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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:
From October 23, 2017 to October 27, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
Gas chromatography mass spectrometry (GCMS) method
Details on sampling:
To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, whole test vessels were analysed test substance using the gas chromatography mass spectrometry method. At the start of the test additional sacrificial vessels with dispensed test solution were taken for whole sample analysis and at the end of the test, replicate vessels of the dilution water control and each test concentration was taken for whole sample analysis.
Vehicle:
yes
Remarks:
AAP medium
Details on test solutions:
Preparation of test solutions
The study was run with a culture medium control and nominal exposure concentrations of 0.004, 0.012, 0.037, 0.117, 0.375 and 1.2 mg/L. A primary stock concentrate of Test substance, with a nominal concentration of 12 mg/L, was prepared by adding a nominal 0.012 g of test substance (actual weight: 0.01197) to 1000 mL of media. The resultant stock was observed to be clear and colourless. The stock solution was used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of concentrate to dilution water. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The test solutions were all observed to be clear and colourless. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3 d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium and under the environmental conditions described for the test. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.
Test type:
not specified
Water media type:
other: AAP-medium
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 ± 2ºC
pH:
7.16 to 7.31
Nominal and measured concentrations:
A 1.2 mg/L test concentration was selected as the highest concentration based on the toxicity demonstrated in a non-GLP range-finding study.
0, 0.004, 0.012, 0.037, 0.117, 0.375 and 1.2 mg/L (Nominal)
0, 0, 0, 0, 0.059, 0.18 and 0.77 mg/L (Mean measured)
Details on test conditions:
Appratus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
ca. 0.14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence limits: 0.037 – 0.27
Remarks:
0.134 mg/L a.i.
Key result
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
ca. 0.097 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Remarks on result:
other: 95% Confidence limits - 0.059 - 0.089
Remarks:
0.0931 mg/L a.i.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.059 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 0.0566 mg/L a.i.
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 0.173 mg/L a.i.
Key result
Duration:
72 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Remarks on result:
other:
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.059 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Remarks on result:
other: 0.0566 mg/L a.i.
Key result
Duration:
72 h
Dose descriptor:
other: ErC10
Effect conc.:
ca. 0.064 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EyC10
Effect conc.:
ca. 0.021 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass

Results:

Analytical data

The limit of quantification of test substance in this study was 0.02 mg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 0 -66% of nominal and at the end were 0 -62% of nominal. The nominal 0.004, 0.012 and 0.037 mg/L test solutions were below the LOQ and as such were not detected at either time point. These concentrations were below the NOEC for growth rate and as such not having a measured analytical value does not impact the result of the study. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 30% and less than 20% at levels greater than the LOQ. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass. The algal cell particle densities measured at each time period are given in below table:

Nominal

concentration of Test substance

(mg/L)

Mean measured concentration of
Test substance

(mg/L)

Replicate

Algal cell particle density

(104cells/mL)

24 h

48 h

72 h

Culture medium control

0

A

2.35

9.81

39.80

B

2.56

10.10

44.00

C

2.70

11.40

43.00

D

2.74

10.50

42.50

E

2.64

10.20

40.30

F

2.43

11.10

48.10

Mean

2.57

10.52

42.95

0.004

0

A

2.26

9.42

41.80

B

2.61

9.63

40.30

C

2.55

10.80

46.80

Mean

2.47

9.95

42.97

0.012

0

A

2.62

10.20

43.30

B

2.63

10.30

43.60

C

2.49

10.10

40.10

Mean

2.58

10.20

42.33

0.037

0

A

2.64

11.30

43.10

B

2.81

11.00

43.90

C

2.36

9.60

38.40

Mean

2.60

10.63

41.80

0.117

0.059

A

2.94

7.58

30.90

B

2.24

10.50

30.20

C

2.51

8.63

32.00

Mean

2.56

8.90

31.03

0.375

0.18

A

0.52

0.93

2.73

B

0.92

1.42

1.03

C

0.61

1.17

1.46

Mean

0.68

1.17

1.74

1.2

0.77

A

0.01

0 (-0.08)

0.19

B

0.00

0 (-0.03)

0 (-0.07)

C

0.04

0 (-0.03)

0 (-0.16)

Mean

0.02

0 (-0.05)

0.06 (-0.01)

Inoculum (Day 0) cell density = 0.533 x 104 cells/mL. All values are quoted to 2 decimal places. Where negative algal yields (shown in parentheses) were calculated due to the subtracted blank particle density being higher than the test vessel particle density, these have been taken as zero (0) for calculation of the mean and statistical analysis.

Growth rates

The growth rate (0 to 72 h) was calculated for each replicate culture.

Nominal concentration of Test substance

(mg/L)

Mean measured concentration of Test substance

(mg/L)

Replicate

0-72 h
Growth rate

(104cells/mL)

Culture medium control

0

A

1.437

B

1.471

C

1.463

D

1.459

E

1.442

F

1.500

Mean

1.462

0.004

0

A

1.454

B

1.442

C

1.491

Mean

1.462

0.012

0

A

1.466

B

1.468

C

1.440

Mean

1.458

0.037

0

A

1.464

B

1.470

C

1.425

Mean

1.453

0.117

0.059

A

1.353

B

1.345

C

1.365

Mean

1.354

0.375

0.18

A

0.545

B

0.218

C

0.335

Mean

0.366

1.2

0.77

A

-a

B

-a

C

-a

Mean

-a

All values are quoted to 3 decimal places.

a - In the nominal 1.2 mg/L test concentration, the growth rates (0-72 h) were not calculable as no cell particles were counted at 72 h (negative cell particle densities were taken as zero).

The growth rates were examined by one-way analysis of variance and t-tests to identify significant differences (p <0.05) from the control. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The mean growth rates, are given in below table, together with the rates expressed as percentages of the control.

Nominal concentration of Test substance

(mg/L)

Mean measured concentration of Test substance

(mg/L)

Mean growth rate/day

(0-72 h)

Mean growth rate/day

95% Cl

Percentage ofcontrol (%)

Culture medium control

0

1.462

1.438-1.486

-

0.004

0

1.462

1.398-1.526

100

0.012

0

1.458

1.419-1.497

100

0.037

0

1.453

1.393-1.513

99

0.117

0.059

1.354

1.329-1.379

93

0.375

0.18

0.366 *

0-0.777

25

1.2

0.77

-a*

-a

-a

* Significant differences (p <0.05) from the control. All biological measurements are quoted to 3 decimal places and percentages to the nearest integer.

a - In the nominal 1.2 mg/L test concentration, the growth rate (0-72 h) was not calculable as no cell particles were counted at 72 h (negative cell particle densities were taken as zero).

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

 

Test substance

(mg/L)

95% confidence limits

NOEC

0.059

-

LOEC

0.18

-

ErC50

0.14

0.12-0.17

ErC20

0.082

0.075-0.091

ErC10

0.064

0.059-0.069

Yield

This response was defined as the biomass at the end of the test minus the starting biomass.

Nominal concentration of Test substance

(mg/L)

Mean measured concentration of Test substance

(mg/L)

Replicate

Yield

(104 cells/mL)

24 h

48 h

72 h

Culture medium control

0

A

1.82

9.28

39.27

B

2.03

9.57

43.47

C

2.17

10.87

42.47

D

2.21

9.97

41.97

E

2.11

9.67

39.77

F

1.90

10.57

47.57

Mean

2.04

10.00

42.4

0.004

0

A

1.73

8.89

41.27

B

2.08

9.10

39.77

C

2.02

10.27

46.27

Mean

1.94

9.42

42.44

0.012

0

A

2.09

9.67

42.77

B

2.10

9.77

43.07

C

1.96

9.57

39.57

Mean

2.05

9.67

41.80

0.037

0

A

2.11

10.77

42.57

B

2.28

10.47

43.37

C

1.83

9.07

37.87

Mean

2.07

10.10

41.27

0.117

0.059

A

2.41

7.05

30.37

B

1.71

9.97

29.67

C

1.98

8.10

31.47

Mean

2.03

8.37

30.50

0.375

0.18

A

0 (-0.01)

0.40

2.20

B

0.39

0.89

0.50

C

0.08

0.64

0.93

Mean

0.16 (0.15)

0.64

1.21

1.2

0.77

A

0.15

0.64

1.21

B

0 (-0.52)

0 (-0.61)

0 (-0.34)

C

0 (-0.53)

0 (-0.56)

0 (-0.60)

Mean

0.05

(-0.30)

0.21

(-0.18)

0.40 (0.09)

Yield values are quoted to 2 decimal places. Where negative algal yields (shown in parentheses) were calculated due to the subtracted blank particle density being higher than the test vessel particle density, these have been taken as zero (0) for calculation of the mean and statistical analysis.

For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by one-way analysis of variance and a t test to identify significant differences (p<0.05) from the control.The EC50 , EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The yield mean values are shown in below table , together with the yields expressed as percentages of the control.

Nominal concentration of Test substance

(mg/L)

Mean measured concentration of Test substance

(mg/L)

Mean yield

(0-72 h)

(104cells/ml)

Percentage of control (%)

Culture medium control

0

42.4

-

0.004

0

42.4

100

0.012

0

41.8

99

0.037

0

41.3

97

0.117

0.059

30.5*

72

0.375

0.18

1.21*

3

1.2

0.77

0.403*

1

* Significant differences (p <0.05) from the control. Mean yield values are quoted to 3 significant figures and percentages to the nearest integer.

The ECxcalculations were based on mean measured test concentrations and the comparison analysis to determine the NOEC and LOEC used nominal and mean measured test concentrations. The results obtained from these statistical analysis, were as follows:

 

Test substance

(mg/L)

95% confidence limits

NOEC

0 (0.037 mg/L nominal value)

-

LOEC

0.059

-

EyC50

0.097

0.087-0.11

EyC20

0.042

0.031-0.057

EyC10

0.021

0.016-0.028

Additional biological data

The microscopic observations, made at test end, showed that for the control and 0.04, 0.012, 0.037 and 0.117 mg/L test concentrations, the cells appeared normal. No cells were visible in the 0.375 and 1.2 mg/L test vessels.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test,cell particle density increase (measured as a surrogate for biomass) was 80.68 over the 72 h for the control.

2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 7%.

3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and in this test, was calculated to be 1.6%.

Based on the study results, it was concluded that the study has fulfilled validity criteria.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOErC, NOEyC, LOErC and LOEyC values for the test substance, 'iso and anteiso C10-40 AAP EDM-ES' with freshwater green algae, were determined to be 0.14 (0.134 mg a.i./L), 0.064 (0.0614 mg a.i./L) 0.097 (0.0931 mg a.i./L), 0.021 (0.02 mg a.i./L), 0.059 (0.0566 mg a.i./L), 0, 0.18 (0.173 mg a.i./L) and 0.059 (0.0566 mg a.i./L), respectively.


Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, 'iso and anteiso C10-40 AAP EDM-ES' (active: 96%), to freshwater green algae (Pseudokirchneriella subcapitata), according to the OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 1.091 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.533 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.004, 0.012, 0.037, 0.117, 0.375 and 1.2 mg/L in test medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a GC-MS method of analysis. Based on whole sample analysis (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0, 0, 0.059, 0.18 and 0.77 mg/L. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The ErC50 and ErC10 values for growth rate were determined to be 0.14 and 0.064 mg/L (i.e., equivalent to 0.134 and 0.0614 mg a.i./L) respectively, whereas the EyC50 and EyC10 values for cell particle density were determined to be 0.097 and 0.021mg/L (i.e., 0.0931 and 0.02 mg a.i./L) respectively. The respective NOEC and LOEC values were determined to 0.059 (0.0566 a.i.) mg/L and 0.18 (0.173 a.i.) mg/L, respectively for growth rate and 0 mg/L (0.037 mg/L; nominal) and 0.059 mg/L (0.0566 mg a.i./L), respectively for cell particle densities. The study results were considered to fulfil the OECD Guideline 201 validity criteria. Therefore, under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, NOErC, NOEyC, LOErC and LOEyC values for the test substance, 'iso and anteiso C10-40 AAP EDM-ES' with freshwater green algae, were determined to be 0.14 (0.134 mg a.i./L), 0.064 (0.0614 mg a.i./L) 0.097 (0.0931 mg a.i./L), 0.021 (0.02 mg a.i./L), 0.059 (0.0566 mg a.i./L), 0, 0.18 (0.173 mg a.i./L) and 0.059 (0.0566 mg a.i./L), respectively (Scymaris, 2018).

Description of key information

Based on the results of the study, the 72 h ErC50, ErC10, EyC50, EyC10, NOErC, NOEyC, LOErC and LOEyC values for the test substance with freshwater green algae, were determined to be 0.14 (0.134 mg a.i./L), 0.064 (0.0614 mg a.i./L) 0.097 (0.0931 mg a.i./L), 0.021 (0.02 mg a.i./L), 0.059 (0.0566 mg a.i./L), 0, 0.18 (0.173 mg a.i./L) and 0.059 (0.0566 mg a.i./L), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.134 mg/L
EC10 or NOEC for freshwater algae:
0.061 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, 'iso and anteiso C10-40 AAP EDM-ES' (active: 96%), to freshwater green algae (Pseudokirchneriella subcapitata), according to the OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 1.091 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.533 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.004, 0.012, 0.037, 0.117, 0.375 and 1.2 mg/L in test medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a GC-MS method of analysis. Based on whole sample analysis (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0, 0, 0.059, 0.18 and 0.77 mg/L. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The ErC50 and ErC10 values for growth rate were determined to be 0.14 and 0.064 mg/L (i.e., equivalent to 0.134 and 0.0614 mg a.i./L) respectively, whereas the EyC50 and EyC10 values for cell particle density were determined to be 0.097 and 0.021mg/L (i.e., 0.0931 and 0.02 mg a.i./L) respectively. The respective NOEC and LOEC values were determined to 0.059 (0.0566 a.i.) mg/L and 0.18 (0.173 a.i.) mg/L, respectively for growth rate and 0 mg/L (0.037 mg/L; nominal) and 0.059 mg/L (0.0566 mg a.i./L), respectively for cell particle densities. The study results were considered to fulfil the OECD Guideline 201 validity criteria. Therefore, under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, NOErC, NOEyC, LOErC and LOEyC values for the test substance, 'iso and anteiso C10-40 AAP EDM-ES' with freshwater green algae, were determined to be 0.14 (0.134 mg a.i./L), 0.064 (0.0614 mg a.i./L) 0.097 (0.0931 mg a.i./L), 0.021 (0.02 mg a.i./L), 0.059 (0.0566 mg a.i./L), 0, 0.18 (0.173 mg a.i./L) and 0.059 (0.0566 mg a.i./L), respectively (Scymaris, 2018).