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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-13 to 2012-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
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:
The test item was analytically verified by GC-MS in the fresh media (0 and 24 h) and old media (after 24 h) of all concentration levels and the control.
After 48 h, samples of the old media, only of the concentration levels 6.25 to 50.0 mg/L and the control were analytical verified, because 100 % adult mortality was observed at the highest concentration level of 100 mg/L.
Sampling from freshly prepared media was carried out immediately after homogenisation. Samples of the old media were taken directly from the test vessels.
Vehicle:
no
Details on test solutions:
PREPARATION
- Pre-treatment of the test item: The test item was completely melted at temperatures up to 70 - 90 °C before weighing out.
- Stock solution: The stock solution (100 mg/L test item was weighed out) was prepared with dilution water one day before start of the exposure phase (0 hours) and water renewal (24 hours).
- Dispersion treatment: The stock solution was stirred with approximately 1100 rpm for 24 hours at room temperature.

APPLICATION
The test vessels were filled up with the test solutions. The daphnids were transferred with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette. The test vessels were closed thereafter immediately to reduce losses of the test item.

CONTROL
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
- Common name: Daphnia magna
- Strain: STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Germany
- Breeder: Dr. U. Noack-Laboratorien, 31157 Sarstedt, Germany
- Age at experimental starting: 2 to 24 h old daphnids, obtained by removing the parental daphnids twice within 22h.
- Culture: 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.
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an alga cell density of > 10^6 cells/mL.
- Feeding during test: no

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:
During the test the incubator temperature was in the range of 18 - 19 °C.
pH:
measured values:
fresh media: between pH 7.63-8.04 (at 0 hr)
fresh media: between pH 7.83-8.02 (at 24 hr)
old media: between pH 7.57-8.20 (24 hr)
old media: between pH 7.76-7.87 (48 hr)
Dissolved oxygen:
measured values:
fresh media: between 8.28 - 8.82 mg O2/L (at 0 hr)
fresh media: between 8.02 - 8.64 mg O2/L (at 24 hr)
old media: between 8.24 - 8.56 mg O2/L (24 hr)
old media: between 8.09 - 8.39 mg O2/L (48 hr)
Salinity:
not applicable
Nominal and measured concentrations:
Nominal test concentration: 6.25, 12.5, 25.0, 50.0, 100 mg/L
Measured concentrations: The recoveries in the fresh media (0 and 24 h) were in the range of 95 to 119% of the nominal values. In the old media (24 and 48 h) the recoveries were in the range of 93 to 112% of the nominal values. Therefore, the endpoints are based on the nominal concentrations of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel/ volume: With regard to the volatility of the test item sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL) filled up with the test solution having nearly no headspace to reduce losses of the test item.
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred afterwards by a pipette.
- Number of daphnids and replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids per concentration level and control
- Test temperature: 18 - 22 °C, ± 1 °C constant
- Illumination: Diffuse light, illumination strength max. 20 µE ∙ m-2 ∙ s-1
- Photoperiod: 16/8 h light/dark cycle

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3

TEST CONCENTRATIONS
- Test concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L (dilution factor 2).
- Range finding study: A non GLP preliminary range finding test was conducted under semi-static conditions with three nominal test item concentrations of 1, 10 and 100 mg/L. The immobilization rates were determined in all test and control groups after 24 and 48 h of exposure.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- At the beginning of the exposure phase (0 h) and water renewal after 24 hours, the water quality parameters (pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured, respectively.
- At the beginning of the exposure phase (0 h) 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 incubator 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.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
43.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits: 41.3 – 45.1 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The concentrations of the test item were analytically verified by GC-MS in the fresh media (0 and 24 h) and old media (24 h) of all test concentrations and the control. After 48 h, samples of the old media were analytical verified only of the concentration levels 6.25 to 50.0 mg/L, because 100 % mortality was observed at the concentration level of 100 mg/L.
In the fresh media (0 and 24 hours) the recoveries were in the range of 95 to 119 % of the nominal values. In the old media (24 and 48 hours) the recoveries were in the range of 93 to 112 % of the nominal values. Therefore, the endpoints given are based on the nominal concentrations of the test item.
The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0, 24 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled
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. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.76 mg/L (CI 1.50 - 2.01 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:
STATISTICAL METHODS OF EVALUATION
EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were determined directly from the observation data, without mathematical calculation.

EC-values and statistical analyses:
EC10- and EC50-values were calculated by sigmoidal dose- response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

Software:
All data were computer processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software: GraphPad Prism5, GRAPHPAD SOFTWARE, INC. and Microsoft® Office Excel, MICROSOFT CORPORATION.

Immobilization Rates of the Definitive Testafter 24 and 48 h ofExposure             

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

test item

IMMOBILIZATION [%]

Nominal

Test Item

Concentration

[mg/L]

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

100

100

100

100

100

100

100

100

100

100

100

 50.0

 60

 60

 80

 80

 70

100

 80

100

 80

 90

 25.0

   0

   0

   0

   0

   0

   0

   0

   0

   0

   0

 12.5

   0

   0

   0

   0

   0

   0

   0

   0

   0

   0

   6.25

   0

   0

   0

   0

   0

   0

   0

   0

   0

   0

Control

   0

   0

   0

   0

   0

   0

   0

   0

   0

   0

MV  = Mean value

Measured Concentrations during the Definitive Test

Sampling date

2012-05-14

0 h

(Fresh medium)

2012-05-15

24 h

(Old medium)

2012-05-15

24 h

(Fresh medium)

2012-05-16

48 h

(Old medium)

Start of analysis

2012-05-14

2012-05-15

2012-05-15

2012-05-16

Nominal

test item

concentration

 

[mg/L]

test item

Meas.

conc.

[mg/L]

%

 

 

Meas.

conc.

[mg/L]

%

 

 

Meas.

conc.

[mg/L]

%

 

 

Meas.

conc.

[mg/L]

%

 

 

100

119

119

107

107

113

113

n.d.

 50.0

 52.9

106

 53.2

106

 50.7

101

50.4

101

 25.0

 25.2

101

 28.0

112

 23.7

 95

23.4

 94

 12.5

 13.8

110

 14.0

112

 14.6

117

11.9

 95

   6.25

   7.07

113

   6.41

103

   6.83

109

5.81

 93

Control

< LOQM

< LOQM

< LOQM

< LOQM

Meas. conc     = Measured concentration of the test item, single injection (dilution factor taken into account)

%                     = Percent of the nominal concentration of the test item

LOQM              = Limit of quantification of the analytical method (0.1 mg test item/L)

n.d.                  = Not determined, due to 100 % mortality after 24 h.

METHOD VALIDATION

- Linearity: 

The analytical system gave linear response in the range of nominal 5 to 75 µg/L of the test item. Linearity (r2) of the calibration curve was > 0.992.

- Limit of Quantification (LOQM):

The limit of quantification (LOQM) of the analytical method was fixed at 0.1 mg test item/L. The 1 x LOQMand 100 x LOQMwere checked by means of accuracy.

- Accuracy, Precision and Specificity:

Mean recoveries of 91 % at 1 x LOQMand 91 % at 100 x LOQMwere obtained indicating accuracy of the analytical method. The corresponding coefficients of variation of 8.10 % and 6.74 % indicated a sufficient precision of the analytical method. Specificity was confirmed because responses of blank samples were < 30 % of the 1 x LOQM.

Validity criteria fulfilled:
yes
Remarks:
The dissolved O2 concentration was > 3 mg/L. In the control group no Daphnia magna were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48 h test period
Conclusions:
Based on the nominal concentrations of the test item, the 48-h EC50 for Daphnia magna was estimated to be 43.2 mg/L (95 % confidence limits: 41.3 – 45.1 mg/L).
The NOEC after 48 h was 25.0 mg/L and the LOEC after 48 h was 50.0 mg/L.

Description of key information

Key data: EC50(48 h) = 43.2 mg/L (nominal); NOEC(48 h) = 25.0 mg/L (nominal); LOEC(48 h) = 50.0 mg/L (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
43.2 mg/L

Additional information

The short-term toxicity test to aquatic invertebrates under semi-static conditions performed according to EU Method C.2., OECD 202 and GLP was rated with RL=1 (reliable without restrictions) and used as a key study. Immobilization to Daphnia magna of the test item was observed at the nominal concentration of 50 mg/L and greater (measured concentrations within 80 - 120% of nominal values).


The EC50 (48 hours) was 43.2 mg/L (95 % confidence limits: 41.3 – 45.1 mg/L), the NOEC after 48 hours was 25.0 mg/L and the LOEC after 48 h was 50.0 mg/L (based on nominal concentrations).