Methoxycyclododecane

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:

22 May - 31 May 2015

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 6.25, 12.5, 25, 50, 100% of a saturated solution. Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 h separate replicates were prepared with and without algae at the beginning of the exposure and incubated under test conditions. Additionally, the adsorption of the test item to glass was analysed after 72 h.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution of 1000 mg/L was prepared four days prior to test start. The test item was placed by pipetting on the surface of the dilution water. Gentle stirring was carried out with a magnetic stirrer at about 25 °C for four days. After 1 h of separation the test item phase was separated by siphon from the homogeneous phase. The homogeneous phase was taken from the bottom and was used for testing. The saturated solution was further diluted with a dilution factor of 2.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Göttingen, Germany
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light amounted to 35-70 µE*m-2*s-1 for 24 h per day
- Age of inoculum: A four days old preculture, prepared in dilution water, was used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+ Mg/L

Test temperature:

22 - 23 °C

pH:

7.91 - 8.06 (0 h)
8.30 - 8.98 (72 h)

Nominal and measured concentrations:

nominal: control, 6.25, 12.5, 25, 50, 100% of a saturated solution
measured: < LOQ, 0.142, 0.293, 0.661, 1.32, 3.10 mg/L (geometric mean measured concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with aluminium tops with PTFE seals
- Material, fill volume: glass, fill volume: 119 mL (flasks without headspace were used)
- Aeration: using a rotary shaker and oscillated at approx. 70 rpm
- Initial cells density: 5024 cells/mL
- Control end cells density: 306857 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 68.4 µE*m-2*s-1 (mean)

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence (exitation: 436 nm, emission: 685 nm).
- Other: Microscopic evaluation of the cells was done after 0 and 72 h. Cells were checked for unusual cell shapes, color differences, differences in chloroplast morphology, flocculation, adherence of algae to test vessels and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, non-GLP in test vessels without headspace

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.661 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.:

1.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.865 - 1.19 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.09 - 2.75 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.681 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.:

1.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.689 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.516 - 0.850 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 1.10 - 1.65 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not observed
- Unusual cell shape: not observed
- Colour differences: not observed
- Flocculation: not observed
- Adherence to test vessels: between 1.6 - 2.1%
- Aggregation of algal cells: not observed
- Any stimulation of growth found in any treatment: yes, 3% stimulation at 0.293 mg/L (geom. mean) and 5% stimulation at 0.142 mg/L (geom. mean) after 72 h.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, solutions were clear and colorless

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 0.613 mg/L (95% CI: 0.589 - 0.638 mg/L, with headspace); 0.774 mg/L (95% CI: 0.711 - 1.08 mg/L, without headspace)
- Other: These results are based on a study performed from 14 Apr - 17 Apr 2015 (with headspace) and 10 Nov - 13 Nov 2014 (without headspace), respectively.

Reported statistics and error estimates:

EC10, EC20 and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett ’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The measured concentrations of the test substance in the fresh media (0 h) were 0.163 - 0.331 - 0.668 - 1.36 - 2.99 mg/L. The measured concentrations in the old media with algae (72 h) were in the range of 76 to 107 % of the initially measured values of all concentrations (0 h). The geometric mean measured concentrations was determined. Additionally, the measured concentrations in old media (72 h) without algae were in the range of 82 - 99% of the initially measured values of all concentrations (0 h).

Table 1: Evaluation after 72 h

Geometric mean measured concentration [mg/L]	Replicate	Growth rate [d-1]	Inhibition of growth rate [%]
3.10	1	0.42	69
	2	0.51	67
	3	0.37	73
	Mean	0.43	68
1.32	1	1.15	16
	2	1.10	20
	3	1.16	15
	Mean	1.14	17
0.661	1	1.38	-1
	2	1.29	6
	3	1.38	0
	Mean	1.35	2
0.293	1	1.42	-4
	2	1.35	1
	3	1.46	-7
	Mean	1.41	-3
0.142	1	1.43	-5
	2	1.42	-4
	3	1.48	-8
	Mean	1.44	-5
Control	1	1.43	-
	2	1.37	-
	3	1.36	-
	4	1.39	-
	5	1.36	-
	6	1.32	-
	Mean	1.37	-

Validity criteria fulfilled:

yes

Conclusions:

The test substance caused inhibition of growth of Pseudokirchneriella subcapitata in a guideline study according to OECD 201 (GLP). An ErC50 (72 h) of 2.47 mg/L and an ErC10 (72 h) of 1.04 mg/L were calculated.

Description of key information

The test substance caused inhibition of growth of Pseudokirchneriella subcapitata in a guideline study according to OECD 201 (GLP). An ErC50 (72 h) of 2.47 mg/L and an ErC10 (72 h) of 1.04 mg/L were calculated.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.47 mg/L

EC10 or NOEC for freshwater algae:

1.04 mg/L

Additional information

One experimental study is available investigating the toxicity of the test substance to aquatic algae (2015). The study was performed according to OECD 201 (GLP) with the freshwater algae Pseudokirchneriella subcapitata. Test concentrations of 6.25, 12.5, 25, 50 and 100% of a saturated solution including a test medium control were tested under static test conditions. The corresponding geometric mean of measured concentrations was 0.142, 0.293, 0.661, 1.32, 3.10 mg/L (GC-MS analysis). An inhibition of growth and yield was recorded. The ErC10 (72 h) and ErC50 (72 h) were calculated at 1.04 mg/L (95% CI: 0.865 - 1.19 mg/L) and 2.47 mg/L (95% CI: 2.09 - 2.75 mg/L) based on growth rate, respectively. For inhibition of yield an EyC10 (72 h) of 0.689 mg/L (95% CI: 0.516 - 0.850 mg/L) and an EyC50 (72 h) of 1.31 mg/L (95% CI: 1.10 - 1.65 mg/L) were calculated. All validity criteria were met.