3,3'-[methylenebis(oxymethylene)]bisheptane

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:

05 March 2019 - 11 May 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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Clear colorless liquid

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control, solvent control and 0.0104 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A single additional sample of the test group containing no algal cells was incubated alongside the test to provide a sample of uninoculated media for analysis at 72 hours.
Further samples were produced in order to comply with the difficult substances directive to determine how much test item remained in solution after centrifugation.
A set of duplicate samples was taken at 0 and 72 hours and stored frozen for further analysis if necessary.
The test samples were analyzed on the day of receipt.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal amount of test item (10.4 mg) was made up to 10 mL in dimethylformamide (1.04 mg/mL). This was further diluted to give a 0.104 mg/mL solvent stock solution. One litre of culture medium was then spiked with 100 μL of the 0.104 mg/mL solvent stock solution (prepared in duplicate). This was then stirred for a total of 5 minutes or 60 minutes using a magnetic stirrer to give final stock solutions of 0.0104 mg/L.

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 under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10e4 to 10e5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

72 hours, under constant illumination and shaking

Post exposure observation period:

n/a

Hardness:

Not specified

Test temperature:

24 ±1 ºC throughout the test

pH:

7.4 - 8.0

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.000104, 0.00104 and 0.0104 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was prepared using a preliminary solution in dimethylformamide.

Details on test conditions:

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, solvent control and 0.0104 mg/L treatment group.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 μL/L of dimethylformamide.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.90 x 10e5 cells per mL. Inoculation of 900 mL of test medium with 7.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10e3 cells per mL and had no significant dilution effect on the final test concentration.
An aliquot (900 mL) of aqueous stock solution was inoculated with algal suspension (7.6 mL) to give the required test concentration of 0.0104 mg/L.The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test conditions:
- Lighting: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)
- Test medium: see Table 1
- pH: The pH of the control, solvent control and 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.
- Test system: constantly shaken at approximately 150 rpm, glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control, solvent control and 0.0104 mg/L treatment group. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator)

Details on the determination of algal biomass:
Cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Samples were taken at 26, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10e3 cells/mL) was taken as the starting cell density.

Determination of growth rates:
The average specific growth rate over the test duration was calculated for each replicate control, solvent control and test item vessel using the nominal inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition, the section by section specific growth rate (Days 0 to 1, 1 to 2 and 2 to 3) was calculated for the control and solvent control cultures and the results examined in order to determine whether the growth rate remained constant.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The growth rate and yield of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.0015 mg/L over the 72-Hour exposure period.
The test concentration of 0.0015 mg/L was the highest attainable test concentration that could be achieved due to the limited solubility of the test item in water and auxiliary solvent (having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline) and the instability of the test item under test conditions. See table 2.

Growth curves:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4.

The pH values of the controls and the test preparation are given in Table 3. Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 7.7 at 72 hours. The pH value of the solvent control cultures was observed to increase from pH 7.5 at 0 hours to pH 7.8 at 72 hours.The pH deviation in the control and solvent control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Other effects:
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test, control and solvent 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. All test, control and solvent control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control, solvent control or test cultures at 72 hours.

Monitoring of test concentrations:
Analysis of the test preparation at 0 hours showed the measured test concentration to be 86% of the nominal concentration. Analysis of the test preparation at 72 hours showed a decline in the measured concentration to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.00052 mg/L. See table 5.

Results with reference substance (positive control):

A positive control (Study number YJ31TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018.
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 to 72 hour) : 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour) : 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L
No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

* In-house data from the last 5 years shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.26) and a mean EyC50 value of 0.66 mg/L (standard deviation = 0.14).

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance was carried out on the growth rate and yield data after 72 hours for the solvent control and the 0.0104 mg/L test concentration to determine any statistically significant differences between the test and solvent control group. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 2 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 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.079	-	1.45E+06	-
	R2	0.077		1.31E+06
	R3	0.071		8.08E+05
	R4	0.078		1.33E+06
	R5	0.075		1.14E+06
	R6	0.074		1.04E+06
	Mean	0.076		1.18E+06
	SD	0.003		2.35E+05
Solvent control	R1	0.069	-	7.26E+05	-
	R2	0.072		9.12E+05
	R3	0.074		1.02E+06
	R4	0.071		8.32E+05
	R5	0.071		8.53E+05
	R6	0.071		8.50E+05
	Mean	0.071		8.66E+05
	SD	0.002		9.75E+04
0.0015	R1	0.076	[7]	1.15E+06
	R2	0.077	[8]	1.27E+06
	R3	0.077	[8]	1.28E+06
	R4	0.078	[10]	1.37E+06
	R5	0.076	[7]	1.17E+06
	R6	0.075	[6]	1.12E+06
	Mean	0.077	[8]	1.23E+06	[42]
	SD	0.001		9.31E+04

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

R = Replicate

SD = Standard Deviation

[ ] = Increase in growth compared to solvent controls

- Not applicable 

Table 3 Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	26 Hours	49 Hours	72 Hours	72 Hours
Control	R1	7.4	3.74E+04	1.97E+05	1.46E+06	7.7
	R2		3.67E+04	1.98E+05	1.32E+06
	R3		3.57E+04	1.57E+05	8.13E+05
	R4		3.47E+04	1.92E+05	1.34E+06
	R5		3.46E+04	1.67E+05	1.14E+06
	R6		3.41E+04	1.69E+05	1.04E+06
	Mean		3.55E+04	1.80E+05	1.18E+06
Solvent control	R1	7.5	3.04E+04	1.43E+05	7.31E+05	7.8
	R2		3.22E+04	1.56E+05	9.17E+05
	R3		3.26E+04	1.64E+05	1.03E+06
	R4		3.45E+04	1.57E+05	8.37E+05
	R5		3.43E+04	1.52E+05	8.58E+05
	R6		3.42E+04	1.56E+05	8.55E+05
	Mean		3.30E+04	1.55E+05	8.71E+05
0.0015	R1	7.5	3.23E+04	1.81E+05	1.16E+06	8
	R2		3.17E+04	1.85E+05	1.27E+06
	R3		3.49E+04	1.97E+05	1.28E+06
	R4		3.36E+04	2.12E+05	1.37E+06
	R5		3.24E+04	1.90E+05	1.18E+06
	R6		3.56E+04	1.83E+05	1.13E+06
	Mean		3.34E+04	1.91E+05	1.23E+06

* 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

R = Replicate

Table 4 - Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.077	0.072	0.087
	R2	0.077	0.073	0.082
	R3	0.076	0.064	0.071
	R4	0.074	0.074	0.084
	R5	0.074	0.069	0.083
	R6	0.074	0.070	0.079
	Mean	0.075	0.070	0.081
Solvent control	R1	0.069	0.067	0.071
	R2	0.072	0.069	0.077
	R3	0.072	0.070	0.08
	R4	0.074	0.066	0.073
	R5	0.074	0.065	0.075
	R6	0.074	0.066	0.074
	Mean	0.073	0.067	0.075

R = Replicate

Table 5 - 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	2	<LOQ	-
	Solvent Control	2	<LOQ	-
	0.0104	2	0.00892	86
P. Rec	0.00115	2	0.00110	96
P. Rec	0.00115	2	0.00116	101
72	Control	2	<LOQ	-
	Solvent Control	2	<LOQ	-
	0.0104	2	<LOQ	-
	0.0104 (no algae)	2	<LOQ	-
P. Rec	0.00107	2	0.00103	96
P. Rec	0.00107	2	0.000922	86

LOQ = Limit of Quantification (0.00052 mg/L)

- = Not applicable

P. Rec = Procedural recovery

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.0015 mg/L. The No Observed Effect Concentration (NOEC) was 0.0015 mg/L.
Exposure of Pseudokirchneriella subcapitata to a measured starting concentration of 0.0089 mg/L (considered to be the limit of solubility of this test item in the test media) resulted in no signs of toxicity being observed.

Description of key information

A test has been performed in 2019 (OECD 201, GLP) after determination of water solubility of 10.4 µg/L and determination of solubility of 2 -ethylhexylal in the algae test media of 8.9 µg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.0015 mg/L. The No Observed Effect Concentration (NOEC) was 0.0015 mg/L.

Exposure of Pseudokirchneriella subcapitata to a measured starting concentration of 0.0089 mg/L (considered to be the limit of solubility of this test item in the test media) resulted in no signs of toxicity being observed.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.5 µg/L

EC10 or NOEC for freshwater algae:

1.5 µg/L