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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 1998 to 10 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

In order to determine the substance concentration, water samples were taken at study start and study end from the concentrations relevant for EC50 determination.

Vehicle:

no

Details on test solutions:

The test substance was weighed into a beaker, mixed with water for dilution, homogenized with the Ultra-Turrax and were then stirred for approximately 24 hours with a magnetic stirrer at a temperature of approx. 50 °C to ensure that the solubility limit of the test substance in the test water was reached. Afterwards the test batches were centrifugated and than 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):

Daphnia magna

Details on test organisms:

Species: Daphnia magna - Clone 5 (IRChA, France)
Origin: Stock culture of Hoechst Marion Roussel Deutschland GmbH, ProTox
Breeding conditions: 100 mL beakers (height 90 mm, diameter 50 mm), filled with 70 mL test water (according to Elendt, M4) served as culture vessels. One daphnid was used per culture vessel. The animals were used for breeding up to an age of 42 days. The reproduction rate and the state of health of the animals were monitored daily apart from weekends. The animals were fed three times a week with monocellular green algae (Scenedesmus subspicatus).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period

Hardness:

2.3 mmol Ca2+ + Mg2+/l

Test temperature:

20.2 – 21.3°C

pH:

7.9 – 8.3

Dissolved oxygen:

6.3 – 9.0 mg/l

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal test concentrations: 0/22/50/100/220 mg/L

Details on test conditions:

The study was conducted in a static system. The test chambers were 250 mL beakers (height 110 mm, diameter 70 mm, water level about 75 mm), each filled with 200 mL test water. The room temperature was about 21 °C. The room was illuminated by fluorescent tubes for 16 hours each day.
After the test concentrations had been prepared and water parameters were recorded, 10 daphnids were used in each test chamber. The daphnids received no feed for the entire study period.

Inspection of the daphnids took place after 24 and 48 hours and involved recording the immobility. Immobile daphnids were removed from the chambers. Those animals not able to swim within 15 seconds after gentle agitation of the test container were considered to be immobile. The water parameters were measured after adding the substance and at the end of the test.

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:

mobility

Details on results:

All tested concentrations were present as clear yellow to yellow-brown solutions.
The substance concentration was determined in the 50,100 and 220 mg/L groups. Due to very low water solubility of the test substance the values determined were only in a range of 0.005 - 0.097 mg/L. Moreover in the 50 mg/L were no immobility occurred a much higher test substance concentration was observed than in the 100 mg/L group were 8 of 20 animals died. Analytical determinations showed that a by product of the test substance results from the solution process, which also is responsible for the orange discolouration of the test batches. A calculation based on comparison of the peak areas showed that the concentration of the by product corresponds with the nominal concentration steps and that it remains at a constant level throughout the exposure period. This leads to the presumption that the observed effects were (might be) caused by the by product and not from the original test substance. However, the immobility values given in this report are based on nominal concentrations of the test substance.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

On the basis of the immobility rate, the EC50 value was determined after 24 by means of the probit analysis (after the method according to UNDER und WEBER, confidence limit according to FIELLER).
Probit analysis could not be carried out after 48 hours because of the immobility rate. As the factor for the concentration steps was 2.2 (third root of 10), no further concentrations were tested. The EC50 was estimated graphically.

IMMOBILITY

 

In the control group no immobility was observed. Under the given study conditions, the following immobility rates of T-9601 were recorded after 24 and 48 hours:

 

Concentration (mg/l)	Immobility
	24 hours		48 hours
	Absolute	%	Absolute	%
22	0/20	0	0/20	0
50	0/20	0	0/20	0
100	1/20	5	8/20	40
220	12/20	60	20/20	100

 

	After 24 hours	After 48 hours
EC0 (mg/l)	50	50
EC50 (mg/l)	198.0	Approx. 115.0
Confidence interval	161.7 – 265.9	-
EC100 (mg/l)	Not determined	220.0

 

Individual values

 

Concentration (mg/l)	Test-chamber No.	Study-start (date)	Hours after study start
			24	48
0	1a	Dec-08-98	0/10	0/10
0	1b	Dec-08-98	0/10	0/10
22	2a	Dec-08-98	0/10	0/10
22	2b	Dec-08-98	0/10	0/10
50	3a	Dec-08-98	0/10	0/10
50	3b	Dec-08-98	0/10	0/10
100	4a	Dec-08-98	0/10	3/10
100	4b	Dec-08-98	1/10	5/10
220	5a	Dec-08-98	5/10	10/10
220	5b	Dec-08-98	7/10	10/10

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test and based on nominal concentrations the EC50 of T-9601 after 48 hours was near 100 mg/L.

Executive summary:

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

 

The present study was conducted in compliance with the requirements of EEC-Guide­line 92/69/EWG Annex part C Methods for the Determination of Ecotoxicity C.2 Acute Toxicity for Daphnia July 31,1992and the OECD-Guideline OECD-Guideline for testing of chemicals, Section 2 - Effects on Biotic Systems Test guideline 202 Daphnia sp., 14-day Reproduction Test(including an Acute Immobilisation Test)Adopted: April 4,1984.This study was conducted in compliance with the Principles of Good Laboratory Practice(GLP).

 

Toxicity of T-9601 was tested in Daphnia magna {water flea) over 48 hours in a static system.

 

The nominal concentrations tested were 22, 50, 100, 220 mg/L and a negative control (0 mg/L).

 

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. Afterwards the test concentrations were centrifugated and than 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.

 

All tested concentrations were present as clear yellow to yellow-brown solutions.

 

The substance concentration was determined in the 50, 100 and 220 mg/L groups. Due to very low water solubility of the test substance the values determined were only in a range of 0.005 - 0.097 mg/L. Moreover in the 50 mg/L where no immobility occurred a much higher test substance concentration was observed than in the 100 mg/L group were 8 of 20 animals died. Analytical determinations showed that a by product of the test substance results from the solution process, which also is responsible for the orange discolouration of the test batches. A calculation based on comparison of the peak areas showed that the concentration of the by product corresponds with the nominal concentration steps and that it remains at a constant level throughout the ex­posure period. This leads to the presumption that the observed effects were (might be) caused by the by product and not from the original test substance. However, the immobility values given in this report are based on nominal concentrations of the test substance.

 

In the control group no immobility was observed. Immobility was observed at 100 and 220 mg/L. The 22 and 50 mg/L group showed no effect in comparison to the negative control.

 

 

Description of key information

Under the conditions of this test and based on nominal concentrations the EC50 of T-9601 after 48 hours was near 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

(48h)

Effect concentration:

>= 100 mg/L