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Toxicity to aquatic algae and cyanobacteria

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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:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.

 

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26.10.2009 - 19.11.2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study without deviation from the guideline followed
Qualifier:
according to
Guideline:
other: "Data requirements for suppporting Registration of Pesticides-Guidelines for Preparation of Study Results Submitted When Applying for Registration of Agricultural Chemicals, 2-7-7"
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The required amounts of the test substance were weighted on an electronic balance. For preparation of stock and test solutions, the required amounts of the test substance were added to flask filled with culture medium. And then, stock and test solutions were prepared using a stirrer.
One thousand mg/L stock solution of the tests substance was diluted with culture medium to yield the desired concentrations. 100 mL of each test solution were placed in test flasks. The test solution was prepared prior to dosing.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): American Type Culture Collection (ATCC, USA)
- Age of inoculum (at test initiation):
- Method of cultivation: Frozen cells which were stored in a deep freezer (- 70 °C) upon receipt were thawed and inoculated in culture medium under aseptic conditions. Then the cells were incubated at temperatures of 23 °C under continous illumination and constantly shaken at approximately 100 rpm using a shaking incubator for 7 days. After incubation a biomass was counted and the suspension was diluted with culture medium to produce the biomass of 1x10exp4 cells/mL. Cells were subcultured continously under the same conditions at intervalls of 3 to 4 days.
Exponentially growing cells which were sub cultered for 4 passages were observed for atypical morphologies, such as flocculation, discolouration, swelling, atrophy and rupture and inoculated in agar medium for long term storage. They were then incubated at a temperature of 23 °C under continous illumination. The culture was maintained under refrigeration (2-8 °C)
Culture medium was inoculated with cells from the colonies on the agar plated from long term storage four days prior to study initiation. A biomass was counted using a particle counter.
The suspension was diluted with culture medium to produce a biomass of 0.7x10exp4 cells/mL. incubated at a temperature of 23 °C under continous illumination and constantly shaken at approximately 100 rpm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.3-22.8 °C
pH:
6.9-7.5 at initiation of test; 7.3-7.5 after 72 h
Nominal and measured concentrations:
Nominal: 0, 100, 177, 316, 562, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks 250-300 mL) plugged with foam stoppers
- Type: closed
- Initial cells density: 1x10exp4 cells/mL
- Control end cells density: 927833 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
Component Concentration in culture medium (mg/L)
NH4Cl 15.0
MgCl2x6H2O 12.0
CaCl2x2H2O 18.0
MgSO4x7H2O 15.0
KH2PO4 1.6
FeCl3x6H2O 0.0640
Na2EDTAx2H2O 0.1
H3BO3 0.185
MnCl2x4H2O 0.415
ZnCl2 0.003
CoCl2x6 H2O 0.0015
CuCl2x2 H2O 0.00001
Na2MoO4x2H2O 0.007
NaHCO3 50



OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: 4440-8880 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A particle counter (Vi-Cell XR, Beckman Coulter, USA) was used for determination of biomass and a Haemacytometer (Superior marienfeld, Germany) was used for cell counting. Samples were collected from all groups included the control group at 24, 48 and 72 hours after initiation of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations (range finding study): 0, 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth rate at 1000 mg/L: 47.1 %
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): At 72 h (after exposure) atypical cell morphology was not observed in the control group and in not more than 177 mg/L dosed group, while atrophy was observed in the 316, 562 and 1000 mg/L dosed groups.
- Flocculation: no
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At 72 h after exposure atypical cell morphology was not observed in the control group and in the not more than 177 mg/L dosed groups, while atrophy was observed in the 316, 562 and 1000 mg/L dosed groups.
- Colour differences: no
- Flocculation: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (growth rate): 1.28 mg/L
- Other: result within historical control range

Average specific growth rate

At initiation of the test and 24, 48 and 72 h after start of exposure the average specific growth rate was calculated using the following equation:

µi-j= (ln Xj– ln Xi) / (tj– ti) (day-1)

µi-j       average specific growth rate from time I to j

Xi       biomass at time i

Xj       biomass at time j

 

Percentage inhibition in average specific growth rate (%)

The following equation was used:

 

% Ir= ((µc- µi) / µc)) x 100

 

% Ir    percent inhibition in average specific growth rate

µc       mean value for average specific growth rate (µ) in the control group

µi       average specific growth rate for the dosed replicate

Biomass during the study

Nominal concentration (mg/L)

Biomass mean values (cells/mL)

0 hr

24 hr

48 hr

72 hr

Control

10000

47500

216500

927833

100

10000

50000

204667

943333

177

10000

46667

167667

528667

316

10000

43333

121000

284333

562

10000

28333

78333

143667

1000

10000

30000

73333

98500

 

Percentage inhibition of average specific growth rate

Nominal concentration (mg/L)

Average specific growth rate (0-72 h) mean values

% Ir(0—72 h)

Control

0.0629

0

100

0.0631

-0.32

177*

0.0551

12.4

316*

0.0465

26.1

562*

0.037

41.2

1000*

0.0318

49.4

*: Significantly different from the control at α = 0.05, 0.01 by Dunnett’s t-test procedure

Validity criteria fulfilled:
yes
Conclusions:
The ErC50 at 72 h after exposure was > 1000 mg/L (the highest concentration). The NOECR at 72 h was calculated 100 mg/L.
Executive summary:

The toxicity of the test substance to the fresh water alga Pseudokirchnerella subcapitata was investigated

in a study according to the guideline "Data requirements for suppporting Registration of Pesticides-Guidelines for Preparation of Study Results Submitted When Applying for Registration of Agricultural Chemicals, 2-7-7". Nominal concentrations of 0, 100, 177, 316, 562 and 1000 mg/L were used for the study. The test was carried out under static conditions with an exposure time of 72 h. Based on growth rate inhibition an ECr50 of > 1000 mg/L and a NOECr of 100 mg/L were obtained.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please see the justification document in chapter “Sophorolipid_Justification for read-across_Ecotox_2015-04-151638581287878619378”.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances, both Sophorolipids have similar environmental fate and pathways, ecotoxicological and toxicological properties based on the underlying identical generic structure. There are only differences in the ratio of the different structures (differentiation between acid and lactone form).
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
GLP compliance:
yes
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal: 0, 100, 177, 316, 562, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): At 72 h (after exposure) atypical cell morphology was not observed in the control group and in not more than 177 mg/L dosed group, while atrophy was observed in the 316, 562 and 1000 mg/L dosed groups.
- Flocculation: no
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At 72 h after exposure atypical cell morphology was not observed in the control group and in the not more than 177 mg/L dosed groups, while atrophy was observed in the 316, 562 and 1000 mg/L dosed groups.
- Colour differences: no
- Flocculation: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (growth rate): 1.28 mg/L
- Other: result within historical control range

Average specific growth rate

At initiation of the test and 24, 48 and 72 h after start of exposure the average specific growth rate was calculated using the following equation:

µi-j= (ln Xj– ln Xi) / (tj– ti) (day-1)

µi-j       average specific growth rate from time I to j

Xi       biomass at time i

Xj       biomass at time j

 

Percentage inhibition in average specific growth rate (%)

The following equation was used:

 

% Ir= ((µc- µi) / µc)) x 100

 

% Ir    percent inhibition in average specific growth rate

µc       mean value for average specific growth rate (µ) in the control group

µi       average specific growth rate for the dosed replicate

Biomass during the study

Nominal concentration (mg/L)

Biomass mean values (cells/mL)

0 hr

24 hr

48 hr

72 hr

Control

10000

47500

216500

927833

100

10000

50000

204667

943333

177

10000

46667

167667

528667

316

10000

43333

121000

284333

562

10000

28333

78333

143667

1000

10000

30000

73333

98500

 

Percentage inhibition of average specific growth rate

Nominal concentration (mg/L)

Average specific growth rate (0-72 h) mean values

% Ir(0—72 h)

Control

0.0629

0

100

0.0631

-0.32

177*

0.0551

12.4

316*

0.0465

26.1

562*

0.037

41.2

1000*

0.0318

49.4

*: Significantly different from the control at α = 0.05, 0.01 by Dunnett’s t-test procedure

Validity criteria fulfilled:
yes
Conclusions:
The ErC50 at 72 h after exposure was > 1000 mg/L (the highest concentration). The NOECr at 72 h was calculated 100 mg/L.

Description of key information

Key study: 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.

Read-across study: ErC50 at 72 h after exposure > 1000 mg/L (the highest concentration). The NOECr at 72 h was calculated 100 mg/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.

 

Further supporting data based on read-across are available. In a read-across study, the toxicity of the source substance to the fresh water alga Pseudokirchnerella subcapitata was investigated in a study according to the guideline "Data requirements for suppporting Registration of Pesticides-Guidelines for Preparation of Study Results Submitted When Applying for Registration of Agricultural Chemicals, 2-7-7". Nominal concentrations of 0, 100, 177, 316, 562 and 1000 mg/L were used for the study. The test was carried out under static conditions with an exposure time of 72 h. Based on growth rate inhibition an ECr50 of > 1000 mg/L and a NOECr of 100 mg/L were obtained.

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