2007) 451–463www.elsevier.com/locate/aqua-online

Aquaculture 270 (

Effect of increasing salinity on physiological response in juvenilescallops Argopecten purpuratus at two rearing temperatures

Gaspar Soria ⁎,1, Germán Merino 1, Elisabeth von Brand 1

Universidad Católica del Norte, Larrondo 1281, CC 117, Coquimbo, IV región, Chile

Received 17 October 2006; received in revised form 15 May 2007; accepted 17 May 2007

Abstract

Argopecten purpuratus can be cultivated using Recirculating Aquaculture Systems (RAS) as a method to increase production.In order to determine physiological response of A. purpuratus under different salinities and temperature conditions, two groups ofjuvenile scallops (small: h=6.5 mm and large: h=25.5 mm) were acclimated and close-cultured at salinities of 34, 38, and 42 g/l,at 16 and 22 °C and fed on Isochrysis galbana and Chaetoceros calcitrans. Survival, shell growth and scope for growth weredetermined at the end of the trials. Survival showed an inverse relationship with temperature and ammonia levels. In small scallopsan increase in salinity at 16 °C increased survival. However, this relationship was not evident at 22 °C. On the other hand, salinitydid not affect survival of large juveniles. Small juveniles had a lower survival (approximately 40%) than larger scallops (up to85%) throughout the trials. Oxygen consumption was not affected by salinity. Small scallops showed similar oxygen consumptionat 16 and 22 °C but in large juveniles higher values were registered at 22 °C. In large juveniles routine consumption at 16 °C washigher (up to 35%) than standard consumption. This pattern was not evident at 22 °C, suggesting that oxygen demand is higherregardless of feeding condition. NH4

+–N excretion rate is inversely related to salinity. Only small juveniles showed a higher NH4+–N

excretion rate at 22 °C. Scope for growth was positive in all treatments, although the upper limit of salinity should not be based only inthis index. Higher scope for growth values at 38 and 42 g/l was related with a reduction in ammonia excretion and high absorptionefficiency. In addition, an increase in salinity produced a reduction in NH3–N proportion and under hypersaline conditions scallopstended to decrease excretion as a way of osmoconformation. This explains our findings of higher survival rates at higher salinities.Even though the scope for growth is positive at 42 g/l, the osmotic stress reduces the survival chances. The data obtained can beconsidered useful information for A. purpuratus culture under controlled conditions.© 2007 Elsevier B.V. All rights reserved.

Keywords: Argopecten purpuratus; Salinity; Temperature; Oxygen consumption; Ammonia excretion; Scope for growth

⁎ Corresponding author. Present address: School of NaturalResources, University of Arizona, Biosciences East 325D, P.O. Box210043, Tucson, Arizona, 85721-0043, United States. Tel.: +1 5203180146.

E-mail addresses: gsoria@email.arizona.edu, triploidia@ucn.cl,rgskua@yahoo.com.ar (G. Soria).1 Tel./fax: +56 51 209970.

0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2007.05.018

1. Introduction

The northern scallop Argopecten purpuratus has ahigh commercial value and its aquaculture in Chile hasincreased significantly during the last decades (von Brandet al., 2006). The success reached in its reproductiveconditioning and spat production in hatchery hassupported that activity (von Brand et al., 2006). Scallopcould be reared using Recirculating Aquaculture Systems

452 G. Soria et al. / Aquaculture 270 (2007) 451–463

(RAS) as an alternative method of production, making asignificant reduction in water requirements through watertreatment and reuse. Design of effective RAS requiresknowledge of physiological aspects of target species inorder to address the range of physiochemical waterparameters.

In RAS processes there is loss of water by evaporationwhich affects water salinity. Variations in salinity andtemperature may change water quality parameters such asdissolved oxygen (DO) andCO2 levels, NH3 toNH4

+ ratio,and pH (Lawson, 1995). Salinity and temperature can alsoaffect survival, growth, and scope for growth, a physio-logical process related to the energy acquisition (ingestionand absorption) and utilization (respiration and excretion)(Bricelj and Shumway, 1991). Although many authorshave documented the effect of decreasing salinity underdifferent temperature regimens (Mercaldo and Rhodes,1982; Strand et al., 1993; Navarro and González, 1998;Laing, 2002; Christophersen and Strand, 2003; Rupp andParson, 2004), available information regarding hypersa-line tolerance of scallops at different temperature is scarce.

Pectinids are osmoconformists (Shumway, 1977;Singnoret-Brailowski et al., 1996) in which hemolymphis close to the osmotic pressure and ionic composition ofseawater and ammonia excretion increases with decreas-ing salinity (Bricelj and Shumway, 1991; Navarro andGonzález, 1998). Nonetheless, scallop species have limitswhere growth and survival aremaximized. In addition, thephysiological effect of salinity can modify or be modifiedby temperature (Kinne, 1964; Paul, 1980).

Knowledge of the physiological response of scallopsunder different salinity and temperature conditions is animportant factor in the cultivation of this species. Themain goal of this study was to determine the effect ofincreasing salinity on survival, shell growth and scopefor growth in A. purpuratus juveniles under twotemperature regimens reared in closed systems. Since,our objective was focused on salinity and temperature wechose the closed system because it allows us to isolate theeffect of the other parameters (like nitrate, nitrite,alkalinity, etc.) which also occur in RAS and, theirphysiological effect on A. purpuratus is not known.Because of the commercial importance of this species,the information reported in this paper will be useful formanaging these parameters under controlled conditions.

2. Materials and methods

2.1. Experimental set up

We obtained juvenile scallops from an experimentalhatchery at Universidad Catolica del Norte, Coquimbo,

Chile (30° LS), where the research was conducted. Wedivided scallops in two groups according to shell height(h): small juveniles (mean h=6.5 mm; SD=0.78) andlarge juveniles (mean h=26.5 mm; SD=1.9) andexposed both groups to three salinity regimes (34, 38,and 42 g/l), at 16 and 22 °C, in accordance with thevalues observed in our RAS with three replicates persalinity treatment. We cultivated scallops in 1 μm-filtered seawater (passed through UV lamps) on plastictrays (25×30×24) in 18, 15 l beakers which in turnwere placed in two water bath containers (250 l,160×64×26 cm), one for each temperature. Thus, thisfactor was pseudoreplicated. We placed 100 smallscallops or 20 large scallops on each tray and waterwas renewed every 24 h, transferring the trays directlyinto a clean beaker containing new seawater at theappropriate salinity and temperature. We obtainedhypersaline water by evaporating seawater. We usedair-stones to provide aeration in order to maintain a DOlevel above 80% saturation. We fed the scallops onIsochrysis galbana and Chaetoceros calcitrans (ratio1:1) in a quantity equivalent to 10% of the scallop's dryweight per day. We divided the daily food allotment intothirds, provided morning, noon, and evening. Weacclimated scallops to different experimental conditionsby increasing salinity and temperature by 1 g/l and 1 °C,respectively, per day. Experiments lasted for 2 months,or ended when we registered significant difference insurvival between treatments.

Wemeasured salinity, temperature, andDO daily usinga hand-sensor (Yellow Spring Incorporated, YSI 85). Wecalibrated the oxygen-meter at room atmosphere.We usedSolórzano's (1969) phenol-hypochlorite method to take aweekly measurement of total ammonia or ammonia (TA)(sum of NH3 and NH4

+). We collected water samples inacid-washed glassware and measured TA immediatelyafter collection. At the time of water collection we used apH meter (calibrated with buffer solutions of pH=7 andpH=10 before use) (Hanna Instruments, HI 9023C) tomeasure water pH. We calculated un-ionized ammonia(NH3) from appropriate temperature, pH, and salinityaccording to Fivelstad (1988). We estimated ionizedammonia (NH4

+) as a difference between ATand NH3. Weexpressed values on a nitrogen basis and wrote them asTA–N,NH3–N, andNH4

+–N (Colt andArmstrong, 1981).

2.2. Survival and growth

Wemeasured initial and final shell height from digitalimages. We transferred trays with scallops to a plasticplate containing seawater at appropriate salinity andtemperature. We used a digital camera (Sony DSC P8;

453G. Soria et al. / Aquaculture 270 (2007) 451–463

resolution=2 megapixel) to photograph juveniles scal-lops (with a 1 mm reference scale). We measured shellheight using the program Image ProPlus 4.0 (MediaCybernetics) for Microsoft® Windows®. This measure-ment method minimized scallop stress due to handling.

We removed and counted dead scallops daily andcalculated survival at the end of the experiments. Wecorrected water level in relation to live scallops in eachbeaker at the moment of water renewal.

2.3. Physiological rates

We calculated absorption of organic matter (A) (mg/min) as:

A ¼ OIR� AE ð1Þwhere: OIR = organic ingestion rate (mg/min); AE =absorption efficiency (%).

We transformed organic matter to energy equivalentsusing the conversion factor 1 mg of organic matter=23(J) (Widdows et al., 1979). We calculated OIR as:

OIR ¼ FR� OF ð2Þwhere: FR = filtration rate (ml/min) and OF = organicfraction (mg/l) in diet (ash free dry weight/dry weight).

We determined filtration rate from an analysis ofmass balance for a closed static system (Coughlan,1964) in which we plotted reduction in algal concen-tration in relation to time. We calculated filtration rate as

FR ¼ Vn

ln C0 � ln Ctð Þ � St

� �ð3Þ

where: FR = filtration rate equivalent to volume of watercleared of particle per unit of time (ml/min); V = volume(ml); n = number of scallops; t: time (min); C0 and Ct:initial and final algae concentration (cell/ml) and s=rateof sedimentation in controls. We used a particle counter(Coulter Counter Z2) fitted with a 100 μm orifice tube tocount particles ranging between 2 and 10 μm. We fedscallops with one third of the daily ration (equivalent to40×103 cells/ml for small scallops and 120×103 cells/ml for large scallops). We used additional beakerswithout scallops (one for each combination of salinityand temperature) as a control.

We estimated absorption efficiency by looking at theratio of organic and inorganic content in diet and feces(Conover, 1966). We calculated absorption efficiencyas.

AE ¼ F � Eð Þ1� Eð ÞF

� �� 100 ð4Þ

where: AE = absorption efficiency; F = organic fractionof the algae (g of ash free dry weight/g of dry weight)and E = organic fraction of feces (g of ash free dryweight/g of dry weight). We collected a sample of eachalgal species as well as feces from each beaker after24 h. We filtered samples through pre-weighed 47 mmfiberglass filters under low vacuum and washed withisotonic ammonium formate (3.4%) to remove salts,dried them at 70 °C until they were a constant weight,weighed and incinerated them at 450 °C for 3 h andweighed them again to obtain the inorganic (dry weight)and organic weights (difference between dry and ashweight) contained in the algae ration and feces.

We determined oxygen consumption rate in smalljuveniles using a Warburg constant-pressure respirom-eter (Umbreit et al., 1957). We did this by placing threejuveniles from each replicate in a sealed Warburgchamber. We fed scallops 1 h before measuring oxygenconsumption (1/3 of daily ration) and took measure-ments every 15 min over a 3 h period. We determinedoxygen consumption in larger scallops directly in the15 l beakers. We fed scallops (1/3 daily ration) andexchanged water after 1 h. We sealed the beakers andused oxygen electrodes (Sensorex CS 511L) to recordDO every 3 min for a period of 6–8 h. We used adatalogger (Campbell Scientific 23 X, 1 mb RAM) tocollect data, which we downloaded to a computer. Wecalibrated DO electrodes in each salinity–temperatureair-saturated seawater combination. We converted signalgiven in mV to ml O2/l according to tables for DO inseawater given in Colt (1984). We did not supplyaeration during DO determination and used a submers-ible pump (Atman 302 6 W Q=20–200 l/min) to mixwater in each beaker. Considering that all scallops werewell fed before DO consumption rate determinations,the measured rate represents the routine metabolism.Additionally, we measured oxygen consumption oncemore on scallops fed 24 h before measurement (fastedscallops). This situation represents the standard metab-olism. We plotted DO values against time for alldeterminations and used linear regression to calculatethe rate of oxygen consumption (slope), represented bydeclining levels of DO. We then multiplied the obtainedoxygen consumption rate by the volume of water in thebeaker and divided it by the number of scallops to yieldoxygen consumption per individual (ml O2/h ind). Werecorded rates of oxygen consumption as ml O2/h g dryweight (dw) and transformed the rates to energy equiva-lents using the conversion factor 1 ml O2=19.9 J (Elliotand Davison, 1975). We sacrificed scallops, measuredshell height, and dried the soft part of each animal at70 °C until a constant dry weight.

454 G. Soria et al. / Aquaculture 270 (2007) 451–463

We determined ammonia excretion by placingscallops in 0.1 l and 0.3 l jars. We used additional jarscontaining seawater but no animals as control treatments.We fed scallops (1/3 daily ration) and replaced seawaterafter 1 h. We determined ammonia and pH following thesame method and materials described previously. Wetook water samples for ammonia determination after a2 h incubation period. We expressed excretion rates inmg NH4

+–N/h g dw and used the conversion factor 1 mgNH4

+–N=24.8 J (Elliot and Davison, 1975) to transformthese rates to energy equivalents using.

After converting all the physiological rate values toenergy equivalents we calculated scope for growth withthe equation given by Widdows (1985) as:

SFG ¼ A� Rþ Eð Þ ð5Þwhere: SFG = scope for growth, A = absorption oforganic matter, R = oxygen consumption and E = ionizedammonia excretion estimates as described above.

In order to test significant effects of salinity andtemperature on survival, shell height, and all physio-logical parameters among treatments, we conductedtwo-way analyses of variance (ANOVA) followed by aHSD-Tukey test. We conducted all statistical tests withα=0.05, using Statistica, Version 6. Before conductingANOVA analyses we arcsine transformed survival andabsorption efficiency values and log transformed shellheight values and tested for homogeneity of variancesand normality (Sokal and Rohlf, 1995).

3. Results

3.1. Experiment 1: small juveniles

The diet fed to small juveniles had a concentrationof 120×106 cells/l per day and corresponded to3.82 mg/l of total dry weight and 3.51 mg/l (92%) of

Table 1Mean values of water quality in small A. purpuratus juveniles experiment

Treatment T (°C) 16

S (g/l) 34 38 42

Temperature (°C) 15.9 (0.1) 15.9 (0.15) 15Salinity (g/l) 34.1 (0.1) 38.1 (0.09) 41DO % 102.5 (1.3) 102.6 (1.3) 102DO (ml/l) 5.8 (0.1) 5.7 (0.12) 5NH4

+–N (mg/l) 0.94 (0.12) 0.39 (0.07) 0NH3–N (mg/l) a 0.026 (3.5) 0.011(1.9) 0pH 8.07 (0.17) 8.09 (0.16) 8

Values in bracket represent standard deviation.a Values of standard deviation×10−3.

organic weight. Mean values of water quality para-meters are shown in Table 1.

3.1.1. Survival and growthSurvival during the acclimatizing period (up to

10 days) was close to 98% in all treatments (Fig. 1).After 25 days, survival ranged between 80 and 90% withno significant differences within groups. At the end of thetrial (day 45), mean final survival was higher at 16 °C thanat 22 °C (p=0.001) and was significantly affected bysalinity (Fig. 1). The group held at 16 °C showed lowsurvival (42%) at 34 g/l and high survival (77%) at 42 g/land the group exposed at 22 °C, survival was near 40%(Fig. 1). There was significant interaction between factors(p=0.038). We clearly observed an inverse relationshipbetween survival and NH3–N concentration (Fig. 2).

We found a significant increase in mean shell heightin all treatments at the end of the trial (Fig. 3). Salinityaffected the final mean shell height ( pb0.001) butneither temperature ( p=0.583) nor the interactionbetween factors affected this parameter ( p=0.854).Final shell height ranged between 9.28 and 10.24 mm.Scallops cultivated at 34, 38, or 42 g/l showed no dif-ferences between temperatures. We observed the lowestvalues (approximately 9.2 mm) when scallops wereexposed to 42 g/l (Fig. 3).

3.1.2. Absorption efficiencySalinity and temperature had no effect on absorption

efficiency in small scallops (Table 2). Values rangedbetween 27.3% and 55.6% and there were no significantdifferences between treatments (pN0.05) (Fig. 4).

3.1.3. Filtration rateLike absorption efficiency, filtration rate was not

significantly affected by salinity or temperature (Table 2).Filtration rate showed values around 304.4 ml/h g dw(SD=107.9) (Fig. 4).

22

34 38 42

.9 (0.2) 21.9 (0.2) 21.9 (0.2) 21.9 (0.1)

.9 (0.1) 34.3 (0.20) 38.2 (0.25) 42.1 (0.21)

.0 (1.1) 95.4 (2.8) 95.1 (2.5) 95.0 (2.08)

.5 (0.1) 4.8 (0.2) 4.7 (0.15) 4.6 (0.17)

.20 (0.08) 1.68 (0.11) 1.21 (0.01) 1.25 (0.09)

.005 (2.1) 0.08 (5.5) 0.051 (0.22) 0.06 (4.5)

.02 (0.06) 7.97 (0.26) 7.98 (0.12) 8.00 (0.14)

Fig. 1. Mean survival of small A. purpuratus juveniles exposed at salinities of 34 (○), 38 (n) and 42 (▲) g/l at 16 °C (a) and 22 °C (b). Vertical linecorresponds to the acclimatizing period. On day 45 at 16 °C the SD were 8.7, 11.8, and 3.5% at 34, 38, and 42 g/l, respectively and at 22 °C they were4.6, 2.1, and 16.0% at 34, 38, and 42 g/l. Different letters indicate significantly different values (Tukey's pb0.05) between treatments.

455G. Soria et al. / Aquaculture 270 (2007) 451–463

3.1.4. Oxygen consumption rateOxygen consumption in small scallops was not

affected by temperature (pN0.88) or salinity (p=0.054)and ranged from 0.68 to 0.91 ml O2/h g dw (Table 2).Juveniles exposed at 16 °C oxygen consumption ratesshowed no differences among salinities. In the group heldat 22 °C the oxygen consumption rate at 34 g/l was higherthan at 38 g/l, and we found no significant differencesbetween the 38 and 42 g/l salinities groups. There were nosignificant differences (pN0.05) among oxygen con-sumption rates measured at 16 and 22 °C when scallopswere exposed to 34, 38, or 42 g/l salinities (Fig. 4).Oxygen consumption quoted by individual showedvalues between 0.016 and 0.021 ml O2/h ind (Table 3).

3.1.5. Ionized ammonia excretion (NH4+–N)

Temperature had a significant affect on NH4+–N

excretion rate. Rates were higher in scallops exposed at

Fig. 2. Relationship between mean final survival and mean NH3–Nlevel measured throughout the trial in small A. purpuratus juvenilesexposed to three salinities (34, 38, and 42 g/l) at 16 °C and 22 °C. EachNH3–N value is based on 15 measurements on five occasions. Verticallines represent standard deviation.

22 °C versus those exposed at 16 °C (p=0.001). Inaddition, NH4

+–N excretion rate was affected by salinity(p=0.004) (Table 2). For the group held at 16 °C, NH4

+–N excretion rate was higher at 34 °C (0.058 mg NH4

+–N/h g dw). We found no significant difference in scallopsheld at 38 and 42 g/l. Scallops exposed at 22 °C did notshow significant differences in NH4

+–N excretion rate atdifferent salinities (pN0.05). Rates ranged between0.139 mg NH4

+–N/h g dw at 34 g/l and 0.101 mg NH4+–

N/h g dw at 42 g/l (Fig. 4). NH4+–N excretion rates per

scallop followed a similar pattern (Table 3).

3.1.6. Scope for growthSalinity significantly affected scope for growth in

small juveniles (p= 0.047). Neither temperature(p=0.06) nor interaction between factors (p=0.416)affected SFG (Table 2). This physiological index rangedbetween 165.2 J/d g dw and 844.4 J/d g dw and showed

Fig. 3. Final mean shell height in small A. purpuratus juvenilesexposed at three salinities (34, 38, and 42 g/l) at 16 °C and 22 °C.Vertical lines represent standard deviation. Different letters indicatesignificantly different values (Tukey's pb0.05) between treatments.

456 G. Soria et al. / Aquaculture 270 (2007) 451–463

a tendency to increase with salinity (Fig. 5). Likesurvival, SFG was inversely related to NH3–N levels(Fig. 6).

3.2. Experiment 2: large juveniles

Scallops received a diet at a concentration of 360×106

cells/l per day with a total dry weight of 11.11 mg/l and anorganic proportion of 10.22 mg/l (92%). Mean waterquality parameters corresponding to this experiment areshown in Table 4.

3.2.1. Survival and growthWe recorded no mortality in the course of the first

10 days (acclimatizing period) with the exception of asingle scallop in the 16 °C and 34 g/l treatment. After thefirst month, mean survival was higher than 95%, for alltreatments combined (Fig. 7). At the end of the trial (onday 65), scallops exposed at 22 °C showed a lower meansurvival than those cultivated at 16 °C (p=0.046). Therewere no significant differences in survival in the groupof scallops held at 16 °C. However, at 22 °C scallopsshowed a higher survival at 38 and 42 g/l. The lowestsurvival value (65.1%) was registered at 22 °C and 34 g/ltreatment. We observed a significant interaction be-tween factors (p=0.008). We observed a moderate de-clining trend when survival was related to NH3–Nlevels (Fig. 8).

Table 2Two-way analysis of variance examining the effects of salinity andtemperature on different physiological variables in small A.purpuratus juveniles

Physiological process df F p value

Absorption efficiency Salinity 2 2.590 0.116Temperature 1 0.288 0.601Interaction 2 0.084 0.920Total 17

Filtration rate Salinity 2 1.689 0.226Temperature 1 1.135 0.308Interaction 2 0.434 0.658Total 17

Oxygen consumption Salinity 2 5.610 0.054Temperature 1 0.021 0.886Interaction 2 5.330 0.006 ⁎

Total 17NH4

+–N excretion Salinity 2 19.312 0.004 ⁎

Temperature 1 158.177 0.000 ⁎

Interaction 2 3.973 0.057Total 17

Scope for growth Salinity 2 3.984 0.047 ⁎

Temperature 1 4.323 0.060Interaction 2 0.946 0.416Total 17

⁎ Significant differences at pb0.05.

Fig. 4. Final mean values of (a) absorption efficiency, (b) filtration rate,(c) oxygen consumption and (d) NH4

+–N excretion in A. purpuratusjuveniles reared at three salinities (34, 38, and 42 g/l) at 16 °C (n) and22 °C (□).Vertical lines represent standard deviation. Different lettersindicate significantly different values (Tukey's pb0.05) betweentreatments.

Table 3Rates (mean; standard deviation) of oxygen consumption and NH4

+–Nexcretion in small A. purpuratus juveniles exposed to three salinities(34, 38, and 42 g/l) at 16 °C and 22 °C

Treatment Oxygenconsumptionml/h ind

Ionizedammonia–Nmg/h ind a

Meanheightmm

Meanweightmg

°C g/l

16 34 0.021 (0.0047) 0.0009 (2.36) 9.8 9.0338 0.017 (0.0031) 0.0003 (1.03) 10.2 10.1242 0.016 (0.0042) 0.0001 (0.99) 9.2 6.87

22 34 0.020 (0.0035) 0.0019 (3.96) 9.7 9.0538 0.016 (0.0027) 0.0011 (3.39) 10.0 10.5042 0.018 (0.0032) 0.0015 (9.02) 9.2 7.65

Rates are shown per individual scallop.a Values of SD×10−4.

Fig. 6. Relationship between scope for growth and mean NH3–Nconcentration measured throughout the trial in small A. purpuratusjuveniles exposed to three salinities (34, 38, and 42 g/l) at 16 °C and22 °C. Each NH3–N value is based on 18 measurements on 6occasions. Vertical lines represent the standard deviation.

457G. Soria et al. / Aquaculture 270 (2007) 451–463

Initial mean shell height was 26.5 mm (SD=1.9) andthere was a significant increase in shell height in alltreatments at the end of the trial. Final mean shell heightranged between 29.6 and 30.8 mm (Fig. 9) and wassignificantly affected by temperature (p=0.001) andsalinity (p=0.032) but not by the interaction of thesetwo variables (p=0.736).

3.2.2. Absorption efficiencySalinity (pb0.001) and temperature (p=0.003) sig-

nificantly affected absorption efficiency in large juve-niles, which ranged between 33.8% and 78.3% (Table 5).Regardless of temperature, absorption efficiency showedlower values at 34 g/l and increased considerably whenscallops were cultivated at 38 and 42 g/l (Fig. 10).

3.2.3. Filtration rateThe filtration rate in large scallops was higher at

22 °C than at 16 °C (pb0.001) and salinity had asignificant effect on filtration rate (p=0.031) (Table 5).

Fig. 5. Mean values of scope for growth in A. purpuratus juvenilesreared at three salinities (34, 38, and 42 g/l) at 16 °C (n) and 22 °C (5).Vertical lines represent standard deviation. Different letters indicatesignificant different values (Tukey's pb0.05) between treatments.

Scallops exposed at 34 g/l showed a lower filtration rateat 16 °C than the group at 22 °C. The filtration rate(1950 ml/h g dw) was highest in the 34 g/l and 22 °Ctreatment; this value was significantly higher than thefiltration rate in the group at 16 °C (Fig. 10).

3.2.4. Oxygen consumptionWe used data corresponding to the lineal phase (up to

150 min) to calculate oxygen consumption rates(Fig. 11). At this point in all treatments mean percentageof DO saturation was near 83% (SD=2.28). Regressionequations are presented in Table 6. Mean oxygenconsumption rates in large A. purpuratus juvenilesshowed values around 0.6 ml O2/h g dw and were higherat 22 °C than at 16 °C (pb0.000). Neither salinity norinteractions between salinity and temperature had anyeffect on mean oxygen consumption rate (pN0.1)(Table 5). There were no significant differences betweenoxygen consumption at 16 and 22 °C when scallopswere cultivated at 34 and 38 g/l. However, at salinity of42 g/l oxygen consumption was higher at 22 °C than at16 °C (p=0.034) (Fig. 10). Oxygen consumption perscallop ranged between 0.107 and 0.093 ml O2/h ind(Table 7).

3.2.5. Ionized ammonia excretion (NH4+–N)

The excretion rate for large scallops ranged between0.212 and 0.339 mg NH4

+–N/h g dw (Fig. 10) and wassignificantly influenced by salinity (p=0.008) andtemperature (p=0.03) (Table 5). There were nosignificant differences in excretion rates measured at16 °C or 22 °C, when scallops were exposed to 34, 38,or 42 g/l. Significant differences of ionized ammoniaexcretion rates were found between treatments at 16 °C

Table 4Mean values of water quality in large A. purpuratus juveniles experiment

Treatment T (°C) 16 22

S (g/l) 34 38 42 34 38 42

Temperature (°C) 15.9 (0.1) 16.0 (0.1) 15.9 (0.1) 22.1 (0.2) 22.0 (0.1) 22.0 (0.2)Salinity (g/l) 33.8 (0.1) 37.9 (0.1) 41.8 (0.2) 34.0 (0.2) 38.1 (0.1) 42.0 (0.2)DO (%) 92.0 (6.5) 91.0 (6.40) 90.3 (5.9) 93.5 (3.9) 92.6 (4.3) 92.0 (4.4)DO (ml/l) 5.18 (0.38) 4.99 (0.36) 4.83 (0.33) 4.8 (0.33) 4.57 (0.23) 4.4 (0.21)pH 7.95 (0.08) 7.95 (0.07) 7.90 (0.10) 8.04 (0.06) 7.99 (0.06) 7.99 (0.06)NH4

+–N (mg/l) 0.97 (0.1) 0.91 (0.02) 0.84 (0.03) 1.47 (0.36) 1.26 (0.09) 1.43 (0.08)NH3–N (mg/l) a 0.022 (2.1) 0.02 (0.5) 0.017 (0.8) 0.068 (1.7) 0.05 (3.7) 0.056 (3.1)

Values in brackets represent standard deviation.a Values of standard deviation×10−3.

458 G. Soria et al. / Aquaculture 270 (2007) 451–463

and 42 g/l and 22 °C and 34 g/l (Fig. 10). Ionizedammonia excretion rates per scallop followed a similartrend to that described above (Table 7).

3.2.6. Scope for growthSalinity (pb0.001) and temperature (p=0.017) affect-

ed the scope for growth, which showed values between2000 and 5400 J/d g dw (Table 5). Like small scallops, thisphysiological index in large scallops showed a tendencyto increasewith salinity at both temperatures. At 16 °C theSFG reached the highest value at 38 g/l (5400 J/d g dw),which was not significantly different from the valuesobtained in the group at 42 g/l at 16 and 22 °C (Fig. 12).SFG did not differ between temperatures when scallopswere exposed at 34 or 42 g/l. However, we did not observethis pattern at 38 g/l (Fig. 12). We found a significantcorrelation but with a low R2 value (0.19) between SFGand NH3–N level (Fig. 13).

3.2.7. Oxygen consumption rates in 24 h fasted and fedscallop juveniles

Wemeasured oxygen consumption rate again in largescallops after a 24 h fast. We used the same criteria

Fig. 7. Mean survival ofA. purpuratus juveniles exposed at salinities of 34 (○), 38correspond to the acclimatizing period.On day 65 at 16 °C the standard deviationswere 7.27, 8.25, and 12.60 at 34, 38, and 42 g/l, respectively. Different letters ind

described for fed scallops to select data. Linearregression for the oxygen rates are shown in Table 6.At 16 °C, oxygen consumption was higher (up to 35%)in fed scallops (0.63 ml O2/h g dw) than in 24 h fastedscallops (0.38 ml O2/h g dw) (p=0.000) (Fig. 14 andTable 8). Conversely, at 22 °C oxygen consumptionrates showed values about 0.6 ml O2/h g dw with nosignificant differences registered among treatments (Fig.14 and Table 8).

4. Discussion and conclusions

Dissolved oxygen (DO) and total ammonia–nitrogen(TA–N) levels are key factors limiting production inaquaculture (Colt and Armstrong, 1981; Lawson, 1995).Although this work did not evaluate the effects of DO andTA–N levels on A. purpuratus, we measured theconcentration values of both parameters throughout thetrials. For A. purpuratus there is a lack of informationabout oxygen consumption requirements at differentlevels of DO. In both experiments, DO levels neverdropped below 90%. In addition, oxygen consumptionrate in large juveniles was stable until DO levels dropped

(n), and 42 (▴) at 16 °C (a) and 22 °C (b). The first 10 days (vertical lines)were 2.75, 12.60, and 4.76%at 34, 38, and 42 g/l, respectively; at 22 °C theyicate significant different values (Tukey's pb0.05) between treatments.

Fig. 8. Relationship between mean final survival and mean NH3–Nconcentration measured throughout the trial in large A. purpuratusjuveniles exposed to three salinities (34, 38, and 42 g/l) at 16 °C and22 °C. Each NH3–N value is based on 18 measurements on sixoccasions. Vertical lines represent standard deviations.

Table 5Two-way analysis of variance examining the effect of salinity andtemperature on different physiological variables in large A. purpuratusjuveniles

Physiological process df F p value

Absorption efficiency Salinity 2 20.082 0.000 ⁎

Temperature 1 12.874 0.004 ⁎

Interaction 2 3.562 0.061Total 17

Filtration rate Salinity 2 4.681 0.031 ⁎

Temperature 1 24.111 0.000 ⁎

Interaction 2 1.403 0.283Total 17

Oxygen consumption Salinity 2 0.088 0.916Temperature 1 27.815 0.000 ⁎

Interaction 2 2.338 0.139Total 17

NH4+–N excretion Salinity 2 5.188 0.009 ⁎

Temperature 1 3.979 0.031 ⁎

Interaction 2 0.228 0.797Total 17

Scope for growth Salinity 2 24.299 0.000 ⁎

Temperature 1 7.698 0.017 ⁎

Interaction 2 4.548 0.034 ⁎

Total 17

⁎ Significant differences at pb0.05.

459G. Soria et al. / Aquaculture 270 (2007) 451–463

to 83% (Fig. 11), suggesting that DO level was not arestrictive factor for respiration. On the other hand, bothionized (NH4

+–N) and un-ionized (NH3–N) ammoniacould be toxic, affecting survival, growth, and physio-logical parameters such as oxygen consumption orammonia excretion (Colt and Armstrong, 1981). Thefraction of TA–N in the un-ionized or ionized form isdependent upon pH, temperature, and salinity; the un-ionized form is proportionally more toxic (Lawson,1995). Concentrations higher than 0.05 NH3–N mg/land 1 mg NH4

+–N/l have been shown to be toxic to mostfishes, crustaceans, and mollusks and these levels shouldnot be exceeded (Colt and Armstrong, 1981; Lawson,1995; Huchette et al., 2003). Our experimental designused closed culture, which would allow level of TA–N toincrease through time between water exchanges. In bothexperiments, NH3–N and NH4

+–N reached values

Fig. 9. Final mean shell height in large A. purpuratus juvenilesexposed at three salinities (34, 38, and 42 g/l) at 16 °C and 22 °C.Vertical lines represent standard deviation. Different letters indicatesignificant different values (Tukey's pb0.05) between treatments.

considered to be toxic (Tables 1 and 4). This allows usto draw some conclusions regarding the relative impor-tance of salinity, temperature, and ammonia as factorsinfluencing scallop's survival, growth and physiologicalparameters.

Survival showed a tendency to decrease with increas-ing temperature, NH3–N and NH4

+–N levels in bothscallop groups. After a 45 day exposure, small juvenileshad lower survival rates (around 40%) than larger scallopsat the same time (up to 85%). Salinity had a clear effect onsurvival in small scallops, where an increase in salinityfrom 34 to 42 at 16 °C was corresponded to an increase insurvival. However, this relationship was not evident at22 °C. On the other hand, salinity did not affect survival oflarge juveniles. These results suggest that small scallopsare more sensitive to changes in salinity than largerjuveniles. In the group of small juveniles, survival couldbe increased by several methods (i.e. increasing waterexchange, reducing scallop density, and properly design-ing biofilters) to avoid NH3–N and NH4

+–N values higherthan 0.02 and 1.0 mg/l respectively. Higher survival ratesin large juvenilesmay indicate a higher tolerance to TA–Nlevel than small juveniles. Nevertheless, determiningwhich factor (salinity, temperature, or TA–N) or synergiceffect is affecting survival requires further study.Nonetheless, our results suggest that small juveniles aremore sensitive to salinity, temperature, and TA–N thanlarge juveniles.

Fig. 10. Final mean values of (a) absorption efficiency, (b) filtrationrate, (c) oxygen consumption, and (d) NH4

+–N excretion in large A.purpuratus juveniles reared at three salinities (34, 38, and 42 g/l) at16 °C (---□) and 22 °C (□).Vertical lines represent standard deviation.Different letters indicate significant different values (Tukey's pb0.05)between treatments.

Fig. 11. Dissolved oxygen against time in large A. purpuratusjuveniles exposed to three salinities at 16 °C and 22 °C. a) 16–34,b) 16–38, c) 16–42, d) 22–34, e) 22–38, and f) 22–42 (°C–g/l).

460 G. Soria et al. / Aquaculture 270 (2007) 451–463

Final mean shell height in both groups of juvenilesshowed a tendency to increase from 34 to 38 g/l with areduction at 42 g/l. Large juveniles had a smaller finalshell height at 22 °C when TA–N level were higher(0.02 mg NH3–N/l and 1 mg NH4

+–N/l). In Haliotislaevigata (Harris et al., 1998) and Haliotis rubra(Huchette et al., 2003) growth rate reductions wereobserved at 0.054 mg NH3–N/l.

Absorption efficiency in small scallop juvenilesranged between 27 and 56% and was not affected bysalinity increments, but this pattern was not observed inlarge juveniles. Absorption efficiency tended to belower at 34 g/l and had higher values at 38 or 42 g/l inboth groups. There is a gap of information related toabsorption efficiency and TA–N levels and furtherstudies are necessary. In A. purpuratus of different shellsizes, absorption efficiency using the same algae species

Table 6Linear regression of DO against time in fed and 24 h fasted large A.purpuratus juveniles exposed to three salinities (34, 38, and 42 g/l) at16 °C and 22 °C

Treatment 1 h fed 24 h fasted

°C g/l Regression equations Regression equations

16 34 DO=5.267−0.195× t

R2=0.99 DO=5.180−0.125× t

R2=0.99

38 DO=5.118−0.201× t

R2=0.98 DO=5.223−0.117× t

R2=0.99

42 DO=5.005−0.175× t

R2=0.99 DO=5.019−0.109× t

R2=0.98

22 34 DO=4.602−0.175× t

R2=0.99 DO=4.731−0.115× t

R2=0.98

38 DO=4.483−0.191× t

R2=0.99 DO=4.503−0.156× t

R2=0.98

42 DO=4.321−0.194× t

R2=0.98 DO=4.360−0.143× t

R2=0.98

DO = dissolved oxygen (ml/l) and t = time (h).

Table 7Rates (mean; standard deviation) of oxygen consumption and ionizedammonia excretion in large A. purpuratus juveniles exposed to threesalinities (34, 38, and 42 g/l) at 16 °C and 22 °C

Treatment Oxygenconsumptionml/h ind

Ionizedammonia–Nmg/h ind

Meanheightmm

Meanweightg

°C g/l

16 34 0.100 (0.006) 0.047 (0.011) 30.23 0.1738 0.107 (0.005) 0.037 (0.009) 30.7 0.1742 0.093 (0.003) 0.036 (0.014) 30.4 0.17

22 34 0.100 (0.010) 0.064 (0.022) 29.5 0.1338 0.098 (0.005) 0.055 (0.021) 30.2 0.1442 0.104 (0.014) 0.052 (0.013) 29.5 0.14

Values are shown per individual. Fig. 13. Relationship between scope for growth and mean NH3–Nconcentration measured throughout the trial in large A. purpuratusjuveniles exposed to three salinities (34, 38, and 42 g/l) at 16 °C and22 °C. Each NH3–N value is based on 18 measurements on sixoccasions. Vertical lines represent standard deviation.

461G. Soria et al. / Aquaculture 270 (2007) 451–463

ranged between 65 and 76% and did not significantlydiffer when scallops were exposed to decreasingsalinities (from 34 to 24 g/l) (Navarro and González,1998). A high absorption efficiency was measured(80%) in adults A. purpuratus at 16 °C and 20 °C(Navarro et al., 2000).

When individual of A. purpuratus of different shellheights was exposed for a period of 1 or 2 weeks todecreasing salinities (from 34 to 24 g/l), oxygen con-sumption increased in relation to catabolism of aminoacid to solve osmoregulatory problems (Navarro andGonzález, 1998). In our experiment, oxygen consump-tion was independent of salinity, suggesting thatscallops were able to adjust their metabolism after along exposure to different salinities (45 and 65 days forsmall and large juveniles, respectively). Differences inoxygen consumption could potentially be measuredwithin the initial weeks of the trials as suggestedNavarro and González (1998). With respect to temper-ature, small scallops showed similar oxygen consump-

Fig. 12. Mean values of scope for growth in large A. purpuratusjuveniles reared at three salinities (34, 38, and 42 g/l) at 16 °C (---□) and22 °C (□). Vertical lines represent standard deviation. Different lettersindicate significant different values (Tukey's pb0.05) betweentreatments.

tion rates at both temperature regimes. Although oxygenconsumption rates in large juveniles were higher at22 °C, a significant decrease (up to 35%) in oxygenconsumption at 16 °C was evident in 24 h fastedscallops, which have reduced oxygen demand inabsence of algae. However, standard and routine oxygenconsumption rates were not significantly different at22 °C, suggesting that oxygen demand is higherregardless of feeding condition. Farías et al. (1997)described a similar pattern, showing that fed adult A.purpuratus scallops have higher oxygen consumptionrates (2.27 ml O2/h ind) than fasted scallops (0.8 ml O2/hind). During the reproductive conditioning of A. pur-puratus with continuous feeding, oxygen consumptionrates did not differ among 16 and 20 °C (Navarro et al.,2000). A similar pattern was described in Chlamysopercularis at 10 and 20 °C, but in A. irradians oxygenconsumption closely tracks changes in temperature(between 5 and 25 °C) (Bricelj and Shumway, 1991).In the abalone H. laevigata, oxygen consumption ratesdid not differ when individuals were exposed up to0.022 mg/l NH3–N (0.74 mg/l TA–N), but ratesincreased significantly when the un-ionized ammonialevel was close to 0.083 mg/l (2.44 mg/l TA–N) (Harriset al., 1998). A similar pattern was observed in Penaeuschinensis (Cheng and Lin, 1998). Based on our study,oxygen consumption in A. purpuratus appears to be notaffected by TA–N levels, as occurred in H. laevigata.

In A. purpuratus, ammonia excretion can varyaccording to diet (Díaz and Martínez, 1992), feedingregimens (Martínez et al., 1995), salinity (Navarro andGonzález, 1998), and temperature (González et al.,2002). In our study NH4

+–N excretion was inverselyrelated to salinity, suggesting that scallops regulate their

Fig. 14. Mean value of oxygen consumption rate in large A. purpuratus juveniles exposed to three salinities at 16 °C (a) and 22 °C (b).□ fed scallopsand 24 h fasted scallops. Vertical lines represent the standard deviation. Different letters indicate significant different values (Tukey's pb0.05)between treatments.

462 G. Soria et al. / Aquaculture 270 (2007) 451–463

osmotic equilibrium by breaking down proteins to formintracellular osmolytes. Navarro and González (1998)have found increasing NH4

+–N excretion rates withdecreasing salinity. These results are in agreement withthose described for other scallops species underdecreasing salinity regimens (Shumway, 1977; Briceljand Shumway, 1991). Small A. purpuratus juvenileshad a higher NH4

+–N excretion rate at higher tempera-tures, although this pattern was not clear in largejuveniles, suggesting higher metabolism. Conversely,for juveniles of the same species (h=14–17 mm)González et al. (2002) found a higher excretion rate at10 °C compared to 18 °C. Other patterns were describedby Navarro et al. (2000), who found similar values ofNH4

+–N excretion at 16 and 20 °C in scallopscontinuously fed during reproductive conditioning.

Scope for growth was positive in all treatments, buthigher values do not represent high survival or final shellheight values. We suggest that interpretation of scope forgrowth values should be complemented with survival andgrowth data. The increasing scope for growth value with

Table 8Two-way analysis of variance examining the effects of salinity andfood (1 h fed or 24 h fasted) on oxygen consumption rates in large A.purpuratus juveniles

Parameter df F p value

16 °C Fed condition 1 140.139 0.000 ⁎

Salinity 2 8.116 0.006 ⁎

Interaction 2 0.012 0.988Total 17

22 °C Fed condition 1 3.785 0.076Salinity 2 0.906 0.430Interaction 2 0.556 0.588Total 17

⁎ Significant differences at pb0.05.

increasing salinity is explained by the reduction inNH4+–N

excretion which, coupled with high level of absorptionefficiency, produced the observed results. In addition,increasing salinity caused a reduction inNH3–Nwhich, inturn, could reduce the toxicity of TA–N (Lawson, 1995).However, at a salinity higher than 38 g/l, osmotic stressbecomes more important, signifying that this salinity isclose to the upper limit. In A. purpuratus, Navarro andGonzález (1998) infer a lower salinity limit of 27 g/l (at12 °C) based on scope for growth and mention that thislimit could be transitory and time dependent.

The physiological results of the present study can beconsidered highly useful for A. purpuratus aquaculturemanagement under controlled hatchery conditions, as wellas inRAS. These results could also be of great use forwaterquality management, especially regarding TA–N levels.

Acknowledgements

The study was supported by a Master's scholarshipfrom InterAmerican Bank of Development-Japan Govern-ment granted toG. Soria and a FONDEFgrant (D 02I1095)received by Dr. E. von Brand. We also thank Mr. HectorGalleguillos, Eng. Mauricio Arco, Gloria Martínez, M.S.,Mr. Raúl Vera, and Magdalena Cisterna, B.S. for fieldassistance and technical support. We wish to express ourthanks to Jennifer N. Duberstein, M.S. for reviewing theEnglish language text and to two anonymous reviewers fortheir useful suggestion made on the manuscript.

References

Bricelj, V.M., Shumway, S.E., 1991. Physiology: energy acquisitionand utilization. In: Shumway, S.E. (Ed.), Scallops: Biology,Ecology and Aquaculture. Elsevier, pp. 305–337.

463G. Soria et al. / Aquaculture 270 (2007) 451–463

Cheng, J.C., Lin, C.Y., 1998. Responses of oxygen consumption,ammonia–N excretion and urea–N excretion of Penaeus chinensisexposed to ambient ammonia at different salinity and ph levels.Aquaculture 136.

Christophersen, G., Strand, O., 2003. Effect of reduced salinity on thegreat scallop, Pecten maximus spat at two rearing temperatures.Aquaculture 215, 79–92.

Colt, J., 1984. Computation of dissolved gas concentration in water asfunctions of temperature, salinity and pressure. American FisheriesSociety. Special Publication, vol. 14. 155 pp.

Colt, J.E., Armstrong, D.A., 1981. Nitrogen toxicity to crustaceans,fish and molluscs. In: Allen, L.J., Kinney, E.C. (Eds.), Proceedingsof the Bio-Engineering Symposium for Fish Culture. Fish CultureSection of the American Fisheries Society, pp. 34–47.

Conover, R.J., 1966. Assimilation of organic matter by zooplankton.Limnol. Oceanogr. 11, 338–345.

Coughlan, J., 1964. The estimation of filtering rate from the clearanceof suspensions. Mar. Biol. 2, 356–358.

Díaz, M.A., Martínez, G., 1992. Efecto de diferentes dietas sobre elbalance energético en juveniles de Argopecten purpuratus L. Rev.Biol. Mar. Valparaíso 27, 167–173.

Elliot, J.M., Davison, W., 1975. Energy equivalents of oxygenconsumption in animal energetics. Oecología 19, 195–201.

Farías, A., Uriarte, I., Varas, P., 1997. Estudio de los requerimientosnutricionales del ostión del Norte Argopecten purpuratus(Lamarck, 1819) durante el acondicionamiento reproductivo.Rev. Biol. Mar. Oceanogr. 32, 127–136.

Fivelstad, S., 1988. Waterflow requirements for salmonids in single-pass and semi-closed land-based seawater and fresh-water systems.Aquac. Eng. 7, 183–200.

González, M.L., López, D.A., Pérez, M.C., Castro, J.M., 2002. Effectof temperature on the scope for growth in juvenile scallops Ar-gopecten purpuratus (Lamarck, 1819). Aquac. Int. 10, 339–349.

Harris, J.O., Maguire, G.B., Edwards, S., Hindrum, S.M., 1998. Effectof ammonia on the growth rate and oxygen consumption ofjuvenile greenlip abalone, Haliotis laevigata Donovan. Aquacul-ture 160, 259–272.

Huchette, S.M.H., Koh, C.S., Day, R.W., 2003. Growth of juvenileblacklip abalone (Haliotis rubra) in aquaculture tanks: effects ofdensity and ammonia. Aquaculture 219, 457–470.

Kinne, O., 1964. The effects of temperature and salinity on marine andbrackish water animals. II Temperature and salinity combinations.Oceanogr. Mar. Biol. 2, 281–339.

Laing, I., 2002. Effect of salinity on growth and survival of kingscallop spat (Pecten maximus). Aquaculture 205, 171–181.

Lawson, T.B., 1995. Water quality and environmental requirements.Fundamental of Aquacultural Engineering, vol. 1. Chapman andHall, London, pp. 12–39.

Martínez, G., Caceres, L.A., Uribe, E., Díaz, M.A., 1995. Effect of thedifferent feeding regimens on larval growth and the energy budgetof the juvenile Chilean scallops, Argopecten purpuratus Lamarck.Aquaculture 132, 313–323.

Mercaldo, R.S., Rhodes, E.W., 1982. Influence of reduced salinity onthe Atlantic bay scallop, Argopecten irradians (Lamarck) atvarious temperatures. J. Shellfish Res. 2, 177–181.

Navarro, J.M., González, C.M., 1998. Physiological responses of theChilean scallop, Argopecten purpuratus to decreasing salinities.Aquaculture 167, 315–327.

Navarro, J.M., Leiva, G.E., Martínez, G., Aguilera, C., 2000. Interactiveeffects of diet and temperature on the scope of growth of the scallopArgopecten purpuratus during reproductive conditioning. J. Exp.Mar. Biol. Ecol. 247, 67–83.

Paul, J.D., 1980. Salinity and temperature in the queen scallopChlamysopercularis. Mar. Biol. 56, 295–300.

Rupp, G., Parson, J., 2004. Effects of salinity and temperature on thesurvival and byssal attachment of the lion's paw scallop Nodipectennodosus at its southern distribution limit. J. Exp.Mar. Biol. Ecol. 309,173–198.

Shumway, S.E., 1977. Effect of salinity fluctuation on the osmoticpressure and Na+, Ca2+ and Mg2+ ion concentrations in thehemolymph of bivalve molluscs. Mar. Biol. 41, 153–177.

Singnoret-Brailowski, G., Maeda-Martínez, A., Reynoso-Granados,T., Soto-Galera, E., Monsalvo-Spencer, P., Valle-Meza, G., 1996.Salinity tolerance of the Catarina scallop Argopecten ventricosus-circularis (Sowerby II, 1842). J. Shellfish Res. 15, 623–626.

Sokal, R., Rohlf, J., 1995. Biometry: the Principles and Practice ofStatistics inBiologicalResearch. Freeman andCo.,NewYork. 850pp.

Solórzano, L., 1969. Determination of ammonia in natural waters bythe phenolhypochlorite method. Limnol. Oceanogr. 14, 799–801.

Strand, O., Solberg, P.T., Andersen, K.K., Magnesen, T., 1993.Salinity tolerance of juvenile scallops (Pecten maximus L.) at lowtemperature. Aquaculture 115, 169–179.

Umbreit, W.W., Burris, R.H., Stauffer, J.F., 1957. The Warburgconstant volume respirometer. Manometric Techniques. BurgessCo., Minneapolis, USA, pp. 1–17.

von Brand, E., Merino, G., Abarca, A., Stotz, W., 2006. Scallop fisheryand aquaculture in Chile. In: Shumway, S.E., Parson, J.G. (Eds.),Scallop, Biology, Ecology and Aquaculture (Developments in Aqua-culture and Fisheries Sciences), vol. 35. Elsevier, pp. 1293–1311.

Widdows, J., 1985. The effects of stress and pollution on marineanimals. In: Bayne, B.L. (Ed.), Physiological Procedures. Praeger,New York, pp. 161–178.

Widdows, J., Fieth, P., Worral, C.M., 1979. Relationship betweenseston, available food and feeding activity in the common musselMytilus edulis. Mar. Biol. 50, 195–207.