Reaction mass of benzenamine, 2-ethyl-N-(2-ethyl-nonylphenyl) nonyl-, branched and benzenamine, 2-ethyl-N-(2-ethyl-nonylphenyl)-, branched

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:

10 November 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

-Purity: >93%
-Description: Dark Amber Liquid
-Storage: Room temperature (15 to 30°C) under nitrogen

Analytical monitoring:

yes

Remarks:

Given that the test substance is a complex multi-constituent substance, the results were based on nominal loading rates.

Details on sampling:

- Concentrations: Control and 100 mg/L loading rate WAF
- Sampling method: Aqueous samples (10 mL) of freshly prepared test media at 0 (fresh media), 24 (old media), 48 (fresh media) and 72 hours (old media) were collected in duplicate, mixed with acetonitrile, then diluted further with test water:acetonitrile (1:1, v/v) as required to bring the response within the calibration range.
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on test solutions:

The substance is a is a multi-constituent substance with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test solutions were prepared as Water Accommodated Fractions (WAFs). At the start of the test, and each media renewal, test substance was weighed onto a glass slide, placed in the test media. Due to the viscous nature of the test substance, and the amount of test substance required, the weighing was split between three glass slides which were then added to the final volume of treated mains water. The preparation was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparation was allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 100 mg/L test concentration. The control solution was prepared in the same manner without the addition of test substance.

The test preparations were observed to be colourless solutions throughout the duration of the test.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Northern Trout (Hebden, Skipton, North Yorkshire, United Kingdom)
- Length at study initiation): 5.2 cm (mean); 4.9 - 5.6 cm
- Weight at study initiation: 1.2 g (mean); 1.0 – 1.6 g

ACCLIMATION
- Acclimation period: 63 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: Fish were fed during acclimation with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish. Uneaten food and debris was siphoned or cleaned from the tanks as required.
- Health during acclimation (any mortality observed): 1.08% mortality observed in the stock batch of fish in the seven days prior to test initiation.

FEEDING DURING TEST
The fish were not fed during the 24 hours before the start of the definitive test and were not fed throughout the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

18.4 mg/L as CaCO3 (typical)

Test temperature:

15.2 - 16.9 ºC, with the exception of the test solution prepared at 0 hour (19.9 ºC). While all other temperature remained with the acceptable range (15 +/- 2°C), the temperature dropped more than 1.0°C during the first 24 hours in both the control and treated vessels.

pH:

6.89 - 7.60

Dissolved oxygen:

9.24 - 10.59 mg/L (>60 % air saturated value, ASV)

Salinity:

Not applicable

Conductivity:

183 uS/cm (typical)

Nominal and measured concentrations:

Nominal: 0 (control) and 100 mg/L loading rate WAF
Measured: Measured concentrations, with the exception of the 48 hour (fresh media) results, were below the limit of quantification of the analytical method (i.e., <0.005 mg/L). The low and high molecular weight components in the freshly prepared test solutions at 48 hours were determined to be 0.0148 mg/L and 0.0115 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L constructed glass aquaria, each fitted with a clear plastic lid (Two sets of test vessels (set A and set B) were used on alternate test media renewal days during the test.)
- Volume of test media: 15 L
- Aeration: yes; sufficient to maintain oxygen concentration
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.56 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Treated tap water; passed through activated carbon filters.
- Alkalinity: 18.4 mg/L (as CaCO3)
- Ca/mg ratio: 6
- Culture medium different from test medium: same
- Intervals of water quality measurement: Dilution water is analyzed bi-annually. At the start of the test and at approximately 24-hour intervals during the test, the media pH, concentration of dissolved oxygen (% air saturation value and mg/L) and the individual test media temperatures were recorded in the freshly prepared and old test media. Continuous temperatures were measured using a digital (min/max) thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hour light: 8 hours dark lighting cycle with a 30 minute transition period
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Fish in each test vessel were observed for mortality and sublethal effects at ca 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: mg/L loading rate WAF

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: mg/L loading rate WAF

Basis for effect:

mortality (fish)

Details on results:

- Behavioral abnormalities: none
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations that might cause a difference between measured and nominal values: The results from the freshly prepared WAFs at 0 and 48 hours showed differences in measured concentrations despite the WAFs being prepared in the same manner. This was considered to be possibly due to the expected low water solubility of the test substance and difficulties associated with the preparation of WAFs.
- Effect concentrations exceeding solubility of substance in test medium: yes

Reported statistics and error estimates:

Statistical analysis was not performed given that no mortality was observed during the test. The LL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) were estimated empirically by observation of the data.

Sublethal observations / clinical signs:

Mortality of fish recorded during the definitive test

Nominal loading	Cumulative number of mortalities recorded*
rate (mg/L)	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0
100	0	0	0	0

 * Seven fish added to each test vessel at test initiation; no sublethal effects were observed.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria for control mortality (less than 10%, actual = 0%) and dissolved oxygen (>60 % ASV, actual >/= 90%) were both satisfied. The test was therefore considered to be valid.

Conclusions:

Based on nominal loading rates, the 96-hour LL50 value was estimated to be >100 mg/L. The NOEL was considered to be 100 mg/L. The test showed no toxicity at the limit of solubility of the test substance in the test media.

Executive summary:

The study was conducted in accordance with the requirements of OECD Guideline No. 203 to assess the toxicity the 96-hour acute toxicity of the substance to rainbow trout (Oncorhynchus mykiss). Test solutions were prepared as Water Accommodated Fractions (WAFs) based on the recommendations of OECD Series on Testing and Assessment, No. 23. 

Based on the results of a rangefinding test, the definitive test was conducted as a ‘Limit Test’ under semi-static conditions (daily renewal) at 15 +/- 2°C (with one noted exception at test initiation) at a single nominal loading rate of 100 mg/L with a concurrent control group, each consisting of a single 20 L constructed glass aquaria containing 15 L of media. Following acclimation to the test conditions for at least 12 days, seven fish were added to each test vessel. TheDaphnia magnain each test vessel were observed for mortality and sub-lethal effects at ca. 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The fish were not fed during the test.

Test solutions were analysed at 0 and 48 hours (fresh media) and at 24 and 72 hours (old media) using high performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of two major constituents (a low molecular weight component and a high molecular weight component) of the test substance. The analytical method was successfully validated at concentrations of 0.0005 to 0.05 µg/mL. Measured concentrations, with the exception of the 48 hour (fresh media) results, were below the limit of quantification of the analytical method (i.e., <0.005 mg/L). The low and high molecularweight components in the freshly prepared test solutions at 48 hours were determined to be 0.0148 mg/L and 0.0115 mg/L, respectively.

Given that the test substance is a multi-constituent substance and analytical monitoring was limited to one low and one high molecular weight component of the test substance, the effect concentrations were based on nominal loading rates.

Based on nominal loading rates, the 96-hour LL50 was determined to be >100 mg/L. The NOEL was determined to be 100 mg/L. The test showed no toxicity at the limit of solubility of the test substance in the test media.

Description of key information

In an OECD Guideline No. 203 study, conducted according to GLP, to assess the toxicity of the substance to the freshwater fish species, Oncorhynchus mykiss, the 96-hour LL50 was determined under semi-static test conditions to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate (Smithers Viscient (ESG) Ltd, ****).