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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:

22-07-2019 to 22-08-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system and in darkness.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: May 2018 ; signature: Nov. 2018

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours).
- Sampling method: At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna (Straus), clone 5
Sex Female
Origin LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint – 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
Reason for selection Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured Daphnids.
Age at test start < 24 hours old
Test water Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202 (1). The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).
Breeding conditions Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The total water hardness was approximately 250 mg.L-1 (as CaCO3).

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 19.7 °C ; 24 hours: Temperature 20.1 °C

pH:

pH: 6.0-9.0, not varying by more than 1.5 units;
See table 1 in "Any other information on results incl. tables".

Dissolved oxygen:

Oxygen: ≥ 60% of the air-saturation value at the end of the test.
See table 2 in "Any other information on results incl. tables".

Nominal and measured concentrations:

- Nominal Loading Rates: 0.41, 0.61, 0.90, 1.33 and 1.97 mg/L
- Measured concentrations: See tables 6.1.3/4 in"Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
Test duration 48 hours
Test type A semi-static test (24h renewals) was performed in order to maintain test concentrations.
Test vessels 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
Number of daphnids 20 per treatment group (including controls), divided into 4 groups of 5
Daphnids
Loading 5 daphnids per vessel each completely filled with test solution and
without headspace
Number of replicates 4 replicates with daphnids per treatment group.
Aeration No aeration of the test solutions occurred throughout the test.
Feeding No feeding
Introduction of daphnids Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST WATER
Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202 (1). The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).

OTHER TEST CONDITIONS
Test environment Controlled environment cabinet (20°C ± 2°C)
Light regime 16h light : 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility: 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.
- pH and dissolved O2 At start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) from all treatment groups.
- Temperature of medium Measured continuously in a temperature control vessel next to the test vessels, over the study period, beginning at the start of the test.
- Additional measurements Absence of undissolved test item in the stock solution was checked by determination of turbidity (ISO 7027, Water quality - Determination of turbidity). Turbidity analysis was not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.

VEHICLE CONTROL PERFORMED: NA

RANGE-FINDING STUDY
- Test concentrations: In this range-finding test, ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 0.32, 1.0, 3.2, 10.0 mg test item.L-1 and to a concentration close to the solubility limit of the test item in test water (20.64 mg.L-1), and to a control. The test system was maintained in closed conditions, in a temperature controlled room (20°C ± 2°C) with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours. During the test the pH, oxygen concentrations and the temperature of the medium were within the optimal ranges for daphnids.
- Results used to determine the conditions for the definitive study: See tables 6.1.3/5 in "Any other information on results incl. tables".

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.761 - 2.664 mg/L ; geometric mean measured concentration

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL:0.900 - 1.178 mg/L ; geometric mean measured concentration

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- On May 14, 2019 (KD19-002; most recent test), the 24h-EC50 was 1.053 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The 24 and 48-hour EC50 values ncluding the 95% confidence interval estimated using the computer program ToxRat using Probit-analysis.

Table 1. pH-values during the final test.

 

		Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
		Control (0)	0.41 (0.41)	0.61 (0.62)	0.90 (0.91)	1.33 (1.36)	1.97 (2.01)
Start t=0 h		8.37	8.36	8.30	8.30	8.34	8.33
t=24 h	Old	8.31	8.25	8.25	8.23	8.27	8.24
	Fresh	8.30	8.31	8.31	8.29	8.27	8.29
End t=48 h		8.18	8.24	8.26	8.28	8.28	8.28

 

Table 2. Dissolved oxygen concentrations (mg.L^-1) during the final test.

 

		Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
		Control (0)	0.41 (0.41)	0.61 (0.62)	0.90 (0.91)	1.33 (1.36)	1.97 (2.01)
Start t=0 h		8.00	7.99	7.95	7.97	7.98	8.00
t=24 h	Old	8.06	7.96	8.02	7.90	7.88	7.96
	Fresh	8.01	8.00	7.96	7.93	7.96	7.96
End t=48 h		8.00	8.10	8.18	8.05	8.03	8.08

All these dissolved-oxygen concentrations correspond to values > 90% of the air-saturation value.

 

Table 3. Acute immobilisation of daphnids after 24 and 48 hours in the final test.

 

Nominal concentration (mg test item.L^-1)	Measured concentration (geometric mean) (mg test item.L^-1)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
				Number	Total %	Number	Total %
Control	Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.41	0.41	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.61	0.62	1 2 3 4	5 5 5 5	0 0 0 0	0	1 1 0 0	10
0.90	0.91	1 2 3 4	5 5 5 5	0 0 0 0	0	0 2 3 4	45
1.33	1.36	1 2 3 4	5 5 5 5	0 1 0 1	10	3 3 3 4	65
1.97	2.01	1 2 3 4	5 5 5 5	1 2 4 3	50	5 5 5 5	100

 

 

Table 4. Concentrations of the test item (mg.L^-1) in test water - Final test.

Nominal concentration (mg.L^-1)	Measured concentration (mg.L^-1)				Geometric mean measured concentrations
	Start (t=0 h)	t=24 hold	t=24 hfresh	End t=48 h	mg.L^-1	% nominal	% initial
Control	Abs.	Abs.	Abs.	Abs.	N.A.	N.A.	N.A.
0.41	0.47	0.43	0.42	0.34	0.41	100	87
0.61	0.71	0.65	0.63	0.50	0.62	102	87
0.90	1.00	0.93	0.92	0.80	0.91	101	91
1.33	1.48	1.36	1.37	1.24	1.36	102	92
1.97	2.20	2.03	2.03	1.80	2.01	102	91

N.A.: not applicable

% = Percent of expected nominal/initial concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.10 mg.L^-1) and the LOD (0.03 mg.L^-1).

 

Table 5 : Acute immobilisation of daphnids after 24 and 48 hours in the range-finding test (static test).

Nominal concentration (mg test item.L^-1)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
			Number	Total %	Number	Total %
Control	1 2	5 5	0 0	0	0 0		0
0.32	1 2	5 5	0 0	0	0 0		0
1.00	1 2	5 5	2 3	50	3 4		70
3.20	1 2	5 5	5 5	100	5 5		100
10.00	1 2	5 5	5 5	100	5 5		100
20.64 (max.solub.)	1 2	5 5	5 5	100	5 5		100

 

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. In addition, dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

Conclusions:

The test item 48h-EC50 was 1.028 mg/L (C.I: 1.761 - 2.664 mg/L) based on geometric mean measured concentrations.

Executive summary:

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna.

Key study: OECD TG 202, 2019 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The criterion measured was the 48 h EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

 Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 0.41, 0.61, 0.90, 1.33 and 1.97 mg.L-1, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) in order to determine if concentrations of the test item were maintained.

 The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). The evaluation of the effects on daphnids was based on the geometric means of the measured concentrations: 0.41, 0.62, 0.91, 1.36 and 2.01 mg.L-1. After 48 hours of exposure, immobilisation rates were 0% at 0.41 mg.L-1, 10% at 0.62 mg.L-1, 45% at 0.91 mg.L-1, 65% at 1.36 mg.L-1 and 100% at 2.01 mg.L-1. Therefore, the highest concentration without effect, and the 48 h no observed effect concentration (NOEC) was 0.41 mg test item.L-1

Under experimental conditions, the 48-hour EC50 value of ST 08 C 19 was estimated to be 1.028 mg.L-1, based upon geometric mean measured concentrations.

Description of key information

EC50 (invertebrates) = 1.028 (C.I. 0.900 - 1.178) mg/L based on geometric mean measured concentrations, 48-hour, freshwater, OECD TG 202, 2019

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.028 mg/L

Additional information

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna.

Key study: OECD TG 202, 2019 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The criterion measured was the 48 h EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

 Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 0.41, 0.61, 0.90, 1.33 and 1.97 mg.L-1, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) in order to determine if concentrations of the test item were maintained.

 The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). The evaluation of the effects on daphnids was based on the geometric means of the measured concentrations: 0.41, 0.62, 0.91, 1.36 and 2.01 mg.L-1. After 48 hours of exposure, immobilisation rates were 0% at 0.41 mg.L-1, 10% at 0.62 mg.L-1, 45% at 0.91 mg.L-1, 65% at 1.36 mg.L-1 and 100% at 2.01 mg.L-1. Therefore, the highest concentration without effect, and the 48 h no observed effect concentration (NOEC) was 0.41 mg test item.L-1

Under experimental conditions, the 48-hour EC50 value of ST 08 C 19 was estimated to be 1.028 mg.L-1, based upon geometric mean measured concentrations.