Simultaneous determination of C4-C9 alkylphenols and bisphenol A in environmental water at the Yellow River Estuary Area in China by gas chromatography-mass spectrometry

A liquid-liquid extraction combined with derivatization and gas chromatography-mass spectrometry (GC-MS) method was developed for the rapid determination of C4-C9 alkylphenols (4-tert-butylphenol, 4-n-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-n-octylphenol, 4-tert-octylphenol, 4-n-nonylphenol and 4-nonylphenol) and bisphenol A in the environmental water. The extraction solvent, extraction time, solvent volume, acidity, salt content and the distillation degree were optimized. After drying and dehydration, the extract was derivatized, detected by GC-MS and quantied by internal standard method. After the dehydration, the extract was derivatized with BSTFA, detected by GC-MS and quantied by internal standard method. The results showed that the relative standard deviations of the relative response factors of the target compounds were less than 20%, the method detection limits were 0.002 µg/L (cid:0) 0.006 µg/L, the relative standard deviations of the three spiked levels were 0.67% (cid:0) 13.7%, and the average recoveries of the actual water samples were 68.0% (cid:0) 122%. The method precision and accuracy were good. The method is simple, rapid, accurate, stable and reliable, which is suitable for the detection of target in water. The contents of C4-C9 alkylphenols and bisphenol A in groundwater, surface water, sewage, waste water and sea water samples in Dongying City located at the Yellow River Estuary were determined by this method. The results showed that the contents of 4-nonylphenol and bisphenol A in these samples were lower than the home and abroad requirements. and rapid analysis was established. Nine C4-C9 alkylphenols (4-tert-butyl phenol, 4-n-butyl phenol, 4-n-pentyl phenol, 4-hexyl phenol, 4-heptyl phenol, 4-octyl phenol, 4-tert-octyl phenol, 4-nonylphenol and 4-nonylphenol) and bisphenol A in water were analyzed. The accurate concentrations of target substances in groundwater, surface water, sewage, waste water and sea water were determined. bisphenol in A liquid-liquid extraction with derivatization and gas chromatography-mass spectrometry (GC-MS) After extraction was the pH value was less and the amount was determination


Introduction
As the typical environmental endocrine disruptors, alkylphenols and bisphenol A have been the focus of attention (Wen et al. 2020;Abu-Alsoud and Bottaro 2021;Liu and Zhao 2020;Murray et al. 2017). There is no domestic standard method to monitor the alkylphenols and bisphenol A in water in China. ASTM, ISO and the Japanese government have developed the corresponding monitoring standard methods (ISO 18857-2 2009;ASTM D7065 2011;ASTM D7574 2009;JIS K 0450-10-10 2006). However, these above monitoring methods were developed early and the research objects are single, which are limited to one or several compounds of 4-tert-octylphenol, nonylphenol and bisphenol A. This is related to the early people's understanding of the use of alkylphenols. The plastic industry developed early, and the nonylphenol and octylphenol were the most widely used in the plastic industry. As a result, the proportion of nonylphenol and octylphenol in the environmental water was high, and the other alkylphenols were not detected or the detection value was very low, which did not attract people's attention. There are many detection methods of alkylphenols and bisphenol A in water, among which the liquid-liquid extraction combined GC-MS method is a more studied method, but the research objects are also concentrated on one or several compounds of 4-tert-octylphenol, nonylphenol and bisphenol A (Selvaraj et al. 2014;Shen et al. 2015;Santhi et al. 2012;Dong et al. 2015;Wang et al. 2013;Martinez and Peñuela 2013). However, with the development of new plastics and liquid crystal products, many new C4-C9 alkylphenols have been widely used. The 4-tert-butylphenol is an important raw material for the production of p-tert-butylphenol formaldehyde resin. The 4-n-pentylphenol is an effective component of disinfectant, food preservative and deodorant. The 4-n-butylphenol, 4-n-hexylphenol and 4-n-heptylphenol are often used as liquid crystal raw materials and intermediates. The wide application of C4-C9 alkylphenols leads to the continuous entry of these new alkylphenols into the environmental water. These C4-C9 alkylphenols are also the endocrine disruptors, which can also affect the performance of human endocrine system. In 1998, the Ministry of environment of Japan listed 4-pentyl phenol and other alkyl phenols as the endocrine disruptors (Ministry of the Environment of Japan 1998). However, there is no standard method for the monitoring of these C4-C9 alkylphenol and bisphenol A in water in China, and there is no standard method developed by the authoritative institutions abroad.
The alkylphenol is a combination of phenol and alkyl chain. Due to the different length of alkyl chain, the physical and chemical properties of these C4-C9 alkylphenols are different, such as nonpolarity, adsorption capacity on the material surface and partition coe cient between aqueous phase and organic phase. The greater the difference of alkyl chain length, the greater the difference of these properties. The quantitative determination of these C4-C9 alkylphenols and bisphenol A in groundwater, surface water, sewage and wastewater, seawater and other actual water samples is an analytical technique for trace or ultra trace mixed components in complex matrix, which is di cult to extract, enrich, separate and detect. Liquid-liquid extraction has the advantages of good separation effect, simple operation and high repeatability, which is the most widely used organic matter extraction and enrichment technology Deng et al. 2018). GC-MS is the most advanced and reliable detection technology (Subuhi et al. 2020;Azzouz et al. 2020). After the derivatization, the polarity and boiling point of these alkylphenols and bisphenol A decreased, the volatility and stability increased, the anti-interference ability increased, and the samples could be stored for a long time for retest (Liu et al. 2019;Tan et al. 2019).
In this study, a liquid-liquid extraction combined with derivatization and GC-MS method with low detection limit, high recovery, good reproducibility and rapid analysis was established. Nine C4-C9 alkylphenols (4-tert-butyl phenol, 4-n-butyl phenol, 4-n-pentyl phenol, 4-hexyl phenol, 4-heptyl phenol, 4-octyl phenol, 4-tert-octyl phenol, 4-nonylphenol and 4-nonylphenol) and bisphenol A in water were analyzed. The accurate concentrations of target substances in groundwater, surface water, sewage, waste water and sea water were determined. Materials And Methods

Solution preparation and standard curve
Water treatment steps: Measured 500 ml water sample into the separating funnel, added 100 µl substitute working solution and mixed well. Then added 10 g sodium chloride, shaked to complete dissolution, added 30 ml dichloromethane, shaked violently for 10 min, standed for 5-10 min, separated layers, collected organic phase, extracted again for 1-2 times, combined organic phase and dehydrated with anhydrous sodium sulfate, and concentrated organic phase to 0.5 ml. Transfered the concentrated sample to a 1 ml volumetric ask, washed the concentrated ask with a small amount of dichloromethane, and combined the washing solution into the volumetric ask. Added 100 µl of internal standard working solution and 100 µl of derivatization reagent in turn, and diluted to 1 ml with dichloromethane. The samples were obtained by derivatization at room temperature for 1 h. 1 h, the target compounds were determined from low concentration to high concentration according to the chromatographic reference conditions. The retention time and quantitative ion response values of the standard series of target compounds and corresponding internal standards were recorded. The average relative response factor was used to draw the calibration curve, and the relative standard deviation (RSD) of the relative response factor (RRF) of the standard series of target compounds should not be more than 20%.

Actual water sample collection
According to the relevant environmental protection standards (HJ 91.1 2019; HJ/T 164 2020; HJ/T 493 2009), the sewage, river water, the drainage of wastewater treatment plant and Bohai Sea water were collected in Dongying City. The hydrochloric acid solution was added to adjust the pH of the water sample to be less than or equal to 2. The water sample should be lled with the sample bottle and sealed. It should be stored under 4 ℃ in dark and refrigerated. The extraction and derivation should be completed within 10 days, and the analysis should be completed within 5 days after the extraction.
(2) Mass spectrometry conditions Results And Discussion 2.1 In uence of extraction solvent 500 ml of laboratory water was added into the separating funnel, and then alkylphenol and bisphenol A working solution were added to make the concentration of target substance in the water sample 0.20 µg/L. The effects of different extraction solvents on the recovery of target compounds are shown in Table 2. According to the data in the table, dichloromethane has the best extraction effect.

In uence of extraction times
The effects of different extraction times of dichloromethane on the recovery of target compounds are shown in Table 3. It can be seen from the table that C4-C9 alkylphenols can be extracted completely after one extraction, and bisphenol A can be extracted mainly after two extraction. When dichloromethane is selected as the extraction solvent, the second extraction can meet the requirements.

In uence of extraction time
The in uence of different extraction time on the recovery rate of target compounds is shown in Table 4. It can be seen that the extraction time has a slight effect on the extraction e ciency. With the extension of the extraction time, the recovery rate of the target substance increases slightly. It can be seen from the table that the extraction time of 5 minutes is enough to fully recover the target substance in the water sample.

In uence of acidity in water
The effect of acidity in water on the recovery rate of target compounds is shown in Table 5. It can be seen from the table that when the pH value is higher than 6, the recoveries of 4-nonylphenol and 4-n-octyl phenol decrease, but the recoveries of other target compounds are not affected. Considering that acidic conditions are conducive to inhibiting bacteria in water and preventing bacteria from consuming alkylphenol organics, the pH value of water sample is usually less than 2.   Table 7. It can be seen from the table that adding sodium chloride can indeed improve the recovery rate of the target substance, but the improvement effect is not signi cant. The main reason is that the recovery rate data is high enough without adding salt. After comprehensive consideration, 5g sodium chloride can be added in water sample extraction. In the standard of organic matter determination in water, when liquid-liquid extraction is used, it is related to whether the extraction liquid is dried during the concentration process. For C4-C9 alkylphenols, some of the target compounds are liquid with vapor pressure, and volatilization occurs when the vacuum is appropriate. Some of the solid targets are powder or velvet with low density, and they also have drift loss when the vacuum is appropriate. The in uence of organic phase concentration to 0.5 ml or concentration to dryness and continuous rotary evaporation for different time on the recovery of target substance is shown in Table 8. It can be seen from the table that drying can signi cantly reduce the detection results, especially for some alkylphenols (4-tert-butylphenyl, etc.) which are liquid at room temperature. The experimental results are consistent with the above analysis. Finally, in the process of concentration, the treatment mode without drying was selected. The chromatogram of the standard sample of the target analytes is shown in Fig 1. It can be seen from the gure that under the conditions of reference gas chromatography-mass spectrometry, the peaks of each compound are independent and clear. Due to the use of derivatization treatment, there is basically no tailing phenomenon of phenolic compounds. 4-nonylphenol is a mixed peak of one group of isomers, and other compounds are single peaks.

Standard curve
The linear correlation coe cient of the standard curve of the object to be measured is shown in Table 9. It can be seen from the table that the standard curves of the target compounds are linear. The detection limit of the method was con rmed according to the relevant environmental protection standard method (HJ 168 2020), and seven laboratory blank samples with or without the target objects working solution were analyzed continuously. Because 4-tert-butylphenyl, 4-tert-octylphenyl, 4nonylphenol and bisphenol A were detected in the laboratory blank, the detection limits of these target substances were determined by the method of laboratory blank without standard addition and the detection limits of the remaining analytes were determined by the method of laboratory blank with standard addition. The concentration of these remaining analytes in the laboratory blank sample with standard addition is all 0.010 μg/L. The lower limit of determination is 4 times of the detection limit and both of which are listed in Table 9. These detection limits of the target compounds changes from 0.002 μg/L to 0.006 μg/L and the lower limit of determination from 0.008 μg/L to 0.024 μg/L.

Recovery and precision
Taking the laboratory water without the target analytes as the blank matrix, C4-C9 alkylphenols and bisphenol A standard solutions with three different concentration levels were added respectively. The pretreatment and test were carried out according to the method steps. Each concentration level was determined for six times in parallel. The relative standard deviation results are shown in table 10. Take surface water, sewage and wastewater as sample matrix, add C4-C9 alkylphenols and bisphenol A standard solution with matrix concentration level of 0.5-3 times, repeat the above laboratory test, and the average recovery results are shown in Table 11. The relative standard deviations of the target samples ranged from 0.67% to 13.7%, and the average recovery ranged from 68.0% to 122%, which indicates that the precision and accuracy of the experiment are good.  Note: surface water 1 is drinking water, surface water 2 is yellow river water, surface water 3 is Guangli River water, surface water 4 is Xiaoqing River water, sewage 1 is outlet water of a sewage treatment plant in Dongying City, sewage 2 is outlet water of a sewage treatment plant in Guangrao County, Dongying City, wastewater 1 is outlet water of an industrial wastewater treatment plant in Dongying City, and wastewater 2 is outlet water of an oil processing enterprise in Dongying City, wastewater 3 is from the workshop of an oil processing enterprise in Dongying City, wastewater 4 is from the workshop of a bisphenol A production enterprise in Dongying City, and wastewater 5 is from the workshop of a bisphenol A production enterprise in Dongying City.

Conclusions
(1) Aiming to monitor of C4-C9 alkylphenols and bisphenol A in water, A liquid-liquid extraction combined with derivatization and gas chromatographymass spectrometry (GC-MS) method was used to optimize the extraction solvent, extraction times, extraction time, extraction solvent volume, acidity, salt content, rotary distillation concentration degree and other conditions. After optimization, dichloromethane was used as the extraction solvent, the extraction time was 5 min, the extraction solvent was 30 ml, the pH value was less than 2, and the amount of salt was 5 g.
(2) The method has the advantages of high sensitivity, low detection limit, accurate quanti cation and good precision, which can meet the needs of the determination of C4-C9 alkylphenols and bisphenol A in water.

Supplementary Files
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