# SatePharm Laboratories SafePharm Laboratories Lt

### d P. O. Box 

### 45 Derby DE1 28T United Kingdom Tel: 01332792896 Fax: 01332799018 Website: www.safepharm.co.uk SPLPROJECTNUMBER: 140/1101

ACUTE TOXICITYTO RAINBOW TROUT 

### 1. INTRODUCTION At the request of the Sponsor 

### , Kao Corporation

### , the following study was undertaken to assess the acute toxicity of EMULGENB

・ 

### 66 to rainbow trout. 2. METHODS AND 

### 1¥置ATERIALS 2.1 Test Method OECDGuideline No203 (1

### 992) 

### “Fish

### , Acute Toxicity Test". 2.2 Test Material Description

## , Identification and Storage Conditions Sponsor's identification Description; Date received; Storage conditions E恥fU 

### LGENB-66 light brown

### , slightly turbidviscous liquid 31 December 2001 room temperature 

### , inthe dark 2.3 加 

### lethod of Preparation Direct solution inwater. 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 groupwas sharedwith similar concurrent studies. 2.4 Test Concentrations 18 

### , 32 

### , 56 

### , 100 and 180 m 

### g/ l 2.5 Test Media Renewal Daily. 2.6 Test Species Rainbowtrout 

### (Oncorhynchus mykiss). Fish were received on 13 February 2002 fromthe supplier and maintained in a glass fibre tank with a constant supply of dechlorinated tap water at a temperature of 14 :t I O

### C. Fishwere fed commercial trout pellets daily. Feeding was discontinued approximately 24 hours prior to the start of the test.

Registered in England No. 3009027-2- 

### SPLPROJECTNUMBER: 140/1101

2.7 Length 4.1 土 

### 0.1 cm. 2.8 Weight 0.67 土 

### 0.10 g

### , Loading:0.23 g bodyweightllitre. 2.9 Supplier Brow Well Fisheries 

### Li mited

### , Hebden

### , near Skipton

### , Y

orkshire 

### , UK. 2.10 Mortalities Zero mortalities inthe seven days prior to the start ofthe test. 2.11 Medication None. 2.12 Test Vessels 20 litres. 2.13 Test Water Laboratory tapwater was dechlorinated by passage through an activated carbon filter (Purite Series 500) and pa 同

### y softened (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 requiredtemperature. 2.14 Temperature 14.0 o

### C. 2.15 Aeration Yes. 2.16 Study Dates Range-finding tests: Definitive tes

### t: 5 February 2002 to 25 February 2002 4

March 2002 to 8 March 2002-3- SPLPROJECTNUMBER: 140/1101

3. RANGE-FINDINGTESTS In the initial range-finding test ,日 sh were exposed to aseries of nominal concentrations of 1. 0 , 10 and 100 m 

### g/l. The results are tabulated below: Nominal Cumulative Mortality (Initial Population= 3) Concen 仕ation (m g/ 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 assignedto the definitive tes

### t. This experimental design conforms to a 

### “

Limit test" to confirm that at the maximum test concentration given inthe OECD 厄 EC Test Guidelines 

### , no mortalities or sub-Iethal effects ofexposure were observed. However 

### , in the definitive test 

### , su

かlethal effects of exposure were observed a白 er 24 hours exposure. These effects were loss of equilibrium andthe presence ofmoribund fishwith 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 mgl l. The results are tabulated below: Nominal Cumulative Mortality (I nitial Population= 3) Concen 汀ation (m g/ I) 24 Hours 48 Hours 72 Hours 96 Hours Control 

# 。 。 。 。

18 

# 。 。 。 。 180 

# 。 3 3 4. DEFINITIVETEST 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 assignedto the definitive tes

### t. Eachtest and control vessel contained 7 fish. Cumulative mortality data from the exposure ofrainbow trout for 96 hours to EMULGENB ・66 are 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

ware package (ToxCalc 1999) and the geometric mean method at 24 hours gave the following results based on nominal test concen 仕ations.

### -4- SPLPROJECTNUMBER: 140/1101 Inspection of the mortality data given in Table 1 

### , gave the following results based on nominal test concentrat lO ns: Time (Hours) LC 50 (mgll) 3 >180 6 >180 24 130 48 100 

### 72 59 96 59 Highest test concentration resulting in 0% mortality 

### = 32 m 

### g/ l Lowest test concentration resulting in 100% mortality = 100 m 

### g/ l 

### “

NoObserved Effect Concentration" 

### (N OEC) = 32 m 

### g/ 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 m 

### g/l. These responses were increased pigmentation

### , loss of equilibrium 

### ,

increased pigmentation with loss of equilibrium 

### ,

loss of equilibrium with swollen abdomens andthe presence of moribund fish (see Table 2). 4.3 Observatioos 00 Test Material Solubility The test material preparations were observedto be clear 

### , colourless solutions throughout the duration oftes

### t. 

### 5. REFERENCES Hamilton

### , M A 

### ,

Russo

### , RC and Thurston

### , RV(1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration inToxicity Bioassays. 

### Environ 

### Sci Technol11 

### , 714-719. ToxCalc Version 5.0.23C (1999) 

### , Tidepool Scientific Software 

### , McKinleyville 

### , CA 95519 

### , USA.

*

Concentrations resulting in0% and 100% mortality respectively

### -5- 

### SPLPROJECTNUMBER: 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 (m g/ I) Control

18 32 56 100 180 

### -6- SPLPROJECTNUMBER: 140/1101

Cumulative Mortality Data for RainbowTrout Exposed for 96Hoursto EMULGEN 

### B-66 Cumulative Mortality (I nitial 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 2 1. 5 hours exposure 7 /7 fish were observed with loss of equilibriumand swollen abdomens. Due to animal wel ぬ re implications these fish were killed in extremis and classed as mortalities for the 24 ・Hour time 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 fishwere killed in extremis and classed as mortalities for the 48 ・ Hour time point *** A食 er approximately 52.25 hours 3 /7 fish at 56 m g/ I and 4/4 fish at 100 m g/ I were observed to be moribund. Due to animal welfare implications these fishwere killed in extremis and classed as mortalities for the 72

・ Hour time point-7- 

### SPLPROJECTNUMBER: 140/1101

Table 

### 2 Sub-lethal Effects ofExposure in the Definitive Test Nominal Time (Hours) Concentration Sub-lethal Effects (mgll) 

### 3 6 24 48 72 

### 96 Control Noabnormalities detected



### 18 Noabnormalities detected 

### 32 Noabnormalities detected 

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

### 7 /7 

### 100 Increasedpigmentation and 4/4** AID 料 * loss of equilibrium 

### 180 Noabnormalities detected A/D*

A/D= All fish dead 

### *

After approximately 

### 21.5 hours exposure 

### 7 /7 fish were observed with loss of equilibriumand swollen abdomens. Due to animal welfare implications these fish were killed in ぽ tremis and classed as mortalities for the 

### 24

・Hour time point 判 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 fishwere killed in extremis and classed as mortalities for the 

### 48 ・ Hourt 出le point 

### *** 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 fishwere killed in extremis and classed as mortalities for the 72 ・ Hour time point