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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:
2020-02-11 to 2020-04-29
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)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods. However, from the two highest test concentrations of 50 and 100 mg/L, samples were taken only from the first renewal period, since all daphnids were dead at the observation time at Day 1.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by completely dissolving 100.38 and 100.43 mg of test item in 1000 mL of test water by intense stirring for 15 minutes at room temperature in the dark, on Day 0 and Day 1, respectively. This highest concentration (clear solution) was used to prepare the test medium of the test concentration of 50 mg/L. The test media of the lower test item concentrations were prepared in a series of further dilution steps.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Stage and instar at study initiation: 6-24 hours old and were not first brood progeny
- Method of breeding: bred in IES Ltd Laboratories under temperature and light conditions identical to those of the tests. The cultivation of the parental daphnids is performed in reconstituted water of the quality identical to the water quality used in the test (with respect to pH, main ions, and total hardness).
- Source: the Daphnia Collection of the University of Basel/Switzerland in 2015
- Feeding during test: no

ACCLIMATION
- Acclimation: Test animals were bred under similar conditions as that used during the exposure period, therefore, additional acclimatization before the test was not necessary.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
19 to 20 °C
pH:
7.8 - 7.9
Nominal and measured concentrations:
Nominal: Control, 6.25, 12.5, 25, 50 and 100 mg/L;
Mean Measured: n.a., 4.7, 8.0, 19 and 50 mg/L, n.a.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Type: loosely covered with glass sheets
- Material, size, fill volume: glass, 100 mL, 50 mL test medium
- Volume of solution: 50 mL
- Aeration: no
- Renewal rate of test solution: 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (ISO Test water) according to OECD Guideline No. 202

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle

EFFECT PARAMETERS: immobilsation after 24 and 48 hours of exposure

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
10 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 9.0 - 12 mg/L
Duration:
48 h
Dose descriptor:
other: EC0 / NOEC
Effect conc.:
4.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits: 12 - 15 mg/L
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: no
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses:
- Any observations that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: October 2019 (24-hour EC50: 1.8 mg/L (OECD 202: 24-hour EC50: 0.60-2.1 mg/L).
Reported statistics and error estimates:
The 24- and 48-hour EC50 and the 95 % confidence limits were calculated by Trimmed Spearman-Karber method for estimating median lethal concentrations, since the estimation based on the Probit or logit models was not be valid (p(F) > 0.05).
Statistical analysis was performed using ToxRat Professional®. The 24-hour and 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

Table 1: Effect of the test item on the Mobility of Daphnia magna

Nominal Test Item Concentration (mg/L)

Mean Measured Concentration (mg/L)

No. of Daphnids Tested

No. Of Daphnids/Replicate

Immobilized Daphnids after 24 Hours

Immobilized Daphnids after 48 Hours

No.

[%]

No.

[%]

Control

---

20

5

0

0

0

0

5

0

0

5

0

0

5

0

0

6.25

4.7

20

5

0

0

0

0

5

0

0

5

0

0

5

0

0

12.5

8.0

20

5

0 (2F)

0

0

25

5

0 (1F)

0

5

0 (2F)

0

5

0

0

25

19

20

5

3 (2F)

90

5

100

 

 

5

5

5

 

 

5

5

5

 

 

5

5

5

50

50

20

5

5

100

--

--

5

5

--

5

5

--

5

5

--

100

n.a.

20

5

5

100

--

-

5

5

--

5

5

--

5

5

--

n.a.: Not analyzed, since this concentration was above the 48-hour EC100.

--: All daphnids already dead after 24 hours exposure time.

Values in parenthesis correspond to the number of mobile test animals with adverse effects.

F: Reduced swimming activity compared to the control animals

 

Table 2: 24-hour and 48-hour ECx values

ECx Values

Immobility

Based on Mean Measured Test

Item Concentration

[mg/L]

24-hour EC50

95 %-confidence limits

14

12 - 15

24-hour EC0

8.0

24-hour EC100

50

48-hour EC50

95 %-confidence limits

10

9.0 – 12

48-hour EC0 and

48-hour NOEC

4.7

48-hour EC100

19

Analytical Results:

Table 3: Results for the Range-Finding Test

Sampling Day/Sample Age

[day/hours]

Nominal Concentration of Test Item Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

% from Initially Measured

[%]

0/0

(fresh)

Control

n.d.

126

< LOQ

n.a.

10

42.0

251

10.6

106

100

39.7

2651

105

105

2/48

(aged)

Control

n.d.

126

< LOQ

n.a.

10

23.6

251

5.92

59

10a

36.9

251

9.27

93

100

31.7

2651

84.2

84

LOQ: 0.130 mg test item /L (LOQ in range-finding test)

n.d. = not detected

n.a. = not applicable

a: without daphnia in the dark

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Table 4: Results for Spiked Samples

Nominal Concentration of Test Item

Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg]

Sample Preparation Factor

F

Determined Concentration of Test Item

C

[mg/L]

Recovery Rate

R

[%]

Accuracy (Average Recovery)

[%]

Precision (Relative Standard Deviation of Recovery)

[%]

0

n.d.

102

n.a.

< LOQ

n.a.

-

1.13

10.5

102

1.07

95

95

4.7

1.13

9.96

102

1.02

90

1.13

10.9

102

1.12

99

1.13

11.1

102

1.13

100

1.13

10.1

102

1.03

91

62.6

101

669

67.5

108

110

1.8

62.6

102

669

68.4

109

62.6

106

669

70.8

113

62.6

104

669

69.4

111

62.6

102

669

68.5

109

Acceptance Target:

80 - 120

≤ 10

LOQ: 0.218 mg test item /L

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Table 5: Results for Test Samples

Sampling Day/Sample Age

[day/hours]

Nominal Concentration of Test Item Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

% of Nominal Concentration

[%]

0/0

(fresh)

Control

n.d.

102

< LOQ

n.a.

6.25

63.6

102

6.49

104

12.5

66.1

202

13.3

107

25.50

62.4

402

25.1

100

50

78.0

669

52.1

104

1/24

(aged)

Control

n.d.

102

< LOQ

n.a.

6.25

46.8

102

4.77

76

12.5

44.5

202

8.98

72

25a

54.4

402

21.9

87

25

68.0

402

27.4

109

50

71.9

669

48.1

96

1/0

(fresh)

Control

n.d.

102

< LOQ

n.a.

6.25

64.6

102

6.59

105

12.5

63.0

202

12.7

102

25

62.2

402

25.0

100

2/24

(aged)

control

n.d.

102

< LOQ

n.a.

6.25

19.6/22.6b

102

2.15*

34

12.5

9.24/10.2b

202

1.96*

16

25

21.7/23.7b

402

9.12*

36

LOQ: 0.218 mg test item / L

a: without daphnia, with light

b: retain sample

n.d. = not detected

n.a. = not applicable

* mean value

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test performed according to OECD 202. The 48-hour EC50 was calculated to be 10 mg/L with 95 % confidence limits of 9.0 and 12 mg/L.
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. A semi-static test with test medium renewal after 24 hours was performed to maintain the concentration of the test item in the test medium as far as possible during the test period of 48 hours. Since the test item was instable in the range-finding test under the normal light conditions (16-hour light to 8-hour dark cycle) the main test was performed under reduced light conditions to avoid potential photolytic degradation of the test item.

At the start of the two renewal periods the measured concentrations in the test media of the nominal test concentrations of 6.25 to 50 mg/L were between 100 and 107 % of the nominal values. Thus, the correct dosage of the test item could be verified. During the test periods of 24 hours, the test item concentrations in the test media decreased. At the end of the two renewal periods, the test item concentrations were in the range of 72 to 96 % (Day 0 to Day 1) and 16 to 36 % (Day 1 to Day 2) of the nominal values, demonstrating a slightly to highly decrease of the test item concentrations during the 24-hour renewal periods. The reason for the degradation of the test item concentration mainly in the second renewal period could not be clarified. It can be assumed that an enhanced adsorption to the daphnids or an enhanced degradation by bacteria occurred, which were transferred with the surviving daphnids from the first renewal period.

The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the two geometric means which were determined from the test item concentrations measured at the start and end of each of the two test medium renewal periods. Due to the decrease of the test item concentrations during the 24-hour renewal periods, the biological results were based on the mean measured test item concentrations. All validity criteria were met. The 48-hour NOEC was calculated to be 4.7 mg/L and the 48-hour EC50 was calculated to be 10 mg/L with 95 % confidence limits of 9.0 and 12 mg/L.

Description of key information

The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test performed according to OECD 202.

The 48-hour EC50 was calculated to be 10 mg/L with 95 % confidence limits of 9.0 and 12 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10 mg/L

Additional information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. A semi-static test with test medium renewal after 24 hours was performed to maintain the concentration of the test item in the test medium as far as possible during the test period of 48 hours. Since the test item was instable in the range-finding test under the normal light conditions (16-hour light to 8-hour dark cycle) the main test was performed under reduced light conditions to avoid potential photolytic degradation of the test item.

At the start of the two renewal periods the measured concentrations in the test media of the nominal test concentrations of 6.25 to 50 mg/L were between 100 and 107 % of the nominal values. Thus, the correct dosage of the test item could be verified. During the test periods of 24 hours, the test item concentrations in the test media decreased. At the end of the two renewal periods, the test item concentrations were in the range of 72 to 96 % (Day 0 to Day 1) and 16 to 36 % (Day 1 to Day 2) of the nominal values, demonstrating a slightly to highly decrease of the test item concentrations during the 24-hour renewal periods. The reason for the degradation of the test item concentration mainly in the second renewal period could not be clarified. It can be assumed that an enhanced adsorption to the daphnids or an enhanced degradation by bacteria occurred, which were transferred with the surviving daphnids from the first renewal period.

The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the two geometric means which were determined from the test item concentrations measured at the start and end of each of the two test medium renewal periods. Due to the decrease of the test item concentrations during the 24-hour renewal periods, the biological results were based on the mean measured test item concentrations. All validity criteria were met. The 48-hour NOEC was calculated to be 4.7 mg/L and the 48-hour EC50 was calculated to be 10 mg/L with 95 % confidence limits of 9.0 and 12 mg/L.

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