Santalum austro-caledonicum, 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:

From 2018-01-15 to 2018-02-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD 201 guideline. Some deviations that were considered not to affect the integrity of the study. The validity criteria were successful.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 10, 2017

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test. 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 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 5 L. A magnetic stirring bar was placed in each mixing vessel and test water was added. The loading rates of the test item were weighed on weighing boats that afterwards were placed above each mixing vessel and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water to obtain 5.2 L of test medium, in order to use a maximum volume and to minimise headspace. Then the mixing vessels were closed immediately. 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 directly 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 and colourless.

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

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

between 23.4 and 23.7°C throughout the test (average value: 23.6°C).

pH:

Control: 7.91 at t=0h and 9.70 at t=72h
WAF 1 mg:L: 8.43 at t=0h and 9.72 at t=72h
WAF 3.2 mg/L: 8.30 at t=0h and 9.69 at t=72h
WAF 10.0 mg/L: 8.37 at t=0h and 9.20 at t=72h
WAF 32.0 mg/L: 8.35 at t=0h and 9.02 at t=72h
WAF 100 mg/L: 8.32 at t=0h and 8.80 at t=72h

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10.0, 32.0 and 100 mg/L (loading rate of a WAF)

Details on test conditions:

TEST DETAILS
- Test vessel: 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.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 controls and 3 replicates of each loading rate for counting. Moreover, replicates of each treatment without algae was prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: yes: Original medium of OECD TG 201.
Nutrient Concentration in stock solution
Stock solution 1: macronutrients
NH4Cl 1.5 g.L-1
MgCl2∙6H2O 1.2 g.L-1
CaCl2∙2H2O 1.8 g.L-1
MgSO4∙7H2O 1.5 g.L-1
KH2PO4 0.16 g.L-1
Stock solution 2: iron
FeCl3∙6H2O 64 mg.L-1
Na2EDTA∙2H2O 100 mg.L-1
Stock solution 3: trace elements
H3BO3 185 mg.L-1
MnCl2∙4H2O 415 mg.L-1
ZnCl2 3 mg.L-1
CoCl2∙6H2O 1.5 mg.L-1
CuCl2∙2H2O 0.01 mg.L-1
Na2MoO4∙2H2O 7 mg.L-1
Stock solution 4: bicarbonate
NaHCO3 50 g.L-1

The stock solutions 1 and 3 were sterilised by autoclaving (120 °C, 15 min), then stored in the dark at 4°C. The stock solutions 2 and 4 were sterilised by membrane filtration (mean pore diameter 0.2 µm). The growth medium was prepared by adding an appropriate volume of the stock solutions 1-4 to water. To 500 mL of sterilised water were added:
- 10 mL of stock solution 1
- 1 mL of stock solution 2
- 1 mL of stock solution 3
- 1 mL of stock solution 4
Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: The mean light intensity was of 4788 lux (range: 4608-4893 lux)

EFFECT PARAMETERS MEASURED :
Cell numbers were counted daily by microscope using a counting chamber.

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 3.16): 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L-1.
The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 0.32, 1.00, 3.20 and 10.00 mg.L-1 and to a control. The WAFs were prepared in the dark under closed conditions and by slow-stirring.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

63.816 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% conf limits [41.665 - 118.644 mg/L]

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.188 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf limits [2.461 - 6.866 mg/L]

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.592 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% conf limits [0.453 - 3.262 mg/L]

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.585 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf limits [0.097 - 1.232 mg/L]

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

5.652 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% conf limits [2.603 - 9.204 mg/L]

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

1.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% conf limits [0.318 - 2.052 mg/L]

Details on results:

Biological results
See results above. It should be noted that a slightly more pronounced effect was observed at 1.0 mg.L-1 than at 3.2 mg.L-1. Moreover, more inhibitory effect was expected at 3.2 mg.L-1 regarding the biological results obtained during the range-finding test. However, that was probably due to the preparation of the WAF, as reflected in TOC results, where similar TOC concentrations were determined at 1.0 and 3.2 mg.L-1 in the final test. Nevertheless, this was not considered to affect the integrity of the study or the ELx values, since statistical recalculations (data not shown) excluding the loading rate of 3.2 mg.L-1 showed a similar ErLx value at t=72h (64.428 mg.L-1).

Analytical results
See table 6.1.5/2. The analytical results of this test showed that WAFs concentrations were stable between the start and the end of the test,within the ± 20% of the initial TOC concentrations values, except for the loading rates of 1.0 and 3.2 mg/L with gains of 28 and 35%, respectively. It would be difficult to determine the specific cause of this last observation (measurement uncertainty of the TOC analyser / TOC concentrations values too close to the LOQ?). This probable overestimation constituted a deviation from the test guideline (higher than ± 20% of the initial concentrations values) but was considered not to affect the results and the integrity of the study regardingproperties of the test item and the non-specific analytical method.
It should be noted that 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. Therefore, due to the complex nature of the WAF and since the test item was a multiconstituent 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 21, 2017 (most recent test), the 72h-EC50 was 1.457 mg.L-1 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 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration(mg test item.L^-1)*
		Control	1.0	3.2	10.0	32.0	100.0
t=24h	1	5.6	3.6	4.0	2.0	1.6	0.4
	2	4.0	2.8	4.0	1.6	0.8	0.0
	3	4.8	4.0	3.6	1.6	0.8	0.0
	4	4.0
	5	6.0
	6	6.8
	Mean	5.2	3.5	3.9	1.7	1.1	0.13
	Std. Dev.	1.13	0.61	0.23	0.23	0.46	0.23
	% Inh.	-	17.1	12.0	46.7	69.8	149.4
t=48h	1	26.0	18.8	28.0	3.6	2.4	1.6
	2	30.8	18.0	26.4	4.8	2.8	1.2
	3	26.4	20.0	23.6	4.8	1.6	1.2
	4	24.4
	5	26.0
	6	29.2
	Mean	27.1	18.9	26.0	4.4	2.3	1.3
	Std. Dev.	2.38	1.01	2.23	0.69	0.61	0.23
	% Inh.	-	9.0	1.1	45.7	62.8	75.7
t=72h	1	94.0	68.0	78.8	13.6	10.0	8.0
	2	103.6	72.0	80.0	12.4	10.4	4.8
	3	96.0	60.0	68.0	18.0	11.6	6.0
	4	108.4
	5	89.2
	6	94.0
	Mean	97.5	66.7	75.6	14.7	10.7	6.3
	Std. Dev.	7.09	6.11	6.61	2.95	0.83	1.62
	% Inh.	-	7.2	4.8	36.1	42.0	52.5

* WAF prepared at the given loading rate.

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

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

Validity criteria of the study

Cell density in controls: 195-fold increase within 72 hours. The specific growth rate was determined at 1.76 day^-1, so greater than 0.92 day^-1.

Coefficient of variation:

1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 31.3%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.4%.

Thus the validity criteria were respected in this study.

Table 6.1.5/2: Results of the determination of TOC analysis (mg/L) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)	Relative loss to initial value (t=0h - t=72h)(%)
Control	0.85	1.18	-39
1.0	0.87	1.11	-28
3.2	0.84	1.13	-35
10.0	3.08	3.11	-1
32.0	5.18	5.32	-3
100.0	9.76	9.16	6

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

The pH level in the control varied more than 1.5 units at the end of the test (1.79 units of difference), but this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of more sodium bicarbonate.

All other test conditions (light intensity and temperature) remained within the ranges prescribed by the study plan and the test guideline (temperature: 23°C ± 2°C, constant within 2°C; light intensity: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

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 63.816 mg/L and 4.188 mg/L, respectively. The 72-hour EL10 for growth rate was 1.592 mg/L and for yield was 0.585 mg/L.

Executive summary:

A study was performed to assess the test item SANDALWOOD AUSTROCALEDONIEN OIL for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The method followed was 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).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L and to a control. The inhibition of growth in relation to control cultures was determined in a static test over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the controls and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the startand the end of the test.

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

After 72 hours of exposure and based on nominal loading rates, E_rL_x and E_yL_x values were as follows, under test conditions:

Parameter	Growth rate (mg.L^-1)	Yield (mg.L^-1)
72-hour EL10	1.592	0.585
95% conf. limits	0.453 – 3.262	0.097 – 1.232
72-hour EL20	5.652	1.150
95% conf. limits	2.603 – 9.204	0.318 – 2.052
72-hour EL50	63.816	4.188
95% conf. limits	41.665 – 118.644	2.461 – 6.866

Statistical analyses were performed by the computer program ToxRat

Description of key information

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 63.816 mg/L and 4.188 mg/L, respectively. The 72-hour EL₁₀ for growth rate was 1.592 mg/L and for yield was 0.585 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

63.816 mg/L

EC10 or NOEC for freshwater algae:

1.592 mg/L

Additional information

For that endpoint, a study on the registered substance was available. This study was performed to assess the registered substance for its ability to generate toxic effects on Pseudokirchneriella subcapitata, under static conditions and during 72 hours. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Under the experimental conditions andbased on nominal loading rates,the 72-hour EL₅₀for the parameters growth rate and yield were determined to be 63.816 mg/L and 4.188 mg/L, respectively. The 72-hour EL₁₀for growth rate was 1.592 mg/L and for yield was 0.585 mg/L.

The study presents some deviations concerning analytical results. Indeed, the analytical results of this test showed that WAFs concentrations were stable between the start and the end of the test, within the ± 20% of the initial TOC concentrations values, except for the loading rates of 1.0 and 3.2 mg/L with gains of 28 and 35%, respectively. It would be difficult to determine the specific cause of this last observation (measurement uncertainty of the TOC analyser / TOC concentrations values too close to the LOQ?). This probable overestimation constituted a deviation from the test guideline (higher than ± 20% of the initial concentrations values) but was considered not to affect the results and the integrity of the study regardingproperties of the test item and the non-specific analytical method.

It should be noted that 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. Therefore, due to the complex nature of the WAF and since the test item was a multiconstituent substance, the results were based on the nominal test loading rates and as Effective Loading Rate 50, according to the OECD No. 23.

Moreover, this study had one minor deviation concerning some pH values but it was considered not to affect the results and the integrity of the study. Besides that, the validity criteria were fulfilled. This study was therefore considered acceptable for that endpoint.