Vermifiltration as a stage in reuse of swine wastewater: Monitoring methodology on an experimental farm

Vermifiltration is a new technology using earthworms to process organically polluted water. A pilot associated with a swine facility (piggery) with 66 swine was developed to treat diluted manure, produce earthworms and vermicompost, and reduce air pollution. The aim of the experiment reported here was to devise an integrated method – biological, chemical and physical – for further research and development of vermifiltration in diluted swine manure, and provide some preliminary results. The earthworm population increased by 30% in 4 weeks, indicating the acclimation of the earthworms. A reduction in ammonia emission was observed of about 50% for the whole system. Higher water (+100%), carbon (+70%), and total nitrogen (+80%) gaseous losses were observed compared to conventional breeding on a slatted floor. This methodology can be used for further studies to develop vermifiltration for earthworm and vermicompost production from diluted animal manure, without pollution transfer.     

蚯蚓纤溶酶原激活剂的分离纯化及其性质

为了从赤子爱胜蚓中获得主要表现为纤溶酶原激活活性的蛋白酶,采用盐析、离子交换层析、凝胶过滤层析和疏水吸附层析从蚯蚓组织匀浆中纯化出6种具有纤溶活性的酶组分(F1、F2、F3、F4、F5和F6).它们均为单一多肽链;表观分子量分别为28500、29500、26100、26300、14850和32800;经非还原型SDSPAGE和扫描仪扫描,纯度分别为100%、95.2%、96.5%、93.6%、98%和97.8%;等电点(pI)均不高于3;SDSPAGE后,用Schiff试剂染色显示F5为糖蛋白;纯化的6种酶于20℃～50℃保温1h,酶活力基本不变;F1和F2、F3和F4、F5和F6分别在pH4～10、4～11、7～12范围内稳定;水解BAEE试验及纤溶活性抑制试验表明,F6既是丝氨酸蛋白酶,又是含金属离子的蛋白酶,其它5种酶为胰蛋白酶样的丝氨酸蛋白酶;免疫双扩散试验结果初步表明,F1和F5以及它们和其它4种组分之间无共同的抗原决定簇;用纤维蛋白平板法及以ChromozymU和ChromozymPL为底物测定,除F1外,其余5种酶的纤溶酶原激活活性明显强于其直接纤溶活性.     

An NMR-based metabonomic investigation of the toxic effects of 3-trifluoromethyl-aniline on the earthworm Eisenia veneta

¹H NMR spectra of earthworms Eisenia veneta treated with 3-trifluoromethyl-aniline in a 72-h contact filter paper test have been analysed using pattern recognition techniques to determine the biochemical response. Various strategies for data reduction of the metabolite profile, and illustration by principal components analysis are applied and discussed. The use of mean principal components plots in simplifying group data representation and highlighting the dose-response function is demonstrated. Hierarchical cluster analysis, and cluster significance analysis of the principal components were also used to examine the relative distribution of dose groups. Identification and assignment of metabolite responses to toxicity were found via correlation coefficient-shift plots. As measured by the correlation coefficients alanine was the most significant metabolite, but increased levels of other amino acids such as glycine and asparagine were also observed. Further, elevated levels of glucose, and the citric acid cycle intermediates citrate and succinate were noted as potential biomarkers of toxicity. This work provides a basis for examining the biochemical response of invertebrates to toxins. This should provide a framework to examine toxicity effects of other halogenated aromatic pollutants to earthworms used as environmental monitors.     

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.     

种植Bt水稻后的土壤对赤子爱胜蚓生长发育及酶活性的影响

【背景】转基因作物种植的安全问题一直备受关注。关于Bt蛋白对地下非靶标生物影响的研究是转基因作物安全评价的重要内容。【方法】在转Bt基因水稻收割后的稻田里分别种植豌豆、紫云英和油菜作为后茬作物。分别于2013年1、3和6月3次采集不同后茬作物田中的土壤作为材料,于室内饲养赤子爱胜蚓,4周和7周后,测定蚯蚓的生长发育指标、存活率以及体内酶活性的变化情况。此外,还测定了不同深度土壤中Bt蛋白的含量以及用Bt蛋白直接饲喂的赤子爱胜蚓的存活率。【结果】与种植过非转基因水稻MH63的土壤相比,分别种植过含cry2A和cry1C基因水稻后的土壤对赤子爱胜蚓的生长发育、存活率及体内酶活性无显著影响。1月份和3月份转cry2A基因水稻田以及1月份转cry1C基因水稻田采集的表层土样中的Bt蛋白含量显著高于地下10 cm和地下20 cm土壤中的含量,地下2层土样中的Cry2A蛋白含量之间无差异。3月份转cry1C基因水稻田以及6月份转cry2A和转cry1C基因水稻田的土壤中Bt蛋白的含量均不受土壤深度的影响。种植的后茬作物对土壤中的Bt蛋白无显著消解作用。室内模拟土壤最高Bt蛋白浓度的条件下,Cry2A蛋白处理的蚯蚓存活率为96.7%,Cry1C蛋白处理的蚯蚓存活率为95.0%,两者与对照相比无显著差异。【结论与意义】转cry2A和cry1C基因Bt水稻的种植对蚯蚓的生长发育和体内酶活性无显著影响。本研究为转基因水稻的安全评价提供了一定的依据。     

Local and chemical distribution of phlorotannins in brown algae

The local and chemical distribution of phlorotannins among the Japanese Laminariaceae, Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, was investigated. As a result of light microscopy observations with vanillin-HCl staining, phlorotannins were found to be accumulated within the vegetative cells of the outer cortical layer of the thalli, regardless of the species, stage of growth or organ. Crude phlorotannins comprised about 3.0% of the algal powder for each of the algae. High-performance liquid chromatography (HPLC) showed that the phlorotannins of E. bicyclis were composed of phloroglucinol (0.9%), phloroglucinol tetramer (4.4%), eckol (7.5%), phlorofucofuroeckol A (21.9%), dieckol (23.4%), and 8,8'-bieckol (24.6%), plus some other unknown phenolic compounds (17.3%). The composition of the phlorotannins differed little among the Laminariaceae, except for a significantly larger amount of the tetramer, MW 478, in E. bicyclis.     

6种重金属对赤子爱胜蚓的急性毒性效应与风险评价

【背景】近年来,土壤重金属污染问题日益凸显,对生态环境、食品安全和人体健康构成了严重威胁,其急性毒性尚未明确。【方法】采用滤纸接触法和人工土壤法测定了铜(Cu2+)、锌(Zn2+)、镍(Ni+)、镉(Cd2+)、铅(Pb2+)和锰(Mn2+)等6种重金属对赤子爱胜蚓的急性毒性效应,并参照欧盟指令91/414/EEC标准评价了其环境风险。【结果】滤纸接触法48h测定结果表明,6种重金属对蚯蚓的LC50为3.17(2.53～3.81)～90.42(69.45～140.47)μg.cm-2,其毒性次序为Cu2+>Zn2+>Ni+>Cd2+>Pb2+>Mn2+。人工土壤法14d测定结果表明,6种重金属对蚯蚓的LC50为1347(1236～1453)～6936(6144～8930)mg.kg-1,其毒性次序为Cu2+>Cd2+>Ni+>Zn2+>Mn2+>Pb2+。风险评价结果显示,Cu2+、Zn2+、Ni+、Cd2+、Pb2+和Mn2+等6种重金属的暴露比(toxicity/exposureratio,TER)分别为3.37、4.46、8.68、1428、13.87和5.85。【结论与意义】6种重金属对土壤动物蚯蚓均具有潜在的毒性效应。Cu2+、Zn2+、Ni+、Mn2+等4种重金属对赤子爱胜蚓存在急性毒性风险,而Cd2+和Pb2+对赤子爱胜蚓的急性毒性风险水平是可接受的。该评价结果可为我国制定基于风险的土壤环境质量标准提供依据。     

Toxicity of A Novel Neonicotinoid Insecticide Paichongding to Earthworm Eisenia fetida

Traditional neonicotinoid insecticides are used worldwide. Paichongding (IPP), as a novel neonicotinoid pesticide, has been widely used in China. However, the ecotoxicity of IPP to non-target invertebrates in soil ecosystem has not been reported yet. In this study, acute toxicity of IPP to earthworm Eisenia fetida, as well as the antioxidant response after IPP exposure, was evaluated. In the filter paper contact test, the LC₅₀ at 24 hr and 48 hr for IPP were 14.98 μg/cm² and 7.59 μg/cm², respectively. In artificial soil test, the LC₅₀ (lethal concentration) at 14 days and 28 days for IPP were 541.07 mg/kg and 238.51 mg/kg, respectively. The LC₅₀ of IPP is much higher than that of traditional neonicotinoid insecticides. However, earthworm body weight assessment demonstrated that the growth of earthworm was inhibited by extended exposure to IPP at sublethal doses. The activities of antioxidative enzymes superoxide dismutase and catalase in earthworms were significantly induced after IPP exposure. Malondialdehyde, a biomarker of lipid peroxidation, was also increased after IPP exposure. Although the results indicated that IPP had potentially adverse effect on earthworms, its toxicity was much lower than traditional neonicotinoids.     

Bt毒蛋白Cry1Ac在人造土壤中对赤子爱胜蚓毒理及生化影响

Bt毒素能通过转基因作物的花粉、根和残株进入土壤.为评估转基因作物对土壤动物的影响,本文模拟转基因棉的Bt毒素进入土壤的发生程度,用含不同浓度Bt毒蛋白Cry1Ac的人造土壤处理蚯蚓,测定蚯蚓存活率、重量变化及体内总蛋白含量和过氧化氢酶(CAT)、乙酰胆碱酯酶(AchE)、谷胱甘肽-S-转移酶(GST)和纤维素酶活性.结果表明,Bt毒蛋白对蚯蚓的生物量和生理水平影响均不明显,不存在急毒性和亚致死毒性影响,对蚯蚓比较安全.     

蚯蚓中抗肿瘤蛋白组分的提取分离及其抗肿瘤活性

以赤子爱胜蚓为原料 ,通过蛋白质的丙酮沉淀和凝胶过滤 ,分离得到一组抗肿瘤活性蛋白成分 (简称蚯蚓抽提物 ) ;这组成分富含Fe、Zn、Cu以及Se等微量元素 ,蛋白含量为 6 0 4 3± 2 36 %(n =5 ) .体外实验中 ,通过MTT法和SRB法测定了蚯蚓抽提物对多种人癌细胞株 (HCT 116、SY5Y、K5 6 2、MGc80 3和HeLa)以及正常细胞株 (HEK2 93和COS 7)的抑制杀伤率 .结果表明 ,蚯蚓抽提物对癌细胞的杀伤有一定的选择性 ,受试人癌细胞达到 5 0 %生长抑制率所需作用浓度约 6 0～110mg L ;但是 ,10 0℃煮沸 5min后 ,该抑制活性完全消失 .通过纤维蛋白平板法 ,测得蚯蚓抽提物同时具有纤溶酶和纤溶酶原激活酶的活性 .体外测得丝氨酸蛋白酶抑制剂aprotinin和PMSF能显著抑制蚯蚓抽提物的细胞杀伤活性 .体内实验中 ,蚯蚓抽提物能有效延长荷瘤 (S180 )小鼠的生存时间 ,并使其身体机能得到明显改善 .腹膜内注射剂量为 2 8mg kg和 36mg kg时 ,生命延长率分别为135 3%和 12 3 5 % ,和标准药物 (环磷酰胺 )治疗后结果 (76 5 % )相比 ,有显著性差异