N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

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:

17 May 2019 to 20 Jan 2020

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:

SOURCE OF TEST MATERIAL
- lot/batch No.: E00268-014-212
- Retest date of the lot/batch: 25 March 2020
- Purity: 100%
- Storage condition of test material: Room temperature under nitrogen

Analytical monitoring:

yes

Details on sampling:

10 mL samples were taken at the start of the test (0 hours) and at media renewal at 24 hours from the control and test media preparation vessel for chemical analysis. 10 mL samples were also taken at 24 and 48 hours for chemical analysis from the pooled old test media from the test and control groups.

Vehicle:

no

Details on test solutions:

The stock media were observed to be colourless solutions with test substance adhered to the glass slide and at the bottom of the preparation vessel. The test preparations were observed to be colourless solutions throughout the duration of the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. These cultures were set up originally from dormant eggs (ephippia) supplied by MicroBioTests Inc., Belgium.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

Test temperature:

The temperature during the definitive test differed by more than 1ºC from initial measured temperatures which deviated from the protocol. This deviation was not considered to have had an adverse impact on the integrity of the test given that temperatures remained within the 20 ± 2ºC range specified in the protocol and no immobilisation was observed during the test.

pH:

The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures 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 at 24 and 48 hours. The pH ranged between 7.6 - 8 which meets the criteria (pH of 6-9)

Dissolved oxygen:

The validity criteria for dissolved oxygen (more than or equal to 3 mg/L) was met.

Nominal and measured concentrations:

Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at a single nominal loading rate of 4.0 mg/L.
Measured concentrations at the limit of quantification were observed in the control sample at 24 hours (old media). Re-analysis was not possible as the results obtained were from the frozen duplicate samples due to initial analytical batch failure. The results were considered to be due to possible post-sampling contamination given that measured concentrations above the LOQ were not observed in the 0 hour samples.
Given that the test substance is a complex UVCB the effect concentrations were based on nominal loading rates.

Details on test conditions:

The test was conducted with a 16-hour light: 8-hour dark photoperiod.
The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures 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 at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area. The test vessels were not aerated during the test.
At the end of each exposure period the test water quality measurements were conducted using pooled replicate samples of old test media.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No mortality or immobility was seen at the limit dose

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No mortality or immobility was seen at the limit dose.

The results of the media preparation trial indicated that a prolonged stirring period did not result in increased measured concentrations therefore a stirring period of 48-hours followed by a 1-hour settle period was selected for the conduct of the tests.

Critical Micelle Concentration Test

Initial surface tension and turbidity analyses of the prepared WAFs indicated that the critical micelle concentration was between 1.0 and 10 mg/L loading rate WAF. Serial dilutions were prepared from the 10 mg/L loading rate WAF and surface tension analysis performed. The results indicated that the critical micelle concentration was approximately 5 mg/L. Chemical analysis of the 4, 5 and 6 mg/L concentrations showed similar measured concentrations. It was therefore decided that the maximum loading rate would be 4 mg/L to avoid the possibility of micelle formation during preparation.

Range-finding Test

The results of the range-finding test indicated that effects may not be observed at 4.0 mg/L loading rate WAF.

Chemical analysis of the WAFs at 0 and 48 hours indicated a decrease in measured concentration over the 48-hour test period therefore the test design for the definitive test was amended to use a semi-static design.

The temperature during the range-finding test differed by more than 1ºC from initial measured temperatures which deviated from the protocol. This deviation was not considered to have had an adverse impact on the integrity of the test given that temperatures remained within the 20 ± 2ºC range specified in the protocol and no immobilisation was observed during the test.

Definitive Test

Chemical Analysis

The results of the chemical analysis are presented in Table 4. Example chromatograms of test samples, standard solutions and typical calibration lines are presented in Appendix 3. The limit of quantification (LOQ) was 0.1 µg/L.

Analysis of the test media samples was conducted at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).

Nominal loading rate (mg/L)			Measured concentration (µg/L)
0 Hours (new media)	24 Hours (old media)**		24 Hours (fresh media)**	48 Hours (old media)
Control	<LOQ*	0.118 0.117	<LOQ	<LOQ
4.0	11.4	4.28 4.51	5.56 5.78	3.14

Measured concentrations at the limit of quantification were observed in the control sample at 24 hours (old media). Re-analysis was not possible as the results obtained were from the frozen duplicate samples due to initial analytical batch failure. The results were considered to be due to possible post-sampling contamination given that measured concentrations above the LOQ were not observed in the 0 hour samples.

Given that the test substance is a complex UVCB substance the effect concentrations were based on nominal loading rates only.

Toxicity to Daphnia magna

The percentage immobility data are summarised below.

Nominal loading rate (mg/L)	Number ofDaphnia magnaexposed	% Immobility at 24 hours	% Immobility at 48 hours
Control	20	0	0
4.0	20	0	0

Toxicity values and the corresponding No Observed Effect Loading rate (NOEL) and Lowest Observed Effect Loading rate (LOEL) values are presented in the following table. Values are presented in terms of nominal loading rates.

Effect loading rate (mg/L)
24-hour			48-hour
EL50	>4.0	>4.0
LOEL	>4.0	>4.0
NOEL	4.0	4.0

The 48-hour EL50 value was determined to be greater than 4.0 mg/L loading rate WAF. The corresponding NOEL was determined to be 4.0 mg/L loading rate WAF.

Analytical analysis of media during definitive test

Measured concentration (µg/L)
Nominal loading rate (mg/L)	0 Hours (new media)			24 Hours (old media)**		24 Hours (fresh media)**	48 Hours (old media)
Control		<LOQ*	0.118 0.117		<LOQ		<LOQ
4.0		11.4	4.28 4.51		5.56 5.78		3.14

LOQ = Limit of quantification of the analytical method (0.1 µg/L)

* Results from duplicate analysis of ‘back-up’ samples given that initial results showed measured concentrations >30% of LOQ

** Results from analysis of ‘back-up’ samples, analysed in duplicate, given that initial analytical batch failed due to poor recoveries

Measured concentrations at the limit of quantification were observed in the control sample at 24 hours (old media). Re-analysis was not possible as the results obtained were from the frozen duplicate samples due to initial analytical batch failure. The results were considered to be due to possible post-sampling contamination given that measured concentrations above the LOQ were not observed in the 0 hour samples.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour acute toxicity of the test item to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
Based on nominal loading rates, the 48-hour EL50 value was estimated to be >4.0 mg/L loading rate WAF.
The corresponding NOEL value was 4.0 mg/L loading rate WAF.
All validity criteria were satisfied, therefore the test was considered valid.

Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of the test substance against the mobility of the freshwater planktonic crustacean, Daphnia magna. The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

The test 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 (2019); Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals). Based on the recommendations of this guideline, the test substance was prepared as Water Accommodated Fractions (WAFs).

The test was conducted using semi-static test conditions with renewal of test media at 24 hours.

Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at a single nominal loading rate of 4.0 mg/L. A control group was also included.

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 at 24 and 48 hours. The Daphnia magna were not fed during the test.

Samples of test media were analysed at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).

Nominal loading rate (mg/L)			Measured concentration (µg/L)
0 Hours (new media)	24 Hours (old media)**		24 Hours (fresh media)**	48 Hours (old media)
Control	<LOQ*	0.118 0.117	<LOQ	<LOQ
4.0	11.4	4.28 4.51	5.56 5.78	3.14

Measured concentrations at the limit of quantification were observed in the control sample at 24 hours (old media). Re-analysis was not possible as the results obtained were from the frozen duplicate samples due to initial analytical batch failure. The results were considered to be due to possible post-sampling contamination given that measured concentrations above the LOQ were not observed in the 0 hour samples.

Given that the test substance is a complex UVCB the effect concentrations were based on nominal loading rates.

The EL50 values and the corresponding No Observed Effect Loading rate (NOEL) and Lowest Effect Loading rate (LOEL) values are presented in the following table.

Effect loading rate (mg/L)
24-hour			48-hour
EL50	>4.0	>4.0
LOEL	>4.0	>4.0
NOEL	4.0	4.0

The 48-hour EL50 value was determined to be greater than 4.0 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) was determined to be 4.0 mg/L loading rate WAF.

The validity criteria for control immobility (=10%) and dissolved oxygen (=3 mg/L) were both satisfied. The test is therefore considered valid.

Description of key information

The test 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 (2019); Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals). Based on the recommendations of this guideline, the test substance was prepared as Water Accommodated Fractions (WAFs).

The test was conducted using semi-static test conditions with renewal of test media at 24 hours.

Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at a single nominal loading rate of 4.0 mg/L. A control group was also included.

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 at 24 and 48 hours. The Daphnia magna were not fed during the test.

Samples of test media were analysed at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).

Given that the test substance is a complex UVCB the effect concentrations were based on nominal loading rates.

The 48-hour EL50 value was determined to be greater than 4.0 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) was determined to be 4.0 mg/L loading rate WAF.

The validity criteria for control immobility (=10%) and dissolved oxygen (=3 mg/L) were both satisfied. The test is therefore considered valid.

There is another acute Daphnia study which has been assigned a Klimish rating of 3 due to significant methodological deficiencies leading to inconsistences in the analytical measurements and toxicity values which prevent a reliable classification determination.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4 mg/L