potassium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;dicalcium

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation in support for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation in support for the category approach.

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 *10^4/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] *10^4/mL at 0 hour	[algae] *10^4/mL at 72 hours	Yield (*10^4/mL)	Average yield (*10^4/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.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation in support for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation in support for the category approach.

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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:

22.8 °C – 23.2°C

pH:

7.4-7.8

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: 5943 to 6026 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.:

20.4 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 *10^4/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	1,854	Not applicable		1.8	-
	b		1,855
	c		1,852
	d		1,860
	e		1,773
	f		1,847
3.2 mg/L	a	1	1,378	25,2	19,7	Not required	*
	b		1,565	15,0
	c		1,467	20,3
10.0 mg/L	a	1	0,952	48,3	45,6	Not required	*
	b		1,012	45,1
	c		1,035	43,8
32.0 mg/L	a	1	0,842	54,3	54,7	Not required	*
	b		0,865	53,0
	c		0,793	56,9
100.0 mg/L	a	1	0,768	58,3	59,0	Not required	*
	b		0,732	60,2
	c		0,764	58,5
320.0 mg/L	a	1	0,750	59,2	59,4	Not required	*
	b		0,725	60,6
	c		0,764	58,5

- : 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

 

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

 

Inhibition Growth rate (%) = 62.178*Test item concentration / (4.96818 + Test item concentration)

 

 

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate anErC5₀-(0-72h)= 20.4 mg/L [16.7 – 27.1]value expressed in nominal test item concentration.

 

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate an ErC₁₀-(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] *10^4/mL at 0 hour	[algae] *10^4/mL at 72 hours	Yield (*10^4/mL)	Average yield (*10^4/mL)	Yield inhibition (Iy) (%)
						Per replicate	Average/ replicate
Control	a	1	260,4	259,4	249,7	Not Applicable
	b		261,0	260,0
	c		258,7	257,7
	d		265,3	264,3
	e		203,9	202,9
	f		255,0	254,0
3.2 mg/L	a	1	62,4	61,4	83,4	75,4	66,6
	b		109,3	108,3		56,6
	c		81,6	80,6		67,7
10.0 mg/L	a	1	17,4	16,4	19,2	93,4	92,3
	b		20,8	19,8		92,1
	c		22,3	21,3		91,5
32.0 mg/L	a	1	12,5	11,5	11,2	95,4	95,5
	b		13,4	12,4		95,0
	c		10,8	9,8		96,1
100.0 mg/L	a	1	10,0	9,0	8,6	96,4	96,5
	b		9,0	8,0		96,8
	c		9,9	8,9		96,4
320.0 mg/L	a	1	9,5	8,5	8,4	96,6	96,6
	b		8,8	7,8		96,9
	c		9,9	8,9		96,4

 

The values as EyC₅₀-(0-72h) were calculated from raw data with the scientific software Minitab 17

 

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

 

Yield inhibition (%) = 99.1128 * Test item concentration / (1.38/201 + Test item concentration)

 

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA”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.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 20.4 mg/L [16.7 – 27.1] value expressed in nominal test item concentration.

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” 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 ZnEDTA, ZnDTPA AND ZnHEEDTA" on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

 

The study was carried out in accordance with the study plan n°17/1107F/b dated from October 31, 2017.

There was no deviation and no amendment during this study.

 

The results of this study are summarized below:

Test item                          REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA

 

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 22.8 – 23.2°C ; shaking at 120 rpm; light intensity: 5943 to 6026 Lux

 

Measurement of cell growth  Beckman Coulter Z2 and Malassez counting cell

 

Results

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” on the toxicological endpoint “inhibition of growth rate” indicate an ErC5₀-(0-72h)= 20.4 mg/L [16.7 – 27.1], value expressed in nominal test item concentration.

 

The results obtained for the test item “REACTION MIXTURE OF ZnEDTA, ZnDTPA AND ZnHEEDTA” 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

Information on the short-term toxicity to aquatic algae is available for two category members: metal chelates with copper and zinc. The result is in line with the expectations i.e. that the aquatic toxicity is the result of the free (metal) ion that is complexed and that this ion is much less bioavailable and thus less toxic to aquatic life when complexed with chelating agents. As expected, the order of aquatic toxicity of the metal chelates follows the order of toxicity of the metal ions: copper chelate is more toxic than zinc chelate. Therefore, it is reasonable to assume that the aquatic toxicity of KCa2DTPA will be lower than that of zinc chelate.

The 72-h EC50 for algal growth (OECD 201) was determined to be 20.4 mg/L for the zinc chelate.

Considering that the aquatic toxicity is determined by the metal ion and that K and Ca are orders of magnitude less toxic than Zn, the substance is not considered toxic to algae.