Aquaculture 557 (2022) 738312

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effects of ozonation and foam fractionation on rainbow trout condition and

physiology in a small-scale freshwater recirculation aquaculture system
Manuel Gesto *, Kim João de Jesus Gregersen , Lars-Flemming Pedersen
Section for Aquaculture, The North Sea Research Centre, DTU Aqua, Technical University of Denmark, Willemoesvej 2, 9850 Hirtshals, Denmark

A R T I C L E I N F O A B S T R A C T

Keywords: The combined use of ozonation and foam fractionation significantly improves the water quality in freshwater
Water quality recirculating aquaculture systems (RAS). In a recent study, numbers and total volume of suspended particles
RAS were reduced by 89% and 76%, respectively, water turbidity was improved by 79% and bacterial activity was
Welfare
reduced by 90% compared to control RAS. In the current study, we assessed the effects of those improvements in
Ozone
Foam fractionation
water quality on the welfare status of the rainbow trout juveniles present in the RAS. Ozone and foam frac­
Rainbow trout tionation were applied separately or in combination in a two-by-two factorial design with triplicated individual
RAS. The trial was 8-week long and the welfare of the fish was assessed on week 4 (mid-term) and on week 8
(final). Fish welfare was assessed by using a combination of variables related to physiological stress, oxidative
stress, growth performance, energy metabolism and external damage. Feed amounts were kept constant during
the study to maintain homogeneous water quality conditions through the study, resulting in a decreasing specific
feeding ration during the experiment. Restrictive feeding affected a number of variables related to the metabolic
condition of the fish, but those effects were similar for all treatments. Effects of ozonation, foam fractionation and
their combination on fish welfare were modest. Both ozone and foam fractionation led to improved skin con­
dition of the fish, and seemed to act complementarily when applied together. Furthermore, foam fractionation
had a small significant effect in improving the fish condition factor with respect to the fish in the RAS without
foam fractionation. While the size of the described effects was small, they were the result of short-term exposure
to the improved water quality. Further studies should focus in investigating the long-term effects of ozonation
and foam fractionation on fish welfare in RAS-based aquaculture.

1. Introduction
Recirculating aquaculture systems (RAS) have developed during the
last decades to become an important land-based alternative for
commercial-scale production of fish (Holan et al., 2020). When
compared to other conventional aquaculture methods, RAS contribute
to certain aspects of environmental sustainability by reducing water
consumption and increasing the control of the discharges to the envi­
ronment (Ebeling and Timmons, 2012). The reuse/recirculation of water
requires continuous control of water quality to ensure optimal condi­
tions and proper health and welfare of farmed fish. One of the main
challenges in RAS related to water quality is the buildup of dissolved and
suspended organic matter, mostly derived from fish feces and feed re­
mains (Martins et al., 2010; Schumann and Brinker, 2020). While large
particulate materials can be removed by specific sedimentation and/or
filtration technology, small organic particles accumulate and constitute
an excellent resource of space and nutrients for bacterial growth (de
Jesus Gregersen et al., 2019). An increase in the bacterial carrying ca­
pacity of the water can result in negative effects in the RAS, such as
increased oxygen consumption, carbon dioxide formation, reduced ef­
ficiency of nitrification by the biofilter or hindered action of disinfec­
tants (Chen et al., 2006; Fossmark et al., 2020; Guerdat et al., 2011;
Hess-Erga et al., 2008).
The presence of dissolved and particulate matter could also affect the
health and wellbeing of the farmed fish. Different studies have demon­
strated potential negative effects of suspended solids in salmonids and
other fishes, both in laboratory conditions and in the natural environ­
ment (Chapman et al., 2014; Kjelland et al., 2015; Schumann and
Brinker, 2020). These effects include alterations of gill tissue, growth
inhibition, higher susceptibility to infections, behavioral alterations and
increased mortality rates (Cavanagh et al., 2014; Goldes et al., 1988;
Redding et al., 1987; Sweka and Hartman, 2001). Most of those studies

* Corresponding author.
E-mail address: mges@aqua.dtu.dk (M. Gesto).

https://doi.org/10.1016/j.aquaculture.2022.738312
Received 26 October 2021; Received in revised form 27 April 2022; Accepted 28 April 2022
Available online 2 May 2022
0044-8486/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Gesto et al.
focused on the effects of mobilized sediments, i.e. of mostly mineral
particles, and on levels of suspended solids markedly higher than the
levels found in aquaculture facilities (Schumann and Brinker, 2020). In
relation to RAS, there is a paucity of information about the effects of
organic matter, dissolved or in the form of fine particles, on the physi­
ology, welfare and health status of fish (Schumann and Brinker, 2020).
High levels of organic matter are closely tied to bacterial loads and al­
terations of bacterial communities in RAS, alterations on biofilter per­
formance or increased water turbidity (Fossmark et al., 2020; Liu et al.,
2018; Schumann and Brinker, 2020; Xue et al., 2017). The scarce in­
formation available point to no direct physical effects of organic parti­
cles on fish physiology and performance (Becke et al., 2018, 2019).
However, the potential effects of dissolved and particulate organic
matter on fish health needs to be addressed for specific RAS conditions.
In a recent study, de Jesus Gregersen et al. (2021) showed that both
ozonation and foam fractionation, alone or in combination, had a clear
effect on the water quality of freshwater RAS. Both treatments reduced
the level of suspended solids, organic matter, bacteria and turbidity, and
those effects were complementary when the treatments were applied
together. In this study, we investigated the effects of the treatments used
in that trial on the welfare of the fish, by assessing the fish external
condition (mass, condition factor, external damage to fins and skin) and
the physiological (plasma levels of cortisol, glucose and lactate – com­
mon indicators of stress/disturbance, and liver glucose and glycogen
levels – as a measurement of the liver energy stores and their mobili­
zation) and oxidative stress status (antioxidant enzymes, lipid peroxi­
dation –common indicators of cellular oxidative damage). Our main
hypothesis was that the improved water quality obtained by using ozone
and foam fractionation had a positive effect on fish welfare and general
fish condition. Secondarily, given that ozone is a potent oxidant (Powell
and Scolding, 2018; Schroeder et al., 2011, 2015) that has been proven
to induce deleterious acute effects on exposed fish (Reiser et al., 2011;
Stiller et al., 2020), we also hypothesized that the ozone dosage used
here (effective dose was 7 g O3 kg− 1 feed) was not high enough to induce
any oxidative damage or any other detrimental effect to the fish. In this
regard, ozonation procedures have been shown to induce histopatho­
logical lesions in the gills of exposed fish, likely related to cellular
oxidative damage (Good et al., 2011; Reiser et al., 2011; Ritola et al.,
2002; Stiller et al., 2020). While effects were observed in both saltwater
and freshwater (Powell and Scolding, 2018), ozonation procedures are
of more concern in water containing marine salts due to the ozone-
mediated generation of toxic free bromine oxidants (Grguric et al.,
1994; Stiller et al., 2020).
2. Materials and methods
2.1. Fish
Rainbow trout [Oncorhynchus mykiss (Walbaum, 1792)] juveniles
(approx. 100 g) were kindly provided by Biomar A/S (Hirtshals,
Denmark) and were left to acclimate to the experimental RAS tanks
described below for 12 weeks, before the start of the experiments.
2.2. Experimental setup
Briefly, the experimental setup consisted of a set of twelve inde­
pendent 0.8 m3 pilot scale freshwater RAS (de Jesus Gregersen et al.,
2021). The effects of ozonation and/or foam fractionation on water
quality and fish physiological status was evaluated by performing a two-
by-two factorial experiment. Each of the four tested conditions (CON­
TROL: no ozone, no foam fractionation; OZONE: only ozone; FF: only
foam fractionation; OZONE + FF: both ozone and foam fractionation)
was randomly assigned to three RAS. The FF and OZONE + FF RAS were
fitted with foam fractionators (Sander Fresh Skim 200, Erwin Sander
Elektroapparatebau GmbH, Germany). In the OZONE + FF group, ozone
was directly injected into the fractionator column. In the OZONE group,
2
Aquaculture 557 (2022) 738312
ozone was injected into a fitted bubble column (same size as that of the
foam fractionators). In the FF group, the fractionator column was
injected with air instead of ozone. In the OZONE and OZONE + FF
groups, ozone was provided by three ozone generators (Ozonizer S 500,
Erwin Sander Elektroapparatebau GmbH, Germany), each attached to
one OZONE RAS and one OZONE + FF RAS.
Foam fractionators were operated with a water flow rate of 1500 L
h− 1 and an air flow rate of either 1320 L h− 1 (FF RAS) or 1200 L h− 1 (air)
plus 120 L h− 1 ozonated air (OZONE + FF RAS). Bubble columns were
supplied with 120 L h− 1 ozonated air (OZONE RAS). Hydraulic retention
time within foam fractionators and bubble columns was kept equal (120
s) to ensure equal contact time in both systems. Gas supply was
controlled by flow meters (Key Instruments Variable area flow meter,
Key Instruments, USA). Ozone was injected at a nominal rate of 20 g O3
kg− 1 feed, which resulted in an effective O3 dose of 7 g O3 kg− 1 feed with
a retention time for the water in the rearing tanks of 15 min (de Jesus
Gregersen et al., 2021).
Just before starting the experiment, fish biomass in each tank was
adjusted to approximately 8 kg (average biomass = 8.05 kg, SD = 0.03
kg). Between 32 and 37 fish were allocated in each tank, and a total of
420 fish were used in the experiment. At this time, the average indi­
vidual mass of the fish was 233.0 g (SD = 57.5 g). The experiment was
started by turning on the ozonation and FF in the corresponding
experimental units. During the experiment, fish in each tank were fed a
fixed daily ration of 100 g feed d− 1 (Efico E 920, Biomar, Denmark)
delivered by means of belt feeders through a 8 h period. Each day, 60 L
water was replaced in each RAS, resulting in a feed load of 1.66 kg feed
m− 3. Photoperiod was kept at 14 h light: 10 h dark, with lights on at 7
am.
2.3. Water quality
Water temperature (17–21 ◦ C) and oxygen saturation (85–90%)
were continuously monitored during the experiment (OxyGuard Pacific
system, OxyGuard International A/S, Denmark) and no significant dif­
ferences were found among treatments. Water quality was also daily
monitored in terms of pH (7.0–7.3, daily corrected by adding sodium
bicarbonate as needed), ammonium (< 0.20 mg N L− 1), nitrite (< 0.15
mg N L− 1) and nitrate (< 100 mg N L− 1). Primary solids were collected
in settling columns at the bottom of the tanks that were emptied daily.
The oxidation-reduction potential (ORP) in the water was monitored
using a Hach HQ40d Portable Multi Meter (Hach Lange, USA) fitted with
a Hach ORP probe (IntelliCAL MTC101). ORP fluctuated between 120
and 210 mV during the experiment.
Ozonation and foam fractionation induced consistent changes in the
resulting water quality. All effects on water quality are described in
detail in a previous publication (de Jesus Gregersen et al., 2021). In
summary, by the last three weeks of the trial, ozonation had reduced the
numbers of particles in the water by 83% (from 2.43 million mL− 1 to
0.42 million mL− 1), along with particulate organic matter (54%, from
5.27 mg L− 1 BOD5 to 2.44 mg L− 1 BOD5), bacterial activity (48%, from
0.84 h− 1 to 0.44 h− 1 in terms of H2O2 degradation rates, Pedersen et al.,
2019) and water turbidity (38%, from 7.02 NTU to 4.34 NTU). Foam
fractionation reduced the number of particles (58%, from 2.43 to 1.01
million mL− 1) and their volume (62%, from 0.037 to 0.014 mm3 mL− 1),
particulate organic matter (56%, from 5.27 to 2.33 mg L− 1 BOD5),
bacterial activity (61%, H2O2 degradation rate reduced from 0.84 h− 1 to
0.33 h− 1) and turbidity (65%, from 7.02 NTU to 2.46 NTU). When both
treatments were applied together, they resulted in reductions of particle
numbers (89%, from 2.43 to 0.27 million mL− 1) and particle volume
(76%, from 0.037 to 0.009 mm3 mL− 1), particulate organic matter (84%,
from 5.27 to 0.86 mg L− 1 BOD5), bacterial activity (90%, H2O2 degra­
dation rate reduced from 0.84 h− 1 to 0.08 h− 1) and turbidity (79%, from
7.02 NTU to 1.49 NTU). Besides affecting the levels of bacterial activity,
ozonation (but not foam fractionation) had also an important effect on
the bacterial community composition of the RAS water (Aalto et al.,
M. Gesto et al.
2022).
2.4. Fish sampling
The experiment lasted 8 weeks and fish physiological status was
evaluated after week 4 (mid-term) and at the end of the experiment after
week 8 (final). At each point, four fish were sampled from each tank.
Samplings started at 9 am and lasted less than 2 h. Fish were not fed on
sampling days. Fish were lethally anesthetized in a benzocaine solution
(200 mg L− 1). Each individual was externally observed for size and
anatomical damage (eyes, snout, operculum, skin and fins) and then a
blood sample was collected from the caudal vessels with a heparinized 1
mL syringe. After collecting all blood samples from a tank (completed in
a 2–3 min window since the fish were anesthetized), fish were decapi­
tated and a piece of the liver (1 g approx.) and the second gill arch of the
left side were collected and stored at − 80 ◦ C. Only three out of the four
sampled fish per tank were used for blood and tissue (liver and gill)
collection. The fourth fish was only assessed for size and external
damage observations.
2.5. Condition and external damage
At the end of the trial, the biomass in each tank was quantified. The
biomass gained and the specific growth rate (SGR) of the fish during the
trial was estimated on a tank-basis, based on the initial and final biomass
and on the biomass removed from the tanks during samplings.
The tank-based SGR was calculated with eq. 1:
( )
SGR %d− 1 = [ln (final biomass) − ln (initial biomass) ] × [trial duration (days) ]− 1 × 100%
Fulton's condition factor (K) of fish individuals was calculated using
eq. 2:
K = 100*individual mass (g)*[Fork length (cm) ]− 3
(2)
Anatomical damage at the level of the eye, skin, operculum, snout
and fins (pectoral, pelvic, dorsal and caudal) was scored with a 4-step
scale from 0 (undamaged) to 3 (severe damage) (Noble et al., 2020).
For eyes and skin damage, different types of damage were considered
(for eyes: cataracts, exophthalmia, hemorrhage; for skin: scale loss,
hemorrhage, wounds), but a single score was assigned in each case (the
one corresponding to the type of damage showing the highest severity).
Eye, operculum and snout lesions were only observed in a few in­
dividuals and therefore, the related damage scores were not considered
further in this study.
2.6. Plasma stress markers
A commercial ELISA kit (Ref: 402710, Neogen Europe, Ayrshire, UK)
was used to quantify plasma cortisol concentration. Plasma glucose and
lactate were analyzed with colorimetric kits from Merck (Darmstadt,
Germany; ref.: CBA086) and Megazyme (Bré, Ireland; ref.: K-Late),
respectively.
2.7. Liver and gill homogenization
The tissues, either liver or gill (only filaments, without gill arch),
were finely minced on an ice-cold Petri dish, while still frozen. Minced
tissue was thoroughly mixed and two portions were allocated into
3
Aquaculture 557 (2022) 738312
different tubes for homogenization. First portion (approx. 100 mg for
livers and 75 mg for gill filaments) was homogenized with a Sonopuls
ultrasonic homogenizer (Bandelin, Germany) in exactly 10 volumes of
an imidazole buffer consisting of 50 mM imidazole (pH 7.0), 4 mM
EDTA, 1 mM β-mercaptoethanol, 50 mM sodium fluoride, 120 mM su­
crose and 0.5 mM phenylmethanesulfonyl fluoride (PMSF) (pH 7.5)
(Polakof et al., 2007). After centrifugation (9000 x g, 4 ◦ C, 25 min),
supernatants were immediately divided into three aliquots and stored at
− 80 ◦ C for the later analysis of protein content and catalase and gluta­
thione peroxidase activities. The second portion of tissue (approx. 300
mg for livers and 150 mg for gill filaments) was homogenized by ul­
trasonic disruption in exactly 3 volumes of a 20% trichloroacetic acid
(TCA) and 0.22 mM butylated hydroxytoluene solution. After centrifu­
gation (9000 x g, 4 ◦ C, 25 min), supernatants were immediately divided
into two aliquots and stored at − 80 ◦ C for the later analysis of TBARS
(liver and gill) and glucose and glycogen content (liver only).
2.8. Energy metabolites in the liver
Glucose in liver homogenates was analyzed with colorimetric kits
from Merck (Darmstadt, Germany; ref.: CBA086) before (free glucose)
and after (free + glycogen glucose) glycogen breakdown by α-amylo­
glucosidase. Glycogen levels were quantified as the difference between
both values, expressed as glucosyl units per tissue mass (Keppler and
Decker, 1974). Glycogen-to-glucose molar ratio was calculated to ac­
count for the extent of glucose mobilization from liver glycogen stores
(lower ratio = higher mobilization).
(1)
2.9. Oxidative stress parameters in liver and gill
TBARS was analyzed in liver and gill TCA homogenates using a
method based on previous protocols (Buege and Aust, 1978; He et al.,
2017; Weeks Santos et al., 2019), with modifications. Supernatants and
standards (200 μL) were mixed with 160 μL of 25 mmol L− 1 Tris buffer
(pH 8.0) containing 100 mmol L− 1 thiobarbituric acid (TBA) and 40 μL
of HCl 0.6 mol L− 1. Mixtures were incubated 45 min at 80 ◦ C and then let
to cool on ice for 5 min. After mixing, 250 μL of each mixture were
transferred to a black 96-well plate and the fluorescence intensity (FI)
was measured (CLARIOstar® microplate reader, BMG Labtech, Ger­
many) using 531 nm and 572 nm as excitation and emission wave­
lengths, respectively. A malondialdehyde (MDA) standard curve (FI was
linear from 0.1 to 2.5 μmol L− 1) was used to quantify TBARS as the tissue
content of MDA equivalents in nmol g− 1 wet tissue.
Catalase activity was analyzed by monitoring the decay in the
absorbance of the enzyme reaction mix at 240 nm, related to the
degradation of hydrogen peroxide (Aebi, 1984). GPX activity was
quantified according to Flohé and Günzler (1984). Total protein in so­
lution was quantified by means of the bicinchoninic acid (BCA) assay
(Smith et al., 1985).
2.10. Statistical analysis
A repeated-measures three-way ANOVAs model was used to test the
effects of factors Time (repeated measures: all tanks were tested at both
times midterm and final), Ozone Treatment (absence or presence) and
Foam Fractionation (absence or presence) on the different quantified
variables. Statistical scores for the different factors and variables are
reported in Supplementary Table 1. Furthermore, potential correlations
M. Gesto et al. Aquaculture 557 (2022) 738312

Table 1 Table 2
Initial and final biomass and biomass gain for the different treatments. Individual performance and external damage.
Treatment Initial RAS Final RAS Biomass gain SGR (% Treatment
biomass (kg) biomass (kg)1 (kg) day-1)
No ozone Ozone
0.94 ±
Control 8.04 ± 0.02 14.36 ± 0.20 6.33 ± 0.19 No FF FF No FF FF
0.02
0.96 ± Variable Time Control FF Ozone Ozone Effects
FF 8.07 ± 0.04 14.59 ± 0.12 6.52 ± 0.09
0.01 + FF
0.95 ±
Ozone 8.05 ± 0.04 14.47 ± 0.13 6.42 ± 0.14 individual week 334 275 331 ±
0.02 280 ± 88
mass (g) 4 ± 51 ± 41 100 Time: W8 >
0.94 ±
Ozone + FF 8.06 ± 0.02 14.45 ± 0.16 6.39 ± 0.16 week 395 378 393 ± W4
0.02
8 394 ± 23 ± 13 ± 22 42
Statistical
Fork length week 28 ± 27 ± 28 ±
scores 27 ± 0.8
(cm) 4 2.2 0.9 2.5 Time: W8 >
(One-way
p = 0.629 p = 0.426 p = 0.491 p = 0.560 week 30 ± 30 ± 30 ± W4
ANOVA)
8 29 ± 0.8 0.6 0.4 0.9
Data represent the mean and SD of n = 3 replicate RAS per treatment 1.48 1.34
Condition week 1.41 ± 1.42
± ±
factor K 4 0.04 ± 0.05
0.06 0.04 FF: FF > no
(Pearson's) among the different variables was assessed at the fish indi­ 1.45 1.39 FF
vidual level, irrespective of treatments (time, ozone or foam fraction­ week 1.44 ± ± ± 1.44
ation) or tanks, by constructing a correlation matrix. All statistical 8 0.01 0.06 0.05 ± 0.02
analyses were performed using JASP version 0.14.1. In all analyses,
0.4 Time: W8 >
statistical significance was set at p < 0.05. Skin week 0.8 ±
±
0.3 ± 0.3 ±
W4
damage 4 0.25 0.14 0.25
0.38 Ozone: no
3. Results ozone >
0.8 ozone
week 1.3 ± 0.9 ± 0.6 ± FF: no FF >
3.1. Fish growth performance and mortality
±
8 0.22 0.13 0.26 0.19 FF
1.9
At the end of the 8-week period, biomass was around 180% of the Pectoral week 1.6 ± ± 1.9 ± 1.4 ±
initial biomass in all RAS. No differences were found in the final RAS fins 4 0.30 0.30 0.22 0.17
1.5
biomass, in the biomass gain or in the tank-based SGR, among the
week 1.8 ± ± 1.7 ± 1.7 ±
different treatments (Table 1). There was a clear effect of time on in­ 8 0.14 0.29 0.30 0.42
dividual size (mass and fork length) of sampled fish (Tables 2 and Suppl. 1.3
Table 1), but not on fish condition factor, which was similar between week 1.1 ± ± 0.7 ± 0.9 ±
both samplings at 4 or 8 weeks after the start of the trial. Foam frac­ Pelvic fins 4 0.17 0.29 0.22 0.3
0.9
tionation induced a significant effect on the fish condition factor; Ful­
week 1.2 ± ± 0.8 ± 1.1 ±
ton's condition factor was slightly higher (4%) in the presence of foam 8 0.08 0.08 0.08 0.22
fractionators (p = 0.040) (Table 2 and Suppl. Table 1). Mortality was 1.2
occasional (only one fish died during the study). week 1.2 ± ± 0.9 ± 1.3 ±
Dorsal fin 4 0.17 0.36 0.17 0.3
0.8
3.2. External damage week 1.4 ± ± 1.3 ± 1.2 ±
8 0.22 0.14 0.29 0.17
Skin damage scores were significantly affected by all the three fac­ 1.0 Interact.
week 1.1 ± 0.7 ± 1.3 ±
Caudal fin Ozone x FF:
tors tested (Time, Ozone and Foam Fractionation), but not by any factor
±
4 0.08 0.08 0.17
0.38 Within no FF:
interaction (Table 2 and Suppl. Table 1). Time had a clear effect on the no ozone >
skin of the fish: overall, skin damage scores were increased on week 8 to 1.3 ozone;
a 228% of those observed on week 4 (p = 0.005). The average scores on week 1.8 ± ± 1.1 ± 1.5 ± within ozone:
8 0.08 0.29 0.17 0.14 FF > no FF
week 8 were still of low severity (average skin damage score: 1.000).
5.3
Ozone treatment had a significant effect (p = 0.027) on the condition of Total fin week 4.9 ± ± 4.2 ± 5.0 ±
the skin, being the skin score 36% lower (less damaged) in the tanks with score 4 0.51 0.22 0.58 0.66
ozone. The average skin damage score in the non-ozonated groups was 4.4
still of low severity (0.875 on average). Finally, the condition of the skin week 6.2 ± ± 4.8 ± 5.4 ±
8 0.22 0.55 0.22 0.36
was significantly better (p = 0.048) in the tanks with foam fractionation,
where the skin score was reduced a 32% with respect to the groups Data are means and SD of n = 3 replicated RAS (each RAS value was calculated
without skimmers. Again, the average skin damage score in the groups from four fish individuals).
without foam fractionation was of low severity (average score in those Column “effects” shows significant differences for the three factors and their
groups was 0.853). interactions (Three-way ANOVA): Time (W4, W8), Ozone (ozone, no ozone) and
FF (FF, no FF)
Regarding fin damage scores, while there were no main factor effects
of Time, Ozone or Foam fractionation, an interaction between Ozone
and Foam fractionation was found at the level of the caudal fin (Table 2 These data was not submitted to further analysis.
and Suppl. Table 1): Damage score was higher in the absence of ozone
treatment, but only in those tanks without a skimmer. Also, caudal fin 3.3. Plasma stress markers
damage was higher in the presence of skimmers, but only when com­
bined with ozone treatment. Among the plasma stress markers (shown in Fig. 1), only the plasma
The prevalence of external lesions in eyes, snout/mouth and oper­ levels of glucose were affected in the experiment, while no significant
culum was low: eye lesions present in two out of 96 fish, snout/mouth effects were found for cortisol or lactate. Plasma glucose levels were
lesions in four out of 96 fish and operculum lesions in six out of 96 fish. affected by Time (p = 0.016) and by the interaction of Time and Foam

4
M. Gesto et al. Aquaculture 557 (2022) 738312

12 b
25

10

liver free glucose (µmol g )

plasma cortisol (ng ml )

-1
-1

20
a
8
15
6

10
4

2 5

0 0
week 4 week 8 week 4 week 8

5 160

liver glycogen (µmol glucosyl units g )

-1
a
140
plasma glucose (mmol L )

b
-1

4
120

100
3
80

2 60

40
1
20

0
0
week 4 week 8
week 4 week 8

18 a
5
16
liver glycogen/glucose ratio

14
plasma lactate (mmol L )

4
-1

12
b
3 10

2 6

1 2

0
0 week 4 week 8
week 4 week 8
Control ozone
FF ozone+FF
Control ozone
FF ozone+FF Fig. 2. Glucose levels (free or in the form of glycogen) in the liver of fish reared
in RAS equipped or not with foam fractionation (FF) and/or ozonation. Data
Fig. 1. Plasma stress indicators in fish reared in RAS equipped or not with foam represent the mean (and SEM) of n = 3 RAS. Each RAS value was calculated as
fractionation (FF) and/or ozonation. Data represent the mean (and SEM) of n = the average of the measurements in three fish. Letters indicate significant dif­
3 RAS. Each RAS value was calculated as the average of the measurements in ferences among treatment factors: Liver free glucose was higher on week 8 than
three fish. The different letters indicate that plasma glucose levels were higher on week 4, only in RAS with FF (striped columns, RM three-way ANOVA, sig­
on week 4 than on week 8, only for the RAS lacking FF (columns without nificant Time x FF interaction). The glycogen-to-glucose ratio was lower on
stripes, RM three-way ANOVA, significant Time x FF interaction). week 8 than on week 4 (overall main effect of Time).

5
M. Gesto et al.
Fractionation (p = 0.047) (Fig. 1, Suppl. Table 1) in such a way that
plasma glucose levels on week 8 were lower than on week 4 in the
absence of skimmers, but not in their presence.
3.4. Energy metabolites in the liver
In the liver (Fig. 2), the levels of free glucose were also affected by
Time (p < 0.001) and by the interaction of Time and Foam Fractionation
(p = 0.035): the liver glucose levels were higher on week 8 than on week
4 in the presence of FF but not in its absence (Fig. 2, Suppl. Table 1). A
significant interaction between the effects of FF and Ozone was also
found for liver glucose levels (p = 0.033) but no significant effects were
found in any pairwise comparison in the post-hoc test. Time had also a
significant effect in the liver glycogen-to-glucose ratio, which was lower
on week 8 than on week 4 (p = 0.016) (Fig. 2, Suppl. Table 1).
3.5. Oxidative stress parameters in liver and gill
No alterations were found in the liver (Fig. 3, Suppl. Table 1). In the
gill, TBARS was affected by sampling time, levels being lower at week 8
than on week 4 (p < 0.001). Foam fractionation had an effect on the
antioxidant enzyme activity in the gills (Fig. 3, Suppl. Table1). Gill CAT
activity was higher in the absence of skimmers (p = 0.014). On the
contrary, gill GPX activity was higher in the presence of skimmers (p =
0.024).
3.6. Individual-based correlations
A Pearson's correlation matrix at the individual level is shown in
Fig. 4. Several significant correlations were found between pairs of
variables. Fulton's CF was positively correlated with liver glycogen, but
also with GPX activity in both liver and gill. Liver glucose was positively
correlated with plasma cortisol, and negatively correlated with liver
glycogen, liver CAT activity and liver and gill TBARS. Liver TBARS was
in turn positively correlated with liver glycogen content. Positive cor­
relations were found between plasma lactate and liver glycogen and
liver GPX activity. Finally, GPX activity levels in liver and gill were
positively correlated, and liver GPX activity was negatively correlated
with gill TBARS.
Pectoral fins damage score was positively correlated with pelvic fins
score, which in turn showed a positive correlation with caudal fin score.
Both pectoral and pelvic fins scores, as well as the total fin damage score,
were negatively correlated with fish individual mass. Dorsal fin score
was negatively correlated with liver glucose, and caudal fin score was
negatively correlated with liver GPX activity. Finally, skin damage score
correlated positively with gill CAT activity and negatively with gill
TBARS and the liver glycogen-to-glucose ratio.
4. Discussion
In a previous study, both ozonation and foam fractionation seemed
to work complementarily in improving water quality in this experiment:
ozonation had a greater effect in reducing particle numbers, and foam
fractionation had a larger effect in reducing total particle volume (de
Jesus Gregersen et al., 2021). This suggests that foam fractionation
targeted particles of larger size than those preferentially affected by
ozonation. There is very limited information about the effects of organic
matter, suspended solids or turbidity on fish welfare in RAS systems, but
some positive effects of water treatment on fish performance have been
attributed before to the improved quality of the water (Powell and
Scolding, 2018; Schumann and Brinker, 2020). We hypothesized that
the greatly improved quality of the RAS water in terms of reduced
particle content, organic matter, bacterial load/activity and water
turbidity might have had potential beneficial effects related to fish
performance and welfare. Therefore, we reported here the effects of the
applied treatments on fish physiological condition related to stress and
6
Aquaculture 557 (2022) 738312
energy metabolism, and on fish general condition and external damage
scores.
4.1. Time-related effects
Some of the significant effects found in the assessed variables were
solely the result of the factor time (week 8 versus week 4), with no or
little influence of the applied treatments (ozone or foam fractionation).
Those effects were mainly related to the energy metabolism of the ani­
mals and pointed to a poorer nutritional status on week 8 than on week
4. This likely nutritional effect mostly consisted of reduced plasma levels
of glucose, and a higher mobilization of glycogen stores (increased levels
of liver glucose and reduced liver glycogen/glucose ratio) in the fish at
week 8. These data suggests that fish were mobilizing glycogen stores
through glycogenolysis at a larger extent than on week 4 in order to keep
adequate circulating glucose levels to support fish activity (Enes et al.,
2009; Polakof et al., 2012). A nutritional effect of this kind was not
unexpected, given that the amounts of feed delivered to the tanks were
kept constant in order to keep water quality homogeneous during the
experiment. Therefore, the specific feeding rate (SFR) of the fish was
decreasing progressively during the duration of the experiment: at the
beginning, SFR was 1.24% fish mass day− 1, which decreased to 0.75%
fish mass day− 1 at the end of the trial. In spite of the different extent of
mobilization of liver glycogen stores, the rate of decrease of the SFR was
not enough to induce any effect on the fish condition factor between
week 4 and week 8. Interestingly, we observed that foam fractionation
was able to mitigate this nutritional effect to a certain extent, as dis­
cussed below in section 4.2.
4.2. Effects of ozonation, foam fractionation and their combination
Among all the assessed variables related to performance, external
damage, stress and energy metabolism, the only main effect found for
the ozonation treatment was a moderate improvement in skin condition.
The same effect was observed for the foam fractionation, so it was the
fish in the control group (no ozone and no skimmers) that showed the
poorest skin condition. Etiology of skin damage is diverse and depends
on the type of damage, but has been suggested to relate to both physical
damage and to infectious pathogens (Noble et al., 2020; Noga, 2000),
factors that can act synergistically since any physical wound to the skin
constitutes an open door for pathogen invasion. Most of the observed
skin damage in this study related to scale loss, but also reddened areas
and even small ulcers were observed. Since both ozonation and foam
fractionation reduced skin damage, we hypothesize that the improve­
ment in skin condition is likely the result of the reduced presence of
bacteria, including harmless, pathogenic and opportunistic bacteria, in
the system water (Liu et al., 2018; Moore et al., 1975). In support of this,
the combined use of ozonation and skimmers, which reduced bacterial
activity by 96% when used in simultaneously (de Jesus Gregersen et al.,
2021), resulted in the lowest score for fish skin damage in this study.
Besides the reduction in total bacterial activity, a differential micro­
biome in the RAS water, as a result of the applied treatments, might also
have a role in the prevalence and severity of external lesions. In our
experiment, both ozonation and foam fractionation resulted in reduced
skin damage, while only the ozonation protocol was able to change the
bacterial community composition in the RAS water (Aalto et al., 2022).
Therefore, there is no clear link between bacterial communities in the
water and skin damage in this case. However, water bacterial commu­
nities have been shown to correlate poorly with skin-mucus bacterial
communities in Atlantic salmon (Minniti et al., 2017). Whether there is a
potential link between bacterial communities and skin damage in
aquaculture fish should be the focus of future studies.
In the performed experiment, the improvement of water quality
variables after ozonation and foam fractionation was likely related to
the important reduction of particle number and volume in the RAS
water. The effect of ozonation was apparently larger for particles of
M. Gesto et al. Aquaculture 557 (2022) 738312

1.4 6
a
liver TBARS (nmol MDA eq g )

gill TBARS (nmol MDA eq g )

-1
1.2

-1
5

1.0
4
0.8 b
3
0.6
2
0.4

0.2 1

0.0 0
week 4 week 8 week 4 week 8

200
FF effect:

8 >
liver CAT activity (U mg prot )

gill CAT activity (U mg prot )

-1

-1
150
6

100
4

50
2

0 0
week 4 week 8 week 4 week 8

100 80 FF effect:

>
liver GPX activity (mU mg prot )

gill GPX activity (mU mg prot )

-1

-1

80
60

60

40

40

20
20

0 0
week 4 week 8 week 4 week 8

Control ozone Control ozone

FF ozone+FF FF ozone+FF

Fig. 3. Levels of TBARS and antioxidant enzyme activity (CAT and GPX) in the liver (left panels) and gill (right panels) of fish reared in RAS equipped or not with
foam fractionation (FF) and/or ozonation. Data represent the mean (and SEM) of n = 3 RAS. Each RAS value was calculated as the average of the measurements in
three fish. Gill CAT activity was higher and gill GPX was lower in RAS without FF (columns without stripes, RM three-way ANOVA, overall FF effect). The different
letters indicate that gill TBARS was lower on week 8 than on week 4 (overall main effect of Time).

7
 M. Gesto et al.
8

Fig. 4. Heatmap showing the Pearson's correlation coefficients among the variables measured in fish individuals from all groups of the experiment. Green and red colors indicate positive or negative relationships,
respectively, and the intensity of the color indicates the strength of the relationship. Borders indicate the level of statistical significance of the correlation. Dotted: p < 0.05; fine: p < 0.01; thick: p < 0.001. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Aquaculture 557 (2022) 738312

M. Gesto et al.
smaller size, since ozonation resulted in a 80% reduction in the number
of particles, but only in a 32% reduction in total particle volume. In
general, suspended solids in RAS are mostly organic and are mainly
generated from the feces of the reared fish (Schumann and Brinker,
2020). Literature available on the effects of these organic suspended
solid on fish performance and health is limited but points to minor or no-
effects of these particles on fish welfare (Becke et al., 2018, 2019;
Davidson et al., 2013; Schumann and Brinker, 2020). Still, ozonation has
shown beneficial effects on fish growth performance in different studies
(Davidson et al., 2011, 2021; Powell and Scolding, 2018). The reason
behind the better growth in those studies was unclear, but has been
attributed to long-term effects of improved water quality, including
reduced organic matter and suspended solids (Powell and Scolding,
2018), but also to the improvement of the visual environment (Davidson
et al., 2011, 2021), maybe helping the fish to better capture food items
when reared in less turbid water. In the current study, no positive effect
of ozonation was detected, even when ozonation clearly altered the vi­
sual environment by reducing water turbidity. However, it should be
taken into consideration that feed amounts were restricted here, prob­
ably masking any potential effect of turbidity on feed capture ability of
the fish.
In spite of its beneficial effects on water quality, ozone, as a potent
oxidant, might also constitute a threat to aquatic organisms through
direct exposure or indirectly through its rapid decomposition into free
radicals and other oxidants (Cho et al., 2003; Önnby et al., 2018; Powell
and Scolding, 2018). In this regard, ozone has been shown to induce
oxidative damage and mortality in fish when present in the water
(Powell and Scolding, 2018). A recent study in Atlantic salmon post-
smolts showed that ozone doses resulting in ORP values above 350
mV were acutely harmful for the fish (Stiller et al., 2020). However, the
study was performed in brackish water, in which the formation of
bromine oxidants are likely responsible, at least in part, of the harmful
effects of ozonation (Grguric et al., 1994; Stiller et al., 2020). In the
current study, ORP values in fish rearing units were similar among the
different groups and stayed below 220mv throughout the entire trial,
suggesting that applied ozone decayed fast, probably because of the high
presence of oxidable organic matter in the RAS water. Accordingly, fish
of the ozone-treated groups showed no sign of oxidative stress, based on
the observed activity of antioxidant enzymes and the levels of lipid
peroxidation.
The current experiment has demonstrated that foam fractionation,
traditionally used in saltwater systems to reduce suspended solids, can
be a relevant tool in freshwater systems, too (de Jesus Gregersen et al.,
2021). Foam fractionation was more effective than ozonation in
reducing total particle volume. This points to foam fractionation likely
targeting larger particles than ozonation. Interestingly, the application
of foam fractionation in this study seemed to have had a positive
nutritional effect on the fish, reflected in a mitigation of the effects of the
decreasing SFR during the trial. On week 8, fish in the FF and OZONE +
FF groups showed higher levels of free liver glucose and a lack of
decrease in plasma glucose, as seen in the groups without foam frac­
tionation with respect to week 4. This better ability to mobilize glucose
from the liver might indicate a better nutritional status in the FF groups.
This is further supported by the observed positive general effect of foam
fractionation on fish condition factor. The reason behind a better
nutritional status in the presence of foam fractionation is not known but
it might be related to a lower energetic costs of maintaining homeostasis
in the absence of relatively large particles and their associated bacterial
communities (Davidson et al., 2021; Powell and Scolding, 2018). For
example, in view of the reduced gill catalase activity in the foam frac­
tionation groups, one might speculate that there could have been some
energy saving in the gill antioxidant system, maybe related to a positive
effect of foam fractionation on the presence of oxidants in the water.
Contrary to the skin damage score, fin damage scores were not
affected by time, and the only effect found in fin damage was an inter­
action between ozonation and foam fractionation effects in the caudal
9
Aquaculture 557 (2022) 738312
fin score. Caudal fin condition was improved with ozonation, but only in
the absence of foam fractionation. Foam fractionation worsened caudal
fin condition when applied together with ozonation. In other words,
caudal fin damage was higher in the absence of treatments and when
both treatments were applied together. We think that a behavioral
component related to the visual environment in the tanks could be
behind the observed effects. Turbidity has been shown to affect
salmonid behavior, including both feeding and social behavior (McElroy
et al., 2018; Rowe et al., 2003). In this trial, water was very turbid in the
control group, and turbidity decreased up to 79% when both treatments
were applied together. Both caudal and pectoral fins seem to be the
preferential target of attacks during antagonistic behavior in salmonids
(Davidson et al., 2021; Turnbull et al., 1998), and both fins were actually
showing the highest damage scores in this trial. It is possible that both
low and high water turbidity had favored antagonistic behavior in the
trout, maybe by affecting the distance at which potential competitors
could detect each other. It is also possible that higher bacterial loads had
favored a worse fin condition in the control group (Andrews et al., 2015;
Noble et al., 2012; Turnbull et al., 1996), but maybe a similar effect in
the other fins would have been expected in that case.
4.3. Individual-based correlations
The use of individual correlation analysis can help in understanding
how the effects seen at different levels relate to each other (even when
treatment effects are not considered), particularly when many variables
are assessed. Some correlations found in the present study were trivial
such as those linking Fulton's condition factor with fish individual mass,
the glycogen-to-glucose ratio with glycogen and glucose, or the ones
linking total fin damage scores to specific fin scores. Other correlations,
even when not trivial, were not unexpected, such as the positive cor­
relation between plasma cortisol and hepatic glucose, or the observed
correlations between hepatic glycogen and condition factor (positive
correlation) or hepatic glucose (negative), since they reflect well-known
physiological process such as the increased glucose mobilization from
the liver in relation to the action of stress hormones or to poorer
nutritional conditions (Enes et al., 2009; Vijayan et al., 1991; Wendelaar
Bonga, 1997). The positive correlation found between plasma lactate
and liver glycogen could be the result of the fish directing lactate to­
wards gluconeogenesis or to replenish hepatic glycogen in conditions of
restricted glucose or glycogen availability (Sangiao-Alvarellos et al.,
2005; Vijayan and Moon, 1994). Interestingly, some of the found cor­
relations shed some light on the potential reason behind some of the
results of the trial related to oxidative stress markers. The quantification
of the extent of lipid peroxidation by the TBARS assay is used as an
indicator of oxidative damage in animal tissues. A significant effect of
time was observed in the present study, fish showing higher TBARS
levels on week 4 than on week 8. While this could be interpreted as fish
being under oxidative stress on week 4, a more plausible explanation
arises from the correlation analysis. Several of the correlations indicate
that TBARS levels were positively correlated with the fish nutritional
condition, including a positive correlation between hepatic TBARS and
hepatic glycogen, negative correlations between both hepatic and gill
TBARS and hepatic glucose, or the trend to a positive relationship (p =
0.057) between Liver TBARS and condition factor. Therefore, finding
higher levels of TBARS on week 4 is likely supporting the view of fish
being in a better nutritional condition than on week 8, as discussed in
section 4.1. The link between nutritional status and TBARS is maybe not
unexpected, given that the substrate for lipid peroxidation are lipids,
particularly polyunsaturated fatty acids (Ayala et al., 2014) and it is well
known that tissue lipid content correlates with nutritional status
(Mozsár et al., 2015; Naeem et al., 2016; Salam and Davies, 1994). This,
together with the fact that GPX has a major role in protecting cell
membranes against lipid peroxidation (Jiao et al., 2017; Van der Oost
et al., 2003) might explain the correlations found between GPX activity
and nutritional state-related variables, such as the positive correlation
M. Gesto et al.
between liver and gill GPX, and between both of them and condition
factor. Also, the higher GPX activity found in the fish tanks treated with
foam fractionation, might be related to the higher condition factor in
those groups. In any case, in view of the link between TBARS levels and
the individual nutritional condition, the use of tissue mass or tissue
protein content for normalizing TBARS levels might be questioned, even
when it is by far the most usual way to express TBARS data in current
literature (Defo et al., 2019; Harrysson et al., 2020). We argue that
normalizing TBARS data by tissue lipid content could be a better way to
use TBARS as an oxidative stress indicator, because potential masking
effects due to differential individual nutritional states would be avoided.
Unfortunately, we did not measure the lipid content in our liver and gill
samples, and further tests will be therefore needed to explore the pos­
sibility of using lipid content for normalizing TBARS data in fish studies.
Finally, some of the external damage variables showed negative corre­
lations with performance or nutritional variables, indicating that a
better nutritional condition often occurs together with a lower amount
of external lesions. For example, skin damage score was negatively
correlated with the liver glycogen:glucose ratio and some fin damage
scores, including total fin score, were negatively related to fish mass.
The latter could reflect a higher position of larger fish in social hierar­
chies, in such a way that fish with larger mass would suffer less antag­
onistic displays from conspecifics than smaller fish.
4.4. Conclusions
It was shown in a previous work that ozone and foam fractionation
had important effects in improving water quality in the freshwater RAS
of this experiment in terms of reduced amounts of suspended solids, an
reduced organic matter, bacterial load and turbidity (de Jesus Gregersen
et al., 2021). In this study, we showed that positive effects of the
increased water quality on fish welfare were only mostly detected at the
level of skin damage of the fish. In general, effects of treatments on fish
condition and physiology were of small size and more relevant for foam
fractionation than for ozonation, suggesting that particles of different
size might have effects of different severity on trout physiology. Ozon­
ation was applied in a way that it improved water quality without
having any deleterious effect on the fish. Interestingly, foam fraction­
ation was able to increase water quality beyond the effects of ozonation.
The application of foam fractionation alone had some positive effects on
fish nutritional status and might be a promising tool to improve fish
welfare in freshwater RAS. While positive effect of increased water
quality were modest in this study, it should be taken into account that
the duration of the experiment comprised only a small fraction of the
usual production time in trout aquaculture. Further studies should focus
on investigating the effects of both technologies in the long term on fish
welfare, in order to fully account for their effects on fish performance
and welfare during the whole production cycle.
CRediT authorship contribution statement
Manuel Gesto: Conceptualization, Formal analysis, Data curation,
Investigation, Methodology, Writing – original draft, Visualization. Kim
João de Jesus Gregersen: Conceptualization, Resources, Investigation,
Methodology, Writing – review & editing. Lars-Flemming Pedersen:
Conceptualization, Resources, Methodology, Writing – review & editing,
Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
10
Aquaculture 557 (2022) 738312
Acknowledgements
The study was funded by the European Maritime and Fisheries Fund
(EMFF) and the Danish Ministry of Food, Agriculture and Fisheries
project”RecirkVet” (J.no. 33111-I-17-054) and project Lanozo” (J.no.
33111-I-17-060). We appreciated the assistance of Rasmus F. Jensen,
Ole M. Larsen and Jens H Nedergaard with technical setups and fish
caretaking.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2022.738312.
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