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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
yes
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by dissolving 10.1 mg of test chemical in 100 mL of M7 medium to get the final concentration of 100 mg/L. The remaining test solutions were prepared by dilution from the above stock solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: waterflea
- Stain: Daphnia magna STRAUS
- Method of breeding: own breed
- Source: Daphnia magna were obtained from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM and maintained at ESSEM Compliance Solutions Private Limited, Nagpur.
- maintainance: A population of Daphnia magna of synchronized age structure has been maintained from last two months after its procurement from DTU in the test facility under constant temperature conditions (18 to 22 °C) at a 16 : 8 hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. Media renewal of Daphnia culture was done twice a week and pH, DO, temperature was recorded daily.
- Food type: The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
175 mg/l as CaCO3
Test temperature:
18 - 22°C
pH:
7.4 to 8.0
Nominal and measured concentrations:
Test chemical conc. taken for the study were 0 (control), 1, 2, 4, 8 and 16 mg/l (nominal conc.).
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 ml glass beaker
- Material, size, headspace, fill volume: 20 ml
- 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: M7 medium was used for the maintenance of test animals, preparation of stock solution and the test solutions of chemical.

OTHER TEST CONDITIONS
- Photoperiod: 16 hr light – 8 hr dark
- Light intensity: 1002 to 1250 lux


RANGE-FINDING STUDY
- Test concentrations: Range finding test was performed using 0.1, 1, 10 and 100 mg/L of test concentrations.
- Results used to determine the conditions for the definitive study: EC50 of
7.82 mg/L was obtained.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: details not known
Results with reference substance (positive control):
The 24-h EC50 of reference item (Potassium dichromate) to daphnid, was determined to be 0.65 mg/l.
Reported statistics and error estimates:
The Effective concentration was calculated using the concentration/ percentage response curve. In those cases where the slope of the concentration/percentage response curve was too steep to permit calculation of the EC50, this value was estimated as the geometric mean of EC0 and EC 100.

Table: pH, temperature and DO values.


 


0 Hour






































































































Nominal conc. (mg/l)



Temperature(° C)



DO(mg/l)



pH



control



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.4



R2=8.4



R3=8.4



R4=8.4



R1=7.4



R2=7.4



R3=7.4



R4=7.4



1



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.3



R2=8.3



R3=8.3



R4=8.3



R1=7.3



R2=7.3



R3=7.3



R4=7.3



2



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.2



R2=8.2



R3=8.2



R4=8.2



R1=7.1



R2=7.1



R3=7.1



R4=7.1



4



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.2



R2=8.2



R3=8.2



R4=8.2



R1=7.1



R2=7.1



R3=7.1



R4=7.1



8



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.1



R2=8.1



R3=8.1



R4=8.1



R1=7.0



R2=7.0



R3=7.0



R4=7.0



16



R1=20.1



R2=20.1



R3=20.1



R4=20.1



R1=8.1



R2=8.1



R3=8.1



R4=8.1



R1=7.0



R2=7.0



R3=7.0



R4=7.0



24 Hours






































































































Nominal conc. (mg/l)



Temperature(° C)



DO(mg/l)



pH



control



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.5



R2=8.5



R3=8.5



R4=8.5



R1=7.3



R2=7.3



R3=7.3



R4=7.3



1



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.6



R2=8.6



R3=8.6



R4=8.6



R1=7.0



R2=7.0



R3=7.0



R4=7.0



2



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.3



R2=8.3



R3=8.3



R4=8.3



R1=7.0



R2=7.0



R3=7.0



R4=7.0



4



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.3



R2=8.3



R3=8.3



R4=8.3



R1=7.1



R2=7.1



R3=7.1



R4=7.1



8



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.3



R2=8.3



R3=8.3



R4=8.3



R1=7.1



R2=7.1



R3=7.1



R4=7.1



16



R1=19.2



R2=19.2



R3=19.2



R4=19.2



R1=8.4



R2=8.4



R3=8.4



R4=8.4



R1=7.0



R2=7.0



R3=7.0



R4=7.0



48 hours






































































































Nominal conc. (mg/l)



Temperature(° C)



DO(mg/l)



pH



control



R1=19.1



R2=19.1



R3=19.1



R4=19.1



R1=8.2



R2=8.2



R3=8.2



R4=8.2



R1=7.4



R2=7.4



R3=7.4



R4=7.4



1



R1=19.1



R2=19.1



R3=19.1



R4=19.1



R1=7.8



R2=7.8



R3=7.8



R4=7.8



R1=7.1



R2=7.1



R3=7.1



R4=7.1



2



R1=19.1



R2=19.1



R3=19.1



R4=19.1



R1=8.0



R2=8.0



R3=8.0



R4=8.0



R1=7.0



R2=7.0



R3=7.0



R4=7.0



4



R1=19.1



R2=19.1



R3=19.1



R4=19.1



R1=7.9



R2=7.9



R3=7.9



R4=7.9



R1=7.1



R2=7.1



R3=7.1



R4=7.1



8



R1=19.1



R2=19.1



R3=19.1



R4=19.1



R1=7.7



R2=7.7



R3=7.7



R4=7.7



R1=7.0



R2=7.0



R3=7.0



R4=7.0



16



R1=19.1



R2=-



R3=-



R4=-



R1=7.8



R2=-



R3=-



R4=-



R1=7.0



R2=-



R3=-



R4=-



 


 


 


 


Table: Result of test chemical


 






































































































































Nominal conc. (mg/l)



Number of Daphnids/replicate



Immobility



0h



24h



48h



Cumulative Immobilization



Percent Inhibition



control



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



-



-



1



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



-



-



2



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



-



-



4



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



R1=N5



R2=N5



R3=N5



R4=N5



-



-



8



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=2



R2=N5



R3=1



R4=N5



R1=N3



R2=2



R3=N4



R4=2



7



35



16



5



R1=N5



R2=N5



R3=N5



R4=N5



R1=3



R2=5



R3=5



R4=5



R1=2



R2=0



R3=0



R4=0



20



100



Any abnormal behavior(if any)



Actively moving



Mortality was observed in higher concentration



Mortality was observed in higher concentration



 



 


Validity criteria fulfilled:
yes
Conclusions:
Based on the effect on the immobilization of test daphnids, the effective dose concentration EC50 was determined to be 8.80 mg/L.
Executive summary:

A study was performed to assess the Acute Immobilization of test chemicals to Daphnia Magna STRAUS (Common Name: Water Flea) under static conditions. The study was conducted by OECD Guideline for the testing of chemicals No. 202, Daphnia, Acute Immobilization Test Adopted on 13 April 2004. The main test was conducted under static conditions with the solution unchanged throughout the 48 hours duration.  Daphnia Magna STRAUS of size 0.45 cm from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM and maintained at ESSEM Compliance Solutions Private Limited, Nagpur. A population of Daphnia Magna of synchronized age structure has been maintained for the last two months after its procurement from DTU in the test facility under constant temperature conditions (18 to 22 °C) at a 16: 8-hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum). Media renewal of Daphnia culture was done twice a week and pH, DO, and the temperature was recorded daily. M7 medium was used for the maintenance of test animals, preparation of the stock solution, and the test solutions of chemicals.  A group of 20 Daphnids each recommended was exposed for a period of 48 hours to a test concentration i.e., control,1,2,4,8,16 mg/L of Test chemical dissolved in media.  Range finding test was performed using 0.1, 1, 10, and 100 mg/L of test concentrations. A definitive test was performed and five test concentrations in replicates were selected, which were arranged in geometric series by considering factor 2. The stock solution was prepared by dissolving 10.1 mg of the test chemical in 100 ml of M7 medium to get the final concentration of 100 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The analytical determinations were performed by HPLC. The pre-treated stock solution was then diluted with media to get the required test solutions ranging from concentrations 1.15, 11.5, 115, and 1150 mg/L. The absorbance of the resulting solution was measured using HPLC against the corresponding blank. A standard curve was plotted against concentration versus area and the maximum solubility was determined from the above standard curve. Analytical assessments were performed for selected test concentrations at 0 hours and 48 hours. During the test, a temperature range of 18 - 22°C, was maintained in the test beakers throughout the exposure period. The pH and oxygen values were measured at the beginning of the test and every 24 hours thereafter. The hardness of the media was above 175 mg/L CaCO3 during the test. The Daphnids were exposed to the test chemicals and dissolved in the M7 media. Immobilization was recorded at 24 hours and 48 hours and EC50 was calculated from probit analysis. Reference chemical Potassium dichromate (K2Cr2O7) was tested for 24 hours to assure the test conditions are reliable. After 48 hr of exposure period, in the control, there was no immobilisation of test daphnids or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was 8.2 (i.e., ≥3 mg/l) in control and test vessels. Hence, the test is valid as per OECD guideline 203. Concentrations were measured and found to remain within ±20% of nominal concentrations. Based on the effect on the immobilization of test daphnids, the effective dose concentration EC50 was determined to be 8.8 mg/L. Since the test chemical was readily biodegradable, the test chemical was considered to be not classified as per CLP classification criteria.

Description of key information

Short-term toxicity to aquatic invertebrates:


A study was performed to assess the Acute Immobilization of test chemicals to Daphnia Magna STRAUS (Common Name: Water Flea) under static conditions. The study was conducted by OECD Guideline for the testing of chemicals No. 202, Daphnia, Acute Immobilization Test Adopted on 13 April 2004. The main test was conducted under static conditions with the solution unchanged throughout the 48 hours duration.  Daphnia Magna STRAUS of size 0.45 cm from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM and maintained at ESSEM Compliance Solutions Private Limited, Nagpur. A population of Daphnia Magna of synchronized age structure has been maintained for the last two months after its procurement from DTU in the test facility under constant temperature conditions (18 to 22 °C) at a 16: 8-hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum). Media renewal of Daphnia culture was done twice a week and pH, DO, and the temperature was recorded daily. M7 medium was used for the maintenance of test animals, preparation of the stock solution, and the test solutions of chemicals.  A group of 20 Daphnids each recommended was exposed for a period of 48 hours to a test concentration i.e., control,1,2,4,8,16 mg/L of Test chemical dissolved in media.  Range finding test was performed using 0.1, 1, 10, and 100 mg/L of test concentrations. A definitive test was performed and five test concentrations in replicates were selected, which were arranged in geometric series by considering factor 2. The stock solution was prepared by dissolving 10.1 mg of the test chemical in 100 ml of M7 medium to get the final concentration of 100 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The analytical determinations were performed by HPLC. The pre-treated stock solution was then diluted with media to get the required test solutions ranging from concentrations 1.15, 11.5, 115, and 1150 mg/L. The absorbance of the resulting solution was measured using HPLC against the corresponding blank. A standard curve was plotted against concentration versus area and the maximum solubility was determined from the above standard curve. Analytical assessments were performed for selected test concentrations at 0 hours and 48 hours. During the test, a temperature range of 18 - 22°C, was maintained in the test beakers throughout the exposure period. The pH and oxygen values were measured at the beginning of the test and every 24 hours thereafter. The hardness of the media was above 175 mg/L CaCO3 during the test. The Daphnids were exposed to the test chemicals and dissolved in the M7 media. Immobilization was recorded at 24 hours and 48 hours and EC50 was calculated from probit analysis. Reference chemical Potassium dichromate (K2Cr2O7) was tested for 24 hours to assure the test conditions are reliable. After 48 hr of exposure period, in the control, there was no immobilisation of test daphnids or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was 8.2 (i.e., ≥3 mg/l) in control and test vessels. Hence, the test is valid as per OECD guideline 203. Concentrations were measured and found to remain within ±20% of nominal concentrations. Based on the effect on the immobilization of test daphnids, the effective dose concentration EC50 was determined to be 8.8 mg/L. Since the test chemical was readily biodegradable, the test chemical was considered to be not classified as per CLP classification criteria.


 

Key value for chemical safety assessment

Fresh water invertebrates

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

Additional information

Short-term toxicity to aquatic invertebrates:


A study was performed to assess the Acute Immobilization of test chemicals to Daphnia Magna STRAUS (Common Name: Water Flea) under static conditions. The study was conducted by OECD Guideline for the testing of chemicals No. 202, Daphnia, Acute Immobilization Test Adopted on 13 April 2004. The main test was conducted under static conditions with the solution unchanged throughout the 48 hours duration.  Daphnia Magna STRAUS of size 0.45 cm from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM and maintained at ESSEM Compliance Solutions Private Limited, Nagpur. A population of Daphnia Magna of synchronized age structure has been maintained for the last two months after its procurement from DTU in the test facility under constant temperature conditions (18 to 22 °C) at a 16: 8-hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum). Media renewal of Daphnia culture was done twice a week and pH, DO, and the temperature was recorded daily. M7 medium was used for the maintenance of test animals, preparation of the stock solution, and the test solutions of chemicals.  A group of 20 Daphnids each recommended was exposed for a period of 48 hours to a test concentration i.e., control,1,2,4,8,16 mg/L of Test chemical dissolved in media.  Range finding test was performed using 0.1, 1, 10, and 100 mg/L of test concentrations. A definitive test was performed and five test concentrations in replicates were selected, which were arranged in geometric series by considering factor 2. The stock solution was prepared by dissolving 10.1 mg of the test chemical in 100 ml of M7 medium to get the final concentration of 100 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The analytical determinations were performed by HPLC. The pre-treated stock solution was then diluted with media to get the required test solutions ranging from concentrations 1.15, 11.5, 115, and 1150 mg/L. The absorbance of the resulting solution was measured using HPLC against the corresponding blank. A standard curve was plotted against concentration versus area and the maximum solubility was determined from the above standard curve. Analytical assessments were performed for selected test concentrations at 0 hours and 48 hours. During the test, a temperature range of 18 - 22°C, was maintained in the test beakers throughout the exposure period. The pH and oxygen values were measured at the beginning of the test and every 24 hours thereafter. The hardness of the media was above 175 mg/L CaCO3 during the test. The Daphnids were exposed to the test chemicals and dissolved in the M7 media. Immobilization was recorded at 24 hours and 48 hours and EC50 was calculated from probit analysis. Reference chemical Potassium dichromate (K2Cr2O7) was tested for 24 hours to assure the test conditions are reliable. After 48 hr of exposure period, in the control, there was no immobilisation of test daphnids or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was 8.2 (i.e., ≥3 mg/l) in control and test vessels. Hence, the test is valid as per OECD guideline 203. Concentrations were measured and found to remain within ±20% of nominal concentrations. Based on the effect on the immobilization of test daphnids, the effective dose concentration EC50 was determined to be 8.8 mg/L. Since the test chemical was readily biodegradable, the test chemical was considered to be not classified as per CLP classification criteria.

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