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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:
2017-11-06 to 2017-11-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.800, 0.250, 0.0781, 0.0244, 0.00763 mg/L and control
- Sampling method: taken in the main test at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test item concentrations and control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: mixed with 20 µL formic acid and stored deep-frozen (≤ - 18 °C)

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution: weight necessary amount of test item in volumetric flask and added test medium up to the benchmark, then homogenised by shaking and one minute of sonication. Lower test solutions: dilution appropriate solution with test medium
- Test concentration: 0.800, 0.260, 0.0808, 0.0233, 0.00736 mg/L and control

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Method of cultivation: grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution.
- Culture conditions: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3 to 4 day
- Culturing media and conditions (same as test or not): same as test

Study design

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

Test conditions

Test temperature:
23.0 – 23.3 °C
pH:
7.55 to 7.76 at 0 h and 7.67 to 7.88 at 72 h
Nominal and measured concentrations:
Nominal concentrations: 0.800, 0.250, 0.0781, 0.0244, 0.00763 mg/L
Measured initial concentration: 0.800, 0.260, 0.0808, 0.0233, 0.00736 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps
- Type : closed
- Material, size, headspace, fill volume: volume: 100 mL, fill volume: ~ 50 mL
- Initial cells density: 0.5E+04 cells/mL
- CO2 supplied: by continuous agitation
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, modified AAP-Medium. As the test item contains metal components, the content of Na2EDTA·2H2O in the employed AAP-medium was reduced to 0.100 mg/L (instead of 0.300 mg/L) as recommended by OCSPP 850.4500.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuously from the side
- Light intensity and quality: 75.9 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement at 24, 48 and 72 hours
- Other: morphological appearance: at the end of the test; pH-value: at t = 0 h and t = 72 h; temperature: measured continuously and recorded at hours 0, 24, 48 and 72 h; light intensity: measured once a year, and confirmed for one representative position at test start; test solution appearance: conducted daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a clear dose response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.01 to 1.00 mg/L. The inhibition of growth rate peaked in 126.7 % and the inhibition of yield peaked in 101.4 % at a nominal test item concentration of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, also tested in modified medium

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.07 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.:
0.015 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.:
0.007 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.:
0.069 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.016 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Morphology evaluation: normal for the control and up to and including a nominal test item concentration of 0.0781 mg/L. No cells were observed at 0.250 and 0.800 mg/L nominal test item concentration.
- Physicochemical Data: pH and temperature during the test, light intensity were within the range specified by OECD 201.
- Chemical analysis of test item concentrations: measured initial concentrations: ranged from 93 % to 104 % of nominal; aged samples: between 98 % and 106 % of nominal.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: growth rate 1.23 mg/L, yield 0.685 mg/L, biomass intergral 0.639 mg/L.
Results were considered valid.
Reported statistics and error estimates:
EC50: Probit analysis following Gompertz distribution for growth rate and following normal distribution for yield
NOEC: Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield

Any other information on results incl. tables

Non-GLP range-finding test:

Percentage inhibition of growth rate and yield observed in the non-GLP range-finding test:

Conc.

 [mg/L]

% Inhibition at test end (72 h)

Growth rate

Yield

Control

0

0

0.0100

3.1

12.0

0.100

32.8

72.2

1.00

119.3

101.2

10.0

119.4

101.2

100

126.7

101.4

Main study - Biological results:

Individual cell numbers:

Concentration (mg/L) Cell numbers (10^4/mL) Yield
0h 24h 48h 72h 0h-72h
Control 0.65 1.76 6.99 27.2 26.55
0.00763   1.76 5.99 22.01 21.36
0.0244   1.28 2.75 12.48 11.83
0.0781   0.8 1.61 5.3 4.65
0.25   0.5 0.33 0.49 -0.16
0.8   0.36 0.15 0.15 -0.5

Percentage inhibition of growth rate:

Conc.

% Inhibition of growth rate

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.00763

-1.2

5.7

5.4

0.0244

32.4

39.0

22.9 *

0.0781

80.4

62.0

43.9 *

0.250

127.8

132.2

108.7 *1)

0.800

160.2

163.1

141.8 *2)

1)Value was set to 100 for EC10, 20, 50-calculation

2)Value was omitted for EC10, 20, 50-calculation

* Statistically significant different to the control

Percentage inhibition of yield:

Conc.

% Inhibition of yield

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.00763

0.0

15.8

19.5

0.0244

43.2

66.9

55.4 *

0.0781

86.5

84.9

82.5 *

0.250

113.5

105.0

100.6 *1)

0.800

126.1

107.9

101.9 *2)

1)Value was set to 100 for EC10, 20, 50-calculation

2)Value was omitted for EC10, 20, 50-calculation

* Statistically significant different to the control

Analytical results:

Determined concentration of test item:

Test item nominal

concentration [mg/L]

Sampling

Test item measured

concentration [mg/L]

% of nominal

Geometric

mean [%]

Geometric mean

concentration [mg/L]

Control

0 h fresh

< LOD

-

-

-

72 h aged

< LOD

-

0.00763

0 h fresh

0.00724

95

96.5

0.00736

72 h aged

0.00750

98

0.0244

0 h fresh

0.0226

93

95.5

0.0233

72 h aged

0.0240

98

0.0781

0 h fresh

0.0810

104

103.5

0.0808

72 h aged

0.0805

103

0.250

0 h fresh

0.256

102

104.0

0.260

72 h aged

0.266

106

0.800

0 h fresh

0.810

101

100.0

0.800

72 h aged

0.790

99

Effect values:

NOEC, LOEC, ECx-values and 95 % Confidence Intervals (0 - 72 hours):

 

Test item[mg/L]

 

nominal

measured

(geometric mean)

ErC10(Growth rate)

0.0159

0.0153

95 % confidential limits

1.10 · 10-9– 0.0423

7.36 · 10-19– 0.0437

ErC20

0.0284

0.0280

95 % confidential limits

0.000000613 – 0.0641

7.25 · 10-13– 0.0672

ErC50

0.0685

0.0695

95 % confidential limits

0.00408 – 0.254

0.000296 – 0.356

EyC10(Yield)

0.00482

0.00450

95 % confidential limits

0.00319 – 0.00654

0.00295 – 0.00616

EyC20

0.00805

0.00763

95 % confidential limits

0.00584 – 0.0103

0.00549 – 0.00982

EyC50

0.0215

0.0210

95 % confidential limits

0.0177 – 0.0257

0.0172 – 0.0252

NOEC

0.00763

0.00736

LOEC

0.0244

0.0233

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.0244 mg/L (nominal) and 0.0233 mg/L (measured) and above. The EC10 for growth rate was set at 0.0153 mg/L (measured) and for yield at 0.0045 mg/L (measured). The EC50-value for growth rate (ErC50) was determined to be 0.0695 mg/L (measured). The EC50-value for yield (EyC50) was 0.0210 mg/L (measured).
Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD 201.

Following a static non-GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control, a static main test with nominal test item concentrations of 0.800, 0.250, 0.0781, 0.0244 and 0.00763 mg/L and control was performed.

Six replicates were employed for the control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

All validity criteria met for the study.

Test concentrations were analytically verified using HPLC-MS/MS. The measured initial concentrations of test item in the samples taken at test start were within 80 – 120 %.

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.0244 mg/L (nominal) and 0.0233 mg/L (measured) and above. The EC10 for growth rate was set at 0.0153 mg/L (measured) and 0.0045 mg/L (measured) for yield. The EC50-value for growth rate (ErC50) was determined to be 0.0685 mg/L (nominal) and 0.0695 mg/L (measured). The EC50-value for yield (EyC50) was 0.0215 mg/L (nominal) and 0.0210 mg/L (measured).