Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

A guideline strudy.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.0 mg/L at 24, 48 and 72 hours.
- Sampling method: An aliquot (500 ml) of each test sample was extracted with three portions (3 x 30 ml) of dichloromethane transferring each extract into a 100 mL volumetric flask. The flasks were then adjusted to volume with dichloromethane, giving a final theoretical test material concentration of a nominal 10 mg/L.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary solution in 10% v/v Tween 80-tetrahydrofuran. 200 mg of the test material was dispersed in solvent with the aid of ultrasonic disruption and the volume adjusted to 10 mL to give a 200mg/10mL solvent stock solution. 2 mL of this solvent stock solution was then washed into 500 mL of water prior to dispersion in dechlorinated tap water and the volume adjusted to 20 L to give a 2.0 mg/L test concentration.
- Eluate: Laboratory tap water, dechlorinated by the addition of sodium thiosulphate. Total hardness approximately 100 mg/L as CaCO3
- Differential loading: No
- Controls: 1 control and 1 solvent control (including 100 µL/L auxiliary solvent (10% v/v Tween 80-tetrahydrofuran)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 10% v/v Tween 80-tetrahydrofuran
Concentration of vehicle in test medium: Solvent stock solution : 200mg/10 mL, Final test solution and solvent control 100 µL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: Parkwood Trout Farm, Wigmore, Kent, U.K.
- Length at study initiation: Mean standard lenght: 4.1 cm (SD = 0.2 cm)
- Weight at study initiation: Mean weight: 0.48 g (SD = 0.08g)
- Feeding during test: Discontinued 24 h prior to the test
- Food type: Commercial trout pellets.
- Frequency: Daily until 24 h prior to the test.

ACCLIMATION
- Acclimation period: 7 days prior to the test.
- Acclimation conditions: Same as test.
- Type and amount of food: Commercial trout pellets.
- Feeding frequency:Daily.
- Health during acclimation (any mortality observed): Zero mortalities in the 7 days prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

0

Hardness:

Approximately 100 mg/L as CaCO3

Test temperature:

14ºC

pH:

7.6 - 7.7

Dissolved oxygen:

9.8 - 10 mgO2/L

Nominal and measured concentrations:

0 hours: Nominal: 2mg/L Measured (mean 2 replicates): 1.87 mg/L
24 hours: Nominal: 2mg/L Measured (mean 2 replicates): 1.86 mg/L
96 hours: Nominal: 2 mg/L Measured (mean 2 replicates): 1.745 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria.
- Material, size, headspace, fill volume: 20 L.
- Aeration: Via narrow bore glass tube.
- Renewal rate of test solution (frequency/flow rate): Daily renewal of medium.
- No. of organisms per vessel: 20 animals
- No. of vessels per concentration (replicates): 1 test concentration per duplicate.
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.24 g body weight/litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by the addition of sodium thiosulphate. Total hardness approximately 100 mg/L as CaCO3
The following values correspond to typical water quality characteristics of the zone of water supply throughout 1992. (averages)
- Total organic carbon: 1.8 mg C/L
- Metals: The content of metals of the water supply were within the limits of the respectives prescribed concentrations/values.
- Pesticides: < 0.10 µg/L
- Chlorine: total: < 0.20 mg/L
- Alkalinity: 88mg mg CaCO3/L
- Ca/mg ratio: Calcium 44 mg Ca/L
- Conductivity: 329 µS/cm
- Culture medium different from test medium: No.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark.

EFFECT PARAMETERS MEASURED: Mortalities; Criteria of death: absense of respiratory movement and response to a physical stimulation.
ENVIRONMENTAL MEASURES: pH, dissolvend oxygen concentration and temperature were recorded daily throughout the study.


TEST CONCENTRATIONS
The experimental design conforms to a limit test.
- Range finding study: 3 fish were exposed to each of the following concentrations for a period of 96 hours: 0.10, 1.0, 2.0 and 3.0 mg/L.
- Test concentrations: 2 mg/L
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the concentrations. However a precipitate was observed at 3.0 mg/L approximately 24 hours after addition of the solvent stock solution to water.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

There were no mortalities or other adverse reactions to exposure in 20 fish exposed to a test concentration of 2.0 mg/L for a period of 96 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: In the preliminary dose range finding experiments at higher concentrations (3.0 mg/L) there was marked precipitation after addition of the solvent stock solution to water.

Results with reference substance (positive control):

No positive control required.

Sublethal observations / clinical signs:

Table 1. Cumulative mortality data for Rainbow Trout exposed for 96 hours to Nigrosine Base EX

Nominal concentration (mg(L)	Cumulative mortality (10 fish initial population)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0
2.0 R1	0	0	0	0	0	0
2.0 R2	0	0	0	0	0	0

See the reporting format and data matrix attached.

Validity criteria fulfilled:

yes

Remarks:

The mortality of the control is 0. The constancy of the environmental conditions is mantained troughout the test (Table 2). There is evidence the concentration of the substance was mantained throughout the test and at least 85.3 % of the nominal value.

Conclusions:

There were no mortalities or other adverse reactions in 20 fish exposed to a test concentration of 2.0 mg/L for a period of 96 hours.

Executive summary:

A short-term (acute) toxicity study was conducted according to the OECD guideline 203 with Rainbow Trout (Oncorhynchus mykiss) under GLP conditions. For 96 hours, under semi-static conditions, groups of 10 fish were exposed to a concentration of 2.0 mg/L of Nigrosine Base EX in water with 10 % v/v Tween 80 -tetrahydrofuran as an auxiliary solvent at a final concentration of 100 µL/L. Two replicates of the test concentration, a control group and a solvent control group were included in the study. Measurements of pH, dissolved oxygen concentration and temperature were recorded. An analytical method was developed and validated. There were no mortalities or other adverse reactions in 20 fish exposed to the test concentration of 2.0 mg/L based on nominal concentration. It was concluded that the LC50 for 96 hours of exposure was superior to 2.0 mg/L and the NOEC for 96 hours of exposure >/= 2.0 mg/L

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

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 203. GLP study.

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
See attached reporting format.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from Letter of Access experimental data.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

There were no mortalities or other adverse reactions to exposure in 20 fish exposed to a test concentration of 2.0 mg/L for a period of 96 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: In the preliminary dose range finding experiments at higher concentrations (3.0 mg/L) there was marked precipitation after addition of the solvent stock solution to water.

Sublethal observations / clinical signs:

Table 1. Cumulative mortality data for Rainbow Trout exposed for 96 hours to Nigrosine Base EX

Nominal concentration (mg(L)	Cumulative mortality (10 fish initial population)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0
2.0 R1	0	0	0	0	0	0
2.0 R2	0	0	0	0	0	0

See the reporting format and data matrix attached.

Validity criteria fulfilled:

yes

Remarks:

The mortality of the control is 0. The constancy of the environmental conditions is mantained troughout the test (Table 2). There is evidence the concentration of the substance was mantained throughout the test and at least 85.3 % of the nominal value.

Conclusions:

There were no mortalities or other adverse reactions in 20 fish exposed to a test concentration of 2.0 mg/L for a period of 96 hours.
Applying the read across approach the same effect has been predicted for the substance Solvent Black 5.

Executive summary:

A short-term (acute) toxicity study was conducted according to the OECD guideline 203 with Rainbow Trout (Oncorhynchus mykiss) under GLP conditions. For 96 hours, under semi-static conditions, groups of 10 fish were exposed to a concentration of 2.0 mg/L of Nigrosine Base EX in water with 10 % v/v Tween 80 -tetrahydrofuran as an auxiliary solvent at a final concentration of 100 µL/L. Two replicates of the test concentration, a control group and a solvent control group were included in the study. Measurements of pH, dissolved oxygen concentration and temperature were recorded. An analytical method was developed and validated. There were no mortalities or other adverse reactions in 20 fish exposed to the test concentration of 2.0 mg/L based on nominal concentration. It was concluded that the LC50 for 96 hours of exposure was superior to 2.0 mg/L and the NOEC for 96 hours of exposure >/= 2.0 mg/L.

Applying the read across approach the same effect has been predicted for the substance Solvent Black 5.

Description of key information

Key study: OECD 203. GLP. The 96h-LC50 in fish was determined to be > 2 mg/L (nominal concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2 mg/L

Additional information

Key study: Experimental results: A fish acute toxicity test was performed according to OECD Guideline 203. The 96h-LC50 was determined to be greater than 2 mg/L under test conditions since no mortality was observed in any of the tested concentrations up to the saturation concentration.