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Contribution of water chemistry and fish condition to otolith chemistry:

Comparisons across salinity environments

Article in Journal of Fish Biology · June 2015

DOI: 10.1111/jfb.12672

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Journal of Fish Biology (2015) 86, 1680–1698

doi:10.1111/jfb.12672, available online at wileyonlinelibrary.com

Contribution of water chemistry and fish condition tootolith chemistry: comparisons across salinity environments

C. Izzo*, Z. A. Doubleday, A. G. Schultz, S. H. Woodcock and B. M.Gillanders

Southern Seas Ecology Laboratories, School of Biological Sciences, The University ofAdelaide, Adelaide, SA 5005, Australia

(Received 1 July 2014, Accepted 24 February 2015)

This study quantified the per cent contribution of water chemistry to otolith chemistry using enrichedstable isotopes of strontium (86Sr) and barium (137Ba). Euryhaline barramundi Lates calcarifer, werereared in marine (salinity 40), estuarine (salinity 20) and freshwater (salinity 0) under different temper-ature treatments. To calculate the contribution of water to Sr and Ba in otoliths, enriched isotopes in thetank water and otoliths were quantified and fitted to isotope mixing models. Fulton’s K and RNA:DNAwere also measured to explore the influence of fish condition on sources of element uptake. Water wasthe predominant source of otolith Sr (between 65 and 99%) and Ba (between 64 and 89%) in all treat-ments, but contributions varied with temperature (for Ba), or interactively with temperature and salinity(for Sr). Fish condition indices were affected independently by the experimental rearing conditions, asRNA:DNA differed significantly among salinity treatments and Fulton’s K was significantly differentbetween temperature treatments. Regression analyses did not detect relations between fish conditionand per cent contribution values. General linear models indicated that contributions from water chem-istry to otolith chemistry were primarily influenced by temperature and secondly by fish condition,with a relatively minor influence of salinity. These results further the understanding of factors thataffect otolith element uptake, highlighting the necessity to consider the influence of environment andfish condition when interpreting otolith element data to reconstruct the environmental histories of fish.

© 2015 The Fisheries Society of the British Isles

Key words: Ba isotopes; otolith element uptake; salinity; Sr isotopes; temperature.

INTRODUCTION

The chemical analysis of otoliths has become a valuable tool for reconstructing theenvironmental histories of teleosts (Elsdon & Gillanders, 2003a; Gillanders, 2005;Elsdon et al., 2008). Water and diet are two sources of elements in the otolith (Cam-pana, 1999), with water largely identified as the predominant source in species testedto date (Doubleday et al., 2013). The contribution of water and diet to otolith chemistrycan, however, be influenced by environmental factors, such as temperature and salinity(Webb et al., 2012). Environmental factors directly affect physiological processes, suchas osmoregulation, respiration, metabolism and growth (Buckley et al., 1999; Bœuf &Payan, 2001), and thus the rate of elemental uptake into the otolith (Campana, 1999).

*Author to whom correspondence should be addressed: Tel.: +61 8 8313 7036; email: [email protected]

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© 2015 The Fisheries Society of the British Isles

C O N T R I B U T I O N O F WAT E R T O OT O L I T H C H E M I S T RY 1681

Osmotic regulation is required by all teleosts, irrespective of their external salin-ity environment (McCormick, 2001). Homeostasis in marine fishes is achieved bydrinking seawater, introducing elements into the blood plasma via the intestine.Conversely, in freshwater fishes, osmoregulation and elemental uptake occur viathe gills (Campana, 1999; McCormick, 2001). As regulatory pathways of elemen-tal uptake are likely to differ between the gill and intestinal interface (Melanconet al., 2009), the relative contribution of water to otolith chemistry may also differamong freshwater teleosts and their marine counterparts (Wells et al., 2003; Els-don et al., 2008). This renders direct comparisons of element deposition amongobligate freshwater and marine species tenuous (Doubleday et al., 2013), imped-ing assessments of the relative influence of salinity environment on the elementalotolith uptake (Zimmerman, 2005). This is particularly problematic as otolithelement data are commonly used to reconstruct the migratory patterns of fishesbetween salinity environments (Milton et al., 2000; Elsdon and Gillanders, 2003a;Gillanders, 2005).

Increased ambient water temperature necessitates an increase in food consumption tomeet greater metabolic demands (Bœuf & Payan, 2001; Katersky & Carter, 2007a, b),which in turn increases the availability of dietary sourced elements for incorporation(Webb et al., 2012). Furthermore, increased rates of somatic (and otolith) growthat higher temperatures exert kinetic and physiological controls on otolith elementincorporation, which likely varies among species and life-history stage (Waltheret al., 2010a). Altogether, these temperature-mediated processes have the potentialto increase the uptake of dietary sourced elements into otoliths, reducing the relativecontributions of water to otolith chemistry.

Consideration of condition indices in relation to environmental variables, whichare inherently linked, provides a means of further understanding the influences oftemperature and salinity on sources of otolith element uptake. Fluctuations in ambientwater temperature and salinity have been shown to alter the growth and conditionof fishes (Katersky & Carter, 2007b; Domingos et al., 2013), as well as modify-ing the chemical composition of otoliths (Milton et al., 2008). This is particularlyrelevant for diadromous species that utilize both marine and freshwater habitats astransitions between salinity environments are likely to alter the growth and conditionof the fish due to changes in osmotic regulatory demands (Bœuf & Payan, 2001).While many studies have investigated diadromous migratory patterns using otolithchemistry (Gillanders, 2005), few studies have explored the relative influence offish condition on elemental otolith uptake, which may have an equal or greaterinfluence on otolith chemistry and may in turn affect the accuracy of interpretingthe environmental histories of individual fishes (Sturrock et al., 2012; Walther &Limburg, 2012).

This study examined the effects of temperature and salinity on the per centcontribution of water chemistry to otolith strontium (Sr) and barium (Ba) inbarramundi Lates calcarifer (Bloch 1790). Lates calcarifer is catadromous inthe wild (Milton et al., 2000), providing an opportunity to directly comparethe effects of different salinity environments (i.e. marine, estuarine and freshwater) to contributions of water to otolith chemistry. This study also soughtto better understand how the uptake of elements from the water into otolithsis further influenced by short (RNA:DNA) and long-term (Fulton’s K) indicesof condition.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 1680–1698

1682 C . I Z Z O E T A L.

MATERIALS AND METHODS

E X P E R I M E N TA L D E S I G N

Lates calcarifer fingerlings (c. 10 weeks of age) were obtained from the Robarra Hatchery(www.robarra.com.au) at West Beach and delivered to the University of Adelaide, South Aus-tralia. Fingerlings had been raised at 27∘ C at a salinity of 10. Upon arrival, fingerlings wereheld in a 200 l holding tank filled with dilute seawater (salinity c. 10). Fingerlings were fedon a marine-based 1⋅5 mm floating pellet diet (Lucky Star; www.luckystarfeed.net) through-out the pre and experimental periods. Five days after arrival, fingerlings were batch marked viaimmersion in a 40 l tank filled with an alizarin complexone (C19H15NO8) solution (40 mg l

−1

in bore water) for 24 h (van der Walt & Faragher, 2003), as a way of distinguishing betweenexperimental and hatchery growth. Following marking, fingerlings were randomly distributedamong experimental tanks at a density of 10–15 fish per tank. Fish were gradually acclimatizedto experimental temperatures at a rate of 2∘ C per 24 h, using external heating units to increasetemperature. Acclimation to the different salinity treatments was achieved by raising or low-ering salinities at a rate of 5 every 24 h. Each tank contained 40 l of water, along with a smallsubmerged filter and aerator.

The overall experimental design consisted of three treatment variables: temperature, salinityand isotope enrichment. Water temperature was maintained at two treatment levels: ambientroom temperature (c. 26∘ C) and heated to 30∘ C. Each temperature treatment level had 12tanks which were randomly distributed across three water baths (n= 24 experimental tanksin total). Within each temperature treatment, tanks were separated into three salinity levels:marine (salinity 40), estuarine (salinity 20) and fresh water (salinity 0). Seawater (salinity 40)was sourced from the South Australian Research and Development Institute at West Beach,South Australia. Water in the freshwater tanks was aged (dechlorinated) tap water that had beenaerated for >24 h in a reservoir tank. The estuarine water consisted of 1:1 seawater:freshwatermix. For each combination of temperature and salinity, the four tanks were further split intotwo isotope treatments: water enriched with a combination of 137Ba and 86Sr isotopes anda non-enriched control. There were two replicate tanks for each treatment combination and10–15 replicate fish within each tank.

Water was enriched by dissolving isotopically enriched BaCO3 or SrCO3 (Oak Ridge NationalLaboratories; www.orni.gov) at a concentration of 0⋅03 mg l−1 for 137Ba and 0⋅25 mg l−1 for86Sr. Enrichment concentrations were based on baseline levels of Sr and Ba in the marine water(Sr= 85⋅93 mg l−1 and Ba= 0⋅08 mg l−1). Although baseline concentrations of Sr and Ba variedamong the three water types, a single isotope enrichment concentration was used for all salinitytreatments, ensuring a constant isotope enrichment volume (Webb et al., 2012).

Fish were fed twice daily to satiation and any excess food was removed from all tanks 1 h afterfeeding. Water temperature and salinity were measured twice weekly throughout the experiment.All tanks had weekly 25% water changes, and tanks were topped up between changes to main-tain salinity and isotope enrichment due to evaporation. Additional water isotope enrichmentwas achieved when water was replenished (not when topped up). Ammonia levels were testedregularly to ensure that water quality was maintained.

Fish were exposed to experimental conditions for up to 34 days. At the conclusion of theexperiment, all of the fish were euthanized in ice slurry and a portion of white pectoral muscletissue was removed and frozen at −80∘ C for nucleic acid quantification. Standard lengths (LS± mm) and wet body mass (WB ± gm) were recorded. Fulton’s K indices were based on LS andWB measurements (Mommsen, 1998).

N U C L E I C AC I D Q UA N T I F I C AT I O N

Frozen tissue samples were lysed in a tris-EDTA buffering solution containing 1%N-lauroylsarcosine (Caldarone et al., 2001) and the genomic supernatant was collectedfor RNA:DNA analysis. Nucleic acid quantification was performed using a Qubit 2.0 benchtop fluorometer and the associated DNA and RNA assay kits following prescribed methods(Invitrogen; wwwlifetechnologies.com). Standard curves were calculated using the Qubitfluorometer’s software, based on analysis of supplied reference standards. All nucleic acid



Table I. Operating parameters for the Agilent 7500cs inductively coupled plasma mass spec-trometer (ICP-MS) and the New Wave UP213 laser ablation system with ICP-MS

Solution ICP-MSCollision cell He (5 ml min−1)Cone PtIntegration time 0⋅1 s with three replicates for each isotope

LaserWave length 213 nmMode Q-switchFrequency 5 HzSpot size 30 μmLaser power 80%Carrier Ar (0⋅92 l min−1)

Laser ICP-MSOptional gas He (58%)Cone PtDwell times (in ms) 138Ba (300), 137Ba (400), 86Sr and 88Sr (200), 43Ca (100)

and 115In (50)

quantifications were undertaken within a single session. For each specimen, replicate aliquotsof the genomic extracts were analysed to quantify the DNA and RNA component. The c.v.of the repeated measures of DNA and RNA concentrations were low (c.v.%= 1⋅48 and 0⋅69,respectively). A sample containing no genomic material was also analysed periodically to testfor potential contamination.

WAT E R I S OT O P E A NA LY S E S

Water samples (25 ml) were collected from each tank at the beginning, middle and conclusionof the experiment. Each sample was filtered through a 0⋅45 μm filter and acidified with ultra-pure nitric acid to create a 2% HNO3 solution and frozen until analysis. For the analysis of Srand Ba isotopes, samples from the control freshwater tanks were analysed undiluted, while allother treatments were diluted 10:1, due to differing ambient elemental concentrations betweentreatments.

Water samples were analysed using an Agilent 7500cs inductively coupled plasma mass spec-trometer (ICP-MS; www.chem.agilent.com) (see Table I for operating parameters). All sampleswere analysed for 86Sr, 88Sr, 137Ba, 138Ba and 43Ca for element Ca ratios. Agilent Mass Huntersoftware was used to collect the raw data, which were calibrated against a multi-element stan-dard. The elemental standard was used to measure instrument drift and precision, which wasdeemed to be acceptable (c.v. < 5%). An indium spike was also used to measure elementrecoveries, which were deemed to be acceptable (recoveries of 98–102%). Water element con-centration data were adjusted using a correction factor based on deviations from expected naturalisotopic ratios in the control treatment tanks.

OT O L I T H P R E PA R AT I O N A N D I S OT O P E A NA LY S E S

Sagittal otoliths were removed and cleaned of any adhering tissue before being air-dried. Oneotolith from each fish was embedded in a clear setting epoxy resin (EpoFix; www.struers.com),which was spiked with 40 mg l−1 indium to enable discrimination between the otolith and resinduring analysis. Embedded otoliths were then thin-sectioned (c. 500 μm) using a low-speed saw(Buehler; www.buehler.com) and polished using progressively finer grades of lapping film. Pol-ished sections were mounted onto a microscope slide using indium-spiked thermoplastic glue(Crystalbond; www.crystalbond.com).


1684 C . I Z Z O E T A L.

Otoliths were analysed using a New Wave UP213 laser ablation system connected to anAgilent 7500cs ICP-MS (www.agilent.com) (see Table I for operating parameters). Spotablations were used to sample the marginal edge of the otolith, which represented experimentalgrowth only (between the alizarin mark and the otolith edge). All samples were analysedfor 86Sr, 88Sr, 137Ba, 138Ba and 43Ca to determine elemental ratios, as well as 115In as anindicator of when the laser was ablating epoxy or thermoplastic glue rather than otolithmaterial. After every 10th sample, a glass reference standard (NIST612) was analysed andused to correct for mass bias and machine drift. Similarly, at the beginning and conclusionof each analysis session, an internal CaCO3 standard (MACS-3: United States GeologicalSurvey; http://crustal.usgs.gov/geochemical_reference_standards/microanalytical_RM.html/)was analysed as a measure of precision, which was deemed to be acceptable (c.v. < 6%). Otolithelement concentration data were adjusted using a correction factor based on the deviation ofthe raw concentration data from the known isotopic concentration of the MACS-3 carbonatestandard. The Glitter software package was used to collect the raw data (Griffin et al., 2008).Further data reduction was undertaken in Excel (Microsoft) and all isotope data were smoothedusing a six-point running mean and the mean of the smoothed data used as spot depth profilesdisplayed an overall consistent signature for the experimental portion analysed. To calculatetotal Ba:Ca and Sr:Ca (in mmol mol−1), 138Ba and 88Sr isotopes were ratioed to 43Ca.

P E R C E N T C O N T R I B U T I O N C A L C U L AT I O N S

The per cent contributions of the isotope-enriched water to otolith Sr and Ba was cal-culated using a linear isotope mixing model for individual fish (Kennedy et al., 2000):% Cw = 100{1− [(Rw −Ro)(Rw −Rd)− 1]}, where R is the isotope ratio of interest and %Cwis the per cent contribution of water to the otolith, and the subscripts o, w and d representthe otolith, water and diet. The isotopic ratios of Ba and Sr measured in the control tankswere used for the water component of the equation. The natural isotope values for the diet(88Sr:86Sr= 8⋅38 and 138Ba:137Ba= 6⋅38) were used for the dietary component of the equation.

S TAT I S T I C A L A NA LY S E S

Statistical analysis was conducted using Primer 6.0 (Clarke & Gorley, 2006). For all tests,non-transformed data were fitted to Euclidean distance matrices and performed using unre-stricted permutations of the data. Three-factor and four-factor ANOVA was used to compare Ful-ton’s K and RNA:DNA among treatments, as well as to test for differences in isotope (88Sr:86Srand 138Ba:137Ba) and elemental (Ba:Ca and Sr:Ca) ratios in both water samples and otoliths,analysing for the effects of temperature, salinity and isotope treatment. Similar tests were per-formed on the per cent contributions of both the enriched Sr and Ba otolith chemistry. Additionaltests were performed to determine variations in rearing conditions among tanks. Temperature,salinity and isotope treatment were treated as fixed factors. For fish condition indices and waterdata, tank was treated as a random factor and nested within the other three variables. Despitedifferences in rearing conditions (Table SI, Supporting Information), there were no significantdifferences found in otolith chemistry among tanks, and thus data for replicate tanks were pooledand re-analysed without the nested tank term. If significant differences occurred, then post hocpair-wise tests were conducted to determine where differences occurred. Regressions were usedto determine whether Fulton’s K and RNA:DNA were significantly related to one another, aswell as element:Ca and per cent contribution values for both Sr and Ba.

The influences of fish condition and environmental factors on the contribution of waterchemistry to otolith Ba and Sr were assessed using generalized linear models. All combinationsof temperature and salinity as well as the condition indices, Fulton’s K and RNA:DNA, weresequentially added into an explanatory model set (n= 10). All models were ranked usingAkaike’s information criterion (AIC) corrected for small sample sizes (AICc) (Burnham &Anderson, 2004). The ratio of evidence for the highest ranked model was calculated by dividingthe weighed AICc (wAICc) for the best model by the wAICc of one of the two base modelsthat either included the terms temperature or salinity. If the difference between the secondranked model’s ΔAICc value and that of the best model was


cent contribution values were tested separately. Modelling was performed using R 3.0.1 (RDevelopment Core Team; www.r-project.org). Model fitting was conducted using the glmfunction from the lme4 package 0.999999-2 (Bates et al., 2013).

RESULTS

R E A R I N G C O N D I T I O N S

Rearing conditions in all of the tanks remained relatively stable across treatments(Table SI, Supporting Information), although significant tank effects were found fortemperature and salinity rearing conditions (Table SII, Supporting Information). Posthoc tests revealed that temperature only differed between tanks for both freshwatertreatments, and salinity varied between replicate tanks in the 30∘ C marine treatment.The remaining treatments showed no variation between tanks. For both temperature andsalinity, significant differences among treatments were found, as was expected basedon the experimental design (Table SII, Supporting Information).

WAT E R C H E M I S T RY

Elemental water concentrations remained consistent among control tanks through-out the experiment (Table SI, Supporting Information). Variability was considerablygreater among the water-enriched treatments (Table SI, Supporting Information), dueto the use of a single enrichment concentration for all salinity treatments. Correctedratios of 88Sr:86Sr and 138Ba:137Ba in the water samples for the control treatments wereclose to natural isotope ratios (Table SI, Supporting Information). These values wereused as the control isotopic ratios in per cent contribution calculations.

For water Sr:Ca, a significant interaction between temperature and salinity was found(Table II). The high temperature treatment showed no variation in water Sr:Ca amongsalinities, whereas the low temperature treatment showed significant variation in waterSr:Ca between freshwater and both the estuarine and marine treatments. There was alsoa significant isotope treatment and temperature interaction for Sr:Ca in water (Table II),with post hoc tests identifying variation between isotope treatments at the high tem-perature and between temperatures for the control treatments. There was a significantsalinity effect for water Ba:Ca among all treatments (Table II), with decreasing Ba:Cawith increasing salinity.

For 88Sr:86Sr, a significant interaction was found between salinity and isotope treat-ment (Table II), with pair-wise tests showing that almost all the pairings were signif-icantly different, with the exception of estuarine and marine salinities at the controltreatment level. A similar, isotope and salinity interaction was detected for 138Ba:137Ba(Table II). Pair-wise tests showed salinity differences were between the freshwater andboth the estuarine and marine treatments within the water-enriched tanks; however,no variation was detected for the control treatment. As expected, all salinities showedvariation between the control and enriched water treatments.

OT O L I T H C H E M I S T RY

Corrected ratios of 88Sr:86Sr and 138Ba:137Ba in otolith samples for the controltreatments (based on the MACS-3 carbonate standard) were close to the naturalisotope ratios (Fig. 1). Otolith 88Sr:86Sr was shifted significantly in all water-enriched


1686 C . I Z Z O E T A L.

Tab

leII

.A

NO

VA

ofSr

and

Ba

isot

ope

and

elem

enta

l(in

mm

olm

ol−

1)

ratio

sin

wat

erof

trea

tmen

ttan

ks

Sr:C

a88

Sr:8

6Sr

Ba:

Ca

138B

a:13

7B

a

d.f.

MS

FP

MS

FP

MS

FP

MS

FP

Isot

ope

trea

tmen

t(I)

1<

0⋅00

111

⋅91

<0⋅

0544

⋅75

329⋅

68<

0⋅00

115

772⋅

002⋅

39>

0⋅05

347⋅

4031

063⋅

00<

0⋅00

1Te

mpe

ratu

re(T

)1

<0⋅

001

8⋅86

<0⋅

050⋅

342⋅

53>

0⋅05

1737

0⋅00

2⋅63

>0⋅

050⋅

097⋅

58<

0⋅05

Salin

ity(S

)2

<0⋅

001

0⋅67

>0⋅

0517

⋅80

131⋅

15<

0⋅00

1<

0⋅00

114

6⋅87

<0⋅

001

0⋅10

9⋅17

<0⋅

05I×

T1

<0⋅

001

11⋅5

9<

0⋅05

0⋅20

1⋅45

>0⋅

0561

4⋅41

0⋅09

>0⋅

05<

0⋅00

10⋅

001

>0⋅

05I×

S2

<0⋅

001

2⋅49

>0⋅

0520

⋅62

151⋅

89<

0⋅00

130

309⋅

002⋅

33>

0⋅05

0⋅19

17⋅0

2<

0⋅05

T×

S2

<0⋅

001

3⋅89

<0⋅

050⋅

211⋅

53>

0⋅05

3717

2⋅00

2⋅85

>0⋅

05<

0⋅01

0⋅57

>0⋅

05I×

T×

S2

<0⋅

001

2⋅43

>0⋅

050⋅

060⋅

51>

0⋅05

2284

⋅80

0⋅18

>0⋅

05<

0⋅01

0⋅30

>0⋅

05Ta

nk(I×

T×

S)12

<0⋅

001

0⋅25

>0⋅

050⋅

642⋅

38>

0⋅05

5670

7⋅00

0⋅11

10⋅

011⋅

95>

0⋅05

Res

idua

l48

<0⋅

001

<0⋅

001

<0⋅

001

<0⋅

001



0

0·2

0·4

0·6

0·8

1·0

0

0·5

1·0

1·5

2·0

2·5

3.5(a) (b)

3.0

0C W C W C W C W C W C W

26° C 30° C

2

4

6

8

137 B

a:13

8 Ba

88Sr

:86 S

r

0

0·002

0·004

0·006

0·008

0·010(c) (d)B

a:C

a (m

mol

mol

–1)

Sr:C

a (m

mol

mol

–1)

C W C W C W C W C W C W

26° C 30° C

Fig. 1. Isotopic (a) 88Sr:86Sr and (c) 138Ba:137Ba and elemental (b) Sr:Ca and (d) Ba:Ca in Lates calcariferotoliths (mean+ s.e.), where control (C) and water-enriched (W) treatments are shown for each temperature(26 and 30∘ C). , fresh water (salinity 0); , estuarine (salinity 20); , marine (salinity 40). (a, c) ,natural isotope ratios 88Sr:86Sr= 8⋅38 and 138Ba:137Ba= 6⋅38.

treatments (Table III), with the greatest shift observed in the freshwater treatments[Fig. 1(a)]. A significant interaction between isotope treatment and salinity wasidentified (Table III), with ratios differing between water-enriched treatments for eachsalinity [Fig. 1(a)]. Otolith 138Ba:137Ba was significantly altered in all water-enrichedtreatments (Table III). Shifts in otolith 138Ba:137Ba appeared to be consistent acrosssalinities [Fig. 1(c)], and a significant interaction was identified between isotope andtemperature treatments (Table III). Post hoc tests indicated that there was significantvariation between otolith 138Ba:137Ba of control and water-enriched treatments forboth temperatures, but there was only significant variation between temperatures forwater-enriched treatments [Fig. 1(c)].

For otolith Sr:Ca, significant interactions between isotope treatments and salinity, aswell as isotope treatment and temperature were identified (Table III). Post hoc tests ofthe interaction between temperature and isotope treatment identified that Sr:Ca differedsignificantly across temperatures in the enriched water treatment, but not among thecontrol treatments [Fig. 1(b)]. Pair-wise tests found that Sr:Ca differed between all


1688 C . I Z Z O E T A L.

Tab

leII

I.A

NO

VA

ofis

otop

era

tios

and

elem

enta

lra

tios

(in

mm

olm

ol−

1)

from

Lat

esca

lcar

ifer

otol

iths

acro

ssal

ltr

eatm

ents

.Tan

kda

taha

vebe

enpo

oled

due

toa

non-

sign

ifica

ntef

fect

amon

gre

plic

ate

tank

s(P

>0⋅

5)

Sr:C

a88

Sr:8

6Sr

Ba:

Ca

138B

a:13

7B

a

d.f.

MS

FP

MS

FP

MS

FP

MS

FP

Isot

ope

trea

tmen

t(I)

10⋅

010⋅

06>

0⋅05

114⋅

8910

5⋅91

<0⋅

001

<0⋅

001

1⋅86

>0⋅

0554

5⋅59

447⋅

88<

0⋅00

1Te

mpe

ratu

re(T

)1

2⋅58

15⋅1

7<

0⋅00

11⋅

351⋅

24>

0⋅05

<0⋅

001

25⋅1

0<

0⋅00

17⋅

105⋅

83<

0⋅05

Salin

ity(S

)2

40⋅5

123

8⋅44

<0⋅

001

47⋅8

044

⋅06

<0⋅

001

<0⋅

001

127⋅

58<

0⋅00

10⋅

760⋅

62>

0⋅05

I×

T1

0⋅97

5⋅73

<0⋅

050⋅

900⋅

83>

0⋅05

<0⋅

001

0⋅01

>0⋅

056⋅

305⋅

17<

0⋅05

I×

S2

0⋅50

2⋅98

<0⋅

0544

⋅55

41⋅0

6<

0⋅00

1<

0⋅00

11⋅

69>

0⋅05

1⋅87

1⋅54

>0⋅

05T×

S2

0⋅45

2⋅68

>0⋅

050⋅

440⋅

40>

0⋅05

<0⋅

001

7⋅86

<0⋅

050⋅

260⋅

21>

0⋅05

I×

T×

S2

0⋅01

0⋅09

>0⋅

050⋅

600⋅

55>

0⋅05

<0⋅

001

0⋅46

>0⋅

050⋅

370⋅

30>

0⋅05

Res

idua

l14

30⋅

171⋅

08<

0⋅00

11⋅

22



026° C

Temperature treatment

20

40

60

80

100

Per

cent

con

trib

utio

n (S

r)

(a)

30° C 26° C 30° C 26° C 30° C0

26° C

20

40

60

80

100

Per

cent

con

trib

utio

n (B

a)

(b)

30° C 26° C 30° C 26° C 30° C

Fig. 2. Per cent contribution of water to (a) Sr and (b) Ba in Lates calcarifer otoliths (mean+ s.e.), where tem-perature treatments (26 and 30∘ C) are shown on the x-axis. , fresh water (salinity 0); , estuarine (salinity20); , marine (salinity 40).

salinities at all isotope treatments, with the exception of estuarine and marine treat-ments [Fig. 1(b)].

Otolith Ba:Ca showed contrasting trends to otolith Sr:Ca, with freshwater treatmentshaving higher levels of otolith Ba:Ca compared with estuarine and marine treatments[Fig. 1(d)]. Otolith Ba:Ca showed a significant interaction between temperature andsalinity (Table III). Pair-wise differences in Ba:Ca were found between temperaturesin the freshwater and marine treatments, but not for estuarine salinities [Fig. 1(d)].Conversely, the estuarine and marine treatments did not significantly differ in Ba:Ca atthe 26∘ C temperature treatment, whilst all of the salinities differed from each other atthe 30∘ C temperature treatment [Fig. 1(d)].

C O N T R I B U T I O N F RO M WAT E R

Per cent contributions of water to otolith chemistry for Ba ranged from 64 to 89%and for Sr from 65 to 99% (Fig. 2). A significant effect of temperature on the con-tribution of Ba from water was found (Table IV), whereby per cent contributions forBa increased with increasing temperatures [Fig. 2(b)]. For Sr, there was a significantinteraction between temperature and salinity (Table IV). The per cent contributionof Sr showed a significant difference between temperatures, although only betweenthe estuarine and marine treatments [Fig. 2(a)]. At the elevated temperature, per centcontributions were significantly greater in the estuarine treatment than the marine,but this pattern was reversed at the ambient temperature. There was no significantdifference between freshwater and either marine or estuarine treatments.

F I S H C O N D I T I O N I N D I C E S

Regressions between Fulton’s K and RNA:DNA showed no significant relationship,either among individual temperature and salinity and isotope treatments or when datawere pooled (r2 values ranged from 0⋅0005 to 0⋅1900), suggesting a decoupling of thetwo indices over the duration of the experiment (34 days).


1690 C . I Z Z O E T A L.

Table IV. ANOVA of per cent contribution of water to Sr and Ba in Lates calcarifer otolithsacross all treatments

%Sr %Ba

d.f. MS F P MS F P

Temperature (T) 1 5962⋅70 4⋅97 0⋅05T × S 2 2144⋅10 1⋅79 0⋅05Residual 66 1200⋅30 440⋅25

No tank effects were observed for RNA:DNA values, but a significant differenceamong replicate tanks was identified for Fulton’s K (ANOVA, F12,147 = 2⋅60, P< 0⋅05),with the paired tanks differing in the 26∘ C marine control treatment. Nevertheless,the effects of temperature and salinity on fish condition were significant irrespectiveof the inclusion of tank as a factor. Hence, tank effects were not considered substantialenough to influence statistical analyses and subsequent tests were performed afterpooling replicate tanks within treatments.

RNA:DNA values differed significantly among salinity treatments (Table V),with marine treatments having lower RNA:DNA than the estuarine and freshwatertreatments [Fig. 3(a)]. Fulton’s K was significantly different between temperaturetreatments (Table V), with fish exposed to the low temperature treatment displayinghigher Fulton’s K [Fig. 3(b)].

In general, there was a trend of decreasing element ratios and per cent contributionvalues for both Sr and Ba with increasing fish condition. Regressions among Fulton’sK, RNA:DNA, element ratios and per cent contributions for Sr and Ba showed nosignificant relationships, either within treatments or when the data were pooled (r2

values ranged from 0⋅0001 to 0⋅4600).Alternate models best explained the influences of environmental factors and fish

condition on the incorporation of otolith Ba and Sr (Table VI). For otolith Sr per centcontributions, the model that contained Fulton’s K and the temperature term was thebest ranked explanatory model (Table VI). The ratio of evidence for the inclusion of

Table V. ANOVA of treatment effects for Lates calcarifer RNA:DNA and Fulton’s K

RNA:DNA Fulton’s K

d.f. MS F P MS F P

Isotope treatment (I) 1 0⋅13 2⋅56 >0⋅05 0⋅36 1⋅75 >0⋅05Temperature (T) 1 0⋅01 0⋅11 >0⋅05 1⋅45 7⋅01 0⋅05 0⋅61 2⋅96 >0⋅05I × S 2 0⋅02 0⋅46 >0⋅05 0⋅24 1⋅17 >0⋅05T × S 2 0⋅03 0⋅51 >0⋅05 0⋅16 0⋅76 >0⋅05I × T × S 2 0⋅01 0⋅18 >0⋅05 0⋅18 0⋅86 >0⋅05Residual 159 0⋅05 0⋅21



0C W C W C W C W C W C W

26° C 30° C

0·2

0·4

0·6

0·8

1·0

RN

A:D

NA

rat

io

0

0·5

1·0

1·5

2·0

2·5

3·0(a) (b)

Fulto

n’s

K

C W C W C W C W C W C W

26° C 30° C

Fig. 3. Condition indices of (a) RNA:DNA and (b) Fulton’s K of Lates calcarifer reared under different condi-tions (mean+ s.e.), where control (C) and water-enriched (W) treatments are shown for each temperature(26 and 30∘ C). , fresh water (salinity 0); , estuarine (salinity 20); , marine (salinity 40).

Fulton’s K to the explanatory model was 3⋅60 and 83⋅86 times more likely to explainthe data than the base models containing either temperature or salinity, respectively(Table VI). Conversely, Ba per cent contributions were best represented by the modelcontaining temperature and RNA:DNA, which was 2⋅99 and 44⋅36 times more likely toexplain the data than the model containing temperature or salinity alone, respectively(Table VI).

Table VI. Rankings of the model set testing the influences of environment and Lates calcarifercondition on the per cent contribution of water chemistry to otolith Ba and Sr. The explanatorymodel set consists of combinations of fixed factors: temperature (T), salinity (S), Fulton’s K (K)

and RNA:DNA (R)

%Sr %Ba

Model d.f. ΔAICc wAICc Model d.f. ΔAICc wAICc

T +K 4 0⋅00 0⋅59 T +R 4 0⋅00 0⋅49T 3 2⋅56 0⋅16 T 3 2⋅20 0⋅16T + S+K 6 3⋅84 0⋅09 T +K 4 2⋅28 0⋅16T +R 4 4⋅70 0⋅06 T + S 5 3⋅14 0⋅10S+K 5 5⋅61 0⋅04 T + S+K 6 5⋅39 0⋅03T + S 5 6⋅03 0⋅03 T + S+R 6 5⋅60 0⋅03T + S+R+K 7 6⋅29 0⋅03 S 4 7⋅56 0⋅01T + S+R 6 8⋅33 0⋅01 T + S+R+K 7 7⋅95 0⋅01S 4 8⋅95 0⋅01 S+K 5 9⋅27 0⋅01S+R 5 10⋅76 0⋅00 S+R 5 9⋅65 0⋅00

AICc, Akaike’s information criterion corrected for small sample sizes; wAICc, weighed AICc.


1692 C . I Z Z O E T A L.

0%Sr

Freshwater 0 Estuarine 10–20

Salinity

Marine 30–40

%Ba %Sr %Ba %Sr %Ba

20

40

60

80

100

Per

cent

con

trib

utio

n fr

om w

ater

Fig. 4. Patterns of per cent water contribution (mean+ s.e.) to otolith Sr ( ) and Ba ( ) chemistry among salinityenvironments (see Table SIII, Supporting Information).

DISCUSSION

These results showed that Sr and Ba in the otoliths of L. calcarifer are predomi-nately sourced from the water. For Sr, the contribution of water ranged from 65 to99% among treatments, compared with 64 to 89% for Ba. This is the first study toreport a higher contribution of Sr from water in comparison to Ba. These results aregenerally consistent with previous studies that have investigated the per cent contri-bution of water and diet to otolith chemistry (Table SIII, Supporting Information).Similarly, water has been identified as the primary contributing source of otolith mag-nesium (Woodcock et al., 2012). There is a high degree of species specificity in percent contributions from water (Table SIII, Supporting Information), with values rang-ing from 62 to 88% for Sr, and 59 to 98% for Ba (excluding the contrasting results ofKennedy et al., 2000).

The greater dietary contributions reported by Kennedy et al. (2000) in thefreshwater-reared Atlantic salmon Salmo salar L. 1758 may be enhanced through theuse of a marine-based food, which is likely to have higher levels of Sr compared withthe freshwater rearing environment. This would result in the increased availabilityof dietary sourced Sr in comparison to the water, facilitating increased elementuptake. These findings, while indicating water as the predominant source, show dietcontributes considerably to otolith chemistry, with inferred maximum contributions ofc. 30 and 40% for Sr and Ba, respectively. These findings support an interactive (albeitnon-significant) effect of dietary sourced elements and salinity on per cent contributionvalues, whereby Sr-limited freshwater treatments had the lowest water per cent con-tributions for Sr and the highest for Ba (with a converse pattern shown for the marinesalinity treatment). Differences in dietary contributions highlight the importance ofquantifying background elemental concentrations in water and diet before determin-ing isotope spike levels (Woodcock & Walther, 2014). Furthermore, assumptions



made with regard to contributions of dietary sourced elements to otolith chemistrymay hinder interpretations based on otolith chemistry analysis (e.g. reconstructingfish movements through heterogeneous environments) and requires consideration(Doubleday et al., 2013).

This study directly compared the proportional contribution of water to otolith Sr andBa among marine, estuarine and freshwater environments through the novel use of acatadromous species. When the available per cent water contribution values for otolithSr and Ba are collated and plotted, no clear trends among salinity environments are evi-dent (Fig. 4), suggesting that there are negligible differences in the uptake of elementsvia the gill or the intestinal interfaces. Elemental partition coefficients (a measure of theamount of an element that is fractionated or restricted from ambient water to otoliths)have been calculated for several species of fishes (Bath et al., 2000; Milton & Chen-ery, 2001), and suggest differences between freshwater and marine species (Wells et al.,2003; Melancon et al., 2009). Considerable variations in element partition coefficients,however, exist among and within species, often differing by several orders of mag-nitude, e.g. Sr: 0⋅13–0⋅52 (Elsdon & Gillanders, 2003b, 2005a) and Ba: 0⋅004–0⋅43(Hamer & Jenkins, 2007; Melancon et al., 2009). Within-species variability of partitioncoefficients for Ba and Sr may partly be due to the influence of salinity and temperature,as well as elemental availability (Reis-Santos et al., 2013).

Temperature-mediated shifts in per cent contributions at different salinities suggestthat ambient water temperature may be the primary environmental regulator of otolithelement uptake. This idea is supported by a similar temperature-related pattern ofuptake in black bream Acanthopagrus butcheri (Munro 1949), whereby per cent con-tribution of water to otolith Sr and salinity were negatively related at high temper-atures, but positively related at low and ambient temperatures (Webb et al., 2012).Furthermore, temperature alone affected the per cent contribution of water to otolithBa in L. calcarifer, with elevated temperatures resulting in higher contributions fromwater. Although it could be assumed that at higher temperatures increased energyand metabolic demands would necessitate an increase in food consumption (Momm-sen, 1998), enhancing the relative contribution of dietary sourced elements in otoliths(Webb et al., 2012). Lates calcarifer are eurythermal and are capable of maximizinggrowth over a wide range of temperatures, including temperatures at which the currentexperiment was undertaken (Katersky & Carter, 2007a). Fulton’s K were significantlyhigher at the ambient (26∘ C) than the increased (30∘ C) temperature treatment (as wereRNA:DNA, albeit non-significantly), indicating optimal growth at lower experimentaltemperature conditions. Enhanced fish condition at the ambient temperature treatmentcoincided with lower per cent contributions from water for both Sr and Ba, indicatinga shift in the relative importance of dietary sourced elements into otoliths at the lowertemperature treatment. The relative shift towards an increasing contribution of dietarysourced elements during periods of optimal growth corresponds well with reported neg-ative correlation between growth rates and element partition coefficients in other teleostspecies (Walther et al., 2010a). Conversely, salinity alone affected RNA:DNA of L.calcarifer, which is consistent with the catadromous life cycle of the species, wherebythe growth of juveniles is enhanced by localized freshwater inflows (Staunton-Smithet al., 2004; Robins et al., 2006). These findings provide baseline values for relationsbetween the ambient environment and the condition of juvenile L. calcarifer and canbe used to direct further exploration of the growth of the species.


1694 C . I Z Z O E T A L.

Regression analyses did not detect relationships between fish condition and elementratios nor per cent contribution values for Sr or Ba. The application of generalizedlinear models, however, facilitated the exploration of the influences of environmentalfactors and fish condition on the per cent contributions of water chemistry to otolithchemistry. Ranking the explanatory models indicated that Fulton’s K and RNA:DNA incombination with temperature influenced chemical contributions of water into otoliths.Model ranking also indicated that salinity was less influential to the contribution ofwater to L. calcarifer otoliths, giving further support to the primary importance oftemperature to otolith element uptake.

Fulton’s K and RNA:DNA had different influences on otolith element incorporation.Strontium per cent contributions were best explained by the inclusion of Fulton’s Kwith temperature. Conversely, Ba per cent contributions from water were best explainedthrough the combination of RNA:DNA and temperature. Contrasting influences ofthese two condition indices to Sr and Ba may be linked to the incorporation rates of theelements into otoliths, as incorporation of otolith Ba from water is achieved in as littleas 5–8 days (Munro et al., 2008; Woodcock et al., 2011a, b), compared with >20 d forSr (Elsdon & Gillanders, 2005b). While element incorporation rates probably differamong teleost species, and life stages (e.g. due to differences in rates of otolith accre-tion) (Melancon et al., 2009; Woodcock et al., 2013), these approximate rates of incor-poration are consistent with the relative time periods of growth represented by the twocondition indices measured. RNA:DNA may represent growth within a week (Weberet al., 2003), providing a means of linking environmental conditions at the time of sam-pling to an individual’s growth and survival (Chicharo & Chicharo, 2008). Conversely,Fulton’s K (and other morphological indices) provides integrated measures of conditionon a time scale of weeks or months (Bolger & Connolly, 1989). Decoupling of thesetwo metrics over the experimental period provides further evidence of the differingtime spans represented by these two condition indices (Walther et al., 2010b). Whilethis study examined two metrics of fish condition, there are a suite of other parameters(e.g. rates of metabolism, reproductive timing and respiration) that are likely to sub-stantially influence the uptake of elements into otoliths that require further exploration.This study has shown that for L. calcarifer, endogenous factors (i.e. fish condition)outweigh the influence of salinity on otolith Sr and Ba concentrations and, thus, needto be considered in reconstructing salinity histories for this species. Reducing uncer-tainty due to endogenously derived noise in otolith chemical signals will enhance theaccuracy of reconstructing environmental histories of fishes (Sturrock et al., 2012;Walther & Limburg, 2012).

Disentangling the relative influences of environmental factors and metrics of fishcondition is complex as the two are inherently correlated (i.e. fish are in better conditionwhen exposed to optimal environmental factors). Moreover, genotype and environmentinteractions may impose further controls over physiological traits such as growth rateas well as individual fitness and condition (Stearns, 1989; Gjedrem, 2005). Therefore,physiological processes that regulate otolith element incorporation may be under somegreater level of genetic control (Clarke et al., 2011; Barnes & Gillanders, 2013), addinganother level of complexity in delineating mechanisms that control otolith elementaluptake.

This study aligns well with the growing body of literature that indicates water chem-istry as the predominant source of Sr and Ba in otoliths. The catadromous life history



of L. calcarifer provided further insights into the influence of temperature and salin-ity on the contribution of water to otolith chemistry, with contributions varying due totemperature (Ba) or interactively with temperature and salinity (Sr). The application ofgeneralized linear models provided a novel means of exploring the influence of environ-mental factors and fish condition on element incorporation, with the results indicatingthat contributions of water chemistry to otolith chemistry were primarily effected bytemperature and secondly by fish condition. These findings highlight the necessity toconsider the effects of fish condition in tandem with environmental factors when inter-preting otolith element data to reconstruct the environmental histories of fish.

The authors thank B. Wade and A. McFadden at Adelaide Microscopy for their assistance withthe laser ablation and solution-based ICP-MS. Thanks also to L. Falkenberg and M. McMil-lan for laboratory assistance. We thank the anonymous referees who improved this paper withtheir detailed comments and corrections. This research was funded by an ARC Discovery grant(DP110100716) and Future Fellowship (FT100100767) awarded to B.M.G. All animal handlingand experimental procedures were approved by the Animal Ethics Committee at the Universityof Adelaide (Permit No. S-2011-010).

Supporting Information

Supporting Information may be found in the online version of this paper:Appendix S1. Diet isotope analysis.Table S1 Rearing conditions and water chemistry for all tanks during the experimentalperiod (34 days), showing the mean ± S.E. temperature (Temp), salinity, isotope ratiosand elemental ratios (element:43Ca)Table SII ANOVA of rearing conditions (recorded temperature and salinity) amongtreatment tanksTable SIII Summary of literature assessing the per cent contribution of water tootolith Sr and Ba chemistry based on isotopic variations. Adapted from Doubledayet al. (2013)

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1696 C . I Z Z O E T A L.

Fisheries Science Center Reference Document 01-11, p. 22. Woods Hole, MA: NortheastFisheries Science Center.

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