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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Sept 2017 to 03 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
ISO media was used in the definitive test due to the chelating agent in Elendt M7 and the presence of zinc within the test item.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Remarks:
Annex 7 of the report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1013Q17761
- Purity: 100%
- Purity test date: 26/06/2014
- Expiry date: 25/06/18. Laboratory testing carried out before this date.
- Physical state/appearance: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: a preliminary media preparation trial indicates that a dissolved test item concentration of 1.9 mg/l was obtained from a saturated solution method of preparation.
- Physical state/ appearance: white powder

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


Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations and control
- Sampling method: samples taken at 0hours from the bulk test preparation for each concentration and control. Samples taken at 48 hours from the control and the test groups, from pooled replicates.
- Sample storage conditions before analysis: frozen prior to analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Saturated solution preparation. Solutions were prepared by stirring 110mg of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours, followed by centrifugation at 40000 g for 30 minutes, to give a 100% v/v saturated solution of the test item. This saturated solution was diluted at test concentrations of 1.0, 3.2, 10, and 32% to provide the remaining test concentrations. Solutions were inverted several times to ensure adequate mixing.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): final concentration determined at 1.9mg/l in initial test solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): at the start of the test all control and test solutions were observed to be clear and colourless solutions.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: in-house laboratory culture
- Source: in-house laboratory culture
- Age of parental stock (mean and range, SD): no details
- Feeding during test: no
- Food type: none
- Amount: not applicable
- Frequency: not fed

ACCLIMATION
- Acclimation period: 1 day before the initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the culture and used for testing.
- Acclimation conditions (same as test or not): not stated
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and GEMMA Micro 300 suspension. Amount not stated.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): not stated



Study design

Test type:
static
Water media type:
other: reconstituted water (ISO medium) used for definitive test
Remarks:
Reconstituted water (Elendt M7 medium) range finding test but was not used in the range finding test due to the presence of chellating agent in Elendt M7 media
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Not applicable
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Reconstituted water hardness 250 mg/L CaCO3.
Test temperature:
21 to 22°C
pH:
7.8 to 8.0
Dissolved oxygen:
8.6 to 8.9 mg O2/L
Salinity:
not stated
Conductivity:
not stated
Nominal and measured concentrations:
Nominal test concentrations: 1.0, 3.2 , 10, 32 and 100 % v/v saturated solution.
Geometric mean measured test concentrations: 0.067, 0.041, 0.17, 0.60, 1.8 mg/L. The geometric mean was used due to a decline observed in the 10% saturated solution.
Adjustment of concentrations: The concentration of zinc in the test samples was determined and the equivalent test item concentrations calculated based on a test item zinc content of 62%. Analysis of the test preparations at 0 hours showed measured zinc concentrations to range from less than the LOQ, determined to be 0.051 mg/L, to 1.2 mg/L; equivalent to test item concentrations of less than the LOQ, determined to be 0.082 mg/L, to 1.9 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker, 150ml
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 ml of test solutions
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): not renewed
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4, 20 animals in total
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 4, 20 animals in total
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hours light, 8 hours dark, 20 minute dusk and dawn transitions
- Light intensity: 1097 to 1122 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation or adverse reactions were recorded at 24 and 48 hours. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Cumulative immobilisation and other observational data were recorded. No significant immobilisation was observed at 0.10, 1.0% v/v saturated solution, however immobilisation was observed at concentrations 1.0, 10 and 100% v/v saturated solution. Based on this the concentrations of 1.0, 3.2, 10, 32 and 100% saturated solution were selected for the definitive test.

INITIAL EXPERIMENT:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The results of the initial experiment showed an unusual pattern of immobilization, therefore, a repeat test was required. This is what is detailed in the RSS.
Reference substance (positive control):
yes
Remarks:
potassium dicrhomate, tested under Envigo study number LK67NP

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.64 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
.- Behavioural abnormalities: sublethal effects of exposure were observed at 0.17 and 0.60 mg/l, reduced mobility and a pale appearance.
- Observations on body length and weight: not detailed
- Mortality of control: 5% at 48 hours
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: No observations of this made
See tables 1 and 2 below for details on the results
Results with reference substance (positive control):
- Results with reference substance valid: yes, the results of the positive control with potassium dichromate were within the normal range for this reference item.
- Relevant effect levels: EC50, NOEC, LOEC
- Limit test: No, 5 test concentrations used
- Dose-response test: yes
- ECx: EC50 48 hours 0.75 mg/l, NOEC 0.56 mg/l and LOEC 1.0mg/l
- Other:

Any other information on results incl. tables

Table 1. Cumulative immobilisation data in the definitive test (24 hours)

 

24 hours

Geometric mean measured concentration (mg/l)

Cumulative immobilised daphnia

(initial population, 5 per rep)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5N

5N

5N

0.067

0

0

0

0

0

0

5N

5N

5N

5N

0.041

0

0

0

0

0

0

5N

5N

5N

5N

0.17

1

0

0

0

1

5

4N

5N

5N

5N

0.60

1

1

1

0

3

15

4N

4N

2R, 2N

2R, 3N

1.8

5

5

5

5

20

100

Ai

Ai

Ai

Ai

Table 2. Cumulative immobilisation data in the definitive test (48 hours)

 

48 hours

Geometric mean measured concentration (mg/l)

Cumulative immobilised daphnia

(initial population, 5 per rep)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

1

0

0

0

1

5

4N

5N

5N

5N

0.067

0

0

0

0

0

0

5N

5N

5N

5N

0.041

0

0

0

0

0

0

5N

5N

5N

5N

0.17

1

0

0

0

1

5

4N

5N

5N

1R, 4N

0.60

3

1

2

2

8

40

2N

4P

1R, 1P

1R, 2N

1.8

5

5

5

5

20

100

Ai

Ai

Ai

Ai

R = Replicate

N = Normal

R = Reduced mobility

P = Pale

AI = All daphnia immobilized

* 1 daphnid unaccounted for, considered to have had no impact to the outcome of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity to Daphnia magna was performed in accordance with OECD Guideline 202 and to GLP certification. The solubility of the test substance was low and an EC50 value of 0.64 mg/l was calculated based on the geometric mean measured test concentration.
Executive summary:

The acute toxicity to Daphnia magna was performed in accordance with OECD Guideline 202 and to GLP certification. Preliminary solubility work indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. A dissolved test item concentration of approximately 1.9 mg/l was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Daphnia magna were exposed to nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for 48 hours at a temperature of 21 to 22 °C under static test conditions. The concentration of zinc in the test samples was determined and the equivalent test item concentrations calculated based on a test item zinc content of 62%. Analysis of the test preparations at 48 hours showed measured zinc concentrations to range from less than the LOQ to 1.0 mg/l; equivalent to test item concentrations of less than the LOQ to 1.7 mg/l. Based on the decline observed in the 10% v/v saturated solution test media it was considered appropriate to calculate the results based 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 0.067, 0.041, 0.17, 0.60 and 1.8 mg/l as test substance. The EC50 value of 0.64 mg/l was calculated based on the geometric mean measured test concentration. Reduced mobility and a pale appearance was observed in the test organisms.