PHoto-Fenton ProCeSS For treAting BioLogiCAL LABorAtorY WASteWAter ContAining FormALDeHYDe

Laboratories consume great amounts of hazardous chemicals substances and consequently generate wastewater containing them, for example formaldehyde. This substance is widely utilized to preserve biological samples generating many liters of this residue every year. The present work proposes the use of the photo-Fenton process to treat formaldehyde wastewater using sunlight irradiation. Some aspects were investigated such as the iron source, sample and hydrogen peroxide concentration and also the use of stirred systems. The use of ferrioxalate (0.5 mmol L-1) improved the efficiency of the process in relation to the use of iron nitrate, while at least 1.0 mol L-1 H2O2 is necessary to treat the sample of the 500 mg C L-1. Under these conditions, every formaldehyde detectable was degradeted and 89% of the dissolved organic carbon was removed in two hours of exposure to sunlight. These results are satisfaction considerate for São Paulo State Environmental Agency.


introduction
A large number of biological laboratories use solutions of formaldehyde to preserve anatomic pieces, generating many liters of formaldehyde waste every year. In according to the literature formaldehyde is genotoxic in exposed cells in vitro or exposed tissues in vivo [1,2,3] Although the concentration of formaldehyde is just 3.7% w/w, discharging formaldehyde without treatment can cause serious damage from aquatic environment, due to its carcinogenicity [4] and there is no safe limit to exposition for this class (carcinogenic). Hence, these wastes require an appropriate treatment to avoid risks to the environment and human health.
The adequate treatment of wastes generated by industrial, agricultural or domestic activities is regulated by environmental agencies throughout the world, [5,6] which the levels of specific parameters are limited and checked in the discharged wastes. This is necessary due to large volumes of toxic generated wastes and consequently to the impacts that this cause. The formaldehyde, for example, is able to react with DNA, RNA and proteins, damaging cells and causing the death of microorganisms present in biological wastewater treatment plants [7,8].
Furthermore, the residues generated by Universities and Research Centers offers a special challenge: present specific treatments of different wastes. The ideal solution would be an in situ treatment of these wastes, avoiding storage and references [1]  Artigo Article transportation risks and reducing the number of people in contact with toxic products. It would be also relatively easy and cheap to treat the residues if their compositions are known.
Advanced Oxidation Processes (AOP) can be applied in situ to degrade a great variety of organic pollutants such as pesticides [9,10,11,12], dyes [13,14,15] and chlorophenols [16]. Among the various AOPs, the so-called photo-Fenton process has attracted great interest due to its high efficiency to generate hydroxyl radicals during the decomposition of H 2 O 2 by Fe (II) in acid medium. Associated to UV-Vis irradiation, a considerable increase in the oxidation power is observed mainly due to the photo-reduction of Fe(III) to Fe(II), which can react with H 2 O 2 , establishing a cycle and generating hydroxyl radicals, as shown in equations 1 and 2 [17,18]. 3 y environmental agencies throughout the world, [5,6] which the levels of specific are limited and checked in the discharged wastes. This is necessary due to large volumes of ted wastes and consequently to the impacts that this cause. The formaldehyde, for example, act with DNA, RNA and proteins, damaging cells and causing the death of microorganisms iological wastewater treatment plants [7,8].
hermore, the residues generated by Universities and Research Centers offers a special present specific treatments of different wastes. The ideal solution would be an in situ f these wastes, avoiding storage and transportation risks and reducing the number of people ith toxic products. It would be also relatively easy and cheap to treat the residues if their s are known.
anced Oxidation Processes (AOP) can be applied in situ to degrade a great variety of lutants such as pesticides [9,10,11,12], dyes [13,14,15] and chlorophenols [16]. Among the Ps, the so-called photo-Fenton process has attracted great interest due to its high efficiency hydroxyl radicals during the decomposition of H 2 O 2 by Fe (II) in acid medium. Associated irradiation, a considerable increase in the oxidation power is observed mainly due to the tion of Fe(III) to Fe(II), which can react with H 2 O 2 , establishing a cycle and generating dicals, as shown in equations 1 and 2 [17,18] y researchers have studied the degradation of formaldehyde in aqueous medium using dvanced Oxidative Processes [8,19,20,21]. However, the difficulty is much greater when, the aqueous solution of formaldehyde, the residue presents a high concentration of organic nd interferences, as was the case described above.
(1) 3 y environmental agencies throughout the world, [5,6] which the levels of specific are limited and checked in the discharged wastes. This is necessary due to large volumes of ated wastes and consequently to the impacts that this cause. The formaldehyde, for example, act with DNA, RNA and proteins, damaging cells and causing the death of microorganisms iological wastewater treatment plants [7,8].
hermore, the residues generated by Universities and Research Centers offers a special present specific treatments of different wastes. The ideal solution would be an in situ f these wastes, avoiding storage and transportation risks and reducing the number of people ith toxic products. It would be also relatively easy and cheap to treat the residues if their ns are known.
anced Oxidation Processes (AOP) can be applied in situ to degrade a great variety of lutants such as pesticides [9,10,11,12], dyes [13,14,15] and chlorophenols [16]. Among the Ps, the so-called photo-Fenton process has attracted great interest due to its high efficiency hydroxyl radicals during the decomposition of H 2 O 2 by Fe (II) in acid medium. Associated irradiation, a considerable increase in the oxidation power is observed mainly due to the tion of Fe(III) to Fe(II), which can react with H 2 O 2 , establishing a cycle and generating dicals, as shown in equations 1 and 2 [17,18] y researchers have studied the degradation of formaldehyde in aqueous medium using dvanced Oxidative Processes [8,19,20,21]. However, the difficulty is much greater when, the aqueous solution of formaldehyde, the residue presents a high concentration of organic and interferences, as was the case described above.
(2) Many researchers have studied the degradation of formaldehyde in aqueous medium using different Advanced Oxidative Processes [8,19,20,21]. However, the difficulty is much greater when, along with the aqueous solution of formaldehyde, the residue presents a high concentration of organic compound and interferences, as was the case described above.
Besides the high efficiency of the photo-Fenton process to oxidize a variety of organic compounds, the simplicity of operation and the possibility of using solar light are advantages which can make the implementation easier of in situ and small scale treatment processes, adequate for laboratory wastes.
In this work, the photo-Fenton process was studied for the treatment of aqueous wastes containing formaldehyde generated in a biological laboratory of the Bioscience Institute of the São Paulo State University. The influence of the concentration of the H 2 O 2 and the sample, the iron source (potassium ferrioxalate or ferric nitrate), and the use of stirred systems were evaluated as to their effect on the mineralization of the waste.

Chemicals
Potassium ferrioxalate (FeOx) was prepared and purified as described previously [22]. The aqueous stock solution of FeOx and Fe(NO 3 ) 3 .9H 2 O (Mallinckrodt) were prepared using Millipore Milli-Q water, at a concentration of 0.25 mol L -1 and stored in the dark at room temperature for a maximum of one week. Hydrogen peroxide 30% (w/w) (Synth) was used. Ammonium metavanadate (Vetec) solution was prepared to have a final concentration of 0.060 mol L -1 in H 2 SO 4 at 0.58 mol L -1 (Synth).
The formaldehyde waste sample was collected in a biological laboratory at the University campus having a concentration of 1.2 mol L -1 of formaldehyde and pH value of 6.8. For all experiments, the pH of the samples was adjusted to 2.5 by adding H 2 SO 4 . The reaction was initiated after H 2 O 2 and iron solutions were added, and when they were exposed to irradiation.

experimental conditions
All experiments were realized under magnetic stirring and in batch mode. The sample was exposed to irradiation in open dark glass vessels having a diameter of 9.5 cm, height of 4.5 cm and a total volume of 250 mL [23] under magnetic stirring unless otherwise stated.

Photo-Fenton Process
The experiments were carried out in Araraquara, SP, Brazil (21º S 48º W) under clear sky conditions. The vessels were covered with PVC film to avoid wastage by evaporation. The absorption/reflection of such film is approximately 10% of the solar irradiation in the UVA region. All the experiments were undertaken in winter (July and August), between 10 a.m. and 2 p.m. The solar energy dose accumulated during exposure time and solar irradiance was measured every 20 minutes using a Solar Light CO radiometer (model PMA 2100) in the UVA region (320 and 400 nm) during the experiments. The sensor was positioned horizontally and covered with the same film of the sample to equal the solar energy reception. The total energy dose was also measured during the experiment, which ranged from 13 to 18 J cm -2 for a total exposure period of 2 h.
In the experiments using artificial irradiation two 15 W germicide lamps with a wavelength of 254 nm were used. In this case, the samples were submitted a two hours of irradiation too.

Chemical analysis
The photodegradation process was monitored by measuring the Total Organic Carbon (TOC) concentration using an organic carbon analyzer (TOC 5000A Shimadzu). Samples were collected at various times during the experiment. These collections were done: before the vessels were exposed to the irradiation; every twenty minutes and once the full 120-minute interval was complete. Then the samples were immediately filtered through a 0.45 μm membrane and analyzed. The results are denoted as Dissolved Organic Carbon (DOC). Residual H 2 O 2 was determined using the metavanadate spectrophotometric method [11]. The formaldehyde concentration -before and after photo-Fenton treatment -was determined by colorimetric method using chromotropic acid [24].

Preliminary experiments
Control experiments (residue, residue + FeOx and residue + H 2 O 2 ) in dark and sunlight were done to determine the rate of photolysis of wastewater in study. It was found that the residue and residue + FeOx did not present removal. Even when H 2 O 2 was added the degradation was only 0.5%.
In this preliminary study artificial and sunlight photo-Fenton were utilized for evaluated the degradation of formaldehyde waste. And it was possible to note that using the artificial and solar irradiation was removed 88.8% and 89% of DOC, respectively, see Figure 1. As the results obtained did not present relevant differences, in the rest of the work was used natural irradiation, it could be simpler to make in situ.

Influence of iron source and hydrogen peroxide concentration
Literature indicates ferrioxalate as a suitable candidate for capturing solar energy since it absorbs at between 250-500 nm and the quantum yield for ferrous-ion photo-generation (equation 3) can reach values higher than 1 [25].
Literature indicates ferrioxalate as a suitable candidate for capturing solar energy since it s at between 250-500 nm and the quantum yield for ferrous-ion photo-generation (equation 3) ch values higher than 1 [25].
The improvement of the photo-Fenton degradation of different contaminants such as toluene, 2ne and urea herbicides by using the ferrioxalate complex compared to that of Fe 3+ /H 2 O 2 under rradiation has been previously reported [18,20,25]. In this work, a commercial source of iron s iron nitrate could be simpler, so the use of iron nitrate at a concentration of 0.5 mmol L -1 was red to ferrioxalate for the removal of DOC in formaldehyde wastes. Although the use of alate implies an increase in the carbon content, at the concentration used in this work, it nts an increase of only 36 mg C L -1 , which is very small when compared to the high amount of c matter present in the sample, 500 mg C L -1 . Furthermore, the use of this iron source is more nt when compared to iron nitrate as can be seen in Figure 2. The results with iron nitrate The improvement of the photo-Fenton degradation of different contaminants such as toluene, 2-butanone and urea herbicides by using the ferrioxalate complex compared to that of Fe 3+ /H 2 O 2 under solar irradiation has been previously reported [18,20,25]. In this work, a commercial source of iron such as iron nitrate could be simpler, so the use of iron nitrate at a concentration of 0.5 mmol L -1 was compared to ferrioxalate for the removal of DOC in formaldehyde wastes. Although the use of ferrioxalate implies an increase in the carbon content, at the concentration used in this work, it represents an increase of only 36 mg C L -1 , which is very small when compared to the high amount of organic matter present in the sample, 500 mg C L -1 . Furthermore, the use of this iron source is more efficient when compared to iron nitrate as can be seen in Figure 2. The results with iron nitrate presented a lower efficiency of DOC removal reaching only 55% in 80 minutes, while 87% of DOC removal is obtained for the same experiment time, when FeOx was used.   The higher efficiency of ferrioxalate in relation to iron nitrate may be a consequence of the higher quantum yield of Fe(II) photo-generation when compared to iron nitrate [26]. This result was also observed for the mineralization of formaldehyde present however in a much lower concentration (1 mmol L -1 ; 12 mg C L -1 ) under solar photo-Fenton degradation [20]. This indicates that besides to degrade the formaldehyde, the organic matter present in the waste is also more efficiently oxidized when ferrioxalate is used as iron source. The enhancement of the photo-generation of Fe 2+ improves the formation of the hydroxyl radical by establishing an iron oxidation cycle (equations 1 and 2), which improves the organic matter degradation. Besides the OH radical, the organic radical R • (equation 3) may also react with oxygen and promote the degradation of organic contaminants [27].
Another parameter to be analyzed is the hydrogen peroxide consume. When the H 2 O 2 is completely consumed in the photo-Fenton process, the degradation reaction practically stops, and new additions of the oxidant is necessary. On the other hand, H 2 O 2 can also act as • OH scavenger when high concentrations are present, hindering the photodegradation reaction due to the lower oxidation power of the HO 2 • radical formed (equation 4) [28] Hence is very important to guarantee that it been utilized H 2 O 2 enough to supply the demand, that depends on the organic matter concentration, which is high in this work. Therefore it does not work as hydroxyl radical scavenger. small decrease in the initial H 2 O 2 concentration to 0.9 and 0.8 mol L -1 rease in DOC removal, reaching 71% and 67%, respectively. Although a 2 was still present in the reaction medium, this did not hinder the reaction In contrast, this was necessary to achieve the higher degradation efficiency.

ration of sample
deal concentrations of the formaldehyde waste had been studied: 500, 800 lutions were irradiated in the presence of 0.5 mmol L -1 ferrioxalate and 1 s presents using this iron source) As can be seen in Figure 4, higher DOC 500 mg L -1 sample, reaching 89% in 2 h irradiation while only 19% was taining 1100 mg L -1 organic carbon for the same exposure same time.
onmental legislation recommends an efficiency of COD reduction of the ter of 60 mg L -1 for treated effluent [5], in this case is 55 mg L -1 . Although matter was not reached all formaldehyde was removed, which it was etric method with chromotropic acid. The results showed that after 120 ion, the formaldehyde levels were below the detection limit of the method g that the high toxicity of such wastes is mainly due to the presence of ed method represents a significant hazard reduction, although total c material was not achieved within two hours irradiation.
his work Pereira and Zaiat [29] observed COD removal was around 70% centrations ranging 110 to 1104 mg L-1 . And in according with Aranã and rmaldehyde waste not is a simple task due to the diversity of organic and ey may contain and due to high demand for H 2 O 2 .
plicity and easily application of a non-stirred system, the results obtained ere compared to the results of a system without stirring maintain the was observed that in the unstirred system, a white solid was formed and Formatado: Realce When the iron source was evaluated it was used 1.06 mol L -1 H 2 O 2 , which is a quite high concentration and it resulted in 87% of DOC removal in 80 minutes of irradiation. After measuring the residual H 2 O 2 concentration, it was observed that only 31% of the initial peroxide was consumed after 80 minutes irradiation. Thus the study was done decreasing the H 2 O 2 concentration from 1.06 to 0.73 mol L -1 (Figure 3).  It supposes that similar mineralization can be achieved with a lower H 2 O 2 concentration. However, a small decrease in the initial H 2 O 2 concentration to 0.9 and 0.8 mol L -1 resulted in a significant decrease in DOC removal, reaching 71% and 67%, respectively. Although a considerable amount of H 2 O 2 was still present in the reaction medium, this did not hinder the reaction by scavenging OH radicals. In contrast, this was necessary to achieve the higher degradation efficiency.

Influence of concentration of sample
For determined the ideal concentrations of the formaldehyde waste had been studied: 500, 800 and 1100 mg L -1 and the solutions were irradiated in the presence of 0.5 mmol L -1 ferrioxalate and 1 mol L -1 H 2 O 2 (due to results presents using this iron source) As can be seen in Figure  4, higher DOC removal was found for the 500 mg L -1 sample, reaching 89% in 2 h irradiation while only 19% was obtained for the sample containing 1100 mg L -1 organic carbon for the same exposure same time.  The Brazilian Environmental legislation recommends an efficiency of COD reduction of the 80% or maxim organic matter of 60 mg L -1 for treated effluent [5], in this case is 55 mg L -1 . Although mineralization of organic matter was not reached all formaldehyde was removed, which it was observed using the colorimetric method with chromotropic acid. The results showed that after 120 minutes of the solar irradiation, the formaldehyde levels were below the detection limit of the method (3.3 µmol L -1 ). Considering that the high toxicity of such wastes is mainly due to the presence of formaldehyde, the proposed method represents a significant hazard reduction, although total mineralization of the organic material was not achieved within two hours irradiation.
In agreement with this work Pereira and Zaiat [29] observed COD removal was around 70% when the formaldehyde concentrations ranging 110 to 1104 mg L-1 . And in according with Aranã and co-workers [30] treating formaldehyde waste not is a simple task due to the diversity of organic and inorganic compounds that they may contain and due to high demand for H 2 O 2 .
Considering the simplicity and easily application of a non-stirred system, the results obtained under magnetic stirring were compared to the results of a system without stirring maintain the conditions early defined. It was observed that in the unstirred system, a white solid was formed and precipitated. Considering that the original sample did not presented solid material, this pre-Artigo Article cipitate can be related to either the formation of insoluble degradation products or to the precipitation of proteins and lipids extracted from the anatomic pieces [30]. These long-chain organic compounds may precipitate due to the increase of the ionic strength of the medium since organic acids and various ions can be formed during oxidation. The further degradation of the precipitate was favored at the stirred system probably due to a better absorption of the irradiation which improved the degradation, as previously observed for high absorbance samples [23]. The results in Figure 5 show a slight improvement in the degradation process in the dissolved fraction of the stirred system. This is the reason why subsequent experiments were carried out under stirring.

Conclusions
The present work proposes the application of the photo-Fenton process as an alternative to incineration, which is currently applied to eliminate biological laboratory wastewaters containing formaldehyde. This treatment applies an AOP to that can easily be applied in situ, avoiding storage and transportation risks. The method uses sunlight in order to provide a simple and feasible procedure for the decontamination of this kind of waste.
The results suggested that in the presence of 0.5 mmol L -1 potassium ferrioxalate, 1 mol L -1 H 2 O 2 , and two hours of exposure to sunlight is sufficient to remove at least 85% of the carbon content and also completely oxidize the formaldehyde present in the sample. Considering that the experiments were carried out in the winter, it is expected that better results can be obtained in the summer when solar irradiance is higher. The suggested procedure is simple and feasible to carry out and can be done by the personnel who work in biological laboratories avoiding the storage of waste and its inappropriate discharge.