Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

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:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

EC Number:

276-657-4

EC Name:

Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Cas Number:

72428-99-0

Molecular formula:

C32H13CuN8O4S2.(SO3H)m.(C4H11NO)n.(C8H18N) (3-m-n).C15H17N3

IUPAC Name:

hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution* was tested in a limit test.
- Sampling method: The concentrations of the test item were analytically determined from the limit concentration and the control at the start of the exposure (0 h) and at the end of the exposure (72 h). Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure samples were taken directly from the pooled test replicates.

- Sample storage: The original and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (5 mg/L test item was weighed out) was freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by centrifugation for 10 min at 18533 g. The saturated solution was checked for the presence of dispersed test item (by Tyndall effect via Laser beam).

- Eluate: Dilution water
- Differential loading: None, limit test with the saturated solution (initially measured concentration of 1.11 mg/L)
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
- pH-Controls: None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x 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 °C

pH:

Initial mean measured pH-value
test item concentration
[mg/L] Start; 0 hours End; 72 hours
1.11 7.56 8.28
Control 8.00 8.49

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

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 25 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 4998 cells/mL
- Control end cells density: Mean 920802 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: 4440 - 8880 lux, corresponding to 60 - 120 µE x m^-2 x s^-1, mean value: 7030 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

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: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.684 0.684 ± 0.281
95 % confidence interval 0.657 - 0.714 0.684 ± 0.281
Yield Inhibition
EyC50 0.315 0.314 ± 0.087
95 % confidence interval 0.297 - 0.3355 0.314 ± 0.087

SD = Standard deviation

Reported statistics and error estimates:

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 were determined by calculation of statistical significance of growth rate and yield. The t-test was used for NOEC/LOEC calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. The Equal Variance Test failed for growth rate and yield data. No transformation was found to pass the Equal Variance Test.

Any other information on results incl. tables

Cell Densities

Initial measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.11	1	4998	28514	169673	926014
	2	4998	37004	193193	927573
	3	4998	24334	169309	955944
	4	4998	25101	170141	942560
	5	4998	25746	163912	875292
	6	4998	27973	185431	1023473
	Mean	4998	28112	175277	941809
Control	1	4998	25577	162665	1000256
	2	4998	27007	163410	849779
	3	4998	22229	166217	821625
	4	4998	27617	159208	1026721
	5	4998	29042	177600	992372
	6	4998	23321	153742	834056
	Mean	4998	25799	163807	920802

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Initial measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.11	1		1.74	0		921016	-1
	2		1.74	0		922575	-1
	3		1.75	-1		950946	-4
	4		1.75	-1		937562	-2
	5		1.72	1		870294	5
	6		1.77	-2		1018475	-11
	Mean	(-)	1.75	-1	(-)	936811	-2
Control	1		1.77			995258
	2		1.71			844781
	3		1.70			816627
	4		1.78			1021723
	5		1.76			987374
	6		1.71			829058
	Mean		1.74			915804

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.63	1.85	1.82	1.77	0.117	6.62	7.43
	2	1.69	1.80	1.65	1.71	0.079	4.60
	3	1.49	2.01	1.60	1.70	0.275	16.1
	4	1.71	1.75	1.86	1.78	0.080	4.50
	5	1.76	1.81	1.72	1.76	0.045	2.57
	6	1.54	1.89	1.69	1.71	0.173	10.2
				Mean	1.74
				SD ±	0.03
				CV [%]	1.98

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 1.11 mg/L. The EC50-values of the test item for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.11 mg/L.
All effect levels are given based on initially measured concentration of Savinyl Blue GLS.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) .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 4998 cells/mL. A saturated solution* with a nominal concentration of 5 mg/ Lwas tested.Six replicates were tested for the limit concentration and the control. Environmental conditionswere determined to be within the acceptable limits.

The concentrations of the test item were analytically verified via HPLC-DAD at the start and the end of the exposure of the limit test item concentration and the control. The recovery rate at the end of the exposure was 94 % of the initial measured limit test item concentration. Therefore, all effect values given are based on the initial measured test item concentration of the test item

 

NOEC, LOEC and EC - values (0-72 hours)

               based on initial measured test item concentrations [mg/L]

                               

	Growth Rate Inhibition Initial measured test item concentration [mg/L]
NOEC	1.11
LOEC	> 1.11
ErC10	> 1.11
ErC20	> 1.11
ErC50	> 1.11
	Inhibition of Yield Initial measured test item concentration [mg/L]
NOEC	1.11
LOEC	> 1.11
EyC10	> 1.11
EyC20	> 1.11
EyC50	> 1.11