Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola, partially hydrolysed

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:

2017-09-18 to 2017-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. The samples were analyzed with an LC-MS method. The method was implemented under non-GLP but documented in the raw data and validated. The method validation is not part of this GLP study.
- Concentrations: The control and the samples were stabilized factor 2 with methanol directly after sampling. Further dilutions were made with methanol : algae dilution water (50 : 50) prior to analysis. For dilution steps, see Table 1 in section “any other details on materials and methods incl. tables”
- Sample storage conditions before analysis: All samples were stored at room temperature until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solution: 1000 mg test item/L, corresponding to 472 mg solid content/L, was freshly prepared with dilution water (see Table 2 in section “any other details on materials and methods incl. tables”). The dispersion was shaken until the solution was visually clear. Although care was taken to avoid the formation of foam during preparation a concentration related foam formation was observed in the test media throughout the test period.
Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
Control
Six replicates (without test item) were tested under the same test conditions as the test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day. Nutrient medium Z according to LÜTTGE et al. (1994)
- Culturing media: dilution water (see Table 2 in section “any other details on materials and methods incl. tables”).
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

0.24 mmol Ca+Mg/L (nominal)

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

pH:

8.1 ± 0.2 (dilution water). For details please see Table 7 in section "any other information on results incl. tables".

Dissolved oxygen:

not reported

Salinity:

not reported; freshwater conditions

Conductivity:

not reported

Nominal and measured concentrations:

Nominal test item concentrations: 10.0 - 31.6 - 100 - 316 - 1000 mg/L, corresponding to 4.72 – 14.9 – 47.2 – 149 – 472 mg solid content/L. For details please see Table 8 in section "any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Type: with cotton wool plugs
- size, fill volume: 250 mL, fill volume 100 mL
- Initial cells density: Nominal: approximately 5000 - 10000 cells/mL; Actual: 6839 cells/mL
- Control end cells density: 2771447
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium
OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorimeter; Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH)
- Chlorophyll measurement: daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. Self-fluorescence was observed in the definitive test. A separate replicate for each concentration was prepared without algae and treated the same as the other test replicates. The self-fluorescence of each measuring point was considered for evaluation.
- Other: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.16
- Range finding study: A preliminary range finding test in an open system with headspace under static conditions over a period of 72 hours was conducted at the test facility. A stock solution of 100 mg test item/L was freshly prepared with dilution water. Out of this stock solution three concentration levels were prepared (by serial dilution) with a concentration range of 1.00 - 10.0 - 100 mg/L- Test concentrations: 10.0 - 31.6 - 100 - 316 - 1000 mg/L, corresponding to 4.72 – 14.9 – 47.2 – 149 – 472 mg solid content/L
- Results used to determine the conditions for the definitive study: The dilution levels are based on the results of a preliminary range finding test. For details see Table 11 and 12 in section “any other details on results incl. tables”

Reference substance (positive control):

yes

Remarks:

potassium dichromate, SIGMA, batch number MKBV0900V, purity 99.0 %

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

47.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

47.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 234 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI (185 - 274)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 20.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CI (< 10.0 – 38.2)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 110 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 9.72 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 444 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI (382 - 567)

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 55.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CI (34.9 – 84.4)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 210 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 26.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

983 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: (918 > 1000)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

258 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CI (206 - 335)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 464 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 122 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

For details please see section "any other information on results incl. tables".

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS. For results, please see table 9 in section "any other details on results incl. tables".

Reported statistics and error estimates:

For information on statistics, please see section "any other details on materials and methods incl. tables".

Results from the definitive test

Biological Data

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

Table 3: NOEC, LOEC, EC-values and 95 % Confidence Intervals of the test item (0 - 72 hours) based on nominal test item concentrations and nominal solid content concentration [mg/L]          

	Growth Rate Inhibition
	Nominal test item concentration [mg/L]	Nominal solid content concentration [mg/L]
NOEC	31.6	14.9
LOEC	100	47.2
ErC10	234 (185 - 274)	110 (87.3 – 129)
ErC20	444 (382 - 567)	210 (180 – 268)
ErC50	983 (918 - >1000)	464 (433 - >472)
	Yield Inhibition
	Nominal test item concentration [mg/L]	Nominal solid content concentration [mg/L]
NOEC	31.6	14.9
LOEC	100	47.2
EyC10	20.6 (< 10.0 – 38.2)	9.72 (<4.72 – 18.0)
EyC20	55.7 (34.9 – 84.4)	26.3 (16.5 – 39.8)
EyC50	258 (206 - 335)	122 (97.2 – 158)

Table 4: Cell densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1000	1	6839	12009	36237	125936
	2	6839	11319	41857	134111
	3	6839	10479	36181	126545
	Mean	6839	11269	38092	128864
316	1	6839	16746	145174	1376508
	2	6839	17613	116225	1284164
	3	6839	12410	110671	1176721
	Mean	6839	15590	124023	1279131
100	1	6839	30639	274828	1738001
	2	6839	31549	248225	1891909
	3	6839	35493	271458	1915960
	Mean	6839	32560	264837	1848623
31.6	1	6839	43107	443594	2678757
	2	6839	35072	403730	2378900
	3	6839	40585	363972	2560664
	Mean	6839	39588	403765	2539440
10.0	1	6839	54398	558849	2615342
	2	6839	43654	541890	2705931
	3	6839	53351	530818	2336786
	Mean	6839	50468	543852	2552686
Control	1	6839	49959	368920	2770159
	2	6839	56683	444340	2868590
	3	6839	54027	510690	2953912
	4	6839	44447	414343	2799653
	5	6839	60944	457519	2534441
	6	6839	49933	587597	2701925
	Mean	6839	52666	463902	2771447

Table 5: Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1000	1		0.971	51		119097	96
	2		0.992	50		127272	95
	3		0.973	51		119706	96
	Mean	(+)	0.979	51	(+)	122025	96
316	1		1.77	12		1369669	50
	2		1.75	13		1277325	54
	3		1.72	14		1169882	58
	Mean	(+)	1.74	13	(+)	1272292	54
100	1		1.85	8		1731162	37
	2		1.87	6		1885070	32
	3		1.88	6		1909121	31
	Mean	(+)	1.87	7	(+)	1841784	33
31.6	1		1.99	1		2671918	3
	2		1.95	3		2372061	14
	3		1.98	1		2553825	8
	Mean	(-)	1.97	1	(-)	2532601	8
10.0	1		1.98	1		2608503	6
	2		1.99	0		2699092	2
	3		1.95	3		2329947	16
	Mean	(-)	1.97	1	(-)	2545847	8
Control	1		2.00			2763320
	2		2.01			2861751
	3		2.02			2947073
	4		2.01			2792814
	5		1.97			2527602
	6		1.99			2695086
	Mean		2.00			2764608

Table 6: Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours). SD = Standard deviation, CV = Coefficient of variation.

	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
Control	1	1.99	2.00	2.02	2.00	0.014	0.69	10.9
	2	2.12	2.06	1.87	2.01	0.131	6.52
	3	2.07	2.25	1.76	2.02	0.249	12.3
	4	1.87	2.23	1.91	2.01	0.198	9.88
	5	2.19	2.02	1.71	1.97	0.241	12.2
	6	1.99	2.47	1.53	1.99	0.470	23.6
				Mean	2.00
				SD ±	0.02
				CV [%]	0.88

 

Physicochemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits (Table 7). The test media were visually clear at the start and after 24 hours of the exposure. After 48 and 72 hours of the exposure the test media were concentration related turbid. Although care was taken to avoid the formation of foam during preparation a concentration related foam formation was observed in the test media throughout the test period.

Table 7: Environmental conditions. CV = Coefficient of variation, n = number of measuring points.

Nominal test item concentration		pH-value
[mg/L]		Start; 0 hours	End; 72 hours
1000		7.62	8.11
316		7.75	8.37
100		7.80	8.56
31.6		7.87	9.28
10.0		7.92	9.41
Control		7.95	9.30
Room temperature [°C]:	min.: 22.0	max.: 23.5		mean value: 22.8
Light intensity [lux]	n = 29	mean value: 6037 range of the measured values: 5247 to 6695 equalling -13.1 to 10.9 %		CV [%]: 8.26

 

Measured Exposure Concentrations during the Definitive Test

The concentrations of the solid content of the test item were analytically verified via LC-MS/MS at the start (0 h) and at the end (72 h) of the exposure in all tested concentration levels and the control.

The measured concentrations at the start of the exposure were in the range of 94 and 102 % of the nominal values. At the end of the exposure the measured concentrations of the test item were in the range of 84 to 105 %. For results see Table 8.

The measured concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.                                                                              

Table 8: Measured Concentrations and Percent of the Nominal Concentrations of the Solid Content of the Test Item. s. c. = solid content. Meas. conc. = Measured concentration of the solid content of the test item, dilution factor taken into account. % = Percent of nominal concentration of the solid content. LOQ = Limit of Quantification (10.0 mg/L test item, corresponding to 4.71 mg s. c./L)

Sampling date		0 hours		72 hours
Nominal concentration		Solid content of the test item
Test item [mg/L]	s. c. [mg/L]	Meas. conc. [mg s.c./L]	%	Meas. conc. [mg s.c./L]	%
1000	472	480	102	495	105
316	149	150	100	152	102
100	47.2	46.9	99	47.6	101
31.6	14.9	14.0	94	14.0	94
10.0	4.72	4.55	96	3.99	84
Control		< LOQ		< LOQ

 

Test of the Reference Item

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS.

Table 9: EC50-Values of the Reference Item based on nominal concentrations [mg/L], (0-72 hours). SD= standard deviation.

	Current Study	Valid Range (average ± 3 x SD)
	Growth Rate inhibition
ErC50	0.559	0.754 ± 0.518
95 % confidence interval	0.538 – 0.581
	Yield inhibition
EyC50	0.320	0.398 ± 0.291
95 % confidence interval	0.242 – 0.341

 

ValidityCriteria

The study meets the validity criteria of the guideline.

Table 10: Validity Criteria

Validity Criterium	Required	This study
Increase of the cell growth in the control cultures	Exponentially ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	405-fold (specific growth rate 2.00 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35 %	10.8 %
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7 %	0.88 %

 

Range finding test

A preliminary range finding test in an open system with headspace under static conditions over a period of 72 hours was conducted at the test facility. A stock solution of 100 mg test item/L was freshly prepared with dilution water. Out of this stock solution three concentration levels were prepared (by serial dilution) with a concentration range of 1.00 - 10.0 - 100 mg/L.The concentration levels were visually clear throughout the exposure period. A concentration-related foam formation was observed. In the range finding test, two replicates per concentration and four for the control, were tested.

Table 11: Results of the Range Finding Test (0 - 72 hours). Negative inhibitions = increase of growth.

Nominal test item concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	11	45
10.0	-2	-10
1.00	-2	-13

 Table 12: Measured exposure concentrations during the range finding test. Determination of the test item via LC-MS/MS. %= percent of the nominal test concentration. Meas. Conc. = Measured concentration. LCL = Lowest calibration level = 0.500 mg test item/L                           

Sampling	0 hours Start of the exposure (Samples with algae)		72 hours End of the exposure (Samples with algae)		72 hours End of the exposure (Samples without algae)
Nominal test item concentration [mg/L]
	Meas. Conc. [mg/L]	%	Meas. Conc. [mg/L]	%		Meas. Conc. [mg/L]	%
100	88.2	88	104	104		118	118
10.0	8.78	88	8.92	59		9.13	91
1.00	1.13	113	1.01	101		1.00	100
Control	< LCL

                                                               

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 983 (918 - > 1000) mg test item/L and 258 (206 - 335) mg test item/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 234 (185 - 274) mg test item /L and 20.6 (< 10.0 – 38.2) mg test item /L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg test item/L. Based on solid content concentrations, the following effect values were obtained: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 464 (433 - > 472) mg solid content/L and 122 (97.2 - 158) mg solid content/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 110 (87.3 - 129) mg/L and 9.72 (< 4.72 – 18.0) mg solid content/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 14.9 mg solid content/L.

Executive summary:

The toxicity of Sophorolipids, partially hydrolysed, to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 Method C. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A stock solution and four dilution levels out of the stock solution were tested in a geometrical series with a dilution factor of √10: 10.0 - 31.6 - 100 - 316 - 1000 mg/L, corresponding to 4.72 – 14.9 – 47.2 – 149 – 472 mg solid content/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear at the start and after 24 hours of the exposure. After 48 and 72 hours of the exposure the test media were concentration related turbid. Although care was taken to avoid the formation of foam during preparation a concentration related foam formation was observed in the test media throughout the test period.

The concentrations of the solid content of the test item were analytically verified via LC-MS/MS at the start (0 h) and at the end (72 h) of the exposure in all tested concentration levels and the control. The measured concentrations at the start of the exposure were in the range of 94 and 102 % of the nominal values. At the end of the exposure the measured concentrations of the test item were in the range of 84 to 105 %. The measured concentrations were all within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 983 (918 - > 1000) mg test item/L and 258 (206 - 335) mg test item/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 234 (185 - 274) mg test item /L and 20.6 (< 10.0 - 38.2) mg test item /L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg test item/L.

Based on solid content concentrations, the following effect values were obtained: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 464 (433 - > 472) mg solid content/L and 122 (97.2 - 158) mg solid content/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 110 (87.3 - 129) mg/L and 9.72 (< 4.72 - 18.0) mg solid content/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 14.9 mg solid content/L.

 

 

Description of key information

Based on solid content concentrations, the following effect values were obtained: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 464 (433 - > 472) mg solid content/L and 122 (97.2 - 158) mg solid content/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 110 (87.3 - 129) mg/L and 9.72 (< 4.72 - 18.0) mg solid content/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 14.9 mg solid content/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

464 mg/L

EC10 or NOEC for freshwater algae:

14.9 mg/L

Additional information

The toxicity of Sophorolipids, partially hydrolysed, to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 Method C. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A stock solution and four dilution levels out of the stock solution were tested in a geometrical series with a dilution factor of √10: 10.0 - 31.6 - 100 - 316 - 1000 mg/L, corresponding to 4.72 – 14.9 – 47.2 – 149 – 472 mg solid content/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear at the start and after 24 hours of the exposure. After 48 and 72 hours of the exposure the test media were concentration related turbid. Although care was taken to avoid the formation of foam during preparation a concentration related foam formation was observed in the test media throughout the test period.

The concentrations of the solid content of the test item were analytically verified via LC-MS/MS at the start (0 h) and at the end (72 h) of the exposure in all tested concentration levels and the control. The measured concentrations at the start of the exposure were in the range of 94 and 102 % of the nominal values. At the end of the exposure the measured concentrations of the test item were in the range of 84 to 105 %. The measured concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 983 (918 - > 1000) mg test item/L and 258 (206 - 335) mg test item/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 234 (185 - 274) mg test item /L and 20.6 (< 10.0 - 38.2) mg test item /L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg test item/L.

Based on solid content concentrations, the following effect values were obtained: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 464 (433 - > 472) mg solid content/L and 122 (97.2 - 158) mg solid content/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 110 (87.3 - 129) mg/L and 9.72 (< 4.72 - 18.0) mg solid content/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 14.9 mg solid content/L.

 

Based on reliable data determined with the target substance, the following parameters were used for assessment:

EC50 for freshwater algae: 464 mg/L based on active ingredient

NOEC for freshwater algae: 14.9 mg/L based on active ingredient