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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 02-29, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Test Item name: Yellow LF 6911
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Purity: 93.3 % (organic part excluding water and salts not containing similar chromophore)
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiry date: 12 December 2018
Storage: Room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test. Concentrations were determined using reverse phase HPLC method with VIS detection.

Test solutions

Vehicle:
no
Details on test solutions:
The test solutions used in the test were prepared by mechanical dispersion. A stock solution was first prepared by dissolving an amount of 0.08 g test item in 800 mL dilution water (OECD medium) resulting a nominal concentration of 100 mg/L. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution (see Table 2). The test solutions were prepared just before introduction of algae (start of the experiment).

Preparation of the test solutions:
Nominal concentration [mg/L] Stock Solution [mL] OECD Medium [mL]
100 400 –
50 200 q.s. ad 400
25 100 q.s. ad 400
12.5 50 q.s. ad 400
6.25 25 q.s. ad 400

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: 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 about four days. (The preculture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted algal growth medium (OECD medium, according to OECD 201)
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Test temperature:
The temperature was between 21.7 and 21.9°C measured in the flask and in the range of
21.6 – 22.5°C measured within the climate chamber.
pH:
The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.57 – 8.42 during the experiment.
Dissolved oxygen:
N/A
Salinity:
Stock solution Substance Final concentration in the prepared growth medium
NH4Cl 1 5.0 mg/L
Stock solution 1 MgCl2 5.6 mg/L*
(macro nutrients) CaCl2 × 2 H2O 18.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L

Stock solution 2 FeCl3 × 6 H2O 64.0 µg/L
(iron) Na2EDTA × 2H2O 100.0 µg/L

Stock solution 3
(trace elements) H3BO3 185.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 × 6 H2O 1.5 µg/L
CuCl2 × 2 H2O 0.01 µg/L
Na2MoO4 × 2 H2O 7.0 µg/L


Stock solution 4
(bicarbonate) NaHCO3 50.0 mg/L
* This corresponds to the concentration of 12.0 mg/L of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)
Conductivity:
N/A
Nominal and measured concentrations:
Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control were tested in the main experiment. The nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of Yellow LF 6911 varied between 93 and 107 % of the nominal concentration (i.e. within ± 20 %) during the experiment therefore the biological results are based on the nominal concentrations. In the untreated control group the test item was not detected.
Details on test conditions:
The test was performed with three replicates per test concentration and six replicates in the control group. The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test item solution. The initial cell density was about 104 cells/mL, in each test flask. Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks. The test flasks were covered with air-permeable stoppers. The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
>= 52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 15.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 6.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
>= 37.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 6.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Validity of the Test
The cell density in the control cultures increased by a factor of 60.83 within 72 hours. This corresponds to a specific growth rate of 1.37 day-1.
The mean coefficient of variation (CV) 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: 20.51 % - CV for section-by-section growth rate day 1-2: 13.22 % - CV for
section-by-section growth rate day 2-3: 5.28 % The mean coefficient of variation for section-by-section specific growth rates: 13.01 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.56 %.
All validity criteria were met, therefore the study can be considered as valid.

Analytical Results
Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control were tested in the main experiment.
The nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of Yellow LF 6911 varied between 93
and 107 % of the nominal concentration (i.e. within ± 20 %) during the experiment therefore the biological results are based on the nominal concentrations.
In the untreated control group the test item was not detected.

Biological Results
The influence of the test item Yellow LF 6911 on the growth of Raphidocelis subcapitata is shown in Tables 1, 5-12 and Figures 1-3.
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 by Bonferroni t-Test using TOXSTAT software. After the exposure period of 72 hours average specific growth rates and yield
were significantly different from the control group in the concentrations of 12.5, 25, 50 and 100 mg/L (see Tables 9 and 10). The test concentration
of 6.25 mg/L was determined as the 72-hour NOEC related to the growth rate and yield.
The 72-h ErC50 was determined to be > 100 mg/L (calculated as 500.6 mg/L). The 72-h EyC50 was determined to be 37.5 mg/L.

Microscopic Examinations
Shape of the algal cells No abnormal shape of algal cells was noticed in the control and in the concentrations of 6.25, 12.5, 25 and 50 mg/L during
the whole exposure period. : Thin and swollen cells were observed in the highest concentration of 100 mg/L during the test.

Cell density in control In the control the cell density has increased from nominal N= 1 × 104 cells/mL at the start of the test (0 hours) to
N= 60.83 × 104 cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year. The date of the last study (Study number: 392-201-1333) with the reference item Potassium dichromate was: 01 – 04 March 2016. The 72h ErC50: 0.70 mg/L, (95 % confidence limits: 0.64 – 0.78 mg/L) The 72h EyC50: 0.39 mg/L, (95 % confidence limits: 0.36 – 0.43 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations were calculated for each concentration at each observation period using Excel for Windows 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 by Bonferroni t-Test (α = 0.05; 2-Tailed) using TOXSTAT software. The data were checked for normality by Chi-Square Test,
for homogeneity of variance by Hartley’s and Bartlett’s Test. For determination of the ErCx and EyCx values with 95 % confidence limits,
Probit analysis was performed using TOXSTAT software.

Any other information on results incl. tables

Analytical results:

Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control were tested in the main experiment. The nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of Yellow LF 6911 varied between 93 and 107 % of the nominal concentration (i.e. within ± 20 %) during the experiment therefore the biological results are based on the nominal concentrations. In the untreated control group the test item was not detected.

Biological results

Algal Cell Concentrations (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

Control

 

1

3

12

63

62

 

1

2

10

54

53

 

1

4

12

62

61

 

1

3

11

64

63

 

1

3

12

59

58

 

1

3

10

63

62

Mean value

1.00

3.00

11.17

60.83

59.83

SD

0.00

0.63

0.98

3.76

3.76

6.25

 

1

3

11

49

48

 

1

3

11

58

57

 

1

3

10

57

56

Mean value

1.00

3.00

10.67

54.67

53.67

SD

0.00

0.00

0.58

4.93

4.93

12.5

 

1

3

10

39

38

 

1

3

11

40

39

 

1

3

11

39

38

Mean value

1.00

3.00

10.67

39.33

38.33

SD

0.00

0.00

0.58

0.58

0.58

25

 

1

3

9

39

38

 

1

3

10

30

29

 

1

3

9

31

30

Mean value

1.00

3.00

9.33

33.33

32.33

SD

0.00

0.00

0.58

4.93

4.93

50

 

1

2

9

32

31

 

1

3

10

27

26

 

1

2

8

29

28

Mean value

1.00

2.33

9.00

29.33

28.33

SD

0.00

0.58

1.00

2.52

2.52

100

 

1

2

6

21

20

 

1

1

7

19

18

 

1

1

6

20

19

Mean value

1.00

1.33

6.33

20.00

19.00

SD

0.00

0.58

0.58

1.00

1.00

The Specific Growth Rates during the Test Period of 72 Hours

Concentration[mg/L]

 

Growth Rates (µ)

0 – 24 h

0 – 48 h

0 – 72 h

Control

 

0.0458

0.0518

0.0575

 

0.0289

0.0480

0.0554

 

0.0578

0.0518

0.0573

 

0.0458

0.0500

0.0578

 

0.0458

0.0518

0.0566

 

0.0458

0.0480

0.0575

Mean

0.0450

0.0502

0.0570

SD

0.0092

0.0019

0.0009

6.25

 

0.0458

0.0500

0.0541

 

0.0458

0.0500

0.0564

 

0.0458

0.0480

0.0562

Mean value

0.0458

0.0493

0.0555

SD

0.0000

0.0011

0.0013

12.5

 

0.0458

0.0480

0.0509

 

0.0458

0.0500

0.0512

 

0.0458

0.0500

0.0509

Mean value

0.0458

0.0493

0.0510

SD

0.0000

0.0011

0.0002

25

 

0.0458

0.0458

0.0509

 

0.0458

0.0480

0.0472

 

0.0458

0.0458

0.0477

Mean value

0.0458

0.0465

0.0486

SD

0.0000

0.0013

0.0020

50

 

0.0289

0.0458

0.0481

 

0.0458

0.0480

0.0458

 

0.0289

0.0433

0.0468

Mean value

0.0345

0.0457

0.0469

SD

0.0098

0.0023

0.0012

100

 

0.0289

0.0373

0.0423

 

0.0000

0.0405

0.0409

 

0.0000

0.0373

0.0416

Mean value

0.0096

0.0384

0.0416

SD

0.0167

0.0019

0.0007

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

%

Control

0.0450

0.0

0.0502

0.0

0.0570

0.0

6.25

0.0458

-1.8

0.0493

1.8

0.0555

2.6

12.5

0.0458

-1.8

0.0493

1.8

0.0510

10.6

25

0.0458

-1.8

0.0465

7.4

0.0486

14.8

50

0.0345

23.2

0.0457

9.0

0.0469

17.8

100

0.0096

78.6

0.0384

23.5

0.0416

27.1

 

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration[mg/L]

Yield y and % Inhibition of y

0–72 h

y

%

Control

59.83

0.0

6.25

53.67

10.3

12.5

38.33

35.9

25

32.33

46.0

50

28.33

52.6

100

19.00

68.2

Results of the statistical evaluation based on the growth rates (Bonferroni t-Test)

Concentration[mg/L]

Growth Rates(0–72 h)

Calculated t-value

Difference from Control

Significance

6.25

1.8242

0.0014

n.s.

12.5

7.5693

0.0060

*

25

10.5886

0.0084

*

50

12.7273

0.0101

*

100

19.3950

0.0154

*

Minimal significant difference

0.0023

Percentage of control

4.1

*:     statistically significantly different from the control(Bonferroni t-Test; 2 Tailed;a= 0.05; df = 5.15)

n.s.: statistically not significantly different from the control (Bonferroni t-Test; 2 Tailed;a= 0.05; df = 5.15)

 

 

Results of the statistical evaluation based on the Yield (Bonferroni t-Test)

Concentration[mg/L]

Growth Rates(0–72 h)

Calculated t-value

Difference from Control

Significance

6.25

2.4934

6.1667

n.s.

12.5

8.6932

21.5000

*

25

11.1192

27.5000

*

50

12.7366

31.5000

*

100

16.5104

40.8333

*

Minimal significant difference

7.2878

Percentage of control

12.2

*:     statistically significantly different from the control (Bonferroni t-Test; 2 Tailed;a= 0.05; df = 5.15)

n.s.: statistically not significantly different from the control (Bonferroni t-Test; 2 Tailed;a= 0.05; df = 5.15)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Parameter (0-72 h) Growth rate (µ) [mg/L] Yield (y) [mg/L]
Calculation based on measured concentrations
EC10 15.9 3.4
95 % conf. limits 9.9 – 25.8 2.0 – 5.8
EC20 52.0 7.7
95 % conf. limits 34.4 – 78.8 5.4 – 11.1
EC50 > 100 (500.6 calculated) 37.5
95 % conf. limits > 100 (157.5 – 1590.7 calculated) 29.8 – 47.1
NOEC 6.25 6.25
LOEC 12.5 12.5

Executive summary:

Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals.

EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield).

Parameter (0-72 h)              Growth rate (µ) [mg/L]                                   Yield (y) [mg/L]

Calculation based on measured concentrations

EC10                                                 15.9                                                        3.4

95 % conf. limits                                   9.9 – 25.8                                          2.0 – 5.8

EC20                                                  52.0                                                        7.7

95 % conf. limits                                   34.4 – 78.8                                          5.4 – 11.1

EC50                                                 > 100 (500.6 calculated)                            37.5

95 % conf. limits                                   > 100 (157.5 – 1590.7 calculated)              29.8 – 47.1

NOEC                                                         6.25                                                        6.25

LOEC                                                        12.5                                                         12.5