Considerations on the Toxicity of Brilliant Blue FCF Aqueous Solutions before and after Ozonation

Abstract

The food dyes have a potential toxic effect on aquatic organisms which trigger the necessity of their removal from industrial or urban wastewaters. Many different removal methods were investigated for this purpose, but the ozonation and advanced oxidation processes (AOPs) were successfully applied in this field. However, the majority of studies emphasized that color removal by ozonation process did not report a complete mineralization of the dye and the resulted by-products may have a greater toxicity than the original compound. In this context, the paper presents a comparative ecotoxicity study of the dye Brilliant Blue FCF (BB FCF) before and after ozone treatment. The BB FCF toxic effect, before and after ozonation was investigated on crustacean (Daphnia magna), lethal or inhibitory concentrations for 50% of tested organisms (LC50 / EC50) were used to estimate the effect level. The dye showed no toxicity on crustacean (CL50/CE50>100mg/L) before ozonation. The ozonized solutions presented a high toxicity for crustaceans compared to initial dye due to the by-products occurrence.

Full text

Revista de Chimie
https://revistadechimie.ro
https://doi.org/10.37358/Rev. Chim.1949
Rev. Chim., 71 (4), 2020, 356-365 356 https://doi.org/10.37358/RC.20.4.8075
Considerations on the Toxicity of Brilliant Blue FCF Aqueous
Solutions before and after Ozonation
MARIA MARCVART TIRON1, IRINA EUGENIA LUCACIU2, MIHAI NITA-LAZAR2,
STEFANIA GHEORGHE2*
1Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Analytical
Chemistry and Environmental Engineering, no. 1-7, Polizu Str., 011061, Bucharest, Romania
2National Research and Development Institute for Industrial Ecology ECOIND Bucharest, Biotests- Biological Analyses
Laboratory, 71-73 Drumul Podu Dambovitei Str., 060652, Bucharest, Romania
Abstract: The food dyes have a potential toxic effect on aquatic organisms which trigger the necessity
of their removal from industrial or urban wastewaters. Many different removal methods were
investigated for this purpose, but the ozonation and advanced oxidation processes (AOPs) were
successfully applied in this field. However, the majority of studies emphasized that color removal by
ozonation process did not report a complete mineralization of the dye and the resulted by-products
may have a greater toxicity than the original compound. In this context, the paper presents a
comparative ecotoxicity study of the dye Brilliant Blue FCF (BB FCF) before and after ozone
treatment. The BB FCF toxic effect, before and after ozonation was investigated on crustacean
(Daphnia magna), lethal or inhibitory concentrations for 50% of tested organisms (LC50 / EC50)
were used to estimate the effect level. The dye showed no toxicity on crustacean
(CL50/CE50>100mg/L) before ozonation. The ozonized solutions presented a high toxicity for
crustaceans compared to initial dye due to the by-products occurrence.
Keywords: Brilliant Blue FCF, ozonized by-products, toxicity, aquatic life.
1.Introduction
Synthetic dyes are widely used in many industries such as food, textiles, rubber, paper, plastics and
in other daily used products. About 7.000 to 10.000 different commercial dyes and pigments are
produced annually all around the world. Food manufacturers prefer synthetic dyes, because they
produce a stronger color than natural colorings with a lower cost [1]. Brilliant Blue FCF (BB FCF) is a
synthetic dye component of the triphenylmethane compounds with the molecular formula: C37-H36-
N2-O9-S3.2Na [2]. The most commonly used synonyms in published literature are: Brilliant Blue
FCF, Acid Blue 9, Alphazurine FG, E133, Erioglaucine disodium salt, FD&C; Blue no. 1, etc. [3].
These are used as food dyes in ice-cream, candies, drinks and other sweets.
Also it is often used in cosmetic, textile fields and in different research applications to detect the
water infiltration in soil [4], for microbial fluorescent or blood cell staining [5, 6]. Sometimes, they are
used in medicine for eye surgery and detecting lung aspiration in critically ill patients [7, 8]. In
addition, BB FCF is an inhibitor of the Panx1 channels and it could be useful for the treatment of
Crohn’s, Acquired Immunodeficiency Syndrome (AIDS), melanoma, epilepsy, neurotrauma,
inflammation, stroke and injuries to the central nervous system [9, 10].
Before 1993, the BB FCF dye has been forbidden in eleven European countries but after EU
foundation this has been certified as a safe food additive [11]. The number of BB FCF in Alimentarius
Codex (updated 2019) is E133. In 2017 based on a rat’s intoxication study, the acceptable daily intake
(ADI) for human was established to 0-6 mg/kg body weight [12, 13].
*email: stefania.gheorghe@incdecoind.ro

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In the medicinal uses, BB FCF dye is absorbed only 5% and 95% is excreted. Nowadays there is an
ongoing controversy regarding the safety of this artificial food dye [14]. Numerous studies reported
toxic effects on animals or human such as: convulsion, tumors of gastrointestinal origin and lymphoma
in rodents [8]; increase of hepatic enzymes and bilirubin level in animal model [14]; oxidation of
thyroid peroxidase forming aromatic amines (carcinogens) [15]; purinergic signaling (implicated in
several cellular functions: proliferation of neural stem cells, vascular reactivity, apoptosis and
cytokine secretion, mediating the effects of neural activity during development, neurodegeneration,
inflammation, neuropathic pain and cancer) [10, 16]; attention deficit and hyperactivity in children
[11]; cumulative absorption through lingual mucosa and through skin [17]. An alert of toxicity was
launched by scientists and FDA regarding the BB FCF absorption and its lethal toxicity in three
critically ill patients [18].
The dyes chemicals industry is under Regulation (EC) No 1907/2006 (REACH) [19] and must
meet the criteria concerning the human and animal safety using the standardized methodologies for
testing. The impact assessment of chemicals on the aquatic environment is described in the Regulation
(EU) No 286/2011 [20] amending Regulation (EC) No 1272/2008, for the purposes of its adaptation to
classification, labelling and packaging of chemical substances/mixtures legislation to Global
Harmonized System of United Nations (GHS).
It has been estimated that about 10-15% of all the industrial dyes are released into the wastewater
effluents [18, 21] during the dyeing process. Small amounts of dye can affect aquatic life and the entire
food chain either by direct chemical / biological effect or by reducing the light transmission [15, 21].
Unfortunately, the present technology of WWTP cannot solve this dye issue and new wastewater
treatment technologies are needed to improve the dye removal amount and subsequently to improve
the water quality of the final effluents [22 - 24]. Various methods for BB FCF wastewater purification
have been developed: physical methods - ultra sonication, ultrafiltration, micro- and nanofiltration or
photocatalytic degradation, coagulation - flocculation, sorption, ion exchange membranes,
electrochemical processes; chemical methods - oxidative processes (electrochemical oxidation,
ozonation, advanced oxidation processes - AOPs), precipitation, complexation; biological methods –
aerobic/ anaerobic degradation, use of fungi, algae and microbial fuel cells [14, 21, 25, 26].
The AOPs generating powerful oxidants have emerged as an important and cheaper class of
technologies for the removal of organic contaminants from wastewater, and for remediation of organic
contaminants in polluted soil and groundwater. These oxidation methods use oxidants like K2S2O8,
KBrO3, KIO4, Fenton’s reagent, photo Fenton, H2O2 and ozone [22, 27].
Ozone may oxidize the organic and inorganic compounds from waste waters [27] but the resulted
effluents has been shown to increase the general toxicity of the water for the rainbow trout larvae and
reduce the immune responses in rainbow trout [22, 27-30].
Related to the known structure of the BB FCF dye (Figure 1), a concern is the potential formation
of toxic ozonolysis by-products (Table 1). These by-products would require subsequent post-ozonation
treatment for their removal [26, 31-34].
Figure 1. The Brilliant Blue molecule [35]

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The paper goal was to evaluate the BB FCF ecotoxicity, specifically on aquatic life, before and
after ozonation process. Acute aquatic toxicity [32] is normally determined using a fish model (testing
96-hours, LC50), a crustacean species (testing for 48-hours, EC50) and/or an algal species (testing for
72 or 96-hours, EC50. These species cover a range of trophic levels and taxa and are considered as
surrogate for all aquatic organisms [20].
Our study was focused on the effect of BB FCF on planktonic crustaceans (Daphnia magna). The
efficiency of ozonation process for color removal was evaluate based on the aquatic organism safety.
Table 1. BB FCF ozonation process and its by-products [28, 31-34, 36]
BB FCF oxidation type
Matrix
By-products
Electrochemical oxidation
Aqueous Solution of NaX
Ethanol, Acetaldehyde, Acetic acid, glyoxylic acid,
formic acid
Ozone-based discoloration
Aqueous solution
N-(2-hydroxypropyl) benzene sulfonamide;
5-Sulfooxymethylfurfural;
2-Sulfobenzoic acid;
2-formylbenzenesulfonic acid
Ozone-based discoloration (simple
mass-transfer, non-adjusted
temp/pH)
Waste fruit leather
Carbonyl compounds (hexanal, ethanol,
benzaldehyde, 2-furfural)
Ozone-based discoloration (simple
mass-transfer, non-adjusted
temp/pH)
Wastewater (ozonized fruit leather)
solution / suspension)
Ethanal, propanal, butanal, pentanal, hexanal, octanal,
acetone, 2-heptanone, 2-Furfural,
benzaldehyde
AOP
Wastewater containing food dyes
Aromatic amines, phenolic compounds
2.Materials and methods
2.1Chemicals and equipment’s
The dye BB FCF, analytical grade (CAS 3844-45-9), was purchased from Sigma Aldrich. The
1000 mg/L concentration of the dye stock solution was prepared in distilled water. Ozonation process
was performed on solutions concentrations (prepared from stock solution) in the range of 1 – 50 mg/L
BB FCF. An OZONOSAN Alpha-Plus Generator with medical oxygen was use. The continuous
measurement of ozone concentration (1 – 130 mg O3/L gaseous mixture – g. m) was assured by UV-
Photometer that accessorized the generator. Under a constant oxygen flow rate, the ozone dose was
constant. The batch experiments of 100mL were carried out using HEILDORPH VIBRAMAX 100
orbital shaker. H2SO4 0.1M was use to adjusted the solutions pH. An ORION 290A pH-meter was
used for pH monitoring. The BB FCF final concentrations in the aqueous samples were detected using
a CINTRA 404 UV/VIS Spectrometer. The maximum absorption wavelength of 630 nm was chosen to
be used in the quantitative discoloration of BB FCF.
The laboratory BB FCF toxicity tests with invertebrates (crustaceans as Daphnia) were performed
using reagents and growth media supplied by MicroBiotests Belgium.
2.2 Ozonation process
The experiment of the dye discoloration efficiency using ozonation in term of yield (% RBB) was
performed at ambient temperature using various concentrations of BB FCF, in range of 1 - 50 mg/L.
All solutions were treated with the same dose of ozone (200 mg/L g.m.) for a contact time of 300
seconds, under continuously stirring (200 rpm). The experiment was fulfilled mostly at pH of BB FCF
solution (pH = 7.05), excepted those where the influence of pH was evaluated when the tests were
done at a pH = 4.03. Ozonation of dye samples was carried in glass recipients. Every time a volume of
100 ml of dye solution with different concentrations was added into the glass recipient. Ozone gas
dose was supplied, followed by aeration for 5 min to remove residual ozone.
To evaluate the dye removal efficiency, the following formula was applied:

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%RBB = (1-CiBB/CtBB) x 100 (1)
where
CiBB = the initial dye concentration at time (t) = 0
CtBB = the dye concentration at time (t).
2.3 Ecotoxicity test
The laboratory toxicity tests were executed using dye solutions prepared into a mineral medium, in
the concentration range of 0.1-100 mg/L (starting to a stock solution of 1000 mg dye dissolved in 1000
mL distillated water), to estimate the median effective concentration (EC50) expected to produce a
certain effect of BB FCF in 50% of test organisms, in a given population under a defined set of
conditions. BB FCF - effluents obtained after ozonation process (range of 1 – 50 mg/L) were tested
under various physical and chemical work conditions. The ozonized solutions were tested undiluted
and 50% vol. diluted, with or without adjusted pH using 1 M NaOH or HCl. The median effective
concentration (EC50) was estimated.
The Daphnia magna were directly exposed to the BB FCF solutions in a test batch within a
continuous period of exposure of 48hrs, in accordance with OECD test 202 [37], Table 2.
The test conditions (temperature, pH and dissolved oxygen) were periodically monitored using a
multiparameter WTW tip Multi 350i. The tests were performed in replicates for each tested
concentration and control to ensure statistically relevant results.
Table 2. Toxicity test
Species
Test
Type of test
Endpoint effect
Test period /
incubation
Daphnia magna
OECD 202
DAPTOXKIT F
Static, acute
Mortality / immobilization, LC50 /
EC50
24-48h, 20oC
3.Results and discussions
3.1 Ozonation treatment of BB FCF
Color removal (% RBB) in presence of various experimental conditions was around 96-99% for all
BB FCF concentrations using similar ozone treatment in a neutral or acid pH media (Table 3). In case
of BB FCF concentrations 1, 2.5, 5 and 10 mg/L and acid pH (4.03), the % RBB was ≥ 99.5% (Table 3)
indicating a good discoloration of ozonized solutions [26]. The acute toxicity of all ozonized solutions
was tested in various conditions, such as: pH correction (in the range of 6.5 – 8.5) or 50% vol.
dilutions.
Table 3. Characterization of BB FCF solutions used in toxicity tests and % RBB
BB FCF
Concentration
(mg/L)
Treatment conditions
% RBB
pH
O3
Concentration
(mg/L g. m,)
Contact
Time
(seconds)
1
7.05
200
300
99.92
4.03
200
300
99.94
2.5
7.05
200
300
99.81
4.03
200
300
99.87
5
7.05
200
300
99.68
4.03
200
300
99.74
10
7.05
200
300
98.80
4.03
200
300
99.50
30
7.05
200
300
97.66
4.03
200
300
98.94
50
7.05
200
300
96.82
4.03
200
300
97.04

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3.2 Toxicity of BB FCF dye
The experiments on conventional and alternative test batteries represents a reliable way of
estimation the complex and significant effects of chemicals on the entire food chain [38- 40].
Microbiotests performed with Daphnia or green algae are being applied more and more often to
evaluate the inhibitory effects of chemicals, the current trend in the field of ecotoxicology being the
abandonment or reduced use of vertebrate aquatic organisms (fish) [19, 40, 41, 42].
Daphnia magna (Cladocera) are small aquatic crustaceans commonly called water fleas.
Daphnia was selected for ecotoxicity testing because crustacean are the most sensible aquatic
organisms to pollutants toxicity, particularly useful because of its short life and good and rapid
reproduction. There are still at least two reasons why Daphnia was selected for dye toxicity testing: a)
to avoid any errors of results in the algal growth inhibition test due to the blue color of BB FCF that
may cause interference; b) fish tests supposed a large number of organisms and this is in contradiction
with international recommendations concerning vertebrate use.
All the acute toxicity tests were performed for BB FCF dye solutions in concentration range 0.1-
100 mg/L. The BB FCF aqueous solutions (from 0.1 to 100 mg/L) whiteout any treatment reveled
nontoxic effects for planktonic crustaceans, having only minor lethal/inhibitory effects (5-20%) on
Daphnia magna after 48h of incubation (Table 4). The EC50 >100 mg/l indicate non harmful impact
of the dye on these organisms. Similar results on Daphnia magna were reported for BB FCF between
97 mg/L to >1000 mg/L by the international data bases for chemical registration such as
Ecotoxicology knowledgebase (ECOTOX) [43] and other Environmental Organization [44].
According to Regulation on the classification categories for substances hazardous to the aquatic
environment [20], the BB FCF is classified as nontoxic for crustacean species (Daphnia )(EC50>100
mg/L).
Table 4. Acute toxicity results of BB FCF on Daphnia magna after 48h of contact
Solutions
whiteout treatment (mg/L
BB FCF)
Average of:
Acute effect
(%)
Toxicity
EC50 – mg/L
/ REACH
classification
pH
Dissolved oxygen
mgO2/L
100
7.60
8.62
20
EC50 >100 mg/L
Non toxic
50
7.03
-
20
20
7.35
-
15
10
7.35
-
10
5
7.42
-
10
1
7.38
-
5
0.1
7.32
-
0
Control
(dilution media)
7.14
8.91
0
3.3 Toxicity of BB FCF ozonized solutions
Daphnia toxicity tests were performed for BB FCF solutions treated with ozone in different
conditions (ozone treated solutions pH 7; ozone treated solutions with acidification pH 4; ozone treated
solutions pH 4 with pH correction and 50% vol. diluted ozone treated solutions with pH correction, to
correspond to toxicity test conditions).
Figure 2 presented the results of acute toxicity of BB FCF ozonized solutions on Daphnia. The data
shown an increased toxicity in case of ozonized samples comparing with the untreated BB FCF
solutions. The toxicity results of ozonized samples were 5-10 fold higher than untreated samples. The
ozone treatment corroborated with the acidification process improved the blue color of BB FCF
removal, but increased their toxicity due to the high oxidation that can lead to by-products, potentially
more toxic that the initial compound. The ozonized and acidified solutions at pH 4 reveled a maximum
toxicity (100%) due to acid pH effect on organisms living. Similar results were also reported in other
studies which highlighted an efficient discoloration of the dye and a by-products formation [45-46]. A

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greater ecotoxicity against Artemia salina was observed after ozonation of BB FCF solutions leading
to the conclusion that the occurred by-products are more toxic that their precursors itself [31].
Figure 2. Acute toxicity of ozonized BB FCF solutions
on planktonic crustaceans
The toxicity of ozonized samples decreased when they were 50% diluted, but even so their toxicity
remained higher than untreated solutions (case of 5, 10, 30 and 50 mg/L BB FCF). The same trend of
toxicity was also observed in case of ozonized and acidified solutions with pH correction and 50% vol.
diluted.
BB FCF ozonized solutions and 50% vol. diluted reveled inhibitory /lethal effects between 0% and
100% on Daphnia, the level of toxicity being lower than undiluted samples. The median effective
concentration – EC50(48h) of ozonized and 50% vol. diluted samples was estimated at 4.8 mg/L BB
FCF (Figure 3). This value indicated that BB FCF solutions resulting after ozone treatment have had
acute toxicity effects, with possible long lasting harmful effects to aquatic life.
Our previous studies shown significant toxic effect (80% mortality) of BB FCF solutions in
concentration of 5 mg/L and 10 mg/L ozonized (200 mg O3/L), under stirring condition (200 rpm) and
acidified (pH 4) [26]. The toxicity effects decreased to 45-50%, after diluting these solutions in
proportion of 50% with distilled water. This fact leads us to conclusion that the toxic effects can be
diminished when the ozonized dye solutions do not exceed the concentration of 10 mg/L and are
diluted with water in proportion of 50% (1:1 v/v), in a pH range of 6.5 – 8.5. Also, smaller
concentration of BB FCF (≤ 5 mg/L) subject to ozone treatment and diluted 50% could cause much
lower toxicity to aquatic organisms (10-40% mortality).
510 10
20
50 55
75
100 100 100
60 55
70
80
90
100
0
10
40 45
80
90
50
65
80
100 100 100 100 100 100
0
10
20
30
40
50
60
70
80
90
100
110
1 2.5 5 10 30 50
Effect %
BB FCF concentration (mg/L)
untreated samples ozonized solutions
ozonized solutions acid ified/ pH correction ozonized solutions / 5 0% dilution
ozonized solutions acid ified / pH correction/ 50% dilution ozonized solution ac idified
TOXIC

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Figure 3. EC50(48h) estimation for BB FCF
ozonized solutions 50% vol. diluted
Some studies on BB FCF degradation shown that color removal was not completed by the total
mineralization of dye and the incidence of degradation compounds such as aromatic amines could be a
problem for the living organisms [47]. Considering the Predicted Environmental Concentrations (PEC)
estimated for BB FCF in surface water about <0.1 to 626 μg/L (in accordance with the economic
sector – industrial, food, cosmetics) [44], the estimated toxicity level could be reduced about 100 times
in the field. A PNEC (Predicted No Effect Concentration) value of 0.15 mg/l taking in to consideration
6 species of a taxonomic group [44].
4.Conclusions
The paper presented a comparative ecotoxicity study of the BB FCF dye on Daphnia magna,
before and after the ozone treatment process, meant to eliminate the blue color. The dye at various
aqueous solutions induced no toxicity / inhibitory effects on crustacean species (EC50 >100 mg/L).
The 200 mg O3/L ozone treated solutions of BB FCF highlighted color efficiency removed more
than 90%, but very significant toxic effects on Daphnia magna was observed (perhaps due to the fact
that by-products were more toxic than initial compound).
In order to discharge dye solutions without toxic effects on aquatic life, the ozone treatment of the
BB FCF in concentration ≤10mg/L must be supplemented by other processes such as pH adjustment
and aqueous dilutions greater than 50% vol.
Acknowledgements: The authors thanks for financial support offered by The National Nucleu
Program through contract no 20N/2019, Project code PN 19 04 02 01.
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Manuscript received: 24.12.2019