Environ. Sci. Technol. 1997, 31, 3663-3668
Bile Acids as a New Class of Sewage Pollution Indicator MOHAMED M. ELHMMALI, DAVID J. ROBERTS, AND RICHARD P. EVERSHED* Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 1TS, U.K.
The bile acid content of raw sewage, treated effluent, and estuarine sediment was determined as the first step in the development of bile acids as a new class of sewage pollution indicator. Sediments and particulates from raw sewage and effluent, were solvent extracted, saponified, fractionated by solid phase extraction (SPE) and “flash” column chromatography, derivatized, and analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/ MS). Mass spectral comparisons confirmed unambiguously the presence of bile acids in all the samples studied. The major compounds detected were deoxycholic and lithocholic acids, together with minor components, in a characteristic distribution that reflected the predominantly human origin of the effluent. Bile acids were found to exist in a “bound” form and were released upon saponification. They were found to be quantitatively relatively more resistant to degradation during sewage treatment as compared to coprostanol. Some changes were observed in the abundances of certain minor bile acids, for example, the 3βepimers were much reduced in relative abundance in the treated effluent and sediment as compared to their 3R counterparts. Bile acids offer considerable promise as indicators of specific sources of sewage pollution, particularly if used in conjunction with the widely employed 5β-stanols, e.g., coprostanol.
Introduction The assessment of sewage pollution is of a considerable importance for health, aesthetic, and ecological reasons. Sewage usually contains a variety of pathogenic microorganisms together with many undesirable chemical pollutants, and hence, contamination of potable water supplies is an obvious hazard to health. Determining the extent and source of sewage are important goals in monitoring this type of pollution. While bacteria, such as coliform and streptococci, have been used as sewage pollution indicators (1-4), their use has been questioned mainly due to variations in their survival under different environmental conditions (2, 5). For this reason, advantages exist in the use of characteristic chemical markers of sewage contamination. Sterols and related compounds have been widely used to detect sewage pollution, with most work having focused on coprostanol (5β-cholestan-3β-ol; (6-11). The potential usefulness was first recognized by Murtaugh and Bunch (12) since it is one of the major sterols present in human feces, being formed through biohydrogenation of cholesterol by symbiotic microorganisms living in the small intestine. * Author to whom correspondence should be addressed. Tel: (0)117 9287671; Fax: (0)117 9251295; e-mail: r.p.evershed@ bristol.ac.uk.
S0013-936X(97)00404-5 CCC: $14.00
1997 American Chemical Society
Coprostanol is not produced in significant amounts by lower animals (13), thus the feces of higher animals is the only significant source in nature (13-15). Although it is generally accepted that the detection of coprostanol in sediments is an indication of sewage inputs, there is evidence to suggest that cholesterol may be reduced to 5R- and 5β-stanols in sediments (16, 17). Teshima and Kanazawa (17-19) have suggested that the relative proportion of coprostanol as compared to cholesterol is important in determining the source of sedimentary coprostanol. In a development of this approach, Grimalt et al. (6) proposed the ratio of 5R:5β-stanols to be a more reliable criterion for assessing fecal input. Although coprostanol is readily degraded by aerobic bacteria (7, 11, 13, 20), under anaerobic conditions it is degraded very slowly (21-23). The survival of coprostanol under favorable conditions is evidenced by its detection in archaeological soils and sediments associated with deposition of ancient sewage, i.e., Roman cesspits and drains (24-26) Interestingly, these investigations of fecal material in archaeological sediments have provided preliminary indications of the co-occurrence of bile acids together with the widely used 5β-stanol fecal marker compounds (24, 26, 27). The survival of bile acids and 5β-stanols in ancient soils and sediments is not surprising since they are the main excretory products of body cholesterol (15, 28-30) and possess structures that are conducive to their survival. Normal human feces contains more than 20 different bile acids that are formed from the primary bile acids, cholic and chenodeoxycholic acid (28-32). Two series of bile acids are found in nature, i.e., those containing 24 carbon atoms and those containing 27 or 28 carbon atoms. The former group are derivatives of cholanic acid, whereas the latter are derivatives of cholestanoic acid (33). 5β-Cholanic acid derivatives constitute the largest class of naturally occurring C24 bile acids. Lithocholic acid (3R-hydroxy-5β-cholanoic acid; LC; I), deoxycholic acid (3R,12R-dihydroxy-5β-cholanoic acid; DOC; II), chenodeoxycholic acid (3R,7R-dihydroxy-5β-cholanoic acid; CDOC; III), cholic acid (3R,7R,12R-trihydroxy-5β-cholanoic acid; C; IV), hyodeoxycholic acid (3R,6R-dihydroxy-5β-cholanoic acid; HDOC; V), and ursodeoxycholic acid (3R,7β-dihydroxy-5βcholanoic acid; UDOC; VI) are the major C24 acids (see Figure 1). DOC (II) and LC (I) are the major secondary bile acids found in the feces of humans (and some other animals) with healthy individuals excreting about 140 mg of DOC (II) and 90 mg of LC (I) acid per day (31). In this paper, we explore the potential of bile acids as a new class of pollution indicator. The objectives of the study were to (i) confirm the identities of bile acids in raw sewage effluent from a U.K. city, (ii) study their resistance to sewage treatment, and (iii) attempt to detect their presence in estuarine sediments adjacent to a disused sewage outfall.
Experimental Section Samples. Samples studied were collected from the following: (i) raw and treated effluent sewage sample (10 L) taken from Wessex Water Saltford Recovery Works, Saltford, U.K., which handles the sewage from the city of Bath. This Works operates a secondary biological filter treatment system, and (ii) surface sediment (500 g) collected 20 m from the sewage outfall at Ladye Bay, Severn Estuary, Clevedon, U.K.. This outfall now receives only stormwater, as raw sewage has not been dumped from the outfall since May 1990. Authentic bile acids were obtained from the Sigma Chemical Co. (St. Louis, MO) and dissolved in methanol for use as recovery standards or for internal standardization (see below). Sample Preparation. Liquid effluents (1 L) were filtered through glass-fiber filters (Grade GF/C Whatman, U.K.), and
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FIGURE 1. Bile acid structures referred to in the text. the filters containing trapped particulate matter were Soxhlet extracted in the same manner as the sediment samples, following appropriate addition of internal standards (see below). The freeze-dried sediment samples were ground to a fine powder with a ceramic pestle and mortar and sieved [250 µm; ENDECOTTS (Test Sieves) LTD]. Hyocholic acids [3R,6R,7R-trihydroxy-5β-cholanoic acid; HC (VIII); (see Figure 1); 40 µg of each] was added as the internal standard to 10 g of dried sediment for the quantification of bile acids. The samples were Soxhlet extracted, in a preextracted thimble, with 150 mL of 2:1 v/v dichloromethane (DCM)/methanol (MeOH) for 24 h. After extraction, solvent was removed by rotary evaporation, the concentrated extracts were transferred to a small vial, and the remaining solvent was removed by evaporation under a gentle stream of nitrogen. Samples were saponified by adding 5 mL of 5 M KOH in 90% methanol to the dried extract and heated at 120 °C for 1 h. Liquid/Solid Extraction. After the saponified sample was cooled to room temperature, 10 mL of water was added to the organic extract, which was then acidified to pH 3-4 with 6 M HCl, and the neutral and acidic compounds were extracted with chloroform (3 × 10 mL). The combined chloroform extracts were reduced in volume by rotary evaporation and then passed through an anhydrous sodium sulfate (BDH Co.) column to remove any water that may effect the efficiency of SPE aminopropyl columns. The extracts were transferred to preweighed vials, reduced to dryness under a gentle stream of nitrogen, dissolved in a minimum volume of hexane by ultrasonication, and then applied to an aminopropyl column (500 mg of NH2; Isolute Co.) that had been preconditioned with hexane (6 mL). The neutral sterols were eluted with 6 mL of 2:1 v/v DCM/2-propanol, and the carboxylic acids were recovered by elution with 12 mL of 3% v/v acetic acid in diethyl ether.
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FIGURE 2. Partial GC profiles for the hydroxy acid fractions of raw sewage (a) and treated effluent from the first (b) and final (c) stages in the treatment process. Numbers on the peaks correspond to those listed in Table 1. For full experimental details, see text. Derivatization. The free carboxylic acids recovered from aminopropyl columns were transferred to screw-capped vials, methylated by adding 10 mL of freshly prepared diazomethane in ether, and allowed to stand at room temperature overnight. Methylated acids were then dissolved in 1 mL 2:1 v/v DCM/ hexane and further fractionated on an activated (120 °C, overnight) silica gel column (150 × 8 mm; 0.6 g; 220-440 mesh). The column was eluted with 5 mL of 2:1 v/v DCM/ hexane and 5 mL of 2:1 v/v DCM/MeOH to recover monocarboxylic fatty acid methyl esters and hydroxy carboxylic acid methyl esters (including the bile acids), respectively. All fractions were evaporated to dryness and converted to their trimethylsilyl (TMS) ethers by adding 100 µL of 9:3:1 v/v/v silylating agent [dry pyridine-hexamethyldisilazane-trichlorosilane (Sigma Chemical Co.)], sealed under nitrogen and allowed to stand at 70 °C for 1 h. The excess derivatizing reagents were then removed with a gentle stream of nitrogen, and the derivatized bile acids were diluted with an appropriate volume of hexane prior to the analysis by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Instrumental Analysis. Aliquots (0.5-1 µL) of all samples were injected manually into a Hewlett-Packard 5890 Series II GC using on-column injection. The analytes were separated using a Chrompack CP-SIL-5 CB fused silica capillary column (50 m × 0.32 mm i.d.; 0.12 µm film thickness). The oven was held for 1 min at 40 °C following injection, then temperature programmed from 40 to 230 °C at 20 °C min-1, then to 300 °C at 2 °C min-1, and held at that temperature for 20 min. Hydrogen was used as the carrier gas (10 psi column head pressure), and a flame ionization detector (FID) was used to
TABLE 1. List of Bile Acids and Other Hydroxy Acids Analyzed by GC and GC/MS as Methyl Ester-Trimethylsilyl (Me-TMS) Ether Derivatives peak no.a
Mol Wt
characteristic fragment ions
compound assignment
1 2 3 4 5 6 7 8 9 10 11 12 13f 13f 14 15 16 17 18 19 20 21 22 23
502 456 398 442 470 412 456 470 484 426 470 462 498 462 550 550 498 550 638 550 550 498 476 638
73,129,147,399 73,397,413,441 98,292,325,367 75,395,411,427 73,411,427,455 98,306,339,381 75,409,425,441 117,423,426,455 73,425,441,469 98,320,353,395 75,423,439,455 215,257,357,372 73,439,455,483 &215,257,357,372 208,255,345,370 208,255,345,370 117,451,454,483 255,355,370,460 253,343,368,458 255,355,370,460 255,355,370,460 75,451,467,483 121,231,386,476 147,369,355,458
C18-monoacylglycerol C23 R-OHb C22 DAc C22 ω-OHd C24 R-OHb C23 DAc C23 ω-OHd C24 (ω-1)OHe C25 R-OHb C24 DAc C24 ω-OHd 3β-hydroxy-5β-cholanoic C26 R-OHb 3R-hydroxy-5β-cholanoic (LC; I)g 3β,12R-dihydroxy-5β-cholanoic 3R,12R-dihydroxy-5β-cholanoic (DOC; II) g C26 ω-1OHc 3R,7R-dihydroxy-5β-cholanoic (CDOC; III) g 3R,7R,12R-trihydroxy-5β-cholanoic (C; IV) g 3R,6R-dihydroxy-5β-cholanoic (HDOC; V) g 3R,7β-dihydroxy-5β-cholanoic (UDOC; VI) g C26 ω-OHd 3R-hydroxy-12-oxo-5β-cholanoic (VII) g 3R,6R,7R-trihydroxy-5β-cholanoic (HC; VIII) g
a Peak numbers correspond to those used in Figures 2 and 4. components. g I-VIII refer to structures shown in Figure 1.
b
R-Hydroxy acids. c Diacids.
monitor the column effluent in GC analyses. GC/MS analyses were carried out using a Carlo Erba HRGC 5160 Mega series GC, comprising an on-column injector, coupled to a single stage quadrapole (Finnigan MAT 4500). The GC conditions were similar to those used for the GC analyses. The temperature of the interface between the GC and MS was held at 300 °C. The MS operating conditions were as follows: ion source 170 °C, filament current 0.25 ma, electron voltage 70 eV, m/z range 50-650, scan rate 1 scan s-1, helium was the carrier gas. Data acquisition and processing were carried out using an INCOS data system. Peak assignments were made by comparison of literature mass spectra and comparisons of retention times of authentic compounds, followed by co-injection. Quantification was based on GC/FID peak areas with reference to an internal standard (see above).
Results and Discussion The aim of the investigation was to establish bile acids as a new class of sewage pollution indicator. Initial work focused on determining the concentration and distribution of bile acids in raw sewage effluent and assessment of their survival through a treatment works. Subsequent studies were carried out to establish whether or not they survive in estuarine sediments at a location that would have received substantial amounts of raw sewage in the recent past. Bile acids were extracted, fractionated, and characterized from sediments and sewage effluent using a protocol that combined and modified a range of existing procedures (32, 34-37). Recoveries of the free bile acids LC, DOC, CDOC, C, HDOC, UDOC, and HC through the analytical procedure were between 60 and >80% with mean reproducibilities of better than (10%. The major loss of these acids was found to occur during the saponification step and is probably due to the differing solubility of these acids in water during the solventsolvent partitioning step of this procedure. A full account of the calibration procedures, recoveries, and reproducibilities will be presented in a future publication devoted to the quantification of the bile acids in environmental samples now in preparation. Bile Acid Content of Raw Sewage. Figure 2a shows the partial GC profile for the hydroxy carboxylic acid fraction
d
ω-Hydroxy acids. e (ω-1)-Hydroxy acids. f Co-eluting
(methyl ester-trimethylsilyl derivatives) of the raw sewage effluent entering the Saltford treatment works. The identities of all the major components are summarized in Table 1. This table also lists the major ions seen in the EI mass spectra of all the hydroxy acids identified. The data show that this fraction comprises mainly bile acids, with R-, ω-, and (ω 1)-hydroxy fatty acids as minor constituents; small amounts of diacids also appear in this fraction. The major bile acid is deoxycholic acid (DOC; II), which was readily identified by GC/MS. Although an M•+ was not evident, the [M - 15]+ ion was present at m/z 535. The characteristic base peak of this compound was present at m/z 255, which corresponds to loss of the side chain () 115 amu) and two of the trimethylsilanol () 90 amu) groups, i.e., [M - (2 × 90 + 115)]+. Other characteristic fragment ions are m/z 370, [M - (2 × 90)]+, m/z 460, [M - 90]+, and m/z 345, [M - (90 + 115)]+. The prominent ion at m/z 208 arises by fragmentation of ring C with retention of the side chain, ring D, and an additional carbon (38). The second most abundant bile acid in the sewage was lithocholic acid (LC; I), which co-eluted with the C26 R-hydroxy acid (ca. 1:1 mixture). The lithocholic acid was identified unambiguously by GC/MS through the presence of M•+ 462, together with the base peak at m/z 372 resulting from the loss of a trimethylsilanol group, [M - 90]+. Other characteristic ions appear at m/z 357, [M - (90 + 15)]+; m/z 215, [M - (90 + 157)]+ and m/z 257, [M - (90 + 115)]+. The corresponding 3β-epimers of deoxycholic and lithocholic acids, seen eluting prior to the 3R-isomers, show analogous fragmentation patterns but with the expected differences in the relative abundances of the various characteristic ions. More minor components include the primary dihydroxy bile acids chenodeoxycholic (CDOC, III) and cholic (C, IV). Further minor bile acids include ursodeoxycholic acid (UDOC, VI) and the keto acid 3R-hydroxy-12-oxo-5βcholanoic acid (VII). As far as we are aware, this analysis represents the first reported study of bile acids in sewage effluent. Significantly, the distribution of compounds is seen in the effluent largely mirrors that reported previously from the stool of healthy humans (29). It should be noted that the hyocholic acid (HC, VIII), eluting later in the chromatogram, is the added internal standard, selected as it is not known to
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FIGURE 3. Plots of the concentrations of deoxycholic acid and coprostanol associated with the particulate fractions (>0.7 µm) at different stages in the sewage treatment process. The data show the higher resistance of bile acids as compared with coprostanol to degradation during the treatment process. be produced in significant abundance by healthy humans (29, 39). Resistance of Bile Acids to Sewage Treatment. The partial GC chromatograms for the bile acids recovered from the sewage after the two main treatment steps are shown in Figure 2b,c. The data show that all the bile acids detected in the raw sewage are detectable in the effluent from first and final treatment stages. Moreover, the relative abundances of the individual bile acids referred to above remain largely unchanged following treatment, which is remarkable given the opportunities that would have existed for microbiological degradation during the treatment process. The main difference seen between the composition of the bile acids in the raw sewage and the treated effluent is the reduction in the
abundance of the 3β-isomers of DOC (II) and LC (I) acids. Although the actual loss is difficult to calculate in the case of LC due to the problem of co-elution with the R-hydroxy C26 fatty acid, the relative ratio of DOC to its 3β-isomer decreases from ca. 3:1 in the raw sewage to ca. 5:1 after the first stage of treatment to ca. 7:1 in the final effluent. The trend towards the formation of the 3R-epimer compares with that seen for coprostanol where the concentration of epicoprostanol increases following sludge digestion (7). Quantitative analysis also showed (Figure 3) that there was a 54% reduction in the concentration of DOC (II) acid between the raw sewage and the final treated effluent. Figure 3 also shows the plot of the concentration of coprostanol through the various stages of the treatment works. While the concentration of coprostanol is much higher than that of the bile acids in the raw sewage, it is clear from the data obtained that the coprostanol is much more severely effected by microbiological degradation occurring during the treatment process, showing an 88% reduction in concentration between the raw sewage and the final effluent. The explanation for this is not completely clear although we have evidence indicating that bile acids exist primarily in a “bound” form, only being revealed in significant abundance following saponifcation. This “binding” would appear to offer substantial protection from microbiological attack; however, more work is required to elucidate the nature of this binding, i.e., whether it be chemical bonding or physical adsorption. Detection of Bile Acids in Estuarine Sediments. Figure 4 shows the partial GC profile of hydroxy acid fraction obtained from sediment taken from the Severn Estuary in the area of Ladye Bay adjacent to a disused sewage outfall (now used as a stormwater drain). The analysis of both excavated surface (top 1 cm) and buried sediment (1-20 cm depth) show analogous distributions of compounds. Extracts of both samples are dominated by bile acids and ω-hydroxy acids (the ω-hydroxy C22 component predominating). Figure 5 panels a and c, shows the mass spectra recorded for two of the major bile acids (LC I and DOC II) recovered from the sediment compared with the spectra of authentic lithocholic (Figure 5b) and deoxycholic (Figure 5d) acids recorded under the same GC/MS conditions. The spectra of the sedimentary bile acids are comparable in almost every respect to those of the authentic compounds, thus confirming unambiguously
FIGURE 4. Partial GC profile for the hydroxy acid fraction of the total lipid extract of the Ladye Bay bottom sediment (1-20 cm). Numbers on the peaks correspond to those listed in Table 1. For full experimental details, see text.
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sediment. Thus, we have shown that bile acids represent a new class of sewage pollution indicator. An important feature of using bile acids as pollution indicators stems from the association of specific components and their distributions with the feces of different animals, which may allow different sources of fecal pollution to be distinguished or mixed inputs to be recognized. For example, hyocholic and hyodeoxycholic are produced in substantial amounts by pigs as compared to humans, while the latter produce deoxycholic acid, which is virtually absent in pigs. Thus, while both humans and pigs produce coprostanol as their major 5β-stanol, their feces can be readily distinguished on the basis of their bile acid distributions. We have already extended this idea to distinguish between other groups of animals likely to be significant sources of fecal pollution, i.e., the major domesticated ruminant and non-ruminant animals (27; Elhmmali, Roberts, and Evershed, in preparation). While we have detected all the major bile acids known to be produced by healthy humans in sewage effluent and sediments, further work is required to fully understand the effects on bile acid distribution and abundances of different domestic cleaning agents, sewage treatments, and environmental influences.
Acknowledgments We gratefully acknowledge Wessex Water Saltford Recovery Works, U.K., for supplying samples and providing statistical information. We thank Mr. Jim Carter for his technical assistance and the NERC for providing the mass spectrometry facility at the University of Bristol (Contract Grant F14/6/13). FIGURE 5. Electron ionization mass spectra of peaks 13 (a) and 15 (c) in the partial GC chromatogram shown in Figure 4 as compared with those of the methyl ester-trimethylsilyl ether derivatives of authentic lithocholic (b) and deoxycholic (d) acids, respectively. For full experimental details, see text. the occurrence of bile acids in estuarine sediments for the first time. It is significant that the distribution of bile acids closely resembles that seen in the sewage effluent (see Figure 2a). In addition to DOC (II) and LC (I) acids, other minor bile acids were also detectable in the sediments; these included CDOC (III), 3R-hydroxy-12-oxo-5β-cholanoic acid and HDOC (V). While the DOC (II) and LC (I) acids are present in approximately the same relative abundance as in the sewage effluent, the abundance of the corresponding 3β-isomers is markedly reduced. This trend follows that seen in the treated effluent relative to the raw sewage. The reason for this is unclear, although it is presumed to relate to the susceptability of the different epimers to microbiological degradation. A further minor difference that exists between the composition of the bile acids in the sewage effluent and the sediment extract concerns the presence of HDOC (V) in the Ladye Bay sediment. This component is not excreted in appreciable amounts by healthy humans (29, 39) as shown by the fact that it was undetectable in the raw sewage entering the treatment works. It is notable that HDOC (V) is one of the major bile acids excreted by pigs [the other being LC (I), with DOC (II) being absent (39)]; hence, the presence of this bile acid in the sediment strongly indicates an origin in agricultural runoff from catchment areas into the streams and rivers that feed the Severn Estuary.
Conclusions Bile acids are a major class of steroid present in feces that up to now appear to have been completely overlooked as pollution indicators. The results presented herein show that bile acids (i) are readily detectable in raw sewage, (ii) survive microbiological degradation during treatment, (iii) are relatively more resistant to degradation during treatment than coprostanol, and (iv) are readily detectable in an estuarine
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Received for review May 7, 1997. Revised manuscript received August 12, 1997. Accepted August 12, 1997.X ES9704040 X
Abstract published in Advance ACS Abstracts, October 1, 1997.
