Lead tolerance in Aporrectodea rosea earthworms from a clay pigeon shooting site

Lead tolerance in individuals of the earthworm species Aporrectodea rosea collected from a clay pigeon shooting site was investigated. Lead concentrations in the shooting site soil and the un-shot control site were 6410±2250 and 296±98 mg_Pb kg⁻¹ dry weight, respectively. Of these concentrations 1050±240 and 12±9 mg_Pb kg⁻¹ dry weight were suggested to be available, using ammonium acetate (1 M), respectively. With respect to earthworm body burdens of lead the shooting site earthworms had a body burden of 6.1±1.2 mg_Pb g⁻¹ dry weight while the uncontaminated site earthworms had almost a 1000-times lower body burden of 7.1±9.0 μg_Pb g⁻¹ dry weight. Lead tolerance was assessed in uncontaminated soil that had been augmented with lead, using lead nitrate solutions, to obtain lead concentrations in soil of 0.5, 5 and 50 mg_Pb kg⁻¹ dry weight. Earthworms were exposed for 28 days during which time a semi-qualitative assessment was made of their condition. Results showed no decrease in condition in the shooting site earthworms with increasing exposure time or concentration. In contrast, earthworms collected from an uncontaminated site showed a significant (p<0.05) decrease in condition when exposed to lead concentrations above, and including, a concentration of 5 mg kg⁻¹ dry weight soil. These results suggested lead tolerance in the shooting site earthworms.     

Earthworm activity as a determinant for N2O emission from crop residue

Earthworm activity may have an effect on nitrous oxide (N₂O) emissions from crop residue. However, the importance of this effect and its main controlling variables are largely unknown. The main objective of this study was to determine under which conditions and to what extent earthworm activity impacts N₂O emissions from grass residue. For this purpose we initiated a 90-day (experiment I) and a 50-day (experiment II) laboratory mesocosm experiment using a Typic Fluvaquent pasture soil with silt loam texture. In all treatments, residue was applied, and emissions of N₂O and carbon dioxide (CO₂) were measured. In experiment I the residue was applied on top of the soil surface and we tested (a) the effects of the anecic earthworm species Aporrectodea longa (Ude) vs. the epigeic species Lumbricus rubellus (Hoffmeister) and (b) interactions between earthworm activity and bulk density (1.06 vs. 1.61 g cm⁻³). In experiment II we tested the effect of L. rubellus after residue was artificially incorporated in the soil. In experiment I, N₂O emissions in the presence of earthworms significantly increased from 55.7 to 789.1 μg N₂O-N kg⁻¹ soil (L. rubellus; p<0.001) or to 227.2 μg N₂O-N kg⁻¹ soil (A. longa; p<0.05). This effect was not dependent on bulk density. However, if the residue was incorporated into the soil (experiment II) the earthworm effect disappeared and emissions were higher (1064.2 μg N₂O-N kg⁻¹ soil). At the end of the experiment and after removal of earthworms, a drying/wetting and freezing/thawing cycle resulted in significantly higher emissions of N₂O and CO₂ from soil with prior presence of L. rubellus. Soil with prior presence of L. rubellus also had higher potential denitrification. We conclude that the main effect of earthworm activity on N₂O emissions is through mixing residue into the soil, switching residue decomposition from an aerobic and low denitrification pathway to one with significant denitrification and N₂O production. Furthermore, A. longa activity resulted in more stable soil organic matter than L. rubellus.     

Interactions between residue placement and earthworm ecological strategy affect aggregate turnover and N2O dynamics in agricultural soil

Previous laboratory studies using epigeic and anecic earthworms have shown that earthworm activity can considerably increase nitrous oxide (N₂O) emissions from crop residues in soils. However, the universality of this effect across earthworm functional groups and its underlying mechanisms remain unclear. The aims of this study were (i) to determine whether earthworms with an endogeic strategy also affect N₂O emissions; (ii) to quantify possible interactions with epigeic earthworms; and (iii) to link these effects to earthworm-induced differences in selected soil properties. We initiated a 90-day ¹⁵N-tracer mesocosm study with the endogeic earthworm species Aporrectodea caliginosa (Savigny) and the epigeic species Lumbricus rubellus (Hoffmeister). ¹⁵N-labeled radish (Raphanus sativus cv. Adagio L.) residue was placed on top or incorporated into the loamy (Fluvaquent) soil. When residue was incorporated, only A. caliginosa significantly (p < 0.01) increased cumulative N₂O emissions from 1350 to 2223 μg N₂O-N kg⁻¹ soil, with a corresponding increase in the turnover rate of macroaggregates. When residue was applied on top, L. rubellus significantly (p < 0.001) increased emissions from 524 to 929 μg N₂O-N kg⁻¹, and a significant (p < 0.05) interaction between the two earthworm species increased emissions to 1397 μg N₂O-N kg⁻¹. These effects coincided with an 84% increase in incorporation of residue ¹⁵N into the microaggregate fraction by A. caliginosa (p = 0.003) and an 85% increase in incorporation into the macroaggregate fraction by L. rubellus (p = 0.018). Cumulative CO₂ fluxes were only significantly increased by earthworm activity (from 473.9 to 593.6 mg CO₂-C kg⁻¹ soil; p = 0.037) in the presence of L. rubellus when residue was applied on top. We conclude that earthworm-induced N₂O emissions reflect earthworm feeding strategies: epigeic earthworms can increase N₂O emissions when residue is applied on top; endogeic earthworms when residue is incorporated into the soil by humans (tillage) or by other earthworm species. The effects of residue placement and earthworm addition are accompanied by changes in aggregate and SOM turnover, possibly controlling carbon, nitrogen and oxygen availability and therefore denitrification. Our results contribute to understanding the important but intricate relations between (functional) soil biodiversity and the soil greenhouse gas balance. Further research should focus on elucidating the links between the observed changes in soil aggregation and controls on denitrification, including the microbial community.     

Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management

Earthworms are known to be important regulators of soil structure and soil organic matter (SOM) dynamics, however, quantifying their influence on carbon (C) and nitrogen (N) stabilization in agroecosystems remains a pertinent task. We manipulated population densities of the earthworm Aporrectodea rosea in three maize-tomato cropping systems [conventional (i.e., mineral fertilizer), organic (i.e., composted manure and legume cover crop), and an intermediate low-input system (i.e., alternating years of legume cover crop and mineral fertilizer)] to examine their influence on C and N incorporation into soil aggregates. Two treatments, no-earthworm versus the addition of five A. rosea adults, were established in paired microcosms using electro-shocking. A ¹³C and ¹⁵N labeled cover crop was incorporated into the soil of the organic and low-input systems, while ¹⁵N mineral fertilizer was applied in the conventional system. Soil samples were collected during the growing season and wet-sieved to obtain three aggregate size classes: macroaggregates (>250 μm), microaggregates (53-250 μm) and silt and clay fraction (<53 μm). Macroaggregates were further separated into coarse particulate organic matter (cPOM), microaggregates and the silt and clay fraction. Total C, ¹³C, total N and ¹⁵N were measured for all fractions and the bulk soil. Significant earthworm influences were restricted to the low-input and conventional systems on the final sampling date. In the low-input system, earthworms increased the incorporation of new C into microaggregates within macroaggregates by 35% (2.8 g m⁻² increase; P=0.03), compared to the no-earthworm treatment. Within this same cropping system, earthworms increased new N in the cPOM and the silt and clay fractions within macroaggregates, by 49% (0.21 g m⁻²; P<0.01) and 38% (0.19 g m⁻²; P=0.02), respectively. In the conventional system, earthworms appeared to decrease the incorporation of new N into free microaggregates and macroaggregates by 49% (1.38 g m⁻²; P=0.04) and 41% (0.51 g m⁻²; P=0.057), respectively. These results indicate that earthworms can play an important role in C and N dynamics and that agroecosystem management greatly influences the magnitude and direction of their effect.     

Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils

The concentrations of Zn, Cd, Pb and Cu in earthworm tissues were compared with the total and DTPA-extractable contents of these heavy metals in contaminated soils. Samples were taken from a pasture polluted by waste from a metallurgic industry over 70 y ago. Three individuals of Aporrectodea caliginosa and Lumbricus rubellus and soil samples were collected at six points along a gradient of increasing pollution. Total metal contents of earthworms, soil, and metals extracted by DTPA from the soil were measured. Total heavy metal contents of the soils ranged from 165.7 to 1231.7 mg Zn kg⁻¹, 2.7 to 5.2 mg Cd kg⁻¹, 45.8 to 465.5 mg Pb kg⁻¹ and 30.0 to 107.5 mg Cu kg⁻¹. Their correlations with metals extracted by DTPA were highly significant. Contents of the metals in earthworm tissues were higher in A. caliginosa than in L. rubellus, with values ranging from 556 to 3381 mg Zn kg⁻¹, 11.6 to 102.9 mg Cd kg⁻¹, 1.9 to 182.8 mg Pb kg⁻¹ and 17.9 to 35.9 mg Cu kg⁻¹ in A. caliginosa, and from 667.9 to 2645 mg Zn kg⁻¹, 7.7 to 26.3 mg Cd kg⁻¹, 0.5 to 37.9 mg Pb kg⁻¹ and 16.0 to 37.6 mg Cu kg⁻¹ in L. rubellus, respectively. Correlations between body loads in earthworms with either total or DTPA-extractable contents of soil metals were significant, except for Cd in L. rubellus and Cu in A. caliginosa. Considering its simple analytical procedure, DTPA-extractable fraction may be preferable to total metal content as a predictor of bio-concentrations of heavy metals in earthworms. Biota-to-Soil Accumulation Factor (BSAF) of these four metals are Cd>Zn>Cu>Pb, with range of mean values between: Cd (6.18-17.02), Zn (1.95-7.91), Cu (0.27-0.89) and Pb (0.08-0.38) in A. caliginosa, and Cd (3.64-6.34), Zn (1.5-6.35), Cu (0.29-0.87) and Pb (0.04-0.13) in L. rubellus. The BSAF of Ca, Fe and Mn are Ca>Mn>Fe, with mean values of: Ca (0.46-1.31), Mn (0.041-0.111), Fe (0.017-0.07) in A. caliginosa and Ca (0.98-2.13), Mn (0.14-0.23), Fe (0.019-0.048) in L. rubellus, respectively. Results of principal component analysis showed that the two earthworm species differ in the pattern of metal bioaccumulation which is related to their ecological roles in contaminated soils.     

Do earthworms increase N2O emissions in ploughed grassland?

Earthworm activity has been reported to lead to increased production of the greenhouse gas nitrous oxide (N₂O). This is due to emissions from worms themselves, their casts and drilosphere, as well as to general changes in soil structure. However, it remains to be determined how important this effect is on N₂O fluxes from agricultural systems under realistic conditions in terms of earthworm density, soil moisture, tillage activity and residue loads. We quantified the effect of earthworm presence on N₂O emissions from a pasture after simulated ploughing of the sod (‘grassland renovation’) for different soil moisture contents during a 62-day mesocosm study. Sod (with associated soil) and topsoil were separately collected from a loamy Typic Fluvaquent. Treatments included low (L), medium (M) and high (H) moisture content, in combination with: only soil (S); soil+incorporated sod (SG); soil+incorporated sod+the anecic earthworm Aporrectodea longa (SGE). Nitrous oxide and carbon dioxide (CO₂) fluxes were measured for 62 d. At the end of the incubation period, we determined N₂O production under water-saturated conditions, potential denitrification and potential mineralization of the soil after removing the earthworms. Cumulative N₂O and CO₂ fluxes over 62 d from incorporated sod were highest for treatment HSGE (973 μg N₂O-N and 302 mg CO₂-C kg⁻¹ soil) and lowest for LSG (64 μg N₂O-N and 188 mg CO₂-C kg⁻¹ soil). Both cumulative fluxes were significantly different for soil moisture (p<0.001), but not for earthworm presence. However, we observed highly significant earthworm effects on N₂O fluxes that reversed over time for the H treatments. During the first phase (day 3-day 12), earthworm presence increased N₂O emissions with approximately 30%. After a transitional phase, earthworm presence resulted in consistently lower (approximately 50%) emissions from day 44 onwards. Emissions from earthworms themselves were negligible compared to overall soil fluxes. After 62 d, original soil moisture significantly affected potential denitrification, with highest fluxes from the L treatments, and no significant earthworm effect. We conclude that after grassland ploughing, anecic earthworm presence may ultimately lead to lower N₂O emissions after an initial phase of elevated emissions. However, the earthworm effect was both determined and exceeded by soil moisture conditions. The observed effects of earthworm activity on N₂O emissions were due to the effect of earthworms on soil structure rather than to emissions from the worms themselves.     

Earthworm-mycorrhiza interaction on Cd uptake and growth of ryegrass

The effects of inoculation of earthworms and arbuscular mycorrhiza separately, and in combination, on Cd uptake and growth of ryegrass were studied in soils contaminated with 0, 5, 10, 20 mg of Cd kg⁻¹ soil. Both earthworms and mycorrhiza were able to survive in all the treatments with added Cd. Earthworm activity significantly increased mycorrhizal infection rate of root and ryegrass shoot biomass. Earthworm activity decreased soil pH by about 0.2 units, and enhanced root Cd concentration and ryegrass Cd uptake. Mycorrhiza inoculation increased shoot and root Cd concentration substantially, and at the highest dosage of 20 mg Cd kg⁻¹ decreased biomass of ryegrass. Inoculation of both earthworms and mycorrhiza increased ryegrass shoot Cd uptake at low Cd concentrations (5 and 10 mg Cd kg⁻¹ soil), when compared with inoculation of earthworms or mycorrhiza alone. In conclusion, earthworm, mycorrhiza and their interaction may have a potential role in elevating phytoextraction efficiency in low to medium level metal contaminated soil.     

Effects of earthworms on decomposition and metal availability in contaminated soil: Microcosm studies of populations with different exposure histories

Population-specific differences in the responses of earthworms to simultaneous exposure to Cu and Zn were studied in microcosm experiments. Two populations of Aporrectodea caliginosa tuberculata (Eisen) with different metal exposure histories were chosen for the studies. Microcosms were prepared containing either uncontaminated soil or soils with low or high combined Cu/Zn -concentrations (79/139 or 178/311 mg kg⁻¹ dry mass of soil, respectively). Earthworms from each population were introduced to the microcosm treatments with some microcosms serving as controls without earthworms. One series of microcosms was destructively sampled after 16 weeks incubation in a climate chamber. Survival, growth, reproduction and decomposition by earthworms in each treatment were measured. An additional microcosm series was sampled for soil and earthworm measurements at four weeks intervals to determine temporal changes in the availability of metals in the soils and their accumulation into earthworms. Cu and Zn were sequentially extracted from the soil samples of both microcosm series to estimate mobility and availability of the metals in the soil. Earthworms with long-term exposure history to metal-contaminated soil seemed to tolerate higher soil metal concentrations than earthworms without earlier exposure. Both earthworms and metals affected soil respiration (CO₂ production) and nitrogen mineralization. In addition, earthworms seemed to decrease the mobility and bioavailability of metals in the soil through their burrowing activity.     

DNA damage and effects on antioxidative enzymes in earthworm (Eisenia foetida) induced by atrazine

To evaluate atrazine (2-chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) ecotoxicology in soil, the effect of atrazine on the activity of antioxidative enzymes (superoxide dismutase, SOD; catalase, CAT; and guaiacol peroxidase, POD) and DNA damage induced by atrazine were investigated in earthworms. Atrazine was added to artificial soil at rates of 0, 2.5, 5 and 10 mg per kg of soil. Earthworm tissues exposed to each treatment were collected on the 7th, 14th, 21st, and 28th day of the treatment. Compared to the controls, the CAT activity was stimulated at 2.5 mg kg⁻¹ treatment except on the 14th day, and inhibited at 5, 10 mg kg⁻¹ atrazine except 5 mg kg⁻¹ on the 28th day and 10 mg kg⁻¹ on the 21st day; the overall SOD activity was inhibited, while the POD activities were stimulated by all atrazine concentrations in 28 days. The olive tail moments of single-cell gel electrophoresis of coelomocytes, as an indication of DNA damage, were increased after treatment with different doses of atrazine on the 7th, 14th, 21st, and 28th day, and significant differences were found compared to the controls. In conclusion, atrazine induces oxidative stress and DNA damage on earthworms, and the adverse effects may be the important mechanisms of its toxicity to earthworms.     

Zinc toxicity on N2O reduction declines with time in laboratory spiked soils and is undetectable in field contaminated soils

Denitrification assays in soils spiked with zinc salt have shown inhibition of the N₂O reduction resulting in increased soil N₂O fluxes with increasing soil Zn concentration. It is unclear if the same is true for environmentally contaminated soils. Net production of N₂O and N₂ was monitored during anaerobic incubations (25 °C, He atmosphere) of soils freshly spiked with ZnCl₂ and of corresponding soils that were gradually enriched with metals (mainly Zn) in the field by previous sludge amendments or by corrosion of galvanized structures. Total denitrification activity (i.e. the sum of N₂O+N₂ production rate) was not inhibited by freshly added Zn salts up to 1600 mg Zn kg⁻¹, whereas N₂O reduction decreased by 50% (EC₅₀) at total Zn concentrations of 231 mg Zn kg⁻¹ (ZEV soil) and 368 mg Zn kg⁻¹ (TM soil). In contrast, N₂O reduction was not reduced by soil Zn in any of the field contaminated soils, even at total soil Zn or soil solution Zn concentrations exceeding more than 5 times corresponding EC₅₀'s of the freshly spiked soil. The absence of adverse effects in the field contaminated soils was unrelated to soil NO₃ or organic matter concentration. Ageing (2-8 weeks) and soil leaching after spiking reduced the toxicity of Zn on N₂O reduction, either expressed as total Zn or soil solution Zn, suggesting adaptation reactions. However, no full recovery after spiking was identified at the largest incubation period in one soil. In addition, the denitrification assay performed with sewage sludge showed elevated N₂O release in Zn contaminated sludges (>6000 mg Zn kg⁻¹ dry matter) whereas this was not observed in low Zn sludge (<1000 mg Zn kg⁻¹ dry matter) suggesting limits to adaptation reactions in the sludge particles. It is concluded that the use of soils spiked with Zn salts overestimates effects on N₂O reduction. Field data on N₂O fluxes in sludge amended soils are required to identify if metals indeed promote N₂O emissions in sludge amended soils.     

Influence of selenium on oxidoreductive enzymes activity in soil and in plants

The aim of this greenhouse experiment was the assessment of the influence of H₂SeO₃ at soil concentrations of 0.05, 0.15 and 0.45 mmol kg⁻¹, on the activity of selected oxidoreductive enzymes in wheat (Triticum aestivum). The wheat plants were grown in 2 dm³ pots filled with dust-silt black soil of pH 7.7. Applied H₂SeO₃ caused activation of plant nitrate reductase at all concentrations, but activation of plant polyphenol oxidase at only two lower concentrations. The highest concentration caused inhibition of polyphenol oxidase and peroxidase. Plant catalase activity decreased under the influence of 0.15 and 0.45 mmol kg⁻¹ concentration. After the final analysis Se was quantified in plants and soil. The amounts in plants were: control (unamended soil) 1.95 mg kg⁻¹; I dose (0.05 mmol kg⁻¹) 18.27 mg kg⁻¹; II dose (0.15 mmol kg⁻¹) 33.20 mg kg⁻¹ and III dose (0.45 mmol kg⁻¹) 38.37 mg kg⁻¹, in soil: 0.265 mg kg⁻¹; 3.61 mg kg⁻¹; 10.53 mg kg⁻¹; 30.53 mg kg⁻¹; respectively. Simultaneously, a laboratory experiment was performed, where the activity of soil catalase and peroxidase were tested after 1, 3, 7, 14, 28, 56, and 112 days after Se treatment. Peroxidase activity in soil decreased with increasing Se content, over the whole experiment. The lowest dose of Se caused activation a significant 10% increase in catalase activity, but the influence of others doses was unclear.     

Uptake pathways and toxicity of Cd and Zn in the earthworm Eisenia fetida

The uptake of Cd and Zn by the earthworm Eisenia fetida was determined at varying Ca concentrations and with pre-exposure to different metabolic inhibitors in simulated soil solutions over a 48-h period. The presence of Ca in the solution had complex actions on Cd uptake. At a low Cd concentration of 0.1 μM, Ca (0.1-1 mM) slightly but significantly stimulated Cd uptake, whereas it inhibited Cd uptake at a higher Cd level (10 μM). Pre-exposure to a Ca-channel blocker (Lanthanum) inhibited Cd uptake over a relatively wide range of Cd concentrations, but not Zn uptake, suggesting that the uptake of Zn was not exerted at a Ca channel. N-ethylmaleimide, which specifically binds to sulfhydryl groups, inhibited Zn uptake at both 0.1 and 10 μM, implying that the transport of Zn is carrier-mediated by proteins or other SH-containing compounds. The present study provides evidence that the mechanisms of Cd and Zn uptake in earthworms are pharmacologically different, although both metals have similar geochemical and environmental properties. After 24 h pre-exposure to a sublethal concentration (1.0 μM) of Cd, Zn toxicity for E. fetida was significantly reduced with 48-h LC₅₀ values (with 95% confidence interval) increasing from 145 (105-201) to 316 (212-470) μM Zn. Pre-exposure to Zn (1.0 μM) did not, however, affect Cd toxicity. Pre-exposure to Cd significantly changed the subcellular Zn distribution, with a decreasing fraction of Zn associated with Fraction B (associated with granules and cell membranes), which is believed to be most indicative of toxic pressure and an increased fraction associated with Fraction G (associated with cytosol). This most likely explains the observed Zn tolerance of E. fetida after low level Cd pre-exposure. These results help to understand the uptake mechanism and interactions of Zn and Cd in earthworms.     

The effect of two endogeic earthworm species on zinc distribution and availability in artificial soil columns

The objective of this study was to determine the impact of earthworm bioturbation on the distribution and availability of zinc in the soil profile.Experiments were carried out with Allolobophora chlorotica and Aporrectodea caliginosa in 24 perspex columns (∅ 10 cm), filled with 20-23 cm non-polluted soil (OM 2%, clay 2.9%, pH 0.01 M CaCl₂ 6.4), that was covered by a 3-5 cm layer of aged zinc spiked soil (500 mg Zn/kg dry soil) and another 2 cm non-polluted soil on top. After 80 and 175 days, columns were sacrificed and each cm from the top down to a depth of 15 cm was sampled. Earthworm casts, placed on top of the soil, were collected. Each sample was analyzed for total and CaCl₂-exchangeable zinc concentrations.Effects of earthworm bioturbation were most pronounced after 175 days. For A. chlorotica, total and CaCl₂-exchangeable zinc concentrations in the polluted layers were lower with than without earthworms. Total zinc concentrations in the non-polluted layers were higher in columns with earthworms. Casts of A. chlorotica collected on the soil surface showed slightly higher total zinc concentrations than non-polluted soil. Casts were found throughout the whole column. For A. caliginosa there were no differences in total zinc concentration between columns with and without earthworms. CaCl₂-exchangeable zinc concentrations in the polluted layers were lower for columns with earthworms. Casts were mainly placed on top of the soil and contained total zinc concentrations intermediate between those in non-polluted and polluted soil layers.This study shows that different endogeic earthworm species have different effects on zinc distribution and availability in soils. A. chlorotica transfers soil throughout the whole column, effectively mixing it, while A. caliginosa decreases metal availability and transfers polluted soil to the soil surface.     

Attempts to derive EC50 values for heavy metals from land-applied Cu-, Ni-, and Zn-spiked sewage sludge

We established a field trial to assess the impacts on soil biological properties of application of heavy metal-spiked sewage sludge, with the aim of determining toxicity threshold concentrations of heavy metals in soil. Plots were treated with sludges containing increasing concentrations of Cu, Ni and Zn in order to raise the metal concentrations in the soil by 0-200 mg Cu kg⁻¹, 0-60 mg Ni kg⁻¹ and 0-400 mg Zn kg⁻¹, and were then cultivated and sown in ryegrass-clover pasture and monitored annually for 6 years. All biological properties measured (soil basal respiration, microbial biomass C, and sulphatase enzyme activities), except phosphatase activity, increased in all plots over the duration of the experiment. Consequently, it was only possible to assess effects of heavy metals across time if, each year, all data for each metal were normalised by expressing them as percentages of the activities measured in an un-sludged control plot. When this was done, no significant effects of increasing heavy-metal concentrations on basal respiration, microbial biomass C or respiratory quotient (qCO₂) were observed, although total Cu and soil solution Cu were significantly negatively related to microbial biomass C when it was expressed as a proportion of soil total C. None of the properties measured were affected by increasing Ni concentrations. Phosphatase and sulphatase activities were significantly negatively related to increasing Zn concentrations, but not usually to increasing Cu unless they were expressed as a proportion of total C. A sigmoidal dose-response model was used to calculate EC₂₀ and EC₅₀ values using the normalised data, but generally, the model parameters had very large 95% confidence intervals and/or the fits to the model had small R² values. The factors primarily responsible for confounding these results were site and sample variations not accounted for by the normalisation process and the absence of any data points at metal concentrations beyond the calculated EC₅₀ values. In the few instances where reasonable EC₂₀ values could be calculated, they were relatively consistent across properties, e.g., EC₂₀ for total Zn and phosphatase (330 mg kg⁻¹), total Zn and sulphatase (310 mg kg⁻¹), and EC₂₀ for total Cu and sulphatase (140 mg kg⁻¹) and total Cu and microbial biomass C (140 mg kg⁻¹), when both sulphatase and microbial biomass C were expressed as a proportion of total C. Our results suggest that Cu and Zn at the upper concentrations used in this experiment were possibly having adverse effects on some soil biological properties. However, much higher metal concentrations will be needed to accurately calculate EC₂₀ and EC₅₀ and this may not be easily achievable without many applications of sewage sludge, even if the sludge is spiked with heavy metals.     

Competition between invasive earthworms (Amynthas corticis, Megascolecidae) and native North American millipedes (Pseudopolydesmus erasus, Polydesmidae): Effects on carbon cycling and soil structure

Invasive earthworms can have significant impacts on C dynamics through their feeding, burrowing, and casting activities, including the protection of C in microaggregates and alteration of soil respiration. European earthworm invasion is known to affect soil micro- and mesofauna, but little is known about impacts of invasive earthworms on other soil macrofauna. Asian earthworms (Amynthas spp.) are increasingly being reported in the southern Appalachian Mountains in southeastern North America. This region is home to a diverse assemblage of native millipedes, many of which share niches with earthworm species. This situation indicates potential for earthworm-millipede competition in areas subject to Amynthas invasion.In a laboratory microcosm experiment, we used two ¹³C enriched food sources (red oak, Quercus rubra, and eastern hemlock, Tsuga canadensis) to assess food preferences of millipedes (Pseudopolydesmus erasus), to determine the effects of millipedes and earthworms (Amynthas corticis) on soil structure, and to ascertain the nature and extent of the interactions between earthworms and millipedes. Millipedes consumed both litter species and preferred red oak litter over eastern hemlock litter. Mortality and growth of millipedes were not affected by earthworm presence during the course of the experiment, but millipedes assimilated much less litter-derived C when earthworms were present.Fauna and litter treatments had significant effects on soil respiration. Millipedes alone reduced CO₂ efflux from microcosms relative to no fauna controls, whereas earthworms alone and together with millipedes increased respiration, relative to the no fauna treatment. CO₂ derived from fresh litter was repressed by the presence of macrofauna. The presence of red oak litter increased CO₂ efflux considerably, compared to hemlock litter treatments.Millipedes, earthworms, and both together reduced particulate organic matter. Additionally, earthworms created significant shifts in soil aggregates from the 2000-250 and 250-53 μm fractions to the >2000 μm size class. Earthworm-induced soil aggregation was lessened in the 0-2 cm layer in the presence of millipedes. Earthworms translocated litter-derived C to soil throughout the microcosm.Our results suggest that invasion of ecosystems by A. corticis in the southern Appalachian Mountains is unlikely to be limited by litter species and these earthworms are likely to compete directly for food resources with native millipedes. Widespread invasion could cause a net loss of C due to increased respiration rates, but this may be offset by C protected in water-stable soil aggregates.     

Ecological implications of motor oil pollution: Earthworm survival and soil health

The nontarget effects of fresh and used motor oil were studied in a soil test system involving such criteria as earthworm survival, response of soil dehydrogenase and urease, and nitrification. When earthworms were exposed to motor oil-contaminated soil for 4 weeks, the observed median lethal concentrations (LC₅₀) were 40.33 and 3.88 g kg⁻¹ soil for fresh and used oil, respectively. Only fresh motor oil application increased earthworms' body weight even at the higher dose of 19 g kg⁻¹ soil. Gas chromatography/mass spectrometry revealed that used motor oil contained more of aromatic hydrocarbons and heavy metals than fresh oil. This disparity in the chemical composition might be the factor responsible for the significant toxicity of used motor oil towards earthworms. Activities of soil dehydrogenase and urease were significantly enhanced in presence of both the motor oils, while there was a significant inhibition in nitrification by the used motor oil even at a low concentration of 0.2 g kg⁻¹ soil. This study clearly demonstrated that earthworm survival and nitrification could serve as suitable indices to assess motor oil pollution in soil.     

Critical body residues (CBRs) for ecotoxicological soil quality assessment: copper in earthworms

The term ‘critical body residue’ (CBR) was defined as the lowest observed total body concentration of a contaminant in an organism, which is associated with the occurrence of adverse toxic effects in either individuals or populations of a defined age or stage of development. In this study, internal toxicity thresholds were determined for copper in the clitellated adult stage of earthworms (Lumbricus rubellus and Aporrectodea caliginosa). The objective was to assess the applicability of CBRs as a practical tool in soil quality assessment of contaminated sites and as a means of a sustainable protection of earthworm fauna. Laboratory studies showed that body concentrations of Cu were generally in agreement with the chemically available CaCl₂-extractable fraction in soil, but that there was also some evidence of internal pH-related homeostatic regulation. Toxicological correlates of body Cu concentrations with adverse effects on cocoon production (fecundity) suggested an approximate sublethal internal threshold of about 40 mg kg⁻¹, with mortality occurring at about 60 mg kg⁻¹. Adult L. rubellus sampled from areas with a wide range of metal pollution showed body Cu concentrations with a minimum of 8 mg kg⁻¹ and a maximum of 60 mg kg⁻¹. Beyond this apparent physiological tolerance range, environmental management directed at optimal earthworm population survival may not be sustainable in contaminated fields. Studies of L. rubellus colonizing a metal-contaminated experimental sludge-treated field showed that a reduced rate of colonization can already be associated with an average body Cu concentration of 25 mg kg⁻¹. However, in this particular field situation mixture effects of other metals that were also present in the soil and the occurrence of avoidance behaviour during colonization may have contributed to this low internal toxicity threshold. It is concluded that the CBR approach seems to be a feasible option for use as a tool in a bioavailability-based soil quality assessment, even for essential trace metals like copper, but that further insight may be needed to establish the uncertainty and reliability of the application in environmental quality assessment and decision making.     

Inter- and intra-specific interactions of Lumbricus rubellus (Hoffmeister, 1843) and Octolasion lacteum (Örley, 1881) (Lumbricidae) and the implication for C cycling

Earthworms are important engineering species of many terrestrial ecosystems as they play a significant role in regulating C turnover. The effects of earthworms on moderating C decomposition processes differ across species and with interactions between species, which is not fully understood. We carried out an experiment to study the interactions of Lumbricus rubellus and Octolasion lacteum, and their effects on soil respiration. Laboratory mesocosms were set up using tulip poplar (Liriodendron tulipifera) leaf litter and varying densities of earthworms in single and combined species treatments. CO₂ efflux rate was used as an indicator of C decomposition rates, and measured with CO₂ sensors every five days over one month. L. rubellus induced higher leaf consumption rate and higher CO₂ efflux than O. lacteum; meanwhile O. lacteum grew more than L. rubellus. Both litter consumption rate and growth rate of earthworms decreased with increasing earthworm density. Soil CO₂ efflux increased with increasing earthworm density (from ∼1-2 μg CO₂ g⁻¹ hr⁻¹ with no earthworms to ∼ 4 μg CO₂ g⁻¹ hr⁻¹ with 8 earthworms). Combining the two species had a synergistic effect on leaf litter consumption, and neutralizing effects on soil respiration. The data suggest that the strength of intra- and inter-specific interactions among earthworm ecological groups varies at different absolute and relative densities, leading to altered leaf litter decomposition and C cycling.     

Sublethal effects of imidacloprid on the burrowing behaviour of two earthworm species: Modifications of the 3D burrow systems in artificial cores and consequences on gas diffusion in soil

Earthworms have been termed ‘ecosystem engineers’ (sensu [Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosysem engineers. Oikos 69, 373-386.]) because of the important roles they play in the soil. As a consequence, it is assumed that if earthworms change their behaviour following exposure to pesticides or pollutants this could have a drastic impact on soil functioning. To test this assumption under laboratory conditions, we studied the burrow systems made by two earthworm species (the anecic Aporrectodea nocturna and the endogeic Allolobophora icterica) in artificial soil cores containing imidacloprid, a widely used neonicotinoid insecticide. After 1-month incubation period, the macropores created in the soil core were analyzed by tomography. In order to further characterize transfer properties associated with burrow systems gas diffusion measurements were also carried out. The burrow systems made by the two earthworm species were very different: A. nocturna made more continuous, less branched, more vertical and wider burrows than A. icterica. Some changes to A. nocturna burrow systems were observed after exposure to imidacloprid (they made a smaller burrow system and burrows were more narrow), but only at the highest concentration of imidacloprid used (0.5 mg kg⁻¹). A. icterica worms were more sensitive to imidacloprid and many differences in their burrow systems (length, sinuosity, branching rate and number of burrows) were observed at both concentrations tested (0.1 and 0. 5 mg kg⁻¹). As a consequence, the continuity of the burrow systems made by both species was altered following imidacloprid treatment. Gas diffusion through the A. nocturna soil cores was reduced but no difference in gas diffusion was observed in the A. icterica soil cores.     

Earthworms change the distribution and availability of phosphorous in organic substrates

In laboratory controlled soil microcosms, the distribution and availability of phosphorous (P) were determined in the surface-casts and the burrows-linings of the anecic earthworm L. terrestris and were compared with non-ingested soil. To simulate more realistic earthworm community conditions, a combination of L. terrestris plus the endogeic A. caliginosa was tested. For a 2-month period, the earthworms were given two organic food substrates: rye-grass littered onto the soil surface and sewage sludge mixed with soil. The following treatments were designed: (i) soil alone (S), (ii) soil and sewage sludge (SS), soil and rye-grass litter (SL), and (iv) soil, litter and sludge (SSL). Analyses were performed for P contents (total, available and organic), organic matter content (organic carbon, C_org and total nitrogen, N_tot) and the two acid and alkaline phosphatase activities (AcPA and AkPA). Earthworms enhanced AcPA and were also responsible for additional AkPA in soil. The two AcPA and AkPA increased not only in surface-casts but also in burrows-linings that paralleled with the decrease of organic P in SL and SSL treatments. The stimulation of AcPA began quickly and declined rapidly in casts (from 19 to 8 μmol phenol g⁻¹ dry wt h⁻¹, respectively at week 2 and 8 in the SL treatment) but it was initiated later and maintained at a high level for longer in burrows (more than 10 μmol phenol g⁻¹ dry wt h⁻¹ at week 8 in the SL treatment). Significant positive correlations were found between the AkPA activities and N_tot contents (r=0.95, p=0.001) and to a lesser extend with C_org contents (r=0.76, p=0.05) in casts from the SL treatment, while AcPA significantly correlated with N_tot (r=0.91, p=0.004) but not with C_org (r=0.72, p=0.06). P availability was always highest in casts. However, the available P contents decreased sharply over time in casts and were still low in burrow-linings, suggesting that a large part of inorganic P produced was rapidly immobilized for the microbial growth. Total P content was unchanged except in the SL treatment in which it increased in casts and burrows (ca. 725 μg g⁻¹, at week 4). Organic P was first the highest in casts and then decreased over time (from 168 at week 1 to 140 μg g⁻¹ at week 8 in the SL treatment). This study illustrates that earthworms facilitate P transfer downward increasing a P patchy distribution in the soil, and significantly change the biogeochemical status of P (availability, organic phosphorous pool, AcPA activities) in certain hot spots such as casts and burrow-linings.