Chronic toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil determined using the earthworm (Eisenia andrei) reproduction test

The sublethal and chronic effects of the environmental contaminant and explosive octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in artificial soil were assessed using the earthworm (Eisenia andrei). Based on various reproduction parameters (total and hatched number of cocoons, number of juveniles and their biomass), fecundity was reduced at the different concentrations of HMX tested (from 280.0 +/- 12.3 to 2502.9 +/- 230.0 mg kg-1 dry soil) in spiked artificial soil (LOEC: 280.0 +/- 12.3 mg kg-1 dry soil). The growth of adult E. andrei was also reduced at the different concentrations tested, though no mortality occurred, even at the highest tested concentrations. The number of juveniles produced was correlated with the number of total and hatched cocoons, and the biomass of juveniles was correlated with the number of cocoons. Pooled results of these and earlier studies on explosives (TNT, RDX) using the E. andrei reproduction test confirm that effects of HMX on cocoon production are indicative of some reproductive consequences (number of juvenile and their biomass), whereas adult growth, in general, does not correlate strongly with change in reproduction capacity.     

Sub-lethal toxicity of the antiparasitic abamectin on earthworms and the application of neutral red retention time as a biomarker

The antiparasitic abamectin has been proven effective against both endo- and ectoparasites of farm animals and hence used widely in animal husbandry. It may enter the soil environment with the excreta of treated animals. Very little information is available with regard to the sub-lethal effects of abamectin on soil invertebrates, such as earthworms. The objective of this study was to evaluate the toxic effect of abamectin on earthworms, using Eisenia fetida, by analyzing changes in the survival, growth, reproduction and cocoon hatchability of exposed earthworms. Furthermore, a biomarker of the lysosomal membrane stability, measured by neutral red retention time (NRR-time), was also applied. Abamectin showed significant toxicity on the growth of earthworms with increasing concentrations up to 5mg/kg. The most sensitive parameter was reproduction (cocoons production and hatchability) and NRR-time. The number of cocoons was reduced at concentrations above 0.25mg/kg and no cocoons were present at the highest concentration of 5mg/kg. Cocoons exposed to abamectin exhibited a reduced hatching success at concentrations above 1.5mg/kg. The NRR-time was reduced significantly at exposure concentrations of abamectin above 0.25mg/kg. The change in lysosomal membrane stability showed a good correlation with reproduction and may hence be a potential predicator of the effects on earthworm populations.     

Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil

To examine the potential of a suite of biomarkers as early warning indicators of environmental pollution, sperm count, neutral red retention time (NRRT) and DNA damage were measured in earthworm Eisenia fetida exposed to increasing concentrations of acetochlor in OECD soil. The neutral red retention time of earthworms coelomocytes was sensitive to acetochlor pollution, and decreased significantly when the concentration was more than 10mgkg(-1) after 30 and 60 days of exposure (P<0.05). The reduced neutral red retention time correlated with the soil acetochlor residual. Sperm count decreased significantly at the concentrations of 40 and 80mgkg(-1) after 15 days of exposure (P<0.05). The DNA damage of earthworms coelomocytes increased significantly after 30 days of exposure at the highest concentration (80mgkg(-1); P<0.05). Earthworms were under physiological stress at field dose of acetochlor (10mgkg(-1)). Higher concentrations of acetochlor caused sperm count decrease and DNA damage of earthworms. Such a suite of biomarkers could serve as indicators of the health of the soil environment and to evaluate the toxicity of acetochlor on earthworms or as a means of monitoring soil acetochlor pollution.     

Eisenia fetida increased removal of polycyclic aromatic hydrocarbons from soil

The removal of phenanthrene, anthracene and benzo(a)pyrene added at three different concentrations was investigated with or without earthworms (Eisenia fetida) within 11 weeks. Average anthracene removal by the autochthonous micro-organisms was 23%, 77% for phenanthrene and 13% for benzo(a)pyrene, while it was 51% for anthracene, 47% for benzo(a)pyrene and 100% for phenanthrene in soil with earthworms. At 50 and 100mg phenanthrene kg(-1)E. fetida survival was 91% and 83%, but at 150 mg kg(-1) all died within 15 days. Survival of E. fetida in soil amended with anthracene < or = 1000 mg kg(-1) and benzo(a)pyrene < or = 150 mg kg(-1) was higher than 80% and without weight loss compared to the untreated soil. Only small amounts of PAHs were detected in the earthworms. It was concluded that E. fetida has the potential to remove large amounts of PAHs from soil, but more work is necessary to elucidate the mechanisms involved.     

Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems

The earthworm Eisenia fetida (Annelida: Oligochaeta) was exposed to a geometric series of concentrations of cadmium, copper, lead and zinc in artificial soil using the OECD recommended protocol. Mortality, growth and cocoon production were measured over 56 days to determine LC50 and EC50 values. No observed effect concentrations (NOECs) were also estimated. Furthermore, the percentage of viable cocoons and number of juveniles emerging per cocoon was recorded. Cocoon production was more sensitive than mortality for all the metals, particularly cadmium and copper for which NOEC reproduction values were an order of magnitude lower than those for NOEC mortality. However, there was no significant effect of metals on the viability of cocoons. The weights of earthworms declined in all treatments (including the controls) during the experiment. This was probably due to the lack of suitable food in the OECD standard soil medium used. It was concluded that future experiments should include animal manure in the test medium. The LC50, EC50 and NOEC values determined in this study were compared with concentrations of metals in soils in the vicinity of a smelting works at Avonmouth, southwest England. The 14-day LC50 for zinc in Eisenia fetida was exceeded in soils covering an area of 75 km2 around the works, compared to 4.2 km2 for copper and 4.7 km2 for lead. Soil values for cadmium did not exceed the LC50 value anywhere in the region. Similar estimates of relative effects on reproduction confirmed that zinc is most likely to be responsible for the absence of earthworms from sites close to the Avonmouth works. However, the OECD standard test overestimated the potential effects of metals on populations, since earthworms can be found as close as 1 km from the smelting works. The discrepancy between test and field observations was probably due to the greater availability of the metals in the artificial soil.     

Effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (Eisenia fetida) under laboratory conditions

To assess the toxic effects of phenanthrene on earthworms, we exposed Eisenia fetida to artificial soils supplemented with different concentrations (0.5, 2.5, 12.5, mgkg(-1) soil) of phenanthrene. The residual phenanthrene in the soil, the bioaccumulation of phenanthrene in earthworms, and the subsequent effects of phenanthrene on growth, anti-oxidant enzyme activities, and lipid peroxidation (LPO) were determined. The degradation rate of low concentrations of phenanthrene was faster than it was for higher concentrations, and the degradation half-life was 7.3d (0.5 mgkg(-1)). Bioaccumulation of phenanthrene in the earthworms decreased the phenanthrene concentration in soils, and phenanthrene content in the earthworms significantly increased with increasing initial soil concentrations. Phenanthrene had a significant effect on E. fetida growth, and the 14-d LC(50) was calculated as 40.67 mgkg(-1). Statistical analysis of the growth inhibition rate showed that the concentration and duration of exposure had significant effects on growth inhibition (p<0.001). Superoxide dismutase (SOD) activity increased at the beginning (2 and 7d) and decreased in the end (14 and 28 d). Catalase (CAT) activity in all treatments was inhibited from 1 to 14 d of exposure. However, no significant perturbations in malondialdehyde (MDA) content were noted between control and phenanthrene-treated earthworms except after 2d of exposure. These results revealed that bioaccumulation of phenanthrene in E. fetida caused concentration-dependent, sub-lethal toxicity. Growth and superoxide dismutase activity can be regarded as sensitive parameters for evaluating the toxicity of phenanthrene to earthworms.     

Dynamics of nitrogen in a PAHs contaminated soil amended with biosolid or vermicompost in the presence of earthworms

Nitrogen mineralization in PAHs contaminated soil in presence of Eisenia fetida amended with biosolid or vermicompost was investigated. Sterilized and unsterilized soil was contaminated with PAHs, added with E. fetida and biosolid or vermicompost and incubated aerobically for 70 days, while dynamics of inorganic N were monitored. Addition of E. fetida to sterilized soil increased concentration of NH(4)(+) 100> mg N kg(-1), while concentrations in unsterilized remained <60 mg N kg(-1) except for soil amended with biosolid plus PAHs where it increased to >80 mg kg(-1). Addition of PAHs had no significant effect on concentration of NH(4)(+) compared to the unamended soil, except in the soil added with biosolid. Addition of E. fetida to sterilized soil increased concentration of NO(2)(-) 15> mg N kg(-1) while concentrations in unsterilized soil remained <7.5 mg N kg(-1) except for soil amended with biosolid where it increased to >20 mg kg(-1). Addition of PAHs had no significant effect on concentration of NO(2)(-) compared to the unamended soil. Addition of biosolid and vermicompost increased concentration of NO(3)(-), while addition of E. fetida decreased concentration of NO(3)(-) in biosolid amended soil. It was found that NH(4)(+) and NO(2)(-) oxidizers were present in the gut of E. fetida, but their activity was not sufficient enough to inhibit a temporarily increase in concentrations of NH(4)(+) and NO(2)(-). Contamination with PAHs induced immobilization of N in biosolid or vermicompost amended soil, as did feeding of E. fetida on biosolid or vermicompost.     

Effects of C60 nanoparticle exposure on earthworms (Lumbricus rubellus) and implications for population dynamics

Effects of C₆₀ nanoparticles (nominal concentrations 0, 15.4 and 154 mg/kg soil) on mortality, growth and reproduction of Lumbricus rubellus earthworms were assessed. C₆₀ exposure had a significant effect on cocoon production, juvenile growth rate and mortality. These endpoints were used to model effects on the population level. This demonstrated reduced population growth rate with increasing C₆₀ concentrations. Furthermore, a shift in stage structure was shown for C₆₀ exposed populations, i.e. a larger proportion of juveniles. This result implies that the lower juvenile growth rate due to exposure to C₆₀ resulted in a larger proportion of juveniles, despite increased mortality among juveniles. Overall, this study indicates that C₆₀ exposure may seriously affect earthworm populations. Furthermore, it was demonstrated that juveniles were more sensitive to C₆₀ exposure than adults.     

Toxicity of the anthelmintic abamectin to four species of soil invertebrates

Abamectin is a veterinary medicinal product with high efficiency against parasitics. This study evaluates the sublethal toxicity of abamectin to three groups of soil dwelling organisms (springtails, enchytraeids and earthworms). The reproduction of the two springtail species Folsomia fimetaria and Folsomia candida was significantly affected at abamectin concentrations of 0.25 and 0.5 mg/kg DW, respectively. Adults of especially F. candida were less sensitive. Abamectin had no effect on the survival of enchytraeids, whereas the number of juveniles was reduced at concentrations above 10 mg/kg. The EC10 values for reproduction were in the range of 0.05 mg/kg for springtails and 12.8 mg/kg for enchytraeids. Abamectin also had a significant effect on the reproduction of earthworms with an EC10 value of 0.06 mg/kg. However, no change in the survival and growth of juvenile earthworms was observed following 70 days of exposure.     

Earthworms to assess the innocuousness of spent biomixtures employed for glyphosate degradation

In this study, the innocuousness of different biomixtures employed for glyphosate degradation was tested through Eisenia fetida earthworms. Eight biomixtures were prepared with local materials: alfalfa straw (AS), wheat stubble (WS), river waste (RW) and two different soils (A and B). Each biomixture was divided into two equal portions: one without glyphosate application (control substrate) and the other was sprayed with a commercial glyphosate formulation of 1,000 mg glyphosate a.i. kg^?1 biomixture (applied substrate). The bioassay started when all sprayed biomixtures reached high percentages of glyphosate degradation (spent biomixtures). Three parameters were studied: survival, adults and juveniles biomass and reproduction. The results allowed the identification of three biomixtures (AWS, BWS and BWSRW) for good maintenance and development of E. fetida. In addition, at the end of the bioassay two of the viable biomixtures (AWS and BWS) showed the highest performance of juvenile earthworms compared to a reference soil. The Principal Component Analysis (PCA) indicated that the biomixtures containing high silt and clay percentages and minor density renders higher values of earthworm growth and reproduction. Therefore, these innocuous biomixtures can be used as organic amendments or recycled materials for new treatments on biobeds.     

Ecotoxicity of nickel to Eisenia fetida, Enchytraeus albidus and Folsomia candida

Despite growing concern about the potential adverse effects of elevated nickel concentrations in the environment, only a few toxicity data are available for terrestrial invertebrates. Therefore, chronic toxicity of nickel was assessed for Eisenia fetida, Enchytraeus albidus and Folsomia candida, the three invertebrates for which standard test protocols are available. The 21 d EC50 for the cocoon production of E. fetida was 362 (241-508) mg Ni/kg dry wt. For the reproduction of E. albidus, a 42 d EC50 of 275 (217-346) mg Ni/kg dry wt was observed. The 28 d EC50 for the reproduction of F. candida was 476 (347-671) mg Ni/kg dry wt. The obtained toxicity data were very similar to those of related species reported in literature. Although the presented data can be considered as a step forward in the assessment of the potential risks of nickel in terrestrial environments, further research is needed to evaluate the influence of soil parameters on the toxicity of nickel and to quantify the effect of ageing on bioavailability.     

Effect of herbicides on glutathione S-transferases in the earthworm, <Emphasis Type="Italic">Eisenia fetida</Emphasis>

AIM AND BACKGROUND: Earthworms have been studied as a readily available, easily maintainable and cheap test species for assessing chemical pollution, and may be an alternative to in vivo rodent bioassays. The current investigation aims to characterize detoxification enzymes in Eisenia fetida and stress response against two herbicides with different modes of action, namely, fenoxaprop and metolachlor. METHODS: Herbicides were applied to soil containing earthworms. Animals were then collected, sacrificed and shock-frozen. Extracted protein was analyzed for glutathione S-transferase (GST) activity using CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), pNBC (p-nitrobenzylchloride), PNOBC (p-nitrobenz-o-ylchloride) and selected herbicides. GST isoenzymes were partially purified by affinity chromatography and molecular weights were estimated by SDS-PAGE. RESULTS: In E. fetida protein extracts, GST activity towards model compounds ranked as CDNB>DCNB>PNBOC>PNBC. Fluorodifen was not conjugated at all, but fenoxaprop and metolachlor were conjugated at low rates. Furthermore, the GST isoenzyme pattern changed during the incubation with herbicides, either due to stress or as a defense reaction. After incubation with monochlorobimane, a strong fluorescence of the intestinal tract and the intersegments was observed, indicating organ-specific GST induction. DISCUSSION: According to the author's knowledge, here, for the first time, evidence is presented that E. fetida GST are also capable of conjugating a wider range of xenobiotic substrates. Different forms of GST were observed and changes in GST isoforms due to the herbicide treatment were also noticed. GST conjugation rates varied between different herbicides used in this experiment. It might be assumed that herbicides may well be detoxified by earthworms, to a certain extent, but that they are also potent stress factors influencing the detoxification system of the animal. High doses or long exposure might lead to deleterious effects on earthworms and limit their survival rate. The use of the animals as bioindicators for herbicides and herbicide residues seems very promising, but is surely influenced by the lack of detoxification for some compounds. CONCLUSIONS: Conjugation of several xenobiotics with model substances and herbicides is proven in the earthworm E. fetida. However, E. fetida has only limited capabilities of detoxifying herbicidal compounds. Different isoforms of GST were involved and altered in their activity after treatment. RECOMMENDATIONS AND PERSPECTIVES: The accumulation of GS-conjugates and their determination via fluorescence microscopy is a quick and secure, additional marker for exposure that should be further developed to complement existing biotests. The described methods and endpoints might help to understand the complex reaction of earthworms towards herbicides and lead to an adapted test methodology.     

Effect of two major N-nitroso hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites on earthworm reproductive success

Soil and topical tests were employed to investigate the effect of two N-nitroso metabolites of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) on earthworm reproduction. The lowest observed effect concentration (LOEC) for cocoon production and hatching was 50mg/kg for both hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soil. MNX and TNX also significantly affected cocoon hatching in soil (p<0.001) and in topical tests (p=0.001). The LOECs for cocoon hatching were 1 and 10mg/kg for MNX and TNX in soil, respectively, and 10mg/L in the topical test. Greater than 100mg/kg MNX and TNX completely inhibited cocoon hatching. In soil, the EC20 values for MNX were 8.7 and 8.8mg/kg for cocoon and juvenile production, respectively, compared to 9.2 and 9.1mg/kg for TNX, respectively. The EC20 values for the total number of cocoon hatchlings were 3.1 and 4.7mg/kg for MNX and TNX, respectively, in soil and 4.5 and 3.1mg/L in the topical test. Both MNX and TNX inhibited cocoon production and hatching, suggesting that they may have a negative affect on soil ecosystems at contaminated sites.     

Effects of metals on life cycle parameters of the earthworm Eisenia fetida exposed to field-contaminated, metal-polluted soils

Two control and eight field-contaminated, metal-polluted soils were inoculated with Eisenia fetida (Savigny, 1826). Three, 7, 14, 21, 28 and 42 days after inoculation, earthworm survival, body weight, cocoon production and hatching rate were measured. Seventeen metals were analysed in E. fetida tissue, bulk soil and soil solution. Soil organic carbon content, texture, pH and cation exchange capacity were also measured. Cocoon production and hatching rate were more sensitive to adverse conditions than survival or weight change. Soil properties other than metal concentration impacted toxicity. The most toxic soils were organic-poor (1-10 g C kg(-1)), sandy soils (c. 74% sand), with intermediate metal concentrations (e.g. 7150-13,100 mg Pb kg(-1), 2970-53,400 mg Zn kg(-1)). Significant relationships between soil properties and the life cycle parameters were determined. The best coefficients of correlation were generally found for texture, pH, Ag, Cd, Mg, Pb, Tl, and Zn both singularly and in multivariate regressions. Studies that use metal-amended artificial soils are not useful to predict toxicity of field multi-contaminated soils.     

Bioaccumulation and elimination of avermectin B1a in the earthworms (Eisenia fetida)

The acute toxicity, bioaccumulation, and elimination of avermectin B1a (AVM B1a) in earthworm (Eisenia fetida) were investigated in different exposure systems. The LC50 of AVM B1a on earthworms were 24.1 mg/kg and 17.1 mg/kg, respectively, for 7 and 14 days in artificial soil. The LC50 tested by the filter paper for 2 days was 4.63 microg/cm2. The earthworms were cultivated in artificial soil containing 0.6 mg/kg and 3.0 mg/kg AVM B1a, respectively for bioaccumulation experiments. The AVM B1a residues in earthworms were determined with HPLC-fluorescence method. The results showed that AVM B1a was taken up from the concentrated artificial soil by the earthworms and the steady-state levels were reached after 9-18 days of exposure. On the 18th day, the final concentrations of AVM B1a in the earthworms treated with two different dosages were 107 ng/g and 165 ng/g, respectively; there were not significantly accumulation. About 80.0% and 94.8% of the accumulated AVM B1a were eliminated respectively in two groups within 1 day after they were exposed to AVM B1a-free soil, but a trace amount of AVM B1a was found for a relative long time in earthworms.     

Effects of carbaryl, chlorpyrifos and endosulfan on growth, reproduction and respiration of tropical epigeic earthworm, Perionyx excavatus (Perrier)

Effects of sub-lethal doses of carbaryl (1-Naphthyl-methylcarbamate), chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridinyl-phosphorothioate) and endosulfan (6,7,8,9,10,10-Hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide), respectively a carbamate, an organophosphate and an organochlorine insecticide on growth, reproduction and respiration of the tropical earthworm, Perionyx excavatus (Perrier) were investigated under laboratory conditions. The results showed significant reduction in biomass, production and hatching of cocoon and production of juveniles of the worms exposed to 0.75 to 3.03 mg/kg soil of carbaryl, 0.91 to 3.65 mg/kg soil of chlorpyrifos and 3.75 to 15.0 μg/kg soil of endosulfan corresponding to 12.5 to 50 % of LC(50) value of the respective insecticide for P. excavatus. Endosulfan was found most dangerous among the three insecticides followed by carbaryl and chlorpyrifos. There was no hatching of the worms at endosulfan treatment 5.0 μg/kg soil (25 % LC(50)) or above while the highest dose of carbaryl and chlorpyrifos (50 % of LC(50)) rendered respectively 87.13 and 24.84 % reductions in hatching as compared to control. Chlorpyrifos produced no change in respiration of the worms except at the highest dose, while the worms showed an increase in evolution of CO(2) at all doses of carbaryl and endosulfan. Based on the recommended agricultural dose of each insecticide, it was concluded that application of endosulfan and carbaryl was potentially dangerous to earthworms.     

Assessing the impact of organic and inorganic amendments on the toxicity and bioavailability of a metal-contaminated soil to the earthworm Eisenia andrei

Metal-contaminated soil, from the El Arteal mining district (SE Spain), was remediated with organic (6 % compost) and inorganic amendments (8 % marble sludge) to reduce the mobility of metals and to modify its potential environmental impact. Different measures of metal bioavailability (chemical analysis; survival, growth, reproduction and bioaccumulation in the earthworm Eisenia andrei), were tested in order to evaluate the efficacy of organic and inorganic amendments as immobilizing agents in reducing metal (bio)availability in the contaminated soil. The inorganic amendment reduced water and CaCl₂-extractable concentrations of Cd, Pb, and Zn, while the organic amendment increased these concentrations compared to the untreated soil. The inorganic treatment did not significantly reduce toxicity for the earthworm E. andrei after 28 days exposure. The organic amendment however, made the metal-contaminated soil more toxic to the earthworms, with all earthworms dying in undiluted soil and completely inhibiting reproduction at concentrations higher than 25 %. This may be due to increased available metal concentrations and higher electrical conductivity in the compost-amended soil. No effects of organic and inorganic treatments on metal bioaccumulation in the earthworms were found and metal concentrations in the earthworms increased with increasing total soil concentrations.     

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.     

Effects, transfer, and fate of RDX from aged soil in plants and worms

The objectives of this study were to provide data that can be used to predict exposure-based effects of RDX in aged soil on multiple endpoint organisms representing two trophic levels. These data can be used for defining criteria or reference values for environmental management and conducting specific risk assessment. Dose-response experiments formed the basis for the evaluation of toxic effects and transfer of contaminants from soil into two trophic levels. Long-term exposure tests were conducted to evaluate chronic, sublethal, toxicity and transfer of aged soil-based explosives, with RDX as main contaminant. In these tests, plants were exposed for 55 days in the greenhouse, biomass was determined and residues of explosives parent compounds and RDX metabolites were analyzed using HPLC techniques. Worms were exposed for 28 days (Eisenia fetida) and 42 days (Enchytraeus crypticus) in the laboratory, biomass and number were determined, and tissues were analyzed for explosives compounds. The plants tolerated concentrations up to 1,540 mg RDX kg(-1) soil-DW. Biomass of Lolium perenne was not significantly related to soil-RDX concentration, while biomass of Medicago sativa significantly increased. No screening benchmark for RDX in soil for plants was calculated, since concentrations up to 1,540 mg kg(-1) soil failed to reduce biomass by 20% as required for a LOEC. RDX, RDX-metabolite MNX, and accompanying HMX concentrations in plants were significantly related to concentrations in soil after 55 days of exposure (RDX: R(2) = 0.77-0.89; MNX R(2) = 0.53-0.77; HMX: R(2) = 0.67-0.71). The average bioconcentration factors (BCF) were for RDX 17 in L. perenne and 37 in M. sativa, and for HMX 2 in L. perenne and 44 in M. sativa. The worms also tolerated concentrations up to 1,540 mg RDX kg(-1) soil-DW. Biomass of E. fetida adults decreased with soil-RDX concentration, and a LOEC of 1,253 mg kg(-1) soil-DW was estimated. RDX concentrations in E. fetida were significantly related to concentrations in soil after 28-day exposure (R(2) = 0.88). The average BCF in E. fetida for RDX was 1. Because in response to exposure to RDX-contaminated soil the RDX concentrations in plants increased initially and decreased subsequently, while those in worms increased continuously, RDX in worm tissues may accumulate to higher concentrations than in plant tissues, regardless of the low average BCF for worms.     

Functional and community-level soil microbial responses to zinc addition may depend on test system biocomplexity

The effect of zinc on soil nitrification and composition of the microbial community in soil was investigated using a full factorial experiment with five zinc concentrations and four levels of biological complexity (microbes only, microbes and earthworms (Eisenia fetida), microbes and Italian ryegrass (Lolium multiflorum var. Macho), and microbes, ryegrass and earthworms). After 6 weeks of exposure, the activity of soil nitrifying bacteria was measured and the microbial community structure was characterized by phospholipid fatty acid (PLFA) analysis. Soil nitrification and several PLFA markers were significantly influenced by either zinc addition and/or the presence of earthworms or ryegrass, and one of the most pronounced changes was the increase of fungi and decrease of bacteria with increasing concentrations of zinc. Of particular interest, however, was the potential interaction between the presence of plants and/or earthworms and the effect of zinc, which the factorial study design allowed us to explore. Such an effect was observed in two cases: Earthworms reduced the positive effect of zinc on the fungal biomass (ANOVA, p=0.03), and the effect of earthworms on the soil nitrification activity depended on zinc concentration (ANOVA, p<0.05). The effect of earthworm presence was not very large, but it does show that multispecies tests might give information about metal toxicity or bioavailability that cannot be predicted from single-species tests.