Matricaria recutita, ext.

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:

From 2017-11-17 to 2018-01-04

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

2017-01-10

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Concentration of dissolved organic material was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and test water. The loading rates of the test item were weighed on weighing boats that afterwards were placed above each mixing vessel and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water to obtain 5.0 to 5.5 L of test water (depending on the brim capacity of the bottles) in order to use a maximum volume and to minimise headspace. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: 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.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- 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.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

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

Test temperature:

The temperature of the test medium was situated between 20.8 and 21.1 °C throughout the test (average value: 20.9 °C)

pH:

7.85 - 8.43
pH requirements: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

7.13 - 8.52 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Loading rates: control, 3.2, 5.7, 10.1, 18.0 and 32.0 mg.L-1 .

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- 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 MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. 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=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.78
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 0.32, 1.00, 3.20 and 10.00 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: after 24 hours of exposure, immobilisation rates were 90% at 32 mg.L-1 100% at 100 mg.L-1; after 48 hours of exposure, immobilisation rate was 100% at 32 mg.L-1. Based on the results of a range-finding test, the following nominal concentrations: .2, 5.7, 10.1, 18.0 and 32.0 mg.L-1 (loading rates) were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

33.544 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 25.43-64.98

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

14.534 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 12.11-17.55

Details on results:

The evaluation was based on the nominal WAFs concentrations.
After 24 hours of exposure, immobilisation rates were 0% at 3.2, 5.7 and 10.1 mg.L-1, 30% at 18.0 mg.L-1 and 40% at 32.0 mg.L-1. After 48 hours of exposure, immobilisation rates were 0% at 3.2 and 5.7, 25% at 10.1 mg.L-1, 75% at 18.0 mg.L-1 and 90% at 32.0 mg.L-1.
The 24 and 48-hour EL50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.

Results with reference substance (positive control):

On August 17, 2017 (most recent test), the 24h-EC50 was 0.92 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Biological results

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

Nominal concentration*(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
3.2	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
5.7	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
10.1	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 2 3	25
18.0	1 2 3 4	5 5 5 5	2 1 2 1	30	4 3 4 4	75
32.0	1 2 3 4	5 5 5 5	2 2 4 0	40	4 5 5 4	90

* WAF prepared at the given loading rate.

Analytical results

Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

The analytical results of this test showed that WAFs concentrations were overall stablebetween the start and the end of each exposure period (t=0h-t24h_Old and t=24h_Fresh-t =48h), within the ± 20% of the initial TOC concentrations values. It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.

Therefore, according to the results of the TOC analysis, due to the complex nature of the WAF and since the test item was an UVCB substance, the results were based on the nominal test loading rates.

Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	t=24h Old	Relative loss to initial value (t=0h - t=24hold)(%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (t=24hfresh- t=48h)(%)
Control	1.01	0.88	13	0.96	0.99	-3
3.2	2.81	2.53	10	2.57	2.41	6
5.7	3.69	3.66	1	3.63	3.46	5
10.1	5.71	5.52	3	5.43	5.67	4
18.0	9.10	8.92	2	8.71	8.79	-1
32.0	13.80	13.80	0	14.60	14.50	1

* WAF prepared at the given loading rate.

Water quality parameters and environmental conditions throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L^-1) are presented below. These parameters remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.8 and 21.1 °C throughout the test (average value: 20.9 °C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

 

pH-values during the final test

		Nominal concentration(mg test item.L^-1)*
		Control	3.2	5.7	10.1	18.0	32.0
Start t=0h		8.50	8.17	8.23	8.27	8.26	8.25
t=24h	Old	8.06	7.99	7.97	8.05	8.04	8.01
	Fresh	8.48	8.52	8.54	8.54	8.59	8.57
End t=48h		8.28	8.35	8.32	8.36	8.34	8.34

* WAF prepared at the given loading rate

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

		Nominal concentration(mg test item.L^-1)*
		Control	3.2	5.7	10.1	18.0	32.0
Start t=0h		8.20	8.15	8.16	7.88	7.70	7.73
t=24h	Old	7.75	7.86	7.77	7.54	7.40	7.13
	Fresh	8.38	8.46	8.52	8.04	7.96	7.78
End t=48h		8.06	8.22	8.27	7.82	7.71	7.68

* WAF prepared at the given loading rate.

All these dissolved-oxygen concentrations correspond to values > 80% of the air-saturation value.

 

 

 

Validity criteria of the study:

Controls In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [0₂] Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels. 

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 14.534 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of the registered substance to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL₅₀(Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 3.2, 5.7, 10.1, 18.0 and 32.0 mg.L^-1and to a control.The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

The analytical results of this test showed that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0h-t24h_Old and t=24h_Fresh-t=48h), within the ± 20% of the initial TOC concentrations values. After 48 hours of exposure, immobilisation rates were 0% at 3.2 and 5.7, 25% at 10.1mg.L^-1, 75% at 18.0mg.L^-1and 90% at 32.0mg.L^-1. 

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 14.534 mg.L-1.

Results synopsis:

Test: Toxic effect to Daphnia magna according to OECD guidelines No. 202, using WAFs.

48h-EL50: 14.534 mg/L(95% Confidence limits: 12.110 - 17.550 mg/L)

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 14.534 mg.L-1.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

14.534 mg/L

Additional information

For that endpoint, an experimental study with the registered substance was available. The study was performed to assess the acute toxicity of the registered substance to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL₅₀(Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 3.2, 5.7, 10.1, 18.0 and 32.0mg.L^-1and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

The analytical results of this test showed that WAFs concentrations were overall stablebetween the start and the end of each exposure period (t=0h-t24h_Oldand t=24h_Fresh-t=48h),within the ± 20% of the initial TOC concentrations values.After 48 hours of exposure, immobilisation rates were 0% at 3.2 and 5.7, 25% at 10.1mg.L^-1, 75% at 18.0mg.L^-1and 90% at 32.0mg.L^-1. 

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 14.534 mg.L-1.

The study respected the requirements of the guideline. The validity criteria have been fulfilled. The study was considered acceptable for that endpoint.