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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:
22 May 2017 to 27 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis.
- All samples were stored frozen prior to analysis.
- Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- A nominal amount of test material (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: in house laboratory cultures
- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: no

CULTURE CONDITIONS
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Reconstituted Water – Elendt M7 Medium (Ingredient Final Concentration (mg/L)): H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143, Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075. The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21°C.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
22°C
pH:
6.6 to 8.0
Dissolved oxygen:
8.4 to 8.8 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: 18, 32, 56 and 100 mg/L
Geometric mean measured test concentrations: 15, 28, 51 and 89 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Material, size, headspace, fill volume: 100 mL
- The test vessels were covered to reduce evaporation
- Aeration: no
- Renewal rate of test solution: For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted Water – ISO Medium (Ingredient Final Concentration (mg/L)): CaCl2.2H2O 294, MgSO4.7H2O 123, NaHCO3 65 and KCl 5.8.
- The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 674 to 687 Lux

EFFECT PARAMETERS MEASURED:
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation. Microscopic observations were performed on the immobilised daphnia due to the precipitation of the test material at 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- In the range-finding test, 2 replicates per concentration were prepared, each containing 5 daphnids. The vessels were maintained in a temperature controlled room maintaining the water temperature at 18 to 22°C with a maximum deviation of ±1°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control group were was maintained under identical conditions but not exposed to the test material.
- A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test material under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CI: 27 to 39 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
No significant immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, significant immobilisation was observed at 100 mg/L. A single daphnia was observed to be pale in the 0.10 mg/L test concentration after 24 hours, however, as no effects were observed in the concentrations of 1.0 and 10 mg/L, this was considered to be due to natural causes. Microscopic observations performed on the immobilised daphnids in the 100 mg/L test concentration showed that brown debris was observed to be covering the daphnids.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the freshly prepared 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations of 9.7 and 99 mg/L, respectively. There was a significant decline in the measured concentrations at 48 hours indicating that the test material was not stable under test conditions.

DEFINITIVE TEST
Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test material during the definitive test are given in Table 1.
The No Observed Effect Concentrations after 24 and 48 hours exposure were 51 and 15 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 89 and 28 mg/L respectively.
The slopes and their standard errors of the response curves at 24 and 48 hours were 22 (SE = 0.096) and 5.5 (SE = 0.037) respectively.
Sub-Lethal Effects: No sub-lethal effects of exposure were observed throughout the test. Microscopic observations were performed on all the immobilised daphnia. No undissolved particles were observed to be adhered to the daphnids in the 32 and 56 mg/L test concentrations, however, brown debris was observed to be adhered to the daphnids in the 100 mg/L test concentration, suggesting that the observed toxicity could be due to physical effects.

- Validation Criteria: The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

- Water Quality Criteria: Temperature was maintained at 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 674 to 687 Lux.

- Observations on Test Material Solubility: The freshly prepared test concentrations at 0 and 24 hours were observed to be clear colourless solutions. In the old or expired media at 24 and 48 hours the control, 10 and 18 mg/L test preparations were observed to be clear colourless solutions and the 32 and 56 mg/L test preparations were observed to be clear colourless solutions with undissolved test material on the bottom of the vessel. After 24 hours, the 100 mg/L test preparations were observed to be clear colourless solutions with undissolved test material on the bottom of the vessel and dispersed throughout and at 48 hours they were observed to be clear colourless solutions with test material on the bottom of the vessel and floating on the surface.
Results with reference substance (positive control):
- A positive control study used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were similar to those in the definitive test.
- Analysis of the immobilisation data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results: 24 hour EC50: 0.83 mg/L (95% CI: 0.70 - 0.98 mg/L), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L. 48 hour EC50: 0.64 mg/L (95% CI: Not possible to determine), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L.
- The No Observed Effect Concentration is based upon equal to or less than 10% immobilisation at this concentration.
- The results from the positive control with potassium dichromate were within the normal range for this reference material.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Table 1: Cumulative Immobilization Data and Observations in the Definitive Test

 

Nominal Concentration (mg/L)

Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

24 hours

Control

0

0

0

0

0

0

5N

5N

5N

5N

10

0

0

0

0

0

0

5N

5N

5N

5N

18

0

0

0

0

0

0

5N

5N

5N

5N

32

0

0

0

0

0

0

5N

5N

5N

5N

56

0

0

0

0

0

0

5N

5N

5N

5N

100

3

3

4

3

13

65

2N

2N

1N

2N

 

48 hours

Control

0

0

0

0

0

0

5N

5N

5N

5N

10

0

0

0

0

0

0

5N

5N

5N

5N

18

0

0

0

0

0

0

5N

5N

5N

5N

32

3

3

1

2

9

45

2N

2N

4N

3N

56

3

5

3

5

16

80

2N

A/I

2N

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

A/I = All daphnia immobilised

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the 48 hour EC 50 was 33 mg/L with 95 % confidence limits 27 to 39 mg/L. The NOEC was 15 mg/L and the LOEC was 28 mg/L.
Executive summary:

The short term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. An Acute Immobilisation Test was performed using Daphnia magna.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22 °C under semi-static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

Analysis of the freshly prepared 18, 32, 56 and 100 mg/L test preparations at 0 and 24 hours showed measured test concentrations to range from 16 to 98 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations to range from 14 to 84 mg/L. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 15, 28, 51 and 89 mg/L.

Under the conditions of this study the 48 hour EC 50 was 33 mg/L with 95 % confidence limits of 27 to 39 mg/L. The NOEC was 15 mg/L and the LOEC was 28 mg/L.

Description of key information

Under the conditions of this study the 48 hour EC 50 was 33 mg/L with 95 % confidence limits of 27 to 39 mg/L. The NOEC was 15 mg/L and the LOEC was 28 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
33 mg/L

Additional information

The short term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. An Acute Immobilisation Test was performed using Daphnia magna.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22°C under semi-static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

Analysis of the freshly prepared 18, 32, 56 and 100 mg/L test preparations at 0 and 24 hours showed measured test concentrations to range from 16 to 98 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations to range from 14 to 84 mg/L. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 15, 28, 51 and 89 mg/L.

Under the conditions of this study the 48 hour EC 50 was 33 mg/L with 95% confidence limits of 27 to 39 mg/L. The NOEC was 15 mg/L and the LOEC was 28 mg/L.