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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:
07 February 2019 to 20 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines (OECD 211) and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
02 October 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 11, 33, 100, 300 and 900 µg/L & negative control
- Sampling method: Required volume of composite sample from all the test concentrations were collected on day of sampling and samples were sent to analytical team for analysis. In all cases, analyses were made on a sample from the same solution from all the groups, when freshly prepared and at renewal.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Serial dilution
- Eluate: Test Medium
- Differential loading: N/A
- Controls: Medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
- Other relevant information:
Test item stock of 6 mg/L was prepared by mixing 12 mg of test item in test medium and making up the final volume to 2000 mL using test medium.
Test medium (1 L) was used as negative control . To prepare test concentrations of 11, 33, 100, 300 and 900 µg/L, 1.834, 5.500, 16.667, 50 and 150 mL of test item stock was added and volume was made up to 1 L in a volumetric flask using test medium separately.
Based on the requirement, to prepare test concentrations of 11, 33, 100, 300 and 900 µg/L, 7.334, 22, 66.667, 200 and 600 mL of test item stock was added and volume was made up to 4 L in a volumetric flask using test medium separately. Test medium (4 L) was used as negative control.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: Ecotoxicology, Eurofins Advinus Limited, Bengaluru, India
- Maintenance: Daphnia were maintained in dilution medium (similar to one which was used in the test for acclimatization for at least three weeks as well as preparation of test medium). The test medium in which the Daphnia were maintained was changed at least twice in a week and they are fed with the alga, Pseudokirchneriella subcapitata.
- Neonates: At the start of the test, the animals were less than 24 hours old and were not first brood progeny. They were derived from a healthy stock (i.e., showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). The stock animals were maintained in culture conditions (light, temperature, medium, feeding and animals per unit volume) similar to those used in this test.
Adult gravid daphnids (which are already given their broods) were incubated individually in 100 mL of test medium, a day prior to start of the treatment. Progenies produced were observed (on the day of treatment) for neonate sex and after ascertaining the exclusive production of female progenies then the treatment was planned. Female daphnids aged less than 24 hours were used in the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
Guideline standard exposure duration
Hardness:
250 mg/L as CaCO3
Test temperature:
The test medium temperature was between 19.5 °C and 19.9 °C during the definitive test.
pH:
The pH was within the range 7.35 - 8.03 during the definitive test.
Nominal and measured concentrations:
Nominal Concentrations: 0.5, 5, 50, 100, 500 and 1000 µg/L
Measured Concentrations: 5, 11, 34, 97 and 331 µg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Beaksers
- Material, size, headspace, fill volume: 150 mL capacity, 100 mL test medium per replicate
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A - semi-static
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium used in the test was Selenium medium with selenium and vitamin supplements (Selenium medium). The test medium was prepared using analytical or better grade reagents as per the following composition and procedure:
CaCl2.2H20: 293.8 g/L stolck - 1 mL/L of stock for medium preparation
MgSO4.7H20: 246.6 g/L stock - 0.5 mL/L of stock for medium preparation
NaHCO3: 64.8 g/L stock - 1 mL/L of stock for medium preparation
KCl: 58 g/L stock - 0.1 mL/L of stock for medium preparation
Na2SeO3: 0.0438 g/L stock - 0.05 mL/L of stock for medium preparation
Thiamine hydrochloride: 0.0750 g/L stock, Cyanocobalamine (B12): 0.010 g/L stock, Biotine: 0.0075 g/L stock (combined) - 0.1 mL/L of stock for medium preparation
Water (reverse osmosis water) was used for preparation of stock solutions and test medium. For preparation of stock solutions, each chemical was dissolved separately (except for Vitamin stock solution) in water and made up to 1 L. To prepare the Selenium medium, the stock solutions was mixed (as per the above composition) in approximately 800 mL water and made up the volume to 1 L. Test medium was aerated using aerators until the dissolved oxygen concentration equaled the air saturation value. The prepared test medium was used within 10 days from the date of preparation.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The dissolved oxygen concentration, temperature, pH and hardness were measured once a week in fresh and old media in the control, lowest and highest test item concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light: 8h dark
- Light intensity: 1307 - 1366 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality among the parent animals was recorded daily (± 2 h from treatment start on day 0 exposure) along with the behaviour (n: parent animal normal and b: parent animal abnormal). An animal was recorded as dead (mortality) when it is immobile, i.e. when it was not able to swim, or if there was no observed movement of appendages or post-abdomen within 15 seconds after gentle agitation of the test container.
The offspring produced by each parent animal were preferably removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the parent. The numbers of living offspring were counted but the presence of aborted eggs and dead offspring were recorded. Offspring produced were observed for presence of male neonates (neonate sex) and ephippia.
The measurement of the length of the parent animals (i.e., body length excluding the anal spine) at the end of the test was measured. Time to production of first brood (and subsequent broods), number and size of broods per parent animal, number of aborted broods, and the intrinsic rate of population increase were also evaluated.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0.5, 5, 50, 100, 500 and 1000 µg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
23.14 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95 % CI: 9.23 - 54.52 µg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 11 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 34 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 34 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.

The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively. Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.

Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.

Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).

Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.
Reported statistics and error estimates:
The estimation of EC50 values was not possible, since there was partial (20 and 50 %) mortality of parent animals observed at the tested concentrations.
The statistical analysis of the body length of the parent animals and juvenile production data was carried out using licensed copies of SYSTAT Statistical package version 12.0. The body length of the parent animals and offspring produced by each group was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Significant difference was tested using Dunnett’s test.
Data (total offspring in each group) was square root transformed prior to EC evaluation, since it’s a count data and follows Poisson distribution. The EC10, EC20, EC50 and EC90 value with 95% fiducial limits, and the equation for the dose-response curve was determined by Probit analysis by the method of Finney (1971) using in-house developed and validated computer programme.
Analysis of the number of living offspring per surviving parent animal, as per the OECD 211 Guideline, was not conducted as the data for total offspring in each group produced the most conservative results for hazard assessment.

 Definitive Test - Parent Animal Mortality

Group

Measured Conc. (µg/L)

No. of parent animal at start of test (1 per replicate)

No. of parent animal mortality on Day 21

% Mortality of parent animal on Day 21

G1

Negative control

10

0

0

G2

5

10

0

0

G3

11

10

0

0

G4

34

10

0

0

G5

97

10

2

20

G6

331

10

5

50

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

 Definitive Test - Offspring Production

Group

Measured Conc. (µg/L)

Offspring produced in a group

Total living offspring produced in a group

No. of live parent animals on day 21

No. of living offspring produced per live parent animal

No. of offspring produced in a group

% Reduction in offspring production

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA

10

Mean

SD

G1

 

Negative control

78

84

80

82

87

86

82

86

88

89

842

10

84.2

84.2

3.6

-

G2

5

85

80

82

78

83

78

82

93

87

92

840

10

84.0

84.0

5.3

-0.2

G3

11

78

77

80

83

87

77

80

84

85

88

819

10

81.9

81.9

4.1

-2.7

G4

34

53

61

48

58

48

41

45

48

39

46

487*

10

48.7

48.7

6.9

-42.2

G5

97

40

41

18

29

33

15

49

35

34

38

332*

8

37.4

33.2

10.3

-60.6

G6

331

3

4

0

0

0

0

0

0

0

0

7$

5

1.4

0.7

1.5

-99.2

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The coefficient of variation for control reproductive output is 4.3% (based on total number of living offspring per parent animal alive at the end of the test). *: Statistically significantly (p ≤ 0.05) lower than the respective control group.$:Statistical evaluation not undertaken due to less sample size.

Definitive Test - Broods Production

Group

Measured Conc. (µg/L)

Average

No. of broods

Time to production of first brood and subsequent broods per parent animal

Time between brood production per parent animal

Size of broods per parent animal

First

Second

Third

Fourth

1 - 2

2 - 3

3 - 4

First

Second

Third

Fourth

G1

 

Negative control

4

8.3

12.5

16.6

20.6

4.2

4.1

4

7.6

20.5

26.0

30.1

G2

5

4

8.5

12.5

16.6

20.6

4

4.1

4

8

19.9

25.4

30.7

G3

11

4

8.5

12.5

16.7

20.8

4

4.2

4.1

7.4

19.8

23.4

31.3

G4

34

2.8

12.1

16.5

20.6

No brood

4.4

4.4

No brood

21.8

15.1

14.8

No brood

G5

97

2.6

12.3

16.8

20.5

No brood

4.4

3.8

No brood

14.7

12.6

10.3

No brood

G6

331

0.4

17

21

No brood

No brood

4

No brood

No brood

1.5

2.0

No brood

No brood

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

 Definitive Test - Male Offspring Production and Intrinsic Rate of Population Increase

Group

Measured Conc. (µg/L)

Male offspring produced in a group

Total living offspring produced in a group

Total male offspring produced in a group

No. of male offspring produced in a group

% Male offspring produced in a group

Intrinsic Rate of Population Increase (r)

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

Mean

SD

G1

 

Negative control

2

-

1

-

-

-

-

1

-

-

842

4

0.4

0.6

0.5

0.3255

G2

5

1

1

1

-

-

1

-

2

1

1

840

8

0.8

0.4

1.0

0.3241

G3

11

-

2

1

-

1

-

1

1

3

1

819

10

1.0

0.8

1.2

0.3184

G4

34

37

47

34

40

29

41

32

31

39

39

487

369

36.9

5.4

75.8

0.1191

G5

97

23

30

18

29

25

15

40

22

30

28

332

260

26.0

7.1

78.3

0.0935

G6

331

3

4

-

-

-

-

-

-

-

-

7

7

0.7

1.5

100

#

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L. #: Not evaluated due to absence of healthy offspring in this group.

 Definitive Test - Length of the Parent Animal

Replicate No.

Length of parent animal (mm)

Group & Measured Concentration (µg/L)

G1 & 0

G2& 5

G3& 11

G4& 34

G5 & 97

G6 & 331

1

4.29

4.34

4.31

4.16

3.61

2.67

2

4.36

4.36

4.23

4.20

3.79

2.74

3

4.42

4.37

4.28

4.27

-

2.11

4

4.34

4.26

4.37

4.16

3.89

-

5

4.38

4.39

4.39

4.07

3.91

-

6

4.41

4.36

4.19

4.21

-

2.01

7

4.44

4.31

4.28

4.25

3.81

-

8

4.35

4.38

4.36

4.08

3.72

-

9

4.36

4.33

4.39

4.18

3.95

2.13

10

4.31

4.38

4.33

4.19

3.84

-

Mean

4.37

4.35

4.31

4.18

3.82*

2.33*

SD

0.05

0.04

0.07

0.06

0.11

0.34

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

*: Statistically significantly (p ≤ 0.05) lower than the respective control group.

 Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 02 April 2019 (0 h sample) - Date of analysis: 02 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

5.67530

5.44041

±

0.352

6.5

51.59

49.46

±

3.20

6.5

G2R1-2

5.03552

45.78

G2R1-3

5.61043

51.00

33

G3R1-1

5.96011

6.79191

±

0.955

14

18.06

20.58

±

2.90

14

G3R1-2

6.58016

19.94

G3R1-3

7.83546

23.74

100

G4R1-1

19.5194

22.7458

±

2.83

12

19.52

22.75

±

2.83

12

G4R1-2

23.8937

23.89

G4R1-3

24.8243

24.82

300

G5R1-1

58.8109

68.1128

±

9.42

14

19.60

22.70

±

3.14

14

G5R1-2

67.8758

22.63

G5R1-3

77.6517

25.88

900

G6R1-1

399.1496

483.763

±

79.2

16

44.35

53.75

±

8.80

16

G6R1-2

496.0824

55.12

G6R1-3

556.0570

61.78

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples

Date of receipt: 03 April 2019 (24 h sample) - Date of analysis: 03 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

4.44827

5.71351

±

1.11

19

40.44

51.94

±

10.1

19

G2R1-2

6.16374

56.03

G2R1-3

6.52851

59.35

33

G3R1-1

3.59673

5.51061

±

2.04

37

10.90

16.70

±

6.19

37

G3R1-2

7.66347

23.22

G3R1-3

5.27162

15.97

100

G4R1-1

9.0020

9.9615

±

0.912

9.2

9.002

9.96

±

0.912

9.2

G4R1-2

10.0661

10.07

G4R1-3

10.8163

10.82

300

G5R1-1

47.7794

41.8375

±

7.71

18

15.93

13.95

±

2.57

18

G5R1-2

33.1316

11.04

G5R1-3

44.6016

14.87

900

G6R1-1

99.2060

119.689

±

23.2

19

11.02

13.30

±

2.58

19

G6R1-2

144.916

16.10

G6R1-3

114.943

12.77

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

 

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 04 April 2019 (0 h sample) - Date of analysis: 04 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

3.01164

3.21714

±

0.223

6.9

27.38

29.25

±

2.03

6.9

G2R1-2

3.18527

28.96

G2R1-3

3.45450

31.40

33

G3R1-1

8.19345

7.75416

±

0.745

10

24.83

23.50

±

2.26

10

G3R1-2

6.89394

20.89

G3R1-3

8.17508

24.77

100

G4R1-1

22.8310

21.5255

±

2.48

12

22.83

21.53

±

2.48

12

G4R1-2

23.0777

23.08

G4R1-3

18.6678

18.67

300

G5R1-1

56.6449

57.8544

±

3.76

6.5

18.88

19.28

±

1.25

6.5

G5R1-2

62.0682

20.69

G5R1-3

54.8500

18.28

900

G6R1-1

189.339

219.205

±

26.5

12

21.04

24.36

±

2.95

12

G6R1-2

239.993

26.67

G6R1-3

228.284

25.36

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples

Date of receipt: 05 April 2019 (24 h sample) - Date of analysis: 05 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

0.562052

0.569688

±

0.0334

5.9

5.110

5.179

±

0.304

5.9

G2R1-2

0.540781

4.916

G2R1-3

0.606231

5.511

33

G3R1-1

0.353196

0.408089

±

0.0480

12

1.070

1.237

±

0.150

12

G3R1-2

0.429035

1.300

G3R1-3

0.442036

1.340

100

G4R1-1

1.99137

1.72117

±

0.238

14

1.991

1.721

±

0.238

14

G4R1-2

1.54459

1.545

G4R1-3

1.62756

1.628

300

G5R1-1

3.74099

3.86265

±

0.647

17

1.247

1.288

±

0.220

17

G5R1-2

4.56218

1.521

G5R1-3

3.28477

1.095

900

G6R1-1

61.67154

53.82243

±

8.901

17

6.852

5.980

±

0.989

17

G6R1-2

44.15122

4.906

G6R1-3

55.64455

6.183

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 09 April 2019 (0 h sample) - Date of analysis: 09 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

9.84758

9.83686

±

0.457

4.6

89.52

89.43

±

4.16

4.6

G2R1-2

9.37445

85.22

G2R1-3

10.2885

93.53

33

G3R1-1

30.6872

29.3491

±

1.17

4.0

92.99

88.94

±

3.54

4.0

G3R1-2

28.5317

86.46

G3R1-3

28.8284

87.36

100

G4R1-1

98.7444

92.5732

±

6.90

7.5

98.74

92.57

±

6.90

7.5

G4R1-2

93.8492

93.85

G4R1-3

85.1258

85.13

300

G5R1-1

222.996

251.574

±

25.1

10

74.33

83.86

±

8.37

10

G5R1-2

270.054

90.02

G5R1-3

261.672

87.22

900

G6R1-1

803.163

840.155

±

32.5

3.9

89.24

93.35

±

3.61

3.9

G6R1-2

853.463

94.83

G6R1-3

863.841

95.98

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples           

Date of receipt: 10 April 2019 (24 h sample) - Date of analysis: 10 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

4.66025

4.28493

±

0.550

13

42.37

38.95

±

5.00

13

G2R1-2

3.65314

33.21

G2R1-3

4.54139

41.29

33

G3R1-1

12.4176

14.0324

±

2.10

15

37.63

42.52

±

6.36

15

G3R1-2

13.2742

40.22

G3R1-3

16.4053

49.71

100

G4R1-1

41.8545

40.9656

±

1.01

2.5

41.85

40.97

±

1.01

2.5

G4R1-2

41.1685

41.17

G4R1-3

39.8739

39.87

300

G5R1-1

102.035

112.839

±

10.7

9.5

34.01

37.61

±

3.57

9.5

G5R1-2

113.013

37.67

G5R1-3

123.470

41.16

900

G6R1-1

309.525

370.765

±

62.2

17

34.39

41.20

±

6.91

17

G6R1-2

368.839

40.98

G6R1-3

433.931

48.21

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion: There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 16 April 2019 (0 h sample) - Date of analysis: 17 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

10.3359

9.69719

±

0.685

7.1

93.96

88.16

±

6.23

7.1

G2R1-2

8.97405

81.58

G2R1-3

9.78162

88.92

33

G3R1-1

30.6844

32.1495

±

3.98

12

92.98

97.42

±

12.1

12

G3R1-2

29.1063

88.20

G3R1-3

36.6577

111.1

100

G4R1-1

86.6861

83.9817

±

3.61

4.3

86.69

83.98

±

3.61

4.3

G4R1-2

85.3718

85.37

G4R1-3

79.8873

79.89

300

G5R1-1

263.052

268.811

±

16.0

6.0

87.68

89.60

±

5.34

6.0

G5R1-2

256.465

85.49

G5R1-3

286.916

95.64

900

G6R1-1

775.845

760.511

±

31.0

4.1

86.21

84.50

±

3.45

4.1

G6R1-2

724.793

80.53

G6R1-3

780.893

86.77

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples           

Date of receipt: 17 April 2019 (24 h sample) - Date of analysis: 17 April 2019

Dose (µg/L)

Sample

As a Test Item

% RSD

% Recovery

Mean ± SD

% RSD

Test Item Conc. (µg/L)

Mean ± SD

11

G2R1-1

2.43400

2.80298

±

0.354

13

22.13

25.48

±

3.22

13

G2R1-2

2.83417

25.77

G2R1-3

3.14076

28.55

33

G3R1-1

9.90250

9.80214

±

0.409

4.2

30.01

29.70

±

1.24

4.2

G3R1-2

10.15169

30.76

G3R1-3

9.35223

28.34

100

G4R1-1

32.5757

32.4508

±

4.99

15

32.58

32.45

±

4.99

15

G4R1-2

27.4003

27.40

G4R1-3

37.3763

37.38

300

G5R1-1

81.8398

86.8849

±

6.93

8.0

27.28

28.96

±

2.31

8.0

G5R1-2

84.0283

28.01

G5R1-3

94.7866

31.60

900

G6R1-1

252.120

243.533

±

15.6

6.4

28.01

27.06

±

1.73

6.4

G6R1-2

225.542

25.06

G6R1-3

252.936

28.10

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Validity criteria fulfilled:
yes
Conclusions:
The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.
The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 11 µg/L.
The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.
The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.
The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.
The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.
The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.
Executive summary:

The reproductive effects of the test item, Amyl Cinnamic Aldehyde were studied on the reproductive output of daphnids, Daphnia magna. This test was conducted as a semi-static test with renewal of test medium at every 24 h interval. Mortality, behaviour and offspring produced (neonate sex) of the parent animals were observed daily during the 21 day test.

A range finding test was carried out to determine the test concentrations for the definitive test. Female, Daphnia magna aged less than 24 hours old were exposed to the nominal test item concentrations of 0.5, 5, 50, 100, 500 and 1000 µg/L along with a negative control in test medium (selenium medium). There was no mortality of parent animals in the negative control and any of the test item treated groups. The percent change in reproductive output of parent animals was 1, 3, 1, -41, -52 and -96 % at the tested concentrations of 0.5, 5, 50, 100, 500 and 1000 µg/L, respectively.

In definitive test, Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 11, 33, 100, 300 and 900 µg/L along with a negative control in test medium.

The stability test results concluded that the test item was not stable in the selenium medium after 24 h in the claimed concentrations of 0.5167 and

1035.4 µg/L.

The results of active ingredient analysis of test concentrations of 11, 33, 100, 300 and 900 µg/L showed that overall percent agreement was as below:

Sample

Mean Minimum (%)

Mean Maximum (%)

0 h Fresh

14.46

101.9

24 h Aged

0.9701

51.95

Since the deviation from the measured initial concentration was greater than ± 20%. The results were expressed in terms of the geometric mean concentration. Geometric mean test concentrations are 5, 11, 34, 97 and 331 µg/L (respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L).

There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.

The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively. Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.

Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.

Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).

Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The effects of Amyl Cinnamic Aldehyde on Daphnia magna reproduction are as below:

Reproduction Endpoints

Test Item

(µg/L)

95% Fiducial Limits (µg/L)

Lower

Upper

NOEC

> 11

-

-

LOEC

34

-

-

EC10

23.14

9.23

54.52

EC20

38.25

15.26

90.13

EC50

100.04

39.91

235.74

EC90

432.54

172.56

1019.23

Regression Equation

(Y = A + BX)

Y = 0.97 + 2.02 X

The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.

The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 11 µg/L.

The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.

The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.

The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.

The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

Description of key information

EC10 (reproduction) = 23.14 µg/L; OECD 211; Halappa, 2019

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
23.14 µg/L

Additional information

OECD 211 (Halappa, 2019)

The reproductive effects of the test item to Daphnia magna were assessed according to OECD Guideline 211 (2012).

Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 11, 33, 100, 300 and 900 µg/L along with a negative control in test medium.  The stability test results concluded that the test item was not stable in the selenium medium and a semi-static exposure scenario was used (24 h renewal). Since the deviation from the measured initial concentration was greater than ± 20%, results were expressed in terms of the geometric mean concentration. Geometric mean test concentrations are 5, 11, 34, 97 and 331 µg/L (respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L).

There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.  The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.  Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.  Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.  The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.  Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.  Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).  Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.

The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 11 µg/L.

The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.

The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 34 µg/L.

The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 34 µg/L.

The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.