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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2018-01-08 to 2018-01-11 at the test facility.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
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 analysed with a LC-MS/MS method, implemented under non-GLP and documented in the GLP raw data. The method was validated.
Vehicle:
no
Details on test solutions:
Stock solution, media preparation
The test item was warmed up to 50 °C before used for preparation of the stock solution. A stock solution with a nominal test item concentration of 1000 mg/L was freshly prepared with demineralized water and treated with ultrasound for 30 minutes at 50 °C.

Test concentrations Out of the stock solution five concentration levels were tested in a
(nominal) geometrical series with a dilution factor of 2: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L. The concentration levels are based on the results of a preliminary range finding test.

Control Six replicates (without test item) were exposed under the same conditions as the test loadings.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Not specified
Test temperature:
min.: 22.0 max.: 22.5 mean value: 22.3
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.71 8.01
0.500 7.74 8.18
0.250 7.80 8.71
0.125 7.87 8.96
0.0625 7.96 9.09
Control 8.01 9.14
Room temperature [°C]: min.: 22.0 max.: 22.5 mean value: 22.3

Light intensity
[lux] n = 20 mean value: 4779
range of the measured values: 4517 to 5032
equalling -5.48 to 5.29% CV [%]: 3.37


Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL. Actual: 6839 cells/mL

- Control end cells density: Mean 2842582 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 4779 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
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 saturated solution. A separate replicate of each test item concentration were prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.


Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.111 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: CI (0.0977 - 0.127)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.039 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: CI (0.0249 - 0.0511)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.038 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.074 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.058 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: CI (0.0482 - 0.0693)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: CI (< 0.0143 - 0.0357)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.047 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: CI (0.0389 - 0.0567)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: All test solutions were visually clear throughout the exposure period, any effects directly related to algae growth not taken into account.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA-ALDRICH, 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 2017-10-10 to 2017-10-13. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC- and LOEC-values The NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01 for growth rate and yield.

Levene’s test on variance homogeneity was done with a significance level 0.01 for growth rate and yield.

Welsh-t-test after Bonferroni-Holm was done with a significance level 0.05 for growth rate and yield.



Software The data for the tables in this report were computer-generated and have been rounded for presentation from the fully 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.
- ToxRat, ToxRat Solutions GmbH
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.


Cell Densities

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

1.00

1

6839

11137

14965

8796

2

6839

16395

14385

10002

3

6839

16670

22519

14953

Mean

6839

14734

17290

11250

0.500

1

6839

23573

37337

31438

2

6839

26411

49535

48725

3

6839

24734

48660

43827

Mean

6839

24906

45177

41330

0.250

1

6839

28333

87240

251628

2

6839

31848

100091

380665

3

6839

34703

151904

928470

Mean

6839

31628

113078

520254

0.125

1

6839

32954

198843

1413192

2

6839

34741

238602

2468541

3

6839

36968

337734

1580208

Mean

6839

34888

258393

1820647

0.0625

1

6839

42864

357854

2207191

2

6839

41199

351580

2432844

3

6839

43300

395658

2422779

Mean

6839

42454

368364

2354271

Control

1

6839

42612

432947

3006750

2

6839

44040

354939

2876192

3

6839

49664

432104

2693024

4

6839

47247

441561

2815623

5

6839

41015

456144

2874553

6

6839

43148

393598

2789348

Mean

6839

44621

418549

2842582


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]

[%]

1.00

1

 

0.084

96

 

1957

100

2

 

0.127

94

 

3163

100

3

 

0.261

87

 

8114

100

Mean

(+)

0.157

92

(+)

4411

100

0.500

1

 

0.508

75

 

24599

99

2

 

0.655

67

 

41886

99

3

 

0.619

69

 

36988

99

Mean

(+)

0.594

70

(+)

34491

99

0.250

1

 

1.20

40

 

244789

91

2

 

1.34

33

 

373826

87

3

 

1.64

19

 

921631

68

Mean

(+)

1.39

31

(+)

513415

82

0.125

1

 

1.78

12

 

1406353

50

2

 

1.96

2

 

2461702

13

3

 

1.81

10

 

1573369

45

Mean

(-)

1.85

8

(+)

1813808

36

0.0625

1

 

1.93

4

 

2200352

22

2

 

1.96

3

 

2426005

14

3

 

1.96

3

 

2415940

15

Mean

(+)

1.95

3

(+)

2347432

17

Control

1

 

2.03

 

 

2999911

 

2

 

2.01

 

 

2869353

 

3

 

1.99

 

 

2686185

 

4

 

2.01

 

 

2808784

 

5

 

2.01

 

 

2867714

 

6

 

2.00

 

 

2782509

 

Mean

 

2.01

 

 

2835743

 


Section-by-Section and Average Specific Growth Ratesof 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

Control

1

1.83

2.32

1.94

2.03

0.257

12.7

10.7

2

1.86

2.09

2.09

2.01

0.131

6.5

3

1.98

2.16

1.83

1.99

0.167

8.4

4

1.93

2.24

1.85

2.01

0.202

10.0

5

1.79

2.41

1.84

2.01

0.343

17.0

6

1.84

2.21

1.96

2.00

0.189

9.4

 

 

 

Mean

2.01

 

 

 

 

 

SD ±

0.01

 

 

 

 

CV [%]

0.61

 

SD = Standard deviation CV = Coefficient of variation

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of three components ofTallowBis(2-hydroxyethyl) amineoxide CASno. 61791-46-6were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS/MS.

 

The measured concentrations of the of three components were between 47% and 67% of the nominal value at the start of the exposure (0 hours) and between 35% and 89% of the nominal value at the end of the exposure (72 hours). The geometric mean measured concentration of total active substance was calculated.

 

Measured Concentrations and Recovery Rates of Test Item for Component 346 Da in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

Sampling date

0 hours

72 hours

 

Nominalconcentrationof the

Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6

(346 Da - C16:0)

test item

 

[mg/L]

a.s.

 

[mg/L]

Meas. conc.

a.s.

[mg/L]

[%]

Meas. conc.

a.s.

[mg/L]

[%]

Geometric Mean Measured Concentration of the active substance

[mg/L]

1.00

0.151

0.102

67

0.131

87

0.116

0.500

0.0755

0.0461

61

0.0669

89

0.0555

0.250

0.0378

0.0228

60

0.0261

69

0.0244

0.125

0.0189

0.0107

56

0.0145

77

0.0125

0.0625

0.00944

0.00463

49

0.00481

51

0.00472

Control

< LOQ

< LOQ

 

Meas. Conc.   = measured concentration of the active substance (dilution and weighing factor taken into account)

a.s.                  = active substance

%                     = percentage of the nominal concentration of the active substance

LOQ           = limit of quantification of the analytical method (15 µg test item /L, corresponding to 2.27 µg a.s./L)

Validity criteria fulfilled:
yes
Conclusions:
In this study, Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.111 (0.0977 – 0.127) mg/L and 0.0473 (0.0389 – 0.0567) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0383 mg/L and < 0.0143 mg/L, respectively.
All effect levels presented here are based on active ingredient concentrations.
Executive summary:

The toxicity of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 (Batch no.:1548134) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3from 2018-01-08 to 2018-01-11 at the test facility.The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

 

The test item Tallow Bis (2-hydroxyethyl) amineoxide CAS no. 61791-46-6is an light yellow paste UVCB substance, which is dispersible in water.The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The test item was warmed up to 50 °C before used for preparation of the stock solution. A stock solution of 1000 mg/L was freshly prepared with demineralized water and treated with ultrasound for 30 minutes at 50 °C. Five concentrations were tested in a geometrical series with a dilution factor of 2:0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

All concentration levels and the control were analytically verified by LC-MS/MS at the start and the end of the exposure.The concentration levels of the test item Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 at the start of the exposure (0 hours) were in the range of 47 to 67% of the nominal values.The measured concentrations ofthe three compoundsof the test item at the end of the exposure (72 hours) were in the range of 35 to 89% of the nominal value.

 

Since the test item is labeled as an UVCB and complex mixture, all effect values given are based on the nominal test item concentrations of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 and on the geometric mean measured concentrations of the three compounds of the test item.

NOEC, LOEC, EC-values and 95% Confidence Intervals of Tallow Bis (2-hydroxyethyl) amineoxide CAS no. 61791-46-6(0 - 72 hours)

                  based on geometric mean measured concentrations of the total active substance and the nominal test item concentrations [mg/L]

 

Growth Rate Inhibition
geometric mean measured concentration of the total active substance [mg/L]

Growth Rate Inhibition
Nominal test item concentration [mg/L]

NOEC

0.0383

0.125

LOEC

0.0744

0.250

ErC10

0.0394 (0.0249 – 0.0511)

0.138 (0.0904 – 0.176)

ErC20

0.0584 (0.0482 – 0.0693)

0.198 (0.165 – 0.232)

ErC50

0.111 (0.0977 – 0.127)

0.350 (0.314 – 0.393)

 

Yield Inhibition

geometric mean measured concentration of the total active substance [mg/L]

Yield Inhibition

Nominal test item concentration [mg/L]

NOEC

< 0.0143

< 0.0625

LOEC

0.0143

  0.0625

EyC10

< 0.0143

< 0.0625

EyC20

0.0226 (< 0.0143 – 0.0357)

0.0783 (< 0.0625 – 0.116)

EyC50

0.0473 (0.0389 – 0.0567)

0.155 (0.127 – 0.187)

Description of key information

The toxicity of C16 -18 (evennumbered, C18 unsaturated) alkyl bis(2 -hydroxyethyl) amine oxide to algae was observed to be ErC50 (72h) of 0.111 mg a.i./L and ErC10 (72h) of 0.0394 mg a.i./L based on Geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.11 mg/L
EC10 or NOEC for freshwater algae:
0.039 mg/L

Additional information

Two algae studies have been performed with Tallow Bis(2-hydroxyethyl) amineoxide

The first study is a repetition of the second study as this second study from 1990 was performed at a time when no suitable analytical method was available. The results of the first study are used for risk assessment and C&L as this study result has a higher reliability.

In the study from Klix (2018), the toxicity of Tallow Bis(2 -hydroxyethyl) amineoxide to Pseudokirchneriella subcapitata was determined according to OECD 201 and EU Method C.3 in compliance with GLP. 

In this test triplicate flasks with five concentrations were tested in a geometrical series with a dilution factor of 2: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L nominal test concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h.  

All concentration levels and the control were analytically verified by LC-MS/MS at the start and the end of the exposure. The concentration levels of Tallow Bis(2-hydroxyethyl) amineoxide at the start of the exposure (0 hours) were in the range of 47 to 67% of the nominal values. The test substance is a UVCB substance and during chemical analysis the three main constituents representing 94.9% were quantified. The effect concentrations were corrected to 100% active ingredient for the calculation of the endpoints. The measured concentrations of the three main constituents at the end of the exposure (72 hours) were in the range of 35 to 89% of the nominal value.

Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.111 (0.0977 – 0.127) mg/L and 0.0473 (0.0389 – 0.0567) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0383 mg/L and < 0.0143 mg/L, respectively. All effect levels presented here are based on active ingredient concentrations.

 

In the study from Kroon (1990) the toxicity of C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide to the freshwater algae Pseudokirchneriella subcapitata (formally called Selenastrum Capricornutum) was determined according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Quantification of the applied concentrations was not possible due to the absence of a suitable analytical method. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.55 and 0.41 mg/L test substance, respectively or 0.28 and 0.21 mg a.i./L. The NOEC of 0.2 mg/L test substance or 0.1 mg a.i./L can be used as long term endpoint (Kroon, 1990).

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