| Arsenic (As) as a widespread pollutant caused by human activities is one of the crucial scientific problems of environmental pollution and toxicology. Humic acid (HA) is ubiquitous in aquatic systems which can complex with arsenic. The mobility and toxicity influenced by HA associated with Arsenite [As(Ⅲ)], the most toxic and variable arsenic species, has not been fully examined. Batch experiments were conducted to study the effects of HA on As(Ⅲ) toxicity to three kinds of typical diatoms. The influence of HA on As(Ⅲ) toxicity was assessed by measuring algal growth, chlorophyll a and reactive oxygen species (ROS), whereas As(Ⅲ) mobility across the cell wall was estimated by determining the concentration of intracellular, cell-wall-bound, and free As(Ⅲ) ions in cell media. Isotherms combined with morphological data by scanning electron microscopy (SEM) were applied to describ adsorption of HA on the Navicula sp. cells. Additional Fourier transform infrared spectroscopic (FTIR) and electrophoretic mobility (EPM) analysis were applied to analyze the impact of cell wall-bound HA on the cell wall characteristic. The main results are listed as follows:(1) Humic acid-mediated toxic effect of arsenite to diatoms. The effects of HA on arsenite toxicity varied depending on various combinations of As(Ⅲ)-HA concentrations. We can see the stimulating effect of20mg L-1HA at all As(Ⅲ) concentrations including the As-free control, and the positive role of HA in promoting Chl-a synthesis. The HA-mediated reduction in toxicity was insignificant at high HA concentrations (≥40mg L-1) for its own pronounced adverse effects on both growth rate and Chl-a. Meanwhile, the effects of HA on arsenite toxicity varied depending on different species of diatoms. EC50had an approximate threefold increase for Navicula sp., and fourfold for Nitzschia palea and Cyclotella meneghiniana, respectively. Although, HA itself can slightly increase ROS generation, intracellular ROS in response to10.0μM As(Ⅲ) was decreased by12.6%and5.95%in the presence of10mg L-1and20mg L-1HA, respectively.(2) The complexation of HA-As(Ⅲ) and As concentration distribution at the cell interface influenced by HA. As(Ⅲ)-HA complexing level decreased along with the elevated As (Ⅲ) concentration at a constant HA concentration (20mg L-1). Conditional distribution coefficients (DHA) values are460-3050L kgDOC-1(logDHA=2.66-3.48), and the complexation ratio of As(Ⅲ)-HA varied in a range of0.91-6.00%, with a decreasing as As/DOC increased. When As/DOC exceeds0.50umol/mg, the DHD and the complexation ratio approached a plateau, presumably due to the saturation of complexation sites at high As(Ⅲ) concentrations relative to HA. Meanwhile, three dimensional fluorescence spectrums (EEMs) was used to analyze the fluorescence quenching effect of As(Ⅲ) on HA. Fluorescence intensity of both polymer humus of larger molecular weigh (Ex=330-380nm, Em=420-460) and the aromatic amino acid substances (Ex=250-250nm, Em=430-480nm) were decresed in the presence of arsenic. In the presence of20mg L-1HA, the cell-wall-bound and intracellular arsenic content decreased by20.1%and20.3%, respectively. A significant contrast between the higher intracellular arsenic reduction (20.3%) and lower arsenite complexation (2.10%±0.16%of the total As) indicated complexation alone could not explain the HA-induced reduction in arsenite toxicity and other factors including HA-cell surface interactions may come into play.(3) Aggregation behavior of humic acid at the siliceous interface of diatom cells as well as the interaction mechanism between As(Ⅲ)-HA-diatom cells were explored. The adsorption of humic acid on diatoms is favourable. Within this environmentally relevant HA concentration range (0-20mg L-1), the adsorption fit the linear adsorption model (HAads=KH×[HA], R2=0.998) well, the Henry adsorption constant KH value is2.35±0.55×10-8L cell-1. The adsorption tends to fit the saturation-type Langmuir isotherm at higher HA concentrations. The Langmuir fit (R2=0.995) estimates the adsorption capacity (qm) of3.31×10-6mg cell-1. Morphological data by scanning electron microscopy revealed a protective HA floccule coating on the cell-walls. Additional Fourier transform infrared spectroscopic data suggested the involvement of carboxylic groups during the adsorption of both HA and As(Ⅲ) on the Navicula sp. cell surface. Indeed, a significant increase by38.5%(p<0.01) in the negative surface charge was observed by electrophoretic mobility (EPM) analysis when the HA concentration is increased from0to20mg L-1,further proofing that cell wall-bound HA reduced the adsorption and absorption of arsenic. Collective data from this study suggest that cell-wall-bound HA can moderate As(Ⅲ) toxicity through the formation of a protective floccule coating occupying As(Ⅲ) sorption sites and decreased effective functional groups capable of binding As(Ⅲ). |
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