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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Aug 2015 to 17 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control were sampled.
- Sampling method: Duplicate samples were taken from each treatment before the start of the test and at end of the test. For the 48-hour samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage condition before analysis: The samples were stored deep-frozen (at about -20 °C) immediately after sampling.

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 150.1 mg of the test item in 1500 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours 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) after preconditioning the filter with the dispersion. 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: 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Clone 5
- Age at study initiation: 6-24 hours, not first brood progeny
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

250 mg CaCO3/L

Test temperature:

21 °C

pH:

Start of test: 7.9 - 8.0
End of test: 7.7 - 7.8

Dissolved oxygen:

Start of test: 7.9 to 8.4 mg/L
End of test: 8.6 to 8.8 mg/L

Nominal and measured concentrations:

- Nominal concentrations: undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the undiluted filtrate
- Arithmetic mean measured concentrations: 2.6, 4.4, 9.2, 18, 37 and 75 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type: Closed (covered with glass plates)
- Material, size, headspace, fill volume: 100 mL glass beakers filled with 50 mL of test medium
- Aeration: Prior to test until oxygen saturation but not during test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 10 mL test medium/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341.
- Alkalinity: 0.8 mmol/L
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: No, culture has been bred in reconstituted water of the quality identical to the water quality used in the tests (in terms of pH, main ions, and total hardness) and under temperature and light conditions identical to those in the tests
- Intervals of water quality measurement: At the start and end of test for pH, dissolved oxygen and water temperature. The appearance of test media was visually inspected at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16:8 light/dark cycle with 30 minute transition period
- Light intensity: Approximately between 800 and 1060 Lux

EFFECT PARAMETERS MEASURED: The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Daphnids considered immobile if they did not swim after 15 seconds of gentle agitation.

RANGE-FINDING STUDY: None performed

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, tested twice a year

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL = 23 and 29 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: At the test concentration of 18 mg/L, adverse effects (reduced swimming activity, daphnids trapped at the water surface) were recorded in 6 of 19 remaining mobile test animals.
- Other adverse effects control: During the first 24 hours of the test, no immobilised test organisms were determined in the control and up to and including the highest mean measured concentration of 75 mg/L. After 48 hours of exposure, no immobilised test organisms were determined in the control and up to and including the mean measured concentration of 9.2 mg/L.
- Any observations that might cause a difference between measured and nominal values: At the end of the test, 96 to 129% of the initially measured concentrations were found. No remarkable observations were made concerning the appearance of the test media.
- Effect concentrations exceeding solubility of substance in test medium: All test media were clear solutions throughout the entire test duration.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 48-hour EC50 = 1.0 mg/L (last tested August 2014)
- Other: The sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2014: 0.36 - 1.1 mg/L).

Reported statistics and error estimates:

The 24-hour EC50 of the test item could not be calculated because none of the responses exceeded 50%. The 48-hour EC50 and the 95 % confidence limits were calculated using Weibull Analysis. The NOEC was determined directly from the raw data.

 Effect of test item on the mobility of Daphnia magna

Treatment/dilution	Mean measured test item concentration (mg/L)	No. of daphnids tested	Immobilised daphnids after 24 hours			Immobilised daphnids after 48 hours
			Number	%		Number	%
Control	-	20	0	0		0	0
1:32	2.6	20	0	0		0	0
1:16	4.4	20	0	0		0	0
1:8	9.2	20	0	0		0	0
1:4	18	20	0	0		1 (4A, 2F)	5
1:2	37	20	0	0		20	100
Undiluted filtrate (loading rate 100 mg/L)	75	20	0	0		20	100

Values in parentheses indicate the number of mobile test animals with adverse effects:

A: daphnids trapped at the water surface

F: reduced swimming activity

Validity criteria fulfilled:

yes

Remarks:

No control daphnids showed immobilisation or other signs of disease or stress and dissolved oxygen levels at end of test were ≥ 3 mg/L in the control and test vessels.

Conclusions:

The 48-hour EC50 was calculated to be 26 mg/L with 95 % confidence limits of 23 and 29 mg/L. The 48-hour NOEC was 9.2 mg/L. The results are based on the mean measured test item concentrations calculated as the arithmetic means of the concentrations measured at the start and end of the test.

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004). 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 at room temperature in the dark over three hours. 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. The nominal treatments of 1:32, 1:16, 1:8, 1:4, 1:2 of the undiluted filtrate and the undiluted filtrate (loading rate of 100 mg/L) corresponded to mean measured concentrations of 2.6, 4.4, 9.2, 18, 37 and 75 mg test item/L. Additionally, a control was tested in parallel. The 48-hour EC50 was calculated to be 26 mg/L with 95 % confidence limits of 23 and 29 mg/L. The 48-hour NOEC was 9.2 mg/L.

Description of key information

48-h EC50= 26 mg/L, freshwater invertebrates (Daphnia magna), OECD 202, Eckenstein 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

26 mg/L

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004). 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 at room temperature in the dark over three hours. 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. The nominal treatments of 1:32, 1:16, 1:8, 1:4, 1:2 of the undiluted filtrate and the undiluted filtrate (loading rate of 100 mg/L) corresponded to mean measured concentrations of 2.6, 4.4, 9.2, 18, 37 and 75 mg test item/L. Additionally, a control was tested in parallel. The 48-hour EC50 was calculated to be 26 mg/L with 95 % confidence limits of 23 and 29 mg/L. The 48-hour NOEC was 9.2 mg/L.