Phenanthrene Exposure Research Articles

Acute phenanthrene toxicity to juvenile diploid and triploid African catfish (Clarias gariepinus): Molecular, biochemical, and histopathological alterations

Information on the biological responses of polyploid animals towards environmental contaminants is scarce. This study aimed to compare reproductive axis-related gene expressions in the brain, plasma biochemical responses, and the liver and gill histopathological alterations in diploid and triploid full-sibling juvenile African catfish (Clarias gariepinus). Fish were exposed for 96 h to one of the two waterborne phenanthrene (Phe) concentrations [mean measured (SD): 6.2 (2.4) and 76 (4.2) μg/L]. In triploids, exposure to 76 μg/L Phe increased mRNA level of fushi tarazu-factor 1 (ftz-f1). Expression of tryptophan hydroxylase2 (tph2) was also elevated in both ploidies following the exposure to 76 μg/L Phe compared to the solvent control. In triploids, 76 μg/L Phe increased plasma alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) levels compared to the other Phe-exposed group. It also elevated lactate and glucose contents relative to the other groups. In diploids, however, biochemical biomarkers did not change. Phenanthrene exposures elevated glycogen contents and the prevalence of histopathological lesions in the liver and gills of both ploidies. This study showed substantial differences between diploids and triploids on biochemical and molecular biomarker responses, but similar histopathological alterations following acute Phe exposures.

Histological responses and localization of the cytochrome P450 (CYP2AU1) in Crassostrea brasiliana exposed to phenanthrene

Phenanthrene (PHE) is an abundant polycyclic aromatic hydrocarbon (PAH), widely distributed in aquatic environment. The aim of this study was to evaluate the histological and molecular effects in the native oyster Crassostrea brasiliana(Lamarck, 1819) exposed to 100 and 1000μgL−1 PHE for 1, 5 and 10 days. Histological and chemical analyses were performed to evaluate, respectively, alterations in oyster tissues and bioaccumulation. In situ hybridization (ISH) was used to assess tissue distribution of CYP2AU1, a gene formerly identified as activated by PHE exposure in this species.Quantitative polymerase chain reaction (qPCR) in mantle was carried out to validate ISH data. Oysters bioaccumulated PHE increasingly along the exposure period in both exposure concentrations. Histologic changes, like tubular atrophy in digestive diverticula (digestive gland) and increased number of mucous cells in the mantle were observed in animals exposed to PHE for 10 days. ISH showed the presence of CYP2AU1transcripts in gills, digestive diverticula, mantle, intestine and gonads, but significant differences in transcript detection by ISH between treatments occurred only in gills, mantle and intestine. A positive and significant correlation between tubular atrophy and CYP2AU1hybridization signal was observed in digestive diverticula, suggesting that this gene product might be involved in energetic metabolism in C. brasiliana. Increased mucous cells and CYP2AU1transcript levels were observed in the mantle, where the inner and middle lobes showed higher intensity of hybridization signal. Mantle should be considered as a target organ for CYP2AU1 transcript evaluation and histological alterations in biomonitoring studies. CYP2AU1 signal in female gonads was observed in all follicular cells from different gonadic stages, while in male only the spermatic follicle cells of the wall in the pre-spawning stage showed this signal. ISH was an effective technique to evaluate the effects of PHE exposure and to locate CYP2AU1 transcripts in different tissues of oyster C. brasiliana.

Impact of phenanthrene exposure on activities of nitrate reductase, phosphoenolpyruvate carboxylase, vacuolar H+-pyrophosphatase and plasma membrane H+-ATPase in roots of soybean, wheat and carrot

Crops differ in uptake of polycyclic aromatic hydrocarbons (PAHs). However, the physiological mechanism underlying the differences in PAH uptake among crop species is unclear. As PAH transport by H+-coupled symporters may affect cytosolic pH, and intracellular pH is precisely regulated for metabolism, four enzymes linked with cytosolic pH regulation in root cells were determined at different concentrations of phenanthrene (PHE) and pHs. Activity of nitrate reductase (NRase) is stimulated by PHE and follows the order: wheat>carrot>soybean. The optimum pH of NRase is 7.5 in wheat and carrot, while 6.5 in soybean. The activity of phosphoenolpyruvate carboxylase (PEPCase) is generally decreased by PHE and always lower in soybean than in wheat and carrot. PEPCase peaks its maximum activity at pH 7.0 and wheat shows the highest value with high PHE (1.2mgL−1) at pHs of 7.0 and 8.0 in comparison with carrot and soybean. Vacuolar H+-pyrophosphatase (V-H+-PPase) is promoted by PHE in wheat and carrot, while it does not change significantly in soybean (P>0.05). V-H+-PPase reaches its maximum value at pH 7.5 and follows the order of wheat>carrot>soybean at alkaline medium with high PHE. Plasma membrane (PM) H+-ATPase is enhanced by K+ and follows the order: wheat>soybean>carrot. The orders of activity of these enzymes are not consistent with that of PHE uptake, indicating their various functions. However, in soybean, PM H+-ATPase activity under high PHE and the uptake of PHE are always higher than those in wheat and carrot, suggesting that PM H+-ATPase plays an essential role in PHE uptake. Therefore, the changes of PM H+-ATPase activity seem to be a direct consequence responding to PHE stress and those of NRase, PEPCase and V-H+-PPase might be the downstream accidents regulated by cytosoilc pH. Our study provides insight into the roles of these enzymes in non-photosynthetic tissues and supports their importance in the physiological responses to PHE stress among different crops.

Aryl hydrocarbon receptor protein and Cyp1A1 gene induction by LPS and phenanthrene in Atlantic cod (Gadus morhua) head kidney cells

The objective of this study was to evaluate interactions between environmental toxicants and cod immune cells during inflammation. Phenanthrene is abundant in plant oils (rapeseed, palm, and soya oil) as compared to fish oils, and consequently constitute an undesirable element in plant replacement diets in aquaculture. Phenanthrene was added to head kidney cell cultures, alone or together with LPS (lipopolysaccharide) or poly I: C (polyinosinic acid: polycytidylic acid), and the responses were evaluated in terms of protein and gene expression. The results showed that LPS, poly I: C or phenanthrene, added to the cultures separately, induced aryl hydrocarbon receptor (AhR) protein expression. Phenanthrene treatment in combination with LPS induced AhR protein expression and Cyp1A1 gene transcription, which not was observed combining poly I: C and phenanthrene. Phenanthrene exposure up regulated the transcription of common stress and detoxification enzymes like catalase, caspase 3 and glutathione S-transferase alfa 3 subunit B (GSTAB3), while LPS exposure alone or combined with phenanthrene down regulated GSTAB3 and catalase in cod leukocytes. It seems clear that immune regulation and phenanthrene induced signaling pathways interact; transcriptional down regulation of detoxification and antioxidant enzymes by LPS could indicate that combating bacterial infections is the number one priority in these cells, and that AhR and Cyp1A1 is somehow involved in this signaling cascade. LPS seems to affect the mitogen activated protein kinases (MAPKs) pathways (P-p38 and ERK1/2) thus modulating the AhR protein and Cyp1A1 gene transcription, while phenanthrene possibly activates AhR by ligand binding.

Oxidative stress and DNA damage responses to phenanthrene exposure in the estuarine guppy Poecilia vivipara

Despite ubiquitous phenanthrene contamination in aquatic coastal areas, little is known regarding its potential effects on estuarine fishes. The present work evaluated the response of a large suite of oxidative stress- and DNA damage-related biomarkers to phenanthrene exposure (10, 20 and 200 μg L−1, 96 h) using DMSO as the solvent in estuarine guppy Poecilia vivipara (salinity 24 psu). Phenanthrene affected oxidative stress-related parameters, and decreased antioxidant defenses and reactive oxygen species in the gills and muscle overall. Lipid peroxidation occurred in muscle at 200 μg L−1 phenanthrene. Genotoxicity was increased at 20 μg L−1, while 200 μg L−1 caused a relative decrease in erythrocyte release into the bloodstream. These findings indicated that phenanthrene is genotoxic and can induce oxidative stress, depending on tissue and phenanthrene concentration analyzed. Thus, some of the biomarkers analyzed in the present study are sufficiently sensitive to monitor the exposure of the guppy P. vivipara to phenanthrene in salt water. However, further studies are required for a better interpretation of the dose–response patterns observed.

The impact of biochar on the bioaccessibility of (14)C-phenanthrene in aged soil.

Biochar is a carbon rich product from the incomplete combustion of biomass and it has been shown to reduce bioavailability of organic contaminants through adsorption. This study investigated the influence of 0%, 1%, 5% and 10% of two different particle sized wood biochars (≤2 mm and 3-7 mm) on the bioaccessibility of (14)C-phenanthrene (10 mg kg(-1)) in aged soil. The extent of (14)C-phenanthrene mineralisation by phenanthrene-degrading Pseudomonas sp. inoculum was monitored over a 14 day period in respirometric assays and compared to hydroxypropyl-β-cyclodextrin (HPCD) aqueous extraction. Notably, biochar amendments showed significant reduction in extents of mineralisation and HPCD extraction. Linear correlations between HPCD extractability and the total amount mineralised revealed good correlations, with 2 mm biochar showing a best fit (r(2) = 0.97, slope = 1.11, intercept = 1.72). Biochar reduced HPCD extractability and bioaccessibility of (14)C-phenanthrene to microorganisms in a similar manner. Biochar can aid risk reduction to phenanthrene exposure to biota in soil and HPCD can serve as a useful tool to assess the extent of exposure in biochar-amended soils.

Phenanthrene exposure produces cardiac defects during embryo development of zebrafish (Danio rerio) through activation of MMP-9

Phenanthrene (Phe) is one of the most abundant polycyclic aromatic hydrocarbons in the aquatic environment as a result of human activities. It is widely accepted that Phe has cardiotoxic effects. Even so, knowledge concerning the mechanism(s) of cardiac development toxicity is still limited. In this study, we exposed zebrafish embryos to environmentally relevant concentrations of Phe and then investigated its cardiotoxic effects and the mechanism(s) involved. Some cardiac morphogenetic defects, which was characterized by an abnormally looped and enlarged heart, dilated and thinner ventricular wall, and increased interstitial fibrosis, were observed in the Phe treated groups. The mRNA and protein expression levels of matrix metalloproteinase-9 (MMP-9), as well as the MMP-9 activity, were induced. Moreover, during co-treatment of the zebrafish embryos with MMP-9 inhibitor, the cardiac defects caused by Phe were attenuated. In addition, Phe exposure led to an up-regulation of transforming growth factor β (TGF-β), which plays a crucial role in mediating cardiac fibrosis. Taken together, our data indicated that the exposure to Phe of zebrafish embryos disrupted normal cardiac development, and that the cardiac defects induced by Phe were mediated by the MMP-9, while TGF-β was also involved in these cardiac defects.

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 (2.5mgkg−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.

Transcriptional responses indicate attenuated oxidative stress in the springtail Folsomia candida exposed to mixtures of cadmium and phenanthrene

Since the 'omics revolution', the assessment of toxic chemical mixtures has incorporated approaches where phenotypic endpoints are connected to a mechanistic understanding of toxicity. In this study we determined the effect of binary mixtures of cadmium and phenanthrene on the reproduction of Folsomia candida and investigated the cellular mechanisms underlying this response. Mixture toxicity modeling showed an antagonistic deviation from concentration addition for reproduction effects of the mixtures. Subsequent transcriptional response analysis was done using five mixtures at the modeled 50% effect level for reproduction. The transcription profiles of 86 high throughput RT-qPCR assays were studied by means of partial least squares regression analysis. The first and second principal components (PCs) were correlated with global responses to cadmium and phenanthrene, while correlations with the mixture treatments were found in the higher PCs. Specifically associated with the mixture treatments were a biotransformation phase II gene, four mitochondrial related genes and a gene involved in the biosynthesis of antioxidant selenoproteins. Membrane integrity related gene inductions were correlated with the single phenanthrene treatment but not with the mixtures. Immune and inflammatory response assays did not correlate with any of the mixtures. These results suggest moderated oxidative stress, a higher mitochondrial maintenance and less compromised membrane function in the mixture exposed samples compared to the separate cadmium or phenanthrene exposures. The antagonism found for inhibition of reproduction may partially originate from these differences. Mechanistic studies on mixture toxicity can ultimately aid risk assessment by defining relevant toxicity pathways in organisms exposed to real-world mixture exposures present in the field.

Reduction in the earthworm metabolomic response after phenanthrene exposure in soils with high soil organic carbon content

We evaluated the correlation between soil organic carbon (OC) content and metabolic responses of Eisenia fetida earthworms after exposure to phenanthrene (58 ± 3 mg/kg) spiked into seven artificial soils with OC contents ranging from 1 to 27% OC. Principal component analysis of 1H nuclear magnetic resonance (NMR) spectra of aqueous extracts identified statistically significant differences in the metabolic profiles of control and phenanthrene-exposed E. fetida in the 1% OC soil only. Partial least squares analysis identified a metabolic response in the four soils with OC values ≤11% which was well correlated to estimated phenanthrene porewater concentrations. The results suggest that the higher sorption capability of high OC soils decreased the bioavailability of phenanthrene and the subsequent metabolic response of E. fetida.

1H NMR-based metabolomic analysis of polar and non-polar earthworm metabolites after sub-lethal exposure to phenanthrene

1H nuclear magnetic resonance (1H NMR)-based metabolomics was utilized to elucidate the earthworm sub-lethal toxicity after exposure to the persistent environmental contaminant phenanthrene. Earthworms were exposed to 0.05, 0.2 and 0.4 mg/cm2 of phenanthrene [which correspond to 1/32nd to 1/4th of the 48-h LC50 (concentration that causes 50 % mortality), respectively] via contact tests over 1, 2 and 3 days of dermal contact. 1H NMR-based metabolomic analysis of the polar and non-polar fractions of the earthworm tissue extracts revealed heightened Eisenia fetida toxic responses with both longer exposure times and higher phenanthrene concentrations. Principal component analysis (PCA) of the polar fraction showed significant separation between control and exposed earthworms along PC1 for all phenanthrene concentrations on each day. The PCA of the non-polar fraction showed significant separation between the controls and exposed earthworms for only the first day of exposure. These results suggested that alanine, glutamate, maltose, and fatty acids were potential indicators of phenanthrene exposure. Interruption in energy production due to a deactivation of the succinate dehydrogenase enzyme in the Krebs cycle was also postulated in exposed earthworms. Cross-validated partial least squares-regression models showed that the polar metabolic profile of E. fetida was weakly but significantly correlated to phenanthrene exposure concentrations after day 1 and day 2 of exposure. Overall, this study indicates that with longer exposures, contact time becomes more important than concentration in discriminating between control and exposed earthworms. This study also shows that NMR-based metabolomics has promise as a powerful ecotoxicological tool for elucidating the mode of toxicity of contaminants.

Brassinosteroid improves seed germination and early development of tomato seedling under phenanthrene stress

Polycyclic aromatic hydrocarbons (PAHs) are one of the toxic persistent organic pollutants, have global environmental concern. Seed germination and early seedling development are the initial handicaps for plant establishment in phytoremediation program. Assisted phytoremediation by plant growth regulators may be a potential solution for these problems. Hence, we investigated the effects of seed treatment with various concentrations (0.01, 1.0, 100 nM) of 24-epibrassinolide (EBR) in tomato under graded levels (30, 100, 300 μM) of a three-ring PAH namely phenanthrene (PHE). Delayed and decreased seed germination, reduced length and fresh weight (FW) of shoot and root were observed following 10 days of PHE exposure in a dose dependent manner. Chlorophyll fluorescence study suggested a possible photoinhibition and damage to photosynthetic apparatus under PHE exposure. However, seed treatment with EBR improved seed germination and increased length and FW of shoot and root. In addition, EBR remarkably restored the studied chlorophyll fluorescence parameters towards control levels. Different responses in antioxidant enzymes were observed following PHE exposure, while malondialdehyde (MDA) content was increased in a concentration dependent manner. EBR treatment prior to PHE exposure remarkably increased the activities of antioxidant enzymes over PHE alone, but decreased the MDA contents both in shoot and root of young tomato seedlings. Considering all the studied parameters, seed treatment with 1.0 nM EBR was most effective followed by 100 and 0.01 nM for the improvement of germination and seedling growth under PHE stress in tomato.

Phenanthrene affects cardiovascular function in the developing African clawed frog (Xenopus laevis)

The incomplete combustion of fossil fuels results in deposition of the tricyclic aromatic hydrocarbon phenanthrene into air, soil, and water. This common and persistent contaminant is also present in tobacco smoke, coal tar sealant, and weathered crude oil. Surprisingly, little is known about its toxicity. To gain a better understanding of potential risks to humans, we characterized the effects of phenanthrene exposure using embryos of X. laevis, an important model for vertebrate development. Heart rates and evidence of arrhythmia were recorded during various stages of heart development using an inverted dissecting microscope, Leica™ camera, and the video recording and editing software Camtasia.™ In stage 42 embryos, phenanthrene exposure (5 ppm, 28.5 μM) led to bradycardia with a decrease to 79% of control values at 6 hrs followed by a further reduction to 69% by 10 hrs. After 5 hrs of depuration, heart rates returned to control levels. Arrhythmia was also evident in phenanthrene‐exposed embryos. A 42% increase in interbeat variability was observed following a 10‐hr exposure to 5 ppm. These phenanthrene‐induced effects may be stage‐specific as cardiac function in stage 47 animals, with a mature morphology, and in stage 37 animals was less affected. These findings underscore the need for further characterization of the cardiotoxic effects of this common pollutant.(Supported by NH‐INBRE grant 1P20RR030360‐01)

The growth, photosynthesis and antioxidant defense responses of five vegetable crops to phenanthrene stress

Polycyclic aromatic hydrocarbons (PAHs) are global environmental problem. To better understand the growth and physiological responses to atmospheric PAHs, we investigated biomass, photosynthetic machinery and antioxidant system in pakchoi, cucumber, flowering chinese cabbage, tomato and lettuce under various levels of phenanthrene (PHE) stress. Foliar exposure to PHE for 14d resulted in a dose dependent decrease in growth, photosynthesis and chlorophyll contents. With few exceptions, antioxidant enzymes (superoxide dismutase, guaicol peroxidase, catalase, ascorbate peroxidase and glutathione reductase) were upregulated following exposure to PHE. Dose dependent increase in malondialdehyde contents together with H2O2 accumulation suggested an occurrence of oxidative stress following PHE exposure. However, to some extent, growth and antioxidant defense responses differ from species to species. Difference in defense capacity might result in different tolerance and phytotoxicity among the studied vegetables. Taken together, phytotoxicity of PHE to five vegetables could be sequenced in the following order: pakchoi>cucumber>lettuce>tomato>flowering chinese cabbage.

Brassinosteroid alleviates phenanthrene and pyrene phytotoxicity by increasing detoxification activity and photosynthesis in tomato

The present study was carried out to investigate the effects of exogenously applied 24-epibrassinolide (BR) on growth, gas exchange, chlorophyll fluorescence characteristics, lipid peroxidation and antioxidant systems of tomato seedlings grown under different levels (0, 10, 30, 100 and 300 μM) of phenanthrene (PHE) and pyrene (PYR) in hydroponics. A concentration-dependent decrease in growth, photosynthetic pigment contents, net photosynthetic rate (Pn), stomatal conductance (Gs), maximal quantum yield of PSII (Fv/Fm), effective quantum yield of PSII ( Φ PSII), photochemical quenching coefficient (qP) has been observed following PHE and PYR exposure. By contrast, non-photochemical quenching coefficient (NPQ) was increased. PHE was found to induce higher stress than PYR. However, foliar or root application of BR (50 nM and 5 nM, respectively) alleviated all those depressions with a sharp improvement in the activity of photosynthetic machinery. The activities of guaicol peroxidase (GPOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) as well as content of malondialdehyde (MDA) were increased in a dose-dependent manner under PHE or PYR treatments. Compared with control the highest increments of GPOD, CAT, APX, GR and MDA by PHE/PYR alone treatments were observed following 300 μM concentration, which were 67%, 87%, 53%, 95% and 74% by PHE and 42%, 53%, 30%, 86% and 62% by PYR, respectively. In addition, both reduced glutathione (GSH) and oxidized glutathione (GSSG) were induced by PHE or PYR. Interestingly, BR application in either form further increased enzymatic and non enzymatic antioxidants in tomato roots treated with PHE or PYR. Our results suggest that BR has an anti-stress effect on tomato seedlings contaminated with PHE or PYR and this effect is mainly attributed by increased detoxification activity.

Chronic Exposure to Phenanthrene Influences the Spermatogenesis of Male Sebastiscus marmoratus: U-Shaped Effects and the Reason for Them

Phenanthrene (PHE) is one of the most abundant polycyclic aromatic hydrocarbons in the aquatic environment. This study was conducted to investigate the effects of PHE at environmentally relevant concentrations on testicular development in male Sebastiscus marmoratus. After 50 days exposure, the gonadosomatic indices and percentage of sperm produced showed a U-shaped dose response. The levels of salmon-type gonadotropin releasing hormone, follicle-stimulating hormone, luteinizing hormone mRNA, 17β-estradiol, and γ-glutamyl transpeptidase activity all showed a U-shaped dose response, which clearly demonstrated the U-shaped effects of PHE exposure on spermatogenesis and also elucidated the action pathway. This result would bring a difficulty and a challenge to any risk assessment of PHE exposure to the reproductive health of fishes. Thus far, there has been no ready explanation for a U-shaped dose-response curve, which is well recognized as a hormetic phenomenon for many hormones, drugs, and toxic compounds. In the present study, PHE accumulation in the brain showed an inverse U-shaped increase compared to the control. Glutathione S-transferase activity in the brain showed a U-shaped dose-response, which was related with the PHE accumulation. These results have given a reasonable explanation for the U-shaped dose-response via alteration of biotransformation enzyme activity in the brain.

Atmospheric phenanthrene pollution modulates carbon allocation in red clover (Trifolium pratense L.)

The influence of atmospheric phenanthrene (PHE) exposure (160μgm−3) during one month on carbon allocation in clover was investigated by integrative (plant growth analysis) and instantaneous 13CO2 pulse-labelling approaches. PHE exposure diminished plant growth parameters (relative growth rate and net assimilation rate) and disturbed photosynthesis (carbon assimilation rate and chlorophyll content), leading to a 25% decrease in clover biomass. The root-shoot ratio was significantly enhanced (from 0.32 to 0.44). Photosynthates were identically allocated to leaves while less allocated to stems and roots. PHE exposure had a significant overall effect on the 13C partitioning among clover organs as more carbon was retained in leaves at the expense of roots and stems. The findings indicate that PHE decreases root exudation or transfer to symbionts and in leaves, retains carbon in a non-structural form diverting photosynthates away from growth and respiration (emergence of an additional C loss process).

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.

1H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure

Environmental contextPhenanthrene is a persistent soil contaminant, whose toxic mode of action in earthworms has not been fully examined. We adopt a metabolomics approach, using 1H nuclear magnetic resonance (NMR) spectroscopy, to measure the response of earthworms to sub-lethal phenanthrene exposure. The results indicate that NMR-based metabolomics may be used to monitor responses to sub-lethal levels of contaminants and to delineate their toxic mode of action. Abstract1H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida to sub-lethal phenanthrene exposure. E. fetida were exposed via contact tests to six sub-lethal (below the measured LC50 of 1.6 mg cm–2) concentrations of phenanthrene (0.8–0.025 mg cm–2) for 48 h. Multivariate statistical analysis of the 1H NMR spectra of earthworm tissue extracts revealed a two-phased mode of action (MOA). At exposures below 1/16th of the LC50, the MOA was characterised by a linear correlation between the metabolic response and exposure concentration. At exposures ≥1/16th of the LC50, the metabolic response to phenanthrene appeared to plateau, indicating a distinct change in the MOA. Further data analysis suggested that alanine, lysine, arginine, isoleucine, maltose, ATP and betaine may be potential indicators for sub-lethal phenanthrene exposure. Metabolite variation was also found to be proportional to the exposure concentration suggesting that NMR-based earthworm metabolomics is capable of elucidating concentration-dependent relationships in addition to elucidating the MOA of sub-lethal contaminant-exposure.

Atmospheric phenanthrene transfer and effects on two grassland species and their root symbionts: A microcosm study

The objective of this work was to determine the transfer of phenanthrene (PHE) from air to grassland plants and soil compartments and its effects on the plant growth and symbiotic root microorganisms (arbuscular mycorrhizal fungi and Rhizobium nodules). The experimental procedure exposed Trifolium pratense L. or Lolium perenne L. to atmospheric PHE pollution (150μgm−3) over the course of one month. PHE was transferred from the air to the leaves and to the soil surfaces. In leaves, PHE was mostly absorbed in the inner leaf tissues, representing 92% and 73% of the total PHE amount quantified in leaves, respectively for clover and ryegrass. In soils, most of PHE contamination was recovered in the top layer (0–1cm) and did not readily diffuse into the deep layer (1–10cm). The highest PHE concentration recovered in deep roots (1.8 and 4.5μgg−1 dry weight (DW), respectively for clover and ryegrass) related to the lowest PHE concentration recovered in its associated soil suggested a PHE translocation from shoots to roots within the two plant species. The large PHE amount quantified in clover shoots (124μgg−1 DW) induced a significant diminution by 30% of the shoot biomass whereas root biomass remained stable. Efficient mycorrhizal symbiosis was maintained during exposure whereas the Rhizobium nodule symbiosis was altered in the surface of soil. By contrast, neither biomass accumulation nor symbiotic association was affected in ryegrass, probably due to a lower sensitivity of this species to PHE exposure. Perspectives of carbon allocation and nitrogen nutrition perturbations are suggested in clovers.