Reaction mass of 2-hydroxy-N-(2-(2-hydroxyethoxy)ethyl)-N-(2-hydroxyethyl)-N-methylethan-1-amininium hydroxide and 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methylethanaminium hydroxide

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:

2017-07-21 to 2017-08-03

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D26-3-3-1
- Expiration date of the lot/batch: 2019-05-01
- Purity: 47.4 wt%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

OTHER SPECIFICS:
- Physical state/Appearance: Brown viscous liquid

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
* Range-Finding Test: A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.
* Definitive Test: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis

Vehicle:

no

Details on test solutions:

- Method:
* Range-Finding Test: The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
* Definitive Test: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (7.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- Controls: yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.
- Source: Liquid cultures of Pseudokirchneriella subcapitata were obtained from the 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.
- Pre-culture: 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 10^3 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.

ACCLIMATATION
- Acclimatation period: not relevant

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

Temperature was maintained at 24 ± 1 °C

pH:

The pH value of the control cultures was observed to range from pH 7.5 at 0 hours to pH 7.4 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.
A concentration dependent increase in pH was observed at the start of the test in the range of pH 7.5 at 6.25 mg/L through to pH 9.5 at 100 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustment to the pH was made prior to the addition of the algal cells.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Chemical analysis of the test preparations at 0 and 72 hours showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): 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
- Initial cells density: 5.00 x 10^3 cells per mL
- Control end cells density: 9.80 x 10^6 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
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
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjuste d to 7.5 with 0.1N NaOH or HCl.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: the pH was 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: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Samples were taken at 25, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.
- Effect calculated parameters: specific growth rate and yield.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 2
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 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): After 72 hours there were no abnormalities detected in the control or test cultures.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: 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, 6.25, 12.5, 25 and 50 mg/L test cultures were observed to be green dispersions with white flakes dispersed throughout whilst the 100 mg/L test cultures were observed to be pale green dispersions with white flakes dispersed throughout.
- ErC10 (0 - 72 h): 89 mg/L
- ErC20 (0 - 72 h): >100 mg/L
- ErC50 (0 - 72 h): >100 mg/L
- EyC10 (0 - 72 h): 31 mg/L
- EyC20 (0 - 72 h): 50 mg/L
- EyC50 (0 - 72 h): >100 mg/L
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% 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 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
- ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
- EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L
- No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
- No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
- Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
- Lowest Observed Effect Concentration (LOEC) 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:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

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 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.075		1.07E+06
	R2	0.071		8.25E+05
	R3	0.070		7.79E+05
	R4	0.076	-	1.16E+06	-
	R5	0.072		9.15E+05
	R6	0.075		1.10E+06
	Mean	0.073		9.75E+05
	SD	0.002		1.57E+05
6.25	R1	0.075	[3]	1.14E+06
	R2	0.074	[1]	1.05E+06
	R3	0.070	4	7.75E+05
	Mean	0.073	0	9.86E+05	[1]
	SD	0.003		1.89E+05
12.5	R1	0.075	[3]	1.07E+06
	R2	0.072	1	8.60E+05
	R3	0.072	1	8.99E+05
	Mean	0.073	[0]	9.41E+05	3
	SD	0.002		1.09E+05
25	R1	0.073	0	9.26E+05
	R2	0.070	4	7.90E+05
	R3	0.069	5	7.13E+05
	Mean	0.071	3	8.09E+05	17
	SD	0.002		1.08E+05
50	R1	0.073	0	9.72E+05
	R2	0.071	3	8.21E+05
	R3	0.070	4	7.79E+05
	Mean	0.071	2	8.58E+05	12
	SD	0.002		1.02E+05
100	R1	0.066	10	5.82E+05
	R2	0.065	11	5.53E+05
	R3	0.061	16	3.86E+05
	Mean	0.064	12	5.07E+05	48
	SD	0.003		1.06E+05

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

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Remarks:

The data satisfied the validation criterion given in the OECD Guideline

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance according to OECD guideline 201 (GLP conditions). The analytically confirmed nominal EC50 for growth rate inhibition (72-h ErC50) was > 100 mg/L. The results of the test can be considered reliable without restrictions.

Executive summary:

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

  Results.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>100*	50	100
Yield	>100^*	50	100

*It was not possible to calculate EC50 values as no concentration tested resulted in 50% inhibition of growth rate or yield.

Description of key information

The study of the laboratories ENVIGO (2018), investigating the acute toxicity of the test substance to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. It was not possible to calculate ErC20 or ErC50 values as no concentration tested resulted in greater than 12% inhibition of growth rate. There were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5, 25 and 50 mg/L test concentrations however the 100 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 50 mg/L.

The 72-h EC50 for growth rate was determined to be >100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

50 mg/L