Essential oil of Cistus ladaniferus L (Cistaceae) obtained from stems and leaves by distillation

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:

2016-10-25 to 2016-11-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. The validity criteria were successfully fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 January 2016

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of “CISTUS ORGANIC OIL” were performed in the test solutions (biotic and abiotic).
In order to verify the nominal or initial concentrations of the test item and maintenance of the concentrations during the ecotoxicological testing, chemical analysis of “CISTUS ORGANIC OIL” in algae test solutions test solutions were performed:
- at the beginning of the test (T = 0h)
- at the end of the test (T = 72h)

Schedule analysis at initial time (T0h):
Control Abiotic
10.0 mg/L Abiotic
32.0 mg/L Abiotic
102.0 mg/L Abiotic
328.0 mg/L Abiotic
1050.0 mg/L Abiotic

Schedule analysis at final time (T72h):
Control Biotic Control Abiotic
10.0 mg/L Biotic 10.0 mg/L Abiotic
32.0 mg/L Biotic 32.0 mg/L Abiotic
102.0mg/L Biotic 102.0 mg/L Abiotic
328.0 mg/L Biotic 328.0mg/L Abiotic
1050.0 mg/L Biotic 1050.0 mg/L Abiotic

The measured concentrations of the “CISTUS ORGANIC OIL” should not vary more than ± 20 % of nominal or initial measured concentration during the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was not soluble in the test medium, a special solubilisation protocol was carried out before the experimentation, according to the OECD n°23 guidance document on aquatic toxicity testing of difficult substances and mixtures. This method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20±2°C (these concentrations were prepared by adding directly the test item in flasks containing test medium).

Cultures of exponentially-growing algae were exposed to five test item concentrations in algae test medium (OECD medium) plus a control.
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline.

Preparation of test solutions for the testing
Nominal concentration (mg/L): 10.0 32.0 102.0 328.0 1050.0
Test item (mg) 10.04 32.02 102.04 328.01 1050.06
Final volume (mL): 1000

All the test solutions plus WAF Control (algae test medium) were then filtered through a membrane filter (cellulose acetate 0.45 µm batch G9952942) to remove suspended particles: solutions obtained were named WAF (Water Accommodated Fraction).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
Freshwater unicellular green alga Pseudokirchneriella subcapitata of the chlorococcal order (formerly named Raphidocellis subcapitata) strain number CCAP 278.4 was used in this study.
The algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

Temperature 22.5 – 23.3 °C

pH:

pH at the start: 7.5 - 8.0
pH after 72 h: 7.5 - 7.7

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0; 10.0; 32.0; 102.0; 328.0; 1050.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Initial cells density: 10000 cells/mL at the start of the test
- No. of vessels per concentration (replicates): 3 biotic replicates + 1 abiotic replicate per concentration
- Control: 6 biotic replicates + 2 abiotic replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae test medium (OECD medium); Algae dilution water

OTHER TEST CONDITIONS
- Light intensity and quality: 6096 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The algae growth inhibition in relation to the control was evaluated over a period of 72 hours. The algae cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter (Beckman Coulter Z2) and/or a microscope with a Malassez counting cell.
- The pH of each test concentration was measured at the start and at the end of the test. The pH of the control medium had not increased by more than 1.5 units during the test.

TEST CONCENTRATIONS
- Range finding study: 5; 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Average inhibition growth rate (%): 6.2; 4.8 and 38.4 at 5, 10 and 100 mg/L, respectively at 72 h. Based on the results of the preliminary test, the following test concentrations 10; 32; 102; 328; 1050 mg/L were proposed for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

99.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval (mg/L): [86.6; 112.2]

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See "any other information on results incl. tables"

Results with reference substance (positive control):

The last algae growth inhibition test performed with the reference item K2Cr2O7 (August 23, 2016) gave an ErC50(0-72h) = 0.75 mg/L (0.66 – 0.85), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.

Reported statistics and error estimates:

None

Algae growth inhibition

Raw data,i.e. algae concentrations values expressed as cell number *10⁴per mL of test solutions after 24, 48 and 72 hours of incubation are indicated in the following table.

Nominal test item concentration		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	cv	Count.	Average	cv	Count.	Average	cv
Control	a	4,4	4.1	4.6	27,5	28.5	12.0	107,8	101.0	7.4
	b	4,1			29,4			94,8
	c	4,1			22,5			98,7
	d	3,9			32,6			90,4
	e	4,0			28,6			107,2
	f	3,9			30,6			107,0
WAF 10.0	a	3,3	3.2	3.6	18,2	21.1	13.3	71,4	80.8	13.6
	b	3,1			23,8			92,9
	c	3,1			21,3			78,0
WAF 32.0	a	2,1	1.9	10.8	13,2	11.6	12.3	42,5	39.4	10.5
	b	1,7			11,3			40,9
	c	2,0			10,4			34,7
WAF 102.0	a	1,7	1.7	3.3	3,6	3.5	10.3	9,4	8.9	17.4
	b	1,8			3,8			10,2
	c	1,7			3,1			7,2
WAF 328.0	a	1,7	1.8	3.3	2,3	2.2	5.3	2,5	2.2	13.3
	b	1,8			2,1			2,0
	c	1,8			2,1			2,0
WAF 1050.0	a	0,9	1.1	28.4	1,1	1.1	31.0	1,0	0.9	22.3
	b	1,5			0,8			0,7
	c	1,0			1,5			1,1

CV = Coefficient of variation.

 

Specific growth rate

 

The coefficients of variation for section-by-section specific growth rates in the control cultures are presented in the following table to calculate one of the validity criterion: average value of coefficient of variation (Average CV).

Control replicates		Specific growth rate
		0-24h	Average	CV (%)	24 -48h	Average	CV (%)	48 -72h	Average	CV (%)	0 - 72h	Average	CV per replicate (%)	Average CV
Control	a	1,482	1.402	3.2	1,833	1.943	7.9	1,366	1.268	12.6	1,560	1.538	15,6	23.5%
	b	1,411			1,970			1,171			1,517		27,0
	c	1,411			1,703			1,479			1,531		10,0
	d	1,361			2,123			1,020			1,501		37,6
	e	1,386			1,967			1,321			1,558		22,8
	f	1,361			2,060			1,252			1,558		28,2

CV: Coefficient of variation (%)

Calculation of the E_rC₅₀(0-72h)

Inhibitions of the average specific growth rate in comparison to the Control are presented in the following table:

Nominal test item concentration		Algae *10^4/mL at 0hour	Average growth rate µ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1		Not applicable		1.6	-
	b
	c
	d		1.538
	e
	f
10.0	a	1		7,5	4,8	Not required	ns
	b		1.464	1,8
	c			5,6
32.0	a	1		18,8	20,4	Not required	*
	b		1.224	19,6
	c			23,1
102.0	a	1		51,4	52,6	Not required	*
	b		0.730	49,7
	c			57,2
328.0	a	1		80,1	83,2	Not required	*
	b		0.258	85,0
	c			85,0
1050.0	a	1		100,0	101,5	Not required	*
	b		-0.023	107,7
	c			97,9

- : not relevant

ns : not significantly different compared to the Control, Dunnet test, α =0.05

* : significantly different compared to the Control, Dunnett test, α=0.05

 

The average specific growth rate (µ) was determined according to the OECD guideline.

 

Statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17.2.1 to determined NOEC/LOEC. No significant difference between the control was observed at the lowest concentration 10.0 mg/L.

NOEC = 10 mg/L

LOEC = 32.0 mg/L

 

The figure: specific growth rate inhibiton versus test item is presentedin Annex 3.

 

The equation of the regression (non linear regression) is the following (rounded values):

 

 % inhibition growth rate = 115.476*[concentration] / 130.289+[concentration]

 

Where: [concentration]= concentration of test item (mg/L)

 

From the equation, the ErC₅₀-(0-72h) was calculated and the value is the following:

ErC₅₀-(0-72h)= 99.5 mg/L [86.6 – 112.2] value expressed in nominal concentration

 

The results obtained for thetest item “CISTUS ORGANIC OIL”on the toxicological endpoint “inhibition of growth rate” indicate anErC₅₀-(0-72h)equal to 99.5 mg/L [86.6 – 112.2]value expressed in nominal test item concentration.

 

From the equation, the ErC₁₀-(0-72h) was calculated and the value is the following:

ErC₁₀-(0-72h)= 12.4 mg/L value expressed in nominal concentration

 

The results obtained for thetest item “CISTUS ORGANIC OIL”on the toxicological endpoint “inhibition of growth rate” indicate anErC₅₀-(0-72h)equal to 12.4 mg/Lvalue expressed in nominal test item concentration.

Calculation of the EyC₅₀(0-72h)

Inhibitions of the yield in comparison to the Control are presented in the following table:

Nominal test item concentration		[algae] *10^4/mL at 0 hour	[algae] *10^4/mL at 72 hours	Yield (*10^4/mL)	Average yield (*10^4/mL)	Yield inhibition (Iy) (%)
						Per replicate	Average/ replicate
Control	a	1	107,8	106,8	100.0	Not Applicable
	b		94,8	93,8
	c		98,7	97,7
	d		90,4	89,4
	e		107,2	106,2
	f		107,0	106,0
1.0 mg/L	a	1	71,4	70,4	79.8	29,6	20.2
	b		92,9	91,9		8,1
	c		78,0	77,0		23,0
3.2 mg/L	a	1	42,5	41,5	38.4	58,5	61.6
	b		40,9	39,9		60,1
	c		34,7	33,7		66,3
10.2 mg/L	a	1	9,4	8,4	7.9	91,6	92.1
	b		10,2	9,2		90,8
	c		7,2	6,2		93,8
32.6 mg/L	a	1	2,5	1,5	1.2	98,5	98.8
	b		2,0	1,0		99,0
	c		2,0	1,0		99,0
1 04.3 mg/L	a	1	1,0	0,0	-0.1	100,0	100.1
	b		0,7	-0,3		100,3
	c		1,1	0,1		99,9

 

The figure: Yield inhibition versus test item is presentedin Annex 4.

 

The equation of the regression (non linear regression) is the following (rounded values):

 

 % Yield inhibition e = 107.288*[concentration] / 26.6804+[concentration]

 

Where: [concentration]= concentration of test item (mg/L)

 

From the equation, the EyC₅₀-(0-72h) was calculated and the value is the following:

EyC₅₀-(0-72h)= 23.3 mg/L value expressed in nominal concentration

 

The results obtained for thetest item “CISTUS ORGANIC OIL”on the toxicological endpoint “inhibition of growth rate” indicate anEyC₅₀-(0-72h)equal to 23.3 mg/L value expressed in nominal test item concentration.

The raw data did not permit to determined EyC₁₀-(0-72h) with reliability because it will be lower than 10.0 mg/L (lowest tested concentation).

 

Results for the chemical analysis

The table below summarizes the results achieved and the variation between T0 and T72 hours.

Nominal test item concentration	mg “NPOC”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour	Results at T72 hours
Control Abiotic	< 2	< 2	NA
10 mg/L Abiotic	3.0	4.2	+40.0
32 mg/L Abiotic	7.2	4.4	-38.9
102 mg/L Abiotic	16.9	12.7	-24.9
328 mg/L Abiotic	49.8	38.9	-21.9
1050 mg/L Abiotic	134.2	110.9	-17.4

NA: not applicable

Nominal test item concentration	mg “NPOC”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour	Results at T72 hours
Control Biotic	< 2	2.8	NA
10 mg/L Biotic	3.0	5.7	+90.0
32 mg/L Biotic	7.2	7.5	+4.2
102 mg/L Biotic	16.9	12.7	-24.9
328 mg/L Biotic	49.8	35.6	-28.5
1050 mg/L Biotic	134.2	111.0	-17.3

NA: not applicable

 

Under Abiotic conditions (without algae), the test item “CISTUS ORGANIC OIL” is not considered to be stable for the nominal concentrations 10 – 32 – 102 and 328 mg/L because the measured concentrations of the test item did vary by more than ± 20% of the initial concentrations during the test.For the concentration 1050 mg/L, the deviation between T0 and T72 hours was less than± 20% of the initial concentration.

Under biotic conditions (with algae), the test item “CISTUS ORGANIC OIL” is not considered to be stable for the nominal concentrations 10 – 102 and 328 mg/L. For the two concentrations (32 and 1050 mg/L),the deviation between T0 and T72 hours, the deviation between T0 and T72 hours was less than± 20% of the initial concentration.

According to theses results and for this study, the concentrations are expressed in nominal mg “CISTUS ORGANIC OIL” / L.

Validity criteria

	Criteria for OECD 201	Validity criteria conformity
Increase biomass during 72 hours	Factor of at least 16	Yes (101.0)
Daily section-by-section specific growth rate CV	< 35%	Yes (23.5%)
Average specific growth rate CV during 72 hours	< 7%	Yes (1.6%)

All the validity criteria were successfully fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “CISTUS ORGANIC OIL” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) equal to 99.5 mg/L [86.6 – 112.2] and an ErC10-(0-72h) equal to 12.4 mg/L, both values expressed in nominal test item concentration.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item CISTUS ORGANIC OIL at 10.0; 32.0; 102.0; 328.0 and 1050.0 mg/L over an exposure period of 72 h. The algae growth inhibition in relation to the control was evaluated over a period of 72 h.

The results of this study are summarized below:

Test item: CISTUS ORGANIC OIL

Test system: Pseudokirchneriella subcapitata

Exposure duration: 72 hours

Test concentrations: Control - 10.0- 32.0 – 102.0 – 328.0 and 1050.0 mg/L

Incubator: Temperature 22.5 to 23.3 °C ; shaking at 120 rpm; light intensity: 6096 Lux

Measurement of cell growth:  Beckman Coulter Z2

Results (growth rate inhibition):                                       

Test item (nominal concentration)	ErC10-(0-72h)	ErC50-(0-72h) With 95 % confidence interval [lower – upper]
CISTUS ORGANIC OIL	12.4 mg/L	99.5 mg/L [86.6 – 112.2]

 

For yield inhibition, the results obtained for the test item “CISTUS ORGANIC OIL” indicate an 72h-EyC50 equal to 23.3 mg/L value expressed in nominal test item concentration. The raw data did not permit to determine the 72h-EyC50 with reliability because it will be lower than 10.0 mg/L (lowest tested concentration).

Validity criteria

In the control, the increase of the biomass during 72 hours was determined to be higher than a factor of 16.

The coefficients of variation ofdaily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35 % and 7% in the control.

The validity criteria were successful fulfilled.

Description of key information

Based on a GLP study performed according to the OECD 201 guideline, the results obtained for the test item “CISTUS ORGANIC OIL” on the toxicological endpoint “inhibition of growth rate” indicate an 72h-ErC50 equal to 99.5 mg/L [86.6 – 112.2] and an 72h-ErC10 equal to 12.4 mg/L, both values expressed in nominal test item concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

99.5 mg/L

EC10 or NOEC for freshwater algae:

12.4 mg/L

Additional information

A reliable algal growth inhibition study was available and performed according to OECD Guideline 201 and in compliance with GLP. The freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item CISTUS ORGANIC OIL at 10.0; 32.0; 102.0; 328.0 and 1050.0 mg/L over an exposure period of 72 h.

Results (growth rate inhibirion):                                       

Test item (nominal concentration)	ErC10-(0-72h)	ErC50-(0-72h) With 95 % confidence interval [lower – upper]
CISTUS ORGANIC OIL	12.4 mg/L	99.5 mg/L [86.6 – 112.2]

For yield inhibition, the results obtained for the test item “CISTUS ORGANIC OIL” indicate an 72h-EyC50 equal to 23.3 mg/L value expressed in nominal test item concentration. The raw data did not permit to determine the 72h-EyC50 with reliability because it will be lower than 10.0 mg/L (lowest tested concentration).

The validity criteria were successfully fulfilled. This study was considered acceptable for that endpoint.