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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
Study period:
01 October 2019 - 02 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0 (control), 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L.
- Sampling method: sampling was performed at the beginning of the test (0 hour) on day 0 and at end of test (48 hours) on day 2 from all the test concentrations during main study. Test samples were collected in duplicates from each group and diluted in the corresponding quantity of methanol before being analyzed.
- Sample storage conditions before analysis: All samples were analyzed on the same day of collection from the respective test vessels.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was freshly prepared prior to exposure, by weighing 100 mg of test item in a beaker and diluted in 100 mL of reconstituted water. After complete soluble of test item by mixing well using a glass rod, the test item formulation was transferred into a measuring cylinder and the beaker was rinsed with reconstituted water and transferred again to the measuring cylinder. Rinsing process was repeated until the complete transfer of test contents. Stock solution prepared was kept on magnetic stirrer to maintain homogeneity.Test medium of the selected concentration was prepared by dilution of the stock solution.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): < 24 hours old
- Method of breeding: 25 gravid female daphinds were acclimatized in reconstituted water for 48 hours prior to test. Daphnids was fed with live algal cells at the beginning and on Day 1 of acclimatization. Progenies produced on the first day were not used. Progenies produced on day two (Less than 24 hours old) were transferred into the test containers for being used in the DRF and main studies.
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Dharwad, Karnataka, India.
- Feeding during test: NO

ACCLIMATION
- Acclimation period: 48 hours
- Acclimation conditions (same as test or not): same as test.
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata) of 2 mL per liter.
- Feeding frequency: not specified.
- Health during acclimation (any mortality observed): daphnids derived from healthy stock showing no signs of stress, such as high mortality rate, discolored daphnids, presence of male and ephippia or delay in the production of the first brood, were selected for the treatment.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
220 mg/L as CaCO3
Test temperature:
20.0 to 20.9ºC
pH:
7.08 to 7.38
Dissolved oxygen:
7.33 to 8.39 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: 0.0 (control), 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L.
Measured concentrations for 0.0, 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L (initial time, T0h): -, 0.60, 1.15, 2.20, 5.65, 11.26, 23.29 and 47.60 mg/L.
Measured concentrations for 0.0, 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L (final time, T48h): -, 0.62, 1.34, 2.54, 6.32, 12.88, 26.15 and 52.79 mg/L.

Details on test conditions:
TEST SYSTEM
- Test vessel: rectangular beaker
- Material, size, headspace, fill volume: glass,100 mL fill volume
- Volume of solution: 100 mL
- Aeration: The dilution water was aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.
- Renewal rate of test solution (frequency/flow rate): Not applicable (static test)
- 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: 20 mL of test media per daphnia.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted water as described in the OECD guideline 202.
- Intervals of water quality measurement: Total hardness of the diluent water (reconstituted water) prior to its use for exposure was analyzed. Temperature, pH and dissolved oxygen were measured at the start and at the end of the test (including Control).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light and 08 hours dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Each test vessel was observed for immobilized Daphnids at 24 and 48 hours after the beginning of the test. Daphnids not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition any abnormal behavior or appearance was recorded. As the test media was coloured violet, observation of daphnid was made against the white fluorescent light.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L along with control (semi-static conditons).
- Results used to determine the conditions for the definitive study: No immobility was observed in control and at the tested concentrations of 0.01, 0.1 and 1.0 mg/L during the 48 hours exposure period. At the tested concentrations of 10.0, 50.0 and 100.0 mg/L during the 48 hours exposure period, an immobilisation of 60, 100 and 100% respectively was observed. Based on these results, the main study was conducted at the concentrations of 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L (using a geometric factor of 2.1).





Reference substance (positive control):
yes
Remarks:
(potassium dichromate)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval = [7.58 – 10.15]
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
5.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2.57 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities:
- Mortality of control: Not observed.
- Immobilisation of control: 0%
- Abnormal responses: Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the last toxic response obtained on the reference item K2Cr2O7 (October 16, 2019) indicates that the daphnids sensitivity is correct (within the accepted interval of 0.6–2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD Guideline 202.
- Relevant effect levels: immobilization of 20, 35, 60 and 75% were observed at the tested concentrations of 0.31, 0.58, 1.11 and 2.10 mg/L.
- EC50-24 h: 0.88 mg/L.
Reported statistics and error estimates:
Data were analyzed by Finney’s probit analysis to calculate the slope of the curve and the EC50 with confidence limits. One away ANOVA was performed for 48 hours immobility for the determination of NOEC and LOEC.

Table 1: Physico-chemical parameters of test medium during main study

Group

Concentration (mg/L)

R

Dissolved Oxygen
 (mg/L)

Temperature (°C)

pH

0 h

48 h

0 h

48 h

0 h

48 h

G1

0

R1

8.11

7.98

20.6

20.8

7.11

7.08

R2

8.13

7.86

20.4

20.6

7.14

7.11

R3

8.19

7.81

20.1

20.0

7.16

7.10

R4

8.29

7.82

20.6

20.3

7.15

7.10

G2

0.58

R1

8.18

7.97

20.9

20.3

7.10

7.14

R2

8.24

7.83

20.6

20.1

7.14

7.26

R3

8.21

7.88

20.5

20.1

7.18

7.21

R4

8.09

7.84

20.1

20.4

7.13

7.28

G3

1.22

R1

8.26

7.81

20.3

20.4

7.11

7.10

R2

8.11

7.86

20.1

20.4

7.18

7.13

R3

8.26

7.33

20.4

20.5

7.17

7.12

R4

8.14

7.79

20.3

20.6

7.23

7.20

G4

2.57

R1

8.14

7.70

20.1

20.6

7.24

7.21

R2

8.03

7.81

20.1

20.3

7.26

7.22

R3

8.16

7.92

20.3

20.4

7.29

7.20

R4

8.27

7.98

20.1

20.1

7.21

7.23

G5

5.40

R1

8.11

7.88

20.4

20.9

7.23

7.18

R2

8.09

7.89

20.9

20.3

7.26

7.15

R3

8.14

7.94

20.2

20.6

7.24

7.24

R4

8.32

7.88

20.4

20.4

7.29

7.23

G6

11.34

R1

8.19

7.86

20.2

20.8

7.31

7.33

R2

8.01

7.80

20.3

20.8

7.30

7.35

R3

8.39

7.78

20.1

20.3

7.28

7.36

R4

8.37

7.91

20.8

20.1

7.26

7.32

G7

23.81

R1

8.31

7.92

20.9

20.4

7.21

7.30

R2

8.39

7.95

20.4

20.4

7.22

7.32

R3

8.26

7.84

20.9

20.1

7.25

7.33

R4

8.26

7.89

20.6

20.3

7.26

7.31

G8

50.0

R1

8.36

7.94

20.3

20.3

7.29

7.34

R2

8.38

7.86

20.6

20.6

7.38

7.36

R3

8.28

7.84

20.4

20.8

7.33

7.31

R4

8.22

7.88

20.3

20.4

7.34

7.38

 

Min

 

7.33

20.0

7.08

 

Max

 

8.39

20.9

7.38

Table 2: Physico-chemical parameters of test medium during dose range finding study

Group

Concentration (mg/L)

R

DO
 (mg/L)

Temperature (°C)

pH

 Day 1

Day 1

Day 1

Day 2

 Day 1

Day 1

Day 1

Day 2

 Day 1

Day 1

Day 1

Day 2

Fresh

Spent

Fresh

Spent

Fresh

Spent

Fresh

Spent

Fresh

Spent

Fresh

Spent

0h

24h

24h

48h

0h

24h

24h

48h

0h

24h

24h

48h

G1

0.0

R1

7.87

7.71

7.89

7.65

20.9

20.1

20.6

20.1

7.86

7.71

7.59

7.41

R2

7.85

7.62

7.82

7.67

21.2

20.4

20.5

20.1

7.85

7.70

7.62

7.56

G2

0.01

R1

7.85

7.50

7.81

7.60

20.7

20.2

20.5

20.1

7.57

7.58

7.51

7.45

R2

7.85

7.63

7.80

7.61

20.9

20.1

20.5

20.1

7.58

7.55

7.62

7.51

G3

0.1

R1

7.82

7.61

7.78

7.60

21.0

20.3

20.5

20.0

7.67

7.61

7.60

7.50

R2

7.84

7.65

7.84

7.58

20.9

20.2

20.6

20.1

7.51

7.46

7.65

7.52

G4

1.0

R1

7.80

7.60

7.75

7.54

20.8

20.1

20.4

20.1

7.51

7.49

7.54

7.48

R2

7.82

7.54

7.79

7.54

20.9

20.1

20.5

20.0

7.48

7.39

7.57

7.45

G5

10.0

R1

7.87

7.61

7.72

7.51

20.9

20.0

20.5

20.2

7.54

7.42

7.58

7.40

R2

7.81

7.66

7.73

7.56

20.9

20.2

20.6

20.2

7.56

7.45

7.60

7.39

G6

50.0

R1

7.75

7.55

7.70

7.48

21.0

20.1

20.7

20.1

7.50

7.41

7.61

7.54

R2

7.70

7.47

7.70

7.50

20.9

20.1

20.5

20.1

7.45

7.40

7.55

7.38

G7

100.0

R1

7.71

7.49

7.68

7.51

20.8

20.3

20.6

20.0

7.41

7.32

7.66

7.51

R2

7.70

7.45

7.65

7.49

21.0

20.2

20.4

20.2

7.41

7.34

7.57

7.55

MIN

7.45

20.0

7.32

       MAX

7.89

21.2

7.86

Table 3: Summary of clinical signs and immobility during dose range finding study

Group

Conc.
 (mg/L)

R

No. of Daphnids per replicate at start 0 h

Signs of toxicity and immobility of Daphnids Observed at

24 h

48 h

Toxic signs

I

Toxic signs

I

G1

0.0

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G2

0.01

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G3

0.10

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G4

1.0

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G5

10.0

R1

5

N(1), L(2)

2

N(1), L(1)

1

R2

5

N(2), B(1), L(1)

1

N(1), B(1)

2

G6

50.0

R1

5

B(2)

3

-

2

R2

5

B(2)

3

-

2

G7

100.0

R1

5

B(2)

3

-

2

R2

5

B(1)

4

-

1

I: Immobility; N: Normal; B: Bottom (Localized at bottom of container); L: Lethargy; Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 4: Summary of immobility at different time points of post exposure during dose range finding study

Group

Conc.
(mg/L)

R

Initial No. of Daphnids

Immobility (No.) of Daphnids

Cummulative No.

% of Immobility

24 h

48 h

24 h

48 h

24 h

48 h

G1

0

R1

5

0

0

0

0

0

0

R2

5

0

0

G2

0.01

R1

5

0

0

0

0

0

0

R2

5

0

0

G3

0.1

R1

5

0

0

0

0

0

0

R2

5

0

0

G4

1.0

R1

5

0

0

0

0

0

0

R2

5

0

0

G5

10.0

R1

5

2

1

3

6

30

60

R2

5

1

2

G6

50.0

R1

5

3

2

6

10

60

100

R2

5

3

2

G7

100.0

R1

5

3

2

7

10

70

100

R2

5

4

1

Table 5: Summary of clinical signs and immobility during main study

Group

Conc.
 (mg/L)

R

No. of Daphnids per replicate at start 0 h

Signs of toxicity and immobility of Daphnids Observed at

24 h

48 h

Toxic signs

I

Toxic signs

I

G1

0.0

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

R3

5

N(5)

0

N(5)

0

R4

5

N(5)

0

N(5)

0

G2

0.58

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

R3

5

N(5)

0

N(5)

0

R4

5

N(5)

0

N(5)

0

G3

1.22

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

R3

5

N(5)

0

N(5)

0

R4

5

N(5)

0

N(5)

0

G4

2.57

R1

5

N(5)

0

N(5)

0

R2

5

N(4), B(1)

0

N(3),B(1)

1

R3

5

N(5)

0

N(5)

0

R4

5

N(4), B(1)

0

N(3),B(1)

1

G5

5.40

R1

5

N(3), B(1)

1

N(2), B(1)

1

R2

5

N(3), B(1) L(1)

0

N(2), B(1) L(1)

1

R3

5

N(3), B(1)

1

N(3), B(1)

0

R4

5

N(2), B(1), L(1)

1

N(1), B(1)

2

G6

11.34

R1

5

N(1), B(1), L(1)

2

B(1)

2

R2

5

N(2), B(1), L(1)

1

N(1), L(1)

2

R3

5

N(1), B(2)

2

B(2)

1

R4

5

N(2), B(1), L(1)

1

B(1), L(1)

2

G7

23.81

R1

5

B(2), L(1)

2

-

3

R2

5

B(2), L(2)

1

B(2)

2

R3

5

B(1), L(2)

2

L(2)

1

R4

5

B(2), L(1)

2

-

3

G8

50.0

R1

5

B(2)

3

-

2

R2

5

L(1), B(2)

2

-

3

R3

5

L(2), B(1)

2

-

3

R4

5

B(2)

3

-

2

I: Immobility; N: Normal; B: Bottom (Localized at bottom of container); L: Lethargy; Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 6: Summary of immobility at different time points of post exposure during main study

Group

Conc.
(mg/L)

R

Initial No. of Daphnids

Immobility (No.) of Daphnids

Cummulative No.

% of Immobility

24 h

48 h

24 h

48 h

24 h

48 h

G1

0.0

R1

5

0

0

0

0

0

0

R2

5

0

0

R3

5

0

0

R4

5

0

0

G2

0.58

R1

5

0

0

0

0

0

0

R2

5

0

0

R3

5

0

0

R4

5

0

0

G3

1.22

R1

5

0

0

0

0

0

0

R2

5

0

0

R3

5

0

0

R4

5

0

0

G4

2.57

R1

5

0

0

0

2

0

10

R2

5

0

1

R3

5

0

0

R4

5

0

1

G5

5.40

R1

5

1

1

3

7

15

35

R2

5

0

1

R3

5

1

0

R4

5

1

2

G6

11.34

R1

5

2

2

6

13

30

65

R2

5

1

2

R3

5

2

1

R4

5

1

2

G7

23.81

R1

5

2

3

7

16

35

80

R2

5

1

2

R3

5

2

1

R4

5

2

3

G8

50.0

R1

5

3

2

10

20

50

100

R2

5

2

3

R3

5

2

3

R4

5

3

2

Table 7: Test concentration analysis in test media during main study

 

Day 0 (0 Hour)

Day 2 (48 Hours)

Group

Nominal Concentration

(mg/L)

Conc. Obtained

 (mg/L)

Recovery

%

Nominal Concentration

(mg/L)

Conc. Obtained

 (mg/L)

Recovery

%

G1

0.00

-

-

0.00

-

-

G2

0.58

0.60

102.59

0.58

0.62

106.90

G3

1.22

1.15

93.85

1.22

1.34

109.84

G4

2.57

2.20

85.61

2.57

2.54

98.64

G5

5.40

5.65

104.63

5.40

6.32

116.95

G6

11.34

11.26

99.30

11.34

12.88

113.54

G7

23.81

23.29

97.80

23.81

26.15

109.81

G8

50.0

47.60

95.19

50.0

52.79

105.58

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
In a short-term toxicity test to daphnia magna, the 48h-EC50 of the test substance was found to be 8.77 mg/L.


Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on test item according to OECD guideline 202, following GLP. In a preliminary range finding study the per cent immobilisation of daphnia was 0, 0, 0, 60, 100 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L for a period of 48 h in static conditions in 4 replicates of 5 daphnids per replicate. ISO reconstituted water was used as test medium. No vehicle was used since the test item was found soluble in test medium at the highest concentration tested. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. A validated analytical method based on LC-MS/MS was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered as stable in the testing conditions after 48 hours of exposure as the measured concentrations in samples were maintained within ± 20 per cent of the nominal concentrations. There was no immobilization of daphnia in the negative control and dissolved oxygen was higher than 3 mg/L in control and test vessels at the end of the test. The immobilization of daphnia was 0, 0, 0, 10, 35, 65, 80 and 100% at 48 hours exposure at 0 (control), 0.58, 1.22, 2.57, 5.40, 11.34, 23.81 and 50.0 mg/L. Based on these results, the 48h-EC50 value is 8.77 mg/L with 7.58 to 10.15 mg/L as 95% confidence interval. The NOEC and LOEC over the 48 hours exposure period were calculated to be 2.57 and 5.40 mg/L respectively based on statistical analysis results.

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EC50 of the test substance to Daphnia magna was found to be 8.77 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8.77 mg/L

Additional information