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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:
21 Sep - 23 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006, corrected 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Office of Public Health Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples were taken from an additional vessel (identical conditions) since the test vessels had to remain completly filled (closed system); duplicate samples were taken (9 mL sample to 6 mL acetonitrile) at test start and after 24, 48 and 72 h.
- Sample storage conditions before analysis: deep-frozen (at about -20 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours (closed stirring vessel). After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the filtrate were used as test media.
- Controls: blank water control
- Differential loading: no
- Eluate: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Age of inoculum (at test initiation): 4 days (inoculum culture)
- Method of cultivation: algae were cultivated under the test conditions (standardized conditions according to the test guidelines)

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
23 °C
pH:
7.9 to 8.1 (control)
7.8 to 8.1 (test concentrations)
Nominal and measured concentrations:
nominal: 1:100 dilution, 1:32 dilution, 1:10 dilution, 1:3.2 dilution, undiluted filtrate (loading rate of 100 mg/L)
measured (geometric mean): not analyzed, 0.10 mg/L, 0.18 mg/L, 0.31 mg/L, 1.2 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks, 60 mL, completely filled (without headspace) and tightly sealed with glass stoppers
- Initial cells density: 5000 cells/mL (corresponding to 0.82 x 10E+04 relative fluorescence units)
- Control end cells density: 110.4 x 10E+04 relative fluorescence units
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (AAP medium, modified according to the International Standard ISO 14442 as a closed test system was applied)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared according to the test guidelines, but modified according to ISO 14442 as a closed test system was applied. The concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the pH of the test media as constant as possible.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: LED light, intensity approximately 66 μE s-1 m-2 (range: 64 to 68 μE s-1 m-2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement (duplicate measurement after 24, 48 and 72 h; excitation: 440 nm, emission: 680 nm)
- Appearance: The shape and size of algae was visually inspected at the end of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.1
Range finding study
- Test concentrations: control, 1:100 dilution, 1:10 dilution and undiluted filtrate
- Results used to determine the conditions for the definitive study: After 72 h effects were obsered in the highest test item concentration (undiluted filtrate with a loading rate of 100 mg/L). No effects were observed in the dilutions 1:10 and 1:100.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Remarks:
0.22 - 0.25 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Remarks:
0.14 - 0.16 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI
Remarks:
0.21 - 0.24 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI
Remarks:
0.13 - 0.15 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): no difference in shape and size of the algal cells were observed at the end of the test between the the dilution 1:10 (0.18 mg/L) and the algal cells in the control.
- Any stimulation of growth found in any treatment: stimulating effects were observed after 48 and 72 h at the dilutions 1:100 (n.a.) and 1:32 (0.10 mg/L)
- Any observations that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- Other: Biological and analytical results are summarized within the section "Any other information on results incl. tables"
Results with reference substance (positive control):
- EC50: ErC50 (72 h) = 0.9 mg/L (IES Study Number 20160277 Oct 2016)
- Other: Evaluation was performed twice a year to demonstrate satisfactory test conditions.
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Weibull Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test, Welch t-test or Jonckheere-Terpstra Step-down test, where appropriate. Statistical analysis was performed using ToxRat Professional®.

Validity criteria: In the control, the biomass increased by a factor of 135 over 72 h. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 h was 20%.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 h was 1.2%.

Table 1: Biomass of Algae

Treatment/ Dilution

Mean Measured Concentration

[mg/L]

Rep. no.

Biomass of algae*

24 hours

48 hours

72 hours

Control

---

1

2.7

17.9

107.0

 

 

2

3.0

20.8

111.0

 

 

3

2.8

20.2

104.0

 

 

4

3.3

23.5

119.2

 

 

5

2.8

19.7

116.8

 

 

6

3.3

20.1

104.6

 

 

Mean

3.0

20.4

110.4

 

 

SD

0.3

1.8

6.4

1:100

n.a.

1

3.2

22.5

124.4

 

 

2

3.4

22.7

127.6

 

 

3

2.8

20.3

114.2

 

 

Mean

3.1

21.8

122.0

 

 

SD

0.3

1.3

7.0

1:32

0.10

1

2.7

21.3

119.0

 

 

2

3.0

20.0

116.6

 

 

3

2.9

20.7

105.3

 

 

Mean

2.9

20.7

113.7

 

 

SD

0.1

0.6

7.3

1:10

0.18

1

3.1

18.8

96.4

 

 

2

2.3

19.4

99.9

 

 

3

2.9

16.8

90.4

 

 

Mean

2.8

18.3

95.6

 

 

SD

0.4

1.4

4.8

1:3.2

0.31

1

1.4

0.8

0.6

 

 

2

1.2

0.7

0.4

 

 

3

1.8

0.9

0.3

 

 

Mean

1.5

0.8

0.4

 

 

SD

0.3

0.1

0.2

Undiluted filtrate°

1.2

1

0.7

0.8

0.6

 

 

2

0.7

0.9

0.9

 

 

3

1.1

0.8

0.6

 

 

Mean

0.9

0.8

0.7

 

 

SD

0.2

0.1

0.2

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

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10E+04). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.82 x 10E+04 relative fluorescence units.

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

Table 2: Average Growth Rates (μ)

Treatment /Dilution

Mean Measured Concentration

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

 

 

0-24 h

 

0-48 h

 

0-72 h

 

 

[mg/L]

μ

Ir [%]

μ

Ir [%]

μ

Ir [%]

Control

---

1.290

0.0

1.605

0.0

1.634

0.0

1:100

n.a.

1.342

-4.0

1.641

-2.2

1.668

-2.0

1:32

0.10

1.250

3.1

1.614

-0.5

1.644

-0.6

1:10

0.18

1.213

6.0

1.553

3.3

1.586#

2.9

1:3.2

0.31

0.570*

55.8

-0.015§

100.9

-0.218#

113.3

Undiluted Filtrate°

1.2

0.037*

97.1

0.017§

99.0

-0.059#

103.6

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

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

*: 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).

§: Mean value statistically significantly lower than in the control (according to Jonckheere Terpstra-test one-sided smaller, α = 0.05).

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

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

---

2.2

0.0

19.6

0.0

109.6

0.0

1:100

n.a.

2.3

-7.3

21.0

-7.4

121.2

-10.6

1:32

0.10

2.0

5.7

19.8

-1.4

112.8

-2.9

1:10

0.18

2.0

9.8

17.5*

10.5

94.8#

13.5

1:3.2

0.31

0.6*

70.1

0.0*

100.1

-0.4#

100.3

Undiluted Filtrate°

1.2

0.0*

97.8

0.0*

99.9

-0.1#

100.1

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

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

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

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

Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 4: Analytical Results of Test Samples

Sampling Day/Age of Sample

Dilution Factor of Filtrate of Loading Rate 100 mg Test Item /L

Measured Concentration of Test Item x

Sample Preparation Factor F

Determined Concentration of Test Item c

% of Initially Measured Concentration

[d/h]

 

[mg/L]

 

[mg/L]

 

0/0

Control

n.d.

1.667

< LOQ

 

(fresh)

1:32

0.564

1.667

0.940

 

 

1:10

1.86

1.667

3.09

 

 

1:3.2

1.18

8.333

9.84

 

 

Undiluted filtrate

1.48

16.67

24.6

1/24

Control

n.d.

1.667

< LOQ

(aged)

1:32

0.0671

1.667

0.112

12

 

1:10

0.235

1.667

0.391

13

 

1:3.2

0.147

8.333

1.22

12

 

Undiluted filtrate

0.266

16.67

4.44

18

2/48

Control

n.d.

1.667

< LOQ

 

(aged)

1:32

n.d.

1.667

< LOQ

n.a.

 

1:10

n.d.

1.667

< LOQ

n.a.

 

1:3.2

< 0.033

1.667

< LOQ

n.a.

 

Undiluted filtrate

0.370

1.667

0.616

3

3/72

Control

n.d.

1.667

< LOQ

 

(aged)

1:32

n.d.

1.667

< LOQ

n.a.

 

1:10

n.d.

1.667

< LOQ

n.a.

 

1:3.2

< 0.033

1.667

< LOQ

n.a.

 

Undiluted filtrate

< 0.033

1.667

< LOQ

n.a.

n.d. = no test item detected

n.a. = not applicable

LOQ: 0.056 mg/L

Table 5: Mean measured conc based on Analytical Results of Test Samples

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]

1:100

n.a.

n.a.

n.a.

n.a.

n.a.

1:32

0.94

0.11

<LOQ

<LOQ

0.10

1:10

3.09

0.39

<LOQ

<LOQ

0.18

1:3.2

9.8

1.2

<LOQ

<LOQ

0.31

Undiluted Filtrate°

25

4.4

0.62

<LOQ

1.2

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

LOQ: Limit of quantification (LOQ = 0.056 mg test item/L).

*: If the measured concentration was below LOQ, half of the LOQ (½ LOQ =0.028 mg/L) was used to calculate the mean measured concentration.

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

 

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD 201 with the green algae Pseudokirchneriella subcapitata. It resulted in an measured ErC50 (72 h) of 0.23 mg/L and an ErC10 (72 h) of 0.15 mg/L. The NOEC was determined to be 0.10 mg/L. All effect concentrations were based on the geometric mean measured concentration.

Description of key information

ErC50 (72 h) = 0.23 mg/L (geometric mean measured concentration, OECD 201, Pseudokirchneriella subcapitata)

ErC10 (72 h) = 0.15 mg/L (geometric mean measured concentration, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.23 mg/L
EC10 or NOEC for freshwater algae:
0.15 mg/L

Additional information

There is one GLP guideline study available, which determined the effects of the substance on the growth of unicellular freshwater green algae according to OECD guideline 201.

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 as saturated solution with a loading rate of 100 mg/L following the filtration method. Therefore 100 mg/L test item was mixed in test water, 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 1:3.2, 1:10, 1:32, and 1:100 of the filtrate were used as test media and analytically verified by HPLC-UV analysis. 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. Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the geometric mean measured concentrations from sampling at 0, 24, 48 and 72 hours. Based on these concentrations the ErC50 (72 h) was 0.23 mg/L and the corresponding NOErC/ErC10 (72 h) were 0.10 mg/L and 0.15 mg/L based on the inhibition of growth, respectively. The EyC50 (72 h) was calculated to be 0.22 mg/L based on yield with a a corresponding EyC10 (72 h) of 0.14 mg/L and NOEyC (72 h) of 0.10 mg/L.