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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:
08 Dec - 11 Dec 2014
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 (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 10, 20, 40, 80 and 160 mg/L. Samples were taken at start and end of the study.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Fore preparation of test solutions, the density was taken into account. Appropriate volumes of the test item were pipetted directly into measuring flasks, containing respective volume of the dilution water. After stirring with approx. 1100 rpm for 30 min at room temperature the prepared test solutions were transferred to the test vessels which were tightly closed, without headspace.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium without test item
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE*m-2*s-1 for 24 h per day. Nutrient medium Z according to Lüttge et al. (1994). A three day old pre-culture, prepared in dilution water was used as inoculum.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 - 23 °C
pH:
8.07 - 8.26 (0 h)
8.63 - 9.63 (72 h)
Nominal and measured concentrations:
nominal: control, 10, 20, 40, 80 and 160 mg/L
measured: The measured concentrations in the fresh medium were in the range of 101 - 109% of nominal. The measured concentrations in the old media (72 h) were in the rangeof 93 - 101% of nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed (aluminium tops with PTFE seals
- Size, headspace, fill volume: Size: 119 mL, Headspace: 0 mL, Fill volume: 119 mL
- Agitation: constantly shake nat 70 rpm on a rotary shaker
- Initial cells density: 7085 cells/mL
- Control end cells density: 700992 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: according to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 h light
- Light intensity and quality: 60 - 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell density was measured daily via Chlorophyll-a-fluorescense, excitation: 436 nm, emission: 685 nm. Dilution water was used as background signal.
- Other: Microscopic evaluation of the cells at the start and end of exposure was carried out.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: 62% inhibition of growth rate was observed at 100 mg/L, whereas stimulation of growth was recorded at 1 and 10 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
43.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 35.5-51.5 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
115 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 105-126 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
53.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 49.9-57.2 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.774 mg/L (95% CL: 0.711 - 1.08 mg/L) based on growth rate; 0.502 mg/L (95% CL: 0.400 - 0.583 mg/L)
Reported statistics and error estimates:
Statistical analysis was performed by using GraphPad Prism (GraphPad Software Inc.), SigmaPlot (SPSS Inc.) and Excel (Microsoft Corporation).

The effect concentrations were based on nominal concentrations since the measured concentrations were in the range of 93 - 101% after 72 h.

Table 1: Evaluation after 72 h

Nominal test item concentration [mg/L]

Replicate

Growth rate [d-1]

Inhibition of growth rate [%]

Yield [cells/mL]

Inhibition of yield [%]

160

1

0.497

68

24372

96

2

0.519

66

26510

96

3

0.379

78

14977

98

Mean

0.465

70

21953

97

80

1

1.06

31

164048

76

2

1.05

31

158709

77

3

1.12

27

198748

71

Mean

1.08

29

173835

75

40

1

1.38

10

442848

36

2

1.40

8

465690

33

3

1.40

9

463764

33

Mean

1.39

9

457434

34

20

1

1.45

5

544108

22

2

1.46

5

559247

19

3

1.50

2

631092

9

Mean

1.47

4

578149

17

10

1

1.47

4

573324

17

2

1.49

3

610110

12

3

1.48

3

592447

15

Mean

1.48

3

591960

15

Control

1

1.47

-

570336

-

2

1.53

-

686138

-

3

1.50

-

629299

-

4

1.53

-

690852

-

5

1.57

-

788792

-

6

1.58

-

798022

-

Mean

1.53

-

693907

-

Validity criteria fulfilled:
yes
Conclusions:
This study resulted in an ErC50 (72 h) of 115 mg/L and an ErC10 (72 h) of 43.3 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201).

Description of key information

ErC50 (72 h): 115 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

ErC10 (72 h): 43.3 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
115 mg/L
EC10 or NOEC for freshwater algae:
42.3 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 and GLP under static conditions using Pseudokirchneriella subcapitata. The definitive test concentrations of 10, 20, 40, 80 and 160 mg/L were defined after a range-finding test. HPLC-DAD analysis at the beginning and the end of exposure indicated that the nominal concentrations remained within 80-120% of nominal. Thus, the effect concentrations were based on the nominal values. Inhibition of growth was recorded resulting in an ErC50 of 115 mg/L and an ErC10 of 43.3 mg/L after 72 h.

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