Determination of Trace Metals in the Freshwater Leech Erpobdella octoculata of the Elbe River — Evaluation of the Analytical Protocol

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

K. Friese et al.346 Acta hydrochim. hydrobiol. 31 (2003) 4–5, 346–355

DOI 10.1002/aheh.200300506

Determination of Trace Metals in the FreshwaterLeech Erpobdella octoculata of the Elbe River –Evaluation of the Analytical Protocol

Kurt Friesea,René Frömmichenc,Barbara Wittera,Helmut Müllerb

a UFZ – Center forEnvironmental ResearchLeipzig-Halle,Department for Inland WaterResearch Magdeburg,Brückstr. 3a,39114 Magdeburg, Germany

b Institute for Analytical andEnvironmental Chemistry,Martin Luther University ofHalle,Geusaer Straße,06217 Merseburg, Germany

c Otto-von-Guericke-UniversityMagdeburg,Chair for Process SystemsEngineering,c/o Max-Planck-Institute forDynamics of ComplexTechnical Systems,Sandtorstr. 1,39106 Magdeburg, Germany

Correspondence: R. Frömmichen, E-mail: [email protected]

A composite procedure to determine heavy metals in the freshwater leech Erpobdella octo-culata (L.) (Annelidae: Hirudinea) of the Elbe river has been developed. All stages of the an-alytical process were checked, including the sampling procedure, sample preparation, andvalidation of the analysis by inductively coupled plasma mass spectrometry (ICP-MS) andatomic absorption spectrometry (AAS). The elements chromium, manganese, copper, zinc,cadmium, mercury, arsenic, and lead were determined. To ensure the quality of the results,the CRM NRC Dorm-1 (dogfish muscle) and Nist 1643c (river water) were analyzed on aparallel basis. A first overview on recent contamination of leeches in the river Elbe (Germa-ny) was gained with the newly developed procedure.

Bestimmung von Spurenmetallen im Süßwasseregel Erpobdella octoculata derElbe – Bewertung des analytischen Verfahrens

Es wurde ein Verfahren zur Bestimmung von Schwermetallen in dem Süßwasseregel Er-pobdella octoculata (L.) (Annelidae: Hirudinea) der Elbe entwickelt. Alle analytischen Schrit-te einschließlich der Probenahme, der Probenvorbereitung und der Messung mit induktivgekoppelter Plasma-Massenspektrometrie (ICP-MS) und Atomabsorptionsspektrometrie(AAS) wurden validiert. Die Elemente Chrom, Mangan, Kupfer, Zink, Cadmium, Quecksil-ber, Arsen und Blei wurden bestimmt. Zur Qualitätssicherung wurden die international zerti-fizierten Standardmaterialien CRM NRC Dorm-1 (Hundshaimuskel) and Nist 1643c (Fluss-wasser) in gleicher Weise parallel mit untersucht. Mit der neu entwickelten Methode konnteein erster Überblick über die aktuellen Kontaminationen von Egeln in der Elbe gewonnenwerden.

Keywords: AAS, ICP-MS, Biomonitoring, Biological Sample Digestion, Statistical Analysis

Schlagwörter: AAS, ICP-MS, Biomonitoring, Aufschluss biologischer Proben, StatistischeAuswertung


Trace Metals in Leech of the Elbe RiverActa hydrochim. hydrobiol. 31 (2003) 4–5, 346–355 347

1 Introduction

The overall pollutant load of the river Elbe has fallen consider-ably since 1989, but the metal loads have decreased onlyslightly since then. In the central reach of the Elbe, remobili-zation from the sediment of the still water areas, direct dis-charges and diffuse inputs along the Elbe, Mulde, and Saalerivers are possible reasons why this reduction is so slight [1].Efforts are being made to discover the sources of the pollu-tion by analyzing the aquatic phase and the sediment [2, 3],but this gives no information on the potential hazard to livingorganisms. Information on metal concentrations in the aquat-ic organisms present in the water is needed to permit a realassessment of the risk. The more recent literature suggestsusing certain aquatic organisms such as water-fleas and wa-ter isopods as bioindicators for contamination of the faunawith metals and for monitoring the quality of the water [4–9].This paper describes investigations carried out with the fresh-water leech Erpobdella octoculata as a bioindicator, e.g., as ameans to supply information on the quality of the river water[10, 11].

The leech Erpobdella octoculata is a pollution-tolerant hard-substrate colonizer. Being a highly abundant organism thatremains in one place it meets the requirements for passivebiomonitoring [12–16].The leech’s physiological resistance tometal contamination and its ability to accumulate metals canbe used for the purposes of bioindication [17–20]. An analyti-cal procedure has been developed to determine the overalllevels of the elements chromium (Cr), manganese (Mn), cop-per (Cu), zinc (Zn), cadmium (Cd), mercury (Hg), arsenic(As), and lead (Pb) in the freshwater leech. It incorporatesvarious methods of trace analysis and can be used as a partof routine analysis. The results are intended to establishguidelines for sampling and for preparation of the biologicalmaterial that are suitable for monitoring of ecological proc-esses found in water bodies.

2 Sampling and sample preparation

The samples used were taken from the river Elbe nearMagdeburg, at kilometer 326, in October 1994. The leecheswere taken from their habitat with ceramic tweezers and con-served for transport to the laboratory in HDPE (high-densitypolyethylene) containers filled with 96% ethanol. In the labo-ratory the leeches were cleaned of suspended particles byrinsing thoroughly three times with bi-distilled water andfreeze-dried (48 h, –20°C, 1.03 mbar). The sample materialwas then stored at –20°C to await further preparation (max. 4weeks). The freeze-dried individuals were sorted into threeweight classes (class I: 0...10 mg (59 individuals), class II:10...20 mg (18 individuals), class III: 20...30 mg (7 individu-als)) and homogenized for 45 min in zircon oxide grindingvessels.

Table 1: Heating programs for digestion in the high-pressureasher with 2 mL 65% HNO3 as a reactant solution and in themicrowave oven with 4 mL 65% HNO3 and 0.5 mL 30% H2O2

as a reactant solution.

Heizprogramm für den Aufschluss mit 2 mL 65%iger HNO3

als Reaktionslösung im Hochdruckverascher und mit 4 mL65%iger HNO3 und 0.5 mL 30%igem H2O2 als Reaktions-lösung im Mikrowellenofen.

High-pressure asherStep Temperature

of beginning°C

Temperatureof finishing

°C

Time

min

Pressure

bar

1 24 90 10 1002 90 120 30 1003 280 280 60 100

Microwave oven

Step Power%

Time MINmin

Time MAXmin

Pressurebar

1 100 1 20 2.752 100 1 20 5.53 100 1 20 10

50 mg of each sample were used for digestion in a high-pres-sure asher (2 mL 65% HNO3; Kürner-Analysentechnik GmbH(HPA)) or in a microwave oven (4.5 mL 65% HNO3, 0.5 mL30% H2O2; MDS 2000; CEM (MW)).

Table 1 shows the heating programs used to produce a resi-due-free solution of the sample material. After digestion, thesolution was diluted to 20 mL (HPA) or 50 mL (MW). A part ofeach solution was then stabilized for detecting mercury ac-cording to Jarvis [22]. For several days the samples treated inthis way produced stable and reproducible signals for deter-mining Hg concentrations. To verify the sample preparationthe certified reference material NRC Dorm-1 was digested bythe same method at a weight of 0.2 g for the HPA and 0.5 g forthe MW.

3 Apparatus

An electrothermal atomic absorption spectrometer with Zee-man background correction (ET-AAS 4100 ZL; Perkin Elmer)and a quadrupole mass spectrometer with inductively cou-pled plasma as excitation source (ICP-MS Elan 5000; PerkinElmer/Sciex) were used for element determination. Part of the



digested and diluted sample solutions were used for deter-mining the concentration of Hg in the flow-injection mercuryAAS (FI-HG-AAS,) and part for ascertaining the concentra-tions of all the other elements with the ET- AAS and ICP-MS.Additionally, a total reflection X-ray fluorescence equipment(TXRF EXTRA II; Atomika) was used to determine metalscontents in ethanol used for conservation of the samples.

The concentrations of the elements Cr, Cu, Cd, and Pb weredetermined with the ET-AAS, using the graphite furnace tech-nique. External calibration was carried out with aqueousnitric-acid single-element standards. The ranges were5...20 µg L–1 for Cu and Pb, 2.5...10 µg L–1 for Cr, and 0.5...2µg L–1 for Cd. Table 2 shows the wavelengths, spectral slitwidths, and matrix modifiers used.

Table 2: Standard operating conditions used for determiningof Cr, Cu, Cd, Pb, and Hg by atomic absorption spectrometry(AAS).

Standard-Messbedingungen für die Bestimmung von Cr, Cu,Cd, Pb und Hg mit der Atomabsorptionsspektrometrie (AAS).

Element Wavelength

nm

Spectralbandpass

nm

Matrix modifier

Cr 357.9 0.7 Mg(NO3)2

Cu 324.8 0.7 Pd(NO3)2/Mg(NO3)2

Cd 228.8 0.7 Mg(NO3)2/(NH4)2HPO4

Pb 283.3 0.7 Mg(NO3)2/(NH4)2HPO4

Hg 253.7 0.7 –

The Hg concentration was determined with the AAS by theflow injection method, using a combination of the cold vaporand amalgam techniques (FI-HG-AAS).The system was cali-brated against aqueous single-element standards in a rangeof 0.1...1 µg L–1.

The concentrations of the elements with the mass numbers52Cr, 55Mn, 63Cu, 66Zn, 111Cd, 75As, and 208Pb were determinedwith the ICP-MS in the peak-hopping mode using externalcalibration against nitric-acid aqueous multi-element stand-ards. The ranges were 10...100 µg L–1 for the elements 55Mnand 66Zn, 2...20 µg L–1 for 52Cr, 63Cu and 208Pb, and 1...10 µgL–1 for 111Cd. The mass numbers of all elements were select-ed for minimum isobaric, spectral, and matrix-dependent in-terference. Background interference was minimized bychoosing nitric acid as the main component of the digestingsolutions and using a high dilution factor [21–23]. 103Rh wasused as an internal standard.Table 3 shows the operating pa-rameters for the ICP-MS.

Table 3: ICP-MS operating conditions.

ICP-MS-Messbedingungen.

Nebulizer cross-flowCarrier gas argonRf frequency 35 MHzRf power 1100 W

Gas flow ratesOuter 12 L min–1

Nebulizer 0.93 L min–1

Auxiliary 0.8 L min–1

Sample uptake 1 mL min–1

Dwell time 25 msMeasurement time 1000 ms

TXRF was used to determine the elements in 96% ethanol,which was used to conserve the samples for transport fromthe field into the lab. 20...50 µL was transferred onto quartzglass slides by the aid of a pipette and dried.The sample wasexcited with MoKα radiation for 500 s and 1000 s at 50 kV and38 mA.To quantify the elements, yttrium was used as an inter-nal standard at a concentration of 1 µg L–1.

To verify the results obtained by the various detection meth-ods, the international water standard Nist 1643c was meas-ured on a parallel basis in all analyses.This material does notcontain the element Hg.

4 Reagents

HNO3 was purified to sub-boiled grade in a retort. H2O2 (30%)was of suprapure grade. All other reagents used were of ana-lytical-reagent grade (Merck, Darmstadt, Germany). Stand-ards were prepared and stabilized by dilution of 1 g L–1 singleelement and multi-element stock solutions (Merck) in 1%HNO3 with ultrapure water. Commercially available 10 g L–1

magnesium solution (Mg(NO3)2 · 6 H2O in 17% HNO3) and 10g L–1 palladium solution (Pd(NO3)2 in 15% HNO3) (Merck)were used as chemical modifiers. A stock solution of diammo-nium hydrogenphosphate, also employed as chemical modifi-er, was prepared by dissolution of 5 g L–1 (NH4)2HPO4 in 15%HNO3. Ultrapure water (18 MΩcm) was used throughout.

5 Calculations and statistical analysis

All analyses were accompanied by a blank control and thedetection limits were calculated for each analytical method



Table 5: Results obtained for the analysis of certified reference material NRC Dorm-1 with two different methods of digestion andtwo methods of determination (m = 2, n = 3; wcert ± 95% confidence limits).

Ergebnisse der Analyse des zertifizierten Referenzmaterials NRC Dorm-1 mit zwei verschiedenen Aufschlussverfahren und zweiBestimmungsmethoden (m = 2, n = 3; wcert ± 95% Vertrauensbereich).

AAS ICP-MSMicrowave

ovenHigh-pressure

asherMicrowave

ovenHigh-pressure

asherwcert w s w s w s w s

Element µg g–1 µg g–1 µg g–1 µg g–1 µg g–1

Hg 0.798 ± 0.074 0.740 0.03 0.76 0.04Cr 3.6 ± 0.4 3.0 0.2 3.5 0.2 3.39 0.08 3.5 0.2Cu 5.22 ± 0.33 4.30 0.08 4.3 0.2 4.41 0.01 4.30 0.19Cd 0.086 ± 0.012 0.082 0.001 0.092 0.004 0.10 0.02 0.089 0.005Pb 0.40 ± 0.12 0.32 0.02 0.40 0.15 0.340 0.010 0.40 0.15Mn 1.32 ± 0.26 1.070 0.010 1.4 0.3Zn 21.3 ± 1.0 15.3 0.4 18.2 0.7As 17.7 ± 2.1 15.1 0.1 15.0 0.7

Table 4: Results for determination of the limit of detection foreach element with AAS (using a 20 µL sample loop) and ICP-MS.

Ergebnisse für die Nachweisgrenzen der einzelnen Elementemit AAS (mit 20-µL-Probenschleife) und ICP-MS.

Limit of detection µg L–1

AAS ICP-MS

Hg 0.012Cr 0.20 52Cr 0.12Cu 0.53 63Cu 0.24Cd 0.03 111Cd 0.02Pb 0.61 206Pb 0.05

55Mn 0.0266Zn 1.4575As 0.05

and each element. The blank value method in accordancewith the German Standard Procedure DIN 32645 was usedfor this purpose [24]. The limits of detection were calculatedfrom m = 5 blank values from the digesting solutions. Table 4shows the detection limits when the microwave system CEM2000 and a digesting mixture of 4.5 mL 65% HNO3 and0.5 mL 30% H2O2 were used.

Two random samples (A and B) were taken from each weightclass of the homogenized sample material of Erpobdella

octoculata, digested, and the element concentrations deter-mined in three measurements (n = 3) by the relevant analyti-cal method. In all parametric tests the metal concentrationswere checked for normal distribution by the Kolmogorov-Smirnov test. The homogeneity of the sample material wasverified using the two-tailed unpaired t-test with the elementconcentrations ascertained from the subgroups A and B. Theconcentrations of the elements Cr, Cu, Cd and Pb in Erpob-della octoculata obtained by ET-AAS and ICP-MS were alsochecked for comparability using the two-tailed unpaired t-test[25–28]. Assuming that the sample material was homogene-ous, the total variance was calculated from the variance ofcomparison and the variance of reproducibility of the two ran-dom samples (A and B) [25].

The results given in this paper are rounded, taking the stand-ard deviation into account [29]. Both the interval and the placeof rounding were determined with the aid of the standard de-viation, so that both the result and the error have the samenumber of decimal places.

6 Results and discussion

6.1 Certified reference material

To check for systematic errors the steps of sample prepara-tion and two different digestion processes were carried outwith the certified reference material (CRM) NRC Dorm-1.Theresults for the digestion process in the HPA and the micro-wave oven, using both AAS and ICP-MS, are shown in



Table 6: Results of the independent repeated analysis of certified reference material NRC Dorm-1 used for the three sample prep-aration steps. Method of digestion: high-pressure asher using 2 mL HNO3. Method of determination: FI-HG-AAS for Hg and ICP-MS for other elements (m = 2, n = 3; wcert ± 95% confidence limits).

Ergebnisse der unabhängigen Wiederholungsmessungen des zertifizierten Referenzmaterials NRC Dorm-1 für drei Probenvor-bereitungsschritte. Aufschlussmethode: Hochdruckverascher mit 2 mL HNO3. Bestimmungsmethode: FI-HG-AAS für Hg und ICP-MS für die anderen Elemente (m = 2, n = 3; wcert ± 95% Vertrauensbereich).

Lyophilization Homogenization Storagewcert w s w s w s

Element µg g–1 µg g–1 µg g–1 µg g–1

Hg 0.798 ± 0.074 0.810 0.014 0.77 0.03 0.798 0.004Cr 3.6 ± 0.4 4.5 1.4 3.6 0.3 3.70 0.10Cu 5.22 ± 0.33 4.85 0.15 5.1 0.3 4.85 0.08Cd 0.086 ± 0.012 0.090 0.006 0.092 0.005 0.100 0.016Pb 0.40 ± 0.12 0.5 0.2 0.66 0.08 0.45 0.13Mn 1.32 ± 0.26 1.35 0.13 1.37 0.06 1.46 0.09Zn 21.3 ± 1.0 18.3 0.7 20.0 1.2 19.0 1.6As 17.7 ± 2.1 16.40 0.10 15.30 0.10 16.20 0.10

Table 7: Results of the repeated analysis of the certified reference materials Dorm-1 and Nist 1643c. Method of digestion: micro-wave oven. Method of determination: AAS and ICP-MS (m = 5, n = 3; wcert ± 95% confidence limits).

Ergebnisse der Wiederholungsmessungen des zertifizierten Referenzmaterials Dorm-1 und Nist 1643c. Aufschlussmethode:Mikrowellenofen. Bestimmungsmethode: AAS und ICP-MS (m = 5, n = 3; wcert ± 95% Vertrauensbereich).

Dorm-1 Nist 1643cAAS ICP-MS AAS ICP-MS

wcert w s w s wcert w s w sElement µg g–1 µg g–1 µg g–1 µg g–1 µg g–1 µg g–1

Hg 0.798 ± 0.074 0.81 0.07Cr 3.6 ± 0.4 3.3 0.2 3.4 0.58 19 ± 0.6 19.5 0.5 19 3Cu 5.22 ± 0.33 5.00 0.19 4.70 0.16 22.3 ± 2.8 19.9 0.2 20.7 0.6Cd 0.086 ± 0.012 0.084 0.003 0.08 0.03 12.2 ± 1.1 12.1 0.5 12.2 0.3Pb 0.4 ± 0.12 0.5 0.1 0.5 0.1 35.3 ± 0.9 34.0 1.2 36.0 1.8Mn 1.32 ± 0.26 1.2 0.2 35.1 ± 2.2 37 3Zn 21.3 ± 1 19 3 73.9 ± 0.9 73 2As 17.7 ± 2.1 14.7 0.4 82.1 ± 1.2 78.5 1.2

Table 5. Each step was checked twice (m = 2), and the analy-sis was carried out three times (n = 3).

With both digestion methods it was possible to separate theelements of the reference materials from the matrix sufficient-ly while maintaining clear solutions. After digestion in the HPAa barely visible cloudiness occurred when the solution was di-luted; this precipitated by time and had no negative influenceon the results. It was possible to achieve good percentage re-covery for all the relevant elements by both methods (Table5). Thus, both digestion methods can be used successful

for the analysis of leeches; the choice of one technique or theother may depend on criteria as throughput of samples or thefunds available.

Table 6 shows the element concentrations of the referencematerial Dorm-1 after lyophilization, homogenization, andstorage using the HPA digestion method and ICP-MS for de-tection. The three sample preparation steps were checkedseparately twice (m = 2). Most results lie in the certified range.There are only small deviations for the elements Pb and Asduring homogenization and for Zn during lyophilization. Sys-



tematic errors are therefore not to be expected during samplepreparation.

We chose the microwave system for our further work as it iseasier to use and allows a greater throughput of samples withadequate destruction of the organic matrix.The element con-centrations of the reference material Dorm-1 were finally de-termined after five independent digestion processes in themicrowave system. The CRM Nist 1643c was measured fivetimes both in the ET-AAS and ICP-MS on a parallel basis toverify the analytical methods. The results are shown in Table7. Good results close to the certified values were achieved forall elements.

6.2 The freshwater leech

When aquatic organisms are used as bioindicators, no stepsshould be taken that might lead to contamination with or lossof metals after collection. For this reason the leeches wereconserved in 96% ethanol for 24 h immediately after collec-tion. The specimens were not kept in a tank until the gut wascompletely empty, as this takes a long time and is difficult tocheck [19]. Storage in a tank may also lead to a loss of metalsfrom the tissues as it has been described for other aquatic in-vertebrates [31]. On the other hand, it must be expected thatthe content of the gut may increase the measured concentra-tion when the leeches are NOT kept in a tank until the gut isempty [30–32].

96% ethanol is an excellent medium for random analyses ofthe content of the gut, as the dermal muscular tunic of theleech hardens and is easy to dissect. To provide informationon possible losses of the relevant elements during conserva-tion, the ethanol was analyzed before and after conservationof the biological material. After measuring the concentrationsof the various elements in the 96% ethanol, the results wereconverted to µg g–1 of the dry weight of the biological materialconserved. The results were given a minus sign if contentsdecreased in relation to the biological material. Figure 1shows the result for the elements Cr, Mn, Cu, Zn, As, and Pb(concerning weight class I). The losses that occurred mayhave been simulated by inputs of suspended material adher-ing to the leeches. In sum it may be said that when comparedwith the element concentrations in the leeches the losses ofall the elements except manganese were small and could beignored.

Table 8 shows the element concentrations (mean) and stand-ard deviations (sdv) for subsamples A and B of each weightclass of the homogenized leech material using the AAS andICP-MS methods of detection.The element concentrations ofboth subsamples were tested for comparability using the two-tailed unpaired t-test with n = 3 and α = 0.05. For this purposethe digestion and analyses of the two subsamples were car-

Fig. 1: Trace element values found in the 96% ethanol afterconservation of the leech Erpobdella octoculata.The elementvalues were normalized per g dry weight of the biological ma-terial. (–): Decrease of elements from the leech concerningweight class I.

Spurenelementwerte in 96%igem Ethanol nach Konser-vierung des Egels Erpobdella octoculata. Die Elementgehaltewurden auf das Trockengewicht des biologischen Materialsbezogen. (–) Vorzeichen: Abnahme des Elementes im Egelbezogen auf die Gewichtsklasse I.

ried out on the same day, using the same evaluation method.Table 8 also shows the difference between the mean values(m. diff.), the limits of the t-distribution (t-value), the signifi-cance level for accepting or rejecting the null hypothesis (p-value) and the probability level of the alternative hypothesis(Power). If all the elements and weight classes are consid-ered without differentiation for both detection methods, thenull hypothesis (meanA = meanB) cannot be rejected in about50% of the cases at the α = 0.05 level. With the statisticallysignificant differences (p-value > α; α = 0.05) the probabilitylevel (Power > 0.8) is not in all cases high enough to assumethe alternative hypothesis (meanA meanB) with certainty. Innearly all cases the differences between the two groups areless than one order of magnitude for all the elements. But theyare apparent at a very low n (n = 3) and therefore constitute apowerful statement that cannot simply be ignored. On the oth-er hand the non-significant differences have to be interpretedwith great caution as they constitute a very weak statement ata low n (n = 3).The statistically significant differences are nei-ther weight-class specific nor element-specific. The concen-trations measured are not all greater or smaller in one sub-group. The differences that occur are probably due to a com-bination of small, random errors in the analytical method.

In the case of Cr there is a noticeable difference between thedeterminations with the AAS and those with the ICP-MS. The



Table 8: Two-tailed unpaired t-test for the results of subsamples A and B for each element and each weight class of the leech Er-pobdella octoculata. Microwave oven, n = 3, α = 0.05. p-value: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Mass fraction(mean) and standard deviation (sdv) are given in µg g–1.

Zweiseitiger t-Test für die Ergebnisse der Unterproben A und B für jedes Element und jede Gewichtsklasse (weight class) desEgels Erpobdella octoculata. Mikrowellenofen, n = 3, α = 0.05. p-value: ns, nicht signifikant; * p < 0.05; ** p < 0.01; *** p < 0.001.Massenanteil (mean) und Standardabweichung (sdv) in µg g–1.

AASSubsample A Subsample B

Element weightclass

mean sdv mean sdv m. diff t-value p-value Power

Hg I 1.16 0.04 1.20 0.01 0.04 2.10 nsII 1.23 0.01 1.27 0.02 0.04 3.10 * <

III 1.42 0.03 1.50 0.02 0.08 3.84 * <Cr I 0.74 0.04 0.65 0.03 0.09 3.12 * <

II 2.25 0.18 0.93 0.04 1.32 12.3 *** >III 0.45 0.01 0.48 0.03 0.03 1.09 ns

Cu I 41 3.3 38.4 0.93 2.6 1.45 nsII 64 2.2 65.00 0.04 1 0.87 ns

III 348 3 370 3 22 7.87 ** >Cd I 0.30 0.01 0.25 0.01 0.05 4.90 ** >

II 0.24 0.01 0.25 0.01 0.01 1.23 nsIII 0.21 0.05 0.22 0.04 0.01 1.73 ns

Pb I 6.2 0.3 6.60 0.13 0.4 2.19 nsII 6.60 0.04 6.88 0.09 0.28 4.92 ** >

III 6.50 0.18 6.40 0.16 0.1 0.72 ns

ICP-MSSubsample A Subsample B

Element weightclass

mean sdv mean sdv m. diff t-value p-value Power

Cr I 2.10 0.16 2.10 0.11 0 0 nsII 3.0 0.4 2.45 0.07 0.55 1.94 ns

III 2.35 0.08 2.40 0.1 0.05 0.68 nsCu I 40.0 1.9 42.2 0.6 2.2 1.90 ns

II 64.0 0.7 75.0 0.2 11 26.8 *** >III 356 2 374 3 18 8.55 ** >

Cd I 0.28 0.02 0.24 0.04 0.04 1.55 nsII 0.30 0.02 0.35 0.02 0.05 3.06 * <

III 0.30 0.07 0.29 0.04 0.01 0.21 nsPb I 7.2 0.3 7.9 0.3 0.7 3.42 * <

II 7.80 0.12 8.4 0.2 0.6 4.93 ** >III 6.80 0.15 7.2 0.4 0.4 2.09 ns

Mn I 49.3 0.41 51.3 0.6 2 4.51 * >II 51.1 0.69 53.5 0.7 2.4 4.20 * >

III 47.8 0.43 49.5 1.0 1.7 2.48 nsZn I 930 17 975 15 45 3.74 * <

II 1070 2 1130 14 60 7.51 ** >III 1086 9 1133 9 47 6.64 ** >

As I 6.6 0.7 7.7 0.7 1.1 1.76 nsII 7.1 0.9 7.4 0.3 0.3 0.55 ns

III 4.7 0.5 4.4 0.3 0.3 1.21 ns



Table 9:Two-tailed unpaired t-test for the results of AAS and ICP-MS determinations for the elements Cr, Cu, Cd, and Pb and eachweight class of the leech, Erpobdella octoculata. The mean value (mean) and standard deviation (sdv) were calculated from sub-samples A and B. Method of digestion: microwave oven, n = 6, α = 0.05. p-value: ns, not significant; * p < 0.05; ** p < 0.01;*** p < 0.001; mean mass fraction (mean) and standard deviation (sdv) are given in µg g–1.

Zweiseitiger t-Test für die Ergebnisse der AAS- und ICP-MS-Bestimmungen der Elemente Cr, Cu, Cd und Pb und jeder Gewichts-klasse des Egels Erpobdella octoculata. Mittelwert (mean) und Standardabweichung (sdv) wurden aus den Unterproben A und B.berechnet. Aufschlussmethode: Mikrowellenofen, n = 6, α = 0.05. p-value: ns, nicht signifikant; * p < 0.05; ** p < 0.01; *** p < 0.001;Mittelwert (mean) und Standardabweichung (sdv) in µg g–1.

AAS ICP-MSElement weight

classmean sdv mean sdv m. diff t-value p-value Power

Cr I 0.69 0.06 2.1 0 1.41 54.1 *** >II 1.6 0.9 2.7 0.4 1.3 2.76 * <

III 0.47 0.01 2.37 0.03 1.9 122 *** >Cu I 40 2 41.0 1.5 1 1.19 ns

II 64.4 0.8 69 8 4.6 1.58 nsIII 360 16 365 13 5 0.73 ns

Cd I 0.27 0.03 0.26 0.03 0.01 0.61 nsII 0.245 0.007 0.32 0.03 0.075 5.43 ** >

III 0.215 0.007 0.295 0.007 0.08 19.6 *** >Pb I 6.4 0.3 7.5 0.5 1.1 4.71 ** >

II 6.75 0.19 8.1 0.4 1.35 6.69 *** >III 6.43 0.07 7.0 0.3 0.57 4.14 ** >

analyses with the ICP-MS show higher concentrations. To asmall extent this is also true for the elements Cu, Cd, and Pb.Table 9 summarizes the significance of the differences be-tween the measurements obtained by the two detectionmethods for one sample. Again, the two-tailed unpaired t-testwas used.To make the result of the test clearer, the measure-ments from subsamples A and B were averaged and a totalvariance was calculated according to [25].The boundary con-ditions were set at n = 6 with α = 0.05.The parameters used inTable 9 correspond to the conditions used in Table 8. For Cuthe null hypothesis (meanAAS = meanICP-MS) cannot be reject-ed. Statistically significant differences between the two detec-tion methods were found for all the other elements except Cdin weight class I. The alternative hypothesis (meanAAS

meanICP-MS) can be supported for all cases at a power of over0.8. The power of less than 0.8 for weight class II in the caseof Cr is a result of averaging two dissimilar values from sub-samples A and B.When the measurements with subsample Awere repeated and two similar values from subsamples A andB were averaged, a power of over 0.8 was achieved (notshown). The higher chromium levels resulting from the ICP-MS analysis may have been caused by interference from mo-lecular ions such as 40Ar12C or 36Ar16O close to the detectionlimit, since detection was carried out at a mass number that isvery insensitive to the element [22, 23]. Raised levels werealso found when the international water standard Nist 1643c

was diluted down close to the detection limit. It is not easy tointerpret the different values for the elements Cd and Pb ob-tained by ICP-MS and AAS, since background, isobaric, mo-lecular-ion, and matrix interferences do not occur with theICP-MS in this mass number range, nor is it usual for in-creased concentrations to be measured. On the other handthe reference materials were recovered when the AAS wasused within the range of the certified values.

To summarize, we may say that with certain reservations thetwo methods produce similar results. To determine the ele-ment Cr correctly by the ICP-MS method, the weights of thesamples must be increased. Taking the certified referencematerial NRC Dorm-1 as a standard, the chromium concen-trations were recovered within the certified range by both de-tection methods when the weights were higher.

7 Conclusions

With the certified reference material NRC Dorm-1 a reliablemethod for determination of element concentrations in biolog-ical tissue has been developed. The analytical process alsoworked satisfactory for samples of the freshwater leech Er-pobdella octoculata.



If only few individual specimens of the leech are available,these should be grouped together and not divided into sever-al weight classes, because this is the only reliable way to de-termine the concentrations of all the relevant elements byICP-MS. Homogenization of the sample material probablyhas to be improved to achieve smaller deviations betweenrandom subsamples. A comparison of the two detectionmethods AAS and ICP-MS shows similar results for the ele-ments concerned, with the exception of chromium.

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[Received: 27 November 2002; accepted: 12 May 2003]

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Documents

Determination of Trace Metals in the Freshwater Leech Erpobdella octoculata of the Elbe River — Evaluation of the Analytical Protocol