Simultaneous Determination Of Dissolved Phenanthrene/Methyl Phenanthrenes And The Intermediate Metabolites In Aqueous Solution

Alkylated polycyclic aromatic hydrocarbons(A-PAHs)are a kind of the common derivatives of polycyclic aromatic hydrocarbons(PAHs)widely distributed in the environment.Particularly in crude oil polluted areas and diesel/biodiesel exhaust,the contents of A-PAHs are higher than their parent PAHs(P-PAHs).The toxicity and endocrine disrupting effects of most A-PAHs are stronger than those of P-PAHs.P-PAHs and A-PAHs contamination become a potential serious environmental problem.On the other hand,with the widespread coexistence of pollutants,PAHs combined contamination has become a research hotspot.Microbial degradation is one of the important methods to remove PAHs from the environment.It is essential to investigate the degradation pathways and to identify metabolites of PAHs.There are several studies prove that the toxicity or carcinogenicity associated with the contaminated soils may remain following bioremediation treatment,which is mainly due to the degradation of PAHs may generated more polar and toxic intermediate metabolites.Extensive studies proposed analytical methods for the determination of the PAHs metabolites,such as chromatographic methods(GC-MS,LC-MS),which are highly sensitive and selective.However,these methods inevitably destroy the originally existing forms of PAHs because of complicated and destructive pretreatment procedures,which are disadvantageous for in situ detecting.In addition,studies focus on non-dissolved PAHs.The intermediate metabolites during biodegradation are low concentration,short residence time and difficult to be detected,making it a challenge of in stu determination of metabolites in the degradation process of dissolved PAHs.Phenanthrene(Phe)was chosed as representative of P-PAHs,1-methyl-phenanthrene(1-MPhe)and 3-methylphenanthrene(3-MPhe)were chosed as representative of A-PAHs,1-hydroxy-2-naphthoic acid(1H2NA)and salicylic acid(SA)was respectively chosed as upstream metabolite and downstream metabolite,dimethyl phthalate(DMP)as coexistent contaminant.A novel method for simultaneous determination of Phe and 1H2NA had been established.Then the biodegradation characteristics of Phe,1-MPhe and 3-MPhe and the biodegradation mechanism of Phe in presence of DMP were discussed.Finally,a novel method for simultaneous determination of Phe,1H2NA and SA had been established to further explore the mechanism of biodegradation.The main results are as follows:1.A novel method for simultaneous determination of dissolving Phe(1-MPhe,3-MPhe)and 1H2NA in aqueous solution had been established using derivative synchronous fluorescence spectrometry.The linear dynamic ranges for the determination of Phe(1-MPhe,3-MPhe)and 1H2NA were 4.0?8.O×102 ?gg/L(4.0?2.5×102 ?g/L,4.0?2.5×102 ?g/L)and 4.0?1.2×103?g/L.The detection limits(LOD)of Phe,1-MPhe,3-MPhe and 1H2NA were 0.08 ?g/L,0.07 ?g/L,0.09 ?g/L,0.07 ?g/L,respectively.The recoveries of standard addition were 87.6%?111.6%,and the relatively standard deviations were less than 2.0%.The established method is simple and high sensitivity.LOD of this method was lower than traditional chromatography approaches.2.A novel method for simultaneous determination of Phe,1H2NA and SA in aqueous solution had been established using derivative synchronous fluorescence spectrometry with the double scans method.On the basis of the established simultaneous determination method of Phe and 1H2NA,the synchronous fluorescence spectra of SA in mixture was obtained with ??,l 55 nm in a pH 7.The linear dynamic ranges for the determination of SA were 4.0?8.0×102 ?g/L,with LOD of 0.88 ?g/L.The relatively standard deviation was 4.75%.The LOD of established method was lower than that of HPLC(2.7?g/L).3.Derivative synchronous fluorescence spectrometry was used to study the biodegradation process of dissolved Phe,1-MPhe,3-MPhe.Due to the toxic stress of PAHs,with the increase of the initial concentration of Phe,1-MPhe and 3-MPhe,the conversion rate of 1H2NA showed a decreasing trend.The average biodegradation rates of Phe,1-MPhe and 3-MPhe respectively were 2.00×10"7 mol/L·h-1,1.25×10-7 mol/·h-1,1.67×10-7 mol/L·h-1 at the same initial concentration of 1.00×10-6 Mol/L.The degradation rate trend was:Phe>3-MPhe(?)>1-MPhe(?),and the trend of the conversion rate of 1H2NA was:Phe>1-MPhe>3-MPhe.This showed that different methyl substituent positions allocate different flow on the methyl oxidation pathway branches.The flow on the methyl oxidation of 1-MPhe larger than that of 3-MPhe,indicating that the biodegradation mechanism between 1-MPhe and 3-MPhe was different.4.The biodegradation process of Phe and the influence on physicochemical properties of US6-1 were investigated to explain the effects of DMP on the biodegradation of Phe.The biodegradation rate of Phe by US6-1 was increased in the presence of DMP.The conversion rate of IH2NA was 30.5%during biodegradation of single Phe,and 30.6%during biodegradation of Phe and DMP.DMP did not affect dioxygenase pathway.The growth rates of US6-1 in different culture substrates were as follows:Blank-DMP<Phe-Phe and DMP coexisting,indicating that DMP did not promote the growth of US6-1.Hydrophobic experimental results were DMP(20.8%)<Phe(25.9%)<Phe and DMP coexisting(30.3%).Obviously,cell surface hydrophobicity(CSH)of US6-lin the coexistence of Phe and DMP was higher than that of Phe.For further research,the cell membrane surface fatty acid content of US6-1 was determined.The ratio of total 3-OH fatty acid were:2216E(5.48%)>Phe(5.16%)>Phe and DMP coexisting(4.42%).It meant the lowest lipopolysaccharides content of cell membrane in the coexistence of Phe and DMP,further verifying the largest CSH of US6-1 in the coexistence of Phe and DMP.In short,the presence of DMP induce changes in the composition and content of cell membrane fatty acids of US6-1,increase the hydrophobicity of the cell surface,which in turn to promote the biodegradation of Phe by US6-1.