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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Link to relevant study record(s)

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 with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: test sample form each vessel was taken form top mid and lower surfaces for analysis
- Sample storage conditions before analysis: chemical was immediately analysed after sampling
Vehicle:
no
Details on test solutions:
Details on test solutions
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: The stock solution was prepared by dissolving 100 mg of test chemical in 100 ml of M7 medium to get the final concentration of 1000 mg/L with continous stirring of 21hours followed by filtration and then analytically detected. The final solubility value obtained after analytical detection was 1000.92 mg/L.
- Controls: M7 Medium (Control),
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s)
including control(s)): Not applicable
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: Eggs of Daphnia magna were obtained from MicroBio tests Kleimoer 15B-9030 MARI AKERKE (GENT) BELGIUM. Later test system was sub-cultured in the test facility and same was used for
the study
- Age at study initiation (mean and range, SD): <24 h
- Age of parental stock (mean and range, SD): > 3 weeks
- Feeding during test : No

Others: A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years 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. During the maintenance of test organism, test daphnids were fed with unicel
lular green algae (Selenestrum capricornutum).


ACCLIMATION
- Acclimation period: 48 hours prior test
- Acclimation conditions (same as test or not): Yes (healthy gravid females were acclimatized)
- Type and amount of food: Food was provided during acclimatization.
- Health during acclimation (any mortality observed): healthy no mortality was observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
164 mg CaCO3/L
Test temperature:
19.7°C
pH:
7.4
Dissolved oxygen:
9.0
Conductivity:
0.27 μS/cm
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 mL Beakers
- Type (delete if not applicable): open / closed Loosely covered with glass lid to reduce the loss of
water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glass, volume 20 mL
- Volume of solution: 20 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 5 daphnids/20 mL test medium
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 1337-1346 Lux

RANGE-FINDING STUDY
Based on the available data on the test chemical range finding test was not conducted directly main
study was intaited to draw the conclusion on test substance toxicity.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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
Details on results:
Details on results
- Mortality of control: No mortality in control
- Other adverse effects control: None
- Immobilisation of control: No immobilisation
- Abnormal responses: None in control
Results with reference substance (positive control):
- Results with reference substance valid? Yes valid
EC50: 0.650 mg/L
Lower limit = 0.631 mg/L
Reported statistics and error estimates:
Not applicable

Standard graph for the assessment of test concentration
 
Sr. No
Concentrations mg/L
 
Wavelength (nm)
 
Absorbance
 
Temperature (°C)
1
blank
00 00
25
2
5.0 199 0.02
25
3
10.0 199 0.04
25
4
20.0 199 0.08
25
5
40.0 199 0.17
25
6
60.0 199 0.28
25
7
80.0 199 0.38
25
8
100.0 199 0.49
25
9
120.0 199 0.60
25
10
140.0 199 0.69
25
11
160.0 199 0.78
25
12
180.0 199 0.87
25
13
200.0 199 0.95
25

 

Concentration After Analytical Determination
 
 

0 Hour

0 Hour

48 Hours

48 hours

Sr. No

Concentration’s(mg/L)

Absorbance (mean)

Analytical concentrations

Absorbance (mean)

Analytical concentrations

1 control 00 00 00 00
2 100 0.48 99.37 0.47 98.29

 

 
ANNEX - 1
Nominal conc. (mg/l) Number of Daphnids/replicate Immobility
0h 24h 48h Cumulative Immobilization Percent Inhibition
control 5 R1=0 R2=0 R3=0 R4=0 R1=0 R2=0 R3=0 R4=0 R1= R2= R3= R4= - -
100 5 R1=0 R2=0 R3=0 R4=0 R1=0 R2=0 R3=0 R4=0 R1= R2= R3= R4= - -
Any abnormal behavior(if any) Actively moving Actively moving Actively moving    
ANNEX - 2
0 Hour
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=19.7 R2=19.7 R3=19.7 R4=19.7 R1=8.8 R2=8.8 R3=8.8 R4=8.8 R1=7.4 R2=7.4 R3=7.4 R4=7.4
100 R1=19.7 R2=19.7 R3=19.7 R4=19.7 R1=9.0 R2=9.0 R3=9.0 R4=9.0 R1=8.1 R2=8.1 R3=8.1 R4=8.1
24 Hours
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=19.4 R2=19.4 R3=19.4 R4=19.4 R1=8.9 R2=8.9 R3=8.9 R4=8.9 R1=7.6 R2=7.6 R3=7.6 R4=7.6
100 R1=19.4 R2=19.4 R3=19.4 R4=19.4 R1=8.7 R2=8.7 R3=8.7 R4=8.7 R1=8.2 R2=8.2 R3=8.2 R4=8.2
48 hours
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=21.9 R2=21.9 R3=21.9 R4=21.9 R1=6.9 R2=6.9 R3=6.9 R4=6.9 R1=87.7 R2=7.7 R3=7.7 R4=7.7
100 R1=21.9 R2=21.9 R3=21.9 R4=21.9 R1=6.5 R2=6.5 R3=6.5 R4=6.5 R1=8.2 R2=8.2 R3=8.2 R4=8.2
 
Validity criteria fulfilled:
yes
Conclusions:
The test concentrations were measured and found to remain within ±20% only in the 0th day of nominal. Hence, the EC50 > 100 mg/L was expressed based on nominal concentrations.
Executive summary:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study was conducted using 0 (control), and 100 mg/L concentrations (limit test). 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups , there was no immobilisation was observed in both controla and exposed group. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 100 mg/L of exposure, which were found in acceptable range between 99.37 at zero hours and 98.29 at 48 hours . Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.4 -7.7), temperature (19.7 -21.9 °C), dissolve oxygen (8.8 -6.9 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 μS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to, Daphnia magna was reported to be >100mg/L. The 48-h EC50of reference item(Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Hence, as per CLP classification category the test chemical can be categorized as Not classified category.

Description of key information

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study was conducted using 0 (control), and 100 mg/L concentrations (limit test). 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups , there was no immobilisation was observed in both controla and exposed group. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 100 mg/L of exposure, which were found in acceptable range between 99.37 at zero hours and 98.29 at 48 hours . Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.4 -7.7), temperature (19.7 -21.9 °C), dissolve oxygen (8.8 -6.9 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 μS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to, Daphnia magna was reported to be >100mg/L. The 48-h EC50of reference item(Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Hence, as per CLP classification category the test chemical can be categorized as Not classified category.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study was conducted using 0 (control), and 100 mg/L concentrations (limit test). 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups , there was no immobilisation was observed in both controla and exposed group. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 100 mg/L of exposure, which were found in acceptable range between 99.37 at zero hours and 98.29 at 48 hours . Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.4 -7.7), temperature (19.7 -21.9 °C), dissolve oxygen (8.8 -6.9 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 μS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to, Daphnia magna was reported to be >100mg/L. The 48-h EC50of reference item(Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Hence, as per CLP classification category the test chemical can be categorized as Not classified category.

To evaluate the influence of the test item on the mobility and survival of Daphnia magna an Acute Immobilisation Test according to OECD TG 202, was carried out under static conditions and in compliance with the GLP principles. Five animals in four replicates were exposed for 48 hours to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. In addition, a blank control including the test medium but without the test item was performed. The test item concentrations were analytically verified by using HPLC-UV to be 6.609, 12.740, 26.350, 51.560 and 103.228 mg/L after 48 hours. The test item concentration was thus 97 – 101 % of the nominal at the end of the experimental phase (at the start of the test: 102 –106 % of the nominal). Therefore, all biological results are based on nominal concentrations.The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate (24h EC50 = 1.12 mg/L). All validity criteria of the test guidelines were fulfilled. The mobility of the test animals was recorded after 24 and 48 hours of exposure. As a result, the 48-hour EC50 of the test item was determined to be 94.3 mg/L in nominal (95 % conf. limits: 77.3 – 133.3 calculated). The 48-hour EC10 was found to be 56.1 mg/L in nominal ( 95 % conf. limits: 28.1 – 70.0 mg/L). Based on the outcomes the test chemical can be classified into aquatic chronic category 3 as per CLP classification criteria.