Consequently, many PPCPs/EDCs are routinely found in WWTP products . At the same time, land application of treated wastewater and bio-solids is increasing . Although these compounds are usually detected at trace levels in soils and plant tissues , there is continual input of these biologically active compounds. Better knowledge of the extent and composition of PPCP/EDC accumulation in plants is needed to improve our understanding of the current and future risk to human health.As natural resources are stressed by population growth, urbanization, and climate change, previously under-utilized waste materials such as treated wastewater and bio-solids from wastewater treatment plants are increasingly being explored and used. For instance, about 3.6 × 109 cubic meters of treated wastewater is currently reused in the U.S. for purposes including agricultural and landscape irrigation, and water reuse is growing by 15% a year . Similarly, approximately 6 × 106 metric tons of bio-solids are produced each year in the U.S., of which about 60% is applied to land . Regulations governing such reuses are mostly concerned with pathogens, nutrients, and heavy metals . However, studies over the last two decades have shown that numerous anthropogenic chemicals, such as pharmaceutical and personal care products and endocrine disrupting chemicals , are present in treated wastewater and bio-solids . Many of these chemicals are known to have unintended biological effects on non-target organisms at low levels . Therefore, the beneficial reuse of these waste materials for irrigation or soil amendmentintroduces contaminants into the soil environment and may pose risks to terrestrial ecosystems and human beings through dietary exposure . In general, the fate of a xenobiotic in soil includes complete mineralization ,vertical planting tower conversion to transformation products, and formation of bound residue .
Mineralization of a compound is viewed as complete detoxification, while formation of bound residue is also generally considered a decontamination process . In soil, PPCP/EDCs may undergo microbially-mediated transformations, processes that are greatly influenced by both the soil microbial community and the physico-chemical properties of PPCP/EDCs . The formation of transformation products poses unknown risks as the new products may have biological activity . However, to date, most studies on the fate of PPCP/EDCs in soil have only considered removal of the parent compound while ignoring fate pathways. In this study, with the coupled use of 14C-labeling and chromatographic separation, we quantitatively characterized mineralization and formation of bound residue, as well as disappearance of the parent compound and formation of transformation products, of four commonly occurring PPCP/EDCs, i.e., bisphenol A , diclofenac , naproxen, and nonylphenol , under different soil conditions. Several transformation products of BPA and DCL were also identified. These PPCP/EDCs appear frequently in treated wastewater and bio-solids , but little information is available on their complete fate in soil. More knowledge of the complete fate of PPCP/EDCs in soil may be used to improve risk evaluation for land application of treated wastewater and bio-solids. Agricultural soils were collected from the University of California’s South Coast Research and Extension Center in Irvine, CA and from the University of California’s Hansen Agricultural Center in Ventura, CA . A third soil was collected from a treated wastewater recharge basin at the Riparian Preserve at Water Ranch in Maricopa, AZ . Soils were collected from the surface layer . After air-drying, soil was passed through a 2 mm sieve.
To examine the effect of organic matter, a sub-sample of the Irvine soil was amended with sieved redwood compost at 50% to create the Irvine Amended soil treatment. To understand the role of soil microorganisms, another sub-sample of Irvine soil was autoclaved at 121°C for 45 min on two consecutive days to create the Irvine Sterilized treatment.The field capacity of each soil was determined using the pressure chamber method, where -33 J/kg of hydraulic head was applied to saturated soil . Table 3.1 lists selected soil properties. Soil respirometers were constructed by suspending a 2 mL glass vial in a 40 mL amber glass bottle with a screw-cap lined with a septum. During incubation, 1.0 mL of 1M NaOH solution was deployed in the 2 mL vial to trap 14CO2 from mineralization. A syringe needle was inserted through the septum to enable the sampling and refill of the NaOH solution to monitor mineralization kinetics. A working solution was prepared for each 14C-PPCP/EDC in water. Air-dried soil, equivalent to 10 g dry weight, was placed in the amber bottle and spiked with 0.8 mL of a working solution containing about 3 × 105 dpm radioactivity, making an initial concentration in soil of 12.6 µg/kg for BPA, 69.3 µg/kg for DCL, 46.4 µg/kg for NPX, or 52.8 µg/kg for NP. Deionized water was added to reach field capacity in each soil, which equated to 35% of the total water capacity for Irvine soil and Irvine Sterilized soil, 21% for Irvine Amended soil, 47% for Maricopa soil, and 45% for Ventura soil. Each soil sample was manually mixed to achieve homogenization. The sample bottles were closed, and then NaOH solution was injected into each suspended vial. All soil respirometers were incubated at room temperature . Respirometers were opened briefly on a weekly basis for aeration and deionized water was added gravimetrically as needed to maintain the soil water content. On 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 102, and 112 d after the treatment, the NaOH solution in each respirometer was exchanged with new NaOH solution using a disposable syringe.
The used solution was placed in a 7 mL glass scintillation vial and mixed with 4 mL of Ultima Gold Scintillation Cocktail , followed by measurement of 14C on a Beckman LS 5000TD Liquid Scintillation Counter . On day 0, 3, 14, and 112, three soil samples from each treatment were transferred into a freezer for extraction and analysis of extractable and bound residues. Soil samples were extracted using EPA Method 1694. In brief, soil samples were removed from the freezer and the thawed soil was transferred to a 50 mL polypropylene centrifuge tube. The soil was sequentially extracted with 35 mL of freshly prepared phosphate buffer -methanol twice and 20 mL of methanol once. For each extraction cycle, the centrifuge tubes were mixed at 260 rpm for 1 h on a horizontal shaker and then centrifuged at 2300 rpm for 15 min. The supernatant was decanted into a 100 mL glass flask, from which a 3 mL sub-sample was removed for analysis on LSC todetermine the total extractable 14C residue. The remaining solvent extract was capped and stored at 4 °C until further analysis. After the sequential solvent extraction,vertical hydroponic farming the soil was air-dried in the fume hood and then 1.0 g aliquots were combusted on an OX-500 Biological Oxidizer at 900 °C for 4 min. The evolved 14CO2 was trapped in 15 mL of Harvey Carbon-14 cocktail , followed by measurement on LSC to determine the total bound 14C residue. The recovery of 14C in soil was determined to be 71-110% by combusting spiked soil samples and was used to correct for the actual amount of 14C in soil. The soil extracts were prepared for analysis of parent and transformation compounds by a method modified from Wu et al. . In brief, selected extracts were removed from the refrigerator and mixed with 1200 mL of deionized water, such that methanol was less than 5% of the total solution. The aqueous sample was then passed through a solid phase extraction cartridge at a rate of 5 mL/min. The cartridge was pre-conditioned with 5 mL each of methylene chloride, methanol, and ultra-pure water. A 6 mL sub-sample of the filtrate that passed through the cartridge was collected and analyzed on LSC to determine the presence of any 14C not retained on the solid phase. The cartridges were then dried under nitrogen gas and eluted with 7 mL methanol. The eluent was condensed to 250 µL under a gentle nitrogen flow and transferred to a 2 mL glass vial. The condensing vessel was rinsed with 200 µL of methanol and the rinsate was added to the eluent in the glass vial. A 50 µL aliquot of non-labeled parent standard stock solution was spiked into each vial to make the final sample volume to 500 µL. To characterize the extractable residue, a 50 μL aliquot of the prepared extract was injected into an Agilent 1100 Series high performance liquid chromatography with an ultraviolet detector. A Dionex Acclaim-120 C18 RP column was used for separation at a flow rate of 1.0 mL/min at 35 °C.
Mobile phase A was ultra-pure water acidified with 0.2% acetic acid and mobile phase B was acetonitrile. The ratio of mobile phase A to B was 60:40 for BPA, 50:50 for DCL, 60:40 for NPX, and 25:75 for NP, with corresponding UV wavelengths of 280, 284, 278, and 280 nm, respectively, for positioning the parent compounds. The HPLC eluent was fractionated in 1 min increments using an automated fraction collector . Each fraction was mixed with 4 mL of cocktail for analysis of 14C to monitor the distribution of 14C as a function of run time. To identify transformation products, extracts from BPA and DCL treatments were further analyzed on an ACQUITY ultra-performance liquid chromatography system using an ACQUITY UPLC BEH C18 column at 40 °C. Mobile phase A was 0.001% formic acid in water and mobile phase B was methanol. The following mobile phase program was used: 0 – 0.5 min, 5 – 50% B; 0.5 – 12 min, 50 – 100% B; 12 – 13 min, 100% B; 13 – 16 min, 5% B. Analysis was performed with a Waters Micromass triple quadrupole detector equipped with an electrospray ionization source in the negative mode. Parameters of MS/MS were as follows: source temperature, 120 °C; desolvation temperature, 350 °C; capillary voltage, 3.0 kV; cone voltage, 20 V; desolvation gas flow, 600 L/h; cone gas flow, 50 L/h. Standards were run in scan and daughter modes to identify the most robust transition pattern and cone voltage for each compound, and the optimized parameters are listed in Supplemental Table S3.1. Quantitative analysis was performed in the multiple reaction monitoring mode. All data were processed using MassLynx 4.1 software . The extractable fraction of xenobiotics is often used to represent the bio-available fraction that may illicit biological effects . Incubated soil samples were extracted with solvents to determine the extractable residue of spiked 14C-PPCP/EDCs. Figure 3.2 depicts the extractable residue of treatments after 112 d of incubation. For all compounds in all soils, the extractable residue decreased over the incubation period. For example, in Irvine soil spiked with DCL, the extractable 14C decreased to only 6.6 ± 0.2 % at 112 d. The abundance of extractable 14C varied among the PPCP/EDCs, and the general order was NP > BPA > DCL ≥ NPX. For example, in Ventura soil at 112 d, the extractable fraction was 12.9 ± 0.8% for NP, 9.8 ± 0.3% for BPA, 6.8 ± 0.4% for DCL, and 5.6 ± 0.1% for NPX . The level of extractable residue was generally similar among Irvine, Maricopa, and Ventura soils. After sterilization, the level of extractable residue was consistently higher than in the non-sterilized treatment, suggesting that the dissipation of extractable residue was largely due to microbially- mediated transformations. In addition, compost amendment slightly increased the level of extractable residue in Irvine soil. In Fent et al. , no 14C was detectable in the extract of soil treated with 14C-BPA after 120 d, which was in agreement with the present study, where extractable residue in the unmodified soils was low at the end of incubation . In a clayey silt soil and a silty sand soil, Kreuzig et al. reported 5% and 43% extractable 14C after 102 d of incubation following 14CDCL treatment; the difference between soils was attributed to indigenous microbial activity In this study, only 6.6 – 8.1% of 14C-DCL residue was extractable at the end of incubation. Lin and Gan found that after 84 d of incubation, 5% and 40% of the spiked NPX were recovered as the parent compound from a sandy soil and medium loam soil, respectively, while the extractable fraction was only 3.1 – 5.6% in the current study.