Development Method for Extracting and AnalyzingAntibiotic and Hormone Residues from Treated WastewaterSludge and Composted Biosolids

Michelle Shafrir & Dror Avisar

Received: 2 October 2011 /Accepted: 8 December 2011# Springer Science+Business Media B.V. 2012

Abstract Extraction and analysis methods have beendeveloped for the detection of the following four anti-bacterial agents and two natural estrogens in treatedmunicipal wastewater sludge and commercial compost:sulfamethoxazole (SMX), sulfadimethoxine (SDM), tet-racycline (TET), oxytetracycline (OXY), estrone (E1),and 17β-estradiol (E2). The antibiotics and estrogenswere extracted from secondary sludge and mixed com-post using ultrasonic solvent extraction. Citric acid(pH 4.7) and methanol were used as extraction buffer,followed by tandem-solid-phase extraction cleanup,strong anion exchange+hydrophilic–lipophilic balancefor antibiotics and CarboPrep/NAX for estrogens. Forquantification, two different methods were employed,using HPLC–MS/MS, with an electrospray ionizationsource for antibiotics and an atmospheric-pressurechemical ionization source for estrogens. Recoverieswere 11–31% for the sulfonamides (SMX and SDM)and tetracyclines (TET and OXY) and 30–59% for theestrogens (E1 and E2) over the entire method. Limits ofdetection for the extraction method were in the

nanogram per gram range for dry weight sludge andcompost samples. Neither of the two sulfonamide anti-biotics was detected in secondary sludge or mixed com-post samples. Estrogens were found in compost inamounts of 160±65 ng/g (E1) and 21±3 ng/g (E2),but not in sludge. The tetracyclines, as well as what isbelieved to be the 4-epimer of OXY, were found in bothsludge and compost in amounts of 1.57±0.67 and 2.95±0.42 μg/g (TET), 0.56±0.12 and 6.51±0.52 μg/g(OXY), and 7.60±1.68 and 1.35±0.24 μg/g (4-epi-OXY), respectively. These results indicate thatsorption-prone compounds are not removed during thewastewater treatment process and can persist throughsludge digestion and that the composting process doesnot sufficiently eliminate these particular contaminants.Thus, biosolids (even composted) are an additionalsource of drug residues leaching into the environment,and it must be considered while using biosolids asfertilizer.

Keywords Sorption . Biosolids . Treated wastewatersludge . Compost . Antibiotics . Estrogens .

Pharmaceutical contaminants . Emerging contaminants

1 Introduction

The presence of a broad range of bioactive materials inthe natural and human environments has become thefocus of worldwide attention in recent years as ques-tions have been raised regarding their potential threat

Water Air Soil PollutDOI 10.1007/s11270-011-1049-5

M. Shafrir :D. Avisar (*)The Hydrochemistry Laboratory, Geography and theEnvironment, Tel Aviv University,Tel Aviv 69978, Israele-mail: droravi@post.tau.ac.il

M. Shafrir :D. AvisarThe Porter School for Environmental Studies,Tel Aviv University,Tel Aviv 69978, Israel

to ecological and human health. There is a growingconcern that these contaminants may reach the envi-ronment when manure, treated sewage sludge andwastewater effluent are applied to agricultural lands.Although little is known about the presence of traceorganic contaminants, land application of biosolidshas become an increasingly attractive route of dispos-al. In Israel in particular, where a large percentage ofbiosolids generated by wastewater treatment plants(WWTPs) are composted and applied to agriculturalland as fertilizer, there is increasing fear that pharma-ceuticals and endocrine disruptors, as well as theirmetabolites and degradation products, are leachinginto the environment. Although researchers in othercountries have found evidence of trace pharmaceuticalcontamination in treated sewage sludge, very little isknown about the potential risks associated with treat-ment, composting, and disposal practices currentlyused in Israel.

Antibiotics and endocrine-disrupting compounds(EDCs) are of particular concern given their capacityto harm wildlife, disrupt ecological systems, and causedisease. While antibiotics have been indispensable intreating infectious diseases previously known to killhumans and animals, it has now become clear that thewidespread use of antibiotics is not without problems.The major concern is that their overuse may lead to thedevelopment of new strains of bacteria that are resis-tant to these antibiotics, resulting in the emergence ofuntreatable diseases (Halling-Sørensen et al. 1998;Jørgensen and Halling-Sørensen 2000; Rooklidge2004; Kumar et al. 2005). EDCs are defined as chem-icals that mimic hormones or block their receptors inthe endocrine systems of the living organisms thatcome into contact with them. Steroidal estrogens likeestrone, estradiol, and estriol have been of particularconcern given that even very low concentrations inwater, in the nanogram per liter range, appear to ad-versely affect the reproductive biology of fish andinvertebrates (Jobling et al. 1998; Oberdörster andCheek 2000; Christiansen et al. 2002; Rose et al.2002; Filby et al. 2007; Thorpe et al. 2009). Com-pound stability and mobility play crucial roles in de-termining the final fates of these contaminants.Antibiotics and hormones, as well as their metabolitesand degradation products, either reach WWTPs inmunicipal waste or are released directly into the envi-ronment in animal manure. The products of wastewa-ter treatment, effluent and sludge, are released into the

environment through marine discharge, stream reha-bilitation, groundwater recharge, and agricultural andlandscape irrigation (effluent) or disposed of by ma-rine deepwater disposal, incineration, landfilling, orland application (sludge). Degradation under environ-mental conditions, during the wastewater treatmentprocess or during composting, may eliminate or inac-tivate many of these compounds. Transfer to theaquatic environment, mobility, and, possibly, bioavail-ability are functions of liquid–solid partitioning andthe potential for sorption to soils, clays, and organicmatter such as humic substances.

In 2009, a total of 103,515 metric tons (dry weight)of sewage sludge was generated from 45 municipalWWTPs in Israel. The Shafdan WWTP in RishonLeTzion, the largest WWTP in Israel, produced41,050 dry tons (40%) of untreated sludge whichwas discharged into the Mediterranean Sea. Theremaining sludge, 61,825 dry tons from 44 WWTPs,was land applied. Of this, 5,431 tons (5%) was dis-posed of by landfilling, and 56,394 tons (55%) re-ceived additional sanitation treatment in order to beused for fertilizer/soil improvement, meeting therequirements for class A biosolids with almost unlim-ited use in agriculture. In order to meet the require-ments for class A biosolids classification, it isnecessary to transfer secondary sludge to industrial-scale composting facilities for further treatment. Aer-ated static pile composting is accomplished by amass-ing the sludge along with shredded municipal greenwaste comprised of palm, poinciana, cypress, pine,and other ornamental tree waste at a ratio of approx-imately 3:1 by weight.

Sulfonamide and tetracycline antibiotics have beenfound in microgram per liter and microgram per kilo-gram concentrations in soil, groundwater, and surfacewater samples (Holm et al. 1995; Hirsch et al. 1999;Lindsey et al. 2001; Sacher et al. 2001; Zhu et al.2001; Hamscher et al. 2002; Vogel et al. 2005; Battet al. 2006; Avisar et al. 2009a, b). The tetracyclineantibiotic doxycycline has been found in treatedwastewater sludge at a concentration between 1.3and 1.5 mg/kg (Lindberg et al. 2005), and in theirstudy of estrogens in activated and digested wastewa-ter sludge, Ternes et al. (2002) found concentrations ashigh as 37 ng/kg (estrone), 49 ng/kg (17β-estradiol),and 17 ng/kg (ethinylestradiol). Several of these stud-ies of antibiotics sourced from aquaculture ponds,WWTP effluents, confined animal feeding operations,

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and landfill runoff have found tetracyclines to be fairlyimmobile in soils, though present in surface waters,while sulfonamides appear to be highly mobile andfind their way into aquifers and groundwater reser-voirs. Although a number of studies report efficientremoval of estrogens during the wastewater treatmentprocess (Ternes et al. 1999a; Andersen et al. 2003;Joss et al. 2004), others have documented natural andsynthetic estrogens in effluent and surface water sam-ples in nanogram per liter concentration levels (Shoreet al. 1992, 1993; Belfroid et al. 1999; Baronti et al.2000; Huang and Sedlak 2001; Christiansen et al.2002) as well as in sludge (Ternes et al. 2002). Whilesulfonamides like sulfamethoxazole and sulfadime-thoxine are very water soluble with low chelatingability and high mobility into effluent and surface-and groundwater, tetracyclines are strong chelatorsthat sorb strongly to particles in sewage sludge andsoil, with the potential to build up persistent residues(Lindsey et al. 2001; Boxall et al. 2002; Hamscher etal. 2002; Christian et al. 2003; Gu et al. 2007). And asa result of strong sorption to organic matter, removalof tetracycline during the wastewater treatment pro-cess is primarily accomplished by sorption to activatedsludge without significant removal by biodegradation,making sorption an important route for pollutants intothe environment when the sludge is used as fertilizeron agricultural land (Ternes et al. 2004; Kim et al.2005).

The main goal of this study was to determine theconcentrations of two natural estrogens and four

antibiotics from two different antibiotic classes intreated municipal wastewater sludge and commercialcompost. Evaluation for the above contaminants willlead to a better understanding of the potential for theirrelease into the environment after disposal of treatedsludge or land application of compost.

2 Selected Compounds

2.1 Antibiotics

Four antibiotics from two major antibiotic classes,sulfonamides and tetracyclines, have been selectedfor this research study. Two specific compounds fromeach antibiotic class were chosen: sulfamethoxazole(SMX) and sulfadimethoxine (SDM), and tetracycline(TET) and oxytetracycline (OXY). The sulfonamideand tetracycline antibiotics vary appreciably in theirphysical properties and tendency to sorb to organicmatter. The tetracyclines are relatively hydrophobic,whereas the sulfonamides are considerably polar andhydrophilic. In general, a more hydrophilic compoundwill partition to the solid phase while a more hydro-philic compound will remain in the aqueous phase.Important physiochemical properties of the selectedantibiotics are shown in Table 1.

Log Kow (the octanol–water partition coefficient) isa measure of the equilibrium concentration of a com-pound between octanol and water indicating the po-tential for partitioning onto sludge organic matter (a

Table 1 Important physiochemical properties of selected antibiotics

Compound Solubility in water (mg/L at 25°C) pKa 1ogKow Kd (L/kg) (1° sludge) Kd (L/kg) (2° sludge)

Sulfamethoxazole 610a 1.8, 5.7c 0.9e 400±1.7h 260±1.7h

Sulfadimethoxine 340b 2.4, 6.0b 1.63b

Tetracycline 231a 3.32, 7.78, 9.58d −1.19f 8,400±5,001

Oxytetracycline 600a 3.22, 7.46, 8.94d −1.12g

a Yalkowsky and Dannenfelser (1992)b Thiele-Bruhn (2003)c Feitosa-Felizzola and Chiron (2009)d Sassman and Lee (2005)e Drillia et al. (2005)f Tolls (2001)g Loke et al. (2002)h Joss et al. (2005)

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high Kow indicates that the compound will preferen-tially partition onto organic matter rather than theaqueous phase). However, doubts have been expressedover the usefulness of predicting partitioning behaviorof pharmaceuticals using logKow values (Tolls 2001;Loke et al. 2002). Loke et al. (2002) observed muchhigher sorption of oxytetracycline to manure thanexpected only by the negative logKow (−1.12) valueof the compound. The most common method for esti-mating contaminant sorption is based on the partition(or distribution) coefficient, Kd, a parameter related tothe partitioning of a compound between the solid andaqueous phases. The Kd measure, given in units ofliters per kilogram, is commonly used to describe theextent to which contaminants are sorbed to soil, sed-iment, clay, and sludge particles. The Kd parameter isvalid only for a particular sorbent and applies only tothose aqueous chemical conditions (e.g., adsorbateconcentration, solution/electrolyte matrix) in which itwas measured. In their study of the removal of phar-maceuticals and fragrances in biological wastewatertreatment, Joss et al. (2005) were not able to define aquantitative relationship between structure and activityof pharmaceutical contaminants during the biologicaltransformation associated with municipal wastewatertreatment. They were, however, able to conclude thatfor compounds showing a sorption coefficient (Kd)less than 300 L/kg, sorption onto secondary sludge isnot relevant.

In addition, it may be useful to examine the pKa

values of ionizable pharmaceutical compounds in or-der to better predict their partitioning behavior be-tween organic matter and the aqueous phase. Theacid-dissociation constant, Ka, is an equilibrium con-stant whose magnitude describes the tendency of ion-izable compounds to dissociate. The tetracyclines arecharacterized by three pKa values while the sulfona-mides are characterized by only two pKa values. Un-der acidic conditions at a pH below 1.4, SMX exists asa cation; between pH 1.4 and 5.8, the molecule carriesa neutral charge, and above pH 5.8, it is negativelycharged. At environmental pH ranges (6–8), SMX iseither in its neutral form or exists as an anion and thuslikely will not accumulate in sludge organic matter,being repulsed from the negatively charged groups(hydroxyl and carboxyl) ubiquitously present on hu-mic and fulvic acids. It is therefore expected thatsignificant amounts of SMX will not be found intreated wastewater sludge but rather in effluents. In

contrast, tetracyclines have multiple ionizable func-tional groups and may exist as cations, zwitterions,or anions. At environmentally relevant pH ranges (6–8), the tetracyclines can be expected to be found aseither an anion or net neutrally charged zwitterion witha positive charge present on the dimethylammoniumgroup in both conformations. The presence of a posi-tive charge on the dipolar (or multipolar) tetracyclinemolecule then results in an increased likelihood ofsorption to organic matter.

2.2 Hormones

Two natural estrogens, estrone (E1) and 17β-estradiol(E2), were selected for this research study becausethey are responsible for a considerable part of theendocrine-disrupting effects seen in the aquatic envi-ronment and are released into the wastewater treat-ment process in significant amounts (Desbrow et al.1998; Körner et al. 2001; Snyder et al. 2001). Steroidestrogens are mostly excreted in the form of conju-gates, mainly glucuronides and sulfates. Conjugatedestrogens are analogous in structure except that asulfate and/or glucuronide group is substituted at theC-3 and/or C-17 positions of the parent compound(e.g., 17β-estradiol-3- sulfate, 17β-estradiol-17-sul-fate, 17β-estradiol-3,17 disulfate) (Hanselman et al.2003; Joss et al. 2004). Glucuronide and sulfate con-jugated estrogens are cleaved to steroid estrogens insewers or upon reaching the WWTP, a reaction that iscompleted in less than an hour for the glucuronides,and the substrate present in the raw influent competi-tively inhibits the degradation of E1 and E2 (Baronti etal. 2000; Andersen et al. 2003; Joss et al. 2004). Intheir analysis of steroid estrogens in both water andactivated sludge in the three steps of the activatedsludge treatment, Andersen et al. (2003) found thatestrogens are mainly degraded during denitrification.Steroid estrogens degrade slowly, E2 being oxidizedto E1 in a few hours. E1 is then degraded over severalhours, while the degradation of the synthetic estrogen17α-ethynylestradiol (EE2) takes as much as a fewdays (Ternes et al. 1999b). It has been reported thatconventional municipal WWTPs, mainly based onactivated sludge systems, are not effective in removingestrogens completely because of the relatively highstability of these compounds and insufficient sludgeretention time during the conventional biological

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process (Johnson et al. 2000; Braga et al. 2005; Wanget al. 2005).

Estrogens are moderately hydrophobic compoundsthat do not ionize at normal environmental pH rangesand should be extensively sorbed by sludge particlesand organic matter. The unconjugated steroidal estro-gens E1 and E2 have low solubilities in water and arerelatively lipophilic, as indicated by their logKow val-ues, 3.13 and 4.01, respectively (Table 2). They areweak acids (pKa, 10.77 and 10.71), losing a phenolichydrogen at high pH. At neutral pH, the hormonesbecome progressively less soluble with increasing ion-ic strength (Shareef et al. 2006). Physicochemical datafor the conjugated estrogens are not available in theliterature. However, estrogen conjugates are likely tohave much greater aqueous solubility than unconju-gated estrogens due to their polar glucuronide or sul-fate functional groups (Hanselman et al. 2003). Afterdeconjugation occurs in the WWTP, it is expected thatthe nonionic steroidal estrogens will be absorbed inthe lipid fractions or sorbed onto humic substancesand organic matter (nonionic van der Waals interac-tions) at environmental pH values.

3 Materials and Methods

3.1 Chemicals and Standards

Analytical standards of SMX, SDM, TET, OXY, E1,and E2 were obtained from Sigma–Aldrich (Israel).Analytical grade citric acid monohydrate and sodiumcitrate dihydrate and HPLC-grade solvents (water,methanol, acetonitrile, dichloromethane, hydrochloricacid, and formic acid) were obtained from Bio-LabLtd. (Jerusalem, Israel). Dewatered secondary sludgesamples were collected from the Kolchai HaSharon

WWTP located 40 km north of Tel Aviv, Israel. Com-post samples were obtained from the Compost Orcomposting facilities located in the Jordan Valley.Solid-phase extraction (SPE) for analysis of antibioticswas performed with Strata strong anion exchange(SAX; 1,000 mg/6 mL, Phenomenex) and Oasis hy-drophilic–lipophilic balance (HLB) cartridges (poly(divinylbenzene-co-N-pyrrolidone), 500 mg/6 mL,Waters). SPE for analysis of natural estrogens wasperformed with combo cartridges containing activatedcarbon combined with aminopropyl (500 mg Carbo-Prep 90/500 mg NAX, 6 mL, Resprep).

3.2 Sampling of Sewage Sludge and Compost

The Kolchai HaSharon WWTP, from which secondarysludge samples were obtained for the study describedhere, is located about 40 km north of Tel Aviv andserves residents in the communities of Hof HaSharon,Even Yehuda, Tel Mond, and Kadima. The plantemploys fine-pore aeration paired with activatedsludge for biological treatment of municipal waste-waters. Effluents are passed through a UV filtrationand disinfection system, and the use of a regulationpool and supply station allow for effluent to be usedfor irrigation of nearby orchards. All biosolids pro-duced are used as agricultural fertilizer after furthercomposting at the Compost Or facilities located in theJordan Valley (Tzadikov 2010).

Secondary sludge was obtained from the KolchaiHasharon WWTP in July 2009 at the end of thetreatment process after dewatering. Compost wasobtained from the Compost Or composting facility inMarch 2010 after a 4-week composting regimen. Priorto storage at −32°C, solid samples were lyophilizedand grinded to a 425-μm particle size.

3.3 Experimental

An analytical method with two extraction steps has beendeveloped and validated for the simultaneous determi-nation of natural estrogens and two classes of antibioticpharmaceuticals in finished secondary sludge from ur-ban wastewater before and after composting. Solid sam-ples were extracted using ultrasonic solvent extraction(USE), the resulting liquid solution extracted and con-centrated by two different SPE methods, and analyzedby reverse-phase liquid chromatography tandem mass

Table 2 Important physiochemical properties of selected naturalestrogens

Compound Solubility in water(mg/L at 25°C)

pKa 1ogKow

El 1.30±0.08a 10.77b 3.13c

E2 1.5 l±0.04a 10.71b 4.01c

a Shareef et al. 2006b Lewis and Archer 1979c Ternes et al. 2002

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spectrometry (HPLC–MS/MS). A chart illustrating theprocedure is shown in Fig. 1.

3.4 Extraction Methods

Both solid matrices (sludge and compost) wereextracted following the same protocol. SPE regimensvaried depending on the analyte group of interest.Clean up and pre-concentration of antibiotics wasperformed using a combination of SAX and HLBcartridges. Cartridges were placed in tandem to simul-taneously remove negatively charged humic material(SAX) and retain the antibacterial agents (HLB).Clean up and enrichment of natural estrogens wereaccomplished with combo cartridges containing acti-vated carbon absorbent material combined with ami-nopropyl (CarboPrep/NAX).

During method development, focus was concentrat-ed on the tetracyclines, as strong sorption of thesecompounds to organic matter particles causes difficul-ties when extracting the antibacterial agents fromsludge and compost (Hamscher et al. 2002; Loke etal. 2002; De Liguoro, et al. 2003). The simultaneousextraction of multiple analytes by USE was developedfollowing Babić et al. (1998), Ternes et al. (2002),Jacobsen et al. (2004), Tor et al. (2006), and Andreuet al. (2009). Extractions were performed at roomtemperature as the tetracyclines are converted to theirepi- or anhydroform when heated (Mitscher 1978;Blackwood et al. 1985). While a number of studiesof the tetracycline antibiotics in various solid matriceshave employed EDTA as a chelating agent to seques-ter divalent metal ions (Hirsch et al. 1999; Lindsey et

al. 2001; Lopez de Alda et al. 2003; Castiglioni et al.2005; Andreu et al. 2009), Hamscher et al. (2002)found that agents such as EDTA or strong acids, whichdestroy the chelate-binding properties of tetracyclineto metallic ions, did not affect their recovery rates insoil. Accordingly, none was used in the present study.Methods for SPE and LC–MS/MS were adapted fromJacobsen et al. (2004), Avisar et al. (2009b), andAvisar et al. (2009c).

3.5 Solid Sample Extraction by USE

Extraction of both antibiotics and natural estrogenswas carried out by adding 1 M citrate buffer (pH 4.7,10 mL) to a polypropylene centrifuge tube (50 mL)containing dry sludge or dry compost (2.00 g) andvortexing for 1 min. The samples were sonicated for20 min, centrifuged at 4,800 rpm for 10 min, and thenthe supernatant decanted and filtered through a 0.45-μm glass PVDF filter into separate glass bottles(250 mL), rinsing with water. This procedure wasrepeated two more times, extracting with a solutionof methanol/water (60:40, 10 mL). The combinedextracts were refrigerated overnight and kept coldand in the dark as much as possible.

3.6 SPE—Antibiotics

The aqueous samples, containing approximately12 mL of methanol, were diluted with water to amethanol content below 10% and a buffer concentra-tion of approximately 0.04 M (250 mL). The SAXcartridges were placed on top of the HLB cartridges,

MS/MS Detection

LC Separation/ UV Detection

Clean-up and

Enrichment

Solid Sample

Extraction

Solid Phase Extraction

Antibiotics1. SAX cartridges2. HLB cartridges

Natural EstrogensCARBOPREP/NAXcartridges

Liquid Sample Ultrasonic Solvent Extraction

Solvent1 M Citrate Buffer (pH 4.7)+ Methanol/Water (60/40)

Fig. 1 Analytical approachfor the extraction anddetection of trace antibioticsand natural estrogens intreated wastewater sludgeand compost

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and both columns were conditioned with methanol(5 mL) and 0.04 M citrate buffer (pH 4.7, 5 mL).The diluted samples were passed through both SPEcolumns at a rate of approximately 5 mL/min. Afterextraction, both cartridges were washed with a solu-tion of 0.04 M citrate buffer (pH 4.7) containing 5%methanol (5 mL). The SAX cartridges were removedand discarded and the HLB cartridges dried undervacuum for 15 min. The antibacterial agents were theneluted from the HLB sorbent into glass test tubes withmethanol (5 mL). The extracts were then evaporated atroom temperature under a gentle stream of nitrogenand stored at −32°C. Just prior to instrumental analy-sis, sample extracts were reconstituted in water (1 mL)and sonicated for 10 min at room temperature.

3.7 SPE—Natural Estrogens

The aqueous samples, containing approximately12 mL of methanol, were diluted with water to amethanol content below 10% and a buffer concentra-tion of approximately 0.04 M (250 mL). The Carbo-Prep/NAX cartridges were conditioned with a solutionof dichloromethane/methanol (80:20, 10 mL), metha-nol (5 mL), and water acidified with concentrated HCl(pH 2, 20 mL). The diluted samples were passedthrough the SPE column at a rate of 3 to 5 mL/min.After extraction, both cartridges were washed withwater (50 mL), methanol acidified with formic acid(50 mM, 10 mL), and methanol (5 mL). The estrogenswere then eluted from the sorbent into glass test tubeswith dichloromethane/methanol (80:20, 10 mL). Theextracts were then evaporated at 40°C under a gentlestream of nitrogen and stored at −32°C. Just prior toinstrumental analysis, sample extracts were reconsti-tuted in 1 mL water/acetonitrile (80:20, 1 mL) andsonicated for 10 min at 40°C.

3.8 Analytical Detection

Separation and detection of antibiotics and naturalhormones in the aqueous phase were achieved usingthe HPLC–UV/MS/MS (AGILENT 1100 Series andFINNIGAN LCQ) system. The HPLC (Agilent 1100)system used in the Hydrochemistry Laboratory at TelAviv University is equipped with a micro-vacuumdegasser, a vacuum solvent degassing unit, a binaryhigh-pressure gradient pump, an automatic sampleinjector, a thermostatic column compartment, a cooled

autosampler (4°C), and a UV diode array detector.Mass spectrometry was performed on a FinniganLCQ (Thermo Scientific) ion trap LC-MS with multi-ple MS/MS capacity and employing either an electro-spray ionization (ESI) source or an atmospheric-pressure chemical ionization (APCI) source operatedin the positive mode.

The sulfonamides were separated using a 2.1×250-mm ACE C8 silica-based column with 5-μm pore sizeand 100-Å particle size in combination with a guardcolumn (2.1×4 mm) of the same type. The UV wave-length was monitored at 260, 270, and 280 nm. Themobile phase consisted of a multi-step gradient com-bining: (a) 1% formic acid (FA) in HPLC water(pH 2.3) and (b) 1% FA in acetonitrile (AcN). Likethe sulfonamides, the tetracycline antibiotics were sep-arated using a 2.1×250-mm ACE C8 silica-basedcolumn with 5-μm pore size and 100-Å particle sizein combination with a guard column (2.1×4 mm) ofthe same type. The UV wavelength was monitored at270 and 360 nm. The mobile phase consisted of amulti-step gradient combining: (a) 1% FA in HPLCwater (pH 2.3) and (b) 1% FA in AcN. The massspectrometer was operated in positive ion mode usingan ESI source. The probe temperature was set to 220°C.The flow rate from the HPLC to the MS interface was60 μL/min. Ions in the range of m/z085–500 wereregistered in the conventional scanning mode. Thecapillary temperature was set to 200°C. The sprayvoltage was set to 4.5 kV. The product ion producingthe highest intensity was used for selected reactionmonitoring (SRM) and quantification in MS/MSmode. The ESI source and MS/MS parameters weretuned and optimized regularly. Instrument control anddata quantification were performed with ThermoQuestXcalibur software version 3.1.

The natural estrogens were separated using a 2.1×250-mm ACE Phenyl silica-based column with 5-μmpore size and 100-Å particle size in combination witha guard column (2.1×4 mm) of the same type. The UVwavelength was monitored at 280 nm. The mobilephase consisted of a multi-step gradient combining:(a) 1% FA in HPLC water (pH 2.6) and (b) 1% FA inmethanol (MeOH). The mass spectrometer was oper-ated in positive ion mode using an APCI source. Theprobe temperature was set to 220°C. The flow ratefrom the HPLC to the MS interface was 260 μL/min.Ions in full range of m/z0100–400 were registered inthe conventional scanning mode. The capillary

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temperature was set to 200°C. The product ion pro-ducing the highest intensity was used for SRM andquantification in MS/MS mode.

3.9 Method Validation and Extraction Recoveries

The response for each of the antibacterial agentsdetected in the LC–ESI–MS–MS and LC–APCI–MS–MS methods was evaluated for linearity, and thelimits of detection and quantification (LOD and LOQ)were determined, using calibration curves for the con-centration range 1–5,000 μg/L (antibiotics) or 1–500 μg/L (estrogens). Stock solutions of the antibac-terial agents were prepared in water (antibiotics) orwater/acetonitrile (80:20, estrogens) and stored at 4°C.Duplicate standards were produced at nine concentra-tion levels (1, 5, 10, 25, 50, 100, and 500 μg/L (plus1,000 and 5,000 μg/L for the antibiotics)) from multi-ple separate stock solutions, and multiple calibrationcurves for SMX, SDM, TET, OXY, E1, and E2 wereproduced.

Quantitative analysis of four antibiotics and twonatural estrogens in secondary sludge and compostwas performed by the standard addition method. Re-coveries for the entire USE–SPE–LC–ESI–MS–MSand USE–SPE–LC–APCI–MS–MS procedures weredetermined. Both sludge and compost samples werefortified with SMX, SDM, TET, and OXY or E1 andE2 in duplicates. The fortified samples were extractedand analyzed using the entire procedure. Recoverieswere calculated as the percentage of extracted antibac-terial agent or estrogen compared to spiked level. LODand LOQ were estimated using the signal-to-noiseratios of S/N03 and S/N09, respectively, for sludgeand compost samples containing either 1 μg/g SMX,SDM, TET, and OXY or 0.1 μg/g E1 and E2.

Freeze-dried sludge and sediments were spikedwith either the antibiotics (SMX, SDM, TET, andOXY) or natural estrogens (E1 and E2) which weredissolved in a working solution (0.4 or 0.04 μg/mL inmethanol) to achieve a concentration of 1 μg antibioticper gram solid or 0.1 μg estrogen per gram solid. Afterspiking, the samples were vortexed to homogenize thespiking solution in the sample and to enable sufficientcontact of the either the antibiotics or the estrogenswith the matrix. The spiked samples then were placedin a fume hood overnight at room temperature toensure sorption to sludge and compost constituents.Solvent extraction took place after 24 h (Sithole and

Guy 1987; Ternes et al. 2002) by evaporating under astream of nitrogen at room temperature. The recover-ies were determined in relation to non-enriched sam-ples, which were subjected to the same spikingprocedure but suspended in pure methanol rather thanthe working solution. For the other cleanup steps, theindividual recoveries were determined without matri-ces by spiking the respective solvent with either theantibiotics or the estrogens. Solvent extraction, SPE,and detection by HPLC/MS/MS were performed asdescribed above.

4 Results and Discussion

4.1 Recoveries and Detection Limits of the AnalyticalMethod

Mean recoveries of the analytes in secondary sludgeover the total method ranged between 17.2±2.0% and58.5±0.8% (Table 3) at a spiking level of 1 μg/g(antibiotic) or 0.1 μg/g (estrogen). SPE cleanup recov-eries ranged from 41.1±6.6% to 95% (Table 3), forwhich the recoveries were determined without matrix.Losses in secondary sludge might be explained byincomplete sludge extraction or possibly by matrix in-terference on the SPE columns. The LOQs for thesulfonamides, tetracyclines, and estrogens ranged from1.1 to 17.1 ng/g (Table 4). Analyte LODs and LOQswere determined considering a signal/noise ratio of atleast 9 using fortified extracts from sludge and compost.

Table 3 Recoveries of sulfonamides, tetracyclines, and naturalestrogens from SPE cleanup and total recoveries from sludgeand compost

SPE method Sludge Compost

SMX 53±9a 17±2c 12±2c

SDM 51±13a 21±3c 21±13c

TET 42±8b 22±5c 11±2d

OXY 41±7b 31±2e 12±3d

E1 (95) 30±1d 30±1e

E2 (85) 59±1d 50±1e

a n05b n04c n06d n03e n09

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Linear calibration ranged from 5 to 1,000 ng/mL(antibiotics) or 5 to 500 ng/mL (estrogens). Calibrationcurves were always essentially linear, with correlationcoefficients greater than 0.99.

Mean recoveries of the estrogens at a spiking levelof 1 μg/g (antibiotics) and 0.1 μg/g (estrogens) rangedfrom 10.7±1.9% to 50.3±1.2% (Table 3). As a rule,recoveries for extraction of compost were less thanthose from sludge, possibly due to the creation ofsmaller-weight humic substances during the compost-ing process which may result in higher matrix inter-ferences during SPE cleanup. Without the SPEcleanup, the HPLC–MS/MS measurements of sampleextracts were not reproducible. After just a few injec-tions, the background increased significantly, andpressure in the HPLC system increased dramatically.Despite possible losses, the SPE cleanup is an essen-tial step for the developed method. The LOQs for thesulfonamides, tetracyclines, and estrogens rangedfrom 0.6 to 16.5 ng/g (Table 4), comparable to thoseof sludge extracted samples.

4.2 Native Contamination

In treated secondary sludge from the KolchaiHaSharon WWTP, tetracycline antibiotics, includingthe OXY degradation product 4-epi-oxytetracycline,were found in concentrations ranging from 1.57 to7.60 μg/g (Fig. 2 and Table 5). No sulfonamide anti-biotics or natural estrogens were found in detectablelevels. These results coincide with a number of studiesfinding significant amounts of tetracycline antibioticsin wastewater sludge and, conversely, sulfonamides inwastewater effluents (Hirsch et al. 1999; Castiglioni etal. 2005; Lindberg et al. 2005; Peng et al. 2006; Barber

et al. 2009). The apparent absence of the natural estro-gens 17β-estradiol and estrone in sludge samples isnot inconsistent with findings in the literature thathave detected estrogens in sludge over a wide rangeof concentrations, including those below the LOQreported here (Andersen et al. 2003; Joss et al. 2004;Ternes et al. 2005). Such variation indicates thatestrogen influent concentrations can vary widelybetween different WWTPs and that sorption and/orbiological degradation of estrogens in the WWTPmay be process engineering dependent.

Samples of mixed compost comprised of the dewa-tered secondary sludge from 14 different WWTPs inIsrael and municipal green waste in a ratio of approx-imately 3:1 by weight were analyzed for the presenceof sulfamethoxazole, sulfadimethoxine, tetracycline,oxytetracycline, 17β-estradiol, and estrone. The sul-fonamide antibiotics were not detected. Tetracyclineantibiotics, as well as the OXY degradation product 4-epi-oxytetracycline, were found in concentrations rang-ing from 1.35 to 6.51 μg/g (Fig. 2), as were the naturalestrogens E1 and E2, in concentrations of 160±65 and21±3 ng/g, respectively (Fig. 3 and Table 5). The ab-sence of sulfonamides in compost partially comprised ofsolid sludge material from multiple WWTPs is in agree-ment with the assumption that these compounds willpreferentially partition to effluents during the wastewa-ter treatment process. Because the two natural estrogenswere found in compost but not in sludge from theKolchai HaSharon WWTP, it is difficult to draw con-clusions regarding the composting process as an effi-cient means of removing these contaminants due to themultiple possible sources of estrogens in the initialcompost mix.

Again, due to the number of WWTPs that contrib-ute sludge, it is not known to what extent compostingis able to degrade tetracyclines or natural estrogens,but it does not appear to be complete. It is of interest tonote the difference in ratios of oxytetracycline and its4-epimer in sludge and in compost, approximately1:15 and 5:1 respectively, indicating that the genera-tion of 4-epi-OXY may vary greatly with WWTPconditions and that it may be a significant degradationproduct of OXY in the environment. Additionally, it ispossible that the analytical method did not successful-ly resolve the signals of the tetracycline antibiotics,and the peaks shown in the MS/MS chromatogramsshown below (Fig. 5) are composites of TET or OXYand various other degradation products.

Table 4 LOD and LOQ of fortified sludge and compostextracts

Substance LODsludge(ng/g)

LOQsludge(ng/g)

LODcompost(ng/g)

LOQcompost(ng/g)

SMX 3.6 6.0 0.6 1.2

SDM 1.1 2.1 0.3 0.6

TET 3.8 7.6 8.3 16.5

OXY 1.8 3.7 1.4 2.7

El 0.6 1.1 1.5 2.5

E2 8.5 17.1 7.8 13.7

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4.3 Extraction of Tetracycline Antibiotics from Sludgeand Compost

Given the strong sorption of tetracyclines to naturalorganic matter and their degradation under acidic–basicconditions, extraction and analysis posed a unique chal-lenge. Precautionary measures were taken to limit sam-ple exposure to temperatures exceeding 25°C and toprocess them in as narrow a time frame as possible inorder to prevent analyte degradation. Without identify-ing the specific products, Onji and Tanigawa (1984)report significant degradation (>90% loss) of TET inacidic (1 N HCl) conditions over the course of 4 daysand under the same conditions; OXY is only moderatelydegraded (40% loss). It has been found that tetracyclineswill convert to their anhydro- and epi- degradationproducts under acidic conditions, and epi-tetracycline,in which epimerization occurs at C-4 (Fig. 4), can beformed in mildly acidic (pH 2–6) aqueous conditions(Kühne et al. 2000; Halling-Sørensen et al. 2002). How-ever, it has been reported that this isomer is not formedin significant levels at pH 4 during a same-day extrac-tion with McIlvaine buffer, nor when left in solution forup to 3 days at −20°C in the absence of light (Pena et al.

1999). In addition, a second paper reports epimerizationof chlorotetracycline, but not TET or OXY, whenextracting the residue from animal feed with pH 2 McIl-vaine buffer (Martinez and Shimoda 1988). A pH 4.7citrate buffer was therefore deemed suitable for thedeveloped extraction method.

The second peak that consistently appeared in all ofthe OXY chromatograms of sludge and compost sam-ples indicates the presence of not only oxytetracylinebut also its epimer, 4-epi-oxytetracyline (Fig. 5a and b).Tetracyclines are known to degrade under acidic or basicconditions accelerated in the presence of light and/orheat to a number of defined degradation products whichare described in detail by Halling-Sørensen et al. (2002)and Anderson et al. (2005). The second peak appearingin the OXY chromatogram with a retention time ofapproximately 5.3 min is presumed to belong to the 4-epimer of oxytetracycline because this is the only com-mon degradation product having the same molecularweight as oxytetracyline. Although the MS/MS analysismethod is optimized for OXY, it is assumed that themass spectrometer parameters would be appropriate forthe epimer as well. Andreu et al. (2009) used identicalquantifier SRM (m/z), qualifier SMR (m/z), cone energy,

Table 5 Concentrations of antibiotics and estrogens in treated wastewater sludge and mixed compost (dry weight basis)

Matrix SMX (μg/g) SDM (μg/g) TET (μg/g) OXY (μg/g) 4-epi-OXY (μg/g) El (ng/g) E2 (ng/g)

Sludge BDLa BDLa 1.57±0.67b 0.56±0.12a 7.60±1.68a BDLc BDLc

Compost BDLa BDLa 2.95±0.42c 6.51±0.52b 1.35±0.24b 160±65a 21±3a

BDL below detectable limitsa n09b n06c n03

Fig. 2 Concentrationsof various antibiotics and adegradation product in trea-ted wastewater sludge andmixed compost in micro-grams per gram (dryweight). BDL belowdetectable limits

Water Air Soil Pollut

and collision energy settings in their LC–ESI–MS/MSanalysis of three different tetracyclines and their epimersin soil. Quantification of 4-epi-OXY was determined inrelation to the OXY standard under the assumption thatfragmentation of both OXYand its epimer would there-fore occur at the same intensity and that extraction yieldswould be identical. Conversion of native 4-epi-OXYback to OXY during sample handling was consideredunlikely given that the epi-tetracyclines are reversedback to their active form under specific alkaline condi-tions in the presence of a complexing metal (Mitscher1978). Additionally, while it is possible that other deg-radation products are present in the analyzed sludge andcompost, their presencewould not be revealed due to thespecificity of the MS/MS method. The lack of a secondpeak in the chromatograms of tetracycline (Fig. 5c andd), while not indicating the presence of the tetracyclineepimer, does not necessarily rule it out either. It ispossible that TETand 4-epi-TETwere not resolved welland the 4-epi-TET signal was eclipsed or that the twocompounds had greatly different retention times, and thesignal corresponding to 4-epi-TET was outside the MSanalysis window. The 4-epimer analogs of tetracycline

antibiotics are excreted in feces and urine as one of anumber of metabolites (Capolongo et al. 2002), makingit very likely that where the tetracyclines are found in theenvironment, their 4-epimers will be found as well.

Neither of the sulfonamide antibiotics, sulfame-thoxazole and sulfadimethoxine, were detected in sec-ondary sludge or compost samples. Althoughbiodegradation of sulfonamides has been observedduring primary and secondary wastewater treatment(Pérez et al. 2005), sulfamethoxazole has also beendetected in primary and secondary effluents of waste-water treatment plants with concentrations rangingbetween 30 and 2,000 ng/L (Hartig et al. 1999; Hirschet al. 1999; Vogel et al. 2005). This indicates thatsorption to sludges, humic substances, and organicmatter is less favored and that sulfonamide antibioticswill have a high mobility into surface water.

4.4 Extraction of Natural Estrogens from Sludgeand Compost

The analysis of organic compounds at low levels insludge and compost is challenging due to the matrix

Fig. 4 Conversion of oxytetracycline to 4-epi-oxytetracycline by epimerization of the tertiary amino group located at C-4

Fig. 3 Concentrationsof estrone and 17β-estradiolin treated wastewater sludgeand mixed compost innanograms per gram (dryweight). BDL belowdetectable limits

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RT: 0.00 - 7.98

90

100

RT: 5.15AA: 6975326SN: 1846BP: 442.9

NL:7.17E5m/z= 442.5-443.5 MS ICIS 13OXY fragment

50

60

70

80

90 RT: 4.67AA: 3639125SN: 1158BP: 442.9

4-epi-OXY fragmentm/z 443rt: 5.2 min

m/z 443rt: 4.8 min

0

10

20

30

40

Rel

ativ

e A

bund

ance

RT: 5.94AA: 189821SN: 83BP: 442.9

RT: 6.57AA: 525110SN: 76BP: 442.8

RT: 4.20AA: 233177SN: 66BP: 442.9

RT: 7.11AA: 124454SN: 57BP: 442.8RT: 4.79

4.790NL:3.17E5

(a) sludge

70

80

90

100

4.790

SN: 211

RT: 5.405.399AA: 2309882

3.17E5TIC MS ICIS 16

OXY fragment m/z 443

4-epi-OXY fragmentm/z 443

30

40

50

60

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

SN: 114

RT: 6.556.546AA: 853941SN: 62

RT: 7.107.102AA: 1352688SN: 58

RT: 6.176.170AA: 923587SN: 45RT: 4.09

4.094AA 135986

rt: 4.8 min

0

10

20 AA: 135986SN: 24(b) compost

rt: 5.2 min

RT: 0.00 - 7.50

90

100

RT: 5.155.150AA: 5275811SN: 246

NL:3.00E5TIC MS ICIS 22TET fragment

m/z 427

50

60

70

80

RT: 4.684.684AA: 901150 RT: 6 57RT: 5.96

rt: 5.2 min

0

10

20

30

40 AA: 901150SN: 73

RT: 6.576.566AA: 510621SN: 49

RT: 5.965.956AA: 71024SN: 25RT: 3.59

3.590AA: 132681SN: 14

RT: 6.896.888AA: 60807SN: 13

RT: 4.334.325AA: 67956SN: 13(c) sludge

80

90

100

RT: 5.195.187AA: 14301408SN: 438

NL:5.32E5TIC MS ICIS 22TET fragment

m/z 427rt: 5.2 min

30

40

50

60

70

RT: 6.546.535AA: 2849013SN: 109

RT: 5.865.856AA: 982565SN: 98

RT: 4.41

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Time (min)

0

10

204.414AA: 143691SN: 36

RT: 3.733.733AA: 212668SN: 28

(d) compost

AA: 4555562

Fig. 5 MS/MS chromatograms of TET and OXY extracted from unfortified sludge and compost samples

Water Air Soil Pollut

impurities that have to be removed by cleanup steps.However, the successful detection of the natural estro-gens, estrone and 17β-estradiol, in mixed compost(Fig. 6a and b) shows that biosolids generated by thewastewater treatment process may be a significantsource of release of these contaminants into the envi-ronment. In addition, the apparent absence of E1 andE2 in sludge from a single WWTP indicates thatsludges from different origins may differ enormously.The fate of estrogens in the wastewater treatmentprocess is not clearly known, and a number of studieshave reported conflicting sorption and removal effi-ciencies. Ternes et al. (1999b) found that the naturalestrogens undergo, in addition to sorption effects, bi-ological degradation processes with activated sludge,and Baronti et al. (2000) reported that activated sludgetreatment efficiently removes estriol (E3), estradiol(E2), and ethinylestradiol (EE2), but not estrone

(E1). Accordingly, it has been argued that biologicaldegradation in a common municipal WWTP with anactivated sludge system can appreciably eliminatenatural and synthetic estrogens with only 5–10% ofinfluent estrogens sorbed onto digested sludge(Andersen et al. 2003; Joss et al. 2004). However,it has also been shown that the conventional mu-nicipal wastewater treatment plants, mainly basedon activated sludge systems, are not effective incompletely removing estrogens because of theirrelatively high stability (Johnson et al. 2000; Wanget al. 2005), and natural and synthetic estrogenshave been found in activated and digested sludgesamples in the nanogram per gram range (Ternes etal. 2002). Effluent discharge and biosolid genera-tion of municipal WWTPs can therefore be one ofthe most important sources of endocrine-disruptingcompounds into the environment.

80

90

100

RT: 13.48AA: 10164445SN: 1469BP: 253.1

NL:4.87E5m/z= 251.5-254.5 MS ICIS 54E1 fragment

m/z 253 1

40

50

60

70

RT: 12.66AA: 3600643SN 388

RT: 11.04 RT: 14.65AA 3481517

.1rt: 13.5 min

0

10

20

30:

BP: 253.1AA: 1876650SN: 300BP: 253.1

: SN: 286BP: 253.1

RT: 16.74AA: 142557SN: 29BP: 253.1

(a)

80

90

100

RT: 13.25AA: 94087SN: 139BP: 159.2

RT: 13.58

NL:7.20E3TIC MS IC IS 35

E2 fragment m/z 159.2

30

40

50

60

70

RT: 15.50AA: 33460SN: 50BP: 160.5

RT: 12.50AA: 42682SN: 44BP: 159.0

AA: 25707SN: 42BP: 159.7

RT: 11.09AA: 9105 RT: 19.17

/ 59rt: 13.3 min

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

0

10

20SN: 26BP: 159.0

RT: 19.17AA: 8838SN: 17BP: 159.1

RT: 0.69AA: 5606SN: 11BP: 158.1

(b)

Fig. 6 MS/MS chromatograms of E1 and E2 extracted from unfortified compost samples

Water Air Soil Pollut

The Kolchai HaSharon WWTP is one of 14WWTPs in Israel that contributes treated wastewatersludge to the Compost Or composting facility. Thepresence of these compounds in compost and notsludge highlights two important concerns: one, thatdifferent municipal WWTP processing conditions ex-perience varying levels of success in removing estro-gens and two, that the current composting processdoes not sufficiently degrade and remove these twocommon hormones from biosolids destined for agri-cultural use. While estrogens have been found to bebiodegradable in soils by ubiquitous microorganismsthat require no prior adaptation (Colucci et al. 2001),the mechanisms and extent of removal during com-posting are unknown.

5 Conclusions

Simultaneous extraction of SMX, SDM, TET, OXY,E1, E2, and what is believed to be the OXY degrada-tion product, 4-epi-OXY, from treated secondarysludge and mixed compost was achieved using a sim-ple USE method followed by cleanup and concentra-tion using two different SPE methods and analysis bytwo different LC–MS/MS methods. Recoveries,LODs, and LOQs for the extraction procedure weresatisfactory, demonstrating that the procedure is appli-cable for environmental samples.Within each group, i.e.,sulfonamides, tetracyclines, and natural estrogens, therewas some variation between the recoveries achievedfor the representatives, and interpretation of the appli-cability of the methods for other compounds in eachgroup is difficult. However, the compounds examinedin this study represent a wide range of physicochem-ical properties, and therefore, the method is expectedto be applicable to many of the antibacterial agentsand pharmaceutical compounds present simultaneous-ly in organic matter. The extraction procedure wasoptimized for tetracyclines, and higher recoveriescould probably be achieved with further method mod-ifications in regard to these compounds. However, thisprocedure represents a useful compromise for simul-taneous extraction of all three groups of compoundsfrom sludges and composts.

Acknowledgments The authors gratefully acknowledge theIsrael Ministry of the Environment and the Winikow Fund fortheir financial support.

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