Registration Dossier

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Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-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
Justification for type of information:
Data is from experimental study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Pre-treatment of test chemical: The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution will be filtered by using whatman filter paper no. 42, which will then analytically determine. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Test chemical was weighed and about 1000mg in 1000 ml of test chemical kept for stirring for 48 hours, which was then filtered and the concentration was anlytically determined, and which was obtained to be 0.1 mg/L
- Controls: Test media M7 was directly used as the control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehilce was used during study
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): no applicable
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Age at study initiation (mean and range, SD): < 24 hr old
- Length at study initiation (length definition, mean, range and SD): 0.45 cm
- Source: Eggs of Daphnia magna were obtained from Denmark Technical University and maintained at Unique Ecotox Research Laboratory, Nagpur

ACCLIMATION
- Type and amount of food: Living cells of Selenestrum capricornutum
- Feeding frequency: Feeding of the Daphnia with living cells of Selenestrum capricornutum was done thrice a week.


TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus
- Justification for species other than prescribed by test guideline: Test organism used was as per guidelines
- Age at study initiation (mean and range, SD): <24houes ols
- Length at study initiation (length definition, mean, range and SD): 0.45 cm
- Stage and instar at study initiation: neonate
- Method of breeding: daphnia were maintained in the M7 medium in similar conditions as per the test conditions, gravid females are seperated from the other daphinds for the collection of young neonates.
- Source: MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM
- Age of parental stock (mean and range, SD): 4-5 weeks
- Feeding during test: yes
- Food type: Fresh water algae ( C vulgaris)
- Amount: 0.1 mg C/Daphnia/day
- Frequency: thrice in a week

ACCLIMATION
- Acclimation period: Parent daphnids were acclimitised minimum for the period of 4 weeks in M7 medium before taking taking their neonates for study
- Acclimation conditions (same as test or not): same as test conditions

- Feeding frequency:
- Health during acclimation (any mortality observed): Parent daphnids were healthy.

QUARANTINE (wild caught): None
- Duration:
- Health/mortality:



Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
170 mg/l as CaCO3
Test temperature:
20.1-21.5°C
pH:
7.4-8.1
Dissolved oxygen:
6.7-7.1 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
10 mg/l ( nominal concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: 50 ml
- Aeration: Test vessel were not aerated during the study.
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

TEST MEDIUM / WATER PARAMETERS: M7 medium

OTHER TEST CONDITIONS
- Photoperiod: 16:8 light:dark conditions
- Light intensity: Not exceeding 1000-1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia.


TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): loosley covered to avoid the dust particles to fall into the media
- Material, size, headspace, fill volume: 50 mll beaker, 40 ml of test solutions, 10 ml head space
- Aeration: No aeration during study
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Thrice weekly
- No. of organisms per vessel: 1organims/ vessels.
- No. of vessels per concentration (replicates): 10 vessels per concentrations
- No. of vessels per control (replicates): 10 vessels per control
- No. of vessels per vehicle control (replicates): Not applicable
- Test medium: M7 Media

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16:8 light:dark conditions
- Light intensity: Not exceeding 1000-1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not appicable
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Based on the available information on the short term daphniamagna concentrations for the long term studies are determined
- Test concentrations: 10 mg/L
- Results used to determine the conditions for the definitive study: 1/10 of the EC50 of Daphina immobilisation study was considerd for main study
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: P Value: 0.6509: not significantly different from control offsprings
Details on results:
- Behavioural abnormalities: no behavioural abnormalities were observed
- Observations on body length and weight: no significant difference was observed in the body length was observed in both parents and offsprings
- Other biological observations: first brood with visible eggs in parents was observed on day 10
- Mortality of control: no mortality was observed in control parents and offsprings
- Other adverse effects control: no adverse effects were observed
- Immobilisation of control: no immobilisation
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The statistical comparision was done by student T test method using Sys stat software

Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along w ith the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly.

 

 

NominalConcentration: Control

 

Day

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

 

Medium renewal

Y

-

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

 

pH(new)

7.5

-

-

7.6

-

-

7.7

-

7.4

-

7.8

-

-

7.9

-

7.8

-

8.0

-

-

8.1

-

 

O2

(mg/l)(new)

6.7

-

-

6.5

-

-

6.9

-

6.7

-

6.7

-

-

7.0

-

7.1

-

7.1

-

-

7.0

-

 

Temp(°C)(new)

20.1

-

-

21.1

-

-

21.5

-

21.6

-

20.8

-

-

21.4

-

21.7

-

21.5

-

-

21.1

-

 

Food provided

Y

-

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

 

 

No.of live off spring per parent animal

Total no.of live offspring/

Vessel

V1

-

-

-

-

-

-

-

-

-

-

-

-

-

20

-

21

37

-

-

-

47

20

145

V2

-

-

-

-

-

-

-

-

-

-

6

-

-

-

21

9

-

35

-

-

5

35

111

V3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5

-

-

-

-

-

15

20

V4

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

-

-

19

-

35

10

47

V5

-

-

-

-

-

-

-

-

-

-

-

-

-

15

14

-

-

-

-

-

7

-

55

V6

-

-

-

-

-

-

-

-

-

-

-

-

-

12

29

-

-

41

-

-

4

2

88

V7

-

-

-

-

-

-

-

-

-

-

-

-

-

1

-

1

47

-

-

-

20

5

94

V8

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

-

-

25

7

34

V9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

10

-

10

V10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

8

42

1

-

-

49

-

100

Cumulative Reproductivity

=704;

                            

NominalConcentration:10mg/L

 

Day

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

 

 

Medium renewal

Y

-

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

 

 

pH(new)

7.5

-

-

7.4

-

-

7.7

-

7.5

-

7.7

-

-

7.9

-

7.9

-

7.9

-

-

8.2

-

 

 

O2

(mg/l)(new)

6.7

-

-

6.6

-

-

6.8

-

6.6

-

6.8

-

-

6.8

-

6.7

-

7.1

-

-

6.9

-

 

 

Temp(°C)(new)

20.1

-

-

21.1

-

-

21.5

-

21.5

-

20.8

-

-

21.2

-

21.4

-

21.3

-

-

21.4

-

 

 

Food provided

Y

-

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

Y

-

Y

-

-

Y

-

 

 

 

No.of live off spring per parent animal

Total no.of liveoffspring/

Vessel

 

V1

-

-

-

-

-

-

-

-

-

-

-

-

-

6

2

-

32

-

 

-

29

-

69

 

V2

-

-

-

-

-

-

-

-

-

-

-

-

-

8

-

-

27

-

-

-

27

-

62

 

V3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

14

1

-

37

-

-

21

4

77

 

V4

-

-

-

-

-

-

-

-

-

-

-

-

-

-

18

-

3

26

-

-

-

17

64

 

V5

-

-

-

-

-

-

-

-

-

-

-

-

-

5

12

-

-

25

-

-

8

1

51

 

V6

-

-

-

-

-

-

-

-

-

-

-

-

-

-

15

-

-

20

-

-

17

13

65

 

V7

-

-

-

-

-

-

-

-

-

-

-

-

-

7

-

19

-

34

-

-

16

-

76

V8

-

-

-

-

-

-

-

-

-

-

-

-

-

-

8

-

4

26

-

-

5

16

59

V9

-

-

-

-

-

-

-

-

-

-

-

-

-

9

-

-

22

02

-

-

16

2

51

V10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

CumulativeReproductivity

=574

 

2: mortality in the adults, both control and test item exposed individuals (raw data)

Control:

No.ofDays

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

V1

DeadP

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

DeadO

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

V2

DeadP

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

DeadO

-

-

-

-

-

-

-

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-

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-

V3

DeadP

-

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-

-

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DeadO

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V4

DeadP

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-

-

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DeadO

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-

-

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-

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-

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-

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-

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-

-

-

V5

DeadP

-

-

-

-

-

-

-

-

-

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-

-

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-

-

-

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-

-

-

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DeadO

-

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-

V6

DeadP

-

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DeadO

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V7

DeadP

-

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DeadO

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-

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-

V8

DeadP

-

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-

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-

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-

-

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DeadO

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-

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-

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-

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-

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V9

DeadP

-

-

-

-

-

-

-

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-

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-

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-

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-

-

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DeadO

-

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-

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-

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V10

DeadP

-

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-

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-

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-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

Testconcentration:10mg/L

No.ofDays

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

V1

DeadP

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

DeadO

-

-

-

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-

-

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-

-

-

-

-

-

-

-

-

-

 

 

V2

DeadP

-

-

-

-

-

-

-

-

-

-

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-

-

-

-

-

-

-

-

-

-

DeadO

-

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-

-

-

-

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-

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V3

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V5

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V6

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V7

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V8

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V9

DeadP

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DeadO

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-

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V10

DeadP

-

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-

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M

-

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-

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-

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DeadO

-

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-

-

 

Analysisofweek

 

Sample (AI)mg/l

Range

Week1

New testmedium

10.86

 

 

92.4%to115%

Oldtestmedium

10.44

Week2

New testmedium

9.79

Oldtestmedium

9.24

Week3

New testmedium

10.84

Oldtestmedium

11.50

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l.
Executive summary:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Parent daphina were acclimitised in the similar conditions as maintined on the test. From this, gravid parents were isolated and miantined in the M7 medium offsprings were collected, and these nenonates were used in the study. The test organims was obatined from MicroBio tests Kleimoer 15B-9030 MARIAKERKE(GENT)BELGIUM. The Neonates whose age was less than <24 hours were selected. Test daphnids were fed with living cells of C. vulgaris and it was done thrice a week. The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution was filtered by using whatman filter paper no. 42, which was then analytically determined. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions. the leniarity range selected for concentrations analysis and stock analysis was 5.0, 10, 20, 30, 40, and 50 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. 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. Test chemical conc. used in the defiinite study was 10 mg/l. Thus, limit test was performed at 10 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 20.1 -21.5°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. Each test concentrations has10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus based on the outcomes chemical could not be classified as per CLP classification criteria.

Description of key information

Reproduction study in daphnia magna:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Parent daphina were acclimitised in the similar conditions as maintined on the test. From this, gravid parents were isolated and miantined in the M7 medium offsprings were collected, and these nenonates were used in the study. The test organims was obatined from MicroBio tests Kleimoer 15B-9030 MARIAKERKE(GENT)BELGIUM. The Neonates whose age was less than <24 hours were selected. Test daphnids were fed with living cells of C. vulgaris and it was done thrice a week. The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution was filtered by using whatman filter paper no. 42, which was then analytically determined. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions. the leniarity range selected for concentrations analysis and stock analysis was 5.0, 10, 20, 30, 40, and 50 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. 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. Test chemical conc. used in the defiinite study was 10 mg/l. Thus, limit test was performed at 10 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 20.1 -21.5°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. Each test concentrations has10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus based on the outcomes chemical could not be classified as per CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10 mg/L

Additional information

Reproduction study in daphnia magna:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Parent daphina were acclimitised in the similar conditions as maintined on the test. From this, gravid parents were isolated and miantined in the M7 medium offsprings were collected, and these nenonates were used in the study. The test organims was obatined from MicroBio tests Kleimoer 15B-9030 MARIAKERKE(GENT)BELGIUM. The Neonates whose age was less than <24 hours were selected. Test daphnids were fed with living cells of C. vulgaris and it was done thrice a week. The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution was filtered by using whatman filter paper no. 42, which was then analytically determined. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions. the leniarity range selected for concentrations analysis and stock analysis was 5.0, 10, 20, 30, 40, and 50 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. 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. Test chemical conc. used in the defiinite study was 10 mg/l. Thus, limit test was performed at 10 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 20.1 -21.5°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. Each test concentrations has10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus based on the outcomes chemical could not be classified as per CLP classification criteria.

 

In the first study (from authoritative database) toxicity was measured on the basis of immobilisation. Aim of this study was to determine the effect of test chemical on the reproductive inhibition and immobilisation of daphnia magna. Test conducted in accordance with the OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test and Reproduction Test). Test conducted under the semi-static system for 21 days. Nominal concentration including control, vehicle control (2.94 mg/L), 0.35, 0.60, 1.02, 1.73, 2.94 mg/L (geometric ratio: 1.7) were used. Test vessel filled with 400 ml of solution and 10 daphnia per vessel were studied. After the exposure of 21 days effect were measured. Based on the effect observed on the daphnia magna after the exposure of 21 days with the test chemical, No effect were observed (NOEC) at 0.33 mg/l and EC50 was observed at 0.99 mg/l. Based on the EC50, chemical consider to be toxic and classified as aquatic chronic 2 category. But the test chemical was readily biodegradable in water and thus on that basis chemical classified in aquatic chronic 3 category.

 

First study was supported by the second study from authoritative database. Study was conducted to access the effect of test chemical on the reproduction of aquatic invertebrate daphnia magna by providing the exposure rate of 21days. Test conducted in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test). Based on the readily biodegradability of test chemical and NOEC value, chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Thus based on the overall studies, it was concluded that the test chemical was toxic and the can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.