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Evaluation of advanced oxidative processes in biodiesel wastewater treatment

Article  in  Journal of Photochemistry and Photobiology A: Chemistry · April 2019

DOI: 10.1016/j.jphotochem.2019.01.013

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 Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology A: Chemistry

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

Evaluation of advanced oxidative processes in biodiesel wastewater T

treatment
Gabriel Ferreira da Silva Britoa, Rhaul Oliveirab,c,d, Cesar Koppe Grisoliab,
⁎
Layanne Souza Guirraa, Ingrid Távora Webera,e, Fernanda Vasconcelos de Almeidaa,
a
Instituto de Química, Universidade de Brasília, Asa Norte, 70910-900, Brasília, Distrito Federal, Brazil
b
Laboratório de Genética Toxicológica, Departamento de Genética e Morfologia, Instituto de Ciências Biológicas, Universidade de Brasília, Asa Norte, 70910-900, Brasília,
Distrito Federal, Brazil
c
Faculdade de Tecnologia, Universidade Estadual de Campinas, UNICAMP, 13484-332, Limeira, São Paulo, Brazil
d
Programa de Pós-graduação em Toxicologia e Análises Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, FCF – USP, 05508-000, São
Paulo, Brazil
e
Programa de Pós-graduação em ciências de Materiais, CCEN, Universidade Federal de Pernambuco, CEP, Recife, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: In biodiesel production by alkaline transesterification, a large amount of wastewater containing a very high
Biodiesel wastewater amount of organic matter is generated. In spite of this, biodiesel wastewater is a neglected residue, which very
Advanced oxidative processes often does not receive the correct treatment. In this work, we have evaluated the potential of advanced oxidative
Fenton processes (AOP) in biodiesel wastewater treatment. The efficacy of four AOP, namely Fenton, photo-Fenton,
Photolysis
solar photo-Fenton and solar-photolysis, was tested measuring the total organic carbon removal from this re-
Ecotoxicity
Fish embryo acute toxicity
sidue. As well, the toxicity of the treated residue was investigated by fish embryo tests (FET). Photo-Fenton, solar
photo-Fenton and solar photolysis led to similar organic load reduction, but solar photolysis (8 h of solar ex-
posure) was considered the best treatment, due to the important organic load reduction reached (more than
92%) associated with an ease of operation and a 100% reduction in toxicity for Zebrafish embryos in the dilution
range studied. In the best cases, approximately 6–8% of the organic content in biodiesel wastewater is re-
calcitrant.

1. Introduction three times the volume of biodiesel) [7–10]. Biodiesel wastewater

The use of biodiesel has been promoted as an environmentally-
friendly alternative to petroleum oil due to its lower pollutant emissions
and the renewable character of biodiesel. A number of countries have
promoted the production of biodiesel as a strategic measure to obtain
cleaner energy from renewable sources (biomass). Biodiesel can be
produced with waste animal fats and sewage sludge [1,2], but the main
raw material is vegetable oil [3,4]. In all cases, the most used process of
biodiesel production is alkaline transesterification - an efficient process,
with a high conversion rate of fatty acid esters in a relatively short
length of time [5]. Biodiesel treatment usually begins with decantation
of glycerol (main by-product), followed by washing with water to re-
move impurities such as soaps, catalysts, methanol and mono, di and
triacylglycerols that have not completely reacted [6].
Environmental concern with biodiesel production has increased due
to the large amount of wastewater produced in this process (around
presents very high concentrations of organic carbon (TOC), oils and
greases (O&G) and therefore has a high chemical oxygen demand
(COD) [11]. Biodiesel wastewater is thus considered as a high toxic
effluent, which is an emerging matter of concern [12,13]. In 2016,
Brazilian biodiesel production was estimated in approximately 3 801
million m3 [14], thus, the produced wastewater could reach about 11.4
million m3 [7–10].
Given this situation, the concerns about biodiesel wastewater have
arisen and a number of investigations have been made to treat this
residue. The literature reports on treatments by coagulation, electro-
coagulation, biological, adsorption, microbial fuel cell and, moreover,
their associated treatments, which consist of a combination of two or
more processes [11,15]. Advanced oxidative processes (AOP), also
known as advanced oxidative technologies, comprise a set of techniques
that, under certain conditions, transform organic contaminants into
carbon dioxide, water and any inorganic ions that may be found in a

⁎
Corresponding author.
E-mail address: fernandaalmeida@unb.br (F.V. de Almeida).

https://doi.org/10.1016/j.jphotochem.2019.01.013
Received 8 August 2018; Received in revised form 6 December 2018; Accepted 14 January 2019
Available online 14 January 2019
1010-6030/ © 2019 Published by Elsevier B.V.
G.F. da Silva Brito, et al.
degraded sample [16]. AOP such as ozonation, heterogeneous photo-
catalysis and photo-Fenton are highlighted in the literature for biodiesel
wastewater [11], nevertheless some authors mention the need for fur-
ther experimentation, to develop or improve the management of the
amount of residue that is expected to be produced in the future.
The Fenton process is based on the production of hydroxyl radicals
(HO•) by the catalytic decomposition of hydrogen peroxide in the pre-
sence of ferrous ions in aqueous solution (Eq. (1)) [17–20]. It can be
considered a relatively simple and low cost process that provides high
efficiency in the removal of organic contaminants [21,22]. Despite the
advantages, the Fenton process has drawbacks such as restricted
working pH range, sludge production and the inability to recover the
catalyst [17–19,21,23]. Among the alternatives to the Fenton process,
photo-Fenton uses ultraviolet or visible radiation to improve the for-
mation of hydroxyl radicals (Eq. (2)) [17,20].
Fe 2 + + H2 O2 → Fe 3 + + HO• + OH− (1)
hv
Fe3 + + H2 O → Fe 2 + + HO• + H+ (2)
The use of radiation not only reduces the formation of sludge, but
also increases the speed of the reactions, allowing a more effective
mineralization of the contaminants [17].
In this study, we evaluated the potential of AOP in biodiesel was-
tewater treatment by measuring organic content reduction and toxicity
of the final residue. We compared the effectiveness of Fenton, photo-
Fenton, solar photolysis and solar photo-Fenton in the treatment of
aqueous residue from soybean oil biodiesel, through the evaluation of
total organic carbon (TOC) degradation. Additionally, the treated re-
sidues of the most efficient treatment process were evaluated for toxi-
city using zebrafish embryos as the biosensor. We point out alternatives
to the disposal of biodiesel wastewater in a practical and safe way.
2. Materials and methods
2.1. Synthesis and characterization of biodiesel wastewater
A volume of 500 mL of commercial soybean oil was pre-treated at
80 °C for 3 h. After cooling to room temperature, the oil was mixed with
200 mL of a methanolic (MTEDIA for HPLC) solution of KOH (5 g,
IMPEX, minimal purity 85%). After one hour under constant stirring at
room temperature, the product was poured into a separating funnel and
left to stand for 24 h. This separation phase allows the removal of crude
glycerin before the biodiesel is washed. Initially, 500 mL of crude bio-
diesel was washed with 3 fractions of 250 mL of a 10% v/v phosphoric
acid solution and then with another 3 fractions of the same volume of
distilled water. When finished, the pH of the last wastewater fraction
was equal to the purified water before using.
The wastewater (sum of 6 fractions) was characterized with respect
to total organic carbon (TOC-L Shimadzu), chemical oxygen demand
[24], oil and grease content [25], pH (HANNA pH 21), conductivity
(Adamo MCA 150), dissolved oxygen (OAKTON PCD 650), turbidity
(ALFAKIT) and toxicity to zebrafish embryos [26].
2.2. Advanced oxidative treatments
Four different processes were applied to study the biodiesel waste-
water, namely Fenton, photo-Fenton, solar photo-Fenton and solar
photolysis. Our main goal was to compare the efficiency of the selected
treatment processes and propose safe alternatives for the disposal of
biodiesel wastewater.
The indoor experiments (Fenton and photo-Fenton) were carried out
in triplicate in three sets of glass reactors kept under constant magnetic
stirring with temperature monitoring. Solar experiments (outdoor)
were performed in duplicate and each set of reactors was homogenized
whenever H2O2 was added (every 15 min). For the solar experiments
the radiation was monitored using a radiometer (Solar Light, PMA
86
Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90
2120). These results are shown in the supplementary material (SM
Fig. 1). Before TOC analysis, all samples were topped up to their ori-
ginal volume by adding deionized water (typically 5–10 mL).
2.2.1. Fenton reactions
Preliminary tests, taking 180 min, based on the works by Grčić et al.
[27] and Sabaikai et al. [28], were performed using [Fe2+] ranging
from 20 to 50 mg L−1 and [H2O2] from 100 to 1000 mg L-1. The best
results were achieved using [Fe2+] = 20 mg L−1 and
[H2O2] = 1000 mg L-1. Based on these results, all further experiments
also used [Fe2+] = 20 mg L−1 and [H2O2] = 1000 mg L-1.
Experiments using the Fenton reaction were performed in closed
glass containers wrapped in aluminum foil. A sample of 100 mL from
biodiesel wastewater was taken and then FeSO4.7H2O (FMAIA P.A.)
was added to obtain a ferrous ion concentration of 20 mg L−1. H2O2
(Vetec 30%) was added every 15 min to give a constant concentration
of 1000 mg L−1. The procedure lasted 300 min with TOC monitored
each 30 min.
2.2.2. Photo-Fenton reaction
This reaction was carried out in 100 mL of biodiesel wastewater,
using the same ferrous ions concentration (20 mg L−1) used for Fenton
reaction. Hydrogen peroxide was added every 15 min to give a constant
concentration of 1000 mg L−1. The sample was also irradiated with UV-
light provided by a high-pressure mercury vapor lamp (125 W)
(λ = 254 nm). This procedure lasted 480 min and the TOC was mea-
sured every 30 min.
2.2.3. Solar photo-Fenton
The experiments were carried out using the same conditions as the
photo-Fenton reaction (item 2.2.4) but using solar radiation instead of a
mercury lamp. A sample of 500 mL of biodiesel wastewater was placed
in a glass recipient (29.6 cm × 17.8 cm × 5 cm) and exposed to natural
sunlight (outdoor) after the addition of Fe2+ (20 mg L−1) and periodic
additions of 1.25 mL H2O2 (1000 mg L−1) every 60 min. The experi-
ment lasted 480 min (during May 2016 in the Midwest Brazil). The TOC
content was measured every 60 min.
2.2.4. Solar photolysis
These experiments were conducted similarly to solar photo-Fenton.
They consisted of exposing 500 mL of biodiesel wastewater (inside a
glass recipient of 29.6 cm × 17.8 cm × 5 cm) to sunlight for 480 min
(during May 2016, in the Midwest of Brazil). The TOC content was
measured every 60 min. The solar experiments were continued on the
next day for another 180 min. This step aimed to evaluate if the treat-
ment had already reached its end point.
2.3. Ecotoxicological assays
To evaluate toxicity from biodiesel wastewater before and after
treatments, we submitted the samples to toxicological assays using
Zebrafish as the test organism. Initially, samples of biodiesel waste-
water were tested at two different pH values. The pHs of 4.0 and 7.0
were obtained by adjusting the original sample (pH 1.4) by adding KOH
8 M, to eliminate the pH as a confounding factor. Zebrafish embryos
were exposed to different dilutions (100%; 75%; 50%; 25%; 10%; 1%;
0.1% and 0.01%) at each pH.
Toxicological assays were also performed using treated biodiesel
wastewater, but in this case at a sole pH value of 7 in three dilutions
(1%, 0.1% and 0.01%), for both the photo-Fenton and solar photolysis
treated samples.
2.3.1. Test organisms
Zebrafish were maintained in aquariums with reverse osmosis and
activated carbon filtered water. The fish had been raised in an aquatic
facility (ZebTec - Tecniplast, Italy) with a photoperiod cycle of 12:12 h
G.F. da Silva Brito, et al. Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90

(light:dark) at the University of Brasilia (Brazil). The water parameters Table 1

were strictly controlled: temperature was maintained at 27.0 ± 1 °C, Biodiesel Wastewater Characteristics (mean values ± standard deviation).
conductivity at 750 ± 50 μS cm−1, pH at 7.0 ± 0.5 and dissolved Parametera Results Replicates (n) Literature datac
oxygen ≥ 95% saturation. Adults were fed two times daily with live
brine shrimp nauplii, Artemia sp., and a dry fish flake mix (TetraMin® TOC 40,700 ± 570 mg L−1 3 6,620 – 48,000
COD 24,210 ± 265 mg O2 3 17,750 –
flake).
L−1 160,000
Embryos were routinely bred from bulk spawning of stock fish in a Turbidity 138.42 ± 9.01 NTU 3 –
spawning platform at a 2:1 male to female ratio (iSPAWN, Tecniplast). O&G 2,237 ± 204 mg L−1 4 1,440 – 2,095
The Zebrafish eggs used for the toxicity tests were collected im- pH 1.66 ± 0.03 2 2.9 – 11.1
mediately after mating, rinsed in water, and checked under a stereo- Conductivity 35,620 ± 70 μS cm−1 2 758 – 1,380
Dissolved Oxygen 7.07 ± 0.06 mg L−1 2 –
microscope (Stereoscopic Zoom Microscope – Stemi 2000, Zeiss,
Fish toxicity 0.118 ± 0.005% 3 –
Germany). The unfertilized eggs and those showing cleavage irregula- (LC50)b
rities or injuries were discarded.
a
TOC = total organic carbon, COD = chemical oxygen demand, O&G = oil
and grease, NTU = Nephelometric Turbidity Units.
2.3.2. Fish Embryo Toxicity (FET) tests b
LC50 for the original biodiesel wastewater with pH adjusted for 7 obtained
Fish Embryo Toxicity (FET) tests were performed according to for zebrafish FET test.
OECD guideline no 236 [26] in order to evaluate the toxicity levels of c
(Hincapié-Mejía et al. [30]; Patiño et al. [31]; Costa et al. [13]; Gonçalves
both initial and treated effluents. One zebrafish egg was placed in each et al. [12]).
microplate well, which was then filled up with 2 mL of each test solu-
tion. Each microplate had 24 wells, with 4, known as the internal 3.2. TOC removal efficiency
control group, destined for internal quality verification of the micro-
plate, which was filled with controlled water from the culture medium. Four different AOP were tested and their effectiveness evaluated by
The embryos were observed daily under a stereomicroscope. The test means of organic content removal. The Fenton reaction was very in-
was started immediately after fertilization and was continued and efficient in TOC removal, presenting only 7.5 ± 1.0% after 300 min of
monitored for four days (96 h) under static conditions - controlled treatment. For this reason, the test was discontinued (Supplementary
temperature (26 ± 1 °C) and photoperiods of 12 h light and 12 h dark. material; SM Fig. 2). No data concerning the use of Fenton reaction for
The tests were performed in triplicate. biodiesel wastewater treatment has been found in literature up to the
Before hatching, egg coagulation, otolith formation, general delay date of our study, probably due to its poor effectiveness.
of development, eye and body pigmentation, somite formation, heart- All the other processes presented a significant TOC decrease
beat, edemas, detachment of the tail-bud from the yolk sac and yolk sac (Table 2). Although the best result was obtained using photo-Fenton
absorption were evaluated. After hatching, spine malformation and (93.79 ± 0.20%), no significant difference between the final TOC
posture were also evaluated. All parameters were assessed and quan- percentages was observed at 480 min for photo-Fenton, solar photo-
tified as observed or not observed. Fenton, or solar photolysis (One Way ANOVA p > 0.05). These results
are shown in Fig. 1.
2.3.3. Statistical analysis According to the data of Fig. 1, a threshold was probably reached
The effective concentrations (LC50 and EC50) were calculated using after 480 min of experiment, indicating a small percentage (6–8%) of
a 4 parameter logistic model. A one-way ANOVA was used to detect the recalcitrant organic load. To evaluate this behavior, we extend the solar
differences between the groups for normally distributed data sets. When processes (outdoor) up to 660 min, exposing the wastewater treated for
data did not pass the Kolmogorov–Smirnov normality test and the 480 min to sunlight for another 180 min. As expected, this prolongation
Levene's homogeneity of variance test, a Kruskal–Wallis test was per- in sunlight exposure did not produce any significant additional reduc-
formed. If significant results were found, the Dunnett’s or Dunn's test tion in TOC (p > 0.05) (Supplementary Material, SM Fig. 3), reinfor-
(for parametric or non-parametric data, respectively) was used to detect cing the idea of approximately 8% of recalcitrant organic matter.
significant differences between the tested concentrations and the con- Hincapié-Mejía et al. [30] also realized experiments for degradation
trol (p < 0.05). All analyses were performed using the Sigma Stat 3.5 of biodiesel wastewater by photo-Fenton. The authors studied Fe2+
statistical package [29]. concentration varying from 5 to 28 mg L−1 and H2O2 in a range of
1200–4500 mg L−1. Although the photo-Fenton parameters (UV wa-
velength and Fe2+ and H2O2 concentration) were similar to those used
3. Results and discussion
in our study, the authors used a flow system and added all of H2O2 at
the beginning of the experiment. Their biodiesel wastewater (initial
3.1. Biodiesel wastewater characteristics
TOC value of 48,000 mg L−1) had 27.15% of TOC removed over a 2 h
treatment period, which is considerably lower than what we obtained
In this work, we used biodiesel produced from soy oil by alkaline
with photo-Fenton after the same period (approximately 66% of TOC
transesterification which was washed 3 times with diluted phosphoric
reduction). Their best results were obtained in another experiment with
acid solution and water (3 L wastewater:1 L biodiesel). The biodiesel
biodiesel wastewater diluted over 100 times and resulting in 85.71% of
wastewater was an opaque milky liquid with a high organic content and
TOC removal.
chemical oxygen demand (COD). The physical-chemical parameters of
In fact, the literature reports on biodiesel wastewater as a difficult
biodiesel wastewater are shown in Table 1.
Biodiesel wastewater has been described as an opaque liquid with
high values of TOC, COD, O&G, conductivity and dissolved oxygen Table 2
[12,13,30,31]. Table 1 shows the main parameters of biodiesel waste- Maximum degradation for advanced oxidative processes in biodiesel
wastewater (mean values ± standard deviation).
water used in this work and a concordance with the values found in the
literature. The difference of pH and conductivity values between this Treatments TOC after 480 min (%)
work and those reported in literature can be explained by the acid wash
Photo-Fenton 93.79 ± 0.20
used in this work. Besides this, biodiesel wastewater is also reported to
Solar photolysis 92.95 ± 0.37
have a high toxic effluent, thus becoming a matter of concern to en- Solar photo-Fenton 89.02 ± 1.21
vironmentalists.

87
G.F. da Silva Brito, et al. Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90

Table 3
Effects on hatching rate of zebrafish embryos exposed to crude and treated
samples of biodiesel wastewaters (average values ± standard deviation).
Assays Day 2 Day 3 Day 4

Biodiesel wastewater
0 0.23 ± 0.10 0.98 ± 0.03 1.00 ± 0.00
0.01 0.50 ± 0.10 0.98 ± 0.03 1.00 ± 0.00
0.1 0.73 ± 0.24* 0.92 ± 0.09 1.00 ± 0.00
1 – – –
Photo-Fenton treated
0 0.13 ± 0.03 1.00 ± 0.00 1.00 ± 0.00
0.01 0.11 ± 0.06 1.00 ± 0.00 1.00 ± 0.00
0.1 0.15 ± 0.05 1.00 ± 0.00 1.00 ± 0.00
1 – – –
Solar-photolysis treated
0 0.20 ± 0.05 0.94 ± 0.05 0.96 ± 0.03
0.01 0.18 ± 0.07 0.98 ± 0.03 1.00 ± 0.00
0.1 0.10 ± 0.10 0.95 ± 0.10 1.00 ± 0.00
1 0.46 ± 0.10* 1.00 ± 0.00 1.00 ± 0.00
Solar photo-Fenton treated
0 0.20 ± 0.05 0.94 ± 0.05 0.96 ± 0.03
Fig. 1. Decrease of the total organic carbon content on biodiesel wastewater for 0.01 0.46 ± 0.11 0.98 ± 0.03 1.00 ± 0.00
solar photolysis, photo-Fenton and solar photo-Fenton treatments after 0.1 0.21 ± 0.20 0.94 ± 0.10 1.00 ± 0.00
480 min. Error bars show ± one standard deviation obtained from multiple 1 0.00 ± 0.00* 0.10 ± 0.02 0.54 ± 0.17
samples.
“*” means statistically different from control group, One-way ANOVA, Dunnet
test p < 0.05.
“–” means that all organisms died in the treatment.

3.3. Biodiesel wastewater toxicological profile

Fig. 2. Mortality for zebrafish embryos exposed to biodiesel wastewater and
after photo-Fenton, solar photo-Fenton and solar photolysis treatments (mean
values ± standard error). All samples tested with pH adjustment for 7.0. Error
bars show ± one standard deviation obtained from multiple samples.
effluent to be treated are probably due to its high organic content.
Combined processes are reported as the most efficient way for biodiesel
wastewater treatment [11,15]. Recently, Gonçalves et al. [12], used
coagulation-flocculation followed by membrane filtration and photo-
Fenton reaction during 24 h for one sample of biodiesel wastewater.
Just with these three combined treatments they achieved approxi-
mately 90% of removal in Dissolved Organic Carbon (DOC) and of COD
reduction. Costa et al. [13] also evaluated a combination of coagula-
tion-flocculation and photolysis (UV-C) or H2O2/UV-C for biodiesel
wastewater treatment. Their results achieved 94% of biodiesel waste-
water mineralization after 6 h of irradiation.
No works were found in the literature that used a single process to
achieve a TOC decrease as high as 90%, especially using only solar
radiation. Solar radiation is abundant in tropical regions and can be
useful from the economic and environmental point of view. Moreover,
this kind of radiation does not present a risk for the operator, in com-
parison with UV-C radiation.
To the best of our knowledge, this is the first study using zebrafish
embryos to evaluate biodiesel wastewater toxicity. According to the
literature, toxicity evaluation using Zebrafish has been performed with
biodiesel seed cake from Jatropha curcas L. [32]. In another study, adult
zebrafish were exposed to purified methyl soy biodiesel through acute
toxicological assay and high toxicity was reported [33].
Embryological development in zebrafish can be continually fol-
lowed in live individuals due to chorion transparency. The embryos are
quite sensitive to chemical exposure especially during early stages. This
makes the zebrafish embryotoxicity test ideal for toxicology research,
facilitating the identification of adverse effects of chemical compounds
[34].
According to Andrade et al. (2017), FET experiments at pH values
less than 3.5 and greater than 10.5 yield 100% mortality of zebrafish
embryos. As expected, the FET experiment performed with the original
pH value (1.4) for our biodiesel wastewater sample showed extremely
high toxicity (96h-LC50 0.033 ± 0.003%). On the other hand, the
experiment performed with the same sample after pH adjustment (pH
4.0) showed a slight decrease in toxicity (96h-LC50 0.070 ± 0.006%).
At optimum pH (7.0) the toxicity decreased by 3.6 fold (96 h- LC50
0.150 ± 0.15%), but still represents a very high value. At all tested
pHs, biodiesel wastewater was highly toxic to zebrafish embryos, with
100% mortality in the first hours of exposure at higher concentrations.
These results indicate that the toxicity observed for zebrafish embryos
is not driven by a pH effect (SM Fig. 4).
Other studies with freshwater microcrustaceans organisms, such as
Daphnia Magna [30] and Daphnia pulex [31], have also reported 100%
toxicity for the biodiesel wastewater evaluated according Standard
Methods 8711-2005. Gonçalves et al. [12] used Vibrio Fischeri as the
model organism to evaluate biodiesel wastewater and also reported
100% mortality, with a residue with a pH equal to 9.7. Costa et al. [13]
also used Vibrio Fischeri and reported 89% mortality for biodiesel
wastewater obtained from a biodiesel factory, with a pH of 2.9.
3.4. Treated biodiesel wastewater toxicity
The toxicity of treated wastewater was also evaluated. FET test
performed with biodiesel wastewater showed 100% mortality of the

88
G.F. da Silva Brito, et al.
embryos up to 1% dilution. Thus, toxicity was investigated only for
dilutions equal to or less than 1% of treated biodiesel wastewater. Fig. 2
presents the results of the FET tests performed with biodiesel waste-
water and treated biodiesel wastewater resulting from photo-Fenton,
solar photo-Fenton and solar photolysis processes. All tests were per-
formed with pH adjusted to pH 7.0.
An important reduction in toxicity with respect to zebrafish em-
bryos was observed for solar photolysis and photo-Fenton treatments,
however solar photolysis showed a very encouraging result since a great
reduction in embryos mortality was achieved under much simpler
conditions. For solar photolysis, no significant difference between the
treated biodiesel wastewater and the control could be observed even at
1% (p > 0.05). For solar photolysis, 96h-LC50 could not be calculated
but it was considered to be higher than 1%. In fact, Costa et al. [13] had
already observed that the use of radiation with no other chemical ad-
dition could reduce the organic load with a reduction in toxicity for this
effluent. However, they did not achieve results as good as ours.
The photo-Fenton treatment showed a 96h-LC50 = 0.175% and the
solar photo-Fenton showed a 96h-LC50 = 0.077%, both with no con-
fidence interval determined. Solar photo-Fenton showed an increase in
toxicity when compared to untreated biodiesel wastewater (96h-
LC50 = 0.118 ± 0.005%). Gonçalves et al. [12] used a photo-Fenton
(UV-A) process combined to flocculation and they also observed an
increase in toxicity. As flocculation is not expected to influence the
toxicity of the medium, the authors suggest that this increased toxicity
could have been related to degradation products. On the other hand,
Costa et al. [13] observed a reduction in toxicity of biodiesel waste-
water using coagulation-flocculation followed by UV-C photolysis. They
reported a change from 89% to 22% in the mortality of Vibrio Fischeri.
Hatching is a critical period of zebrafish embryo development and
has been used as a chronic endpoint in fish early-life stage tests [35]. In
addition, hatching rate is also reported to be a reasonable way to assess
the toxicity in receiving waters [36,37]. Table 3 shows the hatching
rate from day 2 (after 48 h) until day 4 (96 h) in biodiesel wastewater,
biodiesel wastewater submitted to photo-Fenton and in solar photolysis
treatments. Fig. 3 shows an overview of zebrafish embryo hatching (A,
B and C) and developmental abnormalities (D, E and F), compared to
control organisms, after exposure to solar photo-Fenton treated bio-
diesel wastewater at 0.1 and 1.0% dilution.
The biodiesel wastewater, on day 2, promoted the coagulation of
100% of embryos at 1% dilution. So, no hatching was observed. Diluted
samples of biodiesel wastewater presented a premature hatching, sig-
nificantly different from the control (p < 0.05), showing that even at
0.01%, the hatching processes were still being affected by the effluent.
Samples treated by photo-Fenton showed a normal hatching for
0.01 and 0.1% dilutions, but, at 1% no hatching was observed because a
coagulation of 100% of embryos also occurred. Samples treated by solar
photolysis showed a prevalent normal hatching, but only a difference
was observed between at 1% dilution and control, on day 2 (p < 0.05).
Despite this, however all hatched organisms survived in all dilutions.
89
Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90
Fig. 3. Zebrafish embryo hatching and developmental abnormalities after ex-
posure to solar photo-Fenton treated biodiesel wastewater. [A] 72 h control
organism normally developed and hatched; [B and C] 72 h unhatched embryos
exposed to 0.1 and 1% diluted wastewater, respectively; [D] 96 h control or-
ganism normally developed; [E] 96 h hatched larvae exposed to 0.1% waste-
water with severe tail deformities; [F] 96 h unhatched embryo exposed to 1%
wastewater with severe tail deformities.
4. Conclusions
This study demonstrates the importance of the treatment of bio-
diesel wastewater – a neglected residue produced in large volumes
when biodiesel is produced from the alkaline transesterification pro-
cess. Considering a possible increase in the production of biodiesel, the
effluent generated represents an important environmental issue in the
near future due to its high content of organic matter and high Toxicity.
Four oxidative processes have been tested with the objective of redu-
cing environmental damage related to the high organic load of biodiesel
wastewater. Among the evaluated processes, photo-Fenton, solar photo-
Fenton and solar photolysis showed an efficient degradation of the
organic load after 480 min, but the solar photolysis represents the most
promising process, considering its simplicity, viability and ease of
adaptation to large scale. In addition, it does not require the use of any
chemical. Considering the results of the solar photolysis process, bio-
diesel effluents showed about 8% of the organic load resistant to de-
gradation, but this organic residue can be considered non-toxic based
on the decrease of the toxicity of 100% for the Zebrafish embryos after
96 h of test in the dilutions evaluated in the Laboratory.
Ethical standards
The experiments are in accordance with the current laws of the
country where they were performed. The study was approved by the
ethics committee, of the University of Brasilia (reference no 100226/
2014).
Acknowledgements
Brazilian Ministry of Education and Ministry of Science and
Technology of Brazil for the scholarship provided to RO by Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES). CKG for CNPq research grant 305741/2015-2. The English
text of this paper has been revised by Sidney Pratt, Canadian, MAT (The
Johns Hopkins University), RSAdip - TESL (Cambridge University).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.01.
013.
References
[1] U. Biermann, U. Bornscheuer, M.A.R. Meier, J.O. Metzger, H.J. Schafer, Oils and
fats as renewable raw materials in chemistry, Angew. Chem. Int. Ed. 50 (2011)
3854–3871, https://doi.org/10.1002/anie.201002767.
[2] B. Bharathiraja, D. Yogendran, R. Ranjith Kumar, M. Chakravarthy, S. Palani,
Biofuels from sewage sludge-a review, Int. J. Chemtech Res. 6 (2014) 974–4290.
[3] A.C. Ahmia, F. Danane, R. Bessah, I. Boumesbah, Raw material for biodiesel
G.F. da Silva Brito, et al.
production. Valorization of used edible oil, Rev. des Energies Renouvelables 17
(2014) 2–335.
[4] F. Ma, M.A. Hanna, Biodiesel production: a review, Bioresour. Technol. 70 (1999)
1–15, https://doi.org/10.1016/S0960-8524(99)00025-5.
[5] H. Fukuda, A. Kondo, H. Noda, Biodiesel fuel production by transesterification of
oils, J. Biosci. Bioeng. 92 (2001) 405–416, https://doi.org/10.1016/S1389-
1723(01)80288-7.
[6] C.M. Quintella, L.S.G. Teixeira, M.Ga. Korn, P.R. Costa Neto, Ea. Torres,
M.P. Castro, Ca.C. Jesus, Cadeia do biodiesel da bancada à indústria: uma visão
geral com prospecção de tarefas e oportunidades para P&D&I, Quim. Nova 32
(2009) 793–808, https://doi.org/10.1590/S0100-40422009000300022.
[7] E. Goldani, L.A.B. De Boni, C.L.C. Frankenberg, M. Cantelli, Tratamento Físico-
Químico da Água de Lavagem Proveniente da Purificação do Biodiesel, Periódico
Tchê Química 4 (2007) (2017).
[8] S. Kumjadpai, K. Ngamlerdpokin, P. Chatanon, P. Lertsathitphongs, M. Hunsom,
Management of fatty acid methyl ester (fame) wastewater by a combined two stage
chemical recovery and coagulation process, Can. J. Chem. Eng. 89 (2011) 369–376,
https://doi.org/10.1002/cjce.20429.
[9] S.C. Low, G.K. Gan, K.T. Cheong, Separation of methyl ester from water in a wet
neutralization process, J. Sustain. Energy Environ. 2 (2011) 15–19.
[10] I.M. Atadashi, M.K. Aroua, A.R.A. Aziz, N.M.N. Sulaiman, Refining technologies for
the purification of crude biodiesel, Appl. Energy 88 (2011) 4239–4251, https://doi.
org/10.1016/j.apenergy.2011.05.029.
[11] V.B. Veljković, O.S. Stamenković, M.B. Tasić, The wastewater treatment in the
biodiesel production with alkali-catalyzed transesterification, Renew. Sustain.
Energy Rev. 32 (2014) 40–60, https://doi.org/10.1016/j.rser.2014.01.007.
[12] B.R. Gonçalves, A.E.H. Machado, A.G. Trovó, Treatment of a biodiesel effluent by
coupling coagulation-flocculation, membrane filtration and Fenton reactions, J.
Clean. Prod. 142 (2017) 1918–1921, https://doi.org/10.1016/j.jclepro.2016.11.
092.
[13] N.M. Costa, V.M. Silva, G. Damaceno, R.M.F. Sousa, E.M. Richter, A.E.H. Machado,
A.G. Trovó, Integrating coagulation-flocculation and UV-C or H2O2/UV-C as al-
ternatives for pre- or complete treatment of biodiesel effluents, J. Environ. Manage.
203 (2017) 229–236, https://doi.org/10.1016/j.jenvman.2017.07.069.
[14] A.F.D. Schutte, L.S. Motta, L.C. Carvalho, M.A. Leal, M.I.L. Oliveira, P.B. Soares,
P.R.M.F. Costa, M.C. Sant’ana, M.E.G. Rocha, R.B. Gomide, Biofuels Bulletin,
(2017).
[15] N.M. Daud, S. Rozaimah, S. Abdullah, H.A. Hasan, Z. Yaakob, Production of bio-
diesel and its wastewater treatment technologies : a review, Process Saf. Environ.
Prot. 94 (2014) 487–508, https://doi.org/10.1016/j.psep.2014.10.009.
[16] C. Loures, M. Alcântara, H. Filho, Advanced oxidative degradation processes: fun-
damentals and applications, Int. Rev. Chem. Eng. 5 (2013) 102–120.
[17] J.M. Poyatos, M.M. Muñio, M.C. Almecija, J.C. Torres, E. Hontoria, F. Osorio,
Advanced oxidation processes for wastewater treatment: state of the art, Water Air
Soil Pollut. 205 (2010) 187–204, https://doi.org/10.1007/s11270-009-0065-1.
[18] M. Cheng, C. Lai, Y. Liu, G. Zeng, D. Huang, C. Zhang, L. Qin, L. Hu, C. Zhou,
W. Xiong, Metal-organic frameworks for highly efficient heterogeneous Fenton-like
catalysis, Coord. Chem. Rev. 368 (2018) 80–92, https://doi.org/10.1016/j.ccr.
2018.04.012.
[19] M. Cheng, G. Zeng, D. Huang, C. Lai, P. Xu, C. Zhang, Y. Liu, J. Wan, X. Gong,
Y. Zhu, Degradation of atrazine by a novel Fenton-like process and assessment the
influence on the treated soil, J. Hazard. Mater. 312 (2016) 184–191, https://doi.
org/10.1016/j.jhazmat.2016.03.033.
[20] M.J. Lima, C.G. Silva, A.M.T. Silva, J.C.B. Lopes, M.M. Dias, J.L. Faria,
Homogeneous and heterogeneous photo-Fenton degradation of antibiotics using an
90
View publication stats
Journal of Photochemistry & Photobiology A: Chemistry 375 (2019) 85–90
innovative static mixer photoreactor, Chem. Eng. J. 310 (2017) 342–351, https://
doi.org/10.1016/j.cej.2016.04.032.
[21] M. Cheng, G. Zeng, D. Huang, C. Lai, Y. Liu, C. Zhang, J. Wan, L. Hu, C. Zhou,
W. Xiong, Efficient degradation of sulfamethazine in simulated and real wastewater
at slightly basic pH values using Co-SAM-SCS /H2O2Fenton-like system, Water Res.
138 (2018) 7–18, https://doi.org/10.1016/j.watres.2018.03.022.
[22] A. Rubio-Clemente, E. Chica, G.A. Peñuela, Petrochemical wastewater treatment by
photo-Fenton process, Water Air Soil Pollut. 226 (2015), https://doi.org/10.1007/
s11270-015-2321-x.
[23] M. Cheng, G. Zeng, D. Huang, C. Lai, Y. Liu, P. Xu, C. Zhang, J. Wan, L. Hu,
W. Xiong, C. Zhou, Salicylic acid–methanol modified steel converter slag as het-
erogeneous Fenton-like catalyst for enhanced degradation of alachlor, Chem. Eng.
J. 327 (2017) 686–693, https://doi.org/10.1016/j.cej.2017.06.153.
[24] American Public Health Association, American Water Works Association, Water
Environment Federation, W.E. Federation, Standard methods for the examination of
water and wastewater, Stand. Methods 541 (1999), https://doi.org/10.2105/AJPH.
51.6.940-a.
[25] EPA, Method 1664, Revision B, (2010).
[26] OECD, Test No. 236: fish embryo acute toxicity (FET) test, OECD Guidel. Test.
Chem. Sect. Vol. 2 OECD Publ., 2013, pp. 1–22, https://doi.org/10.1787/
9789264203709-en.
[27] I. Grčić, S. Papić, D. Mesec, N. Koprivanac, D. Vujević, The kinetics and efficiency of
UV assisted advanced oxidation of various types of commercial organic dyes in
water, J. Photochem. Photobiol. A: Chem. 273 (2014) 49–58, https://doi.org/10.
1016/j.jphotochem.2013.09.009.
[28] W. Sabaikai, M. Sekine, M. Tokumura, Y. Kawase, UV light photo-Fenton de-
gradation of polyphenols in oolong tea manufacturing wastewater, J. Environ. Sci.
Heal. Part A 49 (2014) 193–202, https://doi.org/10.1080/10934529.2013.838873.
[29] SPSS, Sigma Stat for Windows (Version 3.20), (2004).
[30] G.M. Hincapié-Mejía, D. Ocampo, G.M. Restrepo, J.M. Marín, Fotocatálisis
heterogénea y foto-fenton aplicadas al tratamiento de aguas de lavado de la
producción de biodiesel, Inf. Tecnol. 22 (2011) 33–42, https://doi.org/10.4067/
S0718-07642011000200005.
[31] K.V. Patiño, S.M. Arroyave, J.M. Marín, Oxidación electroquímica y ozonización
aplicadas al tratamiento de aguas de lavado de la producción de Biodiesel, Inf.
Tecnol. 23 (2012) 41–52, https://doi.org/10.4067/S0718-07642012000200006.
[32] A.V. Hallare, P.L.S. Ruiz, J.C.E.D. Cariño, Assessment of Jatropha curcas L.
Biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity
(ZFET) test, Environ. Sci. Pollut. Res. 21 (2014) 6044–6056, https://doi.org/10.
1007/s11356-014-2539-y.
[33] J.N. Holanda, A.P. Maciel, R.L. Santos, Avaliação Ecotoxicológica da Água de
Lavagem da Purificação de Biosiesel de Soja Metílico Utilizando Danio rerio Como
Organismo-Teste, Bol. do Lab. Hidrobiol. 25 (2012) 13–20.
[34] A.J. Hill, H. Teraoka, W. Heideman, R.E. Peterson, Zebrafish as a model vertebrate
for investigating chemical toxicity, Toxicol. Sci. 86 (2005) 6–19, https://doi.org/
10.1093/toxsci/kfi110.
[35] A.F.F.M. Schoots, R.C. Meijer, J.M. Denucé, Dopaminergic regulation of hatching in
fish embryos, Dev. Biol. 100 (1983) 59–63, https://doi.org/10.1016/0012-
1606(83)90200-2.
[36] J. Zha, Z. Wang, Acute and early life stage toxicity of industrial effluent on Japanese
medaka (Oryzias latipes), Sci. Total Environ. 357 (2006) 112–119, https://doi.org/
10.1016/j.scitotenv.2005.04.038.
[37] T. Sisman, U. Incekara, Y.S. Yildiz, Determination of acute and early life stage
toxicity of fat-plant effluent using zebrafish (Danio rerio), Environ. Toxicol. 23
(2008) 480–486, https://doi.org/10.1002/tox.20366.