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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2015 to 17 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to OECD 211 guideline, under GLP without deviation and with certificate of analysis included.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
05 March 2015
Test material information:
Composition 1
Specific details on test material used for the study:
Physico-chemical Properties:
Specific Gravity: 0.96-0.98 at 20°C
Vapour Pressure: 0.6 Pa at 25 °C (calculated value)
Solubility in water: Between 332.2 mg.L-1 (slow-stirring method) and 334.8 mg.L-1 (column elution method)
log Kow: 3.09 (HPLC Method)
Analytical monitoring:
yes
Details on sampling:
Single samples for analysis were taken from the control and all test concentrations three times a week at renewal of the test media.
Samples for fresh solutions were taken from each treatment prior to addition of the daphnids and the suspension of algal cells.
Samples for old solutions were taken from the same replicate used for sampling of fresh solutions and were centrifuged at 500 g for 5 min (in order to pellet algal cells) before sampling of the supernatant.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: slow stirring
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA
- Age of parental stock (mean and range, SD): < 24 hours old
- Feeding: Daphnids were fed five days a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1 and at least three times per week
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
None
Post exposure observation period:
Not applicable
Hardness:
Hardness was in the range 214 to 250 mg CaCO3.L-1 and thus above 140 mg CaCO3.L-1 as recommended
Test temperature:
Between 20.4 and 21.3 °C throughout the test (average value: 20.8 °C).
This is within the recommended range 20 +/- 2 °C.
pH:
Between 7.60 and 8.84 throughout the test. This is withint the recommended range 6.0-9.0.
Dissolved oxygen:
Always > 7 mg/L
Salinity:
NA
Nominal and measured concentrations:
Nominal: 0.5, 1.2, 2.7, 6.4 and 15 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass sealed with screw cap test bottles (air-tight) of approximately 100 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: Semi-static, with three media changes per week; at each renewal, a new series of clean and sterilised test vessels were used.
- No. of animals: 10 animals individually held at each test concentration and the control series
- Loading: 1 daphnid per test vessel each completely filled with test solution and with headspace limited as much as possible
- Introduction of Daphnids: Daphnids were introduced into the test medium immediately after filling the test bottles with test solutions

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Offspring: The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. Only the number of living offspring was counted, but the presence of aborted eggs or dead offspring was also recorded.
- Mortality: Mortality among the parent animals was recorded daily, at the same times as offspring were counted.
- Individual length of adults: At the end of the test the length of the adults was measured by microscopy using an ocular micrometer calibrated with a stage micrometer.
- Dissolved O2, hardness, pH: Measured at every media change in fresh and old media, in a single replicate for each treatment group.
- Temperature of Medium: Measured continuously in a vessel next to the test vessels, over the study period, beginning at the start of the test. Moreover, temperature was recorded at every media change in fresh and old media, in a single replicate for each treatment group (during the pH, hardness and dissolved O2 determinations).
- Light intensity: Light intensity was measured once a week.
Test conditions were recorded with suitable instruments and documented in the raw data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.34
- Nominal concentrations: 0.5; 1.2; 2.7; 6.4; 15 mg.L-1
- Based on QSAR predictions and results of an acute toxicity test on D. magna, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the nominal concentrations
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits: n.d. - 3.0 mg test item/L
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
95% confidence limits: n.d. - 4.0 mg test item/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
95% confidence limits: 0.4 - 199.5 mg test item/L
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The effect of a chronic exposure to the test item on the reproduction of Daphnia magna was tested using five concentrations arranged in a geometric series, spaced by a factor of approximately 2.34

Please, see the tables in the next section for the detailed results.

As said previously, toxicity was based on the time-weighted mean concentrations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were determined by the computer program ToxRat

Validity criteria:

MortalityThe mortality of the parent animals did not exceed 20 % at the end of the test in the controls.

Offspring: The mean number of live offspring produced per parent animal surviving in the controls at the end of the test was ≥ 60.

Thus all validity criteria for the performance of the controls have been fulfilled in the present study.

The following tables shows the No. Live Offspring per Replicate and Day:

Controls

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

8

n.d.

n.d.

20

0

0

20

0

n.d.

n.d.

16

0

11

75

2

0

0

10

n.d.

n.d.

17

0

0

22

0

n.d.

n.d.

16

0

3

68

3

0

0

0

n.d.

n.d.

10

17

0

10

21

n.d.

n.d.

8

0

0

66

4

0

0

10

n.d.

n.d.

17

0

0

23

0

n.d.

n.d.

19

0

0

69

5

0

0

11

n.d.

n.d.

21

0

0

26

0

n.d.

n.d.

19

0

0

77

6

0

10

0

n.d.

n.d.

M

M

M

M

M

M

M

M

M

M

-

7

0

0

12

n.d.

n.d.

13

6

0

2

22

n.d.

n.d.

21

0

0

76

8

0

0

0

n.d.

n.d.

15

13

0

0

19

n.d.

n.d.

17

0

0

64

9

0

0

8

n.d.

n.d.

9

16

0

0

20

n.d.

n.d.

18

0

0

71

10

0

0

15

n.d.

n.d.

16

0

0

0

22

n.d.

n.d.

18

0

0

71

 

Mean Offspring Number:70.8

0.4 mg.L-1

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

9

n.d.

n.d.

18

0

0

22

0

n.d.

n.d.

20

0

19

88

2

0

0

9

n.d.

n.d.

17

0

0

2

17

n.d.

n.d.

22

0

0

67

3

0

0

11

n.d.

n.d.

17

0

0

23

0

n.d.

n.d.

19

0

0

70

4

0

0

0

n.d.

n.d.

11

13

0

0

21

n.d.

n.d.

18

0

0

63

5

0

0

0

n.d.

n.d.

6

7

0

0

12

n.d.

n.d.

13

0

0

38

6

0

0

14

n.d.

n.d.

17

0

0

23

0

n.d.

n.d.

17

0

0

71

7

0

0

0

n.d.

n.d.

9

16

0

0

17

n.d.

n.d.

14

0

0

56

8

0

0

0

n.d.

n.d.

15

16

0

0

19

n.d.

n.d.

14

0

0

64

9

0

0

0

n.d.

n.d.

11

17

0

0

25

n.d.

n.d.

17

0

0

70

10

0

0

0

n.d.

n.d.

28

0

0

0

17

n.d.

n.d.

16

0

1

62

 

Mean Offspring Number:64.9

1.1 mg.L-1

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

0

n.d.

n.d.

23

5

0

0

19

n.d.

n.d.

12

0

0

59

2

0

0

0

n.d.

n.d.

9

10

1

0

15

n.d.

n.d.

19

0

0

54

3

0

7

0

n.d.

n.d.

12

0

19

0

0

n.d.

n.d.

20

14

0

72

4

0

0

12

n.d.

n.d.

19

0

0

22

0

n.d.

n.d.

15

0

0

68

5

0

0

0

n.d.

n.d.

8

14

0

0

14

n.d.

n.d.

14

0

1

51

6

0

0

0

n.d.

n.d.

8

9

1

0

15

n.d.

n.d.

13

0

0

46

7

0

0

0

n.d.

n.d.

6

14

0

0

14

n.d.

n.d.

12

0

0

46

8

0

0

0

n.d.

n.d.

7

13

1

0

13

n.d.

n.d.

15

0

0

49

9

0

0

0

n.d.

n.d.

8

14

0

0

18

n.d.

n.d.

19

0

0

59

10

0

0

13

n.d.

n.d.

18

0

0

20

0

n.d.

n.d.

12

0

17

80

 

Mean Offspring Number:58.4

2.6 mg.L-1

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

10

n.d.

n.d.

14

1

0

0

18

n.d.

n.d.

18

0

0

61

2

0

0

0

n.d.

n.d.

9

12

0

0

17

n.d.

n.d.

15

0

0

53

3

0

0

0

n.d.

n.d.

12

10

0

0

13

n.d.

n.d.

10

M

M

-

4

0

0

0

n.d.

n.d.

10

10

0

0

13

n.d.

n.d.

11

M

M

-

5

0

0

0

n.d.

n.d.

22

0

0

0

18

n.d.

n.d.

13

0

0

53

6

0

0

0

n.d.

n.d.

21

5

0

0

19

n.d.

n.d.

12

0

0

57

7

0

0

0

n.d.

n.d.

8

14

0

0

17

n.d.

n.d.

0

9

0

48

8

0

0

0

n.d.

n.d.

11

13

1

0

15

n.d.

n.d.

9

0

0

49

9

0

0

0

n.d.

n.d.

8

12

0

0

17

n.d.

n.d.

0

12

0

49

10

M

M

M

M

M

M

M

M

M

M

M

M

M

M

M

-

 

Mean Offspring Number:52.9

6.0 mg.L-1

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

0

n.d.

n.d.

8

9

1

0

12

n.d.

n.d.

12

0

0

42

2

0

0

0

n.d.

n.d.

9

10

0

0

12

n.d.

n.d.

4

7

M

-

3

0

0

0

n.d.

n.d.

9

10

0

0

13

n.d.

n.d.

8

0

0

40

4

0

0

0

n.d.

n.d.

7

9

0

0

11

n.d.

n.d.

8

1

0

36

5

0

0

0

n.d.

n.d.

8

10

0

0

5

n.d.

n.d.

6

6

M

-

6

0

0

0

n.d.

n.d.

8

10

0

0

11

n.d.

n.d.

8

2

0

39

7

0

0

3

n.d.

n.d.

7

2

0

4

10

n.d.

n.d.

11

0

0

37

8

0

0

0

n.d.

n.d.

10

4

6

0

0

n.d.

n.d.

M

M

M

-

9

0

0

8

n.d.

n.d.

9

0

0

12

0

n.d.

n.d.

8

0

0

37

10

0

0

0

n.d.

n.d.

7

0

10

0

0

n.d.

n.d.

0

0

5

22

 

Mean Offspring Number:36.1

14.1mg.L-1

Day

Replicate

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Total

1

0

0

0

n.d.

n.d.

0

0

M

M

M

M

M

M

M

M

-

2

0

0

0

n.d.

n.d.

0

0

0

0

1

n.d.

n.d.

0

0

0

1

3

0

0

0

n.d.

n.d.

0

0

0

0

0

n.d.

n.d.

0

0

0

0

4

M

M

M

M

M

M

M

M

M

M

M

M

M

M

M

-

5

0

0

0

n.d.

n.d.

0

0

0

0

0

n.d.

n.d.

0

0

0

0

6

0

0

0

n.d.

n.d.

0

0

0

0

0

n.d.

n.d.

0

0

0

0

7

0

0

0

n.d.

n.d.

0

0

0

0

0

n.d.

n.d.

0

0

0

0

8

0

0

0

n.d.

n.d.

0

0

4

0

0

n.d.

n.d.

0

0

0

4

9

0

0

0

n.d.

n.d.

0

0

0

0

0

n.d.

n.d.

0

0

0

0

10

0

0

0

n.d.

n.d.

0

0

0

0

1

n.d.

n.d.

0

0

0

1

 

Mean Offspring Number:0.8

“M”: dead females

n.d.: not determined (weekend)

The next table shows the Parental Daphnia magna lengths at the end of the test after 21 days (mm)

Replicate

Time-Weighted Mean Concentration(mg test item.L-1)

Control

0.4

1.1

2.6

6.0

14.1

1

4.25

4.35

4.10

3.75

3.65

M

2

4.40

4.35

4.00

4.15

M

2.75

3

4.10

4.15

4.20

M

3.50

3.10

4

4.35

4.00

4.35

M

3.85

M

5

3.90

4.70

4.15

3.65

M

3.00

6

M

4.20

4.20

3.75

4.15

3.10

7

4.0

4.30

4.00

4.10

3.85

3.15

8

3.75

3.95

4.10

3.35

M

3.13

9

4.30

4.45

4.20

4.10

3.70

3.00

10

4.60

4.30

4.15

M

3.80

2.80

Validity criteria fulfilled:
yes
Conclusions:
The effects of a chronic exposure to test item NOPOL on the reproduction of Daphnia magna was investigated in this study. The evaluation was based on the time-weighted mean concentrations. Under the experimental conditions and after 21 days, the NOEC value estimated for the most sensitive endpoint cumulative number of offspring was 0.4 mg test item.L-1.
Executive summary:

A study was performed to assess the effects of a chronic exposure to test item NOPOL on the reproduction of the freshwater crustacean Daphnia magna. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 211, "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation No. 440/2008.

Based on the results of an acute toxicity test on D. magna (LPL Project D12 -002; 48h-EC50= 28.8 mg.L-1) and on QSAR predictions (iSafeRat: 21d-NOEC = 1.17 mg.L-1), daphnids were exposed to an aqueous solution of the test item at the required nominal test concentrations of 0.5, 1.2, 2.7, 6.4 and 15 mg test item.L-1and to an untreated control for a total period of 21 days in closed vessels. Each treatment group consisted of 10 replicates each of one Daphnia magna (one individual per replicate). The test media was renewed three times a week and the daphnids were fed five days a week. Effects on reproductive performance were investigated by counting the living offspring produced by each parent animal every day. Mortality among the parent animals was also recorded. The concentrations of the test item were determined by chemical analyses three times a week at renewal of the test media.

Although every effort was made to maintain the concentrations of the test item (renewal of the test solutions, closed conditions with minimum headspace), the loss of the substance was sometimes greater than 20% of the measured initial concentration. The losses were probably caused by adsorption and degradation of the test item by food algae. Since the deviation of the exposure concentrations of the test substance was greater than 20% of the initial concentrations, the results were expressed in terms of time-weighted mean concentrations for the present study: 0.4, 1.1, 2.6, 6.0 and 14.1 mg test item.L-1. The effect of the test item on the reproduction of Daphnia magna after 21 days of exposure was as follows:

Critical effect and threshold concentration on mean cumulative offspring per survivor after 21 days

(mg test item.L-1)

95% confidence limits

(mg test item.L-1)

EC10

1.3

n.d. – 3.0

EC20

2.1

n.d. – 4.0

EC50

4.8

0.4 – 199.5

LOEC

1.1

-

NOEC

0.4

-

The effects of a chronic exposure to test item NOPOL on the reproduction of Daphnia magna was investigated in this study. The evaluation was based on the time-weighted mean concentrations. Under the experimental conditions and after 21 days, the NOEC value estimated for the most sensitive endpoint cumulative number of offspring was 0.4 mg test item.L-1.

According to Regulation EC No. 1272/2008 and according to GHS criteria, the test item has to be classified in chronic category 2. Hazard statement H411 “Toxic to aquatic life with long lasting effects” is required.

Description of key information

A study was conducted according to OECD Guideline 211. After 21 days, the NOEC value estimated for the most sensitive endpoint cumulative number of offspring was 0.4 mg test item.L-1.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.4 mg/L

Additional information

A study was performed to assess the effects of a chronic exposure to test item NOPOL on the reproduction of the freshwater crustacean Daphnia magna. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 211, "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation No. 440/2008.

The effects of a chronic exposure to test item NOPOL on the reproduction ofDaphnia magnawas investigated in this study. The evaluation was based on the time-weighted mean concentrations. Under the experimental conditions and after 21 days, the NOEC value estimated for the most sensitive endpoint cumulative number of offspring was 0.4 mg test item.L-1.

According to Regulation EC No. 1272/2008 and according to GHS criteria, the test item has to be classified in chronic category 2. Hazard statement H411 “Toxic to aquatic life with long lasting effects” is required.