Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May - 11 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen
Analytical monitoring:
yes
Details on sampling:
- Sampling method: for assessing the concentration of the test item at test start, the samples were taken from the test solutions prior to the addition of the algae at test start. At test end the samples were taken from the pooled replicates per treatment level.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation of the test media was performed under sterile conditions as far as possible. The highest test concentration was directly prepared by weighing 80 mg of the test item on a sterile teflon weighing boat. The weighing boat was given into a 1 L graduated glass flask and stirred strongly for 1 hour (with a sterile magnetic stir bar). The highest test concentration (conc. number 5) was diluted with 10-fold concentrated growth medium and ultra pure water under sterile conditions. Details are given below:

Conc. number Nominal conc. [mg/L] Test media/Conc. number 10-fold concentrated growth medium Ultra pure water
5 80.0 1000 mL no. 5 - -
4 40.0 500 mL no. 5 50 mL 450 mL
3 20.0 500 mL no. 4 50 mL 450 mL
2 10.0 500 mL no. 3 50 mL 450 mL
1 5.0 500 mL no. 2 50 mL 450 mL
Control 0 - 100 mL 900 mL
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guideline No. 201

ACCLIMATION
- Acclimation period: Three days prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test
- Culturing media and conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 23 °C
pH:
7.71 (start) to 9.02 - 9.17 (end) (control)
6.40 - 7.73 (start) to 7.01 - 9.15 (end) (test cultures)
Nominal and measured concentrations:
control, 5, 10, 20, 40, and 80 mg/L (nominal)
control, 4.45, 9.07, 17.52, 35.10, and 71.00 mg/L (measured, initial)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): covered, with air-permeable silicone-sponge caps
- Material, size, headspace, fill volume: 250 mL conical glass flasks filled with 100 mL of the test media
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10^4 cells/mL
- Control end cells density: 355 x 10^4
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“.
- Culture medium different from test medium: no; sterilised synthetic OECD medium according to OECD 201 was used as growth medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: continuous
- Light intensity and quality: 7405 - 8147 Lux (OSRAM “day light”)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell concentrations were determined after 24, 48 and 72 h
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The correctness of the electronic counts was checked by microscopically counting following internal standard operation procedures

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 10, 50 and 100 mg/L and 2, 10 and 20 mg/L
- Results used to determine the conditions for the definitive study: Based on on the results in the range-finding study with 90.9% inhibition at a concentration of 100 mg/L and 5% inhibition at 10 mg/L, concentrations for the definitive study were chosen to be 5, 10, 20, 40, and 80 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
43.73 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 38.40 - 50.37
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
17.52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
15.59 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10.84 - 19.57
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
35.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
23.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 21.44 - 25.97
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.89 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 15.04 - 18.38
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
17.52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
35.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Results with reference substance (positive control):
EC50 (growth rate) = 2.91 mg/L (95% confidence limits 2.35 - 3.63 mg/L)
Reported statistics and error estimates:
The test results showing inhibitions around 50% were statistically analysed to determine an EC50, EC10 and EC10 values together with 95 % confidence intervals using Probit-analysis [Finney. D.J.: Statistical Method in Biological Assay. 2nd ed. London. 1984] assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis.
The NOEC and LOEC values for growth rate were determined by the Welch-t test for Inhomogeneous Variances with Bonferroni correction [Wilcoxon. Frank (1945): Individual Comparisons by Ranking Methods. Biometrics Bulletin 1: 80–83; Mann. Henry & Whitney. Donald (1947): On a test of whether one of two random variables is stochastically larger than the other. Annals of mathematical Statistics 18: 50-60] and for yield by the Williams Multiple Sequential t-test [Williams. D.A. (1971) A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics. 27. 103-117; Williams. D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28. 519-531].
The computer program ToxRat [ToxRat® Professional 2.10. ToxRat® Solutions GmbH. Naheweg 15. D-52477 Alsdorf] was used for statistical evaluations.

Chemical analysis:

At test start the measured concentrations of octanoic acid ranged between 87.6 and 90.7% of the nominal values. The concentrations decreased during the test period to levels between 84.3 and 94.0% of the measured initial values or 74.9 and 82.5% of nominal, respectively. Therefore, the test was evaluated using the initial measured octanoic acid concentrations.

Table: Measured concentrations of the test item in the test solutions

Nominal concentrations [µg test item/L]

Measured concentrations

Day 0

Day 3

[µg test item/L]

% nominal

[µg test item/L]

% nominal

% of day 0 measured

Control

0

 

0

 

84.3

5

4.45

88.9

3.75

74.9

85.3

10

9.07

90.7

7.74

77.4

90.0

20

17.5

87.6

15.76

78.8

94.0

40

35.1

87.8

33.00

82.5

90.8

80

71.0

88.8

64.48

80.6

84.3

Definitive test:

Table: Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

Test item
nominal [mg/L]

Test item
measured [mg/L]

% Inhibition
of growth rate

% Inhibition
of yield

Control

0.0

0.0

0

5

4.45

0.2

1.4

10

9.07

1.8

10.1

20

17.5

2.4

12.8

40

35.1

50.8

95.0

80

71.0

66

98.2

Table: Yield (cell number increase) dependent on initial measured concentration of test item and time.

Concentration
[mg test item/L]

Control

4.45

9.07

17.52

35.10

71.00

24 h

 

 

 

 

8.260

7.588

7.556

6.171

5.680

2.536

8.861

7.146

7.213

6.168

4.591

2.475

8.910

6.900

7.400

5.683

4.948

2.835

8.570

7.406

6.986

7.276

4.023

2.188

7.668

-

7.438

9.040

8.831

Mean:

8.447

7.260

7.289

6.325

4.811

2.508

Std.Dev.:

0.605

0.301

0.246

0.675

0.694

0.265

n:

8.000

4.000

4.000

4.000

4.000

4.000

CV:

7.200

4.100

3.400

10.700

14.400

10.600

 

48 h

 

 

 

 

55.840

51.890

54.230

40.670

11.960

4.548

54.470

54.120

49.420

40.400

10.120

4.041

56.650

46.220

55.300

36.850

9.890

3.986

52.940

56.500

50.870

45.960

9.970

3.551

54.430

-

57.410

64.110

56.360

Mean:

56.526

52.182

52.455

40.970

10.485

4.031

Std.Dev.:

3.387

4.398

2.767

3.755

0.988

0.408

n:

8.000

4.000

4.000

4.000

4.000

4.000

CV:

6.000

8.400

5.300

9.200

9.400

10.100

 

72 h

 

 

 

 

 

348.500

340.500

352.500

301.200

28.030

8.330

338.200

359.900

305.400

326.200

14.610

6.660

377.000

338.100

288.200

267.000

13.710

5.930

331.000

359.200

328.400

342.200

14.680

5.000

352.300

-

361.800

379.400

346.900

Mean:

354.387

349.425

318.625

309.150

17.757

6.480

Std.Dev.:

17.304

11.736

27.950

32.776

6.863

1.408

n:

8.000

4.000

4.000

4.000

4.000

4.000

CV:

4.900

3.400

8.800

10.600

38.600

21.700

(Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation). Cell number at test start: 10 000 cells/mL.

Table: Growth rates (1/d) dependent on initial measured concentration of test item and time.

Concentration
[mg test item/L]

Control

4.45

9.07

17.52

35.10

71.00

24 h

 

 

 

 

2.226

2.150

2.147

1.970

1.899

1.263

2.289

2.098

2.106

1.970

1.721

1.246

2.294

2.067

2.128

1.900

1.783

1.344

2.259

2.129

2.078

2.113

1.614

1.159

2.160

-

2.133

2.307

2.286

Mean:

2.244

2.111

2.115

1.988

1.754

1.253

Std.Dev.:

0.066

0.037

0.030

0.090

0.119

0.076

n:

8.000

4.000

4.000

4.000

4.000

4.000

CV:

2.900

1.700

1.400

4.500

6.800

6.000

2.244

2.111

2.115

1.988

1.754

1.253

 

48 h

 

 

2.020

1.984

2.006

1.865

1.281

0.857

2.008

2.005

1.960

1.862

1.204

0.809

2.027

1.927

2.015

1.817

1.194

0.803

1.994

2.026

1.974

1.925

1.198

0.758

2.008

 

2.034

2.088

2.025

Mean:

2.020

1.984

2.006

1.865

1.281

0.857

Std.Dev.:

2.008

2.005

1.960

1.862

1.204

0.809

n:

2.027

1.927

2.015

1.817

1.194

0.803

CV:

1.994

2.026

1.974

1.925

1.198

0.758

2.008

 

 

 

 

 

 

72 h

 

 

2.020

1.984

2.006

1.865

1.281

0.857

2.008

2.005

1.960

1.862

1.204

0.809

2.027

1.927

2.015

1.817

1.194

0.803

1.994

2.026

1.974

1.925

1.198

0.758

2.008

 

2.034

2.088

2.025

Mean:

1.957

1.953

1.921

1.911

0.963

0.666

Std.Dev.:

0.016

0.011

0.029

0.036

0.107

0.062

n:

8.000

4.000

4.000

4.000

4.000

4.000

CV:

0.800

0.600

1.500

1.900

11.100

9.300

1.957

1.953

1.921

1.911

0.963

0.666

(Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
24 Aug - 09 Sep 2010
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP Guideline study. However, due to the massive loss of test substance during the course of the study and noticeable discrepancies in the results the study was rated RL3. Already after 24h measured concentrations of 0.004 mg/L, which is half the limit of detection (LOD), are reported for nominal test concentrations up to 32 mg/L. After 72h measured concentrations for all test concentrations were reported as 0.004 mg/L , i.e. up to 100 mg/L nominal. Results were reported based on the time weight average (TWA). The EC50 for growth rate is given as 31 mg/L and the NOEC as 0.07 mg/L, which means a difference between both values by a factor of approximately 500. These results are considered not to be reliable and useful for assessment of the test item, thus the study was rated RL3.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples of 2 mL were taken from all test concentrations and the control at test start and after 24 and 72 h.
- Sample storage conditions before analysis: in a freezer
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg/L test solution were dissolved in the test medium by applying 15 minutes of magnetic stirring. The lower test concentrations were prepared by subsequent dilution of these 100 mg/L solution.
- Controls: 1 control with 6 replicated
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL 1
- Source: in-house culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C and a light intensity of 66 - 76 µE/m2/s at a wavelength of 400 - 700 nm.

ACCLIMATION
- Acclimation period: 3 days before test start, cells form the algal stock culture were inoculated in culture medium (M2, according to OECD 201) at a cell density of 1* 10E-4.
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.3 - 23.2 °C, measured continuously in a temperature control vessel
pH:
6.6 - 8.2, measured at test start and test end
Nominal and measured concentrations:
nominal: 10, 18, 32, 56 and 100 mg/L
time weighed average: 0.07, 0.09, 0.12 and 4.9 mg/L.
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL all glass, containing 50 mL test solution
- Initial cells density: 1 * 10E4
- Control end cells density: 101.3 *10E4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per control without algae: 1

GROWTH MEDIUM
- Standard medium used: yes (M2 according to OECD 201)

OTHER TEST CONDITIONS
- Adjustment of pH: yes, with 1 N NaOH
- Photoperiod: continuous
- Light intensity and quality: 66 - 76 µE/m2/s using TLD-lamps ("Cool-white", 30 Watt)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counting chamber and microscope, since the test solutin was too turbid to use a spectrophotometer.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The expected EC50 for growth rate reduction was between nominally 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.07 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.02 - 0.63 mg/L
Details on results:
- Observation of abnormalities (for algal test): No abnormalities observed.
Results with reference substance (positive control):
- Results with reference substance valid?: yes
- EC50: 1.1 mg/L

Table 1: Measured concentrations versus nominal concentrations

Nominal concentrations [mg/L] Measured concentrations [mg/L] TWA concentrations [mg/L]
0 h 24 h 72 h
10 9.76 0.004** 0.004** 0.07
18 18.1 0.004** 0.004** 0.09
32 31.4 0.004** 0.004** 0.12
56 55.5 3.7 0.004** 4.9
100 99.9 50.3 0.004** 24
100* 102* 51.3* 0.004*,**  - 

* Without algae

** Half of the detection limit

Table 2: Percentage reduction of growth rate

TWA concentations

mean growth rate

0-24 h

Reduction [%]

24-48 h

Reduction [%]

48-72 h

Reduction [%]

control

0.09148

-

0.05340

-

0.05830

-

0.07

0.09266

-1.3

0.05184

2.9

0.05399

-0.3

0.09

0.08041

12.1

0.05807

-8.7

0.03986

26.0

0.12

0.05488

40.0

0.05772

-8.1

0.04715

12.4

4.9

0.03773

58.8

0.05885

-10.2

0.03388

37.1

24

0.02118

76.9

0.04540

15.0

0.03382

37.2

Table 3: Percentage reduction of growth rate during the test.

TWA concentrations [mg/L] Mean growth rate
µ (0-72 h) Reduction (%)
control 0.06624
0.07 0.06617 0.1
0.09 0.05945 10.2
0.12 0.05325 19.6
4.9 0.04349 34.3
24 0.03347 49.5

Description of key information

EC50 (72 h) = 43.73 mg/L (meas.) based on growth rate (OECD 201)
NOEC (72 h) = 17.52 mg/L (meas.) based on growth rate (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
43.73 mg/L
EC10 or NOEC for freshwater algae:
17.52 mg/L

Additional information

Two studies investigating the toxicity of octanoic acid (CAS 124-07-2) to freshwater algae are available.

The key study (2012) was conducted according to OECD 201 and GLP. The test organism Pseudokirchneriella subcapitata was exposed to nominal test item concentrations of 0 (control), 5, 10, 20, 40, and 80 mg/L (measured: 0, 4.45, 9.07, 17.52, 35.10, and 71 mg/L) for 72 h. The test substance concentrations were monitored during the experiment by GC-MS analysis. The test solutions were sampled prior to the addition of the algae at test start of the experiment while at test end the samples were taken from the pooled replicates per treatment level. The recovery of the test item concentrations at test start ranged between 87.6 and 90.7% of the nominal values and decreased to levels between 74.9 and 82.5% of nominal, respectively. The test showed growth rate inhibitions which resulted in an EC50 (72 h) of 43.73 mg/L and an EC10 (72 h) of 15.59 mg/L based on the measured initial concentrations.

The second available study (2010) was conducted according to OECD 201 with Pseudokirchneriella subcapitata under GLP condition. The test organisms were exposed to nominal test item concentrations of 0, 10, 18, 32, 56 and 100 mg/L. Test concentrations were analytically monitored using a UPLC TQD mass spectrometer. The test item concentrations were measured at the start of the experiment, after 24 h and at the end of the test. The effect concentrations were calculated in terms of TWA of the measured test concentrations. Where substance was not detected, the half of the calculated lowest calibration concentration (0.004 mg/L) was used for the calculation. On this basis a NOEC of 0.07 mg/L and an ErC50 of 31 mg/L was derived. The study revealed very fast loss of test concentrations in the test solutions. The test substance could not be detected at the nominal concentrations of 10 – 32 mg/L, and only 10% and 50% of the 56 mg/L and 100 mg/L nominal concentrations, respectievely could be measured after 24 h exposure. At the end of the exposure period, the test item was not detected in any concentration level. The presence of algae did not have impact of the loss the test item. Tracking the biological effects, the inhibition of the growth rate in the range from 12% to 77% was mainly affected within the first 24 h exposure period, and continually at the high concentration levels within 24-72 h exposure period, at nominal concentrations above 18 mg/L. Thus the calculation method for the effect values based on TWA mean and half of the calculated lowest calibration concentration (0.004 mg/L) is considered to present an extreme overestimation of the chronic effects of octanoic acid to algae and strongly impacted by the very fast loss of the measured concentrations. Based on the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R7b, Appendix R.7.8—1 (2016) and expert judgement the effect values of the study (2010) with Pseudokirchneriella subcapitata cannot be recalculated with the provided data in a statistical valid way and therefore, this study was disregarded and rated as RL3.

Additionally, the calculated effect values in this study report are not confirmed by the further available study on algae toxicity (2012) and the general toxicity profile of fatty acids on algae. Concerning algae sensitivity on water soluble fatty acids, an increasing toxicity is observed with increasing chain length between heptanoic acid and decanoic acid (heptanoic acid: NOErC (72 h) = 32 mg/L; octanoic acid: NOErC (72 h) = 17.52 mg/L; nonanoic acid: NOErC (72 h) = 4.4 mg/L and decanoic acid: NOErC (72 h) = 3.2 mg/L).

In conclusion the study conducted in 2012 with effect values of an EC50 (72 h) of 43.73 mg/L and an EC10 (72 h) of 15.59 mg/L (both measured, initial) was taken into account for further assessment.