rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-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:

05 Aug 2014 to 22 Oct 2014

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:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae).
- Sampling method: For sampling at the end of test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: The samples were stored deep-frozen (at about -20 °C) and protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 100.43 mg of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 μm). The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. The test media were prepared just before the start of the test.
- Controls: Blank control (test water without test item) and positive control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear throughout the entire test duration.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Age of inoculum (at test initiation): 3 day old culture in exponential growth phase
- Method of cultivation: Algae were cultivated in laboratory under standardised conditions according to the test guidelines.
- Culturing media and conditions: The algae were cultivated under the test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

15 mg/L as CaCO3

Test temperature:

22 ˚C

pH:

Test start range = 7.2
End test range = 8.6 - 9.1

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

- Nominal concentrations: The undiluted filtrate with a loading rate of 100 mg/L and dilutions 1:2.2, 1:4.6, 1:10, 1:22 and 1:46 of this undiluted filtrate
- Mean measured concentrations: 1.5, 3.0, 6.6, 14, 30 and 66 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: Closed, covered with a glass dish
- Material, size, headspace, fill volume: 50 mL flask with a fill volume of 15 mL
- Initial cells density: 5000 cells/mL
- Aeration: None, but solution continuously stirred
- 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: Reconstituted test water (AAP medium)
- Metals: 415.0 µg/L MnCl2*4H2O, 3.27 µg/L ZnCl2, 1.43 µg/L CoCl2*6H2O, 0.012 µg/L CuCl2*2H2O, 7.26 µg/L Na2MoO4*2H2O, 160.0 µg/L FeCl3*6H2O
- Other components: 15.0 mg/L NaHCO3, 1.044 mg/L K2HPO4, 14.6 mg/L MgSO4*7H2O, 12.16 mg/L MgCl2*6H2O, 4.41 mg/L CaCl2*2H2O, 25.5 mg/L NaNO3, 186.0 µg/L H3BO3, 300.0 µg/L Na2EDTA*2H2O
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: Temperature and appearance of the test media were recorded daily and pH was recorded at the start and end of test

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: Approximately 5400 Lux (range: 4600 - 5600 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: Fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800) performed daily.
- Other: The shape and size of the algal cells were examined microscopically at the end of the test.

TEST CONCENTRATIONS
- Range finding study: Yes.
- Test concentrations: 1:100, 1:10 and 1:1 undiluted filtrate (loading rate 100 mg/L)
- Results used to determine the conditions for the definitive study: After 72 hours, the inhibition of yield, average growth and AUC were 3.0, 0.7 and 3.0 %, respectively, in the 1:100 dilution, 3.3, 0.6 and 3.3 %, respectively, in the 1:10 dilution, and were 72, 25.1 and 72 %, respectively, in the undiluted filtrate. After 96 hours, the inhibition of yield, average growth and AUC were -3.7, -0.6 and 0.3 %, respectively, in the 1:100 dilution, -0.4, -0.1 and 1.8 %, respectively, in the 1:10 dilution, and were 71, 20.3 and 72 %, respectively, in the undiluted filtrate.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (tested twice per year)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 66 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 121 - 166 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 26 - 36 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 23 - 30 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 66 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 115 - 164 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 22 - 30 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 20 - 28 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): In the control the biomass increased by a factor of 99 over 72 hours.
- Observation of abnormalities (for algal test): No abnormalities were observed.
- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:2.2 and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- EC50: 96-hour EC50 for growth rate: 1.2 mg/L (August 2014, study no. D94966)
- Other: The sensitivity of the test system was within the internal historical range (96-hour EC50 for the growth rate from 2011 to 2014: 0.89 - 1.4 mg/L).

Reported statistics and error estimates:

The 72-hour and 96-hour EC10, EC20 and EC50 values for the inhibition of AUC, average growth rate and yield and their 95 % confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression. The AUC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test (one-sided smaller, α = 0.05).

The biological results were related to the mean measured test item concentrations calculated as the arithmetic means of the concentrations measured at the start and end of the test:

Treatment/Dilution	Mean measured concentration of the test item (mg/L)
1:46	1.5
1:22	3.0
1:10	6.6
1:4.6	14
1:2.2	30
Undiluted filtrate (loading rate 100 mg/L)	66

The biological results of the study, based on the arithmetic mean measured concentrations of the test item are summarised in the table below:

Parameter	72 hours			96 hours
	AUC (biomass)	Growth rate	Yield	AUC (biomass)	Growth rate	Yield
EC50 (mg/L) (95 % CI)	30 (26 - 36)	> 66 (121 - 166)	26 (23 - 30)	26 (22 - 30)	> 66 (115 - 164)	24 (20 - 28)
EC20 (mg/L) (95 % CI)	8.4 (6.5 -10)	36 (34 - 39)	8.2 (6.5 - 9.8)	7.7 (5.9 - 9.4)	40 (36 - 43)	7.1 (5.2 - 9.0)
EC10 (mg/L) (95 % CI)	4.3 (2.9 - 5.7)	18 (16 - 20)	4.4 (3.2 - 5.7)	4.1 (2.8 - 5.4)	21 (18 - 24)	3.8 (2.4 - 5.2)
NOEC (mg/L)	1.5	1.5	1.5	1.5	1.5	1.5
LOEC (mg/L)	3.0	3.0	3.0	3.0	3.0	3.0

Validity criteria fulfilled:

yes

Remarks:

The control biomass increased by a factor > 16 within 3 days, the mean CV of the daily growth rates in the control was < 35 % and the CV of the average specific growth rates in the replicates of the control was < 7 %.

Conclusions:

The 96-hour EC50 values were 26, > 66 and 24 mg/L based on biomass (AUC), growth rate and yield, respectively. The 96-hour NOEC was 1.5 mg/L and the 96-hour LOEC was 3.0 mg/L. Values are based on mean measured concentrations of the test item.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD Guideline 201 (2011). Due to the low water solubility of the test item, a dispersion with a loading rate of 100 mg/L was prepared using ultrasonic treatment for 15 minutes followed by intense stirring by magnetic stirrers over three hours at room temperature in the dark. Then, the dispersion was filtered. The undiluted filtrate with a loading rate of 100 mg/L and dilutions 1:46, 1:22, 1:10, 1:4.6, 1:2.2 of this undiluted filtrate were used as test media. Additionally, a control was tested in parallel. The nominal concentrations of 1:46, 1:22, 1:10, 1:4.6, 1:2.2 of the undiluted filtrate and the undiluted filtrate (loading rate of 100 mg/L) corresponded to arithmetic mean measured concentrations of 1.5, 3.0, 6.6, 14, 30 and 66 mg test item/L, respectively. At 72 hours, the EC50 values were 30, > 66 and 26 mg/L, based on biomass (AUC), growth rate and yield, respectively, the EC20 values were 8.4, 36 and 8.2 mg/L, respectively, and the EC10 values were 4.3, 18 and 4.4 mg/L, respectively. At 96 hours, the EC50 values were 26, > 66 and 24 mg/L, based on biomass (AUC), growth rate and yield, respectively, the EC20 values were 7.7, 40 and 7.1 mg/L, respectively, and the EC10 values were 4.1, 21 and 3.8 mg/L, respectively. The NOEC was 1.5 mg/L and the LOEC was 3.0 mg/L at 72 and 96 hours.

Description of key information

72-h (and 96-h) EC50 > 66 mg/L and NOEC = 1.5 mg/L, freshwater (Pseudokirchneriella subcapitata), OECD 201, Eckenstein 2015

Key value for chemical safety assessment

EC50 for freshwater algae:

66 mg/L

EC10 or NOEC for freshwater algae:

1.5 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD Guideline 201 (2011). Due to the low water solubility of the test item, a dispersion with a loading rate of 100 mg/L was prepared using ultrasonic treatment for 15 minutes followed by intense stirring by magnetic stirrers over three hours at room temperature in the dark. Then, the dispersion was filtered. The undiluted filtrate with a loading rate of 100 mg/L and dilutions 1:46, 1:22, 1:10, 1:4.6, 1:2.2 of this undiluted filtrate were used as test media. Additionally, a control was tested in parallel. The nominal concentrations of 1:46, 1:22, 1:10, 1:4.6, 1:2.2 of the undiluted filtrate and the undiluted filtrate (loading rate of 100 mg/L) corresponded to arithmetic mean measured concentrations of 1.5, 3.0, 6.6, 14, 30 and 66 mg test item/L, respectively. At 72 hours, the EC50 values were 30, > 66 and 26 mg/L, based on biomass (AUC), growth rate and yield, respectively, the EC20 values were 8.4, 36 and 8.2 mg/L, respectively, and the EC10 values were 4.3, 18 and 4.4 mg/L, respectively. At 96 hours, the EC50 values were 26, > 66 and 24 mg/L, based on biomass (AUC), growth rate and yield, respectively, the EC20 values were 7.7, 40 and 7.1 mg/L, respectively, and the EC10 values were 4.1, 21 and 3.8 mg/L, respectively. The NOEC was 1.5 mg/L and the LOEC was 3.0 mg/L at 72 and 96 hours.