copper sulfate;N'-[2-[2-(2-aminoethylamino)ethylamino]ethyl]ethane-1,2-diamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

2017-2018

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 for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation 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: 0, 0.10, 1.00 and 10.00 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:

22.4 °C – 23.1°C

pH:

7.8-8.0

Nominal and measured concentrations:

Based on the results of the preliminary Test, the following test concentrations .0.10 – 0.32 – 1.00 – 3.30 and 10.00 mg test item /L are proposed for the definitive test in agreement with the Sponsor.
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: 6102 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
0.10 – 0.32 – 1.00 – 3.30 and 10.00 mg test item /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.:

5.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The last algae growth inhibition test performed with the reference item K2Cr2O7 (February 12, 2018) gave an ErC50(0-72h) = 1.12 mg/L (1.00 – 1.30), 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 May 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	Average growth rateμ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control a b c d e f	1,753 1,806 1,818 1,785 1,768 1,773	Not applicable		1.4	-
0.10 mg/L a b c	1,456 1,449 1,503	18,4 18,8 15,8	17,6	Not required	*
0.32 mg/L a b c	1,347 1,332 1,370	24,5 25,4 23,3	24,4	Not required	*
1.00 mg/L a b c	1,327 1,288 1,297	25,7 27,9 27,3	26,9	Not required	*
3.30 mg/L a b c	1,115 1,134 1,020	37,5 36,5 42,9	38,7	Not required	*
10.00 mg/L a b c	0,388 0,388 0,292	78,3 78,3 83,6	79,9	Not required	*

- : 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 Minitab17. The NOEC/LOEC were determined as:

NOEC	LOEC
Not determined < 0.10 mg/L	0.10 mg/L

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

 

Inhibition Growth rate (%) = 19.94 + 6.002*Test item concentration

 

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint“inhibition of growth rate” indicate an ErC50-(0-72h) = 5.01 mg/L [4.70–5.30] value expressed in nominal test item concentration.

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint“inhibition of growth rate” indicate an ErC10-(0-72h) lower than 0.10 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	[algae] *104/mL at 72 hours	Yield (*104/mL)	Average yield (*104/mL)	Yield inhibition (Iy) (%)
				Per replicate	Average/ replicate
Control a b c d e f	192,2	191,2	210,5	Not Applicable
	225,5	224,5
	233,4	232,4
	211,9	210,9
	201,4	200,4
	204,4	203,4
0.10 mg/L a b c	78,8	77,8	81,2	63,0	61,4
	77,2	76,2		63,8
	90,7	89,7		57,4
0.32 mg/L a b c	56,9	55,9	56,4	73,4	73,2
	54,4	53,4		74,6
	60,9	59,9		71,5
1.00 mg/L a b c	53,5	52,5	49,0	75,1	76,7
	47,6	46,6		77,9
	48,9	47,9		77,2
3.30 mg/L a b c	28,4	27,4	25,6	87,0	87,9
	30,0	29,0		86,2
	21,3	20,3		90,4
10.00 mg/L a b c	3,2	2,2	1,9	99,0	99,1
	3,2	2,2		99,0
	2,4	1,4		99,3

The value as EyC50-(0-72h) was not able to be calculated from raw data because there is 61.4% of yield inhibiition at 0.10 mg/L (lowest tested concentration).

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint“Yield inhibition” indicate an EyC50-(0-72h) lower than 0.10 mg/L (lowest tested concentration), value expressed in nominal test item concentration.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 5.01 mg/L [4.70 – 5.30] value expressed in nominal test item concentration.
The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “inhibition of growth rate” indicate an ErC10-(0-72h) lower than 0.10 mg/L (lowest tested concentration) value expressed in nominal test item concentration.
The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) lower than 0.10 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 "Copper (Cu) (Copper diethylenetriamine sulfate)" 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°17/1196F/b dated from March 8, 2018. There was no deviation and no amendment during this study.

The results of this study are summarized below:

Test item:

Copper (Cu) (Copper diethylenetriamine sulfate)

Test system

Pseudokirchneriella subcapitata

Exposure duration

72 hours

Test concentrations

Control–0.10–0.32–1.00–3.30 and 10.00 mg/L

Incubator

Temperature 22.4 °C–23.1°C; shaking at 120 rpm; light intensity: 6102 Lux

Measurement of cell growth :

Beckman Coulter Z2 and Malassez counting cell

Results

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 5.01 mg/L [4.70–5.30] value expressed in nominal test item concentration.

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “inhibition of growth rate” indicate an ErC10-(0-72h) lower than 0.10 mg/L (lowest tested concentration) value expressed in nominal test item concentration.

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “Yield inhibition” indicate an EyC50 -(0 -72h) lower than 0.10 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.

Description of key information

Information was available on the toxicity to aquatic algae for a category member.

The results obtained for the test item “Copper (Cu) (Copper diethylenetriamine sulfate)” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) = 5.01 mg/L [4.70 – 5.30] value expressed in nominal test item concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.01 mg/L