Ecotoxicology and Environmental Safety 83 (2012) 89–95

Contents lists available at SciVerse ScienceDirect

Ecotoxicology and Environmental Safety

0147-65

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Abbre

Fenton’

methyl;n Corr

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journal homepage: www.elsevier.com/locate/ecoenv

Use of duckweed (Lemna disperma) to assess the phytotoxicityof the products of Fenton oxidation of metsulfuron methyl

Javeed M. Abdul a, Anne Colville b, Richard Lim b, Saravanamuthu Vigneswaran a,n, Jaya Kandasamy a

a Faculty of Engineering and IT, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australiab Faculty of Science, School of the Environment, Centre for Environmental Sustainability, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia

a r t i c l e i n f o

Article history:

Received 15 March 2012

Received in revised form

13 June 2012

Accepted 15 June 2012Available online 8 July 2012

Keywords:

Advanced oxidation process (AOP)

Breakdown products

Toxicity

Bioassay

Lemna

Sulfonylurea herbicide

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.06.014

viations: AI, Active ingredients; AOPs, Advan

s oxidation; FR, Fenton’s reagent; HA, Humic

SU, Sulfonylurea herbicide; TEq, Toxic Equiv

esponding author. Fax: þ612 9514 2633.

ail address: s.vigneswaran@uts.edu.au (S. Vig

a b s t r a c t

Because of pressure on water supplies world-wide, there is increasing interest in methods of remediating

contaminated ground waters. However, with some remediation processes, the breakdown products are

more toxic than the original contaminant. Organic matter and salinity may also influence degradation

efficiency. This study tested the efficiency of Fenton oxidation in degrading the sulfonylurea herbicide

metsulfuron methyl (MeS), and tested the reaction products for phytotoxicity with the Lemna (duckweed)

bioassay. The efficiency of degradation by Fenton’s reagent (Fe2þ¼0.09 mM; H2O2¼1.76 mM, 4 h)

decreased with increasing initial MeS concentration, from 98% with 5 mg/L MeS, to 63% with 70 mg/L

MeS. Addition of NaCl (10 mM) and organic matter (humic acid at 0.2 and 2.0 mg C/L as Total Organic

Carbon) reduced the efficiency of degradation at low initial MeS concentrations (5 and 10 mg/L), but had

no effect at high concentrations. The residual Fenton’s reagent after Fenton’s oxidation was toxic to Lemna.

After removal of residual iron and H2O2, the measured toxicity to Lemna in the treated samples could be

explained by the concentrations of MeS as measured by HPLC/UV detection, so there was no evidence of

additional toxicity or amelioration due to the by-products or formulation materials.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

The drive to exploit ground water resources stems from persis-tent drought and scarcity of alternative water sources. However,not all ground water is suitable for use due to contamination fromdifferent sources such as industrial and domestic effluents, andagricultural runoff containing residual pesticides and fertilizers.Such waters must be remediated before use. This study examinedthe effectiveness of Fenton Oxidation as a method for removing theherbicide Metsulfuron methyl (MeS) from water.

To remove contaminants such as MeS from water, variousconventional remediation processes such as adsorption, coagula-tion/flocculation, ion-exchange, etc., have been used (Binnie andKimber, 2009). Recently, Advanced Oxidation Processes (AOPs)have gained popularity due to their ability to degrade refractory(not easily bio-degradable) contaminants with faster reaction rates(Huang et al., 1993). In contrast to the physical phase separationof contaminants in conventional processes, AOPs destroy thechemical structure of contaminants and often convert them intosmaller components that are less toxic than the parent compound.Ideally AOPs result in complete mineralization to simple inorganic

ll rights reserved.

ced Oxidation Processes; FO,

acid; MeS, Metsulfuron

alents

neswaran).

compounds such as carbon dioxide and water. However, if theoxidative degradation is incomplete, it can produce breakdownproducts that may be more toxic than the parent compound.Fernandez-Alba et al. (2002) studied the degradation of the pesti-cide methomyl by photocatalysis, and found that the solutionscould still show toxicity even after total disappearance of themethomyl. Zertal et al. (2001) found that phototransformation ofthe herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) atwavelengths shorter than 350 nm gave products that were moretoxic than the parent compound in the Microtoxs assay. Therefore,in testing the effectiveness of a cleanup method for a toxiccompound, it is not sufficient simply to measure the removal ofthe parent compound, but the breakdown products must also beconsidered. Separation and identification of all these breakdownproducts would be an immense task, and in the absence of toxicitydata for the products, will still not indicate whether the toxicity hasbeen removed. In addition, herbicides are usually applied as com-mercially formulated products, and the formulation materials, whilenominally inert, may also contribute to toxicity (Cox and Surgan,2006). The treatment reagents (in treatment processes) may also betoxic. Natural ground water also contains ions and organic mattersuch as humic acids, which may interfere with the FO process (Farreet al., 2007), or may influence the toxicity of the MeS (Gensemeret al., 1998).

Bioassays, in which the final treated product is tested for toxicityto living organisms, measure the net toxicity of residual parentcompounds and their breakdown products plus any toxicity from

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–9590

the formulation materials and reagents used in the treatmentprocess, minus any ameliorating factors in the final treated product.Bioassays, therefore, account for all these sources of toxicity andgive a functional evaluation of the efficacy of the process, withoutthe necessity for separation, identification and toxicity testing of allthe intermediates.

1.1. Fenton’s process

The more common AOP processes use hydrogen peroxide(H2O2), ozone (O3), oxygen (O2) and UV light as bulk oxidants. Acommon species generated in all AOP processes is the hydroxylradical (OH*). OH* radicals are powerful oxidizing agents with areduction potential of 2.73 V, which is second only to fluorine F2

(2.87 V) in the oxidation reduction potential table (Neyens andBaeyens, 2003). OH* radicals react rapidly with most organiccompounds, either by addition to a double bond or by abstractionof a hydrogen atom.

H2O2 is a strong oxidant capable of degrading a wide range oforganic contaminants in water treatment. When H2O2 is usedalone, the reaction rates are low at concentrations usually appliedin water treatment. However, when combined with catalysts suchas transition metals (iron) or UV light, the reaction rates areconsiderably higher. When the catalyst is ferrous iron (Fe2þ) andthe oxidant is H2O2 the AOP is called Fenton’s process or Fenton’soxidation (FO), named after its inventor (H.J.H. Fenton), and theFe2þ/H2O2 combination is called Fenton’s Reagent (FR) (Fenton,1894). The degradation or decomposition products of FR, i.e., H2Oand O2, are non-toxic, which is an added advantage especially forin-situ applications.

The generation of OH* during FO at pH o3 is shown in Eq. 1.

Fe2þþH2O2-Fe3þ

þOH*þOH� (1)

The rate constant for the generation of OH* is 63 M/s (Abdulet al., 2012; Kang et al., 2002). The Fe2þ initiates and catalyses thedecomposition of H2O2, producing hydroxyl radicals (OHn) and, ifit is oxidized to ferric iron (Fe3þ), this is called the chain initiationstep. The Fe3þ reacts with H2O2 and is reduced to Fe2þ (Eq. 2).

Fe3þþH2O2-Fe2þ

þHþþHO2n (2)

Table 1Properties of metsulfuron methyl (MeS) (adapted from Russell et al., 2002).

Chemical structure

Aromatic ring

Aromatic ring

Sulfonylureabridge

Triazine moiety

Sulfonylurea bridge Triazine m

The rate constant for above equation is 0.01 M/s (Duesterbergand Waite, 2006). The oxidation and reduction of Fe2þ (Eqs. 1and 2) generates a continuous stream of OHn radicals. Oxidationand reduction of Fe2þ (and the production of hydroxyl radicals)occur as long as the residual concentration of H2O2 can sustainthe reaction. Even though FO involves a number of reactions, thereactions shown in Eqs. (1) and (2) are generally considered to bethe key reactions of FO. Fe2þ can also react with hydroxyl radicals(which is called scavenging of OHn) as shown in Eq. (3) (Neyensand Baeyens, 2003).

OHnþFe2þ-OH�þFe3þ (3)

The rate constant for above equation is 3.2�108 M/s (Duesterbergand Waite, 2006).

There is also evidence that higher valence compounds of Fe suchas the ferryl ion (FeIVO2þ) may also be formed in the reactionbetween Fe2þ and H2O2, and contribute to the oxidation of organiccompounds during Fenton oxidation via a non-free-radical pathway(Bossmann et al., 1998; Gallard and Laat, 2000). Hug and Leupin(2003) proposed that in the iron-catalyzed oxidation of arsenic byH2O2, OHn radicals were produced at low pH, but at neutral pH themajor oxidant was another species, possibly Fe(IV).

1.2. Sulfonyl urea herbicides

Metsulfuron methyl (MeS), a sulfonylurea (SU) herbicide, wasselected as the test compound for this study; its characteristicsare given in Table 1. In Australia, MeS is marketed by Du Pont asthe formulated product ‘Brushoff’. SUs are a group of herbicideswith around 27 Active Ingredients (AI), used around the world(Russell et al., 2002). However, because of their high watersolubility, low rate of binding to organic matter, and long half-life, particularly in alkaline conditions, there is potential for themto leach into surface and ground water (EXTOXNET, 1996; Sarmahet al., 2000), and hence they frequently contaminate ground andsurface waters worldwide (Barbash et al., 2001; Battaglin et al.,2000; Fletcher et al., 1993).

A number of intermediate products have been identifiedduring AOP degradation of SUs. Rafqah et al. studied degradationof MeS by photocatalysis with TiO2 (Rafqah et al., 2005) anddecatungstate (Rafqah et al., 2008). They proposed a mechanism

Major crop Range of user rates

(g/ha)

pKa

(25 1C)

Koc

(mL/g)

Field half life

(days)

oiety

Cereals, rice 3–7.5 3.3 35 4–71

Vegetables 14–168

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–95 91

involving attacks by hydroxyl radicals on three main targets inthe molecule (see Table 1 for structure): the benzene ring,resulting in several hydroxylated isomers; scission of the sulfonylbridge, producing 2-amino-4-methoxy-6-methyl-1,3,5-triazineand 2-(carbomethoxy)benzene-sulfonamide; and demethylationof the methoxy group on the triazine ring. The triazine moiety wasdegraded to form cyanuric acid, which was resistant to furtherbreakdown under these conditions. Vulliet et al. (2002) found thatphotocatalysis of two other SU herbicides, cinosulfuron and tria-sulfuron, also proceeded to cyanuric acid, and they proposed adegradation pathway by processes similar to that of Rafqah et al. forMeS. Sleiman et al. (2007) followed the degradation of the SUIodosulfuron by photocatalysis with UV and TiO2, and identified upto 20 degradation products. As in the earlier studies, cyanuric acidwas the final product formed. Cyanuric acid has low toxicity toanimals (Hammond et al., 1986); there seem to be no data availablefor toxicity to plants. There are, however, some studies of environ-mental metabolites of MeS that showed low levels of toxicity toaquatic plants (European Commission, 2000).

No studies have been found that examined FO of MeS. Rafqahet al. (2004) studied the degradation of MeS by photoexcitation ofiron(III) aqua complexes, a process involving the formation ofhydroxyl radicals. Several photo products were formed via hydro-xylation, demethylation, sulfonylurea bridge cleavage and tria-zine ring opening processes, leading ultimately to completemineralization of the solution. There are, therefore, many poten-tial intermediates in the FO degradation process, but little or notoxicological data are available to assess whether these present arisk to plants.

1.3. Outline of study

The first part of this study examined the effects of varying[Fe2þ] and [H2O2] on the efficiency of FO of MeS, to determine theappropriate concentrations to produce partial degradation pro-ducts for toxicity evaluation. The toxicity of the FR was investi-gated with and without treatment to remove residual Fe2þ andH2O2. Finally, the study examined the efficiency of FO of MeS at arange of initial concentrations, with and without added NaCl andorganic matter (humic acid, HA), and measured the toxicity of theoxidized solutions to Lemna disperma. The toxicity, determined asToxic Equivalents (TEq) of MeS quantified by the Lemna bioassay,was compared with the concentration of residual MeS measuredby chemical assay with HPLC, to determine whether all toxicitycould be explained by the MeS concentrations, or the toxicitywas higher than expected (suggesting toxicity due to breakdownproducts or reagents), or lower (suggesting some amelioratingfactors).

2. Materials and methods

Reagents: The formulated product Brushoffs (DuPont), nominally 60% MeS,

was used as the source of MeS. All masses of metsulfuron methyl are reported in

terms of AI. Fe2SO4.7H2O (AR grade), H2O2 (AR grade 50%) and Catalase (bovine

liver powder, 2000–5000 unit/mg protein), were obtained from Sigma Aldrich.

Humic acid was obtained from Fluka Chemicals. All other chemicals used were

Analytical Reagent grade.

2.1. Experimental

2.1.1. Evaluation of conditions for advanced oxidation

Advanced oxidation of MeS was studied using FR (FeSO4.7H2O/H2O2). The FO

experiments were performed at room temperature (22 to 24 1C) in amber colored

bottles to avoid any photo-oxidation. The samples were stirred on a multiple stirring

plate. The solution of MeS (10 mg AI/L; 300 mL) was added to bottles containing

different quantities of Fe2þ (as FeSO4.7H2O). The reaction time started at the moment

when H2O2 was added. The pH was adjusted to 2.5 by adding dilute H2SO4 and NaOH

(1 M). H2O2 was then added. Samples (10 mL) were drawn and the reaction stopped

by rapid pH adjustment and catalase treatment as described in Section 2.1.2. The

residual concentration of MeS was determined by HPLC (Jasco PU 2089) and TOC

(Multi N/C 200 analyzer, Analytica Jena AG). The effects of varying Fe2þ (0.045, 0.09,

0.18 and 0.27 mM) with 1.76 mM H2O2, and H2O2 (1.76, 6.6 and 13.2 mM) with

0.18 mM Fe2þ , were assessed over a reaction time of 4 h. These results were used to

determine an appropriate reaction time and concentration of FR for the bioassays.

2.1.2. Toxicity of FR in the absence of MeS

Samples (300 mL) of Milli Q water were treated with FR in the ratios of 0.9 mM

Fe2þ/13.2 mM H2O2, and 1.8 mM Fe2þ/13.2 mM H2O2 for 4 h. Following Fenton

oxidation, samples were either simply adjusted to pH 7, or treated to remove

residual Fe and H2O2, before toxicity testing. This treatment involved adjusting the

sample to pH 9 with NaOH and filtering to remove the Fe oxide precipitates, and

then readjusting to pH 7 and treating with catalase (1 mL of 1% catalase solution)

to remove residual H2O2. Samples were then tested for toxicity with the Lemna

bioassay. The EC50 (concentration of the test solution that will reduce growth in

the Lemna by 50% compared with that of the control) values of FeSO4, Na2SO4 and

H2O2 to Lemna were also determined, to assess whether these components of the

Fenton reagent might contribute to toxicity.

2.1.3. Effect of different [MeS]0 concentrations, and added NaCl and organic matter

(humic acid), on Fenton oxidation of MeS.

The oxidizing efficiency of FR, and the toxicity of the products, were studied

over an [MeS]0 concentration range of 5–70 mg/L at [Fe2þ]0 concentration of

0.09 mM and [H2O2]0 concentration of 6.6 mM. Because the detection limits of the

HPLC analysis of MeS were higher than the detection limit for toxicity of the MeS

to Lemna (EC50¼2 mg/L), it was not possible to measure chemically when

the parent MeS had completely disappeared leaving only reaction products. The

reaction conditions were therefore chosen so the concentration of MeS would be

high enough for HPLC analysis, but the majority of MeS had been degraded by the

Fenton oxidation to maximize the amount of degradation products present.

The effect of ions (NaCl, 10 mM) plus organic matter (humic acid, HA, at

0.2 and 2 mg C/L as TOC) on the degradation efficiency was investigated over an

[MeS]0 concentration range of 5–80 mg/L, with [Fe2þ]0 of 0.09 mM and [H2O2]0 of

6.6 mM. Samples for HPLC/UV analysis of MeS and toxicity testing were collected

after a 4-h reaction time. The samples were treated to remove the Fe and H2O2 as

described in Section 2.1.2, before analysis as described below (Section 2.2.1).

2.2. Analytical methods:

2.2.1. MeS determination by HPLC/UV:

The samples for analysis by HPLC were pre-treated to remove Fe3þ and

residual H2O2 as described in Section 2.1.2. There is strong interference of FR and

catalase (used to quench the residual H2O2) in the measurement of MeS with UV

spectrophotometry at 232 nm (l max for MeS). Thus, an HPLC method was

developed to analyze the residual MeS in the effluent samples oxidized by FR. An

HPLC (Jasco PU—2089 with auto sampler 2055 plus) equipped with UV and

fluorescence detectors was used for this analysis. The aqueous samples were

analyzed directly without any solid phase extraction. The separation of MeS was

performed isocratically on a Luna PFP 5m C18 reversed-phase column 15 cm in

length (purchased from Phenomenex, Australia), with a pre-mixed solution of 45%

H2O and 55% acetonitrile (v/v) and 0.1% formic acid (HCOOH) as the mobile phase.

The absorbance was measured at 232 nm. The retention time of MeS was 3.6 min.

2.3. Phytotoxicity estimation by the Lemna bioassay

2.3.1. Lemna culture

Stock cultures of L. disperma were maintained under sterile conditions in 500-mL

Erlenmeyer flasks containing 200 mL sterile SIS Lemna growth medium, prepared

according to the OECD protocol (OECD/OCDE, 2006). The concentrations of nutrients

added per litre were: NaNO3, 85 mg/L; KH2PO4, 13.4 mg/L, MgSO4.7H2O, 75 mg/L;

CaCl2.2H2O, 36 mg/L; Na2CO3, 20 mg/L; H3BO3, 1 mg/L, MnCl2.4H2O, 0.2 mg/L;

Na2MoO4.2H2O, 0.01 mg/L; ZnSO4.7H2O, 0.05 mg/L; CuSO4.5H2O, 0.005 mg/L; Co(N-

O3)2.6H2O, 0.01 mg/L; FeCl3.6H2O, 0.84 mg/L; Na2-EDTA.2H2O, 1.4 mg/L; MOPS (3-[N-

morpholino]propane-sulfonic acid,), 490 mg/L. The medium was adjusted to pH

7.0þ/�0.1 with 0.1 M HCl or 0.1 M NaOH if necessary, and filter-sterilized (0.2 mm

pore size, mixed cellulose esters membrane). The flasks were placed on white

polypropylene trays under daylight-type fluorescent lights at an intensity of

70–80 mmoles photons/s/m2, at a temperature of 2472 1C.

2.3.2. Lemna bioassays

Lemna bioassays were carried out by a method modified from (OECD/OCDE,

2006) and the methods used by CSIRO (Monique Binet, pers. comm.). Samples

were diluted with ultrapure water if necessary to bring them within a suitable

toxicity range, nutrients were added in the concentrations given in Section 2.3.1,

and the pH was adjusted to 7.0þ/� 0.1 with 0.1 M NaOH or 0.1 M HCl. A two-fold

serial dilution of the sample/nutrient mixture was then prepared by dilution with

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–9592

Lemna growth medium. A control solution of Lemna growth medium, and

standards of MeS in Lemna medium at nominal MeS concentrations of 12 mg/L,

6 mg/L, 3 mg/L, 1.5 mg/L, 0.75 mg/L, 0.325 mg/L, were tested in parallel with the test

samples.

Ten-mL aliquots of the test solutions were placed in 20-mL clear glass

scintillation vials. Four replicate vials were prepared from each test dilution, and

single colonies of Lemna, each with three fronds, were selected from the stock

cultures and randomly allocated to the treatment vials. The vials were covered with

cling film with two small holes punched into the film to limit evaporation while still

ensuring that air could circulate. The vials were placed on white polypropylene trays

under the same conditions as the stock cultures (Section 2.3.1). The number of

fronds in each vial was recorded on days 0 and 7. The pH, temperature, conductivity

and dissolved oxygen (DO) of all test solutions were measured before pH adjust-

ment, after pH adjustment at the beginning, and on termination of the experiment.

All tests met the acceptability criteria: seven divisions per initial frond in the

controls (21 fronds per vial) over the week’s exposure, and temperature maintained

between 2472 1C. The pH and conductivity of the solutions changed as expected

due to growth (nutrient uptake) and metabolism (pH changes due to respiratory and

photosynthetic activity) of the Lemna, but the physicochemical conditions were

always within acceptable ranges for growth.

2.3.3. Calculations and statistical analysis

In the Lemna bioassays, the toxicity of each solution was estimated as Toxic

Equivalents (TEqs): the calculated concentration of MeS that would produce

reduction in Lemna growth equal to the observed value in the sample. Growth

was determined as the increase in fronds over seven days. Toxic equivalents were

calculated using an adaptation of the method described in Leusch et al. (2006). For

each vial, the increase in number of fronds was determined, and expressed as a

proportion of the increase in number of fronds in the control. The EC50 values (the

amount of sample or standard that will reduce growth in the Lemna by 50%

compared with growth in the control) were calculated using a 4-parameter

logistic equation (Eq. 4), which was fitted using the Solver function in Microsoft

Excel; the fit was checked visually.

y¼minþ(max�min)/(1þ10exp((logEC50� logX) n slope)) (4)

where min¼0 (the minimum value of the standard sigmoid curve where no

growth occurred), max¼1 (the maximum value for the controls in the standard

sigmoid curve, EC50 is the concentration (for the standard curve) or the volume of

sample (for test samples) giving 0.5 of the maximum control growth, and slope is

the slope of the curve at the point of inflection.

For samples, the EC50 value was calculated as the volume of sample causing 50%

inhibition of growth in the Lemna bioassay, and for standards, the EC50 value was

calculated as the mass of MeS causing 50% inhibition (Fig. 1). The EC50 value of the

MeS standard was divided by the EC50 value of the sample to calculate the toxicity

of the samples as TEqs as ng MeS/mL sample (¼mg MeS/L). Note that this does not

indicate that the cause of the toxicity was due solely to MeS; it is simply a

calculation of what concentration of MeS would produce an equivalent toxicity to

the sample.

Samples with sufficient toxicity to determine an EC50 value could be

quantified. The quantification limit therefore equaled the MeS EC50 value, which

averaged 1.92 (SD 0.26) mg MeS/L. Samples with toxicity sufficient to produce a

statistically significant reduction in Lemna growth, but less than 50% reduction,

were reported as Below Quantification Limits. Samples that were not significantly

different from the controls (p40.05) were reported as Below Detection Limits.

The TEq and MeS concentrations were compared by Paired samples t-tests, using

the statistical package SPSS V 17.

3. Results and discussion

3.1. Evaluation of conditions for advanced oxidation

Preliminary experiments demonstrated that FR at concentrationsof 0.45 mM Fe2þ and 1.8 mM H2O2 could completely degrade 10 mg/L MeS over 4 h, resulting in no detectable toxicity or measurable MeSby HPLC/UV). To determine the required FR concentrations to givepartial removal of the parent MeS, the effects of Fe2þ and H2O2

concentrations on the degradation of MeS were examined in trials inwhich 1) the initial concentrations of Fe2þ ([Fe2þ]0) were varied at aconstant initial H2O2 concentration [H2O2]0, and 2) the [H2O2]0

concentrations were varied at a constant [Fe2þ]0. The residual MeSover time was measured by HPLC.

3.1.1. Effect of [Fe2þ]0

The effect of concentration of [Fe2þ]0 on the degradation ofMeS (10 mg/L) at an initial [H2O2]0 concentration of 1.76 mM isshown in Fig. 2a. The degradation rate (percentage breakdown) ofMeS was a function of [Fe2þ]0. This implies that the [Fe2þ]0

establishes the extent of decomposition of the oxidant (H2O2) andthe production of hydroxyl radicals (Eq. 1) that are likely involvedin MeS degradation. The concentration of FR is critical for efficientdegradation of organics in the Fenton’s process.

At [Fe2þ]0¼0.045 mM the degradation of MeS was 40% att¼5 min and the remaining 60% of MeS was degraded in 4 h,whereas with [Feþ2]0¼0.18, the degradation of mM was 80% att¼5 min and it took 1 h for the degradation to reach more than95% (Fig. 2). There was a slight increase (o5%) in the % break-down when [Fe2þ]0 was increased from 0.18 to 0.27 mM. There-fore, 0.18 mM [Fe2þ]0 was used to determine the effects of H2O2

(Section 3.1.2).

3.1.2. Effect of [H2O2]0

The effect of [H2O2]0 on the degradation of MeS (10 mg/L) wasinvestigated at [Fe2þ]0¼0.18 mM and at [H2O2]0 concentrationsof 1.76, 6.6 and 13.2 mM. The percentage of MeS removal isshown in Fig. 2b. The degradation of MeS increased slightly withincreasing [H2O2]0 concentrations. At t¼15 min, the degradationof MeS with [H2O2]0¼1.76, 6.6 and 13.2 mM was 93.5%, 96% and98.5%, respectively. An increase in [H2O2]0 concentration by7.5 times (from 1.76 mg/L to 13.2 mM) increased MeS removalby only 3% (from 96 to 99%) at t¼30 min. The difference indegradation of MeS decreased at longer reaction times and wasnegligible at 60 min. Therefore, the effect of [H2O2]0 on thedegradation of MeS was not as critical as [Fe2þ]0. Catalkaya andKargi (2009), in their study of advanced oxidation and miner-alization of the herbicide simazine by FR, also found that [Fe2þ]0

had a greater effect on the degradation rate (percentage break-down) than the [H2O2]0.

The Fenton degradation of MeS for the toxicity assays wascarried out with [Fe2þ]0¼0.09 mM and [H2O2]0¼6.6 mM, whichgave approximately 99% degradation after 4 h. Under theseconditions the ratio of breakdown products to residual parentcompound is high, and since the parent compound was not fullydegraded, it was likely that the breakdown products were also notfully degraded, maximizing the probability of detecting any toxicbreakdown products.

3.2. Toxicity of FR in the absence of MeS

Blank solutions (no MeS) treated with FR, followed by pHadjustment to pH 7.5, showed toxicity up to 10–15 times theEC50 value of MeS (Fig. 3, pH adjusted only). There was a heavyprecipitate of iron oxides, and the dissolved oxygen in some of thetest solutions was also high (up to 125% saturation) at thebeginning of the test, and still above 100% saturation at the endof the test), although the toxicity did not consistently correlatewith the initial iron or peroxide concentrations, or dissolved oxygenlevels. When the treated blank solutions were adjusted to pH 9 andfiltered to remove iron precipitates, then readjusted to pH 7 andtreated with catalase to remove residual H2O2, the toxicity wasreduced to below detection limits (Fig. 3, Filtration/catalase treated).Na2SO4 was not toxic to Lemna at concentrations up to 3.6 mM,so sulfate is unlikely to contribute to the toxicity. The EC50 values ofFeSO4 and H2O2 to Lemna were 1.8 mM and o3 mM, respectively,so both these compounds could produce some toxicity, but the freeradicals and oxidizing species such as ferryl ions produced duringFO are also likely to be highly toxic to Lemna (Arora et al., 2002).It seemed probable that all of these factors contributed in varying

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.5 1 1.5 2 2.5

Pro

port

ion

of c

ontr

ol m

ax

log MeS (ng)

Standard EC50

0.0

0.2

0.4

0.6

0.8

1.0

1.2

-1.6 -1.2 -0.8 -0.4 0

Pro

port

ion

of c

ontr

ol m

ax

log sample volume (mL)

Sample EC50

Fig. 1. Method of quantifying Toxic Equivalents (TEq) of MeS. (a) A standard MeS curve, showing growth plotted as the proportion of the maximum increase in frond

number in the control sample, against ng MeS in the test vial. (b) A sample response curve, showing growth plotted as the proportion of the maximum increase in frond

number in the control sample, against volume of sample in the test vial. The TEq of the sample is calculated by dividing the standard EC50 (ng MeS causing 50% decrease in

growth) by the sample EC50 (mL of sample causing 50% decrease in growth), to give the TEq in ng/mL (¼mg/L) MeS equivalents (modified from Leusch et al., 2006).

Fig. 2. Effect of H2O2 and Fe2þ on MeS degradation.

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–95 93

degrees to the toxicity of the FR, either by direct toxicity of Fespecies, H2O2 or free radicals, or indirectly by adsorption or pre-cipitation of nutrients such as phosphate from the culture medium.In response to these results, subsequent samples for bioassays weretreated with filtration and catalase addition to remove toxicity dueto residual Fenton reagents.

3.3. Effect of different [MeS]0 concentrations, and added organic

matter and NaCl, on Fenton oxidation of MeS

Over an [MeS]0 concentration range of 5–70 mg/L, with[Fe2þ]0 concentration of 0.09. mM and [H2O2]0 of 6.6 mM, thedegradation of MeS decreased with increasing [MeS]0, while therewas a corresponding increase in the toxicity of the final solution(Fig. 4). FR was very effective in MeS degradation since smallquantities of FR (Fe¼0.09 mM: and H2O2 6.6 mM) were sufficientto degrade up to 10 mg/L MeS by more than 97% in 4 h. Thedegradation at higher [MeS]0, i.e., 40 and 70 mg/L, was delayed,and the efficiency decreased to 73% and 63%, respectively.

The oxidizing efficiency of FR ([Fe2þ]0¼0.09 mM and [H2O2]0¼

6.6 mM) was also studied over an [MeS]0 concentration range of5–80 mg/L, with addition of 10 mM NaCl and organic matter(Humic Acid, HA) at 0.2 and 2 mg C/L. These HA concentrationsbracket the median concentration (0.78 mg/L) of dissolvedorganic carbon detected in groundwater in a study of 28 aquifersin New South Wales, Australia (Lategan et al., 2010). At 10 mMNaCl, the water had a measured conductivity of approximately1100 mS/cm. Water with this conductivity is classified as low-to-moderate salinity, and would be suitable as drinking water forlivestock, and irrigation water for many crops except thoseregarded as sensitive to salinity (DERM, 2011). These additionsof NaCl and HA therefore represent environmentally-relevantconcentrations for groundwater.

At low [MeS]0 (5 mg/L and 10 mg/L), addition of 10 mM NaCland either 0.2 or 2 mg C/L HA reduced the efficiency (percentagedegradation) of the FR by 7–14% after 4 h compared with thetreatments without NaCl or HA, resulting in a 5–7-fold increase inthe measured residual MeS (Fig. 5). Humic acid has been found toeither increase (Fan et al., 2011; Vione et al., 2004) or decrease(Wang and Lemley, 2004) the rate of Fenton degradation ofcontaminants, but in our study there was no dose-response effectassociated with the different HA concentrations, so the effectmust be due to the addition of the NaCl. Chloride has been shownto inhibit Fenton oxidation of organic compounds (Aplin and Waite,2000; Lipczynska-Kochany et al., 1995; Lu et al., 2005 , 1997).The inhibition is attributed to two mechanisms: scavenging theOHn radicals to form less reactive Cl2n

� radicals, and the forma-tion of complexes with Fe3þ ions preventing them from reacting

with H2O2 and regenerating the Fe2þ (Eq. 2) (De Laat and Le,2006; Lu et al., 2005). Groundwaters are frequently saline, andinteractions with chloride ions may interfere with the effective-ness of FR in remediating contaminated saline waters. It was not

0

5

10

15

20

25

30

35

Fe 0.9/H2O2 13.2

Fe 1.8/H2O2 13.2

TE

q (a

s µg

/L M

eS)

Fe/H2O2 (mM)

pH adj. onlyFiltration/catalase

BDL BDLMeS EC50

Fig. 3. Toxicity of Fenton oxidation reagents to Lemna. Blank samples were treated

with FR (Fe/H2O2), then either adjusted to pH 7 (pH adj. only), or adjusted to pH 9,

filtered, readjusted to pH 7 and treated with catalase (filtration/catalase), before

Lemna assay. BDL¼Below detection limits.

0.001

0.01

0.1

1

10

100

0

20

40

60

80

100

120

5 10 20 40 70

TE

q (m

g M

eS/L

)

% M

eS r

emov

ed

Initial MeS conc. (mg/L)

% degradation

TEq (mg MeS/L) EC50 MeS0.002mg/L

Fig. 4. Effect of initial MeS concentration on the efficiency of Fenton oxidation

after 4 h. The % degradation of MeS is plotted against the left y axis, and the

toxicity of the final solution against the right axis. For comparison, the EC50 for

MeS (0.002 mg/L) is indicated (arrow). [Fe2þ]0¼0.18 mM, [H2O2]0¼6.6 mM.

1

10

100

1000

10000

100000

5 10 20 40 70 5 10 20 40 80 5 10 20 40 80

Con

cent

ratio

n (µ

g/L

)

Initial MeS (mg/L)

MeS (HPLC)

TEq (Lemna)

OM = 0 mg/L OM = 0.2 mg/L OM = 2 mg/L+ 10mM NaCl + 10mM NaCl

Fig. 5. Comparison of measured concentration of MeS by HPLC analysis with Toxic

Equivalents (TEq) estimated by the Lemna bioassay. Fenton reagent: [Fe2þ]0

0.18 mM, [H2O2]0¼6.6 mM for 4 h. OM¼Organic Matter (humic acid) as TOC.

Control reactions (no MeS) showed no toxicity (data not shown). Error bars on the

bioassay results represent the standard deviation of 4 replicates.

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–9594

surprising that HA at 0.2 and 2 mg C/L had no effect, because theconcentrations of MeS used were very high to maximize theconcentrations of potential breakdown products. At more envir-onmentally-realistic levels of MeS, organic matter may competewith the MeS and reduce the degradation efficiency.

The efficiency of FO decreased with increasing [MeS]0, but for[MeS]0410 mg/L addition of NaCl/HA did not cause any furtherdecrease in efficiency, and the residual [MeS] was similar intreatments with and without NaCl/HA (Fig. 5). The reason forthe reduced effect of NaCl/HA at higher [MeS]0 is unclear. TheMeS at higher concentration may be able to out-compete thechloride for the available OHn. We also speculate that there maybe inhibitory agents in the components of the formulationmaterials in the Brushoffs, or inhibitory breakdown productsof the MeS, that are sufficiently active to mask the effects ofthe relatively low concentrations of NaCl and HA. The formu-lation components are a commercial secret, but many formula-tions contain phosphate ester surfactants, and phosphate isstrongly inhibitory of Fenton oxidation of organic compounds(Lipczynska-Kochany et al., 1995; Lu et al., 1997). Further work,beyond the scope of this study, is required to investigatethis issue.

To determine whether the toxicity in partially-oxidized sam-ples was due only to residual MeS, or whether there were othersources of toxicity or amelioration, the bioassay toxicity equiva-lents (TEq, concentration of MeS that would produce the observedreduction in growth of Lemna) were compared with the concen-trations of MeS as measured by HPLC (Fig. 5). There was nosignificant difference between the measured MeS concentrationsand the TEqs for the corresponding samples (Paired samplest-test: No added NaCl/HA experiment, p¼0.58; NaCl/0.2 mg C/LHA experiment, p¼0.886; NaCl/2 mg C/L HA experiment,p¼0.156). There was no toxicity in the control reactions (withoutMeS), so toxicity due to residual FR had been successfullyremoved by filtration and catalase treatment. This showed thatfollowing appropriate removal of the FR residues, all the toxicityin the samples was accounted for by the concentration of MeSpresent. Therefore, there were no toxic by-products, reagents orformulation materials in the oxidized solution. It should be notedthat the Lemna assay will only measure toxicity to plants, anddoes not preclude toxicity to animals or microorganisms.While the breakdown products following Fenton oxidation havenot been identified, studies of the metabolites during degradationof MeS in the field or laboratory have found sulfonamide deriva-tives (European Commission, 2000), which may affect bacterialpopulations.

4. Conclusions

Fenton oxidation can be extremely efficient at degrading MeS,but in practical applications it is possible that degradation may beincomplete and produce intermediate compounds, and thatresidual Fenton reagents may remain in the water.

This study demonstrated that residual FR is highly toxic toduckweed, L. disperma; the toxicity was attributed to iron species(possibly ferryl ions), H2O2 and oxygen-centered radicals. Any FRresidues must be removed following remediation of contami-nated waters.

The efficiency of Fenton degradation was dependent on theconcentrations of Fe2þ and H2O2, and inversely related to theinitial concentration of MeS over the range 5–80 mg MeS/L.Addition of 10 mM NaCl plus organic matter (HA at 0.2 and2 mg C/L as TOC) reduced the degradation efficiency for lowerconcentrations of MeS (5 and 10 mg/L), probably because chlorideions can interfere with the Fenton reaction. Since many ground-waters are highly saline, this may limit the effectiveness of Fentonremediation of such waters.

In partially-degraded samples after removal of the residual FR,the toxicity to Lemna was attributable solely to the concentrationof undegraded MeS. There was no evidence for toxicity dueto breakdown products or formulation materials for MeS asmarketed in the formulated product Brushoffs.

It is concluded, therefore, that for Fenton remediation ofwaters contaminated with the herbicide Brushoffs, providedthe residual Fe and H2O2 are removed, the measured

J.M. Abdul et al. / Ecotoxicology and Environmental Safety 83 (2012) 89–95 95

concentration of MeS can be used to estimate the efficiency ofremoval of phytotoxicity.

Acknowledgements

Funding of this project was provided by CRC CARE Australia,under the program and milestone CRC Research RemediationTechnologies (Project Number 2-5-05-05/6), and the UTS Chal-lenge Grant Scheme. We thank Monique Binet and Merrin Adams,CSIRO Land and Water, Lucas Heights, NSW for supplying theLemna culture and the use of their bioassay methodology.

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