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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 203 (Jul-1992) guideline without deviation. The substance was a multi-constiuent of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Principles of method if other than guideline:
- Principle of test: Water accomOdated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- Short description of test conditions: Each concentration is prepared sepArately, whereby the test item is added to the medium at a specific loading rate. The material is left until fully dissolved (or as dissolved as possible), then particulate matter is left to settle out of the water column. The remaining substance in the water column of the sample is transferred to the test vessels. This is done at each concentration. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Tyndall's effect is monitored to ensure minimal particulate matter is transferred to the test system. Further, chemical analysis is conducted to confirm that the correct loading has occurred, presence of the test item and to confirm there is a series of varying concentrations.
- although the study was conducted in accordance with the OECD guideline 203 (Fish acute toxicity test, 1992) as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  However, during study performance a deviation to the study design was noted. Here only the 100 mg/L test loading rate was prepared in accordance with the WAF strategy. All other concentrations were made by serial dilution, even though weighing will have been possible. As no effects were seen at the limit dose which was not prepared by serial dilution, i.e. in accordance with OECD series on testing and assessment no. 23, the study is considered valid and the deviation has not impacted the integrity of the study as a limit test.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: N/D

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The test was conducted with a semi-static design with renewal of test medium every 24 hours. All tested concentrations were analysed at 0 fresh, 24 hours fresh and 48 hours aged. All concentrations were tested these were 100, 45.5, 20.7, 9.39, 4.27 mg/L and the control (0 mg/L test item).
- Sampling method: direct from vessel using pippette
- Sample storage conditions before analysis: All samples were stored deep frozen and stabilised with formic acid until they were transferred to the analytical laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accomadated fractions were used as per the OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002). Each concentration was separately prepared. The prerequisite amount of test item was added to the test vessel containing the test medium 1000 mL of test medium for the concentrations of 100, 45.5, 20.7, 9.39 and 4.27 mg/L stock loading rates. The stocks were then stirred for 24 h in the dark at room temperature. The stock solutions were then allowed to settle to allow undissolved test item to sediment or float to the surface for 1 hour. After this the necessary volumes for the test were withdrawn via a Teflon tube from the middle level of the stock solution. Daphnia were then added to each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel. See table 1 for details on test preparations.
- Eluate: N/A
- Differential loading: N/A
- Controls: Elendt M4 medium only (OECD 202, 2004)
- 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 evidence of particulate in the test system. This was analysed using eye and Tyndall's effect.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna Straus, Clone V, was used as the test organism. The animals are continuously bred in the laboratory and were originally purchased in a healthy condition from the Federal Environment Agency in Berlin/Germany.
Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pH-value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.
The daphnids were reared at a temperature of 20 ± 2 °C in a climatic chamber with 16 hours of illumination and 8 hours of darkness. The medium was changed three times per week. A pipette was used to separate the young daphnids from the adults.
Freshly hatched daphnids less than 24 hours old were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Guideline and regulatory recommendation for acute immobilisation test on Daphnia magna
Post exposure observation period:
None
Hardness:
13°dH (232 mg/L as CaCO3)
Test temperature:
19.0 – 19.8 °C
pH:
7.41 – 10.05
Dissolved oxygen:
=>8.8 mg/L
Salinity:
N/A - freshwater
Conductivity:
N/D
Nominal and measured concentrations:
100, 45.5, 20.7, 9.39, 4.27 mg/L, and control.

Test was performed using loading rates and it was only possible to measure one analyte, therefore measured concnetrations of the entire susbtance were not possible. For the measured analyte the measured concentrations were between 83 to 102 %.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with 50 mL medium or medium/test item
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): No
- Renewal rate of test solution (frequency/flow rate): After 0 hours, every 24 hours
- 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): N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium made in line with OECD guideline 202.
- Total organic carbon: N/A
- Particulate matter: No
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/D
- Ca/mg ratio: N/D
- Conductivity: N/D
- Salinity: N/A
- Culture medium different from test medium: No
- Intervals of water quality measurement: Every 24 hours on fresh and aged test preparations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hour light/8 hours dark
- Light intensity: N/D

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EL50 effective loading rate 50, the test item load to the system which causes 50 % mortality of tyhe tested population. The NOELR, the no observed effect loading rate.

VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 20 mg/L
- Results used to determine the conditions for the definitive study: yes, no mortality was observed at 0-1 mg/L, 25 % mortality was seen at 20 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
53.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
20.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
45.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 24 hours of exposure no immobilisation was observed in the control and at 4.27 and 20.7 mg/L. No immobilisation higher than the allowed control immobilisation (i.e. < 10 %) was observed at 9.39 and 45.5.mg/L. 35 % immobilisation was observed at the highest test item loading rate of 100 mg/L.

After 48 hours of exposure no immobilisation was observed in the control and 20.7 mg/L. No immobilisation higher than the allowed control immobilisation (i.e. < 10 %) was observed at 4.27 mg/L. 15 % immobilisation was observed at 9.39 and 45.5 mg/L. At the highest test item loading rate of 100 mg/L all daphnids were immobile.

- Behavioural abnormalities: After 24 hours the mobile daphnids at test item loading rates 4.27 to 100 mg/L were swimming at the water surface. Daphnids at the highest test item loading rate of 100 mg/L showed jerky swimming movements.
After 48 hours the mobile daphnids at test item loading rates 4.27 to 45.5 mg/L were swimming at the water surface.
- Observations on body length and weight: N/D
- Other biological observations:None
- Mortality/immobility of control: None
- Other adverse effects control:
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Furthermore, the data on the measured analyte showed sufficient stability in the test system and showed suitable dosing at each concentration.
Results with reference substance (positive control):
The results with potassium dichromate indicate an EC50 (24 h) of the reference item potassium dichromate between 1.00 and 2.00 mg/L. Since the results are in accordance with the requirements of the OECD guideline 202 (EC50 = 0.6 – 2.1 mg/L)and fall within the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.
Reported statistics and error estimates:
The values for EL50 (48 h) was determined by Weibull analysis using linear max. likelihood regression. The NOELR was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤ 10 % immobilisation).

The EL50 at 48 hours = 53.2 (95 % confidence limit 45.4 - 64.0 mg/L)

Table 2 Immobility of Daphnia magna at 24 hours

 

Nominal test item loading rate [mg/L]

 

Control

4.27

9.39

20.7

45.5

100

 

Immobilised daphnids after 24 h

Replicate 1

0

0

0

0

1

1

Replicate 2

0

0

0

0

0

2

Replicate 3

0

0

1

0

0

1

Replicate 4

0

0

0

0

0

3

Σ

0

0

1

0

1

7

%

0

0

5

0

5

35

Table 3 Immobility of Daphnia magna at 48 hours

 

Nominal test item loading rate [mg/L]

 

Control

4.27

9.39

20.7

45.5

100

 

Immobilised daphnids after 48 h

Replicate 1

0

0

0

0

1

5

Replicate 2

0

1

1

0

0

5

Replicate 3

0

0

0

0

1

5

Replicate 4

0

0

2

0

1

5

Σ

0

1

3

0

3

20

%

0

5

15

0

15

100

Table 4 Temperature of the test system

 

nominal test item loading rate [mg/L]

 

Control

4.27

9.39

20.7

45.5

100

Time [h]

Temperature [°C]

0 fresh

19.1

19.2

19.1

19.1

19.1

19.0

24 aged

19.6

19.7

19.7

19.7

19.8

19.7

24 fresh

19.2

19.3

19.2

19.3

19.3

19.4

48 aged

19.7

19.6

19.7

19.7

19.7

19.5

Mean

19.4

19.5

19.4

19.5

19.5

19.4

Std. dev.

0.3

0.2

0.3

0.3

0.3

0.3

Mean

19.4

Std. dev.

0.3

Table 5 pH of the test system

 

nominal test item loading rate [mg/L]

 

Control

4.27

9.39

20.7

45.5

100

Time [h]

pH

0 fresh

7.73

8.07

8.37

8.87

9.41[KP1] 

10.05[KP2] 

24 aged

8.13

8.05

8.08

8.13

8.47

8.64

24 fresh

7.41

7.85

8.25

8.86

9.41

9.90

48 aged

7.79

7.90

7.94

8.01

8.27

8.19

Mean

7.77

7.97

8.16

8.47

8.89

9.20

Std. dev.

0.30

0.11

0.19

0.46

0.61

0.92

Mean

8.41

Std. dev.

0.68

Table 6 dissolved oxygen concnetration of the test system

 

nominal test item loading rate [mg/L]

 

Control

4.27

9.39

20.7

45.5

100

Time [h]

Oxygen [mg/L]

0 fresh

9.2

9.0

9.0

9.0

9.0

9.0

24 aged

8.8

8.8

8.8

8.8

8.9

8.9

24 fresh

9.2

9.1

9.1

9.1

9.1

9.1

48 aged

8.9

8.9

8.9

8.9

8.9

9.0

Mean

9.0

9.0

9.0

9.0

9.0

9.0

Std. dev.

0.2

0.1

0.1

0.1

0.1

0.1

Mean

9.0

Std. dev.

0.1

Table 7 measured concentration of the analyte dibutylamine

Test item

Sampling

Test item found

nominal [mg/L]

[h]

[mg/L]

% of nominal

Control

0 fresh

<LOD

-

24 fresh

<LOD

-

48 aged

<LOD

-

4.27

0 fresh

3.60

84

24 fresh

3.56

83

48 aged

3.76

88

9.39

0 fresh

9.20

98

24 fresh

8.64

92

48 aged

8.44

90

20.7

0 fresh

18.6

90

24 fresh

19.3

93

48 aged

19.5

94

45.5

0 fresh

40.4

89

24 fresh

39.9

88

48 aged

46.4

102

100

0 fresh

92.4

92

24 fresh

89.9

90

48 aged

93.4

93

 

 

Validity criteria fulfilled:
yes
Conclusions:
According to the results of the test, the EL50 (48 h) for immobilisation was determined to be 53.2 mg/L (nominal). The corresponding NOELR (48 h) was 20.7 mg/L (nominal).
Executive summary:

The objective of this study was to determine the effects of the test item on mobility of Daphnia magna. Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate (EL50), were estimated.

The study was conducted in accordance with the OECD guideline 202 the “Daphnia sp. acute immobilisation test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 100, 45.5, 20.7, 9.39, and 4.27 and 0 (control) mg/L. WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 1 hour. Water quality parameters were measured at the start and end and at 24 hour intervals in both fresh and aged samples. Analytical confirmation of the test substance was performed at each concentration at 0 fresh, 24 hours fresh, 48 hours old. All water quality parameters and study validity criteria were met as per OECD guideline 202. Apart from pH at time 0 and 24 hours fresh in the upper 2 concentrations. It is recommended in the guideline that pH remain within 6-9.  At 0 hours and 24 hours fresh in the 45.5 mg/L test solution systems pH was 9.41.  At 0 hours and 24 hours fresh in the 100 mg/L solution the pH was 10.05 and 9.90. This reduced to within guideline recommendations within 24 hours. The pH was considered transient and of little impact biologically to the daphnids. Although outwith the guideline recommendation it is not considered to effect the integrity of results and is back within the recommended levels within 24 hours. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

According to the results of the test, the EL50 (48 h) for immobilisation was determined to be 53.2 mg/L (nominal). The corresponding NOELR (48 h) was 20.7 mg/L (nominal).

Description of key information

96 h EL50 = 53.2 mg/L to Daphnia magna, OECD 202 (2004) Kummich, F. 2018.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
53.2 mg/L

Additional information

The objective of this study was to determine the effects of the test item on mobility of Daphnia magna. Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate (EL50), were estimated.

The study was conducted in accordance with the OECD test guideline 202 the “Daphnia sp. acute immobilisation test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 100, 45.5, 20.7, 9.39, and 4.27 and 0 (control) mg/L. WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 1 hour. Water quality parameters were measured at the start and end and at 24 hour intervals in both fresh and aged samples. Analytical confirmation of the test substance was performed at each concentration at 0 fresh, 24 hours fresh, 48 hours old. All water quality parameters and study validity criteria were met as per OECD guideline 202. Apart from pH at time 0 and 24 hours fresh in the upper 2 concentrations. It is recommended in the guideline that pH remain within 6-9.  At 0 hours and 24 hours fresh in the 45.5 mg/L test solution systems pH was 9.41.  At 0 hours and 24 hours fresh in the 100 mg/L solution the pH was 10.05 and 9.90. This reduced to within guideline recommendations within 24 hours. The pH was considered transient and of little impact biologically to the daphnids. Although outwith the guideline recommendation it is not considered to affect the integrity of results and is back within the recommended levels within 24 hours. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

According to the results of the test, the EL50 (48 h) for immobilisation was determined to be 53.2 mg/L (nominal). The corresponding NOELR (48 h) was 20.7 mg/L (nominal).