Fenugreek, 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:

2018-01-31 to 2018-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 201 guideline.

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:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

- Frequency of sampling: At start (t=0h) and at the end of the test (t=72h).
- Concentrations: Samples for analysis will be taken from all test concentrations (WAFs) and control.
- Number of samples: Sampling will consist of single samples taken per treatment.
- Analytical monitoring: Concentration of dissolved organic material in the WAFs will be checked by analysis of Total Organic Carbon (TOC) in the control medium and the loading rates. TOC analysis will not be performed in compliance with the OECD GLP principles but will be adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
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 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The 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 22 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 discarded via the drain port. Then the WAFs were 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. The test was carried out without adjustment of the pH.

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

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata, 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 susceptible to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- Stock culture: Algae stock cultures are started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions are 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 are inoculated in test water at a cell density of 1.104 cells.mL-1. The pre-culture is maintained under the same conditions as used in the test. The cell density is 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:

23°C ± 2°C

pH:

Control : 8.39 (start) - 9.63 (end)

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal loading rates: 10, 18, 32, 56 and 100 mg.L-1

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test vessels: 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.
- Cell concentration: An initial cell density of 5.10^3 using an exponentially growing pre-culture.
- Test environment: Controlled environment cabinet (23°C ± 2°C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous magnetic stirring.
- Light regime/intensity: Continuous illumination using cool white light with a light intensity of 60-120 μE.m-2.s-1 (equivalent range: 4440-8880 lux) and shall not vary more than ± 15% from the average light intensity over the incubation area.
- 6 controls and 3 replicates of each loading rate for counting. Moreover, replicates of each treatment without algae was prepared for chemical analyses.

TEST WATER
- 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

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

DATA HANDLING
- Defining exposure concentrations: the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 10, 18, 32, 56 and 100 mg.L-1
- Determination of algal growth inhibition (if applicable): average growth rate and yield inhibition
- Statistical analysis: The software ToxRat® Professional was used to perform statistical analyses. Complementary statistical analyses were performed using another statistical spreadsheet (using Excel®) for the validity criteria of the study.

TEST 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 1.78): 10, 18, 32, 56 and 100 mg.L-1

RANGE-FINDING TEST
This range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg.L-1 and to a control, according two methods of preparation: use of solvent (acetone); and heating/sonication.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

50.803 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

other: Yield

Details on results:

The analytical results of this test showed that WAF concentrations were overall stable between the start and the end of the test, within or closed to the ± 20% of the initial TOC concentrations values.

Results with reference substance (positive control):

On February 26, 2018 (KA18-001; most recent test), the 72h-EC50 was 0.98 mg.L-1 for the parameter growth rate

Reported statistics and error estimates:

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

TheE_rL_xand E_yL_xvalues at the end of the test were as follows:

Parameter	Growth rate (mg.L^-1)	Yield (mg.L^-1)
72-hour EL10	30.303	13.225
95% conf. limits	1.125 – 49.640	0.198 – 25.180
72-hour EL20	63.761	20.991
95% conf. limits	30.123 – 142.972	1.390 – 34.820
72-hour EL50	n.d. (≥ 100)	50.803
95% conf. limits	n.d. – n.d.	28.262 – 132.694

Validity criteria fulfilled:

yes

Conclusions:

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 ≥ 100 mg.L-1 and 50.8 mg.L-1, respectively. The 72-hour EL10 for growth rate was 30.3 mg L-1 and for yield was 13.2 mg.L-1.

Executive summary:

A study is performed under GLP conditions to assess the test item Fenugreek absolute for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The method followed is 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 by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Algal cells are exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 10, 18, 32, 56 and 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures is determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the test loading rates is checked by TOC analysis at the startand the end of the test.

Under the experimental conditions and based on nominal loading rates,the 72-hour EL₅₀for the parameters growth rate and yield were determined to be ≥ 100 mg.L^-1and 50.8 mg.L^-1, respectively. The 72-hour EL₁₀for growth rate was 30.3 mg L^-1and for yield was 13.2 mg.L^-1.

Description of key information

Under the experimental conditions andbased on nominal loading rates,the 72-hour EL₅₀for the parameters growth rate and yield were determined to be ≥ 100 mg.L^-1and 50.8 mg.L^-1, respectively. The 72-hour EL₁₀for growth rate was 30.3 mg L^-1and for yield was 13.2 mg.L^-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

30.3 mg/L

Additional information

To fulfill that endpoint, an experimental study is performed on the registered substance, in accordance with the OECD 201 guideline and under GLP conditions.

The purpose of this study is to assess the test item for its ability to generate toxic effects on Pseudokirchneriella subcapitata during an exposure period of 72 hours, using Water Accommodated Fractions (WAFs).

Under the experimental conditions andbased on nominal loading rates,the 72-hour EL₅₀for the parameters growth rate and yield were determined to be ≥ 100 mg.L^-1and 50.8 mg.L^-1, respectively. The 72-hour EL₁₀for growth rate was 30.3 mg L^-1and for yield was 13.2 mg.L^-1.