D,L- Menthol / D,L-Isomenthol

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:

04 February 2018 to 16 March 2018

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all test concentrations and the control. The samples of the lower dilutions (1:8 and 1:16) were not analysed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.
- Sampling method: Samples were taken at 0 hours and 48 hours.
- Sample storage conditions before analysis: All samples were frozen (at -20 ± 5 °C) immediately after sampling and stored until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was liquefied in a water bath at approximately 50 °C. 100 µL of the melted test item was pipetted into 1000 mL test water at 50°C to prepare the highest concentrated test medium. Ultrasonic treatment was applied to the test medium at 50 °C for 10 minutes, followed by intensive stirring at room temperature for 2 hours until the test media cooled to 21°C. Dilutions for the lower test concentrations were made from the highest concentrated test medium.
- Controls: Test water with the absence of test item was used as a negative control. Potassium dichromate was used as a positive control to test for the sensitivity of test system.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Undissolved material remained after sample preparation. A 0.45 μm membrane filter (pre-conditioned with 200 mL filtrate) was used to filter the insoluble particles in the test media. The test item was clearly dissolved in all tested dilutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Justification for species other than prescribed by test guideline: Not applicable
- Source: Laboratory culture, originally from the Daphnia Collection of the University of Basel/ Switzerland (2015)
- Age of parental stock (mean and range, SD): Not applicable. Test organisms were 6 – 24 hours old and not first brood progeny.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Not specified

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Not specified

Test temperature:

Start: 21 °C
End: 20 °C

pH:

Start: 7.7 - 7.8
End: 7.9 - 8.0

Dissolved oxygen:

Start: 8.0 - 8.5
End: 8.5 - 8.7

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: undiluted filtrate and dilutions of 1:2, 1:4, 1:8 and 1:16
Measured concentrations (0 h): 96.6 mg/L, 47.6 mg/L, 23.5 mg/L, n.a., n.a.
Measured concentrations (48 h): 56.1 mg/L, 23.2 mg/L, 13.7 mg/L, n.a., n.a.
Mean measured concentrations: 74 mg/L, 33 mg/L, 18 mg/L, n.a., n.a.
n.a. = The dilutions at 1:8 and 1:16 were not analyzed since these concentrations were below the 48-hour NOEC determined in this test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type: open
- Material, size, headspace, fill volume: Glass beakers containing 50 mL test solution, covered with glass plates
- Aeration: None during test. The test water was aerated prior to the start of the study until oxygen saturation was reached.
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not included

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (ISO test water) was used for dilutions
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: The light intensity throughout the light period ranged between 15 and 17 μmol m-2 s-1.

EFFECT PARAMETERS MEASURED: Immobilisation determined after 24 and 48 hours.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: Dilution levels 1:1 to 1:100, factor of 10
- Results used to determine the conditions for the definitive study: Yes, based on the range-finding results dilutions of 1:1, 1:2, 1:4, 1:8 and 1:16 were used in the definitive study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 31 - 42 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The 24h-EC50 was 1.30 mg/L. The response observed was within the range given by the guideline (0.60 - 2.1 mg/L).

Reported statistics and error estimates:

The 24-hours and 48-hour EC50 and the 95% confidence limits were calculated by Trimmed Spearman-Karber method for estimating median lethal concentrations. Statistical analysis was performed using ToxRat Professional®.

Table 1. Analytical results for test samples

Sampling Day/ Sample Age	Treatment	Measured Concentration  of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item  c	% of Initially Measured
[day/hours]		[mg/L]		[mg/L]	[%]
0/0	Control	n.d.	0.1	<LOQ	-
(fresh)	Dilution 1:4	23.5	1.0	23.5	-
	Dilution 1:2	47.6	1.0	47.6	-
	Undiluted Filtrate*	9.66	10.0	96.6	-
2/48	Control	0.791**	0.1	<LOQ	-
(aged)	Dilution 1:4	13.7	1.0	13.7	58
	Dilution 1:2	23.2	1.0	23.2	49
	Undiluted Filtrate*	5.6	10.0	56.1	58

* Undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

** Estimated value below the lowest calibration solution

n.d. = no test item detected

LOQ: 0.322 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact Raw Data.

 

Table 2. Test item effect on the mobility of Daphnia magna

Treatment	Mean Measured Test Item Concentration	No. of Daphnids Tested	Immobilized Daphnids after 24 Hours		Immobilized Daphnids after 48 Hours
	[mg/L]		No.	[%]	No.	[%]
Control	Control	20	0	0	0	0
Dilution 1:16	n.a.	20	0	0	0	0
Dilution 1:8	n.a.	20	0	0	0	0
Dilution 1:4	18	20	0	0	0	0
Dilution 1:2	33	20	4	20	9	45
Undiluted filtrate°	74	20	20	100	--	--

--: All daphnids already dead after 24 hours exposure time.

n.a.: Not analyzed since the concentrations were below the 48‑hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

°: Undiluted filtrateof an equilibrated test item suspension with a loading rate of 100 mg/L

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48-hour EC50 value for Daphnia Magna immobilisation was 36 mg/L with a 95% confidence interval between 31 and 42 mg/L.

Executive summary:

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to OECD 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

For preparation of the highest concentrated test medium, the test item was melted at about 50 °C in a water bath before use. 100 µL of the melted test item were pipetted into 1000 mL test water at 50 °C. Then test medium was submitted to ultrasonic treatment at 50 °C for 10 minutes and intensive stirring at room temperature for two hours, until test media cooled down (21 °C). Due to the undissolved particles in the test water, the suspension was filtered after stirring through a

0.45 µm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrate and the dilutions of 1:2, 1:4, 1:8 and 1:16 of the filtrate were used as test media. A control (test water without addition of the test item) was tested in parallel. During the test period of 48 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 49 to 58 % of the initially measured concentrations were found. Therefore, the biological results were related to the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test). In conclusion, the 48-hour EC50 was determined as 36 mg/L based on a mean measured concentrations with a 95% confidence interval between 31 and 42 mg/L.  

Description of key information

In conclusion, the 48-hour EC50 value for Daphnia Magna immobilisation was 36 mg/L with a 95% confidence interval between 31 and 42 mg/L.  

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

36 mg/L

Additional information

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to OECD 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

For preparation of the highest concentrated test medium, the test item was melted at about 50 °C in a water bath before use. 100 µL of the melted test item were pipetted into 1000 mL test water at 50 °C. Then test medium was submitted to ultrasonic treatment at 50 °C for 10 minutes and intensive stirring at room temperature for two hours, until test media cooled down (21 °C). Due to the undissolved particles in the test water, the suspension was filtered after stirring through a

0.45 µm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrate and the dilutions of 1:2, 1:4, 1:8 and 1:16 of the filtrate were used as test media. A control (test water without addition of the test item) was tested in parallel. During the test period of 48 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 49 to 58 % of the initially measured concentrations were found. Therefore, the biological results were related to the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test). In conclusion, the 48-hour EC50 was determined as 36 mg/L based on a mean measured concentrations with a 95% confidence interval between 31 and 42 mg/L.