Fenugreek, 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:

2018-03-12 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 202 guideline.

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:

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), at t=24h (new and old solutions) and at the end of the test (t=48h).
- Number of samples: 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).
- Analytical monitoring: 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:

PREPARATION AND APPLICATION OF TEST SOLUTION
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.
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 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 discarded via the drain port. Then the WAFs were filtered and 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. 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 solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- 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.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Breeding conditions: Daphnids are 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 is performed in all-glass vessel containing test water. Cultures are maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids are 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 is changed at least once per week. These culture conditions allow to maintain the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

No data

Hardness:

approximately 250 mg.L-1 (as CaCO3).

Test temperature:

the temperature of the test medium was situated between 20.7 and 20.9°C throughout the test (average value: 20.8°C)

pH:

Between 6.0-9.0

Dissolved oxygen:

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

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal loading rate: 12.5, 25, 50, 100 and 200 mg/L

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test duration: 48 hours
- Test type: Semi-static, in order to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents.
- Test vessels: 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 treatment group.
- Number of daphnids: 20 per treatment group (control and loading rate of 100 mg.L-1), divided into 4 groups of 5 animals
- 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
- 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 MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water (Elendt M4 medium), as prescribed by 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. 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 treatment group.
- 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.

DATA HANDLING
- Defining exposure concentrations: Effective concentrations were determined considering nominal WAF concentrations (nominal loading rate values).
- 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. The highest concentration without effect and lowest concentration with 100% immobilisation was determined by visual observation.

RANGE FINDING TEST
- Preparation of test solutions: This range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rates of 1, 10, 32 and 100 mg.L-1 and to a control. Two method of preparation for the WAFs have been tried: heating/sonication and solvent conditions. Both conditions showed no daphnids immobilisation at every tested loading rate.
- Results used to determine the conditions for the definitive study: After the range-finding study, it has been decided to perform a limit test at a loading rate of 100 mg test substance/L for the final test. In the definitive test, the acute immobilisation of daphnids after 48h of exposure at the loading rate of 100 mg/L was 55%. Therefore, the study will be performed again, using the same conditions but with the following loading rates: 12.5, 25, 50, 100 and 200 mg/L (no limit test anymore). To do so, an amendement to the initial study plan has been done.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

164.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Details on results:

After 48 hours of exposure, immobilisation rates were 0% at 12.5, 25.0 and 50.0 mg.L-1, 25% at 100.0 mg.L-1 and 60% at 200.0 mg.L-1.

Results with reference substance (positive control):

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

Reported statistics and error estimates:

The EL50 values including the 95% confidence interval using probit analysis were determined by the computer program ToxRat

The EL₅₀values including the 95% confidence interval using probit analysis were determined by the computer program ToxRat (8), and were as follows at each of the observation times:

Time (h)	EL50(mg test item.L^-1)	95% confidence limits (mg test item.L^-1)
24	251.7	181.9 – 901.4
48	164.8	131.1 – 237.9

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
12.5	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
25.0	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
50.0	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100.0	1 2 3 4	5 5 5 5	1 0 0 1	10	1 1 1 2	25
200.0	1 2 3 4	5 5 5 5	2 3 1 1	35	4 3 3 2	60

* WAF prepared at the given loading rate.

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 start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

TOCanalyses indicate that organic compounds in the loading rates of interest were found to be overall stable between the start and the end of each exposure period (t=0h-t24h_Oldand t=24h_Fresh-t=48h),within the ± 20% of the initial TOCconcentrations values.It should be noted that TOC content at 100 mg.L^-1was higher the first day than the one measured during the 2^ndday of the test. This could be explained by the pre-treatment (heating and sonication), probably more effective on the first day on the viscous paste. It can be assumed that a longer time of heating / sonication could be more effective and would conduct to a probable increase of the TOC values, but this could lead to considerable heating and potential consequences for stability as described in the OECD GD No. 23. Moreover, test solutions must be treated in the same way, that’s why the same reasonable time of heating / sonication was applied for all treatments during the preparation of the fresh solutions.

In any case, 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 nominal loading rates and asEffective Loading Rate 50, according to the OECD No. 23.

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	1.10	1.05	5	0.98	1.33	-36
12.5	6.19	5.27	15	6.05	5.29	13
25.0	8.88	10.90	-23	12.30	8.92	28
50.0	23.60	22.70	4	23.50	24.40	-4
100.0	38.50	36.70	5	18.60	19.90	-7
200.0	71.50	67.00	6	66.70	67.90	-2

* WAF prepared at the given loading rate.

Water quality parameters and environmental conditions throughout the test:

pH-values during the final test.

 

		Nominal concentration(mg test item.L^-1)*
		Control	12.5	25.0	50.0	100.0	200.0
Start t=0h		8.55	8.34	8.02	7.91	7.82	7.65
t=24h	Old	8.18	7.89	7.95	7.88	7.80	7.55
	Fresh	8.23	8.01	7.95	7.93	7.85	7.56
End t=48h		7.97	7.25	7.38	7.40	7.37	7.32

* 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	12.5	25.0	50.0	100.0	200.0
Start t=0h		8.60	7.99	7.93	7.81	7.91	7.61
t=24h	Old	8.45	7.80	7.65	7.29	6.85	5.95
	Fresh	8.82	8.43	8.52	8.55	8.77	8.10
End t=48h		8.28	7.10	7.05	6.50	6.18	5.47

* WAF prepared at the given loading rate.

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:

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was 164.8 mg.L-1

Executive summary:

A study is performed under GLP conditions to assess the acute toxicity of test item FENUGREEK ABSOLUTE to Daphnia magna. The method followed is 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 daphnids are exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 12.5, 25, 50, 100 and 200 mg/L and to a control. The immobility of the daphnids is 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 is checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was 164.8 mg.L-1

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was 164.8 mg.L-1

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

164.8 mg/L

Additional information

To fulfill that endpoint, an experimental study is performed on the registered substance, in accordance with the OECD 202 guideline and under GLP conditions.

The purpose of this study is to assess the test item for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours in a closed, semi-static test, using Water Accommodated Fractions (WAFs).

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was 164.8 mg.L-1