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Earthworm populations of highly metal-contaminated soils restored by fly ash-aided phytostabilisation
Ecotoxicology and Environmental Safety 113 (2015) 183–190
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Earthworm populations of highly metal-contaminated soils restored by fly ash-aided phytostabilisation Fabien Grumiaux a,b,n, Sylvain Demuynck a, Céline Pernin a, Alain Leprêtre a a
Université Lille Nord de France, Laboratoire Génie Civil et géo-Environnement (LGCgE) – Lille1, Bât. SN3, 59655 Villeneuve d’Ascq Cedex, France Université Lille Nord de France, Ecole Supérieure du Professorat et de l’Education (ESPE), site d’Arras, 7 bis rue Raoul François, BP 30927, F-62022 Arras Cedex, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 3 July 2014 Received in revised form 26 November 2014 Accepted 2 December 2014
Highly metal contaminated soils found in the North of France are the result of intense industrial past. These soils are now unfit for the cultivation of agricultural products for human consumption. Solutions have to be found to improve the quality of these soils, and especially to reduce the availability of trace elements (TEs). Phytostabilisation and ash-aided phytostabilisation applied since 2000 to an experimental site located near a former metallurgical site (Metaleurop-Nord) was shown previously as efficacious in reducing TEs mobility in soils. The aim of the study was to check whether this ten years trial had influenced earthworm communities. This experimental site was compared to plots located in the surroundings and differing by the use of soils. Main results are that: (1) whatever the use of soils, earthworm communities are composed of few species with moderate abundance in comparison with communities found in similar habitats outside the TEs-contaminated area, (2) the highest abundance and specific richness (4–5 species) were observed in afforested plots with various tree species, (3) ash amendments in afforested plots did not increase the species richness and modified the communities favoring anecic worms but disfavoring epigeic ones. These findings raised the questions of when and how to perform the addition of ashes firstly, to avoid negative effects on soil fauna and secondly, to keep positive effects on metal immobilization. & 2014 Elsevier Inc. All rights reserved.
Keywords: Earthworms Contaminated soil Trace metals Phytostabilisation Fly ashes
1. Introduction Since the 19th century, industrial activities generated a great number of uncontrolled hazardous sites and large areas contaminated by trace elements (TEs) in many European countries such as Netherlands (Ma et al., 1983) and United Kingdom (Spurgeon and Hopkin, 1996). This is also the case of the former coalmining region in the North of France especially around smelters at Noyelles-Godault, Auby and Mortagne du Nord. At Noyelles-Godault, Metaleurop-Nord, a major Pb plant in Europe for about 100 years generated significant amounts of emitted metal containing smoke until its closedown in 2003. According to the Regional Board for Industry, Research and the Environment (DRIRE, 2003), Metaleurop's annual atmospheric emissions in 2002 were still about 1 t of Cd, 17 t of Pb and 32 t of Zn. This industrial activity resulted in a considerable contamination of the surrounding soils n Corresponding author at: Université Lille Nord de France, Laboratoire Génie Civil et géo-Environnement (LGCgE) – Lille1, Bât. SN3, 59655 Villeneuve d’Ascq Cedex, France. Fax: þ 33 3 20436732. . E-mail address: [email protected] (F. Grumiaux).
http://dx.doi.org/10.1016/j.ecoenv.2014.12.004 0147-6513/& 2014 Elsevier Inc. All rights reserved.
for 7 km to the N-NW by 5 km to the N-NE (Frangi and Richard, 1997). This contamination concerned agricultural (Sterckeman et al., 2000, 2002), urban (Douay et al., 2008a) and forest habitat top soils (Douay et al., 2009). The contamination occured mainly in the upper 20–30 cm of the soil but traces of Cd and Zn were found at about 2 m deep (Sterckeman et al., 2000). Due to the large contaminated surface (approximately 35 km2 around the smelter) low-cost and ecologically sustainable remediation methods had to be developed to reduce impacts on human and environmental health. Indeed, the contamination of soils by TEs have not only effects on herbaceous plants and trees (Pruvot et al., 2006; Bidar et al., 2007; Douay et al., 2008b) but also on human health (de Burbure et al., 2006). Among the methods available, phytostabilisation may be used to reduce the mobility, ecotoxicity and dispersion of TEs through the ecosystem (Vangronsveld et al., 2009). Mineral amendments such as fly ashes (FAs) are known to help phytostabilisation of TEs-contaminated soils (Vangronsveld et al., 2009). FAs, industrial by-products originated from combustion processes, are interesting because of their large availability and low-cost. Pandey and Singh (2010) reviewed recently the interests of FAs in soil systems and indicated the possibility of FAs addition
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in degraded soil for improving nutritional and physico-chemical properties. Among positive effects on soils, FAs can increase the levels of pH, particle density, porosity and water holding capacity (Pandey et al., 2009a). Thus, FAs can be used as an economic fertilizer and soil amendment in small-scale/large-scale cultivation of plants that have ornamental, floricultural, horticultural and forestry potential (Pandey et al., 2009b). FAs are also usefull to enrich soil productivity and crop yields for dry tropical nutrient poor soils (Singh et al., 2011; Singh and Pandey, 2013). However, because FAs can contain toxic inorganic compounds as TEs, their addition to uncontaminated soils can provoke accumulation or translocation of these TEs in plants (Pandey et al., 2009a). Regarding their use as amendments in TEs contaminated soils, FAs represent an effective, cost-effective, eco-friendly management option for reclamation of these soils (in Pandey and Singh, 2010). FAs can play significant role in TEs immobilization in soils. Kumpiene et al. (2007) demonstrated that ashes amendment reduced the leaching by 91.1 percent for copper and by 87.1 percent for lead in a Pb- and Cucontaminated landfilled soil. In 1999, a pilot study started in a former agricultural field located nearby the former smelter Metaleurop Nord. This project consisted in testing the growth of five trees species with or without the use of silico-alumineous FAs (Sodelines, FA1) and sulfo-calcic FAs (Soprolines, FA2) as amendments to reclaim soils with high levels of Pb, Cd and Zn. In the experimental site, after eight years of fly ash-aided phytostabilisation, Lopareva-Pohu et al. (2011a) showed that soil pH had decreased on the whole site while organic carbon content increased. The observed change of these parameters influencing TEs mobility was explained by afforestation. Over time, concentrations of 0.01 M CaCl2-extractable metals increased and were correlated with the soil pH decrease. In the amended soils with FA1 and FA2, extractable Cd and Zn concentrations were about 10-fold lower than in the non-amended soil. The results indicated that the two FAs buffered natural soil acidification due to vegetation development, limited trace element mobility and thus could limit their bioavailability (Lopareva-Pohu et al., 2011a). Fly ash amendments strongly decreased TEs availability to three of the five trees species (Alnus glutinosa, Acer pseudoplatanus and Robinia pseudoacacia) and their translocation to aboveground parts (Pourrut et al., 2011). Similar results were obtained for both FAs on herbaceous species under laboratory conditions (Lopareva-Pohu et al., 2011b). Earthworms have a prominent role in soil functioning (Darwin, 1881). They are known to be bio-indicators for evaluating the effects of soil contamination by trace metals and pesticides, but also for instance the effects of agricultural practices and acid rain (Ghilarov, 1978; Bouché, 1972; Eijsackers, 1983; Paoletti et al., 1991). Grumiaux et al. (2007) tested the intrinsic toxicity of FA1 and FA2 under laboratory-controlled conditions using the earthworm Eisenia andrei. Although, a high mortality (between 60 and 100 percent) was registered in the worms exposed to a freshly ash amended artificial soil, probably due to a rise of soil pH value (from 6 to 8.9 and to 9.5 for FA1 and FA2 respectively). The survival was not significantly affected when worms were exposed to 8 weeks old artificial soil treated with fly ashes which pH stabilized around 8. However, both ashes were still responsible for detrimental effects on worm growth rates (decrease of about 20 percent for the both FAs) and cocoons productions (decrease of 43 and 89 percent for FA1 and FA2 respectively). The sulfo-calcic fly ash FA2 was shown to fully reduce cocoon hatching. Regarding the soil of the experimental site, the acute toxicity of 2 years-old FAstreated soils to E. andrei was significantly lower than those of the untreated soil (Grumiaux et al., 2010). In addition, mortality was 3.6–5-fold lower for worms exposed to FAs amended soil, so 2 years ash-treatment of these contaminated soils decreased their toxicity. However, reproductive parameters appeared more
affected in the worms exposed to the soil treated with sulfo-calcic ashes (FA2) than in those exposed to the untreated metal-contaminated soil. Thus, adding such immobilising agents could negatively affect earthworm populations. This was reinforced by the observation of that experimentally exposed Eisenia fetida to FA1 or FA2 amended soils avoided (avoidance rate 470 percent) these soils significantly (Demuynck et al., 2014). At present, still nothing is known in this experimental site about soil fauna, and particularly about earthworms. Previous results obtained under laboratory conditions with experimental exposures of biological models (Grumiaux et al., 2007, 2010, Demuynck et al., 2014) revealed contrasting physiological effects. In one hand, ash addition should reduce TEs bioaccumulation and reduce worm mortality but in another hand it should alter reproductive output and growth, and modify worm behavior. These findings suggest a possible impact on field earthworm population. So, the aim of the present study was to check whether a ten years trial of fly ash-aided phytostabilisation on highly metal contaminated soils of an experimental site influenced in situ earthworm communities. The study included the comparison of this site with plots located in the surroundings and differing by the use of soils.
2. Materials and methods 2.1. Plots The experimental site is described in details in previous works (Bidar et al., 2007; Lopareva-Pohu et al., 2011a). The site (50°26′N, 3°01′E) is located at Evin-Malmaison, 600 m northeast of Metaleurop-Nord (Noyelles-Godault, North of France) downwind of dominating wind directions (Fig. 1). The experimental site was set up in 1999 on a former agricultural field of about 10,000 m2, which had been cultivated with maize and wheat. It was divided in 2000 into 3 plots of about 3000 m2. The first one (R) was not amended. The two other plots (F1 and F2) were amended respectively with silico-aluminous fly ashes (Sodelines, FA1) and sulfo-calcic fly ashes (Soprolines, FA2) at a rate of 23.3 kg/m2 then plowed up to a 25- to 30-cm soil depth where the most part of the TEs contamination occurred. These two fly ashes (Table 1), provided from Surschiste Ldt (Mazingarbe, France) were previously described (Lopareva-Pohu et al., 2011a). They were produced by fluidisedbed combustion of bituminous coal (Carling thermal power plant) and lignite (Gardanne thermal power plant) respectively. The experimental site (R, F1 and F2) was planted in 2000 with various tree species (R. pseudoacacia L., A. glutinosa L., Quercus robur L., A. pseudoplatanus L. and Salix alba L.) and sown with an herbaceous mixture (Festuca ovina L., Lolium perenne L., Bromus catharticus Vahl and Trifolium repens L.). Three additional plots located in the surroundings of the experimental site and representing different soil uses were also considered in the study to determine the influence of ground covers on earthworm communities. A plot (MV) which had been cultivated with maize and wheat until 2009 was considered as the absolute reference (i.e., agricultural soil without fly ash-aided phytostabilisation and earthworm communities negatively impacted by plowing and TEs contamination). A plot (BS) which is a meadow since 1999, located in the South of the experimental site and an ash tree wood (FO), located in the West have been also sampled. Characteristics of soils are given in Table 2. According to Lopareva-Pohu et al. (2011a), the soil of the experimental site is a silt-loamy redoxic neoluvisol (clay contents, 13–21 percent; silts 55–62 percent; sands 22–26 percent) with a neutral pH, developed from loess. It lies on a truncated paleoneoluvisol developed from clay. During winter, the groundwater level reaches 60 cm in
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Fig. 1. Geographic location of the experimental site and plots where earthworms were sampled. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
Table 1 Characteristics of silico-alumineous and sulfo-calcical fly ashes (Lopareva-Pohu et al., 2011a). Parameter (unit)
Silico-alumineous ashes FA1 (Sodelines)
Sulfo-calcic ashes FA2 (Soprolines)
pH H2O CaCO3 total (g/kg) CEC (cmol þ /kg) CaO (g/kg) MgO (g/kg) K2O (g/kg) Na2O (g/kg) P2O5 (g/kg) S H2O (g/kg) Cd (mg/kg) Cu (mg/kg) Pb (mg/kg) Zn (mg/kg)
9.9 21 5.1 28.4 2.93 0.91 0.18 0.09 5.5 1.0 81 142 257
12.6 67 6.4 184.5 1.99 0.21 0.06 0.24 13.9 0.4 38 39 85
Note: Coal FAs were from Surschiste Ltd. (Mazingarbe, France).
depth. As for agricultural soils around Metaleurop-Nord, the major part of contamination by Cd, Pb and Zn is limited to the plowed layer (20–30 cm) (Lopareva-Pohu et al., 2011a). 2.2. Earthworms sampling Earthworm populations were studied in March 2010 when most earthworm species are particularly active (Bouché, 1972). The number of samples taken at each plot was a compromise between statistical considerations and practical considerations of time and
manpower. For the plots of the experimental site whose ground use was modified in 2000 by the plantation of various tree species and by additions of fly ashes (F1 and F2 only) to the soil, a greater sampling effort (8 samples per plot) was performed than in the surrounding plots (4–5 samples per plot) where the ground use was left unchanged and the soil was not amended. The samples were positioned in a square or in line according to the shape of the plot. The sampling was made in the center of the plot or at least 10 m from the edges. Each sample was distant from each other by at least 5 m. A sampling method coupling chemical expulsion with allyl isothiocyanate (AITC) derived from Zaborski (2003) and hand sorting was used within 60 cm 60 cm quadrats. We chose this combination of methods because different studies have shown that chemical expulsion is, in general, more efficient for anecic species than is hand sorting, but that hand sorting is more efficient for endogeic species (in Zaborski, 2003). The soil of each quadrat was irrigated twice with 5 L of the AITC solution (1 g AITC dissolved in 100 mL isopropanol/10 L water) at 10 min intervals after the removal of the vegetation cover and of the litter. For 10 min after each irrigation, all earthworms emerging on the soil surface were collected. Then each soil quadrat was excavated to a depth of 20 cm with a spade and earthworms were collected by hand sorting. Worms were placed into plastic containers with watermoistened filter paper, returned to the laboratory in coolers, stored at 4 °C and then sorted, counted, weighed and fixed in 95 percent ethanol within the next 24 h. Worms were identified to species level using taxonomic key of Bouché (1972) for adults, or using characteristics such as the coloration, the prostomium shape and the size and pattern of setae for juveniles.
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Table 2 Physicochemical characteristics of the field soil. Plots
MVb
BSa,b
FOa,b
Rb
F1b
F2b
pH C/N Corg (g/kg) N tot (g/kg) CaCO3 total (g/kg) CaO (g/kg) MgO (g/kg) K2O (g/kg) SH2O (mg/kg) CEC (cmol þ /kg)
8.20 (0.09) 15.2 (0.4) 18.2 (0.4) 1.20 (0.04) 10.2 (3.3) – – – – 14.9 (1.6)
7.2 12.9 19.1 1.48 2.32 2.7 0.12 0.23 – 12.2
7.6 12.9 16.9 1.32 5.2 4.3 0.06 0.2 – 13.0
7.31 (0.2) 13.5 (0.1) 23.8 (0.7) 1.76 (0.06) 1.5 (0.8) 4.4 (0.4) 0.19 (0.01) 0.30 (0.01) 14 (4) 14.5 (0.5)
7.95 (0.03) 15.5 (0.1) 25.9 (0.6) 1.68 (0.04) 5.8 (0.9) 5.9 (0.2) 0.34 (0.01) 0.41 (0.01) 16 (2) 13.6 (0.2)
7.74 (0.02) 17.5 (0.3) 29.8 (1.1) 1.70 (0.06) 71.6 (6.5) 18.6 (1.0) 0.16 (0.01) 0.40 (0.04) 2257 (277) 14.6 (0.4)
Cd Cu Pb Zn
14.1 (1.4) 37 (1.1) 731 (67) 1000 (88)
5.8 34.8 288 486
14.9 45.3 858 1061
16.6 (0.6) 34 (1.2) 926 (26) 1135 (42)
18.0 (0.3) 41 (1.4) 967 (16) 1211 (26)
17.8 (0.5) 34 (1.7) 900 (30) 1127 (43)
19.5 53.1 27.4
20.3 52.3 27.4
23.4 56.0 20.6
21.5 55.2 23.3
19.6 58.1 22.3
12.9 61.5 25.6
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
Clay (%) Silt (%) Sand (%)
Note: Mean value (bold characters) and standard deviation (value into brackets). (–) no data. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2. a b
A single sample composed of 5 soil cores was analyzed for these two soils. Phytener (2014).
2.3. Statistical analysis Normality of data (density and biomass) was checked with Kolmogorov–Smirnov's tests and homogeneity of variances was tested using Levene's tests. Due to the heteroscedasticity, data analysis was performed using a non-parametric statistical method
(Kruskall–Wallis test) followed by a multiple comparisons method (Conover–Iman). A dendrogram based on cluster analysis using Chi-square distance and Ward's agglomerative method was performed on worm abundance data.
Table 3 Mean number, mean biomass and species of worms collected in the 6 plots of the studied area in March 2010. Plots n
MV 5
FO 4
BS 4
R 8
F1 8
F2 8
Biomass (g/m2) Total earthworms (ind/m2)
1.2 (1.3) 2.2 (2.3)
24.5 (16.8) 40.3 (18.4)
13.8 (2.1) 25.0 (9.9)
26.3 (13.9) 70.1 (38.5)
40.3 (23.6) 75.7 (23.3)
47.4 (22.9) 117.4 (34.5)
9.0 (7.6) 3.5 (5.3) 12.5 (10.8)
29.5 (23.3) 16.0 (12.6) 45.5 (29.4)
5.6(8.4) 1.0 (1.4) 6.6 (9.1)
12.5 (7.6) 10.4 (15.1) 22.9 (20.3)
Lumbricus castaneus
ad juv tot
0 0 0
2.8 (2.3) 0 2.8 (2.3)
Dendrobaena rubida
ad juv tot
0 0 0
0 0 0
0 0 0
0.3 (1.0) 0 0.3 (1.0)
0 0 0
0 0 0
Lumbricus rubellus
ad juv tot
0 0 0
0 0 0
0 4.2 (5.3) 4.2 (5.3)
2.4 (2.8) 6.6 (7.8) 9.0 (10.2)
1.7 (2.5) 6.9 (13.6) 8.7 (12.8)
2.8 (3.6) 4.9 (5.7) 7.6 (7.4)
Aporrectodea rosea
ad juv tot
0 0 0
0 0 0
0 0 0
0 0.7 (1.3) 0.7 (1.3)
0.3 (1.0) 1.0 (2.1) 1.4 (2.1)
0 0.7 (1.3) 0.7 (1.3)
Lumbricus terrestris
ad juv tot
0 2.2 (2.3) 2.2 (2.3)
3.1 (1.8) 11.5 (11.6) 14.6 (11.8)
4.8 (4.6) 54.2 (28.8) 59.0 (26.6)
3.8 (2.9) 82.3 (22.0) 86.1 (22.0)
Species number
1
3.5 (3.5) 34.0 (18.3) 37.5 (17.9) 2
6.3 (3.5) 2.1 (2.7) 8.3 (3.2) 3
5
4
4
Note: Mean value (bold characters) and standard deviation (value into brackets). n: number of samples, ad: adults, juv: juveniles, tot: total (adults þ juveniles). MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
F. Grumiaux et al. / Ecotoxicology and Environmental Safety 113 (2015) 183–190
3. Results Total number of worms, total weight of worms, and number of earthworm species are presented in Table 3. Five species of earthworms were identified, in varying proportions from a total of 338 individuals (16.3 percent adults, 83.7 percent juveniles) for F2, 218 individuals (16.5 percent adults, 83.5 percent juveniles) for F1, 202 individuals (50.5 percent adults, 49.5 percent juveniles) for R, 58 individuals (15.5 percent adults, 84.5 percent juveniles) for FO, 36 individuals (61.1 percent adults, 38.9 percent juveniles) for BS and 4 individuals (100 percent juveniles) for MV. The five species found were two epigeic (litter-dwellers), Dendrobaena rubida, Lumbricus castaneus; one epi-anecic (litter/soil-dweller), Lumbricus rubellus; one endogeic (soil-dweller), Aporrectodea rosea and one anecic, Lumbricus terrestris. Most worms were collected during irrigation with AITC. Only the soil-dwelling individuals were captured during manual sorting. The agglomerative hierarchical clustering method performed on worm density data separated 2 groups of plots (Fig. 2). In the first group L. castaneus was the dominant species (plots R and BS). L. terrestris was the dominant species in the second group (plots F1, F2, FO and MV). Sites F1 and F2 where the other species L. castaneus, L. rubellus and A. rosea were also present were separated from the plots MV and FO. 3.1. Effects of the plant cover and soil cultivation of the plot Data and statistical results are given in Tables 3 and 4. The abundance data obtained in each plot (Table 3) were transformed into density values. Regarding the agricultural plot MV, density as well as biomass was very low with only 2 ind/m2 and 1 g/m2 respectively. In addition, one single species L. terrestris was found. Density and biomass were higher in plots that had not been cultivated since 1999: the meadow BS (25 ind/m2, 14 g/m2), the ash tree wood FO (40 ind/m2, 25 g/m2) and the phytostabilised plot R (70 ind/m2, 26 g/m2) but significant differences were only found for the woody plots FO and R. Plot R was the only one containing five species. 3.2. Effects of the ash treatment Data and statistical results are given in Tables 3 and 4. The earthworm biomass in the ash-aided phytostabilised plots F1 and
Fig. 2. Dendrogram based on cluster analysis using Chi-square distance and Ward's agglomerative method performed on worm abundance data. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
187
F2 (40 g/m2 and 47 g/m2 respectively) was higher than in the phytostabilised plot R (26 g/m2) but the difference was not significant. Density was significantly higher (p o0.05) in plot F2 (117 ind/m2) containing sulfo-calcic ashes than in the two other plots R and F1 (70 ind/m2 and 76 ind/m2 respectively). Regarding the density of each species, differences between plots were found only for L. castaneus and L. terrestris. Density of L. castaneus was higher in the phytostabilised plot R than in the ashaided phytostabilised plots F1 and F2 but only significant in F1 (p o0.05). Density of L. terrestris (juveniles and total number of individuals) was significantly higher in F1 and F2 than in R (p o0.001).
4. Discussion 4.1. Soil contamination The soils of the studied plots were all classified as silt-loam according to the USDA triangle. The soils of plots BS, FO and R showed a nearly neutral pH value. The pH value (7.7) of the agricultural plot MV must be due to calcic amendments performed in the past. These soil characteristics are suitable for the establishment of earthworm communities (see Bouché, 1972). Ashes addition to the soil resulted in an increase of soil pH (7.9 and 7.7), CaCO3, exchangeable Ca2 þ , Mg2 þ , soluble S and total S contents in both F1 and F2 soils. Cd, Cu, Pb and Zn contents in soils (Table 1) were respectively around 75, 2.5, 20 and 11-fold the references values determined by Baize and Sterckeman (2001) for Northern France (median values). In addition, Pb, Zn and Cd concentrations in these soils clearly exceeded the upper limits (300, 300 and 3 mg/kg, respectively) established by the European Directive 86/ 278/ EEC (Council of the European Communities, 1986) for the addition of sludges as amendments to agricultural soils. Therefore, the soils studied must be considered as polluted soils from a legal standpoint. This high TEs contamination may have hampered the establishment of earthworm communities (e.g. Nahmani and Lavelle, 2002). 4.2. Effect of soil contamination and soil management on earthworm communities Data were obtained from a single location (i.e., without a real replication of the plots according to their proper use). Nevertheless, plots tested offered the main advantage to be located at short distances one with each other or even were adjacent. The plots surrounding the experimental area share with R, F1 and F2 quite similar level of TEs, the historic use of the soil (agriculture) and natural soil parameters values. So, only the treatment received by the soil and the ground cover differ. This choice allowed hypothesizing that differences seen for earthworm communities were more related to the proper use of each plot than to soil types. In addition, this comparison is justified since it allows determining the effect of the ground cover introduced in 2000 in the experimental area (plantation with a tree mix) on the earthworm community. However, because of this possible bias, interpretation of the results has to be taken with care and its significance should be essentially considered with a value at a regional scale of the NordPas de Calais coal-mining region. Earthworms communities from the plots studied with the exception of F1 and F2 (see 4.3 for the comments on these plots) appeared quite poor. Species richness was from 1 to 5 species and earthworm density was ranged from 2.2 to 70.3 ind/m2. The highest values were found in the tree plantations (R particularly) while the lowest concerned the plot still used as cropping soil (MV). These last results were not surprising since earthworm
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Table 4 Statistical results concerning the effect the plant cover and soil cultivation and the effect of the ash treatment. Plot
Effects of the plant cover and soil cultivation
Effects of the ash treatment
P value
MV
FO
BS
R
P value
R
F1
F2
ad juv tot
o0.01 o0.01 o0.01 o0.01 o0.01
a a a a a
b b a a ab
ab ab ab ab bc
b b b b c
0.13 o 0.05 o 0.05 o 0.05 o 0.05
a a b b b
a a a a a
a b ab ab ab
Dendrobaena rubida
ad juv tot
0.65 – 0.65
a – a
a – a
a – a
a – a
0.37 – 0.37
a – a
a – a
a – a
Lumbricus rubellus
ad juv tot
0.06 0.06 0.06
a a a
a a a
a a a
a a a
0.81 0.60 0.98
a a a
a a a
a a a
Allolobophora rosea
ad juv tot
– 0.3 0.3
– a a
– a a
– a a
– a a
0.37 0.99 0.74
a a a
a a a
a a a
Lumbricus terrestris
ad juv tot
o0.05 o0.05 o0.01
a a a
ab b b
b a ab
b ab b
0.76 o 0.001 o 0.001
a a a
a b b
a b b
Biomass Total earthworms Lumbricus castaneus
Note: In the same lane, letters (abc) indicate homogeneous groups. ad: adults, juv: juveniles, tot: total (adults þ juveniles). Data analysis was performed using a nonparametric statistical method (Kruskall–Wallis test) followed by a multiple comparisons method (Conover–Iman). ad: adults, juv: juveniles, tot: total (adults þ juveniles). MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
populations in cultivated land are generally lower than those found in undisturbed habitats (Paoletti, 1999; Curry et al., 2002). However, in the plot MV both density and species richness were extremely low. Agricultural activities have negative effects on invertebrates (Hendrix and Edward, 2004). Direct killing, increased predation, soil compaction, use of biocides, food and moisture limitations and decreasing organic matter contents are factors known to reduce earthworm populations in intensive cropping systems (in Metzke et al., 2007). Ponge et al. (2013) also recorded an increase of earthworm species richness along a gradient of decreasing agricultural intensification (from crop to meadow) in French Brittany. This increase of richness in our plots would be due to an increased availability of trophic resources. Organic matter, surface litter and dead roots are known to constitute part of the diet of various earthworm species (Sims and Gerard, 1999). Both the species richness and the density recorded in our plots were lower than those reported for similar habitats by authors working in locations without high TEs contamination of the soil. It is noteworthy that Nahmani and Rossi (2003) found 392, 271 and 52 ind/m2 and 6, 8 and 2 species in unpolluted grassland, tree plantation and cultivated soils respectively at a distance of only some tens of kilometers of our zone of study. At a shorter distance of the studied site (about 3 km to the northwest) 634 ind/m2 and 13 species were recorded in an unpolluted meadow (Restbiodiv, 2012) while only 25 ind/m2 and 3 species were recorded in the polluted meadow BS. The particular location of the studied plots under the dominant winds of the lead smelter Metaleurop-Nord that was active for almost a century can explain the poor earthworm communities. Earthworm populations are known to be reduced by high concentrations of TEs in soils (Bengtsson et al., 1983; Spurgeon and Hopkin, 1996; Nahmani and Lavelle, 2002). Studies around smelters or other sources of pollutants (urban traffic) suggest that earthworm density and biomass may be reduced by moderate TEs concentrations in soils (Pizl and Josens, 1995).
In our plots, the earthworm communities were composed of only five species. We note the absence of endogeic worms like A. rosea (excepted for the plot R), Aporrectodea caliginosa and Allolobophora chlorotica which are usually found in agricultural soil (Decaëns et al., 2003; Pelosi et al., 2009), and also reported in unpolluted meadow and tree plantation in Nord-Pas de Calais coal-mining region (Nahmani et al., 2003). Three of the five species found, L. terrestris, L. castaneus and L. rubellus, are known to be tolerant to metal pollution as shown by Spurgeon and Hopkins (1996) at sites closed to smelting works. This resistance could be explained by a more active calciferous glands in their gut (Fraser et al., 2011) than in sensitive worms like A. chlorotica, A. rosea or A. caliginosa which are absent or present at very low density (A. rosea) in our sites. However, among the differences between these species, it is noticeable that L. rubellus and L. castaneus consume material rich in relatively undecomposed plants whereas A. caliginosa and A. chlorotica feed mainly on welldecomposed organic detritus (Edwards and Bohlen, 1996). Consequently, the absence of the last two species in the communities could perhaps result from a negative effect of the soil metal contamination on the litter microbial degradation. The particular resistance towards TEs and the ecological needs of Dendrobaena rubida could explain its presence in the plot R. Indeed, D. rubida was shown to be insensitive to soil with large amount of lead (Ireland, 1975) and is considered as a woodland species (Sims and Gerard, 1999). Although the metal contamination may have limit the establishment of complete earthworms communities around the old smelter, we observed the establishment of populations belonging to the three ecological groups (epigeic, anecic and endogeic species) distinguished by Bouché (1972) in the mixed afforested plot R.
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4.3. Effects of fly ash-aided phytostabilisation Among remediation techniques, phytoremediation has attracted attention as a low-cost and ecologically sustainable alternative to physicochemical methods, applicable to large areas and accepted by local populations (Mench et al., 2010). However, the long-term influence of the vegetation could result in soil acidification, due to cation uptake, H þ release and plant exudates (Jobbagy and Jackson, 2003). This change in soil pH condition could influence the earthworm community by selecting acid-tolerant earthworms as Dendrobaena octaedra or D. rubida and disfavor acid-intolerant species as A. caliginosa, A. chlorotica or A. rosea (see Edwards and Bohlen, 1996). In our study, the pH of the three plots of the experimental decreased since 2001 (LoparevaPohu et al., 2011a). However, the pH values remaining above the neutrality, the afforestation should not have prevented the establishment of the acid-intolerant species such as A. rosea in the three plots R, F1 and F2. The formation of a leaf layer and the preservation of the humidity in dense pluri-specific afforested plots as R, F1 and F2 are likely responsible for the establishment of populations of epigeic species as L. castaneus and L. rubellus. The species L. castaneus is especially favored by wet and organic rich soils (Sims and Gerard, 1999). Density of epigeic worms L. castaneus was significantly higher in the phytostabilised plot R than in the ash-aided phytostabilised plot F1. Epigeic populations seem to have difficulty to colonize these plots amended with ash. Demuynck et al. (2014) have demonstrated in laboratory avoidance tests that E. fetida avoided significantly the soils treated with fly ashes (soils F1 and F2) and preferred untreated soil R. They concluded that this practice did not allow the recovery of the soil quality since the habitat function for earthworms was strongly affected. However, since this last species prefers organic rich and more acid soils, other species could be influenced differently. L. castaneus, L. rubellus and some Dendrobaena species were found in ash deposits with high pH and elevated TEs levels (Satchell and Stone 1972, in Eijsackers, 2010 ). Eijsackers et al. (1983) did not observe in a field study the colonization of 8-years ash deposits by L. castaneus. However, these data concerned fuel fly ash deposits but not coal fly ash-treated soils. Grumiaux et al. (2007) measured the toxic impact of ashes on earthworms after their integration into the soil and attributed it to the increase of soil pH. According to Riehl et al. (2010), detrimental effects on worms could be linked to soil pH but also to changes in soil texture and structure and to changes in soil water retention following the addition of ashes to the soil. Regarding positive effects of ashes, Grumiaux et al. (2010) have demonstrated that ash amendment in contaminated soil could prevent worm mortality and could decrease trace metals bioaccumulation. However, ashes have detrimental effect on the reproduction of E. andrei (Grumiaux et al., 2007, 2010). This could explain the low proportion of L. castaneus juveniles in the plot F1. Populations of epigeics in these plots were perhaps been more affected (death of individuals, horizontal migrations to evade bad conditions) by the consecutive increase of the soil pH following the addition of ashes, than the anecics which had the opportunity to withdraw by moving deeper inside the soil. The anecic species L. terrestris which abundance was higher in F1 and F2 than in R is known to be not very sensitive to pH (Satchell, 1955 in Edwards and Bohlen, 1996). The low species abundance and biomass of epigeic worms (L. castaneus) or epi-anecic (L. rubellus) in the ash-amended plots F1 and F2 could also be explained by the respective richness of FA1 and FA2 in magnesium and calcium. Fragoso and Lavelle (1992) showed that the percentage dominance of epigeic species in an earthworm community was negatively correlated with the amounts of these elements. So, the use of ash treatments to reduce the mobility of TEs in contaminated plots did not seem to be in
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agreement with the establishment of a diversified epigeic community. Anecic worms L. terrestris, which can move deeper inside the soil under the layer containing ashes (20–30 cm) did not seem to be impacted by ash amendments. However, these findings raise the questions of when and how to perform the addition of ashes to avoid negative effects on soil fauna. Regarding the period of the year, it would be better to perform the addition of ashes when worms become quiescent in deeper soil coiled in small cells. To avoid or limit the effects on epigeics, the direct use of ashes could be substituted by the use of previously prepared mixtures of ashes with soil or vegetable mold. Experimental trials could be performed to solve these questions. At last, it would be interesting to study the metal body burdens of the different ecological categories of earthworms found in the sites to validate the information obtained previously on E. andrei of a positive effect of ashes on the reduction of the bioavailability of the metals present in the soil for the earthworms. Fly ash addition to soil can cause modification of worm behavior. Indeed, in the presence of ash deep burrows can be increased or decreased depending on the species and on the amount of ash application (Yunusa et al., 2009). A comparison of the effect of the soil bioturbation generated by the activity of the worms on the TEs availability in the soils treated or not with ashes could also be interesting to undertake. We conclude that, the metallic pollution around the old smelter limits the establishment of rich worm communities. In the TEs contamination context of the present study, plantations with various tree species appeared beneficial for the establishment of earthworm communities composed of populations belonging to three ecological categories. Ash amendments increase metal immobilization in the soil and can prevent the acidification of the soil in the case of afforested plots. Consequently, ashes could be beneficial for earthworms. Ashes addition performed 10 years ago on metal-contaminated soils planted with a tree mix favored anecic earthworms but disfavored epigeics.
Acknowledgments The authors wish to thank the “Etablissement Public Foncier Nord-Pas de Calais” and the industrial partners (APINOR Ltd., EDF, Surschiste) for the settlement of the experimental site, and the French Environment and Energy Management Agency (“ADEME” Angers, France), (Grant no. 0972C0053) for the financial support of the research program Phytener.
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Earthworm populations of highly metal-contaminated soils restored by fly ash-aided phytostabilisation Fabien Grumiaux a,b,n, Sylvain Demuynck a, Céline Pernin a, Alain Leprêtre a a
Université Lille Nord de France, Laboratoire Génie Civil et géo-Environnement (LGCgE) – Lille1, Bât. SN3, 59655 Villeneuve d’Ascq Cedex, France Université Lille Nord de France, Ecole Supérieure du Professorat et de l’Education (ESPE), site d’Arras, 7 bis rue Raoul François, BP 30927, F-62022 Arras Cedex, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 3 July 2014 Received in revised form 26 November 2014 Accepted 2 December 2014
Highly metal contaminated soils found in the North of France are the result of intense industrial past. These soils are now unfit for the cultivation of agricultural products for human consumption. Solutions have to be found to improve the quality of these soils, and especially to reduce the availability of trace elements (TEs). Phytostabilisation and ash-aided phytostabilisation applied since 2000 to an experimental site located near a former metallurgical site (Metaleurop-Nord) was shown previously as efficacious in reducing TEs mobility in soils. The aim of the study was to check whether this ten years trial had influenced earthworm communities. This experimental site was compared to plots located in the surroundings and differing by the use of soils. Main results are that: (1) whatever the use of soils, earthworm communities are composed of few species with moderate abundance in comparison with communities found in similar habitats outside the TEs-contaminated area, (2) the highest abundance and specific richness (4–5 species) were observed in afforested plots with various tree species, (3) ash amendments in afforested plots did not increase the species richness and modified the communities favoring anecic worms but disfavoring epigeic ones. These findings raised the questions of when and how to perform the addition of ashes firstly, to avoid negative effects on soil fauna and secondly, to keep positive effects on metal immobilization. & 2014 Elsevier Inc. All rights reserved.
Keywords: Earthworms Contaminated soil Trace metals Phytostabilisation Fly ashes
1. Introduction Since the 19th century, industrial activities generated a great number of uncontrolled hazardous sites and large areas contaminated by trace elements (TEs) in many European countries such as Netherlands (Ma et al., 1983) and United Kingdom (Spurgeon and Hopkin, 1996). This is also the case of the former coalmining region in the North of France especially around smelters at Noyelles-Godault, Auby and Mortagne du Nord. At Noyelles-Godault, Metaleurop-Nord, a major Pb plant in Europe for about 100 years generated significant amounts of emitted metal containing smoke until its closedown in 2003. According to the Regional Board for Industry, Research and the Environment (DRIRE, 2003), Metaleurop's annual atmospheric emissions in 2002 were still about 1 t of Cd, 17 t of Pb and 32 t of Zn. This industrial activity resulted in a considerable contamination of the surrounding soils n Corresponding author at: Université Lille Nord de France, Laboratoire Génie Civil et géo-Environnement (LGCgE) – Lille1, Bât. SN3, 59655 Villeneuve d’Ascq Cedex, France. Fax: þ 33 3 20436732. . E-mail address: [email protected] (F. Grumiaux).
http://dx.doi.org/10.1016/j.ecoenv.2014.12.004 0147-6513/& 2014 Elsevier Inc. All rights reserved.
for 7 km to the N-NW by 5 km to the N-NE (Frangi and Richard, 1997). This contamination concerned agricultural (Sterckeman et al., 2000, 2002), urban (Douay et al., 2008a) and forest habitat top soils (Douay et al., 2009). The contamination occured mainly in the upper 20–30 cm of the soil but traces of Cd and Zn were found at about 2 m deep (Sterckeman et al., 2000). Due to the large contaminated surface (approximately 35 km2 around the smelter) low-cost and ecologically sustainable remediation methods had to be developed to reduce impacts on human and environmental health. Indeed, the contamination of soils by TEs have not only effects on herbaceous plants and trees (Pruvot et al., 2006; Bidar et al., 2007; Douay et al., 2008b) but also on human health (de Burbure et al., 2006). Among the methods available, phytostabilisation may be used to reduce the mobility, ecotoxicity and dispersion of TEs through the ecosystem (Vangronsveld et al., 2009). Mineral amendments such as fly ashes (FAs) are known to help phytostabilisation of TEs-contaminated soils (Vangronsveld et al., 2009). FAs, industrial by-products originated from combustion processes, are interesting because of their large availability and low-cost. Pandey and Singh (2010) reviewed recently the interests of FAs in soil systems and indicated the possibility of FAs addition
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in degraded soil for improving nutritional and physico-chemical properties. Among positive effects on soils, FAs can increase the levels of pH, particle density, porosity and water holding capacity (Pandey et al., 2009a). Thus, FAs can be used as an economic fertilizer and soil amendment in small-scale/large-scale cultivation of plants that have ornamental, floricultural, horticultural and forestry potential (Pandey et al., 2009b). FAs are also usefull to enrich soil productivity and crop yields for dry tropical nutrient poor soils (Singh et al., 2011; Singh and Pandey, 2013). However, because FAs can contain toxic inorganic compounds as TEs, their addition to uncontaminated soils can provoke accumulation or translocation of these TEs in plants (Pandey et al., 2009a). Regarding their use as amendments in TEs contaminated soils, FAs represent an effective, cost-effective, eco-friendly management option for reclamation of these soils (in Pandey and Singh, 2010). FAs can play significant role in TEs immobilization in soils. Kumpiene et al. (2007) demonstrated that ashes amendment reduced the leaching by 91.1 percent for copper and by 87.1 percent for lead in a Pb- and Cucontaminated landfilled soil. In 1999, a pilot study started in a former agricultural field located nearby the former smelter Metaleurop Nord. This project consisted in testing the growth of five trees species with or without the use of silico-alumineous FAs (Sodelines, FA1) and sulfo-calcic FAs (Soprolines, FA2) as amendments to reclaim soils with high levels of Pb, Cd and Zn. In the experimental site, after eight years of fly ash-aided phytostabilisation, Lopareva-Pohu et al. (2011a) showed that soil pH had decreased on the whole site while organic carbon content increased. The observed change of these parameters influencing TEs mobility was explained by afforestation. Over time, concentrations of 0.01 M CaCl2-extractable metals increased and were correlated with the soil pH decrease. In the amended soils with FA1 and FA2, extractable Cd and Zn concentrations were about 10-fold lower than in the non-amended soil. The results indicated that the two FAs buffered natural soil acidification due to vegetation development, limited trace element mobility and thus could limit their bioavailability (Lopareva-Pohu et al., 2011a). Fly ash amendments strongly decreased TEs availability to three of the five trees species (Alnus glutinosa, Acer pseudoplatanus and Robinia pseudoacacia) and their translocation to aboveground parts (Pourrut et al., 2011). Similar results were obtained for both FAs on herbaceous species under laboratory conditions (Lopareva-Pohu et al., 2011b). Earthworms have a prominent role in soil functioning (Darwin, 1881). They are known to be bio-indicators for evaluating the effects of soil contamination by trace metals and pesticides, but also for instance the effects of agricultural practices and acid rain (Ghilarov, 1978; Bouché, 1972; Eijsackers, 1983; Paoletti et al., 1991). Grumiaux et al. (2007) tested the intrinsic toxicity of FA1 and FA2 under laboratory-controlled conditions using the earthworm Eisenia andrei. Although, a high mortality (between 60 and 100 percent) was registered in the worms exposed to a freshly ash amended artificial soil, probably due to a rise of soil pH value (from 6 to 8.9 and to 9.5 for FA1 and FA2 respectively). The survival was not significantly affected when worms were exposed to 8 weeks old artificial soil treated with fly ashes which pH stabilized around 8. However, both ashes were still responsible for detrimental effects on worm growth rates (decrease of about 20 percent for the both FAs) and cocoons productions (decrease of 43 and 89 percent for FA1 and FA2 respectively). The sulfo-calcic fly ash FA2 was shown to fully reduce cocoon hatching. Regarding the soil of the experimental site, the acute toxicity of 2 years-old FAstreated soils to E. andrei was significantly lower than those of the untreated soil (Grumiaux et al., 2010). In addition, mortality was 3.6–5-fold lower for worms exposed to FAs amended soil, so 2 years ash-treatment of these contaminated soils decreased their toxicity. However, reproductive parameters appeared more
affected in the worms exposed to the soil treated with sulfo-calcic ashes (FA2) than in those exposed to the untreated metal-contaminated soil. Thus, adding such immobilising agents could negatively affect earthworm populations. This was reinforced by the observation of that experimentally exposed Eisenia fetida to FA1 or FA2 amended soils avoided (avoidance rate 470 percent) these soils significantly (Demuynck et al., 2014). At present, still nothing is known in this experimental site about soil fauna, and particularly about earthworms. Previous results obtained under laboratory conditions with experimental exposures of biological models (Grumiaux et al., 2007, 2010, Demuynck et al., 2014) revealed contrasting physiological effects. In one hand, ash addition should reduce TEs bioaccumulation and reduce worm mortality but in another hand it should alter reproductive output and growth, and modify worm behavior. These findings suggest a possible impact on field earthworm population. So, the aim of the present study was to check whether a ten years trial of fly ash-aided phytostabilisation on highly metal contaminated soils of an experimental site influenced in situ earthworm communities. The study included the comparison of this site with plots located in the surroundings and differing by the use of soils.
2. Materials and methods 2.1. Plots The experimental site is described in details in previous works (Bidar et al., 2007; Lopareva-Pohu et al., 2011a). The site (50°26′N, 3°01′E) is located at Evin-Malmaison, 600 m northeast of Metaleurop-Nord (Noyelles-Godault, North of France) downwind of dominating wind directions (Fig. 1). The experimental site was set up in 1999 on a former agricultural field of about 10,000 m2, which had been cultivated with maize and wheat. It was divided in 2000 into 3 plots of about 3000 m2. The first one (R) was not amended. The two other plots (F1 and F2) were amended respectively with silico-aluminous fly ashes (Sodelines, FA1) and sulfo-calcic fly ashes (Soprolines, FA2) at a rate of 23.3 kg/m2 then plowed up to a 25- to 30-cm soil depth where the most part of the TEs contamination occurred. These two fly ashes (Table 1), provided from Surschiste Ldt (Mazingarbe, France) were previously described (Lopareva-Pohu et al., 2011a). They were produced by fluidisedbed combustion of bituminous coal (Carling thermal power plant) and lignite (Gardanne thermal power plant) respectively. The experimental site (R, F1 and F2) was planted in 2000 with various tree species (R. pseudoacacia L., A. glutinosa L., Quercus robur L., A. pseudoplatanus L. and Salix alba L.) and sown with an herbaceous mixture (Festuca ovina L., Lolium perenne L., Bromus catharticus Vahl and Trifolium repens L.). Three additional plots located in the surroundings of the experimental site and representing different soil uses were also considered in the study to determine the influence of ground covers on earthworm communities. A plot (MV) which had been cultivated with maize and wheat until 2009 was considered as the absolute reference (i.e., agricultural soil without fly ash-aided phytostabilisation and earthworm communities negatively impacted by plowing and TEs contamination). A plot (BS) which is a meadow since 1999, located in the South of the experimental site and an ash tree wood (FO), located in the West have been also sampled. Characteristics of soils are given in Table 2. According to Lopareva-Pohu et al. (2011a), the soil of the experimental site is a silt-loamy redoxic neoluvisol (clay contents, 13–21 percent; silts 55–62 percent; sands 22–26 percent) with a neutral pH, developed from loess. It lies on a truncated paleoneoluvisol developed from clay. During winter, the groundwater level reaches 60 cm in
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Fig. 1. Geographic location of the experimental site and plots where earthworms were sampled. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
Table 1 Characteristics of silico-alumineous and sulfo-calcical fly ashes (Lopareva-Pohu et al., 2011a). Parameter (unit)
Silico-alumineous ashes FA1 (Sodelines)
Sulfo-calcic ashes FA2 (Soprolines)
pH H2O CaCO3 total (g/kg) CEC (cmol þ /kg) CaO (g/kg) MgO (g/kg) K2O (g/kg) Na2O (g/kg) P2O5 (g/kg) S H2O (g/kg) Cd (mg/kg) Cu (mg/kg) Pb (mg/kg) Zn (mg/kg)
9.9 21 5.1 28.4 2.93 0.91 0.18 0.09 5.5 1.0 81 142 257
12.6 67 6.4 184.5 1.99 0.21 0.06 0.24 13.9 0.4 38 39 85
Note: Coal FAs were from Surschiste Ltd. (Mazingarbe, France).
depth. As for agricultural soils around Metaleurop-Nord, the major part of contamination by Cd, Pb and Zn is limited to the plowed layer (20–30 cm) (Lopareva-Pohu et al., 2011a). 2.2. Earthworms sampling Earthworm populations were studied in March 2010 when most earthworm species are particularly active (Bouché, 1972). The number of samples taken at each plot was a compromise between statistical considerations and practical considerations of time and
manpower. For the plots of the experimental site whose ground use was modified in 2000 by the plantation of various tree species and by additions of fly ashes (F1 and F2 only) to the soil, a greater sampling effort (8 samples per plot) was performed than in the surrounding plots (4–5 samples per plot) where the ground use was left unchanged and the soil was not amended. The samples were positioned in a square or in line according to the shape of the plot. The sampling was made in the center of the plot or at least 10 m from the edges. Each sample was distant from each other by at least 5 m. A sampling method coupling chemical expulsion with allyl isothiocyanate (AITC) derived from Zaborski (2003) and hand sorting was used within 60 cm 60 cm quadrats. We chose this combination of methods because different studies have shown that chemical expulsion is, in general, more efficient for anecic species than is hand sorting, but that hand sorting is more efficient for endogeic species (in Zaborski, 2003). The soil of each quadrat was irrigated twice with 5 L of the AITC solution (1 g AITC dissolved in 100 mL isopropanol/10 L water) at 10 min intervals after the removal of the vegetation cover and of the litter. For 10 min after each irrigation, all earthworms emerging on the soil surface were collected. Then each soil quadrat was excavated to a depth of 20 cm with a spade and earthworms were collected by hand sorting. Worms were placed into plastic containers with watermoistened filter paper, returned to the laboratory in coolers, stored at 4 °C and then sorted, counted, weighed and fixed in 95 percent ethanol within the next 24 h. Worms were identified to species level using taxonomic key of Bouché (1972) for adults, or using characteristics such as the coloration, the prostomium shape and the size and pattern of setae for juveniles.
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Table 2 Physicochemical characteristics of the field soil. Plots
MVb
BSa,b
FOa,b
Rb
F1b
F2b
pH C/N Corg (g/kg) N tot (g/kg) CaCO3 total (g/kg) CaO (g/kg) MgO (g/kg) K2O (g/kg) SH2O (mg/kg) CEC (cmol þ /kg)
8.20 (0.09) 15.2 (0.4) 18.2 (0.4) 1.20 (0.04) 10.2 (3.3) – – – – 14.9 (1.6)
7.2 12.9 19.1 1.48 2.32 2.7 0.12 0.23 – 12.2
7.6 12.9 16.9 1.32 5.2 4.3 0.06 0.2 – 13.0
7.31 (0.2) 13.5 (0.1) 23.8 (0.7) 1.76 (0.06) 1.5 (0.8) 4.4 (0.4) 0.19 (0.01) 0.30 (0.01) 14 (4) 14.5 (0.5)
7.95 (0.03) 15.5 (0.1) 25.9 (0.6) 1.68 (0.04) 5.8 (0.9) 5.9 (0.2) 0.34 (0.01) 0.41 (0.01) 16 (2) 13.6 (0.2)
7.74 (0.02) 17.5 (0.3) 29.8 (1.1) 1.70 (0.06) 71.6 (6.5) 18.6 (1.0) 0.16 (0.01) 0.40 (0.04) 2257 (277) 14.6 (0.4)
Cd Cu Pb Zn
14.1 (1.4) 37 (1.1) 731 (67) 1000 (88)
5.8 34.8 288 486
14.9 45.3 858 1061
16.6 (0.6) 34 (1.2) 926 (26) 1135 (42)
18.0 (0.3) 41 (1.4) 967 (16) 1211 (26)
17.8 (0.5) 34 (1.7) 900 (30) 1127 (43)
19.5 53.1 27.4
20.3 52.3 27.4
23.4 56.0 20.6
21.5 55.2 23.3
19.6 58.1 22.3
12.9 61.5 25.6
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
Clay (%) Silt (%) Sand (%)
Note: Mean value (bold characters) and standard deviation (value into brackets). (–) no data. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2. a b
A single sample composed of 5 soil cores was analyzed for these two soils. Phytener (2014).
2.3. Statistical analysis Normality of data (density and biomass) was checked with Kolmogorov–Smirnov's tests and homogeneity of variances was tested using Levene's tests. Due to the heteroscedasticity, data analysis was performed using a non-parametric statistical method
(Kruskall–Wallis test) followed by a multiple comparisons method (Conover–Iman). A dendrogram based on cluster analysis using Chi-square distance and Ward's agglomerative method was performed on worm abundance data.
Table 3 Mean number, mean biomass and species of worms collected in the 6 plots of the studied area in March 2010. Plots n
MV 5
FO 4
BS 4
R 8
F1 8
F2 8
Biomass (g/m2) Total earthworms (ind/m2)
1.2 (1.3) 2.2 (2.3)
24.5 (16.8) 40.3 (18.4)
13.8 (2.1) 25.0 (9.9)
26.3 (13.9) 70.1 (38.5)
40.3 (23.6) 75.7 (23.3)
47.4 (22.9) 117.4 (34.5)
9.0 (7.6) 3.5 (5.3) 12.5 (10.8)
29.5 (23.3) 16.0 (12.6) 45.5 (29.4)
5.6(8.4) 1.0 (1.4) 6.6 (9.1)
12.5 (7.6) 10.4 (15.1) 22.9 (20.3)
Lumbricus castaneus
ad juv tot
0 0 0
2.8 (2.3) 0 2.8 (2.3)
Dendrobaena rubida
ad juv tot
0 0 0
0 0 0
0 0 0
0.3 (1.0) 0 0.3 (1.0)
0 0 0
0 0 0
Lumbricus rubellus
ad juv tot
0 0 0
0 0 0
0 4.2 (5.3) 4.2 (5.3)
2.4 (2.8) 6.6 (7.8) 9.0 (10.2)
1.7 (2.5) 6.9 (13.6) 8.7 (12.8)
2.8 (3.6) 4.9 (5.7) 7.6 (7.4)
Aporrectodea rosea
ad juv tot
0 0 0
0 0 0
0 0 0
0 0.7 (1.3) 0.7 (1.3)
0.3 (1.0) 1.0 (2.1) 1.4 (2.1)
0 0.7 (1.3) 0.7 (1.3)
Lumbricus terrestris
ad juv tot
0 2.2 (2.3) 2.2 (2.3)
3.1 (1.8) 11.5 (11.6) 14.6 (11.8)
4.8 (4.6) 54.2 (28.8) 59.0 (26.6)
3.8 (2.9) 82.3 (22.0) 86.1 (22.0)
Species number
1
3.5 (3.5) 34.0 (18.3) 37.5 (17.9) 2
6.3 (3.5) 2.1 (2.7) 8.3 (3.2) 3
5
4
4
Note: Mean value (bold characters) and standard deviation (value into brackets). n: number of samples, ad: adults, juv: juveniles, tot: total (adults þ juveniles). MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
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3. Results Total number of worms, total weight of worms, and number of earthworm species are presented in Table 3. Five species of earthworms were identified, in varying proportions from a total of 338 individuals (16.3 percent adults, 83.7 percent juveniles) for F2, 218 individuals (16.5 percent adults, 83.5 percent juveniles) for F1, 202 individuals (50.5 percent adults, 49.5 percent juveniles) for R, 58 individuals (15.5 percent adults, 84.5 percent juveniles) for FO, 36 individuals (61.1 percent adults, 38.9 percent juveniles) for BS and 4 individuals (100 percent juveniles) for MV. The five species found were two epigeic (litter-dwellers), Dendrobaena rubida, Lumbricus castaneus; one epi-anecic (litter/soil-dweller), Lumbricus rubellus; one endogeic (soil-dweller), Aporrectodea rosea and one anecic, Lumbricus terrestris. Most worms were collected during irrigation with AITC. Only the soil-dwelling individuals were captured during manual sorting. The agglomerative hierarchical clustering method performed on worm density data separated 2 groups of plots (Fig. 2). In the first group L. castaneus was the dominant species (plots R and BS). L. terrestris was the dominant species in the second group (plots F1, F2, FO and MV). Sites F1 and F2 where the other species L. castaneus, L. rubellus and A. rosea were also present were separated from the plots MV and FO. 3.1. Effects of the plant cover and soil cultivation of the plot Data and statistical results are given in Tables 3 and 4. The abundance data obtained in each plot (Table 3) were transformed into density values. Regarding the agricultural plot MV, density as well as biomass was very low with only 2 ind/m2 and 1 g/m2 respectively. In addition, one single species L. terrestris was found. Density and biomass were higher in plots that had not been cultivated since 1999: the meadow BS (25 ind/m2, 14 g/m2), the ash tree wood FO (40 ind/m2, 25 g/m2) and the phytostabilised plot R (70 ind/m2, 26 g/m2) but significant differences were only found for the woody plots FO and R. Plot R was the only one containing five species. 3.2. Effects of the ash treatment Data and statistical results are given in Tables 3 and 4. The earthworm biomass in the ash-aided phytostabilised plots F1 and
Fig. 2. Dendrogram based on cluster analysis using Chi-square distance and Ward's agglomerative method performed on worm abundance data. MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
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F2 (40 g/m2 and 47 g/m2 respectively) was higher than in the phytostabilised plot R (26 g/m2) but the difference was not significant. Density was significantly higher (p o0.05) in plot F2 (117 ind/m2) containing sulfo-calcic ashes than in the two other plots R and F1 (70 ind/m2 and 76 ind/m2 respectively). Regarding the density of each species, differences between plots were found only for L. castaneus and L. terrestris. Density of L. castaneus was higher in the phytostabilised plot R than in the ashaided phytostabilised plots F1 and F2 but only significant in F1 (p o0.05). Density of L. terrestris (juveniles and total number of individuals) was significantly higher in F1 and F2 than in R (p o0.001).
4. Discussion 4.1. Soil contamination The soils of the studied plots were all classified as silt-loam according to the USDA triangle. The soils of plots BS, FO and R showed a nearly neutral pH value. The pH value (7.7) of the agricultural plot MV must be due to calcic amendments performed in the past. These soil characteristics are suitable for the establishment of earthworm communities (see Bouché, 1972). Ashes addition to the soil resulted in an increase of soil pH (7.9 and 7.7), CaCO3, exchangeable Ca2 þ , Mg2 þ , soluble S and total S contents in both F1 and F2 soils. Cd, Cu, Pb and Zn contents in soils (Table 1) were respectively around 75, 2.5, 20 and 11-fold the references values determined by Baize and Sterckeman (2001) for Northern France (median values). In addition, Pb, Zn and Cd concentrations in these soils clearly exceeded the upper limits (300, 300 and 3 mg/kg, respectively) established by the European Directive 86/ 278/ EEC (Council of the European Communities, 1986) for the addition of sludges as amendments to agricultural soils. Therefore, the soils studied must be considered as polluted soils from a legal standpoint. This high TEs contamination may have hampered the establishment of earthworm communities (e.g. Nahmani and Lavelle, 2002). 4.2. Effect of soil contamination and soil management on earthworm communities Data were obtained from a single location (i.e., without a real replication of the plots according to their proper use). Nevertheless, plots tested offered the main advantage to be located at short distances one with each other or even were adjacent. The plots surrounding the experimental area share with R, F1 and F2 quite similar level of TEs, the historic use of the soil (agriculture) and natural soil parameters values. So, only the treatment received by the soil and the ground cover differ. This choice allowed hypothesizing that differences seen for earthworm communities were more related to the proper use of each plot than to soil types. In addition, this comparison is justified since it allows determining the effect of the ground cover introduced in 2000 in the experimental area (plantation with a tree mix) on the earthworm community. However, because of this possible bias, interpretation of the results has to be taken with care and its significance should be essentially considered with a value at a regional scale of the NordPas de Calais coal-mining region. Earthworms communities from the plots studied with the exception of F1 and F2 (see 4.3 for the comments on these plots) appeared quite poor. Species richness was from 1 to 5 species and earthworm density was ranged from 2.2 to 70.3 ind/m2. The highest values were found in the tree plantations (R particularly) while the lowest concerned the plot still used as cropping soil (MV). These last results were not surprising since earthworm
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Table 4 Statistical results concerning the effect the plant cover and soil cultivation and the effect of the ash treatment. Plot
Effects of the plant cover and soil cultivation
Effects of the ash treatment
P value
MV
FO
BS
R
P value
R
F1
F2
ad juv tot
o0.01 o0.01 o0.01 o0.01 o0.01
a a a a a
b b a a ab
ab ab ab ab bc
b b b b c
0.13 o 0.05 o 0.05 o 0.05 o 0.05
a a b b b
a a a a a
a b ab ab ab
Dendrobaena rubida
ad juv tot
0.65 – 0.65
a – a
a – a
a – a
a – a
0.37 – 0.37
a – a
a – a
a – a
Lumbricus rubellus
ad juv tot
0.06 0.06 0.06
a a a
a a a
a a a
a a a
0.81 0.60 0.98
a a a
a a a
a a a
Allolobophora rosea
ad juv tot
– 0.3 0.3
– a a
– a a
– a a
– a a
0.37 0.99 0.74
a a a
a a a
a a a
Lumbricus terrestris
ad juv tot
o0.05 o0.05 o0.01
a a a
ab b b
b a ab
b ab b
0.76 o 0.001 o 0.001
a a a
a b b
a b b
Biomass Total earthworms Lumbricus castaneus
Note: In the same lane, letters (abc) indicate homogeneous groups. ad: adults, juv: juveniles, tot: total (adults þ juveniles). Data analysis was performed using a nonparametric statistical method (Kruskall–Wallis test) followed by a multiple comparisons method (Conover–Iman). ad: adults, juv: juveniles, tot: total (adults þ juveniles). MV: agricultural plot, BS: meadow, FO: ash tree wood, R: afforested plot with various tree species, F1: afforested plot with various tree species amended with silico-aluminous fly ashes FA1, F2: afforested plot with various tree species amended with sulfo-calcical fly ashes FA2.
populations in cultivated land are generally lower than those found in undisturbed habitats (Paoletti, 1999; Curry et al., 2002). However, in the plot MV both density and species richness were extremely low. Agricultural activities have negative effects on invertebrates (Hendrix and Edward, 2004). Direct killing, increased predation, soil compaction, use of biocides, food and moisture limitations and decreasing organic matter contents are factors known to reduce earthworm populations in intensive cropping systems (in Metzke et al., 2007). Ponge et al. (2013) also recorded an increase of earthworm species richness along a gradient of decreasing agricultural intensification (from crop to meadow) in French Brittany. This increase of richness in our plots would be due to an increased availability of trophic resources. Organic matter, surface litter and dead roots are known to constitute part of the diet of various earthworm species (Sims and Gerard, 1999). Both the species richness and the density recorded in our plots were lower than those reported for similar habitats by authors working in locations without high TEs contamination of the soil. It is noteworthy that Nahmani and Rossi (2003) found 392, 271 and 52 ind/m2 and 6, 8 and 2 species in unpolluted grassland, tree plantation and cultivated soils respectively at a distance of only some tens of kilometers of our zone of study. At a shorter distance of the studied site (about 3 km to the northwest) 634 ind/m2 and 13 species were recorded in an unpolluted meadow (Restbiodiv, 2012) while only 25 ind/m2 and 3 species were recorded in the polluted meadow BS. The particular location of the studied plots under the dominant winds of the lead smelter Metaleurop-Nord that was active for almost a century can explain the poor earthworm communities. Earthworm populations are known to be reduced by high concentrations of TEs in soils (Bengtsson et al., 1983; Spurgeon and Hopkin, 1996; Nahmani and Lavelle, 2002). Studies around smelters or other sources of pollutants (urban traffic) suggest that earthworm density and biomass may be reduced by moderate TEs concentrations in soils (Pizl and Josens, 1995).
In our plots, the earthworm communities were composed of only five species. We note the absence of endogeic worms like A. rosea (excepted for the plot R), Aporrectodea caliginosa and Allolobophora chlorotica which are usually found in agricultural soil (Decaëns et al., 2003; Pelosi et al., 2009), and also reported in unpolluted meadow and tree plantation in Nord-Pas de Calais coal-mining region (Nahmani et al., 2003). Three of the five species found, L. terrestris, L. castaneus and L. rubellus, are known to be tolerant to metal pollution as shown by Spurgeon and Hopkins (1996) at sites closed to smelting works. This resistance could be explained by a more active calciferous glands in their gut (Fraser et al., 2011) than in sensitive worms like A. chlorotica, A. rosea or A. caliginosa which are absent or present at very low density (A. rosea) in our sites. However, among the differences between these species, it is noticeable that L. rubellus and L. castaneus consume material rich in relatively undecomposed plants whereas A. caliginosa and A. chlorotica feed mainly on welldecomposed organic detritus (Edwards and Bohlen, 1996). Consequently, the absence of the last two species in the communities could perhaps result from a negative effect of the soil metal contamination on the litter microbial degradation. The particular resistance towards TEs and the ecological needs of Dendrobaena rubida could explain its presence in the plot R. Indeed, D. rubida was shown to be insensitive to soil with large amount of lead (Ireland, 1975) and is considered as a woodland species (Sims and Gerard, 1999). Although the metal contamination may have limit the establishment of complete earthworms communities around the old smelter, we observed the establishment of populations belonging to the three ecological groups (epigeic, anecic and endogeic species) distinguished by Bouché (1972) in the mixed afforested plot R.
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4.3. Effects of fly ash-aided phytostabilisation Among remediation techniques, phytoremediation has attracted attention as a low-cost and ecologically sustainable alternative to physicochemical methods, applicable to large areas and accepted by local populations (Mench et al., 2010). However, the long-term influence of the vegetation could result in soil acidification, due to cation uptake, H þ release and plant exudates (Jobbagy and Jackson, 2003). This change in soil pH condition could influence the earthworm community by selecting acid-tolerant earthworms as Dendrobaena octaedra or D. rubida and disfavor acid-intolerant species as A. caliginosa, A. chlorotica or A. rosea (see Edwards and Bohlen, 1996). In our study, the pH of the three plots of the experimental decreased since 2001 (LoparevaPohu et al., 2011a). However, the pH values remaining above the neutrality, the afforestation should not have prevented the establishment of the acid-intolerant species such as A. rosea in the three plots R, F1 and F2. The formation of a leaf layer and the preservation of the humidity in dense pluri-specific afforested plots as R, F1 and F2 are likely responsible for the establishment of populations of epigeic species as L. castaneus and L. rubellus. The species L. castaneus is especially favored by wet and organic rich soils (Sims and Gerard, 1999). Density of epigeic worms L. castaneus was significantly higher in the phytostabilised plot R than in the ash-aided phytostabilised plot F1. Epigeic populations seem to have difficulty to colonize these plots amended with ash. Demuynck et al. (2014) have demonstrated in laboratory avoidance tests that E. fetida avoided significantly the soils treated with fly ashes (soils F1 and F2) and preferred untreated soil R. They concluded that this practice did not allow the recovery of the soil quality since the habitat function for earthworms was strongly affected. However, since this last species prefers organic rich and more acid soils, other species could be influenced differently. L. castaneus, L. rubellus and some Dendrobaena species were found in ash deposits with high pH and elevated TEs levels (Satchell and Stone 1972, in Eijsackers, 2010 ). Eijsackers et al. (1983) did not observe in a field study the colonization of 8-years ash deposits by L. castaneus. However, these data concerned fuel fly ash deposits but not coal fly ash-treated soils. Grumiaux et al. (2007) measured the toxic impact of ashes on earthworms after their integration into the soil and attributed it to the increase of soil pH. According to Riehl et al. (2010), detrimental effects on worms could be linked to soil pH but also to changes in soil texture and structure and to changes in soil water retention following the addition of ashes to the soil. Regarding positive effects of ashes, Grumiaux et al. (2010) have demonstrated that ash amendment in contaminated soil could prevent worm mortality and could decrease trace metals bioaccumulation. However, ashes have detrimental effect on the reproduction of E. andrei (Grumiaux et al., 2007, 2010). This could explain the low proportion of L. castaneus juveniles in the plot F1. Populations of epigeics in these plots were perhaps been more affected (death of individuals, horizontal migrations to evade bad conditions) by the consecutive increase of the soil pH following the addition of ashes, than the anecics which had the opportunity to withdraw by moving deeper inside the soil. The anecic species L. terrestris which abundance was higher in F1 and F2 than in R is known to be not very sensitive to pH (Satchell, 1955 in Edwards and Bohlen, 1996). The low species abundance and biomass of epigeic worms (L. castaneus) or epi-anecic (L. rubellus) in the ash-amended plots F1 and F2 could also be explained by the respective richness of FA1 and FA2 in magnesium and calcium. Fragoso and Lavelle (1992) showed that the percentage dominance of epigeic species in an earthworm community was negatively correlated with the amounts of these elements. So, the use of ash treatments to reduce the mobility of TEs in contaminated plots did not seem to be in
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agreement with the establishment of a diversified epigeic community. Anecic worms L. terrestris, which can move deeper inside the soil under the layer containing ashes (20–30 cm) did not seem to be impacted by ash amendments. However, these findings raise the questions of when and how to perform the addition of ashes to avoid negative effects on soil fauna. Regarding the period of the year, it would be better to perform the addition of ashes when worms become quiescent in deeper soil coiled in small cells. To avoid or limit the effects on epigeics, the direct use of ashes could be substituted by the use of previously prepared mixtures of ashes with soil or vegetable mold. Experimental trials could be performed to solve these questions. At last, it would be interesting to study the metal body burdens of the different ecological categories of earthworms found in the sites to validate the information obtained previously on E. andrei of a positive effect of ashes on the reduction of the bioavailability of the metals present in the soil for the earthworms. Fly ash addition to soil can cause modification of worm behavior. Indeed, in the presence of ash deep burrows can be increased or decreased depending on the species and on the amount of ash application (Yunusa et al., 2009). A comparison of the effect of the soil bioturbation generated by the activity of the worms on the TEs availability in the soils treated or not with ashes could also be interesting to undertake. We conclude that, the metallic pollution around the old smelter limits the establishment of rich worm communities. In the TEs contamination context of the present study, plantations with various tree species appeared beneficial for the establishment of earthworm communities composed of populations belonging to three ecological categories. Ash amendments increase metal immobilization in the soil and can prevent the acidification of the soil in the case of afforested plots. Consequently, ashes could be beneficial for earthworms. Ashes addition performed 10 years ago on metal-contaminated soils planted with a tree mix favored anecic earthworms but disfavored epigeics.
Acknowledgments The authors wish to thank the “Etablissement Public Foncier Nord-Pas de Calais” and the industrial partners (APINOR Ltd., EDF, Surschiste) for the settlement of the experimental site, and the French Environment and Energy Management Agency (“ADEME” Angers, France), (Grant no. 0972C0053) for the financial support of the research program Phytener.
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