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Sample enrichment for bioanalytical assessment of disinfected drinking water: concentrating the polar, the volatiles, the unknowns Daniel Stalter, Leon Peters, Elissa O’Malley, Janet Yat-Man Tang, Marion Revalor, Maria Jose Farre, Kalinda Watson, Urs von Gunten, and Beate I. Escher Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00712 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 8, 2016

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Environmental Science & Technology

Sample enrichment for bioanalytical assessment of disinfected drinking water: concentrating the polar, the volatiles, the unknowns Daniel Stalter,1,2* Leon I. Peters,1,‡ Elissa O’Malley,1 Janet Yat-Man Tang,1 Marion Revalor,3 Maria José Farré,3,§ Kalinda Watson,1 Urs von Gunten,2,4 Beate I. Escher1,5,6

1

National Research Centre for Environmental Toxicology (Entox), The University of

Queensland, Brisbane, Australia 2

Eawag, Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, Switzerland

3

Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane,

Australia 4

School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique

Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland 5

Department of Cell Toxicology, UFZ – Helmholtz Centre for Environmental Research,

Leipzig, Germany 6

Environmental Toxicology, Center for Applied Geosciences, Eberhard Karls University,

Tübingen, Germany ‡

current address: Chromatography and Mass Spectrometry Division, Thermo Fisher

Scientific, Bremen, Germany and School of Geography, Earth and Environmental Sciences, University of Birmingham, UK. §

current address: Catalan Institute for Water Research, ICRA, Universitat de Girona, Spain

*corresponding author: [email protected]; Überlandstrasse 133, 8600 Dübendorf, Switzerland; tel. +41 58 765 6828, fax +41 58 765 50 28.

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TOC/ABSTRACT ART

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ABSTRACT

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Enrichment methods used in sample preparation for the bioanalytical assessment of

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disinfected drinking water result in the loss of volatile and hydrophilic disinfection by-

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products (DBPs) and hence likely tend to underestimate biological effects. We developed

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and evaluated methods that are compatible with bioassays, for extracting non-volatile and

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volatile DBPs from chlorinated and chloraminated drinking water to minimize the loss of

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analytes. For non-volatile DBPs, solid-phase extraction (SPE) with TELOS ENV as solid phase

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performed superior compared to ten other sorbents. SPE yielded >70% recovery of non-

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purgeable adsorbable organic halogens (AOX). For volatile DBPs, cryogenic vacuum

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distillation performed unsatisfactorily. Purge and cold-trap with crushed ice serving as

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condensation nuclei achieved recoveries of 50–100% for trihalomethanes and

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haloacetonitriles and approximately 60–90% for purged AOX from tap water. We compared

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the purgeable versus the non-purgeable fraction by combining purge-and-trap extraction

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with SPE. The purgeable DBP fraction enriched with the purge-and-trap method exerted a

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lower oxidative stress response in mammalian cells than the non-purgeable DBPs enriched

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with SPE after purging, while contributions of both fractions to bacterial cytotoxicity was

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more variable. 37 quantified DBPs explained almost the entire AOX in the purge-and-trap

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extracts but 15

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min, removed any condensed nitrogen from the trap, added the crushed ice, and attached

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the trap to the purge-and-trap apparatus. In the event the cold trap became blocked by ice

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from transferred water vapor we paused the process, removed the blockage with a steel

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rod, and continued. After purging, the cold trap was removed and its content was

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transferred into a glass vial to melt the ice at room temperature. The liquid extract was then

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transferred to a headspace-free vial before analysis on the same day. To avoid

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photochemical degradation of DBPs, the purge and trap procedure was performed in a fume

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hood with minimal light and the light was turned on only during set-up and control.

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AOX analysis. The procedure performed in our laboratory has been described

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previously including limits of detection as well as recoveries of DBPs.29-31 AOX was analyzed

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as adsorbable organic chlorine (AOCl), bromine (AOBr), and iodine (AOI). To avoid the loss of

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volatile analytes during AOX analyses, we used a 10 mL gas-tight glass syringe to aliquot the

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water in the absence of headspace and to load the sample without contact with ambient air

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onto the activated carbon cartridges.

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Non-purgeable AOX recovery with SPE. We benchmarked the recoveries obtained

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with SPE against the non-purgeable AOX fraction primarily because we here intended to

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focus on non-volatile DBPs and because purgeable compounds will likely be lost during the



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blow-down of the eluates.5 We defined the non-purgeable AOX fraction as the AOX present

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after purging 12 mL of sample at 40°C with 200 mL/min nitrogen for 30 minutes, which

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yielded AOXafter purging. This purging method resulted in similar quantities of purgeable AOX

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analogous to that of the subsequently applied purge-and-trap method for the enrichment of

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volatile DBPs (Figure S4) and hence we used the term AOXafter purging for both methods in the

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following equations. The SPE extract was added to ultrapure water before the AOX was

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quantified as AOXSPE at an enrichment factor (EFAOX) of 1 (i.e., 10 μL of 10,000-times

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enriched SPE extract was added to 99.99 mL of ultrapure water; equation 1).

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EFAOX = EFextraction ×

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Vextract (µL) Vultrapure water for dilution of the extract before AOX analysis (µL) + Vextract (µL)

(1)

The enrichment factor of the extraction step (EFextraction) is the ratio of extracted

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water sample volume to resulting extract volume. Recovery of non-purgeable AOX by SPE

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was calculated by equation 2.

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non-purgeable AOX recoverySPE =

AOXSPE (mol) AOXafter purging (mol)

(2)

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Purgeable AOX recovery with the cold trap. We benchmarked the recoveries

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obtained with the purge-and-trap procedures against the purged AOX fraction with a focus

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on volatile DBPs (i.e., AOXbefore purging − AOXafter purging). The cold trap extract, captured in

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ultrapure water, was 5-times diluted with ultrapure water before the AOX was quantified

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and then back-calculated to the concentration in the extract. Subsequently, we determined

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the recovery of purgeable AOX with equation 3.

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purgeable AOX recoverycold trap =

AOXcold trap (mol) AOXbefore purging (mol) − AOXafter purging (mol)


(3)

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For the purge-and-trap method we also investigated Milli-Q water spiked with

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trihalomethanes (trichloromethane, tCM; tribromomethane, tBM; triiodomethane, tIM) as

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well as haloacetonitriles (HANs: dichloroacetonitrile, dCAN; dibromoacetonitrile, dBAN).

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These compounds were measured as AOCl, AOBr, and AOI before purging, after purging,

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and in the cold trap extracts to calculate the AOX recoveries (equation 3).

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AOX mass balance. Finally, we evaluated the consecutive extraction of purgeable

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(cold trap, first extraction step) and non-purgeable DBPs (SPE, second extraction step) from

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municipal tap water samples. We applied a mass balance approach (Figure 1, equation 4) to

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determine the fraction (f) of the total AOX enriched in the SPE extract ( fnon-purgeable AOX SPE ,

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equation 5), lost during SPE ( fnon − purgeable AOXlost , equation 6), trapped in the cold trap (

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fpurgeable AOXcold trap , equation 7), and lost during cold-trapping ( fpurgeable AOXlost , equation 8).

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AOXbefore purging = AOXSPE + (AOXafter purging − AOXSPE ) + AOXcold trap + (AOXbefore purging − AOXafter purging − AOXcold trap)

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fnon-purgeable AOXSPE =

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fnon-purgeable AOXlost =

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fpurgeable AOXcold trap =

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fpurgeable AOXlost =

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AOX SPE (mol) AOXbefore purging (mol) AOXafter purging (mol) − AOX SPE (mol) AOXbefore purging (mol)

AOXcold trap (mol) AOXbefore purging (mol)

AOXbefore purging (mol) − AOXafter purging (mol) − AOX cold trap (mol) AOXbefore purging (mol)

(4)

(5)

(6)

(7)

(8)

Bioanalytical testing of water samples & extracts. We analyzed the biological response of extracts with the Aliivibrio fischeri bioluminescence inhibition assay (Microtox)



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as described previously16, 32 because of its high sensitivity to DBPs.5, 8, 33 We also applied the

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AREc32 assay to test for oxidative stress response as this endpoint is highly responsive to

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DBPs33 and because the assay is based on a human cell line (MCF-7).34 The cold trap extracts

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were tested in a headspace-free setup to minimize the loss of analytes.16

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The 10,000-fold enriched non-volatile SPE extracts were spiked to the bioassay

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medium at a dilution factor of 100, resulting in a maximum relative enrichment factor (REF,

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equation 9) of 100. enrichment factor of the extracts dilution factor in the bioassay

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relative enrichment factor REF =

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Each extract was analyzed in 8-point 2-fold dilutions in two to three independent

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(9)

experiments with two to four replicates each. The cold trap extracts were up to 133-times enriched in Milli-Q water (dependent

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upon the amount of water vapor trapped in the cold trap). For the AREc32 assay we added

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800 μL of the cold trap extract to 100 μL 10-times concentrated assay medium plus 100 μL

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fetal bovine serum (FBS) before adding this mix headspace-free to the cells of a 96-well

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microplate,16 resulting in a dilution factor in the bioassay of 1.25 and REFs of up to 106. For

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the Microtox assay we mixed the cold trap extracts with 10% per volume of 10-times

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concentrated Microtox buffer, serially diluted the extracts using Microtox buffer, and added

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66.67% per volume of this mix to the bacteria in growth medium. This resulted in a bioassay

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dilution factor of 1.67 and REFs of up to 80. Higher REFs are theoretically possible but would

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require a reduced amount of crushed ice in the cold trap or a higher purged sample volume.

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The higher the effect in the extract (i.e., the lower the effect concentration, EC) the higher the extraction efficiency of toxicologically relevant DBPs. Additionally, the


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assessment of biological effects in procedural blanks by use of ultrapure water is important

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to exclude procedural artifacts potentially caused by toxic impurities leaching from SPE

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cartridges.35

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To quantitatively compare the effect of the volatile and non-volatile DBP extracts

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and to assess extraction efficiencies we calculated bioanalytical equivalent concentrations

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relative to the reference compounds (BEQ, equation 10).36 We used phenol as the reference

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compound for the Microtox assay (EC50: 3.2 mM, Figure S5A) to calculate phenol-EQs and

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we used t-butylhydroquinone (tBHQ, ECIR1.5: 0.9 μM, Figure S5B) as the reference compound

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for the AREc32 assay to calculate tBHQ-EQs.

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BEQ (M) =

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EC reference compound (M) EC sample (REF )

(10)

Recovery of selected DBPs. To assess the extraction efficiency of polar DBPs we

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selected HAAs as a representative class of hydrophilic and fully ionized DBPs.37 We

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determined the recoveries by SPE of monochloroacetic acid (mCAA), dichloroacetic acid

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(dCAA), trichloroacetic acid (tCAA), monobromoacetic acid (mBAA), and dibromoacetic acid

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(dBAA) spiked to tap water. Furthermore, the recoveries of additional DBPs

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(trihalomethanes, haloacetonitriles, haloketones, haloacetaldehydes, and haloacetamides)

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were determined in DBP-spiked tap water and in water samples from three different water

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treatment plants (WTPs).8 Recoveries of the selected DBPs were calculated by dividing the

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measured concentration in the extract by the concentration in the sample before extraction.



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RESULTS & DISCUSSION

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Extraction of non-volatile DBPs

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Comparison between freeze-drying, LLE, and SPE. After initial experiments, detailed

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in the SI (page S12), both freeze-drying and LLE were not further considered because SPE

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was an either similar or more effective extraction procedure, which provided samples with

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lower matrix co-extraction.3

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Comparison of various SPE phases. 11 different solid phases for SPE were evaluated

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at pH 1, 1.5, or 7 (the latter for ion-exchange sorbents WAX, MAX, and mixed-bed; Table 1)

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and showed diverse AOX recoveries (Figure 2A for the optimum pH per sampling phase, all

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data present in SI, Figures S6 and S7), while no AOX was detected in the procedural blanks.

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The AOI concentration was in most cases below the limit of detection ( HLB (51%) ≈ Lichrolut (49%) ≈ StrataX (49%) ≈ ENV+ (48%)> mixed-bed (36%) ≈

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WAX (34%) ≈ XAD8/2 (33%) > MAX (27%) ≈ CC-HLB (27%) (Figure 2). XAD resins, which are

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frequently applied for bioanalytical assessment of disinfected water samples,7, 20, 21, 26, 38, 39

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revealed relatively poor recovery of AOX. However, for the solvent exchange to methanol

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we blew down the ethyl acetate eluate from the extraction with XAD to dryness under a

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stream of nitrogen gas before redissolving it in methanol (SI, section: More details on the

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solid-phase extraction SPE) to avoid effects from the solvent in the bioassay. This might have

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increased the loss of volatile and semi-volatile compounds. HLB-CC, HLB, TELOS ENV,

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Lichrolut, StrataX, and ENV+ delivered similarly good recoveries (50–70% recovery),

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presumably because the sorbent materials are all based on polystyrene-divinylbenzene or

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polydivinylbenzene (Table 1). CC-HLB (coconut charcoal on top of HLB (Supelco)) was


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considerably less effective for AOX extraction than HLB-CC. Extraction was most efficient

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when HLB-CC was eluted upside-down to avoid re-adsorption of analytes eluted from HLB

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by the coconut charcoal (Figure S7). The low recovery by SPE with CC-HLB indicated that

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AOX sorbed to CC was poorly eluted with MeOH or MTBE (Figure S7). Except for CC-HLB,

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biological effects of the extracts followed the pattern of AOX recovery: higher AOX recovery

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was associated with higher cytotoxicity. After extraction with WAX, MAX, and mixed-bed

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cartridges the samples showed lowest cytotoxic effects confirming poor recovery of DBPs at

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pH 7. Cytotoxicity of the procedural blanks was highest after HLB-CC and CC-HLB extraction

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(EC50 ≈ 10 REF), indicating that cytotoxic compounds leached off the carbon, deeming CC

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incompatible with bioanalysis. Blank effects were comparably high for HLB, StrataX, and

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ENV+ (EC50 ≤ 100 REF). TELOS ENV delivered one of the highest AOX recoveries and lowest

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blank effects (EC50 ≥ 500 REF; Figure 2A).

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pH dependent DBP recovery. We assessed the effect of pH for DBP recovery with

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Oasis HLB, Lichrolut, StrataX, and TELOS ENV and extracted one tap water sample at pH 1,

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1.5, 2, and 3. TELOS ENV was not evaluated at pH 1 because an irreversible color change

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indicated sorbent degradation. TELOS ENV showed a better performance in terms of AOX

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recovery at pH 1.5 (up to 70% for AOCl) as well as low blank toxicity (Figure 2B) compared to

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HLB, Lichrolut, and StrataX (Figure S8). Generally, the lower the pH the higher was the AOX

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recovery and biological effect in the sample (Figures 2B and S8). This observation is

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consistent with the presence of ionized DBPs, such as HAAs, which require protonation to

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effectively sorb to the solid phase in their neutral form. Acidification of drinking water

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samples before enrichment is common practice to increase the extraction efficiency and

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samples are often acidified to pH 1 for SPE7 or even pH 0.5 for LLE.9, 40 However, a low pH



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can cause the degradation of SPE sorbents and may reduce the recovery of analytes.41 This

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might explain slightly reduced AOX recoveries for Oasis HLB and StrataX at pH 1 (Figure S8).

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According to previous studies, acidification is not likely to produce artifacts in

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genotoxicity assays42 and compounds prone to basic hydrolysis like HANs will be well

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preserved at low pH.43 However, changes in dissolved organic matter composition of

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acidified water samples have been previously reported.44 Due to the risk of sample

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alteration through acidification, extraction at neutral pH would be desirable. However, the

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AOX recoveries after SPE at pH 7 with a mixed-bed anion- and cation-exchange cartridge

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with polystyrene-divinylbenzene sorbents were poor (1000 REF. Displayed are the arithmetic means of two independent

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experiments ± standard deviation (A) or results from one experiment (B).

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Figure 3. A) Concentration of AOCl, AOBr, and AOI in samples before purging (bef. pur.),

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after purging (aft. pur., with salting-out) and in the cold trap extracts (133-times enriched);

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B) % of AOX purged from the samples (% purged) and % of the purged AOX retained in the

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cold trap (purgeable AOX recoverycold trap, equation 3). Samples analyzed: Milli-Q water

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spiked with THMs (orange), Milli-Q water spiked with HANs (green), tap water TW (blue).

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Displayed is the arithmetic mean of two independent experiments per sample ± standard

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deviation.

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Figure 4. A–J) Fractions of chlorinated (AOCl) and brominated (AOBr) organic compounds

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lost or captured with the respective enrichment methods (without salting-out) in five tap

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water (TW) samples (Table S2), calculated according to equations 5–8; 100% refers to the


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AOX concentration in the original sample before extraction; K–L) Bioanalytical equivalent

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concentrations of reference compounds in the bacterial cytotoxicity assay (K, Microtox,

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phenol-EQ) and in the human-cell based oxidative stress assay (L, AREc32, t-

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butylhydroquinone-EQ) in the purgeable fraction (purge-and-trap extracts) versus the non-

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purgeable fraction (SPE extracts) of five tap water samples, calculated according to equation

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10. Each sample was analyzed in two independent replicates and each replicate in three to

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four sub-replicates in two independent experiments (displayed is the mean ± standard

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deviation; the latter for K and L only). The bars in K and L are stacked.



Figure 1. AOX mass balance (equations 5–8) and experimental measures of AOX in this study. 262x208mm (150 x 150 DPI)

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Figure 2. A) Non-purgeable AOX recoverySPE (equation 2) from a representative tap water sample by use of different SPE sorbents (Table 1) and cytotoxicity of resulting extracts as well as procedural blanks; B) pH dependent AOX recovery with TELOS ENV; EC50: 50% effect concentration of a tap water extract and a procedural blank extract in the Aliivibrio fischeri bioluminescence inhibition assay as relative enrichment factor (REF). In case no EC50 is displayed, the value was >1000 REF. Displayed are the arithmetic means of two independent experiments ± standard deviation (A) or results from one experiment (B).



Figure 3. A) Concentration of AOCl, AOBr, and AOI in samples before purging (bef. pur.), after purging (aft. pur., with salting-out) and in the cold trap extracts (133-times enriched); B) % of AOX purged from the samples (% purged) and % of the purged AOX retained in the cold trap (purgeable AOX recoverycold trap, equation 3). Samples analyzed: Milli-Q water spiked with THMs (orange), Milli-Q water spiked with HANs (green), tap water TW (blue). Displayed is the arithmetic mean of two independent experiments per sample ± standard deviation. 87x46mm (600 x 600 DPI)


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Fractions of chlorinated (AOCl) and brominated (AOBr) organic compounds lost or captured with the respective enrichment methods (without salting-out) in five tap water (TW) samples (Table S2), calculated according to equations 5–8; 100% refers to the AOX concentration in the original sample before extraction; K–L) Bioanalytical equivalent concentrations of reference compounds in the bacterial cytotoxicity assay (K, Microtox, phenol-EQ) and in the human-cell based oxidative stress assay (L, AREc32, t-butylhydroquinoneEQ) in the purgeable fraction (purge-and-trap extracts) versus the non-purgeable fraction (SPE extracts) of five tap water samples, calculated according to equation 10. Each sample was analyzed in two independent replicates and each replicate in three to four sub-replicates in two independent experiments (displayed is the mean ± standard deviation; the latter for K and L only). The bars in K and L are stacked. 200x183mm (300 x 300 DPI)


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