Aquacultural Engineering 60 (2014) 77–83

Contents lists available at ScienceDirect

Aquacultural Engineering
journal homepage: www.elsevier.com/locate/aqua-online

Water treatment with crushed marble or sodium silicate mitigates

combined copper and aluminium toxicity for the early life stages of
Atlantic salmon (Salmo salar L.)
Carlos A. Pessot a , Åse Åtland b,∗ , Helge Liltved b ,
Maria Gabriela Lobos c , Torstein Kristensen b
a
NIVA Chile S.A. Norwegian Institute for Water Research Chile, Del Salvador 264, Of. 306, Puerto Varas, Chile
b
NIVA Norwegian Institute for Water Research, Gaustadalléen 21, NO-0349 Oslo, Norway
c
University of Valparaiso, Department of Chemistry and Biochemistry, Science Faculty, Av. Gran Bretaña 1111, Playa Ancha, Valparaíso, Chile

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

Article history: Monitoring programmes have demonstrated seasonal and short term variation of inlet water quality in
Received 25 July 2013 Chilean salmon hatcheries. Potentially toxic levels of copper (Cu) and aluminium (Al) in many cases may
Accepted 3 April 2014 explain observed increased mortalities during the early life-stages of Atlantic salmon. Therefore, there is
a need for efficient water treatments methods. Two alternative water treatment methods were tested to
Keywords: reduce the toxicity of Cu and Al in fresh water production systems (egg to first feeding): crushed marble
Atlantic salmon
filtration treatment (MF) and sodium silicate addition (SA) treatment. To mimic the observed natural
Water quality
conditions an experiment was set up with short term (2–3 days) pulses of elevated Cu (20 and 30 ␮g L−1 )
Metal toxicity
Fish welfare
and Al (80 and 130 ␮g L−1 ) concentrations. The effect of the two treatments was tested during 3 distinct
Water stabilization developmental periods of the salmon, from 401 to 424, 551 to 567, and 729 to 736 accumulated thermal
Chilean aquaculture units (ATU). Treatments were compared with a positive control group with metal addition (PC), and a
negative control group (NC) exposed to untreated water with no metal addition. Compared to the groups
that received metal pulses with no treatment (PC), mortalities were significantly reduced by both silicate
addition and marble filtration. Both at 896 and 1013 ATU marble filtration (MF) group showed the lowest
mortality. The present study suggests that crushed marble filtration and sodium silicate addition is well
suited to treat Al and Cu pulses in the relevant concentration range tested. This constitutes a low cost
and robust alternative for freshwater aquaculture facilities that need to improve the quality of their inlet
water, and may consequently decrease mortality and improve fish welfare for Atlantic salmon.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction on the more important aquaculture species in Chile and Norway:

Toxicity of waterborne Cu and Al to salmonid fishes has
been well documented (Spear and Pierce, 1979; Pagenkopf, 1983;
Gensemer and Playle, 1999; Santore et al., 2001; EPA, 2007). Pres-
ence of Cu in the aquatic environment is a result of both the
natural geochemistry and anthropogenic inputs. The metal is essen-
tial for metabolic processes, but can be acutely toxic to fish in
concentrations varying from 10 to 1000 ␮g L−1 (Spear and Pierce,
1979). Most of the experimental work regarding Cu toxicity and
toxic concentrations on salmonids is based on studies of Rainbow
trout (Onchorhynchus mykiss), and very little work has been done
∗ Corresponding author at: Norwegian Institute for Water Research (NIVA), Gaus-
tadalléen 21, NO-0349 Oslo, Norway. Tel.: +47 901 47 510; fax: +47 22 18 52 00.
E-mail address: aase.aatland@niva.no (Å. Åtland).
Atlantic salmon (Salmo salar L.). Al toxicity is a documented prob-
lem for smolt-production both in Chile and Norway (Kristensen
et al., 2009). Al is the third most abundant element on earth, but
has no known biological function in vertebrates. The concentration
of free Al in water depends on pH. At a low pH, Al exists as Al3+
ions and hydrolysed as Al(OH)2 + and Al(OH)3 . Al is more toxic for
fish at a pH around 5.0–5.5 as Al hydroxides and above pH 7.5 as
aluminates Al(OH)4 − . Whereas the smolt farms in Norway mainly
have the challenge of elevated concentrations of Al due to acidifi-
cation, the higher pH and buffering capacity of the inlet waters in
Chile (Kristensen et al., 2009) suggests that the problem for fish is
mainly related to the aluminate toxicity.
Organic carbon (TOC) and Ca concentration are the two main
modulators of both Cu and Al toxicity. Dissolved organic car-
bon acts by complexing free metal ions, and thereby reducing
their toxicity (Playle et al., 1993; Matsuo et al., 2004). Ca acts

http://dx.doi.org/10.1016/j.aquaeng.2014.04.001
0144-8609/© 2014 Elsevier B.V. All rights reserved.
78 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83

protective by controlling the permeability of the gill membrane

and the integrity of the ionoregulatory function (Hunn, 1985; Hollis
et al., 1997; Richards and Playle, 1999).
Land-based smolt production in Chile is located mainly (around
60%) in Chiles regions IX (Araucanía region) and X (Los Lagos)
(Infante, 2007). These regions are characterized by a humid-
temperate climate with no distinct dry season but with higher
rainfall during the winter. The most commonly used inlet water
source is spring waters (48%), followed by river water inlets (37%)
and to smaller extent water from wells (15%) (Åtland and Bjerknes,
2009). Spring waters are characterized by very low concentra-
tions of TOC (<1 mg C L−1 ) and in many cases the concentrations
of TOC below the detection limit (0.2 mg C L−1 ). A survey of inlet
water quality in Chilean land-based smolt production facilities has
revealed that about 20 percent of sites had Cu-concentrations that
could potentially be toxic to salmonid fish (>5 ␮g L−1 ). Cu concen-
trations were influenced by ice melting during spring as well as
earthquakes (Pessot, 2010). During spring melting, the observed
increase in Cu concentration was coincidental with decreased Ca
concentrations. This effect has been also observed for Al (Åtland and
Bjerknes, 2009). Al concentrations in Chile are generally low (mean
39.6 ␮g L−1 ), and lower than observed in inlet waters to Norwe-
gian smolt farms (Kristensen et al., 2009). However, extreme at-site
variation was observed, with episodes of total Al concentrations in
inlet waters >300 ␮g L−1 observed in Chile (Kristensen et al., 2009).
Consequently, effective and economically viable water treatment
technologies to deal with Cu and Al problems are high needed to
ensure fish health. Based on the literature as well as previous expe-
rience on developing water treatment methods for Al (Teien et al.,
2006; Kroglund et al., 2001a,b), we decided to test two main meth-
ods: crushed marble filtering and dosing of liquid sodium silicate.
Crushed marble is a water treatment technology which neutral-
izes acid water by adding dissolved calcium carbonate (CaCO3 ) and
raising the pH (US Environment Protection Agency 2004). Mar-
ble is a metamorphic rock composed of re-crystallized carbonate
mainly calcite (polymorphic calcium carbonate) and dolomite (cal-
cium magnesium carbonate), this structure and composition may
allow liberation of Ca and Mg to the water. This is a positive effect
considering the effect Ca and Mg has on sodium exchange over the
gills as well as Al and Cu displacement from target sites in this
organ (Cuthbert and Maetz, 1972; Laurén and McDonald, 1985;
Pagenkopf, 1983; Evans et al., 2005).
Soluble silicate reacts and traps polyvalent metal ions effectively
to produce hydroxyl Al-silicates which are not gill reactive (Exley
et al., 1991, 1997; Teien et al., 2006), and this water treatment tech-
nology is commonly applied in salmonid freshwater production
sites.
The aim of the present experiment was to study the effect of
short-term pulse exposures to a mixture of Cu and Al in naturally
low TOC water quality on the survival and on the development in
the early stages of Atlantic salmon/egg stage until start-feeding),
and furthermore to develop a practical and economically viable
water treatment method to reduce the combined toxicity of Al and
Cu.
2. Materials and methods
2.1. Study area and time of the experiment
The study took place during a period of 81 days (June
25th–September 14th, 2010) and was carried out at the facility of
the salmon company Granja Marina Tornagaleones, located in the
Araucanía region, Southern Chile at the northern slopes of the Vil-
larrica volcano (39◦ 19 22 S, 72◦ 04 46 W). Previous studies on this
site as well as in other farms in the same area have shown a high
Fig. 1. Test design, water inlet (A), stock solutions of Cu and Al (B), addition of
sodium silicate solution (C), retention system for mixing (D), peristaltic pump for
addition of solutions (E), filter for CaCO3 (F), egg trays (G). After 1st pulse tray number
3 was switched with tray number 6 to obtain PC with only 1st pulse (PC1, tray
number 6) and PC with only 2nd and 3rd pulse (PC2, tray number 3). After day 66
(896 ATU) 7 groups were formed in order to generate a group treated in MF only
until day 66 and then with SA: NC (group trays 1 and 2; group tray 6); PC (group
trays 5 and 5; group tray 3); SA (group trays 7, 8 and 9; group tray 10); MF (group
tray 11 and 12).
degree of variability on metal concentrations and also low hard-
ness, which is characteristic for water qualities in this area (Åtland
and Bjerknes, 2009).
2.2. Test materials
Al sulphate (Al2 (SO4 )3 × 18H2 O) (Winkler AL-0660) and Cu sul-
phate (CuSO4 × 5H2 O) (Winkler CO-0660) were added in 3 modules
(Fig. 1) in order to replicate the occurrence of elevated metal con-
centration under natural conditions.
Addition of silicate (SiO2 ) was in the range of 1–2 mg L−1 of sili-
cate (equivalent to 2–4 mg L−1 sodium silicate Na2 SiO3 ). The silicate
was added from a stock solution of 3.07% sodium silicate (Na2 SiO3 )
(see Fig. 1, C) with a flow of 1 ml min−1 (see Fig. 1, E peristaltic
pump). The final concentration was 1.5 mg L−1 of silicate expressed
as SiO2 , equivalent to 3.0 mg L−1 sodium silicate.
Treatment with crushed marble was done by installing a cylin-
drical container where marble particles (3–6 mm in size) were
added to a final volume of 45 L (container of 50 cm diameter, 90 cm
height). Inlet water (in the bottom of the tank) went directly to this
solid phase at 12 L min−1 , as indicated previously, and then coming
out from the top to trays in the module.
2.3. Experiment design and schedule
Eggs/fry were exposed to 3 consecutive pulses, with inter-
mittent periods of stable water conditions. The modules were
(i) Negative control (NC) untreated and without any addition of
metals, (ii) Positive control (PC) with the addition of metals (Al and
Cu), (iii) addition of metals (Al and Cu) treated with silicate (SA)
and (iv) addition of metals (Al and Cu) treated with crushed mar-
ble (MF). Two additional Positive controls were included: Positive
control with only 1 pulse (initial pulse) (PC1) and Positive control
with 2 pulses (2nd and 3rd Pulse) (PC2). The experimental setup
was based on four modules (Fig. 1) corresponding to the different
treatments with 3 replicates. Inlet water flow in each treatment was
12 L min−1 . Each module consisted of 3 trays (40 cm length; 40 cm
width and 10 cm deep, each); making a total of 12 trays numbered
consecutively from 1 to 12 following the previously indicated order
 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83
Fig. 2. (A) Tray 6 was moved to position of tray 3 and tray 3 to position of tray 6 in order to obtain populations with one and two pulses respectively. (B) After Episode 3,
tray 10 from MF was set on the line of SA obtaining a new treatment group (tray 10).
of modules: Module NC, trays 1–3; module PC, trays 4–6 (from PC1
and PC2 were obtained later); module SA, trays 7–9 and module
MF, trays 10–12). Each module consisted of 3 trays (30 cm × 30 cm)
containing 1680 eggs each, with a total of 5040 eggs per test. The
eggs were received at 363 accumulated thermal units (ATU).
As described previously, modules PC, SA and MF (Fig. 1) received
a total of 3 different Al/Cu pulses. Al and Cu were episodically added
to the 3 treatment groups at days 4–7 (Episode 1, 401–424 ATU),
24–26 (Episode 2, 551–567 ATU) and 47–48 (Episode 3, 729–736
ATU) to nominal concentrations (i.e. concentrations in the modules
after adding pulses) of 80 ␮g L−1 Al/20 ␮g L−1 Cu for Episodes 1 and
2, and 130 ␮g L−1 Al/30 ␮g L−1 Cu for Episode 3.
After the first pulse, tray number 3 from module NC was moved
to module PC, and tray number 6 from module PC was moved to
module NC. Trays 4 and 5 received pulses 1, 2 and 3. Tray 3 received
pulses 2 and 3, and tray 6 received pulse 1 (Fig. 2A).
In order to compare the differential effect over mortality of
crushed marble versus silicate treatment on biological material,
once 1st feeding was starting after day 66 (corresponding to 896
ATU) specimens (fry) were rearranged into the following groups in
each module: Module NC (group 1–2 from trays 1 and 2, group 6
from tray 6); module PC (group 4–5 from trays 4 and 5, group 3 from
tray 3); module SA (group 7–8–9 from trays 7, 8 and 9; group 10
from tray 10 coming from marble treatment); module MF (group
11–12 from trays 11 and 12) (Fig. 2B). This way group 10 from orig-
inal marble treatment (MF) was exposed to silicate (SA) in order to
compare marble treated specimens (fry) after first feeding (group
79
11–12) with those that finished marble treatment after the first
feeding (group 10).
2.4. Sampling
Water samples were taken from inlet waters (after treatments
MF and SA when correspond) from day 0 (370 ATU, after 1 day eggs
were received) until day 67 (912 ATU). No artificial heating of water
was applied during the experiment, so ATUs is increased based only
the natural temperatures and time. Control module samples were
taken every 3rd day and every day during Cu/Al pulses.
Water samples taken from the NC included metal analyses (Al
and Cu) and also basic water variables related to metal toxicity
modulation: pH, turbidity, total organic carbon (TOC), Ca and Mg.
Water samples from the 3 different treatments (PC, SA and MF)
were only tested for Al and Cu.
Biological samples (3 replicates of 2 g each) were taken to ana-
lyse total Al and Cu concentrations in complete specimens (eggs
and fry). Samples of eggs at Day 0 and Day 5 were analysed at
the laboratory of the Norwegian University of Life Sciences, Oslo,
Norway, with parallel analyses at the Department of chemistry and
biochemistry at the University of Valparaiso, Chile. Samples from
Day 17 and onwards were analysed at the University of Valparaiso.
Metal determination in biological samples consisted of acid, oxi-
dant digestion at 200 ◦ C for 2 h and detection by Inductive Coupled
Plasma (ICP-AES). Certified reference material DOGFISH DORM-2
from National Research Council Canada was used as reference.
80 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83
2.5. Biological protocols
Egg and fry mortalities were followed on a daily basis for of
each treatment until 424 ATU (1 module, 3 trays each, Fig. 1) for
NC, PC, MF and SA, and also after rearranging trays. After the 1st
pulse 2 additional treatments were summed to the previous ones
(PC1 with 1 Al/Cu pulse and PC2 with 2 Al/Cu pulses). This way
starting from 432 ATU 6 different treatments were followed: NC
(trays 1 and 2); MF (trays 10, 11 and 12); SA (trays 7, 8 and 9);
PC (trays 4 and 5, 3 Al/Cu pulses); PC1 (tray 6, 1 Al/Cu pulse); PC2
(tray 3, 2 Al/Cu pulses). Dead eggs (observation of characteristic
dead opaque and white coloured eggs) and fry were removed and
counted. Deformed organisms and organisms exhibiting abnormal
behaviour were recorded on a daily basis. After sampling eggs and
fry, the samples were frozen at −20 ◦ C. The egg samples were ana-
lysed based on dry weight and results expressed as wet weight (24 h
at 60 ◦ C). The metal concentrations (of Al and Cu) were calculated
based on dry weight and then reported as ␮g g−1 (wet weight),
based on water content of samples. Around the time of hatching,
the time and number of hatched larvae were recorded every day.
The number of fry in each group at the end of the experiment was
recorded, and length and weight measured.
2.6. Chemical analysis
Water temperature was measured daily. Upon sampling of
organisms, water samples for analysis of total organic carbon (TOC),
pH and turbidity of the water were taken. Analyses of Al, Cu, Ca and
Mg concentrations were done by ICP-MS (Induced Coupled Plasma
with mass detector).
2.7. Data analysis
Difference between the different treatments in metal and Ca
concentration in water was tested by Analysis of variance (one-way
ANOVA).
To determine if there were significant differences between
treatments after 912 ATU (from first feeding), the Homogeneity
Test for Regression Coefficients was applied (Gerome C.R. Li, 1964).
The test establishes differences between treatments based on the
slopes presented by different treatments on a defined range of time
(from 912 to 1013) for the dependent variable (mortality).
3. Results
3.1. Background water quality in NC and water Ca concentrations
in treatments
Background water quality monitored during the study
showed stable conditions. Average and standard deviations
were, for pH (7.64 ± 0.09), Turbidity as NTU (0.46 ± 0.22), TOC
(0.21 ± 0.06 mg C L−1 ) and Hardness, based on Ca and Mg con-
centration (16.9 ± 0.59 as mg L−1 CaCO3 ). When comparing Ca
concentrations in water for the different modules, no differ-
ences were detected between PC, NC and SA (4.4 ± 0.04 mg L−1 ,
4.4 ± 0.09 mg L−1 and 4.4 ± 0.11 mg L−1 , respectively). However,
treatment with marble showed an increased in Ca concentration to
7.1 ± 0.37 mg L−1 compared to PC, SA and NC. Results from the MF
module show that crushed marble increased the Ca concentration
in water. This difference was highly significant (ANOVA, P < 0.05).
The Ca concentration over a period of 31 days showed no trends
and stayed stable.
Fig. 3. (A) Achieved aluminium concentrations in each module. (B) Achieved copper
concentrations in each module. These concentrations were obtained after treatment
and represent actual concentrations to which eggs and fry were exposed in each case.
3.2. Al and Cu concentration in water
Fig. 3A and B shows the Al and Cu concentrations in the water
samples. Al and Cu analyses were done at the dates of the metal
pulses. Average concentrations of Al and Cu in 1st, 2nd, and 3rd
pulse in the PC were 128, 140 and 207 ␮g L−1 for Al and 12.0,
15.5 and 26.0 ␮g L−1 for Cu respectively. For Al, the first and sec-
ond pulse the target (nominal values) was 80 ␮g L−1 and for third
pulse the target (nominal value) was 130 ␮g L−1 . For Cu, first and
second pulse target (nominal values) were 20 ␮g L−1 and for the
third pulse the target (nominal value) was 30 ␮g L−1 . In the case of
Al, differences between the nominal values and the obtained con-
centrations are explained by the natural background levels of Al
in the water. The average concentration of Al in the water source
was 11 ± 6.84 ␮g L−1 whereas maximum concentration recorded
was 31 ␮g L−1 . Also some loss of water coordinated molecules
from (Al2 (SO4 ) × 18H2 O) could have affected this. Both conditions
explain why the final concentrations were somewhat higher than
calculated.
3.3. Al and Cu in biological material
Samples showed similar trends for both the metals Al and Cu. An
increase in metal accumulation was observed until 522 ATU (initial
sac fry stage) after the 1st metal pulse, whereas a decrease of the
metal concentrations to background levels (average and standard
deviations were 1.21 ± 0.95 ␮g g−1 ; for Al and 1.01 ± 0.17 ␮g g−1 for
Cu, wet weight), was observed after 559 ATU even though a 2nd and
3rd metal pulse were applied at 559 and 736 ATU.
3.4. Accumulated mortalities in the different treatments
Fig. 4 shows mortality trends in the 6 different treatments (NC,
PC, MF, SA, PC1 and PC2). The actual timing of the Al and Cu pulses
is indicated in the graph, described as “1st Pulse”, “2nd Pulse” and
“3rd Pulse”. The figure also shows the time period when hatch-
ing was recorded at 522 ATU. No significant difference in mortality
was observed between the different treatments as a response to
the 1st pulse of Al and Cu before hatching (corresponding to 401
ATU) (F0.5 > F under Test for Regression Coefficients). However after
hatching and the 2nd pulse of Al and Cu at 567 ATU, it is possible to
observe a first significant difference (F0.5 < F under Test for Regres-
sion Coefficients) in mortality between the eggs that received Al/Cu
pulse without water pre-treatment and those that received water
 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83
Fig. 4. Plot of accumulated mortality at different ATU for each different treatment
and time when aluminium and copper pulses were applied. Negative control (NC);
marble filtration (MF); sodium-silicate addition (SA); positive control 1 pulse (PC1);
positive control 1 pulse (PC1); positive control 2 pulses (PC2); positive control 3
pulses (PC3).
pre-treatment (MF and SA) and NC. The difference in mortality sig-
nificantly increased (F0.5 < F under Test for Regression Coefficients)
at 676 ATU (see Fig. 4). At the end of 3rd pulse 736 ATU, treatment
receiving 1st pulse stabilized and showed similar trends as MF and
SA or NC. At 896 ATU the lowest mortality was observed in fry of MF
treatment. Even though this group was exposed to pulses of Al/Cu,
they performed better than the control, which was not exposed to
these metals.
The accumulated mortality was investigated after first feeding
(Fig. 5) and showed a first group formed by NC, MF, SA and PC1;
and a second group formed by PC2 and PC. This difference is sta-
tistically significant (F0.5 = 2.2540; F = 10.68), considering slopes of
curves from 912 to 1013 ATU.
In order to differentiate the effect of marble treatment when
it is applied only until before first feeding from the effect when
it is applied beyond this point (as shown in Fig. 1), tray 10 from
MF was installed in the module of SA treatment, while trays 11
and 12 stayed under MF treatment. At the end of the period (1013
Fig. 5. Plot of accumulated mortality after first feeding, from 896 ATU to 1013
ATU. Test for Regression Coefficients show statistically significant difference
(F0.5 = 2.2540; F = 10.68) among the slopes of curves in the range from 912 to 1013
ATU, demonstrating a decrease in mortality on the group that was kept on mar-
ble pre-treatment MF 1013 compared to group from MF treatment transferred to
silicate treatment (SA).
81
ATU), MF, had lower levels of mortality (identified as “MF 1013”
in Fig. 5), this is statistically significant (F0.5 = 2.2540; F = 10.68).
Those in MF until 896 ATU and then change to SA (identified as “MF
896” in Fig. 5) showed same mortality as NC or SA. Test for Regres-
sion Coefficients show there is statistically significant difference
(F0.5 = 2.2540; F = 10.68) among the slopes of curves in the range
from 912 to 1013 ATU, indicating that there is a significantly lower
mortality on the group that was kept on marble pre-treatment.
4. Discussion
The present study clearly demonstrated that the effect of com-
bined Al and Cu pulses depend on the life stage of the fish, and
causes a more profound negative effect and higher mortalities after
hatching. Pulses of Al/Cu applied before hatching did not increase
mortality. Other studies demonstrate that sensitivity in early life
stages of fish may differ according to specific moment of exposure,
caused by different mechanisms of toxicity for Al (Yamamoto and
Kobayashi, 1996; Keinänen et al., 2003; Eddy and Talbot, 1983),
and also for Cu (Brauner and Wood, 2002; Davies et al., 2005;
Atli et al., 2006; Craig et al., 2007; Pandey et al., 2008). In later
developmental stages (after hatching), the mechanism of Al tox-
icity is mainly related to its negative effect on the structure and
function of the gill (Rosseland and Staurnes, 1994), being highly
influenced by pH (Kroglund et al., 2002; Fivelstad et al., 2004;
Lacroix, 1989; Watt et al., 2000). Under the current experimen-
tal conditions (concentrations and exposure time) the main toxic
effects on fish of Cu could be related to problems with gill ammonia
excretion (Beaumont et al., 2003).
The present experiment shows that MF and SA treatments have
a positive effect, avoiding increased mortality caused by Al and Cu
pulses. Silicate showed a good effect (same trend as NC) as a con-
sequence of its capacity to form complexes with Al (Witters et al.,
1990; Lacroix et al., 1993), and thereby avoiding it from binding
to primary target sites in gill tissue. Compared to NC and SA, fry
treated with MF presents even lower mortalities. Part of the later
effect may be explained firstly by the reduction of actual concentra-
tion of Cu in water (in a lesser extent Al concentration), in addition
to the protective effect of increased Ca concentration.
Trial treated with Al and Cu and no pre-treatment (PC treat-
ment), showed similar trends (Al and Cu in tissue, complete
specimens) as those described by other authors for Atlantic salmon
(Rombough and Garside, 1982), Brown trout (Salmo trutta) (Haugen
et al., 2007) and Rainbow trout (Onchorynchus mykiss= (Beattie and
Pascoe, 1978) meaning that there was an increase in concentra-
tion of Cu and Al from 501 to 522 ATU, corresponding to the time
when the eggs finished hatching. No accumulation of Al and Cu
was observed beyond this point in fry after 2nd and 3rd pulse in any
module or treatment tested. After hatching, no metal accumulation
was observed in the biological material. Correspondingly, Cu con-
centrations in the control, without any addition of metals, showed
a permanently low concentration during the whole studied period.
This was not seen for Al in the control module which showed the
same trend as PC treatment. Previous result was not consequent
neither with Al concentration in water or with mortalities of this
group, and no definitive cause or explanation was found for this
result.
Compared to PC treatment, the module receiving SA treatment
showed difference in Al concentration in biological material (com-
plete specimens), but no significant difference in Cu concentration.
This result suggested that, under tested concentrations, silicate
did not reduce effectively the concentration of free Cu in water.
As described (Witters et al., 1990; Lacroix et al., 1993), according
to Exley et al. (1997) and Teien et al. (2006), this technology
is based on transformation of chemical form. Even though Cu
82 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83
concentrations were lower than Al, SA treatment was not able to
reduce the accumulation of this metal in the eggs before hatching
(from 501 to 522 ATU). This could be related to a low affinity
constant of this metal to silicate and higher relative affinity
constant to biological moieties in eggs or fish. An important factor
to be considered is the high pH this trial was carried out (which is
representative for Chilean salmon hatcheries).
MF treatment when compared to PC, showed lower concentra-
tions for Al or Cu in the period from 501 to 522 ATU, and higher
Cu concentrations than NC in the same period. Biological tissues
(complete specimens) from MF treatment showed concentrations
of Al and Cu lower than those observed for the silicate treatment.
These results are also in accordance with Al and Cu concentration
in water samples, showing lower concentrations for MF than for PC
treatment. Results from the MF treatment demonstrated the capac-
ity of the system to significantly decrease metal concentrations in
water (ANOVA, P < 0.05) but also significantly increasing the con-
centration of Ca (ANOVA, P < 0.05) compared to the inlet water (see
Fig. 3A and B). These results (Fig. 3A) show that water treated with
crushed marble (MF) caused a decrease in the actual concentration
of Al. This difference was statistically significant under a P < 0.05 by
ANOVA when compared with PC treatment. The final concentra-
tion of Al in water treated with silicate (SA) showed even a slightly
higher concentration than NC when no pulse where added and
also higher when pulses were added compared to trays exposed to
pulses and no silicate or marble treatment. This is explained at some
extent by the presence of Al (38 mg L−1 ) in the original stock solu-
tion of silicate. In the case of Cu, MF treatments showed, the same
as for Al, it decreased the actual concentration of Cu. This difference
was statistically significant (P < 0.05, ANOVA) when compared with
PC.
This suggests that the positive effect of marble in terms of reduc-
ing the toxic effect of Cu and Al from 501 to 522 ATU, is given by
a dual effect, including adsorbing Al and Cu plus liberation of Ca
to the media. This dual effect becomes more evident starting from
676 ATU and afterwards being the MF treated group the one with
lowest mortalities, even lower than NC.
At 567 ATU, after the second pulse of Al and Cu was applied,
it was possible to see that NC and pre-treated groups (MF and SA)
showed lower mortalities than to Cu Al treated groups (PC, PC1 and
PC2). The rates of mortality allow us to distinguish a separation of
trends in the range from 676 to 736 ATU for the different treat-
ments: lower mortalities are shown by NC, pre-treated (MF and
SA) and PC1 (eggs receiving only the first pulse of Al and Cu) and
higher mortalities are shown by Cu Al Treatments (PC and PC2). In
this range of ATU there was significant difference for every treat-
ment at P < 0.05. This trend suggested that, even though there was
no accumulation of metals after hatching (522 ATU), the presence
of metals had a stronger toxic effect after hatching. This would be
a consequence, as described before, of the absence of the protec-
tive function of the chorion and change in the target sites of Al
and Cu to more sensitive structures and functions (like the gills).
This is supported by the fact that tray receiving only the first pulse
(401–424 ATU) with no pre-treatment showed a reduced mor-
tality rate after hatching (522 ATU) compared to those with no
pre-treatment receiving the second pulse of Al and Cu (551–567
ATU). The rate of mortality changed on the group that was split at
896 ATU - fry treated until 896 ATU and then transferred to sili-
cate increased their rate of mortality ending up at the same level of
mortality as the silicate pre-treated system, and fry kept on mar-
ble pre-treated water maintained a lower mortality ending up (at
1013 ATU) with the lowest mortality of all 7 groups tested. This
difference is attributed to the fact that MF treatment adds Ca to the
media, which is in accordance to what described by Brauner and
Wood (2002) and Davies et al. (2005) regarding interference of Cu
on Ca and Mg target sites.
5. Conclusion
The present study confirmed that marble filtration as well as
sodium silicate water treatment can be an effective, low cost and
robust alternative for hatcheries facing elevated levels of Al and Cu
in their inlet waters. The water requirement during early life stages
in salmonid aquaculture is relatively low, making this technology
feasible at this life-stage. The experiment is actually carried out in
a commercial facility, albeit not on a full scale egg group. A 10–20
times up-scaling of the marble filter or sodium silicate dosing is
quite feasible in terms of costs and/or space requirements.
There was an effective decrease of Al and Cu levels in the marble
filtrated water which in part can explain the better survival of eggs
and fry exposed to this treatment, but it is also shown that increas-
ing the concentration of Ca by marble treatment can decrease the
background levels of mortality. This effect could be responsible
for the better performance after first feeding, regardless of the
presence of toxic concentrations of Al and Cu. After hatching, as
described previously, no accumulation was observed, but the toxic
effect was stronger clearly suggesting that this is a more sensitive
state possibly given by the Al and Cu targets in biological tissue.
Sodium silicate treatment also confirmed to be an effective tech-
nology to reduce adverse effect driven by Al and Cu in water. Low
decrease in water Al concentration by MF, suggests that a com-
bined application of marble and silicate should be studied in order
to validate this as an option to control combined metal toxicity.
Acknowledgments
Special thanks to Granja Marina Tornagaleones represented by
Ignacio Ochagavía whom allowed the information obtained dur-
ing this project to be published in this article. Also thanks the
company for facilitating its installations and valuable time that
Andres Streuly, Mauricio Schulz, Cristián San Martin, Beatriz Gar-
rido, Roberto Michell and Claudio Bernales dedicated to this project.
Thanks to research assistant Anne May Ilestad for her help in run-
ning the experiment, and to Rafael Pessot for his support in the
statistical analysis of the data. This research was supported by funds
received from CORFO (Corporación de Fomento para la Producción),
Ministry of Economy, Chile, Reference No. O9IERI-5623 and Granja
Marina Tornagaleones.
References
Åtland, Å., Bjerknes, V., 2009. Calidad de Agua para el Cultivo de Smolts en Chile. In:
Åtland, Å., Bjerknes, V. (Eds.), Book. , first ed, 139 pp. NIVA Chile, Puerto Varas,
Chile. ISBN 978-956-8861-00-1.
Atli, G., Alptekin, Ö., Tükel, S., Canli, M., 2006. Response of catalase activity to Ag+ ,
Cd2+ , Cr6+ , Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus.
Comp. Biochem. Physiol. C143, 218–224.
Beattie, J.H., Pascoe, D., 1978. Cadmium uptake by rainbow trout, Salmo gairdneri
eggs and alevins. J. Fish Biol. 13, 631–638.
Beaumont, M.W., Butler, P.J., Taylor, E.W., 2003. Exposure of Brown trout (Salmo
trutta) to sublethal concentration of copper in soft acidic water: effects upon
gas exchange and ammonia accumulation. J. Exp. Biol. 206, 153–162.
Brauner, C.J., Wood, C.W., 2002. Ionregulatory development and the effect of chronic
silver exposure on growth, survival, and sublethal indicators of toxicity in early
life stages of rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B172,
153–162.
Craig, P.M., Wood, C.M., McClelland, G.B., 2007. Oxidative stress response and gene.
Regul. Integr. Comp. Physiol. 293, 1882–1892.
Cuthbert, A.W., Maetz, J., 1972. The effect of calcium and magnesium on sodium
fluxes through the gills of Carassius auratus L. J. Physiol. (Lond.) 221, 633–643.
Davies, T.D., Pickard, J., Hall, K.J., 2005. Acute molybdenum toxicity to rainbow trout
and other fish. J. Environ. Eng. Sci. 4, 481–485.
Eddy, F.B., Talbot, C., 1983. Formation of perivitelline fluid in Atlantic salmon (Salmo
salar) in fresh water and in solutions of metal ions. Comp. Biochem. Physiol. C75,
1–4.
EPA, 2007. Aquatic Life Ambient Freshwater Quality Criteria – Copper. 2007 Revision.
EPA-822-R-07-001.
Exley, C., Chappel, J.S., Brichall, J.D., 1991. A mechanism for acute aluminium toxicity
in fish. J. Theor. Biol. 151, 417–428.
 C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83
Exley, C., Pinnegar, J.K., Taylor, H., 1997. Hydroxyaluminosilicates and acute alu-
minium toxicity in fish. J. Theor. Biol. 189, 133–139.
Evans, P.H., Piermarini, P.M., Choe, K.P., 2005. Multifunctional fish gill: dominant
site of gas exchange, osmoregulation, acid-base regulation and excretion of
nitrogenous waste. Physiol. Rev. 81 (1), 97–177.
Fivelstad, S., Olsen, A.B., Stefansson, S., Waagbo, R., Kroglund, F., Colt, J., 2004. Lack of
long-term dublethal effects of reduced freshwater pH alone on Atlantic salmon
(Salmo salar) smolts subsequently transferred to seawater. Can. J. Fish. Aquat.
Sci. 61 (4), 511–518.
Gensemer, R.W., Playle, R.C., 1999. The bioavailability and toxicity of aluminium in
aquatic environments. In: Logan, T.J. (Ed.), Crit. Rev. Environ. Sci. Technol. 29,
315–450.
Gerome C.R. Li, 1964. Introduction to Statistical Inference, second ed. Edward
Brothers Inc., Ann Arbor, MI, USA.
Haugen, T., Kristensen, T., Kroglund, K., Håvardstun, J., Kleiven, E., 2007. Study of
the effect of cadmium on the early life stages of brown trout (Salmo trutta) at
different levels of water hardness NIVA Report, Serial No. 5468-2007, 47 pp.
ISBN 978-82-577-5203-3.
Hollis, L., Muechen, L., Playle, R.C., 1997. Influence of dissolved organic matter on
copper binding, and calcium on cadmium binding, by gills of rainbow trout. J.
Fish Biol. 50, 703–720.
Hunn, J.B., 1985. Role of calcium on gill function in freshwater fish. Comp. Biochem.
Physiol. A82, 543–547.
Infante, R., 2007. Autorregulación con Estándares de Calidad en toda la Cadena: Siges
y APL. Salmon, Chile.
Keinänen, M., Tigerstedt, C., Kalax, P., Vourinen, P.J., 2003. Fertilization and
embryonic development of whitefish (Coragenus lavaretus lavaretus) in acidic
low-ionic-strength water with aluminum. Ecotoxicol. Environ. Saf. 55, 314–329.
Kristensen, T., Åtland, Å., Rosten, T., Urke, H., Rosseland, B.O., 2009. Important
influent water quality parameters at freshwater production sites in 2 salmon
producing countries. Aquacult. Eng. 41, 53–59.
Kroglund, F., Teien, H.C., Rosseland, B.O., Salbu, B., 2001a. Time and pH dependent
recovery from aluminium in mixing zones between acid and non-acid rivers.
Water Air Soil Pollut. 130, 905–910.
Kroglund, F., Teien, H.C., Rosseland, B.O., Salbu, B., 2001b. Water quality dependent
recovery from aluminium stress in Atlantic salmon smolt. Water Air Soil Pollut.
130, 911–916.
Kroglund, F., Wright, R.F., Burchart, C., 2002. Acidification and Atlantic Salmon: Crit-
ical Limits for Norwegian Rivers. Norwegian Institute for Water Research, Oslo,
Report nr 111, 61 pp. Oslo, Norway. ISBN 82-577-4151-5.
Lacroix, G.L., 1989. Ecological and physiological responses of Atlantic salmon in
acidic organic rivers of Nova Scotia, Canada. Water Air Soil Pollut. 46, 375–386.
Lacroix, G.L., Petersen, R.H., Belfey, C.S., Martin-Robichaud, D.J., 1993. Aluminium
dynamics on gills of Atlantic salmon fry in presence of citrate and effects on
integrity of gill structure. Aquat. Toxicol. 27, 373–402.
Laurén, D.J., McDonald, D.G., 1985. Effects of copper on branchial ionoregulation in
the rainbow trout, Salmo rairdneri Richardson. J. Comp. Physiol. 155, 635–644.
83
Matsuo, Y.O., Playle, R.C., Val, A.L., Wood, Ch.M., 2004. Physiological action of dis-
solved organic matter in rainbow trout in presence and absence of copper:
sodium uptake kinetics and unidirectional flux rates in hard and softwater.
Aquat. Toxicol. 70, 63–81.
Pagenkopf, G.K., 1983. Gill surface interaction model for trace-metal toxicity to
fishes: role of complexation, pH and water hardness. Environ. Sci. Technol. 17,
342–347.
Pandey, S., Ahamd Ansar, R., Ali, M., Kaur, M., Hayat, F., Ahmad, F., Raisuddin, S.,
2008. Effects of exposure to multiple trace metals on biochemical, histological
and ultrastructural features of gills of freshwater fish, Channa punctate Bloch.
Chem. Biol. Interact. 174, 183–192.
Pessot, C., 2010. Early effects of metals in Brown trout, potential implications in in
Chilean salmon industry on the scope of local fresh water qualities. In: Open
Seminar, April 23rd 2010, Puerto Montt, Chile.
Playle, R.C., Dixon, D., Burnison, K., 1993. Copper and cadmium binding to fish gills:
modification by dissolved organic carbon and synthetic ligands. Can. J. Fish.
Aquat. Sci. 50, 2667–2677.
Richards, G.J., Playle, R.C., 1999. Protective effect of calcium against the physiological
effect of exposure to a combination of cadmium and copper in rainbow trout
(Oncorhynchus mykiss). Can. J. Zool. 77 (7), 1035–1047.
Rombough, P.J., Garside, E.T., 1982. Cadmium toxicity and bioaccumulation in
eggs and alevines of the Atlantic salmon, Salmo salar. Can. J. Zool. 60,
2006–2014.
Rosseland, B.O., Staurnes, M., 1994. Physiological mechanism for toxic effects and
resistance to acidic water: an ecophysiological and ecotoxicological approach.
In: Steinberg, C.E.W., Wright, R.G. (Eds.), Acidification of Freshwater Ecosystems
Implications for the Future. Wiley, pp. 227–246.
Santore, R.C., Di Toro, D.M., Paquin, P.R., Allen, H.E., Meyer, J.S., 2001. A biotic
ligand model of the acute toxicity of metals. II. Application to acute cop-
per toxicity in freshwater fish and daphnia. Environ. Toxicol. Chem. 20,
2397–2402.
Spear, P.A., Pierce, R.C., 1979. Copper in the aquatic environment: chemistry dis-
tribution and toxicology. National Research Council of Canada, Report No.
16454.
Teien, H.C., Kroglund, F., Åtland, Å., Rosseland, B.O., Salbu, B., 2006. Sodium silicate
as. alternative to liming-reduced aluminium toxicity for Atlantic salmon (Salmo
salar L.) in. unstable mixing zones. Sci. Total Environ. 358, 151–163.
US, 2004. Environmental Protection Agency Guidelines for water reuse EPA/625/R-
04/108, September.
Watt, W.D., Scott, C.D., Zamora, P.J., White, W.J., 2000. Acid toxicity levels in Nova
Scotia rivers have not declined in synchrony with the decline in sulphate levels.
Water Air Soil Pollut. 118 (3–4), 203–229.
Witters, H.E., Van Puymbroeck, S., Vangenechten, J.H.D., Vanderborght, O.L.J., 1990.
The effects of humic substances on the toxicity of aluminium to adult rainbow
trout, Oncorhynchus mykiss (Walbaum). J. Fish Biol. 37, 43–53.
Yamamoto, T.S., Kobayashi, W., 1996. Formation of enveloping layer of the chum
salmon egg in isotonic salt solutions. J. Fish Biol. 49, 895–909.