N-2-hydroxyethyllactamide

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:

09 Aug - 07 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

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)

Remarks:

Department of Health -The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

N-2-hydroxyethyllactamide

EC Number:

226-546-1

EC Name:

N-2-hydroxyethyllactamide

Cas Number:

5422-34-4

Molecular formula:

C5H11NO3

IUPAC Name:

N-2-hydroxyethyllactamide

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L from the bulk test preparation
- Sampling method: duplicate samples were taken at test start and from the pooled replicates at 72 hours for quantitative analysis. An additional non-inoculated 100 mg/L test sample that had been incubated alongside the inoculated test preparations was taken for analysis at 72 hours to determine the stability of the test item under experimental conditions
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg were dissolved directly in culture medium and the volume adjusted to 1 liter. An aliquot (950 mL) of this stock solution was inoculated with algal suspension (5.1 mL) to give the required test concentration of 100 mg/L.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 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 in the laboratory under constant aeration and illumination at 21 ± 2 °C. 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 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: There were no abnormalities detected in any of the control or test cultures.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 ºC

pH:

test start 7.4
test end 7.7

Nominal and measured concentrations:

Nominal: control and 100 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Initial cells density: 5 x 10^3 cells per mL
- Control end cells density: 6.71 x 10^5 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test medium was prepared with reverse osmosis purified deionized water Elga Optima 15+ or Elga Purelab Option R-15 BP
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was determined at itest start and test end. The temperature was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density determined using a Coulter® Multisizer Particle Counter
- Chlorophyll measurement: no

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: the concentration of the definitive is based on the results of the range-finding test. Results in detail are summarized within table 1 in section "Any other information on material and methods incl. tables"

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

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 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.
- Other: Results of the range-finding test and the definitive test are summarized in tables 1 - 4 in section "Any other information on results incl. tables"

Results with reference substance (positive control):

Reference: ErC50 (0 – 72 h) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L; No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L

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: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.14E+03	6.24E+05	-	-
	R2	5.95E+03	6.22E+05
	Mean	5.54E+03	6.23E+05
0.10	R1	6.14E+03	6.74E+05	2	[9]
	R2	6.26E+03	6.82E+05
	Mean	6.20E+03	6.78E+05
1.0	R1	5.90E+03	6.48E+05	2	[5]
	R2	6.34E+03	6.61E+05
	Mean	6.12E+03	6.54E+05
10	R1	6.08E+03	7.35E+05	0	[19]
	R2	6.51E+03	7.53E+05
	Mean	6.30E+03	7.44E+05
100	R1	6.03E+03	6.58E+05	2	[5]
	R2	6.08E+03	6.47E+05
	Mean	6.06E+03	6.53E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 and R2 = Replicates 1 and 2

[Increase in growth compared to controls ]

Analytical results:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 97% to 99% of nominal and so the results are based on nominal test concentrations only (Table 3).

Validation Criteria:

The cell concentration of the control cultures increased by a factor of 134 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and the coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3%.

Table 2 : Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.071		8.15E+05
	R2	0.068		6.49E+05
	R3	0.068		6.79E+05
	R4	0.068	-	6.79E+05	-
	R5	0.064		5.01E+05
	R6	0.068		6.75E+05
	Mean	0.068		6.66E+05
	SD	0.002		1.00E+05
100	R1	0.068	0	6.58E+05
	R2	0.068	0	6.60E+05
	R3	0.069	[1]	7.01E+05
	R4	0.068	0	6.69E+05
	R5	0.067	1	6.12E+05
	R6	0.067	1	6.10E+05
	Mean	0.068	0	6.52E+05	2
	SD	0.001		3.51E+04

* In accordance with the OECD test guideline only the mean value for yield is calculated

R1 -R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Table 3: Analytical Results for Test Samples

Time point [hours]	Nominal Concentration of Test Item in Test Sample cnom [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample c [mg/L]	Percentage of Nominal Concentration [%]
0	Control	1	<LOQ	-
	100	100	97.8	98
72	Control	1	<LOQ	-
	100	100	99.1	99
	100 no algae	100	97.2	97

LOQ = Limit of Quantification

- = not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes