Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

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:

(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:

QSAR calculation, based on generally accepted model (ECOSAR), supported by publication based on slightly different input data. Details reported in the QPRF document attached to background materials.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs, R.6, May 2008

GLP compliance:

no

Analytical monitoring:

not required

Test organisms (species):

other: Green alga (arbitrary species, QSAR calculation)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 7.9 mg/L

Basis for effect:

growth rate

Previous EC₅₀ estimate (Bowmer et al., 1998, based on calculated log P_ow of 3.168): 11 mg/L.

Recent EC₅₀ estimate (ECOSAR, based on measured log P_ow of 3.3): 7.9 mg/L.

The latter is adopted in a worst-case approach.

Validity criteria fulfilled:

yes

Conclusions:

The algal toxicity of 2-ethylhexyl-S-lactate is predicted by QSAR calculation to result in a 96-h ErC50 of 7.9 mg/L.

Executive summary:

The algal toxicity of 2-ethylhexyl-S-lactate was predicted by QSAR calculation (ECOSAR), resulting in a 96 -h E_rC₅₀ of 7.9 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-04 to 2019-07-26

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:

28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No. 266/2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 1802000003
CAS No.: 186817-80-1
Purity (certified): 98 %

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.
- Sampling method: At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples from pooled replicates.
- Sample storage conditions before analysis: All samples were immediately extracted and the extracts were stored at room temperature if necessary. Until the start of the analysis the prepared samples were stored on an autosampler.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: Stock solution, media preparation: 80.0 mg test item/L were freshly prepared with dilution water. The solution was stirred for 10 minutes (room temperature, 1100 rpm) with a magnetic stirrer. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Preparation of the test item solutions: Out of the stock solution six concentrations were prepared in a geometric series with a dilution factor of √10: 0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 mg/L, corresponding to the geometric mean measured test item concentration of: 0.136 – 0.417 – 1.53 – 5.48 – 20.5 – 64.1 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
- Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
- Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567–5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994)

ACCLIMATION
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): OECD TG 201 medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

33 mg CaCO3/L

Test temperature:

21.5 to 22.0 °C

pH:

7.73 to 9.41

Conductivity:

137 µS/cm

Nominal and measured concentrations:

Nominal: 0.253 - 0-800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L
Geometric mean measured test item concentration of: 0.136 – 0.417 – 1.53 – 5.48 – 20.5 – 64.1 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Fill volume: 100
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): n.a.
- Initial cells density: Nominal: approximately 5E+03–1E+04 cells/mL; actual: 5416 cells/mL
- Control end cells density: 1959999 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n.a.

GROWTH MEDIUM
- Standard medium used: yes (Nutrient medium Z according to LÜTTGE et al. (1994))

TEST MEDIUM / WATER PARAMETERS
- Ultrapure water: Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).
- Test medium: OECD TG 201 medium):
NH4Cl: 15 mg/L
MgCl2 × 6 H2O: 12 mg/L
CaCl2 × 2 H2O: 18 mg/L
MgSO4 × 7 H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3 × 6 H2O: 0.064 mg/L
Na2EDTA × 2 H2O: 0.1 mg/L
H3BO3: 0.185 mg/L
MnCl2 × 4 H2O: 0.415 mg/L
ZnCl2: 3.0E-03 mg/L
Na2MoO4 × 2 H2O: 7 x 10-3 mg/L
CoCl2 × 6 H2O: 1.5E-03 mg/L
CuCl2 × 2 H2O: 1.0E-05 mg/L
NaHCO3: 50 mg/L
pH: 8.1 ± 0.2

- Total organic carbon: < 2 mg C/L
- Alkalinity: 0.8 mmol/L
- Conductivity: 137 µS/cm
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each concentration level and the control. At the end of exposure, the pH-values were measured from pooled samples of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: mean value: 5577 lux
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Inhibition of yield, Inhibition of growth rate
- Determination of cell concentrations: The cell density was measured daily via chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentrations of 100 mg/L. A separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours were taken into account in the evaluation.
- The cell density was measured daily via chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentrations of 100 mg/L. A separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours were taken into account in the evaluation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10: 0.253 - 0-800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L (nominal)

RANGE FINDING STUDY
-Test concentrations: 1, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

inhibition

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

inhibition

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

inhibition

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

inhibition

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: 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

Reported statistics and error estimates:

- EC-values and statistical analyses: EC10-,EC20- and EC50- values with confidence intervals of yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

- The NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01. Levene’s test on variance homogeneity was done with a significance level of 0.01. Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for growth rate. Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for growth rate.Monotonicity of Concentration/Response was tested by non-parametric trend analysis with a significance level of 0.05 for yield.Step-down Jonckheere-Terpstra Test Procedure was performed with a significance level of 0.05 for yield.

- Software: The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures. Calculations were carried out using software:
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Version 3.3.0, ToxRat Solutions GmbH

Cell densities are listed in Table 1. Evaluations of growth rate inhibition and yield inhibition are given in Table 2 (calculated based on cell density values). Section-by-section and average specific growth rate are given in Table 3.

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the geometric mean measured test item concentrations.

Table1:      Cell densities

Geometric mean measured test item concentration[mg/L]	Replicate no.	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
64.1	1	5416	4435	4384	2770
	2	5416	6007	4369	3337
	3	5416	5438	4732	2653
	Mean	5416	5293	4495	2920
20.5	1	5416	9761	20419	96341
	2	5416	11019	27030	130319
	3	5416	9551	25813	106218
	Mean	5416	10110	24421	110959
5.48	1	5416	21225	170027	1177304
	2	5416	19185	156095	1179592
	3	5416	19971	148102	1201450
	Mean	5416	20127	158075	1186115
1.53	1	5416	27049	213448	2084463
	2	5416	25156	209563	1641183
	3	5416	27665	228645	1940078
	Mean	5416	26623	217219	1888575
0.417	1	5416	27531	241436	1957160
	2	5416	30788	244318	1935936
	3	5416	28734	242147	1962320
	Mean	5416	29018	242634	1951805
0.136	1	5416	26317	236568	1635098
	2	5416	28495	242643	2013050
	3	5416	32803	237970	1952930
	Mean	5416	29205	239060	1867026
Control	1	5416	29182	239572	2110656
	2	5416	25097	185771	1590607
	3	5416	33324	287775	1980339
	4	5416	26336	244771	2003511
	5	5416	29943	255071	2119369
	6	5416	29021	222972	1955512
	Mean	5416	28817	239322	1959999

 

Table 2: Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration[mg/L]	Replicate no.	Growth rate [d–1]		Inhibition of growth rate [%]	Yield [cells/mL]		Inhibition of yield [%]
64.1	1		-0.224	100		-2646	100
	2		-0.161	100		-2079	100
	3		-0.238	100		-2763	100
	Mean	(+)	-0.208	100	(+)	-2496	100
20.5	1		0.960	51		90925	95
	2		1.06	46		124903	94
	3		0.992	49		100802	95
	Mean	(+)	1.00	49	(+)	105543	95
5.48	1		1.79	9		1171888	40
	2		1.80	9		1174176	40
	3		1.80	8		1196034	39
	Mean	(+)	1.80	8	(+)	1180699	40
1.53	1		1.98	-1		2079047	-6
	2		1.91	3		1635767	16
	3		1.96	0		1934662	1
	Mean	(-)	1.95	1	(-)	1883159	4
0.417	1		1.96	0		1951744	0
	2		1.96	0		1930520	1
	3		1.96	0		1956904	0
	Mean	(-)	1.96	0	(-)	1946389	0
0.136	1		1.90	3		1629682	17
	2		1.97	-1		2007634	-3
	3		1.96	0		1947514	0
	Mean	(-)	1.95	1	(-)	1861610	5
Control	1		1.99			2105240
	2		1.89			1585191
	3		1.97			1974923
	4		1.97			1998095
	5		1.99			2113953
	6		1.96			1950096
	Mean		1.96			1954583

 

Table 3: Section-by-section and average specific growth rates of the control group (0–72 hours)

Replicate No.	Specific growth rate [d^-1]			Mean (0–72 hours)	SD ±	CV [%]	Mean CV [%]
	section-by-section
	0–24 hours	24–48 hours	48–72 hours
1	1.68	2.11	2.18	1.99	0.266	13.4	13.6
2	1.53	2.00	2.15	1.89	0.321	16.9
3	1.82	2.16	1.93	1.97	0.173	8.8
4	1.58	2.23	2.10	1.97	0.343	17.4
5	1.71	2.14	2.12	1.99	0.243	12.2
6	1.68	2.04	2.17	1.96	0.255	13.0
			Mean	1.96
			SD ±	0.04
			CV [%]	1.79
SD = Standard deviation CV = Coefficient of variation

 

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC10 of Pseudokirchneriella subcapitata exposed to 2-ethylhexyl-S-lactate was determined to be 6.18 (5.43–7.02) mg/L (geometric mean of measured concentrations).

Executive summary:

In a 72-hour acute toxicity study, cultures of Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 were exposed to 2-ethylhexyl-S-lactate at measured concentrations of 0, 0.136, 0.417, 1.53, 5.48, 20.5, and 64.1 mg/L (geometric mean measured) under static conditions in accordance with OECD test guideline 201. The initially measured concentrations of 2-ethylhexyl-S-lactate were in the range of 90 % to 105 % and by the end of exposure after 72 hours in the range of 28 % to 72 % of nominal. All effect values given are based on the geometric mean measured concentrations of 2-ethylhexyl-S-lactate.

The NOEC and EC50 values based on growth rate were 1.53 and 21.0 mg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 1 to 100 %.

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algal growth inhibition test.

Results synopsis

Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4

Test type: Static

Growth rate inhibition

72-h NOEC: 1.53 mg/L

72-h ErC₁₀: 6.18 mg/L (95 % C.I.: 5.43–7.02 mg/L)

72-h ErC₅₀: 21.0 mg/L (95 % C.I.: 20.2–21.8 mg/L)

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification

Qualifier:

no guideline followed

Principles of method if other than guideline:

Structure-activity-relationship (SAR)
SARs were used calculating the toxicity. A reliable SAR from the scientific literature for non-polar narcosis is provided for comparison.

GLP compliance:

no

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

Scenedesmus capricornutum

Test type:

other: SAR

Water media type:

freshwater

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

SAR prediction

Details on results:

SAR was derived from the following test data:
log EbC50 = -0 77 (log Kow) + 0.84
n = 7
r² = 0.79

ErC50 lactates:
log E.C50 = - 0.97 (log Kow) + 1 .83
n = 7
r = 0.93

ErC50 non-polar narcotics
log ErC50 = - 0.92 (log Kow) + 1 .40
n = 5
r² = 0.97

Source: Calamari, C., S. Galassi, F. Setti and M. Vighi (1983). Toxicity of selected chlorobenzenes to aquatic organisms. Chemosphere, 12, No. 2, 253-262.Where S. capricornutum is concerned, the ErC50 (reproduction based on cell counts) for the alkyl lactate esters is remarkably similar to a SAR for non-polar narcotic substances (chloro benzenes, re-calculated by Clements & Nabholz, 1984 with data from Calamari et al., 1983).

Validity criteria fulfilled:

not specified

Conclusions:

The predicted 72 to 96-h ErC50 for S. capricornutum equals 11 mg/L.

Executive summary:

Based on a SAR prediction the ErC50 for Selenastrum capricornutum was calculated to equal 11 mg/L.

Description of key information

In a study according to OECD test guideline 201 under GLP, the inhibition of 2-ethylhexyl-S-lactate to the growth of green algae resulted in a 72-h E_rC₅₀ of 21.0 mg/L, a 72-h E_rC₁₀ of 6.18 mg/L, and a NOE_rC of 1.53 mg/L in R. subcapitata. These effect values are expressed as geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

21 mg/L

EC10 or NOEC for freshwater algae:

6.18 mg/L

Additional information

QSAR calculation of the algal toxicity of 2-ethylhexyl-lactate (ECOSAR, 2015) gave a 96-h E_rC₅₀ of 7.9 mg/L.

Furthermore, in a publication by Bowmer et al. (1998) a SAR prediction of 11 mg/L is presented for the E_rC₅₀ in Selenastrum capricornutum (recent name: Raphidocelis subcapitata).

These results are considered as supportive data only, since constituting theoretical calculations, suffering from limited reporting of validity criteria.