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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:
June - July 2020
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:
March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
6 g NaCl was added to 100 mL of a solution in algal medium (Blank control / 6 / 19.2 mg/L nominal); then, the solution was extracted two times with the dichloromethane (4 mL and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) solution (100 mg/L in dichloromethane) and the solution was measured via GC/MSD.
2 g NaCl was added to 10 mL of a solution in algal medium (60 (0 h) / 192 mg/L nominal); then, the solution was extracted two times with the dichloromethane (4 mL and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) solution (100 mg/L in dichloromethane) and the solution was measured via GC/MSD.
After 72 h, to 20 mL test solution of 60 mg/L 2 g NaCl was added; then, the solution was extracted two times with the dichloromethane (4 mL and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) solution (100 mg/L in dichloromethane) and the solution was measured via GC/MSD.
At the start of the test, 2 g NaCl was added to 10 mL of a solution in algal medium (600 mg/L nominal); then, the solution was extracted two times with the dichloromethane (4 mL and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane. 2 mL of this solution were collected in a 10 mL flask, afterwards, 500 µL ISTD (internal standard) solution (100 mg/L in dichloromethane) was added, the flask was filled up to 10 mL with dichloromethane and the solution was measured via GC/MSD.
At the end of the test, 2 g NaCl was added to 10 mL of a solution in algal medium (600 mg/L nominal); then, the solution was extracted two times with the dichloromethane (4 mL and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane. 5 mL of this solution were collected in a 10 mL flask, afterwards, 500 µL ISTD (internal standard) solution (100 mg/L in di-chloromethane) was added, the flask was filled up to 10 mL with dichloromethane and the solution was measured via GC/MSD.
Vehicle:
no
Details on test solutions:
Based on further pre-tests as already described before, a saturated solution was prepared for the second experiment. This was done by mixing the real load of 599.043 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring vigorously for 25 hours. Afterwards, the solution was centrifuged (10 min, 4500 rpm). The lower treatments (6 / 19.2 / 60 / 192 mg/L nominal) were prepared by dilution of this saturated solution with algal medium.
Positive Control: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test. The results of the last reference study (202007R301) with the positive control K2Cr2O7, performed under GLP conditions in May 2020, are presented here:
72h ErC50: 1.00 mg/L, 95% confidence interval: 0.97 – 1.03 mg/L
72h EyC50: 0.35 mg/L, 95% confidence interval: 0.32 – 0.39 mg/L
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Selection of the test system was made following the proposal of the guidelines.
Specification: Unicellular freshwater green alga, genus Desmodesmus, species subspicatus, SAG Strain Number 86.81, Taxonomic position Chlorophyta - Chlorophyceae
Origin and Culture: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.4 – 22.1 °C
pH:
7.7 - 8.9
Nominal and measured concentrations:
6 / 19.2 / 60 / 192 / 600 mg/L nominal concentration
Details on test conditions:
On each sampling day, validity of calibration was controlled by measuring QC samples (10 mg/L). At the start of the 2nd experiment, the mean recovery rate of the QC sample lay inside the demanded range (101.1 %) and was not taken into account. At the end of the 2nd experiment, the mean recovery rate of the QC sample lay outside the demanded range (104.3 %) and was taken into account.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
19.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Details on results:
Cell Numbers: The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in table F1.
Light Intensity: The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux.
pH values: In the table F2, the pH values measured at the start and the end of the test are stated, ranging from 7.7 to 8.9.
Microscopical Observations: In table F3 the appearance of the algae at the end of the test is stated. Up to a nominal concentration of 192 mg/L algae appeared healthy, whereas at 600 mg/L (nominal) no cell were visible anymore.
Analytical Determination: At the start and at the end of the test, the content of the test item in the test solutions was determined using GC/MSD-Method.
The measured concentrations lay between 7 % and 8 % of the nominal concentrations at the beginning of the test and 8 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see OECD 201 (§ 39)).
The details are given in tables F4, F5 and F6, respectively.
Growth Rate, Yield: From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in table F7.
Inhibition: The mean inhibition values were calculated for the Growth Rate µ and the Yield as shown in table F8.
Results with reference substance (positive control):
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test. The results of the last reference study (202007R301) with the positive control K2Cr2O7, performed under GLP conditions in May 2020, are presented here:
72h ErC50: 1.00 mg/L, 95% confidence interval: 0.97 – 1.03 mg/L
72h EyC50: 0.35 mg/L, 95% confidence interval: 0.32 – 0.39 mg/L
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. The following results were determined in this study:
NOEC (Growth Rate) 72 h: 4.80 mg/L
NOEC (Yield) 72 h: 4.80 mg/L
LOEC (Growth Rate) 72 h: 14.20 mg/L
LOEC (Yield) 72 h: 14.20 mg/L
72h ErC10: 8.03 mg/L, 95 % confidence interval 7.18 mg/L – 8.81 mg/L
72h EyC10: 4.64 mg/L, 95 % confidence interval 3.03 mg/L – 5.90 mg/L
72h ErC50: 19.51 mg/L, 95 % confidence interval 18.47 mg/L – 20.65 mg/L
72h EyC50: 9.04 mg/L, 95 % confidence interval 7.45 mg/L – 10.71 mg/L

Table F1: Cell Number/mL

Nominal Conc. in mg/L

Parameter

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

Mean

2800

13213

59803

399413

SD

0

1620

17668

111827

6

Mean

2800

13173

33380

368240

SD

0

1011

11579

13932

19.2

Mean

2800

12773

76213

508093

SD

0

412

9717

58410

60

Mean

2800

11227

71127

437787

SD

0

1027

17448

148483

192

Mean

2800

8213

22880

79207

SD

0

578

721

4694

600

Mean

2800

4460

4007

4720

SD

0

783

196

1035

SD = Standard Deviation

 

Table F2: pH values

Nominal Concentration in mg/L

0 h

72 h

Blank control

7.7

8.7

6

7.8

8.9

19.2

7.8

8.9

60

7.8

8.5

192

7.8

8.0

600

7.8

7.8

 

Table F3: Microscopical Observations

Nominal Concentration in mg/L

Normal and Healthy Appearance of the Algae

Blank control

Yes

6

Yes

19.2

Yes

60

Yes

192

Yes

600

No cells visible

 

Table F4: Measured Concentrations at 0 h

NominalConc.

Intensity

Measured Conc.

QC recovery and mean accuracy recovery

Dilution factor

Calculated Conc.

Mean Calculated Conc.

mg/L

--

mg/L

%

--

mg/L

mg/L

QC sample (10 µg/L)

50280

37286

98.8

--

10.00

--

48857

36063

99.2

--

10.00

Blank control

101

36094

--

--

0.43

0.43

88

36078

--

--

0.43

6

16635

41044

81.3

0.1

0.42

0.43

15295

35914

81.3

0.1

0.44

19.2

70308

37026

81.3

0.1

1.64

1.61

75751

41385

81.3

0.1

1.59

60

18717

41895

81.3

--

4.60

4.63

17064

37645

81.3

--

4.66

192

65094

42252

81.3

--

13.66

13.70

66898

43180

81.3

--

13.73

600

36531

35448

81.3

5

47.93

47.84

37284

36319

81.3

5

47.76

QC sample (10 µg/L)

50581

35730

103.1

--

10.00

--

53119

37498

103.2

--

10.00

LOQ = 0.1 mg/L

Mean recovery rate of the accuracy of 81.3 % was taken into account. Mean recovery rate of the QC sample lay inside the demanded range (± 3 %) and was not taken into account.

 

Table F5: Measured Concentrations at 72 h

NominalConc.

Intensity

Measured Conc.

QC recovery and mean accuracy recovery

Dilution factor

Calculated Conc.

Mean Calculated Conc.

mg/L

--

mg/L

%

--

mg/L

mg/L

QC sample (10 µg/L)

46760

33145

102.8

--

10.00

--

46910

32817

104.0

--

10.00

Blank control

51

34152

--

--

0.42

0.42

53

32818

--

--

0.42

6

15799

33801

84.8

0.1

0.46

0.46

15731

32872

84.8

0.1

0.47

19.2

66845

34537

84.8

0.1

1.60

1.60

63369

32825

84.8

0.1

1.59

60

38577

34132

84.8

0.5

4.98

5.01

36363

31818

84.8

0.5

5.03

192

57275

32092

84.8

--

14.89

14.75

56943

32591

84.8

--

14.62

600

92029

31278

84.8

2

45.70

45.63

92826

31661

84.8

2

45.57

QC sample (10 µg/L)

43987

30613

104.5

--

10.00

--

45572

31180

106.1

--

10.00

LOQ = 0.1 mg/L

Mean recovery rate of the accuracy of 81.3 % was taken into account. Mean recovery rate of the QC sample lay outside the demanded range (± 3 %) and was taken into ac-count.

 

Table F6: % of Nominal and Geometric Mean

NominalConcentration

% of Nominal

Geometric Mean

mg/L

0 h

72 h

mg/L

Blank control

--

--

--

6

7

8

0.4

19.2

8

8

1.6

60

8

8

4.8

192

7

8

14.2

600

8

8

46.7

 

Table F7: Growth Rate µ, Yield

Nominal Conc. in mg/L

Parameter

Growth Rate (0-72 h)/d

Yield (0-72 h) [Cell Concentration/mL]

Blank control

Mean

1.64

396613

SD

0.09

111827

6

Mean

1.63

365440

SD

0.01

13932

19.2

Mean

1.73

505293

SD

0.04

58410

60

Mean

1.67

434987

SD

0.13

148483

192

Mean

1.11

76407

SD

0.02

4694

600

Mean

0.17

1920

SD

0.07

1035

SD = Standard Deviation

 

Table F8: Inhibition Values

Nominal concentration in mg/L

% Inhibition

Growth Rate (0-72 h)

Yield (0-72 h)

Blank control

0

0

6

1.0

7.9

19.2

- 5.4

- 27.4

60

- 1.6

- 9.7

192

32.2

80.7

600

89.7

99.5

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

Validity criteria fulfilled:
yes
Conclusions:
The test substance was tested according to OECD 201 for toxicity to algae (desmodesmus subspicatus) and an ErC50 (72h) of 19.51 mg/L was determined. The NOEC for yield and growth rate in this study was found being 4.80 mg/L, both.
Executive summary:

Two experiments and one pre-test were performed. The 1st experiment was aborted, because no test item could be measured during the analytical determination at the start of the test. Therefore, an additional pre-test was performed to find out how the substance can best be brought into solution and a second experiment was conducted. The results of the 1st experiment are not stated in the report.

The second experiment was performed using 5 concentrations ranging from 6 to 600 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield (biomass at the end of the exposure period minus the biomass at the start of the exposure period)were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations at 192 and 600 mg/L nominal for growth rate and yield. The test solutions were stirred for 25 hours to guarantee that a sufficient amount of test item has had gone into solution. At the start and at the end of the test, the content of the test item in the test solutions without algae was determined using GC/MSD-Method.

The measured concentrations lay between 7 % and 8 % of the nominal concentrations at the beginning of the test and 8 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see OECD 201 (§ 39)).

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item 1,3-Diiodopropane were determined:

NOEC (Growth Rate) 72 h: 4.80 mg/L

NOEC (Yield) 72 h: 4.80 mg/L

LOEC (Growth Rate) 72 h: 14.20 mg/L

LOEC (Yield) 72 h: 14.20 mg/L

72h ErC10: 8.03 mg/L, 95 % confidence interval 7.18 mg/L – 8.81 mg/L

72h EyC10: 4.64 mg/L*, 95 % confidence interval 3.03 mg/L – 5.90 mg/L

72h ErC50: 19.51 mg/L, 95 % confidence interval 18.47 mg/L – 20.65 mg/L

72h EyC50: 9.04 mg/L, 95 % confidence interval7.45 mg/L – 10.71 mg/L

 

*According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control using a hypothesis test. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.

Description of key information

The test substance was tested according to OECD 201 for toxicity to algae (desmodesmus subspicatus) and an ErC50 (72h) of 19.51 mg/L was determined. The NOEC for yield and growth rate in this study was found being 4.80 mg/L, both.

Key value for chemical safety assessment

EC50 for freshwater algae:
19.51 mg/L
EC10 or NOEC for freshwater algae:
4.8 mg/L

Additional information