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Aquacultural Engineering 60 (2014) 77–83

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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:


Atlantic salmon (Salmo salar L.). Al toxicity is a documented prob-
Toxicity of waterborne Cu and Al to salmonid fishes has lem for smolt-production both in Chile and Norway (Kristensen
been well documented (Spear and Pierce, 1979; Pagenkopf, 1983; et al., 2009). Al is the third most abundant element on earth, but
Gensemer and Playle, 1999; Santore et al., 2001; EPA, 2007). Pres- has no known biological function in vertebrates. The concentration
ence of Cu in the aquatic environment is a result of both the of free Al in water depends on pH. At a low pH, Al exists as Al3+
natural geochemistry and anthropogenic inputs. The metal is essen- ions and hydrolysed as Al(OH)2 + and Al(OH)3 . Al is more toxic for
tial for metabolic processes, but can be acutely toxic to fish in fish at a pH around 5.0–5.5 as Al hydroxides and above pH 7.5 as
concentrations varying from 10 to 1000 ␮g L−1 (Spear and Pierce, aluminates Al(OH)4 − . Whereas the smolt farms in Norway mainly
1979). Most of the experimental work regarding Cu toxicity and have the challenge of elevated concentrations of Al due to acidifi-
toxic concentrations on salmonids is based on studies of Rainbow cation, the higher pH and buffering capacity of the inlet waters in
trout (Onchorhynchus mykiss), and very little work has been done 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
∗ Corresponding author at: Norwegian Institute for Water Research (NIVA), Gaus- modulators of both Cu and Al toxicity. Dissolved organic car-
tadalléen 21, NO-0349 Oslo, Norway. Tel.: +47 901 47 510; fax: +47 22 18 52 00. bon acts by complexing free metal ions, and thereby reducing
E-mail address: aase.aatland@niva.no (Å. Åtland). 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
Fig. 1. Test design, water inlet (A), stock solutions of Cu and Al (B), addition of
increase in Cu concentration was coincidental with decreased Ca
sodium silicate solution (C), retention system for mixing (D), peristaltic pump for
concentrations. This effect has been also observed for Al (Åtland and addition of solutions (E), filter for CaCO3 (F), egg trays (G). After 1st pulse tray number
Bjerknes, 2009). Al concentrations in Chile are generally low (mean 3 was switched with tray number 6 to obtain PC with only 1st pulse (PC1, tray
39.6 ␮g L−1 ), and lower than observed in inlet waters to Norwe- number 6) and PC with only 2nd and 3rd pulse (PC2, tray number 3). After day 66
gian smolt farms (Kristensen et al., 2009). However, extreme at-site (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
variation was observed, with episodes of total Al concentrations in
trays 5 and 5; group tray 3); SA (group trays 7, 8 and 9; group tray 10); MF (group
inlet waters >300 ␮g L−1 observed in Chile (Kristensen et al., 2009). tray 11 and 12).
Consequently, effective and economically viable water treatment
technologies to deal with Cu and Al problems are high needed to
degree of variability on metal concentrations and also low hard-
ensure fish health. Based on the literature as well as previous expe-
ness, which is characteristic for water qualities in this area (Åtland
rience on developing water treatment methods for Al (Teien et al.,
and Bjerknes, 2009).
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- 2.2. Test materials
izes acid water by adding dissolved calcium carbonate (CaCO3 ) and
raising the pH (US Environment Protection Agency 2004). Mar- Al sulphate (Al2 (SO4 )3 × 18H2 O) (Winkler AL-0660) and Cu sul-
ble is a metamorphic rock composed of re-crystallized carbonate phate (CuSO4 × 5H2 O) (Winkler CO-0660) were added in 3 modules
mainly calcite (polymorphic calcium carbonate) and dolomite (cal- (Fig. 1) in order to replicate the occurrence of elevated metal con-
cium magnesium carbonate), this structure and composition may centration under natural conditions.
allow liberation of Ca and Mg to the water. This is a positive effect Addition of silicate (SiO2 ) was in the range of 1–2 mg L−1 of sili-
considering the effect Ca and Mg has on sodium exchange over the cate (equivalent to 2–4 mg L−1 sodium silicate Na2 SiO3 ). The silicate
gills as well as Al and Cu displacement from target sites in this was added from a stock solution of 3.07% sodium silicate (Na2 SiO3 )
organ (Cuthbert and Maetz, 1972; Laurén and McDonald, 1985; (see Fig. 1, C) with a flow of 1 ml min−1 (see Fig. 1, E peristaltic
Pagenkopf, 1983; Evans et al., 2005). pump). The final concentration was 1.5 mg L−1 of silicate expressed
Soluble silicate reacts and traps polyvalent metal ions effectively as SiO2 , equivalent to 3.0 mg L−1 sodium silicate.
to produce hydroxyl Al-silicates which are not gill reactive (Exley Treatment with crushed marble was done by installing a cylin-
et al., 1991, 1997; Teien et al., 2006), and this water treatment tech- drical container where marble particles (3–6 mm in size) were
nology is commonly applied in salmonid freshwater production added to a final volume of 45 L (container of 50 cm diameter, 90 cm
sites. height). Inlet water (in the bottom of the tank) went directly to this
The aim of the present experiment was to study the effect of solid phase at 12 L min−1 , as indicated previously, and then coming
short-term pulse exposures to a mixture of Cu and Al in naturally out from the top to trays in the module.
low TOC water quality on the survival and on the development in
the early stages of Atlantic salmon/egg stage until start-feeding), 2.3. Experiment design and schedule
and furthermore to develop a practical and economically viable
water treatment method to reduce the combined toxicity of Al and Eggs/fry were exposed to 3 consecutive pulses, with inter-
Cu. 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
2. Materials and methods Cu), (iii) addition of metals (Al and Cu) treated with silicate (SA)
and (iv) addition of metals (Al and Cu) treated with crushed mar-
2.1. Study area and time of the experiment ble (MF). Two additional Positive controls were included: Positive
control with only 1 pulse (initial pulse) (PC1) and Positive control
The study took place during a period of 81 days (June with 2 pulses (2nd and 3rd Pulse) (PC2). The experimental setup
25th–September 14th, 2010) and was carried out at the facility of was based on four modules (Fig. 1) corresponding to the different
the salmon company Granja Marina Tornagaleones, located in the treatments with 3 replicates. Inlet water flow in each treatment was
Araucanía region, Southern Chile at the northern slopes of the Vil- 12 L min−1 . Each module consisted of 3 trays (40 cm length; 40 cm
larrica volcano (39◦ 19 22 S, 72◦ 04 46 W). Previous studies on this width and 10 cm deep, each); making a total of 12 trays numbered
site as well as in other farms in the same area have shown a high consecutively from 1 to 12 following the previously indicated order
C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83 79

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 11–12) with those that finished marble treatment after the first
and PC2 were obtained later); module SA, trays 7–9 and module feeding (group 10).
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 2.4. Sampling
eggs were received at 363 accumulated thermal units (ATU).
As described previously, modules PC, SA and MF (Fig. 1) received Water samples were taken from inlet waters (after treatments
a total of 3 different Al/Cu pulses. Al and Cu were episodically added MF and SA when correspond) from day 0 (370 ATU, after 1 day eggs
to the 3 treatment groups at days 4–7 (Episode 1, 401–424 ATU), were received) until day 67 (912 ATU). No artificial heating of water
24–26 (Episode 2, 551–567 ATU) and 47–48 (Episode 3, 729–736 was applied during the experiment, so ATUs is increased based only
ATU) to nominal concentrations (i.e. concentrations in the modules the natural temperatures and time. Control module samples were
after adding pulses) of 80 ␮g L−1 Al/20 ␮g L−1 Cu for Episodes 1 and taken every 3rd day and every day during Cu/Al pulses.
2, and 130 ␮g L−1 Al/30 ␮g L−1 Cu for Episode 3. Water samples taken from the NC included metal analyses (Al
After the first pulse, tray number 3 from module NC was moved and Cu) and also basic water variables related to metal toxicity
to module PC, and tray number 6 from module PC was moved to modulation: pH, turbidity, total organic carbon (TOC), Ca and Mg.
module NC. Trays 4 and 5 received pulses 1, 2 and 3. Tray 3 received Water samples from the 3 different treatments (PC, SA and MF)
pulses 2 and 3, and tray 6 received pulse 1 (Fig. 2A). were only tested for Al and Cu.
In order to compare the differential effect over mortality of Biological samples (3 replicates of 2 g each) were taken to ana-
crushed marble versus silicate treatment on biological material, lyse total Al and Cu concentrations in complete specimens (eggs
once 1st feeding was starting after day 66 (corresponding to 896 and fry). Samples of eggs at Day 0 and Day 5 were analysed at
ATU) specimens (fry) were rearranged into the following groups in the laboratory of the Norwegian University of Life Sciences, Oslo,
each module: Module NC (group 1–2 from trays 1 and 2, group 6 Norway, with parallel analyses at the Department of chemistry and
from tray 6); module PC (group 4–5 from trays 4 and 5, group 3 from biochemistry at the University of Valparaiso, Chile. Samples from
tray 3); module SA (group 7–8–9 from trays 7, 8 and 9; group 10 Day 17 and onwards were analysed at the University of Valparaiso.
from tray 10 coming from marble treatment); module MF (group Metal determination in biological samples consisted of acid, oxi-
11–12 from trays 11 and 12) (Fig. 2B). This way group 10 from orig- dant digestion at 200 ◦ C for 2 h and detection by Inductive Coupled
inal marble treatment (MF) was exposed to silicate (SA) in order to Plasma (ICP-AES). Certified reference material DOGFISH DORM-2
compare marble treated specimens (fry) after first feeding (group 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, Fig. 3. (A) Achieved aluminium concentrations in each module. (B) Achieved copper
the time and number of hatched larvae were recorded every day. concentrations in each module. These concentrations were obtained after treatment
and represent actual concentrations to which eggs and fry were exposed in each case.
The number of fry in each group at the end of the experiment was
recorded, and length and weight measured.
3.2. Al and Cu concentration in water

2.6. Chemical analysis 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
Water temperature was measured daily. Upon sampling of pulses. Average concentrations of Al and Cu in 1st, 2nd, and 3rd
organisms, water samples for analysis of total organic carbon (TOC), pulse in the PC were 128, 140 and 207 ␮g L−1 for Al and 12.0,
pH and turbidity of the water were taken. Analyses of Al, Cu, Ca and 15.5 and 26.0 ␮g L−1 for Cu respectively. For Al, the first and sec-
Mg concentrations were done by ICP-MS (Induced Coupled Plasma ond pulse the target (nominal values) was 80 ␮g L−1 and for third
with mass detector). 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-
2.7. Data analysis centrations are explained by the natural background levels of Al
in the water. The average concentration of Al in the water source
Difference between the different treatments in metal and Ca was 11 ± 6.84 ␮g L−1 whereas maximum concentration recorded
concentration in water was tested by Analysis of variance (one-way was 31 ␮g L−1 . Also some loss of water coordinated molecules
ANOVA). from (Al2 (SO4 ) × 18H2 O) could have affected this. Both conditions
To determine if there were significant differences between explain why the final concentrations were somewhat higher than
treatments after 912 ATU (from first feeding), the Homogeneity calculated.
Test for Regression Coefficients was applied (Gerome C.R. Li, 1964).
The test establishes differences between treatments based on the
3.3. Al and Cu in biological material
slopes presented by different treatments on a defined range of time
(from 912 to 1013) for the dependent variable (mortality).
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
3. Results 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
3.1. Background water quality in NC and water Ca concentrations Cu, wet weight), was observed after 559 ATU even though a 2nd and
in treatments 3rd metal pulse were applied at 559 and 736 ATU.

Background water quality monitored during the study 3.4. Accumulated mortalities in the different treatments
showed stable conditions. Average and standard deviations
were, for pH (7.64 ± 0.09), Turbidity as NTU (0.46 ± 0.22), TOC Fig. 4 shows mortality trends in the 6 different treatments (NC,
(0.21 ± 0.06 mg C L−1 ) and Hardness, based on Ca and Mg con- PC, MF, SA, PC1 and PC2). The actual timing of the Al and Cu pulses
centration (16.9 ± 0.59 as mg L−1 CaCO3 ). When comparing Ca is indicated in the graph, described as “1st Pulse”, “2nd Pulse” and
concentrations in water for the different modules, no differ- “3rd Pulse”. The figure also shows the time period when hatch-
ences were detected between PC, NC and SA (4.4 ± 0.04 mg L−1 , ing was recorded at 522 ATU. No significant difference in mortality
4.4 ± 0.09 mg L−1 and 4.4 ± 0.11 mg L−1 , respectively). However, was observed between the different treatments as a response to
treatment with marble showed an increased in Ca concentration to the 1st pulse of Al and Cu before hatching (corresponding to 401
7.1 ± 0.37 mg L−1 compared to PC, SA and NC. Results from the MF ATU) (F0.5 > F under Test for Regression Coefficients). However after
module show that crushed marble increased the Ca concentration hatching and the 2nd pulse of Al and Cu at 567 ATU, it is possible to
in water. This difference was highly significant (ANOVA, P < 0.05). observe a first significant difference (F0.5 < F under Test for Regres-
The Ca concentration over a period of 31 days showed no trends sion Coefficients) in mortality between the eggs that received Al/Cu
and stayed stable. pulse without water pre-treatment and those that received water
C.A. Pessot et al. / Aquacultural Engineering 60 (2014) 77–83 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
Fig. 4. Plot of accumulated mortality at different ATU for each different treatment stages of fish may differ according to specific moment of exposure,
and time when aluminium and copper pulses were applied. Negative control (NC);
caused by different mechanisms of toxicity for Al (Yamamoto and
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 Kobayashi, 1996; Keinänen et al., 2003; Eddy and Talbot, 1983),
pulses (PC3). 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
pre-treatment (MF and SA) and NC. The difference in mortality sig-
function of the gill (Rosseland and Staurnes, 1994), being highly
nificantly increased (F0.5 < F under Test for Regression Coefficients)
influenced by pH (Kroglund et al., 2002; Fivelstad et al., 2004;
at 676 ATU (see Fig. 4). At the end of 3rd pulse 736 ATU, treatment
Lacroix, 1989; Watt et al., 2000). Under the current experimen-
receiving 1st pulse stabilized and showed similar trends as MF and
tal conditions (concentrations and exposure time) the main toxic
SA or NC. At 896 ATU the lowest mortality was observed in fry of MF
effects on fish of Cu could be related to problems with gill ammonia
treatment. Even though this group was exposed to pulses of Al/Cu,
excretion (Beaumont et al., 2003).
they performed better than the control, which was not exposed to
The present experiment shows that MF and SA treatments have
these metals.
a positive effect, avoiding increased mortality caused by Al and Cu
The accumulated mortality was investigated after first feeding
pulses. Silicate showed a good effect (same trend as NC) as a con-
(Fig. 5) and showed a first group formed by NC, MF, SA and PC1;
sequence of its capacity to form complexes with Al (Witters et al.,
and a second group formed by PC2 and PC. This difference is sta-
1990; Lacroix et al., 1993), and thereby avoiding it from binding
tistically significant (F0.5 = 2.2540; F = 10.68), considering slopes of
to primary target sites in gill tissue. Compared to NC and SA, fry
curves from 912 to 1013 ATU.
treated with MF presents even lower mortalities. Part of the later
In order to differentiate the effect of marble treatment when
effect may be explained firstly by the reduction of actual concentra-
it is applied only until before first feeding from the effect when
tion of Cu in water (in a lesser extent Al concentration), in addition
it is applied beyond this point (as shown in Fig. 1), tray 10 from
to the protective effect of increased Ca concentration.
MF was installed in the module of SA treatment, while trays 11
Trial treated with Al and Cu and no pre-treatment (PC treat-
and 12 stayed under MF treatment. At the end of the period (1013
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.
Fig. 5. Plot of accumulated mortality after first feeding, from 896 ATU to 1013 This result suggested that, under tested concentrations, silicate
ATU. Test for Regression Coefficients show statistically significant difference did not reduce effectively the concentration of free Cu in water.
(F0.5 = 2.2540; F = 10.68) among the slopes of curves in the range from 912 to 1013
As described (Witters et al., 1990; Lacroix et al., 1993), according
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 to Exley et al. (1997) and Teien et al. (2006), this technology
silicate treatment (SA). 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 5. Conclusion
reduce the accumulation of this metal in the eggs before hatching
(from 501 to 522 ATU). This could be related to a low affinity The present study confirmed that marble filtration as well as
constant of this metal to silicate and higher relative affinity sodium silicate water treatment can be an effective, low cost and
constant to biological moieties in eggs or fish. An important factor robust alternative for hatcheries facing elevated levels of Al and Cu
to be considered is the high pH this trial was carried out (which is in their inlet waters. The water requirement during early life stages
representative for Chilean salmon hatcheries). in salmonid aquaculture is relatively low, making this technology
MF treatment when compared to PC, showed lower concentra- feasible at this life-stage. The experiment is actually carried out in
tions for Al or Cu in the period from 501 to 522 ATU, and higher a commercial facility, albeit not on a full scale egg group. A 10–20
Cu concentrations than NC in the same period. Biological tissues times up-scaling of the marble filter or sodium silicate dosing is
(complete specimens) from MF treatment showed concentrations quite feasible in terms of costs and/or space requirements.
of Al and Cu lower than those observed for the silicate treatment. There was an effective decrease of Al and Cu levels in the marble
These results are also in accordance with Al and Cu concentration filtrated water which in part can explain the better survival of eggs
in water samples, showing lower concentrations for MF than for PC and fry exposed to this treatment, but it is also shown that increas-
treatment. Results from the MF treatment demonstrated the capac- ing the concentration of Ca by marble treatment can decrease the
ity of the system to significantly decrease metal concentrations in background levels of mortality. This effect could be responsible
water (ANOVA, P < 0.05) but also significantly increasing the con- for the better performance after first feeding, regardless of the
centration of Ca (ANOVA, P < 0.05) compared to the inlet water (see presence of toxic concentrations of Al and Cu. After hatching, as
Fig. 3A and B). These results (Fig. 3A) show that water treated with described previously, no accumulation was observed, but the toxic
crushed marble (MF) caused a decrease in the actual concentration effect was stronger clearly suggesting that this is a more sensitive
of Al. This difference was statistically significant under a P < 0.05 by state possibly given by the Al and Cu targets in biological tissue.
ANOVA when compared with PC treatment. The final concentra- Sodium silicate treatment also confirmed to be an effective tech-
tion of Al in water treated with silicate (SA) showed even a slightly nology to reduce adverse effect driven by Al and Cu in water. Low
higher concentration than NC when no pulse where added and decrease in water Al concentration by MF, suggests that a com-
also higher when pulses were added compared to trays exposed to bined application of marble and silicate should be studied in order
pulses and no silicate or marble treatment. This is explained at some to validate this as an option to control combined metal toxicity.
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 Acknowledgments
as for Al, it decreased the actual concentration of Cu. This difference
was statistically significant (P < 0.05, ANOVA) when compared with Special thanks to Granja Marina Tornagaleones represented by
PC. Ignacio Ochagavía whom allowed the information obtained dur-
This suggests that the positive effect of marble in terms of reduc- ing this project to be published in this article. Also thanks the
ing the toxic effect of Cu and Al from 501 to 522 ATU, is given by company for facilitating its installations and valuable time that
a dual effect, including adsorbing Al and Cu plus liberation of Ca Andres Streuly, Mauricio Schulz, Cristián San Martin, Beatriz Gar-
to the media. This dual effect becomes more evident starting from rido, Roberto Michell and Claudio Bernales dedicated to this project.
676 ATU and afterwards being the MF treated group the one with Thanks to research assistant Anne May Ilestad for her help in run-
lowest mortalities, even lower than NC. ning the experiment, and to Rafael Pessot for his support in the
At 567 ATU, after the second pulse of Al and Cu was applied, statistical analysis of the data. This research was supported by funds
it was possible to see that NC and pre-treated groups (MF and SA) received from CORFO (Corporación de Fomento para la Producción),
showed lower mortalities than to Cu Al treated groups (PC, PC1 and Ministry of Economy, Chile, Reference No. O9IERI-5623 and Granja
PC2). The rates of mortality allow us to distinguish a separation of Marina Tornagaleones.
trends in the range from 676 to 736 ATU for the different treat-
ments: lower mortalities are shown by NC, pre-treated (MF and
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