Elemental analysis of Dikerogammarus villosus samples for river water monitoring

Microchemical Journal 73 (2002) 99–111

Elemental analysis of Dikerogammarus villosus samples for river water monitoring ´ ´ a, N. Oertelb, E. Szabo´ a, E. Szurdokic, Gy. Zaray ´ a,*, M. Ovari ´ a,d K. Barkacs a

¨ ¨ University, P.O. Box 32, H-1518 Budapest 112, Department of Chemical Technology and Environmental Chemistry, L. Eotvos Hungary b ´ ¨ Hungary Hungarian Danube Research Station, Hungarian Academy of Sciences, Javorka S. 14, H-2131 God, c Botanical Department, Hungarian Natural History Museum, P.O. Box 222, H-1476 Budapest, Hungary d ¨ ¨ University, Research Group of Environmental and Macromolecular Chemistry, Hungarian Academy of Sciences, L. Eotvos P.O. Box 32, H-1518 Budapest 112, Hungary Received 27 February 2002; accepted 8 March 2002

Abstract Dikerogammarus villosus (amphipod crustacean) samples were collected from the River Danube. The applicability of artificial substrates (gravel and clay filled containers translocated at the bottom of the river) and the effect of the applied colonization periods of short (3 weeks) and long (up to 30 weeks) terms were tested. Ag, As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb and Zn concentrations of the amphipods were measured by inductively coupled plasma mass spectrometry (ICP-MS) after the digestion of some milligram samples. It was established that the artificial substrates did not influence the bioaccumulative processes of the elements investigated, and the short-term colonization period turned out to be sufficient for biomonitoring. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Dikerogammarus villosus; Biomonitoring; River water; Artificial substrates; Elemental analysis; ICP-MS

1. Introduction The goal of biological monitoring is to give reliable and proper information on the possible effects of chemicals present in the waters due to human activities, to enable the protection of the aquatic ecosystems and particularly to provide the required scientific guidance in legislation and enforcement w1x. Most of the monitoring efforts, *Corresponding author. Tel.: q36-1-209-0555; fax: q36-1209-0602. ´ E-mail address: [email protected] (K. Barkacs).

started in the 1960s, concerned chemical- and physical parameters. Monitoring programs gave sensitive indications of the concentration levels for substances studied, without essential biological information, while pollution implies deleterious effects, usually assessed in relation to a biological system w2,3x. Emphasis has been laid therefore also on monitoring biological variables on the basis of more reasons, such as: biological monitoring systems can be cheap; require less sophisticated instruments; and as a most important fact, reflect the integrated impact of pollution load.

0026-265X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 2 . 0 0 0 5 6 - 5

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Nevertheless, also chemical monitoring must be simultaneously carried out to reveal causative factors. The determination of micro-pollutants in the biological tissues by physico-chemical methods can characterize the water quality, having some advantages compared to the direct analysis of water. The micro-components concentration data thus obtained, serve as time integrated concentrations of bioavailable pollution. Furthermore, these organisms, used for analysis, fulfil a pre-concentration process of contaminants, similar to the capabilities of the solid phase micro-extraction techniques, sparing thus a pre-treatment step of chemical analysis, namely the enrichment of micro-pollutants in the sample matrix. The selection of a proper species as a biomonitor organism is therefore a very important prerequisite of monitoring, regarding that the pollutants’ concentrations of the water andyor of the sediment phases may be reflected in the organisms’ tissues depending on the characteristics of the given species investigated. In stream waters mainly, some different species (algae and macroinvertabrates) and also communities (periphyton) are widely used as indicators of pollutants w4–8x. Within the most widespread indicator species, the macroinvertebrates have been playing an out-

coming role by their activities carried out in organic matter cycle of the water bodies, reflecting the conditions of both water and sediment phases. Among macroinvertebrates the Gammaridae species, as for instance Gammarus fossarium and G. pulex, have been often tested as potential monitor organisms of the water quality due to their high metal accumulation capacities w9–11x. The characteristic elemental concentrations in the tissues cited in the literature range both according to the qualities of the elements measured, and according to the elemental composition of the water. At differently polluted areas of Great Britain, e.g. the actual elemental concentrations in G. pulex varied for Al, Fe, Mn, Ni and Zn in the ranges of 150– 600, 360–1400, 45–200, 4.5–10.5, 125–250 mgy g, respectively w6x. Mercury and mainly Cd accumulation data of this species, both among natural and artificial experimental conditions, have been more frequently studied, pointing out the correlations in the level of metal concentrations of the organism tissues and those of the water-phase w10,12,13x. A lot of data are demonstrated for Cd, at different sampling spots of having altering water compositions, varying from less than 1.0 even up to 40–50 mgyg Cd concentrations in the organisms, while in all cases the enrichment (accumu-

Table 1 ¨ river 1669 km, 3 October 2000) Concentration of major, minor and trace elements of amphipods determined by ICP-MS (at God, Element

Mean concentration (mgyg)

R.S.D. (%)

Element

Mean concentration (mgyg)

R.S.D. (%)

Ag Al As Ba Bi Ca Cd Co Cr Cu Fe Hg K Li

1.02 510 3.82 123 0.021 108 000 0.305 0.721 1.78 93.6 1620 0.469 4450 0.781

11.4 19.3 24.8 15.4 32.3 10.2 24.2 18.1 21.4 23.7 22.3 17.8 13.7 15.8

Mg Mn Mo Na Ni Pb Rb Sb Se Sn Sr U V Zn

4090 75.8 0.502 4300 4.15 16.5 4.88 0.128 7.23 0.732 312 0.063 1.39 128

14.5 11.1 13.6 19.7 32.3 11.5 20.1 22.4 34.0 18.2 14.0 19.7 17.4 20.9

Mean concentration (expressed in mgyg dry amphipod sample weight) and relative standard deviation data calculated on the basis of five replicates.

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Fig. 1. Concentration of some minor elements in amphipods after 3 weeks of colonization on clay (h) and gravel (j) substrates in 1999.

lation) factors of this element in Gammaridae species was found to be almost the same, i.e. 104 magnitude w12,14x. Also the effects of other elements (such as Zn, Ca and Mg) onto the uptake of this toxic trace component have been investigated in Gammaridaes w12,15,16x. In the River Danube the concentration levels of the toxic elements were also controlled by the bioaccumulative capacity of living organisms both in the case of active and passive monitoring since the early 1980s. For these investigations those species were selected, which were supposed to be present in a high amount and in the littoral zone of the river, and being also able to enrich the contaminants. Among the applied species, most frequently algae of periphyton communities (Cladophora glomerata), and also macroinvertabrates were chosen for analytical pollution survey w4,6,17x. The experience of investigations carried out by the macroinvertabrates (and also by the other indicator species) in smaller surface water bodies, can not be applied directly in large rivers like the Danube, having huge and significantly altering

water load, and having been impacted by varying industrial, agricultural and communal contamination sources of both point and diffuse characters. In such streams the pre-selected sampling spots are barely suitable for sampling during the most critical spring and early summer periods, because of the frequent floods. During these periods, there is almost no possibility of reaching the sampling area, and of getting sufficient sample quantities. To override these problems the use of artificial substrates seemed to be a promising solution w5x. Artificial substrate samplers provide means of sampling in many locations, where other samplers are not effective. They provide representative samples of native communities mainly in rivers and streams using: periphyton (algae); macroinvertebrates (insects larvae, amphipods); and provide also an equally diverse fauna with less variability among samples, thus being applied as an effective method of biological monitoring and evaluation of water pollution w3,5,18x. However, it has to be investigated, whether the artificial substrates, as new habitat for the biota, were comparable to the natural ones. The basic problem is, whether they

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Fig. 2. Concentration of some trace elements in amphipods after 3 weeks of colonization on clay (h) and gravel (j) substrates in 1999.

showed no special selectivity, thus being able to indicate the real pollution effects and serve representative samples, suitable ones for further biological and physico-chemical investigations w16,18x. This type of sampling method, using artificial substrates translocated into the environment, making the sampling strategies easier, avoiding the uncertainties, can be taken as a semi-active monitoring form (combining the elements of both passive and active biomonitoring), and thus it may become also suitable for standardization. From the available artificial substrates two different ones were applied during our experiments, selected upon previous tests results w19x. In addi-

tion to the substrates, different colonization periods have also been compared during a 2-year long experimental period by detailed biological evaluation. Because of its dominant role as shredder and detritus feeder, and of its abundance, Dikerogammarus villosus (Amphipoda Crustacea) was chosen for the recent investigation to follow the effects of the different experimental conditions. The size of these amphipods is generally approximately 8–25 mm, their average life cycle 1–2 years, and their ‘really state’ accumulation period is taken to be approximately 3 weeks. Some elemental concentrations in the dry tissue of this species were determined earlier at the similar

´ et al. / Microchemical Journal 73 (2002) 99–111 K. Barkacs

¨ From these measured data sampling spot, at God. some concentrations and enrichment factors (accumulation factors, EF) were the following: Ag, Cd, Co were present in approximately 10–25 mgyg mean concentration values in Gammaridaes; and their EF were in the magnitude of 105; Pb in 80 mgyg concentration and EF of 104; Cr and Cu in mean concentrations of 10–20 mgyg and EF of 103; Hg and Ni in mean concentrations of 2 and 10 mgyg and EF of 102 w6x. For determination of inorganic micro-pollutants in biological tissues (also in macroinvertebrates), because of the low concentrations measured in the special sample matrix, generally up to date instrumental analytical techniques of high sensitivity and selectivity, such as atomic absorption spectrometry (AAS) w6x, inductively coupled plasma emission spectrometry (ICP-AES) w12x or in some cases also inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray spectrometry (TXRF) w20,21x have been applied. For the elemental analysis the solute form of the biological samples are generally used, produced from the investigated tissues by pretreatment steps of drying and digestion. The digestion has been carried out most frequently by nitric acid with or without hydrogen peroxide, and by microwaveassisted wet digestion techniques both in vapor and liquid phases w6–12,20,21x. In our experiments for the determination of polluting elements present in amphipods ICP-MS was chosen as elemental analytical method, because of its multielement capability and low detection limits (ngyl–pgyl concentration range). 2. Materials and methods 2.1. Sampling The sampling site was located in the littoral zone of the River Danube at river 1669 km, at ¨ north to Budapest. This Danube section has God, been impacted by both industrial and communal pollution. Two types of artificial substrates were here applied: clay granulates and gravels (latest characteristic for and derives from the riverbed itself) w19x. Sampling was carried out every 3 weeks between the beginning of May (the 9th

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May) and the end of November (30th November) in the years of both 1999 and 2000, at every sampling time ensuring samples derived from the two different substrates and at the same time representing two different colonization strategies, also. There were thus samples colonized during the previous 3 weeks preceding the sampling time in question, and other ones colonized for longer periods. Therefore, the samples investigated are signed as being from short-term colonization (artificial substrate was contacted with the water for only 3 weeks, every third week new artificial substrates were located in the river in these cases) and from long-term ones (the contact timeyexposure of these samples was longer than 3 weeks, while sampling frequencies of every 3 weeks were maintained, thus the time periods of these substrates being left in the river water were gradually increasing from the 3rd up to the 30th weeks). Summarizing, the samples were taken during a 210-day long yearly experimental period according to the following schedule: ● Short-term colonizationysampling weeks: No.1–3, 4–6, 7–9, 10–12, 13–15, 16–18, 19– ¯ ¯ ¯ 28–30; 21, 22–24, 25–27, ● Long-term colonization y sampling weeks: No. 1–3, 1–6, 1–9, 1–12, 1–15, 1–18, 1–21, 1– ¯ 24,¯1–27, 1–30. 2.2. Sample preparation After sampling, the organisms found on a given substrate were washed down from the surface of the substrate through a sieve. Amphipods were selected for the analytical tests, specimens of 10– 12 mm length were chosen. They were cleaned by removing surface contamination by fine brushing and rinsing (by distilled water), and were then carried into the laboratory in polyethylene bags and placed into a deep-freezer. (The weight of each wet sample was approx. 400 mg.) Before analysis the organisms were dried in an oven at 105 8C until reaching a constant weight (approx. 3 h).The homogenization of the dried samples was made in an agate mortar. From a sample, approximately 15–20 mg dried tissue was weighed and transferred into quartz micro tubes, and decomposed using 0.5 ml cc. HNO3 (Suprapur

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grade, E. Merck, Darmstadt, Germany). The digestion was carried out in a heating block at 80 8C for 3 h in the sealed quartz tubes. Three independent replicates were made for each sample. After decomposition the samples were filled up with deionized water to 6.0 ml, thus adjusting an average matrix concentration of 2.5 mgyml. The sample solutions were mixed also with standard solutions (as internal standards); after homogenization the solute samples contained 50 ngyml concentration of the standard elements (45Sc, 103 Rh, 175Lu), made from single element standard solutions obtained from E. Merck. Blank samples, as well as the applied chemicals, were regularly controlled by ICP-MS measurements for determining and excluding the impurities of non-sample origin. 2.3. ICP-MS measurements Element concentrations were determined using HP 4500 Plus type (Hewlett Packard) ICP-MS equipment. The operating parameters were as follows: Forward power: 1400 W; reflected power -1 W; RF frequency: 27.12 MHz; plasma gas (Ar): 15 lymin; auxiliary gas (Ar): 1.20 lymin; aerosol gas (Ar): 0.96 lymin; blend gas (Ar): 0.16 lymin; sample uptake rate: 0.3 mlymin; sampling depth: 8.4 mm; spray chamber temperature: 2 8C; sampler cone: Ni 1.0 mm; skimmer cone: Ni 0.7 mm; nebulizer type: V-groove; torch: Fassel-type; data acquisition: peak jumping. Analytical isotopes were: 7Li; 9Be; 11B; 23Na; 25 Mg; 27Al; 39K; 43Ca; 44Ca; 51V; 53Cr; 55Mn; 57Fe; 59 Co; 60Ni; 63Cu; 65Cu; 66Zn; 67Zn; 68Zn; 69Ga; 71 Ga; 75As; 82Se; 85Rb; 88Sr; 98Mo; 107Ag; 109Ag; 111 Cd; 112Cd; 114Cd; 118Sn; 120Sn; 121Sb; 123Sb; 135 Ba; 137Ba; 200Hg; 202Hg; 205Tl; 206Pb; 207Pb; 208 Pb; 209Bi; 238U. The spectral interferences of 75 As originated from the chloride content of the samples were corrected mathematically according to the US EPA 200.8 standard. 2.4. Statistical analysis For the evaluation of the experimental data rows the following non-parametric methods were

applied: Mann–Whitney U-test and Spearman rank correlation. 3. Results and discussion To estimate both the elemental composition of amphipods and also to determine the analytical performance (reproducibility of elemental measurements) of the ICP-MS method for this organism, amphipods were sampled and analyzed once in a larger amount during the experimental period. The analytical reproducibility tests were carried out in this case applying five replicates. The dry mass of an amphipod varied in our tests in the range of 8.0–9.0 mg, giving approximately 27% of the wet total mass. In the amphipods, 50 different elements were detected by ICP-MS in significantly different amounts. The measured element concentrations ranged according to the element quality from ngyg up to 100 mgyg concentrations, as it is shown in Table 1. From the detected 50 elements this table demonstrates the concentration data of 28 selected ones, that is some characteristic macro, minor and mainly trace components. Besides the mean element concentration values, the determined relative standard deviation data (R.S.D.) are also enclosed here. It can be seen that among the macro-components (as Na, K, Ca, Mg) Ca was present at the highest concentration level (approx. 100 mgyg in dry tissue, compared to the other ones being present in an average of 4–5 mgyg concentrations). Also a relatively high concentration was found for Fe (1600 mgyg dry tissue), and approximately 100 mgyg or somewhat higher values were measured for Al, Ba, Cu, Mn, Sr and Zn. Some element concentrations were determined to be in the range of 1–10 mgyg in the amphipods, such as As, Cr, Ni, Pb, Rb, Se and V, some other ones were present at even lower concentration levels, that is around and below 1 mgyg. These latter data were characteristic for Ag, Bi, Cd, Co, Hg, Li, Mo, Sb, Sn and U. The reproducibility of the concentration measurements for macro elements amounted to 10–15%, and for minor and trace elements to 15– 25%. Summarizing, most of the 28 elements here represented, showed an average R.S.D. value around or below 20%.

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Fig. 3. Concentration of some minor elements in amphipods after 30 weeks of colonization on clay (h) and gravel (j) substrates in 1999.

For the detailed representation and evaluation of the element concentration data got during the whole experimental period, 12 elements are selected here from the detected ones. Thus, the variations in the amphipods element concentrations according to the applied sampling strategies, are in this paper followed by controlling their Ag, As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb and Zn concentrations. From the data rows, Figs. 1 and 2 demonstrate an example, showing some element concentrations measured in the amphipods after the first 3 weeks of colonization on the two different artificial substrates. Concentrations of the minor elements, such as Ba, Cu, Mn, Zn, and a trace element Ni are shown in Fig. 1, while the concentrations of the other selected trace elements, that is Ag, As, Cd, Co, Cr, Cu, Hg and Pb can be followed in Fig. 2. Both figures demonstrate the mean element concentrations in the amphipods derived from the two substrates and also the determined standard deviation values. Similarly, Figs. 3 and 4 illustrate the concentration data of the same investigated elements, but in the samples tested after a long-term

(30 weeks) colonization period. Comparing the data of Figs. 1 and 3, it can be seen that no real concentration changes could be observed during the experimental period for the minor elements. However, the substrates also did not influence significantly the measured concentrations of these elements. The trace element concentrations for the same test materials, that is the data of samples taken after 3 and 30 weeks of colonization and from both substrates, show also no real alterations according to the applied substrate types (Figs. 2 and 4). All the concentration data of these figures indicated that the concentrations of the elements measured in amphipods, were in the same ranges during the whole experimental period and close to those, which were got during—the previously detailed— reproducibility test (Table 1.). However, within the ranges, the absolute concentration of each detected element, had been more (mainly in the case of trace elements) or less, but varying in time during the two years long sampling period, as it can be followed in Figs. 5 and 6. In these figures

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Fig. 4. Concentration of some trace elements in amphipods after 30 weeks of colonization on clay (h) and gravel (j) substrates in 1999.

the data trends of two selected characteristic trace elements are demonstrated: that is the measured Ag and Cd concentrations are illustrated here in the function of the sampling time during the 2 years, showing the data of the long-term colonization sampling periods got on the two artificial substrates. While the concentration data varied significantly according to the sampling time, there were no real concentration differences found for these elements according to the quality of the artificial substrates. The comparison of these concentration data rows for the two substrates were also tested by mathematical statistical evaluations. As the P-level values of the Mann–Whitney paired-sample test show in the Table 2, the data rows can be taken as identical ones, except three cases indicated also in Table 2. Table 3 represents also some Ag and Cd concentration data of amphipods, but comparing their concentration values here according to the sam-

pling strategies of short- and long-terms, and also according to the different two experimental years. It can be seen, that these concentrations were also not differing in the function of the colonization periods, but were significantly affected by the sampling time, thus by experimental years. In addition to the concentration data of the previously selected two (toxic- and) trace elements, some more element concentrations are also represented in Table 3. The average measured concentration values of the selected elements for a year term, that is mean concentration values for a given element in the amphipods according to the experimental conditions, are listed here. For a given element also the measured minimum and maximum concentration values, as well as relative standard deviations are indicated in the table. The R.S.D. data represented here, reflect the variances in the reproducibility of the measured element concentrations in this biological matrix, while they

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Fig. 5. Ag concentration of amphipods during long-term colonization on clay (h) and gravel (j) substrates (sampling frequency: sampling on every third week started at the beginning of May and finished at the end of November each year, sampling No. 1–10 in 1999, No. 11–20 in 2000).

were calculated on the basis of the three independent replicates determined for all single samples, and taking all these R.S.D. values of samples into account belonging to a given evaluated data row. The R.S.D. data may be taken thus, as characteristic ones for the common effect of biological sample diversity and the analytical reproducibility onto the concentration data range of a given element determined in amphipod tissues. These R.S.D. values generally turned out to be somewhat higher (in some cases similar), than those calculated in the analytical reproducibility test for the same elements (see data shown in Tables 1 and 2). However, even for the trace elements, these R.S.D. values, depending slightly on the element qualities, remained generally below 30% (they were on average less than 25%, with the exception of the values got for Cd, Cr, Ni and Pb), reflecting an average acceptable reproducibil-

ity of the elemental concentration measurements for the amphipods. Altogether regarding each element, the data of Table 3 suggest no real significant differences in the investigated elemental concentrations, when different artificial substrate qualities or altering colonization times were applied. The element concentration data rows of the whole experimental period were evaluated for each element also by mathematical statistical tools, applying the pairedsampled test according to Mann–Whitney. These evaluations were carried out mainly to check, whether the element concentrations were significantly differing comparing the two different substrates among similar sampling condition (shortand long-term separately), and also these conditions themselves were statistically compared. The P-level data (parameter of the test) turned to be higher than 0.05, signing in this case that no

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Fig. 6. Cd concentration of amphipods during long-term colonization on clay (h) and gravel (j) substrates (sampling frequency: sampling on every third week started at the beginning of May and finished at the end of November each year, sampling No. 1–10 in 1999, No. 11–20 in 2000).

Table 2 Paired-sample testing by rank according to Mann–Whitney U-test Element

1999 Short-term clay-gravel P-level

1999 Long-term clay-gravel P-level

2000 Long-term clay-gravel P-level

1999 Clay Short-long terms P-level

1999 Gravel short-long terms P-level

Ag As Ba Cd Co Cr Cu Hg Mn Ni Pb Zn

0.5338 0.3098 0.9271 0.1265 0.4367 0.9126 0.2002 0.4754 0.3650 0.3506 0.4421 0.2452

0.8795 0.8855 0.7791 0.1455 0.8556 0.6492 0.9035 0.1871 0.5047 0.1724 0.4761 0.6712

0.6492 0.7047 0.4575 0.3875 0.6276 0.9396 0.9637 0.4951 0.4216 0.7617 0.8735 0.2194

0.8954 0.2059 0.9672 0.6106 0.2710 0.1833 0.0416* 0.1833 0.3750 0.0938 0.0301* 1.0000

0.3716 0.8397 0.9328 0.0175* 0.2587 0.3716 0.6612 0.3324 0.5552 0.7616 0.7107 0.7744

Evaluating the differences among elemental concentration data rows measured within altering experimental conditions. * Indicates the data rows, where P-level is -0.05.

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Table 3 Element concentrations of amphipods during the experimental period of the years 1999–2000 as a function of the sampling strategies Element

Ag (s) Ag (l)* Ag (ll)* As (s) As (l) As (ll) Ba (s) Ba (l)* Ba (ll)* Cd (s) Cd (l)* Cd (ll)* Co (s) Co (l)* Co (lI)* Cr (s) Cr (l) Cr (ll) Cu (s) Cu (l) Cu (ll) Hg (s) Hg (l)* Hg (Il)* Mn (s) Mn (l) Mn (ll) Ni (s) Ni (l)* Ni (ll)* Pb (s) Pb (I)* Pb (II)* Zn (s) Zn (l) Zn (ll)

Substrate clay

Substrate gravel

Mean conc. (mgyg)

Min. conc. (mgyg)

Max. conc. (mgyg)

R.S.D. (%)

Mean Conc. (mgyg)

Min. Conc. (mgyg)

Max. conc. (mgyg)

R.S.D. (%)

0.559 0.575 0.437 2.49 2.86 2.71 108 105 76.8 0.251 0.243 0.173 0.975 0.949 0.709 2.44 2.10 2.08 100 110 111 0.224 0.259 0.113 168 140 110 4.09 8.26 4.89 3.13 2.82 2.12 93.8 97.6 102

0.051 0.213 0.238 1.06 1.77 1.71 78.3 74.9 45.3 0.122 0.063 0.071 0.633 0.461 0.393 0.499 0.757 1.03 53.5 75.5 64.9 0.099 0.141 0.026 63.6 55.7 53.2 1.99 2.31 2.26 1.66 1.09 0.925 58.4 79.0 59.1

1.11 1.20 0.949 3.79 5.52 4.29 140 166 107 0.587 0.788 0.314 1.445 2.21 1.38 4.90 4.62 5.42 164 153 161 0.342 0.555 0.209 181 188 221 7.06 41.8 10.2 7.68 7.84 4.51 129 162 123

23.6 24.0 22.0 13.7 12.9 20.2 10.5 12.7 14.7 34.6 30.1 25.4 24.1 23.6 26.5 27.8 26.0 33.8 17.6 12.5 17.5 17.0 22.1 36.3 21.1 27.0 26.0 26.7 28.5 23.7 33.5 38.6 39.1 19.2 11.1 9.2

0.525 0.589 0.416 2.99 2.84 2.85 131 106 75.7 0.221 0.254 0.205 1.07 0.948 0.665 2.62 2.21 1.95 113 118 115 0.269 0.239 0.130 120 120 98.5 4.29 5.62 4.99 2.88 2.79 2.04 103 106 116

0.062 0.229 0.139 1.41 1.63 1.64 77.3 61.4 41.1 0.002 0.069 0.088 0.532 0.409 0.387 0.326 0.373 1.11 62.6 71.2 70.8 0.124 0.120 0.018 60.1 54.5 54 1.83 1.99 2.94 0.942 0.812 0.852 75.7 79.2 78.2

1.09 1.26 1.22 7.56 8.19 4.21 158 243 118 0.643 0.619 0.466 2.17 2.71 1.21 9.01 5.39 3.62 209 314 213 0.79 0.57 0.319 171 236 226 10.5 19.5 9.15 7.76 7.71 7.26 141 303 247

15.0 26.7 23.1 16.3 25.3 13.6 19.6 20.9 15.0 23.5 26.6 14.1 20.9 23.9 13.4 32.5 29.7 20.2 12.7 25.6 16.6 35.8 23.8 33.1 18.6 29.3 22.2 31.0 33.5 18.0 31.5 30.8 31.8 9.7 20.7 17.3

Mean-, minimum-, maximum concentrations of the elements and RSD values (calculated for characterizing the reproducibility of the elemental analysis within the selected data rows, each consisting of 30 samples) are compared according to the quality of the artificial substrates. Data obtained from short-term colonization in 1999 are signed beside the element quality by (s), from longterm colonization in 1999 by (l), and in 2000 by (ll). * Indicates significant difference in the element concentrations of the 2 different years.

significant differences existed between the substrates neither in short-, nor in long term colonization during both experimental years (higher than 95% level of significance of the similarities for each data row was found). In addition to this, also

no significant differences were found by the statistical trial among the elemental compositions of the amphipods derived from short- and long-term sampling (Table 3). Comparing the data rows obtained, that is the accumulation levels for the elements

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determined, the only significant difference was found in time and between the two different years (I, II) for some elements, e.g. for Ag, Ba, Cd, Co, Hg, Ni and Pb, as indicated in Table 3. Furthermore, the statistical evaluations of our experimental data rows could point out, that significant correlation between the concentrations of Cd and Zn in the tissues of amphipods existed among all sampling circumstances (P-levels calculated were the followings: for short-term colonization data: 0.00069; for long-term data: 0.00002 and 0.00579 in the 2 different years, respectively). This phenomena is in accordance with the experience having cited relating Cd toxicity tests carried out for other Gammaridaes w15x. Summarizing, on the basis of the measured element concentration data, in any case no significant enrichment differences were observed in the amphipods element concentration in the function of the investigated sampling circumstances. That is, having controlled the accumulation differences of toxic elements on the two artificial substrates applied, and according to the colonization period’s length, the data do not reflect significant alterations. It also can be seen, that there is no particular advantage in analyzing the elemental concentrations of amphipods collected by gravel as natural, river bed substrate, and from the substrates having translocated for long-term into the river water (longer than 3 weeks). 4. Conclusion ICP-MS spectrometry turned to be a suitable multielement method for determining the concentration of major, minor and trace elements in amphipods. Fifty elements were detected in amphipods, and the most significant 12 of them, mainly trace- and toxic components were regularly and also statistically evaluated in the function of the experimental conditions. These elements, in spite of the biological matrix diversity, and being present in the range of 1–10 or less than 1 mg elementyg dry tissue concentrations, showed an appropriate analytical reproducibility by the ICP-MS measurements, characterized by relative standard deviations of less or approximately 20%, and also less

than 30%, when the element concentration data of all samples, analyzed during the whole experimental period, were evaluated. Regarding the analytical data row got during the 2-year long experimental period, the applicability of amphipods, as dominant macroinvertebrates for monitoring toxic- and trace elements in streamwaters, was straightened, and a significant correlation regarding the Cd and Zn concentrations of the amphipods was also found. Comparing the different artificial substrates as well as the colonization periods of short- and long-terms, the samples thus collected in the River Danube showed no statistically observable alterations regarding the measured metal concentrations of the amphipods. On the basis of both the chemical analytical data and the biological observations, we concluded, that the time saving short term colonization period can be applied as a sufficient semi-active biomonitoring method comparing to the long-term one. We concluded also, that the use of the artificial substrates—containers filled by clay granulates— brought advantages in stream water biomonitoring tests, while the sampling remained relevant to the natural conditions. Acknowledgments The authors gratefully acknowledge the financial support of the Hungarian National Science Foundation (OTKA) T035005 and T025419 and ´ express special thanks to BALINT ANALITIKA Ltd.(Budapest) for the ICP-MS measurements and also for the technical assistance. References w1x W. Slooff, Biological Effects of Chemical Pollutants in the Aquatic Environment and Their Indicative Value. Utrecht, 1983, 191. w2x D. De Zwart, Monitoring Water Quality in the Future, vol. 3,, Biomonitoring. RIVM, Bilthoven, The Netherlands, 1995. w3x N. Oertel, Application of biomonitoring techniques in pollution control. Final Report. Community’s Action for Cooperation in Sciences and Technology with Central and Eastern European Countries, Ref.: ERB3511 PL922924, Prop. Number: 12924, 1993. w4x N. Oertel, Sci. Total Environ. 2 (1993) 1293–1304.

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