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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:
15 May 2015 - 26 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentration levels
- Sampling method: Samples were taken after 0 and 72 h of exposure. Separate samples without algae were prepared to analyse the test substance at the beginning of the test. For the analysis after 72 h separate replicates were prepared with and without algae at the beginning and incubated under the same test conditions. Additionally the adsorption to the glass was analysed after 72 h.
- Sample storage conditions before analysis: If necessary, all samples were stored at 6 ± 2 °C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 1 mL test item/L (nominal: 962.1 mg/L) was prepared once with de-mineralized water four days prior to the start of the exposure. The test item was placed by pipetting on the surface of the demineralized water. The solution was stirred slowly to avoid formation of emulsion (magnetic stirrer, room temperature for 3 d at 30 °C ± 2 °C. After pre-incubation, the temperature of the solution was reduced to the test temperature of 21 - 24 °C for 24 h ± 1 h. The test item phase was separated by siphon from the bottom and was used for testing with further dilution. The saturated solution was checked after stirring via laser beam (Tyndall effect) for un-dissolved test item (formation of an emulsion). Thereafter, the components of the dilution water were added to the solution.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
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
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35-70 µE*m-2+s-1 for 24 h per day. A four days old pre-culture was prepared in dilution water and used as inoculum.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 22 °C (mean: 21.5 °C)
pH:
8.07 - 8.12 (0 h, treatments), 7.95 - 8.86 (72 h, treatments)
8.10 (0 h, control), 8.87 (72 h, control)
Nominal and measured concentrations:
nominal: control, 10, 18, 32, 56 and 100% of saturated solution
geometric mean measured concentration: control, 1.34, 2.43, 4.45, 8.23, 14.4 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel
- Type: closed
- Material, size, headspace, fill volume: glass, 119 mL, headspace: 0 mL, fill volume: 119 mL
- Aeration: no, constantly shaken (oscillated at approx. 70 rpm)
- Initial cells density: approx. 5x10E+03-E+04 cells/mL (nominal), actual: 6305 cells/mL
- Control end cells density: 535430 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, Nutrient medium Z according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured continuously; Light intensity was measured prior to start of exposure; pH was measured in one additional replicate per concentration and control

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 73.9 µE*m-2*s-1 (mean), 64.2 - 81.3 µE*m-2*s-1 (range of measured values), CV: 6.86%

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: Chlorophyll-a-fluorescence measurement (excitation: 436 nm, emission: 685 nm). Dilution water was used as a background signal. No self-fluorescence was found in a preliminary range finding test at saturated solution.
- Microscopic evaluation: Microscopic evaluation of cells was performed at start and end of incubation. 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
Range finding study
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: 1 mg/L: 0% inhibition of growth, 10 mg/L: -1% inhibition of growth, 100 mg/L: 100% inhibition of growth
Reference substance (positive control):
yes
Remarks:
potassium dichromate (tested: 13-16 Oct 2015 and 10-13 Nov 2015)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Remarks:
7.18 - 7.95 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 14.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI
Remarks:
4.37 - 6.07 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI
Remarks:
8.11 - 9.57 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological abnormalities were observed.
- Any stimulation of growth found in any treatment: yes, 1% stimulation of growth at 1.34 mg/L and 2% stimulation of growth at 2.43 mg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, test solutions were clear and colorless
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: ErC50 (72 h): 0.564 mg/L (with headspace, 95% CI: 0.546 - 0.582 mg/L), 1.15 mg/L (without headspace, 95% CI: 0.922 - 1.32 mg/L)
Reported statistics and error estimates:
ECx-values were calculated by sigmoidal dose-response regression. 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 NOEC/LOEC 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 alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Table 1: Evaluation of growth after 72 h

Geometric mean measured test item concentration [% of saturated solution]

Replicate

Growth rate [d-1]

Inhibition of growth rate [%]

14.4

1

0.774

47

2

0.752

49

3

0.749

49

Mean

0.758 (+)

49

8.23

1

1.27

14

2

1.29

12

3

1.29

13

Mean

1.28 (+)

13

4.45

1

1.45

2

2

1.46

1

3

1.47

0

Mean

1.46 (-)

1

2.43

1

1.47

0

2

1.49

-1

3

1.53

-4

Mean

1.50 (-)

-2

1.34

1

1.47

1

2

1.49

-1

3

1.51

-2

Mean

1.49 (-)

-1

Control

1

1.44

 

2

1.50

3

1.51

4

1.45

5

1.47

6

1.48

Mean

1.47

(+): statistically different compared to the control group
(-): not statistically different compared to the control group

Table 2: Measured concentration and percentage if the initially measured concentrations of the test substance in fresh medium (0 h) and old medium (72 h) with algae

Nominal test item concentration [% of saturated solution]

 

Geometric mean measured concentration [mg/L]

Measured concentration 0 h [mg/L]

Measured concentration 72 h [mg/L]

[%]

100

15.5

13.3

86

14.4

56

8.23

8.23

100

8.23

32

4.71

4.21

89

4.45

18

2.49

2.38

96

2.43

10

1.35

1.33

99

1.34

control

< LOQ

< LOQ

-

LOQ: Limit of Quantification (0.3 mg/L test item, enrichment and dilution factor taken into account)
%: Percent of the initially measured concentration of the test item (0 h)

The amount of test item adsorbed to the test vessel was < 1%.

Validity criteria fulfilled:
yes
Conclusions:
The study resulted in an ErC50 (72 h) of > 14.4 mg/L and an ErC10 (72 h) of 7.6 mg/L based on the geometric mean of the measured concentrations (OECD 201).

Description of key information

ErC50 (72 h): > 14.4 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

ErC10 (72 h): 7.6 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
14.4 mg/L
EC10, LC10 or NOEC for freshwater algae:
7.6 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 (GLP) under static conditions using the freshwater algae Pseudokirchneriella subcapitata. Dilutions of 10, 18, 32, 56 and 100% of a saturated solution were chosen based on the results from a preliminary range-finding test. It was ensured by an appropriate test design that the substance does not volatilize from the test vessels (closed vessels; no headspace). Inhibition of growth was recorded resulting in an ErC10 (72 h) of 7.6 mg/L and an ErC50 (72 h) > 14.4 mg/L. All effect values were based on the geometric mean of measured concentrations.