Investigating The Ecotoxicity Of Environmental Samples Using Vibrio Qinghaiensis Sp. Q67and Their Origin

The bioassay implemented on environmental samples has become the importantsupplement of conventional water environment monitoring. It could reflect thecomprehensive toxicity effect of contaminants on the organisms, environment and evenhuman. However, compared with chemical and instrumental analysis, bioassay isrelatively new and needs improvement. It is difficult to conduct a biological toxicitytest using organisms with long life cycle in a common laboratory. Hence, luminescentbacteria were widely applied in the bioassay due to the advantages of short culture timeand easy operation. Of the luminescent bacteria used in bioassay, freshwaterluminescent bacterium, Vibrio qinghaiensis sp. Q67(Q67), found in China, does notneed to maintain a salty environement. So, the bioassay using Q67shows a profitableperspective. Like other luminescent bacteria toxicity test, it is still not suit to detectenvironmental samples with low toxicity and is easy to be disturbed by coexitinginorganic compounds. In order to expand the scope of application and to improve theaccuracy, a series of studies, from the choice of pretreatment methods to theoptimization of Q67toxicity test, were conducted, and the origins of biological toxicityof wastewaters were investigated based on these established methods.This study is part of the project of National Natural Science Foundation of China.For the samples mainly containing organic contaminants, a method combining the Q67acute toxicity assay with solid phase extraction (SPE) was developed. A specifictoxicity assay combining Q67chronic toxicity test with reverse osmosis (RO) wasdeveloped to detect the samples containing both organic and inorganic contaminantsand to eliminate the stimulation caused by inorganic compounds. The two methods were applied on various environmental samples and were proved to be effective.Moreover, the origins of biological toxicity for domestic wastewaters and industrialwastewaters with different contents of contaminants were studied via the Q67toxicityassay and chemical analysis. The main work and achievements are as follows:(1) As to environmental samples mainly containing organic containments, themethod combining Q67acute toxicity assay with SPE was developed. The organicsubstances in water sample were effectively enriched by optimizing the operation ofSPE. The optimized condition of microplate Q67acute toxicity assay was determinedin terms of start relative light unit (RLU), bacterial suspension volume, exposuretimeand pH value. The start RLU was3×106~4×106; Volume of both testing sampleand Q67suspension was100μL respectively; Exposure time was15min; PH value ofthe testing sample was controlled in the range of5~10. The results indicated that theestablished SPE-Q67acute toxicity assay showed high sensitivity and goodreproducibility for samples contaminated by organic toxicants.(2) To the environmental samples in which the toxicity of inorganic compounds isequally important, a method combining Q67chronic toxicity test with RO wasestablished. The RO concentrated method was established using total organic carbon(TOC) as evaluation index for the organic compounds and electrical conductivity (EC)for inorganic ions, which achieved to simultaneously and effectively concentrateorganic and inorganic contaminants. Growth characteristics and uptake of nutrients ofQ67were discussed in the process of prolonging exposure time, and10h exposuretime was determined for evaluating comprehensive toxicity and eliminating thedisturbances caused by nutrient salts. The results denoted that the RO-Q67chronictoxicity assay applied on the environmental samples containing organic and inorganiccontaminants, was characterized by high sensitivity and good reproducibility, andcould expand the application scope of Q67toxicity assay.(3) The established two Q67toxicity assays were applied on the study of thevariation of toxicity in the process of domestic wastewater treatment. According toSPE-Q67acute toxicity assay, Q67acute toxicity of wastewater in terms of toxicityimpact index (TII50) reduced from50.51%in the influent to1.47%after secondarytreatment, suggesting that the acute toxicity of domestic wastewater could be effectively reduced by activated sludge treatment process along with the biochemicaldegradation of organic pollutants. But the final chlorination resulted in a slight TII50increase, indicating that disinfection by-products produced during the reaction betweenchlorine and humic-like microbial products were toxic to organisms. After activatedcarbon adsorption treatment, the acute toxicity of secondary effluent was furtherreduced along the decrease of the concentration of organic compounds. According toRO-Q67chronic toxicity assay of domestic wastewater, chronic toxicity caused byboth inorganic and organic contaminants was decreased from47.62%in the influent to6.21%in the secondary effluent, suggesting that secondary treatment aimed to reduceorganic contaminants was not efficient to remove the toxicants with chronic toxicity.(4) By means of the fraction of organic compounds, the toxicity of organic matterfractions obtained from domestic wastewater was evaluated. Using large-volume SPEcolumn, the organic matter in the domestic wastewater was classified into three distinctfractions: non-polar, medium-polar and polar, by organic eluants with different polarity.The acute toxicity assay of the three fractions demonstrated that the acute toxicity ofdomestic wastewater was mainly caused by polar and medium-polar fractions.Furthermore, the acute toxicity induced by medium-polar fraction of unit mass wasgreatest. Using three kinds of resin, the organic compounds in domestic wastewaterwere classified into six fractions by the properties of polarity, alkalinity, acidity andneutral, and the chronic toxicity of these fractions was further identified. The resultsshowed that organic matter in raw sewage and secondary effluent was mainly thehydrophilic neutrals and the hydrophobic acids. However, the hydrophilic neutralsmade the greatest contribution to the chronic toxicity of raw sewage, and thehydrophobic acids had the greatest contribution to the chronic toxicity of secondaryeffluent.(5) The toxicity of industrial wastewaters (coking, electroplating, dyeing,gas-field and oil-field wastewaters) with different contents of heavy metals and organiccontaminants were investigated and their toxicity origins were exploited using Q67toxicity assay in combination with chemical analysis. With commonly existing heavymetals (such as Cu, Hg, Cr, Zn, Cd, Pb) in wastewaters as the research objectives toanalyze acute toxicity, the concept of EC50,metalwas proposed to express the acute toxicity of heavy metals. With commonly existing organic compounds in wastewatersas the research objectives to analyze acute toxicity, the concept of EC50,TOC(in terms ofTOC) was proposed to express the acute toxicity of organic compounds. Withindustrial wastewaters as research objectives to analyze acute toxicity, EC50,%(in termsof dilution percentage) was proposed to express the acute toxicity of industrialwastewaters. The results showed that EC50,%was a robust index to compare the acutetoxicity of different wastewaters, namely, the smaller the EC50,%, the stronger the acutetoxicity. The contribution of organic contaminants in industrial wastewater to acutetoxicity were judged by comparing the TOC concentration of industrial wastewatersafter dilution for obtaining their EC50,%with the EC50,TOCof organic compounds(especially the organic compounds with high toxicity). Comparing the concentration ofheavy metals in industrial wastewater after dilution for obtaining their EC50,%with thecorresponding EC50,metal, the contribution of heavy metals in industrial wastewater toacute toxicity were judged by. Based on above analysis and comparison, the toxicityand its origins for industrial wastewaters were comprehensively evaluated. This couldprovide a theoretical foundation for selecting a suitable treatment method to control thebiological toxicity of wastewaters.