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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:
2015-08-03 to 2015-08-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP and OECD Guideline conform study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified via HPLC-DAD after 0 hours (start of the exposure) and 72 hours (end of the exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels. The stock solution of 200 mg/L was prepared by direct addition of the test item to the dilution water and agitation.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
0.24 mmol Ca+Mg/L.
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C
pH:
7.59-8.31
Dissolved oxygen:
no data
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
5 concentrations were tested in a geometrical series with a dilution factor of 2.5: 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L (nominal).
Measured concentrations: 5.14 - 12.9 - 32.4 - 79.3 - 196 mg/L. The measured concentrations of the test substance in the fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Type: open
- Test volume: 100 mL
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, current: 6153 cells /mL
- Control end cells density: mean 2856212 cells/mL
- No. of vessels per concentration (replicates): 3 for each concentration
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day
- Light intensity and quality: 35-70 µE /(m^2 *s)

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test at the test item concentration of 200 mg/L.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The 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: 2.5
Range finding study
- Test concentrations: 0.100, 1.00, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
80 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.:
94.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
109 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: none
- Unusual cell shape: none
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no

Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.613 mg/L (gowth rate), 0.281 mg/L (yield)
Reported statistics and error estimates:
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Welshs test on significant differences was done with a significance level 0.05 for yield.
William’s test on significant differences was done with a significance level 0.05 for growth rate.
All calculations were done with ToxRat Professional (ToxRat Solutions GmbH).

Table. 1: Cell densities:

 

Nominal test item concentration

Replicate

 

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

200

1

6153

7953

< LOQ

< LOQ

2

6153

6404

2970

< LOQ

3

6153

8069

3958

< LOQ

Mean

6153

7475

n.a.

n.a.

80

1

6153

40037

348949

2244348

2

6153

31652

327185

2284739

3

6153

32135

410101

2074502

Mean

6153

34608

362078

2201196

32

1

6153

36450

477306

2326511

2

6153

39555

423366

2325256

3

6153

44277

419164

2503632

Mean

6153

40094

439945

2385133

12.8

1

6153

40505

446197

2091750

2

6153

35471

430830

1898949

3

6153

38074

508038

2117590

Mean

6153

38017

461688

2036096

5.12

1

6153

37043

384104

1966310

2

6153

48072

505310

2325695

3

6153

33439

419415

2863708

Mean

6153

39518

436276

2385238

Control

1

6153

48467

532531

2892182

2

6153

54118

579226

2797099

3

6153

47630

588132

2777342

4

6153

45202

511582

2847024

5

6153

43669

507192

2984757

6

6153

43597

484957

2838870

Mean

6153

47114

533937

2856212

 

< LOQ = below the limit of quantification (2736 cells/mL)

n.a. = data not determinable

 

Table 2: Evaluation after 72 hours:Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item concentration

[mg/L]

Replicate

No.

Growth rate

[d-1]

Inhibition of growth rate

[%]

Yield

[cells/mL]

 

Inhibition of yield

[%]

200

1

n.a.

100

n.a.

100

2

n.a.

100

n.a.

100

3

n.a.

100

n.a.

100

Mean

(+) n.a.

n.a.

(+) n.a.

n.a.

80

1

1.97

4

2238195

21

2

1.97

4

2278586

20

3

1.94

5

2068349

27

Mean

(+)1) 1.96

4

(+) 2195043

23

32

1

1.98

3

2320358

19

2

1.98

3

2319103

19

3

2.00

2

2497479

12

Mean

(+) 1) 1.99

3

(+) 2378980

17

12.8

1

1.94

5

2085597

27

2

1.91

7

1892796

34

3

1.95

5

2111437

26

Mean

1.93

6

(+) 2029943

29

5.12

1

1.92

6

1960157

31

2

1.98

3

2319542

19

3

2.05

0

2857555

0

Mean

(+)1) 1.98

3

(-) 2379085

17

Control

1

2.05

 

2886029

 

2

2.04

2790946

3

2.04

2771189

4

2.05

2840871

5

2.06

2978604

6

2.05

2832717

Mean

2.05

 

2850059

 

 

< LOQ = below the limit of quantification (2736 cells/mL)

1) Inhibition < 10% is considered not biologically significant

 

n.a. = data not determinable

 

Table 3: Measured Concentrations and Percentage of the Nominal Concentration of the test substance in Fresh Medium (0 hours) and Old Medium (72 hours)

 

 

 

Nominal test item concentration [mg/L]

0 hours fresh medium without algae

72 hours fresh medium with algae

Measured test item concentration [mg/L]

[%]

Measured test item concentration [mg/L]

[%]

200

196

98

192

96

80

79.3

99

78.1

98

32

32.4

101

31.8

99

12.8

12.9

101

< LOQ 1) 2)

5.12

5.14

100

5.26

103

Control

< LOQ

< LOQ

 

Meas. Conc. = Measured concentration of the test item (dilution factor taken into account)

% = Percent of the nominal concentration of the test item

LOQ = Limit of Quantification (1.00 mg/L test item)

1) Percent of the nominal concentration of the test item = Limit of Quantification (1.00 mg/L test item)

The test item could not recovered in the sample 12.8 mg/L on 2015-08-06 (<SYSQL). Because of this the sample 12.8 mg/L was reanalysed on 2015-08-07 (3 replicates)

2) = not plausible, a sampling error is assumed

Table 4: Inhibition of growth rate and yield of algae (mean of 3 replicates)

Nominal test item conc. [mg/L], geom. mean

% Inhibition at 72 h (growth rate)

% Inhibition at 72 h (yield)

200

100

100

80

4

23

32

3

17

12.8

6

29

5.12

3

17

Validity criteria fulfilled:
yes
Conclusions:
In a key static growth inhibition test, the fresh algae Desmodesmus subspicatus was exposed over 72 hours to the test substance according to OECD 201 (2011) under GLP conditions. The ErC50 was determined to be 109 mg/L, the ErC10 was determined to be 94.1 mg/L and the NOEC was determined to be 80 mg/L.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011). The aim of the study was the determination of NOEC, LOEC, EC10-, EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A stock solution of 200 mg/L was prepared by direct addition of the test item to the dilution water and agitation. Five concentrations were tested in a geometrical series with a dilution factor of 2.5 (nominal): 5.12 - 12.8 - 32.0 - 80.0 - 200 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 test media were clear throughout the test period. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of the exposure. The measured concentrations in the fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations. Potassium dichromate was tested as a reference item and an EC50 value of 0.613 mg/L was obtained for this substance. For the test substance, an EC50 (72 h) of 109 mg/L based on growth rate, an NOEC (72 h) of 80 mg/L and an EC10 of 94.1 mg/L based on growth rate were determined.

Description of key information

The 72h-ErC50 was determined to be 109 mg/L, the 72h-NOEC was determined to be 80 mg/L and the 72h-ErC10 was determined to be 94.1 mg/L in a study conducted on the fresh algae Desmodesmus subspicatus.

Key value for chemical safety assessment

EC50 for freshwater algae:
109 mg/L
EC10 or NOEC for freshwater algae:
94.1 mg/L

Additional information

In a key static growth inhibition test according to OECD 201 (2011) and GLP, the fresh algae Desmodesmus subspicatus was exposed over 72 hours to the test substance with a static system. Based on a preliminary test, five concentrations 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L in the definitive test were tested. The test concentrations were monitored via HPLC-DAD at the start and the end of the exposure. The measured concentrations of test itemthe fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations.The ErC50 was determined to be 109 mg/L, the 72h-ErC10 was determined to be 94.1 mg/L and the 72h-NOEC was determined to be 80 mg/L. All effect values given are based on the nominal test item concentrations and the validity criteria were fulfilled.

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