Ozonation of ibuprofen: A degradation and toxicity study

Science of the Total Environment 466–467 (2014) 957–964

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Ozonation of ibuprofen: A degradation and toxicity study M.J. Quero-Pastor ⁎, M.C. Garrido-Perez, A. Acevedo, J.M. Quiroga Department of Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cádiz, Cadiz, Spain

H I G H L I G H T S • • • •

In this study have been studied degradation of ibuprofen with ozone. We have optimized different operating variables. We have a model that allows us to predict the percentage of ibuprofen degradation. The ozonation of the ibuprofen generates compounds toxic for Selenastrum capricornium.

a r t i c l e

i n f o

Article history: Received 13 May 2013 Received in revised form 18 July 2013 Accepted 18 July 2013 Available online xxxx Editor: Adrian Covaci Keywords: Ibuprofen Ozone Pilot plant Toxicity Selenastrum capricornium

a b s t r a c t This paper presents the results obtained in the degradation of ibuprofen by ozonation. This study aims to evaluate the degradation of ibuprofen by ozonation once the operating variables have been optimized, investigating the degradation and degradation efficiency of the compound and assessing the toxic effect of ibuprofen and of the intermediate compounds generated during oxidative treatment. Work was carried out to optimize the four operating variables: pH, conductivity, hydraulic retention time and the use of a maze of pipes to enhance contact between the ozone and the drug. All the trials were conducted in a purpose-built pilot-scale reactor. Analyses of the compound were carried out after solid–liquid phase extraction on high resolution liquid chromatography (HPLC). Working under optimal operating conditions (pH = 9, HRT = 20 min and 12 ± 2 gN/m3), a degradation value of 99% was obtained, although degradation efficiency or mineralization of the compound was not achieved. The toxicity of ibuprofen and its intermediate compounds formed during the oxidative process was likewise studied. This toxicity was found to increase with increasing initial concentrations of the compound, with the intermediate compounds thus formed being more toxic than the starting compound. © 2013 Published by Elsevier B.V.

1. Introduction Emerging pollutants are compounds whose presence in the environment is not new, although concern about their possible consequences is due to the fact that little is known about their presence or impact on different environmental niches and on humans. This group includes pharmaceuticals. The routes of entry of these pollutants in aquatic and terrestrial ecosystems may have different origins: emissions from production centers, the disposal of surplus household drugs, human and animal excreta, etc., which are detected in wastewater. Numerous studies show that removal of these compounds is not possible via the conventional treatments employed at wastewater treatment plants, as the technologies used there are not effective enough (Al-Ahmad et al., 1999; Andreozzi et al., 2004; Carballa et al., 2004; Heberer, 2002). Some studies report the presence of a great variety of pharmaceuticals in surface waters ⁎ Corresponding author at: Polígono Rio San Pedro s/n 11510, Puerto Real, Cádiz, Spain. Tel.: +34 661173982. E-mail address: [email protected] (M.J. Quero-Pastor). 0048-9697/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.scitotenv.2013.07.067

(Fent et al., 2006; Heberer, 2002; Kolpin et al., 2002; Ternes et al., 1998) as well as the inability to remove them at drinking-water processing plants. Ternes et al. (1998) reveal that not all drugs are quantitatively removed. These processes have been investigated on a laboratory and pilot scale in Germany (Ternes et al., 2002). The widespread occurrence of these products may pose a problem, as these waters are used as a resource to produce drinking water (Von Gunten, 2003). The first findings regarding the environmental impact of pharmaceuticals were reported by Garrison et al. (1976) and Hignite and Azarnoff (1977). These authors detected clofibric acid at low concentration values (ng/l) in treated wastewater in the United States. Studies conducted in 1981 in Great Britain revealed the presence of pharmaceuticals in rivers at concentrations of up to 1 μg/l (Richardson and Bowron, 1985). Rogers et al. (1986) identified ibuprofen and naproxen in wastewater on Iona Island (Vancouver, Canada). Ibuprofen, or 2-(4-isobutyl phenyl) propionic acid (IBP), is the first of the non-steroidal anti-inflammatory drugs (NSAIDs) derived from propionic acid to be marketed in most countries. It is used primarily in musculoskeletal treatments and secondarily as a broad spectrum analgesic. This drug is extensively used worldwide (Zwiener and Frimmel,

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2000) and is marketed in the form of 400 to 600 mg tablets or capsules. It is worth noting that, in 2005, this drug occupied 17th place on the list of most prescribed drugs in the United States (Richards and Cole, 2006). In Spain, consumption of pharmaceutical drugs has increased in recent years, reaching that of other hazardous substances such as pesticides (Daughton and Ternes, 1999). Ibuprofen has been detected in numerous studies, which have reported concentrations of 0.05 to 0.28 mg/l in surface water (Heberer and Stan, 1996). Considering all the above facts, it is vital to develop processes with a considerable potential for removing pharmaceuticals. Through the use of advanced oxidation processes (AOP), good results have been achieved in removing different types of persistent organic pollutants (POPs) present in industrial wastewaters. Although some studies (Sudhakaran et al., 2013) have shown that the use of combining a natural system with an advanced treatment (oxidation) process may provide benefits for the removal of organic micropollutants, the first and most widespread application of these technologies has been in the disinfection stage of drinking-water treatment (Camel and Bermond, 1998; Rodríguez Vidal, 2003). One of the most advanced oxidation processes currently used is ozone treatment. Ozonation has proved to be one of the most efficient techniques for removing emerging pollutants, including pharmaceuticals, in wastewater and drinking-water treatments (Hua et al., 2006; Huber et al., 2005; Ternes et al., 2002, 2003; Westerhoff et al., 2005; Zwiener and Frimmel, 2000). The use of ozone to treat water is limited by two factors: its solubility (Kosak-Channing and Helz, 1983), and the kinetics of its reactions, which are extremely fast in some cases, but slow in others (Sotelo et al., 1989). As regards the former limitation, the solubility of ozone is highly influenced by variables such as pH, temperature, ionic strength and the presence of substances susceptible of being oxidized in the medium (Hoigne, 1998; Muthukumara et al., 2004; Sotelo et al., 1989; Von Gunten, 2003). The decomposition of ozone in water can be accelerated by increasing the pH or by the addition of hydrogen peroxide (Von Gunten, 2003). At low pH or neutral values, it is known that the radical reactions involved in ozone treatment are lower (Hoigne and Bader, 1976). At these pH values, the predominant reaction is the direct one: ozone is available as molecular ozone (Muthukumara et al., 2004; Wu et al., 2012). At alkaline pH, ozone decomposes into secondary oxidants such as OH•, HO2•, HO3• and HO4•; OH• being the most important due to its high oxidative potential (Muthukumara et al., 2004). At pH 8–9, molecular ozone and hydroxyl radicals are in equilibrium (Alder and Hill, 1950; Hoigne et al., 1985; Staehelln and Hoigné, 1982). There is no evidence to date that low concentrations of ibuprofen have adverse effects on human health. Nonetheless, it would be convenient to invoke the principle of precaution and leave drinking water free from this compound, thus minimizing any potential risk that it may cause in the long term (Von Gunten, 2003). Its adverse effects on biological systems are known, however, and are irreversible. Richards and Cole (2006) studied the toxic response of frog embryos to different concentrations of ibuprofen via the Frog Embryo Teratogenesis Assay-Xenopus. The results showed inhibition in embryo growth at concentrations of 30 mg/l after 96 h of exposure. None of the embryos survived at concentrations above 70 mg/l. However, it should be noted that these concentrations have never been found in the environment. Results like the above have generated growing concern in the scientific community, faced with the possibility that certain drugs or combinations of drugs may cause damage to human health for decades, seeing as water, unlike most specific foods, is consumed in considerable amounts daily. If we add to this fact the lack of water quality regulations which take into consideration the presence of pharmaceuticals at trace levels or in which safety limits for drugs in water are established, the situation becomes even more alarming. Raising the awareness of the authorities with respect to this issue is thus necessary. Given all these problems, the main objective of this study is to assess and optimize the degradation of ibuprofen by ozonation. For this

purpose, the operating variables have been optimized and degradation and degradation efficiency of the compound has been studied. The toxic effects of ibuprofen and of its intermediate compounds released during ozone treatment have likewise been studied. The ultimate goal is to apply this knowledge to the treatment of natural waters so as to improve water quality. 2. Materials and methods 2.1. Reagents Ibuprofen was provided in the solid state by the company Sigma Life Science (Madrid, Spain) at a purity exceeding 98%. The reagents and solvents employed, all of analytical or HPLC grade, were supplied by Merck (Madrid, Spain). 2.2. Oxidative treatment Trials were conducted in a pilot-scale plant (Fig. 1) made of inert materials resistant to the action of ozone (polyvinyl chloride, glass and stainless steel). Ozone was generated by a GZ15 ozone generator (Fig. 1, V) equipped with high frequency lamps, providing a production capacity of up to 15 g/h of ozone with ambient oxygen supply (Fig. 1, I). The ambient oxygen is purified by passing it through a silica gel filter (Fig. 1, II) and an activated carbon filter (Fig. 1, II) arranged in series. The ozone is mixed with the aqueous solution using a Venturi device (Fig. 1, X) and subsequently passes through a micro-bubble generator (Fig. 1, XI), a mixing maze of pipes (Fig. 1, XII) to promote contact between the ibuprofen and the ozone, and a reactor (Fig. 1, XIII) (contact column height: 2 m, working volume: 50 l). The setup is also equipped with a visual mechanical flow meter (Fig. 1, VIII) supplied by the company Korus (Cadiz, Spain) to measure the flow of water entering the maze of pipes, and a Mini Hicon Ozone Analyzer (Fig. 1, VI) operating in continuous mode, acquired from the company Hicon (Alaska, USA). Water is pumped into the reactor (Fig. 1, XIV) from a 25 liters capacity external reservoir by an Ebara M6 Fesx centrifugal pump (Fig. 1, VII). The entire facility was constructed by the ozone engineering company Zonosistem, Ingeniería del Ozono (Cadiz, Spain). 2.3. Trials Given the low solubility of ibuprofen in water, we proceeded as follows in order to inject it into the reactor: 20 l of water (Zwiener and Frimmel, 2000) was taken and adjusted to different working pHs (between 7 and 9) with a solution of sulphuric acid and sodium hydroxide, using sodium chloride to adjust the conductivity of the water. The water was pumped into the reactor from an external reservoir (Fig. 1 (XV)) at an input flow rate of 1250 l/h. A stock solution of 100 g/l of IBP in methanol was employed because this compound is only partially soluble in water. The volume of the stock solution of IBP needed to obtain concentrations of 0.1, 1.0 and 10 mg/l for the trials was added to the aqueous solution at the top of the reactor (Fig. 1 (XIII)). Once the water and the pollutant entered the reactor, it was operated under continuous closed-circuit mode. The water–IBP mixture was stirred for 20 min (the time estimated in previous trials to obtain a homogeneous solution in the system) at 2800 rpm. Once the water–IBP mixture had been stirred, ozone treatment commenced by injecting a constant dose (12 ± 2 gN/m3) throughout the trial in order to maintain a fixed concentration, bearing in mind the consumption which takes place in the oxidation of the compound. The working temperature was 25 ± 2 °C. 2.4. Test variables In an initial study, 19 trials were performed in order to know which of the following variables: pH, conductivity, hydraulic retention time

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959

VI I

III

V IV

II

XII VII

XIII

VIII IX

IX

XI

XIV

X

XV

I Input of air II Filters III Flowmeter IV Compressor V Ozone generator

VI Gaseous Ozone meter VII Pump VIII Flowmeter IX Rotameter X Venturi device

XI M-B generator XII Ozonitation maze XIII Ibuprofen input XIV Ozonitation column XV Output / Input water

Fig. 1. Diagram and legend of the ozone plant.

(HRT) or the use of the mixing maze of pipes, had a greater effect on ibuprofen degradation. The working ranges of the studied variables were: 5 to 20 min for hydraulic retention time; 7 to 9 for pH; 200 to 1500 μS/cm for conductivity; and the use or not of the mixing maze. The variables were fixed as follows. The chosen HRT was the maximum established in the literature (Bahr, 2007; Huber et al., 2003; Meunier et al., 2006; Petrovic et al., 2004; Ried and Mielcke, 2006; Zwiener and Frimmel, 2000), along with prior tests and calculations that verify the efficiency of the oxidation of micro-pollutants in drinking water for human consumption (including ibuprofen). The minimum HRT value was chosen to ensure passage of the mixture throughout the system. The working range of the pH and conductivity variables was that required by Council Directive 98/83/EC, 1998, establishing health criteria for the quality of water for human consumption in Europe. The final variable studied was the use of the mixing maze, a set of pipes added to the facility in order to promote contact between the ozone and ibuprofen. Once the variables with a major significant effect were known, 14 new trials were conducted to determine the optimal values of these variables. The relationship between the set of experimentally controllable factors and the observed results was evaluated using the response surface methodology (RSM) (Muthukumara et al., 2004). 2.5. Sampling Water samples for the determinations of ibuprofen concentration, pH, conductivity, temperature, dissolved oxygen and dissolved organic carbon were always performed at the beginning and end of each treatment. In some trials, samples were also taken during the oxidative process. The residual ozone in the samples was removed by adding sodium thiosulfate (Zwiener and Frimmel, 2000). The initial sampling enabled the actual starting concentration of the trial to be determined. In the trials conducted under optimal conditions, mass spectrometry analysis was used to detect the intermediate compounds formed during the treatment. 2.6. Sample analysis To quantify the amount of drug present, the samples to analyze were acidified to pH = 2 with sulphuric acid and subsequently passed through a LiChrolut EN 40–120 μm SPE solid–liquid extraction cartridge

following the method developed by Zwiener and Frimmel (2000), though using 2 ml instead of 1 ml fractions of acetone to recover the compound. The acetone was subsequently evaporated and the ibuprofen dissolved in a mixture of methanol/water (70:30), as this is the mixture used in HPLC quantification of ibuprofen. Under these conditions, recovery of the compound ranged between 80% and 90% with a maximum standard deviation of 5%. Samples were stored at a temperature of −80 °C until their analysis. Identification and quantification of the drug was carried out following the methodology reported by Mendez Arriaga et al. (2009) on high resolution liquid chromatography (HPLC), purchased from the company Bekman Coulter (Madrid, Spain), equipped with a UV detector at a wavelength of 220 nm and using a C18 column (5 μm, 15 × 0.46) supplied by Teknokroma (Barcelona, Spain). The operating conditions are shown in Table 1. The drug concentration was obtained from the quantification of the areas of the peaks detected by HPLC. The calibration curve covered a concentration range of 1 to 100 mg/l (R2 ≥ 0.999). The study of temperature, pH, conductivity and dissolved ozone allowed the evolution of these variables to be determined throughout the oxidative process. The temperature was controlled in the trials by means of a multiparameter probe purchased from Mettler Toledo (Barcelona, Spain) seeing as the temperature was not regulated in the pilot plant. pH measurements were made using a Crison Table 1 Method for determination of ibuprofen. Operating conditions Filtered sample volume (ml) pH Material of extraction (SPE) Methodology SPE Recovery HPLC Columns Flow Injection Methodology of HPLC Detector Reading (min)

200 2.0 Lichrolut EN (40–120 μm) Zwiener and Frimmel (2000) Solution: Methanol/H2O Commercial enterprise: Bekman Coulter Commercial enterprise Teknokroma and model C18 (5 μm, 15 × 0.46). 1.5 ml/min 20 μl Mendez Arriaga et al. (2009) U.V. 15

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GLP-22 pH meter (Madrid, Spain). The conductivity of the sample was measured with a calibrated Crison GLP-22 conductivity meter (Madrid, Spain). The ozone dissolved in water was quantified using a Merck kit (Code 1.00607.0001) and a Spectroquat Pharo 300 supplied by Merck (Madrid, Spain). Total organic carbon (TOC) was determined on a Shimadzu TOC5000 total organic carbon analyzer. TOC concentrations were calculated as the difference between total carbon (TC) and inorganic carbon (IC). Hydroxy-ibuprofen was identified using a Waters UPLC-QTOF (SYNAPT G2) mass spectrometry system. 2.7. Toxicity Ecotoxicological assessment of pharmaceuticals in the fresh water environment is an emerging area of research worldwide. Statements can be found in the literature to the effect that drugs may become more toxic compounds than the parent compound when low ozone doses are employed (Ashbolt, 2004). Toxicity trials were carried out with inocula from the species Selenastrum capricornium. This is a crescent-shaped unicellular green alga (Chlorophyta) with a volume of approximately 40 to 60 μm3. This species of alga was chosen because it can be found in eutrophic or oligotrophic epicontinental aquatic systems and due to being one of the organisms commonly used by the US Environmental Protection Agency (EPA) and that of Canada (Environment Canada). This assay can be used by itself or as part of a battery to estimate the potential phytotoxicity of fresh surface water or groundwater. The assay with the S. capricornium alga presented in this paper is a modification of the standard method published by the Environment Canada. The changes refer to the use of smaller volumes (4 ml in 10 ml borosilicate glass vials) (Garrido-Perez et al., 2008; Lopez-Galindo et al., 2010). Initial concentrations of 0.1, 1 and 10 mg/l IBP were used in the toxicity tests, the solutions subsequently being treated with ozone under optimal conditions. Samples were taken at 0, 5, 10, 15 and 20 min to analyze their toxicity (C0, C5, C10, C15 and C20). Two kinds of samples were considered at time zero: the first comprising ibuprofen dissolved in water (A), and the second made up of ibuprofen dissolved in water adjusted to the optimal pH and stirred for 20 min (B). This phase coincides with C0, the initial treated sample. All times were studied in triplicate using the methodology of Garrido-Perez et al. (2008) and LopezGalindo et al. (2010). Given the effect of exposure to toxic pollutants on the reproduction, growth rate, etc. of algal populations, the toxicity of the compound is determined by comparing normal growth observed in a pollutant-free system (Algal Blank) with growth of the alga in the polluted sample. When the toxicity results do not provide progressive data between tested concentrations, the toxicity is expressed as the percentage inhibition instead of CE50 (the concentration that reduces algal growth by 50% compared to the control) (Lopez-Galindo et al., 2010). The percentage of cell growth inhibition for each test substance concentration is calculated as the difference between the area under the control growth curve and the area under the growth curve for each test substance concentration. 3. Results and discussion 3.1. Preliminary trials and optimization of ozone treatment A series of preliminary trials were conducted to select the variables showing the most significant effect on the system. A statistical software application running on the Windows platform, Modde 9.0, was used to determine the number of trials to carry out. This program proposed 19 trials using the D-optimal model with 3 central points. The results are shown in Table 2. From these tests and the analysis of the results applying the statistical application Anova, it was concluded that the variables HRT and pH

Table 2 Conditions of work and results of 19 previous tests. Essay

HRTa (min)

pHinitial

Conductivityinitial (μS/cm)

Maze of pipes

Degradation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

5.0 5.0 5.0 5.0 20.0 20.0 20.0 20.0 5.0 5.0 5.0 5.0 20.0 20.0 20.0 12.5 12.5 12.5 12.5

7.0 9.0 7.0 9.0 7.0 9.0 7.0 9.0 7.0 9.0 8.0 9.0 7.0 9.0 8.0 8.0 8.0 8.0 8.0

200 200 200 200 200 200 200 200 1500 1500 1500 1500 1500 1500 1500 850 850 850 850

Yes Yes No No Yes Yes No No Yes Yes No No Yes Yes No No Yes No Yes

51 62 69 65 98 99 85 98 63 68 52 89 89 89 96 96 96 97 96

a

HRT = hydraulic retention time.

showed a high significant value, while conductivity and the use or not of the maze had little influence on the process, thus allowing these latter variables to be left as constants in the trials. After selecting the variables that have the greatest influence on the process, 14 trials were carried out (Table 3) to determine their optimal values. From these tests and the analysis of the results applying the statistical application Anova, it was concluded that the optimal values to obtain the highest degradation (99%) with the fastest kinetics were HRT = 20 min and pH = 9. A 2K factorial design was used for the analysis of variance employing the statistical application Anova as this is the most widely-used model in analytical studies (Fernandez-Carballido et al., 2004; Persson Stubberud and Astrom, 1998). A 2K factorial model is obtained, i.e. an exponential-quadratic function with 2 factors, 2 central points and the replicated design (Eq. (1)), whose coefficients of variance and regression are given in Table 4. Both the hydraulic retention time and the pH of the medium were significant (p b 0.05) under the study conditions. 2

Y ¼ cte þ β1 THR þ β2 pH þ β3 THR þ β4 pHTHR

ð1Þ

This is a predictive model, selected from the models proposed by the Anova application, which enables the percentage of ibuprofen degradation in the system to be determined. The adequacy of the model was evaluated by testing the lack of fit. Table 3 Conditions of work and results of 14 optimization tests. Essay

HRTa (min)

pHinitial

Degradation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

5.0 5.0 20.0 20.0 12.5 12.5 12.5 5.0 5.0 20.0 20.0 12.5 12.5 12.5

7.0 9.0 7.0 9.0 8.0 8.0 8.0 7.0 9.0 7.0 9.0 8.0 8.0 8.0

56 62 98 99 98 98 95 51 77 99 99 96 98 96

a

HRT = hydraulic retention time.

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961

O3(mg/l)

Table 4 The analysis of variance ratios and regression model (Anova).

Tª (ºC) 25

0.60

Model parameters R2 SSmodel pH THR pH ∗ THR THR2 pb pH THR pH ∗ THR THR2 Standard errorresidual b0 pH THR pH ∗ THR THR2

0.95 128.00 2812.50 128.00 942.88 0.01725 2.55 ∗ 10−7 0.01725 2.44 ∗ 10−5 3.885 −65.81 10.66 14.13 −0.53 −0.29

20 0.40 15

10 0.20 5

Dissolved ozone Temperature

0

0.00 0

2

4

6

10 12 14 8 HRT (minutes)

16

18

20

b0 = intercept, pH = initial value of pH, HRT = hydraulic retention time (minutes).

Fig. 2 is obtained by plotting the percentage degradation values obtained experimentally versus those predicted by the predictive model. The high correlation coefficient (R2 = 0.97) and the fact that an increase of one unit in the x variable is accompanied by an increase of 1.01 in the y variable give an idea of the goodness of fit of the obtained model.

Fig. 3. Evolution of temperature and dissolved ozone in water [IBP]0 = 1 mg/l, pH0 = 9, HRT = 20 min, [O3]0 = 12 ± 2 gN/m3.

(Hoigne and Bader, 1976, 1985). These results with respect to the evolution of ozone have also been obtained by other authors (Persson Stubberud and Astrom, 1998; Oh et al., 2003). 3.3. Evolution of pH during the oxidation of ibuprofen

3.2. Influence of temperature on the concentration of dissolved ozone An increase in system temperature of around 1 °C was observed for every 5 min of treatment in all the trials. This is due to the passage of water through the pumping system (Fig. 3). Therefore, there can be a difference of up to 4 °C in the temperature of the solution between the start and end of the test. This increase in temperature causes variations in the concentration of dissolved ozone. Fig. 3 shows the evolution of ozone in the system. An increase in ozone concentration in the water can be seen during the first minutes. This results in a higher ozone transfer rate than the ozone degradation rate (0–5 min). A decrease in the concentration of ozone in solution is then produced despite continuing to inject a constant dose of gaseous ozone. This decrease is due to the previously mentioned increase in temperature that occurs in the medium resulting in a decrease in solubility (Persson Stubberud and Astrom, 1998) and a higher ozone degradation rate because an increase in temperature brings about a decrease in the dissolved ozone concentration. This occurs due to a drop in the liquid phase driving force and to a higher ozone decomposition rate (Sotelo et al., 1989). Also, the rate of decomposition of ozone can be lowered by decreasing the pH of the solution

% degradation model

100

80

60

40

Due to its electron configuration, ozone has different types of reactions in water, forming free radicals. These free radicals propagate through elementary step mechanisms to produce hydroxyl radicals, which are extremely reactive with any organic species and with some inorganic species present in water. At alkaline pH, ozone decomposes into secondary oxidants such as OH•, HO2•, HO3• and HO4•. At high pH values, therefore, the radical reactions occurring during ozone treatment are greater (Rodríguez Vidal, 2003). Degradation rates of 99% are achieved under optimal working conditions (pH0 = 9 and HRT = 20 min) (Table 3). These results are comparable to those obtained by Rosal et al. (2008), who obtained similar degradations under the same pH conditions. Zwiener and Frimmel (2000) obtained similar degradations at neutral pH, combining the use of ozone with hydrogen peroxide. If the aim is to work at the lowest pH, ozone treatment must therefore be combined with another advanced oxidation process or the concentration of the ozone dose must be increased in order to achieve the degradation rates obtained here (99%). Fig. 4 shows the evolution of pH during oxidation under optimal conditions. A decrease in pH can be observed during the stirring process, varying between 8 and 9. Once the oxidation process starts (C0), the pH tends to decrease approximately 0.8 units under optimal conditions as the test progresses. This result is in line with those obtained by Coelho et al. (2010), who reported decreases of 5 units in pH at 60 min of treatment working with initial IBP concentrations of 200 mg/l and gaseous ozone concentrations similar to those employed in this study in 1 l capacity reactors. One possible explanation may be related to Le Chatelier's Law, according to which an increase in temperature throughout the oxidative process will displace pH values. 3.4. Evolution of the oxidation process with hydraulic retention time (HRT)

20

0 0

20

40

60

80

% degradation experimental Fig. 2. Goodness-of-fit for the obtained model.

100

Fig. 5 shows the evolution of total organic carbon and the percentage of degraded compound versus HRT in trials conducted under optimal conditions. As regards TOC, it can be seen that there are no appreciable variations throughout the process. This indicates that degradation efficiency of ibuprofen does not occur and the compound is transformed into

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pH 9.4

Median 25%-75% Non-Outlier Range

9.0

Outliers Extremes

8.6

8.2

7.8

7.4

Stages of treatment

7.0 A

C0

C5

C10

C15

C20

HRT ([IBP] = 200 mg/l, [O3] = 8.7–15.3 g/m3, pHinitial = 6.3 and 1 l of sample). Other studies on wastewaters reported fourfold lower HRTs for similar anti-inflammatory drug degradations (99%), although they employed an ozone contribution 4 times higher than that used in this paper (45.9 gN/m3) (Rosal et al., 2008). As mineralization was not produced in the present study, the samples were analyzed under optimal conditions by mass spectrophotometry. The IBP oxidation process has been previously studied by other authors (Huber et al., 2005; Ternes, 1998; Von Gunten, 2003) and its degradation pathway has been established (Zwiener et al., 2002). Studies have shown the formation of carboxy-ibuprofen and hydroxyibuprofen as IBP metabolites present in both wastewater and natural waters (Buser et al., 1999; Ternes et al., 1998). Results show the presence of the intermediate compound, hydroxy-ibuprofen, in all treatment phases, which arises from the initial sample after pH adjustment and prior stirring (20 min). This is in keeping with studies by Zwiener et al. (2002), who reported that the hydroxy-ibuprofen metabolite was formed from IBP under aerobic conditions in activated sludge sewage.

Fig. 4. Evolution of pH vs stages of treatment [IBP]0 = 1 mg/l, pH0 = 9, HRT = 20 min, [O3]0 = 12 ± 2 gN/m3.

3.5. Toxicity study other simpler molecules, without becoming mineralized. We likewise studied the oxidation of methanol (a blank experiment with only methanol), the evolution of total organic carbon in this treatment with ozone (12 ± 2 gN/m3) also being found to remain constant. As regards the evolution of the percentage of degraded compound, it can be seen in Fig. 5 that this percentage increases as the oxidation process evolves. The maximum degradation was 99% at 20 min of treatment (12 ± 2 gN/m3 gaseous O3). Ternes et al. (2003) obtained similar results, detecting no IBP after 18 min of treatment (10–15 mg/l ozone). The degradation of pharmaceuticals by indirect ozonation is very fast, with most compounds presenting high second-order kinetic constants (Persson Stubberud and Astrom, 1998). There are studies with ibuprofen that show low rate constants with direct ozonation. This can be explained by the absence of reactive groups and an aromatic ring that is only slightly activated (Huber et al., 2003). Because of the low rate constant, direct reactions with ozone will play a minor role during ozonation processes and the oxidation of this compound will be caused mainly by OH• radicals originating from ozone decay. The global kinetic constant obtained in the trials conducted in this study is 0.250 min−1, with a correlation coefficient of 0.986. These results are comparable to those reported by Coelho et al. (2010), who obtained 86% degradations employing a threefold higher

Degradation (%)

COT (mg/l)

The toxicity study was carried out with two objectives in mind: firstly, to determine the toxicity of ibuprofen and secondly, to determine the toxicity of the sub-products generated during the treatment. 3.5.1. Toxicity of ibuprofen Fig. 6 shows the percentage of inhibition in S. capricornium cultures for buffer solutions containing different concentrations of dissolved IBP (0.1, 1 and 10 mg/l) (A) and for the same concentrations with optimal pH adjustment (pH = 8.5–9) and stirring of the system for 20 min (B). No toxic effect of ibuprofen on the studied species was observed in its initial phase of dissolution (A). After the stirring and pH adjustment process (B), the percentage inhibition increased (Fig. 6). This may be due to the initial release of the hydroxy-ibuprofen metabolite. As can be seen in Fig. 6, the highest average inhibition obtained was around 5%, corresponding, as expected, to the highest studied ibuprofen concentration in phase B (10 mg/l). These results constitute an improvement on those obtained by Cleavers (2004), who reported inhibition rates of less than 5% in tests with the alga Scenedesmus subspicatus

Inhibition % 30

Median 25%-75% Non-Outlier Range

100

5.0

80

4.0

60

3.0

40

2.0

Outliers

20

Extremes

10

0

-10

Degradation of IBP (%)

20

1.0 COT (mg/l)

0

0.0 0

2

4

6

8

10

12

14

16

18

20

-20

-30

A

(0.1)

A

(1)

A

(10)

B

(0.1)

B

(1)

B

Stages of treatment

(10)

HRT (minutes) Fig. 5. Evolution of percentage of degradation and total organic carbon vs hydraulic retention time. [IBP]0 = 1 mg/l, pH0 = 9, HRT = 20 min, [O3]0 = 12 ± 2 gN/m3.

Fig. 6. Percent inhibition in crops of Selenastrum capricornium ibuprofen patterns (A0.1, A1, A10) and the samples after stirring process and pH adjustments (B0.1, B1, B10) at three different concentrations 0.1, 1 and 10 mg/l. Xy (X: treatment step, y = [IBP]0).

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5

10

15

20

25

1

0

Stages of C 0.1 (0 )

C 0.1 (5 ) C 0.1 (10)

C 0.1 (15 ) C 0.1 (20)

treatment

Inhibition %

5

10

15

20

25

2

0

Stages of C 1 (0 )

C 1 (5 )

C 1 (10 )

treatment

C 1 (1 5 ) C 1 (20)

Inhibition %

3

4. Conclusions

10

20

3.5.2. Toxicity of ibuprofen and its intermediate compounds at various stages of treatment Following the analysis of toxicity of IBP in isolation, we proceeded to study the toxicity of the solutions resulting from the oxidation process at different HRT. As previously shown (Fig. 5), ozone oxidation does not produce mineralization of the compound, given that the initial and final TOC is approximately the same and one of the intermediate compounds thus formed was identified. It was hence considered of interest to study the toxicity of the intermediate compounds that were formed, seeing as the degradation of many other organic compounds produces more toxic intermediate compounds than the compound from which they derive (Ashbolt, 2004). Furthermore, the mixture of ibuprofen and its intermediate compounds may produce different types of effects (antagonistic, synergistic, etc.) on the ecotoxicology of species. To analyze this process, different samples were taken at different points in the IBP oxidation process and at different initial concentrations. The results are shown in Fig. 7. For test concentrations of IBP ≤ 1 mg/l (Fig. 7, (1)–(2)), toxicity increases as the oxidative treatment proceeds up until 20 min, subsequently decreasing from that point on. This decrease may be caused by the generation of less toxic intermediates. The maximum percentage of inhibition obtained in this study interval was 16% (HRT = 15 min). Cleavers (2004) obtained similar inhibition percentages in a toxicity study with IBP (S. subspicatus) for one hundred-fold higher concentrations (102.7–155.5 mg/l). At concentrations of IBP = 10 mg/l (Fig. 7, (3)), toxicity increases as the oxidative process proceeds. Cleavers (2004) reports that, at pH ranges between 7.5 and 8.5, substances mainly dissociate in proportion to their polar form. It should be noted that the concentrations studied in this paper are in the order of 100–1000 times those found in residues in natural water. Hence, acute IBP toxicity in the natural environment cannot be concluded.

Inhibition %

30

Chodat (SAG 86.81 = UTEX 2594), employing IBP concentrations of 72.9 mg/l and a no-observed-effect concentration (NOEC) of 32 mg/l. Furthermore, Ferré et al. (2001) obtained inhibition percentages of 50% for lower concentrations (EC50 = 19.1 mg/l, Vibrio fischeri, Microtox). The latter authors studied the biological effects of ibuprofen on dermatophyte fungi and Gram-positive bacteria, evidencing its high toxicity on these organisms, as well as a high potential against microbial activity in fungi and an inhibitory effect on bacterial growth (Ferré et al., 2001). Other studies (Cleavers, 2004) report toxicity in Daphnia and algae in natural aquatic environments.

963

Median

It will be necessary to increase the concentration of gaseous ozone used or to combine ozonation with other advanced oxidation processes to degrade these released compounds to other, less toxic compounds. Furthermore, in order to improve the environmental risk assessment of ibuprofen and its metabolites, this study should be extended by

0

25%-75%

-10

Non-Outlier Range Outliers

-2

Extremes

-30

• The operating variables of pH, conductivity, HRT and the use of a maze to improve contact between dissolved ozone and the drug were optimized. The most significant variables were found to be pH and HRT. • A predictive model was obtained that allows us to predict the percentage of IBP degradation as a function of pH and HRT under the tested experimental conditions. • Under the tested experimental conditions, mineralization of the compound was not achieved. One of the intermediate compounds thus formed was identified by mass spectrophotometry. • Based on the results of toxicity tests with S. capricornium, the compound released during ozonation is more toxic than the parent compound.

Stages of C 1 (0)

C 1 (5)

C 1 (10) C 1 (15) C 1 (20)

treatment

Fig. 7. Evolution of the toxicity of ibuprofen in the study phases (C0, C5, C10, C15, C20) with Selenastrum capricornium at three concentrations 0.1 (1), 1 (2) and 10 mg/l (3). Xy (X: treatment step, y = [IBP]0).

conducting standard tests on bacteria, invertebrates and fish following established guidelines. Conflict of interest There is no conflict of interest.

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Acknowledgments The authors wish to express their gratitude to Mr Alvaro Valenzuela Romero, at the company Zonosistem Ingeniería del Ozono, S.L., for his contributions to the carrying out of this study. This work was partially funded by the Spanish Ministry of Education and Science via R&D Project CTM2008-04940/TECNO.

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