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Ecotoxicological information

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 February 13, 2018 to March 23, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
LC-MS/MS
Details on sampling:
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 h) and at the end of the exposure (72 h) 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
10.0 mg test item/L was freshly prepared with dilution water. The dispersion was shaken until the solution was visually clear.

Test concentrations Five concentration levels were tested in a geometrical series with a
(nominal) dilution factor of √10: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 µg/L. The concentration levels are based on the results of a preliminary range finding test (non-GLP).

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


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
Reason for the selection of the test organism: Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.
Origin: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 h per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).



Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21.5 to 22.5
pH:
7.60 to 9.37
Nominal and measured concentrations:
0, 0.100, 0.316, 1.00, 3.16, 10.0 μg/L (nominal)
0, 0.108, 0.212, 0.729, 2.30, 5.46 μg/L (mean measured)
Details on test conditions:
(A) Experimental Procedure
Test substance: Oleyltrimethylammonium chloride CAS no. 10450-69-8

Stock solution, media preparation
10.0 mg test substance/L was freshly prepared with dilution water (see Table 2). The dispersion was shaken until the solution was visually clear.

Test concentrations (nominal)
Five concentration levels were tested in a geometrical series with a dilution factor of √10: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 µg/L. The concentration levels are based on the results of a preliminary range finding test (non-GLP)

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

Reference Substance
Potassium dichromate was tested as a reference substance (for details of the results, please refer to part 8.1.4).

Test method
Static procedure

Duration of the test
72 h

Replicates
3 replicates per concentration level and 6 for the control were tested

Test container and pre-treatment
Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used.

Test volume
100 mL

Dilution water
According to the guidelines

Preculture
A three days old preculture, prepared in dilution water, was used as inoculum.

Initial cell density
Nominal: approximately 5 x 103 - 104 cells/mL
Actual :5523 cells/mL

Application
Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.

Incubation
The flasks were positioned randomly and repositioned daily.

Temperature (target)
Nominal range: 21 - 24°C, controlled at ± 2°C

Agitation
Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

Light intensity (target)
Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime
24 h/day light

Light homogeneity (target)
Within ± 15% over incubation area


(B) Type and Frequency of Measurements: Biological Parameters

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. No self-fluorescence was found in the preliminary range finding test at the concentration levels of 10.0 mg/L.

Microscopic evaluation
Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

(C) Physical Chemical Properties
The pH-value at the start of the exposure was measured in one additional replicate of each test substance concentration and the control. At the end of the exposure, it was measured in a pooled sample of each test substance concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

(D) Chemical Analysis of the Test Substance Concentrations
Determination of All concentration levels and the control were analytically verified the test substance via LC-MS/MS at the start (0 h) and at the end of the exposure (72 h) 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.

(E) Calculations
All calculations were based on the fluorescence values of each replicate.

Cell density out of the fluorescence values
The fluorescence values were related to cell density values according to a calibration curve.
x = 4.939 · Fq + 584
x = cell density [cells/mL]
Fq = mean fluorescence
Coefficient of correlation r2 = 0.9930
The growth rate, the growth rate inhibition, the yield and the inhibition of yield after 72 h were calculated from the cell density values according to the formulas listed below.

Growth rate µ = (ln (Nn) - ln (N0)) / (tn - t0)
where,
µ = growth rate of the cell density (1/day) Nn = biomass after tn days in cells/mL
N0 = biomass at t0 in cells/mL
t0= time of beginning of test
tn= time of nth measurement after beginning of test

Growth rate inhibition, Iµt = ((µc - µt) / µc) · 100%
where,
Iµt = growth rate inhibition in %
c= growth rate of the control after n days
t= growth rate of the test concentrations after n days

Yield, Y = Nn – N0
where,
y= yield
0 = nominal number of cells/mL at the beginning of the test
n = measured number of cells/mL at time n

Yield inhibition, Iy = (YC – YT) / YC · 100%
Where,
Iy= percentage inhibition
YC= mean value for yield in the control group
YT= value for yield for the treatment replicate

(F) Evaluation
EC-values and statistical analyses
EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 h was calculated by third order polynomial regression.

(G) NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The value (acceptable probability of incorrectly concluding that there is a difference) is a =0.05. The Normality Test failed for the calculation of growth rate. Therefore, the data were transformed (Y = 10Y).

(H) 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.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.729 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and biomass
Remarks on result:
other: i.e., equivalent to 0.67µg a.i./L
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 2.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and biomass
Remarks on result:
other: i.e., equivalent to 2.11µg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 4.98 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI (4.66 - 5.36); i.e., equivalent to 4.58 µg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 3.28 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI (3.07 - 3.47); i.e., equivalent to3.01µg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 2.8 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: i.e., equivalent to 2.57 µg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.108 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: i.e., equivalent to <0.099 µg a.i./L
Details on results:
The measured concentrations of the test item were between 66% and 112% of the nominal value at the start of the exposure (0 h) and between 37% and 105% of the nominal value at the end of the exposure (72 h).
- 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 (possible turbidity 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 h. The reference substance toxicity is in the valid range following test facility SOP. ErC50 and EyC50 were determined to be 0.535 mg/L (nominal) (95% CI: 0.523 to 0.547) and 0.320 mg/L (nominal) (95% CI: 0.249 to 0.339), respectively.

Results

Cell Densities

Geometric mean measured test substance concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 h

24 h

48 h

72 h

5.46

1

5523

9506

16626

50004

2

5523

7802

26459

110378

3

5523

14349

55812

50952

Mean

5523

10552

32966

70445

2.30

1

5523

30803

292553

1489292

2

5523

32525

224652

1663639

3

5523

32371

251085

1603581

Mean

5523

31900

256097

1585504

0.729

1

5523

37977

278329

1962498

2

5523

31297

234204

1860162

3

5523

39824

303745

2117879

Mean

5523

36366

272093

1980180

0.212

1

5523

37864

289501

1791658

2

5523

40333

303192

1984180

3

5523

38246

290498

1924023

Mean

5523

38814

294397

1899954

0.108

1

5523

32739

257071

1888956

2

5523

35740

304999

2011839

3

5523

36021

254493

1919924

Mean

5523

34833

272188

1940240

Control

1

5523

43356

346299

2440396

2

5523

36705

349934

2199965

3

5523

36451

307656

2042460

4

5523

39200

310768

2352531

5

5523

38177

313188

2038904

6

5523

37686

308022

2060883

Mean

5523

38596

322645

2189190

 

Evaluation after 72 h

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

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[µg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

5.46

1

 

0.734

63

 

44481

98

2

 

1.00

50

 

104855

95

3

 

0.741

63

 

45429

98

Mean

(+)

0.824

59

(+)

64922

97

2.30

1

 

1.87

6

 

1483769

32

2

 

1.90

5

 

1658116

24

3

 

1.89

5

 

1598058

27

Mean

(+)

1.89

5

(+)

1579981

28

0.729

1

 

1.96

2

 

1956975

10

2

 

1.94

3

 

1854639

15

3

 

1.98

1

 

2112356

3

Mean

(-)

1.96

2

(-)

1974657

10

0.212

1

 

1.93

3

 

1786135

18

2

 

1.96

2

 

1978657

9

3

 

1.95

2

 

1918500

12

Mean

(+)*

1.95

2

(+)*

1894431

13

0.108

1

 

1.95

2

 

1883433

14

2

 

1.97

1

 

2006316

8

3

 

1.95

2

 

1914401

12

Mean

(-)

1.95

2

(+)*

1934717

11

Control

1

 

2.03

 

 

2434873

 

2

 

2.00

 

 

2194442

 

3

 

1.97

 

 

2036937

 

4

 

2.02

 

 

2347008

 

5

 

1.97

 

 

2033381

 

6

 

1.97

 

 

2055360

 

Mean

 

1.99

 

 

2183667

 

* =statistically significant, but with a higher concentration level showing no harmful effect/statistical significance

     compared to control.

 

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 h)

 

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

2.06

2.08

1.95

2.03

0.068

3.34

6.04

2

1.89

2.26

1.84

2.00

0.226

11.3

3

1.89

2.13

1.89

1.97

0.140

7.12

4

1.96

2.07

2.02

2.02

0.056

2.75

5

1.93

2.11

1.87

1.97

0.120

6.09

6

1.92

2.10

1.90

1.97

0.110

5.59

 

 

 

Mean

1.99

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.31

 

SD = Standard deviation CV = Coefficient of variatio

Measured Exposure Concentrations during the Definitive Test

The concentrations of test substance were determined in fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS. The measured concentrations of the test substance were between 66% and 112% of the nominal value at the start of the exposure (0 h) and between 37% and 105% of the nominal value at the end of the exposure (72 h).

Measured Concentrations and Percent of Nominal Concentration of the Test substance in Fresh Medium (0 h) and Old Medium (72 h) with Algae

Sampling date

0 h

72 h

 

Nominalconcentrationof the test substance

g/L]

Test substance

Meas. conc.

g/L]

[%]

Meas. conc.

g/L]

[%]

Geometric mean measured Concentration of the test substance

[µg/L]

10.0

8.12

81

3.67

37

5.46

3.16

2.55

81

2.08

66

2.30

1.00

0.804

80

0.661

66

0.729

0.316

0.208

66

0.217

69

0.212

0.100

0.112

112

0.105

105

0.108

Control

< LOQ

< LOQ

 

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

% = percentage of the nominal concentration of the test substance

LOQ = limit of quantification of the analytical method (0.0513 µg test substance /L)

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be 4.98, 3.28, 0.729 and 2.30 μg/L, respectively.
Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, Oleyl TMAC (active: 91.9%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with inoculum culture to give a nominal cell density of 0.5 × 104 to 0.5 × 105 cells/mL. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.100, 0.316, 1.00, 3.16 and 10.0 μg/L in culture medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a LC/MS MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.108, 0.212, 0.729, 2.30 and 5.46 μg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50, ErC10 values (for growth rate) and EyC50, EyC10 value (Cell particle density) were calculated to be 4.98 (4.66 – 5.36), 2.4 (1.99-2.76) and 3.28 (3.07 – 3.47), < 0.108 μg/L respectively. The NOEC and the LOEC values were determined to be 0.729 μg/L and 2.30 μg/L, respectively, for both growth rate and cell particle densities. The study had met all validity criteria. Under the study conditions, 72 h ErC50, ErC10, EyC50,EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be 4.98, 2.8, 3.28, <0.108, 0.729 and 2.30 μg/L, respectively (i.e., equivalent to 4.58, 2.57, 3.01, <0.099, 0.67 and 2.11 μg a.i./L, respectively) (Volker, 2018).

Description of key information

The 72 h ErC50, ErC10, EyC50,EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be 4.98, 2.4, 3.28, <0.108, 0.729 and 2.30 μg/L, respectively (i.e., equivalent to 4.58, 2.2, 3.01, <0.099, 0.67 and 2.11 μg a.i./L, respectively)

Key value for chemical safety assessment

EC50 for freshwater algae:
4.58 µg/L
EC10 or NOEC for freshwater algae:
2.2 µg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, Oleyl TMAC (active: 91.9%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with inoculum culture to give a nominal cell density of 0.5 × 104 to 0.5 × 105 cells/mL. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.100, 0.316, 1.00, 3.16 and 10.0 μg/L in culture medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a LC/MS MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.108, 0.212, 0.729, 2.30 and 5.46 μg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50, ErC10 values (for growth rate) and EyC50, EyC10 value (Cell particle density) were calculated to be 4.98 (4.66 – 5.36), 2.4 (1.99-2.76) and 3.28 (3.07 – 3.47), < 0.108 μg/L respectively. The NOEC and the LOEC values were determined to be 0.729 μg/L and 2.30 μg/L, respectively, for both growth rate and cell particle densities. The study had met all validity criteria. Under the study conditions, 72 h ErC50, ErC10, EyC50,EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be 4.98, 2.4, 3.28, <0.108, 0.729 and 2.30 μg/L, respectively (i.e., equivalent to 4.58, 2.2, 3.01, <0.099, 0.67 and 2.11 μg a.i./L, respectively) (Volker, 2018).