a method for trace element determination of single daphnia specimens using total reflection x-ray...

Ž .Spectrochimica Acta Part B 56 2001 2209�2217

A method for trace element determination of singleDaphnia specimens using total reflection X-ray

fluorescence spectrometry �

Margarete Magesa,�, Stefan Woelflb, Wolf v. Tumpling juna¨aUFZ Center for En�ironmental Research Leipzig-Halle, Department of Inland Water Research Magdeburg, Bruckstrasse 3a,¨

D-39114 Magdeburg, GermanybUni�ersidad Austral de Chile, Instituto de Zoologia, Casilla 567, Valdi�ia, Chile

Received 30 January 2001; accepted 23 May 2001

Abstract

Ž .Two new preparation techniques for total-reflection X-ray fluorescence TXRF element determination of singleŽ �1.freshwater crustacean specimens dry weight: 3�40 �g ind have been developed and tested using Daphnia pulex

from a deep, oligotrophic freshwater lake located in southern Chile. Dry method: Specimens were washed with 0.2�m filtered lake water and frozen in liquid nitrogen. The freeze-dried Daphnia specimens were weighed using anultra-fine microbalance and placed on quartz glass carriers for TXRF analysis. Wet method: Specimens were washedwith 0.2 �m filtered lake water and placed on quartz glass carriers for TXRF analysis and dried in air. The dry weightwas determined using the previously established body length�dry weight relationship. Method validation for both thedry and the wet preparation method in combination with TXRF spectrometry for the element determination in smallsingle freshwater crustaceans showed that both methods can be used for routine investigations. There were nosignificant differences between the dry and the wet methods concerning the elements Ca, K, Fe, Zn, Br, P, Cu, butthe determination of Mn, S and Sr revealed significant differences between the two methods. It seems that the drymethod yields more precise results, but the wet method is easier to handle in the field when samples cannot be fixedwith liquid nitrogen. � 2001 Elsevier Science B.V. All rights reserved.

Keywords: Trace element analysis; Daphnia; Total reflection X-ray fluorescence; Bioaccumulation; Contamination

� This paper was presented at the 8th Conference on Total Reflection X-Ray Fluorescence Analysis and Related Methods,Vienna, Austria, September 2000, and is published in the Special Issue of Spectrochimica Acta Part B, dedicated to that conference.

� Corresponding author. Fax: �49-391-8109305.Ž .E-mail address: mages@gm.ufz.de M. Mages .

0584-8547�01�$ - see front matter � 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S 0 5 8 4 - 8 5 4 7 0 1 0 0 3 2 5 - 1

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�22172210

1. Introduction

Element analyses of zooplankton is importantfor investigating the uptake and the transfer ofsubstances along the trophic chain in order todefine the role of these organisms in bio-

� �geochemical pathways 1,2 . Heavy metals as toxicsubstances along the trophic chain are importantparameters for understanding the main processesin aquatic systems. Ecotoxicological tests of con-taminants often involve the microcrustacean

� �Daphnia as a bioindicator 3 , because it is verycommon and important in freshwater ecosystemsthe world over. In this way element determinationin Daphnia provides information on the bioavail-ability of elements in freshwater, which is impor-tant for the characterization of the water quality� �4 .

To determine heavy metal accumulation inaquatic biota, ICP-MS, ICP OES and AAS are

� �commonly applied 4,5 . The use of these analyti-cal techniques normally requires larger quantities

Žof biological sample material often more than 0.5.mg . Biotest experiments of microcrustaceans like

Žsingle Daphnia specimens individual dry weight.�100 �g are therefore hardly possible using

standard procedures. Moreover, element analysesof natural zooplankton populations are often doneon unspecified size-fractionated plankton sampleswithout distinguishing between species, size or

� �larval stage 5 . This is due to the lack of asuitable quick and economical method for multi-element trace analysis for single microcrus-taceans.

Total reflection X-ray fluorescence analysisŽ .TXRF is a relatively economical micro-analyti-cal method allowing simultaneous multielementanalyses with detection limits in the low picogram

� �range 6 . It is a suitable method to analyze verysmall amounts of homogeneous sample material

� �in the pico to microgram range 6,7 . This appliesalso to element determination in biological mate-rial, but because of the inhomogeneous elementdistribution within a single species and the dif-ficulty of handling live material, special prepara-tion procedures are required.

Thus the development of two preparation tech-niques � the dry and the wet method � for

single freshwater microcrustaceans was an analyt-ical challenge and necessity. The elaboration ofthese two techniques is described in the article.

2. Materials and methods

2.1. Sampling sites and sampling

Ž .Daphnia pulex body size: 0.5�2.5 mm wassampled from the North Patagonian Lake Rinihue˜Ž .southern Chile . Lake Rinihue is a typical, deep˜

Ž �1 .softwater lake conductivity 25 �S cm with low� �nutrient content and low productivity 8 . Sam-

pling was done during summer 2000 using aŽmetal-free zooplankton net aperture: 20 cm; net

.size: 200 �m . The zooplankton samples werestored at 4�C, transported as soon as possible tothe laboratory and prepared according to the dryand wet procedures given in Fig. 1.

2.2. Dry preparation

After collection, Daphnia specimens werewashed three times in 0.2-�m pre-filtered lake

Ž .water, put into small Teflon bottles 7-ml volumeand frozen in liquid nitrogen. After freeze drying,individual Daphnia specimens were selected andmeasured to the nearest 0.04 mm in length under

Ž .a stereomicroscope ZEISS, Stemi11 at 40�magnification. The dry weight was determined

Žusing an ultrafine balance Sartorius Ultramicro.S4, sensitivity 0.1 �g and corrected for humidity

� �9,10 :

Ž . Ž .DW �DW � 1�AH �CF 10 x x

where DW is the dry weight of Daphnia extrap-0olated to 0% air humidity, DW is the measuredxweight, AH is the relative atmospheric humidityxat the time of weighing, and CF is an empirical

Ž .correction factor 0.00293�0.00013 . The humid-ity varied between 33.1 and 44.3%.

ŽAfter weighing, single Daphnia specimens dry.wt.: 3�40 �g were put onto quartz glass carriers

together with 10 �l pure water and air dried.Finally, 5 ng gallium as internal standard wasadded and the individual Daphnia specimens were

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�2217 2211

Fig. 1. Overview of the wet and dry preparation methods developed for single Daphnia specimens.

digested with 10 �l HNO suprapur while the3quartz glass carriers were placed on a hot plateand dried once more. The element concentrationswere determined by TXRF. The final elementconcentration of the sample was corrected for theelement concentrations of the 10-�l pure waterand the 10-�l HNO suprapur previously added3to the sample on the glass carrier.

2.3. Wet preparation

Single Daphnia specimens were washed threetimes with 0.2-�m filtered lake water and putonto quartz glass carriers together with 10 �l0.2-�m filtered lake water. After air drying, thebody length was measured under a microscopeŽ .ZEISS Axioplan II at 25� magnification using

Ž .a video image analyzing system Kontron, ZEISS .The dry weight of Daphnia was calculated usingthe body length�dry weight relationship previ-ously established on the freeze dried specimens

Ž .and given in Eq. 2 :

Ž �1 . 2.5696DW �g ind �6.20�L0

2 Ž .r �0.91, N�130 2

where DW is the dry weight corrected to 0% air0humidity and L is the body length in mm. How-ever, comparisons of length measurements of thesame specimen after freeze drying and after wetpreparation revealed that the wet prepared speci-mens were 3% smaller then freeze-dried speci-mens. Therefore, the final corrected dry weightwas calculated as follows:

Ž . Ž . Ž .DW �g �DW �g � f 3cor . uncor .

where DW is the corrected dry weight in �g,cor.DW is the uncorrected, calculated dry weightuncor.

Ž .in �g and f is the correction factor 1.0816� .After the preparation of Daphnia on the carri-

ers, the further sample preparation follows theŽ .previously described dry method Fig. 1 .

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�22172212

2.4. Instrumentation

A total reflection X-ray fluorescence spec-Žtrometer Extra IIA Atomika Instruments Ltd.,

.Oberschleissheim�Munich, Germany was used.The spectrometer was equipped with an 80-mm2

Ž .Si Li detector having a resolution of 168 eV, Mo-and W-finefocus tubes operated at 50 kV and 38

ŽmA with attenuation filters 50 �m Mo and 1000.�m Al for Mo-excitation and a computer con-

trolled multichannel analyzer system combinedwith a spectrum deconvolution program. Only Moexcitation was used. The measurement time was500 s.

2.5. Method �alidation

A validation is required whenever an entirelynew method is developed. The following valida-tion procedures were performed to demonstratethe accuracy and precision of the dry and wetmethods.

In order to test the recovery rate of higherquantities of reference material prepared accord-ing to the classical method, 0.5�0.74 mg samplesof reference material CRM414 were weighed on aS4 Sartorius microbalance and put into PFA mi-

Ž .crovials 7 ml, Savillex, Canada . The correctionto dry mass was made according to Quevaviller et

� � Žal. 1 . After adding 500 �l HNO 65%, supra-3. �1 Ž .pur and 100 �g g Ga ICP-standard solution

as internal standard the sample was digested withŽa Mars 5 plus microwave CEM GmbH, Kamp-

.Lintfort, Germany . TXRF measurements weredone on triplicates of 10 �l of the digested refer-ence material.

For the determination of the recovery rate ofspiked reference material, it was spiked with a

Žmixed element standard K, Ca, Cr, Mn, Fe, Ni,.Cu, Zn, Sr and Pb in addition to the gallium

standard and measured by TXRF.

2.5.1. Determination of the reco�ery rate and( )precision of small amounts 10�50 � g of unspiked

and spiked reference material on glass carrierThe accuracy of the measurements was checked

by parallel measurements of certified zooplankton� �material CRM 414 1,11 . To test the direct mi-

crodigestion on the quartz carriers, 12.3�51.9 �gof the reference material CRM 414 was weighed

Žon an ultra fine microbalance Sartorius Ultrami-.cro S4, sensitivity 0.1 �g , transferred to the quartz

carriers, fixed with 10 �l ultrapure water andfinally air dried. Analogous to the dry method,the reference material was digested with 10 �l

Ž .HNO 65%, suprapur and 5 ng gallium as inter-3nal standard and measured by TXRF.

For the determination of the recovery rate ofspiked reference material it was spiked; a mixedelement standard was also spiked to the referencematerial and measured by TXRF.

2.5.2. Determination of the reco�ery rate of spikedDaphnia prepared according to the dry method

In order to test the efficiency of the directmicrodigestion of Daphnia prepared on the glass

Žcarrier, single Daphnia specimens 14.1�33.8 �g.dry wt., n�10 were prepared according to the

dry method and measured by TXRF. Afterwards,the same Daphnia specimens were spiked with anelement mix of K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Srand Pb and analyzed again by TXRF. The recov-ery rate was determined by resting the unspikedDaphnia from the spiked Daphnia.

2.5.3. Pro�ing the non-significance of the differencebetween the dry and wet methods

� �According to Doerffel 12 , the significance�non-significance of the difference between twoindependent normally distributed series of analy-ses x ...x and y ...y can be shown using the1 m 1 m‘extended t-test’. This test is based on the hy-pothesis that both series originate from the samepopulation. Following this hypothesis the differ-ences d � y � x deviate non-systematicallyj j jaround zero. The extended t-test therefore provesthat the averaged difference originates from thepopulation � �0. The significance of the dif-Dference between the two series is proved if t�t . Otherwise both series originate from theŽP,f.same population and the difference between the

Ž .series methods in our case is not significant. Allthe statistical analyses were done using the SAS-statistical package and Excel.

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�2217 2213

3. Results and discussion

3.1. Reco�ery rate of standard reference materialCRM414

The TXRF analyses were validated by mea-surements on 517�739-�g samples of certifiedreference material of plankton CRM 414 contain-

� �ing �97% cladoceran Daphnia magna 10 . Therecovery rates of the investigated elements were

Ž .between 83 and 126% Table 1 . The relativestandard deviation of the replicates was �9%Ž .exception Br: 16% which is similar or betterthan other authors found in comparable studies

� �on biological material, e.g. biofilm 7,13 . Ourresults confirm the statement of Quevauviller et

� �al. 1 who found that 0.5 mg reference materialCRM414 was sufficiently homogenous for ele-ment analysis.

To realize the method validation in a rangecomparable to the dry weight of single Daphniaspecimens, a second series of 13.5�57.2 �g sam-ples of reference material CRM 414 was prepareddirectly on the glass carriers. As shown in Table

1, the recovery rates for these samples are similarto the larger 0.52�0.74-mg samples which wereprepared according to the classical method. Thesevalues are reasonably good, varying between 86and 118% for all elements with the exception of

Ž .Cr 194% . Also, the relative standard deviationof the replicates was lower than 20%, an accept-able value.

The spiking experiments with small amounts ofreference material CRM414 on glass carriers

Ž .showed similar recovery rates 87�141%, Table 2Ž .in comparison to the unspiked samples Table 1 .

This confirms that the direct digestion of thereference material on the glass carriers efficientlydestroyed the organo metal bonding, allowinggood TXRF analysis of the elements.

3.2. Reco�ery rate of spiked Daphnia

The next step was to test whether the directdigestion method also yielded acceptable recoveryrates with Daphnia. Indeed, the results shown inTable 3 reveal that the recovery rates of spikedDaphnia are very similar to those of the spiked

Table 1ŽTXRF analysis of reference material CRM414 European Community Bureau reference material 414, trace elements in plankton.with �98% of the cladoceran Daphnia magna

Element Reference values of BCR 414 TXRF: 12.3�51.9 �g samples TXRF: 517�739 �g samples

Mean conc. S.D. Average conc. Average dev. R Mean conc. S.D. Rr r�1 �1 �1 �1�1 �1Ž Ž . Ž . Ž . Ž . Ž . Ž . Ž .�g g �g g �g g �g g % �g g �g g %

cS 6800 200 6051 1077 89 6391 161 94b,cK 7550 170 6813 989 90 6533 329 87cCa 65 000 200 62 858 8360 97 60 507 3446 93

aCr 23.8 1.2 46 21.2 194 24 2 99aMn 299 12 257 34.1 86 277 19 93bFe 1850 190 1884 407 102 1958 5 106

aNi 18.8 0.8 22 4.4 118 16 3 83aCu 29.5 1.3 28 3.6 95 27 2 92

aZn 112 3 116 18.2 104 116 4 103cRb 11.6 0.2 14 2.0 117 15 1 126

bSr 261 25 218 26.7 84 253 17 97

Ž . Ž .The TXRF values are from samples 12.3�51.9 �g n�6 and 517�739 �g n�2 . R �recovery rate �ratio between measuredrand referenced concentrations.

a Certified.b Indicative.c Informative value.

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�22172214

Table 2Ž . Ž .TXRF analysis of 20�60 �g reference material CRM414 n�9 European Community Bureau reference material 414 which was

spiked with an element mix of K, Ca, Cr, Mn, Fe, Ni, Cu, Zn and Sr

Element Reference values Spike mix Spiked reference materialof CRM 414 20�60 �g samples

Mean conc. S.D. Abs. elem. Average conc. Average conc. Average dev. Rr�1 �1 �1�1 �1Ž . Ž . Ž . Ž . Ž . Ž . Ž .�g g �g g content ng �g g �g g �g g %

b,cK 7550 170 26 1036 7828 766 104cCa 65 000 200 108 4296 65 355 5003 101

aCr 23.8 1.2 0.6 22 34 10.5 141aMn 299 12 2.6 103 261 29.1 87bFe 1850 190 9.7 386 1926 401 104

aNi 18.8 0.8 0.25 10 22 4.6 119aCu 29.5 1.3 0.22 9 31 6.4 106

aZn 112 3 0.22 112 111 16.4 99bSr 261 25 2.4 94 224 23 86

Ž . Ž .Spike values are given 1 as absolute element concentration in ng added per sample; and 2 as average concentration persample in �g g�1 dry wt. R �recovery rate �ratio between measured�spiked value and referenced concentrations.r

a Certified.b Indicative.c Informative value.

reference material CRM 414. They are in the� �acceptable range of biological materials 7 . Thus

we conclude that the direct microdigestionmethod is suitable for the preparation of Daphniaon glass carriers, an important prerequisite forthe application of the wet and the dry methods.

3.3. Dry �s. wet methods

The two preparation techniques, the dry andwet method, were developed for the preparationof single microcrustaceans like Daphnia in orderto improve the classical preparation method whichuses 10�100� times more biological material forelement analyses compared to the wet and drymethods. The wet method is designed for fieldstudies where liquid nitrogen is difficult or impos-sible to handle and where dry weight�body length

� �relationships are already available 14,15 . In con-trast, the dry method was developed as a moredirect, quantitative method for element analysesof microcrustaceans when all necessary labora-tory facilities are available.

Our experiences confirm that the wet method iseasy to handle in the field. But care should betaken in handling live Daphnia because physio-logical stress and, as a consequence, leaching

Ž .processes of certain elements e.g. P, K, S must� �be kept as low as possible 16 . Instead of pure

water we therefore used prefiltered lake water forall washing steps.

In comparison to the wet method, the drymethod was much more time consuming becauseof the freeze drying and the weighing steps. Atotal of 2�3 days were needed to prepare andmeasure 50�100 specimens by the dry method.

The element concentrations as measured bythe wet and the dry methods are given in Fig. 2showing the results in the range of 3�40 �g drywt. We did not calculate arithmetic means, be-cause it is not clear how the element content isrelated to the dry weight. Linear relationships, as

� �found for example for Ca 17 , seem to exist formost of the investigated elements, but this shouldbe tested separately. The results of the extendedt-test show that for most of the investigated ele-ments there are no significant differences between

Ž .the dry and the wet methods Table 4 . Significantdifferences were only obtained for S, Mn and Sr.In part, this might be related to a higher elementbackground of the wet method compared to thedry method: The specimens were prepared with10 �l lake water, instead of pure water as in thedry method. However, this background was com-

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�2217 2215

Table 3Ž . Ž .TXRF analysis of single D. pulex from Lake Rinihue Chile 14.1�33.8 �g samples, n�10 which was spiked with an element mix˜

of K, Ca, Mn, Fe, Ni, Cu, Zn, Sr and Pb

Element Spike mix Daphnia Spiked Daphnia-spike

Abs. element content Average conc. Average conc. Average conc. Rr�1 �1�1Ž . Ž . Ž . Ž . Ž .ng �g g �g g �g g %

K 26 1469 9195 9579 104Ca 108 6087 45 619 46 719 102Mn 2.6 145 63 70 111Fe 9.7 546 429 444 104Ni 0.25 14 7.7 6.1 80Cu 0.22 13 8.5 10.2 120Zn 0.22 159 120 130 108Sr 2.4 134 126 133 105Pb 0.28 16 6.7 7.3 109

Ž . Ž .Spike values are given 1 as average element concentration in ng added per sample; and 2 as mean concentration per sample�1 Ž .in �g g dry wt. R �recovery rate �ratio between spiked Daphnia-spike �unspiked Daphnia.r

Table 4Comparison of the wet and the dry method for element determination in single Daphnia specimens: results of the ‘extended t-test’

Element t t t Difference between methods95,25 99,25

P 1.34 2.06 2.79 Non-significantS 2.30 2.06 2.79 SignificantK 0.91 2.06 2.79 Non-significantCa 1.77 2.06 2.79 Non-significantMn 3.03 2.06 2.79 SignificantFe 1.34 2.06 2.79 Non-significantCu 1.10 2.06 2.79 Non-significantZn 1.57 2.06 2.79 Non-significantBr 0.27 2.06 2.79 Non-significantSr 2.38 2.06 2.79 Significant

Table 5Ž .Element concentrations of HNO suprapur, pure water and 0.2 �m filtered lake water washing water as measured by TXRF3

Ž .mean�1� S.D., n�5

Ž .Element HNO suprapure H O pure Lake Rinihue Chile˜3 2�1 �1 �1Ž . Ž . Ž .�g l �g l �g l

K 29.8�3.6 �6.96 867�69Ca 66.8�6.6 13.2�2.34 4974�365Cr �0.9 �2.0 �4.78Mn �1 n.d. �4.96Fe 4.8�0.5 0.93�0.38 9.17�1.19Ni 3.7�0.3 �0.41 �1.78Cu �1.3 1.13�0.18 �2.3Zn �2 1.42�0.21 5.65�1.21Br �0.7 �0.25 4.14�1.02Rb n.d. �0.14 2.67�0.60Sr �0.9� �0.21 18.96�1.59Pb �1.2 �0.64 �2.51

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�22172216

Ž . Ž .Fig. 2. Element content of Daphnia pulex from Lake Rinihue Chile prepared according to the wet method black squares and the˜Ž .dry method white squares as measured by TXRF.

( )M. Mages et al. � Spectrochimica Acta Part B: Atomic Spectroscopy 56 2001 2209�2217 2217

parably low in the range of �5% of the absoluteelement content of Daphnia, although the lakewater had much higher element concentrations

Ž .compared to pure water Table 5 . The crucialpoint of the wet method is the indirect determi-nation of the dry weight using a previously es-tablished relationship between body length anddry weight. Even if the body length�dry weightrelationship is obtained from the same microcrus-tacean population, relatively small errors in lengthmeasurement can cause considerable differences

Ž .in dry weight estimations see Section 2 . Al-though these errors are often only in the range of

� �1�2% as found by us and other authors 18 , thiscauses variations in dry weight and, as a conse-quence, in the element concentration of approxi-mately 8�9%. If body length�dry weight relation-

� �ship are taken from the literature 14,15 , one canexpect much higher errors. Thus, we recommendusing the wet method only for a first, quick ele-ment screening of microcrustaceans where confi-dent body length�dry weight relationships of thesame population are available. In all other casesthe dry method gives more precise results and istherefore preferable.

In cases where the detection limits of certaintrace elements such as Ni and Cr could not bemeasured satisfactorily by either method, the in-homogenities of the biological material on theglass carriers resulting in a high backgroundshould be reduced. In order to achieve this reduc-tion improvements of the dry method, e.g. the useof a cold plasma asher, will be tested in furtherstudies.

4. Conclusions

The dry method as a quantitative standardmethod is a good alternative to the classicalmethod for highly sensitive element analyses forcrustaceans and allows for the first time the mul-tielement detection of individual microscrus-tacean specimens. The wet method is more suit-able for qualitative field studies where shockfreezing techniques are not available or samplesare difficult to handle. Because the dry weightmust be calculated from known body length�dry

weight relationships the wet method yields lessexact information on the element content in sin-gle Daphnia specimens than the dry method. It istherefore preferable to use the dry method. Abetter optimization method might improve thedetection limits of some elements such as Ni andCr.

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