The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).     

Effect of Pseudokirchneriella subcapitata (Chlorophyceae) exudates on metal toxicity and colloid aggregation

The effects of Pseudokirchneriella subcapitata exudates on the acute toxicity of metals (Cd, Cu, Pb and Zn) were studied using (1h) algal photosynthesis inhibition tests. The metal concentrations tested were 30, 60, 120, 250 and 500microgL(-1) for Cd and Zn; and 500, 1000, 2000, 3000, 4000microgL(-1) for Cu and Pb. It was established that P. subcapitata exudates markedly decreased metal toxicity. This effect was ascribed to: (i) the presence of organic ligands that bind metals and reduce the concentration of free ionic metal, and/or (ii) interaction of exudates with the cell surface with a subsequent decrease in metal uptake. The effects of the exudates on colloid aggregation were also examined using two different types of single particle counters (SPC). Exudates facilitated the colloid removal, likely via acceleration of aggregation (bridging). The results clearly demonstrate that algal exudates play an important role in the biogeochemical cycling of metals in natural surface water: (1) by reducing free metal concentrations and toxicity to living organisms and (2) by favoring colloid aggregation leading to the removal of colloid-bound metals (colloidal pumping). Such results highlight one potential application of the algae in the remediation of metal-contaminated waters. The results also suggest that current algal toxicity testing protocols, in particular long-term and static tests, may underestimate metal toxicity because of the presence of algal exudates.     

Assessing the ecotoxicity of vinyl chloride using green alga P. subcapitata, nematode C. elegans, and the SOS chromotest in a closed system without headspace

The ecotoxicity of vinyl chloride (VC) was evaluated using green alga, nematode, and the SOS chromotest. The green alga and nematode tested were Pseudokirchneriella subcapitata, and Caenorhabditis elegans, respectively. Because of the tendency of VC to escape from an aqueous exposure medium to the air phase, all tests in the present study were performed in a closed system without headspace to minimize the losses of VC. Previous studies on VC toxicity were performed in an open system or closed system with headspace. VC inhibits the growth of P. subcapitata. The 48-h IC50 value for P. subcapitata exposed to VC was calculated to be 5.15 mg/L. The survival of C. elegans was not influenced at concentrations of up to 60 mg/L; however, VC has an adverse effect on the reproduction of C. elegans. In a stress-related gene expression test using C. elegans, a significant and concentration-dependent expression of heat shock protein 16.2 was observed, indicating that VC induces the stress of C. elegans at the genetic level. The results of the SOS chromotest using Escherichia coli PQ37 showed an IF_max value of 1.11, indicating that VC is not genotoxic. The present study demonstrated that VC has an adverse effect on the algal growth and reproductive and genetic levels of C. elegans. A closed system without headspace is an effective method of testing the aquatic toxicity of volatile organic compounds such as VC.     

Rapid quantification of bacteria in activated sludge using fluorescence in situ hybridization and epifluorescence microscopy

A rapid quantification method for bacteria in activated sludge has been developed, based on fluorescence in situ hybridization (FISH) and epifluorescence microscopy. Samples are hybridized on slides and analyzed by direct microscopic observation. Abundance categories were designed based on digital images of the target organisms. These rating systems were developed for the filamentous bacteria Microthrix parvicella and for different morphotypes of nocardioform actinomycetes, but can easily be adapted to other types of microorganisms. Due to the quantification by direct microscopic observation, this method is suitable for samples that are difficult to be processed by semi-automated image analysis techniques, such as samples containing fluorescent debris, cells of different fluorescence intensities and target organisms that need partial enzymatic digestion prior to FISH. In contrast to commonly used rating systems consisting of photographs, the newly developed categories allow to quantitatively compare results of different categories and different organisms. The uncertainties of the results were calculated by a non-parametric bootstrap procedure; a thorough uncertainty analysis was performed including sample variability and operator subjectivity.     

Surface-retained organic matter of Microcystis aeruginosa inhibiting coagulation with polyaluminum chloride in drinking water treatment

Algogenic organic matter produced by the excess growth of cyanobacteria in semi-closed water areas causes coagulation inhibition in drinking water production. In this study, hydrophilic substances of Microcystis aeruginosa, which were mainly composed of lipopolysaccharide (LPS) and RNA, were prepared, and the involvement of these cyanobacterial hydrophilic substances in coagulation inhibition was investigated. As a result, it was found that the negatively charged hydrophilic substances with a molecular weight higher than 10 kDa have a significant role in coagulation inhibition. Further fractionation of cyanobacterial hydrophilic substances revealed that surface-retained organic matter (SOM), including LPS, could exhibit a potent inhibitory effect on the coagulation using polyaluminum chloride (PACl), presumably because of the direct interaction of hydrophilic SOM with cations originated from PACl, which could impede the hydrolysis of the coagulant.     

Accumulation and fate of green fluorescent labeled Escherichia coli in laboratory-scale drinking water biofilters

Biological filters combining microbial activity and rapid sand filtration are used in drinking water treatment plants for enhanced biodegradable organic matters (BOM) removal. Biofilms formed on filter media comprised of bacteria enclosed in a polymeric matrix are responsible for the adsorption of BOM and attachment of planktonic microorganisms. This study investigated the removal of Escherichia coli cells injected into laboratory-scale biofilters and the role of biofilm in retaining the injected E. coli. Green fluorescent protein was used as a specific marker to detect and quantify E. coli in the biofilms. About 35% of the total injected E. coli cells were observed in the filter effluents, when initial cell concentrations were measured at 7.4 x 10(6) CFU/mL and 1.6 x 10(7) CFU/mL in two separate experiments. The results from real-time PCR and plate count analysis indicated that 95% of the E. coli retained inside the filters were either non-viable or could not be recovered by colony counting techniques. Injected cells were unevenly distributed inside the filter with more than 70% located at the top 1/5 of the filter. Images obtained from an epifluorescent microscope showed that E. coli cells were embedded inside the biofilm matrix and presented mainly as microcolonies intertwined with other microorganisms, which was consistent with findings from standard plate count methods and qPCR.     

Establishment of an Alert Level Framework for cyanobacteria in drinking water resources by using the Algae Online Analyser for monitoring cyanobacterial chlorophyll a

The Algae Online Analyser (AOA) fluorometer simultaneously distinguishes four different phytoplankton groups by their specific fluorescence spectra and thus allows for real-time in-situ chlorophyll a measurements per algal group. This AOA was used for monitoring cyanobacterial chlorophyll a in the drinking water at the Bronis?awow Bay abstraction point in Sulejow Reservoir (Poland). The main goal of this research was to develop an early warning method for the detection of cyanobacterial biovolume in the source water, in order to establish an Alert Level Framework for the drinking water abstraction point in Sulejow Reservoir. A positive correlation between cyanobacterial biovolume, as determined by conventional methods, and cyanobacterial chlorophyll a, as measured by the AOA, was found (p < 0.05). The results of this study were used to determine threshold values for the Alert Level Framework, based on cyanobacterial chlorophyll a concentrations in the source water of Sulejow Reservoir. The presented threshold values are determined specifically for this abstraction point, but the principles can be applied to other locations.     

Estimating true human and animal host source contribution in quantitative microbial source tracking using the Monte Carlo method

Cultivation- and library-independent, quantitative PCR-based methods have become the method of choice in microbial source tracking. However, these qPCR assays are not 100% specific and sensitive for the target sequence in their respective hosts’ genome. The factors that can lead to false positive and false negative information in qPCR results are well defined. It is highly desirable to have a way of removing such false information to estimate the true concentration of host-specific genetic markers and help guide the interpretation of environmental monitoring studies. Here we propose a statistical model based on the Law of Total Probability to predict the true concentration of these markers. The distributions of the probabilities of obtaining false information are estimated from representative fecal samples of known origin. Measurement error is derived from the sample precision error of replicated qPCR reactions. Then, the Monte Carlo method is applied to sample from these distributions of probabilities and measurement error. The set of equations given by the Law of Total Probability allows one to calculate the distribution of true concentrations, from which their expected value, confidence interval and other statistical characteristics can be easily evaluated. The output distributions of predicted true concentrations can then be used as input to watershed-wide total maximum daily load determinations, quantitative microbial risk assessment and other environmental models. This model was validated by both statistical simulations and real world samples. It was able to correct the intrinsic false information associated with qPCR assays and output the distribution of true concentrations of Bacteroidales for each animal host group. Model performance was strongly affected by the precision error. It could perform reliably and precisely when the standard deviation of the precision error was small (≤0.1). Further improvement on the precision of sample processing and qPCR reaction would greatly improve the performance of the model. This methodology, built upon Bacteroidales assays, is readily transferable to any other microbial source indicator where a universal assay for fecal sources of that indicator exists.     

Characterization of antibacterial polyethersulfone membranes using the respiration activity monitoring system (RAMOS)

Membranes with antibacterial properties were developed using surface modification of polyethersulfone ultrafiltration membranes. Three different modification strategies using polyelectrolyte layer-by-layer (LbL) technique are described. The first strategy relying on the intrinsic antibacterial properties of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(ethylenimine) (PEI) exhibits only little antibacterial effects. The other two strategies contain silver in both ionic (Ag⁺) and metallic (Ag⁰) form. Ag⁺ embedded into negatively charged poly(sodium 4-styrene sulfonate) (PSS) layers totally inhibits bacterial growth. Ag⁰ nanoparticles were introduced to the membrane surface by LbL deposition of chitosan- and poly(methacrylic acid) - sodium salt (PMA)-capped silver nanoparticles and subsequent UV or heat treatment. Antibacterial properties of the modified membranes were quantified by a new method based on the Respiration Activity Monitoring System (RAMOS), whereby the oxygen transfer rates (OTR) of E. coli K12 cultures on the membranes were monitored online. As opposed to colony forming counting method RAMOS yields more quantitative and reliable data on the antibacterial effect of membrane modification. Ag-imprinted polyelectrolyte film composed of chitosan (Ag⁰)/PMA(Ag⁰)/chitosan(Ag⁰) was found to be the most promising among the tested membranes. Further investigation revealed that the concentration and equal distribution of silver in the membrane surface plays an important role in bacterial growth inhibition.     

The burden of drinking water-associated cryptosporidiosis in China: the large contribution of the immunodeficient population identified by quantitative microbial risk assessment

A comprehensive quantitative microbial risk assessment (QMRA) of Cryptosporidium infection, considering pathogen removal efficiency, different exposure pathways and different susceptible subpopulations, was performed based on the result of a survey of source water from 66 waterworks in 33 major cities across China. The Cryptosporidium concentrations in source water were 0-6 oocysts/10 L, with a mean value of 0.7 oocysts/10 L. The annual diarrhea morbidity caused by Cryptosporidium in drinking water was estimated to be 2701 (95% confidence interval (CI): 138-9381) cases per 100,000 immunodeficient persons and 148 (95% CI: 1-603) cases per 100,000 immunocompetent persons, giving an overall rate of 149.0 (95% CI: 1.3-606.4) cases per 100,000 population. The cryptosporidiosis burden associated with drinking water treated with the conventional process was calculated to be 8.31 × 10⁻⁶ (95% CI: 0.34-30.93 × 10⁻⁶) disability-adjusted life years (DALYs) per person per year, which was higher than the reference risk level suggested by the World Health Organization (WHO), but lower than that suggested by the United States Environmental Protection Agency (USEPA). Sixty-six percent of the total health burden due to cryptosporidiosis that occurred in the immunodeficient subpopulation, and 90% of the total DALYs was attributed to adults aged 15-59 years. The sensitivity analysis highlighted the great importance of stability of the treatment process and the importance of watershed protection. The results of this study will be useful in better evaluating and reducing the burden of Cryptosporidium infection.     

Effect of nitrite concentration on the distribution and competition of nitrite-oxidizing bacteria in nitratation reactor systems and their kinetic characteristics

Genus Nitrospira and Nitrobacter species are the key nitrite-oxidizing bacteria (NOB) in nitrifying wastewater treatment plants. It has been hypothesized that genus Nitrospira are K-strategists and can exploit low amounts of nitrite more efficiently than Nitrobacter. In contrast, Nitrobacter species are r-strategists that can grow faster than Nitrospira. To elucidate the K/r hypothesis and to analyze the effect of substrate (nitrite) concentration on the competition and distribution of the two NOB, two different reactor types were employed for nitrite oxidation (nitratation) and NOB growth. The continuous biofilm airlift reactor (CBAR) maintained low nitrite concentration due to the complete oxidation of nitrite in continuous operation while the sequencing batch reactor (SBR) was kept in a relatively high nitrite concentration environment due to a cyclic substrate concentration profile. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) showed that both Nitrobacter species and genus Nitrospira were present in the CBAR and the SBR. Quantitative FISH analyses of the CBAR showed that Nitrospira occupied 59% of the total bacteria while Nitrobacter occupied only 5%. On the other hand, Nitrobacter, occupying 64%, was the dominant NOB in the SBR, and only 3% of total bacteria belonged to genus Nitrospira. Nitrite oxidation kinetics and quantitative FISH analyses revealed that the specific nitrite oxidation activities of Nitrobacter and Nitrospira are 93.8 and 10.5 mg/g NOB h, respectively, and the specific activity of Nitrobacter is about 9 times higher than that of Nitrospira. In conclusion, the results confirm the K/r hypothesis and the distribution of Nitrobacter and Nitrospira is likely to depend mainly on nitrite concentration. It seems that nitrite load and starvation conditions do not give a direct effect on the distribution of NOB.     

Results of field investigations into the impact of intermittent sewage discharges on the microbiological quality of wild mussels (Mytilus edulis) in a tidal estuary

Field surveys were designed to examine the effects of sewage contamination from storm overflow effluent on faecal coliform and Escherichia coli concentrations in the flesh of wild mussels (Mytilus edulis). Bags containing 30 mussels each were fixed at known inter-tidal locations and retrieved at intervals following discharge from a nearby combined sewer overflow (CSO). Concentrations of faecal coliform bacteria and E. coli were measured in the shellfish flesh and in samples of overlying water prior to collection of the mussel samples. Faecal coliform and E. coli concentrations in shellfish increased rapidly after CSO discharge. E. coli concentrations exceeded the European shellfish hygiene class C limit of 46,000 100g(-1), and decayed during subsequent CSO discharge-free periods. The concentration and depuration response was independent of the magnitude of CSO spill volume. First-order exponential decay functions were fitted to the data. Decay rates were lower than those found in corresponding microcosm experiments. This relates to the repeated pattern of inundation and exposure associated with the tidal cycles in the estuary. Relationships between E. coli and faecal coliform concentrations in the shellfish and overlying water samples were relatively weak (r<0.60), a pattern often seen with data from uncontrolled environmental experiments.     

On the development of an urban passive thermal comfort system in Cairo,Egypt

This paper investigates how urban form can be designed to act as a passive thermal comfort system in Cairo. The system utilizes two main elements, the urban fabric form, with its green structure, and thermal comfort adaptation by introducing urban green scene stimulation with the time of exposure to the urban environment. The courtyard form on a large scale is used to study comfort levels, the effect of compactness on solar access and the local radiant heat island potential in a theoretical neighborhood design using a medium population housing concept as required by Egyptian urban planning laws. Urban canyons in a grid network, with three mid latitude orientations 15°, 45° and 75° from the E–W axis, were examined, with one canyon having virtually no shade. Other canyons had a green structure containing two types of native Egyptian trees. Numerical simulations using ENVI-met were performed for hot climate conditions. Although some very hot conditions were recorded, there were evident examples of more acceptable comfort levels and cooling potential for some orientations and degrees of urban compactness due to the clustered form with green cool islands and wind flow through the main canyons. Some design guidance on how to form urban passive cooling systems is presented.     

Effect of moderate pre-oxidation on the removal of Microcystis aeruginosa by KMnO4-Fe(II) process: significance of the in-situ formed Fe(III)

This study developed a novel KMnO₄-Fe(II) process to remove the cells of Microcystis aeruginosa, and the mechanisms involved in have been investigated. At KMnO₄ doses of 0-10.0 μM, the KMnO₄-Fe(II) process showed 23.4-53.3% higher efficiency than the KMnO₄-Fe(III) process did. This was first attributed to the moderate pre-oxidation of M. aeruginosa by KMnO₄, achieved by dosing Fe(II) after a period of pre-oxidation, to cease the further release of intracellular organic matter (IOM) and the degradation of dissolved organic matter (DOM). The extensive exposure of M. aeruginosa to KMnO₄ in KMnO₄-Fe(III) process led to high levels and insufficient molecular weight of DOM, inhibiting the subsequent Fe(III) coagulation. Additionally, Fe(II) contributed to lower levels of the in-situ formed MnO₂, the reduction product of KMnO₄ which adversely affected algae removal by Fe(III) coagulation. However, the in-situ formed Fe(III), which was derived from the oxidation of Fe(II) by KMnO₄, in-situ MnO₂, and dissolved oxygen, dominated the remarkably high efficiency of KMnO₄-Fe(II) process with respect to the removal of M. aeruginosa. On one hand, in-situ formed Fe(III) had more reactive surface area than pre-formed Fe(III). On the other hand, the continuous introduction of fresh Fe(III) coagulant showed higher efficiency than one-off dosage of coagulant to destabilize M. aeruginosa cells and to increase the flocs size. Moreover, the MnO₂ precipitated on algae cell surfaces and contributed to the formation of in-situ formed Fe(III), which may act as bridges to enhance the removal of M. aeruginosa.     

Effect of chromium on the level of IL-12 and IFN-gamma in occupationally exposed workers

Chromium may affect humoral and cellular immunity, acting on T lymphocytes as well as on granulocytes and monocytes cells. Cytokines play an important role in the immune balance. In this study, the level of IL-12 and IFN-γ were evaluated in the sera and PHA/LPS stimulated culture supernatant of human PBMCs of healthy volunteers and occupationally exposed chromium workers. All the workers were highly exposed to chromium having mean of 104.65 ± 77.21 µg/dL (range 23.7-316.8 µg/dL). A suspension of exposed and unexposed human PBMC (0.5 × 10⁶ cells/ml) prepared and cultured in RPMI-1640 supplemented with 10% FCS for 18 h in the presence or absence of LPS (10 ng/ml) which used for stimulation of IL-12 and IFN-γ. The level of IL-12 and IFN-γ were evaluated in the sera samples as well as LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers. In these chromium exposed workers the level of IL-12 was 433.66 ± 197.49 pg/ml and 983.45 ± 330.99 pg/ml in LPS stimulated culture supernatant of normal individuals and highly chromium exposed workers, which was significant (P < 0.05). Although the level of IL-12 was (78.61 ± 61.03 pg/ml to 146.52 ± 46.37 pg/ml) elevated in unstimulated culture supernatant of h-PBMCs of chromium exposed individuals as compared to control, but it was not significant. This observation also suggests that a significant increase in IFN-γ production in LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers as compared to control. However, IFN-γ level have a significant positive correlation between blood chromium level (r = 0.833, t = 6.3872, P 0.05) and exposure time (in years) (r = 0.8916, t = 8.3540, P 0.05) of the occupationally exposed workers.     

Effect of water composition, distance and season on the adenosine triphosphate concentration in unchlorinated drinking water in the Netherlands

The objective of our study was to determine whether water composition, distance to the treatment plant and season significantly affect the adenosine triphosphate (ATP) concentration in distributed drinking water, in order to resolve the suitability of ATP as an indicator parameter for microbial regrowth. Results demonstrated that the ATP concentration in distributed water averaged between 0.8 and 12.1 ng ATP L⁻¹ in the Netherlands. Treatment plants with elevated biofilm formation rates in treated water, showed significantly higher ATP concentrations in distributed drinking water and ATP content was significantly higher in the summer/autumn compared to the winter period at these plants. Furthermore, transport of drinking water in a large-sized distribution system resulted in significantly lower ATP concentrations in water from the distal than the proximal part of the distribution system. Finally, modifications in the treatment significantly affected ATP concentrations in the distributed drinking water. Overall, the results from our study demonstrate that ATP is a suitable indicator parameter to easily, rapidly and quantitatively determine the total microbial activity in distributed drinking water.     

Comparison of natural estrogen removal efficiency in the conventional activated sludge process and the oxidation ditch process

The presence of natural estrogens, 17beta-estradiol (E2), estrone (E1) and estriol (E3), as well as estrogenic activity in wastewater influents and secondary effluents were investigated in 20 full-scale wastewater treatment plants in Japan. In all of the influent samples, natural estrogens were detected at concentrations above the minimum limits of detection (0.5ng/L). The concentrations of natural estrogens detected in the effluent of oxidation ditch plants were generally lower than previously reported values. On the other hand, in the conventional activated sludge plants, increments of E1 during biological treatment were frequently observed although E2 and E3 were removed effectively in the process. The removal rates of natural estrogens or estrogenic activity show no observed statistical relationship with the solids retention time (SRT) and the hydraulic retention time (HRT). However, the plants with high SRT or HRT generally showed high and stable removal of both natural estrogens and estrogenic activity.     

Effects of ozone and ozone/peroxide on trace organic contaminants and NDMA in drinking water and water reuse applications

An ozone and ozone/peroxide oxidation process was evaluated at pilot scale for trace organic contaminant (TOrC) mitigation and NDMA formation in both drinking water and water reuse applications. A reverse osmosis (RO) pilot was also evaluated as part of the water reuse treatment train. Ozone/peroxide showed lower electrical energy per order of removal (EEO) values for TOrCs in surface water treatment, but the addition of hydrogen peroxide increased EEO values during wastewater treatment. TOrC oxidation was correlated to changes in UV₂₅₄ absorbance and fluorescence offering a surrogate model for predicting contaminant removal. A decrease in N-nitrosodimethylamine (NDMA) formation potential (after chloramination) was observed after treatment with ozone and ozone/peroxide. However, during spiking experiments with surface water, ozone/peroxide achieved limited destruction of NDMA, while in wastewaters net direct formation of NDMA of 6-33 ng/L was observed after either ozone or ozone/peroxide treatment. Once formed during ozonation, NDMA passed through the subsequent RO membranes, which highlights the significance of the potential for direct NDMA formation during oxidation in reuse applications.     

Removal of arsenic from water: Effect of calcium ions on As(III) removal in the KMnO4–Fe(II) process

A novel KMnO₄–Fe(II) process was developed in this study for As(III) removal. The optimum As(III) removal was achieved at a permanganate dosage of 18.6 μM. At the optimum dosage of permanganate, the KMnO₄–Fe(II) process was much more efficient than the KMnO₄–Fe(III) process for As(III) removal by 15–38% at pH 5–9. The great difference in As(III) removal in these two processes was not ascribed to the uptake of arsenic by the MnO₂ formed in situ but to the different properties of conventional Fe(III) and the Fe(III) formed in situ. It was found that the presence of Ca²⁺ had limited effects on As(III) removal under acidic conditions but resulted in a significant increase in As(III) removal under neutral and alkaline conditions in the KMnO₄–Fe(II) process. Moreover, the effects of Ca²⁺ on As(III) removal in the KMnO₄–Fe(II) process were greater at lower permanganate dosage when Fe(II) was not completely oxidized by permanganate. This study revealed that the improvement of As(III) removal at pH 7–9 in the KMnO₄–Fe(II) process by Ca²⁺ was associated with three reasons: (1) the specific adsorption of Ca²⁺ increased the surface charge; (2) the formation of amorphous calcium carbonate and calcite precipitate that could co-precipitate arsenate; (3) the introduction of calcium resulted in more precipitated ferrous hydroxide or ferric hydroxide. On the other hand, the enhancement of arsenic removal by Ca²⁺ under acidic conditions was ascribed to the increase of Fe retained in the precipitate. FTIR tests demonstrated that As(III) was removed as arsenate by forming monodentate complex with Fe(III) formed in situ in the KMnO₄–Fe(II) process when KMnO₄ was applied at 18.6 μM. The strength of the “non-surface complexed” As–O bonds of the precipitated arsenate species was enhanced by the presence of Ca²⁺ and the complexation reactions of arsenate with Fe(III) formed in situ in the presence or absence of Ca²⁺ were proposed.