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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-05-22 to 2019-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Six concentrations were tested in a geometrical series with factor of 2.5 in the concentration range of 2.05 - 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L, corresponding to the geometric mean measured test item concentration of 1.13 - 1.94 - 6.44 - 20.5 - 61.3 - 165 mg/L
- Sampling method: At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples were taken from pooled replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
Stock solution: 200 mg test item/L, freshly prepared with dilution water.
Dispersion treatment: The stock solution was agitated until the solution was visually clear and check via laser beam for Tyndall effect, which was negative.
Test concentrations: Out of the stock solution six concentration levels were tested in a geometrical series with a dilution factor of 2.5: 2.05 - 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L corresponding to the geometric mean measured test item concentration of 1.13 - 1.94 - 6.44 - 20.5 - 61.3 - 165 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
Control: Six replicates (without test item) were tested under the same test conditions as the test item replicates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
Synonyms Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
Reason for the selection Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.
of the test organism
Origin Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
Cultivation at test facility Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.
Culture medium Nutrient medium Z according to LÜTTGE et al. (1994)
Acclimation period: No
Culturing media and conditions (same as test or not): Nutrient medium Z for culture and test medium according to OECDTG201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
37 mg CaCO3/L
Test temperature:
21 - 24 °C controlled at ± 2°C
pH:
Control:
Start (0h): 8.06
End (72h): 8.92
Conductivity:
143 µS/cm
Nominal and measured concentrations:
Nominal concentration measured concentration

0 h:
Control < LOQ < LOQ (1.5 mg/L)
200 mg/L 189 mg/L
12.8 mg/L 12.6 mg/L
5.12 mg/L 5.04 mg/L
72 h:
Control < LOQ < LOQ
200 mg/L 144 mg/L
12.8 mg/L 3.30 mg/L
5.12 mg/L < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: 5416 cells/mL
- Control end cells density: 1661286 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3
GROWTH MEDIUM
- Standard medium used: Nutrient medium Z according to LÜTTGE et al. (1994)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECDTG201
- Total organic carbon: < 2 mgC/L
- Alkalinity: 0.8 mmol/L
- Conductivity: 143µS/cm
- Culture medium different from test medium: yes
- Intervals of water quality measurement: at start(0h) and at the end (72h) of study
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes in the highest test concentration of 200 mg/L. Both adjusted and non-adjusted highest concentrations were tested.
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 1; 10; 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
25.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 1.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1.13 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 observed.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other:
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- ErC50: 0.701 mg/L (CI; 0.660 – 0.750)
- Other: EyC50 = 0.353 mg/l (CI; 0.312 – 0.394)
based on nominal conccentrations.

Evaluation after 72 hours: 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

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

165 (pH control)

1

 

0.38

80

 

11347

99

2

 

-0.02

101

 

-312

100

3

 

0.48

75

 

17398

99

Mean

 

0.28

85

 

9478

99

165

1

 

n.a.

100

 

n.a.

100

2

 

n.a.

100

 

n.a.

100

3

 

n.a.

100

 

n.a.

100

Mean

(+)

n.a.

100

(+)

n.a.

100

61.3

1

 

0.49

75

 

17831

99

2

 

0.47

75

 

16882

99

3

 

0.51

73

 

19365

99

Mean

(+)

0.49

74

(+)

18026

99

20.5

1

 

1.80

6

 

1194052

28

2

 

1.77

7

 

1080190

35

3

 

1.81

5

 

1231195

26

Mean

(+)

1.79

6

(+)

1168479

29

6.44

1

 

1.86

3

 

1416325

14

2

 

1.86

2

 

1438426

13

3

 

1.87

2

 

1477759

11

Mean

(+)1

1.86

2

(+)

1444170

13

1.94

1

 

1.85

3

 

1383077

16

2

 

1.83

4

 

1323346

20

3

 

1.83

4

 

1310495

21

Mean

(+)1

1.84

4

(+)

1338973

19

1.13

1

 

1.84

3

 

1360830

18

2

 

1.88

1

 

1533498

7

3

 

1.90

0

 

1620732

2

Mean

(-)

1.88

2

(+)

1505020

9

Control

1*

 

1.75

 

 

1034625

2

 

1.91

 

 

1649162

3

 

1.94

 

 

1822268

4

 

1.93

 

 

1738392

5

 

1.91

 

 

1645949

6

 

1.86

 

 

1423578

Mean

 

1.91

 

 

1655870

negative value = growth stimulation                            n.a. = not applicable

*) Outlier, according to Grubb’s test for outliers, this replicate was excluded from the calculations and evaluation
 1= statistically significant but biologically not relevant (inhibition < 5%)

Validity criteria fulfilled:
yes
Conclusions:
In this study, the effect values of the test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follow based on geometric mean measured test item concentrations: The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 46.7 (45.1 - 48.0) mg/L and 25.5 (22.4 - 39.9) mg/L, respectively. The NOEC for growth rate and yield were 6.44 mg/L and < 1.13 mg/L, respectively
Executive summary:

The toxicity of the test itemto the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3.The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5416 cells/mL. A stock solution with a nominal concentration of 200 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear.The stocksolutionwas checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.Six concentrations were tested in a geometrical series with a factor of 2.5:2.05 - 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L, corresponding to the geometric mean measured test item concentration of 1.13 - 1.94 - 6.44 - 20.5 - 61.3 - 165 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The measured concentrations of the test item were in the range of 83 to 103% of the nominal values at the start of the exposure (0 hours) and < LOQ to 72% of the nominal values at the end of the exposure (72 hours). All effect values given are based on the geometric mean measured test item concentrations.The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 46.7 (45.1 - 48.0) mg/L and 25.5 (22.4 - 39.9) mg/L, respectively. The NOEC for growth rate and yield were 6.44 mg/L and < 1.13 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Remarks:
NOEC EC50 calculation based on nominal values instead of geometric mean concentration. Insufficient documentation of analytical methods of the study.
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
84 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 90% to 118% of nominal.

After 72 hours there was a marked decline in measured test concentrations, which were in the range of 20% to 29% of nominal with the exception of the 6.25 and 100 mg/l test concentrations, which showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed. The results of the analytical work conducted indicated that the test material was stable in culture medium over the exposure period and hence this decline was considered to be due to adsorption of the actively growing algal cells.

As such the algal cells were exposed to the nominal concentrations of the test material throughout the test period.
Therefore the nominal concentrations were used for calculating the Effects Concentrations. Exposure of Scenedesmus subspicatus to the test material gave an EbC50 (72 h) value of 66 mg/l; 95 % confidence limits 58 - 75 mg/l and ErC50 (0 - 72 h) value of 84 mg/l. The No Observed Effect Concentration was 6.25 mg/l.

Validity criteria fulfilled:
not applicable
Conclusions:
2,4,6-tris[(dimethylamino)methyl]phenol is harmful to algae.

Executive summary:

Green algae (Scenedesmus subspicatus) were exposed to 6.25, 12.5, 25, 50 and 100 mg/l of 2,4,6-tris[(dimethylamino)methyl]phenol (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 degrees C. Exposure of green algae to the test material gave an EbC50 (72 h) value of 66 mg/l; 95 % confidence limits 58 - 75 mg/l and ErC50 (0 - 72 h) value of 84 mg/l. The No Observed Effect Concentration was 6.25 mg/l.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 90% to 118% of nominal. After 72 hours there was a marked decline in measured test concentrations, which were in the range of 20% to 29% of nominal with the exception of the 6.25 and 100 mg/l test concentrations, which showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed. The results of the analytical work conducted in the absence of algae indicated that the test material was stable in culture medium over the exposure period and hence this concentration decline was considered to be due to adsorption to the actively growing algal cells.

Description of key information

After a CCH decision a new Freshwater Alga and Cyanobacteria, Growth Inhibition Test (Noak, 2019) was conducted.

The toxicity of the test item to the unicellular freshwater green algae Pseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3.The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5416 cells/mL. A stock solution with a nominal concentration of 200 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear. The stocksolution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Six concentrations were tested in a geometrical series with a factor of 2.5:2.05 - 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L, corresponding to the geometric mean measured test item concentration of 1.13 - 1.94 - 6.44 - 20.5 - 61.3 - 165 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The measured concentrations of the test item were in the range of 83 to 103% of the nominal values at the start of the exposure (0 hours) and < LOQ to 72% of the nominal values at the end of the exposure (72 hours). All effect values given are based on the geometric mean measured test item concentrations. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 46.7 (45.1 - 48.0) mg/L and 25.5 (22.4 - 39.9) mg/L, respectively. The NOEC for growth rate and yield were 6.44 mg/L and < 1.13 mg/L, respectively.

These results are supported by another OECD 201 study (Vryenhoef, 2004) where Green algae (Scenedesmus subspicatus) were exposed to 6.25, 12.5, 25, 50 and 100 mg/l of the test item (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 degrees C. Exposure of green algae to the test material gave an EbC50 (72 h) value of 66 mg/l (nominal); 95 % confidence limits 58 - 75 mg/l and ErC50 (0 - 72 h) value of 84 mg/l (nominal). The No Observed Effect Concentration was 6.25 mg/l (nominal). The measured EbC 50 was 2.8 mg/L. The reason for the difference between nominal and measured concentrations was not quite clear, therefore the new study was conducted (Noak, 2019).

Key value for chemical safety assessment

EC50 for freshwater algae:
46.7 mg/L
EC10 or NOEC for freshwater algae:
25.1 mg/L

Additional information