Commiphora myrrha, 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:

10 -13 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 202 with GLP statement. All validity criteria were fulfilled.

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

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

5 March 2015

Specific details on test material used for the study:

- Relative Density: 1.196 (pycnometer method, OECD Guideline 109, EU Method A.3)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the loading rate of 100 mg/L at the start of the test (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).
Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration. Regarding properties of the test item, COD 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
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for the fresh media at t=0 h and t=24 h) were prepared under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. 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 50 °C, followed by sonication for ca. 10 minutes. Based on experience on similar substances, these steps (heating and sonication) were essential 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 1 hour before use. The first 100 mL were removed via the drain port. Given that a non-negligible number of particles from the non-soluble fraction of the test item was observed through the water column of the mixing vessel at the loading rate of 100 mg/L, each treatment was filtered before addition into test tubes. After filling and introduction of daphnids the test tubes were sealed immediately. 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- 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.
- 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
- 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 three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia/day. 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

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Total water hardness was approximately 250 mg/L (as CaCO3)

Test temperature:

20.7 - 20.9 °C throughout the test (average value: 20.8 °C)

pH:

pH: 8.01 (t=0 h); 7.88 (t=48 h)

Dissolved oxygen:

Dissolved Oxygen Concentrations: 8.05 mg/L (t=0 h); 7.58 mg/L (t=48 h)

Salinity:

No data

Nominal and measured concentrations:

Nominal concentration: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps.
- Type: Closed
- 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 hours (because of the properties of the test item).
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Chemical analyses were taken from additional replicates in order to avoid disturbing the daphnids at t=24 h and to prevent potential test item losses on opening test tubes
- Loading rate: 5 daphnids per vessel each completely filled with test solution and without headspace
- Introduction of daphnids: Daphnids were introduced into the test medium immediately after filling the test vessels with filtered test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), 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=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) from all treatment group.
Temperature of medium: Measured continuously in a vessel next to the test vessels, over the entire study period, beginning at the start of the test.

RANGE-FINDING STUDY
- A range-finding test was conducted to determine the range of concentrations for the definitive test. In the (semi-static) range-finding test, ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 1.0, 10, 100 and 1000 mg/L (loading) and to a control. The immobility of the daphnids was determined by visual observation after 24 and 48 hours.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at any concentration in the range-finding test. Based on the results of a range-finding test, a limit test was performed at 100 mg/L (loading).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg/L, confirming the observations of the range-finding test (where WAFs were not filtered).

Results with reference substance (positive control):

On January 8, 2016 (most recent test at this time), the 24 h-EC50 was 1.05 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24 h-EC50: 0.6 to 2.1 mg/L) at this time.

Reported statistics and error estimates:

No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours in the final test

 

Nominal concentration (mg/L)*	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
100	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0

* WAF prepared at the given loading rate.

Analytical results:

Concentration of dissolved organic material in the control and the loading rate of 100 mg/L was checked by analysis of the Chemical Oxygen Demand (ST-COD) at start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that few organic compounds were found in the WAFs at 100 mg/L, mainly the first day. Nevertheless, these results show that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0 h-t=24 h Old and t=24 h Fresh-t=48 h). It should be noted that COD values at 100 mg/L were higher the 2nd day than the ones found the first day of the test. This could be explained by the pre-treatment (heating and sonication), probably more effective the second day on the paste pellet. It can be assumed that a longer time of heating / sonication could be more effective and would conduct to a probable increase of the COD 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. Thus, since the study was carried out using WAFs of a natural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing, the results were based on the nominal WAFs concentrations.

Table 6.1.3/2: Concentrations of the test substance in test water - results of the determination of the COD analysis in the final test

	COD (mg O2/L)
Nominal concentration* (mg test item/L)	Start (t=0h)		T=24h Old		T=24h Fresh		End (t=48h)
		Mean		Mean		Mean		Mean
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Control	<LOQ		<LOQ		<LOQ		<LOQ
100	6	6	9	9	27	27	26	26
100	6		8		26		25

 * 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 [O2]: The 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 concentrations, the 48-hour EL50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg/L (loading).

Executive summary:

This study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the test substance to Daphnia magna, under semi-static conditions.

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 at a nominal loading rate of 100 mg/L and to a control. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start, at t=24 h (new and old solutions) and at the end of the test.

 

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that few organic compounds were found in the WAFs the first day at 100 mg/L, and ca. 26-27 mg O2/L the second day of the experimentation. Nevertheless, these results show that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0 h t=24 h Old and t=24 h Fresh-t=48 h). After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg/L, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested loading rates (up to 1000 mg/L).

 

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg/L (loading).

Description of key information

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

WAF, 48h-EL50 (Daphnia magna) > 100 mg/L (loading).

Additional information

One key study is available to assess the 48h acute toxicity of the registered substance to Daphnia magna, under semi-static conditions, according to the OECD Guideline 202 with GLP statement. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test substance at a nominal loading rate of 100 mg/L 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 WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start, at t=24 h (new and old solutions) and at the end of the test. Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that few organic compounds were found in the WAFs the first day at 100 mg/L, and ca. 26-27 mg O2/L the second day of the experimentation. Nevertheless, these results show that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0 h t=24 h Old and t=24 h Fresh-t=48 h). After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg/L, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested loading rates (up to 1000 mg/L). Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg/L (loading).