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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
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: all the test concentrations were analytical determined

- Sample storage conditions before analysis: sample was analysed immediatly after sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test chemical was dissolved in test medium of required concentrations and observed for any visible particles
- Controls: Test medium control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): No
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): There was no evidence, undissolved test chemical in the exposure medium
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: fresh water algae
- Strain: ATCC® 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux

ACCLIMATION
- Acclimation period: 3 days prior to experiment
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: no deformities observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.2 to 22.6 °C
pH:
7.68 to 7.74
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 150 ml and 250 ml
- Aeration: no aeration during study
- Initial cells density: 10000
- Control end cells density: 79.54*10^4
- No. of organisms per vessel: 15*10^5
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6


GROWTH MEDIUM
- Standard medium used: yes, Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006)


TEST MEDIUM / WATER PARAMETERS
The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. This medium was equilibrated with carbon dioxide by keeping it in laminar hood for two hours. chemicals used to prepare are mentioned in tables section

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: contious light
- Light intensity and quality:7038 to 7100
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell count was done using a haemocytometer (microscopic observation). Algal cells from at least four squares (with each 0.1 mm3 volume) in the center of slide were counted. The arithmetic mean cell count was multiplied with 104 factor to express the cell concentration per m


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: test concentrations were taken as per requirements of guidelines
- Range finding study: yes conducted
- Test concentrations: 0.01, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: alagal biomass was calculated at each concentration to finalise test concentrations in definitive study
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.737 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CI- 1.761-3.9537
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.255 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CI- 0.80749-1.81297
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.835 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 0.53718-1.20606
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.225 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: 95 % CI- 0.2544-1.9892
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.672 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95 % CI 0.13957-1.0913
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.491 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: 95 % CI- 0.10198-0.79734
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormal cells in control
- Unusual cell shape: NO
- Colour differences: NO
- Flocculation: NO
- Adherence to test vessels: NO
- Aggregation of algal cells: NO
Results with reference substance (positive control):
- Results with reference substance valid :Valid The study was conducted using potassium dichromate, for the period of 72 hours, and all the conditions were maintained as per the main study.
- ErC50: 0.6425 (95% C-0.2836-1.3601)
- ErC20: 0.3196 (0.1405- 0.6786)
- ErC10:0.2214 (0.0973-0.4700)
- EyC50: 0.2130(0.1664-0.2735)
- EyC20: 0.0786 (0.0613-0.1008)
- EyC10:0.0466 (0.0364-0.0598)
Reported statistics and error estimates:
The inhibition of growth was determined by calculating the ErC and EyC (the index “r” refers to growth rate and the index “y” refers to yield) by Probit analysis of Finney (1978) using a validated computer program.

TABLE 1.         Range Finding Test - Test Conditions

pH of Test Solutions

 

Group

Treatment (mg/L)

pH

Beginning of the Test

End of the Test

G1

Negative control

7.64

7.65

G2

0.01

7.65

7.67

G3

0.1

7.64

7.66

G4

1

7.66

7.67

G5

10

7.68

7.69

G6

100

7.69

7.71

 

Treatment days

Temperature (°C)

Light intensity (lux)

Average ± % SD

Maximum

Minimum

Average ± % SD

Day 1

22.2

22.5

22.4

±

0.4

6980

6970

6989 ±1.5

7100

7000

Day 2

22.2

22.4

7000

6980

6900

7000

Day 3

22.4

22.6

6950

6900

7150

7050

Day 4

22.5

22.3

6800

7000

7200

6850

 

TABLE 1.         Range Finding Test - Average Cell Biomass

Group

Treatment

(mg/L)

Average cell counts

(× 104cells/mL)

% Inhibition at end of test

0

hour

24 hour

48 hour

72 hour

G1

Negative control

1

6

14.75

72.5

-

G2

0.01

1

6

14.75

72.5

0

G3

0.1

1

6.25

15

73

-0.17*

G4

1

1

4.5

9.75

46.5

10.35

G5

10

1

3.5

6.5

2

83.81

G6

100

1

2.5

1.75

0

100.00

*: to be considered as 0

 

Percent reduction

=

(Mean value of negative control) – (Value for the treatment group)

× 100

Mean[YB1] [c2] value of negative control

Temperature and Light Intensity Measurements of Algal Growth Chamber

Treatment days

Temperature (°C)

Light intensity (lux)

Average ± % SD

Minimum

Maximum

Average ± % SD

Day 1

22.4

22.3

22.4 ± 0.4

7150

7050

7069 ± 1

7050

7100

Day 2

22.4

22.2

7050

7100

7100

7150

Day 3

22.5

22.3

7100

7000

6900

7150

Day 4

22.6

22.4

6950

7100

7050

7100

TABLE 1.         Definitive Test Results - Algal Cell Biomass

Group No.

Test Concentration (mg/L)

Replicate

Average Cell Counts

(x 10^4 cells/mL)

Yield (x 10^4 cells/mL) at 72 hours

0 hour

24 hour

48 hour

72 hour

G1

Negative Control

R1

1

5.75

17.50

79.75

78.75

R2

1

5.75

17.25

79.50

78.50

R3

1

5.75

17.50

80.25

79.25

R4

1

5.75

17.50

81.50

80.50

R5

1

5.75

17.50

81.00

80.00

R6

1

5.75

17.50

81.25

80.25

Average

1

5.75

17.46

80.54

79.54

G2

0.5

R1

1

6.00

17.50

80.75

79.75

R2

1

5.50

17.25

81.25

80.25

R3

1

6.00

17.75

80.75

79.75

Average

1

5.83

17.50

80.92

79.92

G3

1.0

R1

1

4.00

14.75

57.00

56.00

R2

1

4.75

15.50

57.00

56.00

R3

1

5.50

16.00

57.25

56.25

Average

1

4.75

15.42

57.08

56.08

G4

2.0

R1

1

3.75

8.00

10.00

9.00

R2

1

4.25

8.75

10.00

9.00

R3

1

3.50

9.00

9.75

8.75

Average

1

3.83

8.58

9.92

8.92

G5

4.0

R1

1

2.50

4.50

4.00

3.00

R2

1

2.75

3.75

4.25

3.25

R3

1

2.50

3.75

4.25

3.25

Average

1

2.58

4.00

4.17

3.17

G6

8.0

R1

1

2.00

1.25

1.75

0.75

R2

1

2.25

1.25

2.00

1.00

R3

1

1.50

1.50

1.75

0.75

Average

1

1.92

1.33

1.83

0.83

G7

16.0

R1

1

1.00

1.00

1.25

0.25

R2

1

1.00

1.00

1.00

0.00

R3

1

1.00

1.00

1.25

0.25

Average

1

1.00

1.00

1.17

0.17

 

 

 

 


TABLE 2.         Definitive Test Results - Percent Growth Rate Inhibition

Group

Test Concentration (mg/L)

Average Cell Counts

(x 10^4 cells/mL)

Natural log

(average cell counts)

Growth rate

Growth rate Inhibition (%)

0 hours

72 hours

0 hours

72 hours

 

 

G1

Negative control

1

80.54

9.21034

13.59909

0.06095

-

G2

0.5

1

80.92

9.21034

13.6038

0.06102

-0.11*

G3

1.0

1

57.08

9.21034

13.25479

0.05617

7.84

G4

2.0

1

9.92

9.21034

11.50489

0.03187

47.71

G5

4.0

1

4.17

9.21034

10.63826

0.01983

67.47

G6

8.0

1

1.83

9.21034

9.81466

0.00839

86.23

G7

16.0

1

1.17

9.21034

9.36734

0.0022

96.42

*: to be considered as0

 

 

Percent inhibition in average specific growth rate

=

(Mean value for average specific growth rate in the negative control) – (Average specific growth rate for the treatment replicate)

× 100

Mean value for average specific growth rate in the negative control

 


TABLE 3.         Definitive Test Results - Percent Yield Inhibition

Group No.

Test Concentration (mg/L)

Average Cell Counts (x 10^4 cells/mL)

Yield

(x 10^4 cells/mL) at 72 hours

Yield Inhibition (%)

0 hour

72 hours

G1

Negative control

1

80.54

79.54

-

G2

0.5

1

80.92

79.92

-0.47*

G3

1.0

1

57.08

56.08

29.49

G4

2.0

1

9.92

8.92

88.79

G5

4.0

1

4.17

3.17

96.02

G6

8.0

1

1.83

0.83

98.95

G7

16.0

1

1.17

0.17

99.79

*: to be considered as 0

 

Percent inhibition in yield

=

(Mean value for yield in the negative control) – (Value for yield for the treatment replicate)

× 100

Mean value for yield in the negative control


TABLE 4.         Definitive Test- Specific Growth Rate: Negative Control

Replicate

Specific Growth rate

Replicate-mean specific growth rate

S.D.

% CV

 

0 h- 24 h

24 h - 48 h

48 h - 72 h

 

R1

1.75

1.11

1.52

1.46

0.32

22.20

 

R2

1.75

1.10

1.53

1.46

0.33

22.64

 

R3

1.75

1.11

1.53

1.46

0.33

22.22

 

R4

1.75

1.11

1.54

1.47

0.33

22.24

 

R5

1.75

1.11

1.53

1.46

0.33

22.22

 

R6

1.75

1.11

1.54

1.47

0.33

22.24

 

Mean ± SD (% CV)

1.75 ± 0 (0)

1.11 ± 0 (0.37)

1.53 ± 0.01 (0.49)

1.46 ± 0 (0.2)

-

22.30

 

 

Mean coefficient of variation (% CV) for section by section specific growth rates

22.30

The coefficient of variation (% CV) of average specific growth rates during the whole test period in replicate control cultures

0.20

 

 


TABLE 5.         Definitive Test- Specific Growth Rate: Treatment Group

Group

Test Concentration (mg/L)

Replicate

Specific Growth Rate

0 h - 24 h

24 h – 48 h

48 h - 72 h

G2

 

 

 

 

 

0.5

R1

1.79

1.07

1.53

R2

1.71

1.14

1.55

R3

1.79

1.09

1.51

Average

1.76

1.10

1.53

SD

0.05

0.04

0.02

% CV

2.62

3.28

1.31

G3

 

 

 

 

 

1.0

R1

1.39

1.30

1.35

R2

1.56

1.18

1.30

R3

1.71

1.06

1.28

Average

1.55

1.18

1.31

SD

0.16

0.12

0.04

% CV

10.31

10.17

2.75

G4

 

 

 

 

 

2.0

R1

1.32

0.76

0.22

R2

1.45

0.72

0.13

R3

1.25

0.95

0.08

Average

1.34

0.81

0.14

SD

0.10

0.12

0.07

% CV

7.57

15.17

49.50

G5

 

 

 

 

 

4.0

R1

0.92

0.58

-0.11

R2

1.01

0.31

0.13

R3

0.92

0.40

0.13

Average

0.95

0.43

0.05

SD

0.05

0.14

0.14

% CV

5.47

31.97

277.13

G6

 

 

 

 

 

8.0

R1

0.69

-0.47

0.34

R2

0.81

-0.59

0.47

R3

0.41

0

0.15

Average

0.64

-0.35

0.32

SD

0.21

0.31

0.16

% CV

32.24

-88.25

50.29

G7

 

 

 

 

 

16.0

R1

0

0

0.22

R2

0

0

0

R3

0

0

0.22

Average

0

0

0.15

SD

0

0

0.13

% CV

-

-

86.60

 

Validity criteria fulfilled:
yes
Remarks:
An increase in cell concentration in control by a factor of 79.54 The mean CoV for section-by-section specific growth ratesin in control during the test was 22.30 %, The CoV of average growth rate between replicate cultures of negative control was 0.20%
Conclusions:
The median effective concentration of test chemical to test organism P. Subcapitata based on growth rate and yeild was evaluated to be
At 72 hours EC values in mg/L (fiducial limits)
ErC50 2.737 (1.761-3.9537)
ErC20 1.255 (0.80749-1.81297)
ErC10 0.8349 (0.53718-1.20606)
EyC50 1.225 (0.2544-1.9892)
EyC20 0.6721 (0.13957-1.0913)
EyC10 0.491 (0.10198-0.79734)
LOEC 1.0
NOEC 0.5
Executive summary:

The test chemical was studied for its effects on fresh water algae P. Subcapitata by following OECD 201 test guidelines. The test organism was obtained from ATCC (American type culture collection) 22662, which was recommended as per guideliners. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux.   Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006) The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculatingRaphidocelis subcapitataof continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 217.25 x 104/mL during range finding and 231 x 104/mL during definitive test. All test conditions during range finding and definitive test was similar such as exposure period (72h), test containers (250 ml conical flask), pH(7.64 -7.74), temperature (22.2 -22.6°C), continues light intensity (6963 -7100 lux) shaking of incubator (150 rpm). Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L, each single replicateTest item exhibited 0, 0, 10.35, 83.81 and 100 % Inhibition at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h, respectively. based on the results obtained theDefinitive test was carried out using 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (factor of 2.0) along with a negative control. At test concentration of the test concentrationsof0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L, the observed inhibition in the percent growth rate of algal biomass was 0, 7.84, 47.71, 67.47, 86.23 and 96.42 %; and the percent yield inhibition was 0, 29.49, 88.79, 96.02, 98.95 and 99.79 %, respectively when compared to that of the negative control during the test period at 72 h

The end point values for growth rate and yield 72 hours are presented below based on nominal concentration.

 

At 72 hours

EC values in mg/L (fiducial limits)

ErC50

2.737 (1.761-3.9537)

ErC20

1.255 (0.80749-1.81297)

ErC10

0.8349 (0.53718-1.20606)

EyC50

1.225 (0.2544-1.9892)

EyC20

0.6721 (0.13957-1.0913)

EyC10

0.491 (0.10198-0.79734)

LOEC

1.0

NOEC

0.5

The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 103.47 to 117.02 %(RSD was 2.27 to 5.88 %)at the start and 93.03 to 114.59 %(RSD was 0.29 to 4.69 %)at the end of the test (72 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%).No measurable concentration of test item was recorded in the negative control samples.

Based on outcomes on median effective concentration, test chemical cannot be classified as per CLP classification criteria, since chemical is readily biodegradable

Description of key information

Short term toxicity to algae:

The test chemical was studied for its effects on fresh water algae P. Subcapitata by following OECD 201 test guidelines. The test organism was obtained from ATCC (American type culture collection) 22662, which was recommended as per guideliners. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux.  Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006) The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculatingRaphidocelis subcapitataof continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 217.25 x 104/mL during range finding and 231 x 104/mL during definitive test. All test conditions during range finding and definitive test was similar such as exposure period (72h), test containers (250 ml conical flask), pH(7.64 -7.74), temperature (22.2 -22.6°C), continues light intensity (6963 -7100 lux) shaking of incubator (150 rpm). Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L, each single replicateTest item exhibited 0, 0, 10.35, 83.81 and 100 % Inhibition at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h, respectively. based on the results obtained theDefinitive test was carried out using 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (factor of 2.0) along with a negative control. At test concentration of the test concentrationsof0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L, the observed inhibition in the percent growth rate of algal biomass was 0, 7.84, 47.71, 67.47, 86.23 and 96.42 %; and the percent yield inhibition was 0, 29.49, 88.79, 96.02, 98.95 and 99.79 %, respectively when compared to that of the negative control during the test period at 72 h

The end point values for growth rate and yield 72 hours are presented below based on nominal concentration.

 

At 72 hours

EC values in mg/L (fiducial limits)

ErC50

2.737 (1.761-3.9537)

ErC20

1.255 (0.80749-1.81297)

ErC10

0.8349 (0.53718-1.20606)

EyC50

1.225 (0.2544-1.9892)

EyC20

0.6721 (0.13957-1.0913)

EyC10

0.491 (0.10198-0.79734)

LOEC

1.0

NOEC

0.5

The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 103.47 to 117.02 %(RSD was 2.27 to 5.88 %)at the start and 93.03 to 114.59 %(RSD was 0.29 to 4.69 %)at the end of the test (72 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%).No measurable concentration of test item was recorded in the negative control samples.

Based on outcomes on median effective concentration, test chemical cannot be classified as per CLP classification criteria, since chemical is readily biodegradable

Key value for chemical safety assessment

EC50 for freshwater algae:
2.737 mg/L
EC10 or NOEC for freshwater algae:
0.835 mg/L

Additional information

The test chemical was studied for its effects on fresh water algae P. Subcapitata by following OECD 201 test guidelines. The test organism was obtained from ATCC (American type culture collection) 22662, which was recommended as per guideliners. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux.  Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006) The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculatingRaphidocelis subcapitataof continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 217.25 x 104/mL during range finding and 231 x 104/mL during definitive test. All test conditions during range finding and definitive test was similar such as exposure period (72h), test containers (250 ml conical flask), pH(7.64 -7.74), temperature (22.2 -22.6°C), continues light intensity (6963 -7100 lux) shaking of incubator (150 rpm). Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L, each single replicateTest item exhibited 0, 0, 10.35, 83.81 and 100 % Inhibition at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h, respectively. based on the results obtained theDefinitive test was carried out using 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (factor of 2.0) along with a negative control. At test concentration of the test concentrationsof0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L, the observed inhibition in the percent growth rate of algal biomass was 0, 7.84, 47.71, 67.47, 86.23 and 96.42 %; and the percent yield inhibition was 0, 29.49, 88.79, 96.02, 98.95 and 99.79 %, respectively when compared to that of the negative control during the test period at 72 h

The end point values for growth rate and yield 72 hours are presented below based on nominal concentration.

 

At 72 hours

EC values in mg/L (fiducial limits)

ErC50

2.737 (1.761-3.9537)

ErC20

1.255 (0.80749-1.81297)

ErC10

0.8349 (0.53718-1.20606)

EyC50

1.225 (0.2544-1.9892)

EyC20

0.6721 (0.13957-1.0913)

EyC10

0.491 (0.10198-0.79734)

LOEC

1.0

NOEC

0.5

The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 103.47 to 117.02 %(RSD was 2.27 to 5.88 %)at the start and 93.03 to 114.59 %(RSD was 0.29 to 4.69 %)at the end of the test (72 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%).No measurable concentration of test item was recorded in the negative control samples.

Based on outcomes on median effective concentration, test chemical cannot be classified as per CLP classification criteria, since chemical is readily biodegradable.