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 aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation 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:

- Sampling method: Aqueous samples (10 mL) of freshly prepared test media (0 hour) and pooled expired test media (48 hours) were collected in duplicate, mixed with acetonitrile, then diluted further with Elendt M4 media:acetonitrile (1:1, v/v) as required to bring the response within the calibration range.
- Sample storage conditions before analysis: Frozen storage stability (nominally -20°C) was tested over 7 days, but failed to meet acceptability criteria. Samples were therefore processed on the day of collection.
- Sample processing: Prior to analysis, the samples were mixed with 0.2% formic acid in acetonitrile. Sample extracts were further diluted into the calibration range with 0.1% formic acid in (1:1,V/V) acetonitrile:Elendt M4 medium, if required.

Vehicle:

no

Details on test solutions:

The substance is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) 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 substance was prepared as Water Accommodated Fractions (WAFs).

At the start of the test and media renewal at 24 hours, the test substance was weighed onto a glass slides and placed in 1000 mL of Elendt M4 test medium. The preparations were 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 loading rate WAF. A control group 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):

Daphnia magna

Details on test organisms:

- Test organism: juvenile Daphnia magna (Straus), less than 24 hours old
- Source: parental laboratory cultures, held and maintained at the test facility (culture was initiated from ephippia obtained from MicroBio Tests Inc., Belgium)
- Age of parental stock: less than 24 hours old
- Food type: cultures fed daily with a concentrated suspension of Chlorella vulgaris
- Feeding during test: none

Test type:

static

Water media type:

other: Elendt M4 medium

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Not specified.

Test temperature:

20 +/- 2ºC; constant within 1ºC in the test vessels. The temperature monitored continuously in an adjacent vessel varied by more than 1ºC, but was not considered to have adversely affected the integrity of the test since no immobilization was observed.

pH:

7.61 – 8.43

Dissolved oxygen:

8.47 – 9.60 mg/L (90 - 99% air saturation value)

Salinity:

Not specified.

Conductivity:

Not specified.

Nominal and measured concentrations:

Nominal: 0 (control) and 100 mg/L loading rate WAF
Measured: Measured concentrations in the 0 (fresh media) and 24 hour (old) test solutions were below the limit of quantification of the analytical method (i.e., <0.01 mg/L). Measured concentrations in the 24 (fresh media) and 48 hour (old) test solutions were determined to be 0.124 mg/L and 0.0476 mg/L, respectively, for the low molecular weight component and 0.147 mg/L and 0.0520 mg/L, respectively, for the high molecular weight component.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL (nominal) glass, tall form beakers covered with clear lids
- Test solution volume: 50 mL (nominal)
- Aeration: not specified
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium prepared from trace element stock solutions and reverse-osmosis (RO) water in accordance with OECD Guideline No. 202.

OTHER TEST CONDITIONS
- Intervals of water quality measurement: The pH, dissolved oxygen and temperature were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media (pooled replicates) at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
- Adjustment of pH: none
- Photoperiod: 16-hour light : 8-hour dark
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Statistical analysis was not performed given that no immobilisation was observed during the test. The EL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) were estimated empirically by observation of the data.

RANGE-FINDING STUDY
- Test concentrations: 0, 1.0, 10 and 100 mg/L (duplicate vessels for each)
- Results used to determine the conditions for the definitive study: The range-finding test result indicated that no toxic effects would be observed from exposure of the test organisms to a 100 mg/L loading rate WAF.

Reference substance (positive control):

yes

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: nominal mg/L loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: nominal mg/L loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: nominal mg/L loading rate WAF

Basis for effect:

mobility

Details on results:

- Behavioral abnormalities: No immobilisation was observed in the test group after 24 or 48 hours of exposure. All daphnids were observed to be mobile and submerged in the test solution at 24 hours. Two mobile daphnids were observed to be submerged in the test solution at 48 hours; the remaining 18 were observed to be mobile at the surface.
- Immobilization in negative control: None; all mobile and submerged throughout the test.
- Other adverse effects in negative control: None
- Any observations (e.g. precipitation) 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

Results with reference substance (positive control):

- Reference substance: potassium dichromate
- Test design: same test organism, medium, exposure conditions and replication as this definitive test
- Results with reference substance valid? Yes; EC50 result was within the expected range state in the OECD Guideline No. 202 (0.6 - 2.1 mg/L).
- Relevant effect levels: 48-hour EC50
- Dose-response test: yes; treatment concentrations were 0 (control), 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50: 1.366 mg/L
- NOEC: 1.0 mg/L

Reported statistics and error estimates:

Statistical analysis was not performed given that no immobilisation was observed during the test.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria for control immobility (/=3 mg/L) were both satisfied. The test is therefore considered valid.

Conclusions:

No immobilization was observed during the 48-hour exposure period. The 48-hour EL50 value was therefore considered to be >100 mg/L loading rate WAF. 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.

Executive summary:

The study was conducted in accordance with the requirements of OECD Guideline No. 202. 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 range-finding test the definitive test was conducted at a nominal loading rate of 100 mg/L under static-renewal conditions, with a concurrent control group. At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed for mobility at 24 and 48 hours. The Daphnia magna 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 0.0002 to 0.05 µg/mL. Measured concentrations in the 0 (fresh media) and 24 hour (old) test solutions were below the limit of quantification of the analytical method (i.e., <0.01 mg/L). Measured concentrationsin the 24 (fresh media) and 48 hour (old) test solutionswere determined to be 0.124 mg/L and 0.0476 mg/L, respectively, for thelow molecularweight component and 0.147 mg/L and 0.0520 mg/L, respectively, for thehigh molecularweight component.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.

No immobilization was observed over the 48-hour exposure period. The 48-hour EL50 value was therefore determined to be >100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate WAF. 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. 202 study, conducted according to GLP, the acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated under static conditions and the 48-hour EL50 value was determined 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 WAF (Smithers Viscient (ESG) Ltd, ****).