N-2-hydroxyethylacetamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 2017 to 29 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-2-hydroxyethylacetamide

EC Number:

205-530-8

EC Name:

N-2-hydroxyethylacetamide

Cas Number:

142-26-7

Molecular formula:

C4H9NO2

IUPAC Name:

N-2-hydroxyethylacetamide

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Due to analytical issues, no results were obtained from the analysis of the test samples taken from the range-finding test.

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: N/A
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Remarks:

The test item was dissolved directly in culture medium.

Details on test solutions:

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10⁴ to 10⁵ cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Culturing conditions and media were the same as the test conditions.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

8.7

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type : closed, plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation
- Aeration: N/A
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5E03 cells/mL.
- Control end cells density: 10^5 to 10^5 cells/mL
- No. of vessels per concentration (replicates): duplicate
-- No. of vessels per control (replicates): duplicate

GROWTH MEDIUM
- Standard medium used: yes, The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Culture medium:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 380 to 730 nm

EFFECT PARAMETERS MEASURED : 72 hour growth rate and yield data
- Determination of cell concentrations: After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter
- Other: All test and control cultures were inspected microscopically at 72 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on this information a single test concentration of 6 replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.

Temperature was maintained at 24 ±1 ºC throughout the test.

At the start of the test all test cultures were observed to be clear colorless solutions. After the 72-Hour test period all test cultures were observed to be green dispersions.

Results with reference substance (positive control):

The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 8.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

At the start of the test all control cultures were observed to be clear colorless solutions. After the 72-Hour test period all control cultures were observed to be green dispersions.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Table 1: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 - 72 hours	% Inhibition	0 - 72 hours	% Inhibition*
Control	R1	0.077	-	1.27E+05	-
	R2	0.083		1.97E+05
	R3	0.084		2.07E+06
	R4	0.082		1.85E+06
	R5	0.081		1.67E+06
	R6	0.082		1.80E+06
	Mean	0.082		1.77E+06
	SD	0.002		2..81E+05
100	R1	0.085	[4]	2.19E+06
	R2	0.078	5	1.41E+06
	R3	0.078	5	1.36E+06
	R4	0.079	4	1.43E+06
	R5	0.080	2	1.61E+06
	R6	0.076	7	1.19E+06
	Mean	0.079	3	1.53E+06	14
	SD	0.003		3.51E+05

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 (yield and growth rate) values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

The study was performed to determine the effect of the test item on the growth of the green alga, Pseudokirchneriella subcapitata, using OECD 201 and EU method C.3 with concentrations of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 114% to 115% of nominal and so the results are based on nominal test concentrations only. Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.