According to the sample types (water, sediment, or sludge), sterol concentrations were expressed in μg/L and in μg/g of dry weight (d.w.) for aqueous samples (water and sludge) and solids (sediments and dewatered sludge), respectively. Since the grain-size distribution could significantly affect the sterol concentrations from the sediments [32
], concentrations of the total sterols in sediments were normalized to the total organic carbon, denoted as μg/mg of TOC. Urban samples from the Norman WWTP showed significantly higher amounts of sterols than rural samples from the Illinois River Basin, and the two sites showed distinctive sterol profiles.
3.1. The Norman WWTP
Total sterol concentrations in the Norman WWTP samples ranged from 194 μg/L and 1625 μg/L for water and 12,724 μg/L and 74,099 μg/L for sludge. The difference in concentrations between the water and sludge could be explained by the hydrophobic nature of sterols. Due to their hydrophobic property, discharged sterols tend to associate with particulates and are progressively removed from the water during clarification that leads to a higher concentration of sterols in the sludge. The highest sterol concentration occurred in the primary sludge at 74,099 μg/L, where the solid material as well as domestic vegetable oil and grease accumulate. After undergoing anaerobic digestion, a large amount of organic matter was degraded and/or consumed by the anaerobic bacteria, and the concentration of total sterols was 5117 μg/g in the dewatered sludge of the Norman WWTP (Table 2
Sterols were encountered in lower concentrations in the water than in the sludge. The influent and the primary effluent of the Norman WWTP exhibited similar sterol concentrations, namely 1615 μg/L and 1580 μg/L, respectively (Table 2
). After undergoing the biological treatment and final clarification, the concentration of the total sterols was reduced by a factor of 8 and was 194 μg/L in the final effluent.
Despite the significant differences in sterol concentrations before and after treatments, all of the water, or all of the sludge, samples showed similar sterol distributions, but the water and sludge samples themselves were different (Figure 2
). Water samples, including the activated sludge (high water content), were characterized by a predominance of cholesterol, coprostanol and sitosterol, representing more than 70% of the total sterols (Figure 2
a). All of the sludge samples, excluding the activated sludge, exhibited high proportions of coprostanol, epicoprostanol, and 24-ethylcoprostanol, representing about 60% of the total sterols (Figure 2
Cholesterol is one of the vital components in the cell membranes of vertebrate organisms and some marine algae species [17
]. It is also found, to a lesser extent, in some vascular plants [19
]. Coprostanol is formed from the hydrogenation of cholesterol in the gut facilitated by reducing bacteria. It is found as one of the major sterols in the feces of numerous warm-blooded animals. The presence of high proportions of cholesterol and coprostanol are typical of omnivore feces signatures, such as human and porcine. Moreover, McCalley, et al. [33
] pointed out that the conversion of cholesterol to coprostanol could also occur during sludge digestion, and a higher proportion of coprostanol relative to cholesterol would be found in digested sludge. This is consistent with that was observed in the Norman WWTP samples, where only the digested sludge showed higher amounts of coprostanol associated with relatively low amounts of cholesterol. During the wastewater treatment, concentrations of coprostanol and cholesterol were reduced from 385 μg/L and 547 μg/L in the influent to 25 μg/L and 42 μg/L, respectively, in the final effluent. Concentrations of coprostanol and cholesterol were still relatively high in the final dewatered sludge, i.e., 1489 μg/g and 313 μg/g, respectively (Table 2
Epicoprostanol, an isomer of coprostanol, is another biodegradation product of cholesterol. It was found below the detection limit in all of the water samples but represented one of the major sterols in sludge samples. The differences in concentration between the water and sludge samples could be explained by the lipophilic property of epicoprostanol. However, according to McCalley, et al. [33
], conversion of epicoprostanol does not occur extensively during intestinal digestion but is more effective under the sludge digestion process, due to the enforced and prolonged bacterial activity in the digestion tank. Epicoprostanol could be formed either by the dehydrogenation of cholesterol via coprostanone as an intermediate, or by epimerization of coprostanol during sludge digestion. Therefore, it has been proposed that epicoprostanol is more useful for distinguishing between contamination from treated and untreated waste. Consequently, in the Norman WWTP samples, the occurrence of epicoprostanol (below detection limit in water but high amounts in the sludge) is a result of the sludge treatment rather than human feces input.
β-Sitosterol is essentially synthesized by vascular plants and represents the major sterol in plant material. In the Norman WWTP water samples, it was present in relatively high proportions and represented about 20% of the total sterols. High proportions of β-sitosterol in urban wastewater could originate from domestic food products (cooking oil or vegetables), and/or from the washout of urban vegetation. Concentrations of β-sitosterol in water decreased after the wastewater treatment and were reduced by a factor of 7 compared to the influent.
24-Ethylcoprostanol, an anaerobic degradation product of β-sitosterol, represents one of the major sterols in the digested sludge but showed much lower concentrations in the water samples (Table 2
). It is known as the major fecal sterol of ruminants, such as cow and sheep, but could also be present in relatively high concentrations in human feces, just slightly lower than coprostanol [21
]. During the wastewater processes, about 88% of 24-ethylcoprostanol was removed from the water.
Besides the regular sterols, sulfur-containing steroids, like thiosteranes, were also identified in many of these samples. Two thiosteranes, cholestane-3β-thiol and 24-ethylcholestane-3β-thiol, were quantified in low concentrations in the final sludge products of the Norman WWTP (Table 2
). A good correlation between the concentration of cholestane-3β-thiol and coprostanone was observed, which suggested the thiosteranes were possibly formed from stanones, such as coprostanone, in the presence of H2
S and HS−
during anaerobic reduction process and are ultimately found in digested sludge. Due to the stability of these compounds under anoxic and suboxic conditions, it is proposed that these thiosteranes could be used as novel tracers for the environmental input of sewage products or land application of sewage sludge.
3.2. The Illinois River Basin
Because of their hydrophobic properties, sterols tend to associate with particulates in water and deposit on the surface of sediment layer. In the Illinois River Basin, only the uppermost surface sediment (of about 0.5 cm) was sampled; the results obtained from sediment were considered as comparable to those obtained from water.
In comparison to the urban samples, described above, samples from Illinois River Basin contained much lower concentrations of sterols varying between 1 and 7 μg/g d.w. sed. or 0.35 and 1.20 μg/mg of TOC. The Tenkiller Lake sample showed an exception, with sterol concentrations up to 37.98 μg/g d.w. sed. or 3.22 μg/mg of TOC. All of the Illinois River Basin samples showed similar sterol distributions (Figure 2
c). β-Sitosterol was the major sterol with proportions varying between 32% and 53%. Cholesterol and campesterol were found as the second and third most abundant sterols, and their relative proportions varied between 15% and 28% and 10% and 21%, respectively. As previously mentioned, these sterols are synthesized by a large range of living organisms (including plants and animals), and are ubiquitous in the environment. Their occurrence in the Illinois River samples does not reveal any specific information on their source. Other sterols, in particular fecal stanols, like coprostanol, epicoprostanol and 24-ethylcoprostanol, which contain specific source information, were found in relatively low concentrations in the studied samples. However, high amounts of phytosterols (β-sitosterol and campesterol) and cholesterol in parallel with low amounts of the fecal stanols in the Illinois River samples do not necessarily indicate the absence of fecal contamination. As pointed out by Leeming, et al. [21
], low amounts, or the absence, of intestinal microflora in the gut of several warm-blooded animals may result in low amounts of ingested sterols being transformed into stanols during the digestion process. The non-transformed sterols are discharged and found as the major sterols in these animal feces, whereas the fecal stanols are found as minor compounds. Related animals having low amounts of intestinal microflora are dogs and several avian herbivores like chicken [34
In an attempt to refine the source of contamination, principal component analysis was performed on the sterol distributions of the Illinois River Basin samples and several ”pure” feces samples, such as pig, human, domestic pets, WWTP water, cow, chicken, horse. The latter were considered as potential sources of contamination in the study area. Statistical analysis was performed using the sterol distribution of different pure fecal sources. Different groups were discriminated based on the proportion of cholesterol (87%, F1 axis) and of β-sitosterol plus coprostanol (55% + 28%, F2 axis). Each group corresponds, a posteriori, to specific group of animals having similar diets and the ability to convert sterols into stanols (Figure 3
). Indeed, herbivores, like cows, buffalo, sheep, horse, comprise the first group, and their feces are characterized by high proportions of C29
-sterols and C29
-stanols. The second herbivore group, comprising by avian herbivore such as chicken, swan, and rosella, differs from the others by the presence of a high proportion of C29
-sterols and low amounts of C29
-stanols. Several carnivore avian species (including magpie and seagull), as well as dogs, constituted the third group. The latter group showed higher proportion of C27
-sterols and lower proportion of C29
-sterols. Other omnivores, such as humans and pigs, differed from the others by having a relatively high proportion of C27
-stanols and C29
-stanols. The Illinois River samples showed similar fecal sterol distributions to those of the avian herbivore group. This similarity indicates a potential contribution from the fecal material of swan, rosella and chicken into the Illinois River Basin. However, due to the low amount of fecal stanols and the ability of stanol formation during the incorporation of sterols into the sediments [36
], interpretation of PCA results should be undertaken with caution.
In order to confirm the interpretation obtained from PCA, compound specific isotope analysis (CSIA) was performed on several selected sterols, in particular phytosterols. CSIA applied to phytosterols is based on the differences in carbon isotope signatures between C3
]. Indeed, during plant photosynthesis, atmospheric 12
C carbon dioxide is preferentially incorporated by plants, leading to the formation of sterols that are isotopically lighter than atmospheric carbon dioxide [38
]. This fractionation is more important for C3
plants, due to the two different photosynthetic pathways employed by these plants, which is the Calvin cycle for C3
plants and the C4
-dicarboxylic acid pathway for C4
plants. Therefore, C3
plants are generally more depleted in 13
C (ranges −33‰–−22‰) than C4
plants (around −20‰–−10‰) [39
]. Among the current terrestrial vegetation, C3
plants dominate and represent 85% of plant species including all trees and most temperate and cold species. C4
plants comprise a minority of terrestrial plant species, less than 4%, and are mostly concentrated in the tropical and subtropical areas [42
]. The most common C4
plant species are corn, sorghum, millet and sugarcane. Hence, determination of the δ13
C values of selected phytosterols is a reliable tool to discriminate between livestock feedlot sources (i.e., cattle) and wild animals (i.e., deer) as sources of fecal pollution, since in the former, the animals are mainly fed with corn (C4
plants) whereas the latter consume mainly grass (C3
Previously, Chikaraishi, et al. [43
] defined the δ13
C variation ranges of several selected phytosterols in C3
plants. By comparison with the data published by the authors, the phytosterols detected in the Illinois River Basin samples showed intermediate isotopic values between C3
plants. As the land in the Illinois River Basin is mainly covered by C3
plants, in which the δ13
C is more depleted than C4
plants, the shift towards heavier δ13
C values measured in Illinois samples should refer to the C4
plant contribution. As C4
plants were not cultivated in the study area, but corn is widely used as feed in the poultry farms, the shift towards heavier δ13
C in Illinois River Basin samples was most likely the result of chicken manure input from surrounding chicken farms.
3.3. Sterol Fingerprint as Source of Fecal Contamination Indicator
The decrease in concentration and the change in distribution of the sterols, in particular coprostanol, cholesterol, epicoprostanol and thiosteranes, from input to output of the Norman WWTP indicate the effective removal of the fecal material at the Norman WWTP. However, human fecal signature could still be clearly recognized in the final products (including effluent and dewatered sludge), which is characterized by a predominance of coprostanol. As the final effluent is directly discharged into the Oklahoma river system after the plant, and the final sludge is generally used for agriculture field application, the sterol fingerprints obtained from the output of the WWTP can provide qualitative insights on the impacts of urban sewage on the regional environmental quality. Moreover, use of a combined approach of sterol fingerprint and the carbon CSIA also provides a powerful tool for source tracking in some non-point source pollution sites, such as the Illinois River Basin.
In addition to sterol concentrations and distributions, several sterol ratios were also useful for source discrimination. Examples given in Table 3
were previously determined to discriminate human feces contamination and were applicable to different sample matrices like water, sediment and/or sludge. Ratio calculations were performed, based on the results obtained by Biache, et al. [29
], on the Norman WWTP samples. The results (ratios R1 to R7 reported in Table 3
) confirmed that human feces contamination was dominant in the Norman WWTP samples, including the final effluent and dewatered sludge.
Sterol ratios could also be used to distinguish different animal feces, such as pig, cow, chicken, horse and deer from a cross-polluted water or sediment from rural sites. For instance, sitostanol/coprostanol was used by Derrien,
et al. [48
] to discriminate cow (1.5–3.3) and pig (0.2–1.0) feces. Coprostanol/(Coprostanol + 24-ethylcoprostanol) × 100 permits differentiation between human (>73), herbivore (<38) feces and mixtures of the same (38–73) [45
]. The ratio of (coprostanol+epicoprostanol)/cholesterol was used by Standley,
et al. [22
] to discriminate human (<0.01) and cattle/horse/deer (>0.1) feces. Compared to CSIA, which provides relevant results but involves the use of highly precise and costly measurement by isotope ratio mass spectrometer (IRMS), calculation of sterol ratios is cheaper, faster, and can provide a rapid and relatively reliable preliminary interpretation on the source of fecal contamination.
Fecal material input to the environment is permanent, although the presence of fecal material does not necessarily have to be related to contamination. For a better environmental quality assessment, it is also necessary to associate the FIB interpretation, from which the presence and level of fecal contamination can be determined. Theoretically, for a known fecal source, the quantity of fecal sterols in water should reflect the quantity of fecal material released into the water. Hence, the sterol concentration could also be potentially used to indicate fecal contamination. Numerous studies have demonstrated a potential correlation between sterol fingerprints and FIBs [25
]. For example, it has been shown that coprostanol concentrations of 60 and 400 ng/L seem to correspond to the defined primary and secondary contact limits for thermo-tolerant coliforms and entercococci [49
]. However, Isobe, et al. [52
] also pointed out that this correlation, in particular between coprostanol concentrations and E. coli
density, could be affected by seasonal and geographic variation effects. Indeed, in the current environment, FIBs and molecular MST markers could degrade at different rates due to their differential fate and transport [54
], and the half-life of coprostanol under aerobic conditions is <10 days [55
]; therefore, a reliable correlation between coprostanol (or other fecal stanols) and FIBs should be expected under limited conditions such as the feces being fresh or recently released. The relationship between FIBs and sterol fingerprints seems very complex and need to be more thoroughly investigated in further monitoring programs.