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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:
2017-10-24 to 2017-10-27
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No. / Lot.: S7101247
CAS No.: 873-75-6
Aggregate state at RT: Solid
Colour: White
Molecular Weight: 187.03 g/mol
Purity: 100.0% (GC)
Expiry date: 2020-08-31 (minimum shelf life)

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Three replicate samples (5 mL per replicate) were taken from each test concentration from the control at the start and at the end of the test.
- Sample storage conditions before analysis: None

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions used in the test was prepared by mechanical dispersion without using of any solubilising agent. For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.08 g test item in 800 mL OECD medium using approx. 10 min ultrasonication. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution. After the formulation procedure algal cells were immediately introduced into test solution.
- Controls: Negative control
- Evidence of undissolved material: No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): Exponentially growing pre-culture, set up four days prior to the test start.
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
The temperature in the flask was in the range of 21.9 – 22.6 °C and within the climate chamber between 21.2 and 22.9 °C.
pH:
The range of the pH was 7.86 – 8.82 during the study. The pH of the control cultures did not vary by more than 1.5 units during the test.
Dissolved oxygen:
Not measured
Salinity:
Not applicable
Conductivity:
Not measured
Nominal and measured concentrations:
Nominal concentrations: 3.6, 8.2, 18.9, 43.5 and 100.0 mg/L
Measured mean concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Size, fill volume: 250 mL nominal volume, covered with air-permeable stoppers, 100 mL fill volume
- Aeration: Continuously shaken by a laboratory orbital shaker
- Initial cells density: 104 cells/mL in each test flask
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (according to OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 Int water purification system)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity and quality: 7874 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: Yes. In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
- Test concentrations: 0 (control), 0.1, 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
30.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 2.6 - 45.8 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
38.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 7.7 - 56.1 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 32.3 - 119.6 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
43.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
95% conf. limits: 1.1 - 25.4 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
19.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 3.2– 33.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
34.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 16.5 - 86.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
43.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Cell Density in Control: The cell density in the control has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to 52.0 x 104 cells at the end of the test (72 h).
- Shape of the Algal Cells: No morphological abnormalities were observed in the control and in the concentrations of 3.6 and 8.2 mg/L during the test. Thin and swollen cells were observed together with the normal cells in the concentrations of 18.9 and 43.5 mg/L. Swollen cells were observed in the highest concentration of 100.0 mg/L.
- Any stimulation of growth found in any treatment: Yes, at 8.2 mg/L in nominal after 24, 48 and 72 hours of exposure, slight stimulation of growth compared to the control was observed (yield and growth rate).
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

VALIDITY OF THE STUDY
Cell density in the control cultures increased by a factor of more than 16 (by a factor of 52.00) within 72 hours. This corresponds to a specific growth rate of 1.31 day-1.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 30.67 %
- CV for section-by-section growth rate day 1-2: 33.82 %
- CV for section-by-section growth rate day 2-3: 5.40 %

The mean coefficient of variation for section-by-section specific growth rates: 23.30 %.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test.

- CV for average specific growth rate day 0-3: 3.58 %


All validity criteria were met, therefore the study can be considered as valid.
Results with reference substance (positive control):
72-h ErC50 = 0.82 mg/L (95 % confidence limits: 0.57 – 1.25 mg/L)
72-h EyC50 = 0.51 mg/L (95 % confidence limits: 0.45 – 0.57 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (µ) and yield (y) were calculated using EXCEL for Windows software. The ECx values and their confidence limits for growth rate were calculated using Logit analysis and for yield were calculated using Probit analysis by SPSS software. For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test ( = 0.05; 2-sided) by SPSS software.

Table 1: Test item concentration in samples

Sampling date Nominal concentration (mg Test Item/L) Mean of the measured concentrations (mg Test Item/L) Measured concentration in percentage of the nominal Relative Standard Deviation % (n=3)
24 October 2017 control not detected - -
3.6 3.32 92 0
8.2 7.92 97 1
18.9 18.4 97 0
43.5 41.86 96 0
100 96.56 97 1
27 October 2017 control not detected - -
3.6 3.04 84 3
8.2 7.33 89 7
18.9 18.17 96 2
43.5 42.15 97 1
100 98.08 98 1

 

Table 2: Algal cell number (× 104cell/mL) during the test period of 72 hours and Yield

Concentration
[mg/L]

 

Number of Cells (× 104/mL)

Yield

0 h

24 h

48 h

72 h

Untreated control

 

1

4

8

40

39

1

4

11

58

57

1

2

13

59

58

1

4

13

54

53

1

3

11

50

49

1

2

11

51

50

Mean

1.00

3.17

11.17

52.00

51.00

SD

0.00

0.98

1.83

6.90

6.90

3.6

 

1

3

12

49

48

1

2

10

53

52

1

4

7

55

54

Mean

1.00

3.00

9.67

52.33

51.33

SD

0.00

1.00

2.52

3.06

3.06

8.2

 

1

5

14

68

67

1

3

12

66

65

1

3

13

62

61

Mean

1.00

3.67

13.00

65.33

64.33

SD

0.00

1.15

1.00

3.06

3.06

18.9

 

1

3

8

39

38

1

3

9

39

38

1

2

14

49

48

Mean

1.00

2.67

10.33

42.33

41.33

SD

0.00

0.58

3.21

5.77

5.77

43.5

 

1

2

8

21

20

1

4

8

31

30

1

2

7

20

19

Mean

1.00

2.67

7.67

24.00

23.00

SD

0.00

1.15

0.58

6.08

6.08

100.0

 

1

1

1

1

0

1

1

1

1

0

1

1

1

2

1

Mean

1.00

1.00

1.00

1.33

0.33

SD

0.00

0.00

0.00

0.58

0.58

 

 

Table 3: The specific Growth Ratesduring the test period of 72 hours

Concentration
[mg/L]

 

Growth Rates (µ)

0 – 24 h

0 – 48 h

0 – 72 h

Untreated control

 

0.0578

0.0433

0.0512

0.0578

0.0500

0.0564

0.0289

0.0534

0.0566

0.0578

0.0534

0.0554

0.0458

0.0500

0.0543

0.0289

0.0500

0.0546

Mean

0.0461

0.0500

0.0548

SD

0.0141

0.0037

0.0020

3.6

 

0.0458

0.0518

0.0541

0.0289

0.0480

0.0551

0.0578

0.0405

0.0557

Mean

0.0441

0.0468

0.0550

SD

0.0145

0.0057

0.0008

8.2

 

0.0671

0.0550

0.0586

0.0458

0.0518

0.0582

0.0458

0.0534

0.0573

Mean

0.0529

0.0534

0.0580

SD

0.0123

0.0016

0.0007

18.9

 

0.0458

0.0433

0.0509

0.0458

0.0458

0.0509

0.0289

0.0550

0.0541

Mean

0.0401

0.0480

0.0519

SD

0.0098

0.0061

0.0018

43.5

 

0.0289

0.0433

0.0423

0.0578

0.0433

0.0477

0.0289

0.0405

0.0416

Mean

0.0385

0.0424

0.0439

SD

0.0167

0.0016

0.0033

100.0

 

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0096

Mean

0.0000

0.0000

0.0032

SD

0.0000

0.0000

0.0056

 

 

Table 4: Growth Rates (µ) and percentage inhibition of µ during the test period

Concentration
[mg/L]

Growth Rates (µ)and % Inhibition ofµ

0–24 h

0–48 h

0–72 h

µ

%

µ

%

µ

%

Untreated control

0.0461

0.0500

0.0548

3.6

0.0441

4.3

0.0468

6.5

0.0550

-0.3

8.2

0.0529

-14.6

0.0534

-6.8

0.0580

-6.0

18.9

0.0401

13.0

0.0480

4.0

0.0519

5.2

43.5

0.0385

16.5

0.0424

15.2

0.0439

19.9

100.0

0.0000

100.0

0.0000

100.0

0.0032

94.1


 

Table 5: Yield (y) and percentage inhibition of y during the test period

Concentration
[mg/L]

Yield y and % Inhibition of y

0–72 h

y

%

Untreated control

51.00

3.6

51.33

-0.7

8.2

64.33

-26.1

18.9

41.33

19.0

43.5

23.00

54.9

100.0

0.33

99.3

 

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC10, EC20, EC50 for growth were determined to be 30.9, 38.5 and 56.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 14.3, 19.4 and 34.5 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 43.5 mg/L. The 72-h NOEC for growth and yield was determined to be 18.9 mg/L. All validity criteria were met. The results are based on nominal test item concentrations. In conclusion, the obtained results showed that the test item had inhibitory effects on the growth of Raphidocelis subcapitata.

Executive summary:

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), an algal growth inhibition test according to OECD Guideline 201, EU Method C.3 and US EPA OPPTS 850.5400 was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed to nominal concentrations of 3.6, 8.2, 18.9, 43.5 and 100.0 mg test item/L (based on a preliminary study) over several generations under defined conditions. The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. The analytically measured test item concentrations remained within ± 20 % of the nominal concentration during the test period of 72 hours therefore biological results are based on the nominal concentrations. The results of the statistical evaluation (Dunnett’s test (a= 0.05; 2-sided)) showed that the 0-72 h average specific growth rate and yield was statistically significantly different from the untreated control at the concentrations of 43.5 and 100.0 mg/L and not significantly different at the concentrations of 3.6, 8.2 and 18.9 mg/L. Statistically significant negative inhibition (growth stimulation) was observed in the concentration of 8.2 mg/L for Yield, however no concentration response pattern was noticed and therefore this was considered to be not a treatment related effect. The cell density in the control has increased from nominal N = 1 × 104cells/mL at the start of the test (0 hours) to 52.0 x 104cells at the end of the test (72 h). No morphological abnormalities were observed in the control and in the concentrations of3.6 and 8.2 mg/Lduring the test. Thin and swollen cells were observed together with the normal cells in the concentrations of 18.9 and 43.5 mg/L. Swollen cells were observed in the highest concentration of 100.0 mg/L.In result, the 72-h EC10, EC20, EC50 for growth were determined to be 30.9, 38.5 and 56.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 14.3, 19.4 and 34.5 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 43.5 mg/L. The 72-h NOEC for growth and yield was determined to be 18.9 mg/L. In conclusion, the obtained results showed that the test item had inhibitory effects on the growth of Raphidocelis subcapitata. All validity criteria of the guidelines were met.

Description of key information

The 72-h EC10 and EC50 for growth were determined to be 30.9 and 56.0 mg/L, respectively. The 72-h EC10 and EC50 for yield were determined to be 14.3 and 34.5 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 43.5 mg/L. The 72-h NOEC for growth and yield was determined to be 18.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
56 mg/L
EC10 or NOEC for freshwater algae:
30.9 mg/L

Additional information

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata, an algal growth inhibition test according to OECD Guideline 201, EU Method C.3 and US EPA OPPTS 850.5400 was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed to nominal concentrations of 3.6, 8.2, 18.9, 43.5 and 100.0 mg test item/L (based on a preliminary study) over several generations under defined conditions. The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. The analytically measured test item concentrations remained within ± 20 % of the nominal concentration during the test period of 72 hours therefore biological results are based on the nominal concentrations. The results of the statistical evaluation (Dunnett’s test (a= 0.05; 2-sided)) showed that the 0-72 h average specific growth rate and yield was statistically significantly different from the untreated control at the concentrations of 43.5 and 100.0 mg/L and not significantly different at the concentrations of 3.6, 8.2 and 18.9 mg/L. Statistically significant negative inhibition (growth stimulation) was observed in the concentration of 8.2 mg/L for Yield, however no concentration response pattern was noticed and therefore this was considered to be not a treatment related effect. The cell density in the control has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to 52.0 x 10^4 cells at the end of the test (72 h). No morphological abnormalities were observed in the control and in the concentrations of 3.6 and 8.2 mg/L during the test. Thin and swollen cells were observed together with the normal cells in the concentrations of 18.9 and 43.5 mg/L. Swollen cells were observed in the highest concentration of 100.0 mg/L. In result, the 72-h EC10, EC20, EC50 for growth were determined to be 30.9, 38.5 and 56.0 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 14.3, 19.4 and 34.5 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 43.5 mg/L. The 72-h NOEC for growth and yield was determined to be 18.9 mg/L. In conclusion, the obtained results showed that the test item had inhibitory effects on the growth of Raphidocelis subcapitata. All validity criteria of the guidelines were met (reference 6.1.5-1).