SatePharm 

Laboratories 

SafePharm Laboratories Ltd 

P.O. Box 45 

Derby DE1 28T United Kingdom Tel: 01332792896 Fax: 01332799018 

Website: www.safepharm.co.uk 

SPL PROJECT NUMBER: 140/1101 

ACUTE TOXICITY TO RAINBOW TROUT 

1. INTRODUCTION 

At the request of the Sponsor, Kao Corporation, the following study was undertaken to assess the acute 

toxicity of EMULGEN B・66to rainbow trout. 

2. METHODS AND 1¥置ATERIALS

2.1 Test Method 

OECD Guideline No 203 (1992)“Fish, Acute Toxicity Test". 

2.2 Test Material Description, Identification and Storage Conditions 

Sponsor's identification 

Description; 

Date received; 

Storage conditions 

E恥fULGENB-66

light brown, slightly turbid viscous liquid 

31 December 2001 

room temperature, in the dark 

2.3 加lethodof Preparation 

Direct solution in water. Amounts oftest material (360,640, 1120,2000 and 3600 mg) were each separately 

dissolved into a final volume of 200 ml of dechlorinated tap water, with the aid of ultrasonication for 

approximately 5 minutes, prior to dispersing into a final volume of 20 litres of dechlorinated tap water to 

give the 18, 32, 56, 100 and 180 mgll test concentrations respectively. 

Data from the control group was shared with similar concurrent studies. 

2.4 Test Concentrations 

18, 32, 56, 100 and 180 mg/l 

2.5 Test Media Renewal 

Daily. 

2.6 Test Species 

Rainbow trout (Oncorhynchus mykiss). Fish were received on 13 February 2002 from the supplier and 

maintained in a glass fibre tank with a constant supply of dechlorinated tap water at a temperature of 

14 :t IOC. Fish were fed commercial trout pellets daily. Feeding was discontinued approximately 24 hours 

prior to the start of the test. 

Registered in England No. 3009027 
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SPL PROJECT NUMBER: 140/1101 

2.7 Length 

4.1土 0.1cm. 

2.8 Weight 

0.67土 0.10g, Loading:0.23 g bodyweightllitre. 

2.9 Supplier 

Brow Well Fisheries Limited, Hebden, near Skipton, Y orkshire, UK. 

2.10 Mortalities 

Zero mortalities in the seven days prior to the start of the test. 

2.11 Medication 

None. 

2.12 Test Vessels 

20 litres. 

2.13 Test Water 

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and 

pa同ysoftened (EIga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of 

approximately 100 mgll as CaCu3. After dechlorination and softening the water was then passed through a 

series of computer controlled plate heat exchanges to achieve the required temperature. 

2.14 Temperature 

14.0oC. 

2.15 Aeration 

Yes. 

2.16 Study Dates 

Range-finding tests: 

Definitive test: 

5 February 2002 to 25 February 2002 

4 March 2002 to 8 March 2002 
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SPL PROJECT NUMBER: 140/1101 

3. RANGE-FINDING TESTS 

In the initial range-finding test,日shwere exposed to a series of nominal concentrations of 1.0, 10 and 

100 mg/l. The results are tabulated below: 

Nominal Cumulative Mortality (Initial Population = 3) 

Concen仕ation(mg/I) 24 Hours 48 Hours 72 Hours 96 Hours 

Control 。 。 。 。

1.0 。 。 。 。

10 。 。 。 。

100 。 。 。 。

Based on the results of the range-finding test, a single test concentration (two replicates of 7 fish each) 

of 100 mgll plus a control were assigned to the definitive test. 

This experimental design conforms to a “Limit test" to confirm that at the maximum test concentration given 

in the OECD厄ECTest Guidelines, no mortalities or sub-Iethal effects of exposure were observed. 

However, in the definitive test, suかlethaleffects of exposure were observed a白er24 hours exposure. These 

effects were loss of equilibrium and the presence of moribund fish with swollen abdomens. These sub-Iethal 

effects we問 consideredto be due to the variability of a biological system at a threshold concentration. 

Therefore, in order to define a test range, a second range-finding test was conducted at test concentrations of 

18 and 180 mgll. The results are tabulated below: 

Nominal Cumulative Mortality (Initial Population = 3) 

Concen汀ation(mg/I) 24 Hours 48 Hours 72 Hours 96 Hours 

Control 。 。 。 。

18 。 。 。 。

180 。 3 3 

4. DEFINITIVE TEST 

4.1 Results 

Based on the results of the second range-finding test, test concentrations of 18, 32, 56, 100 and 180 mgll 

plus a control were assigned to the definitive test. Each test and control vessel contained 7 fish. 

Cumulative mortality data from the exposure of rainbow trout for 96 hours to EMULGEN B・66are given in 

Table 1 and sub-lethal effects of exposure are given below. 

Analysis of the mortality data given in Table 1, by the trimmed Spearman-Karber method (Hamilton et al 

1977) at 48, 72 and 96 hours using the ToxCalc computer so食warepackage (ToxCalc 1999) and the 

geometric mean method at 24 hours gave the following results based on nominal test concen仕ations.
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SPL PROJECT NUMBER: 140/1101 

Inspection of the mortality data given in Table 1, gave the following results based on nominal test 

concentratlOns: 

Time (Hours) LC50 (mgll) 

3 > 180 

6 > 180 

24 130 

48 100 

72 59 

96 59 

Highest test concentration resulting in 0% mortality = 32 mg/l 

Lowest test concentration resulting in 100% mortality = 100 mg/l 

“No Observed Effect Concentration" (NOEC) = 32 mg/l 

4.2 Sub-lethal Effects 

95% Confidence Limits (mgll) 

. 

閉

100・ 180*

84 -130 

47 -73 

47 -73 

Sub-lethal effects were observed at test concentrations of 56, 100 and 180 mg/l. These responses were 

increased pigmentation, loss of equilibrium, increased pigmentation with loss of equilibrium, loss of 

equilibrium with swollen abdomens and the presence of moribund fish (see Table 2). 

4.3 Observatioos 00 Test Material Solubility 

The test material preparations were observed to be clear, colourless solutions throughout the duration oftest. 

5. REFERENCES 

Hamilton, M A, Russo, RC and Thurston, RV (1977) Trimmed Spearman-Karber Method for Estimating 

Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol11, 714-719. 

ToxCalc Version 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, USA. 

* Concentrations resulting in 0% and 100% mortality respectively 
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SPL PROJECT NUMBER: 140/1101 

ごと 3仏三日六 臥 TE:........... .1.. a . .M~.R .~~.Q 

21 間関 2002

DATE: ......... 
Table 1 

Nominal 

Concentration 

(mg/I) 

Control 

18 

32 

56 

100 

180 

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SPL PROJECT NUMBER: 140/1101 

Cumulative Mortality Data for Rainbow Trout Exposed for 96 Hours to 

EMULGEN B-66 

Cumulative Mortality (Initial Population: 10) 

3 Hours 6 Hours 24 Hours 48 Hours 72 Hours 96 Hours % Mortality 

。 。 。 。 。 。 。

。 。 。 。 。 。 。

。 。 。 。 。 。 。

。 。 。 。 3*** 3 43 

。 。 。 3** 7*** 7 100 

。 。 7* 7 7 7 100 

* After approximately 21.5 hours exposure 7/7 fish were observed with loss of equilibrium and swollen abdomens. 

Due to animal welぬreimplications these fish were killed in extremis and classed as mortalities for the 24・Hourtime 

point 

** After approximately 29 and 45.5 hours 2/7 fish and 1/5 fish were observed to be moribund. Due to animal welfare 

implications these fish were killed in extremis and classed as mortalities for the 48・Hourtime point 

*** A食erapproximately 52.25 hours 3/7 fish at 56 mg/I and 4/4 fish at 100 mg/I were observed to be moribund. Due 

to animal welfare implications these fish were killed in extremis and classed as mortalities for the 72・Hourtime point 
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SPL PROJECT NUMBER: 140/1101 

Table 2 Sub-lethal Effects of Exposure in the Definitive Test 

Nominal Time (Hours) 

Concentration Sub-lethal Effects 

(mgll) 3 6 24 48 72 96 

Control No abnormalities detected 

18 No abnormalities detected 

32 No abnormalities detected 

56 Loss of equilibrium 1/4*** 1/4 

Increased pigmentation 7/7 

100 Increased pigmentation and 4/4** 

AID料 *

loss of equilibrium 

180 No abnormalities detected A/D* 

A/D = All fish dead 

* After approximately 21.5 hours exposure 7/7 fish were observed with loss of equilibrium and swollen abdomens. 

Due to animal welfare implications these fish were killed inぽ tremisand classed as mortalities for the 24・Hourtime 

point 

判 Afterapproximately 29 and 45.5 hours 2/7 fish and 1/5 fish were observed to be moribund. Due to animal welfare 

implications these fish were killed in extremis and classed as mortalities for the 48・Hourt出lepoint 

*** After approximately 52.25 hours 3/7 fish at 56 mgll and 4/4 fish at 100 mgll were observed to be moribund. Due 

to animal welfare implications these fish were killed in extremis and classed as mortalities for the 72・Hourtime point