4-(1,4-dioxa-spiro[4.5]dec-8-yl)-cyclohexanone

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:

1997-12-02 till 1998-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted June 7, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted December, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

15 November 1989

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the test samples of 10 mLwere taken from three concentrations (10, 32 and 180 mg/L) and the negative control at the start and the end of the test. The samples were frozen until analysis.

Vehicle:

no

Details on test solutions:

Stock solutions were prepared at 100 and 180 mg/L in ISO medium. Prior to dilutions, the stock solutions were treated with ultrasonic waves for approximately 15 min and cooled afterwards from a temperature of 29°C to 22.2°C. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAp 278/4
- Source: not available
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: on agar

ACCLIMATION
- Acclimation period: no, same as test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22.5 - 23.0°C

pH:

8.5 - 8.0

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56, 100 and 180 mg/L
Mean measured concentrations: 10.21, not measured, 33.15, not measured, not measured, 174.5 mg/L (Details in Table 1 below)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels
- Material, fill volume: glass, 100 mL
- Aeration: no
- Initial cells density: 10,000 cells/mL
- Control end cells density: 554,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: ISO medium according to OECD 202

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLS lamps of 18 Watt (Philips, Spain), 7500 - 8000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: counting chamber at beginning
- Determination of cell densities: spectrophotometric measurement at 720 nm (Lambda, Perkin-Elmer, Illinois, USA; 5 cm cuvette)
Temperature was measured every day, pH at beginning and end of test

TEST CONCENTRATIONS
- 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.:

173.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

44.7 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.:

32 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.:

83.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

26.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Validation criteria:
In the controls, cell density increased by an average factor of > 16 within 3 days. Analysis of samples showed that the actual exposure concentrations remained above 80 % relative to the initial concentrations. Further, all test conditions remained within the ranges prescribed by the protocol.

Results with reference substance (positive control):

The 72-hour EC50 for growth inhibition (EC50; 72h) was 0.60 mg/L. The 72-hour EC50 for growth rate reduction (EC50; 72h) was 1.48 mg/L. These results were in agreement with what was expected on the basis of historical data: The historical ranges of the 72h EC50 for growth inhibition lie between 0.32 and 1.8 mg/L and the 72h EC50 for growth rate reduction lie between 0.56 and 3.2 mg/L.

Table 1: Concentrations of the test item in test medium

Time [hours]	Concentration [mg/L]
	Nominal	Analysed*	Relative to nominal [%]
0	0	n.d.	n.a.
	10	10.6	106
	32	34.9	109
	180	170	94
	180^1	176	98
72	0	n.d.	n.a.
	10	9.82**	98
	32	31.4	98
	180	179	100
	180^1	187**	104

*Mean of duplicate analysis. The maximum deviation between the responses was calculated for each sample and was < 10%

**Single analysis; one chromatogram was integrated incorrectly

¹Without algae

n.d. not detected

n.a. not applicable

 

Table 2: Mean cell densities during test

Concentrations (mg/L)	Mean cell densities during exposure (x 10^4cells /mL)
	0h	24h	48h	72h
0	1.00	4.55	16.5	55.4
10	1.00	4.70	17.4	58.1
18	1.00	4.99	16.3	53.3
32	1.00	4.94	15.2	49.4
56	1.00	4.89	11.8	33.9
100	1.00	4.46	7.8	14.5
180	1.00	3.97	6.2	6.8

 

Table 3: Percentage reduction of growth rate at different time intervals

Concentrations (mg/L)	Growth rate (cells/mL/h1):
	24-72h mean	Reduction (%)	0-72h mean	Reduction (%)
Blank - control	0.05208		0.05573
10.00	0.05234	-0.5	0.05637	-1.2
18.00	0.04930	5.3	0.05518	1.0
32.00	0.04796	7.9	0.05415	2.8
56.00	0.04016	22.9	0.04881	12.4
100.00	0.02445	53.0	0.03705	33.5
180.00	0.01088	79.1	0.02641	52.6

 

Validity criteria fulfilled:

yes

Conclusions:

An aqueous solution of the test item in an static system revealed an aquatic toxicity towards algae: Growth rate after 72h: EC50: 173.4 mg/L, EC10: 44.7 mg/L, NOEC: 32 mg/L. Biomass after 72h: EC50: 83.2 mg/L, EC10: 26.8 mg/L, NOEC: 32 mg/L.

Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. After a range-finding test, a final test was performed exposing exponentially growing algal cultures to the test item in concentrations of 10, 18, 32, 56, 100 and 180 mg/L, increasing with a factor of 1.8. The initial cell density was 10E04 cells/mL. The total test period was 72 hours. Samples for analysis of actual test concentrations were taken at the start and the end of the test period. Analysis of the samples taken during the final test showed that the actual concentrations were in agreement with nominal concentrations (94-109% relative to nominal). All validation criteria were fulfilled. Under the conditions of the present study the test item inhibited cell growth and reduced growth rate of this fresh water algae species significantly at 56 mg/L and higher. The EC50 for cell growth inhibition (EC50; 72h) was 83.2 mg/L with a 95 % confidence interval ranging from 73.2 to 95.2 mg/L. The EC10 for cell growth inhibition (EC10; 72h) was 26.8 mg/L with a 95 % confidence interval ranging from 22.0 to 30.6 mg/L. The EC50 for growth rate reduction (EC50; 72h) was 173.4 mg/L with a 95 % confidence interval ranging from 149.5 to 207.0 mg/L. The EC10 for growth rate reduction (EC10; 72h) was 44.7 mg/L with a 95 % confidence interval ranging from 38.0 to 49.4 mg/L. The NOEC for cell growth inhibition and growth rate reduction was 32 mg/L.

Description of key information

An aqueous solution of the test item in an static system revealed an aquatic toxicity towards algae: Growth rate after 72h: EC50: 173.4 mg/L, EC10: 44.7 mg/L, NOEC: 32 mg/L. Biomass after 72h: EC50: 83.2 mg/L, EC10: 26.8 mg/L, NOEC: 32 mg/L (reference 6.1.5 -1).

Key value for chemical safety assessment

EC50 for freshwater algae:

173.4 mg/L

EC10 or NOEC for freshwater algae:

44.7 mg/L

Additional information

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. After a range-finding test, a final test was performed exposing exponentially growing algal cultures to the test item in concentrations of 10, 18, 32, 56, 100 and 180 mg/L, increasing with a factor of 1.8. The initial cell density was 10E04 cells/mL. The total test period was 72 hours. Samples for analysis of actual test concentrations were taken at the start and the end of the test period. Analysis of the samples taken during the final test showed that the actual concentrations were in agreement with nominal concentrations (94-109% relative to nominal). All validation criteria were fulfilled. Under the conditions of the present study the test item inhibited cell growth and reduced growth rate of this fresh water algae species significantly at 56 mg/L and higher. The EC50 for cell growth inhibition (EC50; 72h) was 83.2 mg/L with a 95 % confidence interval ranging from 73.2 to 95.2 mg/L. The EC10 for cell growth inhibition (EC10; 72h) was 26.8 mg/L with a 95 % confidence interval ranging from 22.0 to 30.6 mg/L. The EC50 for growth rate reduction (EC50; 72h) was 173.4 mg/L with a 95 % confidence interval ranging from 149.5 to 207.0 mg/L. The EC10 for growth rate reduction (EC10; 72h) was 44.7 mg/L with a 95 % confidence interval ranging from 38.0 to 49.4 mg/L. The NOEC for cell growth inhibition and growth rate reduction was 32 mg/L (reference 6.1.5 -1).