Reaction products of diphenylamine with nonene, branched

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23/03/2003 to 28/03/2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

Ar-nonyldiphenylamine

EC Number:

248-295-7

EC Name:

Ar-nonyldiphenylamine

Cas Number:

27177-41-9

Molecular formula:

C21H29N

IUPAC Name:

4-nonyl-N-phenylaniline

Test material form:

liquid: viscous

Remarks:

pale brown viscous liquid

Details on test material:

Reaction products of Benzenamine, N-phenyl, and Nonene, branched EC 253-249-4; CAS no.: 36878-20-3, followed by purification (distillation) with a 96% mono-alkyl content.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark
- state: pale brown viscous liquid
- Solubility in water: 0.0113 mg/l
- Reactivity of the test material with the incubation material used (e.g. plastic ware):
Data suggested that the test material would adsorb to glassware (glass bottles were pre-conditioned with test concentration)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): dilution in dimethylformamide
- Final concentration of a dissolved solid, stock liquid or gel: 2.825 mg/25 ml

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: solvent control, each replicate test (0.0.113 mg/l)
- Sampling method: duplicates
- Sample storage conditions before analysis: frozen

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2.2 ml of the 2.825 mg/25 ml solvent stock solution (inverted before) was dispersed in 22 l of dechlorinated tap water and stirred using a flat bladed mixer for approx. 1 min to give the 0.0113 mg/l test concentration
- Controls: chemical analysis of samples after 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): solvent control = 100 µl/l dimethylformamide
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): recovery analysis indicated a significant proportion of test material was present as dispersed test material
- Other: test vessels and sample bottles were pre-conditioned with the test concentration of test material 24 h prior test start

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: Brow Well Fisheries Limited, Hebden, Yorkshire, UK
- Age at study initiation (mean and range, SD): juvenile, not further specified
- Length at study initiation (length definition, mean, range and SD): not individually identified
- Weight at study initiation (mean and range, SD): not individually identified
- Receiving date: 20. February 2003

ACCLIMATION
- Acclimation period: 19 d
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: commercial trout pellets
- Feeding frequency during acclimation: not specified; discontinued approx. 24 h before test start
- Health during acclimation (any mortality observed): no mortality in 7 d prior test

FEEDING DURING TEST (as applicable)
no feeding during test

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

water: approx. 100 mg/l [CaCO3] (used for medium preparation)

Test temperature:

12.4 to 14.1 °C

pH:

7.6 - 8.0

Dissolved oxygen:

9.4 mg O2/l

Nominal and measured concentrations:

nominal: 0.0113 mg/l
time- weighted mean measured: 0.0013 mg/L after 96h

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass vessels, not further specified
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): not specified
- No. of vessels per vehicle control (replicates): not specified
- Biomass loading rate: 0.45 g bodyweight/litre (static volume)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water was dechlorinated (active carbon filter) and partly softened (Water softener), temperature was adapted
- Total organic carbon: 1 mg/l
- Pesticides: < 0.010 µg/l
- Chlorine: 0,156 mg/l (total)
- Alkalinity: 102 mg/l
- Calcium: 54.00 mg/l
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Photoperiod: (light/dark) 16/8 h, 20 min dawn to dust transition

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and sub-lethal effects (recorded at: 3, 6, 24, 48, 72 and 96 h); death = absence of both respiratory movement and response to physical stimulation

TEST CONCENTRATIONS
- Justification for using less concentrations: maximum concentration of 0.0113 mg/l corresponds to the media solubility (water solubility) of test material and was used for a limit test
- Range finding study: yes
- Test concentrations: 0.00113 and 0.0113 mg/l
- Results used to determine the conditions for the definitive study: mortality of one fish after 72 h (probably due to natural causes) for the concentration of 0.0113 mg/l

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: based on the concentration of centrifuged test media to give a "worst case" scenario

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.011 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: no
- Observations on body length and weight: (end oft the test) length = 4.2 +- 0.2 cm; weight 0.90 +- 0.17 g
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: decline in measured test concentrations, probably due to adsorption to particulates, dispersed material or test vessels

Any other information on results incl. tables

Sublethal observations / clinical signs:

Chemical analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured concentrations to range from 98% to 107% of nominal for the uncentrifuged samples and from 15% to 24% of nominal for the centrifuged samples. A marked decline was shown in the measured test concentrations for the 24-Hour old media at 24, 48, 72 and 96 hours with concentrations observed to range from 30% to 47% of nominal for the uncentrifuged samples and from 3% to 17% of nominal for the centrifuged samples.

The decline in test concentrations could be due to loss of material due to adsorption to particulates, dispersed material or the test vessel.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media to give a "worst case" analysis of the data.

The 96-Hour LC50 based on the time-weighted mean measured test concentrations of the centrifuged test media was greater than 0.0013 mg/l and correspondingly the No Observed Effect Concentration was 0.0013 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test with rainbow trout (Oncorhynchus mykiss) according to OECD 203, the test material lead to an acute toxicity of LC50 of >0.00113 mg/l and a NOEC of > 0.00113 mg/l. (based on the time-weighted mean measured test concentrations)

Executive summary:

A semi-static study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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.

A Determination of the Physico-chemical Properties study conducted on the test material showed that the water solubility value of the test material was 0.0113 mg/l. Therefore preliminary dosing trials were conducted in order to determine the most appropriate method of test media preparation. The results of the preliminary dosing trails indicated that due to the physico-chemical properties of the test material, the most commonly used methods for preparation of test media for use in Ecotoxicology tests were not suitable for this test material.

It was therefore considered that in order to maintain consistency with the reported water solubility value of the test material, the maximum concentration employed in the study would be 0.0113 mg/l. In order to enable the accurate and consistent preparation of this test concentration, it was considered that using a preliminary solution in auxiliary solvent to spike test medium was the most suitable method of preparation.

Following a preliminary range-finding test fish were exposed, in two groups of ten, to an aqueous dispersion of the test material, at a single concentration of 0.0113 mg/l for a period of 96 hours at a temperature of 12.4 to 14.1ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results: The 96-Hour LC50 based on nominal test concentrations was greater than 0.0113 mg/l and correspondingly the No Observed Effect Concentration was 0.0113 mg/l.

The results of the preliminary dosing trials indicated that at a test concentration of 0.0113 mg/l prepared using a preliminary solution in auxiliary solvent to spike test medium a significant proportion of undissolved/dispersed test material would be present. Samples taken from the test preparations were therefore analysed untreated and after centrifugation (10 000 G, 30 minutes) in order to give an indication of the dissolved and hence bioavailable test material concentration.

Chemical analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured concentrations to range from 98% to 107% of nominal for the uncentrifuged samples and from 15% to 24% of nominal for the centrifuged samples. A marked decline was shown in the measured test concentrations for the 24-Hour old media at 24, 48, 72 and 96 hours with concentrations observed to range from 30% to 47% of nominal for the uncentrifuged samples and from 3% to 17% of nominal for the centrifuged samples.

The decline in test concentrations could be due to loss of material due to adsorption to particulates, dispersed material or the test vessel.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media to give a "worst case" analysis of the data.

The 96-Hour LC50 based on the time-weighted mean measured test concentrations of the centrifuged test media was greater than 0.0013 mg/l and correspondingly the No Observed Effect Concentration was 0.0013 mg/l.