Evaluation Of Toxicity Data To Green Algae And Ecotoxicity Of TiO₂Nanoparticles On Pseudokirchneriella Subcapitata

In this thesis, evaluation of toxicity data to green algae was conducted in NortheastNormal University, and study on ecotoxicity of TiO2nanoparticles on the freshwater greenalga Pseudokirchneriella subcapitata was carried out in Biotechnology Research Institute,National Research Council Canada (NRC).Algae, as primary producers, play an important role in ecosystem. Therefore, studyingthe toxic effects of chemicals on algae provides valuable information regarding the risks thatchemicals might pose to environment. Quantitative Structure-Activity Relationship (QSAR) isa useful method to assess the environmental hazards of chemicals. The quality of the toxicitydata is of great importance for the development of QSAR models for chemicals’ toxicity toalgae. So far, however, a number of algal toxicity QSAR models in the literature weredeveloped basing on toxicity data without considering the response endpoints, exposureperiods and species sensitivity. It is unkown whether these factors affect algal toxicity dataand hence the quality of QSAR models. This thesis systematically evaluates algal toxicitydata basing on response endpoints, exposure periods and algal species sensitivities.TiO2nanoparticles are currently one of the nano-materials with the largest productionand the widest application in the world. The potential risk of TiO2nanoparticles toenvironment and human health has raised increasing concern. Investigating toxicity of TiO2paritcles is very important. Furthermore, understanding the toxicity mode of TiO2will behelpful for modification of TiO2particles in order to reduce the TiO2toxicity, and thedevelopment of new nontoxic TiO2nano-materials. In this thesis, a freshwater green alga P.subcapitata was used as a test organism to characterize the toxicity of TiO2particles. Wediscussed the relationship between toxicity of TiO2particles to algal growth and theirphysic-chemical properties, and tested the hypothesis that TiO2nanoparticle toxicity tofreshwater green algae is attributed to shading effect or reactive oxygen species (ROS)production during photolysis. This study provides valuable information for the riskassessment of TiO2nanoparticles.The investigation results were summarized as follows:(1)2323algal toxicity data (log1/EC50) in different toxicity response endpoints for1081compounds to26algal species within different exposure periods (14min,24,48,72,96and168h) were used to evaluate the quality of the toxicity data to green algae. We evaluated theexperimental uncertainty of algal toxicity data from different sources, explored the effects ofresponse endpoint, exposure periods, and algal sensitivities to the toxicity data, and analyzedthe relationship between toxicity and hydrophobicity for non-polar narcotics and polar narcotics, respectively. Analysis of72h toxicity to algae showed that the closed test had thesame sensitivity as the open test for most of the test compounds, but a significant differencewas observed for a few compounds. The overall average difference for all compounds rangesfrom0.15to0.43log units between toxicity endpoints (yield–growth rate). The relationshipsbetween exposure periods of24h,48h,72h and96h indicated that48h exposure period isthe most sensitive for algal growth inhibition test, and its sensitivity is0.25/0.14log unitsgreater than72/96h exposure periods. Interspecies relationships showed that some algalspecies have very close sensitivity (e.g. P. subcapitata and Chlorella pyrenoidosa or Chlorellavulgaris and Scenedesmus obliquus, respectively), whereas some species have significantlydifferent sensitivity (e.g. P. subcapitata and S. obliquus). Relationships between toxicity andhydrophobicity demonstrated that no difference was observed for non-lolar narcotics withindifferent exposure periods (24h,48h,72h, and96h) or response variables (yield and growthrate). For polar narcotics, in contrast, algal toxicity is dependent on algal species and is relatedto the response variables and exposure period. We cannot expect significant QSAR modelsbetween algal toxicity and descriptors without considering species sensitivity, exposureperiods and response endpoints.(2) Basing on the above-mentioned results, P. subcapitata,96h, and yield, as morefavorable algal species, exposure period, and response variable, respectively, were selected toinvestigate the toxicity of TiO2particles to green algae. The results will provide valuable datafor QSAR studies of metal oxide toxicity.We examined the toxicity of several TiO2particlesamples to96h growth of freshwater green algae P. subcapitata, and characterized thephysico-chemical properties of these TiO2nanoparticle samples. Results showed that thetoxicity of TiO2particles to P. subcapitata was influenced by their selected physicochemicalproperties: particle size, crystalline phase, and ζ-potential of aggregates in suspensions. Thenano-sized anatase TiO2particles, with larger specific surface area (SSA), showed strongertoxicity than their bigger counterparts. Anatase TiO2nanoparticles, whose diameter was16.2nm, exerted slightly stronger toxicity than rutile TiO2nanoparticles with the similar diameter,15.5nm. At the same concentration (w/v), the ζ-potentials of aggregates of three TiO2particlesamples in algal medium were-21.3,-27.3, and-43.7mV, respectively. The EC50values ofthese three samples inhibiting the96h growth of P. subcapitata were0.14,0.65, and1.39mg/L, respectively. It indicated that the toxicity of TiO2particles decreased with increasingelectronegativity of TiO2particles. Coating or modification on the surface of TiO2particleshas been reported elsewhere to reduce or block the toxicity of TiO2particles to mammaliancells. In contrast, it was found in this thesis that the algal growth inhibition toxicity of acoated TiO2particle sample was extremely high (96h EC50=3.10μg/L). It suggested that thedifference between organism species should be taken into account when surface treatment isperformed in order to block or decrease the toxicity of TiO2particles. (3) The present thesis investigated the possibility that TiO2nanoparticle toxicity in P.subcapitata involves shading effect or ROS production. We tested the shading effect of TiO2nanoparticles on algal growth using a modified experiment basing on a design reported inliterature, and the result showed that TiO2nanoparticle toxicity to green algae P. subcapitatais not due to the blocking of light available for photosynthesis of the cells.2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to mark ROS production inthe mixture of algae and different concentrations of TiO2nanoparticles after3h of irradiationunder different light conditions. Results showed that TiO2nanoparticles increased ROSproduction in the presence of UV, and the production of ROS increased as TiO2concentrationincreased. When UV was filtered out, only the highest test concentration of TiO2couldcatalyze the ROS production. Abiotic experiment, using methylene blue as the indicator ofROS, also showed that UV irradiation triggered the catalytic ROS generation in the presenceof TiO2nanoparticles, and therefore accelerated the fading of methylene blue compared tovisible light. Toxicity studies indicated that exposure to TiO2nanoparticles decreased algalgrowth following3h pre-exposure to UV/visible light and visible light, and EC50s were6.3(95%confidence interval (CI):3.9-10.3) and8.7(95%CI:5.4-14.7) mg/L, respectively. Itsuggested that TiO2nanoparticles were still toxic to the growth of P. subcapitata under visiblelight, and UV irradiation did not increase the toxicity of TiO2nanoparticles to the algalgrowth. Moreover, the addition of high dose of ROS scavenger agentN-tert-butyl-α-phenylnitrone (BPN)(100mg/L) did not influence the toxicity of TiO2nanoparticles (0.2mg/L) to the growth of P. subcapitata. The present study suggests that thegrowth inhibitory effects of TiO2nanoparticles in algae P. subcapitata occurred atconcentrations lower than those that can elevate ROS production, and that ROS generation isneither directly involved with nor increases the growth inhibitory toxicity of TiO2nanoparticles in P. subcapitata. Further research should be conducted to increase our currentunderstanding of the nanotoxicology of TiO2and other metal oxides.