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

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:

20 May 2019 - 13 December 2019

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)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

At the start of the test (0 hours), 10 mL samples of freshly prepared test media were taken from the control and each test media preparation vessel for chemical analysis.
At 24, 48 and 72 hours, 10 mL samples were also taken for chemical analysis from the pooled test media from the test and control groups and from the blank (non-inoculated) vessels.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

no

Details on test solutions:

At the start of the test, amounts of test substance (1.24, 1.27, 2.48, 1.04 and 2.07 mg) were separately added to 10000, 5000, 5000, 1000 and 1000 mL, respectively, of EC medium. The test substance was weighed onto a glass slide which was suspended at the top of the beaker. The preparations were stirred for ca 48 hours followed by a ca 1 hour settlement period after which the final media were syphoned from the mid-section (aqueous phase) of the vessels. A control treatment was prepared in the same manner with EC medium only.

Test organisms (species):

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

Details on test organisms:

The test organism, Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (Strain 278/4), was originally obtained from the Culture and Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.
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 × 104 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Algae cell counts were performed at 24, 48 and 72 hours during the test.

Test temperature:

The incubator temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital thermometer.

pH:

The pH of the test and control solutions ranged from 7.91 and 7.97 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with R. subcapitata. The pH in the control increased by a maximum of 0.70 units.

Nominal and measured concentrations:

Given that the test substance is a complex UVCB the effect concentrations were based on nominal loading rates.
The results of the range-finding test suggested that the 72-hour EL50 values for yield and specific growth rate were likely to be between 0.20 and 2.0 mg/L and that the corresponding NOEL value would be 0.20 mg/L, based on nominal loading rates.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

The media preparation results 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 3 mg/L. Chemical analysis of the 2, 3 and 4 mg/L concentrations showed similar measured concentrations. It was therefore decided that the maximum loading rate would be 2 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 be observed between 0.20 and 2.0 mg/L loading rate WAF.

Chemical analysis of the WAFs at 0 and 72 hours indicated a decrease in measured concentration over the 72-hour test period therefore additional samples were taken at 24 and 48 hours for the definitive test. In addition, analysis of the non-inoculated samples indicated possible adsorption to algae therefore, for the definitive test, additional non-inoculated vessels were prepared for the definitive test and sampled for chemical analysis alongside the inoculated vessels.

The results of the range-finding test suggested that the 72-hour EL₅₀valuesfor yield and specific growth rate were likely to be between 0.20 and 2.0 mg/L and that the corresponding NOEL value would be 0.20 mg/L, based on nominal loading rates. 

Definitive Test

Chemical Analysis

Analysis of the test media samples was conducted on fresh media at 0 hours and old media at 24, 48 and 72 hours. In addition, samples were taken from non-inoculated vessels at 24, 48 and 72 hours for chemical analysis.

The analytical results at 0 hours did not show a concentration dependent response. However, given that each individual WAF was prepared separately, variability between the preparations can occur. The results are therefore considered to be a reflection of the concentration in each individually prepared WAF. The results from the 24 hour inoculated vessels showed an initial decrease in measured concentration compared to the 0 hour results. However, the 48 and 72 hour results indicated that the test concentrations remained stable following the initial decrease in measured concentration after the first 24-hour period.

The results from the 24, 48 and 72 hour non-inoculated vessels showed measured concentrations to be comparable to the corresponding inoculated vessels indicated that adsorption to the algal cells did not occur during the test.

Parameter			Toxicity Values (Nominal Loading Rate - mg/L)		Statistical Test
			72 hours	Confidence limits (LCL-UCL)
Growth Rate		ErL10	0.34	0.33 – 0.34	Linear Interpolation (ICPIN)
		ErL20	0.43	0.41 – 0.44
		ErL50	0.79	0.74 – 0.84
		NOEL	0.25	NA	Nonparametric-Multiple Comparison
Yield		EyL10	0.28	0.28 – 0.28	Linear Interpolation (ICPIN)
		EyL20	0.32	0.31 – 0.32
		EyL50	0.42	0.41 – 0.43
		NOEL	0.25	NA	Nonparametric-Multiple Comparison
NA	Not applicable
LCL	Lower confidence limits
UCL	Upper confidence limits

Analysis of test media during definitive test

Inoculated Test Media				Measured concentration (µg/L)
Nominal Loading Rate (mg/L)		0 hours (new media)		24 hours (old media)	48 hours (old media)		72 hours (old media)
Control	<LOQ		<LOQ			<LOQ	<LOQ
0.125	22.6*		6.16			5.77	6.08
0.25	7.72*		1.81			0.844	0.617
0.50	27.7*		8.99			6.16	8.01
1.0	13.5		7.02			6.04	3.02
2.0	155*		11.7			13.7	4.49
LOQ = Limit of quantification (0.05µg/L)
* Result of repeat analysis following further dilution as initial results were above the calibration line

Non-inoculated Test Media				Measured concentration (µg/L)
Nominal Loading Rate (mg/L)		24 hours (old media)	48 hours (old media)			72 hours (old media)
Control	<LOQ			<LOQ	<LOQ
0.125	7.59			3.37	1.59
0.25	2.49			0.968	0.262
0.50	8.75			4.86	1.40
1.0	3.99			4.00	3.64
2.0	11.7			12.3	6.46
LOQ = Limit of quantification (0.05µg/L)

Validity criteria fulfilled:

yes

Conclusions:

The effects of the test item on the growth of the unicellular green alga, Raphidocelis subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).
Based on nominal loading rates, the 72-hour EyL50 and ErL50 values were calculated to be 0.42 mg/L and 0.79 mg/L, respectively.
The corresponding NOEL values for yield and specific growth rate after 72 hours were 0.25 mg/L.
All validity criteria were met therefore the test was considered valid.

Executive summary:

The objective of the study was to determine the effects of the test substance on the growth of the freshwater alga,Raphidocelis subcapitata,duringa 72-hour growth inhibition toxicity test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011).

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 theOECD 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 study was conducted using a static test design with preparation of test media at 0 hours only.

Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at nominal loading rates of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. A control group was also included.

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 × 10⁴ algae cells/mL and incubated at 21 – 24°C (under 4440 – 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals. 

Samples of test media were analysed at0 (fresh media), 24, 48 and 72 hours (old media). In addition, samples were taken from non-inoculated vessels at 24, 48 and 72 hours for chemical analysis.

The analytical results at 0 hours did not show a concentration dependent response. However, given that each individual WAF was prepared separately, variability between the preparations can occur. The results are therefore considered to be a reflection of the concentration in each individually prepared WAF. The results from the 24-hour inoculated vessels showed an initial decrease in measured concentration compared to the 0-hour results. However, the 48 and 72-hour results indicated that the test concentrations remained stable following the initial decrease in measured concentration after the first 24-hour period.

The results from the 24, 48 and 72-hour non-inoculated vessels showed measured concentrations to be comparable to the corresponding inoculated vessels indicating that adsorption to the algal cells did not occur during the test. 

 

Based on nominal loading rates, the 72-hour E_yL₅₀and E_rL₅₀values were calculated to be 0.42 mg/L and 0.79 mg/L, respectively.

The corresponding NOEL values for yield and specific growth rate after 72 hours were 0.25 mg/L.

All validity criteria were met therefore the test was considered valid.

Description of key information

Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at nominal loading rates of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. A control group was also included.

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 × 10⁴ algae cells/mL and incubated at 21 – 24°C (under 4440 – 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals. 

Samples of test media were analysed at0 (fresh media), 24, 48 and 72 hours (old media). In addition, samples were taken from non-inoculated vessels at 24, 48 and 72 hours for chemical analysis.

The analytical results at 0 hours did not show a concentration dependent response. However, given that each individual WAF was prepared separately, variability between the preparations can occur. The results are therefore considered to be a reflection of the concentration in each individually prepared WAF. The results from the 24-hour inoculated vessels showed an initial decrease in measured concentration compared to the 0-hour results. However, the 48 and 72-hour results indicated that the test concentrations remained stable following the initial decrease in measured concentration after the first 24-hour period.

The results from the 24, 48 and 72-hour non-inoculated vessels showed measured concentrations to be comparable to the corresponding inoculated vessels indicating that adsorption to the algal cells did not occur during the test.

The 72-hour yield (E_yL_x) and growth rate (E_rL_x) toxicity values, with corresponding no observed effect loading rates (NOEL), are presented in the table below:   

Parameter			Toxicity Values (Nominal Loading Rate - mg/L)		Statistical Test
			72 hours	Confidence limits (LCL-UCL)
Growth Rate		ErL10	0.34	0.33 – 0.34	Linear Interpolation (ICPIN)
		ErL20	0.43	0.41 – 0.44
		ErL50	0.79	0.74 – 0.84
		NOEL	0.25	NA	Nonparametric-Multiple Comparison
Yield		EyL10	0.28	0.28 – 0.28	Linear Interpolation (ICPIN)
		EyL20	0.32	0.31 – 0.32
		EyL50	0.42	0.41 – 0.43
		NOEL	0.25	NA	Nonparametric-Multiple Comparison
NA	Not applicable
LCL	Lower confidence limits
UCL	Upper confidence limits

 

Based on nominal loading rates, the 72-hour E_yL₅₀and E_rL₅₀values were calculated to be 0.42 mg/L and 0.79 mg/L, respectively.

The corresponding NOEL values for yield and specific growth rate after 72 hours were 0.25 mg/L.

All validity criteria were met therefore the test was considered valid.

There is another aglae inhibition 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

EC50 for freshwater algae:

0.79 mg/L

EC10 or NOEC for freshwater algae:

0.25 mg/L