Matricaria recutita, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-11-13 to 2018-01-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Experimental study performed according to OECD 201 guideline. All validity criteria were fulfilled. There were two minor deviations (concerning some pH and temperature values). These deviations did not affect the integrity of the study. Thus the study is considered valid for that endpoint

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test. 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 were prepared under closed conditions and by slow-stirring.
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 5.2 to 5.7 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 23 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.

Controls: Test water without test substance but treated in the same way as the test substance solutions (WAFs).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata,
Strain: CCAP 278/4
Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture : Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.104 cells.mL-1. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

Controlled environment cabinet (23°C ± 2°C)

pH:

Start(t=0 h): 7.97 to 8.31
End (t=72 h): 8.23 to 9.98

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 3.16): 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 controls and 3 replicates of each loading rate for counting. Moreover, a replicate of each treatment without algae was prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: Yes; Original medium of OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1, according to the study plan.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Continuous illumination with a light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Temperature of Medium: Measured continuously in the growth chamber, over the study period.
- Light Intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates: 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.495 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 0.695-2.375

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

2.775 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 1.590-3.967

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.053 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 6.810-12.033

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.515 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf. limits: 0.284-0.720

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf. limits: 0.499-0.996

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.657 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf. limits: 1.347-2.020

Details on results:

See above.

Results with reference substance (positive control):

On August 21, 2017 (most recent test), the 72h-EC50 was 1.457 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The software ToxRat® Professional was used to perform statistical analyses.

Biological results

Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴)

	Replicate	Nominal concentration(mg test item.L^-1)
		Control	1.0	3.2	10.0	32.0	100.0
t=24h	1	2.8	2.8	3.2	2.0	2.0	1.2
	2	4.0	4.0	3.6	1.6	1.6	1.6
	3	4.0	4.0	2.8	2.4	2.4	1.6
	4	4.4
	5	4.8
	6	4.0
	Mean	4.0	3.6	3.2	2.0	2.0	1.5
	Std. Dev.	0.67	0.69	0.40	0.40	0.40	0.23
	% Inh.	-	5.1	10.4	33.6	33.6	48.3
t=48h	1	16.4	9.2	7.6	3.2	0.4	0.0
	2	13.2	12.4	6.8	4.8	0.8	0.0
	3	14.8	8.8	7.2	6.0	1.2	0.0
	4	18.4
	5	18.0
	6	16.8
	Mean	16.3	10.1	7.2	4.7	0.8	0.0
	Std. Dev.	1.97	1.97	0.40	1.40	0.40	0.00
	% Inh.	-	13.8	23.3	36.7	89.2	146.3
t=72h	1	83.2	77.6	12.0	7.6	1.2	0.0
	2	95.6	64.8	18.4	14.0	0.8	0.0
	3	93.6	54.0	24.8	12.0	0.8	0.0
	4	82.4
	5	96.4
	6	88.8
	Mean	90.0	65.5	18.4	11.2	0.9	0.0
	Std. Dev.	6.18	11.81	6.40	3.27	0.23	0.00
	% Inh.	-	6.3	31.4	40.7	88.3	131.0

* WAF prepared at the given loading rate.

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

% Inh.: %Inhibition of growth rate relative to the control determined by the computer program ToxRat

Analytical results

The results of analysis of the samples taken during the final test are described in the Table I of the appended Analytical Report.Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at the start and at the end of the test.

The analytical results of this test showed that WAFs concentrations were overall stable between the start and the end of the test, within or close to 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 onthe nominaltest 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)	End (t=72h)	Relative loss to initial value (t=0h - t=72h)(%)
Control	1.94	1.90	2
1.0	1.09	1.24	-14
3.2	2.07	2.52	-22
10.0	5.28	5.40	-2
32.0	13.80	14.40	-4
100.0	41.70	44.90	-8

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

The pH level in the control varied more than 1.5 units at the end of the test (1.96 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of more sodium bicarbonate.

pH-values during the final test

	Nominal concentration(mg test item.L^-1)*
	Control	1.0	3.2	10.0	32.0	100.0
Start t=0h	8.02	7.97	8.16	8.09	8.21	8.31
End t=72h	9.98	9.66	9.22	9.08	8.52	8.23

* WAF prepared at the given loading rate.

The mean light intensity was of 5124 lux (range: 4970-5450 lux), which remained within the ranges prescribed by the study plan (range: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

Temperatures were situated between 23.2 and 23.9°C throughout the test (average value: 23.8°C), and should be complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C). However, the temperature values recorded between t=38h and t=42.5h were outside these requirements (up to 27°C), but the cause was probably due to a problem related to the temperature sensor at this time. This minor deviation was considered not to affect the results of the test as no impact was observed on controls throughout the duration of the test (all validity criteria of the test have been fulfilled).

Validity criteria of the study

Cell density in controls : 180-fold increase within 72 hours. The specific growth rate was determined at 1.73 day^-1, so greater than 0.92 day^-1.

Coefficient of variation:  1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 20.8%.

2.The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.3%.

Thus the validity criteria were respected in this study.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item Blue Chamomilla to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.053 mg.L-1 and 1.657 mg.L-1, respectively. The 72-hour EL10 for growth rate was 1.495 mg L-1 and for yield was 0.515 mg.L-1.

Executive summary:

A study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended byCommission Regulation (EU) 2016/266(2) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 1.0, 3.2, 10.0, 32.0 and 100.0mg.L^-1and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the controls and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the start and the end of the test. 

The analytical results of this test showed that WAFs concentrations were overall stablebetween the start and the end of the test, within or close to the ± 20% of the initial TOC concentrations values.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.053 mg.L-1 and 1.657 mg.L-1, respectively. The 72-hour EL10 for growth rate was 1.495 mg L-1 and for yield was 0.515 mg.L-1

Results synopsis:

Test: Toxic effect to Pseudokirchneriella subcapitata accordding to OECD guidelines No. 201, using WAFs.

72h-EL50 grouth rate: 9.053 mg/L (95% Confidence limits: 6.810 - 12.033 mg/L)

72h-EL50 yield: 1.657 mg/L (95% Confidence limits: 1.347 - 2.020 mg/L)

Description of key information

The toxic effect of the registered substance to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.053 mg.L-1 and 1.657 mg.L-1, respectively. The 72-hour EL10 for growth rate was 1.495 mg L-1 and for yield was 0.515 mg.L-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

9.053 mg/L

EC10 or NOEC for freshwater algae:

1.495 mg/L

Additional information

For that endpoint, an experimental study with the registered substance was available. The study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended byCommission Regulation (EU) 2016/266(2) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test itemover a range of nominal loading values of 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L^-1 and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the controls and the WAFs was checked by analysis of Total Organic Carbon (TOC)at the startand the end of the test. 

The analytical results of this test showed that WAFs concentrations were overall stable between the start and the end of the test, within or close to the ± 20% of the initialTOCconcentrations values.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.053 mg.L-1 and 1.657 mg.L-1, respectively. The 72-hour EL10 for growth rate was 1.495 mg L-1 and for yield was 0.515 mg.L-1.

All validity criteria were fulfilled. Except two minor deviations (concerning some pH and temperature values) that did not affect the integrity and the results of the study, the requirements of the guideline were respected. Thus the study is considered acceptable for that endpoint.