J. Environ. Eng. Manage., 17(6), 377-383 (2007)
ECO-TOXICITY OF A SOAP COMPONENT (SODIUM OLEATE) AND A SYNTHETIC
DETERGENT COCKTAIL USING GREEN PARAMECIA ASSAYED IN NATURAL
WATER SAMPLES FROM EAST ASIA
Kaishi Goto, Cun Lin, Takashi Kadono, Manabu Hirono,
Kazuya Uezu and Tomonori Kawano*
Graduate School of Environmental Engineering
The University of Kitakyushu
Fukuoka 808-0135, Japan
Key Words: Eco-toxicology, fatty acid salts, green paramecia, water hardness
ABSTRACT
A variety of ciliated and flagellated protozoan species have been used as bio-indicators for ecotoxic impacts of chemicals especially in aquatic environments such as rivers, ponds, lakes and
wetlands. It has not been widely concerned that quality of water drastically alters the toxic actions of
various chemicals against various aquatic organisms including fishes, algae, and other microbes.
Previously, we have proposed the use of natural waters sampled from the environments or synthetic
water preparations mimicking the natural waters in addition to the tests in standard laboratory waters,
when examining the toxicity of certain chemicals in aquatic organisms. Here, we employed green
paramecia (Paramecium bursaria, F1-1b strain) widely habitable in freshwaters such as the rivers,
lakes and ponds, for assessing the acute toxicity of detergents under various water conditions.
Since the outflow of detergents from residential and industrial waste waters into aquatic
environments may have considerable impacts on ecosystems, sodium oleate, a soap component, and
a commercial dish washing liquid were used as two typical model detergents and their toxicities
were assayed in a variety of water samples. Waters were sampled mostly from the rivers and lakes in
Japan, China, and Taiwan (totally 81 samples). LC50 for sodium oleate ranged from 42 to 700 ppm
(w/v) with mean of 240 ± 162 ppm (w/v), while LC50 for the commercial kitchen detergent used here
was constantly as low as around 14-89 ppm (w/v) with mean of 53 ± 11 ppm (w/v). Typical assay
results using waters from three Japanese rivers showed that toxicity of the soap was higher in the upstream water with poor minerals and much lower in the down-stream water rich in minerals. There
was a close relationship between the water hardness and soap toxicity. In contrast, there was no link
between the toxicity of synthetic kitchen detergent and the water hardness. Alteration of the soap
toxicity by water conditions may be due to the cation-dependent detoxification (forming metallic
soaps) as we observed the precipitation of sodium oleate in the hard waters. Among cations,
contribution of Ca2+ to the soap detoxification in natural waters was shown to be much greater than
that of Mg2+. This was further supported by the fact that addition of a Ca2+-specific chelator to river
waters resulted in enhanced soap toxicity.
INTRODUCTION
The toxic impacts of various chemicals to
aquatic and terrestrial organisms have been documented in the ecotoxicology database of the U.S. Environmental Protection Agency [1]. There a wide variety of animals (categorized into mammals, birds,
reptiles, amphibians, fishes, mollusks, crustaceans, insects, spiders, worms, and other invertebrates) and
*Corresponding author
Email: [email protected]
plants (categorized into flowering plants, trees, shrubs,
ferns, moss, algae, and fungi) are covered. To understand the toxicity of chemicals at cellular level, mammalian cell cultures play roles as model biomaterials
[2]. In addition to such model organisms and biomaterials, a variety of ciliated [3,4] and flagellated [3,5,6]
protozoan species have been used as eco-toxicity bioindicators for a variety of chemicals especially in the
aquatic environments such as rivers, ponds, lakes and
J. Environ. Eng. Manage., 17(6), 377-383 (2007)
378
wetlands. Usually the toxic impacts of chemicals to
aquatic organisms such as protozoa habitable in fresh
waters are assayed in the media made up with laboratory waters such as distilled water or ultra-pure water
after addition of some necessary nutrients [4,7]. However, such water conditions hardly reflect the actual
environments surrounding the organisms. Therefore
we have recently proposed the use of a series of
natural waters directly sampled from the various natural basins or a series of synthetic water preparations
mimicking the natural waters (by adding some key
minerals at varying concentrations) in addition to the
tests in standard laboratory waters, when examining
the toxicity of certain chemicals in aquatic organisms
[8-10].
Our previous studies have shown that viability of
two paramecium species (Paramecium bursaria and
Paramecium caudatum) in the presence of eight natural fatty acid salts (soap components) can be drastic
cally altered depending on the quality of the waters
used [8,9]. Tested soap components were sodium and
potassium salts of oleate, CH3-(CH2)7-CH=CH(CH2)7-COONa/K; palmitate, CH3-(CH2)14-COONa/K;
myristate, CH3-(CH2)12-COONa/K; and laurate, CH3(CH2)10-COONa/K. Tested waters were low mineral
waters, tap waters and river waters. All fatty acids
salts showed altered toxicities in Paramecium species
depending on the cation contents, especially that of
Ca2+ [8,9].
P. bursaria known as green paramecia is widely
habitable in freshwaters such as the rivers, lakes and
ponds [11]. As shown earlier [3,12], P. bursaria is
suitable for the study of chemical toxicities in fresh
water environments uniquely representing the behaveiors of both algae and ciliates [13-15], since P. bursaria is a photosynthetic unicellular organism evolutionary acquiring Chlorella-like endosymbiotic green
algae in its protozoan cytoplasm most likely as a consequence of host cell’s adaptation to the photodependent oxidative stress driven by the algal photosynthesis [14,16].
Here again, we employed P. bursaria for large
scale assessment of water-dependent changes in the
acute toxicity of detergents using 81 different waters
mostly sampled from natural basins in Japan, China
and Taiwan.
MATERIALS AND METHODS
1. Organism
As previously reported, a P. bursaria strain F11b was used [8], shown in Fig. 1. The primary culture
of F1-1b was propagated in the medium made up with
a yeast extract-based nutrition mixture EBIOS (1 tablet L-1; Asahi food & Healthcare, Tokyo), after inoculation with the food bacterium Klebsiella pneumoniae,
under a light cycle of 12 h light and 12 h dark with ca.
Fig. 1. Microscopic image of a single cell of mature
green paramecia (Paramecium bursaria). Strain,
F1-1b.
3500 lux of natural-white fluorescent light at 23 °C as
described [8]. The bacterized medium was prepared
by inoculating the medium with K. pneumoniae one
day prior to inoculation with ciliate cells. For toxicity
tests, different waters (see below) were used for preparing the EBIOS media. Prior to preparation of the
EBIOS medium, sterilization of natural waters or
dechlorination of tap waters were carried out by autoclaving (121 °C, 20 min).
2. Detergents
A soap component, sodium oleate was obtained
from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). A synthetic commercial detergent cocktail
“Family Compact” for kitchen-use was obtained from
a local market. The dish washing liquid used here was
chosen from the “Family” brand series of Kao Company. While precise compositions of the detergent
cocktails are not open to public, following information
was provided by the company. This product contains
33% (w/v) of synthetic detergent cocktails composed
of sodium alkylpolyoxyethylene sulfates (AES), alkylhydroxylsulfobetaine, alkylamineoxide, polyoxyethylene alkyl ethers (POAE), and alkylglycoside.
This product is one of the most sold kitchen detergents
in Japan. Therefore it can be used as a typical sample
for studying the eco-toxic impacts of synthetic household detergents to the environments. Detergents were
first dissolved in distilled water. Then various water
samples were used for secondary dilutions up to the
required concentrations.
3. Water Samples
Waters were directly sampled by the authors
from the rivers, lakes, fountain and local tap waters in
Goto et al.: Eco-toxicity of Detergents in Natural Waters
379
mula: 100.1·{[Ca] (mg L-1)/40.1 + [Mg] (mg L-1)/24.3},
where 100.1, 40.1 and 24.3 represent the molecular
weight of CaCO3, the atomic weight of Ca and the
atomic weight of Mg, respectively.
For studying the relationship between the water
hardness and the toxicity of fatty acid salts, a Ca2+
chelator, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,
N',N'-tetraacetic acid (EGTA, Wako Pure Chemical
Industries, Osaka, Japan); CaCl2 (as the source of calcium ion) or MgCl2·6H2O (as the source of magnesium ion) was added to the water samples when required.
5. Toxicity Assay
Fig. 2. Water sampling points in East Asia.
The points where natural waters were sampled are marked with
closed squares. Between August 2005 and July 2006, waters
were sampled from Nagara River (4 points, Gifu and Mie,
Japan), Yamakuni River (4 points, Oita, Japan), Kinokawa
River (3 points, Wakayama, Japan), Ota River and its branch (3
points, Hiroshima, Japan), Jintsu River (Toyama, Japan), Kiso
River (3 points, Gifu, Japan), Ibi River (4 points, Gifu, Japan),
Lake Shinji and Hii River streams (3 points, Shimane, Japan),
Onga River and its branch (6 points, Fukuoka, Japan), Kuma
River (4 points, Kumamoto, Japan), Sendai River (4 points,
Kagoshima and Miyazaki, Japan), Oyodo River (4 points,
Miyazaki, Japan), Yoshino River and its branch (7 points,
Kochi and Tokushima, Japan), Kagano Jingu fountain (Gifu,
Japan), Ki-Lung River (Taipei, Taiwan), Tainan city canal
(Tainan, Taiwan), Cheng-Ching Lake (Kaoshung, Taiwan),
Kaoshung city tap water derived from Chane-Ching Lake
(Kaoshung, Taiwan), Beijing city canal, Xiamen University
campus water (Xiamen, China), Biliu River (12 points, Dalian,
China), and Dalian city tap water originated from Biliu River.
the rural and urban areas in western Japan (61 samples); Beijing, Dalian and Xiamen in China (16 samples); and Taipei, Kaoshung, and Tainan in Taiwan (4
samples) (Fig. 2). In addition, ultra-pure water was
used as the typical laboratory water.
Minerals and ions in the waters were detected
with an inductively coupled plasma-atomic emission
spectroscopy (ICP-AES; Optima 4300 DV, Perkin
Elmer) and an ionic chromatograph (DX-120, Dionex).
Data on Mg and Ca contents were used for estimation
of water hardness. In addition, no presence of toxic
elements such as heavy metals in all samples was confirmed prior to the toxicity tests.
4. Modification of Water Hardness
The water hardness equivalent to the concentration of CaCO3 (mg L-1) was calculated from the for-
For toxicity assays, paramecia in the stationary
phase were washed once with the EBIOS medium
made up with the ultra-pure water and then washed
twice with the EBIOS media made up with different
waters to be used. The tests with P. bursaria were carried out on 12-well microplates. Each well on the
plates was filled with 0.9 mL of EBIOS media harboring 100 paramecium cells plus 0.1 mL of detergent solutions. Then the cells were incubated for 12 h at 23
°C under continuous dark condition, and the number
of living cells was counted at the end of incubation
under a stereomicroscope (SMZ645; Nikon, Tokyo).
RESULTS AND DISCUSSION
Fatty acid salts (often being sodium salts) are
soap components massively used in households and
industries since 18th century [17]. Lately soaps have
been largely replaced by synthetic detergents such as
linear-alkylbenzenesulfonates, AES and POAE [17].
These detergents act as surface-active agents and thus
possibly damage the bio-membranes of aquatic organisms [18]. Therefore, the outflow of detergents as in
residential and industrial waste waters into aquatic environments may have considerable impacts on ecosystems.
In the present study, toxicities of two typical
house-hold detergents representing the two different
types of surface-active agents massively emitted from
the urban area to the water environments, namely a
soap component, sodium oleate and a synthetic commercial detergent cocktail for kitchen-use (a dish
washing liquid) were assayed in a variety of water
samples using P. bursaria known as green paramecia
as a model protozoan species habitable in diversified
fresh waters.
In general, LC50 values for sodium oleate recorded in 81 different waters ranged from 42 ppm
(w/v) (Kamiyanagi Bridge on Kyobashi River, Hiroshima, Japan) to 700 ppm (w/v) (Chen Tun along
Biliu River, Dalian, China) with mean of 240 ± 162
ppm (w/v), while the LC50 values for the synthetic detergent recorded in the same set of water samples were
380
J. Environ. Eng. Manage., 17(6), 377-383 (2007)
Fig. 3. Typical data for the toxicities of two detergents tested in different river waters.
Paramecium cells were incubated with various concentrations of sodium oleate (Na Oleate, open circles) and a kitchen detergent
(Detergent, closed circles) dissolved in river waters for up to 12 h and the survival rates were determined by counting live cells under
a microscope. Water hardness differed from sample to sample. Mineral compositions (hardness as CaCO3, Ca, and Mg, mg L-1):
Oyodo R. up-stream (hardness, 41; Ca, 12; Mg, 3), Oyodo R. middle-stream (hardness, 61; Ca, 16; Mg, 5), Oyodo R. down-stream
(hardness, 304; Ca, 30; Mg, 56), Ibi R. up-stream (hardness, 30; Ca, 10; Mg, 1), Ibi R. middle-stream (hardness, 47; Ca, 16; Mg, 2),
Ibi R. down-stream (hardness, 59; Ca, 18; Mg, 4), Onga R. up-stream (hardness, 2; Ca, 1; Mg, not detectable), Onga R. middlestream (hardness, 126; Ca, 40; Mg, 6), Onga R. down-stream (hardness, 118; Ca, 36; Mg, 7).
constantly as low as around 14 ppm (w/v) (Shuang
Tan along Biliu River, Dalian, China) to 89 ppm (w/v)
(De Sheng along Biliu River, Dalian, China) with
mean of 53 ± 11 ppm (w/v). Since actual content of
surface-active agents in the kitchen detergent cocktail
was 33% of total volumes used, apparent LC50 values
for the detergent cocktail may be 1/3 of the given values.
Typical assay results with water samples from
three Japanese major rivers namely Oyodo River (Miyazaki prefecture, Japan), Ibi River (Gifu prefecture,
Japan), and Onga River (Fukuoka prefecture, Japan)
are shown in Fig. 3. We found that toxicity of the soap
tested was scored to be higher (LC50, 49-180 ppm, w/v)
in the up-stream river waters with low mineral composition (hardness, 2-41 mg L-1) and was scored to be
much lower (LC50, 430-590 ppm, w/v) in the downstream water with higher mineral contents (hardness,
59-304 mg L-1). As expected, the soap toxicities determined in the moderately mineralized waters were
scored as moderate (LC50, 180-440 ppm, w/v). In contrast, the toxicity of the synthetic detergent cocktail
for kitchen-use was likely unchanged in all waters
used (48-56 ppm, w/v in up-stream waters; 50-60 ppm,
w/v in middle-stream waters; 50-52 ppm, w/v in
down-stream waters). This may be reflecting the advantage of the synthetic surface-active agents to be active in various waters, but on the other hand, such
property may be the disadvantage hardly being detoxified by minerals in the water environment.
Since it is likely that water hardness actively
lowers the soap toxicity, we plotted the recorded LC50
values against the hardness of waters used (Fig. 4a).
As expected, there was a tight relationship (R2 = 0.558)
between the water hardness and soap toxicity (Fig. 4a).
However, there was no link (R2 = 0.044) between the
toxicity of synthetic detergents and the water hardness
(Fig. 4b).
Alteration of the soap toxicity by water conditions may be due to the cation-dependent detoxification (by forming metallic soaps) as we observed the
precipitation of sodium oleate in the hard waters [8,9].
As the water hardness was calculated from the Ca and
Mg contents in the waters used, we further examined
the contribution of Ca (Fig. 4c) and Mg (Fig. 4e) to
detoxification of sodium oleate. Interestingly, contribution of Ca to the soap detoxification (R2 = 0.680)
was shown to be much greater than that of Mg (R2 =
0.339). This is consistent with our previous report on
the toxicities of several soap components in P. bursaria, examining the effects of mineral additions (either CaCl2 or MgCl2) [8]. When fatty acid salts are
added to the hard water, insoluble and inert precipitates lacking surfactant activity are formed as a consequence of Ca- or Mg-dependent formation of metallic
soaps by replacing the Na or K attached to the fatty
acid [19]. On the other hand, as expected from the
weak impact of water hardness to the toxicities of synthetic detergents, no relationship between the toxicity
of synthetic detergent and contents of divalent cations
(Ca, R2 = 0.005; Mg, R2 = 0.069) was suggested (Figs.
4d and 4f).
Goto et al.: Eco-toxicity of Detergents in Natural Waters
(a)
(b)
(c)
(d)
(e)
(f)
381
Fig. 4. Relationship between toxicities of detergents and mineral contents in the waters used.
Data for sodium oleate (a,c,e) and a kitchen detergent (b,d,f) are compared. LC50 values for detergents at 12 h recorded in different
waters were plotted against water hardness (a,b), calcium content (c,d) and magnesium content (e,f). Open diamonds, water samples
from western Japan; closed diamonds, water samples from China and Taiwan.
For studying the relationship between the water
hardness and the toxicity of fatty acid salts, EGTA, a
Ca2+ chelator, CaCl2 (as the source of calcium ion) or
MgCl2 (as the source of magnesium ion) was added to
the waters used, since alterations of the soap toxicity
by water conditions were likely due to the cationdependent detoxification mechanism (forming metallic soaps). As the contribution of Ca2+ to the soap detoxification in natural waters was shown to be much
greater than that of Mg2+, effect of a Ca2+-specific
chelator (1 mM EGTA) on detoxification of sodium
oleate was tested using P. bursaria and water samples
from Oyodo River and Onga River, shown in Fig. 5.
Addition of EGTA resulted in enhanced soap toxicity
in most water samples. Enhancements in the soap toxicity were greatest in downstream waters in both rivers. These results further supported the view that calcium in the natural waters readily detoxifies the soaps
depending on its concentration.
To date, impacts of various chemicals to a variety of aquatic organisms in fresh waters, including
bacteria [20], green algae [21], protozoa [3,4,12],
crustaceans [22], and fishes [23], have been documented. However, certain chemicals surely behave
differently in different waters and thus toxicities of
such chemicals in different environmental water con-
ditions must be reconsidered as propounded in our
previous works conducted with ciliates P. bursaria
and P. caudatum and cultured in different waters
[8,9,24]. In addition, we have recently demonstrated
the altered fish toxicity of some fire-fighting chemicals using a tiny fish Oryzias latipes, kept in various
waters including laboratory pure water, local tap water,
fresh river water, brackish river water and sea water
[10,24]. We can conclude that the present work is a
successful large scale demonstration using a number
of water samples collected from the natural basins in
East Asia, further confirming the water-dependent
changes in the toxicity of a typical soap component
but not of synthetic detergent cocktail, using a single
organism capable of living in diverse natural water
conditions.
Lastly, we would like to make a brief note on the
background and the aim of our research activities related to soaps’ eco-toxicological nature. Our group
has been engaged to the development of novel firefighting agents by focusing on the use of eco-friendly
materials [25]. Among the candidate materials, some
sorts of soap-related chemicals were chosen as key
components. In order to estimate the actual impacts of
related chemicals to the eco-systems and the environments, especially their impacts on living organisms
382
J. Environ. Eng. Manage., 17(6), 377-383 (2007)
Fig. 5. Lowered toxicity of sodium oleate to green paramecia after removal of calcium ion with a calcium-specific
chelator.
Prior to toxicity assay for sodium oleate, EGTA, a calcium-specific chelator (1 mM) was added to the waters sampled from Oyodo
River and Onga (Hikosan) River. Open circles, controls (without chelator); Closed circles, EGTA-treated (1 mM).
composing flora and fauna, a ciliate P. bursaria was
chosen as one of the model organisms reflecting the
responses of both algae and protista. Through the present study, our understanding of the eco-toxicity of
fatty acid salts (soap components) and synthetic detergents in relation to water quality was further confirmed.
4.
ACKNOWLEDGEMENTS
This work was supported in part by Shabondama
Soap Company and Furukawa Techno-Materials.
Minerals were assayed with an ICP-AES and an ionic
chromatograph at the Instrumentation Center, The
University of Kitakyushu.
5.
REFERENCES
1. U.S.
Environmental
Protection
Agency,
Ecotoxicology
(ECOTOX)
Database,
http://www.epa.gov/ecotox (2003).
2. Bradlaw, J.A., Evaluation of drug and chemical
toxicity with cell culture systems. Fund. Appl.
Toxicol., 6(4), 598-606 (1986).
3. Kawano, T., T. Kosaka, and H. Hosoya, Impact of
a sulfonylureic herbicide on growth of
6.
7.
photosynthetic and non-photosynthetic protozoa.
In: E. Lichtfouse, J. Schwarzbauer, and D. Robert
(Eds). Environmental Chemistry - Green
Chemistry and Pollutants in Ecosystems. SpringerVerlag, Berlin, Germany, pp. 495-504 (2005).
Miyoshi, N., T. Kawano, M. Tanaka, T. Kadono, T.
Kosaka, M. Kunimoto, T. Takahashi, and H.
Hosoya, Use of Paramecium species in bioassays
for
environmental
risk
management:
Determination of IC50 values for water pollutants, J.
Health Sci., 46(6), 429-435 (2003).
Kawano, T., T. Kadono, K. Fumoto, T. Kosaka,
and H. Hosoya, Flow cytometric analysis of
extreme euglenoid movements in Euglena gracilis:
Effects of a sulfonylureic herbicide and hydrogen
peroxide. ITE Lett. Batter. New Technol. Med.,
5(3), 297-302 (2004).
Duval, E., S. Coffinet, C. Bernard, and J. Briand,
Effects of two cyanotoxins, microcystin-LR and
cylindrospermopsin, on Euglena gracilis. In: E.
Lichtfouse, J. Schwarzbauer, and D. Robert (Eds).
Environmental Chemistry - Green Chemistry and
Pollutants in Ecosystems. Springer-Verlag, Berlin,
Germany, pp. 659-671 (2005).
Takahashi, T., M. Yoshii, T. Kawano, T. Kosaka,
Goto et al.: Eco-toxicity of Detergents in Natural Waters
and H. Hosoya, A new approach for the
assessment of acrylamide toxicity using a green
paramecium. Toxicol. in Vitro, 19(1), 99-105
(2005).
8. Kadono, T., K. Uezu, T. Kosaka, and T. Kawano,
Altered toxicities of fatty acid salts in green
paramecia cultured in different waters. Z.
Naturforsch. C, 61(7-8), 541-547 (2006).
9. Kadono, T., K. Uezu, and T. Kawano, Confirming
the altered toxicities of fatty acid salts in
Paramecium caudatum cultured in different waters.
ITE Lett. Batter. New Technol. Med., 7(6), 606609 (2006).
10. Lin, C., T. Kadono, K. Yoshizuka, K. Uezu, and T.
Kawano, Assessing the eco-toxicity of novel soapbased fire-fighting foam using medaka fish
(Oryzias latipes, Red-orange variety) adopted to
river and sea water conditions. ITE Lett. Batter.
New Technol. Med., 7(5), 499-503 (2006).
11. Kosaka, T., Life cycle of Paramecium bursaria
syngen 1 in nature. J. Protozool., 38(2), 140-148
(1991).
12. Tanaka, M., Y. Ishizaka, H. Tosuji, M. Kunimoto,
N. Nishihara, T. Kadono, T. Kawano, T. Kosaka,
N. Hosoya, and H. Hosoya, A new bioassay
system for detection of chemical substances in
environment using green paramecia, Paramecium
bursaria. In: E. Lichtfouse, J. Schwarzbauer, and
D. Robert (Eds). Environmental Chemistry - Green
Chemistry and Pollutants in Ecosystems. SpringerVerlag, Berlin, Germany, pp. 673-680 (2005).
13. Kadono, T., K. Shiota, M. Tanaka, T. Kawano, T.
Kosaka, and H. Hosoya, Effect of symbiotic algae
on the growth kinetics in dark-grown Paramecium
bursaria. Endocytob. Cell Res., 15(1), 63-70
(2004).
14.Kadono, T., T. Kawano, H. Hosoya, and T. Kosaka,
Flow cytometric studies of the host-regulated cell
cycle in algae symbiotic with green paramecium.
Protoplasma, 223(2-4), 133-141 (2004).
15. Tanaka, M., M. Murata-Hori, T. Kadono, T.
Yamada, T. Kawano, T. Kosaka, and H. Hosoya,
Complete elimination of endosymbiotic algae from
Paramecium bursaria and its confirmation by
diagnostic PCR. Acta Protozool., 41(3), 255-261
(2002).
16.Kawano, T., T. Kadono, T. Kosaka, and H. Hosoya,
Green paramecia as an evolutionary winner of the
oxidative symbiosis: A hypothesis and supportive
data. Z. Naturforsch. C, 59(7-8), 538-542 (2004).
17. Wolf, R., D. Wolf, B. Tüzün, and Y. Tüzün, Soaps,
shampoos, and detergents. Clin. Dermatol., 19(4),
383
353-397 (2001).
18. Kalmanzon, E., E. Slotkin, R. Cohen, and Y.
Barenholz, Liposomes as a model for the study of
the mechanism of fish toxicity of sodium dodecyl
sulfate in sea water. Biochem. Biophys. Acta,
1103(1), 148-156 (1992).
19. Kirsner, R.S. and C.W. Froelich, Soaps and
detergents: Understanding their composition and
effect. Ostomy Wound Manag., 44(3A, suppl.),
62S-70S (1998).
20. Omura, T., M. Kiyono, and H. Pan-Hou,
Development of a specific and sensitive bacteria
sensor for detection of mercury at picomolar levels
in environment. J. Health Sci., 50(4), 379-383
(2004).
21. Aruoja, V., I. Kurvet, H.C. Dubourguier, and A.
Kahru, Toxicity testing of heavy-metal-polluted
soils with algae Selenastrum capricornutum: A soil
suspension assay. Environ. Toxicol., 19(4), 396402 (2004).
22. Kramer, K.J.M., R.G. Jak, B. van Hattum, R.N.
Hooftman, and J.J.G. Zwolsman, Copper toxicity
in relation to surface water-dissolved organic
matter: biological effects to Daphnia magna.
Environ. Toxicol. Chem., 23(12), 2971-2980
(2004).
23. Njiwa, J.R.K., P. Muller, and R. Klein, Life cycle
stages and length of zebrafish (Danio rerio)
exposed to DDT. J. Health Sci., 50(3), 220-225
(2004).
24. Kawano, T., Uses of medaka fish, protozoa, algae
and semi-aquatic plants for assessing the
ecological impacts of aquatic pollutants: A Case
for rice paddy field micro-environment exposed to
fire-fighting agents. Proc. Japan-Taiwan Joint Int.
Symp. Environ. Sci. Technol. Environ. Chem.
Biosci. Manag., Kitakyuhsu, Fukuoka, Japan, pp.
57-60 (2007).
25. Mizuki, H., K. Uezu, T. Kawano, T. Kadono, M.
Kobayashi, S. Hatae, Y. Oba, S. Iwamoto, S.
Mitumune, M. Owari, Y. Nagatomo, H. Umeki and
K. Yamaga, Novel environmental friendly firefighting agent. J. Environ. Eng. Manage., 17(6),
403-408 (2007).
Discussions of this paper may appear in the discussion section of a future issue. All discussions should
be submitted to the Editor-in-Chief within six months
of publication.
Manuscript Received: May 8, 2007
Revision Received: June 4, 2007
and Accepted: June 14, 2007