Journal of Analytical Toxicology, Vol. 16, May/June 1992
Determinationof Arsenic in Fish by HydrideGeneration Atomic AbsorptionSpectrometry M. Navarro*, H. L 6 p e z , and M.C. L 6 p e z
Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, E- 18012 Granada, Spain M. S d n c h e z
Department of Analytical Chemistry" Faculty of Sciences, University of Granada, E- 18071 Granada, Spain
Abstract I A method utilizing microwave dissolution in a closed Teflon bomb followed by hydride generation atomic absorption spectrometry has been used for the routine analysis of arsenic in fish species from the coast of the province of Granada, Spain. The technique was evaluated for decomposing biological standard reference material (BCR mussel tissue) for arsenic analyses. The precision of the results ranged from 2.78 to 6.99%. The recovery percentages ranged from 96.33 to 100.00%. The values determined in the samples indicated slight contamination by this element in the zone. The concentrations of arsenic in fish ranged from 0.396 to 45.716 I~g/g.
Introduction The greatest sources of As pollution are human activity and modern technology. The prolonged intake of even low concentrations of As can cause serious toxic effects (1,2). This has led to growing interest in analyzing As levels in foodstuffs in recent years (3-7). It has been shown that fish is one of the greatest sources of As in the diet, to such an extent that it can reach more than 52% of dietary As intake (7). For that reason it can act as a biological indicator of marine pollution. The zone we have studied receives fresh water from the agricultural areas inland through several irrigation channels and small rivers. The sea is the receiver of urban and industrial waste water from the zone of Port and village of Motril. These factors could increase the level of As in the fish of this zone. Critical steps in any chemical analysis are the decomposition and subsequent preparation of the sample for measurements. Currently, the most widely accepted technique involves accelerated digestion by placing the sample in a sealed receptacle and subjecting it to acid and high pressure in a microwave oven (8-13). One of the most common procedures for the determination of "Address for correspondence:Dr. Miguel Navarro,Dpto. Nutrici6n y Bromatologfa,Facultad de
Farmaeia,Universidadde Granada,E-18012 Granada,Spain.
As in fish is hydride generation atomic absorption spectrometry (14-17). We have used this method to determine As levels in marine fish species found abundantly along the Mediterranean Sea coast of the province of Granada (Spain). The fish studied are consumed locally in large quantities and it is thus important to know their As content in order to identify any hazards before fish reach the consumer.
Experimental
Equipment. Digestions were carried out with a Parr Instrument Company 4782 microwave acid digestion bomb heated in a commercially available Moulinex FM-460 microwave oven (600 W). Measurements of the As were performed with a PerkinElmer 2380 atomic absorption spectrophotometer equipped with the Perkin-Elmer MHS-10 hydride generator. Signals (peak height mode) were recorded on an Omniscribe D-5000 Bausch & Lomb recorder. Reagents and standards. All solutions were prepared with ultrapure water with a specific resistivity of 18 Mf~ cm obtained by filtering distilled water through a Millipore Milli-Q RO15 purifier immediately before use. A stock solution containing 1000 mg/L of As was prepared dissolving arsenic (III) oxide in 2.5 mL of 25% (w/v) KOH and further neutralization with 20% (w/v) HC1. Finally, it was diluted to volume with 1.5% (w/v) HCI. Others reagents used were 70% (w/v) nitric acid (Merck Suprapure), 32% (w/v) hydrochloric acid (Merck), and vanadium pentoxide (Merck). For the hydride generation, the solution was 3% (w/v) sodium tetrahydroborate (Merck) in 1% (w/v) sodium hydroxide (Merck). This solution was filtered before use and stored in a refrigerator. All reagents were of analytical grade. The Community Bureau of Reference-Commission of the European Communities (CBR-CEC) certified material reference (CRM) used was mussel tissue (CRM 278), with a certified As content of 5.900 _+0.200 pg/g. Procedure. All different fish species were each analyzed in triplicate. The lyophilized samples (200 mg) were treated with 2.5 mL HNO 3 and 50 lag of V205 as a catalyst in the microwave acid digestion bomb. Mineralization was complete in 90 s with the oven at its highest setting. The digests were cooled and the re-
Reproduction (photocopying)of editorial content of this journal is prohibited without publisher's permission.
169
Journal of Analytical Toxicology. Vol. 16, May/June 1992
suiting solutions were diluted to a total volume of 25 mL with ultrapure water. Different aliquots were transferred to 50-mL volumetric flasks and adjusted to volume with a 1.5% (w/v) HCI solution. Previously. the method of standard additions was pertormed. Finally, a 10-mL aliquot was taken and analyzed for total As by hydride generation atomic absorption spectrometry,
ducible. Seven determinations in three different species were treated statistically as described by Stiel (19), and the results of the precision tests are summarized in Table It. We analyzed the flesh of 32 of the most common fish species in the studied area. The concentrations of As ranged from 0.396 to 45.716/ag/g, and the results for all species tested are shown in Table IIl.
Results Discussion The calibration graph was prepared in the range from 0.5 to 4.0 ~g/L. These solutions, like the blank (HNO3, V205) were subjected to the same acid digestion bomb treatment as the samples. The method of standard additions was performed, adding amounts of As ranging from 0.0 to 4.0 lag/L to four sample fractions of 200 mg. The comparison between the plotting of the calibration graph and the standard addition method indicates that there is matrix interference, so all the determinations have been made by the standard addition method. The sensitivity found, equivalent to 0.0044 units of absorbance, was 0.094/agfL. For instrumental conditions used in sample analyses, our calculated analytical detection limit (18) was 0.146 t.tg/L. Concentrations of As in all samples exceeded the analytical detection limit. The accuracy of the method was tested with recovery assays (Table l). Tests with CRM 278 gave a value of 5.795 + 0.131 IJg/g (n = 13). The technique was found to be both precise and repro-
Table I. Arsenic Recoveries (%) Obtained in the Analysis of Three Fish Samples As present (~tg)
As added (~tg)
As found (~g)
As recoveries
No. 21
5.768 5.768 5.768 5.768
0.000 1,031 2.062 3.093
5.374 6.595 7.634 8.536
93,17 97.00 97.50 96.33
No. 30
45.694 45.694 45.694 45.694
0.000 18.856 37.712 56.568
45.716 62.312 81.179 97.568
100.05 96.53 97.32 95.41
0.428 0.428 0.428 0.428
0.000 0.122 0.244 0.366
0,419 0,550 0.664 0.761
97.90 100.00 98.81 95.84
Sample
No. 31
(%)
Table II. Statistical Analysis of the Results of Determinations of As Concentration in Fish by Hydride Generation Atomic Absorption Spectrophotometry Sample
Mean As concentration* (l~g/g)
RSD (%)
No. 21 No. 30 No. 31
5.768 • 0.3092 45.694 • 1.2681 0.428 • 0.0299
5,36 2.78 6.99
9All values are the mean of seven determinations with standard deviation
170
A microwave acid digestion bomb technique for decomposition of fish samples was used. Decompositions were complete in 90 s, and the results of the analyses were good. The method is less complicated and time consuming than conventional dissolution techniques. It can be concluded that neither significant contamination nor loss of As occur during the digestion of the samples in the closed Teflon bomb. The accuracy of this method was tested with recovery experiments, the results of which are listed in Table I. Results of all analyses of CRM 278 for As were within the certified concentration range. This fact confirms that this digestion procedure gives good results for the determination o f total As in the samples we have used. The limit of detection and sensitivity were comparable to those reported by other authors and adequate for the range of concentrations of the samples analyzed in this study. The levels of As obtained are slightly higher in relation to those established by other authors in other areas ( 14+15,20). This fact could indicate the existence of a slight pollution in the fish. Nevertheless, the intake of this fish would not be harmful for health, because the levels of As are low and the main part of As is organic, which is less toxic, and is easily and rapidly eliminated in the urine (13).
Acknowledgments We would like to thank the Department of Analytical Chemistry of the University of Granada (Spain) for their assistance, and Ms. Karen Shashok for translating the original manuscript into English.
Table III. Concentrations of As in Fish (Fresh Weight) as Determined with Hydride Generation Atomic Absorption Spectrometry Sample As Sample As Sample As Sample As no. (~g/g) no. (pg/g) no. (l~g/g) no. (pg/g) 1 2 3 4 5 6 7 8
0.927 1.003 0.648 1.754 O.675 1.380 1.456 3.302
9 10 11 12 13 14 15 16
9.644 0.951 3.417 2.281 1.721 2.458 0.606 1.020
17 18 19 20 21 22 23 24
0.666 0.934 1.923 6.67O 5.768 2,747 12.584 20.O77
25 26 27 28 29 3O 31 32
2.935 1.379 0.396 1.021 2.694 45.694 0.428 2.705
Journal of AnalyticalToxicology,Vol. 16, May/June1992
References 1. J.M. Concon. In Food Toxicology, Part B: Contaminants and Additives, Marcel Dekker, New York, 1988, pp. 1082-88. 2. J.J. Saady, R.V. Blanke, and A. Poklis, Estimation of the body burden of arsenic in a child fatally poisoned by arsenite weedkiller. J. Anal ToxicoL 13:310-12 (1987). 3. B. Welz and G. Schlemmer. Determination of arsenic, selenium and cadmiun in marine biological tissue samples using a stabilized temperature platform furnace and comparing deuterium arc with Zeeman-effect background correction atomic absorption spectrometry. J. Anal Atom. Spectrom. 1: 119-21 (1987). 4. R.T. Hunter. Determination of diagnostic levels of arsenic in animal tissue: Collaborative study. J. Assoc. Off. Anal Chem. 69:493-95 (1986). 5. W. Hershey and T.S. Oostdyk. Determination of arsenic and selenium in environmental and agricultural samples by hydride generation atomic absorption spectrometry. J. Assoc. Off. Anal. Chem. 71: 1090-93 (1988). 6. E.L. Gunderson. FDA total diet study, April 1982-April 1984, dietary intakes of pesticides, selected elements and other chemicals. J. Assoc. Off. Anal. Chem. 71:1200-1209 (1988). 7. B.W. Van Dokkum, R.H. de Vos, T. Muys, and J.A. Wesstra. Minerals and trace elements in total diets in the Netherlands. Brit. J. Nutr. 61:7-15 (1989). 8. A. Van Eenbergen and E. Brunix. Losses of elements during sample decomposition in an acid digestion bomb. Anal Chim. Acta 98:405-406 (1978). 9. R. Uhrberg. Acid digestion bomb for biological samples. Anal. Chem. 54:1906-1908 (1982). 10. K. Okamoto and K. Fuwa. Low-contamination digestion bomb method using a Teflon double vessel for biological materials.
Anal Chem. 56:1758-60 (1984). 11. H.M. Kingston and L.B. Jassie. Microwave energy for acid decomposition at elevated temperatures and pressures using biological and botanical samples. Anal Chem. 58:2534-41 (1986). 12. R.A. Stripp and D.C. Bogen. The rapid decomposition of biological materials by using a microwave acid digestion bomb. J. Anal ToxicoL 13:57-59 (1989). 13. M. Bettinelli, V. Baroni, and N. Pastorelli. Microwave oven sample dissolution for the analysis of environmental and biological materials. Anal Chem. 58:159-74 (1989). 14. H. Norin, M. Vahter, A. Christakopoulos, and M. SandstrSm. Concentration of inorganic and total arsenic in fish from industrially polluted water. Chemosphere 14:325-34 (1985). 15. M. Burow and M. Stoeppler. Ingestion/excrection experiments for arsenic in fish. Trace Elem. Anal Chem. Med. BioL 4:145-50 (1987). 16. T. Kaise, K. Hanaoka, S. Tagawa, T. Hirayama, and S. Fukui. Distribution of inorganic arsenic and methylated arsenic in marine organisms. App. Organomet. Chem. 2:539-46 (1988). 17. W.G. Brumbaugh and M.J. Walther. Determination of arsenic and selenium in whole fish by continuous-flow hydride generation atomic absorption spectrophotometry. J. Assoc. Off. Anal. Chem. 72:484-86 (1989). 18. G.L Long and J.D. Winefordner. Limit of detection: A closer look at the IUPAC definition, AnaL Chem. 55:713A-15A (1983). 19. R.G. Stiel, Principles and Procedures of Statistics, McGrawHill, London, 1982. 20. M. Jaffar and M. Ashraf. Arsenic concentrations in the edible muscle of commercial marine fish species from the Arabian Sea. Fish. Res. 7:11-16 (1989). Manuscript received April 29, 1991; revision received September 11, 1991.
171
Determinationof Arsenic in Fish by HydrideGeneration Atomic AbsorptionSpectrometry M. Navarro*, H. L 6 p e z , and M.C. L 6 p e z
Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, E- 18012 Granada, Spain M. S d n c h e z
Department of Analytical Chemistry" Faculty of Sciences, University of Granada, E- 18071 Granada, Spain
Abstract I A method utilizing microwave dissolution in a closed Teflon bomb followed by hydride generation atomic absorption spectrometry has been used for the routine analysis of arsenic in fish species from the coast of the province of Granada, Spain. The technique was evaluated for decomposing biological standard reference material (BCR mussel tissue) for arsenic analyses. The precision of the results ranged from 2.78 to 6.99%. The recovery percentages ranged from 96.33 to 100.00%. The values determined in the samples indicated slight contamination by this element in the zone. The concentrations of arsenic in fish ranged from 0.396 to 45.716 I~g/g.
Introduction The greatest sources of As pollution are human activity and modern technology. The prolonged intake of even low concentrations of As can cause serious toxic effects (1,2). This has led to growing interest in analyzing As levels in foodstuffs in recent years (3-7). It has been shown that fish is one of the greatest sources of As in the diet, to such an extent that it can reach more than 52% of dietary As intake (7). For that reason it can act as a biological indicator of marine pollution. The zone we have studied receives fresh water from the agricultural areas inland through several irrigation channels and small rivers. The sea is the receiver of urban and industrial waste water from the zone of Port and village of Motril. These factors could increase the level of As in the fish of this zone. Critical steps in any chemical analysis are the decomposition and subsequent preparation of the sample for measurements. Currently, the most widely accepted technique involves accelerated digestion by placing the sample in a sealed receptacle and subjecting it to acid and high pressure in a microwave oven (8-13). One of the most common procedures for the determination of "Address for correspondence:Dr. Miguel Navarro,Dpto. Nutrici6n y Bromatologfa,Facultad de
Farmaeia,Universidadde Granada,E-18012 Granada,Spain.
As in fish is hydride generation atomic absorption spectrometry (14-17). We have used this method to determine As levels in marine fish species found abundantly along the Mediterranean Sea coast of the province of Granada (Spain). The fish studied are consumed locally in large quantities and it is thus important to know their As content in order to identify any hazards before fish reach the consumer.
Experimental
Equipment. Digestions were carried out with a Parr Instrument Company 4782 microwave acid digestion bomb heated in a commercially available Moulinex FM-460 microwave oven (600 W). Measurements of the As were performed with a PerkinElmer 2380 atomic absorption spectrophotometer equipped with the Perkin-Elmer MHS-10 hydride generator. Signals (peak height mode) were recorded on an Omniscribe D-5000 Bausch & Lomb recorder. Reagents and standards. All solutions were prepared with ultrapure water with a specific resistivity of 18 Mf~ cm obtained by filtering distilled water through a Millipore Milli-Q RO15 purifier immediately before use. A stock solution containing 1000 mg/L of As was prepared dissolving arsenic (III) oxide in 2.5 mL of 25% (w/v) KOH and further neutralization with 20% (w/v) HC1. Finally, it was diluted to volume with 1.5% (w/v) HCI. Others reagents used were 70% (w/v) nitric acid (Merck Suprapure), 32% (w/v) hydrochloric acid (Merck), and vanadium pentoxide (Merck). For the hydride generation, the solution was 3% (w/v) sodium tetrahydroborate (Merck) in 1% (w/v) sodium hydroxide (Merck). This solution was filtered before use and stored in a refrigerator. All reagents were of analytical grade. The Community Bureau of Reference-Commission of the European Communities (CBR-CEC) certified material reference (CRM) used was mussel tissue (CRM 278), with a certified As content of 5.900 _+0.200 pg/g. Procedure. All different fish species were each analyzed in triplicate. The lyophilized samples (200 mg) were treated with 2.5 mL HNO 3 and 50 lag of V205 as a catalyst in the microwave acid digestion bomb. Mineralization was complete in 90 s with the oven at its highest setting. The digests were cooled and the re-
Reproduction (photocopying)of editorial content of this journal is prohibited without publisher's permission.
169
Journal of Analytical Toxicology. Vol. 16, May/June 1992
suiting solutions were diluted to a total volume of 25 mL with ultrapure water. Different aliquots were transferred to 50-mL volumetric flasks and adjusted to volume with a 1.5% (w/v) HCI solution. Previously. the method of standard additions was pertormed. Finally, a 10-mL aliquot was taken and analyzed for total As by hydride generation atomic absorption spectrometry,
ducible. Seven determinations in three different species were treated statistically as described by Stiel (19), and the results of the precision tests are summarized in Table It. We analyzed the flesh of 32 of the most common fish species in the studied area. The concentrations of As ranged from 0.396 to 45.716/ag/g, and the results for all species tested are shown in Table IIl.
Results Discussion The calibration graph was prepared in the range from 0.5 to 4.0 ~g/L. These solutions, like the blank (HNO3, V205) were subjected to the same acid digestion bomb treatment as the samples. The method of standard additions was performed, adding amounts of As ranging from 0.0 to 4.0 lag/L to four sample fractions of 200 mg. The comparison between the plotting of the calibration graph and the standard addition method indicates that there is matrix interference, so all the determinations have been made by the standard addition method. The sensitivity found, equivalent to 0.0044 units of absorbance, was 0.094/agfL. For instrumental conditions used in sample analyses, our calculated analytical detection limit (18) was 0.146 t.tg/L. Concentrations of As in all samples exceeded the analytical detection limit. The accuracy of the method was tested with recovery assays (Table l). Tests with CRM 278 gave a value of 5.795 + 0.131 IJg/g (n = 13). The technique was found to be both precise and repro-
Table I. Arsenic Recoveries (%) Obtained in the Analysis of Three Fish Samples As present (~tg)
As added (~tg)
As found (~g)
As recoveries
No. 21
5.768 5.768 5.768 5.768
0.000 1,031 2.062 3.093
5.374 6.595 7.634 8.536
93,17 97.00 97.50 96.33
No. 30
45.694 45.694 45.694 45.694
0.000 18.856 37.712 56.568
45.716 62.312 81.179 97.568
100.05 96.53 97.32 95.41
0.428 0.428 0.428 0.428
0.000 0.122 0.244 0.366
0,419 0,550 0.664 0.761
97.90 100.00 98.81 95.84
Sample
No. 31
(%)
Table II. Statistical Analysis of the Results of Determinations of As Concentration in Fish by Hydride Generation Atomic Absorption Spectrophotometry Sample
Mean As concentration* (l~g/g)
RSD (%)
No. 21 No. 30 No. 31
5.768 • 0.3092 45.694 • 1.2681 0.428 • 0.0299
5,36 2.78 6.99
9All values are the mean of seven determinations with standard deviation
170
A microwave acid digestion bomb technique for decomposition of fish samples was used. Decompositions were complete in 90 s, and the results of the analyses were good. The method is less complicated and time consuming than conventional dissolution techniques. It can be concluded that neither significant contamination nor loss of As occur during the digestion of the samples in the closed Teflon bomb. The accuracy of this method was tested with recovery experiments, the results of which are listed in Table I. Results of all analyses of CRM 278 for As were within the certified concentration range. This fact confirms that this digestion procedure gives good results for the determination o f total As in the samples we have used. The limit of detection and sensitivity were comparable to those reported by other authors and adequate for the range of concentrations of the samples analyzed in this study. The levels of As obtained are slightly higher in relation to those established by other authors in other areas ( 14+15,20). This fact could indicate the existence of a slight pollution in the fish. Nevertheless, the intake of this fish would not be harmful for health, because the levels of As are low and the main part of As is organic, which is less toxic, and is easily and rapidly eliminated in the urine (13).
Acknowledgments We would like to thank the Department of Analytical Chemistry of the University of Granada (Spain) for their assistance, and Ms. Karen Shashok for translating the original manuscript into English.
Table III. Concentrations of As in Fish (Fresh Weight) as Determined with Hydride Generation Atomic Absorption Spectrometry Sample As Sample As Sample As Sample As no. (~g/g) no. (pg/g) no. (l~g/g) no. (pg/g) 1 2 3 4 5 6 7 8
0.927 1.003 0.648 1.754 O.675 1.380 1.456 3.302
9 10 11 12 13 14 15 16
9.644 0.951 3.417 2.281 1.721 2.458 0.606 1.020
17 18 19 20 21 22 23 24
0.666 0.934 1.923 6.67O 5.768 2,747 12.584 20.O77
25 26 27 28 29 3O 31 32
2.935 1.379 0.396 1.021 2.694 45.694 0.428 2.705
Journal of AnalyticalToxicology,Vol. 16, May/June1992
References 1. J.M. Concon. In Food Toxicology, Part B: Contaminants and Additives, Marcel Dekker, New York, 1988, pp. 1082-88. 2. J.J. Saady, R.V. Blanke, and A. Poklis, Estimation of the body burden of arsenic in a child fatally poisoned by arsenite weedkiller. J. Anal ToxicoL 13:310-12 (1987). 3. B. Welz and G. Schlemmer. Determination of arsenic, selenium and cadmiun in marine biological tissue samples using a stabilized temperature platform furnace and comparing deuterium arc with Zeeman-effect background correction atomic absorption spectrometry. J. Anal Atom. Spectrom. 1: 119-21 (1987). 4. R.T. Hunter. Determination of diagnostic levels of arsenic in animal tissue: Collaborative study. J. Assoc. Off. Anal Chem. 69:493-95 (1986). 5. W. Hershey and T.S. Oostdyk. Determination of arsenic and selenium in environmental and agricultural samples by hydride generation atomic absorption spectrometry. J. Assoc. Off. Anal. Chem. 71: 1090-93 (1988). 6. E.L. Gunderson. FDA total diet study, April 1982-April 1984, dietary intakes of pesticides, selected elements and other chemicals. J. Assoc. Off. Anal. Chem. 71:1200-1209 (1988). 7. B.W. Van Dokkum, R.H. de Vos, T. Muys, and J.A. Wesstra. Minerals and trace elements in total diets in the Netherlands. Brit. J. Nutr. 61:7-15 (1989). 8. A. Van Eenbergen and E. Brunix. Losses of elements during sample decomposition in an acid digestion bomb. Anal Chim. Acta 98:405-406 (1978). 9. R. Uhrberg. Acid digestion bomb for biological samples. Anal. Chem. 54:1906-1908 (1982). 10. K. Okamoto and K. Fuwa. Low-contamination digestion bomb method using a Teflon double vessel for biological materials.
Anal Chem. 56:1758-60 (1984). 11. H.M. Kingston and L.B. Jassie. Microwave energy for acid decomposition at elevated temperatures and pressures using biological and botanical samples. Anal Chem. 58:2534-41 (1986). 12. R.A. Stripp and D.C. Bogen. The rapid decomposition of biological materials by using a microwave acid digestion bomb. J. Anal ToxicoL 13:57-59 (1989). 13. M. Bettinelli, V. Baroni, and N. Pastorelli. Microwave oven sample dissolution for the analysis of environmental and biological materials. Anal Chem. 58:159-74 (1989). 14. H. Norin, M. Vahter, A. Christakopoulos, and M. SandstrSm. Concentration of inorganic and total arsenic in fish from industrially polluted water. Chemosphere 14:325-34 (1985). 15. M. Burow and M. Stoeppler. Ingestion/excrection experiments for arsenic in fish. Trace Elem. Anal Chem. Med. BioL 4:145-50 (1987). 16. T. Kaise, K. Hanaoka, S. Tagawa, T. Hirayama, and S. Fukui. Distribution of inorganic arsenic and methylated arsenic in marine organisms. App. Organomet. Chem. 2:539-46 (1988). 17. W.G. Brumbaugh and M.J. Walther. Determination of arsenic and selenium in whole fish by continuous-flow hydride generation atomic absorption spectrophotometry. J. Assoc. Off. Anal. Chem. 72:484-86 (1989). 18. G.L Long and J.D. Winefordner. Limit of detection: A closer look at the IUPAC definition, AnaL Chem. 55:713A-15A (1983). 19. R.G. Stiel, Principles and Procedures of Statistics, McGrawHill, London, 1982. 20. M. Jaffar and M. Ashraf. Arsenic concentrations in the edible muscle of commercial marine fish species from the Arabian Sea. Fish. Res. 7:11-16 (1989). Manuscript received April 29, 1991; revision received September 11, 1991.
171
