Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

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:

Testing was conducted between 11th August 2014 and 2nd September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with
chloromethyl)oxirane (TK30174)
Physical state/ Appearance: Clear colorless liquid
Batch: AAB1584600
Purity: Not provided
Expiry Date: 31 October 2015
Storage Conditions: Room temperature in the dark
Intended use/ Application: Resin for coating systems

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

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, 3.2, 1.0, 0.32, 0.10 and 0.032 mg/L. An aliquot (500 mL) of each of the 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L stock solutions was separately inoculated with algal suspension (2.6 mL) to give the required test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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. 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 constant illumination at 21 ± 1 °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 10E3 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 10E4 - 10E5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Hardness:

Not applicable

Test temperature:

The temperature within the incubator was recorded daily. Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

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 pH value of the control cultures (see Table 2*) was observed to increase from pH 8.1 at 0 hours to pH 8.5 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.

* For table 2 please see the any other information on results section (including tables)

Dissolved oxygen:

Not applicable

Salinity:

Not applicable as a fresh water study was conducted

Nominal and measured concentrations:

Range finding test: The following nominal concentrations were used 0.10, 1.0, 10 and 100 mg/L

Definitive test:
Nominal concentrations: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Measured concentrations: The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (mg/L) Geometric Mean Measured Test Concentration (mg/L) Expressed as a % of the Nominal Test Concentration
0.032 0.025 78
0.10 0.082 82
0.32 0.25 79
1.0 0.80 80
3.2 2.5 78
10 7.7 77

Details on test conditions:

EXPERIMENTAL PREPARATION:
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, 3.2, 1.0, 0.32, 0.10 and 0.032 mg/L. An aliquot (500 mL) of each of the 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L stock solutions was separately inoculated with algal suspension (2.6 mL) to give the required test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

TEST SYSTEM
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.72 x 10E5 cells per mL. Inoculation of 500 mL of test medium with 2.6 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: warm white lighting at 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

VALIDATION CRITERIA:
The results of the test are considered valid if the following performance criteria are met:

1) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

2) The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

3) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

RANGE FINDING STUDY:
- Test concentrations: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

7.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

7.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range finding Test:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1*.

The results showed no effect on growth rate at the test concentration of 0.10 mg/L. However, growth rate was observed to be reduced at 1.0, 10 and 100 mg/L. Effects on yield were observed at all test concentrations employed.

Based on this information test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 91% to 175% of nominal. Examination of the data could find no obvious cause for the test concentrations obtained in excess of 120% of the nominal value.

* For table 1 please see the any other information on results section, including tables under the sub header range finding study.

Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 86% to 94% of nominal. A decline in measured test concentration was observed at 72 hours in the range of 67% to 72% of nominal.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (mg/L) Geometric Mean Measured Test Concentration (mg/L) Expressed as a % of the Nominal Test Concentration
0.032 0.025 78
0.10 0.082 82
0.32 0.25 79
1.0 0.80 80
3.2 2.5 78
10 7.7 77

The following results were determined from the data:
 
 Inhibition of Growth Rate
ErC10 (0 - 72 h)   :   6.3 mg/L
ErC20 (0 - 72 h)   :   7.1 mg/L
ErC50 (0 - 72 h)   :   9.0 mg/L*
  
 Inhibition of Yield:
EyC10(0 - 72 h)   :   2.7 mg/L
EyC20(0 - 72 h)   :   2.8 mg/L
EyC50(0 - 72 h)   :   3.4 mg/L

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on Test Item Solubility:
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, 0.025, 0.082, 0.25, 0.80 and 2.5 mg/L test cultures were observed to be pale green dispersions whilst the 7.7 mg/L test cultures were observed to be very pale green dispersions.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item. The positive control was conducted between 21 October 2013 and 24 October 2013.

A positive control (Harlan Study Number 41303826) 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) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 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:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations 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).

Geometric Mean Measured Test Concentrations:
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 - R3 pooled:

GM = √C0 x C1
Where:

GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

Range finding Study:

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	5.33E+03	1.25E+06	-	-
	R2	6.35E+03	1.22E+06
	Mean	5.84E+03	1.23E+06
0.10	R1	5.87E+03	1.08E+06	3	23
	R2	4.98E+03	8.25E+05
	Mean	5.42E+03	9.52E+05
1.0	R1	5.65E+03	3.72E+05	22	69
	R2	6.10E+03	3.89E+05
	Mean	5.87E+03	3.81E+05
10	R1	7.38E+03	4.38E+04	68	97
	R2	6.75E+03	3.66E+04
	Mean	7.06E+03	4.02E+04
100	R1	9.02E+03	1.08E+04	97	100
	R2	8.02E+03	9.31E+03
	Mean	8.52E+03	1.00E+04

Definitive Test:

Growth Data:

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

 

Table 2     Cell Densities and pH Values in the Definitive Test:

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	5.31E+03	2.32E+04	1.16E+05	6.15E+05	8.5
	R2		5.78E+03	2.04E+04	1.26E+05	6.58E+05
	R3		5.46E+03	1.78E+04	8.90E+04	4.78E+05
	R4		5.49E+03	1.55E+04	1.08E+05	5.31E+05
	R5		5.15E+03	1.26E+04	1.01E+05	5.21E+05
	R6		6.32E+03	1.92E+04	1.46E+05	6.97E+05
	Mean		5.58E+03	1.81E+04	1.14E+05	5.83E+05
0.025	R1	8.1	6.42E+03	1.57E+04	1.29E+05	8.74E+05	8.5
	R2		5.76E+03	1.74E+04	1.22E+05	8.45E+05
	R3		6.27E+03	2.10E+04	1.28E+05	7.31E+05
	Mean		6.15E+03	1.81E+04	1.27E+05	8.17E+05
0.082	R1	8.1	6.43E+03	1.77E+04	1.10E+05	6.68E+05	8.5
	R2		5.50E+03	2.02E+04	1.28E+05	8.48E+05
	R3		6.96E+03	1.96E+04	1.49E+05	7.80E+05
	Mean		6.30E+03	1.92E+04	1.29E+05	7.65E+05
0.25	R1	8.0	5.74E+03	1.91E+04	1.23E+05	8.14E+05	8.4
	R2		6.32E+03	1.32E+04	1.21E+05	6.94E+05
	R3		5.44E+03	1.75E+04	1.41E+05	7.06E+05
	Mean		5.83E+03	1.66E+04	1.28E+05	7.38E+05
0.80	R1	8.0	5.78E+03	1.56E+04	1.36E+05	7.60E+05	8.4
	R2		5.20E+03	1.55E+04	1.01E+05	7.44E+05
	R3		6.18E+03	1.85E+04	1.55E+05	8.24E+05
	Mean		5.72E+03	1.65E+04	1.31E+05	7.76E+05
2.5	R1	7.9	6.19E+03	9.52E+03	8.55E+04	5.69E+05	8.4
	R2		5.55E+03	9.78E+03	1.18E+05	5.93E+05
	R3		6.05E+03	9.55E+03	1.02E+05	5.87E+05
	Mean		5.93E+03	9.62E+03	1.02E+05	5.83E+05
7.7	R1	7.9	6.43E+03	3.95E+03	2.23E+04	1.60E+05	8.2
	R2		6.83E+03	3.95E+03	2.79E+04	1.28E+05
	R3		6.90E+03	2.48E+03	2.32E+04	1.68E+05
	Mean		6.72E+03	3.46E+03	2.45E+04	1.52E+05

Table 3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test:

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.064	0.067	0.069
	R2	0.059	0.076	0.069
	R3	0.053	0.067	0.070
	R4	0.047	0.081	0.066
	R5	0.039	0.087	0.068
	R6	0.056	0.084	0.065
	Mean	0.053	0.077	0.068

R₁- R₆= Replicates 1 to 6

Table 4     Inhibition of Growth Rate and Yield in the Definitive Test:

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.067		6.09E+05
	R2	0.068		6.52E+05
	R3	0.063		4.73E+05
	R4	0.065	-	5.25E+05	-
	R5	0.065		5.16E+05
	R6	0.069		6.91E+05
	Mean	0.066		5.78E+05
	SD	0.002		8.60E+04
0.025	R1	0.072	[9]	8.68E+05
	R2	0.071	[8]	8.39E+05
	R3	0.069	[5]	7.25E+05
	Mean	0.071	[7]	8.10E+05	[40]
	SD	0.002		7.55E+04
0.082	R1	0.068	[3]	6.62E+05
	R2	0.071	[8]	8.42E+05
	R3	0.070	[6]	7.73E+05
	Mean	0.070	[6]	7.59E+05	[31]
	SD	0.002		9.11E+04
0.25	R1	0.071	[8]	8.08E+05
	R2	0.069	[5]	6.87E+05
	R3	0.069	[5]	7.00E+05
	Mean	0.070	[6]	7.32E+05	[27]
	SD	0.001		6.62E+04
0.80	R1	0.070	[6]	7.54E+05
	R2	0.069	[5]	7.39E+05
	R3	0.071	[8]	8.18E+05
	Mean	0.070	[6]	7.70E+05	[33]
	SD	0.001		4.17E+04
2.5	R1	0.066	0	5.63E+05
	R2	0.066	0	5.87E+05
	R3	0.066	0	5.81E+05
	Mean	0.066	0	5.77E+05	0
	SD	0.000		1.26E+04
7.7	R1	0.048	27	1.53E+05
	R2	0.045	32	1.21E+05
	R3	0.049	26	1.61E+05
	Mean	0.047	28	1.45E+05	75
	SD	0.002		2.16E+04

From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata(CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Response Variable EC10 (mg/L) EC20 (mg/L) EC50 (mg/L) NOEC (mg/L) LOEC (mg/L)
Growth Rate 6.3 7.1 9.0* 2.5 7.7
Yield 2.7 2.8 3.4 2.5 7.7

Executive summary:

Introduction:

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 0.032, 0.10, 0.32, 1.0, 3.2 and 10 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 hours showed measured test concentrations to range from 86% to 94% of nominal. A decline in measured test concentration was observed at 72 hours in the range of 67% to 72% of nominal.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

 

Response Variable	EC10(mg/L)	EC20(mg/L)	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	6.3	7.1	9.0*	2.5	7.7
Yield	2.7	2.8	3.4	2.5	7.7

*Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate.

Description of key information

Pseudokirchneriella subcapitata 72 hr EC 50 = 9.0 mg/l,  EC10 = 6.3 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

9 mg/L

EC10 or NOEC for freshwater algae:

6.3 mg/L