4-trans-(4-trans-Propylcyclohexyl)-cyclohexancarbonic acid

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:

2018-04-17 to 2018-06-27

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:

- Sampling method: At the start of exposure and at the renewal of the test solutions (0 and 24 hours), sampling was carried out after preparation of the saturation levels. At renewal and at the end of exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels.

- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution (100 mg test item/L were weighed out) was freshly prepared with culture medium one day prior to the start of the exposure (day -1) and prior to the renewal of the test solutions (day 0 hours). The stock solution was stirred for 24 ± 1 hour at approx. 1100 rpm at room temperature. After a separation phase of 1 hour at room temperature, the test solution was removed from the bottom of the flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. Since undissolved test item was detected, a filtration step was added.
Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used as the highest dilution level and as the stock solution for the preparation of further dilution levels. During filtration, the filter was always kept covered.
The saturated solution and further 5 dilution levels of the saturated solution in a geometric series with a separation factor of 4, prepared by dilution of the saturated solution with dilution water, were tested as follows: 0.0977, 0.391, 1.56, 6.25, 25.0, 100 % of the saturated solution
The test item concentrations mentioned above were selected based on the results of a non-GLP preliminary range finding test.

- Controls: Dilution water without test item incubated under the same semi-static conditions as the test group.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus (clone 5)
- Justification for species other than prescribed by test guideline: Daphnia magna is the preferred species in accordance with the test guideline.
- Stage and instar at study initiation: Less than 24 hours old
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

0 h: 267 mg CaCO3/L
24 h: 249 mg CaCO3/L

Test temperature:

19.3 - 20.2 °C

pH:

0 h: 8.39
24 h: 7.75

Dissolved oxygen:

0 h: 9.29 mg/L
24 h: 8.84 mg/L

Conductivity:

0 h: 624 µS/cm
24 h: 619 µS/cm

Nominal and measured concentrations:

Nominal conc.: 0.0977, 0.391, 1.56, 6.25, 25.0 and 100 % of the saturated test item solution.

Geometric mean measured concentration: < LOQ, 0.350, 0.129 and 0.671 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Volume of solution: 20 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 Iux

EFFECT PARAMETERS MEASURED: The number of immobilized Daphnia was recorded after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 % of the saturated solution
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

294 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 129 - 671 µg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

294 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 129 - 671 µg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

671 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

671 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

152 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 129 - 671 µg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

152 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 129 - 671 µg/L

Details on results:

The evaluation is based on the two highest concentration levels, which caused 0 and 100% immobilization. The validity criteria of the test guideline were fulfilled.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Validity Range: EC50 (24 hours): 0.6 mg/L - 2.4 mg/L, according to AQS P 9/2 (clone 5), EC50 (24 hours): 0.6 mg/L - 2.1 mg/L, according to OECD 202 (clone A)
- Relevant effect levels: EC50: 1.89 mg/L (%95 % CI: 1.00 – 4.00 mg/L)

Table 1: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each).

Saturation level of the saturated solution [%]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	100	100	100	100	100	100% mortality after 24 hours
25.0	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
1.56	0 ^	0	0	0	0	0	0	0	0	0
0.391	0	0	0	0	0	0	0	0	0	0
0.0917	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Table 2: Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Saturation level of the saturated solution [%]	NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	5 /5	5 /5	5 /5	5 /5	20/20	5 /5	5 /5	5 /5	5 /5	20/20
25.0	0 /5	0 /5	0 /5	0 /5	0 / 20	0 /5	0 /5	0 /5	0 /5	0 / 20
6.25	0 /5	0 /5	0 /5	0 /5	0 / 20	0 /5	0 /5	0 /5	0 /5	0 / 20
1.56	0 /5	0 /5	0 /5	0 /5	0 / 20	0 /5	0 /5	0 /5	0 /5	0/20
0.391	0 /5	0 /5	0 /5	0 /5	0 / 20	0 /5	0 / 5	0 /5	0 /5	0/20
0.0917	0 /5	0 /5	0 /5	0 /5	0 / 20	0 /5	0 /5	0 /5	0 /5	0/20
Control	0 /5	0 /5	0 /5	0 /5	0/20	0 /5	0 /5	0 /5	0 /5	0 / 20

 

Table 3: Measured Concentrations of the Test Item during the Definitive Test

Sampling	Fresh media, 0 hours			Old media, 24 hours			Fresh media, 24 hours			Old media, 48 hours
Nominal concentration of the test item [mg/L]	Test Item
	Meas. Conc. [mg/L]	%	Meas. Conc. [mg/L]		%	Meas. Conc. [mg/L]		%	Meas. Conc. [mg/L]		%	Geometric mean measured concentration [mg/L]
100	0.496	0	0.909		1	Not determined, due to 100% mortality after 24 hours						0.671
25.0	0.126	1	0.193		1	0.118		0	0.0967		0	0.129
6.25	< LOQ		< LOQ			< LOQ			< LOQ			0.0350
1.56	< LOQ		< LOQ			< LOQ			< LOQ			0.0350
0.391	< LOQ		< LOQ			< LOQ			< LOQ			0.0350
0.0977	< LOQ		< LOQ			< LOQ			< LOQ			0.0350
Control	< LOQ		< LOQ			< LOQ			< LOQ

Meas. Conc. = measured concentration of the test item, dilution factors taken into account

% = percentage of the nominal concentration of the test item

LOQ = limit of quantification (0.070 mg test item/L)

 

Table 4: EC₅₀-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate based on nominal concentrations [mg/L], (0 - 24 hours)

	Current Study	Valid Range
EC50	1.89 mg/L	0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits	1.00-4.00 mg/L

 

Validity Criteria

The study was performed according to OECD Guideline 202 (2004). The validity criteria were fulfilled:

- In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10% of the daphnids in the control).

- The dissolved O2 concentration in the 24-hours old media at the renewal of the test solutions and at the end of the exposure was s 7.46 mg/L (required: > 3 mg/L in the 24-hours old media) in all tested saturation levels and the control.

Validity criteria fulfilled:

yes

Conclusions:

Based on geometric mean measured concentrations of the test material, the 48 h-EC50 was determined to be 294 µg/L (0.294 mg/L) (95% confidence limits: 129 – 671 µg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004). The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution of the test item with a nominal loading of 100 mg test item/L and further five saturation levels (diluted from saturated solution, nominal: 0.0977 to 25 %) prepared in a geometric series with a separation factor of 4. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each saturation level and the control. The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all saturation levels and in the control. The measured concentrations of the test item in the saturated solution were 496 µg/L (0.496 mg/L) in the fresh media at the start of the exposure (0 hours) and 909 µg/L (0.909 mg/L) in the old media at the renewal of the test solutions (24 hours). Since 100 % mortality was observed in the saturated solution, no further measurements were made after 24 hours. The next saturation level (25 %) caused no effects on Daphnia magna. The measured concentrations in the saturation level 25 % were 126 and 118 µg/L (0.126 and 0.118 mg/L) in the fresh media (0 and 24 hours) and 193 and 96.7 µg/L (0.193 and 0.0967 mg/L) in the corresponding old media (24 and 48 hours). The lowest four saturation levels are not relevant for evaluation of the ECx-values, since no biological effects occurred. The measured concentrations of the test item in the four lowest saturation levels (0.0977 to 6.25 % of the saturated solution were below the LOQ (70 µg/L). All validity criteria were met. The geometric mean measured concentrations of the saturated solution and the following saturation level (25 %) were calculated to be 671 and 129 µg/L (0.671 and 0.129 mg/L). All effect concentrations are based on the geometric mean measured concentrations of the test item. The evaluation is based on the two highest concentration levels, which caused 0 and 100% immobilization, thus, the 48 h-EC₅₀ was determined to be 294 µg/L (0.294 mg/L) (95 % confidence limits: 129 – 671 µg/L).

Description of key information

The acute toxicity to the freshwater invertebrate Daphnia magna has been investigated according to OECD Guideline 202 in a semi-static test. The test substance concentration was monitored during the test. Based on geometric mean measured concentrations of the test material, the 48 h-EC₅₀ was determined to be 294 µg/L (0.294 mg/L) (95% confidence limits: 129 – 671 µg/L) (reference 6.1.3-1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.294 mg/L

Additional information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004). The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution of the test item with a nominal loading of 100 mg test item/L and further five saturation levels (diluted from saturated solution, nominal: 0.0977 to 25%) prepared in a geometric series with a separation factor of 4. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each saturation level and the control. The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all saturation levels and in the control. The measured concentrations of the test item in the saturated solution were 496 µg/L (0.496 mg/L) in the fresh media at the start of the exposure (0 hours) and 909 µg/L (0.909 mg/L) in the old media at the renewal of the test solutions (24 hours). Since 100 % mortality was observed in the saturated solution, no further measurements were made after 24 hours. The next saturation level (25%) caused no effects on Daphnia magna. The measured concentrations in the saturation level 25% were 126 and 118 µg/L (0.126 and 0.118 mg/L) in the fresh media (0 and 24 hours) and 193 and 96.7 µg/L (0.193 and 0.0967 mg/L) in the corresponding old media (24 and 48 hours). The lowest four saturation levels are not relevant for evaluation of the EC_x-values, since no biological effects occurred. The measured concentrations of the test item in the four lowest saturation levels (0.0977 to 6.25% of the saturated solution were below the LOQ (70 µg/L). All validity criteria were met. The geometric mean measured concentrations of the saturated solution and the following saturation level (25%) were calculated to be 671 and 129 µg/L (0.671 and 0.129 mg/L). All effect concentrations are based on the geometric mean measured concentrations of the test item. The evaluation is based on the two highest concentration levels, which caused 0 and 100% immobilization, thus, the 48 h-EC₅₀ was determined to be 294 µg/L (0.294 mg/L) (95% confidence limits: 129 – 671 µg/L).