Ying Liu, Lianlian Sun

Nice Group Co., Ltd, China

Wanxu Wang

China Research Institute of Daily Chemical Industry, China

Zhiping Du

Resuorces and Environment Engineering Research Institute,Shanxi University, china

Introduction

Nonylphenol ethoxylates (NPnEO), usually a mixture with EO chain length ranging from 6 to 20,are highly cost effective surfactants with excellent performance and, consequently, they are widely used in industrial, institutional, commercial and household applications such as detergents, emulsifiers, wetting and dispersing agents, antistatic agents, demulsifiers and solubilisers.[1]Due to their extensive use, NPnEO are widespread and abundant in the environment,leading to many environmental problems.[2,3]Thus，the biodegradation behavior of NPnEO in the environment has raised public concern. Studies showed that NPnEO under both aerobic and anaerobic conditions can be biodegraded to more persistent and estrogenic metabolites consisting of NP monoto triethoxylates (NP1EO, NP2EO and NP3EO),nonylphenoxy carboxylates (NPnEC) and nonylphenol(NP).[4-6]

The problem of pollution by NPnEO usually originates from sewage treatment plants (STPs) by collecting and treating wastewater containing soluble NPnEO from municipalities as well as industry,they become a major source of NPnEO and their biodegradation products released to the environment .[5]Due to the continuous flow activated sludge simulation test (CFASST), which simulates the conditions prevailing in a sewage treatment plant, study of the biodegradation of NPnEO in the test can lead to a better understanding of their environmental fate. However,reports on the biodegradation behavior of NPnEO in CFASST are rare,[6,7]especially, there has been no detailed information about the biodegradation pathway of the benzene ring in NPnEO.

In this study, NP10EO, as a typical NPnEO, was subjected to continuous flow activated sludge simulation test and its biodegradation products were analyzed by Electrospray ionization-mass spectrometry (ESI-MS).The effect of hydraulic retention time (HRT) on the primary biodegradation and ultimate biodegradation was studied. The possible biodegradation pathways of NPnEO, especially the biodegradation pathway of the benzene ring in NPnEO, were proposed on the basis of the ESI-MS analysis.

Experimental procedure

Materials and apparatus

NP10EO, with an average of ten EO units, was obtained from Rhodia company. Peptone and beef extract were purchased from Beijing Aoboxing Biotech Company. All other reagents used were of analytical grade. Doubledistilled water was used.

Activated sludge used in the experiments originated from Taiyuan’s 4thsewage treatment plant, which handles typical municipal sewage.

The Husmann plant[8]which was built by China Research Institute of Daily Chemical Industry, was used.The volume of the aeration vessel was 3 L. The UV-1600 UV Spectrophotometer was bought from Beijing Rayleigh Analytical Instrument Company. An Agilent 6410 mass spectrometer with electrospray ionization and a quadrupole mass analyzer was used for the ESIMS analysis.

Synthetic sewage.The following were dissolved in 1L of water: peptone, 160 mg; meat extract, 110 mg; urea,30 mg; NaCl, 7 mg; CaCl2·2H2O, 4 mg; MgSO4·7H2O,2 mg; K2HPO4, 28 mg; NaHCO3, 98 mg; and the test surfactant, 30 mg. These concentrations correspond to ISO11733.[8]Chemical oxygen demand (COD) for the synthetic sewage with or without the surfactant was about 360 mg/L or 300 mg/L. The synthetic sewage was freshly prepared each day using tap water. Prior to its use for preparing the synthetic sewage, tap water was allowed to stand for 24 h in order to reduce its free chlorine concentration.

Procedures

The continuous flow activated sludge simulation test in the Husmann plant.Two plants were started by filling the aeration vessel with activated sludge having a concentration of suspended solids of 1 g/L.The synthetic sewage (without surfactant) was applied at the rate of 1 L/h (HRT 3 h) and 0.5 L/h (HRT 6h).The synthetic sewage containing NP10EO (30 mg L-1)was supplied after a 3-days delay. The duration of the experiments was 23 days. The temperature, pH,sludge retention time (SRT) and concentration of suspended solids and dissolved oxygen (DO) were controlled during the experiments. Temperature was kept in the range of 22±3°C. The pH was between 7.0 and 7.5. The concentration of suspended solids of activated sludge (MLSS) was controlled in the range of 2～2.5 g/L by removing the surplus activated sludge in the aeration vessel. The concentration of dissolved oxygen (DO) was between 3 and 4 mg/L, sludge retention time (SRT) was 7 days. Those parameters were measured in accordance with the Standard Method for Examination of Water and Wastewater.[9]Another experiment at the same operation conditions without adding NP10EO was performed at the same time as “blank” .

The samples of treated sewage were collected and analyzed every second day of the experiment, starting from the day NP10EO was supplied.

Determination of concentration of residual surfactant(modified CTAS method [10]).50 ml of wastewater and 50 mL of distilled water were added to a 250 ml separatory funnel, followed by 15 mL of ammonium cobalt thiocyanate reagent (620 g of NH4CNS and 280 g of Co(NO3)2· 6H2O dissolved in 1 L of distilled water) and 35.5 g of NaCl were added. After 15 mins, the mixture was extracted with 15 mL of chloroform three times. After layer separation, the chloroform extract was collected into a 50 mL calibrated flask and pure chloroform was added to the mark. The absorbance of the chloroform extract was determined at 319 nm in a 1 cm quartz cell. A blank sample was carried out simultaneously.The degree of primary biodegradation (η) of the test surfactant was calculated from the equation: η =[(ρo?ρt)/ ρo]×100%, where ρois the initial mass concentration,ρtis the mass concentration of t days, and ρ is calculated from the working curve between absorbance and mass concentration of the test surfactant.

Determination of ultimate biodegradation (COD method [11]).Ultimate biodegradation was determined from the percentage elimination of COD. For ultimate biodegradation, COD was measured for the influent and the effluent from the test and the blank units. COD was determined at least in duplicate to measure the ultimate biodegradation.

Ultimate biodegradation degree (η) of the test surfactant was calculated from the equation: η =[(CODo?CODt)/ CODo]×100 %, where CODois COD for the influent, CODtis COD for the effluent at t days.

Electrospray ionization-mass spectrometry analysis.Mass spectrometric analyses were performed to monitor the molecular weight distributions of NP10EO and its biodegradation intermediates.

Separation of the sample from the influent and effluent was performed as described in ISO 2268.[12]The sample was redissolved in 5 mL methanol for the ESI-MS assay.Because a lot of NaCl was added in the sample separation procedures, the ion peaks in the mass spectrum obtained were assigned as M + Na+ions.

ESI-MS determinations were performed by operating the mass spectrometer in the positive-ion mode. The mass spectra were acquired over the scan range m/z 50–1,000.The following operation parameters were used: source temperature, 350 °C; desolvation temperature, 400 °C;desolvation gas flow rate, 12 L/min; cone gas flow rate,50 L/h; ESI capillary voltage, 3.5 kV.

Results and Discussion

NP10EO primary biodegradation

The primary biodegradation of ethoxylated groups of NP10EO was determined. Curves illustrating the degree of primary biodegradation of NP10EO are shown in Figure 1.

The results indicate that the two curves were almost identical. Whether the HRT was 3 hours or 6 hours,it was clear that significant degradation of NP10EO occurred in the two systems after the beginning of the surfactant spiking, the degree of primary biodegradation of NP10EO was in excess of 90% after 6 days. Similar results in an aerobic batch system have also been observed by other researchers.[13,14]From 6 days to 20 days, biodegradation of NP10EO was steady. The average degrees of primary biodegradation between those days were 96.5 % (HRT 3 h) and 96.2 %(HRT 6 h), respectively. This indicates that the primary biodegradation of NP10EO readily occurred and could be almost complete; increasing the hydraulic retention time from 3 hours to 6 hours had no influence on the primary biodegradation of NP10EO.

Figure 1. Extent of primary biodegradation of NP10EO over time

NP10EO ultimate biodegradation

Figure 2 shows the ultimate biodegradation degree of NP10EO over time. The results indicate that a quick degradation of NP10EO occurred after the beginning of the surfactant spiking. After 6 days, biodegradation reached a plateau phase. The average removal rates of NP10EO in the plateau phase were 84.8% (HRT 3 h)and 87.3% (HRT 6 h), respectively, indicating that most NP10EO can be degraded ultimately. With longer HRT, the removal of NP10EO increased slightly.

Figure 2. Extent of ultimate biodegradation of NP10EO over time

The biodegradation products of NPnEO are more lipophilic than their parent compounds and tend to be adsorbed on sludge and sediments.[15]But due to limitations resulting from lab conditions, the concentrations of the metabolites in the sludge have not been determined in the experiment, thus the actual biodegradation degree was lower than that noted above.

Biodegradation pathways of NPnEO

In order to investigate the molecular weight distributions of nonylphenol ethoxylates and the biodegradation products, ESI-MS analyses were performed to samples extracted from the influent and effluent. Figure 3, 4 and 5 illustrate the mass spectrum of NP10EO in the influent and biodegradation products in the effluent at 20th days (HRT 3 h and 6 h),respectively. According to the results, each peak for the mass spectrum was assigned (Table 1).

Figure 3. ESI-MS spectrum of NP10EO in influent

Figure 4. ESI-MS spectrum of biodegradation products in effluent (HRT 3 h)

Figure 5. ESI-MS spectrum of biodegradation products in effluent (HRT 6 h)

The MS spectrum (Figure 3) shows that NPnEO formed abundant sodiated molecular ions (m/z: 419, 463,507, 551,595, 639, 683, 727, 771, 815, 859, 904, 948, and 992). It illustrates the distribution of the NP10EO mixture,containing NPEO oligomers with an EO group ranging from 4 to 17 units.

Table 1. Peak assignments

As can be seen from Figure 4, the principal biodegradation products formed ions m/z: 331, 375, 419, 463, 507, 551,595 and m/z: 301, 345, 389, 433, 477, 521, 565, 609, 653,697, 741, 785, 829, 873, 917. They were formerly assigned as M + Na+ions of NPnEO (n=2～8) and NPnEC (n=0～14),respectively. Some ions (m/z: 217, 261, 305, 349, 437; 365,409, 453) at low abundance were observed too, they were assigned as Carboxyalkylphenol ethoxylates (CAPnEO).Some molecular ions (m/z: 99, 129, 141, 155, 193, 225)lower than 300 were found which could be discussed as follows.

A comparison of the mass spectrum obtained from the effluent at HRT 6 h (Figure 5) with HRT 3h (Figure 4)showed that the main ions peak were basically identical,just the abundance was different. This means that the biodegradation products were basically the same, whether HRT was 3h or 6h.

As mentioned above, the main biodegradation products were: NPnEO (n=2～8), NPnEC (n=0～14) and Carboxyalkylphenol ethoxylates (CAPnEO, n=0～6). The disappearing of the long EO chain NPnEO (n=4～17) and the appearance of the short chain NPnEO (n=2～8) means that the EO chain shortening from NP10EO to shorter chain polyethoxylates took place, which was also proved by detection of HO(CH2CH2O)2H. But surprisingly,NP1EO, which always was found by other researchers,[4-7]has not been detected. This might be due to the low water solubility and high lipophilicity of NP1EO,[15]which was mainly adsorbed to sludge. NP was not been found either; this was in accordance with the generally accepted NPnEO degradation pathways.[16]The detection of NPnEC with different EO chains means that the terminal alcoholic group of NP10EO was oxidized (ω-oxidation)to the corresponding NPnEC, with shortening of the EO chain, or the formed NPnEC undergoes β-oxidation.The existence of CAPnEO indicates that the alkyl chain was broken down with the oxidation of the terminal methyl group (ω-oxidation) to the carboxylic acid, then the carboxylic acid can undergo β-oxidation and the two carbon fragments enter the tricarboxylic acid cycle as acetylCo-A.[17]The results found were in agreement with the NPnEO metabolic pathway evidenced by other authors.[16-19]

Carboxyalkylphenol polyethoxycarboxylates (CAPnEC)were regarded as the dominant metabolites in the aerobic treatment systems, in recent studies.[20,21]However, CAPnEC were not found in the effluent. As Jonkers et al.[20]and Di Corcia et al.[21]reported, CAPnEC started to accumulate after the short NPnEC formed during the biotransformation of NPnEO. HRTs (3–6h) in the study might not be enough for the formation and accumulation of CAPnEC. Similar results were also observed by Zhang et al.[14]

The mechanism of breakdown of NPnEO involved a degradation of the straight alkyl chain, polyoxyethylene chain and, finally, the benzene ring. But little evidence was observed of any degradation of the benzene ring in previous research.[17]

In this study, there exist some molecular ions (m/z:99, 129, 141, 155, 193, 225) lower than 300. They might be the product of the benzene ring degradation. Due to the fact that NPnEO had a similar molecular structure to linear alkylbenzene sulphonate (LAS), and the mechanism of breakdown of LAS had been studied rather thoroughly,[22]the possible biodegradation pathway of the benzene ring in NPnEO was assumed according to the biodegradation mechanism of the benzene ring in LAS (Figure 6).[22]As mentioned above, the straight alkyl chain of NPnEO could break down with ω/β-oxidation to carboxylic acid, and the polyoxyethylene chain of NPnEO could break down with EO-fission and ω/β-oxidation, so the assumption begun with HOOC(CH2)a-C6H4-OH (Figure 6).

Figure 6. The assumed biodegradation pathway of the benzene ring in NPnEO

According to the assumption, the possible sodiated molecular ions might occur in the mass spectrum . In the results, it was found that some peaks (m/z: 99, 129, 141,155, 193, 225) in the mass spectrum were in accordance with the possible sodiated molecular ions peaks calculated from the assumption (Table 1). That is to say, the proposed assumption was basically confirmed.

As mentioned above, the possible biodegradation pathways of NPnEO, especially the biodegradation pathway of the benzene ring, are shown in Figure 7.

Figure 7. The possible biodegradation pathways of NPnEO

Conclusions

The NPEO-bearing sewage was treated in the continuous flow activated sludge simulation test. The results reported in this paper demonstrated that the primary biodegradation of NP10EO was very easy and most NP10EO could be biodegraded ultimately,the increase in HRT had no influence on the primary biodegradation and had little influence on the ultimate biodegradation. These findings have significant environmental implications in terms of the biodegradation and assessment of NPnEO contamination in STPs.

The possible biodegradation pathways of NPnEO,especially the biodegradation pathway of the benzene ring in NPnEO, were proposed. To our knowledge, this is the first report on the biodegradation pathway of the benzene ring in NPnEO.

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