[No public or meaningful name is available]

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:

18-08-2014 to 18-09-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: clear colourless liquid

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: 0 (control),
0.46, 1.0, 2.2, 4.6 and 10 mg/L prepared as WAF
0 (control), 0.32, 0.88, 1.6, 4.2 and 9.0 mg/L based on geometric mean measured average concentrations, respectively; controls: Test medium without test item or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test 0-hours from the freshly prepared solutions.
2. The end of the test from the 48-hour old solutions, volume: 4 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were used on day of sampling; reserve samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 4 mL were taken for possible analysis if needed.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: Not applicable
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The ISO medium hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 19.8 and 21.4°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 7.5-8.0, not varying by more than 1.5 unit at the end of the test.

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen: >= 3 mg/l at the end of the test.

Nominal and measured concentrations:

Range-finding test:
0 (control), 1.0, 10 and 100 mg/L prepared as WAF
Definitive test:
0 (control), 0.46, 1.0, 2.2, 4.6 and 10 mg/L prepared as WAF
0 (control), 0.32, 0.88, 1.6, 4.2 and 9.0 mg/L based on geometric mean measured average concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass closed
- Type (delete if not applicable): closed ; static
- Material, size, headspace, fill volume: Glass, 80 mL fill volume
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 80 mL of test solution
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare ISO Medium M7
- Culture medium different from test medium: No, ISO Medium M7, renewed by 50% twice per week.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 16 hours light; 8 hours dark photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), 24 hours and at 48 hours.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1.0, 10 and 100 mg/L prepared as WAF
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 5.1 to 8.3 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - mg/L

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: None.
- Mortality of control: No mortalities.
- 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.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 0.90 mg/L. 48h-EC50 was 0.43 mg/L with a 95% confidence interval between 0.39 and 0.48 mg/L
- Other: The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of test organism was in agreement with the historical data.

Table 1. pH and oxygen (mg/L) concentrations during the final test

Concentration Test Item Average (mg/L) (based on WAF in [ ] brackets)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control [0]	7.5	8.8	7.8	8.9
0.32 [0.46]	7.7	8.7	7.9	8.9
1.0 [0.88]	7.9	8.6	7.9	8.9
1.6 [2.2]	7.8	8.7	7.9	8.9
4.2 [4.6]	7.9	8.6	8.0	8.9
9.0 [10.0]	7.7	8.6	8.0	8.9

  

Table 2. Number of introduced daphnids and incidence of immobility in the final test

Concentration Test Item Average (mg/L)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.32	A B C D	5 5 5 5	0 0 0 (2) 0	0	0 0 0 0	0
0.88	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.6	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
4.2	A B C D	5 5 5 5	1 (1) 1 1 1	20	3 2 2 2	45
9.0	A B C D	5 5 5 5	4 4 4 3	75	5 5 5 5	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 4.3 mg/L (C.I: – mg/L) based on average exposure concentrations that were analytically confirmed.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding/limit test in the 0.1 to 100 mg/L range, an identically conducted definitive study was completed in 100 mL closed glass flasks with a 80 mL fill volume under static conditions.Preparation of test solutions started with individually prepared loading rates that were magnetically stirred for two days. The resulting suspensions were left to settle for an hour, were after the Water Accommodated Fractions (WAFs) were siphoned from the middle of the settled suspensions. The WAFs were all clear and colourless and were used for testing. Preparation of test solutions started with the highest loading rate of 100 mg/L (combined limit/range-finding test) or 10 mg/L (final test). The definitive test WAF loading rates were 0 (control), 0.46, 1.0, 2.2, 4.6 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations of 0.37, 1.0, 1.9, 4.8 and 10.0 mg/L in the WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. Concentrations measured after 48 hours decreased by approximately 20 to 30% below the initial measured concentrations. The range tested based on average exposure concentrations corresponded to 0.32, 0.88, 1.6, 4.2 and 9.0 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The results demonstrated 45% immobility at 4.2 mg/L and 100% immobility at 9.0 mg/L average exposure concentration. 95% confidence intervals could not be determined. Under the conditions of this study, the 48h-EC50 based on geometric mean measured average exposure concentrations was 4.3 (C.I. – ) mg/L.

Description of key information

EC50-48h (invertebrates) = 4.3 mg/L (95% CL: – mg/L) based on geometric mean measured average exposure concentrations, 48-hour, freshwater, OECD 202, 2014

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.3 mg/L

Additional information

Key study : OECD TG 202, 2014 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding/limit test in the 0.1 to 100 mg/L range, an identically conducted definitive study was completed in 100 mL closed glass flasks with a 80 mL fill volume under static conditions.Preparation of test solutions started with individually prepared loading rates that were magnetically stirred for two days. The resulting suspensions were left to settle for an hour, were after the Water Accommodated Fractions (WAFs) were siphoned from the middle of the settled suspensions. The WAFs were all clear and colourless and were used for testing. Preparation of test solutions started with the highest loading rate of 100 mg/L (combined limit/range-finding test) or 10 mg/L (final test). The definitive test WAF loading rates were 0 (control), 0.46, 1.0, 2.2, 4.6 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations of 0.37, 1.0, 1.9, 4.8 and 10.0 mg/L in the WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L, respectively. Concentrations measured after 48 hours decreased by approximately 20 to 30% below the initial measured concentrations. The range tested based on average exposure concentrations corresponded to 0.32, 0.88, 1.6, 4.2 and 9.0 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The results demonstrated 45% immobility at 4.2 mg/L and 100% immobility at 9.0 mg/L average exposure concentration. 95% confidence intervals could not be determined. Under the conditions of this study, the 48h-EC50 based on geometric mean measured average exposure concentrations was 4.3 (C.I. – ) mg/L.