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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:
05 July to 22 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Storage condition of test material: Protect from light, refrigerator.
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, quadruplicate samples were taken from the test media of all test concentrations at the start of the test (without algae), after 24, 48 and 72 hours and at the end of the test (containing algae). At the same sampling times, quadruplicate samples were also taken from the control.
For the sampling after 24, 48 and 72 hours and at the end of the test, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.
A second series of samples from Day 1 to 4 were filtered (Nylonfilter, 0.45 μm) before extraction and measurement.
Additionally, one of the quadruplicate additional test medium samples from the test concentration of 22 mg/L was analysed after 24, 48 and 72 hours and at the end of the test.
From the control samples (extracted samples), one of the quadruplicate samples was analysed on Day 0, 3 and 4. From the control samples (filtered and extracted samples), one of the quadruplicate samples was analysed on Day 1, 3 and 4.
Vehicle:
no
Details on test solutions:
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.02 mg of the test item completely in 1000 mL of test water using intense stirring for 15 minutes at room temperature in the dark. The test medium of the highest test concentration was used in a series of dilutions with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test and as far as possible in the dark under red light.PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.02 mg of the test item completely in 1000 mL of test water using intense stirring for 15 minutes at room temperature in the dark. The test medium of the highest test concentration was used in a series of dilutions with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test and as far as possible in the dark under red light.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Method of cultivation: standardized conditions according to the test guidelines

ACCLIMATION
- Culturing media and conditions: An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
Test type:
static
Water media type:
other: Reconstituted test water (AAP Medium)
Limit test:
no
Total exposure duration:
96 h
Hardness:
15 mg/L as CaCO3
Test temperature:
22 °C
pH:
7.4 - 9.2
Nominal and measured concentrations:
Nominal concentrations: 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Reconstituted test water (AAP Medium))

OTHER TEST CONDITIONS
- Light intensity and quality: 5600 Lux (range: 5070 to 5990 Lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm).

TEST CONCENTRATIONS
- Range finding study: Yes (data not shown here)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 102 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 110 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 113 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 105 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 101 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Extracted sample
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Direct analysis of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Corrected for algal uptake of Filtered+ Extracted samples
Remarks on result:
other: 95% CI

Analytical Results for the Biological In-Life Phase

Extracted test samples

The measured concentrations of test item in the extracted test samples of the nominal test concentrations of 1.0 to 100 mg/L were between 85 and 115% of the nominal values at the start of the test . During the test period of 96 hours, a decrease of test item concentration in the test media occurred. The concentrations ranged from 88 to 105% on Day 1, from 38 to 110% on Day 2, from below the LOQ to 92% on Day 3 and from below the LOQ to 91% on Day 4.

The biological results were related to the mean measured test item concentrations in the extracted samples (calculated for 72 hours as the geometric means of the concentrations measured at the start, after 24, 48 and 72 hours and calculated for 96 hours as the geometric means of the concentrations measured at the start, after 24, 48, 72 and 96 hours):

 

Table 6.1.5/1: Analysis of extracted test samples

Nominal test concentration (mg/L)

Mean measured concentration of the test item (geometric mean)

72 hours

96 hours

Mean Measured Concentration (mg/l) *

Concentration Corrected for Algal Uptake (mg/l) °

Mean Measured Concentration (mg/l) *

Concentration Corrected for Algal Uptake (mg/l) ^

1.0

0.46*

0.57*

0.37*

0.55*

2.2

0.87*

1.2*

0.62*

1.1*

4.6

2.2

2.8

1.3*

2.5*

10

6.6

7.6

3.1*

6.3*

22

19

21

12

18

46

42

46

34

42

100

102

110

100

113

*: for measured concentration below LOQ, the ½ LOQ was used to calculate the mean measured concentration (LOQ = 0.313 mg/L and ½ LOQ = 0.1565 mg/L).

°: Mean measured concentrations calculated by geometric mean of the Day 0 values and the 24, 48 and 72 hour values which were corrected for influence of algal presence (5, 4 and 19% for 24, 48 and 72-hour values, respectively).

^:Mean measured concentrations calculated by geometric mean of the Day 0 values and the 24, 48, 72 and 96 hour values which were corrected for influence of algal presence (5, 4, 19 and 35% for 24, 48, 72 and 96-hour values, respectively).

 

Filtered and Extracted test samples

The measured concentrations of test item in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 85 and 115% of the nominal values at the start of the test. During the test period of 96 hours, a decrease of test item concentration in the test media occurred. The measured concentrations of test item in the filtered and extracted test samples of the nominal test concentrations of 1.0 to 100 mg/L were 79 to 86% on Day 1, 31 to 85% on Day 2, below the LOQ to 82% on Day 3 and from below the LOQ to 46% on Day 4.

The biological results were also related to these mean measured test item concentrations (calculated for 72 hours as the geometric means of the concentrations measured at the start, after 24, 48 and 72 hours and calculated for 96 hours as the geometric means of the concentrations measured at the start, after 24, 48, 72 and 96 hours)

 

Table 6.1.5/2: Analysis of filtered extracted test samples

Nominal test concentration (mg/L)

Mean measured concentration of the test item (geometric mean)

72 hours

96 hours

Mean Measured Concentration (mg/l) *

Concentration Corrected for Algal Uptake (mg/l) °

Mean Measured Concentration (mg/l) *

Concentration Corrected for Algal Uptake (mg/l) ^

1.0

0.43*

0.62*

0.35*

0.59*

2.2

0.81*

1.3*

0.58*

1.2*

4.6

2.0

3.0

1.2*

2.6*

10

5.4

7.2

2.7*

6.2*

22

16

21

9.3

18

46

36

44

27

39

100

88

105

78

101

*:  for measured concentration below LOQ, the ½ LOQ was used to calculate the mean measured concentration (LOQ=0.313 mg/L and ½ LOQ=0.1565 mg/L).

°:  Mean measured concentrations calculated by geometric mean of the Day 0 values and the 24, 48 and 72 hour values which were corrected for influence of algal presence (20, 19 and 25% for 24, 48 and 72-hour values, respectively).

^: Mean measured concentrations calculated by geometric mean of the Day 0 values and the 24, 48, 72 and 96 hour values which were corrected for influence of algal presence (20, 19, 25 and 38% for 24, 48, 72 and 96-hour values, respectively).

 

Analytical Results of the Additional Stability Test

In the parallel stability samples with nominal 22 mg/L, the following concentrations were measured

 

Table 6.1.5/3: Analytical Results of the Additional Stability Test

Incubation Conditions

 

0 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Light + Algae (Extracted)

Conc. (mg/L)

25.3

21.6

19.4

11.7

1.84

% of Day 0

100

85

77

46

7.3

Light + Algae (Filtered + Extracted)

Conc. (mg/L)

25.3

17.8

15.7

10.2

0.99

% of Day 0

100

70

62

40

3.9

Light without Algae

Conc. (mg/L)

25.3

22.8

20.4

16.5

10.5

% of Day 0

100

90

81

65

42

Darkness without Algae

Conc. (mg/L)

25.3

22.6

20.0

15.1

8.84

% of Day 0

100

89

79

60

35

 

Loss due to direct aqueous photolysis was insignificant for test item over the 96-hours exposure period. The results do, however, suggest that algal uptake and interaction may be responsible for up to 20, 19, 25 and 38% of uptake and/or degradation of the test item over the 24, 48, 72 and 96 hour incubation periods.

The degradation of the test item may be the result of the combination of an abiotic degradation and absorption onto the algae.

 

Biological In-Life Phase of the Study (Toxicity Testing)

The impact of the test item on the growth of the algae is shown in Tables below.

 

Table 6.1.5/4: Biomass of Algae

Nominal test item concentration (mg/L)

Rep. no.

Biomass of algae*

24 hours

48 hours

72 hours

96 hours

Control

1

3.4

14.8

61.1

208.5

2

3.4

14.2

65.7

213.4

3

2.9

15.0

56.6

196.4

4

3.2

15.5

62.8

201.9

5

3.3

15.1

66.2

210.9

6

3.3

14.7

57.6

194.0

Mean

3.2

14.9

61.7

204.2

SD

0.2

0.4

4.0

8.0

1.0

1

3.1

14.7

59.2

196.4

2

3.1

15.8

64.9

195.5

3

3.0

14.3

58.6

200.1

Mean

3.1

14.9

60.9

197.3

SD

0.1

0.8

3.5

2.4

2.2

1

3.1

13.0

56.1

171.4

2

3.0

14.9

62.7

198.8

3

3.3

15.5

57.9

158.3

Mean

3.1

14.5

58.9

176.2

SD

0.1

1.3

3.4

20.7

4.6

1

3.2

13.1

52.7

178.4

2

2.9

13.5

52.4

175.2

3

2.3

12.1

47.1

174.5

Mean

2.8

12.9

50.7

176.0

SD

0.4

0.7

3.1

2.1

10

1

3.1

12.6

45.0

166.3

2

3.2

12.9

50.3

170.7

3

3.0

13.1

45.3

168.0

Mean

3.1

12.9

46.9

168.3

SD

0.1

0.3

3.0

2.2

22

1

3.0

12.1

40.9

142.2

2

3.0

11.9

37.9

145.7

3

2.8

11.3

38.3

130.5

Mean

2.9

11.7

39.0

139.5

SD

0.1

0.4

1.6

8.0

46

1

2.7

11.5

39.3

125.4

2

2.7

11.2

33.3

118.3

3

2.5

9.7

31.7

116.8

Mean

2.6

10.8

34.8

120.2

SD

0.1

1.0

4.0

4.6

100

1

1.3

3.6

10.9

38.1

2

1.2

3.1

10.2

36.1

3

1.2

3.6

11.5

38.7

Mean

1.2

3.4

10.9

37.7

SD

0.0

0.3

0.7

1.4

SD:Standard deviation

 *:  The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.80 x 103 relative fluorescence units.

 

Table 6.1.5/5: Section-by-Section Growth Rates (μ)

Nominal test item concentration (mg/L)

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

0-24 h

24-48 h

48-72 h

72-96 h

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

Control

1.402

0.0

1.525

0.0

1.420

0.0

1.198

0.0

1.0

1.356

3.2

1.574

-3.2

1.405

1.1

1.177

1.8

2.2

1.360

3.0

1.536

-0.7

1.406

1.0

1.092

8.9

4.6

1.241

11.5

1.543

-1.2

1.368

3.7

1.245

-3.9

10

1.354

3.4

1.429

6.3

1.290

9.2

1.280

-6.8

22

1.303

7.0

1.387

9.0

1.201

15.5

1.273

-6.2

46

1.195

14.7

1.407

7.7

1.169

17.7

1.244

-3.8

100

0.445

68.2

1.007

34.0

1.158

18.4

1.245

-3.9

  

The test item had a significant inhibitory effect on the growth rate μ and yield Y of the algae after the test periods of 72 and 96 hours at the nominal concentration of 4.6 mg/L and at all higher test concentrations.

 

Table 6.1.5/6: Average Growth Rates (μ)

Nominal test item concentration (mg/L)

Average growth rate μ (day-1) and inhibition of μ (Ir)

0-24 h

0-48 h

0-72 h

0-96 h

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

Control

1.402

0.0

1.463

0.0

1.449

0.0

1.386

0.0

1.0

1.356

3.2

1.465

-0.1

1.445

0.3

1.378

0.6

2.2

1.360

3.0

1.448

1.1

1.434

1.0

1.349

2.7

4.6

1.241

11.5

1.392*

4.9

1.384*

4.5

1.349#

2.7

10

1.354

3.4

1.391*

4.9

1.358*

6.3

1.338#

3.5

22

1.303

7.0

1.345*

8.1

1.297*

10.5

1.291#

6.9

46

1.195#

14.7

1.301*

11.1

1.257*

13.2

1.254#

9.6

100

0.445#

68.2

0.726*

50.4

0.870*

39.9

0.964#

30.5

*:    mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, α <= 0.05)

#:   mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller, α = 0.05)

 

Table 6.1.5/7: Yield (Y)

Nominal test item concentration (mg/L)

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

0-96 h

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Control

1.402

0.0

1.463

0.0

1.449

0.0

1.386

0.0

1.0

1.356

3.2

1.465

-0.1

1.445

0.3

1.378

0.6

2.2

1.360

3.0

1.448

1.1

1.434

1.0

1.349

2.7

4.6

1.241

11.5

1.392*

4.9

1.384*

4.5

1.349#

2.7

10

1.354

3.4

1.391*

4.9

1.358*

6.3

1.338#

3.5

22

1.303

7.0

1.345*

8.1

1.297*

10.5

1.291#

6.9

46

1.195#

14.7

1.301*

11.1

1.257*

13.2

1.254#

9.6

100

0.445#

68.2

0.726*

50.4

0.870*

39.9

0.964#

30.5

*:    mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, α <= 0.05)

#:   mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller, α = 0.05)

 

The 72 and 96-hour NOEC based on the growth rate μ were determined to be 2.2 mg/L, since up to and including this nominal test concentration the growth rate of the algae after 72 and 96 hours were not significantly lower than in the control.

The NOEC and ECX-values for growth rate are summarized in the conclusion. Additionally, the corresponding values for yield are given.

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 46 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

In the control, the biomass increased by a factor of 77 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 5.8%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.5%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

The mean coefficient of variation of the daily growth rates in the control during 96 hours was 11%. The coefficient of variation of the average specific growth rates in the replicates of the control after 96 hours was 0.7%. Thus, the validity criteria mentioned above were also fulfilled after 96 hours.

The coefficient of variation of the fluorescence values in replicate control cultures during 72 and 96 hours was 6.5 and 3.9%, respectively. According to OPPTS 850.5400, the coefficient of variation must not exceed 20%. Thus, the validity criterion was fulfilled. Consequently, the test was considered to be valid.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

The pH rose in the control from 7.5 at test start to 9.1 at test end, according to the OECD guideline (test duration: 72h), the pH of the control medium should not increase by more than 1.5 units during the test. The increase in pH during 96 hours test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred continuously during the test. Since all validity criteria concerning the growth are fulfilled the test is considered to be valid.

The pH of the test media was in the range of 7.4 to 9.2 during the test period. The water temperature during the test was maintained at 22 °C.

 

Table 6.1.5/8: pH Values in the Treatments

Nominal test item concentration

pH values

Start

End

Control

7.5

9.1

1.0

7.5

9.1

2.2

7.5

9.1

4.6

7.5

9.2

10

7.5

9.1

22

7.4

9.0

46

7.4

8.9

100

7.4

8.8

 

Table 6.1.5/9: Water Temperature During the Test Period

 

Temperature [°C]

Day 0 (Start)

22

Day 1

22

Day 2

22

Day 3

22

Day 4 (End)

22

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the mean measured concentration the biological results are as following:
The EC50 of extracted sample by direct analysis was >102 and >100 mg/L, for 72 and 96 hours, respectively. The EC50 of extracted sample and Corrected for algal uptake was >110 and >113 mg/L, for 72 and 96 hour, respectively. The EC50 of Filtered + Extracted sample by direct analysis was >88 and >78 mg/L, for 72 and 96 hours, respectively. The EC50 of Filtered + Extracted sample and Corrected for algal uptake was >105 and >101 mg/L, for 72 and 96 hours, respectively.

The EC20 of extracted sample by direct analysis was 45 and 58 mg/L, for 72 and 96 hours, respectively. The EC20 of extracted sample and Corrected for algal uptake was 49 and 69 mg/L, for 72 and 96 hours, respectively. The EC20 of Filtered + Extracted sample by direct analysis was 39 and 45 mg/L, for 72 and 96 hours, respectively. The EC20 of Filtered + Extracted sample and Corrected for algal uptake was 48 and 63 mg/L, for 72 and 96 hours, respectively.

The EC10 of extracted sample by direct analysis was 23 and 26 mg/L, for 72 and 96 hours, respectively. The EC10 of extracted sample and Corrected for algal uptake was 25 and 33 mg/L, for 72 and 96 hours, respectively. The EC10 of Filtered + Extracted sample by direct analysis was 20 and 20 mg/L, for 72 and 96 hours, respectively. The EC10 of Filtered + Extracted sample and Corrected for algal uptake was 25 and 32 mg/L, for 72 and 96 hours, respectively.

The NOEC of extracted sample by direct analysis was 0.8 and 0.62 mg/L, for 72 and 96 hours, respectively. The NOEC of extracted sample and Corrected for algal uptake was 1.2 and 1.1 mg/L, for 72 and 96 hours, respectively. The NOEC of Filtered + Extracted sample by direct analysis was 0.81 and 0.58 mg/L, for 72 and 96 hours, respectively. The NOEC of Filtered + Extracted sample and Corrected for algal uptake was 1.3 and 1.2 mg/L, for 72 and 96 hours, respectively.

The LOEC of extracted sample by direct analysis was 2.2 and 1.3 mg/L, for 72 and 96 hours, respectively. The LOEC of extracted sample and Corrected for algal uptake was 2.8 and 2.5 mg/L, for 72 and 96 hours, respectively. The LOEC of Filtered + Extracted sample by direct analysis was 2.0 and 1.2 mg/L, for 72 and 96 hours, respectively. The LOEC of Filtered + Extracted sample and Corrected for algal uptake was 3.0 and 2.6 mg/L, for 72 and 96 hours, respectively.
Executive summary:

The impact of the test item on the growth of the freshwater green algal speciesPseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD 201 and the EU Method C.3 (Algal Inhibition test), as well as according to the EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II).

The nominal concentrations of the test item of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control. Additional flasks containing the test medium of the test concentration of nominal 22 mg/L were set up. Two different incubation conditions, “Light without algae”, and “Darkness without algae” were applied in order to evaluate the influence of the presence of light and algae on the stability of the test item in the test medium.

Three replicates per treatment with nominal 5000 algal cells/mL were exposed to the test item during 96 hours under continuous lighting at 22°C. The algal biomass in the samples was determined by fluorescence measurement. At the end of the test, the shape and size of the algal cells were visually inspected, and, no adverse effect was observed.

Samples were analyzed using GC-FID. Two analytical procedures (measurements in extracted and in filtered plus extracted test samples) were used to evaluate a possible adsorption of the test item onto the algae.

The measured concentrations of test item in the extracted test samples of the nominal test concentrations of 1.0 to 100 mg/L were between 85 and 115% of the nominal values at the start of the test. During the test period of 96 hours, a decrease of test item concentration in the test media occurred. The concentrations ranged from 88 to 105% on Day 1, from 38 to 110% on Day 2, from below LOQ to 92% on Day 3 and from below LOQ to 91% on Day 4.

The biological results were related to the mean measured test item concentrations in the extracted samples (calculated for 72 hours as the geometric means of the concentrations measured at the start, after 24, 48 and 72 hours and calculated for 96 hours as the geometric means of the concentrations measured at the start, after 24, 48, 72 and 96 hours).

The measured concentrations of test item in the filtered and extracted test samples of the nominal test concentrations of 1.0 to 100 mg/L were 79 to 86% on Day 1, 31 to 85% on Day 2, below the LOQ to 82% on Day 3 and from below the LOQ to 46% on Day 4.

The biological results were also related to the mean measured test item concentrations calculated for 72 hours as the geometric means of the concentrations measured at the start, after 24, 48 and 72 hours and calculated for 96 hours as the geometric means of the concentrations measured at the start, after 24, 48, 72 and 96 hours).

Loss due to direct aqueous photolysis was insignificant for test item over the 96-hours exposure period. The results do, however, suggest that algal uptake and interaction may be responsible for up to 20, 19, 25 and 38% of uptake and/or degradation of the test item over the 24, 48, 72 and 96 hour incubation periods. The degradation of the test item may be a result of the combination of an abiotic degradation and adsorption onto the algae.

The biological results were based on mean measured concentrations of the test item test item in the extracted samples and in the filtered plus extracted samples.

Based on the mean measured concentration the biological results are as following:

The EC50 of extracted sample by direct analysis was >102 and >100 mg/L, for 72 and 96 hours, respectively. The EC50 of extracted sample and Corrected for algal uptake was >110 and >113 mg/L, for 72 and 96 hour, respectively. The EC50 of Filtered + Extracted sample by direct analysis was >88 and >78 mg/L, for 72 and 96 hours, respectively. The EC50 of Filtered + Extracted sample and Corrected for algal uptake was >105 and >101 mg/L, for 72 and 96 hours, respectively.

 

The EC20 of extracted sample by direct analysis was 45 and 58 mg/L, for 72 and 96 hours, respectively. The EC20 of extracted sample and Corrected for algal uptake was 49 and 69 mg/L, for 72 and 96 hours, respectively. The EC20 of Filtered + Extracted sample by direct analysis was 39 and 45 mg/L, for 72 and 96 hours, respectively. The EC20 of Filtered + Extracted sample and Corrected for algal uptake was 48 and 63 mg/L, for 72 and 96 hours, respectively.

 

The EC10 of extracted sample by direct analysis was 23 and 26 mg/L, for 72 and 96 hours, respectively. The EC10 of extracted sample and Corrected for algal uptake was 25 and 33 mg/L, for 72 and 96 hours, respectively. The EC10 of Filtered + Extracted sample by direct analysis was 20 and 20 mg/L, for 72 and 96 hours, respectively. The EC10 of Filtered + Extracted sample and Corrected for algal uptake was 25 and 32 mg/L, for 72 and 96 hours, respectively.

 

The NOEC of extracted sample by direct analysis was 0.8 and 0.62 mg/L, for 72 and 96 hours, respectively. The NOEC of extracted sample and Corrected for algal uptake was 1.2 and 1.1 mg/L, for 72 and 96 hours, respectively. The NOEC of Filtered + Extracted sample by direct analysis was 0.81 and 0.58 mg/L, for 72 and 96 hours, respectively. The NOEC of Filtered + Extracted sample and Corrected for algal uptake was 1.3 and 1.2 mg/L, for 72 and 96 hours, respectively.

 

The LOEC of extracted sample by direct analysis was 2.2 and 1.3 mg/L, for 72 and 96 hours, respectively. The LOEC of extracted sample and Corrected for algal uptake was 2.8 and 2.5 mg/L, for 72 and 96 hours, respectively. The LOEC of Filtered + Extracted sample by direct analysis was 2.0 and 1.2 mg/L, for 72 and 96 hours, respectively. The LOEC of Filtered + Extracted sample and Corrected for algal uptake was 3.0 and 2.6 mg/L, for 72 and 96 hours, respectively.

Description of key information

Under the experimental conditions and based on geometric mean measured concentrations (filtered + extracted samples, direct analysis), the 72-hour ErC50 and 72-hour ErC10 values for the parameters growth rate were >88 mg/L and 20 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
88 mg/L
EC10 or NOEC for freshwater algae:
20 mg/L

Additional information

The impact of the test item GR-87 -6984 -4 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3, as well as according to the OPPTS Guideline No. 850.5400 (Public Draft, April 1996).

The nominal concentrations of the test item of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control. Additional flasks containing the test medium of thetest concentration of nominal 22 mg/Lwere set up. Two different incubation conditions, “Light without algae”, and “Darkness without algae” were applied in order to evaluate the influence of the presence of light and algae on the stability of the test item in the test medium.

Samples were analyzedusing GC-FID.Two analytical procedures (measurements in extracted and in filtered plus extracted test samples) were used to evaluate a possible adsorption of the test item onto the algae.

The biological results based on growth rates are summarised below:

- 72h-EC50 (geometric mean measured concentrations): >88 mg/L

- 96h-EC50 (geometric mean measured concentrations): >78 mg/L

- 72h-EC10 (geometric mean measured concentrations): 20 mg/L

- 96h-EC10 (geometric mean measured concentrations): 20 mg/L