Assessing the toxic effect of nitrapyrin on nitrogen transformation in soil

A 28 d N transformation test was developed according to the OECD guideline 216. In the laboratory-based test, a suitable soil was amended with powdered plant meal as an organic N source. Soil samples of 1 kg treated with five concentrations of nitrapyrin (2-chloro-6-(trichloromethyl)-pyridine), in the range 1.0-100 mg kg⁻¹ dry weight were incubated for 28 d at 20±2 °C. A dose response was produced and the N mineralisation EC₅₀ (95% C.I.) for nitrapyrin was 3.1 (1.9-4.3) mg kg⁻¹ dry soil. The determined EC₅₀ was compared with literature figures for similar end points but using different methodology.     

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.     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.     

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.     

Cyanobacterial inoculation of heated soils: effect on microorganisms of C and N cycles and on chemical composition in soil surface

Physiological groups of soil microorganisms, total C and N and available nutrients were investigated in four heated (350 °C, 1 h) soils (one Ortic Podsol over sandstone and three Humic Cambisol over granite, schist or limestone) inoculated (1.5 μg chlorophyll a g⁻¹ soil or 3.0 μg chlorophyll a g⁻¹ soil) with four cyanobacterial strains of the genus Oscillatoria, Nostoc or Scytonema and a mixture of them.Cyanobacterial inoculation promoted the formation of microbiotic crusts which contained a relatively high number of NH₄⁺-producers (7.4×10⁹ g⁻¹ crust), starch-mineralizing microbes (1.7×10⁸ g⁻¹ crust), cellulose-mineralizing microbes (1.4×10⁶ g⁻¹ crust) and NO₂⁻ and NO₃⁻ producers (6.9×10⁴ and 7.3×10³ g⁻¹ crust, respectively). These crusts showed a wide range of C and N contents with an average of 293 g C kg⁻¹ crust and 50 g N kg⁻¹ crust, respectively. In general, Ca was the most abundant available nutrient (804 mg kg⁻¹ crust), followed by Mg (269 mg kg⁻¹ crust), K (173 mg kg⁻¹ crust), Na (164 mg kg⁻¹ crust) and P (129 mg kg⁻¹ crust). There were close positive correlations among all the biotic and abiotic components of the crusts.Biofertilization with cyanobacteria induced great microbial proliferation as well as high increases in organic matter and nutrients in the surface of the heated soils. In general, cellulolytics were increased by four logarithmic units, amylolytics and ammonifiers by three logarithmic units and nitrifiers by more than two logarithmic units. C and N contents rose an average of 275 g C kg⁻¹ soil and 50 g N kg⁻¹ soil while the C:N ratio decreased up to 7 units. Among the available nutrients the highest increase was for Ca (315 mg kg⁻¹ soil) followed by Mg (189 mg kg⁻¹ soil), K (111 mg kg⁻¹ soil), Na (109 mg kg⁻¹ soil) and P (89 mg kg⁻¹ soil). Fluctuations of the microbial groups as well as those of organic matter and nutrients were positively correlated.The efficacy of inoculation depended on both the type of soil and the class of inoculum. The best treatment was the mixture of the four strains and, whatever the inoculum used, the soil over lime showed the most developed crust followed by the soils over schist, granite and sandstone. In the medium term there were not significant differences between the two inocula amounts tested.These results showed that inoculation of burned soils with alien N₂-fixing cyanobacteria may be a biotechnological means of promoting microbiotic crust formation, enhancing C and N cycling microorganisms and increasing organic matter and nutrient contents in heated 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.     

Survival of rhizobia in soil is sensitive to elevated zinc in the absence of the host plant

The survival of free-living rhizobia in soil is sensitive to elevated heavy metals in soil and can explain adverse effects of metals on symbiotic nitrogen fixation in soils. A survival experiment was set-up to derive critical cadmium (Cd) and zinc (Zn) concentrations in a range of field-contaminated soils in the absence of their host plant (Trifolium repens L.). Soils applied with metal salts or sewage sludge >10 years ago were sampled and were inoculated with Rhizobium leguminosarum bv. trifolii (10⁸ cells g⁻¹ soil) and incubated outdoors for up to 6 months. The most probable number (MPN) decreased 1-2 orders of magnitude in uncontaminated soils during the incubation. There was no significant effect of total metal concentrations on rhizobia survival in soils contaminated with Cd salts or with high Ni/Cd sewage sludge with highest Cd concentrations between 18 and 118 mg Cd kg⁻¹. In contrast, survival was strongly affected in soils contaminated by sewage sludge, where Zn was the principal metal contaminant. Neither total Cd nor soil solution Cd was large enough to attribute these effects to Cd when compared with the soil series, where Cd salts had been applied. The MPN decreased at least one order of magnitude above total Zn concentrations of 233 mg Zn kg⁻¹ (soil pH 5.6) and 876 mg Zn kg⁻¹ (soil pH 6.3). The EC50s of log MPN were 204 and 604 mg Zn kg⁻¹, respectively, and were lower than those for the symbiotic nitrogen fixation measured in the pot trial on the same soils (respectively 602 and 737 mg Zn kg⁻¹). This study corroborates the evidence that symbiotic nitrogen fixation is affected by Zn in the field when Zn decreases the free-living population of rhizobia to below a critical threshold.     

Enzyme activities and microbial biomass in coastal soils of India

Soil salinity is a serious problem for agriculture in coastal regions, wherein salinity is temporal in nature. We studied the effect of salinity, in summer, monsoon and winter seasons, on microbial biomass carbon (MBC) and enzyme activities (EAs) of the salt-affected soils of the coastal region of the Bay of Bengal, Sundarbans, India. The average pH of soils collected from different sites, during different seasons varied from 4.8 to 7.8. The average organic C (OC) and total N (TN) content of the soils ranged between 5.2-14.1 and 0.6-1.4 g kg⁻¹, respectively. The electrical conductivity of the saturation extract (ECe) of soils, averaged over season, varied from 2.2 to 16.3 dSm⁻¹. The ECe of the soils increased five fold during the summer season (13.8 dSm⁻¹) than the monsoon season (2.7 dSm⁻¹). The major cation and anion detected were Na⁺ and Cl⁻, respectively. Seasonality exerted considerable effects on MBC and soil EAs, with the lowest values recorded during the summer season. The activities of β-glucosidase, urease, acid phosphatase and alkaline phosphatase were similar during the winter and monsoon season. The dehydrogenase activity of soils was higher in monsoon than in winter. Average MBC, dehydrogenase, β-glucosidase, urease, acid phosphatase and alkaline phosphatase activities of the saline soils ranged from 125 to 346 mg kg⁻¹ oven dry soil, 6-9.9 mg triphenyl formazan (TPF) kg⁻¹ oven dry soil h⁻¹, 18-53 mg p-nitro phenol (PNP) kg⁻¹ oven dry soil h⁻¹, 38-86 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 213-584 mg PNP kg⁻¹ oven dry soil h⁻¹ and 176-362 mg PNP g⁻¹ oven dry soil h⁻¹, respectively. The same for the non-saline soils were 274-446 mg kg⁻¹ oven dry soil, 8.8-14.4 mg TPF kg⁻¹ oven dry soil h⁻¹, 41-80 mg PNP kg⁻¹ oven dry soil h⁻¹, 89-134 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 219-287 mg PNP kg⁻¹ oven dry soil h⁻¹ and 407-417 mg PNP kg⁻¹ oven dry soil h⁻¹, respectively. About 48%, 82%, 48%, 63%, 40% and 48% variation in MBC, dehydrogenase activity, β-glucosidase activity, urease activity, acid phosphatase activity and alkaline phosphatase activity, respectively, could be explained by the variation in ECe of saline soils. Suppression of EAs of the coastal soils during summer due to salinity rise is of immense agronomic significance and needs suitable interventions for sustainable crop production.     

Earthworm colonisation of abandoned arable soil polluted by copper

The distribution, density and biomass of earthworms were investigated at the copper polluted site, Hygum (Denmark). In 1994, shortly after farming of the area was abandoned, only four earthworm species were present and their distribution was restricted to areas where copper concentration did not exceed 200 mg kg^?1 dry soil. Sixteen years later (in 2010), without any agricultural activity, ten species of earthworms were found, in particular, epigeic species were present where soil copper concentrations reached >1000 mg kg^?1 dry soil.     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Microbial colonisation of roots as a function of plant species

Fifteen plants species were grown in the greenhouse on the same soil and sampled at flowering to obtain rhizosphere soil and root material. In both fractions, the data on fungal and bacterial tissue obtained by amino sugar analysis were compared with the total microbial biomass based on fumigation-extraction and ergosterol data. The available literature on glucosamine concentrations in fungi and on muramic acid concentrations in bacteria was reviewed to prove the possibility of generating conversion values for general use in root material. All microbial properties analysed revealed strong species-specific differences in microbial colonisation of plant roots. The root material contained considerable amounts of microbial biomass C and biomass N, reaching mean levels of 10.9 and 1.4 mg g⁻¹ dry weight, respectively. However, the majority of CHCl₃ labile C and N, i.e. 89 and 55% was root derived. The average amount of ergosterol was 13 μg g⁻¹ dry weight and varied between 0.0 for Phacelia roots and 45.5 μg g⁻¹ dry weight for Vicia roots. The ergosterol content in root material of mycorrhizal and non-mycorrhizal plant species did not differ significantly. Fungal glucosamine was converted to fungal C by multiplication by 9 giving a range of 7.1-25.9 mg g⁻¹ dry weight in the root material. Fungal C and ergosterol were significantly correlated. Bacterial C was calculated by multiplying muramic acid by 45 giving a range from 1.7 to 21.6 mg g⁻¹ dry weight in the root material. In the root material of the 15 plant species, the ratio of fungal C-to-bacterial C ranged from 1.0 in mycorrhizal Trifolium roots to 9.5 in non-mycorrhizal Lupinus roots and it was on average 3.1. These figures mean that the microbial tissue in the root material consists on average of 76% fungal C and 24% bacterial C. The differences in microbial colonisation of the roots were reflected by differences in microbial indices found in the rhizosphere soil, most strongly for microbial biomass C and ergosterol, but to some extent also for glucosamine and muramic acid.     

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.     

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.     

Microbial activity in pig slurry-amended soils under semiarid conditions

Soil amendment with manures from intensive animal industries is nowadays a common practice that may favorably or adversely affect several soil properties, including soil microbial activity. In this work, the effect of consecutive annual additions of pig slurry (PS) at rates of 30, 60, 90, 120 and 150 m³ ha⁻¹ y⁻¹ over a 4-year period on soil chemical properties and microbial activity was investigated and compared to that of an inorganic fertilization and a control (without amendment). Field plot experiment conducted under a continuous barley monoculture and semiarid conditions were used. Eight months after the fourth yearly PS and mineral fertilizer application (i.e. soon after the fourth barley harvest), surface soil samples (Ap horizon, 0-15 cm depth) from control and amended soils were collected and analysed for pH, electrical conductivity (EC), contents of total organic C, total N, available P and K, microbial biomass C, basal respiration and different enzymatic activities. The control soil had a slightly acidic pH (6.0), a small EC (0.07 dS m⁻¹), adequate levels of total N (1.2 g kg⁻¹) and available K (483 mg kg⁻¹) for barley growth, and small contents of total organic C (13.2 g kg⁻¹) and available P (52 mg kg⁻¹). With respect to the control and mineral fertilized soils, the PS-amended soils had greater pH values (around neutrality or slightly alkaline), electrical conductivities (still low) and contents of available P and K, and slightly larger total N contents. A significant decrease of total organic C was observed in soils amended at high slurry rate (12.3 g kg⁻¹). Compared with the control and mineral treatments, which produced almost similar results, the PS-amended soils were characterized by a higher microbial biomass C content (from 311 to 442 g kg⁻¹), microbial biomass C/total organic C ratio (from 2.3 to 3.6%) and dehydrogenase (from 35 to 173 μg INTF g⁻¹), catalase (from 5 to 24 μmol O₂ g⁻¹ min⁻¹), BAA-protease (from 0.7 to 1.9 μmol  g⁻¹ h⁻¹) and β-glucosidase (from 117 to 269 μmol PNP g⁻¹ h⁻¹) activities, similar basal respirations (from 48 to 77 μg C-CO₂ g⁻¹ d⁻¹) and urease activities (from 1.5 to 2.2 μmol  g⁻¹ h⁻¹), and smaller metabolic quotients (from 6.4 to 7.7 ng C-CO₂ μg⁻¹ biomass C h⁻¹) and phosphatese activities (from 374 to 159 μmol PNP g⁻¹ h⁻¹). For example, statistical analysis of experimental data showed that, with the exception of metabolic quotient and total organic C content, these effects generally increased with increasing cumulative amount of PS. In conclusion, cumulative PS application to soil over time under semiarid conditions may produce not only beneficial effects but also adverse effects on soil properties, such us the partial mineralization of soil organic C through extended microbial oxidation. Thus, PS should not be considered as a mature organic amendment and should be treated appropriately before it is applied to soil, so as to enhance its potential as a soil organic fertilizer.     

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.     

Community composition and activities of denitrifying bacteria from adjacent agricultural soil, riparian soil, and creek sediment in Oregon, USA

We examined denitrifying bacteria from wet soils and creek sediment in an agroecosystem in Oregon, USA that received inputs of nitrogen (N) fertilizer. Our objective was to determine the variation in denitrifying community composition and activities across three adjacent habitats: a fertilized agricultural field planted to perennial ryegrass, a naturally vegetated riparian area, and creek sediment. Using C₂H₂ inhibition, denitrifying enzyme and N₂O-reductase activities were determined in short-term incubations of anaerobic slurries. A key gene in the denitrification pathway, N₂O reductase (nosZ), served as a marker for denitrifiers. Mean denitrifying enzyme activity (DEA) was similar among habitats, ranging from 0.5 to 1.8 μg N g⁻¹ dry soil h⁻¹. However, the ratio of N₂O production, without C₂H₂, to DEA was substantially higher in riparian soil (0.64±0.02; mean±standard error, n=12) than in agricultural soil (0.19±0.02) or creek sediment (0.32±0.03). Mean N₂O-reductase activity ranged from 0.5 to 3.2 μg N g⁻¹ dry soil h⁻¹, with greater activity in agricultural soil than in riparian soil. Denitrifying community composition differed significantly among habitats based on nosZ terminal-restriction fragment length polymorphisms. The creek sediment community was unique. Communities in the agricultural and riparian soil were more closely related but distinct. A number of unique nosZ genotypes were detected in creek sediment. Sequences of nosZ obtained from riparian soil were closely related to nosZ from Bradyrhizobium japonicum. Although nosZ distribution and N₂O-reductase activity differed among habitats, relationships between activity and community composition appeared uncoupled across the agroecosystem.     

Possibilities to reduce rice straw-induced global warming potential of a sandy paddy soil by combining hydrological manipulations and urea-N fertilizations

Global warming potential (GWP) of sandy paddy soils may be reduced by trade-offs between N₂O, CH₄ and CO₂ emissions. Laboratory experiments using either rice straw (1% or 0.5%) or together with urea-N (25 or 50 mg N kg⁻¹ soil) at various levels of soil water were carried out for 30 days each, to test this assumption. Waterlogging combined with urea-N increased total N₂O emissions, with greater release upon rewaterlogging (7.4 mg N kg⁻¹ soil) than experienced by removing waterlogging only. Rice straw±urea-N either emitted small amounts of N₂O or resulted in negative values at all water levels, including saturated and aerobic. Total CH₄ fluxes declined with the decreased water levels and amount of rice straw (<193 mg C kg⁻¹ soil), and also for CO₂ with the latter (<1340 mg C kg⁻¹ soil), and rewaterlogging had little influence on both. N₂O under rewaterlogged and waterlogged±urea-N, CH₄ under waterlogged with rice straw, and CO₂ for the remainder were the major contributors to GWP. Results show that waterlogging following aerobic decomposition of rice straw (1%) with urea-N, applied either at the beginning or at the end of the aerobic conditions, could decrease GWP by 56-64% and 32-42% over the sole addition of rice straw (1% and 0.5%) under waterlogged and saturated conditions, respectively.     

Unusually high levels of bio-available phosphate in the soils of Ogasawara Islands, Japan: Putative influence of seabirds

Ogasawara Islands are important ecosystems sustaining many indigenous spices. To clarify the indigenous soil environments of Ogasawara Islands, we studied the chemistry of the soils. Many surface soils were low in bio-available P (0 to 0.55 g P₂O₅ kg⁻¹, average: 0.04 g P₂O₅ kg⁻¹ as Bray II P, n = 22), but several soils were found to contain extremely large amounts of bio-available P (1.36 to 6.98 g P₂O₅ kg⁻¹, average: 2.93 g P₂O₅ kg⁻¹, n = 5). From soil profile analyses, the authors concluded that the extremely large amount of bio-available P could not be explained by the effects of parent materials with high P contents nor the effect of fertilizations by human activity, but the effects of natural seabird activities in the past could be the cause. The soil profiles with large amounts of bio-available P indicate deep migration of soil materials from A horizons, which could be a result of intensive mixing of upper horizons by seabird activities. The intensive mixing was supported by the low mechanical impedance of the horizons for the P-accumulating soils (8.17 ± 2.54 kg cm⁻², n = 8) than those for the non-P-accumulating soils (17.46 ± 3.52 kg cm⁻², n = 36). It is likely that in the past seabirds, such as shearwaters, made burrows in the soils for nesting and propagating and inadvertently transported a large amount of P from the sea to the soils, resulting in the extremely large amounts of bio-available P in the present soils.     

Dynamics of microbial biomass and nitrogen supply during primary succession on blastfurnace slag dumps in dry tropics

This paper reports the role of microbial biomass in the establishment of N pools in the substratum during primary succession (till 40-year age) in Blastfurnace Slag Dumps, an anthropogenically created land form in the tropics. Initially in the depressions in the slag dumps fine soil particles (silt+clay) accumulate, retaining moisture therein, and providing microsites for the accumulation of microbial biomass. In all sites microbial biomass showed distinct seasonality, with summer-peak and rainy season-low standing crops. During the summer season microbial biomass C ranged from 18.6 μg g⁻¹ in the 1-year old site to ca. 235 μg g⁻¹ in the 40-year old site; correspondingly, microbial biomass N ranged from 1.22 to 40 μg g⁻¹. On sites 2.5-years of age and younger, the microbial biomass N content accounted for more than 50% of the organic N in the soil, whereas the proportion of microbial biomass N was ca. 7% of organic N in 40-year old site. The strong correlation between microbial biomass and total N in soil indicated a significant role of microbes in the build-up of nitrogen during the initial stages of succession in the slag dumps. Though the organic N pool in the soil was low (594 mg kg⁻¹) even after 40 years of succession, the available N (NH₄-N and NO₃-N) contents in the soil were generally high through the entire age series (ca. 16-32 μg g⁻¹) during the rainy season (which supports active growth of the herbaceous community). The high mineral-N status on the slag dump was related with high N-mineralization rates, particularly in the young sites (20.6 and 13.9 μg g⁻¹ month⁻¹ at 1 and 2.5-year age). We suggest that along with the abiotic factors having strong effect on ecosystem functioning, the microbial biomass, an important biotic factor, shows considerable influence on soil nutrient build-up during early stages of primary succession on the slag dumps. The microbial biomass dynamics initiates biotic control in developing slag dumps ecosystem through its effect on nitrogen pools and availability.