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The use of a portable total reflection X-ray fluorescence spectrometer for trace element determination in freshwater microcrustaceans (Daphnia)
Spectrochimica Acta Part B 59 (2004) 1265 – 1272 www.elsevier.com/locate/sab
The use of a portable total reflection X-ray fluorescence spectrometer for $ trace element determination in freshwater microcrustaceans (Daphnia) ´ va´ri a, Wolf v. Tu¨mpling Jr. a, Francisco Encina c Margarete Mages a,*, Stefan Woelfl b, Miha´ly O a
UFZ Centre for Environmental Research Leipzig-Halle, Department of Inland Water Research Magdeburg, Bru¨ckstrasse 3a, D-39114 Magdeburg, Germany b Instituto de Zoologı´a, Universidad Austral de Chile, Casilla 567, Valdivia, Chile c Facultad de Ciencias, Universidad Cato´lica de Temuco, Escuela de Ciencias Ambientales, Montt 056, Temuco, Chile Received 30 January 2004; accepted 2 April 2004 Available online 15 July 2004
Abstract The suitability of a newly developed, portable total reflection X-ray fluorescence (TXRF) spectrometer (PicoTAX, Roentec, Berlin, Germany) to analyze trace elements in biological material was tested and compared with a stationary instrument (Spectrometer 8030 C, FEI, Munich, Germany). For that, single freshwater microcrustacean specimens (Daphnia spec.) with dry weights ranging between 1.6 and 18.8 Ag individual 1 were prepared according to the dry method and analyzed with both instruments. Additionally, for orientation purposes, freshly collected Daphnia were prepared in field according to the wet method directly on the glass carrier and analyzed using the portable PicoTAX. For method validation, certified reference material (CRM 414, plankton) was analyzed. The results of the in-field measurements demonstrate that the PicoTAX yields very fast and sufficiently sensitive in-field measurements on the element content of minute biological samples. For As, Cu, K, Mn, Ni and Sr, a good correlation was found between the two spectrometers. Only for Ca, Fe, Pb and Zn the comparison with the results of the stationary equipment has shown significant differences. D 2004 Elsevier B.V. All rights reserved. Keywords: Trace element analysis; Daphnia; Zooplankton; Total reflection X-ray fluorescence; Portable TXRF; Field experiments
1. Introduction Elemental analysis of zooplankton is a very important task to understand the uptake and accumulation processes of trace elements into the trophic chain [1]. Trace elements at elevated concentrations can be bioaccumulated, resulting negative changes in the aquatic ecosystem [2]. Mining activities often introduce untreated wastewater into rivers. This is an important reason for high heavy metal loads in surface waters. Various aquatic biota at the beginning of the trophic chain, like biofilms [3], zooplankton [4,5] and benthic invertebrates $ This paper was presented at the 10th Symposium on Total Reflection X-Ray Fluorescence Analysis and the 39th Discussion Meeting on Chemical Analysis (TXRF-2003) held in Awaji Island, Hyogo, Japan, in September 2003, and is published in the special issue of Spectrochimica Acta Part B, dedicated to that conference. * Corresponding author. Tel.: +49-391-810-9310; fax: +49-391-8109150. E-mail addresses: [email protected] (M. Mages), ´ va´ri), [email protected] (S. Woelfl), [email protected] (M. O [email protected] (W. v. Tu¨mpling).
0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2004.05.006
[6,7] have already been investigated for trace element uptake and kinetics, and used for biomonitoring. The zooplankton of the Hungarian section of the Tisza river system has been investigated since the late 19th century. These investigations were intensified in the past two decades. Detailed periodical Rotatoria and Crustacea surveys among the Tisza and its tributaries have been published [8]. Zooplankton investigation at the Tisza-to´ include quantitative and qualitative surveys, monitoring of seasonal dynamics and characterisation of mosaic patterns and biodiversity [9]. The cyanide pollution in the year 2000 has destroyed most of the zooplankton in the Szamos and Tisza stream; approximately 70 – 80% of Rotatoria and Cladocera species were killed. The regrowth of the zooplankton community started quite soon, already a few days after the spill. Nevertheless, the zooplankton abundance changed significantly compared to the results of the previous 10 –15 years [10]. Unfortunately, no investigation of the trace element concentrations is known until now. Only higher organisms (e.g. fishes) were investigated [11].
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Trace element determinations in aquatic biota are commonly carried out by inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES) or atomic absorption spectrometry (AAS) [12,13]. All these methods often require digestion of the sample. Larger amounts of sample material are needed and contamination originating from the reagents and vessels cannot be avoided in all cases. On the other hand, electrothermal vaporization (ETV) is a quite common technique allowing direct introducing of solid samples into the spectrometer [14]. For the direct trace element analysis of solid biological samples, total reflection X-ray fluorescence spectrometry (TXRF) is very suitable [15 – 17]. Since the organic matrix can increase determination limits considerably, it is often necessary to separate analytes from this matrix. This can be carried out almost under contamination-free conditions, if cold plasma ashing (CPA) in low pressure oxygen atmosphere is used [18]. Recently, a portable TXRF spectrometer was introduced, allowing in-field trace element analyses. First applications have already been reported [19]. Determination of trace elements in river water was carried out. However, this technique has also some limitations, since during in-field investigations contamination originating from airborne dust may occur, and the detector area of the portable TXRF spectrometer is significantly smaller, than those of stationary spectrometers. The aim of this work was to investigate trace elements in single freshwater zooplankton specimens from the Tisza river in Hungary, both in the field with a portable TXRF spectrometer, and in the laboratory with a stationary TXRF spectrometer.
2. Materials and methods 2.1. Sampling and sample preparation Daphnia from the river Tisza (Hungary) were collected from the partly artificial reservoir named Tisza-to´ during summer 2003. The sampling site (Kisko¨re) is located at the main basin, just above the roller dam. A map of the sampling region is shown in Fig. 1. The zooplankton sampling was carried out from a boat with a metal-free zooplankton net (aperture: 20 cm; net size: 100 Am; Hydrobios, Kiel, Germany). The samples were subsequently prepared in the field: first, the whole sample amount was washed three times with 0.2 Am filtered lake water in order to remove stuck suspended matter [17]. After this, single Daphnia specimens were selected using a stereomicroscope. Two methods for the element analyses were applied: (1) To get an overview for orientation purposes in field, freshly collected Daphnia were prepared according to the wet method directly on the glass carrier [17]. The Daphnia were subsequently digested directly on the sample carrier plate with 5 Al HNO3 (Suprapure grade,
Fig. 1. Map of Eastern Hungary, with the sampling point at Kisko¨re.
E. Merck, Darmstadt, Germany) on a hot plate at 100 jC. The HNO3 was previously dopped by Ga standard solution (1 mg l 1), in order to make internal standardization possible. The trace elements were determined using the portable PicoTAX (Fig. 2). (2) For the investigation using the dry method [17] in the laboratory, Daphnia were transferred in PTFE special vials (Savillex, USA), frozen in liquid nitrogen and transported in a Dewar bottle. In addition to the zooplankton, a surface water sample from that sampling location was taken. One part of it was filtered through a cellulose ester filter with a pore size of 0.2 Am (Millipore, France) in the field. Both the filtered (dissolved fraction) and non-filtered (total) subsamples were stabilized with 65% nitric acid, and subsequently analyzed in the field for a first screening and to determine the trace element concentrations in the washing water of the zooplankton samples. The results are published in Ref. [20]. 2.1.1. Preparation in the laboratory Daphnia prepared according to the dry method were first freeze-dried (Alpha 1, Christ, Osterode, Germany) at the laboratory for 24 h at 20 jC. The single lyophilised specimens of Daphnia with dry weights between 3 and 19 Ag individual 1 were prepared according to the modified dry method [17,21]. For this, Daphnia were weighed using an ultrafine balance (S4, Sartorius, Germany). The weights were humidity corrected according to the following formula [17,22]: DW0 ¼ DWx =ð1 þ AHx *CFÞ
ð1Þ
where DW0 is dry weight of Daphnia extrapolated to 0% air humidity, DWx is the measured weight, AHx is the relative
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2.3. Quality control 2.3.1. In-field control After the installation of the TXRF at the sampling site and a warming-up of 30 min, first the gain correction was carried out, and, using previously prepared carrier plates containing Sr, Pb, and Ni, the signal stability was checked. Since there is not any suitable equipment available to avoid contamination originating from the air, blank carrier plates were prepared with Ga internal standard solution and placed beside the sample preparation station, in order to determine the influence of airborne dust. The exposition time was exactly the same as the sample preparation time. In order to minimize the contamination, quartz plates were covered by a glass Petri dish during sample droplet drying (Fig. 3). Fig. 2. In-field measurement with the portable TXRF spectrometer PicoTAX.
atmospheric humidity at the time of the weighing and CF is an empirical correction factor (0.00293 F 0.00013). The weighed Daphnia were fixed with 2.5 Al pure water on the quartz carrier plates, air-dried, and the length measured with a stereomicroscope (Stemi 11, Zeiss, Germany). Subsequently, the Daphnia were digested directly on the carrier plate with 5 Al HNO3 (Suprapure grade, E. Merck) on a hot plate at 100 jC. The HNO3 was previously dopped by Ga standard solution (1 mg l 1) for an internal standardization. To remove the organic matrix, the Daphnia specimens were ashed in a cold plasma asher (Plasma-System 100, Technics Plasma, Kirchheim, Germany). Specific operation parameters were: oxygen pressure 1 mbar; oxygen purity factor 4.5 (99.995% oxygen), microwave power 300 W, reaction time 2 h. From the ashed Daphnia, photos were taken as a tool to select outliers (e.g. not centered sample spot, inhomogeneity after digestion).
2.3.2. Method to control in the laboratory with certified reference material The quality control of the stationary analysis, especially of the microdigestion directly on the carrier plates, was carried out using certified reference material (BCR 414, plankton) [23,24] with the following two ways: (1) The reference material was weighed analogously to the real Daphnia samples. About 1.6 to 18.8 Ag material was weighed onto the quartz carrier plates. Using a stereo magnifying glass, the sample spot was checked to be within the detector area. According to the Daphnia, the material was digested on the plate with a mixture of HNO3 and Ga internal standard, as described in Section 2.1.1 [17]. Although exclusively ceramic tools (scalpels, tweezers) and weighing paper were used for weighing, it was not possible to avoid completely static charge, even using an ionization fan (Sartorius). Thus, the quantitative transfer of the powdered material from the weighing paper onto the quartz carrier plates was highly complicated. For this reason, the following alternative method was also tested. (2) Microamounts of the certified zooplankton material were transferred without weighing onto the carrier plates
2.2. Instrumentation Daphnia were analyzed with two instruments. For the infield investigation, the portable TXRF spectrometer PicoTAX (Roentec) with a power consumption of 180 W and, for the laboratory analyses, in comparison, the stationary TXRF spectrometer 8030 C (FEI) with 3.5 kW was used. While a water-cooled Mo tube at 50 kV and 55 mA was selected for the investigation with the stationary TXRF, the portable TXRF works with an air-cooled Mo tube operated at 40 kV and 1 mA. Similar is it for the detector systems. The stationary spectrometer needs liquid nitrogen for the cooling of the Si(Li) detector and has a significant better resolution (148 eV at 5.9 keV) in comparison to the portable TXRF with a Peltier cooled X-flash detector (160 eV resolution). The stationary and the portable TXRF spectrometers had detector areas of 80 and 10 mm2, respectively.
Fig. 3. Sample preparation in the field.
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and prepared according to Section 2.1.1. Nevertheless, no internal standard was added in this method, but the quantification was carried out using the certified Zn mass fraction data. Zn was chosen because of the optimal signal intensity. All prepared reference samples were analyzed for comparison by both spectrometers. 2.4. Statistical judge and assessment To make a statement whether both TXRF techniques for element determination are equivalent or whether the differences between the methods are significant, the comparison of two data series according to the method of Doerffel [25] has been applied. This test is based on the hypotheses that, in case of equivalence of the analytical series, the differences between the measured results for each sample of both analytical techniques differ irregularly around zero. The significance was tested using the ‘‘extended t-test’’ [26] on the assumption of a normal distribution of the results of measurement. The linear regression as a simple model has been tested to correct the method based on and evaluated the value significantly over or underestimation.
3. Results and discussion 3.1. Quality assurance 3.1.1. In-field control Since in-field analyses of trace element amounts often cannot be carried out in a clean working environment, and the amount of analytes in the samples is quite low, it is always necessary to do one’s best to avoid excess contamination, and to determine this contamination. Fig. 4 shows a
Table 1 In-field analytical results of the blank carrier plate, the washing water of the zooplankton samples and a single Daphnia specimen Element
Blank in field amount (ng)
Washing water (filtered river water) (Ag/L)
Daphnia in field amount (ng)
K Ca Cr Mn Fe Ni Cu Zn As Sr Pb
3.44 3.03 < 0.066 0.167 5.59 < 0.027 < 0.024 0.095 < 0.017 0.056 0.053
3730 53,100 < 1.6 9.1 6.04 2.36 2.06 1.16 3.50 223 < 1.5
152 530 < 0.331 2.15 23.4 < 0.138 0.260 1.67 0.132 1.96 0.231
spectrum of a blank carrier plate, which was exposed to air during sample preparation. The corresponding absolute element amounts are listed in Table 1. In comparison to a real zooplankton sample, it can be assumed that from the most abundant contaminant elements (K, Ca, and Fe) only Fe cannot be neglected. From the trace elements, Mn, Zn, and Pb must be also considered, since the blank values exceed 5% of the detected amounts in the real samples. 3.1.2. Recovery rate of the certified reference material BCR 414 For quality control checks, certified reference material BCR 414 has been used. Testing the material under similar condition as for the investigation, only ca. 0.025 mg of reference material have been used for the analytical procedure. Tests with weighed material using Ga as an internal standard came up to the result that there is a wide recovery range for both spectrometers between 75% and 111%, in
Fig. 4. Spectrum of a blank carrier plate after air exposition.
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Fig. 5. Recovery of the investigated elements in the BCR 414 certified reference material using the weighing method and Ga as internal standard.
average around 90% (Fig. 5). This range of the rates can be mainly explained by difficulties transferring the sample material from the weighing paper onto the quartz plate. As a result of the reason mentioned above, a part of the very fine powdered sample lied outside the detection area, but the added internal Ga standard solution lied completely within the detector area for both equipments. Furthermore, the sample inhomogeneity itself is an additional reason for the wide recovery range. The relative standard deviation of the recoveries ranges between 10% and 20% (n = 15) and the material is certified to be homogeneous for sample weights of more than 1 mg [23]. To avoid the rather complicated weighing and transferring procedures of the reference material as an important fact for the wide recovery range, samples were prepared without weighing and Ga addition. Since an internal standard element is needed for the quantification, Zn was chosen
as internal standard, and its certified mass fraction was used for calculation. More reasons why Zn as internal standard was chosen: (i) its signal intensity was optimal, (ii) its recoveries were found to be very similar with both instruments, so that its distribution inside the sample can be assumed to be quite homogeneous, and (iii) Zn is not a strongly bound element, so that one can calculate Zn in total dissolution. The recoveries obtained with this method were found to be better than with Ga internal standard (Fig. 6). The relative standard deviations of the recoveries decreased. On the basis of these results, it can be established that the direct digestion of the certified reference material on the carrier plate was complete. Using this method, it could not be proved that the weighing process can comply with the quality requirements, since the weighing of the finely powdered CRM has a high uncertainty because of the static charge. Because it does not
Fig. 6. Recovery of the investigated elements in the BCR 414 certified reference material without weighing, and using Zn as internal standard.
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Fig. 7. Spectrum of a single Daphnia specimen prepared in the field according to the wet method.
occur in the case of real single Daphnia specimen, which are not powdered, the uncertainty of weighing could be neglected. 3.2. Results of the in-field determination In-field measurements according to the wet method. Fig. 7 shows a TXRF spectrum of Daphnia prepared according to the wet method. It demonstrates that the PicoTAX yields very fast and sufficient sensitivity for infield measurements on the element content of minute biological samples. The corresponding absolute analyte amounts are in Table 1. The element concentrations of the washing water (filtered river water) are also shown there. The element contents of this water were neglected, since this was the living medium of the Daphnia specimen, and it could be supposed that the zooplankton are in equilibrium with the water. The washing in distilled water could cause a desorption of the elements because of the less osmotic pressure. On the basis of field investigation results of biological samples, there is a direct possibility to select polluted
Table 2 Test results for the intercomparison Element
f
t
t(0.99,f )
Method differences
As Ca Cu Fe K Mn Ni Pb Sr Zn
11 60 7 60 60 60 40 40 60 60
2.16 5.59 2.66 6.78 2.51 0.45 0.12 10.77 0.78 3.50
3.11 2.66 3.5 2.66 2.66 2.66 2.7 2.7 2.66 2.66
non-significant significant non-significant significant non-significant non-significant non-significant significant non-significant significant
f = degree of freedom, t = t value, t(0.99,f ) = t value for a significance level of 99%.
Fig. 8. Significant correlation of the results from both spectrometers for Sr and Cu.
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detection could be modified. This assumption might be right, since there were not any similar differences for Fe at the quite homogeneous certified reference material. This can be proved by future investigations of the element distributions using raster electron microscopy. Since Ca is contained mostly by the shell of the Daphnia, the differences cannot be explained by inhomogeneities. Whether the differences result from the smaller detection area of the PicoTAX should be checked by further investigations.
4. Conclusions
Fig. 9. Nonsignificant correlation of the results from both spectrometers for Zn.
sampling sites. Using an internal standard as Ga, the relative amount of accumulated elements can be obtained. A semiquantitative mass fraction determination can also be carried out, when using the length –mass relationship [17]. However, this has the prerequisite that the above mentioned relationship has been established for the investigated zooplankton species. 3.3. Results of the in-lab determination The intercomparison results from both instruments are shown in Table 2. Nonsignificant differences between the two techniques were found for As, Cu, K, Ni, Mn and Sr. As, Sr and Cu results obtained by both spectrometers are shown in Fig. 8 and demonstrate the extreme good correlation of the measured results for nonsignificant method differences. Significant differences between both methods were obtained for Zn, Pb, Ca and Fe. In comparison to the total analytical uncertainty, the method difference of ca. 5% for Zn is acceptable for biological individual investigation as shown in Fig. 9. For Pb, analytical results of both spectrometers are around the limit of quantification. Higher uncertainty values have to be accepted and the reason for the significant method difference. In the case of Ca and Fe, inhomogeneities in the sample and the difference between the detection area could be the reasons for the significant differences. The extremely high differences between the results of both spectrometers for Ca and Fe could not be explained yet. It can be assumed, that in the case of Fe, there are considerable inhomogeneities within the samples (e.g. ingested particles or metal bearing organs), so that the
The intercomparison has shown that the PicoTAX is suitable for the determination of various trace elements in biological material with small amounts (single specimens in the Ag range). On the basis of the results, it can be concluded that this equipment is suitable for direct in-field analysis of biological material. It was demonstrated that care should be taken for the correct preparation of the sample on the glass carrier when using the portable TXRF spectrometer. Thus, it is necessary to optimize further the preparation of the material for the above mentioned method, and to check the trace element distribution within the measurement area, as well as the sample spot using a raster electron microscope.
Acknowledgements This research has been supported by a Marie Curie Fellowship of the European Community programme Improving Human Research Potential under contract number HPMF-CT-2002-01740. The authors are also grateful for the financial support of the DLR International Bureau, Germany (Project No. HUN 01/001) and the Te´T Foundation in Hungary (Project No. D-17/01).
References [1] C.Y. Chen, R.S. Stemberger, B. Klaue, J.D. Blum, P.C. Pickhardt, C.L. Folt, Limnol. Oceanogr. 45 (2000) 1525 – 1536. [2] C. Barranguet, M. Plans, E. van der Grinten, J.J. Sinke, W. Admiraal, Environ. Toxicol. Chem. 21 (9) (2002) 1955 – 1965. [3] C. White, G.M. Gadd, FEMS Microbiol. Lett. 183 (2000) 313 – 318. [4] R.Q. Yu, W.X. Wang, Limnol. Oceanogr. 47 (2002) 495 – 504. [5] J. Kahle, G.P. Zauke, Aquat. Toxicol. 59 (2002) 115 – 135. [6] A. Trontelj, P. Ponikvar-Zorko, Water Sci. Technol. 37 (8) (1998) 235 – 241. [7] M.J. Marque´s, E. Martı´nez-Conde, J.V. Rovira, Water Air Soil Pollut. 148 (2003) 363 – 388. [8] P. Gulya´s, I. Bancsi, K. Zsuga, Misc. Zool. Hung. 10 (1995) 21 – 47 (Budapest). [9] K. Zsuga, Int. Rev. Hydrobiol. 83 (1998) 199 – 202. [10] International Commission for the Protection of the Danube River, Joint Danube Survey: Investigation of the Tisza River and its Tributaries, in: B. Csa´nyi (Ed.), Final Report, VITUKI Institute for Water Pollution Control, Budapest, Hungary, 2002, pp. 66 – 67.
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[11] E. Fleit, Gy. Lakatos, Toxicol. Lett. 140 – 141 (2003) 323 – 332. [12] K.L. Holding, R.A. Gill, J. Carter, Environ. Geochem. Health 25 (1) (2003) 87 – 93. [13] A. Sanz-Medel, Spectrochim. Acta Part B 53 (1998) 197 – 211. ´ va´ri, Gy. Za´ray, J. Hassler, Microchem. J. 73 (1 – 2) (2002) [14] M. O 125 – 130. [15] R.P. Petterson, Spectrochim. Acta Part B 53 (1998) 101 – 115. [16] H. Miesbauer, G. Ko¨ck, L. Fu¨reder, Spectrochim. Acta Part B 56 (2001) 2203 – 2207. [17] M. Mages, S. Woelfl, W. von Tuempling, Spectrochim. Acta Part B 56 56 (2001) 2209 – 2217. [18] S. Woelfl, M. Mages, F. Encina, Spectrochim. Acta Part B 58 (2003) 2157 – 2168. ´ va´ri, W. von Tuempling, Spectrochim. [19] M. Mages, S. Woelfl, M. O Acta Part B 58 (2003) 2127 – 2136.
´ va´ri, M. Mages, S. Woelfl , W. von Tuempling Jr., K. Kro¨pfl, [20] M. O Gy. Za´ray, Spectrochim. Acta Part B 59 (2004) 1173 – 1181. ´ va´ri, F. Encina, [21] S. Woelfl, M. Mages, S. Mercado, L. Villalobos, M. O Anal. Bioanal. Chem. 378 (4) (2004) 1088 – 1094. [22] I. Vila, M. Contreras, V. Montecino, J. Pizarro, D.D. Adams, Adv. Limnol. 55 (2000) 31 – 44. [23] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, Fresenius J. Anal. Chem. 345 (1993) 12 – 17. [24] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, The Commission of the European Communities, Community Bureau of Reference, Brussels-Luxembourg, EUR 14558 EN, 1993. [25] K. Doerffel, Statistik in der Analytischen Chemie, Deutscher Verlag fu¨r Grundstoffindustrie, Leipzig, 1990, pp. 120 – 122. [26] J. Zar, Biostatistical Analysis, 2th ed., Prentice-Hall, 1984.
The use of a portable total reflection X-ray fluorescence spectrometer for $ trace element determination in freshwater microcrustaceans (Daphnia) ´ va´ri a, Wolf v. Tu¨mpling Jr. a, Francisco Encina c Margarete Mages a,*, Stefan Woelfl b, Miha´ly O a
UFZ Centre for Environmental Research Leipzig-Halle, Department of Inland Water Research Magdeburg, Bru¨ckstrasse 3a, D-39114 Magdeburg, Germany b Instituto de Zoologı´a, Universidad Austral de Chile, Casilla 567, Valdivia, Chile c Facultad de Ciencias, Universidad Cato´lica de Temuco, Escuela de Ciencias Ambientales, Montt 056, Temuco, Chile Received 30 January 2004; accepted 2 April 2004 Available online 15 July 2004
Abstract The suitability of a newly developed, portable total reflection X-ray fluorescence (TXRF) spectrometer (PicoTAX, Roentec, Berlin, Germany) to analyze trace elements in biological material was tested and compared with a stationary instrument (Spectrometer 8030 C, FEI, Munich, Germany). For that, single freshwater microcrustacean specimens (Daphnia spec.) with dry weights ranging between 1.6 and 18.8 Ag individual 1 were prepared according to the dry method and analyzed with both instruments. Additionally, for orientation purposes, freshly collected Daphnia were prepared in field according to the wet method directly on the glass carrier and analyzed using the portable PicoTAX. For method validation, certified reference material (CRM 414, plankton) was analyzed. The results of the in-field measurements demonstrate that the PicoTAX yields very fast and sufficiently sensitive in-field measurements on the element content of minute biological samples. For As, Cu, K, Mn, Ni and Sr, a good correlation was found between the two spectrometers. Only for Ca, Fe, Pb and Zn the comparison with the results of the stationary equipment has shown significant differences. D 2004 Elsevier B.V. All rights reserved. Keywords: Trace element analysis; Daphnia; Zooplankton; Total reflection X-ray fluorescence; Portable TXRF; Field experiments
1. Introduction Elemental analysis of zooplankton is a very important task to understand the uptake and accumulation processes of trace elements into the trophic chain [1]. Trace elements at elevated concentrations can be bioaccumulated, resulting negative changes in the aquatic ecosystem [2]. Mining activities often introduce untreated wastewater into rivers. This is an important reason for high heavy metal loads in surface waters. Various aquatic biota at the beginning of the trophic chain, like biofilms [3], zooplankton [4,5] and benthic invertebrates $ This paper was presented at the 10th Symposium on Total Reflection X-Ray Fluorescence Analysis and the 39th Discussion Meeting on Chemical Analysis (TXRF-2003) held in Awaji Island, Hyogo, Japan, in September 2003, and is published in the special issue of Spectrochimica Acta Part B, dedicated to that conference. * Corresponding author. Tel.: +49-391-810-9310; fax: +49-391-8109150. E-mail addresses: [email protected] (M. Mages), ´ va´ri), [email protected] (S. Woelfl), [email protected] (M. O [email protected] (W. v. Tu¨mpling).
0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2004.05.006
[6,7] have already been investigated for trace element uptake and kinetics, and used for biomonitoring. The zooplankton of the Hungarian section of the Tisza river system has been investigated since the late 19th century. These investigations were intensified in the past two decades. Detailed periodical Rotatoria and Crustacea surveys among the Tisza and its tributaries have been published [8]. Zooplankton investigation at the Tisza-to´ include quantitative and qualitative surveys, monitoring of seasonal dynamics and characterisation of mosaic patterns and biodiversity [9]. The cyanide pollution in the year 2000 has destroyed most of the zooplankton in the Szamos and Tisza stream; approximately 70 – 80% of Rotatoria and Cladocera species were killed. The regrowth of the zooplankton community started quite soon, already a few days after the spill. Nevertheless, the zooplankton abundance changed significantly compared to the results of the previous 10 –15 years [10]. Unfortunately, no investigation of the trace element concentrations is known until now. Only higher organisms (e.g. fishes) were investigated [11].
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Trace element determinations in aquatic biota are commonly carried out by inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES) or atomic absorption spectrometry (AAS) [12,13]. All these methods often require digestion of the sample. Larger amounts of sample material are needed and contamination originating from the reagents and vessels cannot be avoided in all cases. On the other hand, electrothermal vaporization (ETV) is a quite common technique allowing direct introducing of solid samples into the spectrometer [14]. For the direct trace element analysis of solid biological samples, total reflection X-ray fluorescence spectrometry (TXRF) is very suitable [15 – 17]. Since the organic matrix can increase determination limits considerably, it is often necessary to separate analytes from this matrix. This can be carried out almost under contamination-free conditions, if cold plasma ashing (CPA) in low pressure oxygen atmosphere is used [18]. Recently, a portable TXRF spectrometer was introduced, allowing in-field trace element analyses. First applications have already been reported [19]. Determination of trace elements in river water was carried out. However, this technique has also some limitations, since during in-field investigations contamination originating from airborne dust may occur, and the detector area of the portable TXRF spectrometer is significantly smaller, than those of stationary spectrometers. The aim of this work was to investigate trace elements in single freshwater zooplankton specimens from the Tisza river in Hungary, both in the field with a portable TXRF spectrometer, and in the laboratory with a stationary TXRF spectrometer.
2. Materials and methods 2.1. Sampling and sample preparation Daphnia from the river Tisza (Hungary) were collected from the partly artificial reservoir named Tisza-to´ during summer 2003. The sampling site (Kisko¨re) is located at the main basin, just above the roller dam. A map of the sampling region is shown in Fig. 1. The zooplankton sampling was carried out from a boat with a metal-free zooplankton net (aperture: 20 cm; net size: 100 Am; Hydrobios, Kiel, Germany). The samples were subsequently prepared in the field: first, the whole sample amount was washed three times with 0.2 Am filtered lake water in order to remove stuck suspended matter [17]. After this, single Daphnia specimens were selected using a stereomicroscope. Two methods for the element analyses were applied: (1) To get an overview for orientation purposes in field, freshly collected Daphnia were prepared according to the wet method directly on the glass carrier [17]. The Daphnia were subsequently digested directly on the sample carrier plate with 5 Al HNO3 (Suprapure grade,
Fig. 1. Map of Eastern Hungary, with the sampling point at Kisko¨re.
E. Merck, Darmstadt, Germany) on a hot plate at 100 jC. The HNO3 was previously dopped by Ga standard solution (1 mg l 1), in order to make internal standardization possible. The trace elements were determined using the portable PicoTAX (Fig. 2). (2) For the investigation using the dry method [17] in the laboratory, Daphnia were transferred in PTFE special vials (Savillex, USA), frozen in liquid nitrogen and transported in a Dewar bottle. In addition to the zooplankton, a surface water sample from that sampling location was taken. One part of it was filtered through a cellulose ester filter with a pore size of 0.2 Am (Millipore, France) in the field. Both the filtered (dissolved fraction) and non-filtered (total) subsamples were stabilized with 65% nitric acid, and subsequently analyzed in the field for a first screening and to determine the trace element concentrations in the washing water of the zooplankton samples. The results are published in Ref. [20]. 2.1.1. Preparation in the laboratory Daphnia prepared according to the dry method were first freeze-dried (Alpha 1, Christ, Osterode, Germany) at the laboratory for 24 h at 20 jC. The single lyophilised specimens of Daphnia with dry weights between 3 and 19 Ag individual 1 were prepared according to the modified dry method [17,21]. For this, Daphnia were weighed using an ultrafine balance (S4, Sartorius, Germany). The weights were humidity corrected according to the following formula [17,22]: DW0 ¼ DWx =ð1 þ AHx *CFÞ
ð1Þ
where DW0 is dry weight of Daphnia extrapolated to 0% air humidity, DWx is the measured weight, AHx is the relative
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2.3. Quality control 2.3.1. In-field control After the installation of the TXRF at the sampling site and a warming-up of 30 min, first the gain correction was carried out, and, using previously prepared carrier plates containing Sr, Pb, and Ni, the signal stability was checked. Since there is not any suitable equipment available to avoid contamination originating from the air, blank carrier plates were prepared with Ga internal standard solution and placed beside the sample preparation station, in order to determine the influence of airborne dust. The exposition time was exactly the same as the sample preparation time. In order to minimize the contamination, quartz plates were covered by a glass Petri dish during sample droplet drying (Fig. 3). Fig. 2. In-field measurement with the portable TXRF spectrometer PicoTAX.
atmospheric humidity at the time of the weighing and CF is an empirical correction factor (0.00293 F 0.00013). The weighed Daphnia were fixed with 2.5 Al pure water on the quartz carrier plates, air-dried, and the length measured with a stereomicroscope (Stemi 11, Zeiss, Germany). Subsequently, the Daphnia were digested directly on the carrier plate with 5 Al HNO3 (Suprapure grade, E. Merck) on a hot plate at 100 jC. The HNO3 was previously dopped by Ga standard solution (1 mg l 1) for an internal standardization. To remove the organic matrix, the Daphnia specimens were ashed in a cold plasma asher (Plasma-System 100, Technics Plasma, Kirchheim, Germany). Specific operation parameters were: oxygen pressure 1 mbar; oxygen purity factor 4.5 (99.995% oxygen), microwave power 300 W, reaction time 2 h. From the ashed Daphnia, photos were taken as a tool to select outliers (e.g. not centered sample spot, inhomogeneity after digestion).
2.3.2. Method to control in the laboratory with certified reference material The quality control of the stationary analysis, especially of the microdigestion directly on the carrier plates, was carried out using certified reference material (BCR 414, plankton) [23,24] with the following two ways: (1) The reference material was weighed analogously to the real Daphnia samples. About 1.6 to 18.8 Ag material was weighed onto the quartz carrier plates. Using a stereo magnifying glass, the sample spot was checked to be within the detector area. According to the Daphnia, the material was digested on the plate with a mixture of HNO3 and Ga internal standard, as described in Section 2.1.1 [17]. Although exclusively ceramic tools (scalpels, tweezers) and weighing paper were used for weighing, it was not possible to avoid completely static charge, even using an ionization fan (Sartorius). Thus, the quantitative transfer of the powdered material from the weighing paper onto the quartz carrier plates was highly complicated. For this reason, the following alternative method was also tested. (2) Microamounts of the certified zooplankton material were transferred without weighing onto the carrier plates
2.2. Instrumentation Daphnia were analyzed with two instruments. For the infield investigation, the portable TXRF spectrometer PicoTAX (Roentec) with a power consumption of 180 W and, for the laboratory analyses, in comparison, the stationary TXRF spectrometer 8030 C (FEI) with 3.5 kW was used. While a water-cooled Mo tube at 50 kV and 55 mA was selected for the investigation with the stationary TXRF, the portable TXRF works with an air-cooled Mo tube operated at 40 kV and 1 mA. Similar is it for the detector systems. The stationary spectrometer needs liquid nitrogen for the cooling of the Si(Li) detector and has a significant better resolution (148 eV at 5.9 keV) in comparison to the portable TXRF with a Peltier cooled X-flash detector (160 eV resolution). The stationary and the portable TXRF spectrometers had detector areas of 80 and 10 mm2, respectively.
Fig. 3. Sample preparation in the field.
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and prepared according to Section 2.1.1. Nevertheless, no internal standard was added in this method, but the quantification was carried out using the certified Zn mass fraction data. Zn was chosen because of the optimal signal intensity. All prepared reference samples were analyzed for comparison by both spectrometers. 2.4. Statistical judge and assessment To make a statement whether both TXRF techniques for element determination are equivalent or whether the differences between the methods are significant, the comparison of two data series according to the method of Doerffel [25] has been applied. This test is based on the hypotheses that, in case of equivalence of the analytical series, the differences between the measured results for each sample of both analytical techniques differ irregularly around zero. The significance was tested using the ‘‘extended t-test’’ [26] on the assumption of a normal distribution of the results of measurement. The linear regression as a simple model has been tested to correct the method based on and evaluated the value significantly over or underestimation.
3. Results and discussion 3.1. Quality assurance 3.1.1. In-field control Since in-field analyses of trace element amounts often cannot be carried out in a clean working environment, and the amount of analytes in the samples is quite low, it is always necessary to do one’s best to avoid excess contamination, and to determine this contamination. Fig. 4 shows a
Table 1 In-field analytical results of the blank carrier plate, the washing water of the zooplankton samples and a single Daphnia specimen Element
Blank in field amount (ng)
Washing water (filtered river water) (Ag/L)
Daphnia in field amount (ng)
K Ca Cr Mn Fe Ni Cu Zn As Sr Pb
3.44 3.03 < 0.066 0.167 5.59 < 0.027 < 0.024 0.095 < 0.017 0.056 0.053
3730 53,100 < 1.6 9.1 6.04 2.36 2.06 1.16 3.50 223 < 1.5
152 530 < 0.331 2.15 23.4 < 0.138 0.260 1.67 0.132 1.96 0.231
spectrum of a blank carrier plate, which was exposed to air during sample preparation. The corresponding absolute element amounts are listed in Table 1. In comparison to a real zooplankton sample, it can be assumed that from the most abundant contaminant elements (K, Ca, and Fe) only Fe cannot be neglected. From the trace elements, Mn, Zn, and Pb must be also considered, since the blank values exceed 5% of the detected amounts in the real samples. 3.1.2. Recovery rate of the certified reference material BCR 414 For quality control checks, certified reference material BCR 414 has been used. Testing the material under similar condition as for the investigation, only ca. 0.025 mg of reference material have been used for the analytical procedure. Tests with weighed material using Ga as an internal standard came up to the result that there is a wide recovery range for both spectrometers between 75% and 111%, in
Fig. 4. Spectrum of a blank carrier plate after air exposition.
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Fig. 5. Recovery of the investigated elements in the BCR 414 certified reference material using the weighing method and Ga as internal standard.
average around 90% (Fig. 5). This range of the rates can be mainly explained by difficulties transferring the sample material from the weighing paper onto the quartz plate. As a result of the reason mentioned above, a part of the very fine powdered sample lied outside the detection area, but the added internal Ga standard solution lied completely within the detector area for both equipments. Furthermore, the sample inhomogeneity itself is an additional reason for the wide recovery range. The relative standard deviation of the recoveries ranges between 10% and 20% (n = 15) and the material is certified to be homogeneous for sample weights of more than 1 mg [23]. To avoid the rather complicated weighing and transferring procedures of the reference material as an important fact for the wide recovery range, samples were prepared without weighing and Ga addition. Since an internal standard element is needed for the quantification, Zn was chosen
as internal standard, and its certified mass fraction was used for calculation. More reasons why Zn as internal standard was chosen: (i) its signal intensity was optimal, (ii) its recoveries were found to be very similar with both instruments, so that its distribution inside the sample can be assumed to be quite homogeneous, and (iii) Zn is not a strongly bound element, so that one can calculate Zn in total dissolution. The recoveries obtained with this method were found to be better than with Ga internal standard (Fig. 6). The relative standard deviations of the recoveries decreased. On the basis of these results, it can be established that the direct digestion of the certified reference material on the carrier plate was complete. Using this method, it could not be proved that the weighing process can comply with the quality requirements, since the weighing of the finely powdered CRM has a high uncertainty because of the static charge. Because it does not
Fig. 6. Recovery of the investigated elements in the BCR 414 certified reference material without weighing, and using Zn as internal standard.
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Fig. 7. Spectrum of a single Daphnia specimen prepared in the field according to the wet method.
occur in the case of real single Daphnia specimen, which are not powdered, the uncertainty of weighing could be neglected. 3.2. Results of the in-field determination In-field measurements according to the wet method. Fig. 7 shows a TXRF spectrum of Daphnia prepared according to the wet method. It demonstrates that the PicoTAX yields very fast and sufficient sensitivity for infield measurements on the element content of minute biological samples. The corresponding absolute analyte amounts are in Table 1. The element concentrations of the washing water (filtered river water) are also shown there. The element contents of this water were neglected, since this was the living medium of the Daphnia specimen, and it could be supposed that the zooplankton are in equilibrium with the water. The washing in distilled water could cause a desorption of the elements because of the less osmotic pressure. On the basis of field investigation results of biological samples, there is a direct possibility to select polluted
Table 2 Test results for the intercomparison Element
f
t
t(0.99,f )
Method differences
As Ca Cu Fe K Mn Ni Pb Sr Zn
11 60 7 60 60 60 40 40 60 60
2.16 5.59 2.66 6.78 2.51 0.45 0.12 10.77 0.78 3.50
3.11 2.66 3.5 2.66 2.66 2.66 2.7 2.7 2.66 2.66
non-significant significant non-significant significant non-significant non-significant non-significant significant non-significant significant
f = degree of freedom, t = t value, t(0.99,f ) = t value for a significance level of 99%.
Fig. 8. Significant correlation of the results from both spectrometers for Sr and Cu.
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detection could be modified. This assumption might be right, since there were not any similar differences for Fe at the quite homogeneous certified reference material. This can be proved by future investigations of the element distributions using raster electron microscopy. Since Ca is contained mostly by the shell of the Daphnia, the differences cannot be explained by inhomogeneities. Whether the differences result from the smaller detection area of the PicoTAX should be checked by further investigations.
4. Conclusions
Fig. 9. Nonsignificant correlation of the results from both spectrometers for Zn.
sampling sites. Using an internal standard as Ga, the relative amount of accumulated elements can be obtained. A semiquantitative mass fraction determination can also be carried out, when using the length –mass relationship [17]. However, this has the prerequisite that the above mentioned relationship has been established for the investigated zooplankton species. 3.3. Results of the in-lab determination The intercomparison results from both instruments are shown in Table 2. Nonsignificant differences between the two techniques were found for As, Cu, K, Ni, Mn and Sr. As, Sr and Cu results obtained by both spectrometers are shown in Fig. 8 and demonstrate the extreme good correlation of the measured results for nonsignificant method differences. Significant differences between both methods were obtained for Zn, Pb, Ca and Fe. In comparison to the total analytical uncertainty, the method difference of ca. 5% for Zn is acceptable for biological individual investigation as shown in Fig. 9. For Pb, analytical results of both spectrometers are around the limit of quantification. Higher uncertainty values have to be accepted and the reason for the significant method difference. In the case of Ca and Fe, inhomogeneities in the sample and the difference between the detection area could be the reasons for the significant differences. The extremely high differences between the results of both spectrometers for Ca and Fe could not be explained yet. It can be assumed, that in the case of Fe, there are considerable inhomogeneities within the samples (e.g. ingested particles or metal bearing organs), so that the
The intercomparison has shown that the PicoTAX is suitable for the determination of various trace elements in biological material with small amounts (single specimens in the Ag range). On the basis of the results, it can be concluded that this equipment is suitable for direct in-field analysis of biological material. It was demonstrated that care should be taken for the correct preparation of the sample on the glass carrier when using the portable TXRF spectrometer. Thus, it is necessary to optimize further the preparation of the material for the above mentioned method, and to check the trace element distribution within the measurement area, as well as the sample spot using a raster electron microscope.
Acknowledgements This research has been supported by a Marie Curie Fellowship of the European Community programme Improving Human Research Potential under contract number HPMF-CT-2002-01740. The authors are also grateful for the financial support of the DLR International Bureau, Germany (Project No. HUN 01/001) and the Te´T Foundation in Hungary (Project No. D-17/01).
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