Degradation of sulfonamides in aqueous solution by membrane anodic Fenton treatment

Article abstract of DOI:10.1021/jf904066a

Two agricultural antibiotics used heavily in agriculture, sulfamethazine and sulfadiazine, were degraded in an aqueous system by anodic Fenton treatment (AFT), an advanced oxidation technique that has been shown to be effective in degrading various pesticides but has not been applied to antibiotics. The effects of the H₂O₂/Fe²⁺ ratio, Fe²⁺ delivery rate, and initial contaminant concentration on the degradation of sulfamethazine by AR were determined. The optimal H₂O ₂/Fe₂₊ ratio was determined to be 10:1, and the optimal Fe₂₊ delivery rate was found to be between 38.9 and 54.4 μM min^-1. Under these conditions, sulfamethazine was completely degraded within 10 min at a range of concentrations (18-250 μM)commonly found in manure lagoons, contaminated rivers, and groundwater. Using the same optimal conditions, the effect of pH on the degradation of sulfadiazine by AFT was analyzed, and 100 μM sulfadiazine was degraded within 6-8 min of treatment at a range of pH values (3.1-7.1) that could potentially be found in aquatic environments. Degradation products and pathways were proposed for both compounds, and it was inferred that AFT degradation products of sulfadiazine and sulfamethazine are unlikely to retain the bacteriostatic properties of their parent compounds. An aquatic toxicity test employing Lemna gibba confirmed that AFT removes the bacteriostatic properties of sulfamethazine and sulfadiazine during degradation.

Full text of DOI:10.1021/jf904066a

1068 J. Agric. Food Chem. 2010, 58, 1068–1076
DOI:10.1021/jf904066a
Degradation of Sulfonamides in Aqueous Solution by
Membrane Anodic Fenton Treatment
K
ATHERINE
NEAFSEY, XIA
Z
ENG
,
AND
ANN T. LEMLEY*
Graduate Field of Environmental Toxicology, FSAD, MVR Hall, Cornell University, Ithaca,
New York 14853-4401
Two agricultural antibiotics used heavily in agriculture, sulfamethazine and sulfadiazine, were
degraded in an aqueous system by anodic Fenton treatment (AFT), an advanced oxidation
technique that has been shown to be effective in degrading various pesticides but has not been
applied to antibiotics. The effects of the H2O2/Fe2þ ratio, Fe2þ delivery rate, and initial contaminant
concentration on the degradation of sulfamethazine by AFT were determined. The optimal H2O2/
Fe2þ ratio was determined to be 10:1, and the optimal Fe2þ delivery rate was found to be between
38.9 and 54.4 μM min-1. Under these conditions, sulfamethazine was completely degraded within
10 min at a range of concentrations (18-250 μM) commonly found in manure lagoons, contami-
nated rivers, and groundwater. Using the same optimal conditions, the effect of pH on the
degradation of sulfadiazine by AFT was analyzed, and 100 μM sulfadiazine was degraded within
6-8 min of treatment at a range of pH values (3.1-7.1) that could potentially be found in aquatic
environments. Degradation products and pathways were proposed for both compounds, and it was
inferred that AFT degradation products of sulfadiazine and sulfamethazine are unlikely to retain the
bacteriostatic properties of their parent compounds. An aquatic toxicity test employing Lemna gibba
confirmed that AFT removes the bacteriostatic properties of sulfamethazine and sulfadiazine during
degradation.
KEYWORDS: Sulfamethazine; sulfadiazine; anodic Fenton treatment; hydrogen peroxide; oxidation;
wastewater; antibiotics; agricultural waste; Lemna gibba
INTRODUCTION
agriculture and has been detected in agricultural waste and in
the environment (6, 7); its similarity in chemical structure to
sulfamethazine makes it an ideal candidate for comparative
degradation analysis.
Seventy percent of the 23000 tons of antibiotics that are used
each year are used as agricultural growth promoters, because they
improve nutrient uptake in the gastrointestinal tract (1). Large
quantities of unmetabolized antibiotics enter the soil as a result of
the application of agricultural manure to fields to improve soil
fertility (1, 2). Antibiotics and their metabolites that do not
strongly adhere to soil particles may leach into groundwater or
enter surface water via runoff. Their presence in the soil and
aquatic environment at subtherapeutic concentrations may pro-
mote the growth of antibiotic-resistant bacteria and may alter
ecosystemstructurebyreducing thenumbers ofsoil bacteria (1,3).
Sulfonamides are a class of bacteriostatic antibiotics frequently
used in agriculture. They weakly adsorb to soil particles, have
the potential to leach into groundwater, and have been detected in
agricultural manure lagoons and in the aquatic environ-
ment (1, 2, 4). Sulfamethazine has been detected in European
and U.S. manure lagoons at concentrations of approximately
143.7 and 1.4 μM, respectively (1, 5). Concentrations of sulfa-
methazine below the therapeutic range of approximately 1.8-
7.2 μM pose the greatest risk for supporting the growth of
resistant organisms in the environment (1). Because the average
concentration of sulfamethazine in U.S. rivers (∼1.8 ꢀ 10-4 μM)
is below this range, sulfamethazine may promote resistance in the
aquatic environment (1). Sulfadiazine is also used heavily in
Sulfonamides are structurally similar to p-aminobenzoic acid
(PABA), a compound used by bacteria to synthesize folic acid, an
important component inDNA synthesis (Figure 1). Bycompeting
with PABA for binding sites on dihydropteroate synthase
(DHPS), an enzyme necessary in folic acid synthesis, they inhibit
the growth of bacteria (8). Sulfonamides and PABA both have a
substituent on the primary carbon, a sulfonyl group (SO2) in the
case of sulfonamides and a carbonyl group (COOH) in the case of
PABA, as well as a primary amine group (NH2) on the aromatic
ring. The aromatic primary amine group is very active in the first
step in folic acid synthesis, and its activity, and thus the bacterio-
static activity of the sulfonamide, is affected by the properties of
the substituent on the primary carbon (9).
Advancedoxidation treatmentsthat producehydroxyl radicals,
such as those involving ozonation, hydrogen peroxide plus UV
light, and ferric ion plus UV light, have been well studied (10-13).
One oxidation treatment that shows promise for on-site degrada-
tion of contaminants in water uses a combination of ferrous ion
and hydrogen peroxide, known as Fenton’s reagent. Systems
employing Fenton’s reagent are simple to operate and require
reasonably priced equipment (10). The kinetics of degradation
pubs.acs.org/JAFC
Published on Web 12/22/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1069
Electricity was supplied by a BK Precision DC power supply 1610 at
0.05 A. Simultaneously, a hydrogen peroxide solution of 0.311 M,
prepared by dilution of a known concentration and determined by
titration in an acidic solution using a standard potassium permanganate
solution (23), was delivered to the anodic half-cell by a Stepdos 08S pump
(Chemglass, Inc.) at a rate of 0.5 mL min-1 (21). The addition of hydrogen
peroxide to the anodic half-cell is known as the Fenton reaction (eq 3) (21),
which produces hydroxyl radicals that degrade the compound.
Figure 1. Structures of sulfamethazine and PABA. Note the similar C1
substituents and aromatic NH2 groups on both compounds.
anode : Fe f Fe2þ þ 2e-
ð1Þ
ð2Þ
of several pesticides by Fenton’s reagent has been well
researched (10, 14-16), and Fenton’s reagent has been incorpo-
rated into several treatment scenarios, such as photo-Fenton
treatment (17), electrochemical Fenton treatment (18), cathodic
Fenton treatment (19), and anodic Fenton treatment (20). Anodic
Fenton treatment (AFT) is one of the most promising of these
Fenton treatments, as it is highly efficient, has a fairly neutral
effluent, and can easily be applied to contaminated water (20).
This research extends the application of AFT, which has been
shown to be effective in degrading various pesticides (21, 22), to
sulfonamide antibiotics in an aqueous solution. The overall goal
is to develop treatments that will degrade antibiotics in agricul-
tural waste before their application to fields and to remediate
water that is already contaminated.
The specific objectives of this research are (i) to determine the
kinetics of AFT-mediated degradation of sulfamethazine and
sulfadiazine in an aqueous system, (ii) to optimize the AFT
system for these two compounds, (iii) to propose degradation
pathways for the tested sulfonamide antibiotics on the basis of
their degradation products, and (iv) to use the aquatic plant
Lemna gibba as a model for testing the bacteriostatic properties of
the degradation products in the AFT effluent.
cathode : 2H2O þ 2e- f H2 þ 2OH-
Fe2þ þ H2O2 ¼ Fe3þ þ OH þ OH-
ð3Þ
•
Because the ratio of the delivery rate of hydrogen peroxide to the
delivery rate of ferrous ion can affect degradation rate and efficiency (21),
all initial experiments were performed at a fixed H2O2/Fe2þ ratio of
10:1, based on optimization studies (vide infra). Experiments were run at
the ambient temperature, ∼20 °C. Power was delivered when the first
drop of hydrogen peroxide solution reached the anodic half-cell. Samples
(1.0 mL) of the solution from the anodic half-cell were collected at specific
times over 15 min of treatment and put into glass high-performance liquid
chromatography (HPLC) vials containing 0.1 mL of methanol, which
quenched the hydroxyl radical and prevented further degradation of the
compound (21). The treatments were repeated in triplicate, and the
samples were analyzed using HPLC and LC-MS.
Optimization of AFT Degradation of Sulfamethazine and Sulfa-
diazine. To determine the effect of the H2O2/Fe2 ratio on AFT degrada-
tion of sulfamethazine, AFT was performed with various H2O2/Fe2þ
ratios between 1:1 and 20:1 (21). In addition, the effect of the Fe2þ delivery
rate on degradation was determined by applying AFT to sulfamethazine
with Fe2þ delivery rates between 7.89 and 78 μM min-1. To determine the
effect of initial concentration on degradation rate, AFT was applied to
sulfamethazine solutions with concentrations between 18 and 250 μM at a
MATERIALS AND METHODS
Chemicals. Sulfamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)
benzenesulfonamide) (99%) and sulfadiazine (4-amino-N-(2-pyrimidinyl)
benzenesulfonamide) (99%) were purchased from Sigma-Aldrich
(St. Louis, MO). Hydrogen peroxide (30%, analytical grade) and sodium
chloride (crystal) were purchased from Mallinckrodt-Baker, Inc. (Paris,
KY). Acetonitrile (HPLC grade), water (HPLC grade), and NaOH (98%)
were purchased from Fisher Chemicals (Fair Lawn, NJ). Phosphoric acid
(85%, analytical grade) and hydrochloric acid (36.5-38%, analytical
grade) were purchased from Mallinckrodt-Baker, Inc. (Phillipsburg, NJ).
Membranes. The anion exchange membrane was purchased from
Electrosynthesis. The electrical resistance of the anion exchange mem-
H2O2/Fe2þ ratio of 10:1 and a Fe2þ delivery rate of 38.9 μM min-1
.
Unlike sulfamethazine, sulfadiazine required the addition of acid to
dissolve in water, which resulted in a stock solution pH of 2.3. Because
AFT typically works best at pH 3.1 (20), the effect of initial pH on the
degradation rate of sulfadiazine was studied by applying AFT to sulfa-
diazine solutions with initial pH values between 3.1 and 7.1. Due to the
structural similarity of sulfamethazine to sulfadiazine, the optimal condi-
tions for AFT degradation were also used for sulfadiazine. The initial pH
of each solution was adjusted by adding 1 M HCl and was measured using
an Accumet Basic AB15 pH-meter (Fisher Scientific).
brane in 1 M NaCl solution at 25 °C is 8 ohm cm-2
.
Determination of Sulfamethazine and Sulfadiazine Concentra-
tions. The concentration of sulfamethazine in each sample was quantified
using an Agilent HPLC 1100 series with a diode array detector set at
254 nm and an Ultra C18 column (4.6 mm ꢀ 250 mm) with a particle size
Toxicity Test Cultures. Duckweed, L. gibba (G-3), was obtained
from an axenic test culture purchased from the Canadian Phycological
Culture Centre at the University of Waterloo (Waterloo, ON, Canada).
Cultures were propagated according to a standard method (23) but were
grown in autoclaved Hoagland’s media, prepared from a Hoagland-
modified basal salt mixture that was purchased from PhytoTechnology
Laboratories (Shawnee Mission, KS) dissolved in HPLC grade water.
Cultures were grown at 23 °C under 24 h fluorescent light (6800 lx), which
was measured by a Reed LM-81LX digital light meter purchased from
Calright Instruments (San Diego, CA).
˚
of 5 μm and pore size of 100 A. The mobile phase consisted of 75:25 water
(adjusted to pH 3 with phosphoric acid)/acetonitrile. The injection volume
was 20 μL, and the flow rate was 1 mL min-1. Sulfamethazine had a
retention time of 5.7 min under these conditions. The concentration
of sulfadiazine was quantified using an Agilent HPLC 1200 series
with a diode array detector set at 254 nm and the same mobile phase
described above. A Zorbax Eclipse XDB-C18 column (4.6 mm ꢀ 150 mm)
with a particle size of 5 μm was used (rapid resolution liquid chromato-
graphy (RRLC)). The injection volume was 25 μL, and the flow was
0.5 mL min-1. Sulfadiazine had a retention time of 4.9 min under these
conditions.
Determination of Sulfamethazine and Sulfadiazine Degradation
Products and Pathways. Each sulfonamide was treated by membrane
AFT according to the established optimal conditions, and eachsamplewas
prepared as described above. The sample was analyzed by an Agilent LC
1200 series with 6130 quadrapole LC-MS. The LC-MS method consisted
of MM-ESþAPCI with a positive polarity scan m/z 70 to m/z 350, with the
molecule fragmenter voltage set at 110 V. The internal energies and dipoles
of the degradation products were analyzed using ab initio equations in the
GAMESS program in the ChemBio3D Ultra program to determine the
most likely degradation pathways. In order to calculate the internal energy
Degradation of Sulfonamides by Membrane Anodic Fenton
Treatment. The AFT setup consisted of a 600 mL glass anodic half-cell
containing 400 mL of 100 μM sulfamethazine or sulfadiazine connected by
an ion exchange membrane to a 600 mL cathodic half-cell containing
400 mL of 0.08 M NaCl (21). Sulfamethazine was added to the anodic half-
cell from a stock solutionof 1000 μM sulfamethazine in distilled water, and
sulfadiazine originated from a stock solution of 1000 μM sulfadiazine in
distilled water acidified with hydrochloric acid (for dissolution purposes).
Both solutions were stirred with magnetic stir bars. When electrolysis
was applied to the anodic half-cell at a current of 0.05 A, the iron anode
(a 2 cm ꢀ 10 cm ꢀ 0.2 cm iron plate) was oxidized and ferrous ion was
delivered to the anodic half-cell at a rate of 38.9 μM min-1. At the same
time, in the cathodic half-cell, water was reduced on a 1 cm ꢀ 10 cm
graphite cathode to hydrogen gas and hydroxide ions (eqs 1 and 2) (21).

1070 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Neafsey et al.
and dipole of the most likely molecular orientation of each product, energy
and geometry of the product was either minimized or its transition state
was optimized using closed shell HF with a basis set of 3-21G before the
calculations were performed.
Testing the Toxicity of AFT Effluent to L. gibba. To test whether
AFT removed the bacteriostatic properties of sulfamethazine and sulfa-
diazine, toxicity tests using the aquatic plant L. gibba (duckweed) were
performed. Sulfonamides elicit toxic effects by targeting the folate syn-
thesis pathway in bacteria as well as in plants (24), and exposure to
sulfonamides decreases L. gibba wet weight (25). Our aim was not to
determine the LC50 of the AFT effluent, but rather to use the test as a
method for determining the point during AFT at which the effluent is no
longer toxic to L. gibba. This would indicate whether the bacteriostatic
properties of the sulfonamide were removed during AFT.
The procedure for the L. gibba tests was based on a previously
established protocol for testing the toxicity of various compounds to
L. gibba (26, 27). Experimental units and controls (33 10-mL culture
dishes) were arranged on large trays according to a randomized complete
block design with 30 experimental units (10 treatments with 3 replicates)
and 3 control replicates.
To establish that toxicity to L. gibba was due solely to the presence of
the sulfonamide, AFT was run with 444 μM sulfamethazine, with 487 μM
sulfadiazine, or without a sulfonamide (control). These concentrations
would produce concentrations in the culture dishes equivalent to the
published EC50 value for sulfamethazine and would allow for a clear
demonstration of a decrease in effluent toxicity from the beginning of AFT
to the end (25). There is no published EC50 value for sulfadiazine, so AFT
was performed with an initial concentration similar to that of sulfametha-
zine due to the similarity in structure between the two compounds.
Controls contained AFT effluent without a sulfonamide to ensure that
the hydrogen peroxide, iron, and methanol present in the AFT effluent
throughout the experiment did not affect L. gibba growth.
The pH values of the anodic half-cell solutions were adjusted accord-
ingly before AFT so that the pH of the final growth medium would be in
the recommended pH range (27). Effluent (4 mL) was collected at 0, 2, 4, 6,
and 15 min, and 0.4 mL of methanol was added. All experimental units and
controls were prepared under a laminar flow hood. Experimental units
consisted of 10 mL of Hoagland’s medium and 0.1 mL of the AFT
effluent/methanol solution. Controls consisted of 10 mL of Hoagland’s
medium with 0.1% methanol, as 0.2% methanol enhances L. gibba growth
rates (28). Two L. gibba plants with three fronds each were placed in each
culture dish, and plants were transferred daily to corresponding dishes
containing fresh solutions. Tests were performed at 23 °C under 24 h cool,
fluorescent light (6800 lx) for 7 days. On day 7, plants from each dish
were removed, blotted dry, and weighed. Wet mass was the only end
point measured, as it was previously found to be the only significant
L. gibba end point affected by exposure to sulfonamides (25). The total
number of fronds in each control was counted to ensure the validity of the
experiment (26, 27).
Statistical Analyses. Sigmaplot 9.0 (Systat Software, Inc., Richmond,
CA) was used for model fitting. Toxicity test data were analyzed using
SPSS. Residual normality and homogeneous variance satisfied the assump-
tions of a one-way analysis of variance (ANOVA). An ANOVA using
Dunnett’s test was performed on the wet weights of experimental units and
controls to determine significant effects (p < 0.05) for each tested
sulfonamide.
Figure 2. Degradation of 100 μM sulfamethazine by AFT with a Fe2þ
deliveryrate of 38.9μMmin-1 and atvariousH2O2/Fe2þ ratios, by0.311 M
H2O2 alone and by 3.108 M H2O2 alone. Data were fit by the modified AFT
model (eq 5).
compounds (eq 4), which fits the degradation trend of many
pesticides (20).
!
½Cꢁ
1
ln
¼ - Kλπων02t2
ð4Þ
½Cꢁ0t
2
In this model, K represents a combination of the second-order
rate constants of the Fenton reaction (eq 2) and the degradation
of the compound by hydroxyl radicals (20). [C]t represents the
concentration of the compound at time t, [C]0 is initial concen-
tration, and λ is the lifetime of the hydroxyl radical (min) (20).
π is the lifetime of Fe2þ (min), ν0 is the delivery rate of Fe2þ by
electrolysis (μM min-1), and ω is a constant determined from the
ratio of the delivery rate of H2O2 to the delivery rate of Fe2þ and
from the degradation rate of H2O2 (20). The model assumes that
the concentration of Fe2þ in the anodic half-cell is constant,
hydrogen peroxide accumulates in the anodic half-cell, both the
Fenton reaction and the reaction between the hydroxyl radical
and the organic compound follow second-order kinetics, and the
concentration of hydroxyl radicals is proportional to the rate at
which they are generated (20).
The AFT model described above did not fit the degradation
kinetics of sulfamethazine well, so a second previously established
model called the modified AFT model (eq 5) that describes the
degradation of triazines was applied (22).
!
ꢀ
ꢁ
½Cꢁ
a
¼ - ln
b2
a
t
½Cꢁ0t
a þ bt
b
ln
-
ð5Þ
where
RESULTS AND DISCUSSION
1
ηKD-Fe3þ
and b ¼
Kλπων0
a ¼
2
Kλπων0
Degradation of Sulfonamides. AFT completely degraded sulfa-
methazine in an aqueous solution with an H2O2/Fe2þ ratio of
10:1 within 6 min (Figure 2). Controls were run by applying either
electricity alone as a source of Fe2þ (figure not shown) or
hydrogen peroxide alone to anodic half-cell solutions of
100 μM of the sulfonamide. There was no significant degradation
under either of these conditions, confirming that oxidation is due
solely to AFT.
This model shares many of the same parameters as the AFT
model, but there are two important differences. η is a constant less
than 1 and represents the ratio of the concentration of ferric iron
to total iron during the AFT (22). κD-Fe3þ is the equilibrium
½D-Fe3þꢁ
constant of
where [D-Fe3þ] is the concentration of
½Dꢁfree½Fe3þꢁ
Kinetic Model of Degradation. It was initially hypothesized that
the degradation of sulfamethazine could be explained by the
established AFT kinetic model for the degradation of organic
the compound bound to ferric ion and [D]free is the concentration
of the unbound compound (22). With these parameters, the
model accounts for the ability of compounds with unsaturated

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1071
heterocyclic rings containing nitrogen, such as sulfamethazine
and sulfadiazine, to form a complex with the ferric ion generated
in eq 2, making the compound less easily degraded by the
hydroxyl radical (22). As ferric ion accumulates in the system
and complexes with the compound, the amount of the compound
available for degradation, and thus the amount of the compound
that is degraded, decreases (22). This model fits the degradation of
sulfamethazine well, with a regression coefficient >0.999
(Figure 2).
Effect of H2O2/Fe2þ Ratio and Fe2þ Delivery Rate on Degrada-
tion of Sulfamethazine. The effects of the initial concentration of
the compound, the Fe2þ delivery rate, and the H2O2/Fe2þ ratio
on the time to complete degradation were assessed (21). The
degradation of sulfamethazine at various H2O2/Fe2þ ratios fit the
modified AFT model well, with regression coefficients of >0.999
(Figure 2). The degradation of sulfamethazine at various Fe2þ
delivery rates also fit the revised AFT model well, with regression
coefficients of >0.999 (Figure 3). Consistent with the model, the
time at which the compound was completely degraded decreased
as the Fe2þ delivery rate increased (Figure 3).
Figure 3. Degradation of 100 μM sulfamethazine by AFT at various Fe2þ
delivery rates at the optimum H2O2/Fe2þ of 10:1. Data were fit by the
modified AFT model (eq 5).
When AFT is optimized as a potential water remediation
process, the cost-effectiveness of reagents and the environ-
mental consequences of high concentrations of reagents should
be considered in addition to the time to complete degradation of
the compound. With these factors in mind, the optimum H2O2/
Fe2þ ratio for the degradation of sulfamethazine was inferred to
be the lowest ratio at which 100% of the initial concentration of
the compound was degraded in the least amount of time. The
H2O2/Fe2þ ratio that fulfilled this requirement was 10:1, at
which 100 μM sulfamethazine was degraded in 6 min (Figure 2).
This ratio is consistent with the optimum H2O2/Fe2þ ratio
determined for other compounds (21). The two Fe2þ delivery
rate conditions that resulted in the lowest times to complete
degradation of sulfamethazine were 54.4 μM (6 min) and
38.9 μM (8 min) and required hydrogen peroxide concentrations
of 0.435 and 0.311 M, respectively (Figure 3). Therefore, an
optimal Fe2þ delivery rate for the degradation of sulfametha-
zine would be in the range of 38.9-54.4 μM min-1, because it
would require a moderate voltage and hydrogen peroxide
concentration and would degrade the compound in a reasonable
amount of time.
Figure 4. Degradation of sulfamethazine by AFT at various initial con-
centrations at the optimum H2O2/Fe2þ of 10:1 and with a Fe2þ deliveryrate
of 38.85 μM min-1. Data were fit by the modified AFT model (eq 5).
Figure 5. Degradation of 100 μM sulfadiazine at various pH values at an optimum H2O2/Fe2þ of 10:1 and with a Fe2þ delivery rate of 38.9 μM min-1. Data
were fit by the modified AFT model (eq 5).

1072 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Neafsey et al.
Figure 6. Proposed degradation pathways of sulfamethazine by hydroxyl radical attack in AFT.
Effect of Concentration on Degradation of Sulfamethazine. For
AFT to be considered an effective and reasonable method for
treating water contaminated with sulfonamides, it must be able to
degrade sulfonamides at the various concentrations commonly
found in the environment. Anodic Fenton treatment at the
optimum H2O2/Fe2þ ratio and a fixed Fe2þ delivery rate of
38.85 μM min-1 was applied to sulfamethazine at initial concen-
trations between 18 and 250 μM, a range that is within that of
accurate concentration analysis by HPLC and encompasses
the concentration (143.7 μM) detected in European manure
lagoons (1). Because the time to complete degradation decreased
with lower initial concentrations (Figure 4), it can be inferred that
AFT would rapidly degrade sulfamethazine at initial concentra-
tions below the range tested, such as those found in groundwater
in Europe and in U.S. rivers (1).
the model compound to study the effects of initial pH on
degradation, as it requires acidified water to dissolve, resulting
in a stock solution pH of 2.3. In the Fenton reaction, the pH of the
anodic half-cell drops to 2-3, which is advantageous to degrada-
tion because the acidity prevents the formation of iron precipi-
tates that would interfere with the reaction (29). The modified
AFT model fit the degradation of sulfadiazine with different
initial pH values with regression coefficients of >0.99 (Figure 5).
The time at which sulfadiazine completely degraded was ∼8 min
for solutions with pH 3.1 and 7 and ∼6 min for solutions with
pH 4.3-6.3. The sulfadiazine solution with an initial pH of
5.1 completely degraded in the least amount of time, followed
by the solutions with pH 4.3 and 6.3. Therefore, the optimum
initial pH for the degradation of sulfadiazine by AFT is pH ∼5.1.
Overall, it was found that sulfadiazine degraded within 8 min in a
solution at a near-neutral pH and that AFT can rapidly degrade
sulfadiazine at a range of pH values typically found in aquatic
environments (Figure 5).
Effect of pH on Degradation of Sulfadiazine. AFT must also be
able to rapidly degrade sulfonamides in water under a variety of
pH values found in the environment. Sulfadiazine was chosen as

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1073
pathway involves a hydroxyl radical attack at the carbon-
nitrogen bond of the benzene ring, which produces two products
with m/z 124 and m/z 110 (Figure 6). The difference in the sum of
the internal energies indicates the difference in the C-N bond
dissociation energies and the tendency for the bond to break. The
sum of the internal energies of the two products in the first
pathway in their radical forms is 1.5 kcal/mol lower than that of
the second pathway, a value high enough to suggest that the
pathways may not occur simultaneously. This difference in
internal energy, coupled with evidence in the mass spectrum that
hydroxyl radicals substitute hydroxy groups for hydrogens on
products of the first pathway (m/z 141, m/z 125), indicates that
the first pathway is likely to be favored. The hydroxyl radical-
induced degradation of diclofenac, a compound similar in struc-
ture to the extrusion product with m/z 215, involves the same two
pathways postulated above (32). This indicates that cleavage at
either side of the central nitrogen and the addition of a hydroxyl
group to one of the resulting products is a plausible step in the
degradation of m/z 215. The observation of a molecular ion with
m/z 246 suggests that another pathway occurs concurrently, in
which hydroxy groups are added to a methyl group of the
nitrogen-substituted ring of the SO2 extrusion product with m/z
215, forming a carbonyl group. Further ab initio analysis (data
not shown) indicated that the most energetically favorable con-
formation for two hydroxyl radicals to add to the structure is as a
carboxyl group on one of the methyl groups of the nitrogen-
substituted ring.
Sulfadiazine. A degradation pathway for sulfadiazine can also
be proposed on the basis of mass spectra and ab initio analysis
(Figure 8). A molecular ion peak, assumed to be the parent
compound sulfadiazine, was observed at m/z 251 during the first
8 min of AFT; it decreases in peak area as the reaction proceeds
(data not shown). A molecular ion peak at m/z 187 was observed
during the first 8 min and is assumed to be the product of SO2
extrusion (30, 31). The peak area of m/z 187 follows a similar
trend as that of m/z 215 in sulfamethazine (Figure 7B). A
molecular ion with m/z 125 was observed between 0 and 8 min,
and a molecular ion with m/z 219 was observed between 0 and
1.5 min. As with the products of sulfamethazine, these two
probable degradation products do not have quantifiable peak
areas, indicating that they are consumed by hydroxyl radicals in
the system. As with sulfamethazine, there are two possible
breakdown pathways from the SO2 extrusion product. In the
first pathway, a hydroxyl radical attacks the carbon-nitrogen
bond of the dinitrogen-substituted ring of the SO2 extrusion
product, forming two products with m/z 109 and m/z 97. In the
second pathway, a hydroxyl radical attacks the carbon-nitrogen
bond of the benzene ring of the SO2 extrusion product, forming a
product with m/z 110 and a product with m/z 96. The difference in
the sum ofthe internalenergies ofthe two products intheir radical
forms between both pathways is 0.227 kcal/mol. This value is not
high enough to be significant, so it is likely that both pathways
occur. As with the extrusion product of sulfamethazine, the
extrusion product of sulfadiazine with m/z 187 is similar in
structure to diclofenac (32). Because diclofenac degrades accord-
ing to the two mechanisms described above, the proposed
degradation pathways for the molecular ion with m/z 187 are
plausible. The observation of the molecular ion with m/z 219
suggests that another pathway occurs concurrently, in which
hydroxy groups are added directly to the nitrogen-substituted
ring of the SO2 extrusion product with m/z 187.
Figure 7. (A) Peak area of the sulfamethazine degradation product with
m/z 215 throughout AFT. (B) Peak area of the sulfadiazine degradation
product with m/z 187 throughout AFT.
Initial Degradation Products and Proposed Degradation Pathways
of Sulfamethazine and Sulfadiazine. Sulfamethazine. A degradation
pathway for sulfamethazine can be proposed on the basis of mass
spectra and abinitioanalysis ofthe byproducts ofAFT (Figure6).
A molecular ion peak at m/z 279 was observed in the MS
spectrum during the first 8 min of the AFT, which can be
attributed to the parent compound, sulfamethazine; it decreases
in peak area as the reaction proceeds (data not shown). A
molecular ion peak at m/z 215 was observed in the spectrum
from 0 to 4 min of the AFT, which appears to be the product of
SO2 extrusion, a phenomenon frequently exhibited by sulfona-
mides (30, 31). The area of the m/z 215 peak increases from the
beginning of the treatment to 2 min and decreases rapidly after
4 min (Figure 7A). This indicates that SO2 extrusion is continu-
ously occurring throughout the first 2 min of the AFT. A
molecular ion with m/z 125 was observed during the first 6 min
of the AFT; a molecular ion with m/z 141 was observed between 4
and 8 min; and a molecular ion with m/z 246 was observed
between 2 and 4 min. These three molecular ions do not have
measurable peak areas, indicating that, although they are
probable degradation products, they are readily consumed by
hydroxyl radicals and therefore do not accumulate in the system.
Two likely pathways of degradation of the extrusion product with
m/z 215 were analyzed using ab initio analysis. The first involves a
hydroxyl radical attack at the carbon-nitrogen bond of the
dinitrogen-substituted ring, which produces two products with
m/z 109 and m/z 125 (Figure 6). The observed peak at m/z 125
could be either the product with m/z 109, to which a hydroxy
group has been added, or the product with m/z 125. The second
Removal of Bacteriostatic Activity by AFT: Toxicity Test with
L. gibba. The success of AFT depends not only on the complete
removal of sulfamethazine from the system but also on the removal
of any degradation products with bacteriostatic properties.

1074 J. Agric. Food Chem., Vol. 58, No. 2, 2010
Neafsey et al.
Figure 8. Proposed degradation pathways of sulfadiazine by hydroxyl radical attack in AFT.
Table 1. p Values Associated with Dunnett’s t Test, Which Determines
Whether the Wet Weights of L. gibba in Experimental Units (AFT with and
without Sulfonamide) Are Significantly (p < 0.05) Lower than Control
ring and hydroxy groups are added. The replacement of the active
sulfonyl group with a primary amine group or hydroxy group may
decrease the activity of the aromatic primary amine and the overall
bacteriostatic activity of the compound (9). The addition of
hydroxy groups and/or carbonyl groups and their corresponding
electron orbitals, as well as the removal of the large electron orbital
of the sulfonyl group may also decrease the sulfonamide’s bacter-
iostatic activity, because the shape of the degradation product
would likely interfere with its ability to bind to bacterial enzymes
involved in folate synthesis (9).
To test whether AFT removed the bacteriostatic properties of
sulfonamide during degradation, L. gibba was exposed to AFT
effluent from various time points in the reaction for 7 days. The
goals were to determine whether exposure to the sulfonamide-
containing AFT effluent at the beginning of the reaction (0 min)
would result in significantly lower wet weights than the control
and whether exposure to effluent near the end of the reaction
(15 min) would result in wet weights that are not significantly
different from the control.
treatment
p value
treatment
p value
AFT run without
sulfamethazine
0 min
AFT run without
sulfadiazine
0 min
0.997
0.902
0.985
0.657
0.981
1.000
1.000
1.000
1.000
1.000
2 min
4 min
2 min
4 min
6 min
15 min
6 min
15 min
AFT run with
sulfamethazine
0 min
AFT run with
sulfadiazine
0 min
0.000
0.006
0.012
0.270
0.936
0.000
0.000
0.000
0.000
0.998
2 min
4 min
2 min
4 min
6 min
15 min
6 min
15 min
As mentioned previously, SO2 resembles the carbonyl group in
PABA (Figure 1) and confers reactivity to the aromatic amine (9).
In the proposed degradation schemes, SO2 is removed from the
The validity of the experiment was confirmed by the observa-
tion of at least a 7-fold increase in total frond number in all
controls at the end of the experimental period (27). Experimental

Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1075
units exposed to sulfonamide-free AFT effluent did not have
significantly lower wet weights than controls at any time during
AFT (Table 1). Therefore, any toxicity observed in experimental
units exposed to sulfonamide-containing AFT effluent is due only
to the presence of the sulfonamide.
21-25, 2000; Society of Environmental Toxicology and Chemistry:
Brighton, U.K., 2002; pp 199.
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Experimental units exposed to sulfamethazine-containing
AFT effluent from 0, 2, and 4 min had significantly lower wet
weights than the control, whereas those exposed to effluent from
6 and 15 min did not (Table 1). These observations are in
accordance with previously established wet weight EC50 values
for sulfamethazine in L. gibba. The initial concentration of
sulfamethazine in the anodic half-cell was 1122 μg/L, and the
published EC50 is 1277 μg/L. The concentration in the anodic half-
cellat 15min was10μg/L, and thepublishedEC10 is 381 μg/L (25).
Therefore, although AFT did not completely degrade the initial
concentration of sulfamethazine within 15 min, the concentration
of active sulfamethazine after 15 min of degradation was too low
to elicit a significant toxic response.
Similarly, experimental units exposed to sulfadiazine-containing
AFT effluent from 0, 2, 4, and 6 min had significantly lower wet
weights than the control, whereas those exposed to effluent at
15 min did not. The initial concentration of sulfadiazine in the
anodic half-cell was 1107 μg/L, and the concentration at 15 min
was 118 μg/L. Although EC50 values have not been established for
sulfadiazine, it was found in a separate experiment that exposure to
579 μg/L resulted in significantly (p < 0.05) lower wet weights than
the control, whereas exposure to 46 μg/L did not. The results of
these toxicity tests support the hypothesis that AFT removes the
bacteriostatic properties of sulfamethazine and sulfadiazine during
degradation.
Conclusions. AFT fully degraded 100 μM sulfamethazine in
aqueous solution at a Fe2þ delivery rate under optimal
conditions within 6 min and is expected to degrade sulfa-
methazine at concentrations found in contaminated rivers
and groundwater within 10 min. AFT completely degraded
100 μM sulfadiazine under optimal conditions at a range
of pH values likely to be found aquatic environments within
6-8 min of treatment.
During AFT, the sulfonyl group was removed and hydroxy
groups were added to the extrusion product, which was frag-
mented and modified, producing degradation products with
potentially reduced bacteriostatic capabilities. Toxicity tests with
L. gibba indicate that AFT removes the bacteriostatic properties
of sulfamethazine and sulfadiazine during degradation.
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ACKNOWLEDGMENT
We sincerely appreciate the assistance of Dr. Jagdish Tewari
in the operation of the LC-MS and Dr. Anthony Hay and
Dr. Stephen Winans for providing laboratory space and guidance
for the experiments with Lemna gibba.
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Received for review August 11, 2009. Accepted November 23, 2009.