Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

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:

2017-01-17 to 2017-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

EC Number:

279-091-6

EC Name:

Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

Cas Number:

79135-90-3

Molecular formula:

C24H18N6O18S3.3Na

IUPAC Name:

trisodium 3-({4,6-bis[(3-carboxy-2-hydroxy-5-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-5-carboxy-4-hydroxybenzene-1-sulfonate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via UPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:

no

Details on test solutions:

test item stock solution, media preparation
125.9 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the stock solution was clear.

Test concentrations
One limit concentration of 125.9 mg/L (100 mg a.i./L) were tested. The limit concentration was based on the results of a preliminary range finding test.
 
Control
Six replicates (without test item) were exposed under the same conditions as the test item replicates.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item Concentration Nominal a.i. Concentration pH-value
[mg/L] [mg/L] Start; 0 hours End; 72 hours
125.9 100 7.87 8.50
Control Control 8.01 9.28

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal loading 125.9 mg test item/L, corresponding to 100 mg active ingredient/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2089001 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5681 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the concentration 125.9 mg test item/L.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 125.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other:The test media were clear throughout the exposure phase. Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values given are based on the nominal active ingredient concentration of Anodal SH-1 hc dried.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.764 ± 0.513
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEC, LOEC and statistical analyses
NOEC/LOEC were determined by calculation of statistical significance of growth rates and yield. As a standard, a t-test was used for NOEC/LOEC calculations. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.

Any other information on results incl. tables

Cell Densities

Nominal test item Concentration	Nominal a.i. Concentration	Replicate	Cell Density [cells/mL]
[mg/L]	[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
125.9	100	1	6305	42374	429578	1963960
		2	6305	37041	370356	2085458
		3	6305	41509	445173	2041144
		4	6305	46377	395772	2105135
		5	6305	40390	391736	1933289
		6	6305	31656	446424	1882716
		Mean	6305	39891	413173	2001950
Control	Control	1	6305	45359	552238	2193708
		2	6305	43837	469923	2093746
		3	6305	36296	485935	2144263
		4	6305	41497	460170	2129266
		5	6305	38543	384045	1999592
		6	6305	34923	430232	1973431
		Mean	6305	40076	463757	2089001

 

 

 Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item Concentration		Nominal a.i. Concentration		Replicate		Growth rate			Growth rate inhibition		Yield			Inhibition of yield
[mg/L]		[mg/L]		No.		[d^-1]			[%]		[cells/mL]			[%]
125.9		100		1				1.91		1				1957655		6
			2				1.93		0				2079153		0
			3				1.93		0				2034839		2
			4				1.94		0				2098830		-1
			5				1.91		1				1926984		7
			6				1.90		2				1876411		10
			Mean		(-)		1.92		1		(-)		1995645		4
Control		Control		1				1.95						2187403
			2				1.94						2087441
			3				1.94						2137958
			4				1.94						2122961
			5				1.92						1993287
			6				1.92						1967126
			Mean				1.93						2082696

            Negative inhibition = growth stimulation

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.97	2.50	1.38	1.95	0.560	28.7	24.8
	2	1.94	2.37	1.49	1.94	0.439	22.7
	3	1.75	2.59	1.48	1.94	0.580	29.8
	4	1.88	2.41	1.53	1.94	0.440	22.7
	5	1.81	2.30	1.65	1.92	0.338	17.6
	6	1.71	2.51	1.52	1.92	0.525	27.4
				Mean	1.93
				SD ±	0.01
				CV [%]	0.71

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition of growth of the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item. The nominal test item EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 125.9 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours based on the nominal test item concentration were 125.9 mg/L, respectively.
All effect values given are based on nominal test item concentration.

Executive summary:

The toxicity of the test item (Batch no.:CHA0127492) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3 from2017-01-17 to 2017-01-20 at the test facility. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution with a nominal loading of 125.9 mg test item/L, corresponding to 100 mg active ingredient/L, was tested. Six replicates were used for the limit concentration and the control. The environmental conditions were within the acceptable limits.

 

The concentration of the test item were analytically verified by UPLC-DAD at the start and the end of exposure of the limit concentration and the control.

The measured concentration of the test item in the fresh media (0 h) were 95% of the nominal values at the limit concentration level. The measured concentrations in the old media (72 h) were 94% of the nominal values.

Therefore, all effect values given are based on the nominal test item concentration.

 NOEC, LOEC, EC-values and 95% Confidence Intervals ofAnodalSH-1hc dried(0 - 72 hours)

based on the nominal test item concentration [mg/L]

 

	Inhibition of Growth Rate
NOEC	125.9
LOEC	> 125.9
ErC10	> 125.9
ErC20	> 125.9
ErC50	> 125.9
	Inhibition of Yield
NOEC	125.9
LOEC	> 125.9
EyC10	> 125.9
EyC20	> 125.9
EyC50	> 125.9