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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:

27 July - 30 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with the OECD 202 (2004) guideline, without deviation, under GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Principles of method if other than guideline:

According to guideline

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 29 July till 30 September 2021, date of signature 14 January 2022

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for immediate quantitative analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary. An aliquot of each sample was analysed directly or diluted with media.

Vehicle:

no

Details on test solutions:

Method: A nominal amount of test item (222 mg) was dissolved in test water and the volume adjusted to 2 L to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.
- Controls: Test water without test substance but treated in the same way as the test substance solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): < 24 hours old at the test initiation.
- Stage and instar at study initiation: First instar Daphnids.
- Method of breeding: Adult female 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 °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. 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
- Source: In house laboratory cultures from Labcorp (Labcorp Early Development Laboratories Ltd)
- Feeding during test: No feeding during test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L CaCO3

Test temperature:

20 to 22 °C

pH:

6.6-8 at the start of the experiment, 7.8 at 48 hours

Dissolved oxygen:

8.5-8.6 mg oxygen/L at the beginning of the experiment, 8.9-9 mg oxygen/L at 48 hours.

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

During the range finding and the definitive experiment nominal concentrations all stayed within the acceptable range of 80-120% (92-117% in the range finding test, 99-118% in the definitive test).
For full data see tables 6.1 and 6.2 in the Any Other Information on Materials and Methods inc Tables section.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150-mL glass vessels containing approximately 100 mL of test medium each, these were covered to reduce evaporation.
- Type: Closed
- Aeration: No aeration of the test solutions occurred throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable. A static test was performed (stability of the test item was demonstrated in closed and static conditions during the range-finding test).
- 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): Not applicable.
- Biomass loading rate: 5 daphnids per vessel.
- Introduction of daphnids: Daphnids were added to the vessels containing test preparations at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (Elendt M4 medium), as prescribed in OECD TG 202.

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary it was adjusted using NaOH or HCl.
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 200-1200 LUX, measured using an RS light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Eventual immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg test item/L (test water).
- Results used to determine the conditions for the definitive study: Reduced mobility was observed in two daphnids at the test concentration of 100 mg/L, No immobilization was observed at the test concentrations of 1.0 and 10 mg/L, however; 70% immobilization was observed at 100 mg/L. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range finding and definitive tests can be seen in tables 6.3, 6.4 and 6.5 in the Any Other Information incl. tables section.
One Daphnia exposed to a test concentration of 18 mg/L was immobilized. There was no immobilization in 20 daphnids exposed to test concentrations of 10, 32, 56 and 100 mg/L for a period of 48 hours.
Exposure of Daphnia magna to the test item gave No Observed Effect Concentrations after 24 and 48 hours exposure of greater than 100 mg/L.
Reduced mobility was observed at test concentrations of 18 and 100 mg/L and a single Daphnia was observed to be trapped at the surface at the test concentration of 32 mg/L.

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.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were EC50 of 0.86 at 24h, 0.5 at 48h, which are within the normal range for this reference item. (The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L)

Reported statistics and error estimates:

No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 6.3.Cumulative Immobilization Data and Observations in the Range-finding Test
Nominal concentrations (mg ai/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	0	0	5 N	5 N	0	0	5 N	5 N
100	0	0	5 N	5 N	4	3	1 N	2 R

 

Tables 6.4 and 6.5.

Cumulative Immobilization Data and Observations in the Definitive Test

Table 6.4

Nominal concentrations (mg ai/L)	24 Hours
	Cumulative immobilised Daphnia (Initial population: 5 per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	1	0	0	0	1	5	4 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

Table 6.5

Nominal concentrations (mg ai/L)	48 Hours
	Cumulative immobilised Daphnia (Initial population: 5 per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	1	0	0	0	1	5	4 N	3 R 2 N	5 N	4 R 1N
32	0	0	0	0	0	0	5 N	1 T 4 N	5 N	5 N
56	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	1R 4N	5 N	5 N	5 N

 

Rep = Replicate

N = Normal

R = Reduced mobility

T = Trapped at surface

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid as 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

Conclusions:

Reduced mobility was observed at test concentrations of 18 and 100 mg/L and a single Daphnia was observed to be trapped at the surface at the test concentration of 32 mg/L. However, the number of Daphnids affected was not high enough to establish an effect.

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of the test substance to Daphnia magna, according to OECD Test Guideline 202 and EU Method C.2, with GLP compliance.

Analysis of the test concentrations at 0 and 48 hours showed the measured test concentrations ranged between 99% and 118% of nominal concentrations, therefore the results were based on the nominal concentrations.

In the test, one Daphnia exposed to a test concentration of 18 mg/L was immobilized. There was no immobilization in 20 daphnids exposed to test concentrations of 10, 32, 56 and 100 mg/L for a period of 48 hours.

Reduced mobility was observed at test concentrations of 18 and 100 mg/L and a single Daphnia was observed to be trapped at the surface at the test concentration of 32 mg/L.

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.

Description of key information

48h-EC50 (Daphnia magna) > 100 mg/L; OECD TG 202 and EU Method C.2.

Additional information

This study was performed to assess the acute toxicity of the test substance to Daphnia magna, according to OECD Test Guideline 202 and EU Method C.2, with GLP compliance.

Analysis of the test concentrations at 0 and 48 hours showed the measured test concentrations ranged between 99% and 118% of nominal concentrations, therefore the results were based on the nominal concentrations.

In the test, one Daphnia exposed to a test concentration of 18 mg/L was immobilized. There was no immobilization in 20 daphnids exposed to test concentrations of 10, 32, 56 and 100 mg/L for a period of 48 hours.

Reduced mobility was observed at test concentrations of 18 and 100 mg/L and a single Daphnia was observed to be trapped at the surface at the test concentration of 32 mg/L.

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.