4-[4-(3,4-Difluorophenyl)-cyclohexyl]-cyclohexanone

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:

November 17, 2003 - October 13, 2004

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:

adopted April 4, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Annex to Commission Directive 92/69/EEC of 31st July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packing and labeling of dangerous substances
(Official Journal No. L 383 A, December 29th, 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Preparation of the Test Item:
The test medium (reconstituted water and test material) was freshly prepared. A stock preparation with a test material concentration of 100 mg/L was prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. After that the formulation was given through a suction filter (pore size >10 - <16 µm) and a single use syringe filter (pore size 0.2 µm). The filtrate was used for the stock preparation. The stock preparation was diluted with reconstituted water to the different test media concentrations.

Analytical monitoring:

yes

Details on sampling:

Concentrations:
50, 59, 71, 84 and 100 mg/L

Sampling method:
Samples were taken and analyzed by HPLC from control and test media from both exposure periods, i.e. 0 - 24 hours and 24 - 48 hours. The maintenance of the test item concentration was checked by analysis of freshly prepared samples and after a period of 24 hours.

Sample storage conditions before analysis:
Samples were analyzed directly without storage.

Vehicle:

yes

Details on test solutions:

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.84
- Total hardness: 250 mg CaCO3/L (calculated)
- O2-Concentration: 98.6%

Preparation of the Test Item:
The test medium (reconstituted water and test material) was freshly prepared. A stock preparation with a test material concentration of 100 mg/L was prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. After that the formulation was given through a suction filter (pore size >10 - <16 µm) and a single use syringe filter (pore size 0.2 µm). The filtrate was used for the stock preparation. The stock preparation was diluted with reconstituted water to the different test media concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna Straus
Culture conditions: The clone is bred in the laboratories of Merck KGaA.
Parental daphnids are used for reproduction until they are about 6 weeks old. Thereafter, they are replaced by neonates.
Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2°C and a 16 hour light and 8 hour dark regime to ensure similar conditions as in the experiment. Offspring are removed from the vessels at least twice per week.
Feeding: The parental daphnids are fed ad libitum with unicellular green algae Desmodesmus subspicatus once per week.
Age: Offspring less than 24 hours old were used for the study.
Acclimation period: same as test

Feeding during test: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg CaCO3/L (calculated)

Test temperature:

21°C

pH:

7.44 - 7.84

Dissolved oxygen:

94.6 - 98.6 %

Nominal and measured concentrations:

Nominal concentrations: 50, 59, 71, 84 and 100 mg/L
Measured concentrations: 0.31, 0.34, 0.44, 0.59 and 0.77 mg/L (mean analytical concentrations)

Details on test conditions:

EXPOSURE:
At the start of the experimental phase, 5 Daphnia magna were placed into vessels with 10 mL of reconstituted water (4 vessels control group) or test medium (4 vessels per test material group). They were not fed and the media were not aerated during the exposure.

The test was performed as a static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
50 mg/L: 20 daphnids
59 mg/L: 20 daphnids
71 mg/L: 20 daphnids
84 mg/L: 20 daphnids
100 mg/L: 20 daphnids

CONCENTRATION(S)
A range-finding test was carried out with a concentration of 100 mg/L test material. Two Daphnia magna showed immobilization after 48 hours.

Therefore, the study was performed as a static test in an open system with the following nominal concentrations:
50, 59, 71, 84 and 100 mg/L

For the control, reconstituted water (ELENDT M4 medium) was used.

VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.84
- Total hardness: 250 mg CaCO3/L (calculated)
- O2-Concentration: 98.6 %
- Culture medium different from test medium: no
- Intervals of water quality measurement: 48h


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark

References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990

Reference substance (positive control):

no

Remarks:

No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the analytically determined concentrations.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 values were calculated with the analytically determined concentrations.

Details on results:

Under the given conditions of this study the 48 h EC50 value was 0.35 mg/L.

Results with reference substance (positive control):

Potassium dichromate:
24 h EC50 0.78 (0.68 – 0.88) mg/L
48 h EC50 0.64 (0.57 – 0.71) mg/L

Under the given experimental conditions, the test material Art. 104864 (Potassium dichromate) showed a 24 h EC50 value which was within the range of the published data of 0.6 to 2.4 mg/L (c.f. DIN EN ISO 6341).

Table 1: Nominal, corresponding mean analytical concentrations

	Analytical Results
	0h		48h		Mean
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%
50 mg/L	0.25	0.5	0.37	0.7	0.31	0.62
59 mg/L	0.32	0.5	0.36	0.6	0.34	0.58
71 mg/L	0.44	0.6	0.44	0.6	0.44	0.62
84 mg/L	0.60	0.7	0.58	0.7	0.59	0.70
100 mg/L	0.67	0.7	0.86	0.9	0.77	0.77

Table 2: Immobility of Daphnia after 24 h and 48 h

	Number of daphnids immobilized / exposed		% of daphnids immobilized
Nominal	24 hours	48 hours	24 hours	48 hours
0 mg/L	0/20	0/20	0%	0%
50 mg/L	0/20	0/20	0%	0%
59 mg/L	0/20	9/20	0%	40%
71 mg/L	0/20	19/20	0%	90%
84 mg/L	5/20	20/20	25%	100%
100 mg/L	18/20	20/20	90%	100%

Validity criteria fulfilled:

yes

Conclusions:

The acute immobilization study with Daphnia magna was performed according to OECD 202. Under the given conditions the 48h EC50 value was 0.35 mg/L (reference 6.1.3-1).

Executive summary:

The objective of this study was to determine the acute toxicity of the test material using Daphnia magna. For this purpose, young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. The study comprised of four vessels per concentration containing five Daphnia magna, i.e. 20 Daphnia per concentration (test medium group) and control group. The Daphnia were observed for immobilization/deaths up to 48 hours. Daphnia magna were exposed to the vehicle and to test material concentrations of nominal 50, 59, 71, 84, and 100 mg/L in an open static system. The limit of quantification of the analytical method was 0.02 mg/L. Due to the low water solubility of the test material, the analytically determined test material concentrations ranged between 5–6 % and 4–6 % immediately after preparation of the medium and 48 hours thereafter, respectively. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % throughout the experimental phase of the study. Therefore the test was performed as a static test in open vessels. The EC50 values were calculated with the mean analytical concentrations due to the low solubility of the test material. The nominal concentrations of 59 to 100 mg/L led to immobilized daphnia; 50 mg/L showed slight inhibition of the mobility in two daphnia at the end of the experimental phase. The EC50 values with the 95 % confidence intervals and the no observed effect concentration (NOEC) concerning the analytical data were: 24 h EC50: 0.65 (0.61 – 0.69) mg/L; 48 h EC50: 0.35 (0.34 – 0.37) mg/L; NOEC < 0.31 mg/L. Under the given conditions of this study the 48 h EC50 value was 0.35 mg/L and the NOEC below 0.31 mg/L.

Description of key information

The acute immobilization study with Daphnia magna was performed according to OECD 202. Under the given conditions the 48h EC50 value was 0.35 mg/L (reference 6.1.3-1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.35 mg/L

Additional information

The objective of this study was to determine the acute toxicity of the test material using Daphnia magna. For this purpose, young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. The study comprised of four vessels per concentration containing five Daphnia magna, i.e. 20 Daphnia per concentration (test medium group) and control group. The Daphnia were observed for immobilization/deaths up to 48 hours. Daphnia magna were exposed to the vehicle and to test material concentrations of nominal 50, 59, 71, 84, and 100 mg/L in an open static system. The limit of quantification of the analytical method was 0.02 mg/L. Due to the low water solubility of the test material, the analytically determined test material concentrations ranged between 5–6 % and 4–6 % immediately after preparation of the medium and 48 hours thereafter, respectively. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % throughout the experimental phase of the study. Therefore the test was performed as a static test in open vessels. The EC50 values were calculated with the mean analytical concentrations due to the low solubility of the test material. The nominal concentrations of 59 to 100 mg/L led to immobilized daphnia; 50 mg/L showed slight inhibition of the mobility in two daphnia at the end of the experimental phase. The EC50 values with the 95 % confidence intervals and the no observed effect concentration (NOEC) concerning the analytical data were: 24 h EC50: 0.65 (0.61 – 0.69) mg/L; 48 h EC50: 0.35 (0.34 – 0.37) mg/L; NOEC < 0.31 mg/L. Under the given conditions of this study the 48 h EC50 value was 0.35 mg/L and the NOEC below 0.31 mg/L (reference 6.1.3 -1).