Competition between polyphosphate- and glycogen-accumulating organisms in enhanced-biological-phosphorus-removal systems: effect of temperature and sludge age.

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.     

A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus Accumulibacter Phosphatis as PAO and Candidatus Competibacter Phosphatis as GAO). The method showed to be potentially suitable to estimate the PAO and GAO populations regarding the total PAO-GAO biomass. It could be used, not only to evaluate the performance of EBPR systems, but also in the calibration of potential activated sludge mathematical models, regarding the PAO-GAO coexistence.     

Functionally relevant microorganisms to enhanced biological phosphorus removal performance at full-scale wastewater treatment plants in the United States

The abundance and relevance ofAccumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen-accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P(rel)/HAc(up)) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P(rel)/HAc(up) and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to-phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.     

剩余污泥水解酸化液磷去除的影响因素研究

城市污水厂剩余污泥水解酸化后可产生高浓度挥发性有机酸(VFAs),其中的乙酸和丙酸是增强生物除磷(EBPR)工艺的有利基质.但水解酸化液中含有大量的磷,如不进行处理就作为碳源回用到污水处理工艺中,势必增加除磷负荷.利用鸟粪石沉淀法可以去除污水中的磷.对城市污水厂剩余污泥水解酸化液形成鸟粪石的影响因素进行了试验研究.结果表明,在最佳工艺条件下,正磷和总磷的去除率分别可达92.5%和83.8%.     

The mechanism of enhanced biological phosphorus removal washout and temperature relationships.

In this study, the combined effects of temperature and solids retention time (SRT) on enhanced biological phosphorus removal (EBPR) performance and the mechanism of EBPR washout were investigated. Two pilot-scale University of Cape Town (South Africa) systems fed with synthetic wastewater were operated at 5 and 10 degrees C. The results showed that the phosphorus removal performance was optimum at total SRT ranges of 16 to 24 days and 12 to 17 days for 5 and 10 degrees C, respectively, and steady-state phosphorus removal was greater at the lower temperature. Higher SRT values of up to 32 days at 5 degrees C and 25 days at 10 degrees C slightly reduced EBPR performance as a result of increased extent of endogenous respiration, which consumed internally stored glycogen, leaving less reducing power for poly-hydroxy alkanoate (PHA) formation in anaerobic stages. The phosphorus-accumulating organism (PAO) washout SRTs of the systems were determined as 3.5 days at 5 degrees C and 1.8 days at 10 degrees C, considerably less than the washout SRTs of nitrifiers. Polyphosphorus, the main energy reserve of the EBPR bacterial consortium, was not completely depleted, even at washout points. The inability of EBPR biomass to use glycogen to generate reducing power for PHA formation was the major reason for washout. The results not only suggest that glycogen mechanism is the most rate-limiting step in EBPR systems, but also that it is an integral part of EBPR biochemistry, as proposed originally by Mino et al. (1987), and later others (Pereira et al., 1996, Erdal et al., 2002; Erdal, Z. K., 2002). The aerobic washout SRT values (2.1 and 1.2 days for 5 and 10 degrees C, respectively) of this study did not fit the linear line for PAO washout developed by Mamais and Jenkins (1992). Perhaps this was because the feeds used during this study were chemical-oxygen-demand-limited (acetate-based synthetic feed), whereas the feeds used for their study were phosphorus-limited (external acetate added to domestic wastewater), resulting in different ratios of PAOs and nonPAOs in the biomass.     

Mass balance assessment of triclosan removal during conventional sewage treatment

The antimicrobial agent triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol; TCS) is a member of a larger group of polychlorinated binuclear aromatic compounds frequently associated with adverse environmental and human health effects. Whereas the structure and function of TCS would suggest significant resistance to biotransformation, biological wastewater treatment currently is considered the principal destructive mechanism limiting dispersal of and environmental contamination with this compound. We explored the persistence of TCS in a typical full-scale activated sludge US sewage treatment plant using a mass balance approach in conjunction with isotope dilution liquid chromatography electrospray ionization mass spectrometry (ID-LC-ESI-MS) for accurate quantification. Average influent and effluent concentrations (mean +/- SD) of 4.7+/-1.6 and 0.07+/-0.06 microg 1(-1), respectively, revealed an apparent (liquid-phase) removal efficiency of 98+/-1%. However, further analyses demonstrated that the particle-active TCS (80+/-22% particle-associated in influent) was sequestered into wastewater residuals and accumulated in dewatered, digested sludge to concentrations of 30000+/-11000 microg kg-1. Overall, 50+/-19% (1640+/-610 g d-1) of the disinfectant mass entering the plant (3240+/-1860 g d-1) remained detectable in sludge, and less than half of the total mass (48+/-19%) was biotransformed or lost to other mechanisms. Thus, conventional sewage treatment was demonstrated to be much less effective in destroying the antimicrobial than the aqueous-phase removal efficiency of the plant would make believe. Furthermore, study findings indicate that the common practice of sludge recycling in agriculture results in the transfer of substantial quantities of TCS to US soils used, in part, for animal husbandry and crop production.     

Pentachlorophenol association with fulvic acids from recycled wastes

The apparent water solubility of pentachlorophenol was measured at pH=6 and at 25 degrees C in pure water, aqueous solutions of three salts (NaCl, KNO(3) and CaCl(2) at 0.010, 0.10 and 1.0M) and in aqueous solutions of three fulvic acids samples extracted from a natural soil (sFA), composted sewage sludge (csFA) and composted livestock's material (lsFA). A solubility enhancement method was developed for the measurement of partition coefficients (K(oc), L/kg organic carbon). Pentachlorophenol associates strongly with the fulvic acid samples and the calculated K(oc) were the following (averages and standard deviations): (sFA) (211+/-22) x 10(2), (csFA) (253+/-26) x 10(2), (lsFA) (235+/-10) x 10(2). For comparison purposes the K(oc) for pyrene were also calculated for the three FA samples and were the following: (sFA) (119+/-10) x 10(2), (csFA) (239+/-21) x 10(2), (lsFA) (92+/-10) x 10(2). The analysis of variance (one-way ANOVA) of the effect of the type of FA sample on the solubilization of pentachlorophenol and pyrene shows that this factor causes significant differences on the aqueous solubilization of these two organic substances.     

Process performance and polyphosphate-accumulating organism-glycogen-accumulating organism communities in an anaerobic/anoxic/oxic system operated with different carbon sources

A pilot plant anaerobic/anoxic/oxic (A2/O) system fed with domestic wastewater was operated to examine the effect of varying different types of carbon source (acetic acid, propionic acid, and glucose), added as a complement to the wastewater, on the (1) process performance and (2) microbial population. The operational condition that lead to a significant removal of total nitrogen (82%) was achieved with acetic acid. When the complementary carbon source was propionic acid, an improved removal efficiency of orthophosphate (97%) was observed. Because this finding was concurrent with higher polyphosphate-accumulating organism (PAO) population fractions detected using fluorescent in situ hybridization analysis (41.9 +/- 3.0%), it suggests that members of PAO populations that were able to reduce nitrate gained importance over PAO members that could not, thus improving the denitrifying phosphorus removal.     

Recovery of polyhydroxyalkanoate from activated sludge in an enhanced biological phosphorus removal bench-scale reactor.

A sequencing batch reactor was used to study the possibility of harvesting polyhydroxyalkanoate (PHA) from enhanced biological phosphorus removal (EBPR) processes without compromising treatment quality. Because, in EBPR, the highest PHA concentrations are observed after exposure of the sludge to anaerobic conditions, PHA accumulation was evaluated with collection of waste activated sludge (WAS) at the end of the anaerobic stage, in addition to the traditional removal after the aerobic stage. The system achieved good phosphorus removal, regardless of the point of WAS collection. When sludge was harvested at the end of the anaerobic stage, the PHA content of the sludge ranged from 7 to 16 mg PHA/100 mg mixed liquor volatile suspended solids. Although this level of PHA production is below levels obtained with pure cultures, the demonstrated ability to harvest PHA, while simultaneously satisfying phosphorus removal in an EBPR process, is a key initial step towards of the use of wastewater treatment plants for PHA production.     

Responses of indicator bacteria to forest soil amended with municipal sewage sludge from aerated and non-aerated ponds

The survival of faecal and total coliform bacteria and Streptococcus faecalis (indicator bacteria) were monitored in experimental plots receiving various amounts of biological or dephosphatation municipal sewage sludge. Biological sludge was applied on coniferous and mixed regenerative forest soils. The results show that except for total coliforms, there was little significant treatment effect on the indicator bacteria numbers in all of the sites. In most cases, there was a significant time effect, indicating that bacterial populations vary over time, according to environmental factors such as temperature, moisture or nutrient level conditions. In total coliform bacteria, populations varied over time but also in function of treatment levels. The present study suggests that even if the standards of Québec (Canada) for sludge application are respected, there may be some risk for bacterial contamination depending on the types of sewage sludge and forest habitat. Although there is only a short-term effect, high slopes can influence the movement of the indicator bacteria and may potentially cause some contamination problems.     

Enhancing the performance of sequencing batch reactors by adding crushed date seeds to remove high concentrations of 2,4-dinitrophenol

Wastewater treatment systems using simultaneous adsorption and biodegradation processes have been successful in treating toxic pollutants present in industrial wastewater. The goal of this investigation was to assess the effectiveness of date seeds in reducing the toxic effects of 2,4-dinitrophenol (DNP) on activated sludge microorganisms. Two identical sequencing batch reactors (SBRs) (4-L glass vessel), each with a 3.5-L working volume, were used. The initial DNP concentrations in the reactor were 50, 75, 100, 250, and 500 mg/L. The reactor amended with date seeds was capable of degrading DNP at significantly greater rates (11 +/- 2.5 mg/L x h) than the control SBR (4 +/- 1.2 mg/L x h) at a 95% confidence level. Date seeds can be added to the mixed liquor of activated sludge treatment plants to remove high concentrations of DNP from wastewater, to protect the treatment plant against toxic components in the influent and enhance the settling characteristics of the mixed liquor.     

Effects of oxalic acid on the regrowth of heterotrophic bacteria in the distributed drinking water

Three laboratory-scale water pipe systems were set up to study the effects of adding oxalic acid on the bacterial regrowth and biofilm formation in the distributed drinking water. The results of water pipe experiment displayed that around 38% carbon in the oxalic acid could be converted to bacterial biomass. The maximum HPCs in biofilm were equal to 3.5x10(4), 3.38x10(5) and 2.8x10(6) CFUcm(-2) while the maximum HPCs of free bacteria were equal to 1.2x10(3), 2.54x10(3) and 3.78x10(4) CFUml(-1) for the blank and with addition of 10 and 50 micrograms OA eq-Cl(-1), respectively. These results imply that the addition of oxalic acid to distributed water has positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in water pipe. This effect is enhanced with addition of high-level oxalic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials (BRPs) of the blank were equal to 4.25x10(3), 1.46x10(4), 4.9x10(4) and 7.54x10(4) CFUml(-1) for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results show that the biological stability of distributed drinking water is the highest in treatment plant effluent, the moderate in the commercial area and mixed area, and the lowest in the residential area.     

16S ribosomal RNA tools identify an unexpected predominance of Paenibacillus-like bacteria in an industrial activated sludge system suffering from poor biosolids separation.

Molecular biology tools targeting 16S ribosomal RNA (16S rRNA) were used to identify a predominant bacterial population in a full-scale dairy wastewater activated sludge system suffering from poor biosolids separation. Gram and acridine orange staining indicated that viable, Gram-positive microorganisms were present in samples removed from the influent waste stream and represented approximately 50% of total cell counts in samples removed from the mixed liquor. Subsequently, the "full-cycle 16S rRNA approach" showed that phylogenetic relatives of Paenibacillus spp., a low guanine-plus-cytosine percent DNA-content, Gram-positive microorganism, represented up to 30% of total 4,6-diamidino-2-phenylindole (DAPI)-stained cell counts in samples of mixed liquor. Although fluorescent in situ hybridizations with 16S rRNA-targeted oligonucleotide hybridization probes identified Paenibacillus-like spp. in samples removed from the influent waste stream, their abundance was less than 10% of total stained cell counts. Results of this study suggest that Paenibacillus-like spp. were present in low abundance in the influent waste stream, increased in relative abundance within the treatment system, and should be examined further as a candidate bacterial population responsible for poor biosolids separation. This study demonstrates that the full-cycle 16S rRNA approach can be used to identify candidate bacterial populations that may be responsible for operational upsets in full-scale activated sludge systems without prior information from cultivation or microscopic analyses.     

PCB contaminated dust on indoor surfaces--health risks and acceptable surface concentrations in residential and occupational settings

Polychlorinated biphenyls (PCBs) have been used in diverse purposes such as indoor paints. Removal of these paints with dust creating techniques, like sandblasting, will result in contamination of building surfaces with PCB-containing dust. Objectives of this study was to analyze the PCB concentrations on surfaces after sandblasting with silica using wipe samples and estimate the resulting health risks and further calculate the risk based acceptable PCB surface concentrations that do not cause incremental lifetime cancer risk higher that 10(-5) or does not cause immunosupression effects in residential use or in occupational settings. Both deterministic and probabilistic approaches were used. The total PCB concentrations on surfaces ranged from 10 to 1100 microg/m(2). Estimated cancer risk was 1.2 x 10(-4) for childhood exposure, 1.3 x 10(-5) for adult residents and 1.5 x 10(-5) for occupational exposure. Probabilistic risk assessment revealed that point estimates were quite reasonable and located between 45th and 79th percentiles on probabilistic distribution of risk. The noncancer risks were calculated as hazard quotients (HQ) which ranged from 3.3 to 35 depending on the exposure scenario. Acceptable surface concentrations based on noncancer effects that are protective for 95% of exposed population were 7 microg/m(2) for residential use, 65 microg/m(2) for residential use if only adults will be exposed and 140 microg/m(2) for occupational use. Preliminary cleanup experiment revealed that when contaminated dust was carefully removed with industrial vacuum cleaner and further washed with terpene containing liquid the surface concentration dropped below the acceptable levels calculated in this study.     

Determination of growth rate and yield of nitrifying bacteria by measuring carbon dioxide uptake rate.

Nitrifier growth parameters--the maximum growth rate (microAmax) and yield (YA)--were estimated by measuring the rate of carbon dioxide uptake and additional rates of oxygen uptake and ammonia (or nitrite) use. Batch tests in a combined titrimetric and offgas analyzer with enriched Nitrobacter and Nitrosomonas cultures and an activated sludge sample were performed. The measured microAmax values for the Nitrobacter and Nitrosomonas cultures were 0.67 +/- 0.03 day(-1) and 0.54 +/- 0.09 day(-1), while the YA values were 0.072 +/- 0.01 g volatile suspended solids (VSS) x g nitrogen (N)(-1) and 0.14 +/- 0.02 gVSS x gN(-1), respectively. For the activated sludge sample, microAmax was observed to increase with pH (microAmax = 0.72 x 3.3(pH-7.1)) over the range 6.8 to 7.1. All microAmax and YA values determined by this method were similar to those previously reported. Compared with other microAmax and YA estimation methods, this method allows for unique microAmax and YA estimations for given conditions from a single experiment.     

An observation on sludge granulation in an enhanced biological phosphorus removal process

A sequencing batch reactor (SBR) seeded with flocculated sludge and fed with synthetic wastewater was operated for an enhanced biological phosphorus removal (EBPR) process. Eight weeks after reactor startup, sludge granules were observed. The granules had a diameter of 0.5 to 3.0 mm and were brownish in color and spherical or ellipsoidal in shape. No significant change was observed in sludge granule size when operational pH was changed from 7 to 8. The 208-day continuous operation of the SBR showed that sludge granules were stably maintained with a sludge volume index (SVI) between 30 to 55 mL/g while securing a removal efficiency of 83% for carbon and 97% for phosphorus. Fluorescent in situ hybridization (FISH) confirmed the enrichment of polyphosphate accumulating organisms (PAOs) in the SBR. The observations of sludge granulation in this study encourage further studies in the development of granules-based EBPR process.     

混合型污水处理厂原位生物强化脱氮中试及微生物群落结构分析研究

针对混合型污水处理厂进水水质波动大、C/N低、总氮稳定达标困难等共性问题,开发出适宜于低C/N、贫营养等苛刻环境条件的高效脱氮菌剂.现场中试结果表明:经过脱氮菌强化后,生化系统脱氮效率提升8.5百分点,出水平均总氮较强化前下降3 mg/L,出水水质稳定性明显提升,生物强化效果显著.在不补加碳源条件下,深度处理出水总氮完...     

Effect of dissolved oxygen on biological nutrient removal by denitrifying phosphorus-accumulating organisms in a continuous-flow system

A laboratory-scale continuous-flow system with an anaerobic/anoxic/aerobic configuration was set up to study the effect of oxygen in the internal recycle stream; of particular interest was its performance of denitrifying phosphorus-accumulating organisms (DPAOs). It was found that, by using a degas device, the dissolved oxygen in the nitrate recycle stream was effectively decreased from 0.1 +/- 0.02 to 0.01 +/- 0.01 mg/L. This provided a favorable condition for DPAOs to grow under an anoxic condition and thus be sustained successfully in the system. When the degas device was removed from the system, the dissolved oxygen concentration in the anoxic reactor increased to 0.1 +/- 0.02 mg/L. The proliferation of the denitrifying glycogen-accumulating organisms (DGAOs) population and deterioration of DPAOs performance was observed. The increased population of DGAO/GAOs, which competed for the carbon source with DPAO/ PAOs, resulted in a poor performance of biological phosphorus removal.     

降低负荷以提高反硝化颗粒污泥的稳定性

在上流式污泥床反应器（USB）内，接种好氧活性污泥，以甲醇为碳源，NO-3为电子受体，经过40多d培养，得到良好的反硝化颗粒污泥，粒径2～3 mm，MLSS为36 g/L，氮去除速率和COD去除速率分别在0.15 g NO₃-N/(g VSS·d)和0.8 g COD/(g VSS·d)。当负荷提高至6.44 g NO₃-N/(L·d)继续运行1周后，观察到反应器内颗粒污泥出现上浮，浓度降低，颗粒粒径多数在3～5 mm，外观呈乳白色。为恢复反应器稳定运行，当负荷降至3.86 g NO₃-N/(L·d)时，上浮现象减轻，当负荷降至2.57 g NO₃-N/(L·d)时，上浮现象消失，颗粒污泥密度由不稳定时的1.0018 g/cm³提高到1.0126 g/cm³，颗粒粘连现象基本消失，污泥氮去除速率在0.19 g NO₃-N/(g VSS·d)，连续运行30 d，系统保持稳定。分析认为，颗粒污泥表面微生物生长速度过快是导致不稳定的主要因素，较长的泥龄有利于系统稳定。     

Biodegradation of endosulfan-contaminated soil in a pilot-scale reactor-bioaugmented with mixed bacterial culture

A novel mixed bacterial culture was enriched from an endosulfan (6, 7, 8, 9, 10, 10 - hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 3, 4-benzo (e) dioxathiepin-3-oxide) processing industrial surface soil. The cultures were successful in the degradation of aqueous phase endosulfan in both aerobic and anaerobic conditions. Using the cultures, endosulfan degradation in silty gravel with sand (GM) was examined via pilot scale reactor at an endosulfan concentration of 0.78 +/- 0.01 mg g(- 1) of soil, and optimized moisture content of 40 +/- 1%. During operation, vertical spatial variability in endosulfan degradation was observed within the reactor. At the end of 56 days, maximum endosulfan degradation efficiency of 78 +/- 0.2% and 86.91 +/- 0.2% was observed in the top and bottom portion of the reactor, respectively. Both aerobic and anaerobic conditions were observed within the reactor. However, endosulfan degradation was predominant in anaerobic condition and the total protein concentration in the reactor was declined progressively down the soil depth. Throughout the study, no known intermediate metabolites of endosulfan reported by previous researchers were observed.