Doxycycline degradation by the oxidative Fenton process

Article abstract of DOI:10.1155/2015/492030

Doxycycline is a broad-spectrum tetracycline occurring in domestic, industrial, and rural effluents, whose main drawback is the increasing emergence of resistant bacteria. This antibiotic could be degraded by the so-called Fenton process, consisting in the oxidation of organic pollutants by oxygen peroxide (H in the presence of Fe²⁺. Experiments were performed according to an experimental Rotational Central Composite Design to investigate the influence of temperature (0-40.0°C), Hconcentration (100-900 mg/L), and Fe²⁺ concentration (5-120 mg/L) on residual doxycycline and total organic carbon concentrations. Whereas the final residual doxycycline concentration ranged from 0 to 55.8 mg/L, the oxidation process proved unable to reduce the total organic carbon by more than 30%. The best operating conditions were concentrations of Hand Fe²⁺ of 611 and 25 mg/L, respectively, and temperature of 35.0°C, but the analysis of variance revealed that only the first variable exerted a statistically significant effect on the residual doxycycline concentration. These results suggest possible application of this process in the treatment of doxycycline-containing effluents and may be used as starting basis to treat tetracycline-contaminated effluents.

Full text of DOI:10.1155/2015/492030

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2015, Article ID 492030, 9 pages
http://dx.doi.org/10.1155/2015/492030
Research Article
Doxycycline Degradation by the Oxidative Fenton Process
Alexandre A. Borghi,1 Milena F. Silva,2 Saleh Al Arni,3
Attilio Converti,4 and Mauri S. A. Palma1
1
˜
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Sao Paulo,
˜
Bloco 16, Avenida Prof. Lineu Prestes 580, 05508-000 Sao Paulo, SP, Brazil
2
´
Biological Science Center, Federal University of Pernambuco, Avenue Prof. Moraes Rego 1235, Cidade Universitaria,
50670-901 Recife, PE, Brazil
3Department of Chemical Engineering, King Saud University, P.O. Box 800, Riyadh 11421, Saudi Arabia
4Department of Civil, Chemical and Environmental Engineering, Pole of Chemical Engineering, Genoa University,
Via Opera Pia 15, 16145 Genoa, Italy
Correspondence should be addressed to Mauri S. A. Palma; msapalma@usp.br
Received 29 November 2014; Accepted 4 February 2015
Academic Editor: Ana Moldes
Copyright © 2015 Alexandre A. Borghi et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Doxycycline is a broad-spectrum tetracycline occurring in domestic, industrial, and rural effluents, whose main drawback is the
increasing emergence of resistant bacteria. is antibiotic could be degraded by the so-called Fenton process, consisting in the
oxidation of organic pollutants by oxygen peroxide (H O ) in the presence of Fe2+. Experiments were performed according to an
2
2
experimental Rotational Central Composite Design to investigate the influence of temperature (0–40.0∘C), H O concentration
2
2
(100–900 mg/L), and Fe2+ concentration (5–120 mg/L) on residual doxycycline and total organic carbon concentrations. Whereas
the final residual doxycycline concentration ranged from 0 to 55.8 mg/L, the oxidation process proved unable to reduce the total
organic carbon by more than 30%. e best operating conditions were concentrations of H O and Fe2+ of 611 and 25 mg/L,
2
2
respectively, and temperature of 35.0∘C, but the analysis of variance revealed that only the first variable exerted a statistically
significant effect on the residual doxycycline concentration. ese results suggest possible application of this process in the
treatment of doxycycline-containing effluents and may be used as starting basis to treat tetracycline-contaminated effluents.
1. Introduction
as there are strains resistant to almost all currently known
antibiotics [5].
e major source of environmental contamination by drugs
is municipal wastewater, either treated or not. However,
additional important contributions are given by the effluents
of chemical and pharmaceutical industries, the improper
disposal of expired drugs, the rural effluents, and the presence
of antibiotics in animal manure used to fertilize the soil [1].
World consumption of antibiotics has increased dramatically
in the last decade, thus contributing to their release into
the environment, either as being in their original form
or as metabolites. Most ingested antibiotics are not fully
metabolized by humans and animals; therefore, about 25
to 75% of them leave the organism in unaltered form [2–
4]. Bacterial resistance is actually now as a major concern,
Tetracyclines are the third most consumed family of
antibiotics, but, owing to their widespread use, a growing
number of bacteria resistant to them have been detected [6–
8]. ey are considered safe antibiotics with a lot of favorable
properties such as broad spectrum of antibacterial activity,
low toxicity, and low cost [9]. However, there are side health
effects, among which the most common are nausea, vomiting,
and diarrhea; in addition, patients who take insulin must
have special monitoring, since tetracyclines may increase its
persistence in the body [10]. Besides their use in humans,
tetracyclines are used in animal therapy to treat infections
and promote growth [9].

2
Journal of Chemistry
Doxycycline is a tetracycline with broad spectrum of
action, which is used, in both human and veterinary
medicine, against a wide range of Gram-positive and Gram-
negative bacteria. Because of its widespread use, its con-
centration can reach 6.7 ꢀg/L in wastewaters and 1.5 mg/kg
in domestic sewage sludge [1]; therefore, it becomes of
paramount importance to set up efficient and inexpensive
techniques to treat these effluents, given the increase in
bacterial resistance to this antibiotic.
C
D
E
Several processes have been proposed for the treatment
of doxycycline-containing effluents, among which there are
charcoal adsorption, UV photolysis, ozonation [4], UV/H O
2
2
photolysis [11], oxidation with H O in the presence of
2
2
Cu2+ as a catalyst [12], photocatalysis [13], ꢁ-radiolysis, and
electron pulse radiolysis of water [9]. A possible alternative
to these expensive processes could be the so-called Fenton
process that basically consists of the generation of hydroxyl
radicals, via dissociation of hydrogen peroxide in aqueous
solution catalyzed by the oxidation of the ferrous ion to the
ferric one. Indeed, hydroxyl radicals are extremely reactive
and nonselective and are widely used to pretreat wastewaters.
e main advantages of the Fenton process are its low cost
and the formation of nontoxic substances (H O and O ) by
A
B
Figure 1: Schematic diagram of the experimental set-up used for
doxycycline oxidation tests by the Fenton process. A = beaker
containing 100 mg/L doxycycline hydrochloride solution; B = ther-
mostatic bath (0–100∘C); C = mechanical stirrer; D = thermocouple;
E = pH meter.
2
2
H O -induced decomposition, whereas the main drawback
2
2
is the production of ferric hydroxide sludge.
e present work aims to study doxycycline degradation
by the Fenton process and to optimize the experimental
conditions of temperature, initial concentration of hydrogen
peroxide, and initial concentration of ferrous ion in order
to minimize the residual concentrations of doxycycline and
total organic carbon at the end of the process.
2.3. Experimental Design. Doxycycline oxidation tests were
performed according to a Rotational Central Composite
Design (RCCD) [14, 15], whose results in terms of residual
doxycycline (ꢂ
) and total organic carbon (TOC) concen-
Dox,ꢀ
trations at the end of the process are listed in Table 1.
2.4. Experimental Procedure. Two hundred mL of 100 mg/L
doxycycline hydrochloride solution was transferred to a
beaker wrapped in aluminum foil in order to protect it from
ambient light, which was then put in a bath thermostated at
the selected temperature (0, 8.0, 20.0, 32.0, or 40.0∘C) and
mechanically stirred at 250 rpm. Once the desired tempera-
ture had been reached, ferrous sulfate and hydrogen peroxide
solutions were added to the medium up to the achievement
of the desired concentrations of Fe2+ (5.0, 28.3, 62.5, 96.7,
and 120 mg/L) and H O (100, 262, 500, 738, and 900 mg/L).
2. Materials and Methods
2.1. Chemicals. Sulfuric acid (98%), hydrogen peroxide
(29%), sodium hydroxide, and ferrous sulfate heptahydrate,
potassium iodide, anhydrous sodium sulfite, sodium molyb-
date, sodium nitrate, methanol, acetic acid, and sodium
acetate were purchased from Synth (Diadema, Sa˜o Paulo,
Brazil) and doxycycline hydrochloride was purchased from
Calbiochem (San Diego, CA, USA). All the chemical com-
pounds, except the solutions, were of high purity grade
reagents.
2
2
Every test started with H O addition, whereupon a timer
was activated and aliquots of the medium were collected at
selected times for analyses.
2
2
2.2. Experimental Set-Up. A schematic diagram of the exper-
imental set-up used for doxycycline degradation tests is
illustrated in Figure 1.
2.5. Chemical Analyses
A beaker (A) containing an aqueous solution of 100 mg/L
doxycycline hydrochloride was immersed in a thermostatic
bath (B), model TE 184 (Tecnal, Sa˜o Paulo, SP, Brazil), for
temperature regulation between 0 and 100∘C. It was equipped
with a mechanical stirrer (C), model RZR 2021 (Heidolph,
Schwabach, Germany), a thermocouple (D), model Gulterm
200 (Gulton, Sa˜o Paulo, SP, Brazil), and a pH meter (E),
model 10 MB (Marte, Sa˜o Paulo, SP, Brazil). Dosages and
samplings were done using 0–20, 20–200, and 100–1000 ꢀL
micropipettes (Finnpipette, Vantaa, Finland) and an analyti-
cal balance, model AL 500 (Marte).
2.5.1. Determination of Doxycycline Concentration. Since
Fe2+ and H O are well known to interfere with the spec-
2
2
trophotometric determination of doxycycline, it was neces-
sary to treat the samples with an aqueous inhibiting solution
consisting of 1.0 M potassium iodide, 1.0 M sodium sulfite,
and 0.1 M sodium hydroxide as described by Mota et al. [16].
For this purpose, afer addition of 1.0 mL of sample to a
test tube containing 1.0 mL of this inhibiting solution, the
resulting solution was mixed and filtered through cellulose
acetate filters with 0.45 ꢀm pore diameter, model N-11106-25

Journal of Chemistry
3
Table 1: Experimental conditions of tests of doxycycline degradation carried out according to the experimental RCCD, coded and uncoded
values of the independent variables, and results of residual doxycycline (ꢂ
) and total organic carbon (TOC) concentrations at the end of
Dox,ꢀ
the Fenton process.
Test
ꢃ1
ꢃ2
ꢃ3
ꢂ
2+ (mg/L)
ꢂ
(mg/L)
262
262
738
738
262
ꢄ (∘C)
ꢂ
(mg/L)
28.6
5.8
18.5
4.3
13.6
TOC (mg/L)
38.4
45.8
45.0
41.7
41.9
Fe
H O
Dox,ꢀ
2
2
1
2
−1
−1
−1
28.3
96.7
8.1
8.1
8.1
8.1
+1
−1
−1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
−1
+1
+1
−1
28.3
96.7
28.3
96.7
28.3
96.7
5.0
120.0
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
+1
−1
−1
−1
+1
+1
31.9
+1
−1
262
738
738
500
500
100
900
500
500
500
500
500
500
500
500
31.9
31.9
31.9
20.0
20.0
20.0
20.0
0.0
40.0
20.0
20.0
20.0
20.0
20.0
20.0
2.6
7.8
3.0
4.4
3.3
40.7
36.0
40.6
47.4
45.3
46.6
38.8
40.7
40.4
40.4
45.6
45.2
41.2
39.2
38.5
41.7
3.0
−1
+1
+1
0
0
+1
+1
0
0
0
0
+1
−1.68
+1.68
0
0
0
0
0
0
0
0
0
0
−1.68
55.8
4.7
21.4
2.6
0.0
0.2
2.0
0.0
0.0
0.4
0.43
0.52
1.21
+1.68
0
0
0
0
0
0
0
0
−1.68
+1.68
0
0
0
0
0
0
Mean results at the central point
Standard deviation at the central point
Variation coefficient at the central point
0.073
ꢁ
= initial concentration of ferrous ion; ꢁ
= initial concentration of hydrogen peroxide; ꢂ = temperature; ꢃ1 = coded values of ꢁ ; ꢃ = coded
2+
2+
H O
2
Fe
Fe
2
2
values of ꢁ
; ꢃ3 = coded values of ꢂ.
H O
2
2
∘
2+
(Sartorius, Go¨ttingen, Germany). A 1.0 mL aliquot of the
filtrate was then transferred into a 5.0 mL volumetric flask
containing 1.0 mL of 0.005 M Na MoO , 0.5 mL of 1.0 M
RCCD (ꢂ
= 25 mg/L, ꢂ
= 611 mg/L, and ꢄ = 35.0 C)
Fe
H O
2
2
were collected afer 10, 30, 60, 90, 120, and 300 min and
submitted to LCMS-IT-TOF analysis to identify doxycycline
degradation products. irty ꢀL of 0.1 M NaOH was added
to 2.0 mL aliquots of the reaction medium to precipitate Fe3+
and neutralize residual H O . e resulting solution was then
2
4
NaNO , and 0.5 mL of 0.01 M acetate buffer, the pH was
3
adjusted to 5.0, and distilled water was added up to a final
volume of 5.0 mL. Afer mixing, the absorbance of this
solution was read at a wavelength of 390 nm using a UV-
Vis spectrophotometer, model 600 S (Femto, Sa˜o Paulo, SP,
Brazil). To determine the residual concentration of doxy-
cycline in samples afer treatment with the Fenton process,
we used doxycycline hydrochloride solutions with known
concentrations as standards.
2
2
filtered through cellulose acetate filters with 0.45 ꢀm pore
diameter, and the pH adjusted to 1.5 with H SO 1 : 5.
2
4
Analyses were made at 40∘C under isocratic conditions
by a liquid chromatograph, model Prominence UFLC (Shi-
madzu, Tokyo, Japan), coupled with a mass spectrometer
LCMS-IT-TOF, and equipped with a column, model Shim-
pack XR-ODS (2.0 × 50 mm, 2 ꢀm). e mobile phase was
a mixture of 50% methanol and 50% acetic acid (0.1%
v/v) aqueous solutions. e mobile phase flow rate was
0.2 mL/min, the injected sample volume 1.0 ꢀL, and the run
time 5 min. In the chromatograms, only peaks with height at
least three times higher than the noise level were ascribed to
degradation products.
2.5.2. Determination of Total Organic Carbon. To prepare
samples for TOC concentration determination, 3.5 mL of the
above inhibiting solution was added to 3.5 mL of sample,
and the resulting mixture was filtered through cellulose
acetate filters. H SO 1 : 5 was then added to the filtrate
up to a pH value in the range between 2.0 and 3.0, and
TOC concentration was finally determined by a TOC-5000A
equipment (Shimadzu, Tokyo, Japan).
2
4
3. Results and Discussion
2.5.3. Identification of Doxycycline Degradation Products.
Samples of the reaction medium from the Fenton process
carried out under the best conditions identified through the
Table 1 lists the results of doxycycline oxidation tests per-
formed according to a Rotational Central Composite Design
(RCCD) in which the concentration of ferrous ion (ꢂ 2+ ),
Fe

4
Journal of Chemistry
100
80
60
40
20
0
10
8
6
4
2
0
10
20
30
40
50
60
10
20
30
40
50
60
T (∘C)
(a)
T (∘C)
(b)
) and (b) total organic carbon (TOC) at the end
Figure 2: Influence of temperature (ꢄ) on residual concentrations of (a) doxycycline (ꢂ
Dox,ꢀ
of the Fenton process.
concentration of hydrogen peroxide (ꢂ
), and tempera-
only the initial concentration of H O exerted a significant
ture (ꢄꢅ were selected as the independHenOt variables, while
influence on the residual doxycycline concentration in sam-
2
2
2
2
the residual concentrations of doxycycline (ꢂ
) and total
ples collected at the end of the Fenton process.
Dox,ꢀ
organic carbon (TOC) were the responses. Although one can
see a large variation in the residual doxycycline concentration
e experimental results shown in Figures 2–4 confirm
those of ANOVA. In particular, temperature did not statis-
tically significantly influence the performance of the Fenton
(0 ≤ ꢂ
≤ 55.8 mg/L), incidentally the lowest values
Dox,ꢀ
(0 ≤ ꢂ
≤ 2 mg/L) were obtained just at the central point
process in terms either of ꢂ
or of TOC (Figure 2).
Dox,ꢀ
Dox,ꢀ
∘
(ꢂ
= 500 mg/L, ꢂ
= 62.5 mg/L, and ꢄ = 20.0 C),
It is likely that doxycycline was completely degraded into
compounds able to react with molybdate in the same way as
the antibiotic, thus interfering with the spectrophotometric
analysis.
H O
Fe2+
2
2
suggesting significant influence of the operating conditions
on such a response.
To investigate these effects in greater detail, the experi-
mental results of both responses were worked out using the
Statistica sofware, version 12 (StatSof, Tulsa, OK, USA),
Instead, ꢂ
was strongly influenced by ꢂ
Dox,ꢀ
H O
2
2
(Figure 3(a)), thereby suggesting that low values of this
variable did not lead to the formation of hydroxyl radicals
in concentration enough to degrade either doxycycline or
its degradation products. On the other hand, as expected,
the residual concentration of TOC (Figure 3(b)) was found
which showed that only ꢂ
had a statistically significant
H O
2
2
effect (ꢆ < 0.05) on ꢂ
, while TOC reduction at the
Dox,ꢀ
end of tests did not exceed 30% and was not significantly
influenced by any of the independent variables. e best
Dox,R
Fe2+
operating conditions to minimize ꢂ
(ꢂ
= 25 mg/L,
to be independent of ꢂ
, owing to the sensitivity of the
analytical methodologyHtOo any organic compound finally
present in the medium, that is, not only to doxycycline
but also to its oxidation products. Finally, the initial
2
2
∘
ꢂ
= 611 mg/L, and ꢄ = 35.0 C) were then identified
H O
2
2
by equating to zero the partial derivatives of the sofware-
generated surface equation
concentration of Fe2+ had a dual effect on ꢂ
, in that
2
Dox,ꢀ
ꢂ
= 0.625 − 3.995ꢃ1 + 0.015ꢃ1 − 7.536ꢃ2
Dox,ꢀ
Fe2+
for ꢂ
< 20 mg/L an increase in this variable led to a
2
2
(1)
remarkable decrease in ꢂ
, while no significant influence
+ 9.341ꢃ2 − 4.540ꢃ3 + 2.875ꢃ3
Dox,ꢀ
was detected beyond this threshold value (Figure 4(a)). Since
the same behavior was al2s+o observed for TOC (Figure 4(b)),
+ 1.844ꢃ1ꢃ2 + 2.676ꢃ1ꢃ3 + 0.783ꢃ2ꢃ3
it is likely that, at low ꢂ
values, the formation of hydroxyl
Fe
using the coded values of the three independent variables,
radicals, as in the first case, was insufficient to promote the
Fe2+
H O
namely, ꢂ (ꢃ1ꢅ, ꢂ 2 (ꢃ2ꢅ, and ꢄ(ꢃ3ꢅ, that is, finding the
2
degradation of both the antibiotic and its oxidation products.
function minimum [17]. Such operational conditions were
then assumed as the starting point to perform a parametric
study, whose results are shown in Figures 2–4 for both
responses.
Because of TOC insensitivity to the selected independent
variables, to shed light on the phenomena responsible for
doxycycline degradation by the Fenton process, samples
collected at different times during t2h+e test carried out under
e obtained results allowed estimating for ꢂ
the
Dox,ꢀ
the best operating conditions (ꢂ
= 25 mg/L, ꢂ
=
regression coefficients listed in Table 2, while those of
the related analysis of variance (ANOVA) are presented in
Table 3.
Fe
H O
2
2
∘
611 mg/L, and ꢄ
=
35.0 C) were analyzed by LCMS-
IT-TOF, whose mean results in terms of molar masses of
doxycycline degradation products are summarized in Table 4,
while Figure 5 illustrates the most significant chromatograms.
Only the linear and quadratic regression coefficients for
ꢃ2 were statistically significant (ꢆ < 0.05), which means that

Journal of Chemistry
5
Table 2: Results of linear (L) and quadratic (Q) regression analyses applied to residual doxycycline concentration data collected at the end
of the Fenton process under different operating conditions.
Regression coefficient
0.625
Standard error
3.688
2.448
2.386
2. 4 4 8
2.386
2.448
2.386
3.198
3.198
3.198
ꢇ
ꢆ value
95% Lower confidence limit
95% Upper confidence limit
8.85
1.45
5.30
Mean
0.169
0.868
0.132
0.995
0.011
−7.59
ꢃ1 (L)
−3.995
−1.635
−9.46
ꢃ1 (Q)
−0.015
−0.005
−5.33
ꢃ2 (L)
−7. 53 6
−3.078
−12.99
−2.08
ꢃ2 (Q)
9.341
3.914
0.002
0.094
0.256
0.575
0.426
0.813
4.02
14.66
ꢃ3 (L)
−4.540
−1.849
−9.98
0.93
ꢃ3 (Q)
2.875
1.204
−2.44
8.19
ꢃ1ꢃ2
1.844
0.578
−5.28
8.98
ꢃ1ꢃ3
2.676
0.828
−4.48
9.78
7.90
ꢃ2ꢃ3
0.783
0.242
−6.35
L = linear regression; Q = quadratic regression; ꢄ = Student’s ꢄ-test; ꢃ1 = codified values of ꢁ ; ꢃ = codified values of ꢁ
; ꢃ3 = codified values of ꢂ.
2+
2
H O
Fe
2
2
10
100
80
60
40
20
0
8
6
4
2
0
200
400
600
800
1000
200
400
600
800
1000
CH O (mg/L)
CH O (mg/L)
2
2
2
2
(a)
(b)
Figure 3: Influence of initial hydrogen peroxide concentration (ꢂ
) on residual concentrations of (a) doxycycline (ꢂ
) and (b) total
H O
Dox,ꢀ
2
2
organic carbon (TOC) at the end of the Fenton process.
10
100
80
60
40
20
0
8
6
4
2
0
0
20
40
60
80
100
0
20
40
60
80
100
CFe (mg/L)
CFe (mg/L)
2+
2+
(a)
(b)
Figure 4: Influence of initial concentration of ferrous ion (ꢂ 2+ ) on final concentrations of (a) doxycycline (ꢂ
) and (b) total organic
Fe
Dox,ꢀ
carbon (TOC) at the end of the Fenton process.

6
Journal of Chemistry
Table 3: (a) Results of the analysis of variance applied to residual doxycycline concentration data collected at the end of the Fenton process
under different operating conditions. (b) Results obtained by the quadratic model.
(a)
Sum of squares
218.87
0.003
775.21
1253.19
279.76
118.65
27.38
Degrees of freedom
Mean square
218.868
0.002
775.219
1253.195
279.760
118.652
27.380
ꢈ-calc.
ꢆ value
ꢃ1 (L)
1
1
1
1
1
1
1
1
1
2.68
0.00
9.50
15.36
0.133
0.998
0.012
0.003
ꢃ1 (Q)
ꢃ2 (L)
ꢃ2 (Q)
ꢃ3 (L)
3.43
1.46
0.33
0.69
0.06
0.094
0.255
0.575
0.426
0.813
ꢃ3 (Q)
ꢃ1ꢃ2
ꢃ1ꢃ3
ꢃ2ꢃ3
56.18
4.805
56.180
4.805
(b)
Variation source
Regression
Residues
Lack of fit
Pure error
Total
Sum of squares
2688.80
817.95
Degrees of freedom
Mean square
ꢈ-calc.
ꢆ value
9
10
5
298.756
81.795
162.975
0.615
3.65
0.0279
814.87
3.07
265.14
0.000005
5
3506.75
19
2
% explained variation (ꢉ )
76.675
3.02
; ꢃ3 = coded values of ꢂ.
Tabled ꢈ 9; 10; 0.05
L = linear regression; Q = quadratic regression; ꢃ1 = coded values of ꢁ ; ꢃ = coded values of ꢁ
2+
2
H O
Fe
2
2
Inten. (×105)
1.1
Inten. (×104)
268.9914
262.8639
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A
186.9955
445.1650
382.8339
B
104.9948
E
D
142.9524
164.9148
1.0
0.5
0.0
69.2411
502.7805
568.7255 633.7545
433.0587
388.8757
164.9483
3 4 45
6541.7328150819(98)
104.9034(9)
53.9372
246.9354
224.9784
531.9307
500.7744
93.3180(4)
129.9646
350.8849(9)
306.8001
722.9971
306.8755
923.9946
816.7676
198.4806
155.0675
75.0 125.0 175.0 225.0 275.0 325.0 375.0 425.0 475.0 525.0
50
150
250
350
450
550
m/z
(b)
650
750
850
950
m/z
(a)
Figure 5: LCMS-IT-TOF results of doxycycline samples submitted to the Fenton process (a) for 60 s, retention time of 41 s, (b) for 180 s,
∘
retention time of 54 s. Operating conditions of the Fenton process: ꢂ 2+ = 25 mg/L, ꢂ
= 611 mg/L, and ꢄ = 35.0 C. A, B, D, and E:
doxycycline degradation products with MM = 444, 104, 154, and 502 gF/emol, respectively.H O
2
2
Based on these results, it is evident that doxycycline,
which has an empirical formula of C H N O and a molar
results suggest that the hydroxyl radical was capable of
promoting either the loss of atoms and functional groups
located at doxycycline molecules periphery or the breakdown
of aromatic rings.
According to these results, we sought to identify the
main degradation products by comparison with structures
proposed by other authors [9, 11, 13] (Figure 6, B–D). In
particular, Yuan et al. [11], who investigated the products of
doxycycline photolysis by UV and UV/H O , identified the
same MM = 104 g/mol compound detected in this work as
propanedioic acid (Figure 6, B), which was the likely result
of the breakdown of rings of the drug molecule. e same
22 24
2
8
mass (MM) of 444 g/mol (Figure 6, A), was completely
degraded by the Fenton process within only 10 min, that is,
already at the beginning of the process, mainly leading to
products with less MM (104–399 g/mol) and even one with
higher MM (502 g/mol) (E) (Table 4). One can believe that
initially formed degradation products suffered an attack by
the hydroxyl radical that caused the formation of unstable
compounds able to react with other molecules present in
the medium, hence allowing the formation of more stable
compounds, while maintaining a high TOC level. ese
2
2

Journal of Chemistry
7
CH3
N
CH3
N
CH3
CH3
OH
OH
CH3
OH
OH
CH3
OH
O
ꢂ
ꢁ
ꢀ
ꢃ
OH
O
OH
OH
OH
O
O
OH
OH
O
OH
O
NH2
O
A (444)
D (502)
H3C
CH3
OH
OH
N
CH3
HO
OH
O
O
OH
B (104)
OH
O
OH
O
C (399)
CH3
N
O
OH
O+
H3C
CH3
N
H3C
HO
CH2
O+
D (154)
D (154)
Figure 6: Molecular structures of doxycycline (A) and its degradation products with molar mass of 104 g/mol (B), 399 g/mol (C), and
154 g/mol (D) [9, 11, 13]. (E) Molecular structure of doxycycline degradation product with molar mass of 502 g/mol.
authors also found a product with MM = 186 g/mol, like
that detected in our work, but did not suggest any molecular
structure for it.
4. Conclusions
In this work, dealing with doxycycline degradation by the
Fenton process, the optimal conditions of initial concentra-
Jeong et al. [9], who analyzed doxycycline degradation
products formed by the action of hydroxyl radicals gen-
erated by the electron pulse radiolysis of water, reported
the presence, among others, of a compound with MM =
399 g/mol and suggested for it the molecular structure shown
in Figure 6, C. On the other hand, Maroga Mboula et al. [13],
2+
tions of ferrous ion (ꢂ ꢅ and hydrogen peroxide (ꢂ 2 ꢅ
and of temperature (ꢄꢅ, able to minimize the resHiduOal
Fe
2
doxycycline (ꢂDox,ꢀꢅ and total organic carbon (TOC) con-
centrations, were identified through a Rotational Central
Composite Design.
A parametric analysis highlighted that the above three
using the heterogeneous photocatalysis with TiO , identified,
2
variables exerted different effects on ꢂ
, but the analysis of
was the only one that statistically
among the degradation products of tetracycline, which differs
from doxycycline only for the presence of an additional OH,
a compound with MM = 154 g/mol, for which they suggested
two possible molecular structures (Figure6, D). Finally, based
on the results of search in the database of ChemSpider (Royal
Society of Chemistry, Cambridge, UK), we are able to propose
for the product with the highest MM (502 g/mol), detected
in samples withdrawn from the medium afer 3, 5, and
10 min, the structure E illustrated in Figure 6, which could
be explained by (a) replacement of the amino group with a
carboxymethyl group, (b) attack of a hydroxyl group to the
ꢊ ring, and (c) formation of a cycle adjacent to the ꢋ ring of
doxycycline.
Dox,ꢀ
variance showed that ꢂ
significantly influencedHitO. e best experimental conditions
2
2
able to minimize both residual doxycycline (1.1 mg/L) and
Fe2+
TOC (32.8 mg/L) concentrations were ꢂ
= 25 mg/L,
∘
ꢂ
= 611 mg/L, and ꢄ = 35.0 C. e TOC results
H O
2
2
showed that the organic load was not significantly reduced,
which indicates that the Fenton process led to the formation
of doxycycline oxidation products sensitive to the TOC
analytical methodology.
A LCMS-IT-TOF investigation allowed identifying the
main degradation products of doxycycline oxidation, which
were mainly constituted by low molar mass products

8
Journal of Chemistry
Table 4: Molar masses of the main doxycycline degradation products identified by LCMS-IT-TOF at different sampling times during tests
carried out under the best operating conditions identified by the RCCD (ꢂ 2+ = 25 mg/L, ꢂ
= 611 mg/L, and ꢄ = 35.0∘C). (A) Doxycycline,
Fe
H O
2
2
(B–E) main doxycycline degradation products.
Sampling time (s)
Retention time (s)
30
60
180
300
600
Molar mass (g/mol)
104 (B)
164
186
240
269
350
—
104 (B)
154 (D)
164
184
244
268
306
350
142
164
262
306
382
444 (A)
—
74
262
339
104 (B)
164
186
240
268
350
—
—
142
262
382
444 (A)
502 (E)
—
104 (B)
186
115
186
240
268
350
—
240
268
306
350
382
432
142
262
306
382
444 (A)
502 (E)
—
41
—
—
—
142
164
262
382
444 (A)
—
—
—
—
—
142
164
262
306
382
404
502 (E)
—
54
72
—
—
—
—
—
—
—
—
—
—
399 (C)
—
—
—
´
(104–399 g/mol) as well as a product with higher molar mass
(502 g/mol) than doxycycline.
[4] J. Rivas, A. Encinas, F. Beltra´n, and N. Graham, “Application of
advanced oxidation processes to doxycycline and norfloxacin
removal from water,” Journal of Environmental Science and
Health, Part A—Toxic/Hazardous Substances and Environmen-
tal Engineering, vol. 46, no. 9, pp. 944–951, 2011.
Conflict of Interests
[5] D. P. Rocha, G. F. Pinto, R. Ruggiero et al., “Coordination of
metals to antibiotics as a strategy to combat bacterial resistance,”
e authors declare that there is no conflict of interests
regarding the publication of this paper.
´
Quımica Nova, vol. 34, no. 1, pp. 111–118, 2011.
[6] C. Gu and K. G. Karthikeyan, “Interaction of tetracycline with
aluminum and iron hydrous oxides,” Environmental Science and
Technology, vol. 39, no. 8, pp. 2660–2667, 2005.
Acknowledgment
e authors are grateful to Professor Claudio Augusto Oller
do Nascimento, Coordinator of the Laboratory of Process
Simulation and Control (LSCP) and of the Center of Chemi-
cal System Engineering (CESQ) of the Department of Chemi-
cal Engineering of Polytechnic School of the University of Sa˜o
Paulo for the use of TOC and LCMS-IT-TOF equipment.
[7] E. C. Pereira-Maia, P. P. Silva, W. B. De Almeida et al.,
“Tetracyclines and glycylcyclines: an overview,” Quimica Nova,
vol. 33, no. 3, pp. 700–706, 2010.
[8] A. A. Borghi and M. S. A. Palma, “Tetracycline: production,
waste treatment and environmental impact assessment,” Brazil-
ian Journal of Pharmaceutical Sciences, vol. 50, no. 1, pp. 1–16,
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