3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 17, 2019 - June 28, 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: control, abiotic control, 10, 328 and 1050 mg/L, were sampled at the start and at the end of the test (0, 72h).
- Sampling method: At the initial time (T0h), all the solutions were analysed taken directly from the starting solutions without algae. At the end of the test (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 freezer.

Vehicle:

no

Details on test solutions:

- Test solution: the test item was soluble in test medium, so a stock solution was prepared by direct addition of the test item.
- Controls: 6 biotic replicates
- Reference substance: potassium dichromate

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:

no

Total exposure duration:

72 h

Test temperature:

22.8 – 23.5 °C

pH:

8.2

Nominal and measured concentrations:

Nominal concentrations: control (0), 10 , 32, 102, 328, 1050 mg/L
Measured concentrations (T0 - 72h): (0), 9.5, 311.4 - 311.8, 1027.0 - 1028.5 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 250 mL flasks, 100 mL culture.
- 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: yes. At the test item preparation stage, the pH of the prepared solutions was measured and adjusted at 8.1 ± 0.2 with NaOH 1 N.
- Photoperiod: continuous ilumination.
- Light intensity and quality: 5681 lux.

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: A preliminary test (non-GLP), was performed at test item concentrations of 0, 10, and 100 mg/L.
- Test concentrations: 0, 10 , 32, 102, 328, 1050 mg/L.
- Results used to determine the conditions for the definitive study: The main study concentrations were selected based on the preliminary test results, where only ca. 3% inhibition was observed at 100 mg/L test item.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

805 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL [694 - 938]

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL [79 - 123]

Key result

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

171 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CL [140 - 212]

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: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

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 (June 11, 2019) gave an ErC50(0-72h) = 1.13 mg/L (1.07 – 1.20), 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 May 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%).

Table 3. Inhibition of the average specific growth rate in comparison to the Control.

Nominal test item concentration (mg/L)		Algae *10^4 /mL at 0 hour	Specific growth rate µ(0-72h)	Inhibition growth rate (% lr)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1	1.708	Not applicable		2.9	-
	b		1.670
	c		1.712
	d		1.708
	e		1.697
	f*		1.705
10	a	1	1.687	0.8	-0.3	Not required	ns
	b		1.700	0.0
	c		1.728	-1.6
32	a	1	1.632	4.0	4.4		*
	b		1.629	4.2
	c		1.617	4.9
102	a	1	1.488	12.5	12.4		*
	b		1.468	13.7
	c		1.510	11.2
328	a	1	1.309	23.0	26.0		*
	b		1.227	27.8
	c		1.231	27.6
1050	a	1	0.842	50.5	54.2		*
	b		0.732	56.9
	c		0.754	55.7

*significantly different compared to control, Dunnet test α = 0.05; ns: not significantly different. According to the Guidance document on aquatic toxicity testing of difficult substances and mixtures OECD n°23, and OECD TG 201, the deviation from the measured initial concentration was within the range of ± 20 %. Therefore, the test results are expressed relative to the nominal test item concentrations.

Table 4. Nominal and measured test item concentrations.

	Measured concentration (mg/L)
Nominal test item concentration (mg/L)	T0	T72h	Deviation (%)
0 (control)	< 0.5	< 0.5	NA
10	9.5	9.5	0.0
328	311.8	311.4	-0.1
1050	1028.5	1027.0	-0.1

Table 5. Inhibitions of the yield in comparison to the Control.

Nominal test item concentration (mg/L)		[algae] x 10^4 /mL at 0 hour	[algae] x 10^4 /mL at 72 hours	Yield (x 10^4/mL)	Average yield (x 10^4/mL)	Yield inhibition (Iy) (%)
						Per replicate	Average/ replicate
Control	a	1	168.0	167.0	163.2	Not Applicable
	b		149.9	148.9
	c		169.8	168.8
	d		168.1	167.1
	e		162.7	161.7
	f*		166.4	165.4
10	a	1	157.6	156.6	165.7	4.0	-1.6
	b		164.0	163.0		0.1
	c		178.5	177.5		-8.8
32	a	1	133.7	132.7	130.4	18.7	20.1
	b		132.7	131.7		19.3
	c		127.9	126.9		22.2
102	a	1	86.8	85.8	86.1	47.4	47.2
	b		81.8	80.8		50.5
	c		92.8	91.8		43.7
328	a	1	50.8	49.8	42.6	69.5	73.9
	b		39.7	38.7		76.3
	c		40.2	39.2		76.0
1050	a	1	12.5	11.5	9.4	93.0	94.3
	b		9.0	8.0		95.1
	c		9.6	8.6		94.7

Table 6. Measured pH values.

Nominal test item concentation (mg/L)	pH at the start (biotic replicates)	pH after 72 hours (biotic replicates)
Control	8.3	7.9
10	7.9	8.1
32	8.3	7.8
102	8.0	7.7
328	8.3	7.7
1050	8.3	7.7

Table 7. 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	1.099	1.136	4.2	1.752	1.794	2.7	2.273	2.171	2.4	1.708	1.700	34.5	30.9
	b	1.065			1.786			2.159			1.670		33.3
	c	1.131			1.849			2.154			1.712		30.7
	d	1.163			1.833			2.129			1.706		29.0
	e	1.194			1.724			2.174			1.697		28.9
	f	1.163			1.817			2.134			1.705		29.0

Table 8. Raw data, i.e. algal concentrations values expressed as cell number *104 per mL of test solutions after 24, 48 and 72 hours of incubation are indicated in the following table.

Nominal test item concentration (mg/L)		[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	3.0	3.1	4.7	17.3	18.8	6.6	168.0	164.2	4.5
	b	2.9			17.3			149.9
	c	3.1			19.7			169.8
	d	3.2			20.0			168.1
	e	3.3			18.5			162.7
	f*	3.2			19.7			166.4
10	a	3.4	3.4	1.7	22.4	23.3	4.1	157.6	166.7	6.4
	b	3.3			24.3			164.0
	c	3.4			23.2			178.5
32	a	2.8	2.9	2.0	13.7	14.5	18.9	133.7	131.4	2.4
	b	2.9			17.6			132.7
	c	2.9			12.3			127.9
102	a	2.0	2.1	5.6	15.8	15.9	1.0	86.8	87.1	6.3
	b	2.0			15.9			81.8
	c	2.2			16.1			92.8
328	a	2.3	2.1	8.2	14.0	11.5	18.8	50.8	43.6	14.4
	b	2.0			9.9			39.7
	c	2.0			10.7			40.2
1050	a	2.2	2.0	10.2	3.7	3.4	7.8	12.5	10.4	18.1
	b	2.1			3.2			9.0
	c	1.8			3.3			9.6

Validity criteria fulfilled:

yes

Remarks:

Biomass increased exponentially by a factor > 16 (164.2), Daily section-by-section specific growth rate CV (30.9%) were ≤ 35%, and Average specific growth rate CV during 72 hours (0.9%) was ≤ 7%.

Conclusions:

The 72h-ErC50 of the test item in algae was 805 mg/L, the 72h-ErC10 was 102 mg/L, and the NOEC was 10 mg/L, all based on inhibition of growth rate.

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 1, 10, and 100 mg/L test item, where almost no inhibition of growth rate was observed at 100 mg/L, Pseudokirchneriella subcapitata was exposed to 10, 32, 100, 328 and 1050 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. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is regularly performed with potassium dichromate. Analytical monitoring was performed using a validated HPLC-UV method. Since the test item was stable in algae medium (deviation from the measured initial concentration was within ± 20 %), the test results expressed relative to nominal test item concentrations. All validity criteria were met. Under test conditions, the 72h-ErC50 of the test item in algae was determined to be 805 mg/L, and the 72h-ErC10 was 102 mg/L (based on growth rate). The EyC50 was 171 mg/L (biomass). The NOEC and LOEC determined for the test item for the endpoint inhibition of growth rate were 10 and 32 mg/L, respectively.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE : EPI-SUITE EPA (USA)

2. MODEL: ECOSAR v1.11

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
- SMILES: COC1=C(C=C(C)C=C1)C(CC(O)=O)C1=CC=CC=C1
- Experimental partition coefficient value: Log Kow: 2.440
- Experimental water solubility value: Wat Sol: 14.2 mg/L

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: The QMRF is available in "Attached justification".

5. APPLICABILITY DOMAIN : The QPRF is available in 'Attached justification'.

6. ADEQUACY OF THE RESULT : The QPRF is available in 'Attached justification'.

Reason / purpose for cross-reference:

other: supporting information

Remarks:

experimental logKow value

Guideline:

other: REACH Guidance on QSARs R.6

GLP compliance:

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

465.8 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated value.

ECOSAR v1.11 predicted that the substance has a 96h-EC50 = 465.8 mg/L in green algae.

According to the prediction, ifthe effect level exceeds the water solubility by 10X, typically no effects at saturation (NES) are predicted. The experimental water solubility value of the test item was found to be 14.2 mg/L (20ºC, pH 5).

Validity criteria fulfilled:

not applicable

Conclusions:

The calculated 96h-EC50 of the test item in algae is 465.8 mg/L.

Executive summary:

ECOSAR v1.11 calculation of the 96h-EC50 in algae was used. According to this method, the substance has a 96h-EC50 = 465.8 mg/L in green algae.

Description of key information

Key study. Method according to OECD 201, GLP study. Under test conditions, the 72h-ErC50 of the test item in algae was determined to be 805 mg/L, and the 72h-ErC10 was 102 mg/L (based on growth rate). The EyC50 was 171 mg/L (biomass). The NOEC and LOEC determined for the test item for the endpoint inhibition of growth rate were10 and 32 mg/L, respectively.

Supporting study. ECOSAR v1.11, accepted calculation method. The substance has a predicted 96h-EC50 = 465.8 mg/L in green algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

805 mg/L

EC10 or NOEC for freshwater algae:

102 mg/L