Cyclohexanepentanol, .beta.-methyl-

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:

2010-11-21 to 2010-11-26

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)

Remarks:

signed 2010-10-20

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At the first sampling interval of 24 h all test solutions and the control were analytically verified by GC-MS in the fresh media (0 h) and old media (24 h). Due to 100 % mortality at the saturated solution and the dilution level of 45.5 %, analytical determinations were done after the water renewal in the fresh media (24 h) and old media (48 h) of the biologically relevant dilution levels of 0.970 to 22.7 % and the control, only. Additionally, the saturated solution used to prepare the test solutions of the second sampling interval at the water renewal (after 24 h) was analytically verified to demonstrate the reproducibility in comparison to the first sampling interval. Sampling of the old media was carried out directly from the test vessels, which contained daphnids.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 10 mL test item/L was prepared with dilution water three days prior to application (0 h) and another one three days prior to the water renewal (24 h). The test item was placed on the surface of the dilution water. A slow stirring procedure in a closed bottle was applied. Gentle stirring (to avoid the formation of an emulsion) was carried out for 72 hours. After a separation phase of at least 1 h the saturated solution was taken from the homogeneous liquid phase.
- Differential loading: The saturated solution and further 6 dilutions of the saturated solution prepared with dilution water were tested in a geometrical series with a dilution factor of 2.2 (according to the guideline the dilution factor should not exceed 2.2): 0.970 - 2.13 - 4.70 - 10.3 - 22.7 - 45.5 - 100 %,
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2 * s-1.
- Feeding during test: no
- Culture feeding, food type: a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an alga cell density of > 10E6 cells/mL
- Amount: ad libitum
- Frequency: at least 5 times per week

ACCLIMATION
- Acclimation period: at least 2 h in dilution water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no data

Hardness:

no data

Test temperature:

18 - 22 °C, ± 1 °C constant
room temperature (measured): 19 - 20 °C

pH:

measured values:
fresh media: between pH 7.03 - 7.63 (at 0 hr), between 6.69 - 7.66 (at 24 hr)
old media: between pH 6.82 - 7.24 (24 hr)
old media: between pH 6.63 - 6.77 (48 hr)

Dissolved oxygen:

measured values:
fresh media: between 8.02 - 9.09 mg/L (at 0 hr), between 8.76 - 9.01 mg/L (at 24 hr)
old media: between 7.15 - 8.32 mg/L (24 hr)
old media: between 7.14 - 8.37 mg/L (48 hr)

Salinity:

ca. 256 CaCO3/L

Nominal and measured concentrations:

geometric mean measured concentrations: 0.112, 0.249, 0.557, 1.24, 2.70, 4.71, 11.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel, material, size, headspace, fill volume: With regard to the volatility of the test item glass flasks (60 mL) with a glass top were filled up with the test item solutions having nearly no headspace to reduce losses of the test item and closed immediately thereafter.
- Type (delete if not applicable): closed
- 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: ISO Test water, according to OECD 202, pH-value: 7.8 +/- 0.2
- Intervals of water quality measurement: Prior to experimental starting (0 h) and water renewal after 24 h the water quality parameters (pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE*m-2 * s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- At the beginning of the test and after 24 h the water quality parameters of the fresh media (pH value, oxygen concentration) were measured in one additional replicate per concentration level and control.
- After 24 h and 48 h the water parameters of the old media were measured in all replicates per concentration level and control.
- The room temperature was recorded throughout the test with a hygrothermograph.
- Immobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2
- Range finding study:
A non GLP preliminary range finding test was conducted under semi-static conditions with the saturated solution of the test item (100 %) and further 2 dilutions of the saturated solution (1 and 10 %) over a period of 48 h. After 24 and 48 hours the numbers of immobilised daphnids were recorded. No immobilization was observed at dilution level 1 %, 10 – 30 % immobilisation at dilution level 10 % and 100 % immobilisation at 100 %. Chemical analysis at 0 and 24 hours fresh media and 24 and 48 hours old media showed measured test concentrations to range from 68 % to 97 %.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, test conducted as a static acute immobilization test (acc. to DIN 38412 L11) with a duration of 24 h once per month. Test concentrations: 0.625 – 1.00 – 1.60 – 2.56 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: 0.392-0.801

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.899 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: 0.673-1.19

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.112 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: --

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.249 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: --

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: --

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: --

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The saturated solution and all dilution levels were visually clear throughout the exposure period. No liquid substance layer was observed at the top of the aqueous phase.
- The EC10- and EC50-values were calculated in the biologically relevant concentration range showing ≥ 0 % and ≤ 100 % effects by sigmoidal dose-response regression

ANALYTICAL METHOD VALIDATION RESULTS
-Specificity:
Values of two blank samples without test item and spiked with 100µL methanol had to be lower than the lowest calibration standard to prove specificity. Specificity of the analytical method was demonstrated because the test item was not detected in the blank samples.
-Linearity:
Linearity of detector response was checked by analysis of standards and plotting a calibration graph of peak area versus concentration. The coefficients of determination were calculated. The analytical system gave linear responses in the range of nominal 1 to 50 µg/L test item. R2 of the calibration curves was ≥ 0.992.
- Repeatability of injections:
While working with SPME it is not possible to use a sample vial more than once. Due to this fact repeatability in the common use is not possible.
- Precision:
A relative standard deviation (RSD) < 20 % was necessary to follow guideline requirements.
- Accuracy:
Five replicates of daphnia dilution water fortified with 5 µg/L test item (1x LOQ) and 1000 µg/L test item (200x LOQ), latter diluted 1:40 with dilution water to the measured concentration and spiked with 100 µL MeOH were prepared and analysed. A mean recovery of 105 % with a rel. standard deviation (RSD) of 4.24 % was obtained for the samples series fortified on 1 x LOQ level indicating sufficient accuracy and precision according to SANCO 3029/99 rev.4 (2000). Furthermore a validation series spiked on 200 x LOQ level fulfilled the requirements of the corresponding guideline (SANCO 3029/99 rev.4) with a mean recovery of 99 % and a RSD of 3.16 %, as well.
- LOQ:
The LOQ of the analytical method was fixed, a minimum signal to noise ratio of 9 was provided.

Results with reference substance (positive control):

- The percentage immobility for the reference item was determined after 24 h.
- The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression to be: EC50: 1.71 mg/L (CI 1.58 - 1.85 mg/L)
- The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium.
- The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were determined directly from the observation data.
EC10- and EC50-values were calculated by sigmoidal dose-response regression. The test concentrations (24 h: 11.4 mg/L, 48 h: 4.71 and 11.4 mg/L) above the EC100-level were excluded from the calculations. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

(n = 20, divided into 4 replicates with 5 daphnids each)

Dilution Level of the Saturated Solution [%]	Test item Geometric Mean Measured Concentration of the Test Item [mg/L]	Immobilisation [%]
		24 h					48 h
		Replicates					Replicates
		1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
100 (Saturated solution)	11.4	100	100	100	100	100	100	100	100	100	100
45.5	4.71	100	100	100	100	100	100	100	100	100	100
22.7	2.70	80	80	80	80	80	100	100	100	100	100
10.3	1.24	60	60	40	60	55	80	80	60	60	70
4.70	0.557	20	20	20	60	30	40	20	20	80	40
2.13	0.249	20	60	20	20	30	60	80	20	20	45
0.970	0.112	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

MV     = Mean value

 

Concentrations and Recovery Rates ofthe test item

Sampling date	2010-11-24 0 h (Fresh medium)	2010-11-25 24 h (Old medium)		2010-11-25 24 h (Fresh medium)	2010-11-26 48 h (Old medium)
Start of analysis	2010-11-24	2010-11-25		2010-11-25	2010-11-26
Dilution Level of the Saturated Solution [%]	test item						Geometric mean Measured concentration [mg/L]
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	RR   [%]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	RR   [%]
100 (Saturated solution)	12.9	10.1	78	13.2^1)	Not determined^1)		11.4
45.5	5.00	4.43	89	Not determined^1)			4.71
22.7	3.09	2.30	74	2.96	2.53	85	2.70
10.3	1.36	1.12	82	1.41	1.09	77	1.24
4.70	0.535	0.550	103	0.665	0.493	74	0.557
2.13	0.282	0.215	76	0.276	0.229	83	0.249
0.970	0.124	0.0970	78	0.133	0.100	75	0.112
Control	< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = Measured concentration, single injection, dilution factor taken into account

RR = Recovery rate related to the initially measured concentration of the test item in the fresh media

LOQ = Limit of quantification of the analytical method (5 µg test item)

1) = Due to 100% mortality no determination; measuring of the saturated solution only to check the concentration at test start

Validity criteria fulfilled:

yes

Remarks:

In the control group no Daphnia magna were immobilized or showed any signs of disease or stress; the dissolved O2 concentration was > 3 mg/L.

Conclusions:

Based on the geometric mean measured concentrations of the test item the 48-hour EC50 for Daphnia magna was determined to be 0.560 mg/L. The NOEC after 48 h was determined to be 0.112 mg/L as the highest tested concentration with no biologically significant effect. The LOEC after 48 h was determined to be 0.249 mg/L as the lowest tested concentration with a biologically significant effect (immobilisation rates of > 10 % are seen to be significant according to the guidelines).

Executive summary:

The toxic effect of the test item to Daphnia magna according to OECD 202 was assessed in a semi-static dose-response test. For the determination of the test concentrations one range-finding test was performed. The test concentrations were verified by SPME-GC-MS analysis.

At the end of the test the toxicity of the test item to Daphnia magna had increased. After 48 hours of exposure no immobilisation of the test animals was observed in the controls and in the geometric mean measured test item concentration of 0.112 mg test item/L. At the geometric mean measured test item concentration of 0.249 mg test item/L, 45 % of the animals were immobile and 70 % of the animals were immobile at the geometric mean measured test item concentration of 1.24 mg test item/L. 

At the highest nominal test item concentrations of 2.70 and 4.71 mg test item/L, all daphnids were immobile.

The 48-hour NOEC was determined to be 0.112 mg test item/L. The 48 -hour LOEC was determined to be 0.249 mg test item/L and the 48-hour EC50 value was calculated to be 0.56 mg test item/L.

 

Description of key information

EC50 (48 h) 0.56 mg/L (geom. mean measured)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

(48 h)

Effect concentration:

0.56 mg/L

Additional information

The short-term toxicity test to aquatic invertebrates (key_Short-term toxicity to aquatic invertebrates_2011_RL1) performed according to OECD 202 and GLP was rated RL=1 (reliable without restrictions) and used as key study. Toxicity to Daphnia after 48 h started at a concentration of 0.249 mg/L (LOEC). The 48 -h EC50 amounts to 0.56 mg/L (geom. mean measured).