Water Air Soil Pollut (2012) 223:1017–1031 DOI 10.1007/s11270-011-0920-8
Sewage Treatment Plants Efficiencies in Removal of Sterols and Sterol Ratios as Indicators of Fecal Contamination Sources Vesna Furtula & Johnny Liu & Patricia Chambers & Heather Osachoff & Chris Kennedy & Joanne Harkness
Received: 29 March 2011 / Accepted: 5 August 2011 / Published online: 18 August 2011 # Springer Science+Business Media B.V. 2011
Abstract This study assessed the efficiency of sewage treatment plants (STPs) in removing sterols based on chemical analyses of both influents and effluents. Samples from 3s and three tertiary plants were collected and analyzed by gas chromatography mass spectrometry for 23 individual sterols including mestranol, norethindrone, equol, estrone, equilin, norgestrel, 17α-ethinylestradiol, 17α-estradiol, 17βestradiol, estriol, dihydrocholesterol (cholestanol), coprostanol, epicoprostanol, cholesterol, desmosterol, campesterol, stigmasterol, β-sitosterol, coprostanone, cholestanone, epicholestanol, stigmastanol, and 24ethylcoprostanol. The percentage of sterols remaining in effluent samples (compared to influent samples) ranged from 0% to 80% and varied among sterol compounds and with STP location and treatment type. V. Furtula (*) : J. Liu : P. Chambers Aquatic Ecosystem Impacts Research Division, Pacific Environmental Science Centre, Environment Canada, 2645 Dollarton Highway, North Vancouver, BC V7H 1B1, Canada e-mail:
[email protected] H. Osachoff : C. Kennedy Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada J. Harkness Urban Systems Ltd., 200—286 St. Paul Street, Kamloops, BC V2C 6G4, Canada
Differences in the efficiency of sterol removal for secondary and tertiary STPs were statistically significant. Although the concentration of sterol compounds differed between influents and effluents, sterol abundances remained the same. The most abundant sterol detected was cholesterol, followed by the fecal sterol coprostanol, and the plant sterols 24-ethylcoprostanol and β-sitosterol. For three STPs, the hormone estrone was detected in effluents at concentrations of 0.03–0.05 μg L−1. Ten sterol ratios specific for human fecal contamination and eight sterol ratios for differentiating among multiple sources of fecal contamination were calculated and showed that 12 ratios for influent and nine ratios for effluent were successful for human fecal source tracking. Based on sterol ratio values in this study, new criteria for identification of human fecal contamination were suggested. Keywords Sterol ratios . Sewage treatment plant . Influent . Effluent . Human fecal contamination
1 Introduction Human, animal, and industrial wastes are significant contributors to the deterioration of surface water quality and have the potential to impact aquatic life and impair the beneficial uses of water bodies. One major waste component contributing to reduced water quality is fecal material that originates from sources
1018
such as municipal waste sewage treatment plants (STPs), stormwater drains, livestock operations, and wildlife. Source tracking methods to identify sources of fecal contamination generally target either chemical or microbial indicators (Gilpin et al. 2003; Hagedorn and Weisberg 2009; Leeming et al. 1997; Tyagi et al. 2009). Individual fecal sterols and various sterol ratios have been utilized for some time as chemical markers for sewage and agricultural contamination (de Castro Martins et al. 2007; Leeming et al. 1996; Zhang et al. 2008). Compared to bacteria, which are highly affected by natural factors such as temperature, salinity, and sunlight (Chou and Liu 2004; Devane et al. 2006), sterols are more resistant to environmental stress, which make them more suitable as indicators (Leeming et al. 1997; Tyagi et al. 2009 and references within). Sterols are a family of compounds with a steroid ring structure and varying functional groups that confer specific characteristics (such as polarity, bioactivity, and lipophilicity) to the molecule. Some sterols are hormones and/or endocrine-disrupting compounds (EDCs) that can have profound effects on aquatic life by affecting growth, development, and reproductive potential (van Aerle et al. 2002; Imai et al. 2007). Sterols such as estrone, 17β-estradiol, estriol, and 17α-ethinylestradiol are considered ubiquitous in untreated wastewater (Kümmerer 2009), while their presence in effluent is dependent on treatment specifics. Effluent from primary treatment of domestic wastewater may retain high levels of steroid estrogens (Desbrow et al. 1998), whereas secondary treatment may enhance removal (Baronti et al. 2000) although the effects of treatment type and method on estrogenicity are not always clear (Servos et al. 2005). Because estrogenic sterol compounds that act like hormones have the potential to impact the aquatic organisms at the STP discharge site and in the downstream environment, the occurrence of such estrogenic sterols in the environment or discharge from STPs may need to be regulated (Orrego et al. 2009). Evaluation of efficiency of sterol removal for STPs could be an important tool to indicate the type of plant that excels at reducing constituent estrogenic sterols. Studies that employ sterols and sterol ratios as markers for tracing anthropogenic pollution have typically employed as the sample matrix: sediments/ sludge (Carreira et al. 2004; Fattore et al. 1996;
Water Air Soil Pollut (2012) 223:1017–1031
Froehner et al. 2009; Marvin et al. 2001; Mayer et al. 2008), feces/manure (Jardé et al. 2007a; Leeming et al. 1996), or filter/residue from filtration of water samples (Devane et al. 2006; Gilpin et al. 2003; Grimalt et al. 1990; Jardé et al. 2007b). The present study applied sterol analysis to the result of direct liquid–liquid extraction of STP influents and effluents, which are complex matrices, in order to characterize sterol composition and calculate sterol ratios for comparison with literature-based sterol ratio values. Since sterols bind to particulate matter, liquid– liquid extraction is advantageous because it enables the evaluation of bound and unbound sterol compounds thereby capturing more sterol compounds than a filtration method (Gilli et al. 2006; Szúcs et al. 2006). The objectives of this study were: (1) determination of sterol composition of influents and effluents from secondary and tertiary STPs; (2) evaluation of the efficiency of sterols removal for a range of STP types; and (3) assessment of the usefulness of sterol ratios to specify the source of fecal contamination. Following liquid–liquid extraction of six STP influents and effluents, 23 sterols (including 10 estrogenic sterols) were analyzed. Based on the data in this study, new criteria for some sterol ratios were suggested for tracking of human fecal contamination.
2 Materials and Methods 2.1 Sampling Sites Influent and effluent samples were obtained from six STPs between November 2009 and June 2010. STPs are located throughout the province of British Columbia (BC), Canada (spread across an area of 220,000 km2). They were chosen to provide a broad overview of different treatment processes within BC and North America, which occupy very different climate zones (from desert to mountainside to lakeside). All six STPs have incoming sewage that is domestic in nature with no agricultural waste, and little, if any, industrial waste. Most STPs in North America primarily treat waste from households (people and pets) and light industry. Industrial operations (including agricultural operations such as large processing plants), other than light industry, typically receive their own operating licenses and
Water Air Soil Pollut (2012) 223:1017–1031
1019
have their own facilities to treat wastewater, or are subject to municipal sewer by-laws requiring treatment prior to disposal in city sewers. The result is that most wastewater to STPs is from domestic sources and runoff. STPs in this study used either secondary or tertiary treatment, although the processes in each STP differ from one another (Table 1). STP-1 has tertiary treatment an extensive lagoon system with very long hydraulic retention times. The process applies treatment for phosphorus (by the addition of alum), disinfection by chlorination, and no treatment for nitrogen. STP-2 has tertiary treatment. It is mechanical plant with short hydraulic retention times and effluent filtration, with treatment for nitrogen and phosphorus removal (all by biological processes) and disinfection by ultraviolet (UV) light. STP-3 is a mechanical plant with short hydraulic retention times with treatment for nitrogen (by biological processes) and no disinfection or phosphorus removal. This site is subject to very high influent concentrations as a result of the extreme measures which have been implemented for water conservation. This plant can operate as either a secondary or tertiary plant; however, it was operating as a tertiary plant when samples were taken. STP-4 provides secondary treatment. It is a basic lagoon system with no treatment for nitrogen or phosphorus and no disinfection. STP-5 has secondary treatment and is mechanical plant with short hydraulic retention times with no treatment for nitrogen and phosphorus; disinfection is by UV. STP-6 provides secondary treatment. It is mechanical plant with short hydraulic retention times. It has significant ammonia treatment (by biological
processes) as the plant is underloaded. There is no treatment for phosphorus and disinfection is by UV. 2.2 Sampling Influent and effluent samples were collected on eight sampling occasion events except June 2010, where only effluent was collected. Influent and effluent samples were all taken as grab samples collected in 1-L heat-treated amber glass bottles sealed with Teflon-lined caps. Samples were preserved by the addition of 10 mL of formaldehyde solution (1% v/v) per liter of sample. Samples were stored at 4°C in the dark and analyzed within 14 days. Quality-control samples (blanks, duplicates, and spikes) were performed with each sample batch. 2.3 Reagents All sterol standards were analytical grade and purchased from Sigma–Aldrich (Toronto, ON) with the exception of equol (Fluka, Toronto, ON), 24ethylcoprostanol (Caledon, Georgetown, ON), epicholestanol, and coprostanone (Steraloids, Newport, RI). Primary standards were made in acetone at a concentration of 1 mg mL−1 and stored in the freezer at −20°C. Acetylated mixture calibration standards of 0.002 to 1 μg mL−1 were made every 2 months and also stored in a freezer at −20°C. The internal standard p-terphenyl-d14 and surrogate βestradiol 16,16,17-d3, both purchased from Sigma– Aldrich (Toronto, ON), were added to every sample. Analytical grade solvents, chemical reagents, and
Table 1 Summary of sewage treatment plant operations Plant
STP 1
Type
Tertiary
Influent flow (m3 day−1)
30,300
Treatment type
Lagoons a
Treatment level BOD
TSS
NH3
NO3
Advanced P removal
Secondary
Secondary
No
No
Yes
STP 2
Tertiary
9,700
BNR
Secondary
Secondary
Yes
Yes
Yes
STP 3
Tertiary
1,000
Modified ASPb
Secondary
Secondary
Yes
Limited
No
STP 4
Secondary
4,800
Lagoons
Secondary
Secondary
No
No
No
STP 5
Secondary
5,500
RBCc
Secondary
Secondary
No
No
No
STP 6
Secondary
900
ASPb
Secondary
Secondary
Yes
No
No
a
Biological nutrient removal plant
b
Activated sludge plant
c
Rotating biological contactor
1020
potassium carbonate were purchased from VWR (Edmonton, AL). 2.4 Sample Preparation Sterols were analyzed by liquid–liquid extraction using an Environment Canada, Pacific Environmental Science Centre method (Environment Canada 2010). This procedure is capable of analyzing 23 sterols simultaneously (Table 2) and utilized 0.8 L of sample. Samples were adjusted to a pH of ~3 with concentrated sulfuric acid and then spiked with 100 μL of 2 μg mL−1 sterol surrogate (β-estradiol 16,16,17-d3) to track recovery. Samples were then extracted with 100 mL of dichloromethane (DCM) by mixing for 2 h. Following extraction, the content of the sample bottle was transferred into a separatory funnel, and the DCM extract (bottom layer) drained through sodium sulfate
Water Air Soil Pollut (2012) 223:1017–1031
before being concentrated to 100 μL under a stream of nitrogen. For cleanup, the extract was loaded onto a column containing 1 g of 1.5% deactivated silica gel (pre-rinsed with 5 mL of 60:40 (v/v) hexane/acetone) and eluted with 7 mL of 60:40 hexane/acetone and 1 mL of hexane. The elutions were concentrated to 100 μL under a stream of nitrogen. The extract was acetylated by adding 200 μL of pyridine and distilled acetic anhydride and kept at room temperature protected from light for at least 12 h. Once acetylated, 1 mL of a 10% (w/w) potassium carbonate solution was added and the extract was re-extracted with 3×2 mL petroleum ether; the petroleum ether extract (top layer) was removed and dried with sodium sulfate before being blown down to near dryness under a stream of nitrogen. The extract was then reconstituted with 200 μL of 0.1 μg mL−1 internal standard (p-terphenyl-d14) and analyzed by gas chromatography mass spectrometry
Table 2 Sterols information Sterol
Common names
Formula
LOQ1 (μg/L)
% Recovery
24α-Methyl-5-cholesten-3β-ol
Campesterol
C28H48O
0.005
90
5α-cholestan-3-one
Cholestanone
C27H46O
0.005
111
Cholest-5-en-3β-ol
Cholesterol
C27H46O
0.009
130
5β-Cholestan-3β-ol
Coprostanol
C27H48O
0.005
93
5β-cholestan-3-one
Coprostanone
C27H46O
0.005
110
3β-cholesta-5,24-dien-3-ol
Desmosterol
C27H44O
0.008
101
3-β-5-β-cholestan-3-ol
Dihydrocholesterol
C27H48O
0.007
93
5α-cholestan-3α-ol
Epicholestanol
C27H48O
0.005
83
Cholest-5-en-3α-ol
Epicoprostanol
C27H48O
0.005
90
1,3,5,7-Estratetraen-3-ol-17-one
Equilin
C18H20O2
0.07
84
3,4-Dihydro-3-(4-hydroxyphenyl)-2H-1-benzopyran-7-ol
Equol
C15H15O3
0.1
85
(17α)-Estra-1,3,5(10)-triene-3, 17-diol
17a-Estradiol
C18H2402
0.01
87
(17β)-Estra-1,3,5(10)-triene-3,17-diol
17b-Estradiol
C18H2402
0.01
107
1,3,5(10)-Estratriene-3,16α,17β-triol
Estriol
C18H24O3
0.01
71
3-Hydroxyestra-1,3,5(10)-trien-17-one
Estrone
C18H22O2
0.02
65
24-Ethyl-5β-cholestan-3β-ol
24-Ethylcoprostanol
C29H52O
0.005
50
19-Norpregna-1,3,5(10)-trien-20-yne-3,17-diol
17a-Ethinylestradiol
C20H24O2
0.1
84
17α-Ethynyl-1,3,5(10)-estratriene-3,17β-diol 3-methyl ether
Mestranol
C21H26O2
0.01
112
13β-Ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one
Norgestrel
C21H28O2
0.07
96
19-nor-17alpha-ethynyl-17beta-hydroxy-4-androsten-3-one
Norethindrone
C20H26O2
0.08
81
5-Stigmasten-3β-ol
b-Sitosterol
C29H50O
0.007
90
24β-Ethyl-5α-cholestan-3β-ol
Stigmastanol
C29H52O
0.007
93
24-Ethylcolesta-5,22E-dien-3β-ol
Stigmasterol
C29H48O
0.007
89
LOQ=3× MDL LOQ limit of quantitation, MDL method detection limit
Water Air Soil Pollut (2012) 223:1017–1031
(GCMS). As part of quality control, two blanks and one spiked sample were extracted and analyzed with each batch of samples. The blanks consisted of ultra-high purity (UHP) water spiked with only surrogate, while the spike consisted of UHP water spiked with 20 μL of 1 μg mL−1 sterol standard mixture (with all sterol compounds as well as surrogate). In addition, two replicates of direct surrogate were added to the batch of samples at the acetylation step of the method. The spiked and direct surrogate samples tracked the performance of the method while the blanks ensured there was no contamination of sample during sample preparation, extraction, and analysis procedures.
1021
ten ratios specific for identification of human fecal contamination (Table 3) and a set of eight ratios for differentiating among various sources of fecal contamination (Table 4). Sterol ratios for different samples were calculated based on the sterol concentrations measured in the STP influents and effluents. To validate the criteria in Tables 3 and 4, calculated sterol ratios were shown to be either a success in identifying human fecal contamination or a failure if not being within the criteria that identifies human fecal contamination. When our calculated sterol ratio failed to fall within the literature criteria for identifying human fecal contamination, we suggested new criteria which were further tested on independent literature data.
2.5 Gas Chromatography Mass Spectrometry Analysis 3 Results and Discussion The prepared extracts were analyzed using Agilent instrumentation, a 7890A gas chromatograph (GC), with pulsed splitless injection (injector temperature 280°C) and a constant helium flow rate of 1.2 mL min−1, coupled with a 5,975 C mass spectrum detector. The temperature gradient for the GC column during the run was as follows: an initial temperature of 70°C was held for 1 min, increased by 30°C min−1 to 180°C, and increased by 5°C min−1 until 310°C and further held at 310°C for 12 min for a total runtime of 42.7 min. The quantification method utilizes an eight-point calibration curve of 0.002, 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, and 1 μg mL−1 sterol calibration standards each with a 0.1 μg mL−1 sterol internal standard (Environment Canada 2010). 2.6 Statistical Analysis To evaluate if efficiency of sterol removal differed between STPs, the percent of sterol removals were arcine sine square root transformed to meet assumptions of normality and heteroscedasticity. One-way ANOVA with Tukey’s post-test was performed using GraphPad Prism version 5.04 for Windows (GraphPad Software Inc., La Jolla, CA, USA), with statistical significance denoted by p0.7
0.3–0.7
0.7
0.3–0.7
0.7
0.3–0.7
0.5
−
1
−
0.5 >0.4
0.3–0.5
0.2
0.15–0.2
1.5
−
1 0.8
Human
Roser et al. 2006
Water sample from agricultural and urban catchment
Human
Shah et al. 2007
Faces, stp effluent, septic tanks
Mix (human and herbivore)
Devane et al. 2006
Sediment and urban stream, rural stream, duck pond;
>75 30–75
1.5
Human
Bethell et al. 1994
Feces and soil
73 38–73
16
Coprostanol/5β-Stigmastanol
>3.7 1.5 0.5
0%
0%
0.7 for 80% (12/15) of their analyzed environmental sediment samples. Others reported values for ratio #1 ranging from 0.61 to 0.88 for samples from sewage-impacted waters in the Mersey Estuary (Fitzsimons et al. 1995). The newly proposed criteria for ratio #1 here of > 0.5 would better fit both of these studies with their two different
5
6
7
1
0.28, 0.19
2
0.07, 0.11, 0.08
3
0.08, 0.07
4
0.17
5
0.05
6
0.06
1
0.42, 0.55
2
1.09, 0.74, 0.82
3
0.60, 0.25
4
0.46
5
0.87
6
0.88
1
1.05, 0.46
2
0.16, 0.45, 0.79
3
0.53, 0.13
4
0.22
5
1.15
6
0.94
1
0.81, 2.36
2
4.29, 2.50, 5.79
3
1.65, 1.23
4 5 6 8
% Failed 30%
≥0.5
0%
30%
>0.5
0%
10%
>0.5
0%
10%
0.8 (Roser et al. 2006). It is evident that for effluent samples, there are six ratios from the first group (#3, 4, 7, 8, 9, and 10) that are applicable for human fecal detection, and to author’s knowledge, this is the highest number of ratios used for confirmation. From the above examples, the newly suggested criteria based on present model system found confirmation throughout the literature.
14
15
16
17
3.4 Differentiating Among Various Sources of Fecal Contamination The second set of ratios compiled from the literature differentiates sources of fecal contamination, including humans (Table 4), and were developed based mainly on agricultural studies. Application of these
18
>0.36
0%
0%
No
0%
100%
>63
0%
0%
No
0%
0%
No
0%
2.54
1
67.4, 70.4
2
65.7, 70.2
3
67.4
4
73
5
67.01
6
71.7
1
10.1
2
% Failed
9.37
3
15.5
4
14.0
5
12.3
6
18.3
1
91.0, 93.8
2
90.4, 92.8
3
93.9
4
93.3
5
92.5
6
94.8
1
0.65, 0.77
2
0.77, 0.80
3
0.88
4
0.88
5
0.87
6
1.01
1
0.45, 0.56
2
0.60, 0.51
3
0.56
4
0.49
5
0.57
6
0.49
1
0.63, 0.75
2
0.75, 0.78
3
0.86
100%
0%
100%
0.5–3.7
0.5
0%
1028
Water Air Soil Pollut (2012) 223:1017–1031
Table 8 (continued) Ratio #
Samples STP
19
New suggested criteria (human)
Ratio value
4
0.86
5
0.84
6
0.98
% Failed
% Failed
Table 9 Ratio of sterols from STP effluent samples in relation to literature criteria for differentiating among various sources of fecal contamination Ratio #
Samples STP
11
100%
0.36
10%
No
90%
>63
0%
No
10%
No
1
0.16, 0.30
2
0.49, 0.825, 1.08
3
0.39, 0.88
4
0.24 1.27
1
0.03, 0.03
2
0.02, 0.03
3
0.02
5
4
0.02
6
1.52
5
0.03
1
0.75, 1.72
6
0.03
2
3.00, 1.75, 2.64
3
1.47, 2.00
4
2.85
5
2.33
ratios to influents resulted in four ratios (#12, 14, 15, and 17) being 100% successful in positive identification of human fecal contamination (Table 8). For effluents, ratio #14 (coprostanol/5β-stigmastanol) gave 100% positive identification of human fecal contamination in all samples tested and ratios #12 and #15 were 90% successful (Table 9). All three ratios (#12, 14, and 15) that were successful for both influents and effluents, involved coprostanol, 24ethylcoprostanol, and 5β-stigmastanol, there by adding two more sterols important for the characterization of sewage. By comparison, STP effluent sterols analyzed by two different laboratories in Italy yielded values for ratio #12 of 0.51 and 0.49, which is much lower than the failing value of 0.75 in the present study (Gilli et al. 2006). For ratio #11, Chou and Liu (2004) actually calculated average values for different sources of wastewater effluents: human (0.913± 0.251), pig (0.224±0.135), cow (0.023±0.001), and duck (0.007±0.001). The number of samples averaged was not stated, only the number of sources has been specified (four for human, nine for pig, and one for cow and duck). All values for influent in our study were above the upper limit of the Chou and Liu (2004) human range; therefore, this ratio did not fail (Table 8). Most of the values for the STP effluents are above the upper limit for the pig criteria (Table 9). The suggest a criteria of >0.36 for human fecal source identification (which is the upper limit for the pig identification range), which would make this ratio applicable for the STPs in the present study. The (coprostanol+epicoprostanol)/cholesterol (ratio #16, Table 5) suggested by Jardé et al. (2007a) failed to identify humans as a source of fecal contamination in
12
6 13
14
42.8, 63.2
2
63.7, 72.6, 75.1
3
59.6, 66.7
4
74.0
5
70.0
6
68.0
2 3 4 5 15
16
17
18
% Failed
30%
2.12
1
1
New suggested criteria (human)
1.58, 4.25
20%
30.0, 6.91, 10.64 4.42, 4.33 5.54 17.3
6
11.4
1
61.2, 81.0
2
96.8, 87.4, 91.4
3
81.6, 81.3
4
84.7
5
94.5
6
92
1
0.53, 0.66
2
1.16, 0.82, 0.89
3
0.65, 0.27
4
0.53
5
0.91
6
0.94
1
0.94, 0.95
2
2.85, 1.22, 0.55
3
1.02, 1.28
4
0.94
5
0.46
6
0.69
1
0.13, 0.14
2
0.05, 0.08, 0.07
3
0.05, 0.04
4
0.13
5
0.04
6
0.05
N/A
60%
100%
0.5–3.7
