Determination of selected persistent organic pollutants in wastewater from landfill leachates

Determination of selected persistent organic pollutants in wastewater from land?ll leachates,using an amperometric biosensor

Philiswa N.Nomngongo a ,J.Catherine Ngila a ,?,Titus A.M.Msagati a ,Bhekumuzi P.Gumbi b ,Emmanuel I.Iwuoha c

a

Department of Applied Chemistry,University of Johannesburg,PO Box 17011,Doornfontein 2028,Johannesburg,South Africa b

School of Chemistry,University of KwaZulu Natal,Westville Campus,P.Bag X54001,Durban 4000,South Africa c

Sensor Lab,Chemistry Department,University of the Western Cape,Bellville 7535,South Africa

a r t i c l e i n f o Article history:

Available online 19August 2012Keywords:

Amperometric biosensor Horseradish peroxidase Enzyme inhibition Leachate

Persistent organic pollutants

a b s t r a c t

Land?ll leachates that contain persistent organic pollutants (POPs)are a big threat to groundwater systems and are projected to have hazardous effects in the long term if proper management strategies of the land?lls are not put in place by those responsible.Monitoring the levels of POPs in land?ll leachates is very crucial.This work presents an amperometric biosensor for determination of selected POPs in land-?ll leachates.The biosensor is based on kinetic inhibition of horseradish peroxidase (HRP).The enzyme was immobilised by electrostatic attachment on a polyaniline-modi?ed Pt electrode surface.Selected POPs inhibited HRP enzyme activity and the decrease in the enzyme activity was used to determine these environmental pollutants.Selected polybrominated diphenyl ethers (PBDEs),polybrominated biphenyls (PBBs)and polychlorinated biphenyls (PCBs)were the analytes of choice because they are commonly found in South Africa water systems.Limits of detection for the amperometric biosensor were established as 0.014,0.018,0.022,0.016and 0.019l g l à1for BDE-100,PBB-1,PCB-1,PCB-28and PCB-101,respectively.The HRP biosensor system gave different linear ranges for;BDE-100(0.424–25.8l g l à1),PBB-1(0.862–13.4l g l à1),PCB-1(0.930–18.1l g l à1),PCB-28(0.730–15.7l g l à1)and PCB-101(0.930–27.1l g l à1).Inhibition studies on HRP biosensor response toward the reduction of H 2O 2in the absence and presence of the selected POPs were carried out to investigate the inhibition kinetics and its mecha-nism.The results obtained indicated that the inhibition mechanism was competitive for PBDEs and non-competitive for biphenyls (PCBs and PBBs).The application of the biosensor was tested on wastewa-ter samples obtained from land?ll leachate for determination of selected POPs.The leachate samples were found to contain PCB-28(0.28±0.03l g l à1)and PCB-101(0.31±0.02l g l à1).The samples were also analysed by GC–MS as a cross-check method and the two sets of results were in close agreement.

ó2012Elsevier Ltd.All rights reserved.

1.Introduction

Current management practice for land?ll leachate in South Africa prioritises the disposal rather than the actual treatment of land?ll leachate.In this regard,current disposal methods for raw leachate include evaporation,irrigation to available land,recirculation back to the land?ll,or disposal to an available sewer line.The develop-ment of a National Waste Management Strategy for South Africa in 1999(DEAT,1999)promulgated a paradigm shift of the land?ll-mind-set to consider end-of-pipe treatment (Joubert et al.,1999),as opposed to mere dilution principles.Tighter national legislation,more stringent disposal by-laws,an environmental demand for treatment-at-source,and the unavailability of the sewer-disposal option,now demand that on-site treatment of land?ll leachate be carried out.Additionally,it is by now widely known that the levels of persistent organic pollutants (POPs)in land?ll leachates can be hundred times higher than the levels found in domestic sewage.Persistent organic pollutants (POPs)in water pose a consider-able risk to the environment and human health even at extremely low concentrations.These pollutants are characterised by marked persistence against chemical or biological degradation,high envi-ronmental mobility and strong tendency for bioaccumulation in the food chain (Katsoyiannis and Samara,2004).Persistent organic pollutants consist of a wide range of compounds which are produced by industrial activities.These include polybrominated diphenyl ethers (PBDEs),polybrominated biphenyls (PBBs)and polychlorinated biphenyls (PCBs),among others.

In view of major concerns regarding the toxicity of PBDEs and PCBs,there is a growing need for innovative ways to monitor these environmental pollutants.It should noted that leachates are a big threat to groundwater systems and are projected to have hazardous

1474-7065/$-see front matter ó2012Elsevier Ltd.All rights reserved.https://www.360docs.net/doc/4e7957084.html,/10.1016/j.pce.2012.08.001

Corresponding author.Tel.:+27115596196;fax:+27115596425.

E-mail address:jcngila@uj.ac.za (J.Catherine Ngila).

effects in the long term if proper management strategies are not put in place by those responsible.Many methods have been proposed for the determination of POPs.The existing methods for detection of POPs utilise gas chromatography,GC(Vonderheide,2009)and high performance liquid chromatography,HPLC(Vonderheide,2009), both coupled to different types of detectors such as electron capture detector,ECD(Wang et al.,2006)or mass spectrometry,MS (Tadeo et al.,2009)for GC;and DAD/UV–Vis(Vilaplana et al., 2009)and MS(Bacaloni et al.,2009)for the HPLC method.Although the conventional chromatography-based methods have high accu-

racy and low detection limits,they are,however,sophisticated and require skilled operators.They also require a sample preparation step before detection,which is time consuming and tiresome.For this reason,rapid and simpler methods are required for analysis of these environmental pollutants.Electrochemical biosensors offer an alternative method.This is because electrochemical techniques are easy to use,require minimal sample preparation and are easily miniaturised.

During the past decade biosensors have been employed as useful monitoring devices in the environmental programmes (Rodriguez-Mozaz et al.,2006;Suwansa-Ard et al.,2005).This is due to the advantages they possess,such as minimising the sample pretreatment,reducing cost and time of analysis as well as display-ing suf?cient sensitivity and selectivity.Recently,attention has turned to the enzyme inhibition-based biosensors(Yang et al., 2008).The latter is used to determine the concentration of inhibi-tors in the sample by measuring the degree of inhibition with low-er limits of detection.Very few studies have explored the biosensor inhibition approach which offers an indirect method for detection of organic pollutants at trace levels to improve detection levels.

This work therefore aims to explore the application of the Pt/PANi/HRP biosensor according to the following objectives:

Enzyme kinetic determination of PBDEs,PBBs and PCBs.

The inhibitory effect of POPs on the electrochemical reduction of hydrogen peroxide(H2O2,substrate)by HRP biosensor.

Determination of POPs in land?ll leachate samples.

Cross-check results obtained from the biosensor with those from a gas chromatography–mass spectrometry(GC–MS) technique.

This study forms part of wastewater management through monitoring of the levels of organic substances in leachates dis-charged by land?lls.

2.Materials and methods

2.1.Reagents

All chemicals used in this work were of analytical grade unless otherwise stated.Dichloromethane(DCM),hexane methanol, 2,20,4,40,6-pentabromodiphenyl ether(BDE-100),octabromodiphe-nyl ether(octaBDE)mixture of isomers,2,4,40-trichlorobiphenyl (PCB-28),2,20,4,5,50-pentachlorobiphenyl(PCB-101),2-chlorobi-phenyl(PCB-1),2-bromobiphenyl(PBB-1)disodium hydrogen phosphate(dehydrated)(99%)and sodium dihydrogen phosphate (hydrated)(99%)were obtained from Sigma–Aldrich(South Africa) Ltd.Hydrogen peroxide(H2O2)(30%v/v)was obtained from Merck Chemical(Pty)Ltd.The H2O2(30%)stock solution was stored in a refrigerator at4°C.The H2O2working solutions were freshly prepared from the stock solution.Phosphate buffer solution (PBS,0.1M,pH7.0)was prepared by mixing appropriate amounts of0.1M NaH2PO4and0.1M Na2HPO4.The pH was adjusted with 0.1M NaH2PO4or0.1M Na2HPO4.The PBS was used as supporting electrolyte for electrochemical measurements.2.2.Instrumentation

All electrochemical experiments were performed using either BAS100W Electrochemical Analyser(Bioanalytical Systems,West Lafayette,IN)or BASi epsilon Electrochemical Analyser equipped with BASi cell stand(Bioanalytical Systems,West Lafayette,IN). A15ml electrochemical cell with a conventional three electrode system consisting of platinum electrode as the working electrode (A=0.0177cm2),platinum wire as the auxiliary electrode,and Ag/AgCl(saturated3M NaCl)electrode as the reference electrode. Supporting electrolyte solution was degassed with argon gas for electrochemical experiments.All measurements were performed at room temperature(20–25°C).All Fourier transform infrared (FT-IR)and ultraviolet–visible(UV–Vis)spectra were recorded on PerkinElmer Spectrum100FT-IR spectrometer equipped with Universal Attenuated Total Re?ectance(ATR)attachment with a diamond crystal and Perkin Elmer Lambda35UV–Vis spectrome-ter,respectively.

Gas chromatography–mass spectrometry analysis was per-formed on Thermo Finnigan Trace GC equipped with Thermo Finn-igan ion trap mass spectrometer detector Polaris-Q(Thermo Electron Corporation,Waltham,MA)equipped with autosampler. The separation of the analytes was achieved using fused silica SGE forte GC capillary column coated with BPX5(stationary phase 5%phenyl polysilphenylene-siloxane,0.25mm i.d.,0.25l m?lm thickness,30m length).The temperatures for the GC–MS interface and ion source were280°C and250°C,respectively.The mass spectrometer was operated in the electron ionisation(EI)mode at70eV and helium(?ow rate1ml minà1)was used as carrier gas.Full-scan data acquisition was performed over the mass range of m/z150–596.The GC oven temperature programme is presented in Table1.

2.3.Immobilisation of horseradish peroxidase

Horseradish peroxidase immobilisation by electrostatic attach-ment followed the steps described by Nomngongo et al.(2011) and Songa et al.(2009).The biosensor was made by depositing 50l l of2.0mg mlà1of horseradish peroxidase(HRP)on a PANI-modi?ed Pt electrode surface.Fig.1shows the schematic diagram for the preparation of the biosensor.

2.4.Spectrometric characterisation of PANi/HRP?lm

After enzyme immobilisation,the?lm was scraped gently from the platinum electrode surface.The ATR required not more than 2mm3of PANi/HRP?lm(to cover the ATR diamond window). The?lm,free HRP and PANi?lm were analysed by FT-IR-ATR spec-trometer.The?lm was then placed on the surface of the ATR crys-tal and the spectrum was recorded within the wave number range of400–4000cmà1.

The PANi/HRP?lm,free HRP and PANi?lm were dissolved in DMF–PBS mixture,PBS and DMF,respectively,and the solutions were analysed with UV–Vis spectrophotometry.The resulting Table1

Gas oven temperature program.

Initial temperature

(°C)

Ramp

(°C minà1)

End temperature

(°C)

Hold time

(min) 80–802

8050140 1.5

140202201

22022800

3003030010

P.N.Nomngongo et al./Physics and Chemistry of the Earth50–52(2012)252–261253

solutions were placed in a quartz cuvette of1cm path length.The UV–Vis spectrum was recorded from200to900nm.

2.5.Electrochemical response to hydrogen peroxide(H2O2)by the Pt/ PANi/HRP biosensor

Amperometric measurements were performed in stirred sys-tems by applying a potential ofà200mV to the Pt/PANi/HRP elec-trode(working electrode).Current–time data were recorded after steady-state current was reached.The difference between the stea-dy-state current(I ss)upon addition of H2O2,and the background current(I0),was reported as current I.After each experiment,the electrode was rinsed with double-distilled water and kept in the working buffer solution at4°C.

2.6.Determination of persistent organic pollutants

2.6.1.Procedure for POPs determination

An aliquot(200l l)of BDE-100stock solution(50mg là1in iso-octane)was?rst dissolved in200l l methanol,then9.8ml PBS was added with the resulting concentration as1.0mg là1.A concentra-tion of0.1mg là1was prepared by further diluting an aliquot of 10ml containing1.0mg là1,in a volumetric?ask.Amperometric measurements for enzyme inhibition by BDE-100were carried out in an electrochemical cell containing2.0ml of0.1M PBS(pH 7.02)and constant concentration H2O2(0.5mM)with continuous stirring.The experiments were carried out atà0.20V against Ag/AgCl(3M NaCl)while allowing the steady-state current to be attained.The desired volume of the inhibitor stock solution(10l of1.0mg là1BDE-100)was then added using a micropipette with continuous stirring until a steady-state current was obtained.After each experiment the enzyme electrode activity was regenerated by rinsing the electrode with double distilled water.Stock solutions (100mg là1)of PCB-1,PCB-28and PCB-101were prepared by dis-solving1.0mg of each compound in100l l methanol then diluting it to10ml in a volumetric?ask using PBS(0.1M,pH7).The stock solution(1000mg là1)of PBB-1was prepared by dissolving75l l in100l l of methanol then diluting it to100ml in a volumetric ?ask using PBS(0.1M,pH7).Working solutions of PCB-1,PCB-28,PCB-101and PBB-1were prepared by serial dilution of the stock solutions in PBS.

2.6.2.Inhibition studies

The inhibition mechanism was studied by investigating the relationship between the Pt/PANi/HRP response current to H2O2 (substrate)concentration in the absence and presence of the inhib-itor.The biosensor’s response to various H2O2concentrations in the presence of an inhibitor was done as follows:the HRP electrode was?rst incubated in PBS containing a known concentration (IC50)of each inhibitor for20min followed by the successive addi-tion of H2O2concentrations.

Selectivity of the biosensor is very important for the construc-tion and application of the biosensor in actual land?ll leachate samples.Amperometric measurements of Cu2+,Fe2+,Cd2+,Pb2+ and phenol as interferents were measured under the same exper-imental conditions.The effect of the interferents on the signal detection was investigated by adding0.5mg là1BDE-100or PCB-101in0.5mM H2O2in PBS,and different concentration levels of interferents.

2.6.

3.Analysis of real samples

Land?ll leachate samples were collected in polyethylene con-tainers from the Mariannhill Land?ll outside Durban(Ethekwini Municipality’s Durban Solid Waste,DSW),and stored in the fridge at4°C.Determination of POPs in the leachate samples was per-formed by two methods,namely amperometric biosensor and GC–MS.The latter was used as a cross-check for the results ob-tained from the fabricated amperometric biosensor.For ampero-metric analysis,determination of POPs in leachate water samples was done using standard addition method.The samples were?l-tered to remove solid particles and then spiked with0.1mg là1 of each standard solution using standard addition method,fol-lowed by amperometric measurements.

For GC–MS analysis,the land?ll leachate sample was?ltered to remove the solid particles.The organic compounds were extracted using C-18solid-phase extraction(SPE)cartridges.Prior to use,the cartridges were activated with5ml methanol followed by5ml water.After sample loading the SPE cartridges were rinsed with 5ml water.The organics were eluted with5ml hexane and the extract was concentrated to about2ml under nitrogen followed by GC–MS analysis.

3.Results and discussion

3.1.Spectrometric characterisation of PANi/HRP Film

The stability of HRP after immobilisation was investigated by FTIR-ATR and UV–Vis spectroscopy.The FTIR-ATR spectra of(A)free HRP,(B)PANI/HRP?lm and(C)PANI?lm are presented in Fig.2: In curve A the absorption bands at1642cmà1and1530cmà1 were assigned to the stretching of amide groups(I and II)of HRP,respectively(Songa et al.,2009;Sun et al.,2004,2007;

Ma et al.,2007).The amide I band(1700–1600cmà1)is due to C@O stretching vibrations of the peptide linkages.The amide II band(1620–1500cmà1)on the other hand results from a combination of N A H in plane bending and C A N stretching vibrations of the peptide groups(Sun et al.,2007;Ma et al., 2007).

254P.N.Nomngongo et al./Physics and Chemistry of the Earth50–52(2012)252–261

It can be observed from curve C that PANI had no absorption peaks at the absorption region of amide I and amide II bands as in the case of HRP and PANi/HRP.Hence,the IR peaks at 1643cm à1and 1533cm à1for PANi/HRP ?lm (curve B)are thus attributed to those of amide I and II bands of HRP,occurring at similar positions as in the free HRP.

The similarities of the two spectra of free HRP and PANi/HRP (curve A and B)showed that HRP retained the essential features of its secondary structure on the surface of Pt/PANI modi?ed electrode.Sun et al.(2004)reported that when HRP is dena-tured,it shows completely different spectral characteristics in the amide I and II regions.

UV–Vis spectroscopy is an effective method to investigate the characteristic structure of proteins (Yin et al.,2009).For this rea-

in the intense peak (Soret peak)of HRP UV–Vis spectroscopy.Fig.3shows UV–Vis spec-PANI (in DMF)and PANi/HRP (in PBS–DMF spectrum of free HRP in curve A shows a 400nm (Yin et al.,2009).The same was spectrum (curve B)at 404nm.The slight PANi/HRP may be due to the interaction HRP during immobilisation (Songa et al.,

2009).The UV–Vis spectrum (curve B)con?rms that the interac-tions of PANI ?lm and HRP do not destroy the structure of the enzyme (Songa et al.,2009).Therefore it can be concluded that the HRP was attached and retained its biological activity after immobilisation on the Pt/PANI modi?ed electrode (Songa et al.,2009;Yin et al.,2009).The UV–Vis spectrum of PANI in curve C does not have an absorption band at 400nm;this shows that the absorption band (404nm)observed in curve B is entirely due to the presence of HRP.

3.2.Amperometric detection of H 2O 2

The principle of an amperometric biosensor was based on the measurement of the current produced when H 2O 2is reduced by HRP at a constant applied potential.The possible mechanism of PANI-mediated HRP reduction of H 2O 2is presented in Fig.4.In the ?rst step of the mechanism,H 2O 2in solution diffuses to the surface of the ?lm where it is reduced by the immobilised HRP and the latter becomes oxidised to form HRP-I (also known as Compound-I).The latter in turn oxidises PANI to give HRP-II (Com-pound II).The HRP resting state,Fe-III is then regenerated via this intermediate of HRP-II,Compound-II.The partially oxidised PANI +(emeraldine)is then electrochemically reduced at the electrode to leucoemeraldine (fully reduced form)yielding an enhanced reduc-tion current (Liu and Ju,2002;Dhand et al.,2011;Iwuoha et al.,1997).The magnitude of the reduction current produced by the electrode reaction depends on the bulk concentration of the sub-strate,H 2O 2.

Amperometric responses of the Pt/PANi/HRP biosensor were investigated by consecutively increasing the concentration of H 2O 2at a working potential of à200mV.Fig.5presents a typical steady-state current–time plot obtained with the fabricated bio-sensor upon successive additions of 10l l of 0.01M H 2O 2into 2.0ml PBS resulting in a calibration plot,given as an inset.It was observed that,upon the addition of H 2O 2into the PBS,the reduc-tion current rises sharply to reach a steady-state value.In addition,the biosensor attained 95%steady-state current within 5s after each addition of 0.010M H 2O 2aliquot.This observation implied a fast response of the fabricated biosensor.

The biosensor’s responses (current signals)were linear to increasing concentration of H 2O 2in the range 0.05mM to 3.17mM with a correlation coef?cient of 0.9991(n =18);sensitiv-ity of 1.75l A mM à1;and a detection limit of 36.8nM (0.0368l M)(estimated as signal-to-noise ratio of 3).

Fig.2.FT-IR-ATR spectra of (A)free HRP;(B)PANi/HRP ?lm;and (C)PANI ?lm.

Fig.3.UV–Vis spectra of (A)free HRP in PBS;(B)PANi/HRP in PBS–DMF mixture;and (C)PANI in DMF.

selected brominated ?ame retardants and biphenyls in model solution

the typical amperometric Pt/PANi/HRP biosensor successive additions of 10l l of 0.01M H 2O 2(r )fol-successive additions of aliquots (l l)of BDE-100standard mg l à1)(i )in PBS.The terms ‘r ’and ‘i ’represents the response signal recorded after the addition of H 2O 2,and (decrease in signal)upon the addition of BDE-100,can be seen that after each addition of BDE-100,the decreased.The decrease in response current after BDE-100indicated that the latter inhibits the activ-the four POPs (PBB-1,PCB-1,PCB-28and PCB-101)in the range 0.1–44.2l g l à1.The linear ranges,lim-(LOD ?3?SD m

1),limits of quanti?cation (LOQ ?10?SD

m )regression coef?cients for each compound are presented the biosensor could be used to perform several for each analyte without showing a decline in cur-suggests that the type of inhibition is reversible as it in the supporting electrolyte in between the inhibitor measure-ments).Thus,the background current of the Pt/PANi/HRP biosen-sor was restored to the original value of PBS in the absence of BDE-100(Songa et al.,2009;Vidal et al.,2008).The response time of the fabricated biosensor was observed to reach 95%of its maximum response after about 5–7s.As shown in Table 2,the

fabricated Pt/PANi/HRP biosensor exhibits a long linear range,relatively high sensitivity and low LODs for all the tested POPs.Table 2shows that the biosensor had the highest sensitivity to-wards the determination of BDE-100in aqueous media.However,due to the high hydrophobicity of the octaBDE,no observable results were recorded.OctaBDE dissolves only in selected hydro-phobic organic solvents (DCM).There is therefore a need to devel-op an organic-phase biosensor for detection of highly hydrophobic https://www.360docs.net/doc/4e7957084.html,anic-phase biosensors are known to offer a favourable

mechanism of PANI-mediated HRP reduction of H 2O 2,where Fe 3+is the ferric HRP in resting state;Fe át

4t@O stands for oxyferryl hydroxyferryl HRP-II (Compound II);and PANI 0/+stands for leucoemeraldine/emeraldine cation radical redox couple (Iwuoha et al.,1997;Fig.5.Amperometric responses of Pt/PANi/HRP to successive additions of 10l 0.05mM of hydrogen peroxide (inset shows the calibration curve).Potential:à0.2V;supporting electrolyte:0.1M PBS (pH 7.02).

1

SD refers to the standard deviation of the blank signal (n =8)obtained in PBS (SD =4.8?10à5l A)m is the slope of the calibration curve.

environment for the detection of water-insoluble analytes and less interference from water-soluble analytes(Wu et al.,2004,2007). Since it was not possible to determine octaBDE in aqueous media, methanol was used as a supporting electrolyte(instead of PBS)for the electrochemical signal measurements.Electrocatalytic reduc-tion of H2O2was found to be very slow and the biosensor was not as stable as in aqueous media.The degree of stability was found to affect both reproducibility and repeatability.Since the biosensor showed the highest sensitivity to BDE-100and very low response to non-aqueous POps,it was concluded that in aque-ous media only hydrophilic PBDEs could be analysed using the fab-ricated biosensor.The determination of the octaBDE mixture of isomers was not successful.

3.3.1.Inhibition studies

The values of the steady-state current before(I0)and after(I i) the additions of an inhibitor were determined from the recorded amperograms similar to that presented in Fig.6.The inhibition per-centage(I%)was calculated as per Eq.(1).In order to obtain the inhibitor’s concentration that causes50%inhibition(IC50), 1.0mg là1was used instead of0.1mg là1.

I%?I0àI i

I0

?100%e1T

where I%is the inhibition percentage;I0is the steady state current before addition of an inhibitor;I i is the steady state current after addition of an inhibitor.

The I%values obtained were used to compare the inhibitory effect of POPs on immobilised HRP enzyme’s activity.A calibration curve constructed by plotting the inhibition percentage of HRP activity against the concentration of BDE-100is shown in Fig.7.

It was observed that the degree of inhibition increased with an

increase in concentration of an inhibitor.From Table3,it can be seen that the sequence of inhibition to HRP activity is as follows: BDE-100>PCB-101>PCB-28>PCB-1>PBB-1.The inhibition per-centages as well as the inhibitor concentration leading to50%inhi-bition(IC50)are presented in Table3.

3.3.2.Investigation of inhibition kinetics and inhibition mechanism

The biosensor’s response to various H2O2concentrations re-corded(Fig.8)in the absence and presence of BDE-100(curve A) were interpreted using the Lineweaver–Burk plots(curve B).In the absence of the0.407mg là1(IC50)BDE-100,a fast response to the additions of different concentrations of H2O2was observed whereas in the presence of0.407mg là1BDE-100,it was slow. The experiment was repeated for other POPs studied in this work and similar observations were made.Sariri et al.(2006a,2006b) and Zaton and De Aspuru,1995suggested that the inhibition of HRP activity by phenyl-containing compounds could be due to the incorporation of the phenyl group into enzyme molecules at the haem periphery thus leading to a decrease in HRP activity. The slope and the y-intercept values of the linear plots were used to calculate the kinetic parameters(apparent Michaelis–Menten constants K app

M

àá

and maximum current(I max).The K app

M

and I max values in the absence and presence of selected POPs are presented in Table4.

Enzyme kinetic parameters(K app

M

and I max)in Table4were used to estimate the inhibition mechanism of POPs to HRP activity.It can be observed from Table4that the presence of BDE-100had a

positive effect on the K app

M

value but the I max value remained unchanged(1.2l A)irrespective of the absence or presence of the inhibitor.This indicates that BDE-100bound to the same HRP ac-tive sites(causing decrease in the amount of free HRP)is available

for H2O2binding,thus increasing the K app

M

(Amine et al.,2006).

Based on these results it was concluded that the inhibition mechanism in this case is competitive inhibition.The latter occurs

Fig.6.Typical amperometric responses of Pt/PANi/HRP biosensor to successive

additions of0.01mM H2O2(r)and BDE-100(i);applied potential ofà0.20V;

supporting electrolyte of0.1M PBS(pH7.02);where‘r’and‘i’represents the

increasing response due to the addition of H2O2and inhibitory effect of BDE-100,

respectively.

Table2

A summary of analytical characteristics and regression parameters for calibration curves for determination of POPs.

POPs Linear range(ppb)Sensitivity(l A ppbà1)LOD(ppb)LOQ(ppb)R2

BDE-1000.424–25.89.38?10à3±0.0050.0140.0480.9983 OctaBDE Not detected––––PBB-10.862–13.47.56?10à3±0.0020.0180.0590.9991 PCB-10.930–18.9 6.24?10à3±0.0040.0220.0720.9966 PCB-280.730–15.78.29?10à3±0.0060.0160.0540.9939 PCB-1010.930–27.1 6.95?10à3±0.0050.0190.0630.9959

Fig.7.Calibration curve showing the inhibition of HRP activity by BDE-100.

when the inhibitor competes with the target substrate for the ac-tive binding sites of the enzyme.A possible inhibition mechanism is given in Fig.9suggesting that BDE-100(inhibitor,I)binds only to free enzyme(E)active sites rather than the enzyme–substrate(ES)

The inhibition constant(K i)is an absolute value dependent only on the inhibitor-enzyme af?nity.Generally,the smaller the K i va-lue,the tighter the binding,and hence the more effective an inhib-itor is.Apparent K i values for the competitive and non-competitive inhibitor were calculated from the relationship reported by Besom-bes et al.(1995)and Tanimoto de Albuquerque and Ferreira(2007) using Eqs.(2)and(3),respectively.

K i?

K app

M

?I

K0app

M

àK app

M

e2TK i?

I0

max

?I

I maxàI0

max

e3Twhere K0app and I0are Michaelis–Menten constant and maximum

Table3

Inhibition percentages and the inhibitor concentration leading to50%inhibition

(IC50).

POPs Degree of inhibition(%)IC50(ppm)

BDE-10053.2±1.90.407±0.291

PBB-150.8±0.970.487±0.132

PCB-151.7±1.30.531±0.154

PCB-2852.3±0.570.542±0.113

PCB-10152.8±1.20.506±0.511

Pt/PANi/HRP biosensor response to successive additions of H2O2in the absence and presence of BDE100,at applied potential ofà0.20V;(B)Lineweaver–Burke HRP response to H2O2in the absence and presence of BDE-100.

Table4

Apparent Michealis–Menten constants and maximum current in the absence and the presence of BDE-100(0.407ppm).

POPs Kinetic parameters

K app

M

(mM)I max(l A)

Absence of inhibitor Presence of inhibitor Absence of inhibitor Presence of inhibitor BDE-100 1.34±0.03 2.27±0.09 1.18±0.02 1.21±0.08 PBB-1 2.97±0.13 3.01±0.07 1.16±0.030.984±0.14 PCB-1 3.03±0.09 2.99±0.15 1.24±0.050.978±0.07 PCB-28 3.05±0.02 3.10±0.11 1.12±0.060.991±0.03 PCB-101 3.01±0.05 3.07±0.06 1.18±0.100.986±0.08

258P.N.Nomngongo et al./Physics and Chemistry of the Earth50–52(2012)252–261

show that phenols did not interfere with the biosensor response.However,Cu 2+,Fe 2+,Cd 2+and Pb 2+do inhibit the activity of HRP.3.5.Determination of selected brominated ?ame retardants (BFRs)and polychlorinated biphenyls (PCBs)in land?ll leachate samples 3.5.1.Pt/PANi/HRP biosensor

Land?ll leachate samples were analysed for POPs in order to demonstrate the applicability of the Pt/PAN/HRP biosensor.The leachate sample was ?rst ?ltered using 0.45l m pore size cellulose acetate ?lter in order to remove insoluble residues.Determination

of POPs in the leachate samples was done by employing the stan-dard addition method.The sample was spiked with a POP standard to obtain a ?nal concentration of 0.10mg l à1and analysed using the Pt/PAN/HRP biosensor.Amperometric measurements were car-ried out as discussed in Section 2.6.1,but for this experiment PBS contained 0.5mM H 2O 2instead of 0.95mM.The concentrations of the detected POPs (PCB-28and PCB-101)were calculated from the calibration curve.Their concentrations were found to be 0.28±0.03and 0.31±0.02l g l à1for PCB-28and PCB-101,respec-tively.The reported concentration values obtained for PCBs were found to be within the allowed maximum contaminant level (MCL)(0.5l g l à1)set by the United States Environmental Protec-tion Agency (Federal Register,1991).

3.5.2.Analysis of POPs by gas chromatography–mass spectrometry The validation of the results obtained by the proposed ampero-metric biosensor was performed using GC–MS as the cross-check standard method.The presence of the target analytes detected by GC–MS in the leachate sample was con?rmed by comparing their mass spectra to the National Institute of Standards and Tech-nology (NIST,MS Search 2.0)2002library.Full-scan Electron Ioni-sation spectra of components found in the leachate sample with

competitive enzyme inhibition.

E+P

Inhibitor

Substrate

non-competitive enzyme

inhibition.

Table 5

The K i values in nM for inhibition of HRP by POPs using H2O2as a substrate.POPs Inhibition constant,K i (nM)BDE-1000.983PBB-111.7PCB-110.5PCB-289.22PCB-101

7.88

P.N.Nomngongo et al./Physics and Chemistry of the Earth 50–52(2012)252–261259

fragmentation indicative of PCB-101,are presented in Fig.11.The mass spectra were assigned according to Safe and Hutzinger (1972),Medina et al.(2009)and Ramos et al.(2007).In the Elec-tron Ionisation spectrum an intense molecular ion(M+)is found at m/z326([C12H535Cl437Cl]+).A less intense molecular ion is observed at m/z324([C12H535Cl5]+).Loss of one(35Cl),two (2?35Cl,in the case of M+at m/z326and324)and four (3?35Cl and37Cl)chlorine atoms leads to formation of the major fragment ions at m/z291([C12H535Cl4]+),256([C12H535Cl237Cl]+), 254([C12H535Cl3]+)and184([C12H535Cl]+),respectively.

In the mass spectrum of PCB-28(Fig.12),the molecular ion was observed at m/z258([C12H735Cl237Cl]+),along with ions at m/z256 ([C12H735Cl3]+),186([C12H7Cl]+)and151([C12H7]+).The fragment ion at m/z186was due to the loss of two chlorine atoms (2?35Cl)(in the case of M+at m/z256)or35Cl37Cl(in the case of M+at m/z258)).The loss of three chlorine atoms(2?35Cl and 37Cl)led to the formation of the fragment ion at m/z151.The type of fragmentation obtained for both compounds was found to be in close agreement(100%)with the data given in the NIST library.

The results obtained with the proposed biosensor corroborated well with those obtained by GC–MS.Both methods recognised (detected)the presence of the two PCB congeners(PCB-28and PCB-101).However,it was necessary to spike the individual PCB cong-eners(each at a time)so as to differentiate their speci?c effects on the HRP activity.Notwithstanding this limitation,the Pt/PANi/HRP biosensor shows a high potential for routine analysis with high throughput for rapid monitoring of POPs in the environment.

4.Conclusions

The application of Pt/PANi/HRP biosensor for the determina-tion of selected POPs has been achieved by enzyme inhibition mechanism.The degree of inhibition was found to increase with the increase in concentration of the inhibitor.The percentage inhi-bition of the investigated inhibitors decreases in the following or-der:53.2%,52.8%,52.3%,51.7%and50.8%for BDE-100,PCB-101, PCB-28,PCB-1and PBB-1,respectively.The inhibition mechanism was found to be of the competitive type for PBDEs but non-com-petitive for PBBs and PCBs.Thus,the amperometric biosensor was fast,sensitive and showed good linear relationship as well as low limits of detection for the determination of selected POPs. Since the amperometric biosensor requires minimal sample prep-aration procedures,the Pt/PANi/HRP biosensor can be used as a preliminary screening method for different halogenated aromatic hydrocarbons before a detailed quantitative analysis is performed. Thereafter,those that give a positive response could then be re-analysed using the GC–MS method for con?rmation.This method could be very useful as a quick test for qualitative monitoring of POPs in wastewaters and leachates discharged by various land?lls. Thus the technique could be used as a management tool for deter-mining the quality of leachate(water)that could have a potential to be reused,hence an effort to integrate water resources.

Acknowledgement

Philiswa Nomngongo is grateful for NRF funding through grant-holder Prof E.I.Iwuoha of Sensor Lab at the University of Western Cape.The Authors wish to acknowledge?nancial support from the Organization for Prohibition of Chemical Weapons(OPCW). References

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