Resinoid of Liquidambar styraciflua (Hamamelidaceae) obtained from exudate by organic solvents extraction

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 November 28th 2017 to March 8th 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD guideline 202 with GLP compliance

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:

Inspection on 4, 5, and 6th of July, 2016, certificate signed on 10th of January 2017.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, protected from light, in a refrigerator (temp 2-8°C), store under Nitrogen in a closed container after first opening.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: loading rates (spaced by a factor of 2.00): 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 mg.L-1.
- Sampling method: 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).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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 of 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 removed 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 and colourless. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnids
- Strain/clone: 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.
- Age at test start : < 24 hours old
- Feeding during test : No feeding

Test type:

semi-static

Water media type:

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

Limit test:

no

Total exposure duration:

48 d

Hardness:

Approximately 250 mg.L-1 (as CaCO3)

Test temperature:

The temperature of the test medium was situated between 20.8 and 20.9°C throughout the test.

pH:

between 7.36 and 8.75

Dissolved oxygen:

Between 7.13 mg/L and 8.67 mg/L.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loading rates : 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: cylindrical glass bottles sealed with screw caps
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 L
- Aeration: No aeration of the test solutions occurred throughout the test
- Renewal rate of test solution (frequency/flow rate): A semi-static test was performed with renewal of test solutions after 24 hours (because of the properties of some constituents of the test item).
- No. of organisms per vessel: 5 daphnids per vessel each completely filled with test solution and without headspace
- No. of vessels per concentration/control (replicates): 4 replicates with daphnids per treatment group

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of the pH.
- Photoperiod: 16h light / 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility of daphnids

RANGE-FINDING STUDY
- Test concentrations: nominal loading rates of 1, 10, 32 and 100 mg.L-1
- Results used to determine the conditions for the definitive study: Immobility observed after 48 hours : 30 % for daphnids treated at 10 mg/L ; 40 % for daphnids treated at 32 mg/L and 50 % for daphnids treated at 100 mg/L.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 20.008 – 31.237 mg/L

Details on results:

See tables in "Any other information on results incl. tables".

The measured water quality parameters (i.e. pH-value, dissolved oxygen, total water hardness and water temperature) were within the acceptable limits during the study.
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.

The results of analysis of the samples taken during the final test are described in the Table 6.1.3/ 4. 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).
Due to the complex nature of the substance, it was difficult to well solubilise the substance in the water medium (heating, sonication, mixing without headspace …). Even if the TOC analyses indicated that few organic compounds were found throughout the test in the WAFs, analytical results showed that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0h-t24hold and t=24hfresh-t=48h), within the ± 20% of the initial TOC concentrations values.
Therefore, according to the results of the TOC analysis, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on the nominal test loading rates and as Effective Loading Rate 50, according to the OECD No. 23

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.

Table 6.1.3/ 1 : Acute immobilisation of daphnids after 24 and 48 hours in the range-finding test (semi-static 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	5 5	0 0	0	0 0		0
1	1 2	5 5	0 0	0	0 0		0
10	1 2	5 5	0 0	0	1 2		30
32	1 2	5 5	1 0	10	4 0		40
100	1 2	5 5	2 1	30	3 2		50

* WAF prepared at the given loading rate.

Table 6.1.3/ 2 : Concentrations of the test item in test water - TOC analysis results (range-finding test)

	TOC (mg.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0h)	t=24h Old	t=24h Fresh	End (t=48h)
Control	1.73	1.83	1.66	1.86
1	2.20	1.93	1.19	1.89
10	3.06	2.50	3.24	2.90
32	6.65	4.90	4.66	3.64
100	10.80	8.44	10.30	8.54

* WAF prepared at the given loading rate.

Table 6.1.3/ 3 : 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
6.25	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
12.50	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 1 0	5
25.00	1 2 3 4	5 5 5 5	2 1 1 2	30	4 5 5 4	90
50.00	1 2 3 4	5 5 5 5	3 1 1 2	35	4 3 3 5	75
100.00	1 2 3 4	5 5 5 5	3 4 3 3	65	5 4 5 4	90
200.00	1 2 3 4	5 5 5 5	4 4 1 4	65	5 5 3 3	90

* WAF prepared at the given loading rate.

 

Table 6.1.3/ 4 : 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	1.32	1.07	19	1.19	1.10	8
6.25	2.69	2.80	-4	2.40	2.12	12
12.50	3.32	3.49	-5	2.39	2.37	1
25.00	5.18	5.43	-5	4.80	4.33	10
50.00	7.44	8.65	-16	6.85	5.73	16
100.00	12.50	12.50	0	10.00	10.20	2
200.00	17.60	17.40	1	9.01	9.95	-10

* WAF prepared at the given loading rate.

 

Validity criteria fulfilled:

yes

Remarks:

The validity criteria have been fulfilled in the present study.

Conclusions:

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 6.25 mg.L-1, 5% at 12.50 mg.L-1, 75% at 50.00 mg.L-1 and 90% at 25.00, 100.00 and 200.00 mg.L-1.
Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value estimated was 25.00 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The OECD guideline No. 202, referenced as Method C.2, was followed. The study was perfomed in closed and semi-static conditions, for 48 hours.

Following a preliminary range-finding test, daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 mg.L^-1 and to a control. The immobility of the daphnids was determined 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 6.25 mg.L^-1, 5% at 12.50mg.L^-1, 75% at 50.00mg.L^-1 and 90% at 25.00, 100.00 and 200.00 mg.L^-1.

The EL₅₀ at each of the observation times was as follows:

Time (h)	EL50(mg test item.L^-1)	95% confidence limits (mg test item.L^-1)
24	82.623	58.491 – 130.823
48	25.000	20.008 – 31.237

Statistical analyses were performed by the computer program ToxRat. Since the probit model was not appropriate at t=48h, the trimmed Spearman-Karber method was used for estimation of the EL₅₀including the 95% confidence interval.

Under the experimental conditions and based on nominal loading rates, the 48 -hour EL₅₀value estimated was 25.00 mg.L^-1.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EL50 (Daphnia magna) = 25.00 mg/L (95% CL: 20.008 – 31.237 mg/L), based on nominal loading rates

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

25 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The OECD guideline No. 202, referenced as Method C.2, was followed. The study was perfomed in closed and semi-static conditions, for 48 hours.

Following a preliminary range-finding test, daphnids were exposed toWater Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00mg.L^-1and to a control.The immobility of the daphnids was determined 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_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 6.25mg.L^-1, 5% at 12.50mg.L^-1, 75% at 50.00mg.L^-1and 90% at 25.00, 100.00 and 200.00mg.L^-1.

The EL₅₀at each of the observation times were 82.623 mg/L (95% CL :  58.491 – 130.823) at 24 h, and 25.00 mg/L (95% CL : 20.008 – 31.237) at 48 h. Statistical analyses were performed by the computer program ToxRat.

Since the probit model was not appropriate at t=48h, the trimmed Spearman-Karber method was used for estimation of the EL₅₀including the 95% confidence interval.

Under the experimental conditions and based on nominal loading rates,the 48 -hour EL₅₀value estimated was 25.00 mg.L^-1 based on nominal loading rates.