| Arsenic is a metalloid element distributing widely in environment.With the economic development and anthropogenic activities such as mining,sewage irrigation,pesticides usage,etc.,large amounts of arsenic pollutants have been released into the environment.Nowadays global arsenic pollution has become a serious problem.Arsenic has a unique chemical structure which makes it possible to exist in different chemical species in environment and organisms.The most common arsenic species reported include arsenite[As(?)],arsenate[As(?)],monomethylarsonate(MMA),dimethylarsinate(DMA),etc.As the toxicity of different arsenic species varies greatly,it is not rigorous in science for the environmental quality assessment and risk evaluation regarding arsenic in traditional ways,i.e.,by only determining total arsenic content in environmental samples.Moreover,the accurate toxicity information cannot be obtained without arsenic speciation.Therefore,it is essential to develop a method for efficiently separation and detection of different arsenic species in environmental samples.On this basis,the toxicological effects of different arsenic species on organisms and related toxicity mechanism should be studied.Soil system is an important component of the ecosystem.Large amounts of arsenic pollutants have been accumulated in the soil environment.The different arsenic species in soil could be accumulated in organisms in many pathways and then induce a variety of toxic effects.Their entry into the food chain via bioaccumulation and bioconcentration can also pose a significant risk to human health.Traditional pollution detection and risk assessment method for soil environment didn't separate different arsenic species,leading to a limited consequence with little knowledge about the real ecological risk.Therefore,it is essential to conduct research programs that focus on the ecological toxicity characteristic and toxicity mechanism of different arsenic species.The research purpose is to develop a method of ecological risk assessment based on different arsenic species,as well as an evaluation index system and an assessment organism pattern for the indication of different arsenic species in soils.Furthermore,the research results could also provide scientific basis for the environmental pollution control and management based on arsenic speciation.In this thesis,four main arsenic species in environment including As(?),As(?),MMA and DMA were studied.Based on previous researches,we developed two innovative arsenic speciation methods,i.e.,derivatization-supercritical fluid extraction(SFE)-gas chromatography(GC)and hydride generation(HG)-cryotrapping(CT)-atomic absorption spectrometry(AAS).The two innovation breakthrough points are improvement of pretreatment technology and development of automatic on-line analysis technology.The new derivatization-SFE-GC method was developed by combining of derivatization-SFE technology and derivatization-GC technology,thus simplified arsenic speciation procedure as well as improved the efficiency.The new HG-CT-AAS method was developed by designing and installing an automatic on-line pre-reduction system and then connecting to the HG-CT-AAS system,thus made the combined automatic operation of the two systems successful.Then,the acute toxicity and subchronic toxicity of the four arsenic species were investigated by using the earthworm Eisenia fetida,which is recommended as a bioindicator species by national and international standards.The bioconcentration and biotransformation of the four arsenic species in the earthworms were also studied.Moreover,several cellular and subcellular level biomarkers,including seven oxidative stress biomarkers(superoxide dismutase,catalase,glutathione peroxidase,glutathione S-transferase,glutathione reductase,reduced glutathione,and lipid peroxidation),metallothioneins(MTs)and lysosomal membrane stability(LMS)were analyzed in the earthworms.Based on the data obtained above,integrated biomarker response(IBR)index was calculated;then dose-response relationships between biomarker responses and chemical analyses were also investigated.The purpose of these analyses was to study the ecological toxicity effects of different arsenic species on Eisenia fetida and the related toxicity mechanism.The main study contents and conclusions are as follows.(1)An innovative arsenic speciation method,derivatization-SFE-GC,was developed.This method integrated the derivatization-SFE method and the derivatization-GC method.The derivatization reaction was occurred in the SFE process;and then the derivatives of different arsenic species in samples were collected.The arsenic derivatives can be injected directly into the GC system for separation and determination.The amount of inorganic arsenic,MMA and DMA can be determined at the same time by using this method.The effects of many parameters during derivatization-SFE process were systematically investigated.The optimum extraction conditions were 30 MPa,100?,10 min static and 25 min dynamic extraction with 5%(v/v)methanol,and surfactant Triton X-100 modified supercritical CO2.Thioglycolic acid methyl ester(TGM)and thioglycolic acid ethyl ester(TGE)were evaluated as derivatization reagents.Results showed that TGE was more effective for arsenic speciation as a derivatization reagent.When the optimum extraction conditions were applied,detection limits in solid matrices were 1.2,0.3 and 0.15 mg kg-1 for inorganic arsenic,MMA and DMA,respectively.The resulting method was fast,selective,and could extract and detect various arsenic species in solid matrices directly.In addition,the recovery experiments proved that the new analytical methodology was accurate and reliable for arsenic speciation with low detection limits,and could minimize the usage of hazardous chemicals and solvents.(2)A new automatic on-line pre-reduction system was developed and optimized.The system was then connected with HG-CT-AAS system in order to accurately determine As(?),As(?),MMA and DMA in environmental samples.First,we used traditional off-line pre-reduction method to test and optimize the species and concentration of reductant.The results showed that the reduction of As(V)can be completed in a few minutes by using 1%(m/v)thioglycolic acid(TGA)as the reductant,thus made it possible to develop an on-line pre-reduction system and connect it with the HG-CT-AAS system.The results of system optimization showed that the concentration of different components in the reduction reaction system can be maintained with the application of air-assisted segmented flow device,thus the reduction reaction and analysis results can be guaranteed.Finally,the calibrations of the four arsenic species were obtained by using the optimized method.The limits of detection were 152 ng L-1 for As(?),146 ng L-1 for As(V),65 ng L-1 for MMA,52 ng L-1 for DMA,respectively,which means that trace level of different arsenic species can be detected.The resulting method was fast,accurate and with low detection limits.By using the automatic on-line pre-reduction system,automatic operation can be achieved.It can not only reduce the uncertainty of manual operations,but also shorten the sample analysis time,which makes this method very applicable for arsenic speciation.(3)The four main arsenic species in soils including As(?),As(?),MMA and DMA were selected.The earthworm Eisenia fetida was selected as bioindicator species.First,we conducted acute toxicity test according to national and international standard methods.The results showed that 14 d median lethal concentration(LC50)for As(?),As(?),MMA and DMA to the earthworm E.fetida were 293.3,352.4,3425.1 and 3730.3 mg kg-1,respectively.The sequence of the 14 d LC50 was in accordance with the toxicity of different arsenic species.The one-tenth of the 14 d LC50 was selected as the concentrations of spiked soils during a 64-day subchronic toxicity test.Results showed that there were significant bioaccumulation of total arsenic in earthworms exposed to As(?)and As(?),while no bioaccumulation of total arsenic was found in earthworms exposed to MMA and DMA.In this study,the biotransformation of different arsenic species in earthworms was also investigated.The results showed that the model of arsenic biotransformation was in accordance with previous hypothesis,i.e.,earthworms could transform highly toxic inorganic arsenic to the less toxic organic species that accumulated in the tissue,then transform organic species to complicated non-toxic arsenic compounds and excrete to the environment.Through this pathway the detoxification and metabolism of arsenic can be achieved.However,the demethylation of organic arsenic species was also observed in this study,leading to the formation of highly toxic inorganic arsenic species that could cause toxicity effects on organisms.Therefore,the MMA and DMA species also have indirect toxicity to earthworms.(4)During the 64-day subchronic toxicity experiment,we analyzed seven oxidative stress biomarkers and MTs in the earthworm E.fetida.The results showed that the changing trend of biomarkers in earthworms exposed to As(?)and As(?)were similar,while the changing trend of biomarkers in earthworms exposed to MMA and DMA were also similar.The variation of biomarkers of inorganic arsenic treatment groups was larger than the organic arsenic treatment groups,indicating that the toxicity of inorganic arsenic is higher than organic arsenic.At the later stage of the experiment,oxidative damage was observed in the earthworms that exposed to inorganic arsenic species,implying that the contamination stress caused by As(III)and As(V)gradually exceeded the protective capacity of the antioxidant defense system.Meanwhile,there was no oxidative damage found in the earthworms that exposed to organic arsenic species,proving that the effective operation of the antioxidant defense system had prevented the oxidative damage.Based on the data obtained above,IBR index was calculated to integrate the eight biomarker responses.The results showed that the toxicity of the four arsenic species was ranked as:As(III)>As(V)>MMA and DMA.The chronic toxicity of MMA and DMA on E.fetida had no significant difference during the 64-day experiment.In the high level organic arsenic exposure groups,the bioaccumulation of MMA and DMA did not cause significant variation of the biomarkers.However,the demethylation of these two organic arsenics lead to the formation and accumulation of highly toxic inorganic arsenic,as well as the significant variation of the biomarkers that showed good correlations with inorganic arsenic contents.Meanwhile,the biomarker responses and inorganic arsenic level in organisms exposed to As(III)and As(V)also had good correlations between each other,proving that the toxicity mechanism of arsenic was as follows:inorganic arsenic could destroy the dynamic equilibrium of reactive oxygen species(ROS)in organisms by inducing the over-production of ROS,thus causing oxidative damage toxicity.Although organic arsenics showed low biotoxicity,they could be transformed into highly toxic inorganic arsenic under the effect of demethylation and accumulated gradually,thus leading to toxic effects in a same way as inorganic arsenic.In conclusion,in order to obtain accurate results of environmental quality assessment or ecological risk assessment,the contents of different arsenic species should be differentiated and determined in environmental samples and organisms,including the organic arsenic species that exhibit low direct toxic effects.(5)The four main arsenic species in soils including As(?),As(?),MMA and DMA were selected.The earthworm E.fetida was selected as bioindicator species.Then a 70-day subchronic toxicity experiment was conducted.The biomarker,lysosomal membrane stability(LMS),was evaluated for toxicity assessment of the four arsenic species.Furthermore,the contents of total arsenic and arsenic species were analyzed in earthworms in order to evaluate the dose-response relationship between arsenic accumulation and biomarker responses.The results of bioaccumulation and bioconcentration of different arsenic species in earthworms were similar as that of chapter 4.The LMS in the earthworms of the four experimental groups exhibited a decreasing tendency with the increase of the exposure time.The results of LMS decrease implied that the toxicity of the four arsenic species could be ranked as As(?)>As(?)>MMA>DMA.The earthworms exposed to inorganic arsenic accumulated large amount of MMA and DMA,while no significant variation of LMS was observed.However,with the generation and accumulation of highly toxic inorganic arsenic that came from the demethylation of the organic arsenic species,the lysosomal membrane in the organisms was damaged gradually,indicating the indirect toxicity of MMA and DMA.Neutral-red retention time(NRRT)was measured to evaluate the LMS response in the earthworms.Results showed that the NRRT values had significant dose-response relationships with As(?)and As(?)contents,while the correlation coefficient between NRRTs and As(?)was higher than that between NRRTs and As(?).The lysosomal membrane in the organisms was more sensitive to As(?)and showed its stability destroyed thereafter,which could be one of the possible explanations for the mechanism that As(?)is more toxic than As(?)for many biological species.In conclusion,this study proved that more detail of environmental information can be obtained when different arsenic species are separated.The findings of this study have important and practical significance as well as application value for toxicity assessment and ecological risk evaluation for arsenic element. |
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