Research Articles
Research Artides
Determination of Optimal Water Solubility of Capsaicin for its Usage
as a Non-toxic Antifoulant
Cafer T u r g u d , Bi-min N e w b y 2, and Teresa J. Cutright 3.
I Adnan Menderes University, 09100 Aydin, Turkey
2.3Department of Chemical Engineering 2 and Department of Civil Engineering a, The University of Akron, Akron OH 44325-3905, USA
* Corresponding author (tcutrigbt@uakron.edu)
DOh htto://dx.doi.or~l 0.1065/esor2003.12.180
Abstract
Background. Capsaicin, a non-toxic natural product antifoulant,
is an attractive alternative to the currently used toxic metal-
based antifoulants for protecting the submerged surfaces. The
problem associated with conducting capsaicin experiments is
its limited aqueous solubility.
Methods. A procedure was developed to obtain the maximum
capsaicin concentration in water. Capsaicin was first dissolved
in one of the three organic solvents, methanol, ethanol, and ac-
etone. The organic solvent was replaced by gradually adding
distilled water and evaporating the organic solvent.
Results. The best solvent for obtaining highest concentration of
capsaicin in water was ethanol. A maximum capsaicin in water
6400 ppm was achieved by using 16 mg capsaicin dissolved in 0.8 rnl
ethanol followed with the addition of 2.5 ml distilled water in 0.2 ml
increments and then slow evaporation of the solvent.
Conclusion. The concentration obtained using our approach is
well above the threshold concentration of capsaicin in deterring
the marine organism attachments.
Keywords: Antifoulants; biocides; capsaicin; non-toxic anti-
foulant; water solubility
1 Introduction
Biofouling on ship-hulls, oil platforms, pipelines, and har-
bor construction equipment has always posed a major prob-
lem for maritime industries. The attached organisms not only
increase the weight and frictional drag of ships, leading to
reduced speeds and increased fuel consumption, but also
result in corrosion related repairs [1,2]. The annual estimated
cost for removing organisms from ship-hulls, platforms and
pipelines is in billions of dollars. Applying an antifouling
coating to the submerged surface can reduce the biofouling
problem. Over the past several decades, many chemicals,
including organo-mercury compounds, lead, arsenic and
DDT were used as antifoulants [3].
Unfortunately, the most effective metal-based compounds
such as tributyl tin (TBT) are not selective in the organisms
that they kill to reduce the number of attached fouled or-
ganisms. Sensitive marine species such as Nucella lapillus,
ESPR - Environ Sci & Pollut Res 11 (1) 7 - 10 (2004)
9 ecomed publishers, D-86899 Landsberg, Germany and Ft. Worth/TX ~ Tokyo ~ Mumbai 9 Seoul 9 Melbourne 9 Paris
Capsaicin
have exhibited a toxic response to TBT concentrations be-
low 1 ng/1 [4]. There have also been reports of oyster shell
deformities, mutagenic effects in gastropod mollusks, and
accumulation in salmon tissue [5]. As such, many govern-
ments have banned the use of TBT and other metal based
antifoulant coatings [2,4,5].
International bans on the use of metal-based antifoulants have
increased interest in the development of alternative biocides.
Most of the current interest centers on natural compounds
that act as nontoxic repellent such as capsaicin. The active
ingredient in capsaicin is obtained by grinding dried ripe fruits
of Capsicum frutescens L. (Chili peppers) into a fine powder.
Once obtained it can be processed via normal manufactured
processes into repellents or coatings. In 1995, the first capsai-
cin antifouling coating composition was patented [6].
Recent research has documented that capsaicin's effective-
ness is dependent on the concentration used, application
method [attached, in solution, etc.), and the species present.
For instance, when 25 lag capsaicin was coated on a 7,5 cm
x 2.5 cm glass slide, it inhibited the microbial growth of
P. solanaius [7]. In another study using similar concentra-
tions, capsaicin was toxic to Desulvibrious vulgaris and
Geobacter metalreducens [8]. However, capsaicin was not
effective for gram positive bacteria even at aqueous con-
centrations greater than 300 lag/ml [7]. Thus even natural
antifoulants can be toxic depending on the concentration
used and species present.
The primary obstacle for using capsaicin as an antifoulant
at the aforementioned concentrations is its limited water
solubility. Even under saturated conditions, capsaicin has
only slight water solubility (~ 60 mg/l). In patent applica-
tion and research studies mentioned above, capsaicin had to
be predissolved in an organic solvent in order to be easily
processed [9]. For instance, previous studies first dissolved
capsaicin in benzene, which can present its own set of envi-
ronmental concerns [10]. The amount of benzene used was
just as toxic to sensitive microorganisms as TBT based com-
pounds. In addition, benzene could interfere with assays used
to evaluate capsaicin as a natural antifoulant. Therefore the
aim of this study was to determine how to obtain elevated
concentrations of capsaicin into an aqueous environment
without the use of toxic solvent.
7
Capsaicin
2 Materials and Methods
2.1 Reagent sources
Capsaicin was purchased from the Aldrich Chemical Co. The
specific formulation contained 65% capsaicin and 35%
dihydrocapsaicin as active ingredients. Ethanol, methanol, and
acetone were purchased from Fisher Scientificin technical grade.
2.2 Saturated solubility study
The solubility of capsaicin in water was determined by a
saturation study. The saturated solution of capsaicin was
prepared by adding 200 mg of capsaicin and 10 ml distilled
water into a glass vial. The mixture was then subjected to
constant stirring for 1 week under ambient conditions. Af-
ter one week, the stirring was stopped and the vial sealed.
The sealed vial was then stored undistributed for two days
at room temperature to allow the un-dissolved capsaicin to
settle to the bottom. After settling, 200 lal of the superna-
tant was extracted and mixed with an equal volume of pure
acetonitrile. The new solution was then analyzed via High
Performance Liquid Chromatograph (HPLC).
2.3 HPLC method
The HPLC (Shimadzu LC-10AT), equipped with a C-18L
column (Waters Scientific), was operated isocratically with
a 50:50 acetonitrile:water mobile phase. The flowrate and
sample volume was 1 ml/min and 10 tal, respectively.
Calibration curves were used to convert chromatogram ar-
eas to mg/1 of capsaicin. The curves were generated using
capsaicin standards (88000 mg/l). Each standard was pre-
pared by dissolving the required amount of capsaicin in
acetontrile, followed by the addition of an equal volume of
HPLC grade water.
2.4 Experimental approach to optimize aqueous solubility
Capsaicin was added to different volumes of organic sol-
vent to achieve final concentrations ranging from 6.5 lag/ml
to 100 mg/ml. The solvents investigated included methanol,
ethanol and acetone. These solvents were selected since they
pose minimum toxicity to microorganisms at low concen-
trations [11,12]. In some instances, methanol and ethanol
can be used as an auxiliary carbon source for microbial
growth. However, too much of either chemical can alter the
microbial community's respiration mode and/or dynamics.
The experimental approach was first conducted with direct
additions of capsaidin to each solvent. This was followed by
small increments of distilled water with mixing and gentle
heating. The evaporation of solvent before and after all water
additions was evaluated, and the amount of total solution
was measured to estimate the quantity of solvent evaporated.
The optimal rate of water additions was also ascertained.
Details and observations regarding the solution clarity, color,
precipitate formation for each combination were recorded.
3 R e s u l t s and Discussion
3.1 Saturated water solubility
HPLC analysis of the capsaicin standards exhibited two main
peaks at retention times of 12.6 and 18.0 minutes. Other
8 ESPR - Environ Sci & Pollut Res 11 (1) 2004
Research Articles
studies [13,14] have reported that these two peaks corre-
spond, respectively, to capsaicin and dihydrocapsaicin, with
relative peak areas of 66.28 % and 29.13%. Based on the
peaks observed in the HPLC chromatograph for the satu-
rated capsaicin solution, the concentration of capsaicin was
determined to be 60 mg/l.
3.2 Optimization of aqueous capsaicin solubility
As indicated by the saturation study, capsaicin has limited wa-
ter solubility. In order to increase its aqueous concentration,
capsaicin is normally pre-dissolved in an organic solvent that is
miscible with water, and then transferred from the organic so-
lution to the aqueous solution. In this study, three organic sol-
vents, methanol, ethanol, and acetone were used. The solubil-
ity parameters for methanol, ethanol, and acetone were found
to be 29.7 (MJ/m3) 1/2,26.2 (MJ/m3) t:z, and 19.7 (MJ/m3) 1:2,
respectively, whereas water had a solubility parameter of 48
(MJ/m3)1/2.Capsaicin, a stable alkaloid, is an organic compound.
The solubility parameter of capsaicin, estimated using the group
contribution method based on its formula (see Fig. 1), its den-
sity (1.15 g/cm3), its molecular weight (M = 305.4 g/mol), and
the group molar attraction constants [15], is 23.7 (MJ/m3)v2.
By comparing this value to the solubility parameters of metha-
nol, ethanol, acetone, and water, ethanol (AS -- 2.5 (MJ/m3) u2)
appears to be the best solvent for capsaicin, followed with ac-
etone, then methanol. Water apparently is an e~remely poor
solvent for capsaicin with a A~5value of 24.5 (MJ/m3)l~.
o
H3CO N~'~~-'~ CH3
HO~ H " CH3
Fig. 1: Chemical structure of capsaicin
The first set of experiments investigated the dissolved cap-
saicin concentrations used by previous researchers in each
of the three solvents [16]. When capsaicin was dissolved in
pure methanol, ethanol or acetone, neither color changes
nor precipitate formation was present, as summarized in the
first three rows of Table 1. After adding 1 ml of distilled
water, capsaicin still remained in solution. However after
the solvent was evaporated, the solution color turned to
white, indicating a slight phase change. When more distilled
water was added, capsaicin started to precipitate out of so-
lution as evidenced by the formation of insoluble particles
and the experiment was terminated. However it is impor-
tant to note that both acetone and methanol systems had
capsaicin particles after only 1 ml of distilled water was
added. The ethanol system did not form precipitates until
after 3 ml of water had been added, confirming the predic-
tion, based on solubility parameter, that ethanol is the best
of the three solvents to be used for dissolving capsaicin.
The next experimental set investigated the effects of addition
of water on the behavior of capsaicin prior to evaporating the
solvent. The exact procedure starting with an initial 10 mg/ml
capsaicin stock solution is oudined in Table 2. The capsaicin
concentration was diluted via increments of distilled water.
Initially, 0.2 ml increment was used until the desired concen-
Research Articles Capsaicin

Table 1: Solubility of capsaicin in different solvents

Color Particles
EtOH Acet MeOH EtOH Acet MeOH
6.5 mg Capsaicin + 1 ml Ethanol No No No No No No
6.3 mg Capsaicin + 1 ml Acetone No No No No No No
4.5 mg Capsaicin + 1 ml Methanol No No No No No No
1 ml distilled water added No No No No No No
Heated and solvent evaporated W W W 9 No No No
1 ml distilled water added W W W No Yes Yes
2 ml distilled water added W W W No Yes Yes
3 ml distilled water added W W W Yes Yes Yes
EtOH: Ethanol, MeOH: Methanol, Acet: Acetone, W: White

Table 2: Solubility of capsaicin in different solvents with water addition prior to solvent evaporation
Color Particles
EtOH Ac~ MeOH EtOH Aeet MeOH
2 mg Capsaicin + 0.2 ml Ethanol No No No No No No
2 mg Capsaicin + 0.2 ml Acetone No No No No No No
2 rng Capsaicin + 0.2 ml Methanol No No No No No No
0.2 ml distilled water added No No No No No No
0.4 ml distilled water added No W W No No No
0.6 ml distilled water added W W W No No No
1.6 ml water-0.5 ml increments W W W No No No
2.6 ml distilled water added W W W No No No
10.6 ml water in 2 ml increments W W W No No No
15.6 ml distilled water added W W W No No No
25.6 ml distilled water added W W W No No No
45.6 ml distilled water added T T No No No No
65.6 ml distilled water added No No No No No No
Heating and evaporation of solvents No No No No No No
W: White, T: Turbidity, EtOH: Ethanol, MeOH: Methanol, Acet: Acetone

tration, the maximum value in Table 1, was obtained. Then
larger increments of distilled water were added. This was fol-
lowed by solvent evaporation at 40~ with gentle mixing.
Using this approach, the final concentration for ethanol and
methanol was 32 tag/ml (1 pg/ml H = I ppm) in distilled water,
which was too low to be utilized for the subsequent antifoulant
assays to determine the effectiveness of capsaicin on antifoul-
ing behaviors or its effects on microbial activities. As men-
tioned earlier, studies where capsaicin was effective used aque-
ous concentrations ranging from 100 to 1000 ppm. Therefore,
a higher concentration stock solution may be needed to ob-
tain a higher capsaicin aqueous concentration.
In the efforts to achieve higher aqueous capsaicin concentra-
tions, we first attempted to generate a concentrated capsaicin-
methanol stock solution. When 100 mg capsaicin was dis-
solved in 1 ml methanol and followed by 1 ml distilled water,
the addition of water precipitated the capsaicin (shown in
Table 3). The same procedure was repeated with lower
amounts of capsaicin until the addition of 1 ml distilled water
did not facilitate observable precipitants. The highest capsaicin
concentration that did not form precipitates after adding water
was 80 mg/ml. Although this approach did achieve the desired
capsaicin concentration, approximately 12% of the methanol
remained. For small-scale experiments, the impact of the re-
sidual methanol could be easily and cost effectively handled.
However, for large scale applications, this methanol concentra-
Table 3: Solubility of capsaicin in methanol
Color Particles
100 mg Capsaicin No No
+ 1 ml Methanol
1 ml distilled water added No Yes
tion would preferentially increase the amount of methanotrophs,
substantially altering the microbial ecosystem [12].
The viability of ethanol was further evaluated since it would
not increase the amount of methanotrophs. The maximum
aqueous capsaicin concentration that could be obtained from
the most concentrated capsaicin-ethanol stock solution (16 mg
of capsaicin in 0.2 ml) was 40 mg/ml (Table 4a). Afterwards,
more dilute stock solutions, 16 mg capsaicin in 0.4 ml etha-
nol (Table 4b) and 16 mg capsaicin in 0.8 ml ethanol (Table 4c)
were used. For the case of using 0.4 ml ethanol, a total of
0.6 ml distilled water was added in 0.2 ml increments, fol-
lowed by two 0.5 ml additions. As shown in Table 4b, the
first 0.2 ml additions resulted in change in solution clarity
(clear to opaque), but insoluble particles formed only after a
0.5 ml distilled water addition was used. Thus, when water is
added in greater than 0.2 ml increments, it may cause capsai-
cin to precipitate out of solution. In order to verify this specula-
tion, a slow increment (0.2 ml) of water addition to the 16 mg
capsaicin in 0.8 ml was conducted, and a total of 3 ml distilled
water was added (Table 4c). Although there was a color

ESPR - Environ Sci & Poliut Res 11 (1) 2004 9

Capsaicin
Table 4: Evaluation of capsaicin solubility in ethanol
(a) 16 mg Capsaioin + 0.2 ml Ethanol
1 ml distilled water added
(b) 16 mg Capsaicin +0.4 mL Ethanol
0.2 ml distilled water
0.2 ml additional distilled water
0.2 ml additional distilled water
0.5 ml additional distilled water
0.5 ml additional distilled water
(c) 16 mg Capsaicin + 0.8 ml Ethanol
Total 3 ml distilled water in 0.2 ml increments
Evaporation of solvent
W: White, EtOH: Ethanol, MeOH: Methanol, Acet: Acetone
change in the solution upon addition of water, no capsaicin
was precipitated out from the solution as water was being
added. These results indicate that a slow addition of water
to a more dilute capsaicin-ethanol solution, under continu-
ous stirring, would be most favorable for generating more
concentrated capsaicin in water.
Ethanol was evaporated via gentle heating (50~ as the fi-
nal step of the approach to generate a high concentration
capsaicin-aqueous solution. As ethanol was evaporated, the
solution went from clear to slightly opaque. However, cap-
saicin did not precipitate out of solution until the concen-
tration of ethanol was less than 2 wt.%. At 2 wt% ethanol
would not be toxic to most aquatic organisms. Maintaining
the ethanol concentration at 2 wt% would prevent the for-
mation of precipitates while minimizing solvent toxicity.
4 Conclusion
Numerous studies have documented the environmental con-
cerns associated with metal-based antifoulants. The attack on
non-target organisms by TBT-based antifoulant paints have
led to complete international ban on their use by 2008. There-
fore environmentally friendly, natural compounds, such as
capsaicin, have become the preferred alternatives. In this study,
we reported a procedure use to increase capsaicin's water solu-
bility by first dissolving capsaicin in organic solvents, such as
methanol, ethanol, and acetone, then gradually replacing the
organic solvent by adding known amount water and evapo-
rating the organic solvent. Using this approach, the exact
amount of capsaicin in water can be determined. Amount the
three solvents used, acetone was found to re-precipitate cap-
saicin easier than the other two. Methanol was not selected as
the final solvent since it has a greater tendency to change the
microbial population. Overall, the best results were obtained
when ethanol was used as the initial solvent. The optimum cap-
saicin concentration in water was found to be 6.4 mg/ml or
6400 ppm. This concentration was obtained by using a 16 mg
of capsaicin dissolved in 0.8 ml ethanol followed with a gradual
addition of distilled water up to 2.5 ml without precipitating
capsaicin. Then, the ethanol was allowed to evaporate slowly
immediately prior to any precipitation occurs in the solution.
This procedure enabled over 98% of ethanol to be evaporated
to avoid the presence of too much ethanol, which could facili-
tate a change in the population dynamics, thus preventing any
negative impacts of ethanol on the aquatic ecosystem.
10
Research Articles
Color - Particles
No No
W Yes
No No
W No
W No
W No
W Yes/No
W Yes
No No
W No
W No
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Received: April 28th, 2003
Accepted: December 11th, 2003
OnlineFirst: December 15th, 2003
ESPR - Environ Sci & Pollut Res 11 (1) 2004