Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

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:

11 October 1993 - 15 October 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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:

no

Details on test solutions:

Method of Preparation
Preliminary solution in 10% v/v Tween 80-tetrahydrofuran. 200 mg of test material was dispersed in solvent with the aid of ultrasonic disruption and the volume adjusted to 10 ml to give a 200 mg/10 ml 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 litres to give a 2.0 mg/l test concentration.

Stability of test concentrations
Ensured by daily renewal of the test media and verified by chemical analysis of the solvent control and the 2.0 mg/l test groups at 0, 24 and 96 hours.
Data relating to the identity, purity and stability of the supplied test material were the responsibility of the Sponsor.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Parkwood Trout Farm, Wigmore, Kent, U.K.
- Length at study initiation (length definition, mean, range and SD): 4.1 cm (SD = 0.2 cm).
- Weight at study initiation (mean and range, SD): 0.48 g (SD = 0.08 g).
- Feeding during test: Commercial trout pellets daily. Discontinued 24h prior to test.
- Food type: Commercial trout pellets.
- Frequency: daily

ACCLIMATION
- Acclimation period: Stock held since 7 September 1993 and acclimatised to test conditions from 4 - 11 October 1993
- Acclimation conditions (same as test or not): Same as test
- Health during acclimation (any mortality observed): No mortalities in the 7 days prior to the start of the test.
- Medication: None

The stock fish were maintained in a glass fibre tank with a "single pass" water renewal system.
Lighting: controlled - 16 hours light and 8 hours darkness cycle.
Loading: 0.24 g bodyweight/litre

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

100 mg/l as CaC03

Test temperature:

14'C

pH:

7.6 - 7.7

Dissolved oxygen:

9.8 - 10.0 mg O2/L

Salinity:

no information

Nominal and measured concentrations:

Highest attainable test concetration: 2.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: 20 L
- Aeration: narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): one way
- Renewal rate of test solution (frequency/flow rate): Daily

- No. of vessels per vehicle control (replicates): 1 test concentration in duplicate plus 1 control and 1 solvent control (including 100 ul/l auxiliary solvent). 20 animals per test concentration
- Biomass loading rate: 0.24 g bodyweight/litre

TEST MEDIUM / WATER PARAMETERS
Laboratory tap water, dechlorinated by the addition of sodium thiosulphate.
Total hardness approximately 100 mg/l as CaCO3.
- Total organic carbon: 1.8 mg C/L
- Particulate matter:
- Metals: IRON UI 62 ug Fe/L, MANGANESE <10 ug/ Mn/l, ALUMINIUM <20 ug Al/L, COPPER 73 ug Cu/L, ZINC 90 ug Zn/l, LEAD 11 ug Pb/L, CADMIUM <0.5 ug Cd/L, silver < 1.0 ug Ag/L, BORON 30ug B/L, BARIUM 40 ug Ba/L, ARSENIC <1 ug As/L, CHROMIUM <5. 50 ug Cr/L, NICKEL <7. Ug Ni/L, MERCURY 0.6 ug Hg/L, ANTIMONY <1.0 ug Sb/l, SELENIUM 1.0 ug Se/L
- Pesticides: <0.10 ug/L
- Chlorine: free 0.10 mg/L, total 0.13 ug/L


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness cycle
- Light intensity: no information

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiratory movement and response to a physical stimulation

TEST CONCENTRATIONS
- Test concentrations: control, solvent control, 2.0 mg/L

Reference substance (positive control):

no

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.
Highest test concentration resulting in 0% mortality: 2.0 mg/l
Lowest test concentration resulting in 100% mortality: > 2.0 mg/l
"No Observed Effect Concentration" (NOEC): 2.0 mg/l.
The "No Observed Effect Concentration" is based upon zero mortalities and the absence of any behavioural reactions to exposure at this concentration.
2.0 mg/l was the highest test concentration that could be obtained due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Other combinations of the various recognised auxiliary solvents and mixtures of Tween 80 (up to 20% v/v Tween 80 in solvent) were used during the range-finding experiments, however, the combination of 10% v/v Tween 80- tetrahydrofuran was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material after addition of the solvent stock solution to water.

Analysis of the test solutions showed that the measured test concentrations were near nominal values throughout the study. Therefore it was considered justifiable to estimate the LC50 values in terms of nominal test concentrations only.

Sublethal observations / clinical signs:

Nominal Concentration (mg/L)	cumulative mortality (initial population: 10)
	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

R1 - R2 = Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

Highest test concentration resulting in 0% mortality: 2.0 mg/l
Lowest test concentration resulting in 100% mortality: > 2.0 mg/l
"No Observed Effect Concentration" (NOEC): 2.0 mg/l.
The "No Observed Effect Concentration" is based upon zero mortalities and the absence of any behavioural reactions to exposure at this concentration. 2.0 mg/l was the highest test concentration that could be obtained due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Executive summary:

The purpose of this study was to determine the 96-h LC50 of Nigrosine Base EX to the rainbow trout (Oncorhynchus mykiss). The LC50 is the calculated concentration of the test substance that produces 50% mortality after prescribed intervals of exposure. A solvent and a control group were used with test concentrations of 2.0 mg/L (in duplicate), the study was conducted at 14 °C using distilled laboratory water, and pH of test vessels averaging 7.7. The test substance was stable in the test system, with final concentrations averaging better than 96% of the starting level. The LC50 was greater than or equal to 2 mg/L (nominal).

There were no mortalities nor any sublethal effects observed at any test level.

Description of key information

Highest test concentration resulting in 0 % mortality: 2.0 mg/L

Lowest test concentration resulting in 100 % mortality: >2.0 mg/L

"No Observed Effect Concentration" (NOEC): 2.0 mg/L

2.0 mg/L was the highest test concentration that could be obtained due to the limited solubility of the test material in water and auxiliary solvent.

Additional information

One key study was chosen for this endpoint, of Klimisch 1 reliability and performed to the OECD Guidelines for Testing of Chemicals (1992) No. 203 "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The tests showed the 96 hour LC50 value was greater than the limit of solubility in water; approximately 2 mg/L.