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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 - 06 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The temperature in the experiment was in a range of 20.9 – 25.7 °C and therefore higher than stated in the study plan. Because normal growth was observed, this can be stated as uncritical.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PM-5927
- Substance type: Whitish/Crystalline powder
- Physical state: solid
- Lot/batch No.: 14-121
- Expiration date of the lot/batch: 01 June 2016
- Storage condition of test material: Room Temperature (20 ± 5 °C); Keep away from light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test item in the test vessels was measured at the start and at the end of the test.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation:
A stock solution containing 100.4 mg/L test item in nutrient medium (demineralised water enriched with minerals but without algae) was prepared. The test concentrations were prepared by dilution of the stock solution in nutrient medium. For this the volumes 0.44 mL, 0.92 mL, 2.0 mL, 4.4 mL, 9.2 mL and 20 mL of the stock solution were added into a 200 mL volumetric flask and filled up with nutrient medium.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: SAG 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in February 2014 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2-8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
20.9 – 25.7 °C
pH:
7.4 - 8.0
Nominal and measured concentrations:
control, 0.22, 0.46, 1.0, 2.2, 4.6, and 10 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: glass flasks, total volume 65 mL
- Initial cells density: 7546 cells/mL
- Control end cells density: 432458 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: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5300 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer; photometric measurement of optical density at every 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2
- Range finding study: yes
Test concentrations: a) 1, 10, and 100 mg/L; b) 0.05, 0.1, 0.5, and 1 mg/L
Results used to determine the conditions for the definitive study: In a first pre-test at the concentrations 1 / 10 / 100 mg/L in all treatments significant inhibition of algal growth could be observed. Therefore a second pre-test using the concentrations 0.05 mg/L, 0.1 mg/L, 0.5 mg/L and 1 mg/L was performed. In the treatments below 1 mg/L no inhibition of algal growth was observed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.20 - 3.40 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.16 - 0.39 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.31 - 0.87 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.16 - 0.39 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Results with reference substance (positive control):
72h ErC50: 1.1 mg/L (95% CL: 0.95 – 1.3 mg/L)
72h EyC50: 0.39 mg/L (95% CL: 0.33 – 0.44 mg/L)
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.1.

Any other information on results incl. tables

Analytical Determination:

The determined concentrations of the cation at the start and the end of the test were in the range of 35% - 99%. The determined concentrations of the anion at the start and the end of the test were in the range of 97% - 113%. The values found in the two lowest treatments after 72 hours for the cation where significantly lower than at the beginning. Nevertheless, the determination of the biological results was based on the nominal concentrations as the values for the anion were very close to the nominal concentrations.

Table 1: Measured concentrations (cation)

Nominal Concentration test item

Measured Concentration (t = 0 h)

Measured Concentration (t = 72 h)

% of Nominal Concentration (t = 0h)

% of Nominal Concentration (t = 72h)

mg/L

mg/L

mg/L

mg/L

mg/L

Blank control

n.d.

n.d.

n.d.

n.d.

0.22

0.18

0.08

81%

35%

0.46

0.40

0.31

87%

66%

1.0

0.98

0.90

98%

90%

2.2

2.10

1.90

95%

86%

4.6

4.50

4.51

98%

98%

10

9.59

9.86

96%

99%

Table 2: Measured concentrations (anion)

Nominal Concentration test item

Measured Concentration (t = 0 h)

Measured Concentration (t = 72 h)

% of Nominal Concentration (t = 0h)

% of Nominal Concentration (t = 72h)

mg/L

mg/L

mg/L

mg/L

mg/L

Blank control

n.d.

n.d.

n.d.

n.d.

0.22

0.22

0.22

99%

101%

0.46

0.52

0.45

113%

97%

1.0

1.07

1.03

107%

103%

2.2

2.16

2.13

98%

97%

4.6

4.62

4.55

100%

99%

10

9.65

9.82

97%

98%

Table 3: Growth rate and yield

Nominal Concentration in mg/L

 

Growth Rate (0-72h) [day-1]

Yield (0-72h) [Cell Concentration/mL]

Blank control

Mean

1.35

424912

 

SD

0.03

36099

0.22

Mean

1.40

500551

 

SD

0.01

19414

0.46

Mean

1.11

205539

 

SD

0.02

15485

1.0

Mean

0.88

102051

 

SD

0.09

31784

2.2

Mean

0.60

38089

 

SD

0.02

2713

4.6

Mean

0.43

20123

 

SD

0.01

1245

10

Mean

0.31

11499

 

SD

0.03

1647

SD = Standard deviation

Table 4: Inhibition values

Nominal concentration in mg/L

% Inhibition

 

Growth Rate (0-72h)

Yield (0-72h)

Blankcontrol

0

0

0.22

-4.04

-17.80

0.46

17.47

51.63

1.0

34.51

75.98

2.2

55.54

91.04

4.6

67.90

95.26

10

77.18

97.29

Table 5: Cell number/mL

Nominal Concentration in mg/L

Parameter

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

Mean

7546

24075

107081

432458

Blank control

SD

0

2783

18086

36099

0.22

Mean

7546

24794

140499

508097

0.22

SD

0

1078

3293

19414

0.46

Mean

7546

23716

90911

213085

0.46

SD

0

2156

3465

15485

1.0

Mean

7546

26591

64680

109597

1.0

SD

0

1245

3887

31784

2.2

Mean

7546

22997

37371

45635

2.2

SD

0

622

1245

2713

4.6

Mean

7546

20123

24075

27669

4.6

SD

0

622

1647

1245

10

Mean

7546

18685

15811

19045

10

SD

0

1245

1647

1647

Table 6: Daily growth rates of the controls

Growth rates

days 0 – 1

days 1 – 2

days 2 – 3

CV of sectional daily growth rates

days 0 – 3

Replicate 1

0.999

1.425

1.563

22%

1.33

Replicate 2

1.050

1.361

1.562

19%

1.32

Replicate 3

1.312

1.386

1.275

4%

1.32

Replicate 4

1.145

1.671

1.261

20%

1.36

Replicate 5

1.190

1.528

1.372

12%

1.36

Replicate 6

1.232

1.540

1.402

11%

1.39

Mean

1.155

1.485

1.406

15%

1.349

Standard deviation

0.116

0.117

0.133

 

0.027

CV

10%

8%

9%

 

2%

CV = Coefficient of variation

Validity:

Increase Factor:

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates:

Mean coefficient of variation of daily growth rates in the blank controls should be 35% at the most. Coefficient of variation of average growth rate during the whole test period should be 7% at the most.

Values and assessment:

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined.

Change of pH:

The pH of the blank control should not fluctuate by more than 1.5 units. The pH changed by 0.5 units in the blank control.

All validity criteria were met. The result of the test can be considered valid.

Applicant's summary and conclusion

Conclusions:
CLP: Aquatic chronic 2, H411 (basis for classification: not rapidly biodegradable and a NOEC (growth rate) of 0.22 mg/L for algae)