PROFICIENCY TESTING: EXPERIENCES FROM CROATIA ON THE ISSUE OF HEAVY METALS DETERMINATION IN MARINE SEDIMENTS

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1 The First International Proficiency Testing Conference Sinaia, România 11 th 13 th October, 2007 PROFICIENCY TESTING: EXPERIENCES FROM CROATIA ON THE ISSUE OF HEAVY METALS DETERMINATION IN MARINE SEDIMENTS Marijana Matek Saric, Judita Grzunov PUBLIC HEALTH INSTITUTE ZADAR, Kolovare 2, Zadar, Croatia Abstract Laboratories may check the accuracy of heavy metal measurements in marine sediments by taking part in an inter-laboratory comparison. Measurement methods for all metals must be validated and laboratories must implement good laboratory practices to avoid the risk of contamination and/or loss of analytes during the pretreatment processes. The risk of contamination should be avoided by using reagents that are supra-pure or that are of analytical grade as well as by using Milli-Q deionized water (<1µSi) for preparing all lab ware and s. Sediment s are dry s, therefore s should be homogenized (shaking 2 min) before weighing. It is important to wait a few minutes and accurately weigh the s in a labeled Teflon vessels before opening the bottles. For moisture content determination at least two s should be prepared (in separated cups, not in digestion vessels). At least one certified reference material should be used and prepared in duplicate for each digestion batch (12 places). The reference digestions are to be prepared in a similar manner as the actual s. It is best to use a reference material that is of similar composition and concentration range as the actual s. At least two blanks should be prepared for each batch of analytes. Sediment s must be treated in closed Teflon vessels with hydrofluoric acid (HF) in combination with nitric acid (HNO 3 ). The concentration of the standard solution must be calculated so that they bracket the concentrations of the s and the reference materials. In order to check the accuracy of the prepared curve an independent standard is prepared. Key words Proficiency testing, inter-laboratory comparison, marine sediment, heavy metals 177

2 1 INTRODUCTION Proficiency testing (PT) is a kind of inter-laboratory comparison used for determination of the competence of individual laboratories for specific tests and to assist in resolving of inter-laboratory differences. Inter-laboratory comparison is the organization, performance and evaluation of tests of the same items or identical portions of an effectively homogeneous material in two or more different laboratories in accordance with predetermined conditions [1, 2, 3]. Laboratories are encouraged to participate in PT to control the accuracy of their results. The procedure for PT is as follows: each participant in a PT receives a prepared by the organizer and has to determine a list of analytes; the results are sent back to the organizer and the collected data are analyzed [4]. A test material distributed by a PT provider must be similar to the material that is routinely analyzed in testing laboratories with respect to the composition of the matrix, the concentration range, and/or the quantity of the analyte. As a general principle, procedures used by testing laboratories participating in the comparison should simulate those used in their routine analytical work [1, 3]. When the number of testing laboratories participating in PT is a large enough (more than 20-30), the assigned value of the test material can be calculated from analytical results of the laboratories as a consensus value (as the mean of the median of the results) while the performance of the participating laboratories is assessed based on the differences between their results and the assigned value. A performance score is calculated for each laboratory using statistical schemes detailed in [1, 2, 3]. The competence of PT schemes providers is assessed according to the guidelines [5]. PT has dramatically pervaded throughout the world of chemical measurement over the last few decades. A lot of nationally and internationally recognized PT providers are active in different analytical fields. This paper describes the inter-comparison exercise in our laboratory (Health Ecology Service of Public Health Institute Zadar) for the determination of trace elements (Cu, Cr, Fe, Mn, Ni and Zn) in a marine sediment (IAEA-158). The was prepared at the International Atomic Agency, Marine Environmental Studies Laboratory, Monaco (IAE-MEL). All participants were requested to determine as many elements as they normally analyze from the following suite: As, Ag, Cd, Co, Cr, Cu, Fe, Hg (total and methyl Hg), Mn, Pb, Se, Sn, Sr, Zn. 2 MATERIAL AND METHODS Measurement methods for all metals must be validated and the laboratories must implement good laboratory practices to avoid the risk of contamination and/or the loss of analytes during pretreatment process. Validation of methods for AAS determination of metals must include validation criteria described in Table 1 [6]. 178

3 Table 1-List of validation criteria Validation parameters Criteria of acceptance Linearity k 0,99 Precision -reputability of measurement RSD 1 % -repeatability of measurement RSD 2 % intermediary precision RSD 5 % Accuracy 100 ± 10 % stability of measurement standard information Detection limit information Quantification limit information The risk of contamination should be avoided by using reagents that are supra-pure or of analytical grade as well as by using Milli-Q deionized water (<1µSi) for preparing all lab ware and s. Water conductivity (Milli-Q deionized and Milli-R) must be controlled daily. Cleaning procedure-the best way to clean Teflon vessels and their caps (used in digestion) is to soak them overnight in a detergent solution (micro solution 2% in tap water) followed by rinsing them thoroughly with tap water, and then with Milli-R water (< 60 µsi). Afterward the vessels should be filled with 5 ml of HNO 3 (conc.), closed and placed in a microwave-oven. The oven program for cleaning may be the same as the digestion program (Table 2). Allow vessels to cool to room temperature, release pressure carefully by opening the valve, and only then open the vessels. Empty the vessels (note that the acid can be recycled for more cycles of cleaning) and rinse them carefully with Milli-Q water. Leave them to dry under a laminar flow hood. Once Teflon vessels are dry, they should be closed and packaged in polyethylene bags to prevent the risk of contamination prior to use. This procedure should in fact be used for cleaning all plastic lab ware in general, but instead of microwave-oven cleaning digestion procedure plastic lab ware in general should be left to stand in 20% (v/v) HNO 3 conc. solution at least 6 h. If affordable, it is always better to use unused/new lab ware. Our laboratory follows the cleaning procedure for cleaning lab ware as described in SOP Cleaning procedure, RU 5.5/1-3 [9, 10]. Weighing-Sediment s are dry s, therefore they should be homogenized (shaking 2 min) before weighing. Before opening the bottle, wait a few minutes and accurately weigh (4 digit analytical balance: linearity ± 0,2 mg; repeatability SD ±0,03 mg) the in labeled teflon vessel. sture content-the material can easily pick up moisture during storage. Therefore it is necessary to determine the water content of the material at the time of analysis. To do so a separate sub- (not the same as used for analysis) is dried to a constant weight at 105 C (usually at least 24 hours). Analytical quality control-at least one certified reference material should be used and prepared in duplicate for each digestion batch (12 places). The digestion of the reference material is prepared in a similar manner as the s. The reference material used should be similar in composition and concentration range to the s used. For the presented study we used IAEA-433 as a quality control. Digestion-At least two blanks should be prepared for each batch of analysis. They should be prepared in a similar manner as a, except that no is added to the digestion vessels. 179

4 Sample digestion is a pretreatment step that may provide contamination risk and/or loss of analytes. Therefore sediment s must be treated in closed Teflon vessels with hydrofluoric acid (HF) in combination with HNO 3. The use of HF is essential because it is the only acid that completely dissolves silicate lattices and therefore releases all material. This digestion procedure is suitable for later determination of metals like Fe, Cu, Cr, Zn, Ni and Mn. All vessels must be closed tightly and the appropriate digestion program must be used (Table 2). Polypropylene graduated tubes with known quantities of boric acid (0,8 g) should be prepared and weight in order to later neutralize HF and HNO 3 used for the digestion of s. Table 2 - Digestion program for determination of metals in marine sediment [7]. Step Time Power Press Temp 1 Temp :05: :05: :05: :05: When the digestion is completed, s should be allowed to cool to room temperature, followed by the release of pressure by carefully opening the valve. Only then vessels can be opened. Samples are to be transferred into the above mentioned polypropylene graduated tubes (already containing dissolved boric acid). The emptied Teflon vessels previously containing the s should immediately be rinsed with Milli-Q water into graduated tubes (at least 3 times). Those graduated tubes must be diluted with Milli-Q water to the mark. Preparation of metal standard solutions for the calibration curve-the calibration curve must be generated from at least 3 points (standard solutions of different concentration) plus a zero calibration. The concentration of the standard solutions must be calculated so that they bracket the concentrations of the s as well as the concentration of the reference material. If the concentration of a exceeds the limit of the calibration curve, the must be either diluted to a concentration within the calibration curve, or the calibration curve must be extended by using an additional and higher concentration standard. If, on the contrary, the concentration of the s is lower than the lowest calibration curve's point, a new calibration curve must be prepared. In order to prepare a calibration curve, add approximately 10 ml of Milli-Q water into a clean polypropylene tube (50 ml) and add all reagents needed to obtain a similar matrix as the matrix of the s (e.g. Boric acid, HNO 3, HF, etc). Add the appropriate quantity of standard solution using a micropipette, dilute to the mark with Milli-Q water and shake well. The thereby produced solution can be kept for a few days if stored in a refrigerator (+4 C). Depending on the element, it may be necessary to prepare an intermediate stock standard solution. In order to check the accuracy of the prepared calibration curve an independent standard is prepared. The concentration of the external calibration verification (ECV) should be within the calibration curve. This solution is prepared as described above but a different stock standard solution (1000 µg/ml) is used [9]. Analytical conditions may change for each element, so it is necessary to first carefully read the analytical methods book of the AAS before starting an analysis [8]. 180

5 3 RESULTS Reporting of results-make at least three, but preferable six independent determinations for each element and report the results as the arithmetic mean and the standard deviation in mg/kg on a dry weight basis for both, (Table 3) and RM (Table 4). Table 3-Table of analyses. Moisture content is 1,47%. Inter-comparison Sample Number 158, number of bottle analyzed No 19 Ele me nts Unit Average weight (mg) Mean Value Standard deviation* Detecti on limit* Quanti fication limit* Lineari ty Repeatabili ty of measurement RSD<2% Cr mg/kg 266,758 62,55 0,989 0,0003 0,0010 0,9997 1,58 Cu mg/kg 266,758 49,93 0,952 0,0003 0,0010 0,9996 1,90 Fe mg/kg 266,758 28,15 0,391 0,0015 0,005 1,0000 1,39 Mn mg/kg 266, ,26 2,487 0,0021 0,007 0,9998 0,66 Ni mg/kg 266,758 39,37 0,441 0,0018 0,006 0,9994 1,12 Zn mg/kg 266, ,55 2,470 0,0003 0,0010 0,9999 1,81 Ele me nts Table 4-Table of analyses of RM. Moisture content is 1,07%. IAEA 433 Unit Average weight (mg) Mean Value Standard deviati on* Lineari ty Repea tability of measu rement RSD<2 % 95% Confide nce Interval (mg/kg) Cr mg/kg 273, ,76 0,850 0,9997 0, (136) Cu mg/kg 273,294 30,582 0,272 0,9996 0,888 30,2-31,4 (30,8) Fe mg/kg 273,294 40,864 0,568 1,0000 1,389 40,3-41,3 (40,8) Mn mg/kg 273, ,26 4,067 0,9998 1, (316) Ni mg/kg 273,294 38,53 0,206 0,9994 0,534 38,7-40,1 (40,8) Accuracy 100±10 % 99,82 99,29 100,15 99,77 97,79 Zn mg/kg 273, ,72 1,414 0,9999 1, (101) 100,71 *Standard deviation and detection limit should be reported in the same unit as results 181

6 4 CONCLUSIONS The accuracy of the results of heavy metals analysis is influenced by several procedures, including: lab ware cleaning, quality of used water, sampling, preparation, processing, and analysis. Measurement methods for all metals must be validated and laboratories must implement good laboratory practices to avoid the risk of contamination and/or loss of analytes during pretreatment process. Simultaneous and paired analysis of RM and test portion is necessary for PT purpose in order to check results. REFERENCES [1] Thompson M, Wood R. International harmonized protocol for proficiency testing of (chemical) analytical laboratories. J AOAC, 76: , (1993) [2] ISO Proficiency testing by interlaboratory comparison. Part 1: Development and operation of proficiency testing schemes. ISO/IEC Guide 43-1, ISO, Geneva, (1997) [3] Kuselman I, Pavlichenko M. Designs of experiment for proficiency testing with a limited number of participants. Accredit Qual Assur, 9: ,(2004). [4] Lafargue E, Biogeaud S, Rutledge DN, Feinberg MH. Proficiency testing schemes: solutions from homogenity control. Accredit Qual Assur, 9: ,(2004) [5] ILAC. Guidelines for the requirements for the competence of providers of proficiency testing schemes. ILAC G13:2000, International Laboratory Accreditation Co-operation (ILAC), (2000) [6] Lazarić K. Validacija analitičkih metoda. In: Validacija metoda, seminar i raspravljaonica; ed. Gašljević V, Hrvatsko mjeriteljsko društvo, str.1-8.(2001). [7] Milestone acid digestion cookbook for Microwave laboratory system MLS 1200 MEGA, January (1996) [8] Atomic Absorption Spectrophotometry cook book, Basic Conditions for Analysis of Principle of Atomic Absorption Spectrometry, Shimadzu (1995) [9] Laboratory Procedure Book, Training workshop on the analysis of trace metals in biological and sediment. International atomic Energy Agency-Marine Environmental Laboratory (2003) [10] SOP, RU 5.5/1-3 Pranje posuđa za kemijske analize, Zavod za javno zdravstvo Zadar, Služba za zdravstvenu ekologiju (2007) 182