(2α,4aα,8α)-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

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:

2016-10-21 to 2016-11-29

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:

Adopted 2006, corrected 2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations tested: undiluted filtrate, 1:3.2, 1:10, 1:32, 1:100 dilutions. The lowest dilution, 1:320, were not analyzed as this concentration was below NOEC.
- Sampling time: at start (0h) and at end of experiment (72h)
- Sample storage conditions before analysis: deep-frozen at -20°C immediately after sampling. The samples are thawed for 1 h at room temperature. After phase seperation, the organic phase was analysed by GC-FID.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
On day 0: 232 mg in 2320 mL test water mixed intensively for 3 h at room temperature
After stirring emulsion was sieved over 0.45 µm membrane filter (Whatman NC45).
Precautions during filtering:
The filter was pre-conditioned with filtrate to avoid loss of dissolved item due to adsorption.
A negative pressure was applied on the filtration unit to avoid losses of the volatile test item.
Further dilution of the undiluted filtrate: 1:3.2, 1:10, 1:32, 1:100, 1:320

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Former name: Selenastrum capricornutum
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, SAG, Institute for plant physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): inoculum culture set up 4 days before start of exposure. Algae were in exponential growth phase.
- Method of cultivation: same as the test conditions

ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test): synthetic test water AAP medium

Test type:

static

Water media type:

freshwater

Remarks:

AAP medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.15 mmol/L

Test temperature:

23°C

pH:

7.5

Nominal and measured concentrations:

The geometric mean test item concentrations corresponding to the following treatments: dilution 1:320, dilution 1:100, dilution 1:32, dilution 1:10, dilution 1:3.2 and undiluted filtrate are respectively: 'not analysed (below NOEC)', 0.17, 0.51, 2.0, 5.6 and 19 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: tightly sealed with glass stopper
- Material, size, headspace, fill volume:
Sample volume: 60 mL
No headspace
- Initial cells density:
- Control end cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP


OTHER TEST CONDITIONS
- Adjustment of pH: 7.5 using NaOH
- Photoperiod: continuous
- Light intensity and quality: LED light, 68 μE s-1 m-2


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement. Daily measurements of the biomass. Measurements were performed in duplicate.
- Other: shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: undiluted filtrate, 1:10 and 1:100 dilution
- Results used to determine the conditions for the definitive study:
inhibition after 72h (average growth rate) was: 1.2 % for 1:100 dilution, 3.8% for 1:10 dilution and 63% in the undiluted filtrate

Reference substance (positive control):

yes

Remarks:

potassium dichromate, tested twice per year.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 4.1-4.7 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 9.1-10 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 1.3-1.6 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 4.1-4.5 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Unusual cell shape: not observed

Results with reference substance (positive control):

Results with reference substance valid? yes
- EC50: 0.9 mg/L

Reported statistics and error estimates:

The 72-hour EC10 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test or Welch t-test where appropriate.
Statistical analysis was performed using ToxRat Professional® .

The test solution concentrations were assessed at start and end of the experiment:

Treatment	Measured concentrations (mg/L)		Geometric mean measured concentration (mg/L)
	0h	72h
Dilution 1:320	n.a.	n.a.	n.a.
Dilution 1:100	0.2	0.15	0.17
Dilution 1:32	0.52	0.5	0.51
Dilution 1:10	2.5	1.6	2
Dilution 1:3.2	5.8	5.4	5.6
Undiluted filtrate (100 mg/L loading rate)	20	18	19

During the test period of 72 hours, the test item concentrations decreased to 63 to 96 % of the initially measured concentrations.

Due to the decrease of concentrations, the biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and the end of the test.

The growth rate (µ) was assessed:

Treatment	Geometric mean measured concentration (mg/L)	Time frame
		0-24h		0-48h		0-72h
		µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	-	1.404	0.00	1.623	0.00	1.658	0.00
Dilution 1:320	n.a.	1.280	8.90	1.649	-1.60	1.667	-0.60
Dilution 1:100	0.17	1.271	9.50	1.642	-1.20	1.657	0.00
Dilution 1:32	0.51	1.410	-0.40	1.541	5.10	1.632	1.60
Dilution 1:10	2	1.576	-12.20	1.540	5.10	1.588	4.20
Dilution 1:3.2	5.6	1.048	25.14	1.322	18.50	1.347	18.70
Undiluted filtrate (100 mg/L loading rate)	19	0.432	69.30	0.309	80.90	0.206	87.60

The yield (Y) was assessed:

Treatment	Geometric mean measured concentration (mg/L)	Time frame
		0-24h		0-48h		0-72h
		Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	-	1.8	0.00	13.7	0.0	79.7	0.0
Dilution 1:320	n.a.	1.5	17.80	14.4	-5.6	81.9	-2.8
Dilution 1:100	0.17	1.4	19.60	14.2	-4.1	79.4	0.3
Dilution 1:32	0.51	1.7	2.20	11.5	15.6	73.6	7.6
Dilution 1:10	2	2.1	-19.70	11.5	15.9	64.4	19.1
Dilution 1:3.2	5.6	1.0	41.40	7.2	47.0	31.0	61.0
Undiluted filtrate (100 mg/L loading rate)	19	0.3	82.80	0.5	96.5	0.5	99.4

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item on the growth of freshwater algae was assessed according to OECD 201 in a 72h static test. The growth rate ErC50 and ErC10 were determined to be 9.5 mg/L and 4.4 mg/L, respectively (measured, geometric mean).

Executive summary:

The impact of the test item on the growth of the freshwater green algal Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to OECD 201. As the substance is supposed to be volatile, the test was performed in a closed system without headspace.

Due to the low water solubility of the test item, the test media were prepared following the filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320 of the filtrate were used as test media.

The concentrations of the undiluted filtrate and the different dilutions were measured at the start (0 h) and at the end (72 h) of the test. During the test period of 72 hours, the test item concentrations decreased to 63 to 96 % of the initially measured concentrations. Due to the decrease of concentrations, the biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and the end of the test.

Description of key information

For this endpoint, one study assessing the impact of the test item on the growth of freshwater algae is available. The study was conducted according to OECD 201 in a 72h static test. The algae, Pseudokirchneriella subcapitata, were exposed to the undiluted filtrate (100 mg/L loading rate) and the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320. The 72h growth rate ErC50 and the ErC10 were determined to be 9.5 mg/L and 4.4 mg/L, respectively (measured, geometric mean).

Key value for chemical safety assessment

EC50 for freshwater algae:

9.5 mg/L

EC10 or NOEC for freshwater algae:

4.4 mg/L