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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:
14 July 2009 - 16 July 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP, test substance could not be maintained in solution, all reasonable steps taken to maintain test substance concentration

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment No. 23
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
MeDONA
IUPAC Name:
MeDONA
Details on test material:
- Name of test material (as cited in study report): MTDID #10078
- Physical state: clear colorless liquid
- Analytical purity: 91.9%
- Lot/batch no.: 140499-21/8
- Stability under test conditions: Hydrolytically unstable and likely to be volatile
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: At 0h and 24h. 4.5 mL aliquots were taken from all test concentration and controls.
- Sample storage conditions before analysis: Not applicable, samples were analysed same day as sampling because MeDONA concentration were not stable during freezing and thawing of samples.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest concentration (loading rate of 100 mg/L) was prepared by spiking underneath the surface a volume of 63 µL test substance to 1L test medium. The aqueous solution was magnetically stirred for 21 hours in a closed glass vessel with minimal headspace in order to prevent any volatilization and to reach maximum solubility. The mixture was then stabilized for one hour and then the water accommodated fraction (WAF) was siphoned off. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the WAF in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material: The final test solutions were all clear and colorless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algae cells from a pure culture on agar. Suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24°C

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Combined limit/range-finding test

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22.2 - 23.0 °C
pH:
7.4 - 9.1
Nominal and measured concentrations:
Water accommodated fraction: 0.1, 1.0, 10 and 100% from a loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:100 mL all-glass, containing 75 mL of test solution. The vessels were airtight closed with a screw-cap that contained a septum.
- Agitation: Algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 23.85E+04 cells/mL
- No. of vessels per concentration (replicates): 3 for 0.1, 1.0 and 10% WAF. 6 for 100% WAF.
- No. of vessels per control (replicates): 6
1 or 2 replicates of each test concentration for analytical sampling purposes. 1 replicate for each concentration without algae.

GROWTH MEDIUM
- Standard medium used: Yes, M1 medium used for stock culture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA))
- Culture medium different from test medium: yes, propagation in M1 medium, test in M2 medium and a pre-culture was established in M2 medium 4 days before test initiation. M2 test medium was slightly modified by adding additional NaHCO2 and adjusting pH to 7.0 to enable better algal growth in a closed system.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 79 to 91 mE/(m²∙s).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beginning of the test, cells were counted with a microscope and counting chamber. Thereafter cell densities were measured using a spectrophotometer with immersion probe (wavelength 720 nm, path length = 10 mm). At the end of the study final microscopic observations were made on the control and highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes, test was a combination limit/range-finding test
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: The ErC50 result corresponds to a 100% water accommodated fraction of MeDONA (loading rate of 100 mg/L).
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes, see Figure 1
- Growth in controls (validity criteria):
- cell density increase through 72-hours: factor of >16 after 48 hours
- % coefficient of variance in average specific growth rate at hour 48: 4% (Table 1)
- % coefficient of variance in section-specific growth rates to hour 48: 8% (Table 2)

Any visual signs of phytotoxicity (abnormalities): No abnormalities were noted in the report.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance has low solubility, is hydrolytically unstable and likely to be volatile.
- Effect concentrations exceeding solubility of substance in test medium: yes
-Other: The pH in the controls at the end of the study was 9.1 which exceeded the upper limit of 9.0. Cells were still in active log growth at study conclusion; therefore the study integrity was not adversely affected
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h ErC50: 1.6 mg/L, 95% C.I. 1.1 - 2.3 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction EC50: 72h for the Pseudokirchneriella subcapitata tested corresponds with this range.
Reported statistics and error estimates:
No EC50 values could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration). EC50 based on growth rate and biomass >100 mg/L

Any other information on results incl. tables

Table 1 Cell Density, Yield Inhibition and Growth Rate Reductions

Test Group¹ (% WAF)

Vessel Number

Cell Densities

(104cells/mL)

Yield

Yield Inhibition (%)

Growth Rate (µ)

Growth Rate Reduction (%)

Exposure Period (hours)

0

24

48

0 - 48h

0 - 48h

0 – 48h

0 – 48h

 

1

1

4.80

27.02

26.02

 

0.06868

 

 

2

1

6.11

27.74

26.74

 

0.06923

 

 

3

1

5.88

26.73

25.73

 

0.06845

 

 

4

1

5.29

24.44

23.44

 

0.06659

 

Control

5

1

5.35

21.78

20.78

 

0.06419

 

 

6

1

4.82

21.36

20.36

 

0.06378

 

 

Mean

 

 

 

23.85

 

0.06682

 

 

 

 

 

 

 

 

CV  4%

 

 

1

1

5.04

26.57

25.57

-7

0.06833

-2

0.10

2

1

4.87

25.76

24.76

-4

0.06768

-1

 

3

1

5.53

25.96

24.96

-5

0.06784

-2

 

Mean

 

 

 

25.10

-5.2

0.06795

-1.7

 

 

 

 

 

 

 

 

 

 

1

1

5.10

28.16

27.16

-14

0.06954

-4

1.0

2

1

4.21

24.31

23.31

2

0.06648

1

 

3

1

5.12

28.19

27.19

-14

0.06956

-4

 

Mean

 

 

 

25.89

-8.6

0.06853

-2.6

 

 

 

 

 

 

 

 

 

 

1

1

6.11

28.40

27.40

-15

0.06972

-4

10

2

1

5.13

28.25

27.25

-14

0.06961

-4

 

3

1

5.07

25.10

24.10

-1

0.06714

0

 

Mean

 

 

 

26.25

-10.1

0.06882

-3.0

 

 

 

 

 

 

 

 

 

 

1

1

2.07

26.75

25.75

-8

0.06847

-2

 

2

1

2.85

28.29

27.29

-14

0.06964

-4

 

3

1

1.93

25.41

24.41

-2

0.06740

-1

100

4

1

2.08

25.74

24.74

-4

0.06767

-1

 

5

1

3.14

26.71

25.71

-8

0.06844

-2

 

6

1

2.22

25.98

24.98

-5

0.06786

-2

 

Mean

 

 

 

25.48

-6.9

0.06825

-2.1

1) Test groups represent percentages of a water accommodated fraction (WAF) prepared at 100 mg/L.

Table 2 Growth rate: section-by-section

Test Group¹ (% WAF)

Vessel Number

Growth rate (µ)

Growth rate reduction (%)

 

 

0-24h

24-48h

0-24h

24-48h

 

1

0.06536

0.07200

 

 

 

2

0.07541

0.06304

 

 

 

3

0.07381

0.06309

 

 

Control

4

0.06941

0.06377

 

 

 

5

0.06988

0.05850

 

 

 

6

0.06553

0.06203

 

 

 

Mean

0.06990

0.06374

 

 

 

CV

6%

7%

 

 

 

The mean CV for sec-by-sec specific growth rate was: 8%

 

1

0.06739

0.06927

4

-9

0.10

2

0.06596

0.06941

6

-9

 

3

0.07126

0.06443

-2

-1

 

Mean

0.06820

0.06770

2.4

-6.2

 

 

 

 

 

 

 

1

0.06789

0.07119

3

-12

1.0

2

0.05989

0.07306

14

-15

 

3

0.06805

0.07108

3

-12

 

Mean

0.06528

0.07178

6.6

-12.6

 

 

 

 

 

 

 

1

0.07541

0.06402

-8

0

10

2

0.06813

0.07108

3

-12

 

3

0.06764

0.06665

3

-5

 

Mean

0.07039

0.06725

-0.7

-5.5

 

 

 

 

 

 

 

1

0.03031

0.10662

57

-67

 

2

0.04364

0.09563

38

-50

 

3

0.02740

0.10740

61

-69

100

4

0.03052

0.10482

56

-64

 

5

0.04768

0.08920

32

-40

 

6

0.03323

0.10249

52

-61

 

Mean

0.03546

0.10103

49.3

-58.5

A) Test groups represent percentages of a water accommodated fraction (WAF) prepared at 100 mg/L.

 

Table 3 Concentration of MeDONA in test medium

Time of Sampling (hours)

Concentration

Percentage of WAFA(%)

AnalyzedC(mg/L)

 

0

< 0.1

 

0

< 0.1

 

0.1

< 0.1

 

0.1

< 0.1

 

1.0

< 0.1

0

1.0

< 0.1

 

10

< 0.1

 

10

< 0.1

 

100

< 0.1

 

100

< 0.1

 

100B

< 0.1

 

100B

< 0.1

 

 

 

 

0

< 0.1

 

0

< 0.1

 

0.1

< 0.1

 

0.1

< 0.1

 

1.0

< 0.1

24

1.0

< 0.1

 

10

< 0.1

 

10

< 0.1

 

100

< 0.1

 

100

< 0.1

 

100B

< 0.1

 

100B

< 0.1

A) A water accommodated fraction (WAF) prepared at the loading rate of 100 mg/L.

B) Without algae

C) Response in the test samples was smaller than the response of the recovery samples at target concentration of 0.1 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Cell density increased in 48 hours by average factor >16. Control cultures mean coefficient of variation sec-by-sec specific growth rates < 35%. Control cultures coefficient of variation of specific growth rates during whole test < 7%.
Conclusions:
The 48-hour ErC50 of MeDONA to Pseudokirchneriella subcapitata is > 100 mg/L. The 48-hour NOErC of MeDONA to Pseudokirchneriella subcapitata is 100 mg/L.
Executive summary:

The toxicity of MeDONA to the green algae, Pseudokirchneriella subcapitata, was assessed in a 48-hour toxicity test conducted according to the OECD 201 (2006) method. Exposure period was reduced to 48 hours due to the study being conducted in completely sealed test chambers.Exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. No reduction in rate or inhibition of yield was recorded up to and including the highest concentration tested of MeDONA after 48 hours. The 48-hour ErC50 of MeDONA to Pseudokirchneriella subcapitata for growth rate was > 100 mg/L, corresponding to the 100% Water Accommodated Fraction (WAF). Concentration levels that might have been toxic for algae could not be reached due to the behavior of MeDONA in water (low solubility, volatile and subject to hydrolysis). This study is considered reliable with restrictions due to the unstable nature of the compound. The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. Therefore, the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis. Results Synopsis

Test Type: static (based on data obtained using OECD 201 methodology)

48-hr ErC50: > 100 mg/L

48-hr NOErC: 100 mg/L

The ErC50 and NOErC results correspond to a loading rate of 100 mg/L MeDONA.