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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
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, corrected July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: the control, the lowest, the highest concentrations and a concentration around the expected EC50 concentration: 0, 3.2, 100 and 320 mg/L
- Sampling method: All the solutions will be analysed taken directly from the starting solutions without algae.
- Sample storage conditions before analysis: All the solutions were kept in refrigerator or freeze in order to be analyzed if necessary or relevant.
Vehicle:
no
Details on test solutions:
According to the sponsor, the test item is soluble in water. The dilution water was the algae test medium, a weakly saline aqueous medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: /
- Strain: CCAP 278.4
- Source (laboratory, culture collection): laboratory
- Age of inoculum (at test initiation): not specified
- Method of cultivation: 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).

ACCLIMATION
no acclimation
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6 – 22.9°C
pH:
7.1-7.7
Nominal and measured concentrations:
According to the results of the preliminary test, we proposed to test five concentrations for this study: 3.2 – 10.0 – 32.0 – 100.0 and 320.0 mg/L.
Measured concentrations: see table
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: initial concentration of 10^4 cells/mL
- Control end cells density:
- No. of organisms per vessel: initial concentration of 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not relevant

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 6035 Lux
- Salinity (for marine algae): not relevant

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
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 under microscope with Malassez counting cell.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
0 - 3.2 – 10.0 – 32.0 – 100.0 and 320.0 mg/L.
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations:
range finding test: 0, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
< 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Results with reference substance (positive control):
The last algae growth inhibition test performed with the reference item K2Cr2O7 (September 18 2017) gave an ErC50(0-72h) = 1.11 mg/L (0.98 – 1.24), 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.

1 . 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

Algae *104/mL at 0 hour

Average growth rate

µ0-72h

Inhibition growth rate (% Ir)

Average inhibition of growth rate (%)

Coefficient of variation

Significance

Control

a

1

1,791

Not applicable

2.0

-

b

1,838

c

1,872

d

1,776

e

1,795

f

1,812

3.2 mg/L

a

1

1,565

13,8

13,7

 

Not required

*

b

1,560

14,1

c

1,577

13,1

10.0 mg/L

a

1

1,128

37,9

40,6

 

Not required

*

b

1,058

41,7

c

1,042

42,6

32.0 mg/L

a

1

0,732

59,7

61,4

 

Not required

*

b

0,672

63,0

c

0,693

61,8

100.0 mg/L

a

1

0,536

70,5

72,5

 

Not required

*

b

0,478

73,7

c

0,478

73,7

320.0 mg/L

a

1

0,355

80,5

80,2

Not required

*

b

0,343

81,1

c

0,377

79,2

- : 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. The NOEC/LOEC were determined as:

NOEC

LOEC

Not determined < 3.2 mg/L

3.2 mg/L

 

A regression model (non linear) was relevant to calcultate an ErC50-(0-72h) . The equation is:

 

Inhibition Growth rate (%) = 82.8045*Test item concentration / (11.7123 + Test item concentration)

 

 

The results obtained for thetest item “REACTION MIXTURE OF CuDTPA and CuHEEDTA”on the toxicological endpoint “inhibition of growth rate” indicate anErC50-(0-72h)= 17.9 mg/L [16.3 – 19.5]value expressed in nominal test item concentration.

 

The results obtained for thetest item “REACTION MIXTURE OF CuDTPA and CuHEEDTA”on the toxicological endpoint “inhibition of growth rate” indicate anErC10-(0-72h)lower than 3.2 mg/L (lowest tested concentration) value expressed in nominal test item concentration.

 

2.  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

[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

215,5

214,5

231,1

 

Not Applicable

b

248,2

247,2

c

275,2

274,2

d

206,3

205,3

e

217,8

216,8

f

229,6

228,6

3.2 mg/L

a

1

109,5

108,5

109,3

 

53,1

52,7

 

b

107,9

106,9

53,7

c

113,5

112,5

51,3

10.0 mg/L

a

1

29,5

28,5

24,4

 

87,7

89,4

 

b

23,9

22,9

90,1

c

22,8

21,8

90,6

32.0 mg/L

a

1

9,0

8,0

7,2

 

96,5

96,9

 

b

7,5

6,5

97,2

c

8,0

7,0

97,0

100.0 mg/L

a

1

5,0

4,0

3,5

 

98,3

98,5

 

b

4,2

3,2

98,6

c

4,2

3,2

98,6

320.0 mg/L

a

1

2,9

1,9

1,9

99,2

99,2

b

2,8

1,8

99,2

c

3,1

2,1

99,1

  

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

 

Yield inhibition (%) = 102.793 * Test item concentration / (2.50896 + Test item concentration)

 

The results obtained for thetest item “REACTION MIXTURE OF CuDTPA and CuHEEDTA”on the toxicological endpoint “Yield inhibition” indicate anEyC50-(0-72h)lower than 3.2 mg/L (lowest tested concentration),value expressed in nominal test item concentration.

Validity criteria fulfilled:
yes
Conclusions:
The results obtained for the test item “REACTION MIXTURE OF CuDTPA and CuHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 17.9 mg/L [16.3 – 19.5] value expressed in nominal test item concentration.

The results obtained for the test item “REACTION MIXTURE OF CuDTPA and CuHEEDTA” on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) lower than 3.2 mg/L (lowest tested concentration), value expressed in nominal test item concentration.

In the control. the increase of the biomass during 72 hours was determined to be higher than factor of 16.
The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successful fulfilled.
Executive summary:

The aim of this study was to determine the effects of the test item "REACTION MIXTURE OF CuDTPA and CuHEEDTA" on the growth of the unicellular green algaPseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

 

The study was carried out in accordance with the study plan n°17/1108F/b dated from Novembre 6, 2017.

There was no deviation and no amendment during this study.

 

The results of this study are summarized below:

Test item                          REACTION MIXTURE OF CuDTPA and CuHEEDTA

 

Test system                     Pseudokirchneriella subcapitata

 

Exposure duration               72 hours

 

Test concentrations            Control – 3.2 – 10.0 – 32.0 - 100.0 and 320.0 mg/L

 

Incubator                          Temperature 22.6 – 22.9°C ; shaking at 120 rpm; light intensity: 6035 Lux

 

Measurement of cell growth  Beckman Coulter Z2 and Malassez counting cell

Results

The results obtained for the test item “REACTION MIXTURE OF CuDTPA and CuHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 17.9 mg/L [16.3 – 19.5], value expressed in nominal test item concentration.

 

The results obtained for the test item “REACTION MIXTURE OF CuDTPA and CuHEEDTA” on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) lower than 3.2 mg/L (lowest tested concentration), value expressed in nominal test item concentration.

 

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

Chelating agents are known to make metal ions less bioavailable in freshwater (Sorvari et al. 1996).

The aquatic toxicity of the Reaction mixture of CuDTPA and CuHEEDTA is thus expected to be lower compared to more bioavailble copper compounds.

Based on an OECD 201 study, the 72-h ErC50 of 17.9 mg/L was determined.

Key value for chemical safety assessment

EC50 for freshwater algae:
17.9 mg/L

Additional information

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