Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfonated

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental part of study was performed in period from 2013-09-12 to 2013-10-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

The inoculum contained a mixed population of mocroorganisms obtained from secondary effluent of the STP of Pardubice processing predominantly municipal sewage.
The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.
The inoculum was in conformity with recomendations of the test guideline.

Duration of test (contact time):

28 d

Initial conc.:

2.09 mg/L

Based on:

test mat.

Initial conc.:

3.4 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The solution of test substance in mineral medium is inoculated with a relatively small number of microorganisms from a mixed population and kept in completely full closed bottles in the dark at constant temperature. Degradation is determined by analysis of dissolved oxygen over a 28-day period.
The following determinations were done simultaneously under the same conditions:
- check of inoculum activity with the reference substance
- toxicity test of the test substance for used inoculum in mixture of the test and reference
substance
- determination of oxygen consumption for nitrification
- determination of blank (inoculated mineral medium)
The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of COD.

TEST CONDITIONS
- Composition of medium:
Solution A: KH2PO4 8.5 g + K2HPO4 21.75 g + Na2HPO4.2H2O 33.4 g + NH4Cl 0.5 g + water up to 1000 mL
Solution B: MgSO4.7 H2O 22.5 g + water up to 1000 mL
Solution C:CaCl2 27.5 + water up to 1000 mL
Solution D: FeCl3.6H20 0.25 g + water up to 1000 mL
water with conductivity less then 5 microS/cm

Mineral medium: 1 mL A + 1 mL B + 1 mL C + 1 mL D + aerated water up to 1000 mL (1 day stay in laboratory)

Inoculated medium:
The mineral medium was strongly aerated for 20 min. and let standing for 20 h at the test temperature. The volume of waste water for the inoculation of mineral medium was chosen 2.1 mL per 1 L of medium (COD of waste water: 50 mg·L-1).
30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 60 mL of modified waste water. The pH value of medium: 7.37.

Test substance:
The stock solution of test substance was prepared in concentration 0.1043 g.L-1 of water. From this solution 120 mL (20 mL.L-1) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L.
The solution prepared in this way contained 2.09 mg.L-1 of test substance, and had COD 3.40 mg.L-1. The pH value of solution: 7.43.

Reference substance:
The stock solution of sodium benzoate was prepared in concentration 1.0000 g·L-1of water. From this solution the 12.6 mL (2.1 mL·L-1) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L. The solution prepared in this way contained 2.10 mg·L-1 of sodium benzoate, and had COD 3.45 mg·L-1. The pH value of solution: 7.37.

Toxicity test
The solution was prepared by dosing 80 mL of stock solution of test substance and 8.4 mL of stock solution of reference substance into inoculated medium and completed with this medium to the volume of 4 L. The mixture prepared in this way contained 2.09 mg.L-1 of test substance and 2.10 mg.L-1 of sodium benzoate and had COD 6.85 mg.L-1. The pH value of mixture: 7.40.

Control (blank) determination
The inoculated mineral medium without test substance was used for the blank determination.
- Solubilising agent (type and concentration if used): no
- Test temperature: 20 +/- 1.0 °C
- pH: 7.4
- pH adjusted: no
- CEC (meq/100 g):
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: large glass bottles of 5 and 10 L for preparation and inoculation medium
- Number of culture flasks/concentration:
2 x 9 bottles with test substance and inoculated medium
2x9 bottles with reference substance and inoculated medium for check of inoculum activity
9 bottles with test substance and inoculated medium for determination of oxidized nitrogens form
2x5 bottles with test and reference substance mixture and inoculated medium for the toxicity test
2x9 bottles with inoculated medium only for the blank determination
- Method used to create aerobic conditions: solutions were prepared from aerated water
- Measuring equipment: oximeter WTW Oxi 730 with membrane probe CellOx 325

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: oxidized nitrogen form

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

7.1

Sampling time:

28 d

Results with reference substance:

Biodegradation with the reference substance satisfied the 10-day window criteria. The Average degradation with the reference substance at the end of the 28 day test period was determined to be 85.2 %.

Table 1: Biodegradation of test material and reference substance

		Biodegradation (%) after period (days)
		0	3	7	10	14	17	21	24	28
Test substance	1	0	-0.5	0.4	-0.5	-1.6	2.2	2.5	2.9	7.5
	2	0	-1.1	1.3	-1.4	-1.9	2.8	0.7	3.2	6.6
	average	0	-0.8	0.9	-1.0	-1.8	2.5	1.6	3.1	7.1
Reference substance	1	0	55.8	76.2	79.6	82.8	81.7	89.1	85.1	85.9
	2	0	54.9	74.8	77.2	78.1	77.7	87.4	84.8	84.5
	average	0	55.4	75.5	78.4	80.5	79.7	88.3	85.0	85.2

 

Table 2: Results of toxicity test

		Biodegradation (%) after period (days)
		0	3	7	10	14
Test + Reference substance	1	0	29.7	38.8	41.1	42.0
	2	0	29.6	38.1	39.2	39.3
	average	0	29.7	38.5	40.2	40.7

 

Table 3: DOC measurements (main test)

	Replicate	Dissolved oxygen (mg/L) after period (days)
		0	3	7	10	14	17	21	24	28
Test substance	1	8.60	8.37	8.13	8.15	8.04	7.90	8.68	8.55	7.82
	2	8.58	8.37	8.08	8.16	8.03	7.86	8.72	8.52	7.83
	average	8.59	8.37	8.11	8.16	8.04	7.88	8.70	8.54	7.83
Reference Substance	1	8.62	6.43	5.58	5.48	5.19	5.16	5.70	5.77	5.07
	2	8.56	6.40	5.57	5.50	5.29	5.24	5.70	5.72	5.06
	average	8.59	6.42	5.58	5.49	5.24	5.20	5.70	5.75	5.07
Blank Experiment	1	8.67	8.38	8.24	8.24	8.09	7.97	8.84	8.75	8.07
	2	8.62	8.38	8.23	8.26	8.05	8.04	8.76	8.71	8.05
	average	8.65	8.38	8.24	8.25	8.07	8.01	8.80	8.73	8.06

 

 

Table 4: DOC measurements (toxicity test)

	Replicate	Dissolved oxygen (mg·L^-1) after period (days)
		0	3	7	10	14
Test + Reference substance	1	8.59	6.29	5.52	5.38	5.14
	2	8.60	6.31	5.58	5.52	5.33
	Average	8.60	6.30	5.55	5.45	5.24
Blank experiment	1	8.67	8.38	8.24	8.24	8.09
	2	8.62	8.38	8.23	8.26	8.05
	average	8.65	8.38	8.24	8.25	8.07

 

Table 5: Nitrification measurements (main test)

Incubation period in days	0	3	7	10	14	17	21	24	28
Concentration of N-NO3^- (mg/L)	0.146	0.142	0.156	0.162	0.155	0.143	0.144	0.154	0.133
Concentration change of N-NO3^- (mg/L)	0	-0.004	0.01	0.016	0.009	-0.003	-0.002	0.008	-0.013
Oxygen equivalent N-NO3^- (mg/L)	0	0.018	0.046	0.073	0.041	0.014	0.009	0.037	0.059
Concentration of N-NO2^- (mg/L)	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Concentration change of N-NO2^- (mg/L)	0	0	0	0	0	0	0	0	0
Oxygen equivalent N-NO2^- (mg/L)	0	0	0	0	0	0	0	0	0
Total oxygen equivalent (mg/L)	0	0.018	0.046	0.073	0.041	0.014	0.009	0.037	0.059

  Oxygen equivalent N-NO₃^- (mg.L^-1) = 4.57 (factor) x increase in nitrate concentration

Oxygen equivalent N-NO₂^- (mg.L^-1) = 3.43 (factor) x increase in nitrite concentration

Total oxygen equivalent (mg.L^-1) =Oxygen equivalent N-NO₃^-  + Oxygen equivalent N-NO₂^- 

Nitrite ion was not detected, so in this case:

Total oxygen equivalent (mg.L^-1) =Oxygen equivalent N-NO₃^-  

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance was not inhibiting for the used inoculum. In this 28-day study of ready biological degradability the degradation of 7.1 % of the test substance was attained in the end of study.  

Executive summary:

The test substance was tested for the ready biological degradability in Closed Bottle Test performed according to: Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008. The results of biological degradation are related to experimentally determined COD values of test and reference substance at the beginning of the test.

The test substance was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out. Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

COD of the test substance in medium at the beginning of the main test: 3.40 mg·L-1.

COD of reference substance in medium at the beginning of the main test: 3.45 mg·L-1.

In parallel to the main test the toxicity test was performed. Based on the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out. The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7-8 at the beginning of the test. The prescribed validity criteria in the test were fulfilled. The test substance was not inhibiting for the used inoculum. In this 28-day study of ready biological degradability the degradation of 7.1 % of the test substance was attained in the end of study.  

Description of key information

The test substance was tested for the ready biological degradability in Closed Bottle Test performed according to: Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008. The results of biological degradation are related to experimentally determined COD values of test and reference substance at the beginning of the test.

The test substance was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out. Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

COD of the test substance in medium at the beginning of the main test: 3.40 mg·L-1.

COD of reference substance in medium at the beginning of the main test: 3.45 mg·L-1.

In parallel to the main test the toxicity test was performed. Based on the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out. The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7-8 at the beginning of the test. The prescribed validity criteria in the test were fulfilled. The test substance was not inhibiting for the used inoculum. In this 28-day study of ready biological degradability the degradation of 7.1 % of the test substance was attained in the end of study.  

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

[Type of water: freshwater]