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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:
28 Oct 2013 – 21 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Würtemberg, Germany
Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken from all test item concentrations and from the control after 0, 24, 48 and 72 hours. The test item concentrations 4.88, 15.6 and 50.0 mg/L were analysed at 0 and 72 hours to verify the actual test concentrations using a validated analytical method
Vehicle:
no
Details on test solutions:
A stock solution (S1) was prepared by directly weighing 500 mg (S1) of the test item into 1000 mL test medium. The further concentrations V1 – V6 were made by diluting the appropriate solutions to give the required test concentrations. All test item solutions were clear and transparent.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Drebs-Weg 1, D-37075 Göttingen, Germany.
- Method of cultivation: The algae were grown semi-continuously under sterile cultures in the laboratory. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures were ordered regularly from the culture collection. Culture conditions were as follows: Illumination: continuously from the top by light tubes, 60 - 120 µEm-2s-1 at cell culture level; Temperature: 21 – 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; Visually healthy cells were used for the test; CO2 supply by shaking on a rotating shaker, 105 rpm.
Cells from this semi-continuous liquid stock culture were used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
20.3 – 24.0 °C
pH:
7.09 – 7.68
Nominal and measured concentrations:
control, 0.149, 0.476, 1.53, 4.88, 15.6 and 50.0 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed; aluminium caps and two baffles
- Material, size, headspace, fill volume: 500 mL Erlenmeyer flasks filled with 150 - 200 mL
- Initial cells density: 0.5 × 10^4 cells/mL
- Control end cells density: 46.23 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: The temperature was recorded after 0, 24, 48 and 72 hours. The pH-values of the test solutions were measured after 0 and 72 hours and the light intensity at test start (0 hours).

OTHER TEST CONDITIONS
- Adjustment of pH: yes, to 7.5 ± 0.1
- Photoperiod: continuously from the top
- Light intensity and quality: 68 - 107 µEm-2s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After 1, 2 and 3 days, the cell growth was determined. Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.
- Determination of cell concentrations: fluorescence detection

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2x
- Range finding study:
Test concentrations: control, 0.100, 1.00, 10.0 and 100 mg/L
Results used to determine the conditions for the definitive study: see "any other information on results incl. tabes" below
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
28.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 18.3 – 51.2
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 13.5 – 37.9
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.796 – 1.46
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.813 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.588 – 1.08
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.476 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.352 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Statistically significant inhibitory effects were observed after 72 hours from the nominal concentration 1.53 mg/L (nominal) (equivalent to 1.13 mg/L actual) and above.
Reported statistics and error estimates:
The statistical evaluation for 72 hours was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic and the homogeneity of variance of the data was performed by calculating the Levene Test. The NOEC and LOEC were determined by using the Dunnett`s t-test for growth rate and Jonckheere-Terpstra test for yield, left sided. The ErC50 and EyC50 values were evaluated using the probit analysis following Gompertz distribution (growth rate) and logistic distribution (yield).

Analytics:

The measured concentrations of niobium in the test item solutions of 4.88, 15.6 and 50.0 mg/L were between 45 - 113% of nominal concentration. Since some of the measured concentrations were below 80% the biological endpoints were evaluated using the nominal and actual measured concentrations based on the arithmetic mean of the geometric mean of each test item concentration.

Table: Concentrations of niobium in the test medium

Test item

Niobium

Sampling [h]

Niobium

nominal conc. [mg/L]

nominal conc. [mg/L]

[mg/L]

% of nominal

Geometric mean

[%]

Test item conc. actual

[mg/L]*

0 (Control)

0

0

n.d.

-

-

-

72

n.d.

-

4.88

0.839

0

0.549

65

68

3.61

72

0.607

72

15.6

2.68

0

3.04

113

71

11.5

72

1.20

45

50.0

8.60

0

8.16

95

82

37.0

72

6.06

70

Analytical mean:

 

74

 

Limit of quantification (LOQ) = 2.33 mg/L test item, corresponds to 0.400 mg Nb/L

*Based on the arithmetic mean of the geometric mean of each test item concentration

Table: Results of the range-finding test:

Test item conc. [mg/L]

Average cell numbers/mL*

0d

1d

2d

3d

0 (Control)

0.5

2.11

8.85

34.03

0.100

0.5

2.19

8.46

29.82

1.00

0.5

1.71

5.11

14.87

10.0

0.5

1.61

3.26

6.68

100

0.5

0.86

0.90

0.94

* Algae counts are divided by 10000. At the start, the cell density was adjusted to 0.5 x 10^4 cells/mL

Table: Main test - Average cell number for each sampling time and concentration:

Test item conc. nominal [mg/L]

Test item conc. actual [mg/L]

Average cell numbers/mL*

0d

1d

2d

3d

0 (Control)

0

0.5

2.86

12.51

46.23

0.149

0.110

0.5

2.47

12.12

43.84

0.476

0.352

0.5

2.56

13.65

49.79

1.53

1.13

0.5

1.92

5.56

13.56

4.88

3.61

0.5

1.88

4.34

9.17

15.6

11.5

0.5

1.77

2.72

4.30

50.0

37.0

0.5

1.88

3.16

5.56

* Algae counts are divided by 10000. At the start, the cell density was adjusted to 0.5 x 10^4 cells/mL

Table: Main test - Percentage inhibition of growth rate

Test item conc. nominal [mg/L]

Test item conc. actual [mg/L]

Inhibition of growth rate [%]

0d - 1d

0d - 2d

0d - 3d

0 (Control)

0

0.0

0.0

0.0

0.149

0.110

8.4

0.7

1.2

0.476

0.352

6.3

-3.1

-2.2

1.53

1.13

22.9

24.7

26.6*

4.88

3.61

24.2

32.8

35.8*

15.6

11.5

27.6

47.1

52.1*

50.0

37.0

24.1

42.3

46.4*

* statistically significant compared to the control

Table: Main test - Percentage inhibition of yield

Test item conc. nominal [mg/L]

Test item conc. actual [mg/L]

Inhibition of yield [%]

0d - 1d

0d - 2d

0d - 3d

0 (Control)

0

0.0

0.0

0.0

0.149

0.110

16.5

3.2

5.2

0.476

0.352

12.7

-9.5

-7.8

1.53

1.13

39.8

57.9

71.4

4.88

3.61

41.5

68.0

81.0*

15.6

11.5

46.2

81.5

91.7*

50.0

37.0

41.5

77.9

88.9*

* statistically significant compared to the control

Description of key information

EC50 (growth rate) (72h) = 28.5 mg/L with Pseudokirchneriella subcapitata (nominal, OECD 201)
EC50 (growth rate) (72h) = 21.1 mg/L with Pseudokirchneriella subcapitata (measured, geometric mean, OECD 201)
NOEC (growth rate) (72h) = 0.476 mg/L with Pseudokirchneriella subcapitata (nominal, OECD 201)
NOEC (growth rate) (72h) = 0.352 mg/L with Pseudokirchneriella subcapitata (measured, geometric mean, OECD 201)

Key value for chemical safety assessment

Additional information

One study investigating the acute toxicity of Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate to algae is available. The study was conducted with six concentrations of the test item in the range from 0.149 to 50 mg/L. The test was performed in a static design according to OECD 201 (CBMM Europe BV, Key, 2014, Pseudokirchneriella subcapitata). Statistically significant inhibitory effects were observed after 72 hours from the nominal concentration 1.53 mg/L (equivalent to 1.13 mg/L actual) and above. Recovery rates were in part low: The measured concentrations of niobium in the test item solutions of 4.88, 15.6 and 50.0 mg/L were between 45 - 113% of nominal concentration. Since some of the measured concentrations were below 80% the biological endpoints were evaluated using the nominal and actual measured concentrations based on the arithmetic mean of the geometric mean of each test item concentration. Thus, the EC50 for growth rate was determined to be 28.5 mg/L and 1.1 mg/L for yield based on nominal concentrations. Based on geometric mean measured concentrations the EC50s were 21.1 and 0.813 mg/L for growth rate and yield, respectively. The NOECs for growth rate were 0.476 and 0.352 based on nominal and geometric mean concentrations, respectively.