Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014.

Study on Relationship Between

ISSN an
0126-2807
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.
Volume 9, Number 3: 169-174, September, 2014
T 2014 Depar tment of Envir onmental Engineer ing
SepuluhNopember Institute of Technology, Surabaya
& Indonesian Society of Sanitary and Environmental Engineers, Jakarta
International peer-reviewed journal Open Access https://www.trisanita.org/jases

This research paper is licensed under the Creative Commons Attribution 3.0 Unported License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

STUDY ON RELATIONSHIP BETWEEN AN ORDINAL SCALE TOXICITY INDEX

LT50-1 AND A RATIO SCALE TOXICITY INDEX LDR50 IN RIVER BASINS
RIYANTO HARIBOWO1,2*, MINAMI YOSHIMURA3, ARIYO KANNO1 and MASAHIKO SEKINE1

1Graduate School of Science and Engineering, Yamaguchi University, Ube, 755-0096, Japan.
2Department of Water Resources Engineering, Faculty of Engineering, Brawijaya University, Malang,
65145, Indonesia.
3Higashi Ward Office, Hiroshima City Hall, Hiroshima, 732-8510, Japan
*Corresponding Author: Phone: +81-90-64021977; Fax: +81-836-85-9019; E-mail: riyanto@yucivil.onmicrosoft.com

Received: 24th March 2014; Revised: 23rd June 2014; Accepted: 30th June 2014

Abstract: The purpose of this study isto show the relationship between an ordinal scale
toxicity index LT50-1and a ratio scale toxicity index LDR50. One water sample per site was
taken during June-December 2012, between 9AM-12PM with the assumption that
household waste was released. Based on the results, minimum 2-3 grades of LT50-1value
is needed to obtain ideal condition to state the existence of relationship between LT 50-1
and LDR50. To obtain value LT50-1 in the river with excessively high toxic or too low trial
and error is needed by adding up hours of observation or by making a lower or higher
concentrate.Finally, equation obtained for the relationship between LDR 50 and LT50-1 is y
= 0.1752x with R = 0.9306. Furthermore, for better accuracy a more profound study is
required. In future work, more data will be collected and identify the land use areas for
each catchment still require to obtain accurate results. However, with anaccurate
equationresult, it can beused for calculating toxicity of unknown concentrations of toxic
compounds; which can subsequently be used to estimate toxic effects in organisms at
any time of exposure for any level of concern.

Keywords: Lethal dilution rate (LDR50), median lethal time (LT50-1), toxicity index

INTRODUCTION

There are many toxicity test methods which have been recommended by ISO, OECD,
USEPA, and other international or national standard organizations. Most of the methods were
established to measure the toxicity of pure single chemical, but not for unknown environmental
water samples with complex components [1, 2]. However, even if the toxicity of environmental
sample is tested, there is no guidance on how to evaluate the water quality in terms of protection
of aquatic living organisms. One effective way for assessing the aquatic safety of water samples
is to expose them to aquatic organisms directly, a method called bioassay [3]. Fish as secondary
or advanced consumer in aquatic food chain, is popularly selected as toxicity test species in
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Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014. Study on Relationship Between an
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.

scientific researches and environmental management [4]. The Ministry of Environment of Japan
collected ecotoxicity data and compared the sensitivities of Japanese Medaka (Oryzias latipes)
with other six fish species recommended by OECD, and the results indicated that the sensitivity of
Japanese Medaka was equal to or a little higher than others surveyed fish species [5,6].
Toxicity test using Medaka early fry and 100-fold concentrated water were proposed to obtain
result quickly and as quantitatively as possible. Conducted only 100-fold concentrated and 48-
hours test and it disclosed toxicity that is the inverse of median effect time and median lethal time
(ET501, LT501). ET501 and LT501 are used instead of EC50 and LC50 scale [7]. Although this
method had an advantage in reducing the amount of time and sampling needed to perform
toxicity tests, it also had a disadvantage that it cannot be handled as concentration. In this
research, we needed an index which can be treated in the same way as concentration.
From this reason, we expressed the toxicity as a lethal dilution rate (LDR50). LDR50 is the
inverse of lethal concentration rate (LCR50) which Liu et al[8] proposed, and defined as the
dilution rate at which 50% of fish survive the acute toxicity test.There, in this research, will be
discussed about the relationship between an ordinal scale toxicity index LT50-1 to a ratio scale
toxicity index LDR50 and show an index for calculating toxicity of unknown concentrations of toxic
compounds in the same characteristic area, which can subsequently be used to estimate toxic
effects in organisms at any time of exposure for any level of concern.

MATERIALS AND METHODS

Study Area
During June December 2012, water samples were collected from three rivers in Japan
which have majority catchment area is residential area (Figure 1). Data taken between 9AM -
12PM with the assumption that household waste was released. First area was located in M
river, 9 point Samples were taken from this river. The river function is to accommodate the flow of
rain water and household waste from the area around the river. The second area is located in Y
river. Flood risk has been increased because the middle zone of the basin has been urbanized
rapidly in recent three decades. The third area was chosen from Z river, where residential,
commercial and industrial sites are heavily concentrated on these regions. Pure chemical
(Nonylphenol and Triclosan) was used as a comparison. From the literature study it is known that
there is a toxic in Z river for both parameters [6]. Acute toxic levels in the Z river for
Nonylphenol is 0.25 ppm, it has exceeded the allowable threshold of 0.24 ppm. While Triclosan is
equal to 0.013 ppm from 0.67 ppm allowed.

Acute Toxicity Test

For LT50-1 acute toxicity test is based on Yamashita [7]. For LDR 50, 10 L of river water was
filtered with 1-m glass filter. Two set of Sep-Pak Plus PS-2 cartridges were set in series [9].
Hydrophobic organic matter was adsorbed at 10 mL.min-1 for each 5 L sampling water, and
desorbed from each cartridge in 10 mL of acetone. Air was injected into the cartridge with a
syringe to drive out the space water. 40 mL acetone solution will be generated, 36 mL will be
used to acute toxicity test and 4 mL will be used for analysis of GC/MS. A 36-mL volume of
acetone solution was evaporated to 200 L under a purge of nitrogen gas. The acetone solution
was diluted to 50 mL with carbon treatment water and then separated into two 25mL portions. In
toxicity test based on Liu et al[10], organic toxicants were 10, 20, 50 and 100-fold concentrated
from the sample. The lethal effect was observed by exposing every ten individuals of 48-72 hours
old larval Medaka to 25 mL of each solution for 48 hours. When there was a striking difference in

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Journal of Applied Sciences in Environmental Sanitation, 9 (3): 169-174.
Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014. Study on Relationship Between an
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.

test results between the two solutions, the test was considered a failure. Toxicity analysis was
calculated using the Probit method [7].

Fig. 1: Basin Areas of Rivers M, Z, and Y

RESULTS AND DISCUSSION

LT50-1 result from sample water

LT50-1 value which will be discussed here is the LT50-1 values at 100 fold condition. Yamashita
[7] explained that ratio of clear stream benthic animalsharply decreased over 0.25 of LT501 and
tolerant fish becomes dominant over 0.3 of LT501. Whereas, average LT50-1 values for M, Y
and Z are 1.6, 0.07 and 0.07 respectively (Table 1). If compare with index from Yamashita [7],
this indicates that the average of safety biological condition on M river is shown high value,
meanwhile in Y and Z river are still in good enough condition, although there is still toxicity
content in water.
At M river, LT50-1 values in the upper stream (M1) were 2; this suggests that the toxic
content was quite high. LT50-1 values for tributaries at the point of M2 and M3 are also very high in
the amount of >2. High toxicity values occurred due there are areas in the upstream of M river
are not covered with wastewater treatment system. Subsequently after going through a process
of purification, the LT50-1 values at downstream it was getting improved at 0.333, 0.095 and 0.014
on the M4, M5 and M6. The presence of sewerage system in this area and decomposition
process in river water has also led to a decrease in the value of LT 50-1. At the point M7, LT50-1
values increase to 0.047, this was probably due to the water coming from the M8 and M9 with
LT50-1 values of 0.019 and >2. At the point of M9, LT50-1 values was high, it was possible since the
industries that threw their waste without prior good treatment.
Sampling point location of Y1 is 96 m above the sampling point of Y2 and Y3. The water
sampling period of Y3, Y4 and Y5 was in September, and Y1 and Y2 in November. Y3 had high
toxicity compared to the others samples. Before toxic substances reach the water sampling point,
decomposition occur, since the basin area of Y3 if compared to Y5 is narrow, the toxic
substances will not completely be decomposed, and it is believed to cause high toxic. The toxicity
of Y3 is higher than Y1, The existence of farmland area probably contribute to toxicity appears,
because in September the agricultural activity was more active.

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Journal of Applied Sciences in Environmental Sanitation, 9 (3): 169-174.
Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014. Study on Relationship Between an
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.

LT50-1 values for the Z river is 0.07 which indicates that there are toxic substances in the
river. With the condition that Z River has high BOD value (5 mg.L-1) especially at the
downstream area [6], toxic conditions in Z river is not too big. Based on literature the high BOD
condition in Z river was not comparable with LT50-1 values which were quite high. In the pure
chemical nonylphenol and triclosan, toxic contained is very high at 100 fold.

Table 1: Result of LT50-1 and LDR50 of the sampling water

LT50-1*
Sampling point LDR50
(10 fold) (20 fold) (50 fold) (100 fold)
M1 0.07 0.02 0.06 0.29 2.00
M2 1.00 2.00 2.00 2.00 2.00
M3 0.09 0.01 0.38 2.00 2.00
M4 0.02 0.01 0.02 0.01 0.33
M5 0.02 0.01 0.02 0.02 0.10
M6 0.01 0.01 0.02 0.02 0.01
M7 0.01 0.01 0.01 0.01 0.05
M8 0.01 0.01 0.01 0.01 0.02
M9 1.00 0.61 1.12 2.00 2.00
Y1 0.02 0.01 0.01 0.01 0.06
Y2 0.02 0.01 0.01 0.01 0.05
Y3 0.05 0.01 0.02 0.04 0.09
Y4 0.02 0.01 0.01 0.02 0.09
Y5 0.01 0.02 0.01 0.01 0.02
Z 0.01 0.01 0.01 0.02 0.07
Nonylphenol 0.03 0.01 0.02 0.43 2.00
Triclosan 0.11 0.05 0.22 0.99 2.00
*The reliable range for LT50-1 value is between 0.02-2.0

Relationship between LT50-1 and LDR50

From various concentrations of toxicity test (10, 20, 50 and 100 fold), not all of them yielded
LT50-1 or LDR50value, this is because of the content of toxic condition of the river is very low or its
toxic content is very toxic high (Table 1). Ideally, for optimum results in determining the
relationship between LT50-1 and LDR50, at least there are should be obtained minimum 2-3 grade
of LT50-1value in each sample taken. From the data obtained for the river whose toxic conditions
were low, the value LT50-1 on average obtained only at 100 fold.
In this research to gain minimum 2-3, of LT50-1 values, for the observations of sample Y1, Y2,
Y3, Y4, Y5, M1, M2, M3 and M4, previously observed only 1,2,3,6,12,24 and 48 hours, added
with observation at 0.5 period of time. Hopefully, with the addition of hour of observation, the
change happening to the condition of fish of river with high toxic will be known quickly. It turned
out that the results obtained from the sample Y1, Y2, Y3, Y4, Y5, M1, M3 and M4, did not match
as expected, due to the condition of the river which does not have a high toxic. But for M2,
despite hours of observations were added, still cannot get value of LT 50-1 due to the extremely
high toxicity.
M river with a total sample of 9 points, only 8 units of LT 50-1 values was obtained. For M4,
M5, and M7, LT50-1 values could be obtained only on the condition of 100 fold. As for the M2, M6
and M8 no LT50-1 values was obtained , because the toxic conditions was extremely low for M6
and M8, and otherwise for M2 have very high toxic. For M9 with high toxic conditions, LT 50-1

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Journal of Applied Sciences in Environmental Sanitation, 9 (3): 169-174.
Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014. Study on Relationship Between an
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.

values were obtained at 10 and 20 fold. In the Y River, from 5 points taken, at Y3 the LT 50-
1values were obtained as much as 3 units and consecutively as much as 2 point of LT 50-1 values

for Y4 and 1 point of LT50-1 values for Y1, Y2 and Y5. As for the I river because of the low toxic
conditions can only 1 point of LT50-1 value could be obtained. For nonylphenol only one LT50-1
values could be obtained namely in the condition of 50 fold. While the LT 50-1 value for triclosan
could be obtained from 10, 20 and 50 fold. LT50-1 value of nonylphenol and triclosan for 100 fold
were very high. In this pure chemistry it needed to be studied further with regard to the dilution
process, especially for nonylphenol.
In Figure 2 by using the linear method, it can be seen that the relationship between the two
parameters was obtained by the equation y = 0.1196x with R = 0.6152. Possibly it was due to the
work mechanism which was independent from toxicity, for pure chemicals (nonylphenol and
triclosan) it looked deviating from the existing plot. Based on these results, pure chemicals
excluded from the analysis. Equation obtained for the relationship between LDR 50 and LT50-1 by
using sampling point that have more than one LT50-1 value (M1, M9, Y3 and Y4) is y = 0.1752x
with R = 0.9306 (Fig. 3). And equation for relationship between LDR50 and LT50-1 for M river is y
= 0.1562x with R = 0.8741 (Fig. 4). On Y river using linier method the equation obtained is y =
0.3981x with R = 0.5759 (Fig.5).Furthermore, for better accuracy a more profound study is
required. In future work, more data will be collected and identify the land use areas for each
catchment still require to obtain accurate results. However, with an accurate equationresult, it can
be used for calculating toxicity of unknown concentrations of toxic compounds; which can
subsequently be used to estimate toxic effects in organisms at any time of exposure for any level
of concern.

Fig. 2: Relationship between LT50-1 and LDR50 using all Fig. 3: Relationship between LT50-1 and LDR50 using
LT50-1 value sampling point that have more than one LT 50-1 value

Fig. 4: Relationship between LT50-1 and LDR50 for M Fig. 5: Relationship between LT50-1 and LDR50 for Y
river river

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Riyanto Haribowo, Minami Yoshimura, Ariyo Kanno and Masahiko Sekine, 2014. Study on Relationship Between an
Ordinal Scale Toxicity Index LT50-1 and a Ratio Scale Toxicity Index LDR50 In River Basins.

CONCLUSIONS

Based on the study above, it is minimum 2-3 grades of LT50-1value is needed to obtain ideal
condition to state the existence of relationship between LT 50-1 and LDR50.To obtain value LT50-1 in
the river with excessively high toxic or too low trial and error is needed by adding up hours of
observation or by making a lower or higher concentrate. Finally, equation obtained for the
relationship between LDR50 and LT50-1 is y = 0.1752x with R = 0.9306. It shows a significant
value of R2, but with a small sample, it might be difficult to obtain statistical evidence of strong
relation.Furthermore, to obtain more accurate results for the relationship between LT50-1 and
LDR50, more data and identify the land use areas for each catchment of the river still
require.Thisresearch is now in progress.

References
1. ECETOC. Environmental hazard assessment of substances. ECETOC Technical Report, vol. 51.
Brussels: European Center for Ecotoxicology and Toxicology of Chemicals; 1993. 92 pp.
2. US Environment Protection Agency. Methods for measuring the acute toxicity of effluents and
receiving waters to freshwater and marine.
3. Wei D B, Kisuno A, Kameya T, Urano K, 2006. A new method for evaluating biological safety of
environmental water with algae, daphnia and sh toxicity ranks. Science of the Total
Environment, 371(1-3): 383390.
4. Zha J, Wang Z., 2005. Acute and early life stage of toxicity of industrial effluent on Japanese Medaka
(Oryzias latipes). Science of the Total Environment 357 (2006) 112 119.
5. MOE of Japan. Results of eco-toxicity tests of chemicals. Tokyo, Japan: Ministry of the Environment
in Japan; 2003. http://www.env.go.jp/chemi/sesaku/02.pdf, 06/06/2006.
6. MOE of Japan. Comparison on ecotoxicities among different test species (in Japanese). Tokyo,
Japan: Ministry of the Environment of Japan; 2002.
http://www.env.go.jp/council/05hoken/y053-02/ref03.pdf, 06/06/2006.
7. Yamashita H, Haribowo R, Sekine M, Oda N, Kanno A., Shimono Y, Shitao W, Higuchi T, Imai T &
Yamamoto K, 2012. Toxicity test using Medaka (Oryzias latipes) early fry and concentrated
sample water as an index of aquatic habitat condition. Environ Sci Pollut Res (2012)
19:2581-2594.
8. Liu, R., Kameya, T., Kobayashi, T., Sugimura, Y., Kubo, T., Sawai, A., (2006). Evaluating the fish
safety level of river water and wastewater with a larval Medaka assay. Chemosphere (in
press).
9. Ishii S, Urano K, Kameya T (2000) General conditions for concentrating trace organic compounds in
water with porous polystyrene cartridges. J Jpn Soc Water Environ 23:301307 (in
Japanese).
10. Liu R, Kameya T, Sawai A, Urano K (2007) Application of a larval Medaka assay to evaluate the fish
safety level in Sagami River, Japan. Environ Monit Assess 130:475482

174
Journal of Applied Sciences in Environmental Sanitation, 9 (3): 169-174.