Riboflavin storage in earthworm chloragocytes and chloragocyte-derived eleocytes and its putative role as chemoattractant for immunocompetent cells

We have shown previously that riboflavin is stored in chloragosomes of chloragocyte-derived earthworm eleocytes what may have an adaptive value for worms vulnerable to soil-derived pathogen invasion. Thus, it is important to determine whether riboflavin stores are also present in earthworm species with low eleocyte counts, especially in the chloragocytes of their chloragogen tissue that surrounds the coelomic site of the intestine, and find out its putative roles in earthworm immunity. Experiments were performed on adult worms from 6 species. Freely floating coelomocytes (consisting of amoebocytes plus species-specific numbers of eleocytes) were extruded by mild electric shock and then chloragocytes from the same anaesthetized worm were mechanically detached into buffer. Both cell suspensions were analyzed by spectrofluorometry for riboflavin content. It turned out that riboflavin is stored in species-specific quantities in all investigated species. Riboflavin storage predominates in free coelomocytes of eleocyte-rich species (Eisenia andrei, Dendrobaena veneta, Allolobophora chlorotica) while in chloragocytes of species with few eleocytes (Lumbricus terrestris, L. rubellus, and Aporrectodea caliginosa). Upon a massive microbial impact, the coelomocytes (both amoebocytes and eleocytes) are involved in the formation of multicellular bodies encapsulating soil-derived pathogens, what is connected with enhanced coelomocyte mobility. Thus the second aim of the present investigation was to check if riboflavin can participate in coelomocyte accumulations. Tests performed in a 48-well chemotaxis chamber revealed that riboflavin behave as chemoattractant for coelomocytes of all investigated earthworm species, which may be one of mechanisms underpinning its immunomodulatory functions.     

Species-specific sensitivity of earthworm coelomocytes to dermal metal (Cd,Cu, Ni,Pb, Zn) exposures: Methodological approach

Earthworm immune-competent cells (coelomocytes) can be adversely affected by soil metal contamination. The aim of the present paper was to perform comparative studies on the coelomocytes of four lumbricid species dermally exposed for 2 days to filter paper soaked with Ni, Cu, Zn, Cd, or Pb chlorides (1 mM). Coelomocytes were expelled by electro-stimulation, counted, and assayed ex vivo by flow cytometry and spectrofluorometry. The coelomocytes of Allolobophora chlorotica and Dendrobaena veneta contain both amoebocytes and riboflavin-storing eleocytes; consequently, they were subjected to assays based on flow cytometric quantification of autofluorescent eleocytes and spectrofluorometric measurement of riboflavin content in coelomocyte lysates. In Al. chlorotica, the number of coelomocytes, the percentage of eleocytes, and the amount of riboflavin were significantly lower in Cu-exposed worms although these cytometric parameters were less affected by Ni, Zn, Cd exposure, and entirely unaffected by Pb exposure. In D. veneta, such cellular effects were also only observed in Cu-exposed worms. The coelomic fluids of Aporrectodea caliginosa and Lumbricus rubellus are constitutionally impoverished of eleocytes; therefore, these species could not be subjected to assays measuring the riboflavin content of these granular cells. Rather, the amoebocytes of Ap. caliginosa and L. rubellus were subjected to flow cytometric measurements of in vitro neutral red uptake (NR). However, the NR uptake assay was technically demanding, requiring a strictly normalized incubation period over all samples to yield useful comparative data. In contrast, the riboflavin content in the coelomocyte lysates of eleocyte-rich species appears to be a robust and convenient immune-function biomarker of environmental stress.     

Soil amendment with municipal sewage sludge affects the immune system of earthworms Dendrobaena veneta

Dendrobaena veneta is an earthworm species capable of consuming a wide range of organic wastes which may be used as a field indicator of municipal sewage sludge applied to land. The aim of the present 8-week laboratory experiment was to check viability, reproduction and the immune system of D. veneta maintained in soil without food additions (control 0s group) or in soil amended with 25% or 50% municipal sewage sludge (25s and 50s groups, respectively). Reproduction and immunity are important physiological functions whose detailed study can provide information on the effects of pollutants. After the 8-week exposure period, earthworm mortality (2 out 20 individuals) was recorded only in the 50s group. Reproduction was high in the 25s group (44 cocoons and 41 juveniles) whereas reproduction was almost completely inhibited both in the food-deprived control 0s group (1 cocoon, 3 juveniles) and in the 50s group containing a high amount of sludge (2 cocoons). Significantly increased numbers of non-invasively extruded coelomocytes were recorded 3 weeks after the start of the experiment in the 50s group, but they dropped to the food-deprived control level by the end of 8 weeks likely due to exhaustion of the immune system coping with sludge-derived microbes and/or toxins. In contrast, numbers of coelomocytes in the 25s group increased gradually reaching the maximum at the end of the experiments. In conclusion, high amounts of municipal sewage sludge are detrimental to worms, inhibiting reproduction and inflicting mortality. A moderate amount of municipal sewage sludge provides a good source of nutrients for D. veneta, supporting their growth and reproduction for at least 8 weeks. Immunological parameters might serve as useful indicators of earthworm exposure to sewage sludge.     

Short-term changes of metal availability in soil. II: The influence of earthworm activity

This study aimed to evaluate short-term earthworm-induced changes in the availability of metals applied to soil directly (metal-spiked) or via an organic matrix (sludge-amended). A laboratory experiment was performed using destructive sampling of microcosms filled with agricultural soil. A concentration gradient of industrial sludge contaminated predominantly with Cr, Cu, Ni, and Zn, and a soil freshly spiked with the same metal concentrations were applied on top of the soil columns. Individuals of Dendrobaena veneta (mimicking a realistic density of 500 earthworms per m²) were introduced in half of the replicates of each treatment. Total and 0.01 M CaCl₂ extractable metal concentrations were measured in soil after 0, 3, 6 and 12 weeks and metal concentrations in earthworms and percolates were measured after 3, 6, and 12 weeks. Earthworm activity did not affect metal availability of any treatment over time, but Ni and Cu concentrations in D. veneta were higher at the highest treatment levels. Earthworm Zn concentrations were similar in all treatments while Cr concentrations increased with increasing soil total metal content only for sludge treatments. Existing relationships of earthworm metal concentrations with total metal content in soil, taken from the literature, were not able to predict the metal levels measured in D. veneta. Results demonstrated that although over 12 weeks earthworm activity did not affect metal availability in soil, their burrowing activities did influence the metal concentrations of percolates over time.     

Development of QSAR's in soil ecotoxicology: Earthworm toxicity and soil sorption of chlorophenols,chlorobenzenes and chloroanilines

Soil adsorption and the toxicity of four chloroanilines for earthworms were investigated in two soil types. The toxicity tests were carried out with two earthworm species, Eisenia andrei and Lumbricus rubellus. LC₅₀ values in mg kg^?1 dry soil were recalculated towards molar concentrations in pore water using data from soil adsorption experiments. An attempt has been made to develop Quantitative Structure Activity Relationships (QSAR's) using these results and data on five chlorophenols and dichloroaniline in four soils and five chlorobenzenes in two soils published previously (Van Gestel and Ma, 1988, 1990; Van Gestel et al., 1991). Significant QSAR relationships were obtained between 1) adsorption coefficients (log K _om ) and the octanol/water partition coefficient (log k _ow ), and 2) LC₅₀ values (in itμmol L^?1 soil pore water) and log K _ow . It can be concluded that both earthworm species tested are equally sensitive to chlorobenzenes and chloroanilines, E. andrei is more sensitive than L. rubellus to chlorophenols.     

Heavy metal tolerance of nematode-trapping fungi in lead-polluted soils

A study was conducted to establish whether the diversity of nematode-trapping fungi in Pb-polluted soils increases or decreases with increasing degree of soil contamination, and whether the fungi from polluted soils exhibit higher tolerance to Pb toxicity than those from unpolluted soils. Five genera containing 28 nematode-trapping fungi were recorded in total from five collection sites highly contaminated by Pb, with the concentration ranging from 306 to 4907 mg kg⁻¹. These fungi fell into seven groups according to their trapping mechanisms. In this area, the most frequent group was the net former of which 16 species were recorded and its occurrence frequency (61.15%) was higher than those of the others. Fungal diversity of NTF was slightly positively correlated with the Pb pollution levels (r = 0.29), which suggested the distribution of nematode-trapping fungi was not restricted by the heavy metal at these sites. The mycelial growth of nematode-trapping fungi which derived from either Pb-polluted soils or from unpolluted soils was completely inhibited by 1.8 mmol of Pb. At the Pb concentration of 1.2 mmol, the inhibition growth rates varied between 18.50 and 22.57% and there was no significant difference in the Pb tolerance of nematode-trapping fungi as to whether the strains derived from Pb pollution soils or unpolluted soils.     

Regenwürmer als Bioindikatoren für Schwermetallbelastungen von Böden unter Freilandbedingungen

The use of earthworms in monitoring soil pollution by heavy metals Total heavy metal contents (HNO₃-soluble) and exchangeable fractions (Ca(NO₃)₂-soluble) of Pb, Zn and Cd were measured in soils, litter layers and earthworms (dry masses) from forest, arable and pasture sites in a transect of the main wind direction and varying distances (1.5, 5.4, 11.4, 15.6 km) to a lead smelter near Bad Ems, Germany. Additionally, cast materials of Lumbricus terrestris-individuals were collected from the surface of the pasture sites. In the observed area total soil contents of Pb and Cd exceeded the C-level and total contents of Zn the B-level of the “Netherland-list”. Heavy metal contents in soils and earthworms decreased with increasing distance to the smelter. Pb showed the best correlation. Correlation between total contents of Pb and Cd in soils and earthworms were significant (rs = 0.66; p < 0.05 and rs = 0.67; p < 0.01, respectively). The uptake of heavy metals by Lumbricus rubellus, L. terrestris and Aporrectodea caliginosa was metal specific rather than species specific with factors of accumulation being <1 (Pb), 2.7–7.6 (Zn) and 19.5–85.5 (Cd). The heavy metal contents of the observed cast materials signified the different transfer of elements from soil material via earthworm individuals to the faeces. In the cast materials the amounts of Pb were high and the amounts of Zn and Cd were low. This indicates a high accumulation rate for Zn and especially for Cd in the tissues of the observed earthworm individuals. The present data support the necessity of ecotoxicological threshold levels.     

Flooding responses of three earthworm species,Allolobophora chlorotica,Aporrectodea caliginosa and Lumbricus rubellus,in a laboratory-controlled environment

To get a better understanding of earthworm’ responses towards flooding, three laboratory experiments were performed with the species Allolobophora chlorotica, Aporrectodea caliginosa and Lumbricus rubellus.Flooding response was determined in a pot experiment, in which the earthworms were incubated for 42 days in flooded or non-flooded soil, with or without heavy metal pollution. To determine moisture preference, earthworms were incubated for 9 days in aquaria with five compartments, containing soil with a moisture gradient (%, w/w), ranging from 35%, 45% (field capacity), 55%, 65% (saturated) to 65%+ (saturated and an extra water layer). Effects on earthworm health were studied by incubating earthworms of each species for 42 days in soil with the same range of moisture contents and determining the dry/wet weight ratio and dry weight gain as an indication of earthworm health.A. chlorotica was tolerant to water, although the worms tended to escape from flooded soil. Their health was significantly lower in the flooded soils (P<0.05). A. caliginosa showed little response to flooding. This species, however, was affected by the heavy metal pollution in the pot experiment. L. rubellus was sensitive towards flooding, with escape and avoidance behaviour being the main mechanism of survival. This species was able to survive when forced to stay in flooded soil for at least 42 days, but this significantly reduced its health (P<0.05).These results suggest that earthworms are able to survive in inundated soils, but there are large differences between species in response to flooding conditions.     

Heavy metal content and toxicity of mine and quarry soils

Purpose

Soils formed in metallic mines and serpentinite quarries, among other unfavourable features, have high levels of heavy metals. They can release into the environment causing surface and subsurface water contamination, uptake by plants, their accumulation in the food chain and adverse effects on living organisms. In this work, we studied the magnitude of the soils’ toxic effects not only on spontaneous plants but also on two species with phytoremediation potential.

Materials and methods

Several soils from two different exploitations were selected: a lead and zinc mine and a serpentinite quarry. Soils were characterized, and the pseudo-total and extractable contents of Co, Cr and Ni in soils from a serpentinite quarry were determined. The Cd, Pb and Zn pseudo-total and extractable contents were determined in soils developed in the Pb/Zn abandoned mine. Using a biotest, the chronic toxicity of the soil samples on higher plants was determined. Festuca ovina L., Cytisus scoparius (L.) Link., Sinapis alba L. and Brassica juncea L. were selected, the first two because they are spontaneous plants in the study areas and the last two because they have heavy metal phytoremediation potential.

Results and discussion

Pseudo-total contents of Co, Cr and Ni in the serpentinite quarry soils and of Zn, Pb and Cd in the Zn/Pb mine soils exceed generic reference levels. CaCl₂ is the reactant that extracts the highest proportion of Co, Cr and Ni in the quarry soils and EDTA the largest proportion of Pb Zn and Cd content in the mine soils. The germination index values based on seed germination and root elongation bioassays revealed increasing plant sensitivity to the mine soils in the following order: B. juncea?<?S. alba?<?F. ovina?<?C. scoparius. The wide range of GI values indicates that the response of test plants to soil heavy metals depended on their concentrations and soil characteristics, especially pH and organic matter content.

Conclusions

The pollution index indicates severe Cd, Pb and Zn contamination in the mine soils, as well as high Cr and Ni and moderate Co contamination in the serpentinite quarry soils. The performed biotests were suitable for identifying toxic soils and showed that the studied soils are toxic to the spontaneous plants, more to C. scoparius than to F. ovina. They also indicate that the mine soils are more toxic than the quarry soils for both species.

     

Lysosomal Fragility in Earthworms (Eisenia Veneta) Exposed to Heavy Metal Contaminated Soils from Two Abandoned Pyrite Ore Mines in Southern Norway

In this study we have investigated the toxicity of soils from thetwo abandoned pyrite mines Ertelien and Konnerud in the southernpart of Norway. Soil samples were collected close to the pitheadof the mines, and earthworms were exposed to different concentrations of the contaminated mine soil mixed with commercial plant soil for a period of 14 days. Life-cycle responses such as growth and survival, as well as the biomarkerresponse lysosomal fragility were measured. The body burdens ofthe four heavy metals Cu, Cd, Zn and Pb were detected in the worms, and the body concentration of the metals generally increased with increasing heavy metal concentration in the soil.The growth and mortality of the earthworms were not influenced when exposed to any of the mine soil concentrations used. The lysosomal fragility of the earthworm coelomocytes measured by the neutral red retention time (NRR-time) was found to be markedly reduced in all the exposed earthworms. This assay seemsto be a sensitive and dose dependent endpoint for the toxicity assessment of heavy metal mixtures in pyrite mine soils.     

Influence of different temperatures on metal tolerance measurements and growth response in bacterial communities from unpolluted and polluted soils

The effects of temperature on the growth rate and metal toxicity in soil bacterial communities extracted from unpolluted and polluted soils were investigated using the thymidine and leucine incorporation techniques. An agricultural soil, which was contaminated in the laboratory with Cu, Cd, Zn, Ni or Pb, and an uncontaminated forest soil were used. Measurements were made at 0°C and 20°C. Leucine incorporation was found to be as sensitive to heavy metals as thymidine incorporation in the short-term trial used to indicate heavy metal tolerance. Similar IC₅₀ values (the log of the metal concentration that reduced incorporation to 50%) were also obtained at 0 and 20°C, independently of the technique used. Metal tolerance could thus be measured using both techniques at any temperature in the range 0–20°C. In the long-term experiment different temperature-growth relationships were obtained on the basis of the rate of thymidine or leucine incorporation into bacterial assemblages from unpolluted and polluted soils, as judged from the minimum temperature values. This could not be attributed to the metal addition alone since different patterns were observed when different metals were added to the soil. Thus, the minimum temperature for thymidine incorporation was similar in Cu-polluted and unpolluted soil, while in soils polluted with Cd and Zn the minimum temperature increased by 2°C, and Ni and Pb additions increased the minimum temperature by 4°C compared to the unpolluted soil. This suggested that heavy metal pollution led to bacterial communities showing different temperature characteristics to those in the corresponding unpolluted soil. Similar observations were deduced from the minimum temperatures required for leucine incorporation. Three groups of bacterial communities were distinguished according to the growth response to temperature in polluted soils, one group in Cu-polluted soil, a second group in soil polluted with Zn and Cd, and a third group in soils polluted with Ni and Pb.     

Phosphatase activity in earthworm faeces

Wormcasts of Allolobophora caliginosa have been shown to have higher phosphatase activity than occurs in uningested soil, resulting in an increase in inorganic P released by mineralization of organic P. This paper describes similar observations on organic wastes which may be used for vermiculture. Besides A. caliginosa, three species which will feed on dung or other organic substrates were investigated, Eisenia foetida, Dendrobaena veneta and Lumbricus rubellus. When cow dung was used as a substrate, background phosphatase activity was too high for the effects of earthworm activity to be detected and a culture medium was therefore developed from sterilized paper waste sludge with added phytin. Phosphatase activity, assayed by a modified Hoffman method, was higher in the presence of all four earthworm species than in controls. It showed two peaks in relation to pH, at 3–5 and 9–10. The former is attributed to microbial activity and the latter to microbial activity or earthworm alkaline phosphatase. After 1 month, cultures of paper waste sludge with phytin contained about twice as much water soluble P when worms were present than in their absence.     

Effects of heavy metals on the litter consumption by the earthworm Lumbricus rubellus in field soils

Aim of this study was to determine effects of heavy metals on litter consumption by the earthworm Lumbricus rubellus in National Park the “Brabantsche Biesbosch”, the Netherlands. Adult L. rubellus were collected from 12 polluted and from one unpolluted field site. Earthworms collected at the unpolluted site were kept in their native soil and in soil from each of the 12 Biesbosch sites. Earthworms collected in the Biesbosch were kept in their native soils. Non-polluted poplar (Populus sp.) litter was offered as a food source and litter consumption and earthworm biomass were determined after 54 days. Cd, Cu and Zn concentrations were determined in soil, pore water and 0.01 M CaCl₂ extracts of the soil and in earthworms. In spite of low available metal concentrations in the polluted soils, Cd, Cu and Zn concentrations in L. rubellus were increased. The litter consumption rate per biomass was positively related to internal Cd and Zn concentrations of earthworms collected from the Biesbosch and kept in native soil. A possible explanation is an increased demand for energy, needed for the regulation and detoxification of heavy metals. Litter consumption per biomass of earthworms from the reference site and kept in the polluted Biesbosch soils, was not related to any of the determined soil characteristics and metal concentrations.     

Heavy metal levels in earthworms of a forest ecosystem influenced by traffic and air pollution

Soils of a forest ecosystem in Berlin (West) are highly polluted by Pb and less polluted by Cd and Cu. Pb levels in earthworm species depend primarily on soil type and only secondarily on the distance from a highway crossing the forest. The dominating species Lumbricus rubellus and Dendrobaena octaedra show different body burdens especially of Cd. Liming the forest soils decreases the Pb concentrations significantly. Only in D. octaedra has body weight been found to be related to Pb; Pb has been increased from 50 mg kg^–1 in small to 250 mg kg^–1 in large specimens; Cu has been regulated at a more or less constant absolute level (about 0.4 pg per specimen). This species obviously regulates or accumulates the three metals in different ways. Centipedes as predators of earthworms in the sites show markedly less pollution of Pb and Cd (about 2.5 and 0.6 mg kg^–1) than the worms, but higher concentrations of the essential Cu (about 40 mg kg^–1). The use of earthworms as indicators of heavy metal pollution should take into account the biology of the various species, the soil type, the type and amount of organic matter as well as chemical parameters such as pH value or basic anions.     

The effect of compost and Bacillus licheniformis on the phytoextraction of Cr,Cu, Pb and Zn by three brassicaceae species from contaminated soils in the Apulia region,Southern Italy

The selection of appropriate plant species is critical in the successful application of phytoremediation techniques. The present study is an attempt to assess the capability of three brassicaceae, Brassica alba (L.) Rabenh, Brassica carinata A. Braun and Brassica nigra (L.) Koch, for the phytoextraction of Cr, Cu, Pb and Zn from an unpolluted and polluted silty loamy soil added with either Bacillus licheniformis BLMB1 or compost or both. Experiments were conducted in a greenhouse in pots filled with the soils. In all experiments metals were shown to accumulate in shoots and roots of plants grown on polluted soils, and both compost and B. licheniformis BLMB1 strain were able to enhance the accumulation of metals, especially Cr. In particular, Cr accumulation in B. alba resulted higher than the Cr threshold for hyperaccumulator plants (1000 mg kg^? 1). This result provides a new plant resource that may have a potential use for phytoextraction of Cr from contaminated soil. However, because of the low bioconcentration factors (< 1) for all studied metals, these species cannot be regarded as suitable for the phytoextraction of excessive Cr, Cu, Pb and Zn from polluted soils. Thus, these species may be used with success only for low metal polluted soils.     

Use of earthworms Eisenia andrei on the bioremediation of contaminated area in north of Tunisia and microbial soil enzymes as bioindicator of change on heavy metals speciation

Purpose

A better understanding is required of the potential of soil biota in controlling the availability and mobility of heavy metals and ascertaining their toxicity. The objectives of this work are to assess, first, the modification of heavy metal speciation induced by earthworms Eisenia andrei and, second, the consequence of this metal speciation change on soil enzyme activities as an easy bioindicator of stress.

Materials and methods

The experiment was conducted on six sites from Jebel Ressas Mines, which are characterized by a gradient heavy metal contamination (Pb, Zn, and Cd). Earthworms E. andrei were introduced in these six soils for 60 days. We had performed heavy metal speciation both in the presence and absence of worms. Modifications of activities of seven enzymes implicated in C, N, and P biochemical cycles were used as a bioindicator of metal stress. We had used the co-inertia statistical method to evaluate the correlation between change in heavy metal speciation induced by earthworms and the enzyme activities in soils.

Results and discussion

Our results suggested that earthworms modified the heavy metal dynamic and speciation. They decrease the amount of metal associated with the most available fraction, such as exchangeable one, and increase the amount of metal bound to the more stable fraction, like Mn and Fe oxide ones. On the same hand, enzyme activities increased in majority of the soils, following earthworm activity, but this effect is dependent on the amount of soil contamination. Moreover, the co-inertia results denote that change in heavy metal speciation significantly influences the soil enzyme activities in Jebel Ressas soils, especially β-glucosidase, urease, deshydrogenase, and fluorescein diacetate hydrolysis (FDA), and can be considered as bioindicators of metal toxicity and biological quality in the contaminated area.

Conclusions

By reducing the availability of heavy metals, the earthworms are useful in the bioremediation of heavy metal contaminated soils. Soil enzymes β- glucosidase, urease, deshydrogenase, and FDA can be used to assess the changes in metal speciation and can let us, therefore, predict if the soils are bioremediated.

     

Lethal critical body residues as measures of Cd,Pb, and Zn bioavailability and toxicity in the earthworm<Emphasis Type="Italic">Eisenia fetida</Emphasis>

Background. Earthworm heavy metal concentrations (critical body residues, CBRs) may be the most relevant measures of heavy metal bioavailability in soils and may be linkable to toxic effects in order to better assess soil ecotoxicity. However, as earthworms possess physiological mechanisms to secrete and/or sequester absorbed metals as toxicologically inactive forms, total earthworm metal concentrations may not relate well with toxicity. Objective  The objectives of this research were to: i) develop LD₅₀s (total earthworm metal concentration associated with 50% mortality) for Cd, Pb, and Zn; ii) evaluate the LD₅₀ for Zn in a lethal Zn-smelter soil; iii) evaluate the lethal mixture toxicity of Cd, Pb, and Zn using earthworm metal concentrations and the toxic unit (TU) approach; and iv) evaluate total and fractionated earthworm concentrations as indicators of sublethal exposure. Methods  Earthworms (Eisenia fetida (Savigny)) were exposed to artificial soils spiked with Cd, Pb, Zn, and a Cd-Pb-Zn equitoxic mixture to estimate lethal CBRs and mixture toxicity. To evaluate the CBR developed for Zn, earthworms were also exposed to Zn-contaminated field soils receiving three different remediation treatments. Earthworm metal concentrations were measured using a procedure devised to isolate toxicologically active metal burdens via separation into cytosolic and pellet fractions. Results and Discussion  Lethal CBRs inducing 50% mortality (LD₅₀, 95% CI) were calculated to be 5.72 (3.54-7.31), 3.33 (2.97-3.69), and 8.19 (4.78-11.6) mmol/kg for Cd, Pb, and Zn, respectively. Zn concentrations of dead earthworms exposed to a lethal remediated Zn-smelter soil were 3-fold above the LD₅₀ for Zn and comparable to earthworm concentrations in lethal Zn-spiked artificial soils, despite a 14-fold difference in total soil Zn concentration between lethal field and artificial soils. An evaluation of the acute mixture toxicity of Cd, Pb, and Zn in artificial soils using the Toxic Unit (TU) approach revealed an LD₅₀ (95% CI) of 0.99 (0.57-1.41) TU, indicating additive toxicity. Conclusions  Total Cd, Pb, and Zn concentrations in earthworms were good indicators of lethal metal exposure, and enabled the calculation at LD₅₀s for lethality. The Zn-LD₅₀ developed in artificial soil was applicable to earthworms exposed to remediated Zn-smelter soil, despite a 14-fold difference in total soil Zn concentrations. Mixture toxicity evaluated using LD₅₀s from each single metal test indicated additive mixture toxicity among Cd, Pb, and Zn. Fractionation of earth worm tissues into cytosolic and pellet digests yielded mixed results for detecting differences in exposure at the sublethal level Recommendation and Outlook  CBRs are useful in describing acute Cd, Pb, and Zn toxicity in earthworms, but linking sublethal exposure to total and/or fractionated residues may be more difficult. More research on detoxification, regulation, and tissue and subcellular partitioning of heavy metals in earthworms and other invertebrates is needed to establish the link between body residue and sublethal exposure and toxicity. Keywords: Bioavailability; Cd; critical body residues; earthworms; metals; Pb; soil; Zn An erratum to this article is available at .     

Exposure of coelomocytes from the earthworm Eisenia hortensis to Cu,Cd, and dimethylbenz[a]anthracene: An in vitro study examining reactive oxygen species production and immune response inhibition

The principal aims of this study were to investigate the in vitro effects of copper (Cu), cadmium (Cd) and the polycyclic aromatic hydrocarbon (PAH) dimethylbenz[a]anthracene (DMBA) on reactive oxygen species (ROS) production and the effect of DMBA on immune function (phagocytosis) in coelomocyte samples isolated from the earthworm Eisenia hortensis. Using a fluorogenic indicator of ROS, dihydrorhodamine 123, we show that earthworm coelomocytes are induced to produce significant increases in ROS when exposed to Cu or Cd at 32–250 μM, an effect diminished by the chelator ethylenediaminetetraacetic acid (EDTA). DMBA also induced significant ROS production in the range of 20–400 μM compared to controls. Testing the effect of DMBA on cellular immune mechanisms, we used a phagocytosis assay to reveal an immunosuppressive effect of DMBA (50–200 μM) on the capacity of hyaline amoebocytes to ingest fluorescent Escherichia coli. These studies illustrate the relative ease and efficiency of these in vitro procedures for testing the toxicological effects of environmental pollutants, including heavy metals and PAHs, using earthworm coelomocytes as indicators of oxidative stress and immune response impairment.     

Impact of five different tillage systems on soil organic carbon content and the density,biomass, and community composition of earthworms after a ten year period

To assess the impact of different types of soil tillage on the density, biomass, and community composition of earthworms, a long-term field study was performed in which soils were tilled in different ways for ten years. This study included five different types of tillage: (i) plough, (ii) grubber, (iii) disc harrow, (iv) mulch sowing, and (v) direct sowing. At the end of the experiment the earthworm density, biomass, and community composition, and the SOC (soil organic carbon) content were determined. The results show that density, biomass, and community composition of earthworm populations varied in relation to the type of soil tillage used. The density of anecic earthworm species decreased when soils were managed by conventional ploughing, relative to reduced tillage practices, whereas conversely the density of endogeic species increased. Additionally, the varying types of soil tillage influenced the abundance and biomass of different earthworm species in different ways. The density of Aporrectodea caliginosa was positively influenced by ploughing, whereas Aporrectodea longa, Lumbricus castaneus, and Satchellius mammalis showed a positive relationship to the grubber and Allolobophora chlorotica to direct sowing. We attribute these changes to modifications in the vertical distribution of SOC and varying potentials for mechanical damage of earthworms by tillage. A decrease in tillage intensity modified the vertical SOC distribution in the topsoil and consequently revealed positive effects on earthworm biodiversity, thus sustaining soil functioning.     

Uptake and bioaccumulation of heavy elements by two earthworm species from a smelter contaminated area in northern Kosovo

As, Cd, Cu, Pb, Sb and Zn concentrations were determined in two earthworm species (Allolobophora rosea and Nicodrilus caliginosus) from a mining and industrial area in northern Kosovo and compared with their contents in the bulk soil and the main soil fractions. Earthworm specimens were collected at fifteen sites located at different distances from a Pb–Zn smelter along a gradient of decreasing contamination. Individuals of A. rosea and N. caliginosus showed similar tissue levels of As, Cd, Cu, Pb, Sb and Zn, suggesting that earthworm species belonging to the same eco-physiological group have a similar propensity to uptake and bioaccumulate heavy elements. Cd, Pb, Sb and Zn concentrations in both earthworm species were positively correlated with the respective total soil contents and generally decreased with distance from the smelter. The bioaccumulation factor (BAF) revealed that Cd and Zn were the only elements bioaccumulated by earthworms. The rank order of BAF values for both species was as follows: Cd > > Zn > > Cu > As = Pb = Sb. The absorption of Cd, Pb, Sb and Zn by earthworms mostly depended on the extractable, reducible and oxidable soil fractions, suggesting that the intestine is likely the most important uptake route. The extractable soil fraction constantly influenced the uptake of these heavy elements, whereas the reducible fraction was important mainly for Pb and Zn. The water soluble fraction had an important role especially for the most mobile heavy elements such as Cd and Zn, suggesting that dermal uptake is not negligible. As a whole, the analytical data indicate that soil fractionation patterns influence the uptake of heavy elements by earthworms, and the extractable fraction is a good predictor of heavy element bioavailability to these invertebrates in soil.