Reaction product of Chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium, sulphuric acid and caustic soda

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please find the attached justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

3 min

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Result of RA Source CAS 1328-53-6 (BASFSE, 2002)

Duration:

3 min

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Result of RA Source CAS 1328-53-6 (BASFSE, 2002)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Remarks on result:

other: Result of RA Source CAS 28901-96-4 (BASFSE, 2015)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Remarks on result:

other: Result of RA Source CAS 28901-96-4 (BASFSE, 2015)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Remarks on result:

other: Result of RA Source CAS 28901-96-4 (BASFSE, 2015); not reported but determined by the assessor

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04-07-91 to 12-07-91

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

April, 1984

Principles of method if other than guideline:

- Sludge from a Busman laboratory apparatus; higher microbiocial inoculum concentration; higher synthetic sewage feed concentration; higher temperature variation

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): FAT-60'149/E
- Batch No.: EN 199474/06
- Expiry date: February 1992
- Stability: guaranteed by the sponsor
- Solubility: miscible
- Storage: room temperature
- Appearance: liquid

Analytical monitoring:

no

Vehicle:

not specified

Test organisms (species):

other: mixed culture of bacteria

Details on inoculum:

Sludge from a Husman laboratory apparatus (2.0 g/L)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

24.8 °C

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1, 3.2, 10, 32, 100 mg/L

Details on test conditions:

EFFECT PARAMETERS MEASURED: O2-consumption, measured with an oxygen-sensitive electrode system: OXI DIGI 530

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

The 3-h IC50 of the reference substance 3,5-Dichlorophenol is: 16.5 mg/L

Reported statistics and error estimates:

The respiration rate of each test concentration was calculated as a percentage of the mean of the two control respiration rates. The results were plotted on a log (concentration)/normal (% inhibition) graph of which the EC50 value was derived.

Consumption rate and inhibitory effects of the test substance and reference

	3h
Test substance (mg/L)	O2-consumption (mg/L/min)	Inhibition (%)
100	1.78	11.44
32	1.88	6.47
10	2.10	0
3.2	2.07	0
1	2.10	0
0.32	-	-
0.1	-	-
0.032	-	-
Blank 1	2.15	2.01
Blank 2	1.87
Positive control 100 mg/L	0.12	94.03
Positive control 32 mg/L	0.32	84.08
Positive control 10 mg/L	1.52	24.38

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

this document is a summary-reprint of a test report

Qualifier:

according to guideline

Guideline:

DIN 38412-27 (Pseudomonas putida Zellvermehrungshemmtest)

Version / remarks:

draft

Principles of method if other than guideline:

The test is used for insoluble, sparingly soluble and strongly coloured substances.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Heliogen Grün 8730; Hexadecachlorkupferphthalocyanin

Analytical monitoring:

not specified

Vehicle:

yes

Remarks:

Tween 80

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the stock solution (10,000 mg/L with 100 mg/L dispersant) was stirred for 17 hours.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Strain: DSM 50026
- Medium: 25 g/L Nutrient broth I; 18 g/L Agar poured into slant-agar tubes
- Maintanance temperature: 24 ± 1 °C
- Duration of growth: 24h
- Storage: 4 °C
- Culturing: the stemculture is taken up in 0.9 % strength NaCI solution. Batches of 200 mL of culture fluid <25 g/L of nutrient broth I) are inoculated with ceII suspension under sterile conditions. The cells are harvested after 16 ± 2 h of shaking on a rotary shaker at 22 ± 2 °C and 200 r.p.m.
- CeII harvest and adjustment of ceII suspending: the cells harvested are seperated by centrifugation for 10 min at 6000 r.p.m. and at 10 °C in a refrigerated centrifuge, washed twice with phosphate buffer and resuspended in buffer. Absorbence
measurements are carried out in 1 cm cells an an aliquot of the ceII suspension at a dilution of 1: 100. A value of 200 TE/F is set at a wavelength of 436 nm against the buffer blank value.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Test temperature:

25 °C

pH:

7.6 - 8.4

Nominal and measured concentrations:

Nominal concentrations: 0, 5000 and 10,000 mg/L

Details on test conditions:

TEST SYSTEM
- Total volume: 100 mL
- Other: 95 mL of the test substance in the given concentrations, 2-3 mL of bacterial suspension (the volume of the suspension must be chosen such that the oxygen consumption of the blank value is 1 .5 - 2.5 mg/L*min).
- Aeration: yes
- Replicates: 2

EFFECT PARAMETERS MEASURED: decline in the concentration of dissolved oxygen, measured in a flow cell

Reference substance (positive control):

not specified

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Validity criteria fulfilled:

yes

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 May 2014 to 06 May 2014 (collection of sludge to end of exposure)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

May, 2008

Principles of method if other than guideline:

The start value of oxygen content for measurement of oxygen consumption was lower than 7 mg/L.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Pigment Additive PAG / Cu-phthalocyanine monosulfonic acid
- Test substance No.: 14/0146-1
- Batch identification: 130001/RA gemahlen (Laborjournalnr. V12424/ RA)
- Identity: The IR spectrum showed the bands expected for the test item.
- Purity: No purity of the test item can be stated by NMR or HPLC.
- Homogeneity: Homogeneous
- Expiry date: 17 Feb 2034
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer
- Date of production: 17 Feb 2014
- Physical state/Appearance: Solid/ blue
- Water solubility: Miscible
- Storage conditions: Ambient at room temperature; protect against humidity

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Application of the test substance: Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.
- Reference substance preparations: 1000.0 mg of the reference substance was added to about 800 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.1 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with deionized water. The stock solution appeared colorless-clear and no undissolved reference substance was visible.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Age: 1 day
- Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
- Collection of the test system: 05 May 2014
- Arrival in the test facility: 05 May 2014

After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.9 - 21.3 °C

pH:

- Start: 7.1 - 7.6
- End: 8.0 - 8.1

Dissolved oxygen:

6.3 - >7 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L
- Measured concentrations: no analytical verification of stock solution.

Details on test conditions:

TEST SYSTEM
- Test vessels: Glas-beakers (nominal volume 1L)
- Test volume: 500 mL
- Sludge concentration: 1.5 g/L Dw
- Test replicates: 6 for the control, 3 for each substance concentration and 2 for each reference substance concentration

TEST MEDIUM / WATER PARAMETERS
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

EFFECT PARAMETERS MEASURED: The consumption rate (mg O2/L*x minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to
calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.
- Duration of the measurement of oxygen consumption: about 8 - 10 min

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

OTHER TEST CONDITIONS
- Adjustment of pH: yes

PREPARATION OF THE TEST
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked and adjusted in the relevant test assays. After addition of 250 mL of inoculum suspension (3 g/L Dw) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. After 3 hours incubation at 20 ± 2 °C the mixtures in the test vessels were placed subsequently for oxygen measuring. The content of oxygen at the start of the measurements was > 7 mg/L with some exceptions; see 'Any other information on materials and methods incl tables'. The total oxygen consumption was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at last. No abiotic control was tested. The oxygen uptake was measured for a period of about 8 to 10 minutes or until the oxygen concentration fell below 2 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: not reported, but determined by the assessor

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No monotone concentration response exists, see 'Any other information on results incl. tables'. Therefore the ECx calculation is not meaningful and the EC10 value is assumed to be greater than 1000 mg/L.

Results with reference substance (positive control):

The 3-h EC50 of the reference substance was 13.6 mg/L.

Reported statistics and error estimates:

The consumption rates were used for the determination of the ECx by the probit method based on Finney [Finney, D.J., Probit Analysis; Cambr. Univ. Press, 3rd ed., 1971] with the software TOXRAT Professional 2.10 [Ratte, M.: ToxRat Professional 2.10 (2001-2010). ToxRat Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany].

Respiration rate in activated sludge after 3.0 h

Treatment (mg/L)	Mean	Std. Dev.	N	% decrease
Control	43.167	3.3714	6	0.0
62.5	43.333	0.5774	3	-0.4
125.0	46.333	2.5166	3	-7.3
250.0	50.667	2.3094	3	-17.4
500.0	47.000	3.0000	3	-8.9
1000.0	44.667	2.5166	3	-3.5

Description of key information

EC50(3h) > 100 mg(L (nominal) for respiration inhibition of sludge (OECD 209, read across)

Additional information

In total, three studies are used to assess the toxicity to microorganisms. One study has been performed with the substance itself and two other studies are performed with structural analogues as part of a weight of evidence approach. The structural analogues used are Pigment Green 7 (CAS 1328-53-6) and Pigment Additive PAG (CAS 28901-96-4).

 

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (BASF 1991). In this study a mixed culture of bacteria (sludge from a Husman laboratory apparatus) was exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10.0, 32.0, and 100 mg/L for 3 hours. Analytical verification of the test concentration was not performed/is not required. The effects on microorganisms were determined by measuring total respiration rates and comparing these to an unexposed control. A slight negative inhibition of respiration rates was observed at the two highest concentrations (6.47% and 11.44%, respectively). From the test a 3-h EC50 of >100 mg/L was derived.

 

The toxicity to microorganisms was also determined with structural analogue Pigment Green 7 (CAS 1328-53-6) in a study according to DIN 38412 part 27 (draft) (BASFSE, 2002). In this study, bacteria (Pseudomonas putida) were exposed to nominal test concentrations of 5000 and 10,000 mg/L for 30 minutes at 25 °C. Analytical verification of the test concentration was not performed/is not required. Oxygen consumption was determined at the end of the test. The 0.5-h EC50, EC10 and EC90 were determined to be >10,000 mg/L.

 

Additionally, the toxicity of Pigment Additive PAG (CAS 28901-96-4) to microorganisms was assessed in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (BASF 2015). In this study activated sludge from a municipal wastewater treatment plant was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. Analytical verification of the test concentration was not performed/is not required. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. No inhibition of respiration rate was observed at any tested concentration. From the test 3-h EC50 and 3-h EC10 values of >1000 mg/L were derived. Although no NOEC was derived in the study report, the complete absence of inhibition of respiration up to the highest tested concentration of 1000 mg/L justifies the NOEC to be determined at ≥1000 mg/L.

 

Overall, in all studies no effects on microorganisms were observed. The 3-h EC50 value is determined to be >100 mg/L.