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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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

Details on sampling:

- Sampling method: Aqueous samples (10 mL) of freshly prepared test media (0 hour) and pooled expired test media (72 hours) were collected in duplicate
- Sample storage conditions before analysis: Frozen storage stability (nominally -20°C) was tested over 9 days, but failed to meet acceptability criteria. Samples were therefore processed on the day of collection.
- Sample processing: Prior to analysis, the samples were diluted 1:1 with acetonitrile containing 0.2% formic acid, then dilute further with OECD medium:acetonitrile (1:1 v/v) containing 0.1% formic acid as required to bring the response within the calibration range.

Vehicle:

no

Details on test solutions:

The substance 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, the test substance was weighed onto a glass slides and placed in flasks containing 1000 mL of OECD 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 preparations were allowed to settle for ca 1 hour, after which the aqueous phase was removed by careful mid-depth siphoning to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. A control group was prepared in the same manner without the addition of test substance.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Oban, UK
- Age of inoculum (at test initiation): Concentrated, axenic liquid slope cultures stored under refrigeration (2-8 °C) for up to 8 months.
- Method of cultivation: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 x 10^4 cells/mL.

Test type:

static

Water media type:

other: OECD medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.8 – 23.0 °C

pH:

7.73 – 8.97

Nominal and measured concentrations:

Nominal concentrations: Control, 0.10, 0.32, 1.0, 3.2, 10 and 100 mg/L loading rate WAF
Measured concentrations in freshly prepared samples (0 hour) ranged from less than the limit of quantification (LOQ; 0.01 mg/L) to 7.81* mg/L, and at 72 hours showed measured concentrations ranging from
* This 100 mg/L LR WAF was observed to be cloudy indicating that undissolved test substance may have been present. This was considered not to have affected the integrity of the test given that no inhibition of growth was observed at this loading rate or in the 10 mg/L LR WAF that showed a measured concentration closer to the expected solubility value.

Details on test conditions:

Nominal concentrations:
Control, 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF

Measured concentrations:
For the low molecular weight component analysis of the freshly prepared samples at 0 hours resulted in measured concentrations of 0.0190, 0.0384, 0.0728, 0.243 and 0.346 mg/L. For the high molecular weight component analysis of the freshly prepared samples at 0 hours resulted in measured concentrations of 0.0172, 0.0372, 0.0751, 0.228 and 0.344 mg/L.

The corresponding old pooled media samples (low molecular weight component) resulted in measured concentrations of 0.0103, 0.0272, 0.0327, 0.0372 and 0.0887 mg/L. The corresponding old pooled media samples (high molecular weight component) resulted in measured concentrations of 0.0105, 0.0209, 0.0354, 0.0399 and 0.110 mg/L.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

other: (for growth rate and yield)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Basis for effect:

other:

Remarks:

(growth rate and yield)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Test media descriptions: The freshly prepared test media at 0 hours in the definitive test appeared as colourless solutions. The appearance of the test media following the 72-hour definitive exposure period was described as green with a non-homogenous hazy dispersion of algae cells.
- Any stimulation of growth found in any treatment: yes
- Any observations that might cause a difference between measured and nominal values: none specified
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

A positive control study was conducted to determine the effects of the reference substance, potassium dichromate, on algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance at nominal concentrations of 0.1, 0.32, 1.0 and 3.2 mg/L, along with a control, for 72 hours at 21-24˚C under static conditions with continuous illumination. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 1.19, 1.21 and 1.93 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate were determined to be 0.32 mg/L.

These results were within the expected toxicity range (growth rate EC50: 0.38 - 2.60 mg/L) for this reference substance and all validity criteria were met; the test is therefore considered valid.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. A single replicate from the control was not included in results calculations due to unusual growth. Statistical analysis confirmed the replicate to be an outlier (Grubbs Extreme Value test). The area under the average specific growth rate (µ) and the final yield over specified time intervals were analysed using Dunnett Multiple Comparison test to determine the no observed effect loading rate (NOEL). Linear interpolation analysis was performed in order to estimate EL10, EL20 and EL50 values. Where possible, 95% confidence limits were calculated for the EL10, EL20 and EL50 values.

Table 1: Percent inhibition of growth rate of Pseudokirchneriella subcapitata

Nominal loading rate	Average specific growth rate, “µ” (cells x 10^-2/mL)				Inhibition of average specific growth  rates relative to the control (%)
(mg/L)	0-24 hours	24-48 hours	48-72 hours	0-72 hours	0-24 hours	24-48 hours	48-72 hours	0-72 hours
Control	8.115	4.080	5.680	5.958	-	-	-	-
1.0	7.368	4.873	5.479	5.907	9	-19	4	1
3.2	7.288	4.661	5.878	5.942	10	-14	-3	0
10	7.496	5.140	5.448	6.028	8	-26	4	-1
32	7.538	5.104	5.464	6.035	7	-25	4	-1
100	7.153	5.241	5.901	6.098	12	-28	-4	-2

 - Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

  

Table 2: Percent inhibition of yield ofPseudokirchneriella subcapitata

Nominal loading rate (mg/L)	Initial cell density (cells/mL)	Yield at 72 hours * (cells/mL)	Mean Final Yield per treatment (cells/mL)	% Inhibition in Final Yield relative to control treatment
Control	1 E04	3277400 # 607760 776560 838220 646060 755280	72.5E04	-
1.0	1 E04	635140 683880 766400	69.5E04	4
3.2	1 E04	685220 697400 752920	71.2E04	2
10	1 E04	793540 701420 780440	75.8E04	-5
32	1 E04	677020 774680 840740	76.4E04	-5
100	1 E04	698880 803100 902240	80.1E04	-11

 * Final yield (cells/mL) = cell density (cells/mL) at 72 hours – initial cell density (cells/mL)

- Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

# Replicate not included in results calculations due to unusual growth. Statistical analysis confirmed the replicate to be an outlier (Grubbs Extreme Value test).

Validity criteria fulfilled:

yes

Remarks:

The control cell densities increased by a factor of 72. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 9.47% and the overall CV for specific growth rate was 3.07%.

Conclusions:

Based on nominal loading rates, the 72-hour EyL50 and the 72-hour ErL50 values were determined to be greater than 100 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) values for yield and specific growth rate after 72 hours were 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.

Executive summary:

The study was conducted in accordance with the requirements of OECD Guideline No. 201 to assess the effects of the test substance on the growth of the freshwater green alga,Pseudokirchneriella subcapitata,duringa 72-hour growth inhibition toxicity test.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 under static conditions at nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 x 10^4 algae cells/mL and incubated at 21 - 24°C with continuous lighting (5260 - 7670 lux) for 72 hours. Cell counts were performed at 24-hour intervals. Water quality was measured at 0 and 72 hours.

Samples of test media were analysed at 0 (new media) and 72 hours (expired test media, pooled by replicate) using liquid chromatography with time-of-flight tandem mass spectrometry detection (LC-TOF/MS) based on quantitation oftwo major constituents (a low molecular weight component and a high molecular weight component)of the test substance.Measured concentrations of the low and molecular weight components in the freshly prepared samples (0 hours) ranged from 0.0190 to 0.346 mg/L and 0.0172 to 0.344 mg/L, respectively. Measured concentrations of the low and molecular weight components in the test solutions at 72 hours ranged from 0.0103 to 0.0887 mg/L and 0.0105 to 0.110 mg/L. Given that the test substance is a complex multi-constituent substance, the results were based on nominal loading rates.

The 0-72 hour ErL50 (growth rate) and the 72-hour EyL50 (yield) values were determined to be greater than 100 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) values for yield and specific growth rate after 72 hours were 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. 201 study, conducted according to GLP, the effect of the test substance on the growth of Pseudokirchneriella subcapitata was investigated and the 72 hour EL50 value based on growth rate 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, ****).