Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

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:

26-30 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline No 201.

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations: samples for analysis were taken from the control and all test concentrations.

Sampling method: samples were taken at the start (t = 0 h) and the end of the test (t = 72 h) and were analysed by HPLC-MS/MS.

Storage: samples were analysed directly after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: a stock solution was prepared with a nominal concentration of 22.2 mg/L for the final test. For this purpose, ca. 100 mg of the test item were carefully added to 4.5 L of test water in a sealed glass vessel of 5 L capacity containing a magnetic stirring bar. The mixing was performed during 24 ± 2 h in the dark. Following mixing, the content of the vessel was allowed to stand undisturbed for 1 h 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 x 10^3 cells/mL/vessel. The final test solutions were all clear and colourless, and remained unchanged during the 72 h 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
Common name: Algae
Strain: Pseudokirchneriella subcapitata, CCAP 278/4
Source (laboratory, culture collection): Museum National d'Histoire Naturelle, Paris, bred in the Laboratory under standardised conditions according to the test guidelines

ACCLIMATION
Acclimation period: 2-4 days
Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

no data

Test temperature:

22.7-23.2 °C

pH:

7.62-9.72

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L
Measured concentrations: 0.753, 1.331, 1.837, 3.386, 5.291 and 9.062 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel: glass flasks (100 mL) sealed with a fritted glass stopper
Type: closed
Shaking: during incubation, the algal cells were kept in suspension by continuous shaking.
Initial cells density: 5000 cells/mL
Control end cells density: 506000 ± 77400 cells/mL
No. of vessels per concentration (replicates): 3
No. of vessels per control (replicates): 6
Moreover, a replicate of each test concentration without algae was prepared in order to assess the abiotic stability of the test substance during the test period and to determine maintenance of actual concentrations by chemical analysis compared to that in the algae samples.

GROWTH MEDIUM
Standard medium used: yes; original medium of OECD TG 201

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water: original medium of OECD TG 201, prepared using sterilised water
Culture medium different from test medium: no
Intervals of water quality measurement: temperature was measured continuously in the growth chamber, over the study period and pH was recorded at the beginning and at the end of the test in one vessel per concentration and the control.

OTHER TEST CONDITIONS
Sterile test conditions: yes
Photoperiod: Continuous illumination
Light intensity and quality: 5852-6460 lux

EFFECT PARAMETERS MEASURED:
Determination of cell concentrations: cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Spacing factor for test concentrations: 1.82

Range finding study
Test concentrations: 0, 0.10, 1.0, 10 and 20 mg/L
Results used to determine the conditions for the definitive study: in the range-finding test after 72 h, no significant effect on algal growth was observed at the nominal exposure concentrations of 0.1 and 1.0 mg/L, while a slight effect was observed at 10 mg/L. Significant algal growth inhibition was found at the concentration of 20 mg/L. Based on these results, test solutions used in the definitive test were prepared to obtain the following nominal concentrations: 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

7.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 6.61–7.74

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

3.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL = 2.80–3.28

Details on results:

Exponential growth in the control: yes
ErC10 (72 h) = 2.59 (2.16–2.96) mg/L (nominal); EyC10 (72 h) = 1.31 (1.09-1.49) mg/L (nominal)
ErC20 (72 h) = 3.66 (3.24-4.02) mg/L (nominal); EyC20 (72 h) = 1.74 (1.53-1.93) mg/L (nominal)
ErC50 (72 h) = 7.11 (6.61-7.74) mg/L (nominal); EyC50 (72 h) = 3.03 (2.80-3.28) mg/L (nominal)
Refer tables 6.1.5/3 and 6.1.5/4 for details

Results with reference substance (positive control):

Results with reference substance valid: yes
Historical data (dated: July 2012): ErC50 (72 h) = 0.96 mg/L

Reported statistics and error estimates:

Effective concentrations were determined using the software ToxRat® Professional.

Table 6.1.5/3: Effects on Yield

Treatment [mg/L]	0-24 h		0-48 h		0-72 h
	Y	%I	Y	%I	Y	%I
Control	1.233	0.0	11.300	0.0	50.100	0.0
0.753	0.967	21.6	8.033	28.9	52.567	-4.9
1.331	2.167	-75.7	10.967	2.9	47.100	6.0
1.837	1.633	-32.4	6.967	38.3	36.833	26.5
3.386	0.433	64.9	4.433	60.8	22.033	56.0
5.291	0.967	21.6	2.967	73.7	10.167	79.7
9.062	-0.100	108.1	0.300	97.3	2.433	95.1

 

Table 6.1.5/4: Effects on Growth Rate

Treatment [mg/L]	0-24 h		0-48 h		0-72 h
	G	%I	G	%I	G	%I
Control	1.209	0.0	1.562	0.0	1.536	0.0
0.753	1.046	13.5	1.412	9.6	1.554	-1.2
1.331	1.665	-37.7	1.566	-0.3	1.517	1.2
1.837	1.424	-17.8	1.349	13.7	1.437	6.4
3.386	0.470	61.1	1.134	27.4	1.267	17.5
5.291	1.067	11.7	0.967	38.1	1.016	33.8
9.062	-2.757	328.0	0.187	88.0	0.568	63.0

Validity criteria fulfilled:

yes

Remarks:

control cultures (0-72 h): cell concentration increased by 101 folds; mean CV for section by section specific growth rate was 28 %; CV for average specific growth rate was 3.4 %

Conclusions:

Under the test conditions, based on measured concentrations of nopyl acetate, the ErC50 (72 h) and EyC50 (72 h) for green algae (Pseudokirchneriella subcapitata) were determined to be 7.11 mg/L and 3.03 mg/L respectively.

Based on the results of this study, nopyl acetate is classified:
- dangerous for the environment according to Directive 67/548/EEC "R51/53: toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment"
- and hazardous to the aquatic environment under Regulation (EC) No 1272/2008 (CLP) - Category Chronic 2.

Executive summary:

An algal toxicity study was performed in accordance with OECD Guideline No 201 and GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata algae were exposed to nopyl acetate at nominal concentrations of 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L (three replicate flasks per test concentration and six replicates for control) for 72 h, under constant illumination and shaking at a temperature of 22.7-23.2 °C. Measurements of biomass, growth inhibition and yield were done by determination of cell densities at 24, 48 and 72 h using a counting chamber.

After 72 h, cell concentration in the control culture increased by 101 folds; mean CV for section by section specific growth rate was 28 and CV for average specific growth rate was 3.4 %. Therefore the control met the validity criteria for the control vessels.

The concentrations of the test item (with and without algae) were measured at the start of the study and at the 72-h incubation period. The concentrations were not satisfactorily maintained within ± 20% of the initial concentrations, thus the results were based on the geometric mean of measured exposure concentrations in the abiotic test.

Therefore, based on measured concentrations of nopyl acetate, the ErC₅₀(72 h) and EyC₅₀ (72 h) for green algae (Pseudokirchneriella subcapitata) were determined to be 7.11mg/L and 3.03 mg/L respectively.

Based on the results of this study, nopyl acetate is classified:

- dangerous for the environment according to Directive 67/548/EEC "R51/53: toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment"

- and hazardous to the aquatic environment under Regulation (EC) No 1272/2008 (CLP) - Category Chronic 2.

Description of key information

An algal toxicity study was performed in accordance with OECD Guideline No 201.
The 72h-ErC50 of nopyl acetate for freshwater algae is 7.11 mg/L.
The 72h-ErC10 of nopyl acetate for freshwater algae is 2.59 mg/L.
Based on the results of this study, nopyl acetate is classified: 
- dangerous for the environment according to Directive 67/548/EEC "R51/53: toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment" 
- and hazardous to the aquatic environment under Regulation (EC) No 1272/2008 (CLP) - Category Chronic 2.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.11 mg/L

EC10 or NOEC for freshwater algae:

2.59 mg/L

Additional information

Key study conducted according to OECD 201 and EU C3 Guidelines under GLP without deviation.

Algae cultures (Pseudokirchneriella subcapitata) were exposed to nopyl acetate at nominal concentrations of 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L over a test period of 72 hours (static test). Concentrations were measured at the start of the study and daily until the end of the test. The concentrations of the test item were not satisfactorily maintained within ± 20% of the initial concentrations throughout the test, thus the EC₅₀ were based on the geometric mean of the measured concentrations in abiotic replicates. The 72h-ErC₅₀ and the 72h-ErC₁₀ obtained in this study can be used as key values for chemical safety assessment.