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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:
From July 01, 2011 to January 24, 2012.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no analytical control and the methodology of exposure is not the WAF as recommended in OECD 23
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Temperatures of the cooling incubator not recorded due to a dysfunction of the recording device.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Remarks:
The substance is a very complex UVCB.
Details on sampling:
No sampling for analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test solutions have been prepared from a stock solution of the test item previously diluted in Algae dilution water. The stock solution was continuously stirred during the preparation of the test solutions.
- Controls: Only the mineral medium (without the tested item)
- Positive control : K2Cr2O7
- Chemical name of vehicle : no vehicule used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
- Other relevant information:
- In the preliminary test : 3 tested concentrations at 10, 100 and 1000 mg/L, under static conditions, for 72h. 2 replicats for the tested concentrations and 3 replicats for the control.
- in the final test : 5 tested concentrations at 20, 60, 180, 540 and 1620 mg/L, under static conditions, for 72h. 3 replicats for each tested concentration and 6 replicats for the control
- Algae Dilution water:
NH4Cl : 15 mg/L; CaCl2, 2H20 : 18 mg/L; KH2PO4 : 1.6 mg/L; Na2EDTA, 2H2O : 0.1 mg/L; MnCl2, 4H2O : 0.415 mg/L; CoCl2, 6H2O : 1.5 µg/L; Na2MoO4, 2H2O : 7 µg/L; MgCl2, 6H2O : 12 mg/L; MgSO4, 7H2O : 15 mg/L; FeCl3, 6H2O : 0.08 mg/L; H3BO3 : 0.185 mg/L; ZnCl2 : 3 µg/L; CuCl2, 2H2O : 0.01 µg/L; NaHCO3 : 50 mg/L.

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 274.4
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae cultures stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).
- Culturing media and conditions (same as test or not): 3 days before the test, a preculture of algae was run in the same conditions as for the test, in order to have a log phase culture for the inoculation.
- Any deformed or abnormal cells observed: Before the inoculation, this preculture was checked under a miscroscope with a haemocytometer in order to check the algae morphology and to measure the cell number.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
NaHCO3 = 50 mg/L
Test temperature:
Not available due to a disfunction of the recording device. However the tests are performed in a cooling incubator.
pH:
At the start between 7.8 - 6.1.
At 72 h between 9.1 - 5.9
Dissolved oxygen:
not reported however the test medium is shaken continously.
Salinity:
not relevant
Conductivity:
not reported
Nominal and measured concentrations:
Nominal concentrations : 20; 60; 180; 540 and 1620 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 erlenmeyer flasks
- Material, size, headspace, fill volume: 100 ml of algae culture
- Aeration: constant shaking at 120 rpm
- Renewal rate of test solution ): static test
- Initial cells density: 10000 algae /ml at the start of the test
- Control end cells density: yes
- No. of vessels per concentration (replicates): 3 replicats
- No. of vessels per control (replicates): 6 replicats

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: continuous illumination
- Light intensity and quality: 5032 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: the algae concentrations were measured at 24h, 48, and 72h under a microscope with a Malassez countijng cell in order to distinguish the test substance particles from the algae cells.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 3 (20; 60; 180; 540; 1620 mg/L
- Range finding study :
- Test concentrations: 10; 100; 1000 mg/L
- Results used to determine the conditions for the definitive study: EC50 estmated between 10 and 1000 mg/l
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
194.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
81.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Results with reference substance (positive control):
ErC50 (0-72h) = 0.91 mg/L (0.77 - 1.1 mg/L). This result conforms well with the historical results of the laboratoryindicating a correct sensitivity of the algae.
Reported statistics and error estimates:
The ErC50 was calculated with the software SIGMAPLOT 8.0 and TOXSTAT.
The Ey50 (0-72h) was calculated with the software SIGMAPLOT 8.0

 Algae concentrations expressed in cell number * E+4/ml after 24h, 48h and 72h of exposure

Nominal test conc. (mg/L)

[Algae]*E+4 at 24 hours

[Algae]*E+4 at 48 hours

[Algae]*E+4 at 72 hours

Count

Average

Count

Average

Count

Average

Dilution water control

4.6

5.3

41.6

35.2

278

246.3

4.7

29

232

5.7

31

240

5.2

39.1

238

5.8

38.6

238

6

31.9

252

20 mg/L

5.3

5.7

39.9

33.3

180

179.3

6.6

31.8

200

5.3

28.1

158

60 mg/L

4.6

4.8

18.5

19.7

170

164.7

4.3

20.6

150

5.6

20.1

174

180 mg/L

3.1

3.4

11.1

11

23.9

20.7

2.7

10.8

17.5

4.3

11

20.6

540 mg/L

3.9

3.4

4.6

5

5.1

5.2

2.6

4.9

4.3

3.8

5.4

6.2

1620 mg/L

1.4

1.6

2.2

2.2

3.9

3.7

1.8

2.5

3.6

1.7

2.0

3.5

 

Calculation of the ErC50 (0-72h)

Inhibition of the specific growth rate for each treatment group

Final test growth rate inhibition data

Test item conc. in mg/L

20

60

180

540

1620

Inhibition of specific growth rate (%)

Replicate a

5.7

6.7

42.4

70.4

75.3

Replicate b

3.8

9.0

48.0

73.5

76.7

Replicate c

8.1

6.3

45.1

66.9

77.3

Inhibition of average specific growth rate

5.8

7.3

45.0

70.1

76.4

 

 

Calculation of the EyC50 (0-72h)

Inhibition of the yield

 

Test item conc. in mg/L

[algae]*E+4/ml at 0 hour

[algae]*E+4/ml at 72 hour

Yield (*E+4)

Yield (mean, E+4)

YIELD INHIBITION (%)

Per replicate

Per mean of replicate

Control

1

278

277

245.3

Not applicable

232

231

240

239

238

237

238

237

252

251

20 mg/L

1

180

179

178.3

27

27.3

200

199

18.9

158

157

36

60 mg/L

1

170

169

163.7

31.1

33.3

150

149

39.3

174

173

29.5

180 mg/L

1

23.9

22.9

19.7

90.7

92

17.5

16.5

93.3

20.6

19.6

92.0

540 mg/L

1

5.1

4.1

4.2

98.3

98.3

4.3

3.3

98.7

6.2

5.2

97.9

1620 mg/L

1

3.9

2.9

2.7

98.8

98.9

3.6

2.6

98.9

3.5

2.5

99

 

Validity criteria fulfilled:
yes
Remarks:
pH change of control < 1.5; Biomass Increase of control > 16; Average specific growth rate CV of controls < 7%, Daily average growth rate CV of controls < 35%.
Conclusions:
Since the validity criteria are fulfilled, the inhibition of the growth rate is chosen as the representative criteria to express the toxicity of the test item to algae. The ErL50 (0-72h) = 194.4 mg/L nominal concentration and the ErL10 = 20 mg/L nominal concentration. So "Saccharomyces cerevisiae cell wall, extracted" does not show any toxicity to algae.
 
Executive summary:

The aquatic toxicity of "Saccharomyces cerevisiae cell wall, extracted" to the green algae Pseudokirchnerellia subcapitata was determined according to the OECD guideline 201 and in compliance with the GLP.

100 ml of medium with an initial algal concentration of 10000 cells/ml were exposed for 72 h , under static conditions, to the following test item concentrations : 20; 60; 180; 540 and 1620 mg/L.

The tested solutions were prepared from a stock solution of the test item, previously diluted in the Algae mineral medium, and continously stirred during the preparation of the test solutions. Althought this procedure is not the recommended methodology for insoluble UVCB, the saturation is likely reached for each ingredient of this UVCB, due to the high concentrations tested.

The flasks were placed in a cooling incubator under a constant shaking at 120 rpm and a continuous illumination (5032 lux). The pH of each tested concentration was measured at the start and the end of the test. No analysis was performed on the tested solutions due to the complex composition of this UVCB. The algae concentrations were counted at 24h, 48, and 72h with a Malassez cell.

All the validity criteria were fulfilled : pH change of control < 1.5; Biomass Increase of control > 16; Average specific growth rate CV of controls < 7%, Daily average growth rate  CV of controls < 35%.

The inhibition of the yield is : EyL50 (0 -72h) = 81.6 mg/L, nominal concentration and the inhibition of the growth rate is : ErL50 (0 -72h) = 194.4 mg/L and ErL10 (0 -72h) = 20 mg/L, nominal concentration. Since the validity criteria are fulffilled, the inhibition to the growth rate is chosen as the representative value of the toxicity of the test item to algae.

This toxicity test to the green algae is classified as Klimish 2 because of the choice of the methodology and the lack of analytical control. Nevertheless it was demonstrated that "Saccharomyces cerevisiae cell wall, extracted" does not show any toxicity to algae.

 

Description of key information

The aquatic toxicity of "Saccharomyces cerevisiae cell wall, extracted" to the green algae Pseudokirchnerellia subcapitata was determined according to the OECD guideline 201 and in compliance with the GLP.

The inhibition of the growth rate is : ErL50 (0 -72h) = 194.4 mg/L and ErL10 (0 -72h) = 20 mg/L. "Saccharomyces cerevisiae cell wall, extracted" does not show any toxicity to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
194.4 mg/L

Additional information

The aquatic toxicity of "Saccharomyces cerevisiae cell wall, extracted" to the green algae Pseudokirchnerellia subcapitata was determined according to the OECD guideline 201 and in compliance with the GLP.

100 ml of medium with an initial algal concentration of 10000 cells/ml were exposed for 72 h , under static conditions, to the following test item concentrations : 20; 60; 180; 540 and 1620 mg/L.

The tested solutions were prepared from a stock solution of the test item, previously diluted in the Algae mineral medium, and continuously stirred during the preparation of the test solutions. Although this procedure is not the recommended methodology for insoluble UVCB, the saturation is likely reached for each ingredient of this UVCB, due to the high concentrations tested.

The flasks were placed in a cooling incubator under a constant shaking at 120 rpm and a continuous illumination (5032 lux). The pH of each tested concentration was measured at the start and the end of the test. No analysis was performed on the tested solutions due to the complex composition of this UVCB.

The algae concentrations were counted at 24h, 48, and 72h with a Malassez cell.

All the validity criteria were fulfilled : pH change of control < 1.5; Biomass Increase of control > 16; Average specific growth rate CV of controls < 7%, Daily average growth rate CV of controls < 35%.

The inhibition of the yield is : EyL50 (0 -72h) = 81.6 mg/L, nominal concentration and the inhibition of the growth rate is : ErL50 (0 -72h) = 194.4 mg/L, nominal concentration. Since the validity criteria are fulfilled, the inhibition to the growth rate is chosen as the representative value of the toxicity of the test item to algae.

This toxicity test to the green algae is classified as Klimish 2 because of the choice of the methodology and the lack of analytical control. Nevertheless it was demonstrated that "Saccharomyces cerevisiae cell wall, extracted" does not show any toxicity to algae.