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Methyl bromide (MB) has been used in a wide range of applications, but since it was determined to be an ozone-depleting compound, it has only been used for pre-shipment and quarantine purposes in trade. Phosphine (PH3) is currently the ideal fumigant as an MB alternative worldwide. However, the development of PH3 resistance in the target insect pest and longer PH3 fumigation treatment times raise questions about the continued use of PH3. This study attempted to shorten treatment time via combination treatment with ethyl formate (EF). Planococcus citri was used as the main quarantine pest in Korea, and the acute toxicity of EF, PH3, and EF + PH3 was determined at every developmental stage. EF treatment at 4 h showed LCT99 values of 45.85~65.43 mg∙h/L, and PH3 treatment at 20 h showed that of 0.13~0.83 mg∙h/L depending on the developmental stage. The efficacy of PH3 decreased after reducing the treatment time, but synergistic effects were observed at all stages of development of P. citri when both fumigants were used simultaneously for 4 h. After combined treatment, dihydrolipoyl dehydrogenase expression and the production of two phospholipids, PI(O-16:0) and PC(18:2), were significantly reduced in treated P. citri adults compared with the control. Therefore, combined treatments might be key to reducing the treatment time and resistance of PH3 in the field.

Ethiopian honey wine, Tej, is spontaneously fermented traditional alcoholic beverage, usually made from honey and “gesho” (Rhamnus prinoides). Till now, limited amount of information is available on the characterization of Tej. Thus, the aim of this paper is to reveal the microbiological diversity and physicochemical properties of Tej samples collected from different areas of Ethiopia. High-throughput sequencing, electrochemical and chromatographic techniques, and spectrophotometric methods were used to achieve these objectives. Although there was a statistical difference in the exact values of physicochemical properties between the collected Tej samples, the pH and titratable acidity values of the samples ranged from 2.8 to 3.8 and from 1.81 to 8.65 g/L, respectively. Similarly, the alcohol and sugar contents of the samples were in the range of 6.36 to 11.34 g/100 mL and from 0.37 to 31.6 g/L, respectively. Moreover, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) values of the samples were in the range of 37.9 to 81.0% and 27.4 to 73.1%, respectively. Furthermore, microbial community structure was predominated by a few fermentative microorganisms. Specifically, the bacterial community structure was dominated by the genera of Lactobacillus (53.15%) and Zymomonas (38.41%). Whereas, the fungal community structure was exclusively dominated by genus of Saccharomyces (99.66%). Additionally, Lactobacillus, Zymomonas and Saccharomyces were the detected core microbiome for the collected Tej samples. Both bacterial and fungal communities had shown no statistically significant differences in alpha diversity analysis based on the area of sample collection. However, the bacterial communities had a statically significant difference in Unweighted Unifrac beta diversity analysis. Generally, the observed shared physicochemical characteristic features and the dominance by certain group of microorganisms might be seen as a boon for the development of direct fermentation system to this traditional alcoholic beverage.

The increase in worldwide trade has caused the quality maintenance of commercialized agriproducts to be crucial in keeping its economic value. In recent years, methyl bromide (MB) has been used dominantly during quarantine and pre-shipment, despite it being an environmental hazard with global repercussions. Through this study, it was shown that Arabidopsis thaliana’s 2 h exposure to the MB treatment displayed no signs of phytotoxicity, whereas its 4 h exposure significantly interfered with growth. The transcriptomic analysis found the molecular modifications in A. thaliana after the MB fumigation with the up-regulation of genes specifically relative to the abiotic and oxidative stress, and the down-regulation of auxin transporter genes. Some important gene expressions were verified by RT-qPCR and their expression patterns were similar. Oxidative stresses via the reactive oxygen species (ROS) in relation to MB phytotoxicity were confirmed with the increased malondialdehyde in MB-4h-treated A. thaliana. Uneven distribution of auxins via lower expression of auxin transporter genes was also determined using UPLC-ESI-QqQ MS. Application of two ROS scavengers such as N-acetyl-cysteine and L -glutathione minimized MB phytotoxic effect in A. thaliana. Therefore, MB caused severe oxidative stress, and alternatives regarding the use of MB should be considered.

Melanogenesis represents a series of processes that produce melanin, a protective skin pigment (against ultraviolet rays), and determines human skin color. Chemicals reducing melanin production have always been in demand in the cosmetic market because of skincare interests, such as whitening. The main mechanism for inhibiting melanin production is the inhibition of tyrosinase (TYR), a key enzyme for melanogenesis. Here, we evaluated gedunin (Ged), a representative limonoid, for its anti-melanogenesis action. Melanin production in vitro was stimulated by alpha-melanocyte stimulating hormone (α-MSH) in B16F10 mouse melanoma cells. Ged reduced α-MSH-stimulated melanin production, inhibiting TYR activity and protein amount. We confirmed this result in vivo in a zebrafish model for melanogenesis. There was no sign of toxicity and malformation of zebrafish embryos during development in all treated concentrations. Ged reduced the number of produced zebrafish embryo pigment dots and melanin contents of embryos. The highly active concentration of Ged (100 µM) was much lower than the positive control, kojic acid (8 mM). Hence, Ged could be a fascinating candidate for anti-melanogenesis reagents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Piperaceae plants consist of about 3600 species, of which about 2000 are Piper plants. Their habitat is distributed across pantropical regions. The representative plant is Piper nigrum, known as black pepper. These plants have been widely used in folk medicine in Korean traditional medicine. This review collected papers identifying and separating the amides obtained from these Piper plants, with a focus on Piper amides potential to control the production and growth of fungal strains that cause plant disease and their insecticidal properties against agricultural pests. Piper amide benefits include antiaflatoxigenic activities, antiparasitic activities, anticancer properties, antiplatelet activities, and anti-inflammatory activities, among other therapeutic properties for the treatment of human diseases. In addition, this review paper provides a total synthesis study on the mass production of Piper amides and their derivatives, with a formulation study for industrial use. This review paper is designed to help inform future studies on Piper amide applications.

In this study, the antimelanogenic activity of piperlongumine in murine B16F10 melanoma cells and zebrafish was investigated, and its mode of antimelanogenic action was elucidated using quantitative reverse transcription-polymerase chain reaction. A melanocyte-stimulating hormone (α-MSH, 200 nM) was used to induce melanin production in B16F10 melanoma cells, and kojic acid (200 μM) was used as a positive control. Piperlongumine had no inhibitory effects on cell growth at the treated concentrations (3 and 6 μM), and it significantly reduced total melanin production. Piperlongumine decreased the expression of Mitf, Tyr, Trp-1, and Trp-2 and tyrosinase activity was also dramatically reduced by the piper amide addition under α-MSH treatment. With these findings, zebrafish embryos were used to confirm antimelanogenic activity of piperlongumine, and it showed the potent antimelanogenic activity at the concentration of 1 μM. Altogether, piperlongumine has potent antimelanogenic activity, and these results support it as a candidate for natural depigmentation agent in a cosmetic and pharmaceutical industries.

Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The purpose of this study was to investigate the prevalence of Staphylococcus aureus (S. aureus) and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems in Korea. Among 200 chicken meat samples, 94 were observed to be positive for S. aureus. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among the operations. Four methicillin-resistant S. aureus isolates (MRSA) were recovered from 3 different operations. A high proportion of the S. aureus isolates were resistant to penicillins (51.2%), tetracycline (38.8%), and ciprofloxacin (CIP; 33.9%). Especially, 3 different operations showed a high number of CIP resistance (45.5∼100%) and multidrug resistance (50.0∼100%). Among 41 CIP-resistant S. aureus isolates, 75.6% showed a double amino-acid exchange of both gyrA and parC, with CIP minimum inhibitory concentrations (MIC) of ≥32 μg/mL. Four MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, exhibiting oxacillin, CIP, and enrofloxacin MIC ranges of 16 to 128, 32 to 64, and 8 to 128 μg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Our findings suggest that S. aureus with resistance to important antimicrobial compounds can now be found in association with integrated broiler operations, providing the data to support the development of a monitoring and prevention program in integrated operations.

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 μg/L and 1459 μg/L, respectively. At the concentration of 2000 μg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC50 value of 354 μg/L. At the concentration of 1000 μg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks.

Plant growth-promoting rhizobacteria (PGPR) affect plant growth through various mechanisms, such as indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and biofilm formation. The aim of the study reported here was to isolate and characterize rhizobacteria that produce quorum-sensing signal molecules and other PGPR-related molecules. A biofilm-forming bacterium, GS2, was isolated from the rhizosphere of a sesame plant and subsequently found to produce two quorum-sensing signal molecules that were identified as N-hexanoyl-L-homoserine lactone (m/z 200) and N-octanoyl-L-homoserine lactone (m/z 228) by liquid chromatography–tandem mass spectrometry analysis. The strain was also found to produce IAA (17.2 μg mL−1), gibberellins (113.7 μg mL−1), and ACC deaminase (9.7 μM α-ketobutyrate mg−1 protein h−1). The strain was identified as Serratia glossinae based on a comparison of 16S rRNA gene sequences. Inoculation of the strain promoted growth of a gibberellin-deficient rice dwarf mutant (Waito-C). Different growth attributes, including shoot and root elongation, chlorophyll content, and plant weight could be attributed to the PGPR characteristics of strain GS2. These results suggest that S. glossinae strain GS2 can serve as a microbial agent that improves plant growth.

α-Endosulfan and some polycyclic aromatic compounds (PAHs) are persistent in the environment and can reach crop products via contaminated agricultural soils. They may even be present as mixtures in the soil and induce mixture toxicity in soil organisms such as earthworms. In this study, the combined toxicities of PAHs with α-endosulfan were determined in Eisenia fetida adults using an artificial soil system. α-Endosulfan and five PAHs were tested for their acute toxicity toward E. fetida in artificial soils. Only α-endosulfan, fluorene, and phenanthrene showed acute toxicities, with LC50 values of 9.7, 133.2, and 86.2 mg kg−1, respectively. A mixture toxicity assay was conducted using α-endosulfan at LC10 and fluorene or phenanthrene at LC50 in the artificial soils. Upon exposure to the mixture of fluorene and α-endosulfan, earthworms were killed in increasing numbers owing to their synergistic effects, while no other mixture showed any additional toxicity toward the earthworms. Along with the acute toxicity results, the biochemical and molecular changes in the fluorene- and phenanthrene-treated earthworms with or without α-endosulfan treatment demonstrated that enhancement of glutathione S-transferase activity was dependent on the addition of PAH chemicals, and the HSP70 gene expression increased with the addition of α-endosulfan. Taken together, these findings contribute toward understanding the adverse effects of pollutants when present separately or in combination with other types of chemicals.

Azoxystrobin (AZ) and pyraclostrobin (PY) are strobilurin fungicides that inhibit fungal mitochondrial respiration. In this study, a representative model, zebrafish (Danio rerio), was used as a test species for acute and developmental toxicity. Survival and malformation rates were observed only PY-treated embryos, with an LC50 value of 77.75 ppb accompanied by a dramatic decrease in hatching rate, while AZ did not show great mortality. Morphological changes were observed in PY-treated embryos with the occurrence of pericadial edema at 25 ppb. A delay in growth was observed after treatment with pyraclostrobin at 50 ppb. Use of genetically engineered Tg(cmlc:EGFP) allowed fluorescence observation during heart development. PY interfered with normal heart development via upregulation of the nppa gene responsible for the expression of natriuretic peptides. Heart function was dramatically reduced as indicated by reduced heart rates. Increased expression of the nppa gene was also seen in AZ-treated embryos. The expression level of cyp24a1 was also up-regulated, while ugt1a1 and sult1st6 were down-regulated after treatment of zebrafish embryos with AZ or PY. Overall, strobilurin fungicides might inhibit normal heart formation and function within the range of concentrations tested.

In this study, fluorene (FL), FL-1-carboxylic acid (FC-1), and FL-9-carboxylic acid (FC-9) were investigated to understand their acute toxicity by measuring inhibitory effects on hatching rates and developmental processes of zebrafish embryos (Danio rerio). For exposure concentrations up to 3000 μg/L, FC-1 alone showed acute toxicity at 1458 μg/L for LC50 value. FC-1 caused yolk sac and spinal deformities, and pericardial edema. Molecular studies were undertaken to understand FC-1 toxicity examining 61 genes after exposure to 5 μM (equivalent to LC20 value of FC-1) in embryos. In the FC-1-treated embryos, the expression of the cyp7a1 gene, involved in bile acid biosynthesis, was dramatically decreased, while the expression of the Il-1β gene involved in inflammation was remarkably increased. In addition to these findings, in FC-1-treated embryos, the expression of nppa gene related to the differentiation of the myocardium was 3-fold increased. On the other hand, cyp1a, cyp3a, ugt1a1, abcc4, mdr1, and sult1st1 responsible for detoxification of xenobiotics were upregulated in FC-9-treated embryos. Taken together, carboxylation on carbon 1 of FL increased acute toxicity in zebrafish embryos, and its toxicity might be related to morphological changes with modification of normal biological functions and lowered defense ability.

This study was performed to understand how chlorpyrifos (CHL) affects zebrafish (Danio rerio) embryos and adults, by exposing this model organism to various concentrations of the insecticide. The 96-h acute toxicity test to determine the effect of CHL on adult zebrafish yielded a LC50 of 709.43μg/L(-1). Small molecular weight proteins less than 25kDa and phospholipids were analyzed with MALDI-TOF MS/MS in order to compare expression patterns, revealing that some peaks were dramatically altered after CHL treatment. Whereas no acute toxicity was detected in the embryo toxicity test, malformation of zebrafish larvae was observed, with many individuals harboring curved spines. In an angiogenesis test on larvae of transgenic zebrafish, CHL did not have an inhibitory effect. Relative gene expression analyses using real-time polymerase chain reaction (RT-PCR) of DNA from zebrafish embryos revealed that different subtypes of cytochrome P450 (CYP450), such as CYP1A and CYP3A, were significantly up-regulated in response to CHL at a concentration of 400μg/L(-1) compared to the control. The expression level of NR1I2, a CYP gene transcriptional regulator, UGT1a1, and MDR1 were all up-regulated in the CHL-treated embryos. Finally, the expression level of acetylcholinesterase (AChE) and catalase (CAT) decreased, whereas that of superoxide dismutase (SOD) did not differ significantly. Our results suggest that the up-regulation of metabolic enzymes including CYP450 and MDR1 may be involved in CHL resistance in zebrafish.

Chlorpyrifos (CHL) is an organophosphate compound that is widely used as an insecticide. Due to its repeated use and high environmental residual property, CHL is frequently passed into aquatic environments by runoff. Consequently, there may be an adverse effect on aquatic vertebrate animals, including fish. Therefore, in this study, we assessed how CHL affected Japanese medaka (Oryzias latipes). The acute toxicity of CHL in adult fish after 96 h of exposure was determined to be 212.50, 266.79, and 412.28 μg L(-1) (LC25, LC50, and LC95, respectively). Acetylcholinesterase (AChE), glutathione S-transferase (GST), and carboxylesterase (CE) activities were obtained from the livers of dead or surviving fish, and the results showed 4.8-fold lower, 4.5-fold higher, and 18.6-fold lower activities for the AChE, GST, and CE, respectively, for 64-h exposure at a concentration of 400 μg L(-1) of CHL. In the embryo toxicity test, curved spines were observed in embryos that were exposed to CHL for 48 h in a concentration-dependent manner. With identification of biomarkers for CHL in the fish, two protein peaks, 5550.86 and 5639.79 m/z, were found to be upregulated. These two proteins can be used as protein biomarkers for CHL contamination in aquatic systems. A phosphatidyl choline with an m/z ratio of 556.32 dramatically decreased after CHL exposure in the fish; thus, it may be considered as a lipid biomarker for CHL. It is assumed as the first report to identify a phospholipid biomarker using a lipidomics approach in fish toxicology. Taken together, these results demonstrated the adverse effects of CHL on Japanese medaka and reveal several candidate biomarkers that can be used as diagnostic tools for determining CHL.

The repeated use of pesticides, and their subsequent residues, has contributed to severe adverse effects on the environment, including risks to human health. Therefore, it is important to assess the quality of the environment to ensure it remains free from pesticide residues. The six pesticides tested in this study showed high mortality on Eisenia fetida with LC 50 values ranging from 7.7 to 37.9 g L −1 . The strongest lethal effect resulted from the organochlorine insecticide endosulfan (LC 50 = 7.7 g L −1 ). Following exposure to the carbamate pesticides, acetylcholinesterase activity in E. fetida decreased dramatically in comparison to the control. Carboxylesterase activity was only lowered in E. fetida exposed to propoxur, when compared to the control. The remaining five pesticides had no significant effect on carboxylesterase activity in E. fetida . In order to discover pesticide-specific biomarkers with differentially expressed proteins after exposure to pesticides, protein patterns of pesticide-treated E. fetida were analyzed using SELDI-TOF MS with Q10 ProteinChips. Protein patterns were compared with their intensities at the same mass-to-charge ratios ( m / z ). All 42 peaks had intensities with associated p -values less than 0.089, and 40 of these peaks had associated p -values of 0.05. Using SELDI-TOF MS technology, selective biomarkers for the six pesticides tested were found in E. fetida ; four proteins with 5425, 5697, 9523, and 9868 m / z were consistently observed in the earthworms following exposure to the carbamates.

Individual and combined toxicities of endosulfan (ENDO) with phenanthrene (PHE) were evaluated using zebrafish (Danio rerio) adults. The 96-h LC50 values for ENDO and PHE were 4.6 μg L−1 and 920 μg L−1, respectively. To evaluate the mixture toxicity, LC10 and LC50 concentrations were grouped into four combinations as ENDO-LC10 + PHE-LC10, ENDO-LC10 + PHE-LC50, ENDO-LC50 + PHE-LC10, and ENDO-LC50 + PHE-LC50, and their acute toxicities were determined. The combination of LC50-ENDO and LC10-PHE exhibited a synergistic effect. In addition, acetylcholinesterase activity decreased in zebrafish bodies exposed to ENDO with or without PHE. Combined treatments induced higher glutathione S-transferase activity compared to individual treatments. Carboxylesterase activity increased in both heads and bodies of ENDO-treated fishes compared with PHE-treated fishes. Using RT-qPCR technique, CYP1A gene expression significantly up-regulated in all combinations, whereas CYP3A was unchanged, suggesting that enzymes involved in defense may play different roles in the detoxification. CYP7A1 gene responsible for bile acid biosynthesis is dramatically down-regulated after exposure to the synergistic combination exposure, referring that the synergistic effect may be resulted from the reduction of bile production in zebrafishes. Among gender-related genes, CYP11A1 and CYP17A1 genes in female zebrafish decreased after treatment with ENDO alone and combination of LC50-ENDO and LC10-PHE. This might be related to a reduction in cortisol production. The overall results indicated that ENDO and PHE were toxic to zebrafish adults both individually and in combination, and that their co-presence induced changes in the expression of genes responsible for metabolic processes and defense mechanisms.

Diverse pesticidal activities of anthothecol derived from Khaya anthotheca (Meliaceae) and three limonoids (gedunin, limonin and obacunone) were determined using six phytopathogenic fungi and four insect pests. Anthothecol showed fungicidal activity to Puccinia recondita with control rates of 90, 76 and 53% at concentrations 0.5, 0.25 and 0.1 mg/mL, respectively, while the other limonoids exhibited little inhibitory effect on the fungal survival. The currently used fungicide mancozeb possessed potent fungicidal activity on P. recondite, with 92.3% controlling effect at a concentration of 0.005 mg/mL. Anthothecol exhibited larvicidal activity to Plutella xylostella with 80, 50 and 30% mortality at concentrations of 1.0, 0.5 and 0.25 mg/mL, respectively. Anthothecol showed selective pesticidal activity on P. recondita and P. xylostella. Structural re-examination of anthothecol was conducted and is reported.

The genus Serratia incorporates many agriculturally important species. Serratia fonticola strain GS2, isolated from a sesame rhizosphere, can produce N -acyl homoserine lactone quorum sensing signal molecules and indole-3-acetic acid (IAA). Here we report the complete genome sequence and phenotypic characteristics based on genomic information of this bacterium. The complete genome sequence of S. fonticola strain GS2 consists of a chromosome of 6.1 Mbp and two plasmids of 132 kbp and 94 kbp. The genome clusters for IAA and N -acyl homoserine lactone biosynthesis were identified in the genome. Subsequently, the Salkowski test and gas chromatography-mass spectrometry (GC–MS) analysis also revealed that the strain GS2 produces indolic compounds (27.1 μg mL −1 ) and IAA (6.7 μg mL −1 ) in its culture broth. HPLC analysis confirmed that the strain GS2 produced the quorum sensing signal molecules N -hexanoyl- L -homoserine lactone and N -octanoyl- L -homoserine lactone. This new information on the genome sequence and phenotype features will inform future ecological studies related to plant-microbe interactions.

Pesticidal activities of 4-[5-(2-cyclopropylaminopyrimidin-4-yl)-4-(4-chloro-2-fluoro-phenyl)thiazol-2-yl]-1-methylpiperidine, designated as Comp I, have been determined against a mosquito larva,Culex pipiens pallens, and a phytopathogenic fungus,Phytophthora capsici. Comp I was used as the leading compound in this study. The compounds were synthesized by reacting them with two functional groups, 3-thiophenyl and 2-thiophenyl groups, instead of 4-chloro-2-fluorophenyl group in Comp I. Other functional groups such as 2-aminothiazole, 2-(1-methylpiperazin-4-yl)thiazole, and 2-(piperazin-4-yl)thiazole were also introduced instead of 2-methylpiperidin-4-yl-thiazole of Comp I. Compounds designated as XIII-6~XV-7 were newly synthesized and their structures were confirmed by1H- and13C-NMR spectroscopy. Mosquito larvicidal activities of all the synthesized compounds againstC. pipiens pallenswere examined and Comp I among them showed the strongest larvicidal activity as 0.513 mM of LC50value. The fungicidal activities of all the synthesized compounds againstP. capsiciwere examined using the whole plant method. Among the XIII-6~XV-7 chemicals, 5-(2-cyclopropylaminopyrimidin-4-yl)-4-(thiophen-2-yl)thiazol-2-amine (VIII-6) showed the most potent antifungal activityin vivo. While the EC50value of the commercial fungicide dimethomorph was 4.26 μM, EC50of VIII-6 was 0.94 μM. Therefore, thiazole derivatives can be considered as viable candidates for the control of mosquito larvae and plant diseases.

Polycyclic aromatic hydrocarbon (PAH) compounds are persistent, carcinogenic, and mutagenic. When PAHs enter agricultural soils through sewage sludge, they pose an environmental risk to soil organisms, including earthworms. Therefore, we aimed to determine the toxic effects of PAHs on earthworms. Five PAHs were used: fluorene, anthracene, phenanthrene, fluoranthene, and pyrene. Only fluorene and phenanthrene exhibited toxicity (LC50 values 394.09 and 114.02 g L(-1), respectively) against the earthworm Eisenia fetida. None of the other PAHs tested in this study enhanced the mortality of adult earthworm until the concentrations reached to 1000 g L(-1). After exposure to PAHs, acetylcholinesterase (AChE) activity in E. fetida decreased in a concentration-dependent manner, and phenanthrene exhibited the strongest inhibitory effect on AChE, followed by fluorene. Activity of a representative detoxifying enzyme, carboxylesterase, was dramatically reduced in E. fetida exposed to all tested PAHs in comparison with that observed in the control test. The remaining glutathione S-transferase activity significantly decreased in E. fetida after exposure to PAHs. To profile small proteins