Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 2 because the study followed a standard guideline of reference (OECD 203), which describes a procedure designed to evaluate this endpoint. The results were reviewed f or reliability and assessed as valid. However the study was not conducted under GLP condition.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

EC Number:

700-615-0

Molecular formula:

BIS: C82H74N14CuS2O6 TRIS: C107H103N17CuS3O9 TETRA: C132H132N20CuS4O12

IUPAC Name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Violet 3B
- Substance type: Organocopper salt
- Physical state: violet powder
- Lot/batch No.: 028942
- Expiration date of the lot/batch: 2013/05/24
- Stability under test conditions: Stable

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations:10, 31.6, 100, 316 et 1000 mg/L.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: The day of the sampling of the sludge, the sludge is centrifuged for 10 min (1100g). The supernatant is eliminated and the sludge is re-suspended in declorinated tap water . The washing and centrifuging process is repeated 2 more times. The sludge solids concentration is then measured in the inoculum and in the test vessels.

The tap water is dechlorinated by stripping for around 24 hours: free chloride concentration < at 30 µg/L.

Study design

Test type:

static

Limit test:

no

Total exposure duration:

180 min

Test conditions

Test temperature:

20°C +/-2 °C

Nominal and measured concentrations:

nominal concentrations: 10, 31.6, 100, 316 et 1000 mg/L, directly weighed into the test vessels.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass bottles
- Material, size, headspace, fill volume: wide-necked glass bottles of 1 liter
- Test vessel for measurment of dissolved oxygen: BDO flask of 250 mL.
- Aeration: yes - filtered compressed air with a flow rate of 0.5 to 1 L/min

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophénol at 4.8, 12 and 30 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

ca. 292 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: witout ATU

Effect concentrations 2

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

ca. 220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: with ATU

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test item poorly soluble

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50-180 min = 12.2 mg/l for the total respiration and EC50-180 min = 9.4 mg/L for the heterotrophic respiration

Any other information on results incl. tables

Total respiration : Sepisol Fast Violet 3B

Time (min)	Blank control n°1	Reference substance			Sepisol Fast Violet 3B					Blank control 2
		4 mg/L	12 mg/L	36 mg/L	10 mg/L	31.6 mg/L	100 mg/L	316 mg/L	1000 mg/L
0	6.9	6.6	7.8	9.0	5.4	6.5	7.0	6.9	7.2	6.6
1	6.5	6.3	7.8	7.9	4.7	5.8	6.4	6.6	7.0	6.0
2	6.2	6.0	7.5	7.9	4.7	5.8	6.4	6.6	7.0	6.0
3	5.9	5.7	7.4	7.8	4.2	5.5	6.4	6.4	6.8	5.6
4	5.5	5.5	7.1	7.8	3.8	5.1	6.2	6.3	6.7	5.3
5	5.1	5.2	7.0	7.7	3.4	4.6	5.8	6.1	6.6	4.9
6	4.7	4.9	6.7	7.6	3.0	4.3	5.7	6.0	6.5	4.6
7	4.3	4.6	6.5	7.6	2.5	4.0	5.4	5.9	6.4	4.3
8	4.0	4.3	6.3	7.6	2.1	3.6	5.2	5.6	6.3	3.9
9	3.6	4.0	6.1	7.5	1.7	3.3	4.9	5.5	6.1	3.5
10	3.2	3.7	5.9	7.5	1.4	2.9	4.8	5.3	6.0	3.2
Respiratory rateRin mgO2/L/h	22.1	170	11.6	3.5	24.1	21.5	13.3	9.4	7.0	20.8
% of inhibition vs blank	/	21%	46%	84%	-12%	0%	38%	56%	67%	/
Mean of the blank controls			21.5
Gap between blank controls			6.1%

CE50= 292 mg/L

CE20 = 67.6 mg/L

CE10 = 31.4 mg/L

Heterotrophic respiration: Sepisol Fast Violet 3B + ATU

Time (min)	Blank control n°1	Reference substance			Sepisol Fast Violet 3B					Blank control 2
		4 mg/L	12 mg/L	36 mg/L	10 mg/L	31.6 mg/L	100 mg/L	316 mg/L	1000 mg/L
0	5.88	7.01	6.73	7.61	6.69	6.38	6.57	7.39	7.55	6.79
1	5.49	6.84	6.34	7.59	6.23	6.02	6.36	7.28	7.46	6.35
2	5.1	6.55	6	7.54	5.85	5.79	6.15	7.09	7.39	6
3	4.69	6.24	5.83	7.51	5.39	5.4	5.9	6.89	7.27	5.62
4	4.33	6.02	5.68	7.43	5.01	4.83	5.69	6.71	7.23	5.2
5	3.97	5.73	5.54	7.38	4.58	4.72	5.49	6.56	7.13	4.87
6	3.65	5.48	5.37	7.33	4.17	4.35	5.28	6.42	6.99	4.45
7	3.12	5.18	5.15	7.28	3.71	3.99	5.05	6.22	6.99	4.07
8	2.91	4.87	5.13	7.22	3.32	3.56	4.85	6.09	6.84	3.65
9	2.57	4.61	5.01	7.17	2.88	3.21	4.63	5.93	6.74	3.27
10	2.19	4.31	4.92	7.11	2.45	2.85	4.38	5.74	6.63	2.81
Respiratory rateRin mgO2/L/h	22.1	16.2	10.9	3.0	25.4	21.2	13.1	9.8	5.5	23.9
% of inhibition vs blank	/	30%	53%	87%	-11%	8%	43%	57%	76%	/
Mean of the blank controls			23
Gap between blank controls			-7.6%

CE50= 220 mg/L

CE20 = 52.4 mg/L

CE10 = 24.7 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of the test item Sepisol Fact Violet 3B vis-à-vis the respiration rate of activated sludge is equal to 292 mg/L for the total respiration and to 220 mg/L for the heterotrophic respiration. The oxygen uptake rate due to the nitrification process is negligible.