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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:
24 April 2017 - 23 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
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: undiluted filtrate (loading rate 100 mg/L) and the dilutions 1:2 to 1:16
- Sampling method: duplicate samples without algae were taken from the test media of all test concentrations and the control at the start of the test and duplicate stability samples with algae were taken, after 24 and 48 hours and at the end of the test (72 hours). Samples at 24 and 48 hours were taken from a set of additional flasks containing the corresponding test medium (with algae) which have been incubated under conditions identical to the test.
- Sample storage conditions before analysis: after adding of acetonitrile (6 mL acetonitrile and 9 mL sample volume), frozen (at -20 ± 5 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Highest test concentration (undiluted filtrate with a loading rate 100 mg/L): 256 μL of the test item in 2300 mL test water, intensive stirring for 3 h in a closed vessel, after stirring filtered through a 0.45 μm membrane filter (pre-conditioned with 200 mL filtrate, negative pressure of the filtration unit reduced to a minimum)
Lower test concentrations: highest test concentration further diluted with test water
- Controls: synthetic test water (AAP Medium)
- Evidence of undissolved material: No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: No. 61.81 SAG
- Source: cultivated at the testing laboratory
- Method of cultivation: under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 3 days before test initiation
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.125 mmol/L
Test temperature:
24 °C
pH:
- pH (0 h): 7.5
- pH (72 h): 7.6 - 7.8
Dissolved oxygen:
no data
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
- Nominal concentrations: undiluted filtrate (loading rate of 100 mg(L) and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the filtrate
- Measured concentrations (geometric mean): 4.7, 1.7, 0.86, 0.44, 0.21 mg/L and n.a.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with glass stoppers
- Material, size, headspace, fill volume: without headspace, 60 mL test solution
- Initial cells density: algal cell density of 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM
- Standard medium used: AAP Medium modified according to ISO 14442 (as a closed test system was applied)
- Detailed composition if non-standard medium was used: The concentration of NaHCO3 was increased to 250 mg/L and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to keep the pH of the test media as constant as possible.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED light with intensity of approx. 72 μE s-1 m-2 (range: 71 to 74 μE s-1 m-2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: undiluted filtrate (loading rate 100 mg/L) and the dilutions 1:2, 1:4, 1:8, 1:16, 1:32


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 3.0 - 3.2 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 1.3 - 1.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 1.5 - 1.7 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.64 - 0.84 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: none
- Unusual cell shape: none
- Colour differences: none
- Flocculation: none
- Aggregation of algal cells: none
- Any stimulation of growth found in any treatment: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.0 mg/L (May 2017)
Reported statistics and error estimates:
EC10, EC20 and EC50 were calculated by Probit Analysis using linear maximum likelihood regression.
LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test or Welch t-test with Bonferroni-Holm-adjustment where appropriate.
Statistical analysis was performed using ToxRat Professional®.

Table 1: Mean measured concentrations (geometric mean)

Treatment /
Dilution

Analytical Measured Concentration
of the Test Item
[mg/L]

Mean Measured
Concentration
(Geometric Mean)*

 

0 hours

24 hours

48 hours

72 hours

[mg/L]

Dilution 1:32

n.a.

n.a.

n.a

n.a

n.a

Dilution 1:16

0.964

0.532

<LOQ

<LOQ

0.21

Dilution 1:8

2.03

1.08

0.290

<LOQ

0.44

Dilution 1:4

4.18

2.15

1.01

<LOQ

0.86

Dilution 1:2

8.07

4.28

2.04

0.116

1.7

Undiluted Filtrate°

16.4

7.79

4.00

0.962

4.7

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L

LOQ: Limit of quantification (LOQ = 0.118 mg test item/L)

*: If the measured concentration was below LOQ, half of the LOQ (½ LOQ = 0.059 mg/L) was used to calculate the mean measured concentration

n.a.: Not analyzed since below NOEC determined in this test

Table 2: Average Growth Rates (µ)

Treatment / Dilution

Mean Measured Concentration [mg/L]

Average Growth Rate µ (day-1) and Inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir[%]

µ

Ir[%]

µ

Ir[%]

Control

---

1.698

0.0

1.740

0.0

1.745

0.0

1:32

n.a.

1.760

-3.7

1.763

-1.3

1.756

-0.7

1:16

0.21

1.581

6.9

1.749

-0.5

1.739

0.3

1:8

0.44

1.673

1.4

1.715

1.5

1.708

2.1

1:4

0.86

1.771

-4.3

1.678

3.6

1.699

2.6

1:2

1.7

1.231*

27.5

1.378#

20.8

1.468#

15.9

Undiluted Filtrate°

4.7

0.448*

73.6

0.517#

70.3

0.419#

76.0

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

*: 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).

n.a.: Not analyzed since below NOEC determined in this test.

 

Table 3: Yield (Y)

Treatment / Dilution

Mean Measured Concentration [mg/L]

Yield Y (x 104) and Inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

Y

Iy[%]

Y

Iy[%]

Y

Iy[%]

Control

---

3.50

0.0

24.62

0.0

145.79

0.0

1:32

n.a.

3.77

-7.8

25.81

-4.8

151.01

-3.6

1:16

0.21

3.05

12.8

25.04

-1.7

143.82

1.3

1:8

0.44

3.38

3.3

23.40

4.9

130.77*

10.3

1:4

0.86

3.83

-9.4

21.64*

12.1

127.07*

12.8

1:2

1.7

1.90*

45.8

11.51*

53.2

63.11*

56.7

Undiluted Filtrate°

4.7

0.44*

87.4

1.67*

93.2

2.03*

98.6

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

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

n.a.: Not analyzed since below NOEC determined in this test.

 

Table 4: Anlytic results for Test Samples

Sampling Day / Sample Age

[d/h]

Loading Rate of 100 mg Test Item/L

Measured Concentration of Test Item

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

% of Initially Measured

[%]

0/0

(fresh)

Control

Dilution 1:16

Dilution 1:8

Dilution 1:4

Dilution 1:2

Undiluted Filtrate°

n.d.

0.578

1.22

2.51

4.84

9.83

1.67

1.67

1.67

1.67

1.67

1.67

<LOQ

0.964

2.03

4.18

8.07

16.4

-

-

-

-

-

-

1/24

(aged)

Control

Dilution 1:16

Dilution 1:8

Dilution 1:4

Dilution 1:2

Undiluted Filtrate°

n.d.

0.319

0.647

1.29

2.57

4.67

1.67

1.67

1.67

1.67

1.67

1.67

<LOQ

0.532

1.08

2.15

4.28

7.79

n.a.

55

53

52

53

48

2/48

(aged)

Control

Dilution 1:16

Dilution 1:8

Dilution 1:4

Dilution 1:2

Undiluted Filtrate°

n.d.

n.d.

0.174

0.608

1.22

2.40

1.67

1.67

1.67

1.67

1.67

1.67

<LOQ

<LOQ

0.290

1.01

2.04

4.00

n.a.

n.a.

14

24

25

24

3/72

(aged)

Control

Dilution 1:16

Dilution 1:8

Dilution 1:4

Dilution 1:2

Undiluted Filtrate°

n.d.

n.d.

n.d.

n.d.

0.0697*

0.577

1.67

1.67

1.67

1.67

1.67

1.67

<LOQ

<LOQ

<LOQ

<LOQ

0.116

0.962

n.a.

n.a.

n.a.

n.a.

1

6

Remark: The analytical samples of the dilution 1:32 were not analyzed as this concentration was below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

LOQ: 0.118 mg test item /L

n.a.: not applicable

n.d.: no test item detected

°: undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

*: Value 2% below the lowest calibration solution, the determined concentration was considered as estimated value.

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

Validity criteria fulfilled:
yes
Conclusions:
The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined as follows:
72h-ErC10 (growth rate) = 1.4 mg/L
72h-ErC50 (growth rate) = 3.1 mg/L
NOEC (growth rate) = 0.86 mg/L
The results are based on geometric mean measured concentrations of the test item.
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled (without headspace) with test medium, minimizing the air-space in the flasks and to avoid losses of test item by evaporation. Due to the low water solubility of the test item, the test media were prepared following the stirring/filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the filtrate were used as test media. Additionally, a control was tested in parallel. During the test period of 72 hours the test item concentrations in the test media decreased below the limit of quantification of the analytical method (LOQ = 0.118 mg/L). Thus, the mean measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48, and 72 hours). Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations resulting in a 72-hour ErC10 (growth rate) = 1.4 mg/L (95 % CI: 1.3 and 1.5 mg/L) and 72 -hour ErC50 (growth rate) = 3.1 mg/L (95 % CI: 3.0 and 3.2 mg/L).

Description of key information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined as follows:

72h-ErC10 (growth rate) = 1.4 mg/L

72h-ErC50 (growth rate) = 3.1 mg/L

The results are based on geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:
3.1 mg/L
EC10 or NOEC for freshwater algae:
1.4 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3.

As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled (without headspace) with test medium, minimizing the air-space in the flasks and to avoid losses of test item by evaporation. Due to the low water solubility of the test item, the test media were prepared following the stirring/filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 µm membrane filter. The undiluted filtrate and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the filtrate were used as test media. Additionally, acontrol was tested in parallel.During the test period of 72 hours the test item concentrations in the test media decreased below the limit of quantification of the analytical method (LOQ = 0.118 mg/L). Thus, the mean measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48, and 72 hours). Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations resulting in a 72-hour ErC10 (growth rate) = 1.4 mg/L (95 % CI: 1.3 and 1.5 mg/L) and 72 -hour ErC50 (growth rate) = 3.1 mg/L (95 % CI: 3.0 and 3.2 mg/L).