Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

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 January 8th, 2019 to January 21st, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.3 – 1.0 – 3.0 – 10.0 - 32.0 - 102.0 - and 328.0 mg/L.
- Sampling method: At the initial time (T0h), all the solutions were analysed taken directly from the starting solutions without algae. For other times (T24h, T48h, T72h), around 1 mL were sampled in one flask for chemical analyses.
- Sample storage conditions before analysis: All the solutions were kept in the refrigerator or freeze.

Vehicle:

yes

Remarks:

OECD medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary works (non GLP) were performed to define the solution preparation protocol and to obtain the maximum achievable dissolved concentration of a substance in the test medium. Test item concentrations were measured by total carbon (TC) after stirring times of 16 and 24 hours. The TC measurements indicated that the equilibrium time was achieved at 24 hours. Considering the density of the test item lower than 1, the decantation was chosen as technical separation. Based on these results, the prepared samples were stirred 24 hours at 20 ± 2°C and at darkness. After this step, the samples were filtered on cellulose acetate (filter porosity: 0,45 μm) after a settling period of 60 minutes.
- Controls: A concurrent untreated control (6 biotic replicates) was examined in parallel to the test item concentrations. Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- Chemical name of vehicle: OECD medium.

Test organisms (species):

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

Details on test organisms:

- Common name: Freshwater unicellular green alga
- Strain: CCAP 278.4
- Source (laboratory, culture collection): Laboratoire de Chimie-Écotoxicologie, FCBA (Institut Technologique Forêt Cellulose Bois Construction Ameublement), Pôle des Laboratoires Bois, Allée de Boutaut – BP 227, 33028 BORDEAUX Cedex - FRANCE
- Method of cultivation: 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).

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.4-23.1

pH:

8.1

Nominal and measured concentrations:

Nominal concentrations: control, 0.3 , 1, 3, 10, 32, 102, 328 mg/L
Measured concentrations (geometrical mean): control, 0.197, 0.282, 0.306, 0.459, 0.532, 0.681 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open / closed: open
- Material, size, headspace, fill volume: 250 mL flasks
- Initial cells density: 10^4 cells/mL
- Control end cells density: 47.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was not adjusted with HCl and NaOH.
- Light intensity and quality: 6193Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman Coulter Z2 and Malassez counting cell at 24, 38 and 72h.

TEST CONCENTRATIONS
- Range finding study: A preliminary test, non GLP, was performed to choose seven concentrations for this final test.
- Test concentrations: 0.3 , 1, 3, 10, 32, 102, 328 mg/L
- Results used to determine the conditions for the definitive study: An inhibition growth rate was observed at 100 mg/L during the preliminary test. The concentrations chosen for the final test were based on these observations. The response variables in the Control and treatment groups were analysed, using a statistical test to compare means with scientific software Minitab.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.282 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.306 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.681 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.455 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.341 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.283 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: stability of the test item.
- Effect concentrations exceeding solubility of substance in test medium: yes, 10, 32, 102, 328 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The last algal growth inhibition test performed with the reference item K2Cr2O7 (October 08, 2018) gave an ErC50(0-72h) = 0.90 mg/L (0.76 – 1.04), 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 201 Guideline) and in accordance with previous data obtained by the laboratory

Reported statistics and error estimates:

Statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17. A significant difference between the control was observed at the concentration 0.306 mg/L.

According to the Guidance document on aquatic toxicity testing of difficult substances and mixtures OECD n°23, 15/12/00 [9] and the OECD 201, the deviation from the measured initial concentration was not within the range of ± 20 %, the calculation and the test results were determined and expressed relative to the geometric mean of the measured concentrations and not determined and expressed in nominal test item concentrations.

Table 1.Inhibitions of the average specific growth rate in comparison to the Control

Geometric mean measured test item concentration		Algae x10^4 /mL at 0 hour	Specific growth rate µ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1	1.600	Not applicable		1.0	-
	b		1.640
	c		1.612
	d		1.599
	e		1.596
	f*		1.618
0.197	a	1	1.674	-3.9	-3.9	Not required	ns
	b		1.658	-2.9
	c		1.689	-4.8
0.282	a	1	1.657	-2.8	-1.8		ns
	b		1.663	-3.2
	c		1.597	0.9
0.306	a	1	1.530	5.0	9.2		*
	b		1.432	11.1
	c		1.413	12.3
0.459	a	1	1.393	13.5	20.0		*
	b		1.213	24.7
	c		1.231	23.6
0.532	a	1	1.044	35.2	38.7
	b		0.985	38.9
	c		0.926	42.5
0.681	a	1	0.969	39.9	35.6		*
	b		1.087	32.5
	c		1.047	35.0

Table 2.Inhibitions of the yield in comparison to the Control are presented in the following table:

Geometric mean measured test item concentration		[algae]   x10^4/mL at 0 hour	[algae] x10^4/mL at 72 hours	Yield x10^4/m	Average yield x10^4/mL	Yield inhibition (Iy) (%)
						Per replicate	Average/ replicate
Control	a	1	121.6	120.6	124.7	Not Applicable
	b		137.0	136.0
	c		126.1	125.1
	d		121.1	120.1
	e		120.0	119.0
	f*		128.4	12704
0.197	a	1	151.9	150.9	150.8	-21,0	-21.0
	b		144.7	143.7		-15,2
	c		158.9	157.9		-26,6
0.282	a	1	144.2	143.2	136.2	-14,8	-9.2
	b		146.8	145.8		-16,9
	c		120.5	119.5		4,2
0.306	a	1	98.5	97.5	79.4	21,8	36.3
	b		73.4	72.4		41,9
	c		69.4	68.4		45,1
0.459	a	1	65.3	64.3	46.8	48,4	62.4
	b		38.0	37.0		70.3
	c		40.2	39.2		68.6
0.532	a	1	22.9	21.9	18.4	82.4	85.2
	b		19.2	18.2		85.4
	c		16.1	15.1		87.9
0.681	a	1	18.3	17.3	21.5	86.1	82.8
	b		26.1	25.1		79.9
	c		23.1	22.1		82.3

Table 3. Raw data, i.e. algal concentrations values expressed as cell number *10^4 per mL of test solutions after 24, 48 and 72 hours of incubation are indicated

Nominal test item concentration		[algae] x10^4 /mL at 24 hours			[algae] x10^4 /mL at 48 hours			[algae] x10^4 /mL at 72 hours
		Count.	Average	CV	Count.	Average	CV	Count.	Average	CV
Control	a	3.0	3.1	5.2	22.3	21.5	9.5	121.6	125.7	5.1
	b	3.4			20.8			137.0
	c	3.0			20.1			126.1
	d	3.1			20.7			121.1
	e	3.0			19.9			120.0
	f	3.0			25.3			128.4
0.3	a	2.3	2.0	12.4	24.3	18.7	28.0	151.9	151.8	4.7
	b	2.0			18.0			144.7
	c	1.8			13.9			158.9
1.0	a	1.9	1.9	3.0	15.8	13.1	18.1	144.2	137.2	10.6
	b	2.0			11.6			146.8
	c	1.9			11.8			120.5
3.0	a	1.8	1.8	3.1	11.0	10.1	8.3	98.5	80.4	19.6
	b	1.8			9.8			73.4
	c	1.9			9.4			69.4
10.0	a	2.2	2.2	0.0	8.9	7.7	13.8	65.3	47.8	31.7
	b	2.2			6.8			38.0
	c	2.2			7.5			40.2
32.0	a	2.1	2.1	0.0	4.4	4.3	4.0	22.9	19.4	17.5
	b	2.1			4.1			19.2
	c	2.1			4.4			16.1
102.0	a	2.1	2.1	7.4	4.4	5.3	15.6	18.3	22.5	17.5
	b	1.9			6.0			26.1
	c	2.2			5.6			23.1
328.0	a	2.1	2.1	/	15.9	12.9	/	131.1	113.8	/
	b	2.3			12.4			88.8
	c	1.9			10.4			121.5

Table 4. Coefficients of variation for section-by-section specific growth rates in the control cultures 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	1.099	1.125	4.5	2.006	1.940	5.8	1.696	1.768	5.4	1.600	1.611	28.8	27.2
	b	1.224			1.811			1.885			1.640		22.1
	c	1.099			1.902			1.836			1.612		27.7
	d	1.131			1.899			1.766			1.599		25.7
	e	1.099			1.892			1.797			1.596		27.1
	f	1.099			2.132			1.624			1.618		31.9

Table 5. pH values and observations

Nominal test item concentration (mg/L)	pH at the start (biotic replicates)	pH after 72 hours (biotic replicates)
Control	8.1	7.8
0.3	7.9	7.7
1.0	8.0	7.8
3.0	8.0	7.6
10.0	7.9	7.6
32.0	7.9	7.7
102.0	7.8	7.5
328.0	7.8	7.7

Validity criteria fulfilled:

yes

Remarks:

Biomass increased exponentially by a factor of a least 16 (125.7), Daily section-by-section specific growth rate CV (27.2%) were ≤ 35%, and Average specific growth rate CV during 72 hours (1.0%) was ≤ 7%.

Conclusions:

The 72h-ErC50 of the test item was > 0.681 mg/L, value expressed in geometric mean measured test item concentration and based on inhibition of growth rate. The EC50 was 0.351 mg/L based on biomass. The EC10 based on growth rate and biomass were 0.341 mg/L and 0.288 mg/L. The NOEC and LOEC determined for the test item or the endpoint inhibition of growth rate were 0.282 and 0.306 mg/L respectively.

Executive summary:

A freshwater algae growth inhibition test was performed on the test item, according to OECD 201, under GLP conditions. Based on the results of a preliminary study performed at concentrations of 10, 50, 100 mg/L test item, where an inhibition of growth rate was observed between at100 mg/L, Pseudokirchneriella subcapitata was exposed to 0.3, 1, 3, 10, 32, 10 and 328 mg/L test item (nominal) or medium (negative control) for 72h under static conditions. Six replicates were run for the control and 3 for the test item concentrations. Analytical monitoring was performed using a validated HPLC method and measurements were taken each day of the study. The deviation from the measured initial concentration was not within the range of ± 20 %, and according to OECD guidelines, the calculation and the test results were determined and expressed relative to the geometric mean of the measured concentrations and not determined and expressed in nominal test item concentrations. The geometric means of the test item measured concentrations were 0.197, 0.282, 0.306, 0.459, 0.532, and 0.681 mg/L. All validity criteria were met. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is regularly performed with potassium dichromate. The 72h-ErC50 of the test item was greater than 0.681 mg/L, value expressed in geometric mean measured test item concentration and based on inhibition of growth rate. The EyC50 was 0.351 mg/L based on biomass. The NOEC and LOEC determined for the test item for the endpoint inhibition of growth rate were 0.282 and 0.306 mg/L respectively and the EC10 based on growth rate and biomass were 0.341 mg/L and 0.288 mg/L.

Description of key information

Key study. Method according to OECD 201, GLP study. The 72h-EC50 of the test item in freshwater algae was determined to be greater than 0.681 mg/L expressed on the measured test item geometric mean concentration based on growth rate. The overall NOEC and LOEC determined for the test item for the endpoint inhibition of growth rate were 0.282 and 0.306 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.681 mg/L

EC10 or NOEC for freshwater algae:

0.341 mg/L

Additional information

A freshwater algae growth inhibition test was performed on the test item, according to OECD 201, under GLP conditions. Based on the results of a preliminary study, Pseudokirchneriella subcapitata was exposed to 0.3, 1, 3, 10, 32, 10 and 328 mg/L test item (nominal) or medium (negative control) for 72h under static conditions. Six replicates were run for the control and 3 for the test item concentrations. The deviation from the measured initial concentration was not within the range of ± 20%, and according to OECD guidelines, the calculation and the test results were determined and expressed relative to the geometric mean of the measured concentrations and not determined and expressed in nominal test item concentrations. The geometric means of the test item measured concentrations were 4.197, 0.282, 0.306, 0.459, 0.532, and 0.681 mg/L. The 72h-ErC50 of the test item was greater than 0.681 mg/L, value expressed in geometric mean measured test item concentration and based on inhibition of growth rate. The EC50 was 0.351 mg/L based on biomass. The NOEC and LOEC determined for the test item for the endpoint inhibition of growth rate were 0.282 and 0.306 mg/L respectively. The EC10 based on growth rate and biomass were 0.341 mg/L and 0.288 mg/L.