ECOTOXICOLOGYANDENVIRONMENTALSAFETY 15,186-194(1988)

Toxicity of an Anionic Detergent, Dodecylbenzene Sodium Sulfonate, to a Freshwater Fish, Rita rita: Determination

of L&, Values by Different Methods

DEBASISH ROY’

Department of Zoology, Banaras Hindu University, Varanasi - 221005, India

Received June 1. I987

LCm values and their 95% confidence limits for various intervals of exposure to an anionic detergent, dodecylbenzene sodium sulfonate, have been determined using recommended meth- ods. The advantages and disadvantages of these methods are discussed in light of the variations in the values. Different visible behaviors of the fish under the intluence of the detergent have d80 been eXpkkRd. 0 1988 Academic Ress, Inc.

INTRODUCTION

Synthetic detergents are the most widely used substances in modern civilization. They are used as washing powders, as dye fasteners, and in the preparation of sham- poos, industrial and household cleaning agents, toothpaste and tooth powder, etc., and thus are one of the major sources of aquatic pollution. The acute toxicity of a variety of organophosphorus and organochlorine compounds to many species of fish has been studied (Konar, 198 1) but no attention has yet been given to acute toxicity of synthetic detergents in India. The determination of LCsO values is useful in acute toxicity testing for certain toxicants to a particular animal species under specific phys- icochemical conditions. Probit and logit methods are very often used in most of the bioassay laboratories for routine analyses of median lethal concentration (LC&, al- though these methods have sufficient statistical deficiencies. The conventional Spear- man-Karber method, comparatively better in a few other aspects, is also employed in laboratories to estimate LCSO values. Hamilton et al. (1977) proposed a trimmed Spearman-Karber method for accurate and precise calculation, either manually or using Fortran, of LCso values and their 95% confidence endpoint intervals. in this paper, an effort has been made to calculate LCsO values and 95% confidence limits of an anionic detergent, dodecylbenzene sodium sulfonate, using various recommended methods and to compare the variations among values, the advantages of one method over another, and the limitations of these methods.

MATERIALS AND METHODS

Live specimens of Rita rita (12-16 cm in length) were collected in January from the River Ganges in the vicinity of Varanasi, India, and were then acclimated to laboratory conditions for 30 days before the experiment was started. During acclima- tization fish were fed on alternate days with minced goat liver. Feeding was, however, stopped 24 hr before the start of the experiment. A static bioassay test was performed

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186

TOXICITY OF DETERGENT ON FISH 187

CONCENTRATIONS (LOG 1

FIG. 1. Determination of 96-hr LCw, value of dodecylknzene sodium sulfonate to Ritn da.

to determine the 96-hr LCsO of an anionic detergent, dodecylbenzene sodium sulfo- nate (&-LAS, Koch-Light Laboratories, Colnbrook, England), to R. rifa following the methods of APHA, AWWA, and WPCF ( 15th ed.). Tap water having a dissolved oxygen content of 6.5 to 8.4 ppm, a pH of 7.1 +_ 0.2, and a hardness of 18 mg/liter at a temperature of 2 1 + 1.8”C was used in this experiment. Batches of 10 fish were kept in different glass aquaria, each containing 30 liters of detergent medium of varying concentration, for observing periodic mortality of fish for calculation of the LGO value. They were kept under constant observation. Visible behaviors, e.g., opercular movement, rapid jerky movement, restlessness, muscle spasm, body torsion, and coughing, at different detergent concentrations were recorded regularly at different time intervals throughout the experiment.

The mortality rate in each aquarium was recorded at intervals of 2,4,6,8, 10, 12, 24,48,72, and 96 hr. Dead fish, if any, detected during the experiment were instantly discarded. The 96-hr LCso values together with 95% confidence limits were deter- mined using the graphic method, the logistic method (Byron and Brown, 1967), the Spearman-Karber method (Byron and Brown, 1967), and the trimmed Spearman- Karber method (Hamilton et al., 1977) using Fortran on an ICL 1904s computer. The toxicity curve was drawn as per the recommended procedure.

RESULTS

On the basis of the mortality of R. rita at various concentrations of the detergent medium, the LCsO is estimated using the following methods.

Graphic method. Figure 1 shows the logarithm of percentage of mortality plotted on the ordinate and the concentration of the detergent plotted on the abscissa. The 96-hr LCso of the detergent to R. rita is 7.16 mg/liter.

Logistic method. Data obtained during the experiment and calculations using this method are summarized in Table 1.

Spearman-Karber estimation. The 96-hr LCso with confidence limits using this method is 6.926279 (7.3461805, 6.5303791) (Table 2). Similarly, for various inter- vals, the L&‘s have been calculated using the Spearman-Karber estimation (Table 3).

188 DEBASISH ROY

TABLE 1

CALCULATION OF L& (96-hr) AND ITS CONFIDENCE INTERVALS BY ASSUMING A LOGISTIC RESFQNSE MODEL AND USING THE AVAILABLE DATA

y=log, w=np x P n P/Cl-P) (1-P) wx WY wx* WXY

0.778 1 0.10 10 -2.19 0.9 0.700 -0.971 0.5448 -1.533 0.8450 0.60 10 0.405 2.4 2.028 0.972 1.713 0.821 0.9030 0.70 10 0.847 2.1 1.8963 1.77 1.712 1.606 0.9542 1.00 10 0 ii 0 0 0 0 1.0 1.00 10 0 0 0 0 0

x = logarithm of concentrations 2 w = x wx = zwy= zwx*= zwxy=

p = proportion of mortality 5.4 4.6243 0.77 1 3.969 0.894

n = number of fish in each tank

X= 2 wx/C w = 4.624314.3 = 0.856

j7= z WY/~ w = 0.77li5.4 = 0.142

b = cz wx)cz wxy) - cz wx)(C WY)

E WME wx2) - cz wxZ)

= 26.053663

U=jj-bF

= -22.159368

(-a/b) = 0.850 antilog 0.850 = 7.079 LCSO = 7.079

(9

(ii)

SE (a/b) = vllb + 112 ~@,@)(l/~ W(X - $)

= 0.0165

95% confidence limits are 7.6383578,6.5614527

Trimmed Spearman-Karber method. The LCsO with 95% confidence limits for different time durations was determined using Fortran with the same data and is summarized in Table 4.

The toxicity curve obtained by plotting the LCsa’s of dodecylbenzene sodium sulfo- nate to R. rita at various time intervals against the exposure time (Fig. 2) is asymp totic, showing a smooth progression of the test.

Change in&h behavior. Under static bioassay test conditions in the laboratory, control fish tend to remain at the bottom of the aquarium, exhibiting little movement or agitation. Initially the water of the aquaria remains clear. However, 1 day after transfer of the fish to a weak detergent medium (0.1 mg/liter), the water gradually becomes turbid due to thread-like slimy material in the aquaria. Subsequently, as the experiment progresses, the turbidity and the slime in the water increase further.

Fish exposed to the detergent medium at 1 mg/liter and in 96-hr LCsO (6.9 mg/ liter) do not show any striking change in their behavior. The turbidity and slime

TOXICITY OF DETERGENT ON FISH 189

TABLE 2

CALCULATION OF LCsO AND ITS CONFIDENCE INTERVALS USING THE SPEARMAN-KARBER ESTIMATION

x = dose 1% d = differ- n = number Percentage of p = proportion ~$1 - p) mg/liter dose ence of animals mortality of mortality n

6.0 0.7781 10 10 0.10 0.009 7.0 0.8450 0.9669

*0.05 8 10 60 0.60 0.024

8.0 0.9030 *os 12

10 70 0.70 0.02 1 9.0 0.9542

*0.0458 10 100 1.0 0

10.0 1.0 10 100 1.0 0

Mean d = 0.055 i Pi = 3.4 2 ‘+ = 0.054 I

iF=Xk-d/2-d; Pi 6)

= 0.8450

LCsO = antllog x = 6.926279

= 0.0127808

95% confidence limits are antilog%+ 2 SE(Z) = (7.3461805,6.530379)

(ii)

conditions of the water in the experimental tanks become relatively greater than those of the control from 1 day on.

Fish kept in an aquarium with detergent at a concentration of 10 mg/liter initially behave like the controls. After 2 hr, however, they start showing the first signs of

TABLE 3

LCsO VALUES AND THEIR 95% CONFIDENCE LIMITS OF VARIOUS DURATIONS OF DETERGENT EXFQSURE TO Rita rita

Duration of exposure (hr) LC50

95% confidence limits

Upper Lower

96 72 48 24 12 10

: 4and2

6.926279 7.0226335 7.2861818 7.3790423 8.3755292 8.8 104887 8.922776 1 9.6050582

>lO

7.3461805 6.5303791 7.4524 174 6.6176349 7.7019306 6.892875 7.7863586 6.9930334 8.8582799 7.91864 9.2623929 8.3806326 9.3229317 8.5387961

10.097712 9.1364398

190 DEBASISH ROY

TABLE 4

LCSO VALUES AND THEIR CORRESPONDING 95% CONFIDENCE LIMITS FOR VARIOUS EXPOSURE DURATIONS USING THE TRIMMED SPEARMAN-KARBER METHOD

Duration of LC% and 95% exposure (hr) confidence limits

Spearman-Karber (% trimmed)

10 20 50

96 LCSO 7.02364 6.96890 6.78748 Lower 6.53059 6.37852 6.78748 Upper 7.55404 7.61393 6.78748

72 LCSO 7.15107 7.13341 7.00000 Lower 6.64310 6.52799 7.00000 Upper 7.69788 7.79498 7.00000

48

24

LGO Lower Upper

Go Lower Upper

7.44514 7.46067 7.48331 6.93050 6.84492 7.4833 1 7.99800 8.13181 I 7.48331

7.57413 7.60904 7.65 172 7.07571 7.038 10 7.65172 8.10766 8.22629 7.65172

restlessness, as indicated by continuous erratic movements: not remaining stationary at any one place and frequently swimming to the surface, gasping for air. This behav- ior is followed by muscle twitching. After the fish have been subjected to such condi- tions for 4-6 hr, their distressed movements slow down and they start floating with the head and tail directed downward, resulting in a marked twisting of the body with intermittent jerky movements. During this period, the fish start to die, as indicated by no opercular movements and no response to gentle prodding and, ultimately, within 8-9 hr, all fish die. Under these test conditions, within 2 hr of exposure, the water becomes very turbid due to large amounts of thread-like slimy substance.

Fish transferred to aquaria containing detergent at a concentration of 100 mg/liter of water immediately start showing distress in the form of very fast, erratic move- ments in all directions. Muscle twitching is quite pronounced. A few fish start floating in a twisted condition and ultimately die after 30 min; within 2-3 hr of exposure aI1 fish are dead (Table 5).

DISCUSSION

The 96-hr LCso values with their 95% confidence limits derived from the same set of fish mortality data, using various recommended procedures, vary: 7.16 using the graphic method, 7.09 (7.6383578, 6.5614527) using the logistic method, 6.926279 (7.346 1805, 5303791) using the Spearman-Karber method, and 6.96890 (6.37852, 7.6 1393) 20% trimmed using the trimmed Spearman-Karber method. These devia- tions in LCsO values prompt discussion of the flaws of various methods in light of routine bioassay tests.

The most widely used probit or logit model for determining the median lethal con- centration (L&-J often describes the relationship between mean mortality and toxi-

TOXICITY OF DETERGENT ON FISH 191

FIG. 2. Toxicity curve based on L&‘s determined with 95% confidence limits for each value.

cant concentration. Ham&on et al. (1977) discussed in detail the advantages of one model over the other. According to these workers, the deficiencies in the probit and logit models are sufficiently important such that these methods should probably not be used for routine analysis of an extended series of bioassay experiments. The method used must have three characteristics, if all analyses are to be based on a single statistical method: it must be reasonably accurate and precise, be programmable so that all calculations can be done on a digital computer, and be robust enough that it will not fail when the data are somewhat unusual.

The Litchfield and Wilcoxon ( 1949) method, which is widely used for acute toxic- ity testing, is a rapid graphic method for finding the maximum likelihood estimate of (r for the probit model. This method may not produce the exact maximum likelihood estimate and is not programmable because it requires lines to be drawn following visual inspection of plotted data.

It is true that the conventional Spearman-Karber procedure does not yield a com- parison of the slopes of the two dose-response functions. But no other procedure will function better than the Spearman-Karber estimation, unless the sample size is very large. The trimmed Spear-man-Karber method (Hamilton et al., 1977) is not subject to the problems of the probit and logit models, has good statistical properties, is easy to use, and is recommended for accurate and precise calculation of LC5e values and their 95% confidence interval endpoints. The trimming is employed in much the same way as in calculating the trimmed mean. The experimentor has to choose a constant, where 0 K L < 50, which is the percentage of extreme values to be trimmed from each tail of the tolerance distribution before estimating w. The trimmed version

TABL

E 5

VAR

IOU

S TW

ES O

F BE

HAV

IOR

S OF

TH

E FI

SH

Ric

a rif

a UN

DER

THE

INFL

UEN

CE

OF

DIF

FER

ENT

CO

NC

ENTR

ATIO

NS

OF

DET

ERG

ENT

Rap

id je

rky

mov

emen

t Re

stles

snes

s M

uscl

e sp

asm

Bo

dy to

rsio

n C

ough

ing

Expe

rimen

tal

Expe

rimen

tal

Expe

rimen

tal

Expe

rimen

tal

Expe

rimen

tal

(con

cn in

mat

er)

(con

cn in

n-&

liter

) (c

oncn

in m

g/lit

er)

(con

cn in

mg/

liter

) (c

oncn

in m

g/lit

er)

Dur

atio

n of

ob

serv

atio

n C

ontro

l 1

6.9

10

100

Con

trol

1 6.

9 10

10

0 C

ontro

l 1

6.9

10

100

Con

trol

1 6.

9 10

10

0 C

ontro

l 1

6.9

10

100

0 hr

-

----

- ---

- -

----

- ---

- -

____

3(

)min

_

---+3

-

---+3

-

---+2

-

---+3

-

----

2hr’j

()fin

_

_ -

- *

- -

- +l

*

- -

- j-2

*

- -

- +1

*

- -

- _

* 4h

r30m

in

- -

- tl

- ---

-

-- +2

-

-- +1

-

---

6hr3

Om

in

- -

- +1

-

- -

- -

- -

- -

- -

- -

--_

8hr3

0min

-

- -

* -

-- IN

-

-- *

- --

* -

-- *

lOhr

30m

in

- -

- -

- --

- --

- --

- --

12hr

30m

in

- -

- -

- --

- --

- --

- --

24hr

30m

in

- -

- -

- --

- --

- --

- --

36hr

-

-- -

-- -

-- -

-- -

-- 48

hr

- --

- --

- --

- --

- --

60hr

-

-- -

-- -

-- -

-- -

-- 72

hr

- --

- --

- --

- --

- --

84 h

r -

-- -

-- -

-- -

-- -

-- 96

hr

- --

- --

- --

- --

- --

Note

. *,

All

fish

died

, +3

, ve

ry st

rong

; +2

, st

rong

; +

1, w

eak.

TOXICITY OF DETERGENT ON FISH 193

of the Spearman-Karber estimation (Hamilton et al., 1977) has properties as good as those of the conventional estimation method, but it is not as sensitive to anomalous responses as the latter. Regarding the percentage of trimming (Cc), Hamilton and his associates recommend & = 10, for a group of experiments where the lowest concen- tration causes approximately 5% mortality or less and/or the highest concentration causes approximately 95% mortality or more.

The reason for no change in the values of the upper and lower extremes of the 95% confidence intervals of the LCsO values of different time intervals (where L = 50) using the trimmed Spear-man-Karber method, could not be explained. Probably such a high degree of trimming (50%) for these particular sets of experimental data exceeds the limitations of the method.

The fish, when transferred to the detergent medium, show erratic and haphazard movements, due perhaps to the pathogenic effects of the toxic detergent, such as fa- tigue or comatose-like behavior. Before death, the fish bends into a twisted shape. It may therefore be presumed that these substances, apart from having toxic effects through contact with the body, also disturb the central nervous system. Similar types of effects, especially the various categories of behaviors, were also found by Bhatta- charaya et al. (1975) while treating Clarius butruchus with different concentrations of endrin. The higher the concentration, the more pronounced the peculiar behav- iors. These authors also observed that after several minutes of exposure, the fish lost equilibrium and remained vertically suspended, with complete cessation of move- ment, and eventually died.

CONCLUSION

This investigation provides a comprehensive overview of the various methods for determining the LCsO values used in regular bioassay tests and discusses the varia- tions, advantages, and limitations of one method compared to the other. The 96-hr LCsO values with their 95% confidence limits derived from the same set of data of fish mortality, using various recommended procedures, show a variation. Among them, the rapid graphic method and the probit and logit methods, due to a number of defi- ciencies, are not found suitable for routine bioassay experiments. The conventional Spearman-Karber method functions somewhat better than any other method only when the sample size is small. The trimmed Spearman-Karber estimation (Hamilton et al., 1977), which has good statistical properties, is reasonably precise and accurate, and has a programming option, appears to be the most advantageous for a series of bioassays.

When the fish are transferred into low detergent concentrations, there is not much change in their behavior, whereas fish placed in higher concentrations show erratic movements, muscle spasms, and body torsion due to toxic effects of the detergent. The sense organs, connected by the nervous system, are in direct contact with the detergent medium, but the involvement of the central nervous system, especially dur- ing the violent bending spasms of the fish, cannot be overlooked.

ACKNOWLEDGMENTS

Financial assistance by the University Grants Commission and the Indian Council of Agricultural Re- search and the concerned Principal Investigator, in the form of a Research Fellowship, is gratefully ac- knowledged.

I94 DEBASISH ROY

REFERENCES

APHA, AWWA, AND WPCF (1980). Standard Methods for the Examination of Water and Waste Water, 15th ed., Washington.

BHAITACHARAYA, S., MUKHERJEE, S., AND BHATTACHARAYA, S. (1975). Toxic effects ofendrin on hepa- topancreas of the teleost fish, Clarias batrachus (Linn.) Indian J. Exp. Biol. 13,185-l 86.

BYRON, W., AND BROH~, J. R. (1967). Quantal-response assay. In Statistics in Endocrinology (J. W. McArthur and T. Colton, Eds.). MIT Press, Cambridge, MA/London.

HAMILTON, M. A., Russo, R. C., AND THURSTON, R. V. (I 977). Trimmed Spearman-Karbcr method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technof. 1 l(7), 7 14-7 19.

KONAR, S. K. ( 198 1). Pollution of water by pesticides and its influence on aquatic ecosystem. Indian Rev. LijeSci. 1, 139-165.

LITCHFIELD, J. T., AND WILCOXON, F. (1949). A simple method of evaluating dose-effect experiments. Pharmacol. Exp. Ther. %,99.