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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 May 2006 to 24 July 2006
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
A saturated solution was tested in a limit test.
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.
The solution (100 mg/L test item was weighed out) was prepared with dilution water one day day prior to application.
The stock solution was stirred with ~ 1100 rpm for 24 h. Undissolved particles were removed by membran filtration (0.45¿m)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany; Breeder: Dr. U. Noack-Laboratorien, Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2-24 h
- Method of breeding:
Culture: In 2-3 L glass vessels with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 ¿E¿m-2 ¿s-1.
Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/l.
- Feeding during test: no feeding during the study

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

Reference:
Elendt, B. P. (1990): Selenium Deficiency in Crustacea, Protoplasma, 154, 25-30
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
245 mg CaCO3/L in dilution water prior to test start
Test temperature:
18 - 22 °C, ± 1 °C constant
pH:
7.67 (saturated solution) and 7.65 (controls) at the start
7.55-7.61 (saturated solution, 4 replicates) and 7.62-7.80 (controls, 4 replicates) at the end (48 h)
Dissolved oxygen:
8.27 (saturated solution) and 8.52 (controls) mg/L at the start
8.26-8.33 (saturated solution, 4 replicates) and 8.43-8.55 (controls, 4 replicates) mg/L at the end (48 h)
Nominal and measured concentrations:
A limit test with a saturated solution was carried out.
The saturated solution and the control after 0 h (new media) and 48 h (old media) were analytically verified via DOC analysis according to DIN EN 1484 (DOC of saturated solution: 1.8 mg/L). Separate replicates without test organisms for the test item analysis after 48 h were prepared and incubated under test conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
- 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: dilution water according to EU Method C.2, Annex

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: diffuse light, illumination range max. 20 ¿E¿m-2 ¿s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation at 24 and 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test with saturated solution (see above)
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: dilutions of the saturated solution of 1:1, 1:10 and 1:100
- Results used to determine the conditions for the definitive study: Based on the absence of any immobilisation, the main study was conducted as a limit test with the saturated solution.
Reference substance (positive control):
yes
Remarks:
potassium dichromate p.a.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item, a saturated solution had been prepared using a nominal concentration of 100 mg/L
Details on results:
No immobilisation after 24 and 48 h with the test substance (saturated solution) or the control.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality:
- EC50 (immobilisation): 1.95 mg/L (95% confidence interval: 1.71-2.16 mg/L)
- Other:
EC50 values determined by sigmoidal dose-response regression.
EC100 (immobilisation): 2.87 mg/L (determined directly from the test results)
Reported statistics and error estimates:
EC10- and EC50-values were only calculated for the reference item by sigmoidal dose-response regression. Calculation of the confidence intervals for EC50 were carried out using standard procedures according to CLOPPER and PEARSON (1934).
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: GraphPad Prism4 (2005), GRAPHPAD SOFTWARE, INC.

Reference:
Clopper and Pearson (1934): Biometrika, 26: 404-413 citied in GraphPad Prism Statistics Guide 4.0.
Validity criteria fulfilled:
yes
Conclusions:
At the saturated solution of the test item no biologically significant effect was determined.
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution of the test item was determined according to OECD 202 (2004) and Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h from 2006-06-13 to 2006-06-15 at DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Straße 1, D-31157 Sarstedt. 20 test organisms were exposed to the saturated solution and the control. Details of the analytical results are given in part 9. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.95 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

At the saturated solution of the test item (Pigment Red 122) no biologically significant effect was determined.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-11-12 - 2004-12-21
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5], based on OECD GLP 1997 (C(97) 186/Final)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
- Test item: Young daphnids of a clone of the species Daphnia magna Straus
- Origin: A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined by the supplier as clone 5. Since that time, the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age: daphnids used for the test were 6-24 hours old and were not first brood progeny.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Water Hardness 2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
19 - 20 °C during the test period. The test was performed in a temperature-controlled room (room temperature continuously monitored):
Control: 20°C (start), 20°C (end)
Treatment: 19°C (start), 20°C (end)
pH:
Control: 7.9 (start), 8.0 (end)
Treatment: 7.9 (start), 7.9 (end)
Dissolved oxygen:
[mg/L]
Control: 9.0 (start), 7.8 (end)
Treatment: 9.1 (start), 7.7 (end)
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration 100 mg/L
Due to poor solubility in water, no analytical determination possible.
Details on test conditions:
- The test medium is identical with ISO Test water (1) as specified in OECD 202
- Feeding: none
- Limit-test according to guideline (poor solubility in water):
The test medium was prepared as follows: Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was prepared by weighing 50.1 mg of the test item into 500 mL of test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring. The dispersion was stirred for 96 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
Then, the dispersion of the test item was filtered through a glass microfibre filter (Whatman GF/C, maximum pore size approximately 1.2 µm) just before the start of the test. The undiluted filtrate of the dispersion with the maximum concentration of dissolved test item was used as test medium.
The limit test was based on the results of a range-finding test and on pre-experiments to determine the solubility of the test item in test water (without GLP).

Reference substance (positive control):
yes
Remarks:
Potassium dichromate, tested at least once a year with the strain used
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Details on results:
The biological results are based on the loading rate of the test item of 100 mg/L. Analytical measurements of the actual test item concentration in the test medium were not possible.
In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours.
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear non-coloured solution throughout the whole test duration.
Results with reference substance (positive control):
The latest result of the positive control test with potassium dichromate in 2004 (48-hour EC50: 0.83 mg/L, RCC Study no. 852355) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2004: 48-hour EC50: 0.55 - 1.1 mg/L).
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
In a 48 hour chronic toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L). The acutal concentration could not be determined due to the low solubility of the submission substance in water and the salt concentration present in the ISO-test medium.
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202, Part I (1984). A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water. Due to the low water solubility of the test item, a dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved or very finely dispersed test item was used as the test medium. Additionally, a control was tested in parallel. No analytical determination of the test item concentration in the test medium was performed since the analysis of the test item in the filtrates - after comprehensive trials - proved impossible due to the very low solubility of the test item in test water and the matrix of the test water. In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours. The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours) and the 48-hour EC0 of the test item to Daphnia magna were determined to be at the loading rate of 100 mg/L. The 48-hour NOEC and the 48 hour EC0 might even be higher but concentrations above the solubility limit of the test item in test water (loading rate of 100 mg/L) were not tested according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than the solubility limit of the test item in test water (loading rate of 100 mg/L) since the test item had no toxic effect on the daphnids up to the highest test item concentration which could be dissolved or very finely dispersed in the test water under the conditions of the test. In conclusion, the test item had no acute toxic effects on Daphnia magna up to its solubility limit in test water under the present conditions of the test.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-03-18 - 2005-04-13
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)
Deviations:
yes
Remarks:
At the sponsor¿s request no test item analysis was carried out.
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German Chemikaliengesetz and directive 88/320/EEC
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
Stock solution:
The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment: The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 µm (RC, SCHLEICHER & SCHUELL).
Limit concentration: Saturated solution (100 mg/L)

CONTROL: Dilution water without test item tested under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna STRAUS (Clone 5), bred in the test facility.

Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)

Breeder: DR. U . NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture: In 2-3 L glass vessels with approximatively 1 .8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE/(m2 ¿ s).

Culture medium: Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

Feeding: At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
not applicable
Hardness:
Total hardness of 266 mg CaCO3/L
Test temperature:
20 +/- 1°C
pH:
Start:
control: 7.8 -- treatment: 7.7

End:
control: 7.83 - 7.94 -- treatment: 7.4 - 7.8
Dissolved oxygen:
[mg/L]
Start:
control: 8.69 -- treatment: 8.87

End:
control: 7.76 - 7.93 -- treatment: 7.91 - 7.98
Salinity:
not applicable
Nominal and measured concentrations:
Due to the poor solubility of the submission substance in water a limit test at 100 mg/L nominal concentration had been performed.
Details on test conditions:
A static preliminary range finding test was conducted as limit test with the saturated solution (NON-GLP-state).
The definitive study was performed as a limit test with saturated solution (100 mg/L) under static conditions to enable the determination of immobilisation after 24 and 48 hours. 2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h. The daphnids were acclimated to the dilution water ca. 2 hours before test start. The daphnids were not fed during the study.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure. Water parameter were measured at begin and end of the study. The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.
*) The information in brackets refer to the test of the reference item
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Details on results:
The percentage immobility was determined for the tested limit concentration and the control after 24 h and 48 h. In both, the control and treatment replicates, no immobilization occured . There is no biologically significant effect neither in the tested limit concentration nor in the control group.
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures. EC-values after 24 h of the reference item in mg/L:
EC10: 1.51
EC50: 1.84 (CI 1.74-1.95)
EC100: 3.2
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: DIN Guideline 38412 L 30.

AQS-Merkblatt: Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenüber Daphnien über Verdünnungsstufen (DIN 38412 - L 30)
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
In a 48 hour chronic toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L).
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L) as limit concentration of the test item, was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h from 2005-03-22 to 2005-03-24 at DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Straße 1, D-31157 Sarstedt. 20 test organisms were exposed to the limit concentration and control. No test item analysis was carried out. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.84 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Percentage Number of Daphnia Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

IMMOBILISATION [%]

 

24 h

48 h

Replicates

Replicates

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

Saturated solution
(100 mg/L)

Control

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

MV = Mean value

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2005-03-18 - 2005-04-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 202 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
At the sponsor¿s request no test item analysis was carried out.
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German Chemikaliengesetz and directive 88/320/EEC
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
Stock solution:
The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment: The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 µm (RC, SCHLEICHER & SCHUELL).
Limit concentration: Saturated solution (100 mg/L)

CONTROL: Dilution water without test item tested under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna STRAUS (Clone 5), bred in the test facility.

Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)

Breeder: DR. U . NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture: In 2-3 L glass vessels with approximatively 1 .8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE/(m2 ¿ s).

Culture medium: Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

Feeding: At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
not applicable
Hardness:
Total hardness of 266 mg CaCO3/L
Test temperature:
20 +/- 1°C
pH:
Start:
control: 7.8 -- treatment: 7.7

End:
control: 7.83 - 7.94 -- treatment: 7.4 - 7.8
Dissolved oxygen:
[mg/L]
Start:
control: 8.69 -- treatment: 8.87

End:
control: 7.76 - 7.93 -- treatment: 7.91 - 7.98
Salinity:
not applicable
Nominal and measured concentrations:
Due to the poor solubility of the submission substance in water a limit test at 100 mg/L nominal concentration had been performed.
Details on test conditions:
A static preliminary range finding test was conducted as limit test with the saturated solution (NON-GLP-state).
The definitive study was performed as a limit test with saturated solution (100 mg/L) under static conditions to enable the determination of immobilisation after 24 and 48 hours. 2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h. The daphnids were acclimated to the dilution water ca. 2 hours before test start. The daphnids were not fed during the study.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure. Water parameter were measured at begin and end of the study. The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.
*) The information in brackets refer to the test of the reference item
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item in water, limit test at nominal concentration of 100 mg/L
Details on results:
The percentage immobility was determined for the tested limit concentration and the control after 24 h and 48 h. In both, the control and treatment replicates, no immobilization occured . There is no biologically significant effect neither in the tested limit concentration nor in the control group.
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures. EC-values after 24 h of the reference item in mg/L:
EC10: 1.51
EC50: 1.84 (CI 1.74-1.95)
EC100: 3.2
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: DIN Guideline 38412 L 30.

AQS-Merkblatt: Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenüber Daphnien über Verdünnungsstufen (DIN 38412 - L 30)
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
In a 48 hour chronic toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L).
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L) as limit concentration of the test item, was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h from 2005-03-22 to 2005-03-24 at DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Straße 1, D-31157 Sarstedt. 20 test organisms were exposed to the limit concentration and control. No test item analysis was carried out. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.84 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Percentage Number of Daphnia Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

IMMOBILISATION [%]

 

24 h

48 h

Replicates

Replicates

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

Saturated solution
(100 mg/L)

Control

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

0


0

MV = Mean value

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2004-11-12 - 2004-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 202 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5], based on OECD GLP 1997 (C(97) 186/Final)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
- Test item: Young daphnids of a clone of the species Daphnia magna Straus
- Origin: A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined by the supplier as clone 5. Since that time, the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age: daphnids used for the test were 6-24 hours old and were not first brood progeny.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Water Hardness 2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
19 - 20 °C during the test period. The test was performed in a temperature-controlled room (room temperature continuously monitored):
Control: 20°C (start), 20°C (end)
Treatment: 19°C (start), 20°C (end)
pH:
Control: 7.9 (start), 8.0 (end)
Treatment: 7.9 (start), 7.9 (end)
Dissolved oxygen:
[mg/L]
Control: 9.0 (start), 7.8 (end)
Treatment: 9.1 (start), 7.7 (end)
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration 100 mg/L
Due to poor solubility in water, no analytical determination possible.
Details on test conditions:
- The test medium is identical with ISO Test water (1) as specified in OECD 202
- Feeding: none
- Limit-test according to guideline (poor solubility in water):
The test medium was prepared as follows: Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was prepared by weighing 50.1 mg of the test item into 500 mL of test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring. The dispersion was stirred for 96 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
Then, the dispersion of the test item was filtered through a glass microfibre filter (Whatman GF/C, maximum pore size approximately 1.2 µm) just before the start of the test. The undiluted filtrate of the dispersion with the maximum concentration of dissolved test item was used as test medium.
The limit test was based on the results of a range-finding test and on pre-experiments to determine the solubility of the test item in test water (without GLP).

Reference substance (positive control):
yes
Remarks:
Potassium dichromate, tested at least once a year with the strain used
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: test item poorly soluble in water
Details on results:
The biological results are based on the loading rate of the test item of 100 mg/L. Analytical measurements of the actual test item concentration in the test medium were not possible.
In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours.
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear non-coloured solution throughout the whole test duration.
Results with reference substance (positive control):
The latest result of the positive control test with potassium dichromate in 2004 (48-hour EC50: 0.83 mg/L, RCC Study no. 852355) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2004: 48-hour EC50: 0.55 - 1.1 mg/L).
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
In a 48 hour chronic toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L). The acutal concentration could not be determined due to the low solubility of the submission substance in water and the salt concentration present in the ISO-test medium.
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202, Part I (1984). A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water. Due to the low water solubility of the test item, a dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved or very finely dispersed test item was used as the test medium. Additionally, a control was tested in parallel. No analytical determination of the test item concentration in the test medium was performed since the analysis of the test item in the filtrates - after comprehensive trials - proved impossible due to the very low solubility of the test item in test water and the matrix of the test water. In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours. The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours) and the 48-hour EC0 of the test item to Daphnia magna were determined to be at the loading rate of 100 mg/L. The 48-hour NOEC and the 48 hour EC0 might even be higher but concentrations above the solubility limit of the test item in test water (loading rate of 100 mg/L) were not tested according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than the solubility limit of the test item in test water (loading rate of 100 mg/L) since the test item had no toxic effect on the daphnids up to the highest test item concentration which could be dissolved or very finely dispersed in the test water under the conditions of the test. In conclusion, the test item had no acute toxic effects on Daphnia magna up to its solubility limit in test water under the present conditions of the test.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
29 May 2006 to 24 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 202 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
A saturated solution was tested in a limit test.
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.
The solution (100 mg/L test item was weighed out) was prepared with dilution water one day day prior to application.
The stock solution was stirred with ~ 1100 rpm for 24 h. Undissolved particles were removed by membran filtration (0.45¿m)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany; Breeder: Dr. U. Noack-Laboratorien, Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2-24 h
- Method of breeding:
Culture: In 2-3 L glass vessels with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 ¿E¿m-2 ¿s-1.
Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/l.
- Feeding during test: no feeding during the study

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

Reference:
Elendt, B. P. (1990): Selenium Deficiency in Crustacea, Protoplasma, 154, 25-30
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
245 mg CaCO3/L in dilution water prior to test start
Test temperature:
18 - 22 °C, ± 1 °C constant
pH:
7.67 (saturated solution) and 7.65 (controls) at the start
7.55-7.61 (saturated solution, 4 replicates) and 7.62-7.80 (controls, 4 replicates) at the end (48 h)
Dissolved oxygen:
8.27 (saturated solution) and 8.52 (controls) mg/L at the start
8.26-8.33 (saturated solution, 4 replicates) and 8.43-8.55 (controls, 4 replicates) mg/L at the end (48 h)
Nominal and measured concentrations:
A limit test with a saturated solution was carried out.
The saturated solution and the control after 0 h (new media) and 48 h (old media) were analytically verified via DOC analysis according to DIN EN 1484 (DOC of saturated solution: 1.8 mg/L). Separate replicates without test organisms for the test item analysis after 48 h were prepared and incubated under test conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
- 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: dilution water according to EU Method C.2, Annex

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: diffuse light, illumination range max. 20 ¿E¿m-2 ¿s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation at 24 and 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test with saturated solution (see above)
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: dilutions of the saturated solution of 1:1, 1:10 and 1:100
- Results used to determine the conditions for the definitive study: Based on the absence of any immobilisation, the main study was conducted as a limit test with the saturated solution.
Reference substance (positive control):
yes
Remarks:
potassium dichromate p.a.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to poor solubility of test item, a saturated solution had been prepared using a nominal concentration of 100 mg/L
Details on results:
No immobilisation after 24 and 48 h with the test substance (saturated solution) or the control.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality:
- EC50 (immobilisation): 1.95 mg/L (95% confidence interval: 1.71-2.16 mg/L)
- Other:
EC50 values determined by sigmoidal dose-response regression.
EC100 (immobilisation): 2.87 mg/L (determined directly from the test results)
Reported statistics and error estimates:
EC10- and EC50-values were only calculated for the reference item by sigmoidal dose-response regression. Calculation of the confidence intervals for EC50 were carried out using standard procedures according to CLOPPER and PEARSON (1934).
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: GraphPad Prism4 (2005), GRAPHPAD SOFTWARE, INC.

Reference:
Clopper and Pearson (1934): Biometrika, 26: 404-413 citied in GraphPad Prism Statistics Guide 4.0.
Validity criteria fulfilled:
yes
Conclusions:
At the saturated solution of the test item no biologically significant effect was determined.
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution of the test item was determined according to OECD 202 (2004) and Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h from 2006-06-13 to 2006-06-15 at DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Straße 1, D-31157 Sarstedt. 20 test organisms were exposed to the saturated solution and the control. Details of the analytical results are given in part 9. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.95 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

At the saturated solution of the test item (Pigment Red 122) no biologically significant effect was determined.

Description of key information

Three Key study with read across substances are available. In one key study, at the saturated solution of the test item no biologically significant effect was determined.

Both results with the read across substances show the same result.

In a 48 hour toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L).

 

In a 48 hour toxicity test (reliability category 1, compliant to GLP) on Daphnia manga Straus performed as a static limit test due to the low solubility of the submission substance, the NOEC (48 h) had been determined to be the nominal concentration of 100 mg/L. The EC50 is higher than the nominal limit concentration (100 mg/L). The actual concentration could not be determined due to the low solubility of the submission substance in water and the salt concentration present in the ISO-test medium.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
> 100 mg/L

Additional information