[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

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:

14 October - 19 November 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)

Version / remarks:

(2006; Annex 5 corrected 28 July 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(amended 2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples (3 mL, in duplicate) were taken from the control and each exposure level at the start of the test and after 24 and 72 hours. In addition, the glass wool containing the undissolved test substance was kept for possible analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Samples were stored in the freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring followed by an one-hour settlement period. The aqueous Water Soluble Fraction (WSF) was siphoned off through glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

ACCLIMATION
- Stock culture medium: M1
- Pre-culture: 3 to 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) 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.

After preparation, volumes of 120 mL test solution were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L (as CaCO3)

Test temperature:

22 - 23 °C

pH:

7.4 - 7.8.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32, and 100% of the WSF prepared at 100 mg/L (based on range-finding test).
- Measured concentrations (TWA): n.d., 0.014, 0.047, 0.12, 0.51 and 1.8 mg/L.

In addition, a nominal concentration of 10 mg/L without algae was measured. The measured concentration was 0.19 mg/L.

The concentrations at the start of the test were at the level of 3.9 – 5.4% of nominal. Analysis of the samples at the end of the test showed that the concentrations decreased to 2.0 – 11% of initial.

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL all-glass airtight capped vessels were used, each containing 120 mL of test preparation for the control and each treatment group. All test vessels were maintained under identical conditions.
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 2 (at 10% of WSF)
- No. of replicates: 1 extra replicate of each test concentration and the control for sampling purposes

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: M2 according to OECD 201. The medium was prepared using reverse osmosis purified deionised water (Milli-RO, Millipore)
- Illumination: continuously using TLD-lamps with a light intensity within the range of 107 - 108 µE.m-2.s-1 while constantly shaking.
- Determination of cell concentrations: Samples were taken at 0, 24 and 72 hours and the cell densities were determined using a microscope and a counting chamber.
- Results used to determine the conditions for the definitive study: The results of the range-finding test showed that growth rate was in the range of the control up to the solution containing 10% of the WSF. In the undiluted WSF growth rate was 17% inhibited. Yield was inhibited in all concentrations tested. The inhibition increased to 50% in the undiluted WSF. Based on this information the test concentrations of the definitive test were defined.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 0.50 - 0.86 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95% C.L.: 0.78 - 1.5 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95% C.L.: 0.052 - 0.22 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Details on results:

- Findings: Growth rate was in the range of the control up to the solution containing 10% of the WSF. In the undiluted WSF growth rate was 17% inhibited. Yield was inhibited in all concentrations tested. The inhibition increased to 50% in the undiluted WSF.
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Microscopic observations: at the end of the test in the highest concentration revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: growth rate: 1.2 mg/L (95% CI: 1.1-1.2 mg/L); test performed September 2015
- Other: >10% growth rate inhibition at and above 0.56 mg/L.

The 72h-ErC50 for the algal culture tested falls within the historical range at the test facility (0.82-2.3 mg/L).

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

No EC50-values could be calculated for growth rate because effects observed in the study were below 50%.

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Table:   Measured concentrations versus nominal concentrations

Nominal Concentration (mg/L)*	Measured concentration (mg/L)
	t=0h	T=24h	t=72h	Average
1.0	0.039	0.016	0.005	0.014
3.2	0.137	0.075	0.005	0.047
10	0.507	0.159	0.010	0.12
10 (without algae)	0.543	0.228	0.059	0.19
32	1.45	0.780	0.067	0.51
100	4.55	2.39	0.484	1.8

* As % of the WSF prepared at 100 mg/L

Inhibition results

Table: Percentage inhibition of growth rate (total test period) during the final test 

Average measured exposure concentration (mg/L)	Inhibition of growth rate
	t=72h
0 (control)	-
0.014	1.7
0.047	0.2
0.12	2.2
0.51	6.5^1, 2
1.8	22.1^1

1: effect was statistically significant

2: effect was biologically insignificant (<10%)

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

The ErC50, ErC10 and NOErC in freshwater green algae (P. subcapitata) were >1.8, 0.70 and 0.51 mg/L, respectively.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: N1. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Soluble Fraction (WSF) prepared at a loading rate of 100 mg/L and 5 dilutions of this WSF (three replicate flasks per concentration) and a control (six replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature between 22 and 23°C. Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring followed by an one-hour settlement period. The aqueous Water Soluble Fraction (WSF) was siphoned off through glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.

Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. Measured concentrations decreased steadily throughout the course of the test: the lowest two concentrations were not detectable anymore at t = 72h. Therefore TWA measured concentrations were calculated and used for expression of endpoints.

Statistically significant inhibition of growth rate was found at and above the TWA measured concentration of 0.51 mg/L. The ErC10 and ErC50 based on TWA measured concentrations were 0.70 and >1.8 mg/L, respectively. The NOErC was set at 0.51 mg/L.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: N1.The method followed that described in the OECD TG No 201. Following a preliminary range-finding test,Pseudokirchneriella subcapitata was exposed to a Water Soluble Fraction (WSF) prepared at a loading rate of 100 mg/L and 5 dilutions of this WSF (three replicate flasks per concentration) and a control (six replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature between 22 and 23°C. Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring followed by an one-hour settlement period. The aqueous Water Soluble Fraction (WSF) was siphoned off through glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless. Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. Measured concentrations decreased steadily throughout the course of the test: the lowest two concentrations were not detectable anymore at t = 72h. Therefore TWA measured concentrations were calculated and used for expression of endpoints. Statistically significant inhibition of growth rate was found at and above the TWA measured concentration of 0.51 mg/L. The ErC10 and ErC50 based on TWA measured concentrations were 0.70 and >1.8 mg/L, respectively. The NOErC was set at 0.51 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.7 mg/L