Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The analogue substance which shares the same functional groups with the target substance also has comparable values for the relevant molecular properties. The results obtained can be used for the read-across approach. Test method according to OECD 202. GLP study.

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control (R1-R4 pooled) and the 2.2 mg/L test group (replicates R1-R2 and R3-R4 pooled) at 0 and 48 hours for quantitative analysis.
- Sampling method: A volume of test was extracted with dichloromethane (3x30mL). The combined extracts were made to volume with dichloromethane (100mL final volume) to give a final theoretical concentration of 11mg/L.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at -20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results for media preparation trials it was considered that the most appropriate method of preparation of test solutions was the use of a saturated solution.
550 mg of the test material was dispersed in 11L of reconstituted water at approximately with the aid of propeller stirring at 1500 rpm at 21ºC for 24 hours. After 24 hours the stirring was stopped and undissolved test material was removed by centrifugation (4000 rpm for 3 minutes) followed by a filtration through Postlip filter papaer (10µm retention size) to give a 100% saturated solution of the test material with a nominal concentration of 2.2 mg/L (based on the results of media preparation trials).
- Eluate: Reconstituted water.
- Controls: Negative solvent control and a positive control: potassium dichromate (0.32, 0.56, 1.0, 1.8 and 3.2 mg/L)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The definitive test included an additional use of filtration through Postlip BW/S filter paper (10 µmretention size) after centrifugation because there was undissolved material not removed by centrifugation. Additional filtration was not performed for the rage-finding test or media preparation trials as undissolved material was not observed in the supernatant after centrifugation.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna.
- Source: Derived from in-house laboratory cultures.
- Age at study initiation: 1st instar, less than 24 hours.
- Method of breeding: Daphnia were mantained in propylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20ºC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minutes dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before the initiation of the test. The young daphnids were removed from culture and used for testing.
- Feeding during test: No.
- Food type: Suspension of algae (Chlorella sp.)
- Frequency: Daily before the start of the test.
The diet was not considered to have any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not required.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3

Test temperature:

20.7 - 20.8

pH:

8.0

Dissolved oxygen:

8.3 - 8.5

Nominal and measured concentrations:

0 hours: Nominal: 2.2 mg/L (R1-R4) Measured (mean, pooled R1-R2 and R3-R4): 0.05625 mg/L
48 hours Nominal: 2.2 mg/L (R1-R4) Measured (mean, pooled R1-R2 and R3-R4): 0.0855 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Jar
- Type: covered to reduce evaporation
- Material, size, headspace, fill volume: Glass
- No. of organisms per vessel: 5 Daphnids.
- No. of vessels per concentration: 4 replicates
- No. of vessels per control: 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water had 25 mL of 4 stock solutions: a) CaCl22H2O=11.76g/L b)MgSO47H2O=4.93g/L c)NaHCO3=2.59g/L and d)KCL= 0.23g/L in a final volume of 1 L. It was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Conductivity: Deionised water used:< 5 µS/cm.
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours darkness with 20 minutes dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED : Immobilisation, Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation. Any immobilisation or adverse reactions to exposure were recorded at 24 and 28 hours after the start of exposure.

TEST CONCENTRATIONS
Limit test
- Range finding study: Based othe results for media preparation trials, the use of a saturated solution was considered the most appropriate method for preparation of test solutions. In the range finding test, 10 Daphnia magna were exposed to a series of nominal concentration of 1.0, 10 and 100 % v/v saturated solution. The conditions were the similar to those used in the main study.
- Results used to determine the conditions for the definitive study: No immobilisation was observed in all the test concentrations, based on this information a limit test at a 100% saturated solution was conducted to confirm that no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.071 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.071 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilisation in 20 daphnids exposed to a mean measured concentration of 0.071mg/L of the test substance for a period of 48 hours
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The definitive test included an additional use of filtration through Postlip BW/S filter paper (10 µmretention size) after centrifugation because there was undissolved material not removed by centrifugation. This can be the reason why measured concentrations are lower than those obtained in media preparation trials.
- Effect concentrations exceeding solubility of substance in test medium: The test showed that there were no toxic effects of saturation.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 48 hours= 1.2 mg/L (95% confidence limits: 1.0 - 1.3)

Table 1. Cumulative Immobilisation data in the definitive test

Nominal Concentration (mg/L)		Cumulative Immobilised Daphnia (Initial population: 5 per replicate)
		24 hours			48 hours
		No. per replicate	Total	%	No. per replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
2.2	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

Table 2. Cumulative Immobilisation data in the rage-finding test

Nominal concentration (mg/L)	Cumulative Immobilised Daphnia (Initial population: 10 per replicate)
	24 hours	48 hours
Control	0	0
0.020	0	0
0.22	0	0
2.2	0	0

Table 3. Cumulative Immobilisation data in the positive control

Nominal control (mg/L)	Cumulative Immobilised Daphnia (Initial population: 10 per replicate)
	3 hours				24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	0	0		0
1.0	0	0	0	0	0	0	0	0	2	3	5	25
1.8	0	0	0	0	8	8	16	80	10	10	20	100
3.2	1	2	3	15	10	10	20	100	10	10	20	100

See the reporting format and data matrix attached.

Validity criteria fulfilled:

yes

Remarks:

In the control no daphnids were immobilised. The dissolven oxygen concentration was mantained throughout the duration of the test (8.4-8.5)

Conclusions:

The 48 hours EC50 (inmobilisation) value for the test substance with Daphnia magna was >0.071 mg/L based on the mean measured exposure concentration.

Executive summary:

A short-term toxicity test on aquatic invertebrates was performed with the Nigrosine Base SAPL according to OECD Guideline 202 under GLP conditions. Preliminary solubility work was conducted and the substance was considered a difficult substance. Therefore media preparation trials were undertaken, the saturated solution was considered the most appropriate method, 2.2 mg/L was the concentration of the saturated solution. A preliminary range-finding study was conducted with 1, 10 and 100% of the satured solution. No immobilisation was seen in any of the concentrations; according to these results, a limit main test was performed with Daphnia magna at 100% of the saturated solution. 20 Daphnia magna(<24 h old, 10 per replicate) were exposed to either a control medium or 100% saturated solution for 48 hours under static conditions. Validity criteria were fulfilled and an analytical monitoring was performed and validated. However there were big differences between the nominal (2.2 mg/L) and mean measured concentration (0.071mg/L), this was suspected to be due to the additional filtration through Postlip BW/S filter paper (10 µm retention size) after centrifugation, which was considered necessary to remove undissolved test material not removed by centrifugation in the preparation of the medium for the main test.

There was no immobility observed within the period of the test, therefore the 48h-EC50 for the test substance was >0.071 mg/L and the correspondingly NOEC was 0.071 mg/L based on the mean measured concentration.

Description of key information

Key study: OECD 202. GLP study. The 48-EC50 in Daphnia magna (basis for effect immobilisation) was determined to be >0.071 mg/L based on the geometric mean measured concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.071 mg/L

Additional information

Key study: Experimental data: A Daphnia acute immobilisation test was performed according to OECD Guideline 202. Based on the observed immobilisation, the 48h-EC50 was determined to be greater that 0.071 mg/L in Daphnia magna under test conditions based on the geometric mean measured concentration since no effects were observed up to the saturation concentration.