Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)

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:

From 2018-02-05 to 2018-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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:

Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)

EC Number:

277-086-3

EC Name:

Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)

Cas Number:

72928-60-0

Molecular formula:

C32H16-τN8Cu(SO3C15H18N3)τ

IUPAC Name:

Bis and tris and tetra [N-(2-methylanilino)-N’-(2-methylanilino) methaniminium][phthalocyaninesulfonato-κ4N29,N30,N31,N32]cuprate(II)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At test facility: 2017-04-19 to 2017-04-27: refrigerator (6 ± 2 °C)/ since 2017-04-27: room temperature, protected from light, in the tightly closed original container; since 2017-11-02 additionally in evacuated desiccator

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
* 0 mg/L (control)
* 0.500 mg/L of test item
* 1.58 mg/L of test item
* 5.00 mg/L of test item
* 15.8 mg/L of test item
* 50 mg/L of test item

- Sampling method: Sampling done at the start of exposure (0 hours) and at the end of exposure (72 hours) with algae.

- Preparation of the samples:
The control and the 2 lowest test item concentrations (0.500 mg/L and 1.58 mg/L) were analyzed undiluted.
The test item concentrations from 5.00 mg/L to 50.0 mg/L were diluted with ultra-pure water prior to analysis.


- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution with a nominal test item concentration of 50.0 mg/L was freshly prepared
with dilution water. This dispersion was stirred at 1100 rpm at room temperature for 24 hours.

The four other concentrations0.500 - 1.58 - 5.00 - 15.8 mg/L were prepared by appropriate dilution of the stock solution.

- Control: Six replicates (without test item)

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata

- Source (laboratory, culture collection):
* Origin :Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

*Cultivation at test facility
- Age of inoculum (at test initiation): Four day old

- Method of cultivation:
* Fresh stocks are prepared on Z-Agar.
* Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 μE ∙ m-2 ∙ s-1 for 24 hours per day.
* Nutrient medium Z according to LÜTTGE et al. (1994)
* Culture apparatus: Climate room

ACCLIMATION
- Acclimation period: No acclimation period as the preculture was incubated under the same conditions as the test replicates.
- Culturing media and conditions (same as test or not): same as test

- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

other: OECD TG 201 medium

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 24°C, controlled at ± 2°C

pH:

8.1 ± 0.2

Nominal and measured concentrations:

Nominal concentrations : 0.500 – 1.58 – 5.00 – 15.8 – 50.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flasks 250mL sterile glass Erlenmeyer flasks sealed with cotton wool plugs.

- Type : closed with cotton wool plugs

- Material, size, headspace, fill volume: 250mL sterile glass Erlenmeyer flasks

- Renewal rate of test solution (frequency/flow rate): no renewal

- Initial cells density: 5523cells/ml

- Control end cells density: yes

- No. of vessels per concentration (replicates): 3 replicates

- No. of vessels per control (replicates): 6 replicates


GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

OTHER TEST CONDITIONS
- Sterile test conditions: no

- Adjustment of pH: yes, for the culture medium

- Photoperiod: 24hours/day light

- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m-2*s-1. Light homogeneity within ± 15% over incubation area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Determination of cell concentrations:fluorimeter (Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH)
- Chlorophyll measurement: Chlorophyll a-fluorescence, (with excitation at 436 nm, emission at 685 nm), used to determine cell density.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: spacing factor of √10

- Range finding study : yes

- Test concentrations: 1, 10 and 100 mg of test item /L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 15.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 50 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.:

ca. 35.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CI :5.144 - > 50.0

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 38.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI :5.55 - > 50.0

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 43.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 6.09 - > 50.0 mg/L

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 15.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 28.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 956% CI : > 0.500 – 50.0

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 35.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 5.25 – 50.0

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 0.535 mg/L (CI 95% 0.523-0.547 mg/l) (based on growth rate)
- EyC50: 0.320 mg/L (CI 95% 0.249-0.339 mg/l) (based on yield)

Reported statistics and error estimates:

ErC50 (based on growth rate) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))


EyC50 (based on yield) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

NOEC/LOEC based growth rate were determined after Wiliams multiple sequential t-test procedure

NOEC/LOEC based on yield were determined after Wiliams multiple sequential t-test procedure

Any other information on results incl. tables

Table 1: Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
50.0	1	5523	6565	4385	2977
	2	5523	5859	5827	<LOQcell density
	3	5523	5402	5906	3831
	Mean	5523	5942	5373	n.a.
15.8	1	5523	22148	229640	1644026
	2	5523	23244	226489	1929155
	3	5523	25415	273340	1829239
	Mean	5523	23602	243156	1800807
5.00	1	5523	27531	269927	2168894
	2	5523	26835	318898	1944614
	3	5523	31517	320769	2055692
	Mean	5523	28628	303198	2056400
1.58	1	5523	17893	165112	1507315
	2	5523	33134	417040	2090759
	3	5523	23785	378803	1997807
	Mean	5523	24937	320318	1865294
0.500	1	5523	18559	173755	1280862
	2	5523	17838	174733	1374209
	3	5523	15497	157239	960123
	Mean	5523	17298	168576	1205065
Control	1	5523	27521	277850	2113231
	2	5523	38222	413504	2311038
	3	5523	20441	298179	1956961
	4	5523	30756	287876	2159164
	5	5523	30090	367295	2045863
	6	5523	17880	275775	1437379
	Mean	5523	27485	320080	2003939

< LOQ_{cell density}= below the limit of quantification (2736 cells/mL)          

n.a. = data not determinable
The cell growth in the control increased 362-fold from 0 to 72 hours:
(2003939 cells/mL) x (5523 cells/mL)^-1= 362-fold (required: at least 16-fold).

Table 2: Evaluation after 72 hours

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

tiNominal test item concentration	Replicate	Growth Rate		Inhibition of Growth Rate	Yield		Inhibition of Yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
50.0	1		-0.21	100		-2546	100
	2		n.a.	100		n.a.	100
	3		-0.12	100		-1692	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
15.8	1		1.90	3		1638503	18
	2		1.95	0		1923632	4
	3		1.93	1		1823716	9
	Mean	(-)	1.93	2	(-)	1795284	10
5.00	1		1.99	-2		2163371	-8
	2		1.96	0		1939091	3
	3		1.97	-1		2050169	-3
	Mean	(-)	1.97	-1	(-)	2050877	-3
1.58	1		1.87	5		1501792	25
	2		1.98	-1		2085236	-4
	3		1.96	0		1992284	0
	Mean	(-)	1.94	1	(-)	1859771	7
0.500	1		1.82	7		1275339	36
	2		1.84	6		1368686	32
	3		1.72	12		954600	52
	Mean	(-)	1.79	9	(-)	1199542	40
Control	1		1.98			2107708
	2		2.01			2305515
	3		1.96			1951438
	4		1.99			2153641
	5		1.97			2040340
	6		1.85			1431856
	Mean		1.96			1998416

n.a. = data not determinable

Table 3:

Section-by-section and average specific growth rates oght 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	1.61	2.31	2.03	1.98	0.355	17.9	24.9
	2	1.93	2.38	1.72	2.01	0.337	16.7
	3	1.31	2.68	1.88	1.96	0.689	35.2
	4	1.72	2.24	2.02	1.99	0.261	13.1
	5	1.70	2.50	1.72	1.97	0.460	23.3
	6	1.18	2.74	1.65	1.85	0.800	43.2
				Mean	1.96
				SD ±	0.06
				CV [%]	2.84

SD = Standard deviation CV = Coefficient of variation

Table 4: Environmental conditions

Nominal test item concentration		pH-values
[mg/L]		Start; 0 hours	End; 72 hours
50.0		7.61	7.65
15.8		7.68	7.98
5.00		7.76	8.23
1.58		7.87	8.44
0.500		7.96	8.59
Control		8.11	8.89
Room temperature [°C]:	min.: 22.0	max.: 22.5		mean value: 22.3
Light intensity [lux]	n = 23	mean value: 5777 range of the measured values: 5077 to 6600 equalling -12.1 to 14.2%		CV [%]: 7.75

CV = Coefficient of variation                             

n = number of measuring points

Table 5 : Measured concentration of test item iun the definitive test with algae

Sampling date	Fresh Media, 0 hours		Old Media, 72 hours
Nominal test item concentration [mg/L]	TEST ITEM
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
50.0	49.7	99	51.5	103
15.8	14.9	94	15.9	101
5.00	4.89	98	5.08	102
1.58	1.51	96	1.54	97
0.500	0.462	92	0.453	91
Control	< LOQ		< LOQ

Meas. conc.  = measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percent of the nominal measured concentration of the test item

LOQ             = limit of quantification of the analytical method (0.300 mg/L of the test item)

Table 6 : EC50 values of the reference item

Based on nominal concentrations [mg/l], 72 hours.

	Current Study	Valid Range (average ± 3 x SD)
	Growth Rate inhibition
ErC50	0.535	0.745 ± 0.524
95 % confidence interval	0.523 – 0.547
	Yield inhibition
EyC50	0.320	0.398 ± 0.291
95 % confidence interval	0.249 – 0.339

     SD = Standard deviation

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, the effect values of te test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on nominal test item concentrations):

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 43.5 (6.09 - > 50.0) mg/L and 36.6 (5.25 – 50.0) mg/L, respectively. The NOEC for growth rate and yield was 15.8 mg/L, respectively.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/ Method C.3 from 2018-02-05 to 2018-02-26, with the definitive exposure phase from 2018-02-20 to 2018-02-23 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The test item is a turquoise blue powder and has a water solubility of 774 mg/L. The study was conducted under static conditions with an initial cell density of 5523 cells/mL. A stock solution and four concentration levels were tested in a geometrical series with a dilution factor of √10: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L.

Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The measured concentrations in the test solutions at the start of the exposure were in a range of 92 to 99% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 91 to 103% of the nominal concentrations.

 

The test item concentrations remained within ± 20% of the nominal concentrations. Therefore, all effect values are given as nominal concentrations.