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Ecotoxicological information

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:
2017-01-31 to 2017-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:


Read-across from an analogue substance.
The analogue substance Turkish Rose Absolute and the registered substance Turkish Rose Concrete have the same botanical origin: Rosa Damascena (Rosaceae). They are both obtained from fresh rose flowers. They follow the same production pattern. They both have the same main constituents and in similar concentrations, regarding their respective SIP. Based on these similarities, both substances are not expected to show different ecotoxicological behaviour. Thereby, results from the experimental OECD 201 study assessing the toxicity of the analogue substance Turkish Rose Absolute on freshwater algae Pseudokirchneriella subcapitata can be used as a read accross in the present dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
14 January 2016
Specific details on test material used for the study:
None
Analytical monitoring:
yes
Details on sampling:
Chemical analyses of “ROSE TURKISH ABSOLUTE” were performed in the test solutions (biotic and abiotic).
In order to verify the nominal or initial concentrations of the test item and maintenance of the concentrations during the ecotoxicological testing, chemical analysis of “ROSE TURKISH ABSOLUTE” in algae test solutions were performed:
-at the beginning of the test (T = 0h)
-at the end of the test (T = 72h)

Schedule analysis at initial time (T0h):
Control Abiotic
4.4 mg/L Abiotic
11.4 mg/L Abiotic
29.6 mg/L Abiotic
77.0 mg/L Abiotic
200.0 mg/L Abiotic

Schedule analysis at final time (T72h):
Control Biotic Control Abiotic
4.4 mg/L Biotic 4.4 mg/L Abiotic
11.4 mg/L Biotic 11.4 mg/L Abiotic
29.6 mg/L Biotic 29.6 mg/L Abiotic
77.0 mg/L Biotic 77.0 mg/L Abiotic
200.0 mg/L Biotic 200.0 mg/L Abiotic

The measured concentrations of the “ROSE TURKISH ABSOLUTE” should not vary more than ± 20 % of nominal or initial measured concentration during the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was not soluble in the test medium, a special solubilisation protocol was carried out before the experimentation, according to the OECD n°23 guidance document on aquatic toxicity testing of difficult substances and mixtures.

Cultures of exponentially-growing algae were exposed to five test item concentrations in algae test medium (OECD medium) plus a control.
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline.

Preparation of test solutions for the testing
This method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20±2°C (these concentrations were prepared by adding directly the test item in flasks containing test medium).

All the test solutions plus WAF Control (algae test medium) were then filtered through a membrane filter to remove suspended particles: solutions obtained were named WAF (Water Accommodated Fraction).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Freshwater unicellular green alga Pseudokirchneriella subcapitata of the chlorococcal order (formerly named Raphidocellis subcapitata) strain number CCAP 278.4 was used in this study.
The algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
Temperature 22.7 – 23.4 °C
pH:
pH at the start: 7.6 - 7.8
pH after 72 h: 7.5 - 7.7
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0; 4.4, 11.4, 29.6, 77.0, 200.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Initial cells density: 10000 cells/mL at the start of the test
- No. of vessels per concentration (replicates): 3 biotic replicates + 1 abiotic replicate per concentration
- Control: 6 biotic replicates + 2 abiotic replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae test medium (OECD medium); Algae dilution water

OTHER TEST CONDITIONS
- Light intensity and quality: 6159 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The algae growth inhibition in relation to the control was evaluated over a period of 72 hours. The algae cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter (Beckman Coulter Z2) and/or a microscope with a Malassez counting cell.
- The pH of each test concentration was measured at the start and at the end of the test. The pH of the control medium had not increased by more than 1.5 units during the test.

TEST CONCENTRATIONS
- Range finding study: 5; 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
175 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval (mg/L): [162 - 180 mg/L]
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
103.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 79.5 - 121.5 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
156 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% CL: 122.0 - 178.0 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
95.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 32.0 - 137.0 mg/L
Details on results:
See "any other information on results incl. tables"
Results with reference substance (positive control):
The last algae growth inhibition test performed with the reference item K2Cr2O7 (January 23, 2017) gave an ErC50(0-72h) = 1.18 mg/L (1.03 – 1.36), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.
Reported statistics and error estimates:
None

Algae growth inhibition

Raw data,i.e. algae concentrations values expressed as cell number *104per mL of test solutions after 24, 48 and 72 hours of incubation are indicated in the following table.

Nominal test item concentration

[algae] *104/mL at 24 hours

[algae] *104/mL at 48 hours

[algae] *104/mL at 72 hours

Count.

Average

cv

Count.

Average

cv

Count.

Average

cv

Control

a

2,9

2.8

7.6

10,5

11.0

11.4

49,6

67.8

15.9

b

3,2

10,4

63,5

c

2,7

9,9

66,9

d

2,6

13,4

77,8

e

2,7

11,3

70,1

f

2,9

10,5

78,9

WAF 4.4

a

3,5

3.4

5.1

14,1

12.8

16.1

62,5

63.8

7.6

b

3,2

10,4

59,8

c

3,5

13,8

69,2

WAF 11.4

a

2,6

3.0

15.3

11,4

13.6

15.8

56,7

59.9

4.8

b

2,9

13,6

62,3

c

3,5

15,7

60,6

WAF 29.6

a

2,8

2.8

2.1

12,4

12.0

14.8

56,0

80.5

33.6

b

2,7

10,1

76,0

c

2,8

13,6

109,5

WAF 77.0

a

2,5

2.5

13.9

13,3

13.4

12.3

64,5

60.7

5.5

b

2,2

11,8

58,5

c

2,9

15,1

59,0

WAF 200.0

a

2,5

2.4

4.2

2,3

2.3

2.5

5,3

3.2

55.4

b

2,4

2,2

2,2

c

2,3

2,3

2,2

CV = Coefficient of variation.

 

Specific growth rate

 

The coefficients of variation for section-by-section specific growth rates in the control cultures are presented in the following table to calculate one of the validity criterion: average value of coefficient of variation (Average CV).

Control

replicates

Specific growth rate

0-24h

Average

CV

(%)

24 -48h

Average

CV

(%)

48 -72h

Average

CV (%)

0 - 72h

Average

CV per replicate (%)

Average CV

Control

a

1,065

1.039

7.2

1,287

1.354

11.9

1,553

1.812

8.6

1,301

1.406

18,8

28.7%

b

1,163

1,179

1,809

1,384

26,6

c

0,993

1,299

1,911

1,401

33,3

d

0,956

1,640

1,759

1,451

29,9

e

0,993

1,432

1,825

1,417

29,4

f

1,065

1,287

2,017

1,456

34,2

CV: Coefficient of variation (%)

Calculation of the ErC50(0-72h)

Inhibitions of the average specific growth rate in comparison to the Control are presented in the following table:

Nominal test item concentration (mg/L)

Algae *104/mL at 0hour

Average growth rate

µ0-72h

Inhibition growth rate (% Ir)

Average inhibition of growth rate (%)

Coefficient of variation

Significance

Control

a

1

1,301

Not applicable

4.0

-

b

1,384

c

1,401

d

1,451

e

1,417

f

1,456

WAF 4.4

a

1

1,378

1,9

1.4

Not required

ns

b

1,364

3,0

c

1,412

-0,5

WAF 11.4

a

1

1,346

4,2

3.0

Not required

ns

b

1,377

2,0

c

1,368

2,7

WAF 29.6

a

1

1,342

4,5

-4.1

Not required

ns

b

1,444

-2,7

c

1,565

-11,4

WAF 77.0

a

1

1,389

1,2

2.6

Not required

ns

b

1,356

3,5

c

1,359

3,3

WAF 200.0

a

1

0,556

60,4

72.2

Not required

*

b

0,263

81,3

c

0,263

81,3

- : not relevant

ns : not significantly different compared to the Control, Dunnet test, α =0.05

* : significantly different compared to the Control, Dunnett test, α=0.05

 

The average specific growth rate (µ) was determined according to the OECD guideline.

Statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17.2.1 to determined NOEC/LOEC. No significant difference between the control was observed at the lowest concentration 77.0 mg/L.

NOEC = 77.0 mg/L

LOEC = 200.0 mg/L

 

The equation of the regression (non linear regression) is the following (rounded values):

 % inhibition growth rate = 3.310 - 0.2479*[concentration] + 0.003017 [concentration]2

Where:

[concentration]= concentration of test item (mg/L)

 

From the equation, the ErC50-(0-72h) was calculated and the value is the following:

ErC50-(0-72h)= 175.0 mg/L [162.0 – 180.0] value expressed in nominal concentration

 

The results obtained for thetest item “ROSE TURKISH ABSOLUTE”on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) equal to 175.0 mg/L [162.0 – 180.0] value expressed in nominal test item concentration.

 

From the equation, the ErC10-(0-72h) was calculated and the value is the following:

ErC10-(0-72h) = 103.6 mg/L [79.5 – 121,5] value expressed in nominal concentration

 

The results obtained for thetest item “ROSE TURKISH ABSOLUTE”on the toxicological endpoint “inhibition of growth rate” indicate anErC10-(0-72h) equal to 103.6 mg/L [79.5 – 121,5]value expressed in nominal test item concentration.

Calculation of the EyC50(0-72h)

Inhibitions of the yield in comparison to the Control are presented in the following table:

Nominal test item concentration

(mg/L)

[algae] *104/mL at 0 hour

[algae] *104/mL at 72 hours

Yield

(*104/mL)

Average yield

(*104/mL)

Yield inhibition (Iy) (%)

Per replicate

Average/ replicate

Control

a

1

49,6

48,6

66.8

Not Applicable

b

63,5

62,5

c

66,9

65,9

d

77,8

76,8

e

70,1

69,1

f

78,9

77,9

WAF 4.4

a

1

62,5

61,5

62.8

7,9

5.9

b

59,8

58,8

12,0

c

69,2

68,2

-2,1

WAF 11.4

a

1

56,7

55,7

58.9

16,6

11.9

b

62,3

61,3

8,2

c

60,6

59,6

10,8

WAF 29.6

a

1

56,0

55,0

79.5

17,7

-19.0

b

76,0

75,0

-12,3

c

109,5

108,5

-62,4

WAF 77.0

a

1

64,5

63,5

59.7

4,9

10.7

b

58,5

57,5

13,9

c

59,0

58,0

13,2

WAF 200.0

a

1

5,3

4,3

2.2

93,6

96.7

b

2,2

1,2

98,2

c

2,2

1,2

98,2

The equation of the regression (non linear regression) is the following (rounded values):

 

 % Yield inhibition = 5.455 – 0.3314*[concentration] + 0.003955 [concentration]2

 

Where:

[concentration]= concentration of test item (mg/L)

 

From the equation, the EyC50-(0-72h) was calculated and the value is the following:

EyC50-(0-72h)= 156.0 mg/L [122.0 – 178.0] value expressed in nominal concentration

 

The results obtained for thetest item “ROSE TURKISH ABSOLUTE”on the toxicological endpoint “inhibition of growth rate” indicate anEyC50-(0-72h)equal to 156.0 mg/L [122.0 – 178.0]value expressed in nominal test item concentration.

 

From the equation, the EyC10-(0-72h) was calculated and the value is the following:

EyC10-(0-72h)= 95.8 mg/L [32.0 – 137.0] value expressed in nominal concentration

 

The results obtained for thetest item “ROSE TURKISH ABSOLUTE”on the toxicological endpoint “inhibition of growth rate” indicate anEyC10-(0-72h)equal to 95.8 mg/L [32.0 – 137.0]value expressed in nominal test item concentration.

 

Results for the chemical analysis

The table below summarizes the results achieved and the variation between T0 and T72 hours.

Nominal test item concentration

mg “NPOC”/L in algae solutions

Variation between T0 and T72 hrs (%)

Results at T0 hour

Results at T72 hours

Control Abiotic

< 2

2.6

NA

4.4 mg/L Abiotic

2.6

< 2

NA

11.4 mg/L Abiotic

4.9

2.9

- 40.8

29.6 mg/L Abiotic

11.8

8.7

- 26.3

77.0 mg/L Abiotic

28.4

27.6

- 2.8

200.0 mg/L Abiotic

71.3

70.0

- 1.8

NA: not applicable

Nominal test item concentration

mg “NPOC”/L in algae solutions

Variation between T0 and T72 hrs (%)

Results at T0 hour

Results at T72 hours

Control Biotic

< 2

3.2

NA

4.4 mg/L Biotic

2.6

3.6

+ 38.5

11.4 mg/L Biotic

4.9

5.3

+ 8.2

29.6 mg/L Biotic

11.8

7.2

- 39.0

77.0 mg/L Biotic

28.4

14.9*

- 47.5

200.0 mg/L Biotic

71.3

70.7

- 0.8

NA: not applicable

 

A weak difference in NPOC content between T0h and T72h causes a large variation (out of specification).

 Under Abiotic conditions (without algae), the test item “ROSE TURKISH ABSOLUTE” is not considered to be stable for the nominal concentrations 11.4 and 29.6 mg/L because the measured concentrations of the test item did vary by more than ± 20% of the initial concentrations during the test.For the concentration 200.0 mg/L, the deviation between T0 and T72 hours was less than± 20% of the initial concentration.

Under biotic conditions (with algae), the test item “ROSE TURKISH ABSOLUTE” is considered to be stable for the nominal concentrations 11.4 and 200.0 mg/L, except the following concentrations 4.4 – 29.6 and 77.0 mg/L.

 According to theses results and for this study, the concentrations are expressed in nominal mg “ROSE TURKISH ABSOLUTE” / L.

Validity criteria

 

Criteria for OECD 201

Validity criteria conformity

Increase biomass during 72 hours

Factor of at least 16

Yes (67.8)

Daily section-by-section specific growth rate CV

< 35%

Yes (28.7%)

Average specific growth rate CV during 72 hours

< 7%

Yes (4.0%)

All the validity criteria were successfully fulfilled.

Validity criteria fulfilled:
yes
Conclusions:
The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) equal to 175.0 mg/L [162.0 - 180.0], and an an ErC10-(0-72h) equal to 103.6 mg/L [79.5 - 121.5], both values expressed in nominal test item concentration.
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item ROSE TURKISH ABSOLUTE at 4.4, 11.4, 29.6, 77.0 and 200.0 mg/L over an exposure period of 72 h. The algae growth inhibition in relation to the control was evaluated over a period of 72 h.

The results of this study are summarized below:

Test item: ROSE TURKISH ABSOLUTE

Test system: Pseudokirchneriella subcapitata

Exposure duration: 72 hours

Test concentrations: Control – 4.4 – 11.4 – 29.6 – 77.0 and 200.0 mg/L

Incubator: Temperature 22.7 to 23.4°C ; shaking at 120 rpm; light intensity: 6159 Lux

Measurement of cell growth: Beckman Coulter Z2

Based on growth rate inhibition, the results are as follows:

Test item

(nominal concentration)

ErC10-(0-72h)

With 95 % confidence interval [lower – upper]

ErC50-(0-72h)

With 95 % confidence interval [lower – upper]

ROSE TURKISH ABSOLUTE

103.6 mg/L

[79.5 – 121,5]

175.0 mg/L

[162.0 – 180.0]

                                  

Based on yield inhibition, the results are as follows:

Test item

(nominal concentration)

EyC10-(0-72h)

With 95 % confidence interval [lower – upper]

EyC50-(0-72h)

With 95 % confidence interval [lower – upper]

ROSE TURKISH ABSOLUTE

95.8 mg/L

[32.0 – 137.0]

156.0 mg/L

[122.0 – 178.0]

Validity criteria

In the control, the increase of the biomass during 72 hours was determined to be higher than a factor of 16.

The coefficients of variation ofdaily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35 % and 7% in the control.

The validity criteria were successful fulfilled.

Description of key information

Based on a read-across from an experimental GLP study performed on the analogue substance Turkish rose absolute according to the OECD 201 guideline, the following results, based on growth rate, have been extrapolated to the registered substance:

-       ErC50-(0-72h) = 175.0 mg/L [95% CI: 162.0 - 180.0 mg/L]

-       ErC10-(0-72h) = 103.6 mg/L [95% CI: 79.5 - 121.5 mg/L]

Key value for chemical safety assessment

EC50 for freshwater algae:
175 mg/L
EC10 or NOEC for freshwater algae:
103.6 mg/L

Additional information

For that endpoint, a study on the registered substance was not available. Therefore, in order to assess the toxicity of the registered substance to freshwater algae, the results from an experimental OECD 201 study performed on the analogue substance Turskish rose absolute have been used.

In this study, the toxic effect of the test item Turskish rose absolute to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a 72h-static limit test using Water Accommodated Fractions. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 761/2009 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Based on growth rate inhibition, the results for the analogue substance were as follows:

Test item

(nominal concentration)

ErC10-(0-72h)

With 95 % confidence interval [lower – upper]

ErC50-(0-72h)

With 95 % confidence interval [lower – upper]

ROSE TURKISH ABSOLUTE

103.6 mg/L

[79.5 – 121,5]

175.0 mg/L

[162.0 – 180.0]

                                  

The validity criteria were fulfilled and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.