Environ. Sci. Technol. 1994, 28,383-387
Determination of Total Selenium and Dissolved Selenium Species in Natural Waters by Fluorometry Dacheng Wang,' Georg Alfthan, and Antti Aro
Department of Nutrition, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland
A method for the determination of total selenium and dissolved selenium species at nanogram levels in natural water samples was developed. Selenite and selenate were separated through a Dowex AG2-X8 column, and dissolved hydrophobic base, acidic, and neutral organic selenium were separated by an XAD-8 column. The elution or a water sample of 50-200 mL with nitric acid addition (2.5 mL/L) was evaporated on an electric plate and digested with HNOS/HC104 (2/1, v/v). After being reduced with HC1 and complexed with 2,3-diaminonaphthalene, the fluorophore was extracted into cyclohexane, and its fluorescence was measured. The recovery of selenium added to 50 mL of river water was 97.9-104%, with an average of 99.9% ( n = 20). The detection limit of the method for water total selenium was 0.35 ng of selenium (blank + 2 SD),and the precision of the method for natural water samples with selenium concentrations ranging from 28 to 115ng/L was 2.1 % . For individual dissolved selenium species, the detection limits were all lower than 1.8 ng.
The speciation methods for selenite and selenate using different instrumentation published previously always involved an oxidation-reduction reaction with acid addition or solvent extraction (6-9). The accuracy of the methods depends on the efficiency of this process. Because a rather large amount of acid is introduced, the detection limit may be affected. Tanzer and Heumann separated selenite and selenate successfully from environmental waters by a Dowex AG1-X8 column, but their method for organoselenium compounds failed to separate hydrophobic basic and neutral organoselenium (IO). The methanol they used to elute this fraction cannot be pumped with peristaltic pumps because of pump-tubing degradation. Air bubbles appearing in the XAD column when methanol is mixed with water may affect elution efficiency. Leenheer demonstrated an excellent method for the isolation and fractionation of dissolved organic carbon from natural waters which can be applied for selenium speciation (11). The purpose of this study was to develop a simple method for natural water total selenium and dissolved hydrophobic organic selenium species determination with low detection limit, good precision, and accuracy.
Introduction Selenium deficiency diseases and toxicity in animals and human beings have been frequently reported during the past decades. Recently, research has focused on the behaviour of selenium in the environment, especially its toxicity in the water ecosystems ( I ) . The range of selenium concentrations in the environmental medium between those associated with deficiency and toxicity is rather narrow, especially in natural water ecosystems. Lindstrom reported that the biomass development, final yield, and growth rate of a bloom-formingalga were maximal a around 50 ng/L selenite-Se (2). He also reported that in Lake Erken, Sweden, blooms and decline of algae coincided with decreases (from >70 to 20 ng/L) and increases (up to >SO ng/L) of bioactive selenium (3). On the other hand, a water selenite concentration of 3000 ng/L may decrease the reproduction of protozoa ( 4 ) . The bioavailability of selenium depends on its chemical forms present in the environment. Advance knowledge about the amount and proportion of different dissolved chemical selenium species will help us to understand the environmental biogeochemical process of selenium in natural waters. Natural water selenium concentrations are usually very low. The median level of selenium concentrations in Finnish groundwaters, rivers, and lakes is only 60-70 ng/L (5). There are numerous methods available for the measurement of total selenium concentration and for the separation and determination of dissolvedselenium species in natural waters, but generally their detection limit is not low enough for samples from low selenium areas, or the sample treatment process is very complicated to perform. Thus, to develop a sensitive, simple, and fast analysis method for the separation and determination of natural water selenium in nanogram levels becomes a critical task. 0013-936X/94/0928-0383$04.50/0
0 1994 American Chemical Society
Experimental Section Apparatus. A Perkin-Elmer Model LS-5 fluorospectrometer, a Simadzu TOC-5000 total organic carbon analyzer with an ASI-5000 autosampler, a Slev 2000-W twin electronic plate, a Liebisch aluminum heating block with 60 holes (diameter 18 mm, depth 65 mm), an Orion Research Model 701-A pH meter with a 6-mm diameter glass electrode, a W 350r W.Memmert, Schwabach water bath, a Peristaltic pump 132100 (Heidelberg, Germany), plastic chromatography column 9 X 300 mm (Pharmacia Fine Chemicals, Uppsala, Sweden) and 14 X 100 mm (BioRad, US.). Glass beakers (250 mL) and glass-stoppered test tubes (16 X 125 mm) were used. All glassware were borosilicate and acid washed. Reagents. The selenium stock standard, 0.5 g/L selenium in 0.1 M HC1, was prepared from analyticalgrade sodium selenite (Merck, Germany). The stock solution was further diluted to 1pg/mL with 0.1 M HCl. In addition, selenate, analytical-grade (BDH, England), and seleno-DL-methionine (Sigma, US.)were used. 2,3-Diaminonaphthalene (DAN) was prepared from a technical-grade product (95-98 9% , Sigma), by dissolving 0.2 g of DAN in 200 mL of 0.1 M HC1. The 0.1 % solution was purified by extracting with 10 mL of cyclohexane five times. The resins used were anion-exchange resin Dowex AG2X8 (Bio-Rad, U.S.), 200-400-mesh, chloride form, and nonionic polymeric adsorbent Amberlite XAD-8 (Sigma, US.), mesh size 20-60. Water was purified by a Millipore water purification device through organic adsorption, deionization, and filtration cartridges. Environ. Sci. Technol., Voi. 28,
No. 3, 1994 383
Water sample
I
c_T7 I
I
I 0.45prn f i l t e r
14XlOOnm
T i d i s s o l v e d Se
I
XAD-8 -Adjust pH 2 9X300mm w i t h HC1 Elute with 0.1M H C l
Elute with 1M HCOOH
Elute with 3M H C 1
FJ Iselenste) I
I
organoselenium Elute with 0 . l M , NaOH
organoselenium Unpack column e x t r a c t w i t h CH,OH
I
I
Neutral organoseleniurn
1
Flgure 1. Sample treatment for the determination of dissolved selenium species in natural waters.
CloH14N~NazOa.2HzO (EDTA), hydroxylammonium chloride, and sodium hydroxide were analytical grade from Merck. The following stock reagents were used: hydrochloric acid 37 % ,nitric acid 65 % ,perchloric acid 70-72 96 ,formic acid 98-100 % ,hydrogen peroxide 30 % ,ammonia solution 25 % , diethyl ether, and acetonitrile; all analytical grade from Merck. Analytical-grade methanol from BDH and HPLC-grade cyclohexane from Rethburn, Scotland were used. Procedure. (I)Preparation of Columns. Dowex AG2X 8 . The resin was purified by sequential extraction with 1 M HCOOH and 3 M HCl and then washed with water until neutral pH was reached. Then the resin was packed in a 100-mm column with an inner diameter of 14 mm. The column was rinsed three times with 15 mL of 1 M HCOOH and 30 mL of water, 15 mL of 3 M HCl, and 30 mL of water. The effluent of the last rinse was measured for selenium as the selenite and selenate fraction blank. The effluent pH was checked before applying the sample. XAD-8. The resin was cleaned as described by Thurman and Malcolm (12). The resin was then extracted with 0.1 M NaOH in an Erlenmeyer flask. Fines were decanted off. The resin was then sequentially extracted with methanol, diethyl ether, acetonitrile, and methanol and stored in methanoluntil used. Before packing the column, methanol was displaced from the resin with distilled water. The resin was packed in a 300-mm column with an inner diameter of 9 mm and then rinsed with about 50 bed volumes of water. The column was washed three times with 10 mL of 0.1 M HC1 and 30 mL of water, 10 mL of 0.1 M NaOH, and 30 mL of water. The effluent of the last wash was measured for selenium as the hydrophobic basic and acidic fractions blank. ( 2 )Separation of Selenite and Selenate. The complete separation procedure for the determination of dissolved selenium species in natural waters is shown in Figure 1. All water samples (200 mL) were first filtered through a 0.40-km Nuclepore polycarbonate filter and pumped through the anion-exchange column at a flow rate of 4 mL/min (C30 bed vol/h). 384
Envlron. Scl. Technol., Vol. 28, No. 3, 1994
Following the sample, the column was washed with 30 mL (2 bed vol) of water. Selenite was eluted with 15 mL (1bed vol) of 1M HCOOH, followed by 30 mL (2 bed vol) of water. Selenate was eluted with 15 mL of 3 M HC1, followed by 30 mL of water. ( 3 ) Separation of Hydrophobic Organic Selenium. Basic Fraction. A water sample (200 mL) was applied on the XAD-8 column a t a flow rate of 4 mL/min (e30 bed vol/h). Following the sample, the column was washed with 30 mL of water. The influent and effluent tubings were then reversed, and the column was turned upside down. The hydrophobic bases were backflush-eluted with 10 mL of 0.1 M HC1, followed by 30 mL of 0.01 M HC1. Acidic Fraction. The effluent from the XAD-8 column was acidified with HC1 to pH 2 and recycled through the XAD-8 column a t 4 mL/min. The nonsorbed portion of the sample was displaced from the resin by 30 mL of 0.01 M HC1. The hydrophobic acids were desorbed by backflush elution with 10 mL of 0.1 M NaOH, followed by 30 mL of water. The effluent was immediately acidified with HC1 to pH 2 to avoid oxidation of humic substances (13). Neutral Fraction. The column was unpacked, and the resin was transferred to a Petri dish where it was allowed to dry a t room temperature. The dried resin was extracted with 1bed vol of methanol. The methanol was transferred to a test tube and evaporated to dryness with nitrogen. The effluents of the different fractions were then evaporated with gentle boiling, and their selenium concentration was determined according to the procedure for total selenium. Before evaporation, a portion of 5 mL was taken from each fraction for TOC measurement. (4) Determination of Total Selenium. To a water sample of 50-200 mL in a 250-mL beaker, 0.25 mL of concentrated nitric acid and a few anti-bumping granules were added. The sample was then gently evaporated on an electric plate, until 2-3 mL remained. The remainder was transferred to tubes, and the beaker was washed twice with water. A few anti-bumping granules and 1.5 mL of nitric acid/perchloric acid mixture (vlv, 2/1) were added, and the tubes were placed in an A1 block at 110 "C for 6 h (or overnight). The temperature was then increased to 150 "C for 1hand to 180 "C for 1h, about 0.5 mL remained in the tubes. Then 4 drops of 30 % HzOz were added after cooling, and the tubes were heated at 150 "C for 10 min. To reduce all selenium to selenite, 0.67 mL of concentrated HC1was added, and the mixture was heated for 10 minutes at 110 "C. After the mixture was cooled, 0.5 mL of 0.2 M Na2-EDTA, 0.5 mL of 1% hydroxylammonium chloride, and about 1mL of 1:l 25% NH40H (v/v) were added to the test tubes, and the pH was adjusted to 1.5-2.0 with 4 M HC1 or NH40H. Then 0.5 mL of 0.1% DAN was added, and the tubes were placed in a 50 "C covered water bath for 20 min and then cooled in cold water. Finally, 2.5 mL of cyclohexane was added; the tubes were capped with glass stoppers and extracted manually for 1min. The fluorescence was measured within 2 h, at an excitation wavelength of 370 nm and an emission wavelength of 518 nm. Two blanks and a standard series of 5,10,15,20,25, and 30 ng of selenium (NaZSeOa) were run through all procedures.
Results and Discussion Preconcentration. As the selenium concentration of natural waters is usually very low, water samples have to
Table 1. Recovery of Selenium Added to 200 mL of Purified Water# NazSeO3
NazSeO4
selenomethionine
10.0
9.64
9.57
Se added (ng) Se found (7%) no acid HCl HNO3
96.8 95.3 98.8 70.6
98.2 98.7 98.1 72.0
Table 3. Within-Series and Between-Series Precision of the Method for Total Selenium Determination in Different Types of Water Samples sample
80.3 51.1 87.3 46.4
river water tap water snowme1t
no. of series
no. of determinations
mean f SD (ng/L)
CV ( % )
1 1 1
Within Series 8 8 8
115 f 2 33.9 f 0.5 28.0 f 0.6
1.8 1.5 2.1
5 5 10 4
Between Series 9 9 20 8
126 f 1 44.0 f 1.4 67.8 f 2.2 137 f 3
0.8 3.2 3.2 2.2
Means of duplicate determinations. Table 2. Recovery of Selenite Added to 50 mL of River Water* Se added (ng) Se found (ng) recovery ( % ) recovery, range ( % ) mean recovery ( % ) a
0 5.68
5 10 11.0 15.6 103 99.5 97.9-104 (n = 20) 99.9 f 1.9 (n = 20)
15 20.5 99.1
20 25.3 98.5
Means of five replicates.
be preconcentrated. A simple method is to evaporate the water sample to a small amount and then digest it with an acid mixture like biological samples and soils. To decrease selenium losses during evaporation, the addition of different acids was tested. With 0.5 mL of acid added to 200 mL of purified water, the best recovery rate was in the sample evaporated with nitric acid, 98.8% for selenite, 98.1 % for selenate, and 87.3 % for seleno-methionine (Table 1). This recovery was satisfactory for both organic and inorganic selenium fractions in water samples. Purified water was chosen because different forms of selenium are more unstable in it than in natural waters. Selenite and seleno-methionine added into purified water stored in a polyethylene bottle at room temperature for 15 days lost 57.1 % and 4 2 . 0 % , respectively. Under the same condition, selenium in well water, river water, and snowmelt water only lost 3.5%, 17.6%, and 24.6 %, respectively (Wang, unpublished data). Just by coincidence, most researchers use nitric acid to preserve natural waters. In the amount commonly added, 2.5 mL of acid/L sample, no acid addition is needed during evaporation. The recovery of selenium (as NazSeO3) added to 50 mL of river water with 0.25 mL of nitric acid was shown in Table 2. The mean f SD recovery in 20 analyses was 99.9 f 1.9%. Precision and Accuracy. Reference water samples with certified or recommended selenium concentrations at nanogram per liter levels are not available. Therefore, the method was verified by the precision for different water samples. The within and between-series precision of the method for river water, tap water and snowmelt water is shown in Table 3. At levels between 28 and 115 ng/L, the CV from eight determinations was 12.1 % The betweenseries precision of the method was slightly higher than the within-series variation, probably because of water selenium losses during storage. The method was also applied to total selenium determination of soil, sediments, and biologicalsamples, starting by adding 1.5 mL of nitric acidlperchloric acid mixture. The mean f SD of total selenium from eight determinations for a certified soil sample SO-4 by Canada Centre for Mineral and Technology was 553 f 12 ng/g with a CV of 2.2 % , compared with the certified value of 490 ng/g. For another soil reference sample, obtained from Institute
.
river water tap water groundwater snowmelt
Table 4. Precision of the Method for Total Selenium Determination in Certified Soil and Interlaboratory Food Reference Materials
CV
sample
n
mean f SD (ng/g)
(%)
recommended value (ng/g)
SO-4 (soil)a S-MS (soilIb EKT-80 (wheat bread)c EKT-82 (wheat flour)c
8 8 8 7
553f 12 225f5 132 f 3 119 f 2
2.2 2.2 2.3 1.7
490 213f70d 133 f 27d 117 f 33d
Reference soil sample, Canada Centre for Mineral and Technology. Reference soil sample,Institute of Radioecologyand Applied Nuclear Techniques,Kosice,formerCzechoslovakia. Interlaboratory referencematerials,Departmentof Food Chemistry and Technology, University of Helsinki. Results of seven Finnish laboratories;six replicates each.
*
Table 5. Detection Limit (Blank + 2 SD) of the Method for Total and Dissolved Selenium Species species
detection limit (ng)
total Se selenite selenate hydrophobicbasic organoselenium hydrophobicneutral organoselenium hydrophobicacidic organoselenium
0.35 0.71 1.0 1.3 1.8 0.93
of Radioecology and Applied Nuclear Techniques, Kosice, former Czechoslovakia, and two wheat product reference materials prepared by the Department of Food Chemistry and Technology, University of Helsinki, for selenium interlaboratory calibration study, the results by the presented method were comparable with the results by seven other laboratories in Finland using different methods (Table 4). Speciation of Selenite and Selenate. The detection limits of the method for total and dissolved selenium species, expressed as blank + 2 SD, were all lower than 1.8 ng of selenium (Table 5). The lowest natural surface water selenium concentration found in Finland was 25.4 ng/L in a lake, even though there was a groundwater sample with 4.1 ng/L of selenium as an extreme example. Thus, the present method was capable of selenium speciation of all natural waters in a 200-mL sample. The separation of 100 ng of selenite (as NazSeO3) and selenate (as NazSeOd added to 100 mL of purified water is shown in Figure 2. The effluent was collected at 1-mL intervals. The recovery was 100 f 3 % and 98.3 f 4.0% (n.= 3), respectively. To confirm the separation efficiency of selenite and selenate, only 100 ng of selenate was added to 100 mL of purified water and run through the column. An amount of 100.7 ng of selenium was found in the 3 M Environ. Sci. Technol., Vol. 28, No. 3, 1994
385
Table 7. Recovery of Added Selenium in XAD-8 Column Effluents species
PH
NazSeO3 NazSeOl selenomethionine
7 7 7 2 2 2
NazSeO3 NazSeOd selenomethionine a
20
Seadded W L )
Sefound (ng/L)
1recovery
25.0 23.0 22.0
24.4 22.2 20.0
97.6 96.5 90.9
25.0 23.0 22.0
24.7 21.5 20.4
98.8 93.5 92.7
(%)
Means of duplicate determinations.
Table 8. Analysis of Dissolved Selenium Species and DOC in River Waters and Lake Water species
Se (ng/LP
fractions
Brook Viikki, Helsinki, January 8, 1993 8.9 f 2.3 68.0 f 2.5 selenate basic organoselenium 0.6 f 0.2 hydrophobic bases neutral organoselenium 6.2 f 1.0 acidic organoselenium 26.0 f 4.3 hydrophobic acids 110 remained in effluent sum of all species determined total DOC determined total Se 123 f 1 Lake Valkea-Kotinenjiirvi, December 18,1992 selenite 3.8 f 0.6 selenate 4.8 f 0.7 basic organoselenium 0.8 f 0.1 hydrophobic bases neutral organoselenium 4.4 f 0.7 acidic organoselenium 32.2 f 2.5 hydrophobic acids sum of all species 45.9 remained in effluent determined total Se 58.3 f 2.3 determined total DOC
DOC (mg/L)b
se1enite
10
ND 3.58 5.49 8.08
0.03 6.62
4.51 11.6
*
Means of four determinations. Means of duplicate determinations; ND, not detected. (I
0
~~
0
10
20 30 40 50 60 70 80 80 Elution volume (ml)
Figure 2. Chromatographic separation of selenite and selenate with an anion-exchange resin.
Table 6. Analysis of Dissolved Selenium Species in Natural Waters (nglL)a sample
total Se
selenite
selenate
humic substance
rain snow 1 snow 2 tap river 1 river 2 lake 1 lake 2
43.5 50.9 55.9 34.7 115 99.7 33.7 89.4
26.3 17.3 16.6 10.9 10.5 13.9 4.4 6.7
14.7 28.5 31.6 20.5 60.2 13.0 3.5 6.5
NDb ND ND ND 37.0 41.6 19.9 48.7
a
Means of duplicate determinations. Not determined.
HC1 effluent, while in the 1 M HCOOH effluent, the selenium was same as in the blank. The results of natural water selenium speciation are shown in Table 6. In two river water samples, the selenate concentration equaled or much exceeded that of selenite. This is in agreement with the findings in Japanese rivers (8). In rainwater, selenite was the more abundant form as reported from the United States (6). Selenate dominated in snowmelt as well as in tap water and groundwater. In lake water, the amount of selenium in humic substances made up to 5559% of the total selenium; selenite and selenate together made up only 1 5 2 3 % . 388
Environ. Sci. Technol., Vol. 28, No. 3, 1994
Speciation of Hydrophobic Bases, Neutrals, and Acids. The method for separation of hydrophobic selenium fractions was adopted from that of Leenheer for isolation and fractionation of dissolved organic carbon from natural waters (11). The recovery of sodium selenite, sodium selenate, and selano-methionine added to 25 mL of purified water at both pH 7 and pH 2 in XAD-8 column effluents were tested in order to ensure that no significant amounts of these forms were retained in the column with hydrophobic bases and the acidic fraction. The recovery results (Table 7) were satisfactory, exceeding 90% for all three compounds. The recovery of seleno-methionine (90.9% at pH 7, 92.7% at pH 2) as a hydrophilic amino acid was relatively lower than that of selenite and selenate, perhaps due to minor decomposition during prolonged storage of the reagent. The present method was applied to a river water sample and a lake water sample. The results for the selenium speciation and the DOC fractions are shown in Table 8. The selenium in the hydrophobic bases fraction was near the detection limit, and so was the organic carbon. Organic bases, including both hydrophobic and hydrophilic bases, constituted only 5% of the surface water DOC ( 1 4 ) . To separate this fraction from surface waters for the determination of selenium, a more sensitive method should be employed (15). Similarily, organic neutrals also constituted about 5 % of the DOC, and the hydrophobic neutral fraction separated by the present method may still include some hydrophobic acids (humic substances) which are retained by the column, as the elution efficiency is not likely to be 100%. Therefore, the selenium value of this fraction may only be considered an estimate. Acidic organoselenium in the fraction of
hydrophobic acids, verified by DOC,was more than 55 % of the total dissolved selenium in lake water. Its bioavailability to algae and its ecological effect for the lake is an interesting question. The sum of the different selenium species was lower than that of the total selenium concentration, 89 7% for river water and 79 % for lake water (Table 8). The reason was that the selenium in the hydrophilic basic, neutral, and acidic fractions was not included. Until the selenium in those fractions can be separated and determined, the sum of different selenium species cannot reach 100% of the total selenium. In conclusion, the present method was simple, sensitive, and accurate; no special instrumentation and reagent were required. It was possible to analyze all natural waters.
Acknowledgments This study was supported by the Ministry of Agriculture and Forestry of Finland.
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(5) Wang, D.; Alfthan, G.; Aro, A.; Kauppi, L.; Soveri, J. In
Trace Elements in Health and Disease; Aitio, A., Aro, A,, Jtirvisalo, J., Vainio, H., Eds.; Royal Society of Chemistry: London, 1991;pp 50-56. (6) Cutter, G.A. Anal. Chim. Acta 1978,98,59-66. (7) Cutter, G.A. Anal. Chem. 1985,57,2951-5. (8) Uchida, H.; Shimoishi, Y.; Toei, K. Environ. Sci. Technol. 1980,14,541-4. (9) Roden, D.R.; Tallman, D. E. Anal. Chem. 1982,54,307-9. (10) Tanzer, D.;Heumann, K. G. Anal. Chem. 1991,63,1984-9. (11) Leenheer, J. A. Enuiron. Sci. Technol. 1981,15, 578-87. (12) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981,15,463-6. (13) Aiken, G.R. In Humic Substances in Soil, Sediments, and Water; Aiken, G. R., Mcknight, D. M., Wershaw, R. L., Maccarthy, P., Eds.; John Wiley & Sons: New York, 1985; Chapter 14. (14) Malcolm, R. L.In Humic Substances in Soil, Sediments, and Water;Aiken, G. R., Mcknight, D. M., Wershaw, R. L., Maccarthy, P., Eds.; John Wiley & Sons: New York, 1985; Chapter 7. (15) Chu, C. H.; Pietrzyk, D. J. Anal. Chem. 1974,46,330-6. Received for review April 27, 1993.Revised manuscript received September 29, 1993. Accepted October 28, 1993." Abstract published in Advance ACS Abstracts, December 15, 1993.
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