Published September, 1994
Brilliant Blue FCFas a Dye Tracer for Solute Transport Studies-
A Toxicological Overview
Markus Flury* and Hannes Fltihler
Abstract
Brilliant Blue FCF(C.I. 42090) was foundto be a useful dye tracer
to stain the flow paths of waterin soil media. Being neutral or anionic,
it is not strongly adsorbed by negatively charged soil constituents.
The dye is used in food because its general toxicity is low. However,
to stain the flow paths of waterin soil, fairly large concentrationsare
requiredto ensure goodvisibility of the tracer. Therefore,toxic effects
cannot be entirely excluded, and assessing the environmentalrisk is
a necessity, especially when lidd research is conductedin an environ-
mental context. A literature review was carried out to compile data
on Brilliant Blue FCFtoxicity. The literature suggests that there is
no carcinogenicity or mutagenicity to rodents. Brilliant Blue FCF
does not accumulatein plants or animals, but degrades slowly in the
environment. Fromthe toxicological point of view the dye can be
considered as a suitable and environmentally acceptable tracer for
studyingsolute transport in soil, especially in the lield.
A BROAD VARIETY OF CHEMICALS are used to trace the
travel paths of solutes in the soil and to estimate
their travel times, but their impact on organisms and
their role in environmental compartmentsis rarely docu-
mented in sufficient depth. For studying water movement
in soil columns, soil profiles, or entire fields, conserva-
tive tracers are preferred, because they are more or less
convected without being sorbed by permanent-charged
soil material. Twosuch chemicals are the anions C1-
and Br-. After application or injection of the tracers,
soil or water samples are taken and analyzed for chemical
concentrations. One disadvantage of using C1- or Br-
as tracers is that the path taken by the water in the soil
cannot be identified exactly. Dyes, on the other hand,
can be used to make the flow paths visible.
In ground and surface water hydrology, fluorescent
dyes have been applied successfully for investigating
travel times and flow paths (Wright and Collings, 1964;
Smart and Laidlaw, 1977; Aldous and Smart, 1988).
Fluorescent dyes have also been used in transport studies
in soils. However,the visibility of these dyes is not as
good in soil as in water (Reynolds, 1966; Omoti and
Wild, 1979a, b). Nonfluorescent dyes were used to study
bypass- and unstable flow in field soils and in laboratory
columns (e.g., Boumaand Dekker, 1978; Smettem and
Collis-George, 1985; Kung, 1988), where the staining
of the flow pathwayswas the prime goal of the tracing.
To stain the flow paths of water in soils, an ideal dye
tracer should have the following characteristics: (i)
should be clearly visible in the soil, (ii) it should have
transport properties similar to that of water (i.e., the
retardation should be low), and (iii) the compound itself
and its derivatives should not be toxic, nor should they
Soil Physics, Institute of Terrestrial Ecology, ETHZiirich, Grabenstrasse
3, 8952 Schlieren, Switzerland. Received 22 July 1993. *Corresponding
author (flury@ito.umnw.ethz.ch).
Published in J. Environ. Qual. 23:1108-1112(1994).
1108
induce processes harmful at later stages. Someof these
requirements are mutually exclusive. To be mobile in
negatively charged soil the compoundshould be very
soluble in water, have a small octanol-water partition
coefficient, and be anionic (Corey, 1968). Staining the
soil matrix with dyes requires, however,a certain retar-
dation because part of the chemical must be left behind
on the solid surfaces. To minimizethe retardation without
losing the visibility, the contrast betweenthe stained and
unstained soil matrix should be strong. To warrant a good
visibility in field soil, a dye should therefore preferably be
blue, green, red, or violet. The requirement of low
toxicity eliminates most otherwise suitable dyes, since
many dyes are toxic.
Our objective was to find an acceptable compromise
betweenmobility, visibility, and toxicity. Brilliant Blue
FCF, which had been used by Andreini and Steenhuis
(1990) and Boll et al. (1992) in laboratory experiments,
turned out to be ideal in this respect. Our experience
with infiltration studies in different soils showedthat the
concentrations of Brilliant Blue FCFrequired for good
visibility in soil are 3 to 5 g L-1. In this contribution
important chemical characteristics and toxicity of Bril-
liant Blue FCFare discussed.
General Information on Brilliant Blue FCF
Chemical and Physical Properties of Brilliant
Blue FCF
Table 1 lists the chemical and physical properties of
Brilliant Blue FCF, and Fig. 1 shows its chemical struc-
ture. Brilliant Blue FCFpossesses sulfonate groups that
can dissociate in aqueoussolution. In the solid phase it
can occur as a Na, NH4,Ba, or AI salt, respectively.
The chemical properties and structural formula are given
only for the Na salt. The physical properties of the NH4
salt are very similar to those of the Na salt (Doa, 1987).
Only the Na and NH4salts are permitted for food color-
ing. Note that at low pH the compoundoccurs largely
in the neutral form and as such becomesmore susceptible
to sorption in soil media than the anionic form.
Use of Brilliant Blue FCF
Brilliant Blue FCFis producedin two grades: technical
grade and food grade. The latter must contain more
than 85 % active colorant (FAO/WHO,1966; Marmion,
1991). The major use of the technical grade is as dye
in toilet bowl cleaners (64-75 % of total production vol-
ume in the USA). Brilliant Blue FCF is furthermore
used in ponds to control weeds, to enhance evaporation,
and to improveaesthetics in parks and golf water hazards
(7-14%). Minor uses are: dye for paper (2%), marker
Abbreviations: C.I., color index; LDs0, lethal dose where 50%of the
test animals die; FDA,Food and Drug Administration; ADI, acceptable
daily intake.
 FLURY& FLOHLER: TOXICITY OF BRILLIANT BLUE FCF
Table 1. Somechemical and physical properties of Brilliant Blue FCF (disodium salt).
Property
Common name Brilliant Blue FCF, C.I. Acid Blue 9~’, FD&C Blue No. 1, C.I. Food Blue 2
Chemical name N-Ethyl-N-[4-[[4-[ethyl[(3-sulfophenyl)methyl]-aminolphenyl](2-
sulfophenyl)methylene]-2,5-cyclohexadien-
1-ylidene]-3-sulfobenzene-
methanaminiumhydroxide inner salt, disodium salt
Classification Triphenylmethane dye
Sum formula C37H~N12Na209-Sa
Color index (C.I.) 42090
Purity Technical grade or food grade
Solubility 200 g L-1 (2°, 25°, 60°C)
pK= 5.83, 6.58
<1.0 × 10-4 (at a pHof 5.7)
Thermalstability >160 °C
Toxicity(rat) -t
Acute oral LDso: >5000 mgkg
Molar mass 792.85 g mo1-1
Absorption maximum 630 nm
Absorptivity# 164 Lg- 1 cm- 1
The nameC.I. Acid Blue 9 refers to the nonfoodgradeof the dye.
BASF,1990, Physikalische Chemie, Dr. E. Brunner.
Kow:octanol-waterpartition coefficient.
Ciba Ltd., Basel, safety data sheet No. TS150/A,1989.
Solvent system: water, buffered with 0.01 Mammonium acetate.
for agricultural products (2 %), componentin inks (1%),
textile dye (woodand leather), indicator dye, biological
stain and colorant in miscellaneous products. All these
data on uses and production volumeare taken from Heath
(1987).
The food grade Brilliant Blue FCFconstitutes 15 to
20%of the total production of the dye (Heath, 1987).
Most of the food grade goes into dairy products, cakes and
confectionary (80 %), pet food ( 10 %), dry mix beverages
(5 %), and pharmaceuticals (5 %) (Long, 1987). Brilliant
Blue FCF is further used for making green hues in
combination with C.I. Food Yellow 4 (C.I. 19140, The
Soc. of Dyers and Colourists [1971a, p. 4385]). Concen-
trations of Brilliant Blue FCFin beverages are reported
as 0.5, 1, and 15 mg L-1 (Noonan and Meggos, 1980).
Toxicity
The toxicity of dyes used as tracers has seldom been
considered. Smart and Laidlaw’s (1977) and Smart’s
(1982) toxicological reviews of fluorescent dyes used
water studies are rare exceptions.
Anexcellent short compilation on toxicity of Brilliant
Blue FCF is given in the Federal Register (Federal
Register, 1988). Since Brilliant Blue FCF is a food
additive, manytoxicological studies have been carried
out. There exist several reviews on the toxicology of
food colors and some of them contain short sections on
Brilliant Blue FCF(e.g., de Meuron, 1955; Radomski,
SO3Na
SOaNa
Fig. 1. Chemical structure of Brilliant Blue FCF. This formula shows
the disodium salt. There exist also other salt forms with NH4, AI,
and Ba (The Soc. of Dyers and Colourists, 1971a, p. 4385).
] 109
References
Merck(1989, p. 208-209)
Merck(1989, p. 208-209)
TheSoc. of DyersandColourists (1971b,p. 2778)
Merck(1989, p. 208-209)
TheSoc. of Dyersand Colourists (1971b,p. 2778)
Marmion(1991, p. 71)
BASF¶
BASF¶
Ciba$
Ciba$
Merck(1989, p. 208-209)
Merck(1989, p. 208-209)
Marmion(1991, p. 249)
1974; Drake, 1975; Khera and Munro, 1979). Toxicolog-
ical data can often be divided in two parts: (i) data
acute toxicity refer to a single application of a chemical
to a test species, and (ii) those on the chronic toxicity
refer to repeated exposure of a test species to a chemical
over a prolonged time. In the following acute, chronic,
and ecotoxicity are discussed.
Acute Toxicity
The acute toxicity of Brilliant Blue FCFwas found
to be low. The subcutaneous LDs0 (lethal dose, where
50%of the test animals are killed) in mice was 4600
mg (kg body weight) -I (Federal Register, 1988). The
acute oral LDs0in adult Wistar rats was reported to be
larger than 2000 mg(kg body weight)-1 (Federal Regis-
ter, 1988). Unpublished data from Ciba Ltd. reported
oral LDs0values for rats larger than 5000 mg(kg body
weight)-1 (Ciba Ltd., Basel, safety data sheet No. T5150/
A, 1989). Borzelleca et al. (1990) summarizedresults
genotoxic studies on mammalsand cultured mammalian
cells, and the authors pointed out that the dye is not
genotoxic.
Chronic Toxicity
Several chronic toxicity studies, in which Brilliant
Blue FCFwas subcutaneously injected in rats weekly,
were summarized by Borzelleca et al. (1990). These
authors concluded that Brilliant Blue FCFis not carceno-
genic when administered subcutaneously.
The absorption of Brilliant Blue FCFin rats, dogs,
and guinea pigs is very small (Hess and Fitzhugh, 1955;
Phillips et al., 1980). The dye administered orally to
rats was almost completely excreted in the feces of the
test animals. Brownet al. (1980) confirmedthat Brilliant
Blue FCFis poorly absorbed by the gastro-intestinal
tract of rats. The mean fecal excretion of the dye was
97%. Intestinal absorption was only 0.27%. Iga et al.
(1971) reported that rats excreted 90%of the intrave-
nously administered dye within 4 h.
Feeding studies with rats, mice, and dogs were corn-
1110 J. ENVIRON.QUAL., VOL. 23, SEPTEMBER-OCTOBER
Table 2. Long-termexposure of rats, mice, and dogs to oral administratioa of Brilliant
Dyelevel Time of
Test animal~ in diet¢ exposure
% d
Rats (male) 5 7, 14, 21
5 21
5 21
Rats 3 525
Rats (female) 3 525
Rats 5 730
Rats 2 9OO
Rats (male) 2 900
Rats (female) 1 900
Rats (female) 2 900
Mice 5 730
Dogs 2 365
Whensex is not indicated, then both male and female animals wereused.
Basedon fresh weightof diet.
piled in Table 2. The results of these studies indicate
that Brilliant Blue FCFhas a low chronic toxicity. Ad-
verse effects were reported only for dietary concentra-
tions of 5 % for male rats and 2 % for female rats. The
no-observed-adverse-effect level in the study of Borzel-
leca et al. (1990) was given by dietary concentrations
of 2% (1072 mg[kg body weight day]-1) for male rats,
1% (631 mg [kg body weight day] -1) for female rats,
and 5% (7354-8966 mg [kg body weight day] -1) for
mice.
Based on such results, the U.S. Food and Drug Admin-
istration (FDA) has included Brilliant Blue FCFas
permitted additive for food, drugs, and cosmetics, except
those used around the eyes (Federal Register, 1982,
1988). The joint FAO/WHO Expert Committee on Food
Additives established an acceptable daily intake (ADI)
for humans for Brilliant Blue FCFof 12.5 mg (kg body
weight) -1 (FAO/WHO,1970). The FDAestimated
ADI of 12.0 mg (kg body weight) -1 (Federal Register,
1982).
Ecotoxicity
The ecotoxicology of Brilliant Blue FCF has been
evaluated mostly with studies in aquatic organisms. We
could not find any data of toxicity to soil organisms.
Table 3 summarizes data on toxicity of Brilliant Blue
FCFto aquatic organisms. The test criteria refer mainly
to the concentration where 50%of the test animals are
killed (LCs0). The acute LCso values reported range from
larger than 100 mgL-1 to larger than 3000 mgL-1 for
fish and amphibians.
Tonogai et al. (1980) determined octanol-water parti-
tion coefficients for several dyes, though Brilliant Blue
FCF was not included. A linear correlation between
partition coefficient and toxicity to fish was found for
different azo, triphenylmethane, and indigoid dyes. The
larger the octanol-water partition coefficient, the larger
was the toxicity of the dye to fish. The increasing number
of sulfonic acid groups makes the compoundmore hydro-
philic and would diminish the octanol-water partition
coefficient. As a result, the fish wouldabsorb less dye
1994
Blue FCFin their diet.
Toxicityeffect References
Growthretardation Tsujita et al. (1979)
Reducedfood intake Tsujita et al. (1979)
Noeffect on liver andcecumweight Tsujita et al. (1979)
Noadverseeffects on growth;no reductionof food Mannellet al. (1962)
consumption;no diminutionof food efficiency
Increase of mortality Mannellet al. (1962)
Nomortality; no effect on hematology;no effect on weight Hansenet al. (1966)
of organs
No effect on appearance,hematology,biochemical Borzellecaet al. (1990)
values, and urinalysis; no carcinogenicity
Nomortality; no growth retardation Borzellecaet al. (1990)
Nomortality; no growth retardation Borzellecaet al. (1990)
Mortality; growth retardation Borzellecaet al. (1990)
Nomortality; no effect on hematology,behavior, Borzellecaet al. (1990)
and morbidity; no carcinogenicity
Nomicroscopiclesions; no clinical signals Hansenet al. (1966)
in their bodies. Comparing the value of the octanol-
water coefficient of Brilliant Blue FCF (Kow< 1.0
10-4 at pH 5.7) with the values indicated by Tonogai
et al. (1980) for other dyes, Brilliant Blue FCFwould
have very low toxicity to fish.
Data on phytotoxicity of Brilliant Blue FCFare avail-
able for aquatic plants. A 30-d chronic study reported
a 98 % reduction of the population of aquatic plants at
a dye concentration of 2 mg L-1 (Federal Register,
1988). This effect is due to light absorbance of the dye
in the red band of the electromagnetic spectrum which
is used for photosynthesis. Brilliant Blue FCFis therefore
used as an algicide in ponds to control aquatic weeds.
Concentrations typically used are 1 mg L-1 (Federal
Register, 1988). The enhancement of light absorbance
due to color addition to water is also exploited to enhance
evaporation of water.
Degradation
Dyes can decomposeby chemical degradation, photo-
degradation, and bio(chemical)degradation. Biodegrada-
tion of colorants takes place slowly, since unfortunately
most synthetic dyes are xenobiotic (Clarke and Anliker,
1980; Zollinger, 1991). Dyes are designed not to be
readily biodegradable. The natural system of microor-
ganisms in rivers and lakes does not contain the enzymes
needed to degrade such compoundsunder aerobic condi-
tions (Zollinger, 1991).
Tonogai et al. (1978a) studied the biochemical decom-
position of Brilliant Blue FCF, under aerobic and anaero-
bic conditions. They mixed 750 mLof sludge with 250
mLof 23.8 g L-1 dye solution and sampled the mixture
daily for measurements. For the aerobic condition, the
mixture was bubbled with air. The chemical decomposed
very slowly under aerobic conditions; only 5% of the
dye was decomposed after 10 d of culture. Brilliant
Blue FCF decomposed more rapidly under anaerobic
conditions; after 6 d the dye content decreased by 25 %.
Photochemical degradation of Brilliant Blue FCFin
aqueous solution can occur in surface waters, such as
lakes or rivers. For most dyes such degradation is likely
 FLURY& FLf0HLER: TOXICITYOF BRILLIANTBLUE FCF 1111

Table 3. Toxicity of Brilliant Blue FCFto aquatic organisms.

Test organisms
Test criteria Exposure Effect
Scientific name Commonname~" "effect" time concentration References
% h -t
mg L
Invertebrates
Daphniasp. Water flea 50%Mortality 48 >1000 FederalRegister (1988)
Daphniacarinata Water flea 50%Mortality 3 >100 Yasuhiro (1984)
Sympetrumfrequens Dragonfly 50%Mortality 48 >100 Yasuhiro (1984)
lndoplanorbis exustus - ~ 50%Mortality 48 >100 Yasuhiro (1984)
Sewagebacterias Reduction
of activity - >300 Ciba (1989)§
Amphibians and Fish
Bufo bufo japanicus Commontoad (Japanese) 50%Mortality 48 >100 Yasuhiro (1984)
Ranabrevipoda porosa - 50%Mortality 48 >100 Yasuhiro (1984)
Cyprinus carpio Carp 50%Mortality 48 >100 Yasuhiro (1984)
Poecilia reticulata Guppy No mortality 48 >500 Ciba (1989)§
Oryziaslatipes Ricefish Nomortality 48 >3000¶ Tonogaiet al. (1978b)
Common namestaken from Grzimek (1970).
Not available.
Ciba Ltd., Basel, safety data sheet No. TS150/A,1989.
¶pH 7.

to progress very slowly because synthetic dyes are de- Conclusions

signed to be very stable to light (Porter, 1973; Pagga
and Brown, 1986; Zollinger, 1991). A field study on
the use of the chemical as an algicide in ponds suggested
that Brilliant Blue FCFhas a degradation half-life of up
to 2 mo (Lynch, 1987). The mechanism of degradation
is probably photolysis. Noonanand Meggos(1980) men-
tioned that the light stability of Brilliant BlueFCFin food
is good. The photostability of the dye on pharmaceuticals
seems to depend on the composition of the background
material (Hajratwala, 1974). As regards photostability,
no general conclusion can be drawn from the literature.
Brilliant Blue FCFin aqueous solution is decomposed
when exposed to strong alkali (Dolinsky, 1955). The
resulting decomposition products are disulfonated dyes,
which are formed by the separation of one sulfonic acid
group or one sulfonated benzyl group from Brilliant Blue
FCF(Dolinsky, 1955; Stein, 1969).
Recommendation for Use as Tracer
The results of the above-mentioned studies show that
Brilliant Blue FCFhas low toxicity. Acute effect concen-
trations to aquatic organisms are larger than 100 mg
L-1 . However, since in solute transport studies larger
concentrations (3-5 g -~) are r equired t o e nsure t he
visibility in the soil, the application of the chemical
cannot be considered as harmless. Due to its long half-
life, especially whenprotected from sunlight the chemical
should be applied with caution. To minimize environmen-
tal risks in the field, the dosage of Brilliant Blue FCF
should be selected such that final concentrations in water
systems-after possible dilution with precipitation or
-~.
groundwater-will be less than 1 mg L
Dyestuffs are readily detectable in water, even in con-
centrations much less than 1 mg L-1. Hence, even at
concentrations, where no toxic effects are to be expected,
the dyestuff can be detected by eye. Therefore, not only
toxicity, but also aesthetic considerations have to be
taken into account (Clarke and Anliker, 1980). Brilliant
Blue FCFappears to be much less toxic than other dyes
typically used in field studies. Therefore, it could be
used in preference in the interest of environmentalsafety.
Anideal color tracer for staining flow paths of water
in soil should meet three criteria: (i) the chemicalshould
be easily visible in the soil, (ii) it should be readily
mobile, and (iii) it should not be toxic. Brilliant Blue
FCFmeets all these criteria. Its blue hue is easily visible
in soil and the dye is moderately mobile in the soil,
since it is neutral or anionic.
The toxicity of Brilliant Blue FCFis low. The acute
toxicity of Brilliant Blue FCFto mammalsranges from
larger than 2000 to larger than 5000 mg (kg body
weight)-~ for rats. Several studies indicated that the dye
is not genotoxic. Rats, mice, dogs, and fish were found not
to accumulatethe dye. No-observable-effect-concentrations
range from 631 mg(kg body weight)-m for female rats to
8966 mg(kg body weight)- ~ for mice. The ADIfor human
is given as 12 mg (kg body weight) -m. The short-term
effect concentrations of Brilliant Blue FCFon aquatic
-~.
organisms are reported to be larger than 100 mg L
Nothing is knownabout the toxicity of Brilliant Blue
FCFto soil organisms. The decomposition of Brilliant
Blue FCFis apparently mainly by photodegradation.
The concentrations required for visibility of the dye
in soil are several orders of magnitude larger than the
toxicity limits. The total amount of the dye should be
selected such that final concentrations in water will be
below 1 mg L-~. However, among several dyes used in
transport studies, Brilliant Blue FCFis one of the least
toxic compoundsand therefore suitable for field and
laboratory experiments. With our knowledgeof its toxi-
cology, Brilliant Blue FCF can therefore be recom-
mendedfor use as a dye tracer in solute transport studies.
Despite its favorable toxicological properties, massive
use of Brilliant Blue FCFin natural systems may pose
some problems. From these concerns we extrapolate that
a toxicological appraisal of other tracers used under the
flag of environmental research would be justified.
Acknowledgments
Wethank Ciba Ltd., Basel, whosupported this study and
whosupplied the Brilliant Blue FCF.Wealso thank Dr. R.
Anliker and Dr. E.A. Clarke, Ecological and Toxicological
1112 J. ENVIRON. QUAL., VOL. 23, SEPTEMBER-OCTOBER 1994

Association of the Dyestuffs Manufacturing Industry, ETAD,

Basel, who gave us helpful recommendations for our search
for a suitable dye tracer, and R. Webster, visiting professor
at our institute, who helped us with the English.