A MEASUREMENT OF MICRONUTRIENTS IN FISH OTOLITH AND SEA WATER

USING A HIGH PERFORMANCE ION CHROMATOGRAPHY-“HPIC”

M. Marini1 , A. Campanelli1 , F. Abballe2

1
National Research Council-Marine Science Institute,
Largo Fiera della Pesca, 2, 60125 Ancona, Italy
2
DIONEX S.r.l., Via della Maglianella 65, 00166 Roma, Italy

Abstract

Fish growth and the relation between growth and environment conditions offer a good

opportunity for measuring the micronutrients in fish otoliths.

The analytical method must involve high sensitivity when attempting to discriminate between

fish growth and environment conditions.

The aim of this paper is to identify a new chromatographic method: High Performance Ionic

Chromatography (HPIC) equipped with a high detection limit in determining important

micronutrients present in sea water and fish otoliths.

The work samples are: coast, off-shore and sediment waters and fish otoliths (Engraulis

encrasicholus, Mullus barbatus, Umbrina cirrhosa, Sciaena umbra, Pagellus erythrinus) in

the Adriatic sea and the Canal of Sicily.

The analytical instrumentation used has been furnished by Dionex: a gradient pump GP50

model, conductivity CD25 model and PEAKNET6 software unit. The method includes an

IONPAC CS12A chromatographic column, 18mM metansolfonoc acid eluent.

The detection limit readings obtained with this method, for fish otolith, are equal to or inferior

to 0.1 ppb for lithium (Li), 59 ppb for sodium (Na), 46 ppb for ammonium (NH4 ), 23 ppb for

potassium (K), 13 ppb for magnesium (Mg), 88 ppb for manganese (Mn), 2.567 ppm for

calcium (Ca) and 13 ppb for strontium (Sr) respectively.

The HPIC method described in this essay resolving overlaps such as Na which Li, and NH4 in

seawater; Ca which Mg and Sr in fish otolith. These elements are an essential constituent

present in otolith for describing the relation between growth and environment conditions.

Considering the final Sr reading the ion retention times of approximately 16 minutes is

relatively short.

Keywords: Fish Otolith, Seawater, Micronutrient, HPIC, Adriatic Sea.

1. Introduction

In the sea, there are continuous fluxes of organic and inorganic substances at different levels

of biotic and abiotic life as well as the production, consumption, degradation, mineralization

and other processes. The scope of this paper is to identify a chromatographic method HPIC

(High Performance Ionic Chromatography) equipped with detection limit (DL) capabilities in

determining important micronutrients present in seawater and fish otoliths.

Otolith of teleost fish are small, calcified structures located within the inner ear, whose

primary function appears to be the detection of sound and balance and/or hearing (Carlstrom,

1963). Their microstructure provides an accurate chronology of growth and life history

transitions (Campana and Neilson, 1985; Jones 1986; Wright 1991; Jones 1992; Yamamoto et

al., 1998; Campana 1999). Early studies of the chemical composition of otolith (Degens et al.,

1969; Dauphin and Dufour, 2003) describe two main components: an inorganic component

containing aragonitic calcium carbonate constituting up to 90 % of the otolith, and an organic

component, composed primarily of protein. Recent research has demonstrated the potential

value of otolith composition analysis for information about individual life- histories. This

potential is based on two assumptions: 1) otolith are metabolically inert, unlikely to be re-

absorbed, and grow throughout the life of the fish (Campana and Neilson 1985); 2) the
calcium carbonate and other elements which make up 90 % of the otolith are mainly derived

from the water and their food, where, the incorporation of different elements, is more

influenced by environmental factors rather than physiological factors (Mugiya, 1984; Gauldie,

1988; Edmonds, et al., 1992; Pontual et al., 1997; Elsdon and Gillanders, 2003).

The use of otoliths as environmental recorders is based on the premise that the differences in

water chemistry among different aquatic environments are manifested in the elementary

composition of otolith (Bagenal, 1974; Radke and Shafer 1992; Jones 1992; Campana and

Gagne 1994). This use also dependent on the observation that otoliths are a-cellular and

physiologically static; once accreted, material is not re-absorbed or metabolically re-worked

and thus the growth of these tissues continues throughout fish life (Campana and Neilson

1985; Mulligan et al., 1983). If these assumptions are correct, calcified structures are

permanent recorders of the influence of endogenous and exogenous factors on their calcium-

protein matrices. The hypothesis was that differences in the environments in which fish in

each population were exposed affected the elements in its calcified structures, which resulted

in chemical compositions specific to each population. The two assumptions are well

supported by an extensive fisheries literature for otoliths (Sauer and Watabe, 1989; Campana

1999). The elemental composition of otoliths is increasingly being examined as a means of

resolving the fine scale stock and population structure of fish. The technique has been applied

to three sets of problems: 1) discriminating between marine and freshwater populations (or

life history stages), 2) determining links between natal rivers or nursery area and adult stocks,

and 3) assessing population structure in marine fish (Thresher 1999).

The most popular instruments for targeted elementary analysis are four: wavelength-

dispersive electron microprobe (WD-EM); energy-dispersive electron microprobe (ED-EM);

proton- induced X-ray emission (PIXE); laser ablation inductively coupled plasma mass

spectrometry (LA-ICPMS). To rigorously compare the sensitivity, accuracy, and precision of

these four analytical tools is reported in Campana et al., 1997. The main conclusions can be

drawn regarding the analytical instruments in current use are reported in Thresher 1999. They

are: 1) probe micro-analysers offer a number of technical advantage over bulk analysers.

Chiefly, comparisons between individuals/stocks can in theory be made at specific fish life

history stage s.

However, the trade-off for spatial resolution and increased information content is that probe

micro-analysers are relatively insensitive. Although both micro-PIXE and LA-ICPMS can

nominally measure precisely elements at concentrations of 1 ppm or less, in practice neither

appears to have that resolution when applied to otolith studies (Campana et al., 1997). Given

the scarcity of elements in otolith at concentrations consistently greater then 1 ppm, current

generation probe micro-analysers severely limit the range of elements that could be factored

into stock analysis, 2) because every analyser assays a different set of elements, no single

analytical instrument is inherently more useful than any other. Hence, micro-PIXE is

extremely sensitive for metal analyses, but it can not “see” alkaline and earthy ions. ICP-AES

and electron probes do well with alkaline and earthy ions, and are nominally sensitive to a

wide range of elements, but have problems resolving overlaps and in practice and in general

are limited for metals. ICP-MS appears to provide a good compromise between ranges of

elements detected and sensitivity but also can accurately assay few elements when present at

low (<10 ppm) concentration and requires bulk treatment. The HPIC method described in this

essay resolving overlaps such as Na which Li, NH4 , in seawater; Ca which Mg, and Sr in fish

otolith. These elements are an essential constituent present in otolith and they are important

for discriminating between marine and freshwater populations, determining links between

natal rivers or nursery area and adult stocks, and assessing population structure in marine fish.

This method has been considered effective in determining alkaline and earthy ions across

different samples such as sea waters and otoliths.

The work samples are: coast waters (eutrophic water), off- shore waters (oligotrophic water),

sediment waters and fish otoliths in the Adriatic sea and Mediterranean sea.

2. Material and Methods

2.1 Sample collection and analysis

The otolith samples are: Engraulis encrasicholus of different ages, about 1 to 3 years, taken

from the Adriatic sea and the Canal of Sicily; Mullus barbatus and Pagellus erythrinus

captured in Gulf of Castellamare, Sicily; Umbrina cirrhosa and Sciaena umbra from the

Adriatic. The samples of Engraulis encrasicholus, Umbrina cirrhosa and Sciaena umbra are

taken from an ISMAR-CNR Ancona collection group, the sample of Mullus barbatus and

Pagellus erythrinus originate from IAMC-CNR of Mazzara del Vallo (TP) grouping. The

weight of the otolith and fish dimension are reported in Table 1.

The seawater samples are: coastal, sediment and off-shore waters of the Adriatic diluited 200

times in order to control the Na interference. The seawater sample, filtered using a GF/F

Wathman filter. In Figure 1 reports sites of the seawater samples.

The otolith sample, Figure 2, has been weighed and fully dissolved in 90 ? L HCl al 37 % than

diluted up to 15 mL with deionizate water.

The analyses of the otolith portion is possible starting from the margin to the centre with

additions of 5 ? L HCl (using micropipette 0.5-10 ? L with 0.1?? L increments) and weighing

after each acid addition which 0.01mg balance Mettler Toledo model AG285. This last

operation is computed by optical microscope Zeiss model Stemi 2000.

2.2 Analytical instrumentation

The analytical instrumentation used has been furnished by Dionex: a gradient pump GP50

model, conductivity CD25 model and PEAKNET6 software unit. The method includes an
IONPAC CS12A chromatographic column, 18mM metansolfonic acid eluent with a flux of 1

ml/min, a sample volume of 100 ? L. A standard solution containing 8 cations was prepared:

Li, Na, NH4 , K, Mg, Mn, Ca, and Sr, with concentration 0.5, 2, 2.5, 5, 2.4, 1.1, 5.1, 4.6 mg/L

respectively, of which Li, Na, NH4 , K, Mg, and Ca in a standard Dionex solution (6 cations II,

pH 3.0 in NO3 , product by Dionex) to which have been added Mn (MnSO4 2H2 0) and Sr

(SrCl2 6H2 O) produced by Merck. From this, 4 standard solutions were prepared (1:10, 1:20,

1:100, 1:1000 in deionized water) in order to determine the calibration rate for each cation.

3. Results and discussion

A five point calibration curve has been calculated for each element. Four of the five points

have been identified at specific concentration levels and through the point of origin (Table 2).

The co-efficient linear correlations are superior to 99 %. The 1:10 c hromatogram standards

are reported in Figure 3 with a retention time of 16.3 minutes for the last element.

In Table 3, we have detection limit values calculated in repeated analyses (10) taken from an

Engraulis encrasicholus otolith sample weighing 2.6 mg.

The detection limit is calculated with this equation: standard deviation * ts(99%).

In Figure 4, a chromatogram of Engraulis encrasicholus otolith shows good peak resolutions

of each of the 8 elements with the following concentration readings (mg/g) values

respectively as: Li (0.046), Na (38.936), NH4 (19.444), K (38.982), Mg (28.773), Mn (0.287),

Ca (2159.277), and Sr (8.464) as example.

In Table 4, the detection limit values have been calculated in repeated analyses (10) of a one

water sample. In Figure 5, 6 and 7, we have a water chromatogram for each sample (sediment,

coastal, and off-shore seawater) originating from different areas, with different concentrations

of dissolved nutrients. In coastal and off-shore water comparisons, there is strong presence of

Sr in coastal waters. In Figure 8, are reported data of Strontium concentration in seawater of

the Adriatic Sea, the readings are: for salinity >38 Strontium <22 mg/L, for salinity <37
Strontium >34 mg/L. This factor can be attributed to the presence of inflowing fresh river

water particular to this Western coast area of the Adriatic sea (Artegiani et al. 1997; Cushman

et al. 2001; Marini et al. 2002; Campanelli et al. 2004).

In the otolith Engraulis encrasicholus Adriatic Sea comparisons, there is a strong influence of

Strontium and Magnesium in the juvenile samples with respect to the adult samples. In Figure

9, Strontium and Magnesium concentration are reports concentration are reporting ranging

from 0.13 to 2.1 mg weight of the fish otolith groupings collected. This is attributed to the

different growth area of the Engraulis encrasicholus: juveniles grow in coastal waters while

adults migrate to off-shore waters (Haedrich, 1983; Able and Fahay, 1998; Cingolani et al.,

2001).

In a study of composed of 50 otoliths representing a population of approximately 2000

specimen, wholly analyzed Engraulis encrasicholus with different age groups and weights

ranging from 0.13 to 2.1 mg, one can observe that the Sr/Ca ratio decreases with increasing

age of the specimen (Fig. 10) and thereby confirming the validity of the Sr/Ca relation in the

study growth conditions with respect to environmental conditions as well as for Engraulis

encrasicholus found in the Adriatic Sea.

4. Conclusions

The method permits in relatively short time (16 min.) the identification of 8 cations that had

been present in seawaters and fish otoliths. The most revealing are the otoliths because of the

limited number of ppb of Sr, an important element in the aim of the study of otoliths. The

detection limit readings of otoliths are best for Li (0.1 ppb), while Strontium and Magnesium

are good (12 ppb), the others are intermediate.

Their reproducibility is very good; the standard deviation varies from 31.6-6 mg/L (Li) to

790.0 E-3 mg/L (Ca) in the otolith while, in water, it varies from 0.18 E-3 mg/L (Li) to 521.7

E-3 mg/L.
The speed and simplicity of the method facilitates the study of marine fish such Engraulis

encrasicholus which require repeated studies of water and fish samples ensures statistically

sound results.

The analytical method of application in Adriatic Engraulis encrasicholus samples, a specie

whose entire lifespan is spent in the sea, demonstrates a decrease in the Sr/Ca ratio with

increasing weight of the otolith is such that in coastal waters the Strontium is greater than in

the off-shore waters, thereby confirming the hypothosis that juveniles grow in proximity to

eutorophic coastline waters.

Acknowledgements. Firstly, I would like to thank Enrico Arneri for the starting input in this

research, followed by thanks to Fabio Badalamenti for the samples of Mullus barbatus and

Pagellus erythrinus, Gulf of Castellamare, and Nando Cingolani for the samples of Engraulis

encrasicholus, Sciaena umbra, Umbrina cirrhosa from the ISMAR-CNR Ancona collection

as well as thanks to Fortunata Donato and Sabrina Colella for her assistance in otolith sample

preparation. Lastly, my thanks go to Carmen Agostini for partecipation in the editing of this

manuscript.

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 A MEASUREMENT OF MICRONUTRIENTS IN FISH OTOLITH AND SEA WATER
USING A HIGH PERFORMANCE ION CHROMATOGRAPHY-“HPIC”

M. Marini1 , A. Campanelli1 , F. Abballe2

1
National Research Council-Marine Science Institute,
Largo Fiera della Pesca, 2, 60125 Ancona, Italy
2
DIONEX S.r.l., Via della Maglianella 65, 00166 Roma, Italy

Mauro Marini,
Istituto di Scienze Marine-Consiglio Nazionale delle Ricerche
Largo Fiera della Pesca, 60125 Ancona,
fax: +39 71 55313
e-mail: m.marini@ismar.cnr.it
 FIGURE CAPTIONS

Figure 1. Locations of samplings areas ( ? = north-western Adriatic: coastal and off-shore

waters, fish samples; ¦ = Gulf of Manfredonia: coastal waters, off-shore waters and sediment

water; ? = Gulf of Castellamare: fish samples; ? = Sicily Canal: fish saples.

Figure 2. Otolith of Engraulis encrasicholus, weight 2,6 mg, in square are evidenced the daily

increments (picture by OPTYMAS 6.5); Cingolani collection, ISMAR-CNR, Ancona.

Figure 3. Standard cromatogram of the eight cations; 1:10 concentration of the master

standard solution.

Figure 4. Cromatogram of sample of the Engraulie encrasiculus (weight 2.6 mg, fish length

140 mm).

Figure 5. Cromatogram of sample of the interstitial sediment seawater of the Adriatic sea.

Figure 6. Cromatogram of sample of the eutrophic seawater of the Adriatic sea.

Figure 7. Cromatogram of sample of the oligotrophic seawater of the Adriatic sea.

Figure 8. The concentration of Sr in seawater comparisons are as follows: in coastal waters

the salinity value is <37 while in off-shore waters the salinity value is >38.

Figure 9. Strontium and Magnesium concentration, from 0.13 to 2.1 mg weigh of the

Engraulis encrasicholus otolith collection (p<0.001).

Figure 10. Elemental Sr, Mg and Ca ratio of Engraulis encrasicholus otolith of the 0.13 to

2.10 mg weight collection (p<0.001).

 39

38

37
salinity

36

35

34
0 10 20 30 40 50 60
Strontium, mg/L
 1600
Sr
1400
Mg
1200 Correlation line, Sr
concentration, mg/g

1000 Correlation line, Mg

800
600

400

200
0
0 0.5
0,5 1 1.5
1,5 2 2.5
2,5
weight otolith, mg
 0,00600
0.006
Sr/Ca
0,00500 Mg/Ca
0.005
Correlation line, Sr/Ca
Correlation line, Mg/Ca
0,00400
0.004
elemental ratio

0.003
0,00300

0.002
0,00200

0.001
0,00100

0.000
0,00000
0 0.5
0,5 1 1.5
1,5 2 2.5
2,5
weight otolith, mg
Table 1.
Weight of otoliths and dimension of fish.
Fish species Fish length Weight otolith
mm mg
Pagellus erythrinus 165.0 40.96
Mullus barbatus 145.0 3.34
Engraulis encrasicholus 145.0 2.60
Engraulis encrasicholus collection 41.6-97.9 0.13-2.10
Umbrina cirrhosa 480.0 1110.96
Sciaena umbra 670.0 910.26
Table 2.
Calibration coefficients of the standard 1:10.
Elements Retention Correlation b a Standard
time, min coefficient, deviation
%
Li 3.77 99.9903 -0.0008 3.1455 0.012
Na 4.50 99.9648 +0.0487 1.6038 0.0427
NH4 5.14 99.1340 +0.0369 0.9130 0.0169
K 6.44 99.6416 +0.00242 0.6899 0.0162
Mg 11.09 99.8224 +0.0208 1.9063 0.0159
Mn 12.49 99.9349 -0.0002 0.1889 0.0005
Ca 13.92 99.9978 +0.0401 1.2518 0.0223
Sr 16.23 99.9579 +0.0007 0.1616 0.0013
Table 3.
Detection Limit (D L=S d * ts,99%) calculated in ten repeated analyses from otolith sample weighting 2.6 mg.
1 2 3 4 5 6 7 8 9 10 Mean SD ts DL

mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L (99%) mg/L

Li 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.0013 0.032E-3 3.25 0.000103

Na 0.4423 0.4345 0.4408 0.4455 0.4531 0.4479 0.4539 0.4502 0.4510 0.5012 0.4520 18.30E-3 3.25 0.05948

NH4 0.0843 0.0932 0.0909 0.0955 0.0961 0.1005 0.0999 0.1034 o.1132 0.1346 0.1011 14.06E-3 3.25 0.045716

K 0.3547 0.3452 0.3514 0.3505 0.3587 0.3512 0.3580 0.3543 0.3494 0.3716 0.3545 7.24E-3 3.25 0.023521

Mg 0.0190 0.0189 0.0199 0.0210 0.0215 0.0208 0.0211 0.0213 0.0221 0.0328 0.0218 3.99E-3 3.25 0.012973

Mn 0.0269 0.0313 0.0323 0.0583 0.0564 0.0530 0.0418 0.0490 0.0642 0.1216 0.0535 27.06E-3 3.25 0.087949

Ca 56.9693 548404 561346 553869 56.4712 55.1803 568271 56.3956 54.9781 56.4199 55.9604 790.02E-3 3.25 2.567575

Sr 0.2361 0.2419 0.2438 0.2449 0.2330 0.2418 0.2449 0.2397 0.2437 0.2412 0.2411 3.89E-3 3.25 0.012663
Table 4.
Detection limit (D L=S D* tStudent,99%) calculated in ten repeated analyses from sea water sample.
1 2 3 4 5 6 7 8 9 10 Mean SD t DL

mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L (99%) mg/L

Li 0.0014 0.0019 0.0013 0.0013 0.0013 0.0013 0.0013 0.0014 0.0013 0.0013 0.0013 0.18E-3 3.25 0.00061

Na 37.8390 37.7758 36.8843 36.4930 36.9697 36.6670 37.5696 36.4882 36.5727 37.0089 37.0268 521.74E-3 3.25 1.69564

NH4 0.0246 0.0280 0.0239 0.0270 0.0278 0.0297 0.0312 0.0357 0.0376 0.0396 0.0305 5.432E-3 3.25 0.01765

K 2.1670 2.2119 2.1168 2.1156 2.1509 2.1491 2.2145 2.1724 2.2065 2.2301 2.1735 41.05E-3 3.25 0.13341

Mg 7.1383 7.1308 6.9416 6.8582 6.9392 6.9198 7.0524 6.9125 7.0194 7.0297 6.9942 94.99E-3 3.25 0.30871

Ca 1.8185 1.8328 1.7702 1.7448 1.7702 1.7720 1.8058 1.7866 1.8222 1.8164 1.7940 29.12E-3 3.25 0.09465

Sr 0.1019 0.0933 0.1038 0.0819 0.1032 0.1058 0.0989 0.1049 0.1047 0.1132 0.1012 8.45E-3 3.25 0.02746