Parabens and Paraben Transformation Products in The Brazos River (Texas, USA) Before and After Wastewater Treatment

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Although little is known about the release of paraben transformation products and fate of transformation products in surface water. This study evaluates both parabens and paraben transformation products in the Brazos River upstream and downstream of a wastewater facility located in Waco, Texas. Concentrations of thirteen compounds were reported in this study, five parent parabens and eight paraben disinfection byproducts. Analyte concentrations were spatially evaluated to determine if release of wastewater effluent effects their concentrations in the river. Two Brazos River tributaries were also sampled to determine if they released parabens and related compounds to the Brazos. Sampling occurred weekly for one year with at least 40 samples collected at each site. Analyses were completed for both yearly and seasonal data. Sites downstream of wastewater treatment outfalls had lower concentrations of methyl paraben during the yearly analysis and across multiple seasons in the seasonal analysis. Para-hydroxybenzoic acid was the compound present in greatest concentration at most sites across most seasons, and spatial changes in para-hydroxybenzoic acid varied by season, with no identifiable trends. Dichlorinated paraben concentrations increased in the river at sites downstream of wastewater treatment. Concentration increases indicate that wastewater effluent contains sufficiently high dichlorinated paraben concentrations to effect concentrations downstream of effluent discharges. Dichlorinated species also persisted in the environment, with no significant decreases at sites further downstream during any season. Methyl paraben concentrations decreased at the site furthest downstream while dichlorinated methyl paraben concentrations remained stable showing that the dichlorinated species degrade slower than their respective parent paraben. Due to the dichlorinated species being released in higher concentrations in effluent than parents and being more resistant to degradation, the dichlorinated parabens are more likely to environmentally relevant than are parent parabens. Parabens disinfection byproducts wastewater treatment emerging contaminants Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Parabens are used as antimicrobials in foodstuff, personal care products and pharmaceuticals. An array of parabens are found in surface water across the globe with methyl paraben (MeP) concentration maxima of 1600 ng/L in Poland (Czarczyńska-Goślińska et al., 2017 ), 6140 ng/L in Brazil (Galinaro et al., 2022 ) and up to 32.6 ng/L in China. (Feng et al., 2019 ) Parabens mostly enter the environment in wastewater effluent after being washed down drains due to their extensive use in personal care products. However, parabens are well removed by wastewater treatment, via transformation. The release of these transformation products in surface water is not well known. However, a study by Li et al ( 2016 ) found that surface water near industrialized areas had higher concentrations of parent parabens but not chlorinated disinfection products. (Li et al., 2016 ) Though while wastewater treatment plants were in the industrialized area, the source of the paraben release was not identified. The three most common tertiary disinfection processes include chlorination, UV disinfection, and ozonation. Therefore, chlorinated and hydroxylated disinfection products are common transformation products released into surface water. Transformation of parabens in river water will primarily produce para-hydroxybenzoic acid (PHBA). Parabens can undergo both acid and base hydrolysis. Acid hydrolysis would only occur at pH values below 4, basic hydrolysis occurs at pH values above 10. (Valkova et al., 2001 ; Xu et al., 2021 ) Therefore, hydrolysis is unlikely to play a major role in paraben transformation in natural rivers with moderate pHs. Photolysis is another degradation pathway that could transform parabens into PHBA and further transformation into phenol. (Gomes et al., 2017 ) Mechanisms of photodegradation include the addition of hydroxyl radicals to the aromatic ring which will transform parabens into hydroxylated compounds such as methyl 3,4-dihydroxybenzoate. (Gao et al., 2016 ) However, parabens have a maximum absorption from 256 to 258 nm, which falls in the UVC range. (Talrose et al., n.d.) There will not be a significant amount of UVC radiation in natural light within the troposphere due to ozone absorption of wavelengths up to 310 nm. (Svobodova et al., 2006 ) Therefore, photodegradation is unlikely to be a major paraben transformation pathway in surface water. The most likely transformation pathway is biodegradation. (Song et al., 2017 ) In wastewater treatment plants, biodegradation is effective at transformation parabens, mostly by transformation into PHBA using esterases and further transformation into phenol via decarboxylases. (Lu et al., 2018 ; Wu et al., 2017 ) Transformation of parabens in river water would vary based on microorganisms present and environmental conditions such as, temperature, pH, and dissolved oxygen concentrations. While further transformation of paraben disinfection byproducts in natural waters has not been well evaluated, chlorinated disinfection byproducts are more persistent than the parents and transformation will occur slower. (Li et al., 2015 ) This can be seen by the lower biodegradation and disinfection rates of chlorinated paraben species in wastewater treatment. Dichlorinated parabens released from effluent could be transformed into dichlorinated PHBA. However, halogenated PHBA has not been evaluated in either wastewater or river water. (Penrose and Cobb, 2022 ) Halogenation could also occur in natural river water where residual chlorine will likely be present due to release from upstream water treatment processes. Parabens are chlorinated via addition to the aromatic ring, followed quickly by a second addition on the aromatic ring. (Yoom et al., 2018 ) However, further chlorination once a paraben has been dichlorinated, is unlikely as even at chlorine concentrations of 10 mg/L seen in wastewater chlorination would takes days to add another chlorine. (Yoom et al., 2018 ) Residual chlorine concentrations released in effluent are much lower after dechlorination with a concentration of 0.050 mg/L (0.3289 nM). Rate constants for MeP are 64.00 M − 1 S − 1 for the addition of one chlorine, 243.0 M − 1 S − 1 for a second addition of chlorine and 1.300 M − 1 S − 1 for a third addition with a chlorine concentration of 113.0 µM. (Mao et al., 2016 ) Given the low residual chlorine concentrations released in wastewater effluent, it is unlikely that diluted residual chlorine concentrations in the Brazos would cause halogenation of parabens in the river. Sorption to sediments is not likely to occur as parabens do not tend to sorb to sediments during primary treatments in wastewater treatment. The average residual chlorine concentration in Waco drinking water during the year studied was 1.45 mg/L, a concentration too low to cause significant transformation into chlorinated paraben before entering wastewater treatment. This study evaluates parabens and paraben transformation products in the Brazos River upstream and downstream of a major wastewater treatment plant in Waco Texas, USA. This study also determined which parabens and released transformation products were transformed in the river by analyzing river sites and two tributaries further downstream. Seasonal differences in analyte concentrations in the river concentrations were also evaluated. To the best of the authors’ knowledge, this is the first study to evaluate the direct impact of effluent release on chlorinated and hydroxylated paraben transformation product concentrations in river water, both directly downstream of release and then further downstream. 2. Methods 2.1. Standards and Supplies Analytical standards included five parent parabens, four chlorinated paraben transformation products, three hydroxylated products, PHBA and five internal standards. Methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), and butyl paraben (BuP) were purchased from AccuStandard. Para-hydroxybenzoic acid 3,4-dihydroxybenzoic acid (DHBA), methyl 3,4 dihydroxybenzoic acid (MePOH), ethyl, 3,4 dihydroxybenzoic acid (EtPOH), methyl 3-chloro-4-hydroxybenzoic acid (ClMeP), methyl 3,5-dichloro-4-hydroxybenzoic acid (Cl 2 MeP), ethyl 3-chloro-4-hydroxybenzoic acid (ClEtP) and ethyl 3,5-dichloro-4-hydroxybenzoic acid (Cl 2 EtP) were purchased from Sigma-Aldrich. Internal standards MeP-d 4 , EtP-d 4 , PrP- 13 C, BuP-d 9 and PHBA-d 4 were all purchased from Cambridge Isotopes. 2.2 Instrumentation An Acquity UPLC (Waters Corp., Milford, MA, USA) coupled to a Xevo TQS mass spectrometer was used for quantification of target products. Chromatographic separation was done using an Aquity UPLC BEH C18 column, 2.1 x 50 mm, 1.7 µm (Waters) with an injection volume of 10 µL and a flow rate of 300 µL/min. The mobile phase consisted of a 0.01% formic acid and acetonitrile gradient that began at a < > ratio and transitioned to < > during < > minutes. Masslynx 4.1 (Micromass, Manchester, UK) software was used to process chromatographic data and Targetlynx (Waters) was used to generate calibration curves and quantify analyte concentrations in sample. Compounds were analyzed in ESI negative mode with both a quantifier ion and a qualifier ion. Fragmentation patterns, operating conditions and method validation are included in Tables S1-S3. 2.3. Determination of Sample Sites Eight sample sites near Waco, Texas were chosen based on accessibility (Fig. 1 ). This is more of a constraint in Texas than in some other locations. Unlike many areas of the world where the public has access to rivers and narrow widths of shoreline, most US landowners have legal authority to prevent access to riverbanks on their properties. Seven sampling sites were along the Brazos River and were selected based upon proximity to a major wastewater treatment plant (WWTP1). All sample sites were either located along publicly accessible bridges, or on the Brazos River at sites along publicly accessible roads. Sampling along the Brazos occurred both upstream and downstream from WWTP1. Two tributaries downstream of WWTP1 were included in the study, with Bull Hide Creek having a smaller wastewater treatment plant (WWTP2) that releases effluent into the tributary. The distance between WWTP2 and the point at which the tributary meets the Brazos is approximately 30 km. The second tributary is located 1.5 km downstream of WWTP 1. WWTP 1 utilizes chlorination as a disinfection treatment process while WWTP2 uses UV disinfection. Three sites were upstream of WWTP 1, three sites were downstream of WWTP1, and two tributary sites were included in the analysis. 2.4. Sample Collection and Solid Phase Extraction River water was collected in 500 mL amber glass bottles as grab samples. Samples were taken weekly over the course of the year, with a few weeks omitted. Sampling at 5 of the sites with bridge access (Sites 1, 2, 3, 5 and 6) occurred every Monday morning, while the remaining sites were taken Monday afternoons or evenings. The sample preparation is similar to the method used by Penrose and Cobb ( 2023 ). Briefly, once the samples were returned to the lab, each sample was spiked with 20 ppb of internal standard, acidified to a pH of 3, filtered, extracted using Oasis HLB cartridges and concentrated using nitrogen evaporation. 2.5. Quality Control Blanks were included to monitor possible contamination throughout the analysis. One spiked blank, and one method blank were included and analyzed with every batch of samples (n = 16). Analyte concentrations detected in method blanks were subtracted from all samples run in the same batch as the blank. Blank spikes were used to normalize concentrations for analyte recovery, with each blank spike being used for the samples run in the same batch as the spike. All samples were spiked with internal standards before extraction to evaluate recovery. Analytes that had no internal standards but are similar in structure to internal standards of the parent compounds used recovery result from the most structurally similar parent compound. A calibration curve was created using 8 calibration standards ranging in concentrations of 0.5 ng/mL to 100 ng/mL for most analytes and concentrations ranging from 5 ng/mL to 1000 ng/mL for PHBA and DHBA. 2.6. Data Analysis 2.6.1 Site Comparisons Concentrations were reported in ng/L with variance reported as confidence intervals. Only weeks where all samples were taken were included in analysis with, 40 weeks over the course of a year for all eight sites. The concentration of each paraben and transformation product was compared upstream and downstream of the major wastewater treatment plant to determine if concentrations in the river changed after the release of wastewater effluent. The site just downstream of wastewater treatment was compared to sites further downstream to see if natural degradation could be occurring as parabens and transformation products move downstream. Analyte concentrations in the tributaries were analyzed to see if any of the changes seen at sites further downstream of wastewater treatment could be related to movement from tributaries into the Brazos River. Of the potential paraben transformation products, only shorter chained transformation products were evaluated, as there are no available standards for longer chained paraben transformation products.(Albero et al., 2012 ) As a previous study showing transformation in wastewater treatment processes in the study area reported concentrations in picomolar units (pM) a table showing the conversion factor between ng/L and pM for each compound in included as Table S4. (Penrose and Cobb, 2023 ) 2.6.2. Seasonal Evaluation Analyte concentrations (Fig. S1 -S4) were evaluated to determine concentration differences between seasons at sites upstream and downstream of WWTPs. Maps were created for seasonal concentrations similarly to the yearly evaluation, these maps were added as supplemental information. (Fig. S5-S8) 2.6.3. Statistical Analysis Neither untransformed or log transformed concentrations were determined to meet the assumptions of ANOVA using Shapiro Wilk’s tests for normality and Bartlett’s tests for homogeneity of variances. Therefore, non-parametric tests were performed. All statistical comparisons were done using Kruskal-Wallis followed by Dunn’s Tests. Maximum likelihood estimations (MLE) were used to treat non-detects before performing statistical analysis. Statistical analyses were done using Microsoft Excel (2016), R (Version 3.4.1), and Sigma Plot (12.0). Maps and map features were made in QGIS 3.14.16 and ARCGIS 10.8.1. 3. Results and Discussion 3.1 Yearly Concentrations in the Brazos River Of the evaluated compounds, MeP, PrP, BuP, Cl 2 MeP, Cl 2 EtP, PHBA and DHBA were determined to be significantly different by site. Of the parent products, MeP was found in the highest concentrations at sites along the Brazos River that are upstream of the wastewater treatment and in the Tehuacana Creek tributary (Fig. 2 ). PrP was detected in higher concentrations than MeP at the sites following release. Most of the differences in BuP concentrations are due to higher concentrations at site 6 located Downstream (south) of the meeting point of Tehuacana Creek and the Brazos River. Concentrations of both dichlorinated paraben transformation products were higher at sites downstream of the effluent release location. While PHBA saw a significant decrease at the site just downstream of the release of wastewater effluent, DHBA did not significantly decrease. The oxidation of PHBA to DHBA could be responsible for the decrease in PHBA and could also explain the stability in DHBA concentrations. The products that were both significantly different between sites and had notable spatial trends, were mapped to visualize differences in analyte concentrations at different points in the Brazos. Of the parent compounds MeP, PrP and BuP concentrations had spatial trends (Fig. 3 ). MeP concentrations decreased significantly from the site upstream of wastewater treatment to the site downstream of wastewater treatment. However, this decrease is unlikely to be solely due to dilution by the release of wastewater effluent, as the small outflow of the wastewater treatment with a flow of 32 ft 3 /sec is unlikely to have a large impact on the Brazos with flow rates ranging from an average of 220 ft 3 /sec in March 2021 to an average of 16,640 ft 3 /sec in June 2021. Flow rates remained above 2000 ft 3 /sec through August before decreasing in September. The higher water flows in the summer months would decrease concentrations due to dilution. Transformation of parabens in the environment is a major contributor to the removal of parabens in surface water. Biodegradation is the most likely route of transformation of parabens in surface water. The differences in microbial communities along the Brazos could be the cause for the sudden change in methyl paraben concentrations seen at the site downstream of wastewater treatment. Biodegradation occurs much more quickly under aerobic conditions than under anaerobic conditions. (Wu et al., 2017 ) A change to more aerobic conditions could cause more degradation to occur between site 3 and site 4. Neither hydrolysis nor photolysis are major pathways in paraben transformation in the environment. (Svobodova et al., 2006 ; Talrose et al., n.d.; Valkova et al., 2001 ; Xu et al., 2021 ) MeP concentrations in the Brazos increased from site 4 to site 6 (p = 0.041), which could be due to the input of MeP from other sources. Considering that site 6 is located near a residential neighborhood, input from recreational activities is one potential source of the observed increase. Desorption from solids released from the wastewater treatment plant is another possibility as parent parabens have been detected in wastewater sludge with MeP concentrations of 89.4 ng/g. (Chen et al., 2017 ) MeP has also been detected in river surface sediment, with a median concentration of 12.4 ng/g. (Feng et al., 2019 ) A study by Feng et al ( 2019 ) found that in the Huai River, mean MeP concentrations were 51.7 ng/L while mean MeP concentrations in the sediment were 11.6 ng/g giving a K d of 0.224 L/g. However, the same study detected MeP concentrations of 8.84 ng/L in surface water and 13.0 ng/g in sediment in the Yellow River. The K d for the Yellow River values was 1.47 L/g. Arfaeinia et al ( 2022 ) found that the median K d between seawater and sediment for MeP was 3.52 g/mL, which is lower than the K d values calculated from the MeP concentrations in the river samples, showing variation by environment. (Arfaeinia et al., 2022 ) Using an average of 0.847 L/g from the two Kd values determined from the Huai River and Yellow River, and with average total dissolved solids of 300 mg/L in effluent at WWTP1, the minimum concentration sorbed to sludge needed to cause the observed concentration change is 783 nmol/kg or 119 ng/g. MeP concentrations detected in wastewater sludge in previous studies show that this concentration change could possibly be due to desorption from wastewater solids after effluent release. While the tributary upstream of site 6 does have detectable MeP concentrations at site 5, a combination of the lower MeP concentration and small size of the tributary means that the tributary is unlikely to have a large impact on the paraben concentration in the Brazos. It is unlikely that the increase in MeP concentrations are due to direct input as the area of the Brazos River is not used for recreation. MeP concentrations decreased further downstream with significantly lower concentrations at site 8 when compared to site 6 (p = 0.045). The observed change in concentration is likely due to natural MeP degradation and dilution in the river. The tributary with the small wastewater treatment plant has a very low MeP concentration which is unlikely to have a large impact on MeP concentration in the Brazos. EtP concentrations were not significantly different between any site in the yearly analysis. This due to the low concentrations of EtP at all sites. The low EtP concentrations were expected due to EtP being used seldom in industry. PrP had similar concentration changes in the river as MeP. PrP decreased at site 4 (site 3 and site 4: p = 0.030) downstream of effluent release before increasing at site 6 (site 4 and site 6: p = 0.015). However, the visual decrease in PrP is smaller than that of MeP, and the differences between site 6 and site 8 were determined to be insignificant (site 6 and site 8: p = 1.00) The lack of decrease shows that PrP degraded at a slower rate than MeP in the Brazos, this matches the transformation rates seen in many treatments and metabolic process, though PrP has been seen to degrade faster than MeP in activated sludge. (Abbas et al., 2010 ; Li et al., 2015 ; Lu et al., 2018 ) The Bull Hide Creek tributary also had a higher concentration of PrP but are still not in high enough concentration to explain the similarities in PrP concentrations in the main channel. Similarity of PrP concentrations in the Brazos River main channel demonstrates a slower degradation rate for PrP than for MeP. BuP concentrations at upstream sites were lower in concentration than either MeP or PrP. This is expected as BuP is used much less often than MeP or PrP in either personal care products or foodstuff. BuP concentrations increased from site 3 to site 4 (p < 0.001), which could be due to release of BuP in wastewater effluent. BuP increased from site 4 to site 6 (p < 0.001), followed by a decrease from site 6 to site 8 (p < 0.001), as MeP had. No notable spatial trends were observed for BzP. Of the transformation products, PHBA, DHBA, Cl 2 MeP and Cl 2 EtP concentrations showed spatial trends (Fig. 4 ). PHBA was the compound detected in highest concentrations at all sites with its highest concentration at 10.30 ng/L (4.850 ng/L to 21.88 ng/L) also having the largest variation amongst the compounds. As with MeP and PrP, PHBA concentrations significantly decreased from site 3 to site 4 (site 3-site 4: p = 0.031). This decrease in PHBA could be due to aerobic biodegradation occurring between site 3 and site 4. In the case of aerobic biodegradation, PHBA can be further degraded into phenol via carboxylases, which does not occur with anaerobic biodegradation. This change in environment is likely due to a dam less than a mile upstream of site 3 that causes aeration of the river water as the water traverses the dam. The short distances between the dam and site 3 could explain the changes are not yet seen at that site. PHBA concentrations increased visually from site 4 to site 6 and from site 6 to site 8. However, these increases were not significant (site 4 and site 6: p = 1.00, site 6 and site 8 p = 1.00). This was unexpected, as PHBA is the common degradation product of all parent parabens and would be expected increase in concentration as parabens degrade. Site 7 had a relatively high concentration of PHBA, when compared to the low concentration of the other analytes at that site with an average concentration of 4.465 ng/L (2.479 ng/L to 8.045 ng/L). This could be due to PHBA being a potential transformation product of UV disinfection. However, the higher concentration in the tributary did not seem to have significant effect on Brazos River PHBA concentrations. While DHBA concentrations showed overall significant difference between sites, the only significant differences were between the Brazos River sites and the tributaries and none of the sites along the river showed any significant differences between each other. Cl 2 MeP concentrations remained consistently low upstream of wastewater effluent release, with no significant differences between sites 1 through 3 (site 1 and site 2: p = 1.00, site 2 and site 3: p = 1.00). However, at site 4, downstream of wastewater treatment, concentrations were notably higher and Cl 2 MeP concentrations were significantly different from site 3 (site 3 and site 4 p = 0.032). Dichlorinated parabens are not used in industry and are not likely to be introduced from other sources between site 3 and site 4 and dichlorinated parabens quantified at upstream sites are likely remaining from release in effluents further upstream. A waterpark with high concentrations of chlorine located along the Bosque River 2 km upstream of the where the Bosque and Brazos meet could introduce chlorine or chlorinated parabens to the upstream sites. The dichlorinated paraben species are released in higher concentrations than the parents in effluent at the major wastewater treatment plant. (Penrose and Cobb, 2023 ) Cl 2 MeP concentrations remained consistent at sites downstream of effluent release, with only small visual decreases and no significant differences between sites 4, 6 and 8 (site 4 and site 6: p = 1.00, site 6 and site 8: p = 1.00). The stability in concentrations at downstream sites shows that Cl 2 MeP degraded at a much slower rate than the parent parabens. Given the low concentrations of Cl 2 MeP in the tributary and water quantity differences between the tributaries and the Brazos River, it is unlikely that either of the tributaries played a significant role in the lack of decreases in Cl 2 MeP at sites 6 and 8. Cl 2 EtP concentrations had similar trends to Cl 2 MeP, with a stable concentration upstream of effluent, an increase at release (site 3 and site 4: p = 0.036), and a stable concentration downstream. Cl 2 EtP concentrations were lower than Cl 2 MeP concentrations due to the higher use of MeP in products resulting in higher concentrations of MeP transformation products in the river. Both dichlorinated paraben products were released from wastewater effluent and entered the Brazos at concentrations high enough to cause a change in the Cl 2 MeP concentrations in the Brazos River. This combined with persistence seen with both compounds show that the dichlorinated species, will be more relevant than their respective parent compounds in the environment. Dichlorinated PHBA is an important transformation product due to the high concentrations in the river. Data has been obtained for both mono and dichlorinated PHBA. However, they were not included in spring quality control due to the standards not being available until after spring analysis and neither are included in this study. While the dichlorinated species were in detectable concentration changes along the river, ClMeP and ClEtP did not have noticeable spatial trends during the yearly analysis, which is due to the low concentrations of ClMeP and ClEtP at all sites. The dichlorinated species are the only compounds evaluated that increase in the Brazos River directly as due to the release of wastewater effluent. Longer chained chlorinated species have not been evaluated due to a lack of available standards. 3.2 Seasonal Differences in Analyte Concentrations Along the Brazos River. 3.2.1 Differences in Analyte Concentrations Between Seasons Of the quantified compounds, PrP BzP, DHBA and ClEtP concentrations were determined to be significantly different between seasons. (Table 1 ). For PrP most of the differences were due to lower concentrations in the summer. Variation in concentrations were generally highest in the summer for multiple compounds, which is likely due to the low concentrations during June and early July and high concentrations in early August. Summer was expected to have the highest concentrations due to increased use of personal care products, particularly sunscreen. However, the study area has a large student population that would be absent during summer. The low concentrations in early summer could also be explained by high water levels during that period due to a large amount of rainfall in late May and early June. High flow rates in May would also cause low analyte concentrations in late Spring. March and April flow rates were lower at an average of 183 ft 3 /sec and 176 ft 3 /sec respectively before increasing due to rainfall in May with average flow in May being 7617 ft 3 /sec. Flow rates were highest in June at 16640 ft 3 /sec and remained higher throughout July and in early August with average flows of 5870 ft 3 /sec in July and 1674 ft 3 /sec in August. August had higher analyte concentrations than the other summer months, the change in analyte concentrations are due to the decreasing water level and return of the student population. The change from low analyte concentrations in early and mid-summer and higher analyte concentrations in late summer caused higher variation in the summer. By September the flow rates were near flows in April at 375 ft 3 /sec, though there were days in September with flow rates up to 1840 ft 3 /sec. High water levels impact the concentrations detected in late spring, but overall did not cause noticeable differences between spring and winter or spring and fall. Summer PrP concentrations were significantly different than winter but not significantly different from fall and spring, with summer having generally lower concentrations than winter. This is likely due to both the high-water levels that persisted from late spring throughout most of summer and the decreased population in the general area during the summer. While ClEtP concentrations showed significant differences by season, adjusted p-values showed no differences between ClEtP concentration between any two specific seasons (Table 2 ). Seasonal differences in DHBA concentration were determined to be caused by low DHBA concentrations in the winter. Table 1 General differences between seasons. Bolded values are significant. Compound P-Values Between Seasons MeP 0.610 EtP 0.803 PrP 0.022 BuP 0.076 BzP 0.019 PHBA 0.746 DHBA 0.029 MePOH 0.184 EtPOH 0.070 ClMeP 0.809 Cl 2 MeP 0.506 ClEtP 0.027 Cl 2 EtP 0.359 Table 2 Significant p-values from seasonal analyses. A lager table that includes non-significant p-values is included as supplemental information. (Table S5) Compound Seasons P-Value MeP Fall and Summer 0.0381 Spring and Summer 0.0088 BzP Fall and Summer 0.0004 Summer and Winter 0.0361 DHBA Fall and Winter 0.0485 Spring and Winter 0.0492 Summer and Winter 0.0453 MePOH Spring and Winter 0.0363 EtPOH Spring and Winter 0.0272 Summer and Winter 0.0487 Cl 2 EtP Fall and Summer 0.0497 Spring and Summer 0.0264 Summer and Winter 0.0076 General differences in concentration by season were determinable for multiple compounds. When evaluating differences in seasonal concentrations at each individual site, differences were much more sporadic only having a few notable differences randomly distributed across sites. (Table 3 ). None of the three upstream sites had any significant differences in MeP, PrP, or PHBA concentrations between seasons, despite those three compounds being found in the highest concentrations at the three sites. Reasons for the lack of differences could be consistent release of these compounds from upstream, though differences would still be expected due to changes in water level. Sites 4, 6 and 8 were expected to have different concentrations of Cl 2 MeP and Cl 2 EtP, as the concentrations released in wastewater effluent upstream of site 4 would vary by season and flow rate through the plant. However, Cl 2 MeP concentrations were only seasonally different at site 3, while Cl 2 EtP was significantly different at site 3 and the two tributaries. No specific compound was seasonally different across all upstream sites or all downstream sites and so no trends could be identified. P-values from Dunn’s tests showing which seasonal concentrations were significantly different at each site are included as supplemental information. Table 3 Seasonal concentration differences at individual sites along the Brazos River. Bolded values are significant. Seasonal P-Values by Site Compound Site 1 Site 2 Site 3 Site 4 a Site 5 Site 6 Site 7 b Site 8 MeP 0.576 0.587 0.752 0.236 0.035 0.564 0.043 0.459 EtP 0.358 0.009 0.196 0.586 0.220 0.224 0.108 0.845 PrP 0.364 0.217 0.089 0.190 0.791 0.438 0.009 0.152 BuP 0.665 0.325 0.141 0.392 0.460 0.137 0.021 0.638 BzP 0.021 0.308 0.227 0.002 0.002 0.144 0.046 0.182 PHBA 0.711 0.068 0.407 0.160 0.579 0.325 0.3372 0.138 DHBA 0.199 0.919 0.499 0.346 0.753 0.768 0.023 0.566 MePOH 0.026 0.562 0.367 0.386 0.649 0.513 0.027 0.580 EtPOH 0.578 0.914 0.612 0.893 0.015 0.118 0.002 0.700 ClMeP 0.124 0.009 0.165 0.501 0.819 0.554 0.456 0.597 Cl 2 MeP 0.809 0.011 0.164 0.118 0.028 0.050 0.560 0.501 ClEtP 0.640 0.015 0.056 0.022 0.226 0.496 0.025 0.780 Cl 2 EtP 0.079 0.161 0.074 0.828 0.086 0.024 0.112 0.486 a. Site 4 is just downstream of WWTP1 b. Site 7 is downstream of WWTP2. 3.2.2 Differences Between Sites Across Seasons In general, concentration differences between sites at each season was consistent with the differences seen in the yearly analysis with some variation between seasons. (Table 4 ) MeP concentrations were generally the highest of the parent parabens at upstream sites with averages of 1.814 ng/L (0.4826 ng/L to 6.817 ng/L) during fall, 1.162 ng/L (0.3133 ng/L to 3.145 ng/L) during spring, 0.8018 ng/L (0.2031 ng/L to 3.165 ng/L) during summer and 0.9434 ng/L (0.1849 ng/L to 4.814 ng/L) during the winter at site 1. MeP concentrations varied by site during every season except summer (Table 4 ) with Dunn’s Tests (Table S6 to Table S10) showing that the differences are specifically between sites 1 and 4 (spring: p = 0.009, fall: p = 0.028, winter: p = 0.022), sites 2 and 4 (spring: p = 0.011, fall: p = 0.009, winter: p = 0.038) and sites 3 and 4 (spring: p = 0.006, fall: p = 0.018, winter p = 0.018). Therefore, concentrations of MeP dropped significantly during three out of four of the seasons moving from site 3 to site 4, which matches the change seen in the yearly analysis. Maps showing spatial changes and bar graphs showing analyte concentrations by site are included as supplemental information. The increase in MeP at site 4 to site 6 seen in the yearly analysis was visually seen across seasons. However, winter (site 4 to site 6: p = 0.041) was the only season with a significant increase, going from an average MeP concentration of 0.0421 ng/L (0.0155 ng/L to 0.1049 ng/L) at site 4 to 0.8998 ng/L (0.3292 ng/L to 2.459 ng/L) at site 6. No significant trends were identified for PrP in any individual season. Visual changes in PrP were similar to that of changes seen in MeP. However, the only significant differences were due to low PrP concentrations in the tributaries with no differences between sites in the main channel, sites and the tributaries, and not between two sites along the Brazos. BuP was the only compound with significant differences between sites in every season (spring: p < 0.001, summer: p < 0.001, fall p < 0.001, winter: p = 0.031). However, BuP concentrations are noticeably high at site 6 with average concentrations of 0.4685 ng/L (0.3928 ng/L to 0.5588 ng/L) in spring, 0.6120 ng/L (0.4716 ng/L to 0.7943 ng/L) in summer, 0.5617 ng/L (0.5023 ng/L to 0.6280 ng/L) in the fall and 0.5239 ng/L (0.4714 ng/L to 0.5822 ng/L) in the winter. The high concentrations of BuP at site 6 are likely responsible for the observed differences, as the only differences in BuP concentrations are between site 6 and other sites. BzP concentrations were significantly different between sites in the fall and winter. However, differences in the fall were between sites 1 and 4 (p = 0.035), sites 4 and 5 (p = 0.009) and sites 5 and 6 (p = 0.0122), while differences in winter were only between site 4 and 5 (p = 0.036). While there are differences, no trends were identified. Table 4 General differences between sites by season. Bolded values are significant. P-Values by Season Compound Spring Summer Fall Winter MeP < 0.001 0.079 < 0.001 < 0.001 EtP 0.016 0.814 < 0.001 0.064 PrP 0.073 0.009 0.001 0.003 BuP < 0.001 < 0.001 < 0.001 0.031 BzP 0.979 0.021 < 0.001 < 0.001 PHBA 0.256 0.052 0.048 0.013 DHBA 0.015 0.001 0.100 0.481 MePOH 0.830 0.086 0.009 0.792 EtPOH 0.811 0.006 0.037 0.050 ClMeP 0.005 0.502 < 0.001 0.006 Cl 2 MeP 0.261 0.008 < 0.001 0.018 ClEtP 0.004 0.284 < 0.001 0.006 Cl 2 EtP 0.169 0.039 < 0.001 0.043 During most seasons, PHBA and DHBA were present in higher concentrations than any of the other analytes, likely due to being natural degradation products of all parent parabens. PHBA concentrations were significantly different between site during the fall and winter while DHBA concentrations were significantly different in spring and summer. However, in all cases, the differences were between non-tributary sites and site 5, meaning the only notable trend is that there are significantly lower concentrations of PHBA at site 5 during fall and winter and DHBA during spring and summer. It is important to note that PHBA was only significantly different during seasons where DHBA was not significantly different, and vice versa. This could be due to DHBA being a hydroxylated product of PHBA. Increases in DHBA concentration would be related to decreases in PHBA concentration, which could be due to transformation of PHBA into DHBA by aerobic microbes using hydroxylases. Transformation from PHBA to DHBA upstream of site 5 during the fall in winter would decrease PHBA concentrations, causing PHBA concentrations at that site to be significantly lower than other sites. Cl 2 MeP and Cl 2 EtP concentrations were significantly different between sites during all seasons except spring (Table 5). Further investigation using adjusted p-values from Dunn’s tests (Table S4) did not show significant differences in Cl 2 MeP concentrations between any individual sites during the summer. Differences in winter followed trends seen in the yearly analysis with a significant decrease from site 3 and site 4 (p = 0.024) and no significant changes at sites downstream of site 4. As with the yearly concentrations, Cl 2 MeP remained consistent from site 4 to site 6 and from site 6 to site 8. Given the lack of statistical differences, the numerical increases are due to the high variation of Cl 2 MeP concentrations during all seasons at all downstream sites. One explanation for the high variation in Cl 2 MeP is inconsistent release in Cl 2 MeP concentrations in wastewater effluent between weeks. During fall there was a numeric change between site 3 and site 4 but no significant change, though there were differences between other upstream and downstream sites showing that there was a change between upstream and downstream sites (site 2 and 4: p < 0.001. site 1 and 6: p = 0.004, site 2 and 6: p < 0.001, site 2 and 8: p = 0.008). Cl 2 EtP changes were spatially similar to Cl 2 MeP. While Cl 2 EtP concentrations were determined to be different during the summer, the only differences were between site 6 and site 7 (p = 0.020) and between site 3 and site 7 (p = 0.010). The lack of differences between upstream and downstream sites of both Cl 2 MeP and Cl 2 EtP in the summer, means that the release of effluent did not have the same effect on river water concentrations that it had in other seasons. This could be due to the high rainfall in early summer that would not only increase water level in the Brazos, thereby increasing dilution of effluent, but also cause high flow rates and reduced retention times in wastewater treatment, reducing transformation from parent to dichlorinated species. As with Cl 2 MeP, Cl 2 EtP also was not significantly different between site 3 and 4 during the fall but were different between other upstream and downstream sites (site 2 and 6: p = 0.034). However, unlike Cl 2 MeP, Cl 2 EtP concentrations were significantly different between individual upstream sites and site 4 during spring (site 2 and 4: p = 0.023, site 3 and 4: p = 0.034). Cl 2 MeP concentrations would be expected to have greater increases due to the higher use of MeP that would result in higher concentrations released in effluent. 4. Conclusions Year-long and seasonal spatial relationships of parabens and paraben transformation products were evaluated at different sites along the Brazos River in relation to a wastewater treatment plant. All sites were located in or near Waco, Texas. MeP and PrP concentrations decreased after release of wastewater effluent, followed by an increase in concentration further downstream. MeP concentrations decreased at the site furthest downstream, which is likely due to natural degradation and dilution. Other parent parabens had similar visual trends, but any differences matching those trends were not significantly different. PHBA was the transformation product quantified in the highest concentration at most sites during most seasons. The dichlorinated parabens are the only compounds that increased directly as a result of wastewater effluent release, meaning that released concentrations high enough to cause increases in the Brazos River. The dichlorinated species also remained in similar concentrations at sites further downstream, showing the relevance of the dichlorinated transformation products in the environment. The Cl 2 MeP and Cl 2 EtP concentrations were not high enough to induce estrogenic effect. However, chlorinated PHBA will be in higher concentrations than either Cl 2 MeP or Cl 2 EtP and no studies have determined estrogenic effects of chlorinated PHBA. While the concentrations in this study are low, this study focuses more on identifying which compounds are being released in effluent and which are transformed in the environment. This could be helpful by identifying analytes that are likely to accumulate in areas downstream of wastewater treatment plants with different tertiary treatment processes. Future research in this area should identify other important transformation products in the environment specifically chlorinated PHBA, or the potential bioaccumulation of the studied transformation products. Research could also be geared towards identifying different toxicological effects of the transformation products. Declarations Author Contributions Michael Penrose, designed experiments, conducted research, collected data, created figures and tables, and wrote the manuscript with input from George Cobb. George Cobb helped design experiments, reviewed methodology, and reviewed/revised the manuscript. Acknowledgements The authors would like to thank Chad Mansfield and Bianca Possamai for their part in sampling along the Brazos and Janelle Oldfather for helping process samples. The authors would also like to thank the Baylor Mass Spectrometry Center for providing access to the LC-MS used in analysis. The authors thankfully acknowledge financial support from the C. Gus Glasscock, Jr. Endowed Fund of Excellence in Environmental Sciences. Funding This work was supported by the C. Gus Glasscock, Jr. Endowed Fund of Excellence in Environmental Sciences. Conflicts of Interest The authors have no conflicts of interest to disclose. Data Availability The data that support the finding of this study are available in an OSF repository at https://osf.io/2gvjp/?view_only=66ace57fc1f64e8f90c3eee1c265808c. References Abbas S, Greige-Gerges H, Karam N, Piet M-H, Netter P, Magdalou J (2010) Metabolism of Parabens (4-Hydroxybenzoic Acid Esters) by Hepatic Esterases and UDP-Glucuronosyltransferases in Man. Drug Metab Pharmacokinet 25:568–577. https://doi.org/10.2133/dmpk.DMPK-10-RG-013 Albero B, Pérez RA, Sánchez-Brunete C, Tadeo JL (2012) Occurrence and analysis of parabens in municipal sewage sludge from wastewater treatment plants in Madrid (Spain). J Hazard Mater Occurrence fate Emerg contaminants municipal wastewater Treat Syst 239–240. https://doi.org/10.1016/j.jhazmat.2012.05.017 Arfaeinia H, Asadgol Z, Ramavandi B, Dobaradaran S, Kalantari RR, Poureshgh Y, Behroozi M, Asgari E, Asl FB, Sahebi S (2022) Monitoring and eco-toxicity effect of paraben-based pollutants in sediments/seawater, north of the Persian Gulf. https://doi.org/10.1007/s10653-021-01197-2 . Environ Geochem Health Chen J, Pycke BFG, Brownawell BJ, Kinney CA, Furlong ET, Kolpin DW, Halden RU (2017) Occurrence, temporal variation, and estrogenic burden of five parabens in sewage sludge collected across the United States. Sci Total Environ 593–594:368–374. https://doi.org/10.1016/j.scitotenv.2017.03.162 Czarczyńska-Goślińska B, Zgoła-Grześkowiak A, Jeszka-Skowron M, Frankowski R, Grześkowiak T (2017) Detection of bisphenol A, cumylphenol and parabens in surface waters of Greater Poland Voivodeship. J Environ Manage 204:50–60. https://doi.org/10.1016/j.jenvman.2017.08.034 Feng J, Zhao J, Xi N, Guo W, Sun J (2019) Parabens and their metabolite in surface water and sediment from the Yellow River and the Huai River in Henan Province: Spatial distribution, seasonal variation and risk assessment. Ecotoxicol Environ Saf 172:480–487. https://doi.org/10.1016/j.ecoenv.2019.01.102 Galinaro CA, Spadoto M, de Aquino FWB, de Souza Pelinson N, Vieira EM (2022) Environmental risk assessment of parabens in surface water from a Brazilian river: the case of Mogi Guaçu Basin, São Paulo State, under precipitation anomalies. Environ Sci Pollut Res 29:8816–8830. https://doi.org/10.1007/s11356-021-16315-x Gao Y, Ji Y, Li G, An T (2016) Theoretical investigation on the kinetics and mechanisms of hydroxyl radical-induced transformation of parabens and its consequences for toxicity: Influence of alkyl-chain length. Water Res 91:77–85. https://doi.org/10.1016/j.watres.2015.12.056 Gomes JF, Leal I, Bednarczyk K, Gmurek M, Stelmachowski M, Diak M, Emília Quinta-Ferreira M, Costa R, Quinta-Ferreira RM, Martins RC (2017) Photocatalytic ozonation using doped TiO2 catalysts for the removal of parabens in water. Sci Total Environ 609:329–340. https://doi.org/10.1016/j.scitotenv.2017.07.180 Li W, Gao L, Shi Y, Wang Y, Liu J, Cai Y (2016) Spatial distribution, temporal variation and risks of parabens and their chlorinated derivatives in urban surface water in Beijing, China. Sci Total Environ 539:262–270. https://doi.org/10.1016/j.scitotenv.2015.08.150 Li W, Shi Y, Gao L, Liu J, Cai Y (2015) Occurrence, fate and risk assessment of parabens and their chlorinated derivatives in an advanced wastewater treatment plant. J Hazard Mater 300:29–38. https://doi.org/10.1016/j.jhazmat.2015.06.060 Lu J, Li H, Tu Y, Yang Z (2018) Biodegradation of four selected parabens with aerobic activated sludge and their transesterification product. Ecotoxicol Environ Saf 156:48–55. https://doi.org/10.1016/j.ecoenv.2018.02.078 Mao Q, Ji F, Wang W, Wang Q, Hu Z, Yuan S (2016) Chlorination of parabens: reaction kinetics and transformation product identification. Environ Sci Pollut Res 23:23081–23091. https://doi.org/10.1007/s11356-016-7499-y Penrose M, Cobb G (2023) Evaluating seasonal differences in paraben transformation at two different wastewater treatment plants in Texas and comparing parent compound transformation to byproduct formation. Water Res 235:11978. https://doi.org/10.1016/j.watres.2023.119798 Penrose M, Cobb G (2022) Identifying potential paraben transformation products and evaluating changes in toxicity as a result of transformation. WER 94:e10705. https://doi.org/10.1002/wer.10705 Song H, Alfiya Y, Dubowski Y, Friedler E (2017) Sorption and biodegradation of propylparaben in greywater by aerobic attached-growth biomass. Sci Total Environ 598:925–930. https://doi.org/10.1016/j.scitotenv.2017.04.032 Svobodova A, Walterova D, Vostalova J, ULTRAVIOLET LIGHT INDUCED, ALTERATION TO THE SKIN (2006) Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150, 25–38. https://doi.org/10.5507/bp.2006.003 Talrose V, Yermakov A, Usov A, Goncharova A, Leskin A, Messineva N, Trusova N, Efimkina M n.d. NIST Standard Reference Data Program, NIST Standard Reference Database Number 69. NIST Chemistry Workbook, Gaithersburg MD, 20899 Valkova N, Lepine F, Valeanu L, Dupont M, Labrie L, Bisaillon J-G, Beaudet R, Shareck F, Villemur R (2001) Hydrolysis of 4-Hydroxybenzoic Acid Esters (Parabens) and Their Aerobic Transformation into Phenol by the Resistant Enterobacter cloacae Strain EM. Appl Environ Microbiol 67:2404–2409. https://doi.org/10.1128/AEM.67.6.2404-2409.2001 Wu Y, Sun Q, Wang Y, Deng C, Yu C-P (2017) Comparative studies of aerobic and anaerobic biodegradation of methylparaben and propylparaben in activated sludge. Ecotoxicol Environ Saf 138:25–31. https://doi.org/10.1016/j.ecoenv.2016.12.017 Xu T, Chen J, Chen X, Xie, Huaijun, Wang Z, Xia D, Tang W, Xie Hong-bin (2021) Prediction Models on p K a and Base-Catalyzed Hydrolysis Kinetics of Parabens: Experimental and Quantum Chemical Studies. Environ Sci Technol 55:6022–6031. https://doi.org/10.1021/acs.est.0c06891 Yoom H, Shin J, Ra J, Son H, Ryu D, Kim C, Lee Y (2018) Transformation of methylparaben during water chlorination: Effects of bromide and dissolved organic matter on reaction kinetics and transformation pathways. Sci Total Environ 634:677–686. https://doi.org/10.1016/j.scitotenv.2018.03.330 Statements & Declarations Supplementary Files SupplementalInformationAECT.docx Cite Share Download PDF Status: Published Journal Publication published 09 Aug, 2023 Read the published version in Archives of Environmental Contamination and Toxicology → Version 1 posted Reviewers agreed at journal 22 May, 2023 Reviewers invited by journal 16 May, 2023 Editor assigned by journal 15 May, 2023 First submitted to journal 12 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2928595","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":200633868,"identity":"209cdc17-1cf3-478f-aef1-b0b93c9f96ad","order_by":0,"name":"Michael Penrose","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie3MvWrDMBDA8RMGeckDaEhJX6DgYEiH9ONVdBjqpXTpkiGDoWBtnV0ofYZMIaPhQF0EfYEM9pKu7ualH0pNwYtFxkL0HwQ66XcAPt9/LOC9i1yUAGE3P5BUpvz77CDQI6zODyBnimPFNhd350rVFb5sJxEFuoHFHLMBMiNOETM392Nj4gjXu+mKeFKASR0kzAXLCQtxywWuia1oFMN+4iCqZfm3Jel7i8903ZEvF+Ha7iwtkTPAjLAjmZMkgpkEi5GJhdS75MlOQOo0HiRvetqwzRUWoao/2uX28vH1gaBZzk+GyL7gs/96WtpDOr7/xvpk4tru8/l8R9kPxmRfmsFbin8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9419-965X","institution":"Baylor University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Penrose","suffix":""},{"id":200633869,"identity":"c6955f56-c645-4737-a547-61d2bff29fb7","order_by":1,"name":"George Cobb","email":"","orcid":"","institution":"Baylor University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"George","middleName":"","lastName":"Cobb","suffix":""}],"badges":[],"createdAt":"2023-05-12 16:41:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2928595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2928595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00244-023-01025-x","type":"published","date":"2023-08-09T21:55:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37190316,"identity":"e581d222-2369-449c-bba2-752e6a882986","added_by":"auto","created_at":"2023-05-18 12:57:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":517780,"visible":true,"origin":"","legend":"\u003cp\u003eWater Sample Site Locations Along the Brazos River Texas, USA\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/792ea5bf0c0362ddbef4ffc7.png"},{"id":37190315,"identity":"326ccfba-d894-4a57-a770-3720418d9e85","added_by":"auto","created_at":"2023-05-18 12:57:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44886,"visible":true,"origin":"","legend":"\u003cp\u003eAnnual analyte concentrations by site along the Brazos River. Asterisks represent differences between sites (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/823725f7b69294e47a4800e3.png"},{"id":37189272,"identity":"fa376d37-5fd6-4882-b239-e2777ac32845","added_by":"auto","created_at":"2023-05-18 12:49:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":410528,"visible":true,"origin":"","legend":"\u003cp\u003eAnnual\u003cstrong\u003e \u003c/strong\u003econcentration maps for parent parabens with spatial trends along the Brazos River. a. Methyl paraben, b. Propyl paraben, c. Butyl paraben\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/2d221e9b7597d34d28da22c3.png"},{"id":37189274,"identity":"4eaca454-75c9-4e7d-8731-9275117c267f","added_by":"auto","created_at":"2023-05-18 12:49:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":514332,"visible":true,"origin":"","legend":"\u003cp\u003eAnnual concentration maps of paraben transformation products along the Brazos River. a. Para-hydroxybenzoic acid, b. 3,4-dihydroxybenoic acid, c. methyl 3,5-dichloro-4-hydroxybenzoate, d. ethyl 3,5-dichloro-4-hydroxybenzoate\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/b17cf97851510c1bc0773b14.png"},{"id":44735765,"identity":"b630a785-8797-4321-8193-62488151681c","added_by":"auto","created_at":"2023-10-16 22:27:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1914141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/69dd7f45-9b39-459a-a960-f9b601289e3a.pdf"},{"id":37189276,"identity":"b8bb4361-f414-45e2-9c5e-feb1f311dfd0","added_by":"auto","created_at":"2023-05-18 12:49:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26833052,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformationAECT.docx","url":"https://assets-eu.researchsquare.com/files/rs-2928595/v1/66e246416a692ee91882b53b.docx"}],"financialInterests":"","formattedTitle":"Parabens and Paraben Transformation Products in The Brazos River (Texas, USA) Before and After Wastewater Treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eParabens are used as antimicrobials in foodstuff, personal care products and pharmaceuticals. An array of parabens are found in surface water across the globe with methyl paraben (MeP) concentration maxima of 1600 ng/L in Poland (Czarczyńska-Goślińska et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), 6140 ng/L in Brazil (Galinaro et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and up to 32.6 ng/L in China. (Feng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Parabens mostly enter the environment in wastewater effluent after being washed down drains due to their extensive use in personal care products. However, parabens are well removed by wastewater treatment, via transformation. The release of these transformation products in surface water is not well known. However, a study by Li et al (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that surface water near industrialized areas had higher concentrations of parent parabens but not chlorinated disinfection products. (Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Though while wastewater treatment plants were in the industrialized area, the source of the paraben release was not identified. The three most common tertiary disinfection processes include chlorination, UV disinfection, and ozonation. Therefore, chlorinated and hydroxylated disinfection products are common transformation products released into surface water.\u003c/p\u003e \u003cp\u003eTransformation of parabens in river water will primarily produce para-hydroxybenzoic acid (PHBA). Parabens can undergo both acid and base hydrolysis. Acid hydrolysis would only occur at pH values below 4, basic hydrolysis occurs at pH values above 10. (Valkova et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Therefore, hydrolysis is unlikely to play a major role in paraben transformation in natural rivers with moderate pHs. Photolysis is another degradation pathway that could transform parabens into PHBA and further transformation into phenol. (Gomes et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) Mechanisms of photodegradation include the addition of hydroxyl radicals to the aromatic ring which will transform parabens into hydroxylated compounds such as methyl 3,4-dihydroxybenzoate. (Gao et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) However, parabens have a maximum absorption from 256 to 258 nm, which falls in the UVC range. (Talrose et al., n.d.) There will not be a significant amount of UVC radiation in natural light within the troposphere due to ozone absorption of wavelengths up to 310 nm. (Svobodova et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) Therefore, photodegradation is unlikely to be a major paraben transformation pathway in surface water. The most likely transformation pathway is biodegradation. (Song et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) In wastewater treatment plants, biodegradation is effective at transformation parabens, mostly by transformation into PHBA using esterases and further transformation into phenol via decarboxylases. (Lu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) Transformation of parabens in river water would vary based on microorganisms present and environmental conditions such as, temperature, pH, and dissolved oxygen concentrations. While further transformation of paraben disinfection byproducts in natural waters has not been well evaluated, chlorinated disinfection byproducts are more persistent than the parents and transformation will occur slower. (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) This can be seen by the lower biodegradation and disinfection rates of chlorinated paraben species in wastewater treatment. Dichlorinated parabens released from effluent could be transformed into dichlorinated PHBA. However, halogenated PHBA has not been evaluated in either wastewater or river water. (Penrose and Cobb, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eHalogenation could also occur in natural river water where residual chlorine will likely be present due to release from upstream water treatment processes. Parabens are chlorinated via addition to the aromatic ring, followed quickly by a second addition on the aromatic ring. (Yoom et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) However, further chlorination once a paraben has been dichlorinated, is unlikely as even at chlorine concentrations of 10 mg/L seen in wastewater chlorination would takes days to add another chlorine. (Yoom et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Residual chlorine concentrations released in effluent are much lower after dechlorination with a concentration of 0.050 mg/L (0.3289 nM). Rate constants for MeP are 64.00 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the addition of one chlorine, 243.0 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for a second addition of chlorine and 1.300 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for a third addition with a chlorine concentration of 113.0 \u0026micro;M. (Mao et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Given the low residual chlorine concentrations released in wastewater effluent, it is unlikely that diluted residual chlorine concentrations in the Brazos would cause halogenation of parabens in the river. Sorption to sediments is not likely to occur as parabens do not tend to sorb to sediments during primary treatments in wastewater treatment. The average residual chlorine concentration in Waco drinking water during the year studied was 1.45 mg/L, a concentration too low to cause significant transformation into chlorinated paraben before entering wastewater treatment.\u003c/p\u003e \u003cp\u003eThis study evaluates parabens and paraben transformation products in the Brazos River upstream and downstream of a major wastewater treatment plant in Waco Texas, USA. This study also determined which parabens and released transformation products were transformed in the river by analyzing river sites and two tributaries further downstream. Seasonal differences in analyte concentrations in the river concentrations were also evaluated. To the best of the authors\u0026rsquo; knowledge, this is the first study to evaluate the direct impact of effluent release on chlorinated and hydroxylated paraben transformation product concentrations in river water, both directly downstream of release and then further downstream.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Standards and Supplies\u003c/h2\u003e \u003cp\u003eAnalytical standards included five parent parabens, four chlorinated paraben transformation products, three hydroxylated products, PHBA and five internal standards. Methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), and butyl paraben (BuP) were purchased from AccuStandard. Para-hydroxybenzoic acid 3,4-dihydroxybenzoic acid (DHBA), methyl 3,4 dihydroxybenzoic acid (MePOH), ethyl, 3,4 dihydroxybenzoic acid (EtPOH), methyl 3-chloro-4-hydroxybenzoic acid (ClMeP), methyl 3,5-dichloro-4-hydroxybenzoic acid (Cl\u003csub\u003e2\u003c/sub\u003eMeP), ethyl 3-chloro-4-hydroxybenzoic acid (ClEtP) and ethyl 3,5-dichloro-4-hydroxybenzoic acid (Cl\u003csub\u003e2\u003c/sub\u003eEtP) were purchased from Sigma-Aldrich. Internal standards MeP-d\u003csub\u003e4\u003c/sub\u003e, EtP-d\u003csub\u003e4\u003c/sub\u003e, PrP-\u003csup\u003e13\u003c/sup\u003eC, BuP-d\u003csub\u003e9\u003c/sub\u003e and PHBA-d\u003csub\u003e4\u003c/sub\u003e were all purchased from Cambridge Isotopes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instrumentation\u003c/h2\u003e \u003cp\u003eAn Acquity UPLC (Waters Corp., Milford, MA, USA) coupled to a Xevo TQS mass spectrometer was used for quantification of target products. Chromatographic separation was done using an Aquity UPLC BEH C18 column, 2.1 x 50 mm, 1.7 \u0026micro;m (Waters) with an injection volume of 10 \u0026micro;L and a flow rate of 300 \u0026micro;L/min. The mobile phase consisted of a 0.01% formic acid and acetonitrile gradient that began at a\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;70:30\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;ratio and transitioned to \u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;25:75\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;during \u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;15\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;minutes. Masslynx 4.1 (Micromass, Manchester, UK) software was used to process chromatographic data and Targetlynx (Waters) was used to generate calibration curves and quantify analyte concentrations in sample. Compounds were analyzed in ESI negative mode with both a quantifier ion and a qualifier ion. Fragmentation patterns, operating conditions and method validation are included in Tables S1-S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of Sample Sites\u003c/h2\u003e \u003cp\u003eEight sample sites near Waco, Texas were chosen based on accessibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is more of a constraint in Texas than in some other locations. Unlike many areas of the world where the public has access to rivers and narrow widths of shoreline, most US landowners have legal authority to prevent access to riverbanks on their properties. Seven sampling sites were along the Brazos River and were selected based upon proximity to a major wastewater treatment plant (WWTP1). All sample sites were either located along publicly accessible bridges, or on the Brazos River at sites along publicly accessible roads. Sampling along the Brazos occurred both upstream and downstream from WWTP1. Two tributaries downstream of WWTP1 were included in the study, with Bull Hide Creek having a smaller wastewater treatment plant (WWTP2) that releases effluent into the tributary. The distance between WWTP2 and the point at which the tributary meets the Brazos is approximately 30 km. The second tributary is located 1.5 km downstream of WWTP 1. WWTP 1 utilizes chlorination as a disinfection treatment process while WWTP2 uses UV disinfection. Three sites were upstream of WWTP 1, three sites were downstream of WWTP1, and two tributary sites were included in the analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Sample Collection and Solid Phase Extraction\u003c/h2\u003e \u003cp\u003eRiver water was collected in 500 mL amber glass bottles as grab samples. Samples were taken weekly over the course of the year, with a few weeks omitted. Sampling at 5 of the sites with bridge access (Sites 1, 2, 3, 5 and 6) occurred every Monday morning, while the remaining sites were taken Monday afternoons or evenings. The sample preparation is similar to the method used by Penrose and Cobb (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, once the samples were returned to the lab, each sample was spiked with 20 ppb of internal standard, acidified to a pH of 3, filtered, extracted using Oasis HLB cartridges and concentrated using nitrogen evaporation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Quality Control\u003c/h2\u003e \u003cp\u003eBlanks were included to monitor possible contamination throughout the analysis. One spiked blank, and one method blank were included and analyzed with every batch of samples (n\u0026thinsp;=\u0026thinsp;16). Analyte concentrations detected in method blanks were subtracted from all samples run in the same batch as the blank. Blank spikes were used to normalize concentrations for analyte recovery, with each blank spike being used for the samples run in the same batch as the spike. All samples were spiked with internal standards before extraction to evaluate recovery. Analytes that had no internal standards but are similar in structure to internal standards of the parent compounds used recovery result from the most structurally similar parent compound. A calibration curve was created using 8 calibration standards ranging in concentrations of 0.5 ng/mL to 100 ng/mL for most analytes and concentrations ranging from 5 ng/mL to 1000 ng/mL for PHBA and DHBA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Data Analysis\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.6.1 Site Comparisons\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eConcentrations were reported in ng/L with variance reported as confidence intervals. Only weeks where all samples were taken were included in analysis with, 40 weeks over the course of a year for all eight sites. The concentration of each paraben and transformation product was compared upstream and downstream of the major wastewater treatment plant to determine if concentrations in the river changed after the release of wastewater effluent. The site just downstream of wastewater treatment was compared to sites further downstream to see if natural degradation could be occurring as parabens and transformation products move downstream. Analyte concentrations in the tributaries were analyzed to see if any of the changes seen at sites further downstream of wastewater treatment could be related to movement from tributaries into the Brazos River. Of the potential paraben transformation products, only shorter chained transformation products were evaluated, as there are no available standards for longer chained paraben transformation products.(Albero et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) As a previous study showing transformation in wastewater treatment processes in the study area reported concentrations in picomolar units (pM) a table showing the conversion factor between ng/L and pM for each compound in included as Table S4. (Penrose and Cobb, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Seasonal Evaluation\u003c/h2\u003e \u003cp\u003eAnalyte concentrations (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S4) were evaluated to determine concentration differences between seasons at sites upstream and downstream of WWTPs. Maps were created for seasonal concentrations similarly to the yearly evaluation, these maps were added as supplemental information. (Fig. S5-S8)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Statistical Analysis\u003c/h2\u003e \u003cp\u003eNeither untransformed or log transformed concentrations were determined to meet the assumptions of ANOVA using Shapiro Wilk\u0026rsquo;s tests for normality and Bartlett\u0026rsquo;s tests for homogeneity of variances. Therefore, non-parametric tests were performed. All statistical comparisons were done using Kruskal-Wallis followed by Dunn\u0026rsquo;s Tests. Maximum likelihood estimations (MLE) were used to treat non-detects before performing statistical analysis. Statistical analyses were done using Microsoft Excel (2016), R (Version 3.4.1), and Sigma Plot (12.0). Maps and map features were made in QGIS 3.14.16 and ARCGIS 10.8.1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Yearly Concentrations in the Brazos River\u003c/h2\u003e \u003cp\u003eOf the evaluated compounds, MeP, PrP, BuP, Cl\u003csub\u003e2\u003c/sub\u003eMeP, Cl\u003csub\u003e2\u003c/sub\u003eEtP, PHBA and DHBA were determined to be significantly different by site. Of the parent products, MeP was found in the highest concentrations at sites along the Brazos River that are upstream of the wastewater treatment and in the Tehuacana Creek tributary (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PrP was detected in higher concentrations than MeP at the sites following release. Most of the differences in BuP concentrations are due to higher concentrations at site 6 located Downstream (south) of the meeting point of Tehuacana Creek and the Brazos River. Concentrations of both dichlorinated paraben transformation products were higher at sites downstream of the effluent release location. While PHBA saw a significant decrease at the site just downstream of the release of wastewater effluent, DHBA did not significantly decrease. The oxidation of PHBA to DHBA could be responsible for the decrease in PHBA and could also explain the stability in DHBA concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe products that were both significantly different between sites and had notable spatial trends, were mapped to visualize differences in analyte concentrations at different points in the Brazos. Of the parent compounds MeP, PrP and BuP concentrations had spatial trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). MeP concentrations decreased significantly from the site upstream of wastewater treatment to the site downstream of wastewater treatment. However, this decrease is unlikely to be solely due to dilution by the release of wastewater effluent, as the small outflow of the wastewater treatment with a flow of 32 ft\u003csup\u003e3\u003c/sup\u003e/sec is unlikely to have a large impact on the Brazos with flow rates ranging from an average of 220 ft\u003csup\u003e3\u003c/sup\u003e/sec in March 2021 to an average of 16,640 ft\u003csup\u003e3\u003c/sup\u003e/sec in June 2021. Flow rates remained above 2000 ft\u003csup\u003e3\u003c/sup\u003e/sec through August before decreasing in September. The higher water flows in the summer months would decrease concentrations due to dilution. Transformation of parabens in the environment is a major contributor to the removal of parabens in surface water. Biodegradation is the most likely route of transformation of parabens in surface water. The differences in microbial communities along the Brazos could be the cause for the sudden change in methyl paraben concentrations seen at the site downstream of wastewater treatment. Biodegradation occurs much more quickly under aerobic conditions than under anaerobic conditions. (Wu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) A change to more aerobic conditions could cause more degradation to occur between site 3 and site 4. Neither hydrolysis nor photolysis are major pathways in paraben transformation in the environment. (Svobodova et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Talrose et al., n.d.; Valkova et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) MeP concentrations in the Brazos increased from site 4 to site 6 (p\u0026thinsp;=\u0026thinsp;0.041), which could be due to the input of MeP from other sources. Considering that site 6 is located near a residential neighborhood, input from recreational activities is one potential source of the observed increase. Desorption from solids released from the wastewater treatment plant is another possibility as parent parabens have been detected in wastewater sludge with MeP concentrations of 89.4 ng/g. (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) MeP has also been detected in river surface sediment, with a median concentration of 12.4 ng/g. (Feng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) A study by Feng et al (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that in the Huai River, mean MeP concentrations were 51.7 ng/L while mean MeP concentrations in the sediment were 11.6 ng/g giving a K\u003csub\u003ed\u003c/sub\u003e of 0.224 L/g. However, the same study detected MeP concentrations of 8.84 ng/L in surface water and 13.0 ng/g in sediment in the Yellow River. The K\u003csub\u003ed\u003c/sub\u003e for the Yellow River values was 1.47 L/g. Arfaeinia et al (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that the median K\u003csub\u003ed\u003c/sub\u003e between seawater and sediment for MeP was 3.52 g/mL, which is lower than the K\u003csub\u003ed\u003c/sub\u003e values calculated from the MeP concentrations in the river samples, showing variation by environment. (Arfaeinia et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) Using an average of 0.847 L/g from the two Kd values determined from the Huai River and Yellow River, and with average total dissolved solids of 300 mg/L in effluent at WWTP1, the minimum concentration sorbed to sludge needed to cause the observed concentration change is 783 nmol/kg or 119 ng/g. MeP concentrations detected in wastewater sludge in previous studies show that this concentration change could possibly be due to desorption from wastewater solids after effluent release. While the tributary upstream of site 6 does have detectable MeP concentrations at site 5, a combination of the lower MeP concentration and small size of the tributary means that the tributary is unlikely to have a large impact on the paraben concentration in the Brazos. It is unlikely that the increase in MeP concentrations are due to direct input as the area of the Brazos River is not used for recreation. MeP concentrations decreased further downstream with significantly lower concentrations at site 8 when compared to site 6 (p\u0026thinsp;=\u0026thinsp;0.045). The observed change in concentration is likely due to natural MeP degradation and dilution in the river. The tributary with the small wastewater treatment plant has a very low MeP concentration which is unlikely to have a large impact on MeP concentration in the Brazos. EtP concentrations were not significantly different between any site in the yearly analysis. This due to the low concentrations of EtP at all sites. The low EtP concentrations were expected due to EtP being used seldom in industry.\u003c/p\u003e \u003cp\u003ePrP had similar concentration changes in the river as MeP. PrP decreased at site 4 (site 3 and site 4: p\u0026thinsp;=\u0026thinsp;0.030) downstream of effluent release before increasing at site 6 (site 4 and site 6: p\u0026thinsp;=\u0026thinsp;0.015). However, the visual decrease in PrP is smaller than that of MeP, and the differences between site 6 and site 8 were determined to be insignificant (site 6 and site 8: p\u0026thinsp;=\u0026thinsp;1.00) The lack of decrease shows that PrP degraded at a slower rate than MeP in the Brazos, this matches the transformation rates seen in many treatments and metabolic process, though PrP has been seen to degrade faster than MeP in activated sludge. (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) The Bull Hide Creek tributary also had a higher concentration of PrP but are still not in high enough concentration to explain the similarities in PrP concentrations in the main channel. Similarity of PrP concentrations in the Brazos River main channel demonstrates a slower degradation rate for PrP than for MeP.\u003c/p\u003e \u003cp\u003eBuP concentrations at upstream sites were lower in concentration than either MeP or PrP. This is expected as BuP is used much less often than MeP or PrP in either personal care products or foodstuff. BuP concentrations increased from site 3 to site 4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which could be due to release of BuP in wastewater effluent. BuP increased from site 4 to site 6 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), followed by a decrease from site 6 to site 8 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as MeP had. No notable spatial trends were observed for BzP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the transformation products, PHBA, DHBA, Cl\u003csub\u003e2\u003c/sub\u003eMeP and Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations showed spatial trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). PHBA was the compound detected in highest concentrations at all sites with its highest concentration at 10.30 ng/L (4.850 ng/L to 21.88 ng/L) also having the largest variation amongst the compounds. As with MeP and PrP, PHBA concentrations significantly decreased from site 3 to site 4 (site 3-site 4: p\u0026thinsp;=\u0026thinsp;0.031). This decrease in PHBA could be due to aerobic biodegradation occurring between site 3 and site 4. In the case of aerobic biodegradation, PHBA can be further degraded into phenol via carboxylases, which does not occur with anaerobic biodegradation. This change in environment is likely due to a dam less than a mile upstream of site 3 that causes aeration of the river water as the water traverses the dam. The short distances between the dam and site 3 could explain the changes are not yet seen at that site. PHBA concentrations increased visually from site 4 to site 6 and from site 6 to site 8. However, these increases were not significant (site 4 and site 6: p\u0026thinsp;=\u0026thinsp;1.00, site 6 and site 8 p\u0026thinsp;=\u0026thinsp;1.00). This was unexpected, as PHBA is the common degradation product of all parent parabens and would be expected increase in concentration as parabens degrade. Site 7 had a relatively high concentration of PHBA, when compared to the low concentration of the other analytes at that site with an average concentration of 4.465 ng/L (2.479 ng/L to 8.045 ng/L). This could be due to PHBA being a potential transformation product of UV disinfection. However, the higher concentration in the tributary did not seem to have significant effect on Brazos River PHBA concentrations. While DHBA concentrations showed overall significant difference between sites, the only significant differences were between the Brazos River sites and the tributaries and none of the sites along the river showed any significant differences between each other.\u003c/p\u003e \u003cp\u003eCl\u003csub\u003e2\u003c/sub\u003eMeP concentrations remained consistently low upstream of wastewater effluent release, with no significant differences between sites 1 through 3 (site 1 and site 2: p\u0026thinsp;=\u0026thinsp;1.00, site 2 and site 3: p\u0026thinsp;=\u0026thinsp;1.00). However, at site 4, downstream of wastewater treatment, concentrations were notably higher and Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations were significantly different from site 3 (site 3 and site 4 p\u0026thinsp;=\u0026thinsp;0.032). Dichlorinated parabens are not used in industry and are not likely to be introduced from other sources between site 3 and site 4 and dichlorinated parabens quantified at upstream sites are likely remaining from release in effluents further upstream. A waterpark with high concentrations of chlorine located along the Bosque River 2 km upstream of the where the Bosque and Brazos meet could introduce chlorine or chlorinated parabens to the upstream sites. The dichlorinated paraben species are released in higher concentrations than the parents in effluent at the major wastewater treatment plant. (Penrose and Cobb, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations remained consistent at sites downstream of effluent release, with only small visual decreases and no significant differences between sites 4, 6 and 8 (site 4 and site 6: p\u0026thinsp;=\u0026thinsp;1.00, site 6 and site 8: p\u0026thinsp;=\u0026thinsp;1.00). The stability in concentrations at downstream sites shows that Cl\u003csub\u003e2\u003c/sub\u003eMeP degraded at a much slower rate than the parent parabens. Given the low concentrations of Cl\u003csub\u003e2\u003c/sub\u003eMeP in the tributary and water quantity differences between the tributaries and the Brazos River, it is unlikely that either of the tributaries played a significant role in the lack of decreases in Cl\u003csub\u003e2\u003c/sub\u003eMeP at sites 6 and 8.\u003c/p\u003e \u003cp\u003eCl\u003csub\u003e2\u003c/sub\u003eEtP concentrations had similar trends to Cl\u003csub\u003e2\u003c/sub\u003eMeP, with a stable concentration upstream of effluent, an increase at release (site 3 and site 4: p\u0026thinsp;=\u0026thinsp;0.036), and a stable concentration downstream. Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations were lower than Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations due to the higher use of MeP in products resulting in higher concentrations of MeP transformation products in the river. Both dichlorinated paraben products were released from wastewater effluent and entered the Brazos at concentrations high enough to cause a change in the Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations in the Brazos River. This combined with persistence seen with both compounds show that the dichlorinated species, will be more relevant than their respective parent compounds in the environment. Dichlorinated PHBA is an important transformation product due to the high concentrations in the river. Data has been obtained for both mono and dichlorinated PHBA. However, they were not included in spring quality control due to the standards not being available until after spring analysis and neither are included in this study. While the dichlorinated species were in detectable concentration changes along the river, ClMeP and ClEtP did not have noticeable spatial trends during the yearly analysis, which is due to the low concentrations of ClMeP and ClEtP at all sites. The dichlorinated species are the only compounds evaluated that increase in the Brazos River directly as due to the release of wastewater effluent. Longer chained chlorinated species have not been evaluated due to a lack of available standards.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Seasonal Differences in Analyte Concentrations Along the Brazos River.\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Differences in Analyte Concentrations Between Seasons\u003c/h2\u003e \u003cp\u003eOf the quantified compounds, PrP BzP, DHBA and ClEtP concentrations were determined to be significantly different between seasons. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For PrP most of the differences were due to lower concentrations in the summer. Variation in concentrations were generally highest in the summer for multiple compounds, which is likely due to the low concentrations during June and early July and high concentrations in early August. Summer was expected to have the highest concentrations due to increased use of personal care products, particularly sunscreen. However, the study area has a large student population that would be absent during summer. The low concentrations in early summer could also be explained by high water levels during that period due to a large amount of rainfall in late May and early June. High flow rates in May would also cause low analyte concentrations in late Spring. March and April flow rates were lower at an average of 183 ft\u003csup\u003e3\u003c/sup\u003e/sec and 176 ft\u003csup\u003e3\u003c/sup\u003e/sec respectively before increasing due to rainfall in May with average flow in May being 7617 ft\u003csup\u003e3\u003c/sup\u003e/sec. Flow rates were highest in June at 16640 ft\u003csup\u003e3\u003c/sup\u003e/sec and remained higher throughout July and in early August with average flows of 5870 ft\u003csup\u003e3\u003c/sup\u003e/sec in July and 1674 ft\u003csup\u003e3\u003c/sup\u003e/sec in August. August had higher analyte concentrations than the other summer months, the change in analyte concentrations are due to the decreasing water level and return of the student population. The change from low analyte concentrations in early and mid-summer and higher analyte concentrations in late summer caused higher variation in the summer. By September the flow rates were near flows in April at 375 ft\u003csup\u003e3\u003c/sup\u003e/sec, though there were days in September with flow rates up to 1840 ft\u003csup\u003e3\u003c/sup\u003e/sec. High water levels impact the concentrations detected in late spring, but overall did not cause noticeable differences between spring and winter or spring and fall. Summer PrP concentrations were significantly different than winter but not significantly different from fall and spring, with summer having generally lower concentrations than winter. This is likely due to both the high-water levels that persisted from late spring throughout most of summer and the decreased population in the general area during the summer. While ClEtP concentrations showed significant differences by season, adjusted p-values showed no differences between ClEtP concentration between any two specific seasons (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Seasonal differences in DHBA concentration were determined to be caused by low DHBA concentrations in the winter.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral differences between seasons. Bolded values are significant.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP-Values Between Seasons\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.610\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.022\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBuP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBzP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.019\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.746\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.029\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMePOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.184\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtPOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.809\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.027\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignificant p-values from seasonal analyses. A lager table that includes non-significant p-values is included as supplemental information. (Table S5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeasons\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFall and Summer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring and Summer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eBzP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFall and Summer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eDHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFall and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0485\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0492\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0453\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMePOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0363\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEtPOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0272\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0487\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFall and Summer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0497\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring and Summer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer and Winter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGeneral differences in concentration by season were determinable for multiple compounds. When evaluating differences in seasonal concentrations at each individual site, differences were much more sporadic only having a few notable differences randomly distributed across sites. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). None of the three upstream sites had any significant differences in MeP, PrP, or PHBA concentrations between seasons, despite those three compounds being found in the highest concentrations at the three sites. Reasons for the lack of differences could be consistent release of these compounds from upstream, though differences would still be expected due to changes in water level. Sites 4, 6 and 8 were expected to have different concentrations of Cl\u003csub\u003e2\u003c/sub\u003eMeP and Cl\u003csub\u003e2\u003c/sub\u003eEtP, as the concentrations released in wastewater effluent upstream of site 4 would vary by season and flow rate through the plant. However, Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations were only seasonally different at site 3, while Cl\u003csub\u003e2\u003c/sub\u003eEtP was significantly different at site 3 and the two tributaries. No specific compound was seasonally different across all upstream sites or all downstream sites and so no trends could be identified. P-values from Dunn\u0026rsquo;s tests showing which seasonal concentrations were significantly different at each site are included as supplemental information.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSeasonal concentration differences at individual sites along the Brazos River. Bolded values are significant.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eSeasonal P-Values by Site\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSite 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSite 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSite 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSite 4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSite 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSite 6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSite 7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSite 8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.035\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.043\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.459\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.845\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBuP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.638\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBzP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.046\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.3372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.023\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.566\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMePOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.386\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.027\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.580\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtPOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.015\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.700\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.597\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.011\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.028\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.501\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.015\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.022\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.025\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.780\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.024\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.486\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ea.\u003c/b\u003e Site 4 is just downstream of WWTP1 \u003cb\u003eb.\u003c/b\u003e Site 7 is downstream of WWTP2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Differences Between Sites Across Seasons\u003c/h2\u003e \u003cp\u003eIn general, concentration differences between sites at each season was consistent with the differences seen in the yearly analysis with some variation between seasons. (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) MeP concentrations were generally the highest of the parent parabens at upstream sites with averages of 1.814 ng/L (0.4826 ng/L to 6.817 ng/L) during fall, 1.162 ng/L (0.3133 ng/L to 3.145 ng/L) during spring, 0.8018 ng/L (0.2031 ng/L to 3.165 ng/L) during summer and 0.9434 ng/L (0.1849 ng/L to 4.814 ng/L) during the winter at site 1. MeP concentrations varied by site during every season except summer (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) with Dunn\u0026rsquo;s Tests (Table S6 to Table S10) showing that the differences are specifically between sites 1 and 4 (spring: p\u0026thinsp;=\u0026thinsp;0.009, fall: p\u0026thinsp;=\u0026thinsp;0.028, winter: p\u0026thinsp;=\u0026thinsp;0.022), sites 2 and 4 (spring: p\u0026thinsp;=\u0026thinsp;0.011, fall: p\u0026thinsp;=\u0026thinsp;0.009, winter: p\u0026thinsp;=\u0026thinsp;0.038) and sites 3 and 4 (spring: p\u0026thinsp;=\u0026thinsp;0.006, fall: p\u0026thinsp;=\u0026thinsp;0.018, winter p\u0026thinsp;=\u0026thinsp;0.018). Therefore, concentrations of MeP dropped significantly during three out of four of the seasons moving from site 3 to site 4, which matches the change seen in the yearly analysis. Maps showing spatial changes and bar graphs showing analyte concentrations by site are included as supplemental information. The increase in MeP at site 4 to site 6 seen in the yearly analysis was visually seen across seasons. However, winter (site 4 to site 6: p\u0026thinsp;=\u0026thinsp;0.041) was the only season with a significant increase, going from an average MeP concentration of 0.0421 ng/L (0.0155 ng/L to 0.1049 ng/L) at site 4 to 0.8998 ng/L (0.3292 ng/L to 2.459 ng/L) at site 6. No significant trends were identified for PrP in any individual season. Visual changes in PrP were similar to that of changes seen in MeP. However, the only significant differences were due to low PrP concentrations in the tributaries with no differences between sites in the main channel, sites and the tributaries, and not between two sites along the Brazos. BuP was the only compound with significant differences between sites in every season (spring: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, summer: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, fall p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, winter: p\u0026thinsp;=\u0026thinsp;0.031). However, BuP concentrations are noticeably high at site 6 with average concentrations of 0.4685 ng/L (0.3928 ng/L to 0.5588 ng/L) in spring, 0.6120 ng/L (0.4716 ng/L to 0.7943 ng/L) in summer, 0.5617 ng/L (0.5023 ng/L to 0.6280 ng/L) in the fall and 0.5239 ng/L (0.4714 ng/L to 0.5822 ng/L) in the winter. The high concentrations of BuP at site 6 are likely responsible for the observed differences, as the only differences in BuP concentrations are between site 6 and other sites. BzP concentrations were significantly different between sites in the fall and winter. However, differences in the fall were between sites 1 and 4 (p\u0026thinsp;=\u0026thinsp;0.035), sites 4 and 5 (p\u0026thinsp;=\u0026thinsp;0.009) and sites 5 and 6 (p\u0026thinsp;=\u0026thinsp;0.0122), while differences in winter were only between site 4 and 5 (p\u0026thinsp;=\u0026thinsp;0.036). While there are differences, no trends were identified.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral differences between sites by season. Bolded values are significant.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eP-Values by Season\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.016\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBuP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.031\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBzP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.048\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.013\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDHBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.015\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.481\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMePOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.792\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtPOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.037\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.502\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eMeP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eEtP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.039\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.043\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDuring most seasons, PHBA and DHBA were present in higher concentrations than any of the other analytes, likely due to being natural degradation products of all parent parabens. PHBA concentrations were significantly different between site during the fall and winter while DHBA concentrations were significantly different in spring and summer. However, in all cases, the differences were between non-tributary sites and site 5, meaning the only notable trend is that there are significantly lower concentrations of PHBA at site 5 during fall and winter and DHBA during spring and summer. It is important to note that PHBA was only significantly different during seasons where DHBA was not significantly different, and vice versa. This could be due to DHBA being a hydroxylated product of PHBA. Increases in DHBA concentration would be related to decreases in PHBA concentration, which could be due to transformation of PHBA into DHBA by aerobic microbes using hydroxylases. Transformation from PHBA to DHBA upstream of site 5 during the fall in winter would decrease PHBA concentrations, causing PHBA concentrations at that site to be significantly lower than other sites. Cl\u003csub\u003e2\u003c/sub\u003eMeP and Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations were significantly different between sites during all seasons except spring (Table\u0026nbsp;5). Further investigation using adjusted p-values from Dunn\u0026rsquo;s tests (Table S4) did not show significant differences in Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations between any individual sites during the summer. Differences in winter followed trends seen in the yearly analysis with a significant decrease from site 3 and site 4 (p\u0026thinsp;=\u0026thinsp;0.024) and no significant changes at sites downstream of site 4. As with the yearly concentrations, Cl\u003csub\u003e2\u003c/sub\u003eMeP remained consistent from site 4 to site 6 and from site 6 to site 8. Given the lack of statistical differences, the numerical increases are due to the high variation of Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations during all seasons at all downstream sites. One explanation for the high variation in Cl\u003csub\u003e2\u003c/sub\u003eMeP is inconsistent release in Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations in wastewater effluent between weeks. During fall there was a numeric change between site 3 and site 4 but no significant change, though there were differences between other upstream and downstream sites showing that there was a change between upstream and downstream sites (site 2 and 4: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. site 1 and 6: p\u0026thinsp;=\u0026thinsp;0.004, site 2 and 6: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, site 2 and 8: p\u0026thinsp;=\u0026thinsp;0.008). Cl\u003csub\u003e2\u003c/sub\u003eEtP changes were spatially similar to Cl\u003csub\u003e2\u003c/sub\u003eMeP. While Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations were determined to be different during the summer, the only differences were between site 6 and site 7 (p\u0026thinsp;=\u0026thinsp;0.020) and between site 3 and site 7 (p\u0026thinsp;=\u0026thinsp;0.010). The lack of differences between upstream and downstream sites of both Cl\u003csub\u003e2\u003c/sub\u003eMeP and Cl\u003csub\u003e2\u003c/sub\u003eEtP in the summer, means that the release of effluent did not have the same effect on river water concentrations that it had in other seasons. This could be due to the high rainfall in early summer that would not only increase water level in the Brazos, thereby increasing dilution of effluent, but also cause high flow rates and reduced retention times in wastewater treatment, reducing transformation from parent to dichlorinated species. As with Cl\u003csub\u003e2\u003c/sub\u003eMeP, Cl\u003csub\u003e2\u003c/sub\u003eEtP also was not significantly different between site 3 and 4 during the fall but were different between other upstream and downstream sites (site 2 and 6: p\u0026thinsp;=\u0026thinsp;0.034). However, unlike Cl\u003csub\u003e2\u003c/sub\u003eMeP, Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations were significantly different between individual upstream sites and site 4 during spring (site 2 and 4: p\u0026thinsp;=\u0026thinsp;0.023, site 3 and 4: p\u0026thinsp;=\u0026thinsp;0.034). Cl\u003csub\u003e2\u003c/sub\u003eMeP concentrations would be expected to have greater increases due to the higher use of MeP that would result in higher concentrations released in effluent.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eYear-long and seasonal spatial relationships of parabens and paraben transformation products were evaluated at different sites along the Brazos River in relation to a wastewater treatment plant. All sites were located in or near Waco, Texas. MeP and PrP concentrations decreased after release of wastewater effluent, followed by an increase in concentration further downstream. MeP concentrations decreased at the site furthest downstream, which is likely due to natural degradation and dilution. Other parent parabens had similar visual trends, but any differences matching those trends were not significantly different. PHBA was the transformation product quantified in the highest concentration at most sites during most seasons. The dichlorinated parabens are the only compounds that increased directly as a result of wastewater effluent release, meaning that released concentrations high enough to cause increases in the Brazos River. The dichlorinated species also remained in similar concentrations at sites further downstream, showing the relevance of the dichlorinated transformation products in the environment. The Cl\u003csub\u003e2\u003c/sub\u003eMeP and Cl\u003csub\u003e2\u003c/sub\u003eEtP concentrations were not high enough to induce estrogenic effect. However, chlorinated PHBA will be in higher concentrations than either Cl\u003csub\u003e2\u003c/sub\u003eMeP or Cl\u003csub\u003e2\u003c/sub\u003eEtP and no studies have determined estrogenic effects of chlorinated PHBA. While the concentrations in this study are low, this study focuses more on identifying which compounds are being released in effluent and which are transformed in the environment. This could be helpful by identifying analytes that are likely to accumulate in areas downstream of wastewater treatment plants with different tertiary treatment processes. Future research in this area should identify other important transformation products in the environment specifically chlorinated PHBA, or the potential bioaccumulation of the studied transformation products. Research could also be geared towards identifying different toxicological effects of the transformation products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMichael Penrose, designed experiments, conducted research, collected data, created figures and tables, and wrote the manuscript with input from George Cobb. George Cobb helped design experiments, reviewed methodology, and reviewed/revised the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Chad Mansfield and Bianca Possamai for their part in sampling along the Brazos and Janelle Oldfather for helping process samples. The authors would also like to thank the Baylor Mass Spectrometry Center for providing access to the LC-MS used in analysis. The authors thankfully acknowledge financial support from the C. Gus Glasscock, Jr. Endowed Fund of Excellence in Environmental Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;C. Gus Glasscock, Jr. Endowed Fund of Excellence in Environmental Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the finding of this study are available in an OSF repository at https://osf.io/2gvjp/?view_only=66ace57fc1f64e8f90c3eee1c265808c.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas S, Greige-Gerges H, Karam N, Piet M-H, Netter P, Magdalou J (2010) Metabolism of Parabens (4-Hydroxybenzoic Acid Esters) by Hepatic Esterases and UDP-Glucuronosyltransferases in Man. 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Sci Total Environ 634:677\u0026ndash;686. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2018.03.330\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2018.03.330\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Parabens, disinfection byproducts, wastewater treatment, emerging contaminants","lastPublishedDoi":"10.21203/rs.3.rs-2928595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2928595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParabens are ubiquitous, being found in surface waters around the world. Although little is known about the release of paraben transformation products and fate of transformation products in surface water. This study evaluates both parabens and paraben transformation products in the Brazos River upstream and downstream of a wastewater facility located in Waco, Texas. Concentrations of thirteen compounds were reported in this study, five parent parabens and eight paraben disinfection byproducts. Analyte concentrations were spatially evaluated to determine if release of wastewater effluent effects their concentrations in the river. Two Brazos River tributaries were also sampled to determine if they released parabens and related compounds to the Brazos. Sampling occurred weekly for one year with at least 40 samples collected at each site. Analyses were completed for both yearly and seasonal data. Sites downstream of wastewater treatment outfalls had lower concentrations of methyl paraben during the yearly analysis and across multiple seasons in the seasonal analysis. Para-hydroxybenzoic acid was the compound present in greatest concentration at most sites across most seasons, and spatial changes in para-hydroxybenzoic acid varied by season, with no identifiable trends. Dichlorinated paraben concentrations increased in the river at sites downstream of wastewater treatment. Concentration increases indicate that wastewater effluent contains sufficiently high dichlorinated paraben concentrations to effect concentrations downstream of effluent discharges. Dichlorinated species also persisted in the environment, with no significant decreases at sites further downstream during any season. Methyl paraben concentrations decreased at the site furthest downstream while dichlorinated methyl paraben concentrations remained stable showing that the dichlorinated species degrade slower than their respective parent paraben. Due to the dichlorinated species being released in higher concentrations in effluent than parents and being more resistant to degradation, the dichlorinated parabens are more likely to environmentally relevant than are parent parabens.\u003c/p\u003e","manuscriptTitle":"Parabens and Paraben Transformation Products in The Brazos River (Texas, USA) Before and After Wastewater Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-18 12:49:06","doi":"10.21203/rs.3.rs-2928595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-05-23T00:15:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-16T19:30:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-15T14:24:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Environmental Contamination and Toxicology","date":"2023-05-12T12:40:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"af398ac4-cf44-45c3-8023-c5f88e928a69","owner":[],"postedDate":"May 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:15:59+00:00","versionOfRecord":{"articleIdentity":"rs-2928595","link":"https://doi.org/10.1007/s00244-023-01025-x","journal":{"identity":"archives-of-environmental-contamination-and-toxicology","isVorOnly":false,"title":"Archives of Environmental Contamination and Toxicology"},"publishedOn":"2023-08-09 21:55:30","publishedOnDateReadable":"August 9th, 2023"},"versionCreatedAt":"2023-05-18 12:49:06","video":"","vorDoi":"10.1007/s00244-023-01025-x","vorDoiUrl":"https://doi.org/10.1007/s00244-023-01025-x","workflowStages":[]},"version":"v1","identity":"rs-2928595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2928595","identity":"rs-2928595","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}