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Horizontal distribution of copper, nickel and enchytraeid worms in polluted soil
ENVIRONMENTAL POLLUTION
Environmental Pollution 104 (1999) 351±358
Horizontal distribution of copper, nickel and enchytraeid worms in polluted soil J. Salminen a,*, J. Haimi b a
Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FIN-15210 Lahti, Finland Department of Biological and Environmental Science, University of JyvaÈskylaÈ, PO Box 35, FIN-40351 JyvaÈskylaÈ, Finland
b
Received 19 May 1998; accepted 8 October 1998
Abstract We studied the horizontal distribution of Cu, Ni and enchytraeid worms (Cognettia sphagnetorum, Vejdovsky, Oligochaeta, Enchytraeidae) in forest soil near a Cu±Ni smelter in SW Finland. Soil samples were taken from a polluted site (2 km from the smelter) and a reference area (8 km from the smelter). We used a hierarchical sampling design in the polluted area for studying possible scale-dependent variability in parameters measured, distance between the samples (dierent scales) being 5, 50 and 500 cm. Distribution of metals was moderately heterogeneous in the polluted soil; coecient of variances (CV), 26% for Cu and 32% for Ni. Instead, distribution of enchytraeids in the area was highly heterogeneous (CV, 135%) and the variability increased with increasing a distance between the samples: CV increased from 67 to 104% from the smallest to the largest sampling scale. Soil metal concentrations did not correlate with enchytraeid densities in the polluted area. However, three out of our four sampling plots having distance of 500 cm between each other had lower enchytraeid density than the reference area. Results may indicate that C. sphagnetorum has quite low sensitivity to metal pollution, at least on the forest stand scale. On the other hand, enchytraeids may have population dynamics connected to patches (sources±sinks) caused by uneven distribution of metals. This can mitigate the eects of metals on their population densities. Due to importance of enchytraeids in decomposition processes and the fact that usually only one enchytraeid species is present in boreal coniferous forests, they may be appropriate for ®eld monitoring in these humus rich soils. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heavy metals; Enchytraeid worm; Heterogeneous distribution; Forest soil; Field monitoring
1. Introduction Usually both abiotic (e.g. porosity, nutrients and pH) and biotic (species composition and population densities) properties of soils vary in a small scale (in centimeters). Thus, soils tend to have patchy habitats for soil dwelling decomposer organisms (Swift et al., 1979). On the other hand, soil organismsÐperhaps excluding earthworms (Mather and Christensen, 1992) and macroarthropodsÐhave low capability to disperse long distances by active locomotion (Usher, 1985). Thus, when a species has became extinct from a patch, it may take a long time before the patch is recolonized (Siepel, 1994). Little is known about passive dispersal of soil animals
* Corresponding author. Department of Ecology and Ecotoxicology, Vrije Universiteit, De Boelelaan 1087, HV Amsterdam, The Netherlands. Tel.: +31-20-4447075; fax: +31-20-4447123; e-mail: [email protected].
by the aid of, for example, water and other animals, but it may help the recolonization. Anthropogenic pollutants, such as heavy metals, may also become heterogeneously distributed in the ®eld soils (in this paper we are dealing only with horizontal variability). Properties of both emission source and recipient environment can cause this variability. Once the metals have deposited, amount and quality of organic matter, pH and soil water content aect sorption and desorption processes of heavy metals and thus their accumulation to biota (Hopkin, 1989). Vegetation may also accumulate metals and hence partly aect their distribution in the soil (metals returning to the soil surface in dead plant material). Field studies on the eects of heavy metals on soil organisms have mainly been done by comparing samples taken at dierent distances from emission points. It has been assumed that metals are more or less evenly distributed within each distance zone. However, there are some studies in which consequences of small-scale
0269-7491/99/$Ðsee front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0269 -7 491(98)00198 -5
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spatial variation in soil metal concentration and subsequent eects on soil organisms have been discussed (Bengtsson et al., 1994; SjoÈgren et al., 1995). If uneven distribution of heavy metals and/or soil organisms takes place, reliability (i.e. accuracy) of monitoring the eects of pollutants on organisms can be low, especially when based on a limited number of samples (Bengtsson and Torstensson, 1988). Thus, when eects of pollutants are monitored in the ®eld, natural patchiness of the soil environment and soil-living organisms should be taken into account. However, we found no studies especially focused on small-scale spatial variability of metals and organisms in the soil. Monitoring ecological eects of soil pollution in the ®eld via changes in soil animal populations does not have a long tradition (van Straalen and Verhoef, 1997). One possible constraint in using soil organisms for monitoring purposes could arise from taxonomical problems, hundreds of species being found in each square meter. However, an enchytraeid worm Cognettia sphagnetorum, Vejdovsky (Oligochaeta, Enchytraeidae) is usually the only abundant enchytraeid species found in northern coniferous forest soils (Nurminen, 1967; Huhta et al., 1986). C. sphagnetorum is a white 1-cm long (adult) species which reproduces asexually by fragmentation (Christensen, 1959; Standen, 1973). It has been shown to be an important species in decomposition and nutrient cycling processes (Standen, 1978; Abrahamsen, 1990). Enchytraeids are known to be sensitive to many anthropogenic disturbances including contamination by heavy metals (Bengtsson and Rundgren, 1982; SjoÈgren et al., 1995). Although it is normally found that enchytraeids exhibit clustered distribution in the ®eld soils (Didden, 1993), Bengtsson and Rundgren (1982) observed that enchytraeids seemed not to be as much aggregated as other soil animals. They concluded that due to their moderately homogeneous spatial distribution, enchytraeids are optimal organisms for monitoring purposes. Moreover, enchytraeids are to be taken into toxicity test protocols as terrestrial decomposer animals (LeÂon and Van Gestel, 1994; Augustsson and Rundgren, 1998). Hence, risk assessment based on standardized toxicity tests and ®eld data on enchytraeids may be reasonable. Here we report results from a ®eld sampling carried out near a Cu±Ni smelter in SW Finland. Concentrations of Cu and Ni, and enchytraeid densities in the soil were determined from each sample. Total number of samples was kept as low as possible (36 samples plus 5 samples from a reference site) because we wanted to estimate the relevance of a pro®table sampling procedure done for monitoring purposes. Hierarchical sampling design was used in order to observe possible scaledependent heterogeneity in the parameters measured (distances between the samples varied from 5 to 500 cm). Our hypothesis was that if the parameters measured
are unevenly distributed, increased distance between samples increases the variability (Bell et al., 1993). Size of an individual soil sample was supposed to form a movement area of an individual worm (15.6 cm; SjoÈgren et al., 1995). Finally, we also discuss the usefulness of enchytraeid (C. sphagnetorum) density as a ®eld monitoring tool in ecological risk assessment of contaminants. 2. Study site Sampling was conducted in metal polluted and unpolluted areas near the town of Harjavalta, Finland (61 190 N 22 90 E). The Cu±Ni smelter has been operated in the area since 1945. Soils near the smelter are polluted with Cu, Ni, Zn and Fe and there is a clear decreasing gradient of soil pollution from the smelter. The gradient is also found in a visually homogeneous Scots pine (Pinus sylvestris) forest growing on podsolic soil pro®le. Drastic changes in vegetation, soil micro¯ora, fauna and physico-chemical properties of the soil have been observed along the increasing pollution gradient (Laaksovirta and Silvola, 1975; Fritze et al., 1989; Vanhala and Ahtiainen, 1994; Haimi and SiiraPietikaÈinen, 1996) as follows: (1) decrease in ground layer vegetation biomass and diversity, (2) decrease in soil microbial biomass and activity and (3) increase in thickness of organic soil layer when comparing areas 8 and 2 km from the pollution source, that is, our polluted and reference sites, respectively. More details about the study area can be found in Haimi and SiiraPietikaÈinen (1996) and references therein. 3. Materials and methods 3.1. Sampling and analyses Sampling was done on 23 October 1996 at distances of 2 and 8 km from the Cu±Ni smelter. At the 2-km site (the polluted site) soil samples were taken according to a hierarchical sampling design where distances of individual samples were controlled. Distance between three individual samples was 5 cm in the small scale, 50 cm in the medium scale (three plots having small-scale samples inside) and 500 cm in the largest scale (four plots, each of them having three medium-scale plots inside) (Fig. 1; for more details, see Section 3.2). From the reference site, ®ve soil samples were taken, the distances between the samples being more than 1 m. Soil samples (organic soil layer only, thickness varied from 3 to 5 cm) were taken with a steel corer (sample area was 0.0025 m2). Enchytraeids were extracted from the soil with wet funnels (O'Connor, 1962) and counted under a microscope (only C. sphagnetorum was found). After
J. Salminen, J. Haimi / Environmental Pollution 104 (1999) 351±358
Fig. 1. A scheme of the sampling design (note hierarchical relationship of the samples) and how coecients of variation (CV) were calculated for samples taken from certain distance between them. Black dots are individual soil samples 1±9 taken within plots A, B, C and D in the polluted area. 5, 50 and 500 cm are the distances between samples.
the extraction, soil samples were oven dried (60 C) and homogenised in a mortar. Total Cu and Ni concentrations were analysed from the samples with atomic absorption spectroscopy after nitrogen acid extraction. Other heavy metals were not analyzed in this study because their in¯uence on soil animals was considered negligible (Fritze et al., 1989; Hopkin, 1989). Metals in the water of wet funnels were analysed after animal extraction: 0.24 mg literÿ1 Cu (SD: 0.205) and 0.11 mg literÿ1 Ni (SD: 0.064) were found in the water. The concentrations meant that 0.1% (Cu) and 0.4% (Ni) of total metal content in the samples were leached before metal analyses. Subsamples of the homogenised samples were burned in an oven (550 C, 5 h) for analysing organic matter content of the soil. 3.2. Data analyses Spearman correlations between Cu and Ni concentrations, organic matter content and enchytraeid densities in the soil within the polluted site were calculated. Student's t-test was used to discover overall differences between the reference and polluted sites in the parameters measured. For analysing possible dierences between the reference site (®ve replicates) and mediumscale plots at the polluted site (four plots A, B, C and D, nine replicates per plot, see Fig. 1) ANOVA was used. Data were normally distributed (Kolmogorow± Smirnov's test). Before ANOVAs, homogeneities of variances were tested with Bartlett-Box's test. Due to heterogeneous variances, data of enchytraeid density were log10+1 transformed. Simple contrast method (with Bonferroni adjusted p-values) was used to compare certain large-scale plots (plots A, B, C and D) in the polluted area to the reference site. Possible scale-dependent variability in the parameters measured within the polluted site was estimated by
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calculating coecient of variances (CV, as percentage) within three samples having constant mutual distances. There were three distance categories: 5, 50 and 500 cm between the samples (Fig. 1). We decided to keep n=12 in all scales. Hence, in the largest scale (500 cm), we randomly chose 12 combinations of the samples from all possible combinations (all nine samples of each mediumscale subplot were used only once in the lottery). Possible dierences in CV between the sampling scales were tested with ANOVA and pairwise comparisons were made by Tukey's HSD test (the variances were homogeneous and the data were normally distributed). Statistical analyses were performed with SPSS for Windows 6.1 statistical package (SPSS, Chicago, IL). 4. Results In the polluted area, sample speci®c Cu concentrations varied between 630 and 2900 mg kgÿ1 of dry soil (mean: 2084 mg kgÿ1; Fig. 2). Ni concentrations varied between 87 and 460 mg kgÿ1 of dry soil (mean: 298 mg kgÿ1; Fig. 3). At the reference site, metal concentrations in the soil were 64 mg kgÿ1 (mean; range: 16±170) and 13 mg kgÿ1 (mean; range: 6±28) for Cu and Ni, respectively (Figs. 2 and 3). Both metal concentrations were signi®cantly higher in the polluted area than at the reference site (t-test, p<0.001 for both metals). In the polluted area, organic matter content of the samples was signi®cantly higher (mean: 71%) than at the reference site (mean: 11%; t-test, p<0.001; Fig. 4). Numbers of enchytraeids did not dier signi®cantly between the sites. There were 27,360 individuals mÿ2 (range: 9200±88,000; n=5) at the reference site and 8489 mÿ2 (range: 400±53,200; n=36) at the polluted site (t-test, p=0.284; Fig. 5). However, when using mediumscale subplots in the polluted area as individual sampling sites and comparing them (Fig. 1) to the reference site in ANOVA, the number of enchytraeids was signi®cantly lower in three medium-scale subplots (A, B and C) than at the reference site (ANOVA F=9.33, p<0.001, df=4 and post hoc contrast's p<0.05). Enchytraeid numbers at the site D did not dier from the numbers at the reference site (Fig. 5). Within the polluted site Cu and Ni concentrations were not correlated with the enchytraeid numbers, but strong correlations between the metal concentrations and soil organic matter content were observed. Enchytraeid numbers were not correlated with soil organic matter content (Table 1). Increasing distance between the samples did not signi®cantly increase CV in the case of soil metal concentrations (ANOVA, p=0.468 for Cu and p=0.214 for Ni; Fig. 6). However, in enchytraeid numbers there was an increasing variation with increasing distance between the samples (ANOVA, F=5.321, p=0.010, df=2).
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Fig. 2. Total Cu concentrations in the soil samples taken at certain distances from a Cu±Ni smelter in SW Finland. `Reference' is the sampling site 8 km from the smelter. Sites A, B, C and D are `medium-scale' (see Fig. 1) samples taken at the distance of 2 km from the smelter. Numbers 1±3, 4±6 and 7±9 refer to the `small-scale' samples.
Fig. 3. Total Ni concentrations in the soil samples taken near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2.
5. Discussion 5.1. Distribution of metals in soil and their eects on enchytraeids Neither Cu nor Ni was as heterogeneously distributed as enchytraeids in the contaminated forest soil studied here (CV was 26% for Cu, 33% for Ni and 135% for enchytraeids for all samples). Variation in soil metal concentrations was lower than assumed on the basis of high variability found in `natural' soil properties (e.g. nitrogen and water content of the soil), especially in forests (Kleb and Wilson, 1997). Possibly, long-term airborne pollution from the smelter has `saturated' the soil by metals, or biological properties of recipient area (canopies of trees, ground layer vegetation, etc.) have
not aected the distribution of metals as much as expected. Although overall variation is quite small in soil metal concentrations, there are patches where toxic eects on enchytraeids should occur (e.g. over 2000 mg Cu kgÿ1 soil) and also patches that are clearly less toxic (1000 mg Cu kgÿ1; Bengtsson and Tranvik, 1989). However, no correlations between population densities of enchytraeids and soil metal concentrations were found. It even seems that there are patches within the polluted area (site D with as high metal concentrations as the other sites) where enchytraeid numbers are as high as at the reference site. Metals may be heterogeneously distributed on a smaller scale than our smallest sampling scale (5 cm), and hence the size of our soil corer may be ecologically (as a foraging area of an individual enchytraeid worm)
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Fig. 4. Organic matter content of the soil samples taken near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2.
Fig. 5. Density of Cognettia sphagnetorum near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2. Table 1 Spearman correlation coecients between soil Cu and Ni concentrations, soil organic matter content and enchytraeid density (n=36)
Enchytraeid density Organic matter content Ni concentration
Cu concentration
Ni concentration
Organic matter content
0.073NS 0.614*** 0.826***
0.092NS 0.806*
0.229NS
NS, non-signi®cant, ***p<0.001.
too large, although behaviourally (escape from hostile area) it seemed reasonable (SjoÈgren et al., 1995). Anderson (1978) has shown that soil living microarthropods can have microhabitats in a scale of
millimeters (around a certain soil particle). However, enchytraeids are relatively big in size (smallest fragments are 0.2 cm and adults 1 cm) and they are capable of moving centimeters in a short time (SjoÈgren
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(presence and dimensions of patches and realized niches) has rarely been studied or described, neither in this study. Data on ®tness and movements of individual worms are lacking, too. Hence, more experiments are needed to show whether sink±source dynamic is present in polluted forest soils. There were positive correlations between soil metal concentrations and organic matter content. This relationship could be a consequence of decreased litter decomposition due to metal contamination (both biomass and activity of microbes have been reduced; Vanhala and Ahtiainen, 1994) and could lead to increased sorption of metals to accumulated litter and humus, and concomitant reduced metal bioavailability and toxicity to soil organisms (Ross, 1994). Lack of correlation between metals and enchytraeids may also indicate that factors other than direct toxicity of metals were controlling the enchytraeid abundance. It has been well documented that vegetation, biomass and activity of microbes, and physico-chemical properties of the soil have changed around the smelter studied here (Laaksovirta and Silvola, 1975; Fritze et al., 1989; Vanhala and Ahtiainen, 1994; Haimi and SiiraPietikaÈinen, 1996). Reduction in microbial food (enchytraeids assimilate microbes together with decaying plant material from the digested soil) and other changes in abiotic and biotic conditions in the soil (e.g. altered soil structure due to changes in litter quality) can indirectly reduce enchytraeid densities. It seems that changes in habitat characteristics, including possible patchiness, in moderate polluted habitats (not the toxicity of the compounds per se) have an important role in the survival of soil dwelling organisms (Posthuma and van Straalen, 1993). 5.2. Use of enchytraeids in ®eld monitoring
Fig. 6. Variability (coecient of variation as percentage, CV) of enchytraeid density (A), soil Cu concentration (B) and soil Ni concentration (C) in three samples taken from constant mutual distances between each other (mean and SD of CV). `All' represent CV calculated over all samples taken from the contaminated area. Note dierent scales on y-axis.
et al., 1995). The patches with lower concentrations can serve as source habitats (less toxic and more available resources) for more contaminated sink habitats (sensu Dias, 1996). Hence, enchytraeid densities in the contaminated area were able to be higher than expected. Unfortunately, real habitat structure of polluted soils
As was pointed out earlier, no direct correlations between soil metal concentrations and enchytraeid densities were found in our data. This observation is consistent with laboratory studies done by Augustsson and Rundgren (1998). They found that in contrast to sublethal responses, mortality of C. sphagnetorum was not a sensitive parameter to Cu contamination in soil with high organic matter content. Haimi and SiiraPietikaÈinen (1996) sampled the same sites as investigated in this study. They found large variation in enchytraeid densities within and between years, as well as spatially. There tended to be more enchytraeids at the site 8 km from the smelter compared to the 2-km site, but this was not true on every sampling occasion (Haimi and Siira-PietikaÈinen, 1996). Because of high enchytraeid densities in the site D, no statistically signi®cant dierence between the polluted and the reference site was observed in the present study when the polluted site was used as one independent variable in the
J. Salminen, J. Haimi / Environmental Pollution 104 (1999) 351±358
analysis. However, three out of the four mediumscale subplots had signi®cantly lower enchytraeid densities than the reference site, indicating that enchytraeids responded to the soil metal contamination. Without our sampling design with medium-scale subplots, the between-site dierences (polluted vs reference) might not have been statistically justi®ed. Contrary to earlier results by Bengtsson and Rundgren (1982) distribution of enchytraeids was highly heterogeneous in the soil (CV was 135% for all samples in the contaminated area and 124% at the reference site). Chalupsky and Leps (1985) found that enchytraeids (17 species from four genera, genus Cognettia was not included) were patchily distributed in natural soils. Others have also had similar observations (Nurminen, 1967; Standen, 1973; Abrahamsen, 1990). According to laboratory experiments done by SjoÈgren et al. (1995), enchytraeids can actively avoid polluted soil in arti®cial metal gradients, and uneven distribution in heterogeneously contaminated ®eld sites can be expected. Hence, to get a reliable assessment for the eects of metals on enchytraeid worms at a certain forest site, a lot of samples should be taken at the site or samples should be taken according to certain sampling design (plot design) to reduce the variance between samples. 6. Conclusion Concentrations of Cu and Ni were quite high and their distribution moderately heterogeneous in the forest area studied. Our results indicate that C. sphagnetorum shows low sensitivity to soil metal contamination when normal sampling resolution is used. It is possible that, in the contaminated area, the presence of patches of lower metal concentrations is mitigating the eects of the metals on enchytraeid population densities. Hence, reduced enchytraeid densities in the contaminated area were observed especially when heterogeneous spatial distribution of enchytraeids was taken into account. Because of patchy distribution of enchytraeids, the use of hierarchical plot design as a sampling method gave more statistical power compared to spatially random sampling. The importance of enchytraeids in decomposition and nutrients cycling together with the lack of taxonomical problems suggests them to be possible monitoring soil animals in northern coniferous forests.
Acknowledgements Metal extractions and analyses were done in the Institute for Environmental Research, University of JyvaÈskylaÈ. Tommi Malinen and Harri HoÈgmander helped with statistics.
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Environmental Pollution 104 (1999) 351±358
Horizontal distribution of copper, nickel and enchytraeid worms in polluted soil J. Salminen a,*, J. Haimi b a
Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FIN-15210 Lahti, Finland Department of Biological and Environmental Science, University of JyvaÈskylaÈ, PO Box 35, FIN-40351 JyvaÈskylaÈ, Finland
b
Received 19 May 1998; accepted 8 October 1998
Abstract We studied the horizontal distribution of Cu, Ni and enchytraeid worms (Cognettia sphagnetorum, Vejdovsky, Oligochaeta, Enchytraeidae) in forest soil near a Cu±Ni smelter in SW Finland. Soil samples were taken from a polluted site (2 km from the smelter) and a reference area (8 km from the smelter). We used a hierarchical sampling design in the polluted area for studying possible scale-dependent variability in parameters measured, distance between the samples (dierent scales) being 5, 50 and 500 cm. Distribution of metals was moderately heterogeneous in the polluted soil; coecient of variances (CV), 26% for Cu and 32% for Ni. Instead, distribution of enchytraeids in the area was highly heterogeneous (CV, 135%) and the variability increased with increasing a distance between the samples: CV increased from 67 to 104% from the smallest to the largest sampling scale. Soil metal concentrations did not correlate with enchytraeid densities in the polluted area. However, three out of our four sampling plots having distance of 500 cm between each other had lower enchytraeid density than the reference area. Results may indicate that C. sphagnetorum has quite low sensitivity to metal pollution, at least on the forest stand scale. On the other hand, enchytraeids may have population dynamics connected to patches (sources±sinks) caused by uneven distribution of metals. This can mitigate the eects of metals on their population densities. Due to importance of enchytraeids in decomposition processes and the fact that usually only one enchytraeid species is present in boreal coniferous forests, they may be appropriate for ®eld monitoring in these humus rich soils. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heavy metals; Enchytraeid worm; Heterogeneous distribution; Forest soil; Field monitoring
1. Introduction Usually both abiotic (e.g. porosity, nutrients and pH) and biotic (species composition and population densities) properties of soils vary in a small scale (in centimeters). Thus, soils tend to have patchy habitats for soil dwelling decomposer organisms (Swift et al., 1979). On the other hand, soil organismsÐperhaps excluding earthworms (Mather and Christensen, 1992) and macroarthropodsÐhave low capability to disperse long distances by active locomotion (Usher, 1985). Thus, when a species has became extinct from a patch, it may take a long time before the patch is recolonized (Siepel, 1994). Little is known about passive dispersal of soil animals
* Corresponding author. Department of Ecology and Ecotoxicology, Vrije Universiteit, De Boelelaan 1087, HV Amsterdam, The Netherlands. Tel.: +31-20-4447075; fax: +31-20-4447123; e-mail: [email protected].
by the aid of, for example, water and other animals, but it may help the recolonization. Anthropogenic pollutants, such as heavy metals, may also become heterogeneously distributed in the ®eld soils (in this paper we are dealing only with horizontal variability). Properties of both emission source and recipient environment can cause this variability. Once the metals have deposited, amount and quality of organic matter, pH and soil water content aect sorption and desorption processes of heavy metals and thus their accumulation to biota (Hopkin, 1989). Vegetation may also accumulate metals and hence partly aect their distribution in the soil (metals returning to the soil surface in dead plant material). Field studies on the eects of heavy metals on soil organisms have mainly been done by comparing samples taken at dierent distances from emission points. It has been assumed that metals are more or less evenly distributed within each distance zone. However, there are some studies in which consequences of small-scale
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spatial variation in soil metal concentration and subsequent eects on soil organisms have been discussed (Bengtsson et al., 1994; SjoÈgren et al., 1995). If uneven distribution of heavy metals and/or soil organisms takes place, reliability (i.e. accuracy) of monitoring the eects of pollutants on organisms can be low, especially when based on a limited number of samples (Bengtsson and Torstensson, 1988). Thus, when eects of pollutants are monitored in the ®eld, natural patchiness of the soil environment and soil-living organisms should be taken into account. However, we found no studies especially focused on small-scale spatial variability of metals and organisms in the soil. Monitoring ecological eects of soil pollution in the ®eld via changes in soil animal populations does not have a long tradition (van Straalen and Verhoef, 1997). One possible constraint in using soil organisms for monitoring purposes could arise from taxonomical problems, hundreds of species being found in each square meter. However, an enchytraeid worm Cognettia sphagnetorum, Vejdovsky (Oligochaeta, Enchytraeidae) is usually the only abundant enchytraeid species found in northern coniferous forest soils (Nurminen, 1967; Huhta et al., 1986). C. sphagnetorum is a white 1-cm long (adult) species which reproduces asexually by fragmentation (Christensen, 1959; Standen, 1973). It has been shown to be an important species in decomposition and nutrient cycling processes (Standen, 1978; Abrahamsen, 1990). Enchytraeids are known to be sensitive to many anthropogenic disturbances including contamination by heavy metals (Bengtsson and Rundgren, 1982; SjoÈgren et al., 1995). Although it is normally found that enchytraeids exhibit clustered distribution in the ®eld soils (Didden, 1993), Bengtsson and Rundgren (1982) observed that enchytraeids seemed not to be as much aggregated as other soil animals. They concluded that due to their moderately homogeneous spatial distribution, enchytraeids are optimal organisms for monitoring purposes. Moreover, enchytraeids are to be taken into toxicity test protocols as terrestrial decomposer animals (LeÂon and Van Gestel, 1994; Augustsson and Rundgren, 1998). Hence, risk assessment based on standardized toxicity tests and ®eld data on enchytraeids may be reasonable. Here we report results from a ®eld sampling carried out near a Cu±Ni smelter in SW Finland. Concentrations of Cu and Ni, and enchytraeid densities in the soil were determined from each sample. Total number of samples was kept as low as possible (36 samples plus 5 samples from a reference site) because we wanted to estimate the relevance of a pro®table sampling procedure done for monitoring purposes. Hierarchical sampling design was used in order to observe possible scaledependent heterogeneity in the parameters measured (distances between the samples varied from 5 to 500 cm). Our hypothesis was that if the parameters measured
are unevenly distributed, increased distance between samples increases the variability (Bell et al., 1993). Size of an individual soil sample was supposed to form a movement area of an individual worm (15.6 cm; SjoÈgren et al., 1995). Finally, we also discuss the usefulness of enchytraeid (C. sphagnetorum) density as a ®eld monitoring tool in ecological risk assessment of contaminants. 2. Study site Sampling was conducted in metal polluted and unpolluted areas near the town of Harjavalta, Finland (61 190 N 22 90 E). The Cu±Ni smelter has been operated in the area since 1945. Soils near the smelter are polluted with Cu, Ni, Zn and Fe and there is a clear decreasing gradient of soil pollution from the smelter. The gradient is also found in a visually homogeneous Scots pine (Pinus sylvestris) forest growing on podsolic soil pro®le. Drastic changes in vegetation, soil micro¯ora, fauna and physico-chemical properties of the soil have been observed along the increasing pollution gradient (Laaksovirta and Silvola, 1975; Fritze et al., 1989; Vanhala and Ahtiainen, 1994; Haimi and SiiraPietikaÈinen, 1996) as follows: (1) decrease in ground layer vegetation biomass and diversity, (2) decrease in soil microbial biomass and activity and (3) increase in thickness of organic soil layer when comparing areas 8 and 2 km from the pollution source, that is, our polluted and reference sites, respectively. More details about the study area can be found in Haimi and SiiraPietikaÈinen (1996) and references therein. 3. Materials and methods 3.1. Sampling and analyses Sampling was done on 23 October 1996 at distances of 2 and 8 km from the Cu±Ni smelter. At the 2-km site (the polluted site) soil samples were taken according to a hierarchical sampling design where distances of individual samples were controlled. Distance between three individual samples was 5 cm in the small scale, 50 cm in the medium scale (three plots having small-scale samples inside) and 500 cm in the largest scale (four plots, each of them having three medium-scale plots inside) (Fig. 1; for more details, see Section 3.2). From the reference site, ®ve soil samples were taken, the distances between the samples being more than 1 m. Soil samples (organic soil layer only, thickness varied from 3 to 5 cm) were taken with a steel corer (sample area was 0.0025 m2). Enchytraeids were extracted from the soil with wet funnels (O'Connor, 1962) and counted under a microscope (only C. sphagnetorum was found). After
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Fig. 1. A scheme of the sampling design (note hierarchical relationship of the samples) and how coecients of variation (CV) were calculated for samples taken from certain distance between them. Black dots are individual soil samples 1±9 taken within plots A, B, C and D in the polluted area. 5, 50 and 500 cm are the distances between samples.
the extraction, soil samples were oven dried (60 C) and homogenised in a mortar. Total Cu and Ni concentrations were analysed from the samples with atomic absorption spectroscopy after nitrogen acid extraction. Other heavy metals were not analyzed in this study because their in¯uence on soil animals was considered negligible (Fritze et al., 1989; Hopkin, 1989). Metals in the water of wet funnels were analysed after animal extraction: 0.24 mg literÿ1 Cu (SD: 0.205) and 0.11 mg literÿ1 Ni (SD: 0.064) were found in the water. The concentrations meant that 0.1% (Cu) and 0.4% (Ni) of total metal content in the samples were leached before metal analyses. Subsamples of the homogenised samples were burned in an oven (550 C, 5 h) for analysing organic matter content of the soil. 3.2. Data analyses Spearman correlations between Cu and Ni concentrations, organic matter content and enchytraeid densities in the soil within the polluted site were calculated. Student's t-test was used to discover overall differences between the reference and polluted sites in the parameters measured. For analysing possible dierences between the reference site (®ve replicates) and mediumscale plots at the polluted site (four plots A, B, C and D, nine replicates per plot, see Fig. 1) ANOVA was used. Data were normally distributed (Kolmogorow± Smirnov's test). Before ANOVAs, homogeneities of variances were tested with Bartlett-Box's test. Due to heterogeneous variances, data of enchytraeid density were log10+1 transformed. Simple contrast method (with Bonferroni adjusted p-values) was used to compare certain large-scale plots (plots A, B, C and D) in the polluted area to the reference site. Possible scale-dependent variability in the parameters measured within the polluted site was estimated by
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calculating coecient of variances (CV, as percentage) within three samples having constant mutual distances. There were three distance categories: 5, 50 and 500 cm between the samples (Fig. 1). We decided to keep n=12 in all scales. Hence, in the largest scale (500 cm), we randomly chose 12 combinations of the samples from all possible combinations (all nine samples of each mediumscale subplot were used only once in the lottery). Possible dierences in CV between the sampling scales were tested with ANOVA and pairwise comparisons were made by Tukey's HSD test (the variances were homogeneous and the data were normally distributed). Statistical analyses were performed with SPSS for Windows 6.1 statistical package (SPSS, Chicago, IL). 4. Results In the polluted area, sample speci®c Cu concentrations varied between 630 and 2900 mg kgÿ1 of dry soil (mean: 2084 mg kgÿ1; Fig. 2). Ni concentrations varied between 87 and 460 mg kgÿ1 of dry soil (mean: 298 mg kgÿ1; Fig. 3). At the reference site, metal concentrations in the soil were 64 mg kgÿ1 (mean; range: 16±170) and 13 mg kgÿ1 (mean; range: 6±28) for Cu and Ni, respectively (Figs. 2 and 3). Both metal concentrations were signi®cantly higher in the polluted area than at the reference site (t-test, p<0.001 for both metals). In the polluted area, organic matter content of the samples was signi®cantly higher (mean: 71%) than at the reference site (mean: 11%; t-test, p<0.001; Fig. 4). Numbers of enchytraeids did not dier signi®cantly between the sites. There were 27,360 individuals mÿ2 (range: 9200±88,000; n=5) at the reference site and 8489 mÿ2 (range: 400±53,200; n=36) at the polluted site (t-test, p=0.284; Fig. 5). However, when using mediumscale subplots in the polluted area as individual sampling sites and comparing them (Fig. 1) to the reference site in ANOVA, the number of enchytraeids was signi®cantly lower in three medium-scale subplots (A, B and C) than at the reference site (ANOVA F=9.33, p<0.001, df=4 and post hoc contrast's p<0.05). Enchytraeid numbers at the site D did not dier from the numbers at the reference site (Fig. 5). Within the polluted site Cu and Ni concentrations were not correlated with the enchytraeid numbers, but strong correlations between the metal concentrations and soil organic matter content were observed. Enchytraeid numbers were not correlated with soil organic matter content (Table 1). Increasing distance between the samples did not signi®cantly increase CV in the case of soil metal concentrations (ANOVA, p=0.468 for Cu and p=0.214 for Ni; Fig. 6). However, in enchytraeid numbers there was an increasing variation with increasing distance between the samples (ANOVA, F=5.321, p=0.010, df=2).
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Fig. 2. Total Cu concentrations in the soil samples taken at certain distances from a Cu±Ni smelter in SW Finland. `Reference' is the sampling site 8 km from the smelter. Sites A, B, C and D are `medium-scale' (see Fig. 1) samples taken at the distance of 2 km from the smelter. Numbers 1±3, 4±6 and 7±9 refer to the `small-scale' samples.
Fig. 3. Total Ni concentrations in the soil samples taken near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2.
5. Discussion 5.1. Distribution of metals in soil and their eects on enchytraeids Neither Cu nor Ni was as heterogeneously distributed as enchytraeids in the contaminated forest soil studied here (CV was 26% for Cu, 33% for Ni and 135% for enchytraeids for all samples). Variation in soil metal concentrations was lower than assumed on the basis of high variability found in `natural' soil properties (e.g. nitrogen and water content of the soil), especially in forests (Kleb and Wilson, 1997). Possibly, long-term airborne pollution from the smelter has `saturated' the soil by metals, or biological properties of recipient area (canopies of trees, ground layer vegetation, etc.) have
not aected the distribution of metals as much as expected. Although overall variation is quite small in soil metal concentrations, there are patches where toxic eects on enchytraeids should occur (e.g. over 2000 mg Cu kgÿ1 soil) and also patches that are clearly less toxic (1000 mg Cu kgÿ1; Bengtsson and Tranvik, 1989). However, no correlations between population densities of enchytraeids and soil metal concentrations were found. It even seems that there are patches within the polluted area (site D with as high metal concentrations as the other sites) where enchytraeid numbers are as high as at the reference site. Metals may be heterogeneously distributed on a smaller scale than our smallest sampling scale (5 cm), and hence the size of our soil corer may be ecologically (as a foraging area of an individual enchytraeid worm)
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Fig. 4. Organic matter content of the soil samples taken near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2.
Fig. 5. Density of Cognettia sphagnetorum near a Cu±Ni smelter in SW Finland. Other explanations, see Fig. 2. Table 1 Spearman correlation coecients between soil Cu and Ni concentrations, soil organic matter content and enchytraeid density (n=36)
Enchytraeid density Organic matter content Ni concentration
Cu concentration
Ni concentration
Organic matter content
0.073NS 0.614*** 0.826***
0.092NS 0.806*
0.229NS
NS, non-signi®cant, ***p<0.001.
too large, although behaviourally (escape from hostile area) it seemed reasonable (SjoÈgren et al., 1995). Anderson (1978) has shown that soil living microarthropods can have microhabitats in a scale of
millimeters (around a certain soil particle). However, enchytraeids are relatively big in size (smallest fragments are 0.2 cm and adults 1 cm) and they are capable of moving centimeters in a short time (SjoÈgren
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(presence and dimensions of patches and realized niches) has rarely been studied or described, neither in this study. Data on ®tness and movements of individual worms are lacking, too. Hence, more experiments are needed to show whether sink±source dynamic is present in polluted forest soils. There were positive correlations between soil metal concentrations and organic matter content. This relationship could be a consequence of decreased litter decomposition due to metal contamination (both biomass and activity of microbes have been reduced; Vanhala and Ahtiainen, 1994) and could lead to increased sorption of metals to accumulated litter and humus, and concomitant reduced metal bioavailability and toxicity to soil organisms (Ross, 1994). Lack of correlation between metals and enchytraeids may also indicate that factors other than direct toxicity of metals were controlling the enchytraeid abundance. It has been well documented that vegetation, biomass and activity of microbes, and physico-chemical properties of the soil have changed around the smelter studied here (Laaksovirta and Silvola, 1975; Fritze et al., 1989; Vanhala and Ahtiainen, 1994; Haimi and SiiraPietikaÈinen, 1996). Reduction in microbial food (enchytraeids assimilate microbes together with decaying plant material from the digested soil) and other changes in abiotic and biotic conditions in the soil (e.g. altered soil structure due to changes in litter quality) can indirectly reduce enchytraeid densities. It seems that changes in habitat characteristics, including possible patchiness, in moderate polluted habitats (not the toxicity of the compounds per se) have an important role in the survival of soil dwelling organisms (Posthuma and van Straalen, 1993). 5.2. Use of enchytraeids in ®eld monitoring
Fig. 6. Variability (coecient of variation as percentage, CV) of enchytraeid density (A), soil Cu concentration (B) and soil Ni concentration (C) in three samples taken from constant mutual distances between each other (mean and SD of CV). `All' represent CV calculated over all samples taken from the contaminated area. Note dierent scales on y-axis.
et al., 1995). The patches with lower concentrations can serve as source habitats (less toxic and more available resources) for more contaminated sink habitats (sensu Dias, 1996). Hence, enchytraeid densities in the contaminated area were able to be higher than expected. Unfortunately, real habitat structure of polluted soils
As was pointed out earlier, no direct correlations between soil metal concentrations and enchytraeid densities were found in our data. This observation is consistent with laboratory studies done by Augustsson and Rundgren (1998). They found that in contrast to sublethal responses, mortality of C. sphagnetorum was not a sensitive parameter to Cu contamination in soil with high organic matter content. Haimi and SiiraPietikaÈinen (1996) sampled the same sites as investigated in this study. They found large variation in enchytraeid densities within and between years, as well as spatially. There tended to be more enchytraeids at the site 8 km from the smelter compared to the 2-km site, but this was not true on every sampling occasion (Haimi and Siira-PietikaÈinen, 1996). Because of high enchytraeid densities in the site D, no statistically signi®cant dierence between the polluted and the reference site was observed in the present study when the polluted site was used as one independent variable in the
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analysis. However, three out of the four mediumscale subplots had signi®cantly lower enchytraeid densities than the reference site, indicating that enchytraeids responded to the soil metal contamination. Without our sampling design with medium-scale subplots, the between-site dierences (polluted vs reference) might not have been statistically justi®ed. Contrary to earlier results by Bengtsson and Rundgren (1982) distribution of enchytraeids was highly heterogeneous in the soil (CV was 135% for all samples in the contaminated area and 124% at the reference site). Chalupsky and Leps (1985) found that enchytraeids (17 species from four genera, genus Cognettia was not included) were patchily distributed in natural soils. Others have also had similar observations (Nurminen, 1967; Standen, 1973; Abrahamsen, 1990). According to laboratory experiments done by SjoÈgren et al. (1995), enchytraeids can actively avoid polluted soil in arti®cial metal gradients, and uneven distribution in heterogeneously contaminated ®eld sites can be expected. Hence, to get a reliable assessment for the eects of metals on enchytraeid worms at a certain forest site, a lot of samples should be taken at the site or samples should be taken according to certain sampling design (plot design) to reduce the variance between samples. 6. Conclusion Concentrations of Cu and Ni were quite high and their distribution moderately heterogeneous in the forest area studied. Our results indicate that C. sphagnetorum shows low sensitivity to soil metal contamination when normal sampling resolution is used. It is possible that, in the contaminated area, the presence of patches of lower metal concentrations is mitigating the eects of the metals on enchytraeid population densities. Hence, reduced enchytraeid densities in the contaminated area were observed especially when heterogeneous spatial distribution of enchytraeids was taken into account. Because of patchy distribution of enchytraeids, the use of hierarchical plot design as a sampling method gave more statistical power compared to spatially random sampling. The importance of enchytraeids in decomposition and nutrients cycling together with the lack of taxonomical problems suggests them to be possible monitoring soil animals in northern coniferous forests.
Acknowledgements Metal extractions and analyses were done in the Institute for Environmental Research, University of JyvaÈskylaÈ. Tommi Malinen and Harri HoÈgmander helped with statistics.
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