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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 29-OCT-2007 to 11-JAN-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of the test item Dimethyl 2-methyl glutarate was measured in the duplicate test media samples from the nominal test concentration of 100 mg/L taken at the start and the end of the test. The samples from the nominal test concentrations of 4.6 to 46 mg/L were not analyzed, since these concentrations were below the 72-hour NOEC. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sampling method: For determination of the actual test item concentrations, the following samples were taken:
Just before the start of the test: duplicate samples from each test medium (without algae), duplicate samples from the control (without algae)
After 72 hours (stability samples): duplicate samples from each test medium (with algae), duplicate samples from the control (with algae)
For sampling of the stability samples, the contents of the replicates of each treatment were combined.
- Sample storage conditions before analysis: Immediately after sampling, all samples were stored deep-frozen (at about -20°C). The stability of the test item in samples under the storage conditions was confirmed in a pre-experiment (non-GLP).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test medium of the highest nominal test item concentrationof 100 mg/L was prepared by dissolving 100.0 mg of the test item completely in 1000 ml of test water using ultrasonic treatment (15 minutes) and intense stirring (15 minutes at room temperature). The test medium was diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).
- Controls: test water without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Scenedesmus subspicatus)
- Strain: No. 61.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)
- Age of inoculum (at test initiation): the algal cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.
- Method of cultivation: cultivated in Harlan laboratories in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions: see method of cultivation above
- Any deformed or abnormal cells observed: data not available
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:
Day 0 (start): 22°C
Day 1: 21°C
Day 2: 23°C
Day 3 (end): 22°C



pH:
At the start of the test, the pH of the test medium and the control was 8.3. At the end of the test, pH values of 8.5 and between 8.3 to 8.7 were measured in the control and the test medium, respectively.

Dissolved oxygen:
not measured

Salinity:
not applicable
Nominal and measured concentrations:
nominal concentrations: 4.6, 10, 22, 46 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50-mL flasks, filled with 15 mL of algal suspension
- Aeration: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used
- Initial cells density: 10000 algal cells per mL of test medium (corresponding to 1.4 relative fluorescence units)
- Control end cells density: 1498571.43 cells/ml (corresponding to 209.8 relative fluorescence units)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (reconstituted test water according to test guidelines)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:
Macro-nutrients: NaHCO3 50.0 mg/L
KH2PO4 1.6 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
Trace elements: H3BO3 185.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 × 6 H2O 1.5 µg/L
CuCl2 × 2 H2O 0.01 µg/L
Na2MoO4 × 2 H2O 7.0 µg/L
FeCl3 × 6 H2O 64.0 µg/L
Na2EDTA × 2 H2O 100.0 µg/L
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: data not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: the measured light intensity was about 6900 Lux (mean value) and was achieved by fluorescent tubes (Philips TLD 36W/840) installed above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A small volume of the test media and the control were taken from all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIOTEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate.
Based on the determination of the cell concentration (biomass), the specific growth rate (µ), the inhibition of growth rate (Ir), the yield (Y) and the inhibition of yield (Iy) were calculated.
- Other: In addition, after 72 hours of exposure, a sample was taken from the control and from the highest test concentration. The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes (non-GLP)
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory test conditions.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 60 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
BIOLOGICAL RESULTS
- Exponential growth in the control (for algal test): Yes (The biomass increased by a factor of 153 over 72 hours).
- Observation of abnormalities (for algal test):
Unusual cell shape and size: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at and up to this test concentration were obviously not affected.
Colour differences: No
Flocculation: No
Adherence to test vessels: No
- 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

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration.

ANALYTICAL MONITORING
The analytically determined concentration of the test item in the test medium of the highest nominal test concentration of 100 mg/L was 96% of the nominal value at the start of the test. At the end of the test, 38% of the nominal concentration was found. Thus the concentration of the test item decreased during the test period of 72 hours under the test conditions, most probably due to degradation of the test item.


Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC 50: The result of the latest positive control test performed in 2007 showed that the sensitivity of the test system was within the historical range of the test laboratory (72-hour EC50 for the growth rate: 0.92 mg/L, range of the 72-hour EC50 for the growth rate from 2000 to 2007: 0.71–1.74 mg/L) (potassium dichromate).
Reported statistics and error estimates:
The 72-hour EC10, EC50 and EC90 values could not be calculated because of the absence of a significant inhibitory effect of the test item on the growth of Pseudokirchneriella subcapitata at the tested concentrations.
For the determination of the LOEC and NOEC, the calculated average growth rate and the mean yield at the test concentrations were tested for significant differences when compared to the control values by Dunnett's tests.

Table 1: Biomass of Algae

 

Nominal test item concentration

(mg/L)

Rep. no.

Biomass of algae*

(relative Fluorescence units)

24 hours

48 hours

72 hours

Control

1

2

3

4

5

6

7.9

7.7

7.2

7.1

7.0

5.9

48.6

46.5

48.1

43.8

38.5

36.1

213.5

215.2

201.8

236.7

204.6

187.2

Mean

SD

7.1

0.7

43.6

5.2

209.8

16.6

4.6

1

2

3

5.6

5.9

7.8

40.9

34.4

47.3

183.5

162.1

206.3

Mean

SD

6.4

1.2

40.9

6.5

184.0

22.1

10

1

2

3

5.7

5.5

7.0

37.0

33.0

44.1

185.9

177.6

194.6

Mean

SD

6.1

0.8

38.0

5.6

186.0

8.5

22

1

2

3

7.3

6.6

6.9

42.0

37.9

42.0

201.4

214.3

234.7

Mean

 SD

6.9

0.3

40.6

2.3

216.8

16.8

46

1

2

3

6.0

5.5

4.7

39.8

35.4

35.8

210.4

182.5

170.0

Mean

 SD

5.4

0.7

37.0

2.5

187.6

20.7

100

1

2

3

5.3

4.4

5.5

41.0

31.2

41.1

197.8

176.6

212.1

Mean

 SD

5.1

0.6

37.8

5.7

195.5

17.9

 

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (duplicate measurements) and is given as relative fluorescence units. At the start of the test,10000algal cells/mL were inoculated, corresponding to 1.4 relative fluorescence units).

Table 2: Growth Rates (µ) and Inhibition of µ (Ir) during the test period

 

Nominal test item concentration

(mg/L)

Growth rate μ (1/day) and inhibition of μ (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir (%)

µ

Ir (%)

µ

Ir (%)

Control

1.65

0.0

1.73

0.0

1.68

0.0

4.6

1.53

6.8

1.69

1.9

1.63

2.7

10

1.48

9.8

1.66

4.0

1.64

2.4

22

1.62

1.4

1.69

1.9

1.69

-0.7

46

1.37*

16.7

1.65

4.6

1.64

2.3

100

1.30*

20.9

1.65

4.2

1.65

1.4

 

Negative percentage inhibition : increase in growth relative to that of control

* mean value significantly lower than in control

(according to a Dunnett’s-test, one-sided, alpha = 0.05)

Table 3: Yield (y) and Inhibition of y (Iy) during the test period

 

Nominal test item concentration (mg/L)

Yield y and inhibition of y

0-24 h

0-48 h

0-72 h

yield

Iy (%)

yield

Iy (%)

yield

Iy (%)

Control

5.8

0.0

42.2

0.0

208.5

0.0

4.6

5.0

12.3

39.5

6.5

182.6

12.4

10

4.7

18.3

36.7

13.2

184.7

11.4

22

5.6

3.2

39.3

7.0

215.4

-3.3

46

4.1*

29.6

35.6

15.7

186.2

10.7

100

3.7*

36.0

36.4

13.8

194.1

6.9

 

Negative percentage inhibition : increase in growth relative to that of control

* mean value significantly lower than in control

(according to a Dunnett’s-test, one-sided, alpha = 0.05)

Table 4: Section-by-section growth rates

 

Nominal test item concentration (mg/L)

Section-by-section growth rates (1/day) and inhibition of the growth rates (Ir)

0-24 h

24-48 h

48-72 h

µ

Ir (%)

µ

Ir (%)

µ

Ir (%)

Control

1.65

0.0

1.81

0.0

1.57

0.0

4.6

1.53

6.8

1.85

-2.4

1.51

4.2

10

1.48

9.8

1.83

-1.4

1.59

-1.2

22

1.62

1.4

1.77

2.3

1.67

-6.2

46

1.37

16.7

1.92

-6.4

1.62

-3.0

100

1.30

20.9

2.01

-11.0

1.65

-4.7

 

Negative percentage inhibition : increase in growth relative to that of control

 

Validity criteria fulfilled:
yes
Remarks:
The control biomass is multiplicated by 153 over 72 hours (threshold >16), the mean coeff. of variation of the daily growth rates was 8.5% (threshold < 35%), and the coeff. of variation of the average specific growth rates was 1.6% (threshold < 7%)
Conclusions:
The test item had no statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (mean measured concentration of the test item of 60 mg/L).
Executive summary:

The influence of the test substance DIMETHYL 2 -METHYL GLUTARATE on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Scenedesmus subspicatus) investigated in a 72 hour static test according toEEC Directive 92/69, C.3 (1992), theOECD Guideline No. 201 (2006).

 

The nominal test item concentrations of 4.6, 10, 22, 46 and 100 mg/L were tested in parallel to a control.

The analytically determined concentration of the test item in the test medium of the highest nominal test concentration of 100 mg/L was 96% of the nominal value at the start of the test. At the end of the test, 38% of the nominal concentration was found. Thus the concentration of the test item decreased during the test period of 72 hours under the test conditions, most probably due to degradation of the test item.

The test item had no statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (mean measured concentration of the test item of 60 mg/L).

The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) was determined to be 100 mg/L (nominal concentration) (mean measured concentration of 60 mg/l).

The 72-hour LOEC (lowest concentration tested with toxic effects after the test period of 72 hours) and the 72-hour EC10 and EC50 values for the growth rate and yield could not be quantified due to the absence of a toxic effect of Dimethyl 2-methyl glutarate at the tested concentrations. Accordinly, these parameters were clearly higher than the nominal concentration of 100 mg/L (mean measured concentration of 60 mg/L).

Description of key information

The 72-hour NOEC of dimethyl 2-methyl glutarate to Pseudokirchneriella subcapitata was > = 60 mg/L (measured concentration) based on the growth rate. The 72-hour LOEC, EC10 and EC50 were clearly higher than the mean measured concentration of 60 mg/L. Hence, Dimethyl 2-methyl glutarate is not harmful for the algal species tested.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
60 mg/L

Additional information

One experimental study, scored as Klimisch 1, is available (Bätscher R., 2008) and selected as a key study.