[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

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

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)

Version / remarks:

March 23 2006, corrected July 28 2011

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

August 24 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15 2000

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II

Version / remarks:

rev.4 11/07/00

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Duplicate samples from the freshly prepared media containing algae were taken from all test concentrations and controls at test start. At the end of the test, samples were taken after pouring together the contents of the test beakers of each treatment (containing algae)
- Sample storage conditions before analysis: Samples extracted immediately after sampling, and extracts stored in a refrigerator.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test medium of the highest concentration of 20 mg test item/L prepared by dissolving 20.4 mg test item in 1020 mL of OECD medium by intense stirring for 10 minutes. Other test concentrations prepared by dilution of the highest test concentration.
- Eluate: OECD medium
- Controls: OECD medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Commercial supplier
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: following test guidelines under standard conditions

ACCLIMATION
- Culturing media and conditions (same as test or not): Same
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

24 mg/L as CaCO3

Test temperature:

21.9 to 23.1ºC

pH:

Test start: 8.1
Test end: 8.8 to 9.5

Nominal and measured concentrations:

Nominal: 0.9, 2.0, 4.3, 9.3 and 20 mg/L
Geometric mean measured: 0.119, 0.237, 2.10, 5.36 and 13.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Contain 50 mL test media to minimise headspace, with glass stoppers
- Initial cells density: 5000 cells/mL
- Control end cells density: 72.408 x 1000 cells/mL (mean). Standard deviation 5.680 x 1000 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: Deionised water with analytical grade salts added (OECD medium)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: 5125 Lux (Range: 4880 to 5460 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Density, yield, and growth rate every 24 hours
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Test concentrations: 0.1, 1, 10 and 20 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate tested at least twice per year (last test February 2016)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 7.33 - 11.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 3.90 - 5.81 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.237 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.:

6.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 3.78 - 10.5 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence interval: 1.71 - 3.89 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence interval: 1.04 - 2.46 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.119 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.721, 1.50 and 0.794 mg/L for yield, growth rate and biomass, respectively, at 72 hours

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and EyC50 and the corresponding EC20 and EC10 values and where possible the 95 %-confidence limits were calculated by non-linear regression. For the 72 hours LOEC and NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).

Summary of Analytical Results

Sample Description	Fresh			Aged			Geometric Mean Measured Concentration
(mg test item/L)	% of nominal^1	RSD (%)	n	% of nominal	RSD (%)	n	mg test item/L
Control	n.a.	n.a.	2	n.a.	n.a.	2	n.a.
0.9	86	11	2	13	1	2	0.119
2	80	18	2	19	0	2	0.237
4.3	81	16	2	29	4	2	2.1
9.3	74	17	2	45	2	2	5.36
20	75	9	2	57	3	2	13.1

1 Mean value of all measured samples per treatment group

italic: Values are partially < LOQ. For values <LOQ, half the LOQ was used for calculation of geometric mean measured concentrations

RSD: Relative standard deviation per treatment group

n: number of samples analysed

n.a.: not applicable

Yield, y, and Percentage inhibition of y during the Test Period

Geometric mean measured concentration	yields, y, (1000 cells/mL) and % inhibition of yield
	0 - 24 hours		0 - 48 hours		0 - 72 hours
(mg test item/L)	y	%	y	%	y	%
Control	1.799	-	15.675	-	71.908	-
0.119	1.301	27.7	14.487	7.6	68.995	4.1
0.237	1.531	14.9	12.494	20.3	64.548	10.2*
2.1	1.148	36.2*	11.42	27.1	59.182	17.7*
5.36	0.841	53.3*	9.044	42.3*	38.943	45.8*
13.1	0.228	87.3*	2.604	83.4*	17.477	75.7*

Negative values in % inhibition indicates an increase in growth relative to the control

* mean value signifcantly different from the control (tested with Bonferroni-Welch t-test (48 h) and Williams t-test (24 h and 72 h), α = 0.05, one-sided

Growth Rates and Percentage inhibition of growth rate during the Test Period

Geometric mean measured concentration	Growth rates, μ, (1/day) and % inhibition of μ
	0 - 24 hours		0 - 48 hours		0 - 72 hours
(mg test item/L)	μ	%	μ	%	μ	%
Control	1.515	-	1.732	-	1.658	-
0.119	1.268	16.3	1.699	1.9	1.645	0.8
0.237	1.376	9.2	1.626	6.1*	1.622	2.2
2.1	1.165	23.1*	1.581	8.7*	1.594	3.8*
5.36	0.983	35.1*	1.474	14.9*	1.456	12.2*
13.1	0.342	77.4*	0.913	47.3*	1.189	28.3*

Negative values in % inhibition indicates an increase in growth relative to the control

* mean value signifcantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EyC50 and EyC10 were calculated to be 6.36 mg test item/L and 1.60 mg test item/L, respectively. The 72-hour ErC50 and ErC10 values were calculated to be > 13.1 mg test item/L and 4.76 mg test item/L, respectively.

Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in a study following OECD guideline 201. Algae were exposed to nominal concentrations of 0.9, 2.0, 4.3, 9.3 and 20 mg test item/L, equivalent to geometric mean measured concentrations of 0.119, 0.237, 2.10, 5.36 and 13.1 mg test item/L alongside a control. The 72-hour EyC50 and EyC10 were calculated to be 6.36 mg test item/L and 1.60 mg test item/L, respectively. The 72-hour ErC50 and ErC10 values were calculated to be > 13.1 mg test item/L and 4.76 mg test item/L, respectively.

The study is a GLP compliant, guideline experimental study available as an unpublished report. There are no restrictions and it is fully adequate for assessment.

Description of key information

The 72-hour ErC50 and ErC10 values were calculated to be > 13.1 mg test item/L and 4.76 mg test item/L, respectively. The 72-hour EyC50 and EyC10 were calculated to be 6.36 mg test item/L and 1.60 mg test item/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

13.1 mg/L

EC10 or NOEC for freshwater algae:

4.76 mg/L

Additional information

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in an OECD 201 guideline test. Algae were exposed to nominal concentrations of 0.9, 2.0, 4.3, 9.3 and 20 mg test item/L, equivalent to geometric mean measured concentrations of 0.119, 0.237, 2.10, 5.36 and 13.1 mg test item/L alongside a control (2016). The study is a GLP compliant, guideline experimental study available as an unpublished report. There are no restrictions and it is fully adequate for assessment.