1H NMR-based metabolomics of time-dependent responses of Eisenia fetida to sub-lethal phenanthrene exposure

¹H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm² of phenanthrene (1/64th of the LC₅₀) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by ¹H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies.     

Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time

Eisenia fetida earthworms were exposed to phenanthrene for thirty days to compare hydroxypropyl-β-cyclodextrin (HPCD) extraction of soil and ¹H NMR earthworm metabolomics as indicators of bioavailability. The phenanthrene 28-d LC₅₀ value was 750 mg/kg (632-891, 95% confidence intervals) for the peat soil tested. The initial phenanthrene concentration was 319 mg/kg, which biodegraded to 16 mg/kg within 15 days, at which time HPCD extraction suggested that phenanthrene was no longer bioavailable. Multivariate statistical analysis of ¹H NMR spectra for E. fetida tissue extracts indicated that phenanthrene exposed and control earthworms differed throughout the 30 day experiment despite the low phenanthrene concentrations present after 15 days. This metabolic response was better correlated to total phenanthrene concentrations (Q² = 0.59) than HPCD-extractable phenanthrene concentrations (Q² = 0.46) suggesting that ¹H NMR metabolomics offers considerable promise as a novel, molecular-level method to directly monitor the bioavailability of contaminants to earthworms in the environment.     

Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

Relative proportions of polycyclic aromatic hydrocarbons differ between accumulation bioassays and chemical methods to predict bioavailability

Chemical methods to predict the bioavailable fraction of organic contaminants are usually validated in the literature by comparison with established bioassays. A soil spiked with polycyclic aromatic hydrocarbons (PAHs) was aged over six months and subjected to butanol, cyclodextrin and tenax extractions as well as an exhaustive extraction to determine total PAH concentrations at several time points. Earthworm (Eisenia fetida) and rye grass root (Lolium multiflorum) accumulation bioassays were conducted in parallel. Butanol extractions gave the best relationship with earthworm accumulation (r² ≤ 0.54, p ≤ 0.01); cyclodextrin, butanol and acetone-hexane extractions all gave good predictions of accumulation in rye grass roots (r² ≤ 0.86, p ≤ 0.01). However, the profile of the PAHs extracted by the different chemical methods was significantly different (p < 0.01) to that accumulated in the organisms. Biota accumulated a higher proportion of the heavier 4-ringed PAHs. It is concluded that bioaccumulation is a complex process that cannot be predicted by measuring the bioavailable fraction alone.     

Bioavailability of residual polycyclic aromatic hydrocarbons following enhanced natural attenuation of creosote-contaminated soil

The impact of residual PAHs (2250 ± 71 μg total PAHs g⁻¹) following enhanced natural attenuation (ENA) of creosote-contaminated soil (7767 ± 1286 μg total PAHs g⁻¹) was assessed using a variety of ecological assays. Microtox™ results for aqueous soil extracts indicated that there was no significant difference in EC₅₀ values for uncontaminated, pre- and post-remediated soil. However, in studies conducted with Eisenia fetida, PAH bioaccumulation was reduced by up to 6.5-fold as a result of ENA. Similarly, Beta vulgaris L. biomass yields were increased 2.1-fold following ENA of creosote-contaminated soil. While earthworm and plant assays indicated that PAH bioavailability was reduced following ENA, the residual PAH fraction still exerted toxicological impacts on both receptors. Results from this study highlight that residual PAHs following ENA (presumably non-bioavailable to bioremediation) may still be bioavailable to important receptor organisms such as earthworms and plants.     

Natural variability and correlations in the metabolic profile of healthy Eisenia fetida earthworms observed using ¹H NMR metabolomics

¹H NMR metabolomics can be used to assess the sub-lethal toxicity of contaminants to earthworms by identifying alterations in the metabolic profiles of contaminant- exposed earthworms in contrast to those of healthy (control) individuals. In support of this method this study sought to better characterize the baseline metabolic profile of healthy, mature earthworms of the species, Eisenia fetida, which is recommended for both acute and sub-lethal toxicity testing for soil contaminants. Profiles of D₂O-buffer extracted metabolites were determined using ¹H NMR spectroscopy and both inter-individual metabolic variability and pair-wise metabolic correlations were assessed. The control earthworm extracts exhibited low overall inter-individual metabolic variability, with a spectrum-wide median relative standard deviation (%RSD = standard deviation/mean × 100) of 14%, which suggests that the metabolic profile of E. fetida earthworms is well controlled in laboratory conditions and supports further use of this organism in environmental metabolomics research. In addition, strong positive correlations were detected between the levels of maltose, betaine, glycine, and glutamate as well as between the levels of lactate, valine, leucine, alanine, lysine, tyrosine, and phenylalanine which had not previously been reported. Since comparison of pair-wise metabolic correlations between control and treated organisms can reveal changes in the underlying pattern of biochemical relationships between the metabolites, identification of these significant metabolic correlations in control earthworms provides an additional characteristic that may be applied to delineate between control and treated earthworms in future NMR-based metabolomic studies.     

Effects of aqueous stable fullerene nanocrystals (nC60) on Daphnia magna: Evaluation of sub-lethal reproductive responses and accumulation

Concerns exist regarding the inadvertent release of engineered nanomaterials into natural systems, and the possible negative ecosystem response that may occur. Understanding sub-lethal effects may be particularly important to determining ecosystem responses as current levels of nanomaterial release are low compared to levels projected for the future. In this work, the sub-lethal effects and bioaccumulation of water stable, nanocrystalline fullerenes as C₆₀, (termed nC₆₀) were studied in Daphnia magna, a globally distributed, parthenogenetic zooplankton. Sub-lethal concentrations were first determined for both mature mother (LD₅₀ = 0.4 mg L⁻¹) and neonate (gestating) daphnids (0.2 mg L⁻¹) in standard 48 h exposure tests. Subsequent experiments focused on the accumulation and effects (at temperatures of 18–28 °C) of nC₆₀, during the D. magna reproductive cycle. The results demonstrate that upon sub-lethal exposure, the mortality rates of gestating daphnids increased with time and developmental stage. The maturation of daughter daphnids was negatively impacted. The mother daphnids were unable to reproduce again after exposure during pregnancy, and differential bioaccumulation occurred as a function of lipid content in the daphnia with the highest accumulation level of 7000 mg kg⁻¹ wet weight. Taken together, these results not only describe the accumulation and sub-lethal effects of nC₆₀ on exposed daphnia, but also highlight the importance of sub-lethal exposure scenarios, which are critical to fully understanding the potential impact of fullerenes and other engineered nanoscale materials on natural systems.     

Metabolic responses of Eisenia fetida after sub-lethal exposure to organic contaminants with different toxic modes of action

Nuclear magnetic resonance (NMR) - based metabolomics has the potential to identify toxic responses of contaminants within a mixture in contaminated soil. This study evaluated the metabolic response of Eisenia fetida after exposure to an array of organic compounds to determine whether contaminant-specific responses could be identified. The compounds investigated in contact tests included: two pesticides (carbaryl and chlorpyrifos), three pharmaceuticals (carbamazephine, estrone and caffeine), two persistent organohalogens (Aroclor 1254 and PBDE 209) and two industrial compounds (nonylphenol and dimethyl phthalate). Control and contaminant-exposed metabolic profiles were distinguished using principal component analysis and potential contaminant-specific biomarkers of exposure were found for several contaminants. These results suggest that NMR-based metabolomics offers considerable promise for differentiating between the different toxic modes of action (MOA) associated with sub-lethal toxicity to earthworms.     

Fractionation and bioavailability of Cu in soil remediated by EDTA leaching and processed by earthworms (<Emphasis Type="Italic">Lumbricus terrestris</Emphasis> L.)

Background, aim, and scope  

Soil remediation with ethylenediamine tetraacetic acid (EDTA) leaching is capable of removing only part of the total metal concentration in the soil, mostly the labile, bioavailable metal species (metal bioavailability stripping). However, reintroduction of remediated soil in the environment exposes the soil to various environmental factors, which could potentially shift nonlabile residual metals back to labile bioavailable forms. We studied the effect of autochthonous earthworm species as model biotic environmental factor on the fractionation and bioavailability of Cu residual in soil after remediation.     

Influence of earthworm mucus and amino acids on tomato seedling growth and cadmium accumulation

The effects on the growth of tomato seedlings and cadmium accumulation of earthworm mucus and a solution of amino acids matching those in earthworm mucus was studied through a hydroponic experiment. The experiment included four treatments: 5 mg Cd L⁻¹ (CC), 5 mg Cd L⁻¹ + 100 mL L⁻¹ earthworm mucus (CE), 5 mg Cd L⁻¹ + 100 mL L⁻¹ amino acids solution (CA) and the control (CK). Results showed that, compared with CC treatment, either earthworm mucus or amino acids significantly increased tomato seedling growth and Cd accumulation but the increase was much higher in the CE treatment compared with the CA treatment. This may be due to earthworm mucus and amino acids significantly increasing the chlorophyll content, antioxidative enzyme activities, and essential microelement uptake and transport in the tomato seedlings. The much greater increase in the effect of earthworm mucus compared with amino acid treatments may be due to IAA-like substances in earthworm mucus.     

Interactions of arsenic and phenanthrene on their uptake and antioxidative response in Pteris vittata L

The interactions of arsenic and phenanthrene on plant uptake and antioxidative response of Pteris vitatta L. were studied hydroponically. The combination of arsenic and phenanthrene decreased arsenic contents in fronds by 30-51%, whereas increased arsenic concentrations 1.2-1.6 times in roots, demonstrating the suppression of arsenic translocation compared to the corresponding treatment without phenanthrene. Under the co-exposure, As(III) concentrations in fronds deceased by 12-73%, and at higher arsenic exposure level (≥10 mg/L), As(V) in fronds and As(III) in roots increased compared to the single arsenic treatment. Arsenic exposure elevated phenanthrene concentrations in root by 39-164%. The co-existence of arsenic and phenanthrene had little impact on plant arsenic accumulation, although synergistic effect on antioxidants was observed, suggesting the special physiological process of P. vitatta in the co-exposure and application potential of P. vitatta in phytoremediation of arsenic and PAHs co-contamination.     

Effects of formalin on some biomarker activities of earthworms pre-exposed to temephos

Despite its negative effects, formalin has been often used for the expulsion of earthworms due to its high efficiency; however it is not known whether it will affect any significant measurable molecular processes in sampled earthworms. The aim of this research was to investigate effects of formalin on the activities of chosen molecular biomarkers in Eisenia andrei earthworms previously exposed to temephos. Additionally, the inhibitory effect of temephos, hitherto evaluated only on laboratory-bred earthworm species, was confirmed on two earthworm species obtained from their natural environment – Dendrobaena octaedra and Lumbricus rubellus. Earthworms were first exposed to the sub-lethal concentration of temephos for 2 h and then to formalin 15 min in order to simulate the sampling procedure. Besides acetylcholinesterase (AChE) inhibition – a known biomarker of exposure to organophosphate insecticides – the concentration of oximes and the activities of catalase (CAT) and efflux pump were measured. Results showed that in all species temephos caused inhibition of AChE and CAT activity. Exposure of E. andrei to formalin caused inhibition of AChE, however after post-exposure to formalin for 15 min significant increase in AChE activity was recorded. Similar results were obtained with the measurement of oximes concentrations. Exposure to only formalin and combination of temephos (2 h) and formalin (15 min) led to an increase in the CAT activity. The obtained results showed that exposure to formalin during the sampling could affect measured molecular biomarkers and also may change effects caused by exposure to temephos.     

Ecotoxicological responses of the earthworm Eisenia fetida exposed to soil contaminated with HHCB

Although polycyclic musks have been shown to cause lethal and sub-lethal effects on organisms, their biochemical toxicity to earthworms is not well understood. In the current study, we investigated the responses of antioxidant systems and lipid peroxidation after exposing Eisenia fetida to soil contaminated with 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran (HHCB). Significant increase in lipid peroxidation level was observed on day 14 at two high concentrations, 50 and 100 mg kg⁻¹. Among antioxidant enzymes, the primary response to chronic HHCB exposure can be attributed to superoxide dismutase (SOD) and catalase (CAT). Of the two enzymes, SOD exhibited more sensitive response to HHCB stress. In addition, these two enzymes could have a combined effect on fighting damage by reactive oxygen species, evidenced by a marked relationship between lipid peroxidation and enzyme activity. On the other hand, dose-dependent inhibition of peroxidase (POD) activity has been observed throughout the test. The results suggest that the variations in investigated parameters of E. fetida could be used as responsive biomarkers for oxidative stress caused by HHCB in a soil environment.     

Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil

A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.     

H NMR-based metabolomics investigation of Daphnia magna responses to sub-lethal exposure to arsenic,copper and lithium

Metal and metalloid contamination constitutes a major concern in aquatic ecosystems. Thus it is important to find rapid and reliable indicators of metal stress to aquatic organisms. In this study, we tested the use of ¹H nuclear magnetic resonance (NMR) – based metabolomics to examine the response of Daphnia magna neonates after a 48 h exposure to sub-lethal concentrations of arsenic (49 μg L⁻¹), copper (12.4 μg L⁻¹) or lithium (1150 μg L⁻¹). Metabolomic responses for all conditions were compared to a control using principal component analysis (PCA) and metabolites that contributed to the variation between the exposures and the control condition were identified and quantified. The PCA showed that copper and lithium exposures result in statistically significant metabolite variations from the control. Contributing to this variation was a number of amino acids such as: phenylalanine, leucine, lysine, glutamine, glycine, alanine, methionine and glutamine as well as the nucleobase uracil and osmolyte glycerophosphocholine. The similarities in metabolome changes suggest that lithium has an analogous mode of toxicity to that of copper, and may be impairing energy production and ionoregulation. The PCA also showed that arsenic exposure resulted in a metabolic shift in comparison to the control population but this change was not statistically significant. However, significant changes in specific metabolites such as alanine and lysine were observed, suggesting that energy metabolism is indeed disrupted. This research demonstrates that ¹H NMR-based metabolomics is a viable platform for discerning metabolomic changes and mode of toxicity of D. magna in response to metal stressors in the environment.     

Toxicological responses of earthworm (Eisenia fetida) exposed to metal-contaminated soils

The aim of this study was to evaluate the toxicological responses of earthworm (Eisenia fetida) induced by field-contaminated, metal-polluted soils. Biochemical responses and DNA damage of earthworm exposed to two multi-metal-contaminated soils in a steel industry park and a natural reference soil in Zijin Mountain for 2, 7, 14, and 28 days were studied. Results showed that three enzyme activities, including superoxide dismutase (SOD), acetylcholinesterase (AChE), and cellulase, in earthworm in metal-contaminated soils were significantly different from those of the reference soil. Cellulase and AChE were more sensitive than SOD to soil contamination. The Olive tail moment of the comet assay after 2-day exposure increased 56.5 and 552.0 % in two contaminated soils, respectively, compared to the reference soil. Our findings show that cellulase and DNA damage levels can be used as potential biomarkers for exposure of earthworm to metal-polluted soils.     

Comparing the desorption and biodegradation of low concentrations of phenanthrene sorbed to activated carbon,biochar and compost

Carbonaceous soil amendments are applied to contaminated soils and sediments to strongly sorb hydrophobic organic contaminants (HOCs) and reduce their freely dissolved concentrations. This limits biouptake and toxicity, but also biodegradation. To investigate whether HOCs sorbed to such amendments can be degraded at all, the desorption and biodegradation of low concentrations of ¹⁴C-labelled phenanthrene (?5 μg L^?1) freshly sorbed to suspensions of the pure soil amendments activated carbon (AC), biochar (charcoal) and compost were compared. Firstly, the maximum abiotic desorption of phenanthrene from soil amendment suspensions in water, minimal salts medium (MSM) or tryptic soy broth (TSB) into a dominating silicone sink were measured. Highest fractions remained sorbed to AC (84 ± 2.3%, 87 ± 4.1%, and 53 ± 1.2% for water, MSM and TSB, respectively), followed by charcoal (35 ± 2.2%, 32 ± 1.7%, and 12 ± 0.3%, respectively) and compost (1.3 ± 0.21%, similar for all media). Secondly, the mineralization of phenanthrene sorbed to AC, charcoal and compost by Sphingomonas sp. 10-1 (DSM 12247) was determined. In contrast to the amounts desorbed, phenanthrene mineralization was similar for all the soil amendments at about 56 ± 11% of the initially applied radioactivity. Furthermore, HPLC analyses showed only minor amounts (<5%) of residual phenanthrene remaining in the suspensions, indicating almost complete biodegradation. Fitting the data to a coupled desorption and biodegradation model revealed that desorption did not limit biodegradation for any of the amendments, and that degradation could proceed due to the high numbers of bacteria and/or the production of biosurfactants or biofilms. Therefore, reduced desorption of phenanthrene from AC or charcoal did not inhibit its biodegradation, which implies that under the experimental conditions these amendments can reduce freely dissolved concentration without hindering biodegradation. In contrast, phenanthrene sorbed to compost was fully desorbed and biodegraded.     

Biomonitoring airborne parent and alkylated three-ring PAHs in the Greater Cologne Conurbation I: Temporal accumulation patterns

Polycyclic aromatic hydrocarbons (PAHs) comprise an important group of air pollutants, with three-ring components (PAH-3) often dominating. Spatiotemporal variation in atmospheric PAH-3 can be analyzed by biomonitoring but high vapour pressure and low octanol-air-partitioning of PAH-3 cause dynamic accumulation on plant surfaces. This study for the first time shows that PAH-3 exhibit systematic accumulation trends on pine needles of 3-48 months of exposure time at six sites in Germany. Correlation of needle exposure time with PAH-3 concentration was r² = 0.83 for phenanthrene and methylphenanthrenes, r² = 0.77 for cyclopenta[def]phenanthrene, r² = 0.60 for dibenzothiophene, r² = 0.57 for dimethylphenanthrenes and r² = 0.32 for retene. Variations in PAH-3 for summer and winter collected needles emphasize vegetation-air-partitioning influence on cumulative PAH-3 loads. PAH-3 ratios calculated for needle cohorts indicate persistence of original PAH patterns thus demonstrating the source-diagnostic potential of pine needle biomonitoring, which is utilized in part II of this study where spatial distribution of PAH-3 is investigated and related to emission sources.     

Sorption of polar and nonpolar organic contaminants by oil-contaminated soil

Sorption of nonpolar (phenanthrene and butylate) and polar (atrazine and diuron) organic chemicals to oil-contaminated soil was examined to investigate oil effects on sorption of organic chemicals and to derive oil–water distribution coefficients (K_oil). The resulting oil-contaminated soil–water distribution coefficients (K_d) for phenanthrene demonstrated sorption-enhancing effects at both lower and higher oil concentrations (C_oil) but sorption-reducing (competitive) effects at intermediate C_oil (approximately 1 g kg⁻¹). Rationalization of the different dominant effects was attempted in terms of the relative aliphatic carbon content which determines the accessibility of the aromatic cores to phenanthrene. Little or no competitive effect occurred for butylate because its sorption was dominated by partitioning. For atrazine and diuron, the changes in K_d at C_oil above approximately 1 g kg⁻¹ were negligible, indicating that the presently investigated oil has little or no effect on the two tested compounds even though the polarity of the oil is much less than soil organic matter (SOM). Therefore, specific interactions with the active groups (aromatic and polar domains) are dominantly responsible for the sorption of polar sorbates, and thus their sorption is controlled by available sorption sites. This study showed that the oil has the potential to be a dominant sorptive phase for nonpolar pollutants when compared to SOM, but hardly so for polar compounds. The results may aid in a better understanding of the role of the aliphatic and aromatic domains in sorption of nonpolar and polar organic pollutants.