitates efficient degassing.
5. DYNAMICS OF CO2 IN AQUACULTURE
Daily variations in levels of dissolved CO2 are likely to be largely dependent

on the type of aquaculture operation, the biomass of fish in the system, feeding
levels and implementation of water treatment installations. Degassers will obvi-
ously play a large role in CO2 dynamics in a facility, but also the type of aeration
used, if any, as well as sludge removal practices. Recirculating facilities, despite
considerable variation in design and specifications, have a great deal of control
over dissolved gases. While dissolved CO2 concentrations may be generally ele-
vated above saturation levels, they tend to be stable. For example, in Danish
trout farms which are constructed as outdoor concrete raceways with fish den-
sities up to 50 kg m3, CO2 levels rarely exceed 10 mg L1, and daily fluctuations
are typically <2–3 mg L1 (personal observation). These facilities operate with-
out dedicated degassers, but instead employ the airlift principle to achieve oxy-
genation of water and for water circulation throughout the farm. The airlift
principle is based on delivering air at depth via diffusors located at the bottom
of a 2–4 m deep U-tube interconnecting rearing units. The addition of air
reduces the overall mass of water on one side of the tube, enabling gravity
to push water through the tube. Although large volumes of air are delivered,
G:L ratios are still modest, but a large number of airlifts are positioned through-
out the farm, which ensures the removal of a large proportion of CO2.
RAS facilities that are structurally enclosed typically use pure oxygen, or a
combination of oxygen and air, for subsurface oxygenation. In this type of
facility a dedicated degasser is required to maintain acceptable levels of dis-
solved CO2.
6. EFFECTS OF DISSOLVED CO2 ON GROWTH
There is a great deal of evidence showing that high levels of dissolved CO2
have a negative impact on growth of fishes. This has been demonstrated for
several species in aquaculture production, predominantly Atlantic salmon

(Fivelstad et al., 2003a,b, 2007, 2018, 1998, 1999; Hosfeld et al., 2008;
Khan et al., 2018; Gil Martens et al., 2006; Mota et al., 2019), but also in rain-
bow trout (Oncorhynchus mykiss) (Danley et al., 2005; Good et al., 2010;
Smart et al., 1979), Atlantic cod (Gadus morhua) (Foss et al., 2006; Moran
and Støttrup, 2011), turbot (Scopthalmus maximus) (Stiller et al., 2015), white
sturgeon (Acipenser transmontanus) (Crocker and Cech, 1996) spotted wolfish
(Anarhichas minor) (Foss et al., 2003) and pike perch (Sander lucioperca)
(Steinberg et al., 2017).
The effects of CO2 on Atlantic salmon performance were reviewed by
Fivelstad (2013). The clear pattern that emerges is that as the severity of
CO2 exposure increases, a reduction in feed intake becomes apparent. Numer-
ous species experience progressive loss in metabolic scope (MS, defined as the
difference between MMR and SMR) as CO2 levels increase (see also
Chapter 6, Vol 37: Lefevre, 2019), but a clear correlation between loss of
MS and reduced appetite is lacking. Recently, Khan et al. (2018) found that
in Atlantic salmon, there is a 35% loss in MS at 20 mg CO2 L1, and a 55% loss
in MS at 30 mg L1, compared to control levels, originating from an inability
to uphold maximum metabolic rates (Fig. 2). Data on effects of CO2 on
growth in nonsalmonids is given in Table 1. With a few exceptions in gilthead
seabream (Ben-Asher et al., 2013), Atlantic cod (Foss et al., 2006), and rain-
bow trout (Danley et al., 2005), CO2 exposure is accompanied by a decrease in
feed intake. Regardless of feed intake, feed conversion ratios are almost
always negatively affected, although exceptions are found for turbot (Stiller
et al., 2015) and spotted wolffish (Foss et al., 2003), while specific growth rates
are consistently reduced.
Based on data from Atlantic salmon, Fivelstad et al. (2015) established a
model describing the relationship between dissolved CO2 concentrations and
changes in specific growth rate (SGR) (Fig. 3A). From this it is apparent that
there is a critical CO2 level close to 15 mg L1, beyond which growth perfor-
mance quickly becomes compromised. The model is specific to parr. Smolts do
not appear to show a breakpoint in their SGR when exposed to increasing CO2
levels, but rather a linear response (Fig. 3B).
A multitude of other factors are also likely to contribute to reduced growth,
including digestive efficiency, overall cost of living, and increased protein oxi-
dation. In attempts to elucidate the role of these mechanisms, a number of
recent publications have approached the issue from a bioenergetic angle,
examining how dissolved CO2 influences factors such as metabolic rate, met-
abolic scope, and instantaneous fuel use (Khan et al., 2018; Methling et al.,
2013; Steinberg et al., 2018; Stiller et al., 2015). A summation of possible,
although not exhaustive, mechanisms are highlighted in Fig. 4.
Fig. 2. Standard metabolic rate (SMR) (A), maximum metabolic rate (MMR) (B) and the associated
linear depressant effect of CO2 on the difference between MMR and SMR; the metabolic scope (MS)
(C) of Atlantic salmon exposed acutely to different [CO2]. Values are measured as the rate of oxygen
consumption (Ṁ O2) in mg O2 kg1 h1. Modified from Khan et al. (2018).
Table 1
Summary of known effects of CO2 exposure and the corresponding experimental conditions on growth performance investigated for nonsalmon species
Species Size [CO2] T Sal (%o) SGR FCR CF FI Duration Comments Ref
Atlantic cod (Gadus morhua) 80 2 10 35 1.7 – 1.07 – 40 1

8 10 35 1.35 – 1.01 – 40
18 10 35 0.75 – 0.97 – 40
33.4 3–4 10 35 1.13 1.08 0.87 833 TFC 63 5
30.5 4–5 10 35 1.15 0.97 0.87 839 TFC 63
31.8 7–9 10 35 0.98 1.11 0.85 837 TFC 63
32.4 10–12 10 35 0.93 1.18 0.84 845 TFC 63
Arctic charr (Salvelinus alpinus) 380 5 7 0 0.75 1.08 1.04 190 TFC 60 2
350 15 7 0 0.65 1.39 0.99 130 TFC 60
320 21 7 0 0.5 1.86 0.97 90 TFC 60
Gilthead seabream (Sparus aurata) 26.5 5.2 22 35 2.96 1.15 – 25.8 TFC 64 a
3
22.7 20.5 22 35 2.60 1.52 – 27.8 TFC 64
18.9 32.3 22 35 2.14 2.14 – 25.2 TFC 64
14.7 56.3 22 35 1.99 2.25 – 23.6 TFC 64
405 7.2 22 35 0.40 2.77 – 249 TFC 71 3
384 22.2 22 35 0.36 2.87 – 224 TFC 71
294 39.3 22 35 0.28 4.32 – 242 TFC +71
Yellowtail kingfish (Seriola lalandi) 164 0.8 26 35 6.05 0.98 – 3080 TFC 32 b,c
4
153 4.6 26 35 5.77 0.98 – 2820 TFC 32
114 23.8 26 35 4.52 1.26 – 2120 TFC 32
Turbot (Scopthalmus maximus) 160 4.9 18 20 1.5 0.7 3.2 1.3% d1 56 6
130 25.8 18 20 1.3 0.7 2.9 1.2% d1 56
100 41.6 18 20 1.0 0.7 2.75 0.9% d1 56
Pikeperch (Sander lucioperca) 447 4.4 23 0 0.89 1.04 – 0.92% d1 58 7
435 14.7 23 0 0.86 1.14 – 0.98% d1 58
420 29.7 23 0 0.78 1.13 – 0.88% d1 58
(Continued )
Table 1 (Continued)
Species Size [CO2] T Sal (%o) SGR FCR CF FI Duration Comments Ref
Spotted wolffish (Anarhichas minor) 33.1 1.1 6 33 1.0 0.69 0.97 235 TFC 70 8
30.0 18.1 6 33 0.9 0.65 0.90 213 TFC 70
30.7 33.5 6 33 0.9 0.62 0.92 174 TFC 70
21.4 59.4 6 33 0.4 0.71 0.92 62 TFC 70
White sturgeon (Acipenser transmontanus) 8.99 0.5 19 0 2.86 – – – 12
5.57 42 19 0 1.17 – – –
Rainbow trout (Oncorhynchus mykiss) 817 8 15 0 1.65 1.25 – – 155 9
849 24 15 0 1.67 1.25 – – 155
200 12 9 0 0.84 1.41 – – 275 10
200 24 9 0 0.84 1.45 – – 275
135 55 9 0 0.69 1.66 – – 275
650 22.1 13 0 1.05 – – 2.5% 84 11
550 34.5 13 0 0.85 – – 2.5% 84
500 48.7 13 0 0.73 – – 2.5% 84
a
TFC was recalculated from FCR and weight gain.
b
TFC recalculated from individuals feed intake, duration and number of fish.
c
CO2 calculated from pH with eq. 7 in Appendix F in National Academies of Sciences, Engineering, and Medicine (2017).
Size refers to final body mass in the experiments (g); T is temperature in °C; SGR refers to specific growth rate in % BM increase per day; FCR is
feed conversion ration (g feed per g weight gain); CF, Fulton’s condition factor; FI, feed intake as either % of body mass per day or total feed con-
sumption (TFC) over the duration of the experiment, duration of the experiment is in days.
1 Moran and Støttrup (2011); 2 Musa and Thorensen (2013); 3 Ben-Asher et al. (2013); 4 Abbink et al. (2011) 5 Foss et al. (2006); 6 Stiller et al.,
(2015); 7 Steinberg et al. (2017); 8 Foss et al. (2003); 9 Good et al. (2010); 10 Smart et al. (1979); 11 Danley et al. (2005); 12 Crocker & Cech (1996).
Fig. 3. Percentage reduction in mean specific growth rate in Atlantic salmon parr compared to
normocapnic (2 mg CO2 L1) values followed the equation SGR (% day1) ¼1.32 + 0.0158 ∙
[CO2] – 0.00093 ∙ [CO2] (n ¼ 193, r2 ¼ 0.50; P < 0.05), which was indexed to 100 at a CO2 concen-
tration of 2 mg L1 (A), and SGR for smolts, which followed the equation SGR ¼ 0.0095
[CO2] + 1.18 (r2 ¼ 0.99) (B). Panel A modified from Fivelstad et al. (2015) and Panel B from
Khan et al. (2018).
Fig. 4. A schematic illustration of possible mechanisms influencing the growth of fish in response
to CO2 exposure. See text for details.
6.1. Feed Intake and Appetite
Appetite in fishes is regulated by a number of complex pathways under the

influence of intrinsic and extrinsic factors (Volkoff et al., 2009). Factors such
as dissolved oxygen, temperature, and photoperiod have received considerable
attention. A rapid and common response to hypoxia is a decrease in feed
intake (Buentello et al., 2000; Chabot and Dutil, 1999; Pedersen, 1987;
Pichavant et al., 2001), which is considered a strategy to conserve or prioritize
oxygen consumption, and which is typically accompanied by a reduction in
voluntary activity (Chabot and Dutil, 1999; Pichavant et al., 2001). The
responsible mechanisms for appetite suppression has received increasing inter-
est, and Bernier et al. (2012) recently found that in common carp (Cyprinus
carpio) an increased expression of the anorexic hormone leptin, appears to
be the main driver of decreased feed intake during hypoxia.
Surprisingly, comparable studies in which the effects of high levels of dis-
solved CO2 on appetite regulating hormones are absent, despite documented
effects of CO2 on feed intake. It is plausible that changes in oxygen availability

caused by elevated CO2 levels could potentially exert a hypoxia-like effect on
hormones that regulate appetite, considering that high CO2 levels reduce both
the oxygen affinity and oxygen carrying capacity of hemoglobin (Jensen
et al., 1993).
Feeding trials conducted under hypercapnic conditions do not show equiv-
ocal results, and in a number of experiments, determination of feed intake is
not feasible (Khan et al., 2018; Moran and Støttrup, 2011). In turbot, fish
exposed to 41.6 mg CO2 L1 had a daily feed intake that was 40% lower than
for fish reared at 4.9 mg CO2 L1 (Stiller et al., 2015). While appetite hormone
levels were not investigated, the authors found that routine metabolic rates
decreased from 80 to 45 mg O2 kg1 h1, suggesting that at high concentra-
tions of dissolved CO2, oxygen availability becomes compromised, despite
normoxic rearing conditions.
6.2. Digestive Function

A possible reason for the observed reduced feed efficiency and growth dur-
ing exposure to unnaturally high CO2 concentrations may be an associated
reduction in digestive performance, possibly due to a decreased ability to
digest macronutrients or a prolongation of the digestive processes. This could
be caused by stress induced impairment of digestive function, as has been
observed in Arctic charr (Salvelinus alpinus) (Olsen & Ringø, 1999) or redis-
tribution of blood flow from the gastrointestinal tract (Farrell et al., 2002).
Acute exposure to hypercapnia (PCO2 of 20 mmHg) in unfed white sturgeon
causes a short-lived near cessation of gastric blood flow (GBF), and although a
partial recovery did occur, a 40% reduction in GBF persisted for at least
20 min (Farrell et al., 2002). During chronic exposure to CO2 fish show an abil-
ity to recover their GBF over time, and do show differences in comparison to
normocapnic individuals. In these experiments animals were unfed, and while
GBF can be recovered under these conditions, it remains unknown to what
extent CO2 exposure might influence the ability of fish to upregulate GBF
by 60–80% following feeding, which has been shown to be the normocapnic
response in rainbow trout (Eliason et al., 2008).
Evidence from feeding experiments in Atlantic cod and European eel show
that the duration of digestion is prolonged during exposure to hypercapnic
conditions (Methling et al., 2013; Tirsgaard et al., 2015). In Atlantic cod,
the duration of the digestive processes (the specific dynamic action, SDA),
in individuals fed a 5% ration of herring fillet increased from 81 h under
normoxic conditions, to 110 h in fish exposed to a PCO2 of 7 mmHg
(Tirsgaard et al., 2015). Similarly, European eel fed 0.5% of their body mass
with commercial eel pellets, increased the duration of their SDA response from
36 to 44 h (Methling et al., 2013). In both instances, this effect appeared, at

least in part, to be caused by a reduction in metabolic scope, which prevented
fish from obtaining peak Ṁ O2 values comparable to the control groups. While
nitrogen excretion rates during digestion were unaffected in eel, this topic
remains to be determined for Atlantic cod and other species. Interestingly,
the SDA coefficient, which represents the fraction of ingested energy that is
used to fuel the digestive processes, was unaffected. This indicates, that
although digestion is prolonged, it does not appear to become more costly.
These observations are somewhat in contrast to the results of Heuer and
Grosell (2016) who measured oxygen consumption rates in isolated intestinal
tissues in Gulf toadfish (Opsanus beta). Here, an acclimation to moderate
hypercapnia (PCO2 ¼ 1.45 mmHg) resulted in an 8% increase in the surface spe-
cific metabolic rate of the anterior intestine, probably associated with the met-
abolic cost of increased secretion rates of HCO 3 . It is relevant to point out that
the anterior intestine of fishes plays a minor role, if any, in the absorption of
nutrients, so that this increased energy expenditure is unlikely to compromise
digestive function. Furthermore, as pointed out by the authors themselves,
minor increases in the oxygen consumption rates of the anterior intestine
may not be reflected in whole animal metabolic rates (Heuer and Grosell, 2016).
The data provided by Methling et al. (2013) demonstrates the significance
of acclimation to chronically and constant CO2 exposure, as is employed in
most studies. Here, the authors included a treatment group with oscillating
PCO2 levels, from which it was apparent that the magnitude of the SDA
response fluctuated in response to changes in CO2 tension (Fig. 5).
Although acute stress is associated with a decrease in lipid digestibility
(Olsen & Ringø, 1999), this does not appear to be the case during chronic
exposure in acclimated fish. Unpublished data from our lab shows that fish
acclimated to 11, 21, and 36 mg CO2 L1 do not show changes in the digest-
ibility coefficients of lipid and protein. Despite significant changes in the abil-
ity of fish to convert feed to growth (FCR) (Fig. 6A), no significant changes in
the digestion of feed energy were observed (Fig. 6B). The net result of CO2
exposure was a near proportional increase in the gross cost of growth, which
increased from 15.5 kJ g1 in the normocapnic group, to 20.7 kJ g1 in the
highest CO2 exposure group (Fig. 6C).
7. SEAWATER TRANSFER
Anadromous species such as Atlantic salmon undergo a parr-smolt trans-

formation in preparation to seaward migration. This entails a number of phys-
iological and morphological adaptations, which have recently been reviewed
Fig. 5. Postprandial oxygen consumption in A. anguilla exposed to hypercapnia and/or low pH. (A) SDA response in control fish under normocapnic
conditions, (B) under hypercapnic conditions at a constant high PCO2 of 60 mmHg, (C) under oscillating PCO2 conditions from 20 to 60 mmHg, and
(D) in a low pH group (6.5) under normocapnic conditions. Eels were acclimated to hypercapnia/low pH for at least 3 weeks. The meal consisted of 0.5%
BW in commercial feed pellets. Data points are hourly averages s.e.m. (N ¼ 6–8). Straight lines represent standard metabolic rates (SMR) s.e.m. as
determined before feeding. Dotted line in B represents the actual CO2 partial pressures during the postprandial phase. Note how fluctuating CO2 levels
causes opposing fluctuations in Ṁ O2 values in panel B. Modified from Methling et al. (2013).
Fig. 6. Feed conversion, energy digestibility and cost of growth (kJ g1) in rainbow trout (100 g)
during a short term growth trial (21 days) in fish acclimated for 3 weeks at different CO2 concen-
trations. Feed intake and digestibility of nutrients were unaffected by CO2, but feed conversion
ratios and cost of growth increase. P.V. Skov, unpublished data.
extensively by McCormick (McCormick, 2012). The Na+/K+ ATPase (NKA),

Na+/K+/2Cl transporter (NKCC) and the cystic fibrosis transmembrane reg-
ulator (CFTR) constitute the three main transport mechanisms responsible for
salt secretion in fishes, of which NKA activity is up-regulated in preparation
for, and in response to, saltwater exposure.
Although limited data is available, there is evidence to suggest that mito-
chondria rich cells (MRCs) and pavement cells of the gill undergo changes
under hypercapnic conditions, to compensate for the associated respiratory
acidosis. To maintain plasma pH, fishes elevate their plasma bicarbonate
concentrations by reducing Cl/HCO–3 exchange (Cameron and Iwama,
1987) (see also Chapter 3, Vol 37: Brauner et al., 2019). This results in a
net decrease in plasma Cl levels, which in salmonids appears to be propor-
tional to the PCO2 levels (see also Chapter 3, Vol 37: Brauner et al., 2019).
Goss et al. (1992a) observed that this was achieved by a large (95%) reduction
in MRC surface area, caused by covering of the adjacent pavement cells
(PVCs). Following 6 h of hypercapnic exposure in brown bullhead (Ictalurus
nebulosus), MRCs recessed into the filament, and the influx rate of sodium

Jin Na + increased almost threefold. The increase in PVC surface area was also
associated with an increase in surface microvilli, indicative of an increased
activity, as well as ultrastructural changes within the PVCs, presumably in
the excretion of acid and uptake of Na+ (Goss et al., 1992b). Similar results
have been observed for Atlantic salmon, in which gill NKA activity decreased
significantly during short-term (24 h) exposure of postsmolts (Seidelin et al.,
2001) as well as long-term (37 days) exposure of smolts (Hosfeld et al.,
2008) to hypercapnia. However, Seidelin et al. (2001) also investigated the
effects of longer exposure times, and observed that after 4 days, gill vacuolar
H+-ATPase mRNA expression, and NKA activity were comparable to con-
trol values. Thus, although the implications of the functional changes associ-
ated with hypercapnia are not entirely clear, and probably related to smolt
status, it is plausible that the modifications to the acid-base regulation and
osmoregulatory function of the gill associated with CO2 exposure may repre-
sent a challenge in the face other osmotic disturbances.
Net pen production of salmonids is typically characterized by the freshwa-
ter life stage occurring on land in recirculated aquaculture systems, following
by a transfer to sea for grow out when fish reach a mass of 200–800 g. In some
instances, it is possible for the RAS facility to gradually increase salinity in the
tanks prior to transfer, but most commonly, the transfer occurs directly from
freshwater to saltwater. Brauner et al. (2000) investigated the effects of previ-
ous hypercapnic exposure in Atlantic salmon on their ability to hyp-
oosmoregulate following transfer to seawater. Contrary to expectations, it
appeared that CO2 exposure in fact facilitated successful transfer, and fish that
had been exposed to CO2 showed no mortalities, and an ability to regulate

plasma Cl and Na+ that was as good, or even better, than control fish. The
authors propose that this observation may be facilitated by the decreased
plasma Cl levels associated with acclimation to high CO2 levels, and that
this may leave fish with a bigger tolerance window for influx of Cl during sea-
water exposure, or that the morphological changes outlined above simply
decrease permeability of the gills, and thus exerts a protective effect (Brauner
et al., 2000).
Despite an ability to successfully adapt to SW following CO2 exposure in
the FW stage, other carry over effects seem to occur. Mota et al. (2019) inves-
tigated the effects of long term (12 weeks) CO2 exposure on the growth perfor-
mance of Atlantic salmon reared in freshwater, and subsequently followed their
growth performance for 6 weeks following transfer to SW under normocapnic
conditions. Exposure to high CO2 concentrations higher than 12 mg L1
resulted in significantly reduced growth trajectories in the last 6 weeks of the
trial. Interestingly, subsequent to transfer to normocapnic SW, fish did not show
an ability to improve their growth, but in fact showed a continuous decline in the
growth performance. Since feed intake was not quantified, changes in appetite
or feed conversion could not be assessed, but no CO2 induced damage, such as
eye cataracts or nephrocalcinosis (see below) were observed, that could provide
an explanation for the reduced growth performance.
8. PATHOLOGICAL EFFECTS OF CO2
8.1. Nephrocalcinosis
Nephrocalcinosis is a pathological condition of the kidney which can be

recognized macroscopically, as white calcareous deposits in the ureters and
tubules, consisting mainly of calcium, phosphorous and magnesium (Smart
et al., 1979). The prevalence of nephrocalcinosis is thought to depend on a
variety of factors, including dietary mineral content, as well as dissolved
CO2 levels in the water. The functional consequences of nephrocalcinosis in
fish have not been determined, but is an area for further research, as tissue
necrosis may ultimately lead to kidney failure (Fivelstad et al., 2018). While
it has been suggested that other tissues such as intestine and gills may compen-
sate for a loss of ionoregulatory function in the kidney during progressive
nephrocalcinosis (Harrison and Richards, 1979), the occurrence of
nephrocalcinosis is certainly a concern for the welfare of fish in aquaculture.
Studies on the prevalence of nephrocalcinosis in Atlantic salmon
exposed to hypercapnia in fresh water have provided unequivocal results.
Hosfeld et al. (2008) quantified the occurrence of nephrocalcinosis exposed to

18 mg L1 CO2 and found that 8% of individuals displayed the pathology after
42 days of exposure. In a study investigating the effects of up to 24 mg L1 CO2
exposure for a period of 57 days, all groups, including a 6 mg L1 control
group, displayed more than 50% occurrence of nephrocalcinosis after 3 months
of rearing (Fivelstad et al., 2003b). Fivelstad et al. (2018), in a 3-month study,
found that nephrocalcinosis did not occur at CO2 concentrations less than
21 mg L1, while studies by Gil Martens et al. (2006), Good et al. (2010),
Fivelstad et al. (2015), and Mota et al. (2018) did not observe pathologies
in fish reared at up to 40 mg L1 dissolved CO2 for periods of up to 135 days.
In contrast, Fivelstad et al. (1999) observed an almost linear correlation
between the frequency of occurrence of nephrocalcinosis and dissolved CO2
levels after 61 days of exposure, showing that in fish exposed to 8, 19 and
32 mg L1, approximately 30, 40, and 80% of individuals were inflicted.
The majority of research on nephrocalcinosis has been conducted in salmo-
nids in fresh water, but a few studies on marine species have shown that high
CO2 concentrations may also lead to calcium deposits in the kidney of sea bass
(Dicentrarchus labrax) (Petochi et al., 2011) and wolfish (Anarhichas minor)
(Foss et al., 2003).
From the data available, it seems that there are other contributing factors
in the formation of nephrocalcinosis, which are, as yet, unidentified. It has
been proposed that elevated bicarbonate concentrations in the urine may be
a contributing factor (Fivelstad et al., 2018), although presumably these would
be comparable between groups exposed to similar CO2 levels, unless the chem-
ical composition of water and body fluids differed between experiments.
Water hardness could potentially be an explanatory factor, if calcium ions
were contributing to increasing hardness levels, which may lead to a net cal-
cium influx across the gill epithelium (Flik et al., 1995), or if branchial or oper-
cular calcium transport mechanisms are affected by exposure to high CO2
levels. In the face of high Ca2+ and bicarbonate levels in the plasma, aqueous
calcium bicarbonate could potentially react with CO2 and water to form pre-
cipitations of calcium carbonate. Exposure to high CO2 levels alone does not
appear to cause an increase in the whole body (Cameron, 1985; Fivelstad et al.,
2018; Gil Martens et al., 2006) or whole blood content of calcium (Good et al.,
2018). Despite this, evidence suggests that an increased rate of bone remo-
deling does occur (Gil Martens et al., 2006), potentially elevating the flux
of free Ca2+ levels in the plasma. Mechanisms by which increased calcium
complexes are formed remains to be investigated further.
Interestingly, if fish are simultaneously exposed to moderate (111%) or severe
(127%) hyperoxia and hypercapnia (18 mgL1), the prevalence of
nephrocalcinosis increased in 33–50% of the individuals (Hosfeld et al., 2008).
The authors did not discuss the possible mechanisms by which this occurred,
but suggested that hyperoxia induced hypercapnia (Wood, 1991) may have
resulted in a more severe CO2 exposure than indicated by the dissolved CO2
levels in the water. Such observations lend further evidence to the notion
that supersaturated oxygen conditions exacerbate the effects of hypercapnia
(Brauner et al., 2000).
8.2. Swim Bladder Inflation

The initial gaseous inflation of the swim bladder of larval fishes is achieved
by gulping air at the water surface, followed by transfer of air to the swim blad-
der via the pneumatic duct (Woolley and Qin, 2010). Although this is consid-
ered the mechanism for swim bladder filling in all species, Elsadin et al. (2018)
suggested that initial filling of the swim bladder in white grouper (Epinephelsu
aeneus) is achieved, at least in part, by means of oxygen unloading from hemo-
globin to the swim bladder via the rete mirabile, and that dissolved CO2 may
impair this crucial step in development. A limited amount of research has been
conducted on the effects of dissolved CO2 on larval fishes in culture. Arguably,
this is because biomass loads are much lower than in grow out production
systems, and it is assumed that good water quality, including dissolved gases,
are easier to maintain. However, aquaculture facilities for larval rearing are
still based on recirculation technology with limited water exchange, and while
biomass may be low, feeding rates are high, and the possibility of CO2 accu-
mulation cannot be ignored. Elsadin et al. (2018) investigated the effects of
three levels of dissolved CO2; 0.8, 5.6, and 28.6 mg L1, on swim bladder infla-
tion rate and volume in white grouper. In the normocapnic treatment, 79% of
all fish had normal swimbladder inflation at 105 days post hatch, only 57% of
fish in the moderate hypercapnic successfully inflated their swim bladder, and
only 42% were capable in the high CO2 group. How CO2 interferes with swim
bladder filling if the process is physostomous, (i.e. by filling the swimbladder
with engulfed atmospheric air via a pneumatic duct in the alimentary canal) is
unknown, but improper swim bladder inflation is a common problem in larval
culture, and the potential role of dissolved CO2 warrants further attention.
Following swim bladder filling, gas regulation is achieved by unloading oxy-
gen to the swim bladder via the rete mirabile, and in this process, dissolved
CO2 appears to have an effect, for reasons as yet unknown. White grouper that
had been exposed to medium and high concentrations of CO2, but had success-
fully inflated swim bladders, showed over a 50% reduction in swim bladder
volume (Elsadin et al., 2018). In the absence of successful swim bladder infla-
tion, fish are unable to properly orient themselves in the water column, tend to
display erratic swimming and elevated metabolic rates, and have much higher
mortality rates (Lund et al., 2014). It is generally assumed that larval fishes are
more sensitive to dissolved CO2, therefore future work on hypercapnia in
aquaculture should also include aspects of larval husbandry, particularly at
more moderate levels relevant for such rearing systems.
8.3. Cataract Formation

Cataracts in the eyes of farmed fish is a major concerns from an economical
and ethical standpoint. Cataracts are a clouding of the lens of the eye that may
initially be small and nucleated, but which can progress into a cortical cataract
affecting the entire lens. The cause of cataracts is not clear, and a multitude of
different possible mechanisms have been suggested, including diet composi-
tion, growth rates, parasite infection, and water quality parameters (Bjerkås
et al., 2004). The transfer of salmon from fresh- to seawater has also been
shown to cause significant increases in cataract formation, indicating that
osmotic disturbance may be a causative agent (Breck and Sveier, 2001).
Only recently have correlations between CO2 exposure and cataract forma-
tion been documented, although it does not appear to apply to all species. In
juvenile Atlantic cod, a positive correlation between not only the prevalence of
cataracts, but also their severity, and dissolved CO2 concentrations, was docu-
mented by Moran et al. (2012). In fish exposed to 18 mg L1 CO2, more than
80% of individuals displayed some sort of eye damage after 55 days of rearing,
and 73% had some degree of cataract formation, with nearly half being severe
cortical cataracts affecting more than 75% of the lens (Fig. 7). In contrast,
fish exposed to 8 and 2 mg CO2 L1 had only 11–14% incidence of cataracts,
which were generally less severe. A study by Neves and Brown (2015) con-
firmed these results in a study where cod were exposed to slightly higher
CO2 levels of 7, 12, and 20 mg L1 for a much longer period of 5 months.
Inspection of fish on a monthly basis revealed that cataract formation contin-
ued to develop, such that in the high CO2 group at the end of the trial all had
cataracts in the most severe form, and that even the lowest CO2 group had a
30% incidence rate affecting 51–75% of the lens. Moran et al. (2012) suggested
a number of possible mechanisms by which dissolved CO2 might induce cat-
aract formation, including CO2 disruption of the pH levels in the eye, ionic
balance of the lens, or alterations to the oxygen delivery to the eye.
In contrast to the results from cod, studies on Atlantic salmon have not
documented any correlation between dissolved CO2 levels as high as 40 mg L1
and the prevalence of cataracts (Mota et al., 2019; Waagbø et al., 2008). Instead,
Waagbø et al. (2008) found a correlation between hyperoxygenated water
and cataract formation, once more highlighting the possible interactive roles
of oxygen and carbon dioxide.
Fig. 7. Prevalence of different eye lesions in Atlantic cod subjected to different CO2 treatments
over a period of 55 days (A), and cataract intensity observed in each treatment. Modified from
Moran et al. (2012).
9. CONFOUNDING WATER QUALITY EFFECTS
For the large majority of research conducted, practical and logistical con-
ditions often dictate that experimental work in aquaculture is largely limited to
studies where CO2 levels fluctuate as the sole variable. More often than not,
multiple water quality parameters change in concert in aquaculture systems.
Given that there is continued pressure to increase the degree of recirculation
and loading density within RAS, new water quality parameters are becoming
relevant, such as the accumulation of metals of dietary origin, levels of dissolved
atmospheric gases, and nitrogenous waste compounds.
9.1. Hypercapnia and Hypoxia
In a modern RAS facility the occurrence of hypoxic events are unlikely, due
to the option of supplemental oxygenation. However, more than 60% of the
global aquaculture production volume still comes from extensive or semi-
intensive pond culture (Bostock et al., 2010) which rely on limited or absent
aeration technology. While some semi-intensive pond systems may employ aer-
ation devices, such as paddle wheel aerators or diffusors, extensively cultured
ponds do not. Instead, these ponds rely on photosynthesis for the addition of
dissolved oxygen to the water during daylight hours, and maintain low fish
densities in the ponds. Following sunset, oxygen becomes severely depleted
from the respiration of fish, plankton and bacteria (Lefevre et al., 2011), while
dissolved CO2 levels progressively increase. Despite this undisputable correla-
tion, surprisingly little attention has been given to the effects of combined
hypoxia and hypercapnia in aquaculture.
Exposure to elevated CO2 levels induces a Bohr shift and Root effect, caus-
ing both a reduced oxygen binding affinity of hemoglobin as well as a reduced
oxygen carrying capacity of the blood, respectively (Jensen et al., 1993; see
also Chapter 3, Vol 37: Brauner et al., 2019). When hypoxia is concurrent,
fish must increase their ventilatory work to compensate for the reduction in
oxygen availability (see also Chapter 3, Vol 37: Brauner et al., 2019).
The response of fish to combinations of hypoxia and hypercapnia are
dependent on the dissolved levels of both gases. In many fish species, moderate
levels of dissolved CO2 lead to either no change (Khan et al., 2018; Thomas
et al., 1983) or only modest increases in Ṁ O2 (Crocker and Cech, 2002). Pre-
sumably this is driven in part by an increase in the cost of ventilation (Gilmour
and Perry, 1994; Perry and Gilmour, 1996). As hypoxia progresses, venous
PO2 decreases, which increases the solubility of CO2 (Permentier et al.,
2017), the so-called Haldane effect, which further reduces the affinity of
hemoglobin for O2 (Jensen et al., 1993).
The effects of combined hypoxia and hypercapnia on hypoxia tolerance

has been demonstrated in the European eel (Cruz-Neto and Steffensen,
1997). Here it was shown that the critical oxygen tension, below which fish
are no longer able to maintain their SMR, increases from 3.4 kPa under nor-
mocapnic conditions, to 6.4 kPa O2 at a PCO2 of 4 kPa. With a simultaneous
decrease in Ṁ O2 with increasing CO2 levels, this shows that both hypoxia tol-
erance and metabolic scope are compromised when hypoxia and hypercapnia
cooccur. This may not necessarily be the universal response of fishes; in white
sturgeon, Ṁ O2 increases almost twofold when hypoxia (PO2 ¼ 12 kPa) and
hypercapnia (PCO2 ¼ 2.7 kPa) occur simultaneously (Crocker and Cech,
2002). Presumably, the experimental conditions and acclimation history of
the animals have a large influence on the outcome of experiments examining
the effects of hypoxia–hypercapnia. The different responses observed in the
studies above may also be a result of the different PCO2 and PO2 levels inves-
tigated. While fish exposed to long-term hypercapnia show an ability to
compensate for respiratory acidosis by accumulation of plasma bicarbonate
(Larsen and Jensen, 1997), this is, in all likelihood, not the case for fish that expe-
rience large fluctuations in PCO2 on a daily basis. The effects of concurrent
hypoxia and hypercapnia under such conditions warrant further attention, spe-
cifically directed towards how it affects fish health, welfare and productivity
under extensive culture. Concurrently, CO2 may also influence the tolerance
of fish to hypoxia, which is the topic of Lefevre (2019: Chapter 6, Vol 37).
9.2. Hypercapnia and Metal Toxicity

All fish have a nutritional requirement for a number of trace metals, includ-
ing magnesium, zinc, selenium, manganese, iron and copper (Antony Jesu
Prabhu et al., 2016), and as in other animal feed, vitamin and mineral premixes
are added to satisfy nutritional requirements. Because fish do not completely
digest their feed, a fraction of dietary metals are lost via fecal material (Bury,
2002). As water exchange rates in recirculating aquaculture systems (RAS)
decrease, there is growing awareness that a number of organic and inorganic
compounds may start to accumulate in the system. In a study by Davidson
et al., (2009) the accumulation of metal ions was examined in RAS rearing
rainbow trout (Oncorhynchus mykiss) with high or low hydraulic retention times
(6.7 vs 0.67 days). Out of the 8 metals investigated, copper increased 10-fold
compared to intake water, while zinc increased 2-fold. Similar results have
been found in RAS with turbot; as water exchange rates decrease, significant
accumulation of zinc, copper, cobalt and manganese was observed (van
Bussel et al., 2014).
Copper toxicity is influenced by several environmental variables, only a
few of which are discussed here. Cu exposure in fish leads to ionoregulatory
disturbances, affecting sodium homeostasis (Lauren and McDonald, 1987).
However, Cu toxicity appears to be higher for fish that live in an anisoosmotic

environment presumably due to a higher requirement for sodium regulation
(Blanchard and Grosell, 2006). Furthermore, the toxicity of Cu is influenced
by water pH. Acute toxicity studies on the streaked prochilod (Prochilodus
lineatus) showed that at pH 4.5 the LC50 increased 6-fold, from 0.016 mg
Cu L1 to 0.1 mg Cu L1 (Carvalho and Fernandes, 2006).
In their study on the interacting effects of Cu and hypercapnia exposure,
Larsen et al. (1997) showed that Atlantic cod exposed to 0.4 mg L1 copper
responded with extracellular acidosis due to a decrease in plasma bicarbonate
concentrations with no change in plasma PCO2. Simultaneous exposure to
hypercapnia (1 kPa) resulted in an aggravation of this acidosis as well as a
large increase in PCO2, and prolonged acid-base recovery compared to fish
that had only been exposed to hypercapnia. These results show that Cu expo-
sure exerts a negative effect on the ability of fish to recover from acute CO2
exposure. On the other hand, hypercapnia exerted a protective effect against
the toxicity of Cu, by reducing the osmotic and ionic (Na+, K+, Cl)
disequilibria caused by copper alone. The exact mechanisms are not detailed,
but the authors suggest that hypercapnia may have caused reduced gill perme-
ability. Alternatively, the PCO2 used (1 kPa) may have reduced water pH
sufficiently to reduce the toxicity of Cu (Carvalho and Fernandes, 2006).
A somewhat similar response was observed for rainbow trout in freshwater
(Wang et al., 1998). Here, the simultaneous exposure to 0.6 mg L1 Cu and
0.8 kPa CO2 also aggravated the extracellular drop in pH and prolonged
recovery, but contrary to Larsen et al. (1997) no protective effects of hypercap-
nia on ionic disturbance from Cu exposure were observed.
Although the accumulation of Cu in RAS with low water exchange does
not reach levels as high as those reported in the studies above, observed levels
of 0.045–0.09 mg L1 (Davidson et al., 2009; van Bussel et al., 2014) may be
cause for concern, and potential toxic effects from long term exposure should
be investigated.
9.3. Carbon Dioxide and Aluminum

Aluminum toxicity in fishes has been extensively reviewed by (Exley et al.,
1991). Briefly, aluminum (Al) is most toxic under acidic conditions, where Al spe-
ciation leads to high solubility in water (as Al(OH)2+). For this reason, toxicity of
Al has been of interest to aquaculture for the past several decades, particularly in
combination with elevated CO2 levels which reduces water pH. This applies in
particular for areas where Al is abundant in the natural water supply due to either
run-off from land or naturally high deposits of aluminum ore in the earth.
Al causes irritation of the gills, and can precipitates on the gills of fish as
aluminum hydroxide, leading to ionoregulatory disturbance and an increase in
gill membrane permeability leading to uptake of protons and Al, respiratory
failure and loss of osmoregulatory capacity (Exley et al., 1991). Stunted fish
growth has been shown to occur for brown trout (Salmo trutta) at Al concen-
trations as low as 20 μg L1 at pH levels of 4.4–5.2, and increased mortality
was observed at 50 μg L1 (Fairman and Sanz-Medel, 1995).
Tench (Tinca tinca) experiencing short-term Al exposure (2 mg L1) at pH 5,
display a rapid drop in arterial PO2, blood oxygen content and saturation, in
addition to a doubling of plasma PCO2 and lactate, as well as a drop in plasma
chloride levels (Jensen and Weber, 1987). This suggests that Al toxicity is a
relevant concern for aquaculture, especially when exposure coincides with
hypercapnia induced reductions in pH. Furthermore, in the streaked prochilod
(Prochilus lineatus) exposed to much lower Al concentrations (0.2 mg L1)
significant and persistent reductions in plasma osmolality were observed after
24 h from decreases in Na+ and Cl, demonstrating the osmoregulatory distur-
bance of Al. After 96 h exposure there was a significant reduction in the number
of both filamentous and lamellar MRC cells. A concomitant elevation in
plasma glucose illustrates that fish exposed to Al suffer from metabolic
disturbances, as was also indicated by the increase in blood hemoglobin concen-
trations (Camargo et al., 2009).
The studies above are more ecotoxicological in nature, and fish in aquacul-
ture may not experience Al concentrations and pH levels in this range. However,
Al can be toxic at higher pH levels, and the water in the microenvironment of
the gill may be as much as 0.5 pH unit lower than the ambient water (Wilson,
2011). See also Wilson (2011) in this series, for a more detailed description of Al
toxicity and mechanisms. Fivelstad (2013) provided a theoretical relationship
between dissolved CO2 levels in culture water and the pH at which Al becomes
toxic, showing that at least in low alkalinity water, Al becomes toxic at levels as
low as 7 mg L1 dissolved CO2, where pH levels drop to 6.2 (Fivelstad et al.,
2003a). Fivelstad et al. (2003a) showed that when Atlantic salmon were reared
in soft water containing 2, 9, or 19 mg L1 CO2 the corresponding pH levels
dropped from 6.6 to 6.0 and 5.7 respectively. This lead to significant increases
in the accumulation of Al in the gills, from 20 μg g1 in the low CO2 treatment to
100 and >140 μg g1 in the medium and high CO2 group. These findings illus-
trate the importance of dissolved Al levels in aquaculture, even at levels consid-
erably lower than conventional toxicology studies, and perhaps particularly for
fish undergoing the parr-smolt transformation.
10. CONCLUSIONS AND PERSPECTIVES
A considerable amount of work has demonstrated the effects of CO2 on

growth in a variety of fish species, although with an overrepresentation of
the salmonids, in the grow-out phase. For the majority of these studies,
experimental conditions have favored exposure to constant levels of dissolved

CO2, and with CO2 as the sole experimental variable. Dissolved CO2 levels
fluctuate on an hourly or daily basis in all aquaculture production systems,
but less so in recirculating systems, than in extensive systems with no water
treatment technology, such as earthen ponds. Fish show remarkable capacity
to acclimate to different environmental conditions. However, acclimation
comes at a metabolic cost, but once achieved may not exert a large expenditure
for the individual, depending on the severity of conditions. Multiple water
quality parameters, when not stable, tend to fluctuate in concert. In pond cul-
ture, dissolved CO2 levels and dissolved oxygen levels fluctuate in a recipro-
cative manner, accompanied by changes in pH, ammonia ionization, and
more. These events happen on a diurnal rhythm, probably never allowing fish
to achieve homeostasis, and induce a large allostatic load. Further research is
required to understand how fish respond to daily fluctuations in multiple water
quality parameters, relative to long-term acclimated individuals. From our
current knowledge on hypercapnia, it is apparent that there are interacting
effects of hyperoxia and hypercapnia. It may be hypothesized that hyperoxia
either aggravates the effects of CO2 by additional respiratory acidosis, or cau-
ses oxidative damage to the gills and their adaptive capacity. In the light of the
use of pure oxygen injection in RAS, this is a highly relevant are for further
research.
There is a remarkable scarcity of work on larval stages of fishes. Aquacul-
turists may perceive larviculture as an aspect of fish husbandry that is
unproblematic in relation to CO2. However, husbandry practices dominated
by low water exchange rates and high input of organic matter (feed), in com-
bination with the increased sensitivity of fish larvae to hypercapnia, may prove
this wrong. Although the mechanisms are not entirely clear, it appears that
even quite low CO2 levels may potentially lead to development issues. In this
respect, aquaculture researchers should join forces with ecologists, who
already have a detailed understanding on the effects of increasing CO2 levels
in relation to climate change science. Such research should aim to include
carry-over effects from hypercapnic exposure during the larval stage to juve-
nile and adult fishes.
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Co2 in Aquaculture

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8

Aquaculture of fishes is increasingly using recirculation technology to con-

bacterial metabolism, before discussing what is currently being considered as

Aquaculture production of fish, molluscs, and crustaceans has increased

2. CO2 SOURCES IN AQUACULTURE

2.1. Fish Metabolism

2.2. Bacterial Metabolism

3. SAFE LEVELS AND WELFARE GUIDELINES

Sensitivity to dissolved CO2 is likely to be highly species specific, and esta-

aquaculture systems. The majority of research on safe levels of CO2 in aqua-

4. FREE CO2, pH AND ALKALINITY

In aquaculture production systems, it is most common to refer to the

4.1. Current Removal and Control Practices

dissolved CO2 increases proportionally with alkalinity (Loyless and Malone,

5. DYNAMICS OF CO2 IN AQUACULTURE

Daily variations in levels of dissolved CO2 are likely to be largely dependent

6. EFFECTS OF DISSOLVED CO2 ON GROWTH

several species in aquaculture production, predominantly Atlantic salmon

Atlantic cod (Gadus morhua) 80 2 10 35 1.7 – 1.07 – 40 1

6.1. Feed Intake and Appetite

Appetite in fishes is regulated by a number of complex pathways under the

effects of CO2 on feed intake. It is plausible that changes in oxygen availability

6.2. Digestive Function

36 to 44 h (Methling et al., 2013). In both instances, this effect appeared, at

Anadromous species such as Atlantic salmon undergo a parr-smolt trans-

extensively by McCormick (McCormick, 2012). The Na+/K+ ATPase (NKA),

had been exposed to CO2 showed no mortalities, and an ability to regulate

8. PATHOLOGICAL EFFECTS OF CO2

Nephrocalcinosis is a pathological condition of the kidney which can be

Hosfeld et al. (2008) quantified the occurrence of nephrocalcinosis exposed to

8.2. Swim Bladder Inflation

8.3. Cataract Formation

9. CONFOUNDING WATER QUALITY EFFECTS

9.1. Hypercapnia and Hypoxia

The effects of combined hypoxia and hypercapnia on hypoxia tolerance

9.2. Hypercapnia and Metal Toxicity

However, Cu toxicity appears to be higher for fish that live in an anisoosmotic

9.3. Carbon Dioxide and Aluminum

10. CONCLUSIONS AND PERSPECTIVES

A considerable amount of work has demonstrated the effects of CO2 on

experimental conditions have favored exposure to constant levels of dissolved

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