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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2020 - 26 January 2021

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:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L loading rate WAF
- Time period: 0 hours (bulk test preparation) and from the pooled replicates (Replicates 1 to 4) at 48 hours
- Sample storage conditions before analysis: stored frozen prior to quantitative analysis

Vehicle:

no

Remarks:

Water accommodated fraction (WAF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Concentrations: Based on the results of the range-finding and initial tests, the following loading rates were assigned to the definitive test: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Method: Nominal amounts of test item (10, 18 and 32 mg) were each separately added to the surface of 10 L of test water to give the 1.0, 1.8 and 3.2 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A 10 and 2.0 mL pipette, covered at one end with Nescofilm was submerged into the vessels, sealed end down to mid—depth within the vessels. A length of Tygon tubing was attached to the 10 mL pipette and the Nescofilm was removed using the 2.0 ml pipette. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 100 mL discarded) to give the 1.0,.8 and 3.2 mg/L loading rate WAFs.

Evidence of undissolved material: Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Dilutions: Dilutions were made from the 1.0 mg/L loading rate WAF to give further test concentrations of 0.32 and 0.56 mg/L loading rate WAF. The 0.32 and 0.56 mg/L loading rate WAFs were inverted several times to ensure adequate mixing and homogeneity.

Analytical monitoring: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Annex 5, appended to 'attached background material').

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Organism: Daphnia magna (as per guideline)
Age at study initiation: less than 24 hours old
Stage at study initiation: first instar Daphnia magna
Method of breeding: Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 CC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
Source: in-house laboratory cultures
Feeding during test: the daphnids received no food during exposure


A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test. Details of the most recent positive control (Covance study number GV41TT) are given in Annex 3 (appended to 'attached background material').

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

According to OECD guideline

Post exposure observation period:

N/A

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

18 to 22 °C

pH:

7.9 ±0.3

Dissolved oxygen:

Not reported

Salinity:

N/A - freshwater

Conductivity:

N/A - freshwater

Nominal and measured concentrations:

Range-finding test: nominal loading rates of 0.10, 1.0, 10 and 100 mg/L.
Initial experiments: loading rates: 1.0, 1.8, 3.2, 5.8 and 10.5 mg/L (tests 1 - 3) 0.32, 0.56, 1.0, 1.8 and
3.2 mg/L (test 4)
Definitive test: loading rates 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel: 150 mL vessels
Type: closed (covered)
Fill volume: 100 mL
Aeration: No
Renewal rate of test solution: The test preparations were not renewed during the exposure period
No. of organisms per vessel: 5
No. of vessels per concentration: 4
No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water: Reconstituted water (Elendt M4) - full details reported in Annex 2 (appended to 'attached background material')

Main Compounds (mg/L)
CaCl2.2H2O 293.8 mg/L
MgSO4.7H2O 123.3mg/L
KCl 5.8mg/L
NaHCO3 64.8 mg/L
NazSiO3.9H2O 10.0 mg/L
NaNO3 0.274 mg/L
KH2PO4 0.143mg/L
K2HPO4 0.184 mg/L



OTHER TEST CONDITIONS
Adjustment of pH: 7.9 ±0.3, adjusted using NaOH or HCl.
Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
Light intensity: between 200 and 1200 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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 RS ILM Light meter. The appearance of the test media was recorded daily.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Chemical Analysis of Test Loading Rates
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (Replicates 1 to 4) at 48 hours and stored frozen prior to quantitative analysis. Duplicate samples of the control and 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L loading rate WAF test
groups were taken and stored frozen for further analysis if necessary. The method of analysis, recovery and test preparation analyses are described in Annex 5 (appended to 'attached background material').

RANGE-FINDING STUDY
Test concentrations: nominal loading rates of 0.10, 1.0, 10 and 100 mg/L.
Results used to determine the conditions for the definitive study: No immobilization was observed at 0.10 and 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF. Chemical analysis of the test preparations at 0 hours (see Annex 5, appended to 'attached background material') showed measured test concentrations of 0.052 to 91 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Water accommodated fraction (WAF)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Water accommodated fraction (WAF)

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (appended to 'attached background material').
No sub-lethal effects of exposure were observed throughout the test.
No immobilization was observed at 0.10 and 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF.
Loading rates of 1.0, 1.8, 3.2, 5.8 and 10.5 mg/L loading rate WAF were selected for the definitive test, this was later adjusted to 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L loading rate WAF following initial tests.
Chemical analysis of the test preparations at 0 hours (see Annex 5, appended to 'attached background material') showed measured test concentrations of 0.052 to 91 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

DEFINITIVE TEST

Chemical Analysis of Test Loading Rates
Chemical analysis of the fresh test preparations at 0 hours (see Annex 5) showed measured test concentrations to range from 0.23 to 2.9 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.22 to 2.9 mg/L.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilization and concentration at 48 hours is given in Figure 1 appended to 'attached background material'.
Visual inspection of the immobilization data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 48 hours based on the nominal loading rates gave the results reported (EL50).
The NOEL rates after 24 and 48 hours exposure were 3.2 and 0.56 mg/L loading rate WAFs respectively. Correspondingly the LOEL rate after 24 hours exposure was not determined and considered to be 1.0 mg/L loading rate WAF after 48 hours exposure.

Sub-Lethal Effects
Trapping at the surface was observed in one Daphnia at a test concentration of 1.0 mg/L (see Table 2 appended to 'attached background material').

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization 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
The results of the water quality measurements are given in Table 3 appended to 'attached background material'.
Temperature was maintained at approximately 20 °C to 22 ºC 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 941 to 969 lux.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of the mixing period the 1.0, 1.8 and 3.2 mg/L preparations were observed to be clear, colorless water column with test item floating at the surface of water. After 23 hours stirring and a 1-Hour standing period the 1.0, 1.8 and 3.2 mg/L preparations were observed to remain as at the start of the mixing. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 1.0, 1.8 and 3.2 mg/L preparations were observed to be clear, colorless solutions. The 0.32 and 0.56 g/L preparations were observed to be clear, colorless solutions for the duration of the test.

Results with reference substance (positive control):

Time Point (Hours) EC50 (mg/L) NOEC (mg/L) LOEC (mg/L)
24 0.90 0.56 1.0
48 0.79 0.56 1.0


NOEC = No Observed Effect Concentration
LOEC = Lowest Observed Effect Concentration


Results with reference substance valid: Yes
Full positive control details can be found in Annex 3 (appended to 'attached background material')

Reported statistics and error estimates:

24 h EL50 is >3.2 mg/L loading rate, confidence limits not calculated due to nature of the data.
48 h EL50 is 0.95 mg/L loading rate, 95% confidence limits 0.59 – 1.5 mg/L Loading Rate WAF

Validity criteria fulfilled:

yes

Conclusions:

Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

At 48 h the EL50 is 0.95 mg/L Loading Rate with 95% confidence limits 0.59 - 1.5 mg/L Loading Rate WAF; the NOEL is 0.56 mg/L; and the LOEL is 1.0 mg/L

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008. Study conducted under GLP Conditions.

 

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).
Following preliminary range-finding and initial tests, 20 daphnids (4 replicates of 5 animals) were exposed to WAFs of the test item over a range of nominal loading rates of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.23 to 2.9 mg/L and from 0.22 to 2.9 mg/L at 48 hours.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Exposure of Daphnia magna to the test item gave the following results:

 

Time Point (Hours)	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	NOEL (mg/L)	LOEL (mg/L)
48	0.95	0.59 - 1.5	0.56	1.0

Description of key information

Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

At 48 h the EL50 is 0.95 mg/L Loading Rate with 95% confidence limits 0.59 - 1.5 mg/L Loading Rate WAF; the NOEL is 0.56 mg/L; and the LOEL is 1.0 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

EL50: 0.95 mg/L Loading Rate WAF

Effect concentration:

0.95 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008. Study conducted under GLP Conditions.

 

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).
Following preliminary range-finding and initial tests, 20 daphnids (4 replicates of 5 animals) were exposed to WAFs of the test item over a range of nominal loading rates of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.23 to 2.9 mg/L and from 0.22 to 2.9 mg/L at 48 hours.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Exposure of Daphnia magna to the test item gave the following results:

 

Time Point (Hours)	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	NOEL (mg/L)	LOEL (mg/L)
48	0.95	0.59 - 1.5	0.56	1.0