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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 with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L.
- Sampling method: Test item was weighed and transferred into a volumetric flask and made up to the required volume using M7 medium and coded as stock. Test solutions of the selected concentrations were prepared by dilution of the stock solution. The solutions were prepared in M7 medium. Volumetric flaks and measuring cylinder were used for solution preparation.
- Sample storage conditions before analysis: sample was analysed immediately after preperation
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: 500 mg of test item was transferred and made up to 500 mL in a volumetric flask using M7 medium (Stock solution). From the stock solution 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L were prepared using M7 medium in a 250 mL measuring cylinder.
- 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 unicellular 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:
no
Total exposure duration:
48 h
Hardness:
164 mg CaCO3/L
Test temperature:
20 °C
pH:
Control 7.2 - 7.0
Dissolved oxygen:
6.8-7.2 mg/L
Salinity:
Not applicable
Conductivity:
0.27 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 21.72,32.58, 48.87, 73.30, 110 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation
VEHICLE CONTROL PERFORMED: Not applicable
- Spacing factor for test concentrations: 1.5

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.:
66.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 CI 62.64 and 70.51
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:
48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT Plus Software, version 8.

 

 

Total inhibition

Concentration (mg/L)

0 hours

24 hours

48 hours

Cumulative Immobilisation

Percent immobilisation

Control (0)

0

0

0

0

0

21.72

0

0

0

0

0

32.58

0

0

1

1

5

48.87

0

1

3

4

20

73.30

0

5

7

12

60

110

0

8

10

18

90

 

 Inhibiton at individual Replicates

 

 

Total inhibition

Concentration (mg/L)

 

0 hours

24 hours

48 hours

Cumulative Immobilisation

Control (0)

R1

0

0

0

0

R2

0

0

0

R3

0

0

0

R4

0

0

0

21.72

R1

0

0

0

0

R2

0

0

0

R3

0

0

0

R4

0

0

0

32.58

R1

0

0

1

1

R2

0

0

0

R3

0

0

0

R4

0

0

0

48.87

R1

0

1

0

4

R2

0

0

0

R3

0

0

2

R4

0

0

1

73.30

R1

0

0

2

12

R2

0

1

3

R3

0

2

1

R4

0

2

1

110

R1

0

2

1

18

R2

0

2

3

R3

0

3

2

R4

0

1

4

 

 

 

 

Physio chemical parameters

 

 

pH

Temperature (°C)

Dissolved oxygen (mg/L)

Concentration (mg/L)

0 hours

48 hours

0 hours

48 hours

0 hours

48 hours

Control (0)

7.2

7.0

20

20

8.6

8.1

21.72

7.0

7.0

20

20

8.5

7.9

32.58

7.0

6.9

20

20

8.8

8.0

48.87

6.9

6.8

20

20

8.6

7.5

73.30

6.9

6.7

20

20

8.4

7.6

110

6.8

6.7

20

20

8.2

7.6

 

Analytical monitoring:

 

Total inhibition

Concentration (mg/L)

0 hours (mg/L)

48 hours (mg/L)

Percent range

Blank

0

0

0

21.72

22.21

21.16

102.25 – 97.42

32.58

34.16

33.48

104.84 – 102.76

48.87

45.96

46.71

94.42 – 95.58

73.30

77.25

74.93

105.38 – 102.22

110

110.12

112.60

100.10 – 102.36

Validity criteria fulfilled:
yes
Remarks:
In the control, there was no daphnid found immobilized or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was ≥ 3 mg/ L in control and test vessels
Conclusions:
The 48-h EC50 of test chemical to daphnid, Daphnia magna was reported to be 66.46 mg/L (CI - 62.64 - 70.51 mg/L).
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 (using a spacing factor of 1.5) was conducted using 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L concentrations. 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 and 21.72 mg/L but 5%, 20%, 60% and 90% immobilisation were observed in the test concentrations of 32.58, 48.87, 73.30, 110 mg/L, respectively. 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 32.58, 48.87, 73.30, 110 mg/L, conc were 97.42%, 102.76%, 95.58%, 105.38%, 102.22%, and 102.36%, respectively, after 48 hours of exposure, which were found in acceptable range. 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 (7.0 -7.2), temperature (20-21 °C), dissolve oxygen (8.6 - 8.1 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 daphnid, Daphnia magna are 66.46. 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. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT plus Software, version 8. Hence, as per CLP classification category the test chemical can be categorized as Chronic Category 3.

Description of key information

Short term toxicity to daphnia:

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 (using a spacing factor of 1.5) was conducted using 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L concentrations. 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 and 21.72 mg/L but 5%, 20%, 60% and 90% immobilisation were observed in the test concentrations of 32.58, 48.87, 73.30, 110 mg/L, respectively. 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 32.58, 48.87, 73.30, 110 mg/L, conc were 97.42%, 102.76%, 95.58%, 105.38%, 102.22%, and 102.36%, respectively, after 48 hours of exposure, which were found in acceptable range. 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 (7.0 -7.2), temperature (20-21 °C), dissolve oxygen (8.6 - 8.1 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 daphnid, Daphnia magna are 66.46. 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. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT plus Software, version 8. which was reported to be 66.46 (64.64 -70.51) Hence, as per CLP classification category the test chemical can be categorized as Chronic Category 3.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
66.46 mg/L

Additional information

Various experimental studies of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across chemical were reviewed for short term toxicity to aquatic invertebrate end point which are summarized as below:

 

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 (using a spacing factor of 1.5) was conducted using 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L concentrations. 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 and 21.72 mg/L but 5%, 20%, 60% and 90% immobilisation were observed in the test concentrations of 32.58, 48.87, 73.30, 110 mg/L, respectively. 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 32.58, 48.87, 73.30, 110 mg/L, conc were 97.42%, 102.76%, 95.58%, 105.38%, 102.22%, and 102.36%, respectively, after 48 hours of exposure, which were found in acceptable range. 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 (7.0 -7.2), temperature (20-21 °C), dissolve oxygen (8.6 - 8.1 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 daphnid, Daphnia magna are 66.46. 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. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT plus Software, version 8. Hence, as per CLP classification category the test chemical can be categorized as Chronic Category 3.

In an experimental study from study report (2019), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 162.82 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 6.25, 13.12, 27.55, 57.85 and 121.48 mg/l, respectively was from the saturated test concentration. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 63.86 mg/L. Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per CLP classification criteria.

 

Another acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2017). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 100 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 1.2, 2.0, 3.5, 5.8 and 10.0 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. EC50 was calculated using non-linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. The dissolved oxygen concentration at the end of the test both in the control and test vessels was evaluated to be ≥ 3 mg/l (i.e, reported as > 8.6 mg/l), indicating that the validity criteria has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 3.6 mg/l (95 % C. I. - 2.7 to 4.8 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

 

In a supporting weight of evidence study, short term toxicity to aq. Invertebrate study was conducted for 24 hrs for assessing the effect of test chemical (Cecilia Labbe et. al; 2005 and authoritative database, 2017). Artemia salina (Brine shrimp) (one-day nauplii) was used as a test organism. Study was performed using 10 test organisms alongwith the test chemical in 10 ml of seawater solution in a static system. Test organism was exposed to different nominal test chemical concentrations (0, 2.5, 4, 10, 25 and 50 mg/l) for a period of 24 hrs. All experiments were performed in triplicates. Lindane was used as a reference substance for the study. Test organism survivors were counted after an exposure period of 24 hrs. The LC50 values were calculated using Finney’s Probit Analysis program. The 24 hr LC50 value of the reference substance lindane was determined to be 7.2 mg/l (95% C. I. = 4.9 to 9.7). On the basis of effect on the mortality of the test organism, the 24 hr LC50 value was determined to be < 2.5 mg/l. Thus, based on the LC50 value, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations.

 

For the test chemical from study report (2017), short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed following the OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 10 g/l was prepared by dissolving test chemical in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0, 1.0, 1.5, 2.2, 3.4 and 5.0 mg/l, respectively. Study was performed using total 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical and other control vessel containing reconstituted water plus acetone without test chemical were also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. The dissolved oxygen concentration at the end of the test both in the control and test vessels was evaluated to be ≥ 3 mg/l (i.e, reported as > 7.4 mg/l), indicating that the validity criteria has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 1.7 mg/l (1.4 to 2.0 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

 

On the basis of the overall results, it can be concluded that the test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.