(1R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-ethanol

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:

24-31August, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. All validity criteria were fulfilled.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2013

Analytical monitoring:

yes

Details on sampling:

- Samples for analysis were taken from the control and all test concentrations. Samples taken from all the concentrations (biotic and abiotic medium) were analysed at the start and the end of the test in order to determine maintenance of actual concentrations.
- Frequency of sampling: At the start (t=0 h) and the end of the test (t=72 h).
- Storage: Samples were stored in a freezer until analysis or analysed directly after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test sample of Nopol formed a clear completely soluble and stable solution when mixed with test water at the concentrations prepared.
A stock solution was prepared with a nominal concentration of 200 mg/L for the final test. For this purpose, 200 mg (nominal) of the test item (actual weight: 203.17 mg) were carefully added to 1 L of test water in a sealed glass vessel of 1 L capacity containing a magnetic stirring bar. The mixing was performed during 24 hours in the dark. Following mixing, the contents of the vessel was allowed to stand undisturbed for 1 hour at room temperature. Then, the stock solution was diluted with test water and a fixed amount of inoculum to obtain the required test concentrations in the flasks and a cell density of 5 x10^3 cells/mL per vessel. The final test solutions were all clear and colourless, and remained unchanged during the 72 hours of incubation (except control vessels and non toxic concentrations of the test item, where solutions became greenish due to algal proliferation).
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d'Histoire Naturelle - 12, rue Buffon, Case No. 19 - 75005 PARIS, bred in the Laboratoires des pyrénées under standardised conditions according to the test guidelines.
- Stock culture: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.

ACCLIMATION
- Acclimation period: 2 to 4 days before the start of the test, cells from the algal stock culture were Inoculated in test water at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

None

Test temperature:

22.7-22.9 °C (average value: 22.8 °C)

pH:

At start of the test pH 7.74-7.96; at end of the test pH 7.82-9.39

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0.1, 0.32, 1.0, 3.16, 10.0, 31.6 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all glass flasks sealed with a fritted glass stopper.
- Type: Closed
- Aeration: Algal suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.
- Initial cells density: An initial cell density of 5 x 10^3 cells/mL using the exponentially growing preculture.
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the controls
- Additionally a replicate of each test concentration without algae was prepared in order to assess potential bioaccumulation and/or adsorption effects of the test Item by P. subcapitata during the test period and to determine maintenance of actual concentrations.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Original medium of OECD TG 201.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity was of 5444 lux (range: 5073-5662 lux).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- pH: At the beginning and at the end of the test in at least one vessel per concentration and the control.
- Temperature of medium: Measured continuously in the growth chamber next to the test vessels, over the study period, beginning at the start of the test.
- Light intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: After 72 hours, no significant effect on algal growth was observed at the nominal exposure concentrations of 0.1 mg/L, while a slight effect was observed at 1.0 mg/L. Significant algal growth inhibition was found at the concentration of 10 mg/L. Concerning the concentration of 100 mg/L, results showed 100% algal growth inhibition. Based on the results of a range finding test, test solutions used in the definitive test were prepared to obtain the following nominal concentrations (spaced by a factor of 3.16): 0.1, 0.32, 1.0, 3.16, 10.0, 31.6 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.934 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL 5.567 – 8.213 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL 0.891 – 2.114 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10.166 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL 8.636 – 11.571 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.573 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL 1.762 – 3.346 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL 19.313 – 23.088 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.224 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL 5.903 – 8.872 mg/L

Results with reference substance (positive control):

In July 2012 (most recent test), the 72h-EC50 was 0.96 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.60 to 1.03 mg/L).

Reported statistics and error estimates:

The evaluation of the effects was based on the average of measured exposure concentrations in the biotic system. The software ToxRat® Professional was used to for the determination of the 72-hour ErCx and EyCx.

Chemical analyses

Chemical analyses revealed test item losses in the presence of algae, suggesting metabolism and/or adsorption effects of the test item by P. subcapitata, since test item concentrations remained broadly stable without algae during the test period (except for some low level concentrations). Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations in the biotic test.

Nominal concentrations: 0.1, 0.32, 1.0, 3.16, 10.0, 31.6 and 100 mg/L

Corresponding average exposure concentrations: 0.026, 0.175, 0.610, 2.394, 7.489, 27.356 and 93.558 mg/L

 

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

 

Exposure duration	Replicate	Nominal concentration (mg/L)
		Control	0.1	0.32	1.0	3.16	10.0	31.6	100
t = 24 h	1	2.4	2.0	2.4	1.2	2.0	0.8	0.8	0.8
	2	2.8	2.0	1.6	1.6	1.2	1.2	0.8	1.2
	3	3.6	2.4	2.4	1.6	1.2	2.0	1.6	0.4
	4	2.0	NA
	5	2.4
	6	2.8
	Mean	2.7	2.1	2.1	1.5	1.5	1.3	1.1	0.8
	Std. Dev	0.5	0.2	0.5	0.2	0.5	0.6	0.5	0.4
t = 48 h	1	6.0	9.6	6.8	5.2	6.0	6.4	2.0	0.4
	2	9.2	7.2	8.0	5.6	7.2	6.0	1.6	0.4
	3	8.8	9.2	6.8	7.6	5.6	5.6	2.4	0
	4	8.4	NA
	5	10
	6	10.8
	Mean	8.9	8.7	7.2	6.1	6.3	6	2	0.3
	Std. Dev	1.6	1.3	0.7	1.3	0.8	0.4	0.4	0.2
t = 72 h	1	38.4	37.6	41.2	36.4	28.4	17.6	2.8	0.4
	2	35.6	33.2	35.2	34.8	26.8	22.4	2.4	0
	3	33.6	34.8	39.6	37.2	24.4	23.6	3.2	0
	4	36.4	NA
	5	35.6
	6	36.0
	Mean	35.9	35.2	38.7	36.1	26.5	21.2	2.8	0.1
	Std. Dev	1.5	2.2	3.1	1.2	2.0	3.2	0.4	0.2

 

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

 

Validity criteria of the study

Cell density in controls: 72-fold increase within 72 hours

Coefficient of variation:

- The mean coefficient of variation for section by-section(0-24 h; 24- 48 h; 48-72 h)specific growth rates in the control cultures was of 20.6%.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.0%.

Thus the validity criteria have been respected in the present study.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50 for the parameters growth rate and yield were determined to be 21.14 mg/L and 7.22 mg/L, respectively.
The 72-hour EC10 for growth rate was 6.93 mg/L and for yield was 1.50 mg/L, based on the geometric means of measured exposure concentrations.

Executive summary:

In an algal growth inhibition study performed according to Guideline OECD 201 and under GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item at the nominal concentrations of 0.1, 0.32, 1.0, 3.16, 10.0, 31.6 and 100 mg/L for 72 h under static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and thus over several algal generations. The concentrations of the test item (with and without algae) were determined by chemical analyses at start and at the 72 h incubation period. 

 

Test item concentrations remained broadly stable without algae during the test period (except for some low-level concentrations). However, chemical analyses revealed test item losses in the presence of algae, suggesting metabolism and/or adsorption effects of the test item by

P. subcapitata. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations in the biotic test, the results were based on the geometric means of measured exposure concentrations.

Corresponding average exposure concentrations: 0.026, 0.175, 0.610, 2.394, 7.489, 27.356 and 93.558 mg/L.

 

Therefore, the 72-hour EC50 for the parameters growth rate and yield were determined to be 21.14 mg/L and 7.22 mg/L, respectively. The 72-hour EC10 for growth rate was 6.93 mg/L and for yield was 1.50 mg/L, based on the geometric means of measured exposure concentrations.

Therefore, the substance is classified in chronic category 3 (H412) according to CLP regulation (EC) No. 1272/2008.

Description of key information

The 72-hour EC50 of Nopol to P. subcapitata for the parameters growth rate was 21.14 mg/L and the 72-hour EC10 for growth rate was 6.93 mg/L (measured concentration).

Key value for chemical safety assessment

EC50 for freshwater algae:

21.14 mg/L

EC10 or NOEC for freshwater algae:

6.93 mg/L

Additional information

In an algal growth inhibition study performed according to Guideline OECD 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item at the nominal concentrations of 0.1, 0.32, 1.0, 3.16, 10.0, 31.6 and 100 mg/L for 72 h under static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and thus over several algal generations. The 72-hour EC50 for the parameters growth rate and yield were determined to be 21.14 mg/L and 7.22 mg/L, respectively. The 72-hour EC10 for growth rate was 6.93 mg/L and for yield was 1.50 mg/L, based on the geometric means of measured exposure concentrations.