Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

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:

31 October 2011 to 02 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

Principles of method if other than guideline:

-

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Stability in water: Not indicated
Solubility in water: ≤24.5 ng/L at 20 °C (determined in NOTOX project 497887)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter was kept for possible analysis of undissolved residue.

Frequency: at t=0 h and t=48 h
Volume: 1 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 1 ml were taken for possible analysis.

Vehicle:

no

Details on test solutions:

The batch of FAT 41043/A tested was a red powder, solid with a purity of 99.8 % and consisted of two isomeric forms. The substance was not completely soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 15-minute period of magnetic stirring combined with a treatment with ultrasonic waves. The obtained aqueous mixture was subsequently magnetically stirred for a period of two days to ensure maximum dissolution in test medium and subsequently filtered through a 0.45 µm membrane filter (rc55, Whatman).The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20 %, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: For the test selection of young daphnids with an age of <24 hours, from parental daphnids of more than two weeks old.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

-

Post exposure observation period:

none

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

19.3 - 20.5 °C

pH:

8.0-8.1

Dissolved oxygen:

8.9-9.3 mg O2/l

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10 and 100 % of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.

Measured concentration: Analysis of the sample taken at the start of the test showed measured concentration of 3.0 µg/l. The measured concentration remained stable during the test (81 % of initial). Therefore, the effect parameters were expressed in terms of initially measured concentrations.

Note that the measured concentration was above water solubility of the test substance which was determined to be ≤24.5 ng/l.

Details on test conditions:

Test duration: 48 hours

Test type: Static

Test vessels: 100 ml, all-glass

Medium: Adjusted ISO medium

Number of daphnids: 20 each for the control and the highest concentration, 10 each for the lower concentrations

Loading: 5 per vessel containing 80 ml of test solution

Number of replicates: 4 for the control and the highest concentration, 2 for the lower concentrations

Light: 16 hours photoperiod daily

Feeding: No feeding

Aeration: No aeration of the test solutions.

Introduction of daphnids: Within 1¼ hours after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 3 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 3 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Details on results:

No immobility was observed at any of the groups tested.

- Behavioural abnormalities: no
- Mortality of control: no

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 were just outside (below) the ranges of the expected historical responses at the different concentrations, i.e. a 48h-EC50 between 0.3 and 1.0 mg/l. Hence, the sensitivity of this batch of D. magna was slightly higher when compared to the historical data collected at NOTOX.

The 24 h EC50 was 0.53 mg/l with a 95 % confidence interval between 0.47 and 0.62 mg/l.

The 48 h EC50 was 0.28 mg/l with a 95 % confidence interval between 0.26 and 0.32 mg/l.

Reported statistics and error estimates:

No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration).

Table1           Acute immobilisation of daphnids after 24 and 48 hours

FAT 41043/A TE % of filtrate prep. at 100 mg/l	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
control	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.1	A  B	5 5	0 (4) 0 (5)	0	0 (4) 0 (1)	0
1.0	A  B	5 5	0 0	0	0 0	0
10	A  B	5 5	0 0	0	0 0	0
100 [3.0]	A  B C D	5 5 5 5	0 0 (4) 0 0	0	0 0 (2) 0 0	0

[] – between brackets: initially measured concentration (µg/l)

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Conclusions:

FAT 41043/A did not induce acute immobilisation of Daphnia magna at 3.0 µg/l after 48 hours of exposure (NOEC).

The 48h-EC50 was beyond the range tested, i.e. exceeded an initially measured concentration of 3.0 µg/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l.

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for daphnids could not be reached.

Description of key information

The short-term toxicity to aquatic invertebrates of FAT 41043/A was investigated according to OECD Guideline Nr. 202, EU Method C.2 and GLP principles. The 48h-EC50 was beyond the range tested, i.e. exceeded an initially measured concentration of 3.0 µg/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l.

Additional information

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for daphnids could not be reached.