Resinoid of Commiphora erythraea (Burseraceae) obtained from the gum by ethanol extraction

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:

25 - 29 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 201 with GLP statement. All val idity criteria were fulfilled.

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

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

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: 1.55 (pycnometer method, OECD Guideline 109, EU Method A.3)

Analytical monitoring:

yes

Details on sampling:

Concentration of dissolved organic material was checked by analysis of the Chemical Oxygen Demand (ST-COD). 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. Duplicate abiotic samples for analysis were taken from the control and the loading rate of 100 mg/L at the start and every day thereafter until the end of the test.

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 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 ± hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5 x 10^3 cells/mL 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):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: 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.
- 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 x 10^4 cells/mL. 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:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

23.2-23.6 °C throughout the test (average value: 23.4 °C)

pH:

8.38 (0 h); 9.60 (72 h)

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Nominal concentration: 100 mg/L (loading rate)

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.
- Type: Closed
- Initial cells density: An initial cell density of 5 x10^3 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 6 replicates
- No. of vessels per control (replicates): 6 replicates
* Moreover, replicates of each treatment without algae will be prepared for chemical analyses.

TEST MEDIUM / WATER PARAMETERS
- Test water: 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.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity was 5042 lux (range: 4754-5301 lux)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0 h and t=72 h).
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
- Range finding study: A range-finding test was conducted to determine the range of concentrations for the definitive test. Algal cells were exposed to the nominal concentrations (in triplicate) of 1.0, 10, 100 and 1000 mg/L (loading) and to a control for 72 hours. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.
- Results used to determine the conditions for the definitive study: Algal growth inhibition at 72 hours was 0.4, 0.3, 0.2 and 0.1% at 1.0, 10, 100 and 1000 mg/L, respectively. 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

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed (quasi-identical growth curves), confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates (up to 1000 mg/L).
Based on these results, no ErCx and EyCx values could be determined due to the absence of effect at the tested loading rate.

Results with reference substance (positive control):

On January 29, 2016 (most recent test), the 72 h-EC50 was 0.84 mg/L 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 72 h-EC50: 0.60 to 1.03 mg/L).

Reported statistics and error estimates:

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

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mLx10⁴)

Time	Replicate	Nominal concentration(mg/L)*
		Control	100
t=24 h	1	2.8	6.4
	2	5.6	4.4
	3	4.4	4.8
	4	6.0	4.4
	5	5.2	4.0
	6	4.0	4.8
	Mean	4.7	4.8
	Std. Dev.	1.18	0.84
	% Inhibition	-	-2.1
t=48 h	1	27.2	24.0
	2	24.0	24.8
	3	25.6	23.6
	4	24.8	24.0
	5	23.2	23.2
	6	25.6	24.4
	Mean	25.1	24.0
	Std. Dev.	1.40	0.57
	% Inhibition	-	1.1
t=72 h	1	85.6	79.6
	2	88.4	75.6
	3	86.4	80.0
	4	86.4	79.2
	5	79.6	80.8
	6	83.6	84.0
	Mean	85.0	79.9
	Std. Dev.	3.06	2.71
	% Inhibition	-	1.2

* WAF prepared at the given loading rate.

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

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 6 replicates of each test concentration.

Std. Dev.: standard deviation

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 the start and every day thereafter until 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 (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 30 mg O2/L in the WAFs at 100 mg/L.

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 6.1.5/2: 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		t=48h		End (t=72h)
		Mean		Mean		Mean		Mean
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Control	<LOQ		<LOQ		<LOQ		<LOQ
100	37	38	30	29	22	20	25	26
100	39		27		18		26

 * WAF prepared at the given loading rate

Physico-chemical parameter values throughout the test

The temperature in the incubator was situated between 23.2 and 23.6 °C throughout the test (average value: 23.4 °C). The mean light intensity was of 5042 lux (range: 4754-5301 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23 ± 2 °C, constant within 2 °C; light intensity: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

 

Validity criteria of the study:

Cell density in Controls: 170-fold increase within 72 hours

Coefficient of Variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 32.7%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 0.7%.

Thus the validity criteria were respected in this study.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were estimated to be higher than 100 mg/L (loading).

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, algal cells (Pseudokirchneriella subcapitata) 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 potential 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 was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start and every day thereafter until the end of the test. Definitive test was performed based on the results of range-finding 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 (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 30 mg O2/L in the WAFs at 100 mg/L. After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates (up to 1000 mg/L). Based on these results, no ErCx and EyCx values could be determined due to the absence of effect at the tested loading rate.

 

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were estimated to be higher than 100 mg/L (loading).

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

WAF, 72h-ErL50 (Pseudokirchneriella subcapitata) > 100 mg/L (loading)

WAF, 72h-NOEC (Pseudokirchneriella subcapitata) > 100 mg/L (loading)

Additional information

One key study is available to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to the OECD Guideline 201 with GLP statement. Following a preliminary range-finder test, the algae 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 potential 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 was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the limit test concentration at the start and every day thereafter until 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 (closed conditions with minimum headspace), COD analyses indicate that organic compounds were found ca. 30 mg O2/L in the WAFs at 100 mg/L, although this is not unusual as non-soluble fractions are excluded from WAFs. After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates (up to 1000 mg/L). Based on these results, no ErCx and EyCx values could be determined due to the absence of effect at the tested loading rate. Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were estimated to be higher than 100 mg/L (loading).