(E)-3-(benzyl(4-((4-nitrophenyl)diazenyl)phenyl)amino)propanenitrile

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 1998 to 22 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study condcuted to EU & OECD test guidance in compliance with GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentrations 0/100 mg/L (nominal concentration)
Reconstituted water, composition according to ISO/DIS 7346/1 was used as water for dilution.
Preparation was carried out in a unit consisting of two Hostalen- lined steel vessels with a capacity of 1000 litres each.
The dilution water was prepared as described and was aerated until oxygen saturation.

Vehicle:

no

Details on test solutions:

The test substance was weighed into a beaker. Water for dilution was added and the mixture homogenized using an Ultra-Turrax. The mixture was transferred quantitatively into a 5 litre glass bottle. The content was stirred for approximately 24 hours at a temperature of approx. 50 °C to ensure that the solubility limit of the test substance in the test water was reached and then filtered off through a cellulose filter (pore size 0.2 μm) to remove undissolved parts of the test substance. Test vessels, filter and any other material coming in contact with the saturated solution were saturated with test substance before use.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: zebra fish, Danio rerio (HAMILTON-BUCHANAN)
Origin: Hoechst Marion Roussel Deutschland GmbH ProTox
Date of hatching: June 23, 1998
Delivery date: August 12, 1998

The fish were kept for 14 days before the start of the study in water for dilution under the following conditions:
Temperature: 22 ± 1 °C
Oxygen content: > 80 % of the saturation value
Duration of light period: 12 hours daily
Population density: < 1 g fish / L water
Feeding : twice daily ad libitum
Food: Tetra Min, Tetra Werke, Melle (Germany)

The body length of 7 representative fish from each batch was measured.
Part of study Batch No. Date n Body length (cm)
Variation range Mean s
1 6/98/L4 Dec-22-98 7 3.2 – 3.5 3.4 ± 0.14

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period

Hardness:

2.1 – 2-3 mmol Ca2+ + Mg2+ / l

Test temperature:

21.8 – 22.2°C

pH:

7.5 – 8.0

Dissolved oxygen:

5.5 – 9.6 mg/l

Salinity:

Not applicable.

Nominal and measured concentrations:

0 and 100 mg/L (nominal concentration)

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 4 litres, were made of glass (length 20 cm, width 15 cm, height 20 cm) and stood in a water bath made of Hostalit Z with a Plexiglas viewing panel. The tem¬perature of the water bath was regulated by a thermostat to 22 + 1 °C. The chambers were illuminated from above from 06.00 a.m to 06.00 p.m. The light intensity directly over the chambers was approximately 700 lux.
The test chamber of the 100 mg/L group was aerated during the whole course of the study, since the maintenance of an oxygen content of at least 60 % of the saturation value was not ensured due to the warming to 50 °C of the test batch 24 hours during preparation of the test concentrations. Aeration was carried out using a glass capillary with a bubble-frequency of 1 - 3 per second.

After the test concentration had been prepared and water parameters recorded, 7 fish were assigned to each test and control chamber. The fish were not fed during the entire study period, inspection of the fish took place after 3, 6, 24, 48, 72 and 96 hours and involved recording the lethality and visible alterations in appearance and behaviour. Dead fish were removed from the chambers. Fish were considered dead when there was a lack of opercular movement and no response to slight mechanical stimulus. The water parameters were measured and recorded before study start and after 0, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The tested concentration was visually present as clear orange solution.
The substance concentration was determined in the test batches relevant for LC50 determination.

Observations
The fish of the 100 mg/L group showed changes in behaviour, swimming behaviour and respiration rate. Some fish showed an orange discoloured integument. Symptoms occurred during the entire exposure time.

Lethality
in this 96-hour acute toxicity study of T-9601 in zebra fish (Danio rerio) no lethality occurred in the 100 mg/L-group (nominal concentration) and in the control group. The tested concentration was above the solubility limit in the test water. A mean concentration value of 0.004 mg/L was determined analytically.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

No data

Sublethal observations / clinical signs:

The following mean value was determined analytically during the study:

Nominal concentration (mg/l)	Measured concentration
	Mean (mg/l)	%
100	0.004	0.004

All values determined were in a range of ± 10% of the value observed at study start.

 

Symptoms – Individual values

0 mg/l (control group)
Hours after study start	Hours------------------------------------------------- >
	3	6	24	48	72	96
Lethality (absolute)	0/7	0/7	0/7	0/7	0/7	0/7
No changes in appearance and behaviour	a	a	a	a	a	a
100 mg/l
Hours after study start	Hours--------------------------------------------------- >
	3	6	24	48	72	96
Lethality (absolute)	0/7	0/7	0/7	0/7	0/7	0/7
Swimming at the bottom	s	s	s	s	s	s
Swimming posture tail heavy	s	s	s	s	s	s
Hyperactivity	-	s	s	s	s	s
Projecting opercula	-	-	s	s	s	s
Irregular respiration	-	-	s	s	s	s
Orange coloured integument	-	-	s	s	s	s

o = one fish

s = some fish

a = all fish

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test the LC50 of T-9601 after 96 hours was > 100 mg/L (based on nominal concentration) and is above the solubility limit of the test substance in the test water.

Executive summary:

The study was conducted to evaluate the toxicity of T-9601to fish following short-term exposure.  

The present study was conducted in compliance with the requirements of EEC-Guideline Guideline 92/69/EWG Annex part C Methods for the determination of ecotoxicity C.1. Acute toxicity on fish Dated: July 31,1992 and the OECD-Guideline for testing of chemicals, 203 Fish, Acute Toxicity Test, Adopted: July 17,1992.This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).  

T-9601 was tested in zebra fish (Danio rerio) over 96 hours in a static system.   A nominal concentration of 100 mg/L and a negative control (0 mg/L) were tested.   The chamber contents were stirred for approximately 24 hours at a temperature of approx. 50 °C to ensure that the solubility limit of the test substance in the test water was reached and then filtered off through a cellulose filter (pore size 0.2 urn) to re­move undissolved parts of the test substance. Test vessels, filter and any other mate­rial coming in contact with the saturated solution were saturated with test substance before use.   The tested concentration was visually present as clear orange solution.   The substance concentration was determined in the 100 mg/L test batch. Because of low water solubility a mean concentration value of only 0.004 mg/L was observed. The concentration values observed remained at a constant level throughout the exposure period. The lethality values given in this report are based on nominal concentrations.  

No lethality occurred up to the solubility limit of the test substance in the test water.   The 100 mg/L group exhibited symptoms but no lethality.

Description of key information

Under the conditions of this test the LC50 of T-9601 after 96 hours was > 100 mg/L (based on nominal concentration) and is above the solubility limit of the test substance in the test water.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Remarks:

(96 h)

Effect concentration:

> 100 mg/L

Additional information

The study was conducted to evaluate the toxicity of T-9601to fish following short-term exposure.  

The present study was conducted in compliance with the requirements of EEC-Guideline Guideline 92/69/EWG Annex part C Methods for the determination of ecotoxicity C.1. Acute toxicity on fish Dated: July 31,1992 and the OECD-Guideline for testing of chemicals, 203 Fish, Acute Toxicity Test, Adopted: July 17,1992.This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).  

T-9601 was tested in zebra fish (Danio rerio) over 96 hours in a static system.   A nominal concentration of 100 mg/L and a negative control (0 mg/L) were tested.   The chamber contents were stirred for approximately 24 hours at a temperature of approx. 50 °C to ensure that the solubility limit of the test substance in the test water was reached and then filtered off through a cellulose filter (pore size 0.2 urn) to re­move undissolved parts of the test substance. Test vessels, filter and any other mate­rial coming in contact with the saturated solution were saturated with test substance before use.   The tested concentration was visually present as clear orange solution.   The substance concentration was determined in the 100 mg/L test batch. Because of low water solubility a mean concentration value of only 0.004 mg/L was observed. The concentration values observed remained at a constant level throughout the exposure period. The lethality values given in this report are based on nominal concentrations.  

No lethality occurred up to the solubility limit of the test substance in the test water.   The 100 mg/L group exhibited symptoms but no lethality.