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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:
02 February 2018 - 19 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0 (control), 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0% w/v
- Sampling method: The test solutions were analysed for active ingredient concentration analysis and stability at 24-h intervals. 310 mL of test sample from each replicates was drawn and divided into equal portion (155 mL in each portion). One portion was sent for analysis and second portion was stored at -20 ºC. The Samples were centrifuged at 2000 rpm in order to remove the algal cells before sending for analysis. Active ingredient concentration in culture medium was determined using validated analysis method [Study Number 228-2-14-19135].
- Sample storage conditions before analysis: not required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary non-GLP experiments were conducted to determine the maximum solubility of test item in reconstituted water. Several sets of mixer of 100 mg of test item and 1000 mL test medium were prepared. Different stirring duration (from 60 to 300 minutes), equilibrium duration, and separation methods (centrifugation vs. equilibrium) were applied. Based on the obtained results, 100% w/v saturated concentration was determined to be prepared by mixing 100 mg of test item with 1 L of reconstituted water followed by continuous stirring on magnetic stirrer for 180 min and 30 min of equilibrium.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Specie: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Method of cultivation: The algae were cultured under standarised conditions, according to OECD 201 in the Ecotoxicological Laboratory of Jai Research Fundation.The pre-culture was prepared 2 days prior to the commencement of the study by transferring 1 mL from the latest sub culture into a new culture vessel. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5 x 10^3 - 10^4 cells/mL). The temperature for pre-culture ranged between 21 - 23 ºC for positive control study and main study.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21 – 23 ºC
pH:
7.19 – 8.06
Nominal and measured concentrations:
Nominal concentrations: 0.0 (control), 0.3, 1.0, 3.1, 9.8, 31.3, and 100.0% w/v saturated concentrations.
Measured concentrations: 0.0 (control), 1.05, 1.05, 1.50, 3.74, 11.43, 40.55 mg/L a.i.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL sterile screw capped conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL capped conical flasks, continuously shaken by a laboratory orbital shaker to keep algae in suspension. Since the test item is volatile, all experiments were carried out with minimal or no head space in the test vessels in order to avoid the substance loss from media. The fill volume was 310 mL. In addition to this, algae medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2).
- Aeration: No
- Initial cells density: 5476 cells/mL
- Control end cells density: The cell density in the control cultures increased by the factor of 127.0 within three days.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, pH was adjusted to 7.0 by addition of HCl, (to maintain CO2 concentration to support algal growth in the absence of headspace) as per Mayer et al., 2000.
- Photoperiod: Continuously illuminated.
- Light intensity and quality: 6527 and 6487 lux. The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell numbers were determined at 24, 48 and 72 hours after starting the test by visual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in test concentration level and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
- Range finding study: Non-GLP research trial study was conducted with test concentrations of 0.0 (control), 0.1, 1.0, 10.0, 50.0 and 100.0% (w/v) saturated concentration prepared at 100 mg/L.
- Results used to determine the conditions for the definitive study: The percent inhibition of growth rates observed were 0.47, 8.85, 23.60, 49.07, and 82.14 at the test concentrations of 0.0 (control), 0.1, 1.0, 10.0, 50.0 and 100.0% w/v saturated concentration prepared at 100 mg/L, respectively. Based on the results of this preliminary study, the test concentrations selected for the main study were 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L (geometric factor 1.4).
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate is tested at least twice a year by CitoxLab)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.97 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.:
2.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.83 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 (for algal test): yes.
- Observation of abnormalities (for algal test): no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
Potassium dichromate was tested in study No. 501-3-07-19136 (04/06/2018) to demonstrate satisfactory test conditions.
- EC50: The 72h ErC 50: 0.90 mg/L; the 72h EbC 50: 0.45 mg/L; the 72h EyC 50: 0.49 mg/L. These values are within the range of laboratory ring test data.
Reported statistics and error estimates:
Based on statistical analysis, there was no significant alteration in biomass (cell growth), yield and growth rate observed for G2 (0.3% w/v saturated concentration). The calculated value of geometric mean measured for both G2 and G3 (0.3% and 1.0% w/v saturated concentrations) was 1.05 mg/L a.i. According to these values, NOEC and LOEC values would be the same and therefore cannot be considered.

Table 1.Geometric Mean of test item (Main Study)

Nominal Concentration

[% Saturated Solution Prepared at 100 mg/L]

Measured Concentration (mg a.i./L)

Geometric Mean

(mg a.i./L)

0 h

24 h

48 h

72 h

72 h

0.0 (Control)

-

-

-

-

-

0.3

1.0500000

1.0500000

1.0500000

1.0500000

1.05

1.0

1.0500000

1.0500000

1.0500000

1.0500000

1.05

3.1

1.5000000

1.5000000

1.5000000

1.5000000

1.50

9.8

4.74537

4.06313

3.63316

2.79959

3.74

31.3

13.91359

13.86161

10.87363

8.14256

11.43

100.0

43.81902

41.03174

38.53571

39.03733

40.55

Key:  h = Hour, GM = Geometric Mean

 

Table 2. Mean Values of Algal Biomass, Yield, Specific Growth Rate and Validity Criteria (Main Study)

A. Mean Values of Algal Biomass, Yield, Specific Growth Rate       

Group

Nominal

Concentration

[% Saturated Solution Prepared at 100 mg/L]

Measured Concentration

[mg a.i./L]

Mean Number of Cells Count/mL at

Specific Growth Rate

(0 – 3)

0 h

24 h

48 h

72 h

Yield

G1

0.0 (Control)

0.0 (Control)

5476

26667

132917

700833

695357

1.62

G2

0.3

1.05

25833

120833

658333

652857

1.60

G3

1.0

1.05

23333

108333

590000

584524

1.56

G4

3.1

1.50

20833

93333

441667

436191

1.46

G5

9.8

3.74

16667

62500

186667

181191

1.17

G6

31.3

11.43

5833

15000

30833

25357

0.57

G7

100.0

40.55

2500

5833

7500

2024

0.09

B. Validity Criteria

Group

Test

Concentration

(mg/L)

Replicate

Specific Growth Rate

Mean % coefficient of variation for

days 0-1, 1-2, and 2-3

0-1

1-2

2-3

0-3

Mean

SD

CV

G1

0.0

(Control)

R1

1.52

1.57

1.76

1.62

1.62

0.13

8.02

R2

1.70

1.47

1.67

1.61

1.61

0.13

8.07

R3

1.61

1.61

1.67

1.63

1.63

0.03

1.84

R4

1.41

1.73

1.7

1.62

1.61

0.18

11.18

R5

1.70

1.58

1.54

1.60

1.61

0.08

4.97

R6

1.52

1.70

1.64

1.62

1.62

0.09

5.56

 

 

Mean

-

1.62

Mean CV : 6.61

 

 

SD

0.01

 

 

CV

0.62

 

* Cell concentration in control

(within the 3 days)

Total Biomass in Control at 72 h

Initial Cell Count

Increased by factor: 127 times

Key: h = Hour,  = significantly lower than control at 1% level (p 0.01), - = Not Applicable

 

Table 3. Percentage (%) Inhibition of Biomass, Growth Rate, and Yield (Main Study)

Group

Nominal

Concentration

[% Saturated Solution Prepared at 100 mg/L]

Measured Concentration

[mg a.i./L]

Percentage (%) Inhibition of

Biomass

(EbC) 0 - 72 h

Growth Rate

(ErC) 0 - 72 h

Yield

(EyC) 0 - 72 h

G1

0.0 (Control)

0.0 (Control)

-

-

-

G2

0.3

1.05

6.88

1.34

6.11

G3

1.0

1.05

16.79

3.56

15.94

G4

3.1

1.50

35.26

9.64

37.27

G5

9.8

3.74

68.00

27.30

73.94

G6

31.3

11.43

95.45

64.69

96.35

G7

100.0

40.55

100.32

94.36

99.71

Key: - = Not Applicable, h = Hour

Note: Growth rate is a logarithmic term. Therefore, EbC and ErC values are not numerically comparable. All values mentioned above are based on the nominal concentrations selected for the study.

 

Table 4. EC10, EC20and EC50Values (Main Study).

A.     Based on Nominal Concentration:

Exposure

Time (72h)

EbCxValue

for Biomass Inhibition

Concentration

(% Saturated Solution Prepared at 100 mg/L)

95% Confidence Interval(% Saturated Solution Prepared at 100 mg/L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

EbC10

0.56

0.39

0.80

y = -0.52 + 1.54x

EbC20

1.08

0.81

1.43

EbC50

3.78

3.06

4.68

Exposure

Time (72h)

ErCxValue for Growth Rate Inhibition

Concentration

(% Saturated Solution Prepared at 100 mg/L)

95% Confidence Limits(% Saturated Solution Prepared at 100 mg/L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

ErC10

2.12

1.19

3.80

y = -1.25 + 1.49x

ErC20

4.19

2.66

6.60

ErC50

15.31

10.89

21.53

Exposure

Time (72h)

EyCxValue

for Yield Inhibition

Concentration

(% Saturated Solution Prepared at 100 mg/L)

95% Confidence Limits(% Saturated Solution Prepared at 100 mg/L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

EyC10

0.62

0.45

0.84

y = -1.18 + 1.75x

EyC20

1.10

0.86

1.41

EyC50

3.32

2.75

4.01

B.     Based on Geometric Mean Measured Concentration: 

Exposure

Time (72h)

EbCxValue

for Biomass Inhibition

Concentration

(mg a.i./L)

95% Confidence Interval (mg a.i./L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

EbC10

0.89

0.66

1.22

y = -4.37+ 2.74x

EbC20

1.29

1.02

1.65

EbC50

2.62

2.11

3.27

Exposure

Time (72h)

ErCxValue for Growth Rate Inhibition

Concentration

(mg a.i./L)

95% Confidence Limits (mg a.i./L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

ErC10

1.97

1.62

2.40

y = -3.56 + 2.21x

ErC20

3.12

2.65

3.68

ErC50

7.51

6.40

8.81

Exposure

Time (72h)

EyCxValue

for Yield Inhibition

Concentration

(mg a.i./L)

95% Confidence Limits (mg a.i./L)

Regression Equation

(y = a + bx)

Lower Limit

Upper Limit

EyC10

0.83

0.47

1.46

y = -3.67 + 2.53x

EyC20

1.23

0.68

2.25

EyC50

2.65

1.40

5.02

 

Table 5. Main Study: pH Values recorded at 0 and 72 h

Group

Test Concentration

(% Saturated Solution Prepared at 100 mg/L)

0 h [Initial]

72 h [Final]

 

R1

R2

R3

R4

R5

R6

R1

R2

R3

R4

R5

R6

G1

0.0 (Control)

7.25

7.37

7.43

7.24

7.20

7.20

8.00

7.98

8.01

7.99

7.98

7.99

G2

0.3

7.20

7.20

7.19

-

-

-

7.93

7.95

7.94

-

-

-

G3

1.0

7.25

7.23

7.23

-

-

-

7.75

7.80

7.79

-

-

-

G4

3.1

7.24

7.26

7.28

-

-

-

8.00

8.05

8.06

-

-

-

G5

9.8

7.27

7.28

7.28

-

-

-

7.94

7.96

7.93

-

-

-

G6

31.3

7.31

7.32

7.31

-

-

-

7.55

7.61

7.68

-

-

-

G7

100.0

7.37

7.42

7.44

-

-

-

7.43

7.40

7.44

-

-

-

Table 6. Algal Cell Count, Yield and Specific Growth Rates (Main Study).Initial Mean Cell Count: 5476 cells/mL

Group

Nominal

Concen-

tration

[mg/L]

Measured Concen-tration

[mg a.i./L]

Rep                 .

Cell Count at

Yield

Specific Growth Rate

24 h

48 h

72 h

Cells/mL

0-1

1-2

2-3

0-3

G1

0.0

(Control)

 

0.0

(Control)

 

R1

25000

120000

700000

694524

1.52

1.57

1.76

1.62

R2

30000

130000

690000

684524

1.70

1.47

1.67

1.61

R3

27500

137500

730000

724524

1.61

1.61

1.67

1.63

R4

22500

127500

700000

694524

1.41

1.73

1.70

1.62

R5

30000

145000

675000

669524

1.70

1.58

1.54

1.60

R6

25000

137500

710000

704524

1.52

1.70

1.64

1.62

G2

0.3

1.05

R1

25000

130000

627500

622024

1.52

1.65

1.57

1.58

R2

22500

120000

697500

692024

1.41

1.67

1.76

1.62

R3

30000

112500

650000

644524

1.70

1.32

1.75

1.59

G3

1.0

1.05

R1

25000

120000

605000

599524

1.52

1.57

1.62

1.57

R2

20000

100000

570000

564524

1.30

1.61

1.74

1.55

R3

25000

105000

595000

589524

1.52

1.44

1.73

1.56

G4

3.1

1.50

R1

25000

105000

490000

484524

1.52

1.44

1.54

1.50

R2

17500

80000

400000

394524

1.16

1.52

1.61

1.43

R3

20000

95000

435000

429524

1.30

1.56

1.52

1.46

G5

9.8

3.74

R1

17500

80000

212500

207024

1.16

1.52

0.98

1.22

R2

15000

50000

162500

157024

1.01

1.20

1.18

1.13

R3

17500

57500

185000

179524

1.16

1.19

1.17

1.17

G6

31.3

11.43

R1

7500

15000

37500

32024

0.31

0.69

0.92

0.64

R2

5000

12500

25000

19524

-0.09

0.92

0.69

0.51

R3

5000

17500

30000

24524

-0.09

1.25

0.54

0.57

G7

100.0

40.55

R1

2500

5000

5000

-476

-0.78

0.69

-0.00

-0.03

R2

2500

7500

10000

4524

-0.78

1.10

0.29

0.20

R3

2500

5000

7500

2024

-0.78

0.69

0.41

0.10

Key:      h = Hour, R = Replicate, SD = Standard Deviation, Rep. = Replicate

Note:   Values are total number of cells counted per mL

Yield = Biomass at the end of Exposure period (72 h) – Biomass at the start of the Exposure period

Validity criteria fulfilled:
yes
Remarks:
(cell density in control increased by the factor of 127>16, mean coefficient of variation for section-by-section specific growth rates in control was 6.61%<35%, coefficient of variation of average specific growth rates in control was 0.62%<7%)
Conclusions:
The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 7.51 mg/L (measured, geometric mean), and the EC10 was 1.97 mg/L (measured, geometric mean), based on growth rate.
Executive summary:

A Freshwater Alga Growth Inhibition Test was performed in accordance with OECD Guideline 201, following GLP. The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a static system. Based on the results of a non-GLP preliminary range-finding test, algae cultures were exposed to 0.0 (control), 0.3, 1.0, 3.1, 9.8, 31.3 and 100% (w/v) saturated solutions of the test item (due to low water solubility) or a control. As the test item was found to be volatile, all tests were performed in 250 mL (metric capacity) screw-capped glass bottles with 310 mL of medium to reduce the headspace and thus avoid substance loss from the test media. Furthermore, the algae medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2), and the pH was adjusted to 7.0 by addition of HCl (to maintain the necessary CO2 concentration to support algal growth in the absence of headspace). The test design included three replicates per test concentration and six replicates for the untreated control. Test concentrations were analytically determined at 24-hour intervals by a validated HPLC-UV method. As the deviation from the initial values was greater than 20% from the nominal, measured concentrations are reported: 1.05, 1.05, 1.50, 3.74, 11.43 and 40.55 mg/L test item (geometric mean). A positive control (potassium dichromate) was performed to ensure the quality of the results. All validity criteria were met. The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 7.51 mg/L (measured, geometric mean), and the EC10 was 1.97 mg/L (measured, geometric mean), based on growth rate.

Description of key information

Key study. Method according to OECD 201. GLP study. The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 7.51 mg/L (measured, geometric mean), and the EC10 was 1.97 mg/L (measured, geometric mean), based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.51 mg/L
EC10 or NOEC for freshwater algae:
1.97 mg/L

Additional information

Key study. Freshwater Alga Growth Inhibition Test according to OECD 201, GLP study. Based on the results of a preliminary range-finding test, exponentially growing cultures of unicellular green alga Pseudokirchneriella subcapitata were exposed to 0.0 (control), 0.3, 1.0, 3.1, 9.8, 31.3 and 100% (w/v) saturated solutions of the test item (due to low water solubility) or a control, over an exposure period of 72 hours in a static system. As the test item was found to be volatile, all tests were performed in screw-capped glass bottles and reduced headspace to avoid substance loss from the test media. Furthermore, the test medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2), and pH was adjusted to 7.0 by addition of HCl (to maintain the necessary CO2concentration to support algal growth in the absence of headspace). The test design included three replicates per test concentration and six replicates for the untreated control. Test concentrations were analytically determined at 24-hour intervals by a validated HPLC-UV method. As the deviation from the initial values was greater than 20% from the nominal, results are based on measured concentrations (geometric mean). A positive control (potassium dichromate) was performed to ensure the quality of the results. All validity criteria were met. The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be 7.51 mg/L (measured, geometric mean), and the EC10 was 1.97 mg/L (measured, geometric mean), based on growth rate.