The Science of the Total Environment, I l l (1992) 201-218 Elsevier Science Publishers B.V., Amsterdam

201

Toxic trace elements in Chilean seafoods: development of analytical quality control procedures I. De Gregori a, D. Delgado a, H. Pinochet"'*, N. Gras b, M. Thieck b, L. Mufioz b, C. Bruhn c and G. Navarrete c ~'Chemical Institute, Catholic University of Valparaiso, P.O. Box 4059, Valparaiso, Chile bNeutron Activation Analysis Laboratory, Nuclear Center La Reina, Chilean Nuclear Energy Commission, P.O. Box 188-D, Santiago, Chile ~lnstrumental Analysis Department, Pharmacy Faculty, University ~[~Concepcion, P.O. Box 273, Concepcion, Chile (Received March 18th, 1991; accepted May 4th, 1991)

ABSTRACT Chile is a well known producer and exporter of shell fish. These seafoods, like other specimens of marine origin, are susceptible to environmental and other contamination by trace elements, including toxicants. Therefore, adequate analytical quality assurance is mandatory before accepting analytical results. In this context, the use of at least two independent methods of determination and validation with certified reference materials (CRM) provides acceptable criteria for judging the reliability of the data. This paper describes sample treatments and analytical procedures for Cd, Cu and Hg determinations in mollusc samples. Three independent analytical techniques, namely differential pulse anodic stripping voltammetry, neutron activation analysis and atomic absorption spectrometry, were used. CRM standards of the 1AEA, NIST and BCR were analyzed to evaluate quality assurance. Following the quality control phase, the concentrations of cadmium, copper and mercury in fresh and canned mollusc samples Tagelus dombeii and Semelle solida (Navajuelas and Almejas chilenas respectively) from different locations were determined. INTRODUCTION

Chile is a country with --~4500 km of continental coastline bordering the south Pacific Ocean, with diverse geographical zones and with ocean waters of different abiotic and biotic characteristics. It is therefore in a favourable position to develop fishing activities, since its waters contain a great variety of marine resources, namely fish, shellfish and seaweeds. Fishing plays an important role in Chile. In 1988 alone, exports reached a total of > 5 million

* Author to whom all correspondence should be addressed. 0048-9697[92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

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J L)E GRE(IORI ET AL.

tons [1], thus placing Chile as one of the most important fish-exporting countries in the world. Environmental pollution is a growing hazard to human health. Chemical industries are proliferating worldwide, and their harmful effects on the environment are increasing yearly [2]. Heavy metals, as harmful environmental pollutants, are the second most important toxicants producing adverse effects in organisms, and have caused concern in the marine environment. Therefore, countries consuming seafood have gradually increased their surveillance regarding maximum levels of metal contamination allowed for these products, as reflected by stricter quality control approaches for seafoods. In turn, these developments have increased the need for the exporting countries to enhance the quality of their products to comply with prescribed standards. Adequate quality assurance is required for every environmental investigation involving analyses of biological materials, probably the most crucial point being reliable sampling and sample treatment [3-7]. In view of this, three Chilean research institutions joined together to develop analytical procedures. These institutions had the required expertise for determining toxic trace elements by at least three different analytical techniques: neutron activation analysis (NAA) [8, 9], anodic and cathodic stripping voltammetry (DPASV) [10-12] and atomic absorption spectroscopy (AAS) [13, 14]. The objective of this collaboration was to establish sampling procedures and protocols, sample preparation and treatment methods, and the application of instrumental methods for the analysis of seafoods such as oysters, mussels, and various clams (razor, pink, etc.). The potential of shellfish analysis for monitoring human health risks from toxic elements such as Cd, Pb and Hg is well documented [15-18]. Here we describe different sample treatment procedures (including sampling, dissection, homogenization, lyophilization and dissolution) that have been investigated in the analysis of toxic trace elements in different Chilean seafoods, and analysis by the three different techniques. In order to assure quality control, reference materials were analyzed; the analysis of real samples was not undertaken until the analytical performances were satisfactory. Interlaboratory comparisons were also carried out in association with these studies. EXPERIMENTAL

Sampling and sample storage Samples of fresh molluscs (Semelle Solida and Tagelus Dombeii, "Almejas'" and "'Navajuelas chilenas'" respectively) were caught manually by diver from three areas off the Chilean coast well known as natural commercial banks for

TOXIC TRACE ELEMENTS IN CHILEAN SEAFOODS

203

these species: Arauco gulf (region VIII), Corral Bay and Ancud Gulf (region X). Samples were also obtained from initial stages of the canning industry's seafood export operations. The specimens were classified according to size. Semelle Solida: > 7.5 cm (large); between 7.4 and 4.6cm (medium); and < 4 . 5 c m (small). Tagelus Dombeii were classified as small ( < 8.4cm) and large (> 8.5 cm). All samples were collected in precleaned containers and stored deep frozen in precleaned plastic bags.

Sample preparation Preparation and treatment of all samples to be analysed was carried out in adequate working areas. Two interconnected rooms were equipped for this purpose at the Nuclear Research Center La Reina, near Santiago. The first room is a "preclean area" designated for changing clothes and initial storage of samples. The second room in the "clean area" has a laminar fumehood which was used for sample treatment. The walls and ceiling of the laboratory are covered with white epoxy paint, the floor is covered with vinyl and the doors and windows are completely sealed with special materials to avoid external contamination [19, 20]. Mollusc samples were thawed in a microwave oven for a few minutes prior to analysis, then opened with a titanium knife and the tissue separated from the shell. Each sample was divided into two subsamples; one was dissected and the visceral tissue was separated from the muscular mass. Samples were crushed and homogenized using a plastic food processor (Moulinex), specially adapted with a high-purity titanium blade which was designed and fabricated in-house. Once this stage was completed, the samples were mixed and homogenized in a food processor equipped with plastic blades. Homogenized samples were then transferred to special plastic flasks and were lyophilized (Lyovac GT2) for 72 h at 12°C and at 0.1 mbar pressure. Moisture loss during lyophilization was determined for all samples. Lyophilized samples were homogenized again, and preserved in desicators at room temperature until the analysis was carried out. The sample treatment scheme is summarized in Fig. 1. All sampling flasks, cells and materials for sample treatment and heavy metal determination were acid cleaned, rinsed with adequate amounts of pure water and stored in polyethylene bags until used. All sampling and handling operations were carried out using plastic implements, quartz or titanium blades and stored with great care in order to prevent contamination. Reagents for treatments, digestions and analytical determinations were of ultrapure quality (Suprapur Merck). The water used was obtained from a Millipore Milli Q water purifying system which provided deionized water with particularly low levels of the trace metals under study.

204

L DE GI~,I~OORI I-~! A L .

SAMPLING

SAMPLE PREPARATION

SHELL REMOVAL ]

HOMOGENIZATION (Grinding -- M i x i n g )

SAMPLE WEIGHING]

FREEZE ( - - 2 0 j C)

POLYETHYLENE

FLAS~

FREEZE DRYING

HOMOGENIZATION (Grinding

-- M i x i n g )

SAMPLE STORAGE]

4__

1

DETERMINATION OF RESIDUAL HUMIDIT9

1__1 SUBSANPLIHG ANALgSIS

FOR

Fig. I. Scheme of general sample treatment.

Voltammetric determination Reference material or tissue subsamples of 0.3-0.5 g minimum lyophylized weight were digested in duplicate at 110°C with HNO3 (4 ml)/H:SO4 (1 ml) in high-pressure decomposition vessels. The digested samples were cooled, transferred to a quartz flask and treated with HNO3 to decompose residual organic matter totally, and then heated to give a white residue. The sample was then dissolved and made up to the appropriate volume with 0.1 M KC1 solution. Aliquots of this solution were analysed by differential pulse anodic stripping voltammetry at a mercury film

TOXICIRACE ELEMENTSIN CHILEANSEAFOODS

205

I SAMPLE WEIGHT }

HUMIDIT9 DETERMINATION ]

SAMPLE PRESSURE DECOMPOS I T I ON HN0~./H=SO, l i ~ ' C ~ R In

DESTRUCTION OF RESIDUAL ORGANIC MATTER HN0:

RASED WITH O.1 M HCI sp

1 I ALIQUOTATIOH I

VOLTAMMETRIC DETERMINATION I

I DPASU on HMD pH 2

DPASU on TFME

/ I

pH 2

{Hg

(I I)

Ig,/l

200

;.11

I I STANDARD ADDITION

Fig. 2. Scheme of general analytical procedure using voltammetric analysis.

electrode formed in situ on a glassy carbon support, or at a hanging mercury drop (HMD) electrode, depending on the concentration of metals in solution. The instrumental conditions employed have recently been described in detail [21]. The general analytical procedure is summarized in Fig. 2. Standard solutions containing 1000 #g/ml of metals were prepared from Merck Titrisol and deionized water from a Milli-Q purification system. Diluted standard solutions were prepared daily. A known sample weight was heated at 100°C for 16 h; results are reported on a dry weight basis.

206

J. D E G R E G O R I

EI AL

Neutron activation analysis Reference materials, samples of 0.3-0.5 g minimum lyophylized weight and standards were placed in clean quartz ampoules and irradiated for 24 h at a thermal neutron flux of 2 x 10~3n/cm2/s. Each sample and standard was irradiated using an iron flux monitor for neutron flux corrections in the different irradiation configurations.

Mercury determination After a decay time of 2 or 3 weeks, the irradiated sample was transferred to a clean polyethylene vial and then counted for l h using gamma-ray spectrometry, recording the 279 keV photopeak produced by the decay of 2o3Hg. A special correction was performed in order to avoid interference from 75Se in the 279 keV gamma-ray considered for the determination of mercury [22].

Copper and cadmium determination Following a 4-5 day decay period, the irradiated samples were decomposed with a mixture of concentrated HNO3 and H202 in the presence of inactive Cu and Cd, and heated until the solution was completely clear. The solution was evaporated to dryness and the residue dissolved in 6 N HC1. This solution was eluted through a chromatographic column containing antimony pentoxide to retain sodium. Na2 SO3 was added to the solution to ensure that all the copper was present as Cu(I), which was then precipitated as CuCNS by addition of a solution of KSCN. The solution was counted for 30min for Cd determination using the peak of 336 keV. The CuCNS precipitate was centrifuged, washed twice with distilled water, re-dissolved in concentrated HNO 3 and the solution counted for 20 min using the 511 keV photopeak [23, 24]. Schemes for the analysis of Hg, Cd and Cu are shown in Figs 3 and 4.

Atomic absorption spectrometry Sample preparation Powdered and homogenized sample (0.5 g) was weighed out accurately into P T F E digestion vessels from acid digestion bombs (Parr 4748). Reagent grade conc. H N O 3 (14ml) (with low Hg content) was added carefully to each sample, and the sample covered with a lid and digested at room temperature for ,-~ 1 h. The PTFE-lined vessels were inserted into the bomb vessels and the screw-on caps were tightened appropriately. The bombs were placed into a clean, preheated oven and sample digestion was performed at 140-145°C for 2.0 h. The bombs were then air-cooled to ambient temperature for 2 h. Sample solutions were transferred quantitatively into volumetric flasks and diluted to

207

TOXICTRACE ELEMENTSIN CHILEANSEAFOODS

I

Weighedand seated samplesinquartzampoutes

Neutron Irradiation ( RECH-1

L

NuclearReactor F

24h Thermal neutron flux lx

Decoyduring

1013 n//cm 2x s

20 days

1 hour counting measuring 27g KeV gamma ray of Hg-203

Fig. 3. Scheme of the analysis of Hg using neutron activation analysis.

25ml with reagent-grade water. Each sample was prepared in duplicate. Blanks were prepared similarly in duplicate, containing the same amount of reagents.

Determination of mercury Mercury determinations were performed in a Perkin Elmer 380 atomic absorption spectrometer equipped with a deuterium arc background corrector, a Perkin Elmer hollow-cathode lamp (i = 6 mA), and a homemade cold-vapor system with a gold amalgamation stage, described elsewhere. The 253.7nm Hg line was used with a slit setting of 0.7 nm. The peak height absorbance mode was used and the integration time was 12 s. The absorption signals were recorded on a Beckman 10" lin-log potentiometric recorder run at 0.1 inch/min. An aliquot of the sample solution (0.2-2.0ml) was introduced into the reagent vessel with a micropipette, and reagent-grade water was added to 10ml. Using micropipettes, additions were made of 0.2ml (or more)

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J. DE GREGORI E'] AL

Weighed and seated samptes in quartz ampoutes

Neutron Irradiation ( RECH-1 Nuclear Reactor

Decay during

F

2Z,h Thermal neutron flux $ 1 x 1013 n/¢m2x /

4 days

I Wef ash degestion of Ii radioactive sample

HNO3- H202- CuCd"carrier"

I Heat to dryness ]

/ ' Redissotu~fion using HCI 6N

HAPI

ElufiOnresmthrough.

Addition of Nu2SO3 and KSCN

d v[ CuSCN(precipitate) ]

Dissotution with HNO33N 30 minutes counting for 115-Cd/115-[n I determination using I gammaroy specfrometr

Determination of 64-fu usingl gamma ray spectrometry ]

Fig. 4. Scheme of the analysis of Cd and Cu using neutron activation analysis.

] O X I C TRACE ELEMENTS IN CHILEAN SEAFOODS

209

5% KMnO4 until a purple color persisted, the solution shaken for 45s, followed by the addition of 1 ml conc. HNO3 and 0.1 ml conc. H2SO4, the solution shaken for a further 20 s, and finally 25/tl 4% NH2OH ° HC1 was added and the solution shaken until colorless. The vessel was immediately connected to the cold-vapor system and a stream of Hg-free air (180 ml/min) passed through the solution for 10s. The reducing solution (10% SnCI~ in 30% v/v HC1, free of Hg) was added to the reagent vessel by a peristaltic pump at a rate of 8.0ml/min for 15 s (1.0ml). The carrier gas caused rapid mixing of the sample with the reducing solution, and the elemental mercury vapor evolved was transferred by the carrier gas to the gold amalgamation unit for collection. Alter 2.5 min of collection, the gold amalgamation unit was heated rapidly to 700°C to remove mercury, and the carrier gas stream transferred the mercury vapor to the absorption cell, until the recorded absorption signal reached a maximum in a few seconds. Simultaneously, the read function of the spectrophotometer was activated. Peak-height absorption signals were then determined by the spectrophotometer.

Determination of copper and cadmium Copper and cadmium determinations were performed using a Perkin Elmer 1100 microprocessor-controlled atomic absorption spectrophotometer equipped with an air-acetylene flame system, a 4-inch single-slot burner head, a deuterium arc background corrector and an Epson FX-800 printer. Hollowcathode lamps of copper (i = 15 mA) and cadmium (i = 4 mA) were used. The 324.8 nm Cu line and the 228.8 nm Cd line were used with a slit setting of 0.76 nm. The deuterium arc background corrector was used in the cadmium determinations. The integration time was 1.0s and the hold measurement mode was used. The gas flow rates were 1.81/min for acetylene (99.998%, I N D U R A ) and 8.0 l/min for auxiliarly compressed air. Determination of lead Lead was determined by electrothermal atomic absorption spectrometry with a L'vov platform, using a Perkin Elmer 1100 microprocessor-controlled atomic absorption spectrophotometer equipped with a deuterium and background corrector, a HGA-400 graphite furnace, a AS-40 autosampler and a lead hollow-cathode lamp. A wavelength of 283.3 nm and a spectral slit width of 0.7 nm (Alt.) were used. The hollow-cathode lamp was set at 8 mA, and background-corrected absorption signals (peak-height mode) were measured with an integration time of 7 s. Argon (99.998%, I N D U R A ) was used as carrier gas and sample aliquots were 10#1. The optimized graphite furnace conditions are listed in Table 1.

210

i DE GREGORI~-:rAL

TABLE 1 Graphite-furnace program for lead determinations with the L'vov platform Program step

Temp. (°C)

R a m p time (s)

Hold time (s)

Argon (ml/min)

Drying (I) Drying (2) Pyrolysis Atomization b Cleaning Cooling

130 200 550 1550 2500 20

20 15 30 0~ I 1

10 20 15 6 3 I0

300 300 300 ~' 40 300 300

~'Miniflow (40 ml/min) after 40 s of pyrolysis, bRead " o n " - - 1 s. ~Maximum power heating mode.

Calibrations Quantitation of metals was made by the standard addition method. Additions were made in duplicate through appropriate aliquots taken from standard solutions prepared daily. Calibration blank absorbances were low and were subtracted via the autozero function of the spectrometer. The mean blank content was subtracted from the sample contents. RESULTS AND DISCUSSION

Analysis of certified reference materials During the early seventies, atomic absorption spectroscopy (AAS), differential pulse polarography (DPASV) and neutron activation analysis (NAA) became the most important methods for the determination of many hazardous trace metals such as Cd, Pb and Hg. Since then, a vast number of determinations has been made in all kinds of environmental materials. Monitoring metal bioaccumulation in organisms requires strict quality control of the analyses. Proper application of quality assurance requires the analysis of certified reference materials (CRM) that match as closely as possible the matrix type and the elemental level of the actual samples [14, 15, 26]. Depending on the matrix type, the instrumental methods AAS, DPASV and NAA are used in conjunction with appropriate digestion or pretreatment procedures which must be performed correctly to avoid the risk of contamination. For this purpose, the use of working standards, the extended participation in interlaboratory intercomparisons and, where possible, calibration against appropriate standard reference materials,

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I O X I C TRACE ELEMENTs IN CHILEAN SEAFOODS

TABLE 2

Results obtained for the CRM analyzed by DPASV CRM

Element [~g/g + CU' (dry weight)] Cd

Pb

Obtained Oyster Tissue,

Certified

% Diff.

2.9

Obtained

Certified

0.48 ± 0.03

0.48 ± 0.04

% Diff.

3.6 ± 0.4

3.5 ± 0.4

0

0.11 + 0.03

0.11 ± 0.01

0

42 ± 9

45 ± 3

-6.7

0.7 ± 0.1

0.75 _+ 0.03

-2.7

1.8 ± 0.2

2.1 ± 0.3

-14.3

9 ± 3

10.2 ± 1.5

-10.8

703 ± 98

714 ± 28

-1.5

0.37 ± 0.08

0.36 ± 0.07

28 ± 3

28.2 ± 1.8

NBS 1566

Orchard Leaves, NBS 1571

Copepoda, MA-AI/TM IAEA

River Sediment, SRM 1645

Estuarine Sediment,

2.8

0.7

SRM 1646

~'Confidence limit.

become necessary [14-18, 25, 26]. One difficulty in this approach is the lack of an appropriate CRM with a similar matrix as the molluscs used in this study, and also with the required concentrations of Cd, Pb, Cu and Hg. Therefore, to overcome this situation, we used various NIST, IAEA and BCR certified reference materials, with different concentrations of these toxic metals, for analytical quality control. The results obtained for the CRMs analyzed by the different techniques are summarized in Tables 2-4. It can be seen that the results for all elements analysed by the different techniques are in agreement with the certified values. The measured value, including its uncertainty, lies within the certified value and its confidence interval (95%), and therefore there is no reason to believe that the difference is significant [27].

International interealibration exercise It is known that cadmium concentrations in canned mussels from Chile (Navajuelas Chilenas) are higher than those found in European mussels

< 1.9

Copepoda, MA-A1/TM IAEA

3.10 + 0.2

t.90 _+_ 0.1

0.38 _+ 0.07

"Confidence limit. **, Recommended values,

Pig Kidney, BCR 186

Rice Flour, NIES CRM 10C

Rice Flour, NIES CRM 10B