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Accumulation of heavy metals by enchytraeids and earthworms in a floodplain
European Journal of Soil Biology 42 (2006) S117–S126 http://france.elsevier.com/direct/ejsobi
Original article
Accumulation of heavy metals by enchytraeids and earthworms in a floodplain P.C.J. Van Vlieta,*, W.A.M. Didden1, S.E.A.T.M. Van der Zeea, W.J.G.M. Peijnenburgb a
Department Soil Quality, Wageningen University, P.O. 8005, 6700 EC Wageningen, The Netherlands b National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Available online 20 September 2006
Abstract The river floodplain ‘Afferdense and Deestsche Waarden’ (ADW) in The Netherlands is diffusely contaminated with several heavy metals. It is, however, unclear whether this mixed contamination exerts any adverse ecotoxicological effects. In November 2000 and May 2001 a field survey was conducted in two areas in the ADW to collect a wide range of data concerning contamination levels, bioavailability, enchytraeids and earthworms and abiotic factors such as lutum and organic matter content, cation exchange capacity (CEC) and soil nutrient concentrations. Earthworms and enchytraeids were also analyzed for heavy metal content. At both sites arsenic and zinc were present in soil at relatively high concentrations (above the Dutch intervention value). In the two areas, both enchytraeids and earthworms accumulated metals. Fridericia ulrikae accumulated more cadmium than Enchytraeus buchholzi and Henlea perpusilla. The earthworm Lumbricus rubellus accumulated larger concentrations of Cr, Cu and Pb than Aporrectodea caliginosa and Allolobophora chlorotica. Dietary, physiological and behavioral characteristics may have contributed to these differences. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Floodplain; Heavy metals; Enchytraeids; Earthworms; Accumulation; Regression
1. Introduction Floodplains of Dutch rivers are moderately to heavily polluted with heavy metals and toxic organic compounds. This is mostly due to historical contamination in the 1960s and 1970s when contaminated suspended matter settled in the floodplains. Although the water quality has improved, there is still a certain level of contamination [1]. Contamination levels in Dutch * Corresponding author. Tel.: +31 317 48 2344; fax: +31 317 48 3766. E-mail address: [email protected] (P.C.J. Van Vliet). 1 Wim Didden sadly deceased on 28 April 2005.
floodplains are above background levels but below the intervention levels prompting remediation [2]. These sites are therewith under a ‘gray veil’ of toxicant stress, where local effects are not evident, possibly due to natural variation. Exposure of organisms to heavy metals in these systems depends on the total metal content, soil composition (in particular environmental variables such as pH, organic matter content, concentrations of competing ions, concentrations of complexing ligands in solution) and the characteristics of the organism itself [3]. It has long been recognized, that only a part of the present heavy metals is biologically available and that availability also depends on the organism of interest. In
1164-5563/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2006.09.005
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the case of enchytraeids and earthworms, a complication is that the exposure to heavy metals in soil involves both oral and dermal routes. It is plausible, that these exposure routes depend differently on the distribution of metals over the soil solution, the fractions adsorbed onto the solid phase, and those present as a chemical precipitate (e.g. [4,5]). In most research on toxic stress of invertebrates by metals, relatively high metal contamination levels in an artificial soil system have been considered. It is unclear whether the principles established by that research can be transferred to more moderate contamination levels as found in Dutch floodplain soils. The reason is that it is not evident whether the contamination levels in Dutch floodplain soils pose a toxic stress or whether perhaps other stressors are more important in affecting the population dynamics of resident organisms. Appreciable species-specific differences in the sensitivity of earthworms to heavy metals contamination have been demonstrated [6,7]. Different earthworm species accumulate different concentrations of heavy metals and do so at different rates when exposed to the same soil (e.g. [8,9]). Furthermore, they may exhibit different degrees of resistance to the adverse effects of contaminant exposure [10,11]. Not much is known about species-specific responses of enchytraeid species to metal concentrations in soil. A field study in contaminated forest sites in Germany found high concentrations of cadmium and lead in enchytraeids, with certain species accumulating higher concentrations than others [12]. Several factors may contribute to the observed differences in accumulation. According to their activities and distribution in soil, three groups of worms may be distinguished, i.e. (1) epigeics that process organic matter on or near the soil surface, (2) endogeics, which live in the mineral soil and feed on humus, and (3) anecics, which transfer materials between the soil and litter habi-
tats [13]. Differences in the food sources may induce differences in exposure if the heavy metal content or its availability differs between these sources. It has been observed for earthworms that selected food sources were exploited in different ways, due to differences in alimentary and/or excretory physiology [14]; furthermore physiological differences in the accumulation of metals by earthworms have been demonstrated [15]. In this paper, we consider the consequences of moderate heavy metal contamination in floodplain soils on three species of earthworms and of enchytraeids. Our aims were to assess the degree with which these organisms accumulate heavy metals in cases of moderate contamination, and to identify and understand speciesspecific differences in bioconcentration. With regard to the accumulation of heavy metals our scope was to determine whether it is possible, even at moderate metal contamination, to relate accumulation by these organisms with heavy metal concentrations and environmental conditions. 2. Material and methods 2.1. Site description The study area is the ‘Afferdensche and Deestsche Waarden’ (ADW) located along the river Waal (Fig. 1). Within this floodplain, two field sites were selected, which are called the Rijswaard and Deel3. The Rijswaard is located between the summer dyke and the river Waal, therefore the lower parts of this area are frequently flooded (about 3–4 times per year). Soil texture ranges from sand to sandy loam and loam. The site is predominantly grassland and grazed by young cattle during summer. Deel3 is located in the area between the summer and winter dykes. The area floods only if the summer dyke overflows (1–2 times per year). After flooding, the water subsides through evaporation and drainage. Texture of Deel3 varies from loam, to silty
Fig. 1. Map indicating the sampling sites in the floodplain ‘De Afferdensche en Deestsche Waarden’ (ADW) in The Netherlands (Scale = 1:50.000). The map to the left shows The Netherlands and the location of the ADW-area.
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clay loam and clay loam. This site includes grassland and is grazed by horses throughout the year, except when the area is flooded. 2.2. Sampling times Sampling took place in November 2000 and May 2001. In Deel3 a transect of eight sampling points, 25 m apart, was sampled. In the Rijswaard a transect of 46 sampling points, 15 m apart, was sampled. At each sampling point, samples for soil chemistry and soil texture determination were collected for the 0–10 and 10–20 cm layers. At each sampling point in Deel3 and every fourth point in the Rijswaard two soil pits of 20 × 20 × 20 cm were excavated for earthworm sampling. From each pit, the earthworms were collected by handsorting and taken back to the laboratory for identification and metal analysis. Enchytraeid samples were taken to a depth of 20 cm and split into two depths (0–10 and 10–20 cm). In Deel3 this was done at each point, and in the Rijswaard at every second point. An additional 0–10 cm sample was also taken from the intermediate points in the Rijswaard. 2.3. Soil chemical analyses After extraction with a 0.01 M CaCl2 solution, pH, Dissolved Organic Carbon (DOC), and available heavy metals (As, Cd, Cr, Cu, Pb, Zn) were determined according to Houba et al. [16]. DOC was measured with a fully automated Skalar TOC/DOC analyzer. Total concentrations of the heavy metals (Cd, Cr, Cu, Pb and Zn) and the element As were determined in an aqua regia extract. The elements As, Cd, Cu, Pb, and Zn were measured with a Perkin–Elmer Elan 6000 ICPMS. The element Cr was measured with a Spectroflame–Spectro ICP-AES. For statistical analysis concentrations were expressed as mmol kg–1 dry weight of soil. Free aluminum and iron compounds, such as goethite and gibbsite were extracted using the Holmgren citrate-dithionite method [17]. Amorphous iron and aluminum were extracted in an ammonium oxalate solution [18]. Extracts were measured with a Spectroflame–Spectro ICP-AES. Total carbon in the soil samples was determined with a Fisons Instruments EA 1108 CHNS-O analyzer, using a Porapak QS column with He as carrier gas. The organic matter content of the soil was calculated from the organic carbon content using a conversion factor of 1.72 [18]. Clay content was determined by sieve and pipette, unbuffered 0.01 M BaCl2 was used to deter-
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mine the cation exchange capacity (CEC) [18]. In November 2000, soil pore water was collected from the collected soil at both sites by centrifugation, and was analyzed for heavy metals and DOC in the laboratory. Concentrations of heavy metals in pore water were expressed in μmol l–1. 2.4. Biological analyses Enchytraeid samples were stored at 4 °C until extraction. For the extraction (modified O’Connor method [19]) each soil sample was split into four layers of 2.5 cm each. Total number of enchytraeids per sample and per depth increment was determined. All live specimens were identified to species. Mature enchytraeid specimens were frozen separately in 96-well plates. Sufficient material was needed for the internal metal analysis. The most common species found were Fridericia ulrikae, Henlea perpusilla and Enchytraeus buchholzi. Several specimens of these species were found in each sample. For internal metal analysis, specimens were pooled by species, sample and sampling depth, and freeze-dried. The freeze-dried material was weighed into Pyrex destruction tubes, to which concentrated nitric acid and perchloric acid were added. The samples were heated at 170 °C until the nitric and perchloric acids were completely evaporated. Samples were dissolved in 750 μl of 15 mmol HNO3 and worm metal concentrations in the digests were measured for As, Cd, Cr, Cu, Pb and Zn using an ICPMS Elan6000. Identified mature earthworms of the species Lumbricus rubellus, Aporrectodea caliginosa and Allolobophora chlorotica were put on filter paper (Schleicher and Schuell 589) to defecate for 48 hours at 15 °C in the dark before being freeze-dried. Earthworms were destructed with the aqua regia method. Concentrations of the elements As, Cd, Cr, Cu, Pb and Zn in the extracts were determined with an ICP-AES. Metal concentrations in earthworms and enchytraeids were expressed on a mmol kg–1 dry weight basis. For the earthworm and enchytraeid species the biotato-soil accumulation factor (BSAF) was calculated. The BSAF is defined as the ratio of the concentration of the metal in the worm tissue and the total concentration of the metal in the soil. 2.5. Statistical analysis SAS/STAT version 8.0 was used for statistical analysis. Before analysis, all site parameters were checked for normality and were transformed if necessary. Out-
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liers (values larger than the mean of the total dataset ± 3 times the standard deviation) were removed from the dataset. One-way ANOVA and t-tests were used to determine significant differences between worm species. Using log-transformed regression analysis the accumulated concentration of heavy metals by the different earthworm and enchytraeid species was expressed as a function of one or more of the following parameters: total metal concentration, available metal concentration and CEC. Regressions were determined for all the data split by species. Concentrations of metals in earthworms were associated with parameter values for the depth at which the earthworms were mostly found in the field. We found A. caliginosa mainly at the 10–20 cm depth, and A. chlorotica and L. rubellus mainly in the 0–10 cm depth increment. Concentrations of metals in the enchytraeid species were associated with the metal concentrations at the depth they were found. The multivariate regressions have the format of: logðMewormÞ ¼ intercept þ a logðAÞ þ b logðCECÞ
3. Results 3.1. Soil characteristics At both sites the concentrations of heavy metals were lowest in the top layer (Table 1). At both sites, concentrations of arsenic and zinc in the 10–20 cm layer were higher than the intervention values set by the Dutch government [2]. Other heavy metals were present in concentrations above the target values [2] but mostly far below the intervention values. Total metal concentrations in the floodplain differed at most by a factor (maximum over minimum concentration) of 14 (Pbtotal = Pb concentration in soil determined by aqua regia) (Table 2). The range in concentrations of As and the heavy metals was very small, implying that variation within the floodplain was limited. The CEC correlated strongly with other soil properties like aluminum and iron hydroxides, clay content and organic matter content (Table 3). This soil parameter is therefore used in the regression analysis. 3.2. Earthworms and enchytraeids
or logðMewormÞ ¼ intercept þ a logðBÞ where, Meworm is the concentration of metal accumulated by the worm in mmol kg–1; A is the total metal concentration (aqua regia) in mmol kg–1; CEC is the cation exchange capacity (cmol(+) kg–1); B is the metal concentration in 0.01 M CaCl2 in mmol kg–1 or in pore water extract in μmol l–1; a and b are the corresponding coefficients. Only significant (P < 0.05) parameters are mentioned. Significance of the regression equation was judged by R2adjusted, which accounts for the number of observations and the number of parameters used, and is an indicator of the variation in the data that is explained by the model.
Average accumulated metal concentrations in earthworms and enchytraeids are shown in Figs. 2 and 3. Concentrations of cadmium in all earthworm and enchytraeid species are higher than the soil concentrations present. Cadmium is a non-essential element with a relatively long half-life [4,20], resulting in high BSAF-values ranging from 4 to 12. A. chlorotica accumulated significantly more As than the other two earthworm species and had the lowest accumulated concentrations of heavy metals (except Cd). L. rubellus accumulated significantly more Cu, Cr, and Pb than A. caliginosa and A. chlorotica (Fig. 2). Accumulated copper concentrations in the three enchytraeid species were much higher than the corresponding soil concentrations, resulting in BSAF values of about 12. H. perpusilla accumulated more Zn and As
Table 1 The mean total arsenic and heavy metal concentrations expressed as a ratio of the corresponding Dutch target values [2] at the two sites and two depths. Determined in November 2000. I/S = Dutch intervention value divided by the Dutch target value for standard soil containing 25% lutum and 10% organic matter. Letters within a column denote significant (P < 0.05) different metal concentrations in the two depths per site. Bold values indicate values larger than the I/S value. Site Rijswaard Deel3 I/S
Depth (cm) 0–10 10–20 0–10 10–20
As 1.4 2.1 1.6 2.5 1.9
a b a b
Cd 3.2 a 5.0 b 4.8 5.4 12
Cu 1.9 a 2.6 b 2.4 2.8 5.3
Cr 1.3 a 1.8 b 1.3 1.3 3.8
Ni 1.3 a 1.5 b 1.3 1.4 6
Pb 1.7 2.6 2.6 3.8 6.2
a b a b
Zn 4.2 5.7 4.6 6.5 5.1
a b a b
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Table 2 Mean, standard deviation, minimum and maximum values of the composite data set for the two sites, two depths and two sampling dates. Total metal concentrations (Metotal) determined in November 2000. Available (CaCl2) metal concentrations determined in November 2000 and May 2001. Porewater metal concentrations determined in November 2000. Variable pH %lutum CEC Days flooded DOC C Astotal Cdtotal Crtotal Cutotal Pbtotal Zntotal As-CaCl2 Cd-CaCl2 Ni-CaCl2 Pb-CaCl2 Cr-CaCl2 Cu-CaCl2 Zn-CaCl2 As-porewater Cd-porewater Cr-porewater Cu-porewater Pb-porewater Zn-porewater
Unit
Mean 7.33 16.04 20.28 25.40 1.55 × 5.40 6.35 × 2.92 × 2.26 × 1.18 × 9.67 × 9.10 × 0.64 × 0.10 × 0.40 × 0.01 × 0.19 × 3.07 × 1.65 × 0.08 × 0.00 × 0.12 × 0.53 × 0.01 × 0.50 ×
percentage cmol(+) kg–1 mol C kg–1 percentage mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol l–1 mol l–1 mol l–1 mol l–1 mol l–1 mol l–1
10–2 10–4 10–5 10–3 10–3 10–4 10–3 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
Table 3 Correlations (Pearson coefficients) between CEC (cmol(+) kg–1), and soil parameters, aluminum and iron amorphous hydroxides (Alox and Feox, respectively), aluminum and iron crystalline hydroxides (Alcd and Fecd, respectively), % soil carbon (%C), and %lutum (%L), at the two sites. Both depth increments were included and combined in the analysis. Sampling date = November 2000. *** = P < 0.001; **** = P < 0.0001 Sites Parameter Alcd Alox Fecd Feox %C %L
0.68 0.64 0.73 0.49 0.59 0.67
Rijswaard **** **** **** *** *** ****
0.88 0.84 0.86 0.73 0.93 0.87
Deel3 **** **** **** *** **** ****
than the two other enchytraeid species. F. ulrikae contained the highest concentrations of Cd and Pb (Fig. 3). Regressions between the accumulated metal concentrations by the earthworm and enchytraeid species and total soil metal concentrations, available metal concentrations (0.01 M CaCl2), porewater metal concentrations and CEC were derived. Significant regressions are shown in Table 4. R2adjusted values were quite small for all regressions; with a minimum of 0.10 (Cr
Stdev 0.19 8.00 7.75 29.56 9.90 × 1.59 2.63 × 1.15 × 5.90 × 4.18 × 4.46 × 3.39 × 0.27 × 0.04 × 0.29 × 0.02 × 0.20 × 1.80 × 1.22 × 0.05 × 0.00 0.05 × 0.20 × 0.01 × 0.26 ×
10–3 10–4 10–5 10–4 10–4 10–4 10–3 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
Min 6.88 0.31 7.06 0 2.65 1.70 1.27 4.45 6.25 2.38 1.41 2.20 0.06 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.24 0.00 0.16
× 10–3 × × × × × × ×
10–4 10–6 10–4 10–4 10–4 10–3 10–6
× 10–6
× 10–6 × 10–6 × 10–6
Max 7.83 34.00 34.97 78 4.07 × 8.80 1.21 × 5.42 × 3.31 × 2.06 × 1.98 × 1.70 × 1.26 × 0.19 × 2.11 × 0.11 × 1.15 × 8.76 × 4.59 × 0.23 × 0.02 × 0.34 × 1.15 × 0.03 × 1.36 ×
10–2 10–3 10–5 10–3 10–3 10–3 10–2 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
and Cu, E. buchholzi) and a maximum of 0.64 (Pb, L. rubellus). CEC was the only variable that significantly improved the regression equation in the case of zinc and the earthworm species L. rubellus (R2adjusted increased from 0.15 to 0.51). Using available concentrations (0.01 M CaCl2) instead of total soil metal concentrations as the independent variable in the regression increased the fit of the Cu-regression equations for E. buchholzi and F. ulrikae, by a factor of 2 and 2.4, respectively. Due to the small range in total soil metal concentrations the derived equations cannot be extrapolated to encompass higher soil metal concentrations. Therefore we used regressions developed for cadmium, copper, lead and zinc by Ma [21] to predict the amount of metals accumulated by earthworms on basis of total concentrations (simple equation), as well as total concentrations combined with pH, organic matter percentage, and earthworm type (extended equation). Our data were not modeled accurately using the simple regression equations. For cadmium, lead and zinc the calculated concentrations were much higher than the measured concentrations (data not shown). The extended Pb regression equation, combining total soil metal con-
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Table 4 Regressions for the different metals and As between accumulated metals by enchytraeids and earthworms, soil (total/available/porewater) metal concentrations and CEC (sites, sampling dates and depths combined). Metal Enchytraeids Cr Cr
Species
Model
R2adjusted
n
Ench Hen
log(Crworm) = 0.429 × log(CrCaCl2) log(Crworm) = 0.353 × log(CrCaCl2)
0.10 0.11
36 26
Cu Cu Cu Cu
Ench Ench Frid Frid
log(Cuworm) = 1.149 – 0.916 × log(Cutotal) log(Cuworm) = –0.817 × log(CuCaCl2) log(Cuworm) = 1.139 – 1.188 × log(Cutotal) log(Cuworm) = –0.924 × log(CuCaCl2)
0.10 0.21 0.12 0.29
36 36 24 24
Zn
Frid
log(Znworm) = 1.793 – 0.966 × log(Zntotal)
0.14
24
Earthworms As
Achl
log(Asworm) = –0.194 + 0.825 × log(Astotal)
0.12
26
Cd Cd
Achl Achl
log(Cdworm) = 0.551 × log(Cdtotal) log(Cdworm) = 0.636 × log(CdCaCl2)
0.21 0.16
26 26
Cr
Achl
log(Crworm) = –2.002 – 0.151 × log(CrCaCl2)
0.18
26
Cu Cu Pb Pb
Acal Lrub Achl Lrub
log(Cuworm) = –0.479 + 0.563 × log(Cutotal) log(Cuworm) = –0.345 + 0.535 × log(Cutotal) log(Pbworm) = –1.553 +1.013 × log(Pbtotal) log(Pbworm) = –0.827 +1.496 × log(Pbtotal)
0.33 0.27 0.19 0.64
21 20 26 20
Zn Zn
Lrub Lrub
log(Znworm) = 0.690 × log(Zntotal) log(Znworm) = 2.418 × log(Zntotal) – 1.245 × log (CEC)
0.15 0.51
20 16
Me = metal; Meworm = accumulated metal by worm in mmol kg–1; Metotal = total metal concentration (aqua regia) in mmol kg–1; MeCaCl2 = metal concentration in 0.01 M CaCl2 in mmol kg–1. Only significant regressions and significant parameters are shown. Ench = Enchytraeus buchholzi; Hen = Henlea perpusilla; Frid = Fridericia ulrikae; Achl = Allolobophora chlorotica; Acal = Aporrectodea caliginosa; Lrub = Lumbricus rubellus.
centrations with pH, organic matter percentage and earthworm type, overestimated the accumulated concentrations for A. caliginosa and A. chlorotica, but underestimated those in L. rubellus (Fig. 4). For most data points the Cd and Zn extended equations underestimated or overestimated the accumulated concentration, respectively. 4. Discussion For the observed accumulation of metals by the organisms, we made a comparison with observations from other studies. The levels that we found for L. rubellus were, for all metals except Pb, higher than the levels found in worms from uncontaminated soil [22]. Zn concentrations in A. caliginosa in our study were higher than levels found in an uncontaminated soil (6.1–10.7 mmol kg–1) [23]. In the cases of A. chlorotica, and the three enchytraeid species, com-
parisons with accumulated metal concentrations in specimens sampled at uncontaminated sites could not be made, due to a lack of published data. Field and laboratory studies have shown that E. buchholzi accumulated cadmium, copper and zinc [24,25]. We can conclude that our data show that earthworms and enchytraeids accumulated metals above background levels in this moderately contaminated soil. Our observations confirm the published consensus that different species accumulate different quantities of metals. Hence, an attempt was made to relate exposure and metal accumulation to the habitat and behavior of the different species. L. rubellus is an epigeic species which is mostly present in the top layer of the soil, whereas A. caliginosa is an endogeic species which occurs mainly in the mineral layer. A. chlorotica is classified as endogeic species but often occurs in the top layer of the soil. Most Fridericia species can be classified as endogeics, while H. perpusilla and E. buchholzi
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Fig. 2. Mean accumulated concentrations (mmol kg–1) and standard errors for As, Cd, Cu, Cr, Pb and Zn for the three earthworm species combined by site and sampling date. Letters above bars denote significant (P < 0.05) differences between species.
can be classified as epigeic as well as endogeic [26]. In this study H. perpusilla was mainly found in the top layer and therefore considered an epigeic species. Higher total concentrations of heavy metals in the floodplain are found in the deeper layers (Table 1). We therefore expected endogeic species like A. caliginosa, F. ulrikae and possibly E. buchholzi, to accumulate larger amounts of metals than epigeic species like L. rubellus and H. perpusilla. Yet, all three enchytraeid species contained very high concentrations of copper, resulting in BSAF-values of around 12. At high concentrations copper is lethal for earthworms [27] and probably also for enchytraeids [28]. No toxicological endpoints measured in natural soil for the three
enchytraeid species are known. The EC50 value for copper (reproduction as endpoint) for Enchytraeus crypticus in artificial soil is 13.7 mmol kg–1 [29]. It is possible that copper concentrations in the floodplain soil (max 2.1 mmol kg–1) were far below the LC50 values for enchytraeids in general, so accumulation of copper occurred without causing major toxicological effects. Of the earthworms, L. rubellus contained the highest concentrations of Cu, Cr and Pb, although even these concentrations were still lower than in the surrounding soil. The endogeic enchytraeid species F. ulrikae contained similar concentrations of cadmium and lead as the epigeic earthworm L. rubellus. For earthworms as well as enchytraeids zinc concentrations in the worms
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Fig. 3. Mean accumulated concentrations (mmol kg–1) and standard errors for As, Cd, Cu, Cr, Pb and Zn for the three enchytraeid species combined by depths, sites and sampling dates. Letters above bars denote significant (P < 0.05) differences between species.
are similar to concentrations in the soil (the BSAF for zinc is about 1). For both taxa, zinc is an essential element and apparently the soil zinc concentrations are too low to induce a regulation mechanism. Earthworm and enchytraeid cadmium BSAF values were much higher than unity. Cadmium accumulation in earthworms may be associated with the induction of metallothioneins, as found in L. rubellus by Mariño et al. [30]. For enchytraeids, cadmium accumulation may be associated with Cd-binding proteins, as was found for E. buchholzi by Willuhn et al. [31]. The data at hand do not point to a strong relationship between total soil metal concentration, accumulated concentration, and the general habitat preferences of the different earthworm and enchytraeid
species. More detailed knowledge on species-specific physiological differences might enhance our understanding of these processes. Bioaccumulation of metals may provide an integrated picture of their bioavailability. In view of the relationship between total and available metal concentrations, we investigated the relationship between metal accumulation, metal availability and CEC. For almost all regressions the explanatory power remained quite low, which was probably due to the small range in metal concentrations, in pH, and in other soil/porewater properties present in the study sites. The equations for metal accumulation by earthworms developed by Ma [21] hardly explained any of the variation that we mea-
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Fig. 4. Comparison of measured metal (Cd, Cu, Pb, Zn) concentrations and concentrations calculated using the extended regression models published by Ma [21] for the three different earthworm species. Acal = A. caliginosa; Achl = A. chlorotica; Lrub = L. rubellus.
sured in the different earthworm species. Although the ranges in total metal concentrations, pH, and organic matter content were small in our sites, large differences in accumulated concentrations were found in the different species. We thus conclude that extrapolation of regression equations which are based on large broadrange datasets to small, limited-range datasets with fairly uniform edaphic properties must be done with great caution. Acknowledgements The authors like to thank Wim Ma and Jos Bodt of Alterra, Green World Research for the collection and metal analysis of the earthworms and Willem Menkveld for assisting with the sampling. We like to thank 2 anonymous reviewers for their comments. This work was financially supported by The Netherlands Science Foundation (NWO) in the framework of the Stimulation
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P.C.J. Van Vliet et al. / European Journal of Soil Biology 42 (2006) S117–S126 between intestinal and dermal uptake of metals in earthworms, Soil Biol. Biochem. 35 (2003) 125–132. A.M.N. Abdul Rida, M.B. Bouché, A method to assess chemical biorisks in terrestrial ecosystems, in: M.H. Donker, H. Eijsackers, F. Heimbach (Eds.), Ecotoxicology of Soil Organisms, Lewis, Boca Raton, 1994, pp. 383–394. A.M.N. Abdul Rida, M.B. Bouché, The eradication of an earthworm genus by heavy metals in southern France, Appl. Soil Ecol. 2 (1995) 45–52. J.E. Morgan, A.J. Morgan, Earthworms as biological monitors of cadmium, copper, lead and zinc in metalliferous soils, Environ. Pollut. 54 (1988) 123–138. B. Morris, A.J. Morgan, Calcium–lead interactions in earthworms: observations on Lumbricus terrestris L. sampled from a calcareous abandoned leadmine site, Bull. Environ. Contam. Toxicol. 37 (1986) 226–233. C.J. Langdon, T.G. Piearce, S. Black, K.T. Semple, Resistance of arsenic-toxicity in a population of the earthworm Lumbricus rubellus, Soil Biol. Biochem. 31 (1999) 1963–1967. D.J. Spurgeon, S.P. Hopkin, The development of genetically inherited resistance to zinc in laboratory-selected generations of the earthworm Eisenia fetida, Environ. Pollut. 109 (2000) 193–201. M. Heck, R.U.G. Weigmann, Blei- und Cadmiumbelastung vond Bodentieren in einem immissionsbeeinflussten Forst in der Nähe von Berling, Z. Ökologie u. Naturschutz 4 (1995) 75–85. M.B. Bouché, Strategies lombriciennes, in: U. Lohm, T. Persson (Eds.), Soil Organisms as Components of Ecosystems, Ecol. Bull. 25, 1977, pp. 122–132. J.E. Morgan, A.J. Morgan, Heavy metal concentrations in the tissues, ingesta and faeces of ecophysiologically different earthworm species, Soil Biol. Biochem. 24 (1992) 1691–1697. D.J. Spurgeon, S.P. Hopkin, The effects of metal contamination on earthworm populations around a smelting works: quantifying species effects, Appl. Soil Ecol. 4 (1996) 147–160. V.J.G. Houba, E.J.M. Temminghoff, G.A. Gaikhorst, W. van Vark, Extraction with 0.01 M CaCl2, Wageningen University, Wageningen, 1999. G.G.S. Holmgren, A rapid citrate-dithionite extractable iron procedure, Soil Sci. Soc. Am. J. 31 (1967) 210–211. V.J.G. Houba, J.J. van der Lee, I. Novozamsky, Soil Analysis Procedures. Other procedures (Soil and Plant Analysis, Part 5B), Wageningen University, Wageningen, 1997.
[19] F.B. O’Connor, Extraction of enchytraeid worms from a coniferous forest soil, Nature 175 (1955) 815–816. [20] D.J. Spurgeon, S.P. Hopkin, Comparisons of metal accumulation and excretion kinetics in earthworms (Eisenia fetida) exposed to contaminated field and laboratory soils, Appl. Soil Ecol. 11 (1999) 227–243. [21] W.-C. Ma, Estimating heavy metal accumulation in Oligochaete earthworms: a meta-analysis of field data, Bull. Environ. Contam. Toxicol. 72 (2004) 663–670. [22] W.-C. Ma, in: Regenwormen als bio-indicators van bodemverontreiniging, Ministerie van VROM, Den Haag, 1983, p. 111. [23] R.F.M.J. Cleven, J.A. Janus, J.A. Annema, W. Slooff, in: Integrated Criteria Document Zinc, RIVM, Bilthoven, 1993, p. 180. [24] U. Elzer, Subletale, Effekte von Blei, Cadmium und deren Kombination auf Enchytraeus buchholzi (Vejdovský 1879) under Laborbedingungen, Newsletter on Enchytraeidae 4 (1995) 63–67. [25] U. Rüther, H. Greven, The effect of heavy metals on enchytraeids. I. Uptake from an artificial substrate and influence on food preference, Acta Bio. Benrodis 2 (1990) 125–131. [26] U. Graefe, R.M. Schmelz, Indicator values, strategy types and life forms of terrestrial Enchytraeidae and other microannelids, Newsletter on Enchytraeidae 6 (1999) 59–67. [27] B. Streit, Effects of high copper concentrations on soil invertebrates (earthworms and oribatid mites): experimental results and a model, Oecologia 64 (1984) 381–388. [28] J. Willuhn, A. Otto, H. Koewius, F. Wunderlich, Subtoxic cadmium-concentrations reduce copper-toxicity in the earthworm Enchytraeus buchholzi, Chemosphere 32 (1996) 2205– 2221. [29] L. Posthuma, R. Baerselman, R.P.M.v. Veen, E.M.D.-v. Breemen, Single and joint toxic effects of copper and zinc on reproduction of Enchytraeus crypticus in relation to sorption of metals in soils, Ecotoxicol. Environ. Saf. 38 (1997) 108–121. [30] F. Mariño, S.R. Stürzenbaum, P. Kille, A.J. Morgan, Cu–Cd interactions in earthworms maintained in laboratory microcosms: the examination of a putative copper paradox, Comp. Biochem. Physiol. 120 (1998) 217–223. [31] J. Willuhn, H.-P. Schmitt-Wrede, H. Greven, F. Wunderlich, Cadmium-induced mRNA encoding a nonmetallothionein 33kDa protein in Enchytraeus buchholzi (Oligochaeta), Ecotoxicol. Environ. Saf. 29 (1994) 93–100.
Original article
Accumulation of heavy metals by enchytraeids and earthworms in a floodplain P.C.J. Van Vlieta,*, W.A.M. Didden1, S.E.A.T.M. Van der Zeea, W.J.G.M. Peijnenburgb a
Department Soil Quality, Wageningen University, P.O. 8005, 6700 EC Wageningen, The Netherlands b National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Available online 20 September 2006
Abstract The river floodplain ‘Afferdense and Deestsche Waarden’ (ADW) in The Netherlands is diffusely contaminated with several heavy metals. It is, however, unclear whether this mixed contamination exerts any adverse ecotoxicological effects. In November 2000 and May 2001 a field survey was conducted in two areas in the ADW to collect a wide range of data concerning contamination levels, bioavailability, enchytraeids and earthworms and abiotic factors such as lutum and organic matter content, cation exchange capacity (CEC) and soil nutrient concentrations. Earthworms and enchytraeids were also analyzed for heavy metal content. At both sites arsenic and zinc were present in soil at relatively high concentrations (above the Dutch intervention value). In the two areas, both enchytraeids and earthworms accumulated metals. Fridericia ulrikae accumulated more cadmium than Enchytraeus buchholzi and Henlea perpusilla. The earthworm Lumbricus rubellus accumulated larger concentrations of Cr, Cu and Pb than Aporrectodea caliginosa and Allolobophora chlorotica. Dietary, physiological and behavioral characteristics may have contributed to these differences. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Floodplain; Heavy metals; Enchytraeids; Earthworms; Accumulation; Regression
1. Introduction Floodplains of Dutch rivers are moderately to heavily polluted with heavy metals and toxic organic compounds. This is mostly due to historical contamination in the 1960s and 1970s when contaminated suspended matter settled in the floodplains. Although the water quality has improved, there is still a certain level of contamination [1]. Contamination levels in Dutch * Corresponding author. Tel.: +31 317 48 2344; fax: +31 317 48 3766. E-mail address: [email protected] (P.C.J. Van Vliet). 1 Wim Didden sadly deceased on 28 April 2005.
floodplains are above background levels but below the intervention levels prompting remediation [2]. These sites are therewith under a ‘gray veil’ of toxicant stress, where local effects are not evident, possibly due to natural variation. Exposure of organisms to heavy metals in these systems depends on the total metal content, soil composition (in particular environmental variables such as pH, organic matter content, concentrations of competing ions, concentrations of complexing ligands in solution) and the characteristics of the organism itself [3]. It has long been recognized, that only a part of the present heavy metals is biologically available and that availability also depends on the organism of interest. In
1164-5563/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2006.09.005
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the case of enchytraeids and earthworms, a complication is that the exposure to heavy metals in soil involves both oral and dermal routes. It is plausible, that these exposure routes depend differently on the distribution of metals over the soil solution, the fractions adsorbed onto the solid phase, and those present as a chemical precipitate (e.g. [4,5]). In most research on toxic stress of invertebrates by metals, relatively high metal contamination levels in an artificial soil system have been considered. It is unclear whether the principles established by that research can be transferred to more moderate contamination levels as found in Dutch floodplain soils. The reason is that it is not evident whether the contamination levels in Dutch floodplain soils pose a toxic stress or whether perhaps other stressors are more important in affecting the population dynamics of resident organisms. Appreciable species-specific differences in the sensitivity of earthworms to heavy metals contamination have been demonstrated [6,7]. Different earthworm species accumulate different concentrations of heavy metals and do so at different rates when exposed to the same soil (e.g. [8,9]). Furthermore, they may exhibit different degrees of resistance to the adverse effects of contaminant exposure [10,11]. Not much is known about species-specific responses of enchytraeid species to metal concentrations in soil. A field study in contaminated forest sites in Germany found high concentrations of cadmium and lead in enchytraeids, with certain species accumulating higher concentrations than others [12]. Several factors may contribute to the observed differences in accumulation. According to their activities and distribution in soil, three groups of worms may be distinguished, i.e. (1) epigeics that process organic matter on or near the soil surface, (2) endogeics, which live in the mineral soil and feed on humus, and (3) anecics, which transfer materials between the soil and litter habi-
tats [13]. Differences in the food sources may induce differences in exposure if the heavy metal content or its availability differs between these sources. It has been observed for earthworms that selected food sources were exploited in different ways, due to differences in alimentary and/or excretory physiology [14]; furthermore physiological differences in the accumulation of metals by earthworms have been demonstrated [15]. In this paper, we consider the consequences of moderate heavy metal contamination in floodplain soils on three species of earthworms and of enchytraeids. Our aims were to assess the degree with which these organisms accumulate heavy metals in cases of moderate contamination, and to identify and understand speciesspecific differences in bioconcentration. With regard to the accumulation of heavy metals our scope was to determine whether it is possible, even at moderate metal contamination, to relate accumulation by these organisms with heavy metal concentrations and environmental conditions. 2. Material and methods 2.1. Site description The study area is the ‘Afferdensche and Deestsche Waarden’ (ADW) located along the river Waal (Fig. 1). Within this floodplain, two field sites were selected, which are called the Rijswaard and Deel3. The Rijswaard is located between the summer dyke and the river Waal, therefore the lower parts of this area are frequently flooded (about 3–4 times per year). Soil texture ranges from sand to sandy loam and loam. The site is predominantly grassland and grazed by young cattle during summer. Deel3 is located in the area between the summer and winter dykes. The area floods only if the summer dyke overflows (1–2 times per year). After flooding, the water subsides through evaporation and drainage. Texture of Deel3 varies from loam, to silty
Fig. 1. Map indicating the sampling sites in the floodplain ‘De Afferdensche en Deestsche Waarden’ (ADW) in The Netherlands (Scale = 1:50.000). The map to the left shows The Netherlands and the location of the ADW-area.
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clay loam and clay loam. This site includes grassland and is grazed by horses throughout the year, except when the area is flooded. 2.2. Sampling times Sampling took place in November 2000 and May 2001. In Deel3 a transect of eight sampling points, 25 m apart, was sampled. In the Rijswaard a transect of 46 sampling points, 15 m apart, was sampled. At each sampling point, samples for soil chemistry and soil texture determination were collected for the 0–10 and 10–20 cm layers. At each sampling point in Deel3 and every fourth point in the Rijswaard two soil pits of 20 × 20 × 20 cm were excavated for earthworm sampling. From each pit, the earthworms were collected by handsorting and taken back to the laboratory for identification and metal analysis. Enchytraeid samples were taken to a depth of 20 cm and split into two depths (0–10 and 10–20 cm). In Deel3 this was done at each point, and in the Rijswaard at every second point. An additional 0–10 cm sample was also taken from the intermediate points in the Rijswaard. 2.3. Soil chemical analyses After extraction with a 0.01 M CaCl2 solution, pH, Dissolved Organic Carbon (DOC), and available heavy metals (As, Cd, Cr, Cu, Pb, Zn) were determined according to Houba et al. [16]. DOC was measured with a fully automated Skalar TOC/DOC analyzer. Total concentrations of the heavy metals (Cd, Cr, Cu, Pb and Zn) and the element As were determined in an aqua regia extract. The elements As, Cd, Cu, Pb, and Zn were measured with a Perkin–Elmer Elan 6000 ICPMS. The element Cr was measured with a Spectroflame–Spectro ICP-AES. For statistical analysis concentrations were expressed as mmol kg–1 dry weight of soil. Free aluminum and iron compounds, such as goethite and gibbsite were extracted using the Holmgren citrate-dithionite method [17]. Amorphous iron and aluminum were extracted in an ammonium oxalate solution [18]. Extracts were measured with a Spectroflame–Spectro ICP-AES. Total carbon in the soil samples was determined with a Fisons Instruments EA 1108 CHNS-O analyzer, using a Porapak QS column with He as carrier gas. The organic matter content of the soil was calculated from the organic carbon content using a conversion factor of 1.72 [18]. Clay content was determined by sieve and pipette, unbuffered 0.01 M BaCl2 was used to deter-
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mine the cation exchange capacity (CEC) [18]. In November 2000, soil pore water was collected from the collected soil at both sites by centrifugation, and was analyzed for heavy metals and DOC in the laboratory. Concentrations of heavy metals in pore water were expressed in μmol l–1. 2.4. Biological analyses Enchytraeid samples were stored at 4 °C until extraction. For the extraction (modified O’Connor method [19]) each soil sample was split into four layers of 2.5 cm each. Total number of enchytraeids per sample and per depth increment was determined. All live specimens were identified to species. Mature enchytraeid specimens were frozen separately in 96-well plates. Sufficient material was needed for the internal metal analysis. The most common species found were Fridericia ulrikae, Henlea perpusilla and Enchytraeus buchholzi. Several specimens of these species were found in each sample. For internal metal analysis, specimens were pooled by species, sample and sampling depth, and freeze-dried. The freeze-dried material was weighed into Pyrex destruction tubes, to which concentrated nitric acid and perchloric acid were added. The samples were heated at 170 °C until the nitric and perchloric acids were completely evaporated. Samples were dissolved in 750 μl of 15 mmol HNO3 and worm metal concentrations in the digests were measured for As, Cd, Cr, Cu, Pb and Zn using an ICPMS Elan6000. Identified mature earthworms of the species Lumbricus rubellus, Aporrectodea caliginosa and Allolobophora chlorotica were put on filter paper (Schleicher and Schuell 589) to defecate for 48 hours at 15 °C in the dark before being freeze-dried. Earthworms were destructed with the aqua regia method. Concentrations of the elements As, Cd, Cr, Cu, Pb and Zn in the extracts were determined with an ICP-AES. Metal concentrations in earthworms and enchytraeids were expressed on a mmol kg–1 dry weight basis. For the earthworm and enchytraeid species the biotato-soil accumulation factor (BSAF) was calculated. The BSAF is defined as the ratio of the concentration of the metal in the worm tissue and the total concentration of the metal in the soil. 2.5. Statistical analysis SAS/STAT version 8.0 was used for statistical analysis. Before analysis, all site parameters were checked for normality and were transformed if necessary. Out-
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liers (values larger than the mean of the total dataset ± 3 times the standard deviation) were removed from the dataset. One-way ANOVA and t-tests were used to determine significant differences between worm species. Using log-transformed regression analysis the accumulated concentration of heavy metals by the different earthworm and enchytraeid species was expressed as a function of one or more of the following parameters: total metal concentration, available metal concentration and CEC. Regressions were determined for all the data split by species. Concentrations of metals in earthworms were associated with parameter values for the depth at which the earthworms were mostly found in the field. We found A. caliginosa mainly at the 10–20 cm depth, and A. chlorotica and L. rubellus mainly in the 0–10 cm depth increment. Concentrations of metals in the enchytraeid species were associated with the metal concentrations at the depth they were found. The multivariate regressions have the format of: logðMewormÞ ¼ intercept þ a logðAÞ þ b logðCECÞ
3. Results 3.1. Soil characteristics At both sites the concentrations of heavy metals were lowest in the top layer (Table 1). At both sites, concentrations of arsenic and zinc in the 10–20 cm layer were higher than the intervention values set by the Dutch government [2]. Other heavy metals were present in concentrations above the target values [2] but mostly far below the intervention values. Total metal concentrations in the floodplain differed at most by a factor (maximum over minimum concentration) of 14 (Pbtotal = Pb concentration in soil determined by aqua regia) (Table 2). The range in concentrations of As and the heavy metals was very small, implying that variation within the floodplain was limited. The CEC correlated strongly with other soil properties like aluminum and iron hydroxides, clay content and organic matter content (Table 3). This soil parameter is therefore used in the regression analysis. 3.2. Earthworms and enchytraeids
or logðMewormÞ ¼ intercept þ a logðBÞ where, Meworm is the concentration of metal accumulated by the worm in mmol kg–1; A is the total metal concentration (aqua regia) in mmol kg–1; CEC is the cation exchange capacity (cmol(+) kg–1); B is the metal concentration in 0.01 M CaCl2 in mmol kg–1 or in pore water extract in μmol l–1; a and b are the corresponding coefficients. Only significant (P < 0.05) parameters are mentioned. Significance of the regression equation was judged by R2adjusted, which accounts for the number of observations and the number of parameters used, and is an indicator of the variation in the data that is explained by the model.
Average accumulated metal concentrations in earthworms and enchytraeids are shown in Figs. 2 and 3. Concentrations of cadmium in all earthworm and enchytraeid species are higher than the soil concentrations present. Cadmium is a non-essential element with a relatively long half-life [4,20], resulting in high BSAF-values ranging from 4 to 12. A. chlorotica accumulated significantly more As than the other two earthworm species and had the lowest accumulated concentrations of heavy metals (except Cd). L. rubellus accumulated significantly more Cu, Cr, and Pb than A. caliginosa and A. chlorotica (Fig. 2). Accumulated copper concentrations in the three enchytraeid species were much higher than the corresponding soil concentrations, resulting in BSAF values of about 12. H. perpusilla accumulated more Zn and As
Table 1 The mean total arsenic and heavy metal concentrations expressed as a ratio of the corresponding Dutch target values [2] at the two sites and two depths. Determined in November 2000. I/S = Dutch intervention value divided by the Dutch target value for standard soil containing 25% lutum and 10% organic matter. Letters within a column denote significant (P < 0.05) different metal concentrations in the two depths per site. Bold values indicate values larger than the I/S value. Site Rijswaard Deel3 I/S
Depth (cm) 0–10 10–20 0–10 10–20
As 1.4 2.1 1.6 2.5 1.9
a b a b
Cd 3.2 a 5.0 b 4.8 5.4 12
Cu 1.9 a 2.6 b 2.4 2.8 5.3
Cr 1.3 a 1.8 b 1.3 1.3 3.8
Ni 1.3 a 1.5 b 1.3 1.4 6
Pb 1.7 2.6 2.6 3.8 6.2
a b a b
Zn 4.2 5.7 4.6 6.5 5.1
a b a b
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Table 2 Mean, standard deviation, minimum and maximum values of the composite data set for the two sites, two depths and two sampling dates. Total metal concentrations (Metotal) determined in November 2000. Available (CaCl2) metal concentrations determined in November 2000 and May 2001. Porewater metal concentrations determined in November 2000. Variable pH %lutum CEC Days flooded DOC C Astotal Cdtotal Crtotal Cutotal Pbtotal Zntotal As-CaCl2 Cd-CaCl2 Ni-CaCl2 Pb-CaCl2 Cr-CaCl2 Cu-CaCl2 Zn-CaCl2 As-porewater Cd-porewater Cr-porewater Cu-porewater Pb-porewater Zn-porewater
Unit
Mean 7.33 16.04 20.28 25.40 1.55 × 5.40 6.35 × 2.92 × 2.26 × 1.18 × 9.67 × 9.10 × 0.64 × 0.10 × 0.40 × 0.01 × 0.19 × 3.07 × 1.65 × 0.08 × 0.00 × 0.12 × 0.53 × 0.01 × 0.50 ×
percentage cmol(+) kg–1 mol C kg–1 percentage mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol kg–1 mol l–1 mol l–1 mol l–1 mol l–1 mol l–1 mol l–1
10–2 10–4 10–5 10–3 10–3 10–4 10–3 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
Table 3 Correlations (Pearson coefficients) between CEC (cmol(+) kg–1), and soil parameters, aluminum and iron amorphous hydroxides (Alox and Feox, respectively), aluminum and iron crystalline hydroxides (Alcd and Fecd, respectively), % soil carbon (%C), and %lutum (%L), at the two sites. Both depth increments were included and combined in the analysis. Sampling date = November 2000. *** = P < 0.001; **** = P < 0.0001 Sites Parameter Alcd Alox Fecd Feox %C %L
0.68 0.64 0.73 0.49 0.59 0.67
Rijswaard **** **** **** *** *** ****
0.88 0.84 0.86 0.73 0.93 0.87
Deel3 **** **** **** *** **** ****
than the two other enchytraeid species. F. ulrikae contained the highest concentrations of Cd and Pb (Fig. 3). Regressions between the accumulated metal concentrations by the earthworm and enchytraeid species and total soil metal concentrations, available metal concentrations (0.01 M CaCl2), porewater metal concentrations and CEC were derived. Significant regressions are shown in Table 4. R2adjusted values were quite small for all regressions; with a minimum of 0.10 (Cr
Stdev 0.19 8.00 7.75 29.56 9.90 × 1.59 2.63 × 1.15 × 5.90 × 4.18 × 4.46 × 3.39 × 0.27 × 0.04 × 0.29 × 0.02 × 0.20 × 1.80 × 1.22 × 0.05 × 0.00 0.05 × 0.20 × 0.01 × 0.26 ×
10–3 10–4 10–5 10–4 10–4 10–4 10–3 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
Min 6.88 0.31 7.06 0 2.65 1.70 1.27 4.45 6.25 2.38 1.41 2.20 0.06 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.24 0.00 0.16
× 10–3 × × × × × × ×
10–4 10–6 10–4 10–4 10–4 10–3 10–6
× 10–6
× 10–6 × 10–6 × 10–6
Max 7.83 34.00 34.97 78 4.07 × 8.80 1.21 × 5.42 × 3.31 × 2.06 × 1.98 × 1.70 × 1.26 × 0.19 × 2.11 × 0.11 × 1.15 × 8.76 × 4.59 × 0.23 × 0.02 × 0.34 × 1.15 × 0.03 × 1.36 ×
10–2 10–3 10–5 10–3 10–3 10–3 10–2 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6 10–6
and Cu, E. buchholzi) and a maximum of 0.64 (Pb, L. rubellus). CEC was the only variable that significantly improved the regression equation in the case of zinc and the earthworm species L. rubellus (R2adjusted increased from 0.15 to 0.51). Using available concentrations (0.01 M CaCl2) instead of total soil metal concentrations as the independent variable in the regression increased the fit of the Cu-regression equations for E. buchholzi and F. ulrikae, by a factor of 2 and 2.4, respectively. Due to the small range in total soil metal concentrations the derived equations cannot be extrapolated to encompass higher soil metal concentrations. Therefore we used regressions developed for cadmium, copper, lead and zinc by Ma [21] to predict the amount of metals accumulated by earthworms on basis of total concentrations (simple equation), as well as total concentrations combined with pH, organic matter percentage, and earthworm type (extended equation). Our data were not modeled accurately using the simple regression equations. For cadmium, lead and zinc the calculated concentrations were much higher than the measured concentrations (data not shown). The extended Pb regression equation, combining total soil metal con-
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Table 4 Regressions for the different metals and As between accumulated metals by enchytraeids and earthworms, soil (total/available/porewater) metal concentrations and CEC (sites, sampling dates and depths combined). Metal Enchytraeids Cr Cr
Species
Model
R2adjusted
n
Ench Hen
log(Crworm) = 0.429 × log(CrCaCl2) log(Crworm) = 0.353 × log(CrCaCl2)
0.10 0.11
36 26
Cu Cu Cu Cu
Ench Ench Frid Frid
log(Cuworm) = 1.149 – 0.916 × log(Cutotal) log(Cuworm) = –0.817 × log(CuCaCl2) log(Cuworm) = 1.139 – 1.188 × log(Cutotal) log(Cuworm) = –0.924 × log(CuCaCl2)
0.10 0.21 0.12 0.29
36 36 24 24
Zn
Frid
log(Znworm) = 1.793 – 0.966 × log(Zntotal)
0.14
24
Earthworms As
Achl
log(Asworm) = –0.194 + 0.825 × log(Astotal)
0.12
26
Cd Cd
Achl Achl
log(Cdworm) = 0.551 × log(Cdtotal) log(Cdworm) = 0.636 × log(CdCaCl2)
0.21 0.16
26 26
Cr
Achl
log(Crworm) = –2.002 – 0.151 × log(CrCaCl2)
0.18
26
Cu Cu Pb Pb
Acal Lrub Achl Lrub
log(Cuworm) = –0.479 + 0.563 × log(Cutotal) log(Cuworm) = –0.345 + 0.535 × log(Cutotal) log(Pbworm) = –1.553 +1.013 × log(Pbtotal) log(Pbworm) = –0.827 +1.496 × log(Pbtotal)
0.33 0.27 0.19 0.64
21 20 26 20
Zn Zn
Lrub Lrub
log(Znworm) = 0.690 × log(Zntotal) log(Znworm) = 2.418 × log(Zntotal) – 1.245 × log (CEC)
0.15 0.51
20 16
Me = metal; Meworm = accumulated metal by worm in mmol kg–1; Metotal = total metal concentration (aqua regia) in mmol kg–1; MeCaCl2 = metal concentration in 0.01 M CaCl2 in mmol kg–1. Only significant regressions and significant parameters are shown. Ench = Enchytraeus buchholzi; Hen = Henlea perpusilla; Frid = Fridericia ulrikae; Achl = Allolobophora chlorotica; Acal = Aporrectodea caliginosa; Lrub = Lumbricus rubellus.
centrations with pH, organic matter percentage and earthworm type, overestimated the accumulated concentrations for A. caliginosa and A. chlorotica, but underestimated those in L. rubellus (Fig. 4). For most data points the Cd and Zn extended equations underestimated or overestimated the accumulated concentration, respectively. 4. Discussion For the observed accumulation of metals by the organisms, we made a comparison with observations from other studies. The levels that we found for L. rubellus were, for all metals except Pb, higher than the levels found in worms from uncontaminated soil [22]. Zn concentrations in A. caliginosa in our study were higher than levels found in an uncontaminated soil (6.1–10.7 mmol kg–1) [23]. In the cases of A. chlorotica, and the three enchytraeid species, com-
parisons with accumulated metal concentrations in specimens sampled at uncontaminated sites could not be made, due to a lack of published data. Field and laboratory studies have shown that E. buchholzi accumulated cadmium, copper and zinc [24,25]. We can conclude that our data show that earthworms and enchytraeids accumulated metals above background levels in this moderately contaminated soil. Our observations confirm the published consensus that different species accumulate different quantities of metals. Hence, an attempt was made to relate exposure and metal accumulation to the habitat and behavior of the different species. L. rubellus is an epigeic species which is mostly present in the top layer of the soil, whereas A. caliginosa is an endogeic species which occurs mainly in the mineral layer. A. chlorotica is classified as endogeic species but often occurs in the top layer of the soil. Most Fridericia species can be classified as endogeics, while H. perpusilla and E. buchholzi
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Fig. 2. Mean accumulated concentrations (mmol kg–1) and standard errors for As, Cd, Cu, Cr, Pb and Zn for the three earthworm species combined by site and sampling date. Letters above bars denote significant (P < 0.05) differences between species.
can be classified as epigeic as well as endogeic [26]. In this study H. perpusilla was mainly found in the top layer and therefore considered an epigeic species. Higher total concentrations of heavy metals in the floodplain are found in the deeper layers (Table 1). We therefore expected endogeic species like A. caliginosa, F. ulrikae and possibly E. buchholzi, to accumulate larger amounts of metals than epigeic species like L. rubellus and H. perpusilla. Yet, all three enchytraeid species contained very high concentrations of copper, resulting in BSAF-values of around 12. At high concentrations copper is lethal for earthworms [27] and probably also for enchytraeids [28]. No toxicological endpoints measured in natural soil for the three
enchytraeid species are known. The EC50 value for copper (reproduction as endpoint) for Enchytraeus crypticus in artificial soil is 13.7 mmol kg–1 [29]. It is possible that copper concentrations in the floodplain soil (max 2.1 mmol kg–1) were far below the LC50 values for enchytraeids in general, so accumulation of copper occurred without causing major toxicological effects. Of the earthworms, L. rubellus contained the highest concentrations of Cu, Cr and Pb, although even these concentrations were still lower than in the surrounding soil. The endogeic enchytraeid species F. ulrikae contained similar concentrations of cadmium and lead as the epigeic earthworm L. rubellus. For earthworms as well as enchytraeids zinc concentrations in the worms
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Fig. 3. Mean accumulated concentrations (mmol kg–1) and standard errors for As, Cd, Cu, Cr, Pb and Zn for the three enchytraeid species combined by depths, sites and sampling dates. Letters above bars denote significant (P < 0.05) differences between species.
are similar to concentrations in the soil (the BSAF for zinc is about 1). For both taxa, zinc is an essential element and apparently the soil zinc concentrations are too low to induce a regulation mechanism. Earthworm and enchytraeid cadmium BSAF values were much higher than unity. Cadmium accumulation in earthworms may be associated with the induction of metallothioneins, as found in L. rubellus by Mariño et al. [30]. For enchytraeids, cadmium accumulation may be associated with Cd-binding proteins, as was found for E. buchholzi by Willuhn et al. [31]. The data at hand do not point to a strong relationship between total soil metal concentration, accumulated concentration, and the general habitat preferences of the different earthworm and enchytraeid
species. More detailed knowledge on species-specific physiological differences might enhance our understanding of these processes. Bioaccumulation of metals may provide an integrated picture of their bioavailability. In view of the relationship between total and available metal concentrations, we investigated the relationship between metal accumulation, metal availability and CEC. For almost all regressions the explanatory power remained quite low, which was probably due to the small range in metal concentrations, in pH, and in other soil/porewater properties present in the study sites. The equations for metal accumulation by earthworms developed by Ma [21] hardly explained any of the variation that we mea-
P.C.J. Van Vliet et al. / European Journal of Soil Biology 42 (2006) S117–S126
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Fig. 4. Comparison of measured metal (Cd, Cu, Pb, Zn) concentrations and concentrations calculated using the extended regression models published by Ma [21] for the three different earthworm species. Acal = A. caliginosa; Achl = A. chlorotica; Lrub = L. rubellus.
sured in the different earthworm species. Although the ranges in total metal concentrations, pH, and organic matter content were small in our sites, large differences in accumulated concentrations were found in the different species. We thus conclude that extrapolation of regression equations which are based on large broadrange datasets to small, limited-range datasets with fairly uniform edaphic properties must be done with great caution. Acknowledgements The authors like to thank Wim Ma and Jos Bodt of Alterra, Green World Research for the collection and metal analysis of the earthworms and Willem Menkveld for assisting with the sampling. We like to thank 2 anonymous reviewers for their comments. This work was financially supported by The Netherlands Science Foundation (NWO) in the framework of the Stimulation
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