identification of cytotoxic compounds in european wastewaters during a field experiment

Analytica Chimica Acta 426 (2001) 265–277

Identification of cytotoxic compounds in Europeanwastewaters during a field experiment

M. Castilloa, M.C. Alonsoa, J. Riua, M. Reinkeb, G. Klöterb,H. Dizerc, B. Fischerc, P.D. Hansenc, D. Barcelóa,∗

a IIQAB-CSIC, Department of Environmental Chemistry, Jordi Girona, 18-26, 08034 Barcelona, Spainb Merck KgaA, Scientific Laboratory Products, Frankfurter Str., 250, 64293 Darmstadt, Germany

c Department of Ecotoxicology, Institute for Ecological Research and Technology, Berlin University of Technology,Keplerstrabe, 4-6, D-10589 Berlin, Germany

Received 26 October 1999; received in revised form 21 January 2000; accepted 27 January 2000

Abstract

Two short term bioluminescence inhibition assays based onVibrio fischeri (Microtox® and ToxAlert® 10 tests) wereused for the estimation of the cytotoxicity of several European wastewaters from Germany, Spain and Sweden during a fieldexperiment carried out in Berlin (Germany) organised by the Environment and Climate program of the European Commission.The various effluents, freshly collected and transported immediately to Berlin, were tested for their cytotoxic potential andwere characterised by an analytical protocol involving the use of sequential solid phase extraction (SSPE) followed by liquidchromatography-mass spectrometry (LC-MS).

The bioluminescence tests showed a relatively high sensitivity concerning cytotoxic effects. Besides the wastewater samplesof a cement industry (K1 to K3), the effluents of all industrial plants demonstrated a high cytotoxic impact on test bacteria. Thehighest toxicity was observed in the tannery raw effluent from Sweden and Spain (SW1 and BAR1, respectively). Cytotoxicitywas investigated in different wastewater samples of two sewage treatment plants (STP) receiving the raw tannery wastewatersof Sweden and Spain mixed with domestic wastewaters and industrial effluents of a German manufacturing plant. A substantialdecrease on the inhibition from 70–80% down to 15–20% was observed when analysing the untreated influent versus thetreated effluent of both STPs, respectively. In addition, genotoxicity with the umu-assay (DIN 38415T3, ISO FIS/DIS13829)was also performed and high genotoxicity was observed when analysing the same untreated wastewater samples that showedthe highest bioluminescence inhibition withV. fischeriwith a good correlation between both tests.

The analytical protocol applied for the evaluation of the toxicity tests involves the use of SSPE using C18 and polymericcartridges (Isolute ENV+) followed by ion pair-electrospray-mass spectrometry (IP-ESI-MS) and by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). By this protocol different analytes includingnon-ionic (polyethoxylates) surfactants, ionic (linear alkyl benzene-sulfonates) surfactants, phenols, benzene-sulfonates andnaphthalene-sulfonates were determined. Combination of this previously developed methodology with toxicity measurementsallowed to detect different groups of pollutants responsible for the toxicity of the studied wastewaters. Evidence of the ex-istence of a correlation between the presence of LAS and cytotoxic effects was observed in those samples where these analytes

∗Corresponding author. Tel.:+34-93-4006118; fax:+34-93-2045904.E-mail address:[email protected] (D. Barcelo).

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(00)00829-1

266 M. Castillo et al. / Analytica Chimica Acta 426 (2001) 265–277

were major pollutants. Synergetic effects were observed for naphthalene-sulfonate derivatives whose presence enhanced thecytotoxicity of other pollutants present in the sample. © 2001 Elsevier Science B.V. All rights reserved.

Keywords:Industrial effluents; Surfactants; Cytoxicity; Genotoxicity; Liquid chromatography; Mass spectrometry

1. Introduction

Commonly, analysis of industrial wastewater con-stituents has to face a complex mixture of variousorganic substances that hamper the extraction andanalysis of the compounds under investigation. Thisis especially true for untreated wastewaters, espe-cially from leather production, that are heavily loadedwith inorganic (e.g. chromium derivatives) and or-ganic constituents, its purification being an importanttask worldwide. Because of the very large numberof chemical pollutants, no useful monitoring andscreening of polluted wastewaters can be based onlyon chemical analysis which does not provide suffi-cient information to asses the ecological risk. Toxiceffects occur generally at high concentrations of sin-gle substances or at low concentrations of a mixtureof substances. Therefore, chemical analysis resultsshould be supported by measurements of toxicity.The US EPA [1] and the European Union require thatan industrial effluent reaches certain limits of non-toxicity before the effluent is discharged into the envi-ronment. In Section 7a of the German Federal WaterAct [2], were defined as those exhibiting toxic, per-sistent genotoxic, mutagenic and carcinogenic effectsas ‘dangerous substances’.

Biotests are used for the discharge control of in-dustrial wastewater and secondary effluents using thepollution unit concept. They can be used to estimatethe function capability of aquatic biotops and as an‘early warning’ system for the monitoring and screen-ing of surface water [3]. Because of the variety ofaquatic organisms and the heterogenecity of condi-tions in aquatic environment, there is no reliable stan-dard biotest developed for detecting chronic effectson the function and structure of aquatic organisms[4]. The methods for cytotoxicity and genotoxicitybased on molecular and cellular levels of physiologi-cal regulation of test organisms such as enzymatic andmetabolic activity of cells are usually highly sensitive,but not very specific [5,6]. Only the combination ofsome biotests with different prokaryotic and eukary-

otic organisms contribute to estimate the large effectof toxicants in surface water [6,7]. Most of thesebiological toxicity tests are expensive, require largesample volumes and usually take more than 24 h.Consequently, the greatest number of recent studieshave dealt with the use of bacterial luminescent assayfor toxicity screening and assessment [8] being rapid,reproducible and cost-effective. The potential of en-hanced chemiluminescence tests as a rapid screeningtechnique for monitoring effluents [9] and sewagetreatment plants (STP) [10] processes has been eval-uated. The sensitivity of the assay is standardised byusing exactly the same photobacterial strain as used inthe well-established national and international toxic-ity testing standards (ISO DIS)Vibrio fischeriNRRLB-111 77 [11]. The umu array (ISO FIS/DIS 13829and DIN 38415T3) for genotoxicity assessment is acost effective and validated assay.

Toxicity assays are slowly being incorporated intoenvironmental monitoring of remediation sites, withfurther chemical characterisation using generallygas chromatographic-mass spectrometric methods(GC-MS) [12,13,14]. The work reported here com-bines toxicity measurements with chemical analysisin order to identify specific chemicals or classes ofchemicals contributing most to the toxicity of a com-plex mixture. The novelty as compared to previousworks is that in this work liquid chromatography-massspectrometric (LC-MS) methods are being used. Thiswill allow to identify polar and highly watering sol-uble compounds that are usually not detected byGC-based methods. In a recent paper [14], it wasfound that compounds of relatively high polarity wereresponsible for the bioluminescence inhibition in tan-nery wastewaters, although nothing was mentionedabout their chemical composition.

In Europe, efforts are being made during the lastyears to develop and to use different bioassays andbiosensors for environmental monitoring. The pur-pose of the two different technical meetings organisedin September 1997 and 1998 was to test the differentbiosensors and bioassays under real-world environ-

M. Castillo et al. / Analytica Chimica Acta 426 (2001) 265–277 267

mental conditions and also to investigate what kindof additional information can biosensors give for thecharacterisation of real environmental samples. Twoof the groups involved in the second technical meet-ing held in Berlin used different types ofV. fischeriwhereas a third group was carrying out the chemicalanalysis. The present work reports the results of thesethree groups and how the chemical data correlateswith cytotoxicity using Microtox® and ToxAlert 10®

systems. Wastewater samples of different nature werefreshly collected from different places in Europe,immediately transported to Berlin for the cytotoxic-ity measurements and afterwards were immediatelytransported to the central laboratory in Barcelona,Spain, to perform the chemical analysis. An analyticalprotocol based on sequential solid phase extraction(SSPE) that allows to identify a broad spectrum ofpolar analytes in wastewaters using LC-APCI-MSand IP-ESI-MS [15] was used.

The goals of this work were (i) to test two differentbioluminescence inhibition assays under real-worldenvironmental conditions for the characterisation of avariety of European wastewaters, like those dischargedfrom either untreated industrial effluents, from mixedindustrial and domestic effluents and from treated ef-fluents of the STP, and (ii) to achieve a high level ofknowledge, by using biotests and advanced LC-MSmethodologies, allowing to identify and quantify po-lar, ionic and highly water soluble organic pollutantspresent in various European wastewaters. To ourknowledge, such a type of field experiment was notperformed before in Europe. In practice, most of thecytotoxicity measurements are carried out at the lab-oratory and very few work has been carried out underfield environmental conditions. In addition, the varietyof the samples simultaneously analysed will permitto achieve a broad perspective on the performanceof the bioluminescence assays and to correlate thebioluminescence inhibition with the chemical data.In addition genotoxicity with the umu-assay was alsoperformed and the data obtained will be a useful addi-tion to the cytotoxic values obtained with Microtox®

and ToxAlert®. The results of this study should serveto obtain a better perspective on the chemicals respon-sible for the wastewater toxicity and also to complywith the European Union Directive 91/271/EC of 21May 1991 concerning urban wastewater treatmentthat was amended by Commission Directive 98/15/EC

of 27 February 1998. This Directive requires the EUMember States to ensure that discharges of urbanwastewater and their effects are monitored.

2. Experimental

2.1. Chemicals and reagents

HPLC-grade water, acetonitrile and methanol wereobtained from Merck (Darmstadt, Germany) and werepassed through a 0.45mm membrane filter beforeuse. Hexane and methylene chloride for organic traceanalysis were also purchased from Merck. Aceticand sulfuric acid pro-analysi grade from Panreac(Barcelona, Spain) and Merck, respectively, wereused. Sodium chloride (NaCl) was supplied by CarloErba (Milano, Italy). All the individual polyethoxy-lated surfactants (polyethylene glycols (PEGs),nonylphenol polyethoxylates (NPEOx) and alkylalco-hol polyethoxylates (CnEOx)) not corresponding toa pure standard but to a mixture of substances withan average number of ethoxy units (x) were fromKao Corporation (Barcelona, Spain). Benzene- andnaphthalene-sulfonates (BS and NPS) were a gift fromAGBAR (Aigües de Barcelona, Barcelona, Spain) andcommercial linear alkylbenzene-sulfonates (CnLAS)were supplied by Petroquımica Española S.A. in asingle mixture standard (not single standard solutionsof each LAS). The proportional composition of thedifferent homologues in the standard mixture is C10(3.9%), C11 (37.4%), C12 (35.4%), C13 (23.1%),C14 (0.2%).

2.2. Sample preparation

Effluent waters were collected as 24 h compositesamples from different points in Europe and were im-mediately shipped by plane at 4◦C to Berlin, the daybefore the technical meeting took place. The samplesanalysed for cytoxicity and genotoxicity were (a) araw tannery wastewater located in the neighbourhoodof Barcelona (Spain) (labelled BAR1) and from a STPreceiving both domestic and industrial waste with asignificant proportion of tannery wastewaters fromthe same city (labelled BAR2 and BAR3, for influentand effluent, respectively), (b) three wastewater sam-ples corresponding to a raw tannery effluent locatedin Sweden (SW1) and the influent and the effluent of


a STP (SW2 and SW3) receiving wastewaters fromthe same tannery, (c) different wastewater samplesfrom the area of Berlin coming from cement industry,labeled K1, K2 and K3 and (d) industrial effluents la-beled BASF1 and BASF2 from BASF Schwarzheide,Germany. After performing the measurements in thefield side, in Berlin, samples were transported at4◦C to the central laboratory, in Barcelona, Spain,and were immediately extracted using the extractionprotocol described ahead.

The extraction and fractionation methodology usedin presently is described as follows: 200 ml of thefiltered samples were extracted following a SSPEapproach [15] based on the use of an octadecyl-silica (C18) sorbent in series with the polymericIsolute ENV+. Medium-polarity organic compoundswere preconcentrated in the C18 cartridge and des-orbed separately using solvents of different polarity(2×5 ml of hexane allowed to obtain fraction A; frac-tion B was eluted from the cartridge with 2×5 mlof dichloromethane/hexane (4:1, v/v) and 2×5 ml ofmethanol/dichloromethane (9:1, v/v) led to fractionC). The most polar organic compounds in the acidifiedwater residue were trapped by the polymeric sorbentand eluted with 1 ml of water containing ion-pairreagent (5 mM TEA and 5 mM acetic acid, pH=6.5)and 9 ml of methanol. Details of the extraction pro-cedure are given elsewhere [15]. The eluates (10 ml)were concentrated by a gentle stream of nitrogen to1 ml.

These SSPE experiments were performed using anautomated sampler processor from Gilson (Villiers-le-Bel, France). This system includes one automatedsample preparation with extraction columns system(ASPEC XL) fitted with an external 306 LC pumpfor the dispensing of samples through the SPE car-tridges and with a 817 switching valve for the selec-tion of samples from Gilson for the preconcentrationstep. The drying step was carried out using a BakerSpe 12G apparatus from J.T. Baker (Deventer, Nether-lands). Average recoveries ranging from 72 to 103%were obtained for a variety of 23 different analytes(see Ref. [15] for details).

2.3. LC-MS analysis

For LC-MS experiments a VG Platform fromMicromass (Manchester, UK) equipped with a stan-

dard atmospheric pressure ionization (API) sourcewhich can be configured for atmospheric pressurechemical ionization (APCI) or pneumatically assistedelectrospray ionization (ESI) was used. The solventwas delivered by a Waters 616 gradient pump sys-tem controlled by a Waters 600 S controller fromWaters-Millipore (US). For APCI experiments, sourceand probe temperatures were set at 150 and 400◦C,respectively, corona discharge voltage was maintainedat 3 kV and the cone voltage was set at 30 V. TheHV lens voltage was set at 0.20 kV. For ESI experi-ments, a voltage of 3.7 kV was applied to the needletip. Nitrogen was used as nebulizing and drying gasat a flow rate of 10 l/h and 300 l/h, respectively. Infull scan mode them/z range was from 70 to 350in negative ion (NI) mode and from 100 to 1000 inpositive ion (PI) mode of ionization.

For the determination of naphthalene- and benzene-sulfonates (NPS and BS) an LC system model 1090 Afrom Hewlett Packard (Palo Alto, USA) was used. Thedetection was accomplished by a model 1040M diodearray detector that was coupled on-line with a massspectrometer HP 1100 HPLC-MSD API-ES all fromHewlett Packard. Nitrogen was used as nebulizing anddrying gas, with a nebulizer pressure of 55 psi, a dryinggas flow rate of 12 l/min and a temperature of 350◦C.The capillary voltage was set at 3500 V and fragmentorvoltage was set at 80 and 150 V. In full scan mode them/z range was from 75 to 350 in negative ion (NI)mode.

Chromatographic conditions for LC-APCI-MSanalysis were as follows: mobile phase of water andacetonitrile/methanol (1:1, v/v) both acidified with0.5% of acetic acid was passed at 1 ml/min through anHypersil Green ENV column (150 mm×4.6 mm i.d.,5mm particle size) from Shandon HPLC (Cheshire,UK). In the case of LC-ESI-MS analysis, a Hyper-sil BDS C-18 analytical column from Shandon(250 mm×2 mm i.d., 5mm particle size) with wa-ter and acetonitrile/water (8:2, v/v) both contain-ing 5 mM triethylamine and 5 mM acetic acid wereused at a flow rate of 0.3 ml/min. In the case ofion pair chromatography (IP)-ESI-MS, the mo-bile phase was composed of water and methanolphase both containing 5 mM triethylamine and 5 mMacetic acid which were passed through a Hyper-sil BDS C-18 analytical column at a flow rate of0.8 ml/min and a post-column addition of 0.2 ml/min


of methanol was necessary. Further information onchromatographic conditions are given elsewhere[15].

LC-MS analysis with an atmospheric pressure che-mical ionization (APCI) interface, showed charac-teristic peaks in PI mode at 133 and 177m/z unitsfor polyethoxylated non-ionic surfactants. In order todistinguish between the different functionalities, thepresence of peaks atm/z 151 and 195 were checkedfor AEOn,x , atm/z271 and 291 for NPEOx and atm/z101 and 145 for PEGx . In addition to this compari-son, non-ionic polyethoxylated surfactants were iden-tified by its molecular ion [M+H]+ and checkingcorrespondence with the following expressions:AEOn,x (CnH2n+1(OCH2CH2)xOH) should corre-spond with MW=14n+44x+18; NPEOx (C9H19–C6H4(OCH2CH2)xOH) with MW=220+44x, PEGx

(H(OCH2CH2)xOH) with MW=18+44x and mono-(H(OCH2CH2)xOCH2COOH) and dicarboxylatepolyethoxylated glycol (HOOC–CH2(OCH2CH2)x-OCH2COOH) withMW=76+44xandMW=134+44x,respectively.

The analysis of LAS was carried out by LC-MSwith electrospray ionization (ESI) using a two-stepapproach: a chromatographic run at a cone voltageof −80 V was first performed in order to check thepresence of the diagnostic ion [CH2=CH-C6H4-SO3]−at m/z 183 and a second run at−20 V permitted tounequivocally identify each of the four C10–13LAS bytheir [M−H]− ion with m/z values of 297, 311, 325and 339, respectively .

For the determination of BS and NPS by IP-ESI-MS,the fragmentor voltage was set at 150 V, commonlosses of the groups HSO2 or HNO2 (for nitrosulfo-nates) were observed, therefore [M−HSO2]− or [M−HNO2]− were used as diagnostic ions. However, whenapplying lower voltages (80 V), the mass spectra isdominated by the presence of the [M−H]− ion for allmonosulfonate compounds. Them/zvalues monitoredfor each target analyte corresponding to [M−H]− ionwere m/z 222 for 1-amino-4-naphthalene-sulfonate,1-amino-6-naphthalene-sulfonate (1-amino-6-NPS)and 1-amino-7-naphthalene-sulfonate (1-amino-7-NPS);m/z 202 for 3-nitrobenzene-sulfonate (3-nitro-BS); m/z 223 for 1-hydroxy-4-naphthalene-sulfonate(1-hydroxy-4-NPS) ;m/z 191 for 4-chlorobenzene-sulfonate (4-Chloro-BS) and 207 for 2-naphthalene-sulfonate (2-NPS).

2.4. Quantitation

External calibration was used for quantitation. Aseries of injections of the different target analytes wereused to obtain the calibration equations and the studiedrange of concentration was close to the amount oftarget compounds in the analyzed wastewaters [15].

Quantitation of polyethoxylated non-ionic surfac-tants was carried out in full scan mode (FS) in PI modechecking the presence of their characteristic peaks inthe spectra (see Section 2.3).

The quantification of LAS was carried out in singleion monitoring (SIM) mode using a two-step approachpreviously described.

For the quantification of BS and NPS by ion pairchromatography (IP)-ESI-MS, the presence of the ions[M−HSO2]− and [M−HSO3]− was checked with afragmentor voltage set at 150 V in SIM mode. At lowervoltages (80 V) the presence of the ions [M−H]− or[M−2H]2− confirmed the corresponding compound.

2.5. Cytotoxicity determination

The determination of toxicity was performed us-ing two commercial toxicity tests: Microtox® andToxAlert® 10. Both of them are based upon the bi-oluminescence inPhotobacterium phosphoreumor V.fischeri(NRRL B-11177). This bioluminescent bacte-ria is integrated with the citric acid cycle and thereforeluminescence activity is proportional to metabolic ac-tivity. Light output can be reduced by chemicals whichscavenge free radicals, for example, anti-oxidants orinhibitors of the enzyme (e.g. cyanides, amines, phe-nols and heavy metals). The reduction of light out-put (inhibition) is usually proportional to the concen-tration of contaminants present [16]. Luminescenceactivity was measured according to German standardmethods for water and wastewater investigation (DIN38412 T34, 1991). The percentage of inhibition (%I)was determined by comparing the response given bya control solution (2% NaCl) to that corresponding tothe sample. Therefore, the parameter of response con-sidered in this paper was the bioluminescence inhibi-tion, defined as %I=[1−(sample light/control light)]×100.

The inhibition of light emission by more than 20%was considered to evidence cytotoxic potential in theanalyzed samples.


2.6. Genotoxicity determination: Umu-assay

The cytotoxic and genotoxic effect of water sam-ples onSalmonella typhimuriumTA1535 pSK1002were tested with the umu-assay (ISO FIS/DIS 13829,DIN 38415 T3, 1994). The genetically engineeredbacteriumS. typhimuriumTA1535 carried the plas-mid pSK1002 with theumuC–lacZ fusion gene. TheumuC operon was induced by genotoxic agents of sam-ples in response to genotoxic lesions in the bacterialDNA. Since theumuC operon is fused with thelacZgene forb-galactosidase, the induction of theumuCoperon can be easily assessed by the determination ofb-galactosidase activity. The comparison of the induc-tion ratio of theumuC gene on exposure to water sam-ples with its spontaneous activation gives a measureof genotoxicity. The inhibition of bacterial growth wasused to calibrate the genotoxicity potential against thecytotoxic effects of water samples. The growing ratiobelow 0.80 or inhibition of biomass more than 20%were considered as a clear cytotoxic effect of watersamples, otherwise the results could be a false positivefor genotoxicity.

The bacterial suspension were exposed to a dilutionseries of samples. The values of the dilution steps bywhich an induction ratio lower than 1.5 (1.3 after ISOFIS/DIS 13829) was measured counts for genotoxicity(LIDU=1.5).

The addition of S9 enzyme fraction simulates themetabolic activity of higher eukaryotic organisms suchas sorptive disguising, accumulation and some phase Ienzyme reactions missing in bacterial cells. Therefore,S9 enzyme fraction can also cause a higher toxicitythan those of precarcinogens.

3. Results and discussion

3.1. LC-MS analysis of target compounds

Table 1 shows the concentration values of linearalkylbenzene, benzene- and naphthalene-sulfonates,non-ionic polyethoxylated surfactants and phthalatesdetermined in different wastewater samples.

Table 1 shows that 1- and 2-naphthalene-monosul-fonate were found in all samples and exhibited thehighest concentration, with values up to 580mg/l.These analytes are generally found at high concentra-

tions in many untreated tannery and textile wastewa-ters [15], being of extreme importance as dye interme-diates, as tanning materials and as hydrotropic agentsin different industrial processes. So these analytescould be chosen as tracers of industrial pollution, sincethey are the most frequently detected compounds inthis work. The results showed that some aromaticsulfonates, like 1- and 2-naphthalene-sulfonate partic-ularly, cannot be totally removed from wastewaters bybiological treatments. Naphthalene-disulfonates, usedmainly in chrome tanning to soften the leather and intextile industry as stabilisers for diazo compounds, arepresent at concentration levels of 30–81mg/l, in thecase of Swedish wastewaters and at much lower valuesin the case of Spanish wastewaters. These compoundsare only present in these wastewaters as a consequenceof their use in the tannery industry and thereby are notpresent in other analysed wastewaters. The fact thatthese compounds are not removed from the wastewa-ter treatment indicates that a specific tailor-made watertreatment, such as adsorption by graphitized carbonor other treatment is needed for their removal [17].High contents of amino-naphthalene-sulfonate andhydroxy-amino-naphthalene-sulfonate were found inwastewaters corresponding to BASF1 and BASF2at 436.6 and 215.9mg/l, respectively. Since we didnot obtain information about the processes of thischemical plant, we cannot draw any conclusion aboutthe specific use of these two analytes that were de-tected at high concentration values in their effluents.The industrial use of these analytes is generally as acoupling components for azo dyes and peroxidationreagents. From the data reported in Table 1, it can beconcluded that naphthalene- and benzene-sulfonatescan be used as industrial pollution tracers as they arenot present in urban wastewaters but in industrial ef-fluents. This fact is clearly shown in samples BAR1to BAR3, that show a decrease in concentration ofthese analytes from raw wastewater (BAR1) or mixedindustrial and domestic wastewater (BAR2) to thefinal and cleaner treated effluent (BAR3).

Concentration of linear alkylbenzene-sulfonatesfrom C10 to C14 (LAS) is reported in Table 1. Four ofthe samples, corresponding to raw tannery and mixedindustrial wastewaters, showed total concentration ofLAS higher than 100mg/l. Although these compoundsare mainly used in Europe as laundry and householddetergents they are also used in tannery industry for


Table 1Concentration (mg/l) of target analytes and coefficient of variation (in brackets) found in the analysed samples using SSPE followed byLC-MSa

Compound Samples

SWE1 SWE2 SWE3 BAR1 BAR2 BAR3 K1 K2 K3 BASF1 BASF2

Linear alkylbenzene-sulfonate (3–4%)C10LAS 78.3 88.2 0.5 24.0 203.9 1.8 2.8 0.0 0.9 0.1 0.3C11LAS 278.5 192.6 1.2 67.0 686.0 7.4 5.5 0.0 2.5 0.7 0.9C12LAS 452.8 170.7 0.6 42.0 441.0 6.5 3.3 1.2 2.8 0.7 0.8C13LAS 175.4 46.0 0.6 18.0 92.5 2.1 7.8 2.7 3.1 0.9 0.7Total LAS 985.0 497.5 2.9 151 1423.5 17.8 19.5 3.9 9.4 2.4 2.6

Non-ionic polyethoxylated surfactant (7–9%)PEG4 110.8 108.7 829C6EOx 52 41.4C7EOx 41.7C8EOx 42.1C9EOx 41.6C10EOx 63.0 41.7C12EOx 64.1 41.8C13EOx 63.2 42.7NPEO6 322.0 282.4

Benzene- and naphthalene-sulfonates (1–11%)1,5-NPDS 3.3 3.62,6-NPDS 32 30 30.0 1.3 0.8 0.82,7-NPDS 85 82 81.71-Hydroxy-3,6-NPDS 6.71-Amino-5-NPS 0.1 18.6 436.61-Amino-6-NPS 2.9 7.4 0.9 0.1 0.1 4.81-Amino-7-NPS 2.51-Hydroxy-6-amino-3-NPS 27.3 13.7 4.3 215.93-Nitro-BS 0.6 0.1 0.9 0.11-Hydroxy-4-NPS 3.9 0.4 0.5 7.04-Methyl-BS 2.7 0.2 0.1 8.4 5.34-Chloro-BS 1.9 1.2 1.31-NPS 64.4 62.2 1.3 408.8 135.7 61.6 1.8 0.8 0.8 13.7 2.32-NPS 304 305.7 2.9 580.0 243.4 96.3 8.1 4.8 4.3 27.0 8.81-Amino-6-NPS 2.9 7.4 0.9 0.1 0.1 4.81-Amino-7-NPS 2.5

a LAS denotes linear alkylbenzene-sulfonates; PEGx denotes polyethylene glycol; CnEOx denotes alkylalcohol polyethoxylate;NPEOx denotes nonylphenol polyethoxylate; NPDS denotes naphthalene disulfonate; NPS denotes naphthalene-sulfonate; BS denotesbenzene-sulfonate.

removing fat from animal skins and consequently,they can be found in industrial effluents. The reporteddata show that total LAS concentration is higher inSwedish tannery effluents than in the Spanish ones.This fact can be attributed to a different practice in dif-ferent European countries based on regulatory issuesand more environmental concern. Several Europeanregulatory agencies from Denmark, Sweden, UK andSwitzerland pose restrictions to the use of alkylphenolethoxylates (APEO) because they are likely to cause

adverse effects into the environment. European envi-ronmental quality standards for APEO will be fixedat a maximum level of 1mg/l in surface waters [18]and this is the reason for the decline of 40% in APEOreported in the Scandinavian countries. APEO areforbidden in Europe to be used as laundry detergentsand, as a general trend, its use is also being restrictedin industrial applications, specially in Scandinaviancountries being replaced by LAS. However, southernEuropean countries like Spain, Italy and Portugal,


have an important tannery industry and are still usingalkylphenol ethoxylates (APEO) since its use in in-dustrial applications has not yet been restricted by theEuropean Union. As a consequence of such practicesand the strict regulation in the Scandinavian countries,absence of nonylphenol ethoxylates was observed inSwedish wastewaters, whereas up to 322mg/l werefound in Spanish wastewaters (BAR1 and BAR2).Comparing the LAS data from samples BAR1 andBAR2, a considerable increase in the LAS concentra-tion levels can be noticed. LAS are used in tanneryprocesses at relatively low concentrations in that area(BAR1) as compared to the high input released by thedischarges of the urban wastewaters correspondingto a population of 200,000 inhabitants (BAR2). Thiseffect is opposed to that observed for benzene- andnaphthalene-sulfonates in the same samples, suggest-ing that LAS could be used as urban pollution markersin the case of Spanish wastewaters whereas aromaticsulfonates are typically originated from industrialprocesses. Different alcohol polyethoxylates are usedin Swedish tannery as demonstrated by the presenceof heptanol, octanol and nonanol polyethoxylates ata concentration level around 40mg/l in its effluents.Regarding polyethylene glycols (PEG), high con-centration values (up to 829mg/l) were found in thesame industrial wastewaters and this is not surprisingsince these analytes are mainly formed as degradationproducts of the above-mentioned polyethoxylatedsurfactants.

Table 2Cytotoxicity of studied samples obtained using the Microtox® and the ToxAlert 10® bioluminescence inhibition assaysa

Sample LID Microtox® LID ToxAlert®b Inhibition Microtox®b Inhibition ToxAlert® (%)

SWE1 4000 10000 n.d.d. 100 (0)SWE2 2000 100 n.d.d. 80 (0.42)SWE3 <2 <2 n.d.d. 20 (0.46)BAR1 80 100 n.d.d. 100 (0)BAR2 <2 10 n.d.d. 70 (0.35)BAR3 <2 <2 n.d.d. 15 (0.40)K1 4 <2 25% 22 (0.58)K2 <2 <2 14% 0 (0.2)K3 <2 <2 19% 5 (0.37)BASF1 64 n.m. n.d.d. n.m.BASF2 8 n.m. n.d.d. n.m.

a Reproducibility of the ToxAlert 10® test is indicated in terms of coefficient of variation (in brackets) of the inhibition value.b n.d.d.: Not determined data; n.m.: not measured.

We should also mention that the absence of alkyl-phenol ethoxylates was observed in those wastewa-ter samples analysed after passage of the biologicaltreatment, like SWE3 and BAR3, and the levels ofLAS were considerably reduced as compared to thesamples analysed before treatment. Overall, it showsthat biological STP can eliminate most of the organicpollution detected, except that originated from aro-matic sulfonates that usually requires a more specifictreatment.

3.2. Cytotoxicity of the analysed samples

The inhibition of bioluminescence of test bacteriaV. fischeriand the LID values (defined as the lowestidentified dilution step of sample showing no relevantinhibition values) obtained using the Microtox® andToxAlert® kits are summarised in Table 2.

From the LID values, it can be observed that com-parable results are obtained with both kits. In thissense, the maximal inhibition of bioluminescencewas observed in raw wastewater from the plant SWEwith a LID value of 4000 and 10,000 obtained withthe Microtox® and the ToxAlert® kit, respectively.A similar toxicity pattern was observed for the othersamples, that is to say, toxicity was decreasing fromSWE1 to SWE3, from BAR1 to BAR3 and theK1 to K3 samples inhibited bacterial luminescenceonly around 25% with LID value ranging from 4to <2.


Fig. 1. Dose effect curve of luminescence inhibition in the ToxAlert 10® instrument when analysing wastewater samples from a Swedishtannery (SWE1), from the tannery mixed with domestic sewage before entering the STP (SWE2) and from the treated effluent after passagethrough the STP (SWE3).

Primary effluents of the industrial plants of thetannery industry in Sweden (SWE1 and 2) and Spain(BAR1 and 2) and the effluents from the BASF weregenerally highly cytotoxic toV. fischeri. In certaincomplex organic samples (especially like SWE1) thedose effect curve of luminescence inhibition oftenshows a very gradual slope (see Fig. 1 obtained withToxAlert®). This toxic effect of the SWE1 sampleincreases over a range of more than four decades. Inall cases, after wastewater treatment the cytotoxic im-pact of secondary effluents from all sampling plantsdecreased clearly up to LID values<2, confirmingthat the treatment was successful.

Comparison of cytotoxic effects with analytical re-sults may lead to find the main organic compounds ofthe samples causing the luminescence inhibition. Ina previous paper from our group [16] the results of atoxicity directed analysis using ToxAlert®, were re-ported. It was found that mainly alcohol and alkylphe-nol ethoxylates and linear LAS were responsible fortoxicity of the studied samples.

Taking into account these previous results and com-paring the data in Tables 1 and 2, it can be observedthat the toxicity found in the Swedish samples (SWE1to SWE3) could be attributed in its major part toLAS. A correlation between total LAS concentrationand inhibition to bioluminescence ofVibrio fischeriwas observed for these samples containing 985, 497and 2.9mg/l of LAS and with inhibition values of100, 80 and 20%, for SWE1, SWE2 and SWE3, re-spectively. It has been reported that when preparing

standard spiked samples at the laboratory with con-centration values of 10 and 20mg/l of LAS inhibitionfactors of 32 and 53%, respectively were obtained[16]. Therefore, it can be considered that LAS aremainly responsible for the cytotoxicity measured inthe mentioned samples. A similar correlation betweenLAS concentration and inhibition of bioluminescenceis observed for samples K1 to K3. In this case, thehighest LAS content (19.5mg/l) corresponded to thehighest cytotoxicity for sample K1, whereas the otherones, with a low content of this contaminant did notexhibit a relevant toxicity value. This effect can be ob-served in Fig. 2 where LAS concentration (mg/l) andinhibition values (%I) are represented. This data isnot surprising, since an EC50 of 25 mg/l was reportedfor LAS using the ToxAlert® kit. Other authors [19]reported an experimental EC50 value obtained withthe Microtox® chronic test of 39.2 mg/l of LAS. Thehighest concentration values of LAS in SWE1 andBAR1 were 0.98 to 1.4 mg/l, respectively.

The correlation between LAS levels and biolumi-nescence inhibition does not exist for the samplestaken in Spain (BAR1 to BAR3). Considering sam-ple BAR1, it should be taken into account that LASconcentration is lower than that found for somepolyethoxylated surfactants (such as polyethylene gly-col and nonylphenol polyethoxylate with a concentra-tion of 829.4 and 282.0mg/l, respectively). It shouldbe taken into account that alkylphenol polyethoxylatesare toxic compounds. So, we can indicate, based onprevious work [16] that the alcohol polyethoxylates


Fig. 2. Correlation between the LAS content (mg/l) and the bioluminescence inhibition measured in samples SWE1 to SWE3 and K1to K3.

used in the tanneries of Spain and detected in BAR1and BAR2 samples [15,16,20] are partly responsi-ble for the measured toxicity, together with LAS.Naphthalene-sulfonates are detected at concentrationsof 408.8 and 580.0mg/l for 1-naphthalene-sulfonateand 2-naphthalene-sulfonate, respectively in BAR1sample, whereas BAR2 and BAR3 samples havelower levels, but still important. It has been observedthat when these compounds are present at high con-centration values (>100mg/l) together with the othercompounds analysed in this work, a synergetic ef-fect favouring the bioluminescence of the sampleis observed [16]. The synergetic effect attributed tonaphthalene-sulfonates derivatives is also observedin samples BASF1 and BASF2. The content of thesesamples only differs in the high concentration of1-amino-5-naphthalene-sulfonate (436.6) in BASF1which promotes toxicity in this sample to a value of 64

in terms of LID, whereas a LID value of 8 correspondsto BASF2. These samples have very low values ofLAS and no alkylphenol ethoxylates were detected.

3.3. Correlation between cytotoxicity and polarity

The work reported here has some general remarksthat should be taken into consideration. First, it isremarkable that the use of advanced analytical meth-ods based on SSPE followed by LC-MS with toxicitymeasurements is an excellent system for characteris-ing classes of toxic pollutants that cause biolumines-cence inhibition in wastewaters. Since the analyticalprotocol that we have used enables to analyse basi-cally polar analytes, it appears that the Microtox®

or ToxAlert® systems are useful to measure toxicityof these analytes and not of the most lipophilic ones.Indeed, it has been recently reported [21] that no


correlation was observed between the most lipophiliccompounds and the results of the Microtox® assay.The authors suggested that these compounds are notthe ones that affect Microtox® bacteria, but theydid not give any indication how to proceed or whichcompounds to measure. The same observation is validwhen looking at the results of the authors of Ref.[14] that reported toxicity in the most polar fractionsof tannery wastewaters after passage through SPEalthough no indication about the analytes responsiblefor the observed toxicity was made. It is obvious thatmost of the literature [12–14,21] intends to correlatethe toxicity observed with compounds identified byGC-MS methods. In this case only the most non-polarcompounds are determined and the most polar onesescape detection. We have published the first paperon the identification of toxicants based on the biolu-minescence inhibition using ToxAlert® and LC-MS[16]. The promising results observed in this first pa-per have been now more extensively demonstratedgoing in two different directions: (i) by using thewell-established Microtox® assay and by comparingthe values with ToxAlert® and (ii) by analysing a vari-ety of wastewater samples of different origin and fromdifferent points of Europe simultaneously, under fieldconditions.

3.4. Genotoxicity

During the field experiment held in Berlin in ad-dition to cytotoxicity that was determined and com-pared with the chemical data, one of the laboratories(Prof. P.D. Hansen) performed the umu-assay. This isa standard method for genotoxicity in Germany thathas been described in the report published by the Eu-ropean Commission [22]. The genotoxicity values ob-tained by this assay correlates well with those of cy-totoxicity. In this sense, high LID values were ob-tained for the untreated wastewater samples BAR1,BASF1 and SWE2, corresponding to 48, 48 and 12,respectively, whereas no genotoxicity values were ob-served in the treated samples, similarly to the othersamples analysed during the field experiment. Thegenotoxicity data were measured against the referencesubstances of the ISO and DIN standards commonlyused for calibration. We observed that genotoxicitydata matches cytotoxicity and genotoxicity is only

observed in the samples exhibiting highest cytotoxic-ity values, with no indication about the genotoxicityof the samples exhibiting low or medium cytotoxicity.The genotoxicity of industrial effluents has been re-ported in the literature and shows that some untreatedindustrial effluents offer, in general, high genotoxicitythat can vary substantially over one order of magnitude[23]. In one of the analysed samples, the genotoxic-ity does not suffer any variation between two differentuntreated samples, BAR1 and BASF1, and in the othercase, SWE2, the variation of genotoxicity is less thanone order of magnitude. The other aspect to consideris the correlation between the chemical substancesand the genotoxicity observed in the samples BAR1,BASF1 and SWE2. In a previous study of genotoxic-ity of different raw and treated water samples, differ-ent types of surfactants like polyethoxylated nonylphe-nols and different alkylbenzene-sulfonates were iden-tified by mass spectrometric methods as the com-pounds present in the most genotoxic fractions of theextracts [24]. In the present study, the chemical analy-sis performed gave either high levels of LAS and aro-matic sulfonates, (SWE2) high levels of polyethoxy-lated nonylphenols, alcohol ethoxylates and aromaticsulfonates (BAR1) and high levels of aromatic sul-fonates (BASF1). However, this is only a hypothesis,at present, since we did not carry out genotoxicitystudies of the single substances or artificial mixturesof these substances. So, whereas cytotoxicity has beensupported with separate experiments in the laboratorywith single substances and artificial mixtures of thesesubstances [16], in the case of genotoxicity this in-formation is not available. But the data suggest thatgenotoxicity is observed in the same complex samplesas those with high cytotoxic effects, so most likely thecomplex mixture of substances present in these sam-ples has both effects, cytotoxicity and genotoxicity.

4. Conclusions

Overall, we should indicate that both biolumines-cence tests showed a relatively high sensitivity con-cerning cytotoxic effects. Evidence of the existenceof a correlation between the presence of LAS and cy-totoxic effects was observed in those samples wherethese analytes were major pollutants. High toxicitywas observed in primary effluents of the Swedish


tannery industry, following tannery effluents fromSpain (Barcelona) and BASF in Germany. Aftertreatment of sewages, the cytotoxicity decreased sig-nificantly in all secondary effluent samples. This wasdue to the fact that major pollutants are efficientlyeliminated in the STP. For example, more than 90%of the LAS are degraded after passing through thewater treatment plant. This behaviour can be ob-served in the results of the data reported from Sweden(SWE2 to SWE3) and from Barcelona (BAR2 toBAR3). A similar behaviour is observed for non-ionicpolyethoxylated surfactants which are efficiently de-graded from the primary to the secondary effluentwhen comparing the data of BAR2 and BAR3. Re-garding naphthalene-sulfonates, although most ofthem exhibit acute toxicity to bacteria in the range of100 mg/l, [25] it should be noticed that some sulfonicacid exhibit acute toxicity to bacteria in the range of30 mg/l, and consequently it exists the possibility thatthe activity of unadapted biological waste water treat-ment plants may be inhibited during times of extremepeak loads as those originating from untreated textileand tannery wastewaters.

The present work has shown that it is possible touse bioluminescence inhibition assays during a fieldexperiment to quantify the major toxicity present indifferent European wastewaters. The toxicity-directedanalysis, with further identification by LC-MS, is auseful approach. By a first screening in the field ofthe wastewater samples using bioluminescence inhi-bition assays it is possible to pre-select the more toxicsamples for further LC-MS analysis and to discardwastewater samples with low or no inhibition becausethey are not toxic to the environment. This approachis time saving and can be of great help to the differ-ent type of industries and regulatory water agenciesto control wastewater discharges and wastewater treat-ment. The approach reported here is cost-effective andconstitutes a rapid method for identifying and quanti-fying cytotoxic compounds.

Acknowledgements

This work has been financed by AccompanyingMeasures of the Environment and Climate Program(1994-1998, ENV4-CT97-6154 and ENV4-CT98-6137) and through WasteWater Cluster (Project SAN-

DRINE ENV-CT98-0801) and CICYT (AMB1999-1705-CE). We thank Dr. J. Büsing, from the EuropeanCommission, for stimulating discussions. We thankMerck and IST for supplying ToxAlert and SPEcartridges, respectively.

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identification of cytotoxic compounds in european wastewaters during a field experiment

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