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Nuclear instruments and Methods in Physics Research B 130 (1997) 396-401 EJSEVIER

Nuclear microscopy study of fish scales

S.M. Tang ‘- * , I. Orlic ‘, K.N. Yu a3’, J.L. Sanchez ‘, P.S.P. Thong a, F. Watt ‘, H.W. Khoo b

’ ~epartnle~~t of Phy.sics, Naii~nu~ Utlii,ersi~ of Si~~ap~~re~ Kent Ridge, Singtlpore 119260, Singapore ’ School qfBiulogicul Sciences, Nutionnl Uniclersity qf Singupure, Kent Ridge. Singapore I I9260. Singupore

Abstract

Fish scales are useful to ichthyologists for purposes of classification, identi~cation, age det~rminatjon and history study. So far, all studies of fish scales, for whatsoever purposes, have relied on the use of optical or electron microscopes to study their form and structure. Employing the NUS nuclear microscope, we measured the concentrations of Sr, Zn, Fe, Mn, Ni, Cu, Cr, As, Se, Br and Pb in the scales of 17 fish species. The objective was to test the hypotheses that the trace elements in scales could be used for species differentiation, and that the fish scale could potentially be used as a monitor for the environment. Our preiimina~ results gave positive indications. 0 1997 Etsevier Science B.V.

Keywordst Nuclear microscopy; Fish scale; Trace elements; Environmental effects; Species differentiation

1. Introduction

Early studies of fish scales were motivated by the belief that they could be used for species classifica- tion [1,21, which turned out to be not very fruitful. Soon, ichthyologists realized that scales of temper- ate-zone fishes have year marks created by seasonal and temperature changes and these marks allow age dete~ination f3,4]. The variation in the external

structure of a fish scale in fact tells a lot about the life history of the fish, such as when it first went to

sea and how many times it has spawned [3]. So far, all studies of fish scales have been con-

fined to scale mo~hology. This paper reports our recent work on the study of trace elements in fish

* Corresponding author. Fax: + 65-777-6126; email:

phytsm~leonis.nus.sg

’ On leave from the City University of Hong Kong.

scales, which was carried out with the folIowing objectives: (a> to investigate if the concentrations of trace elements in fish scales can be used for species differentiation and (b) to investigate if fish scales can potentially be used as a monitor of the environment.

2. Materials and sample preparation

2. I, Specimens studied

Over 60 scales of the following 17 fish species were used in our study: Choerdon anchorago (Sl), Halichoeres bicolor (S2), Scolopsis phaeops (S3), Pentapus setosus (S4), Apogon sp. (S5), Gerres

abbreviatus (S6), Lutjanus chrysotaenia (S7), Up-

eneus tragula (S8), Cepha~ophoi~spachycentron (S9), Goby sp. (SlO), ~lenny sp. (Sl I>, marine Ore-

0168-583X/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO168-583X(97)00202-4

uchro~zjs ~ossu~bicus (S12), fresh water Ore-

uchru~~s ~ossa~zbicus (S 13), Salmo salar 6 141, Dussumieria elopsoides (S 15>, Lutes calcarifer fSl6), Ranio rerio (Sl7). Specimens S 1 -Sl 1 were caught by handline off the southern islands of Singa- pore and S 12 from a coastal estuary. S 13 and S17 were reared in a laboratory of the School of Biologi-

cal Sciences, National University of Singapore. S14 and S15 were imported fish obtained from a super- market. S16 was collected from a food store.

The fish used for the study were all members of the teleost group. Teleost scales exist in two forms:

cycloid and ctenoid. They consist of a layer of

acellular bone and an underlying fibrilary plate of collagenous connective tissue. The main constituent

of the bony layer is hydroxyapatite crystal compris- ing chiefly of calcium phosphate [5]. The lower fibrillar layer has a non-mineralised matrix compris- ing mainly of collagen and also a special protein, isopedine or ichthylepidin [6].

2.2. Salute Freparat~on

The tissues covering the scales were first scraped

off. Washing of the scales in distilled water was then carried out using a ultrasonic cleaner. After washing, scales of larger size ( > 5 mm X 5 mm) were clamped tightly between two metal frames while they were still moist. This was necessary because fish scales curl up when they are dry. For smaller scales (< 5 mm X 5 mm), ALTECO 110 super glue was used to hold them flat on a sheet of perspex. This super glue

was the only one found to be free from any interfer- ing impurity among 12 other brands of adhesive we had tested. All scale samples were coated with a thin

film of carbon by means of a carbon coater to minimize the effect of charge accumulation during irradiation.

3. Experimental method

The scales were analyzed with a 2 MeV proton

beam of spot size w 5 pm at the NUS nuclear microscopy facility 171. Proton Induced X-ray Emis- sion (PIXE) and Rutherford Backscattering Spec- trometry (RBS) measurements were carried out si-

multaneously. The latter was used to obtain informa- tion on the scale matrix and the accumulated charge

received by the scale. The accumulated charges used in our measurements varied between 5- 15 p_C. Typi- cally, a scale was irradiated with a 5 nA beam for 25 min. A 100 pm thick aluminum filter was placed in front of the Si(Li) detector to attenuate the Ca X-ray

intensity, hence enhancing the detection sensibility for the higher energy X-rays from the heavier trace elements. However, this filter absorbed the X-rays

from the lighter elements, such as Cl, S, P, Si, Al and Mg, which are also known to be present in fish

scales. The scan size employed varied from 400

pm X 400 pm to 4000 p,m X 4000 km, depending

on the size of the specimen being measured. With a few p.C of accumulated charge, specimen

damage due to the effects of heating was clearly observed. Since the scales were relatively thick (25-

250 pm>, the damage was normally confined to the side on which the proton beam directly hit. For the bony side, the irradiated area became brownish but there was no obvious scarring or calcination. For the

collagen side, the damage appeared to be more se- vere. The collagenous tissue swelled and its color

changed from milky white to brown after irradiation. However, repeated irradiation of the same area of a scale yielded almost identical spectra, indicating that the damage did not seem to affect the data quality significantly.

4. Measurements and data analysis

At least two scales were taken from each fish specimen for measurements. For the big scales, two

sets of PIXE and RBS spectra were obtained by

scanning both sides with 2 MeV protons. The thick- ness of the big scales varied from 100 p+m to 250

pm which could be considered infinitive thickness

for 2 MeV protons. For the scales of the small fish specimens, one set were glued on a perspex sheet with the bony side facing up and the other with the collagen side facing up so that PIXE and RBS spectra for either side could be obtained. The thick- ness of the small scales varied from _ 25 pm to -lOOi_Lm.

Since the teleost scale has two layers of different matrix and the thickness of each layer varies across

VIII. BIOLOGY

the scale, it is not possible to accurately extract the concentrations of the trace elements in the scale from the PIXE and the RBS spectra without knowing the exact thickness of each layer in the area of irradia- tion. In our spectrum analyses, we assumed that each of the two layers was infinitively thick. Although this assumption was not valid for some of the scales,

especially the small ones, it did give reasonable estimates for the purposes of our experiments. The

RBS spectra were analyzed by using the computer

code RUMP developed by Doolittle [E] and the PIXE spectra by the computer program GUPIX developed

by Maxwell et al. [9].

5. Results and discussion

5.1. Trace elements in fish scales and their distribu-

tions

Besides Sr, Zn was found to be the trace element

present with the highest concentration in all scales

measured and concentrated largely on the bony side. Its concentration in the scales of most species was in

4s60 RBS Specmim

t

(Bony side)

0 200 400 600 800

the range of 25-100 ppm but could be as high as a few hundred ppm in the scales of some species. Present in all scales were also Fe and Mn with concentrations ranging from 5 ppm to 30 ppm. Cr

and Ni were present in many but not all scales with concentrations of a few ppm or less. Cu was also found to be present in many scales, but not all, with

concentration about the same as those of Cr and Ni in most cases, except for two where it was well

above 10 ppm. As, Se and Br were observable in some scales, but with a fairly low concentration of a

few ppm or less. Fig. 1 shows the RBS and the PIXE spectra

obtained from the bony side as well as the collagen side of a Lates calcarifer scale. An interesting ob- servation from the PIXE spectra is that Zn was present with a higher concentration in the bony layer while Cu was found to be more concentrated in the

collagen layer. This appears to be the case for most of the scales we have analyzed. In Fig. 2, the Ca-,

Sr-, Cu- and Zn-maps obtained from a cross-sec-

tional scan of a Lates calcari’r scale show the profiles of these elements in the two layers.

I - 7500

7-n

i 5ooO

$5

I - 230

I 0

PLXE Spectrum (Collagen side)

. 1600

- 12w

150 300 450 600

Channel

Fig. 1. PIXE and RBS spectra obtained from a Z&es calcurffer scale.

SM. Tang et al. / Nucl. Instr. und Meth. in Phys. Res. B 130 (I 9971 396401 399

Zn

Fig. 2. Ca, Sr, Cu and Zn maps and profiles obtained from a 200 pm cross-sectional scan of a Lates cafcariffr scale.

5.2. The Danio rerio experiment

This experiment was performed to check if the amount of trace elements in fish scales is influenced by the aquatic environment’s elemental concentra- tions. The species Danio rerio was chosen for this

experiment. Six adult Dunio rerio were reared for a month in separate aquaria containing normal dechlo- rinated tap water spiked respectively with Feel, CuSO, and ZnSO, of two different concentrations:

1 X 10s5 M and 1 X 10e6 M. After the treatment, they were returned to an aquarium containing normal tap water for about two weeks before they were

sacrificed and their scales obtained for analyses. Three other Danio rerio were kept all the time in normal tap water and were used as control speci- mens.

Two scales from each of the nine Danio rerio were analyzed by proton scanning over an area of 800 pm X 800 pm, one on the bony side and the

VIII. BIOLOGY

400 S.M. Tang et al./ Nucl. Instr. and Meth. ‘in Phys. Res. B 130 f 1997) 396-401

Table I Sr/Ca, Fe/Ca, Zn/Ca and Cu/Ca concentration ratios in control and treated Daniu rerict scales

Scale

s17-1

S17-2

517-3

s17-4

517-5

S17-6

517-7

s17-8

s17-9

Average

Std. dev.

Remark

control

control

control

FeCI 1 X IO-’ M

FeCt i X 10°6 M

ZnSO, I X 10-” M

&SO, I X low6 M

CuS04 I X IO-’ M

CuS04 1 X 1O-6 M

Cont. in bold not

included

Sr/Ca Fe,/& Zn/Ca Cu/Ca

(mg/g) (mg/g) (mg/g) (mglg)

3.2 0.38 5.0 0.043

4.0 0.32 2.7 0.032

3.9 0.3 1 5.8 0.066

4.4 0.54 5.2 0.019

3.2 0.32 4.9 0.090

3.7 0.49 13.6 0.124

3.9 0.26 3.9 0.072

4.2 0.27 3.5 0.290

4.3 0.24 3.8 0.020

3.9 0.32 4.4 0.064

0.4 0.08 1.0 0.033

other on the collagen side. The mean overall thick- ness of these scales was about 35 p+m, which is less

the range of 2 MeV protons. In fact, the PIXE spectra obtained from the opposite sides do not differ

much (less than 10% in Ca X-ray intensity). For the purpose of comparison, we used the average concen- trations obtained from the two sides and normalized the concentrations of all the trace elements to that of Ca. The normalization minimizes the effects of thickness variations within the scaIes and among

different scales. The results are shown in Table 1. For the specimens which had been ‘treated’ with

1 x 10e6 M of FeCl/ZnSO,/CuSO,, there was no evidence of any increase of the treatment elements in

their scales. However, the scales of those specimens

which had been reared in aquaria spiked with 1 X

1O-5 M of FeCl/ZnSO,/CuSO, showed a clear

increase in Fe/Zn/Cu concentration. The Cu con- centration in scale S17-8 was more than four times of that in the control scales. The Zn concentration in scale SI7-6 was about three times of the normal average value. The increase of Fe concentration in S17-4 was, however, only marginal. The rest&s of this experiment indicate that the concen~ations of

Fe, Cu and Zn in fish scales, which have physio- logical function in the fish, respond to the elemental

increase in the aquatic environment. However, it must be mentioned that the concentration of 1 X 10m5

Table 2

Relative concentrations of trace elements in scales of fish species SI-S15

Element Fish species

Cr/Ca

Mn/Ca

Fe/Ca

Ni/Ca

Cu/Ca

Zn/Ca

As/Ca

Se/a

Br/Ca

Sr/Ca

Pb/Ca

Sl

0.053

0.275

0.220

0.006

0.009

0.429

0.001

0.005

0.006

4.366

s2

0.025

0.186

0.064

0.006

0.012

0.42 1

0.001

0.003

0.016

5.219

0.009

s3

0.018

0.079

0.099

0.019

0.019

0.202

0.001

0.009 0.160

4.474

s4 S5 56 s7 S8 s9 SlO Sll s12 s13 s14 S15

0.083

0.071

0.003

0.262

0.004

0.019

2.918

0.008

0.012

0.045

0.058

0.438

0.01 I

4.434

0.109

0.109 0.004

0.004

0.402

0.009 0.005 3.691 0.021

0.017

0.051

0.102

0.009

0.014

0.168

0.001

0.008 0.018 0.027 0.019

0.030 0.094 0.145 0.088

0.053 0.069 0.179 0.312

0.005 0.025 0.018 0.016

0.007 0.006 0.026 0.077

0.136 0.210 0.565 0.481

_ _

0.057 0.017 2.861 4.134

-

0.005 4.094 0.007

0.006 0.022 9.183 0.024

0.010

9.641

0.030

0.015 0.098

0.062 0.149

0.066 0.323

0.003 0.030

0.053 0.078

0.138 0.167

0.007 0.006

0.002

1.092

0.002

2.286

0.017

0.304 0.572

0.084 0.070

0.010 0.002

0.025 0.013

0.993 1.697

0.006 3.724 0.848

Unit: mg/g.

‘--): < 0.001 mg/g.

SM. Tang et af./~~cl. Instr. and Meth. in Pfzys. Res. 3 130 (19971396-401 401

M of Fe/Z/G is higher than the normal concentra- tion in seawater by two orders of magnitude.

5.3. Variation of elemental concentrations among different species

Table 2 shows the relative concentrations of eleven trace elements in the scales of 15 fish species (Sl- S15). S 1-S 11 were caught by handline off the south- ern islands of Singapore. The concentrations of many of the trace elements in the scales of SlO and Sl 1

were distinctly higher than those in the scales of Sl-S9. The concentrations of the trace elements in the scales of Sl -S9 varied, but within relatively narrow ranges. There was no obvious similarity in trace element concentrations among the scales. This implies that species differentiation by using the trace element concentrations in the scale couId be possi- ble.

The concen~ations of most of the trace elements in the scales of the fresh water Oreochromis mossambicus (S13) were higher than those in the scales of the marine Oreochromis mossambicus (S 12). This indicates that concentrations of the trace elements in the fish scale do depend on the condi- tions of the aquatic environment.

The Salmo salar (S14) and the Dussumieria elop- soides (S15) were imported from Norway and Japan, respectively. The concentrations of Zn in the scales of these two species were 2 to 4 times higher than those found in the locaf fish caught around Singa- pore. In the scales of Dussumieria elopsoides, the Sr content was only l/4 of that found in the local fish.

6. Concluding remarks

The result of the Dunio rerio experiment and the difference found between marine Oreochromis mossambic~ scale and fresh water Oreochromjs mossamb~c~s scale in trace element concentrations show that fish respond to changes of elemental con- centrations in the aquatic environment. Our measure- ments revealed that scales of some species contain very high concentration of Zn (such as the scales of Danio rerio, Salmo salar and Dussumieria elop- soides). Some fish scales have high concentrations of Sr (e.g. scales of Goby sp. and Blenny sp.) while

other show unusually low Sr concentration (e.g. scales of Dussummieria eloposoides). All these im- plies the possibility of using the trace elements in scales for species differentiation.

The advantage of using fish scales for the pur- poses of environmental monitoring and species dif- ferentiation as compared to other materials such as otoliths [10,11] is that sample preparation is easier and repeat measurements of the same scale can be done. One major problem in PIXE analysis of fish scales is that the determination of absolute elemental concentrations is not easy because of the scale’s two-layer structure and the irregular variation of layer thickness across the scale.

Acknowledgements

The authors wish to thank Mr. T.H. Ng for his assistance in sample preparation, data acquisition and in preparation of the figures used in this article. Thanks are also due to Mr. T.F. Choo for help he rendered in all the experimental runs.

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El

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