Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Real Decreto 363/1995 (BOE núm. 133 de Junio de 1995).

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test concentrations: 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L, plus two controls.

Preparation of stock and test solutions: A stock solution has been prepared with a concentration of 1 g/L of active matter of the test item. Therefore, 1.025 g of the test item has been dissolved with 100 mL of deionized water in a 100 mL Class A volumetric flask by shaking, so we have a 10250 mg/L stock solution. Because the study material presents an average of 97.54% of active matter, the stock solution contains 1000 mg/L of active matter. The different solutions used in the test have been prepared from this 1000 mg/L stock solution.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Preparation of inoculum for exposure: Based on the known concentration of the of dried activated sludge (7.2 g/L) from a domestic waste water treatment plant, a suspestion is prepared by adding water until the concentration os suspended matter is 4 g/L. Then, 200 ml of this suspended sluge solution, is added to the vessels with the rest of components up to a final volume of 500 mL. Therefore, the final concentration of activated sludge (supended solids) is 1.6 g/L
- Pretreatment: no
- Initial biomass concentration: 4g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20±2 °C

pH:

pH values at initial times:
control (7.23)
10 mg/L (7,25)
32 mg/L (7,22)
100 mg/L (7,16)
320 mg/L (7,12)
1000 mg/L (7,06)
control (7,22)

Nominal and measured concentrations:

Concentration of the test substance: 10 mg/L; 32 mg/L; 100 mg/L; 320 mg/L; 1000 mg/L and two controls.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass
- Aeration: yes, continous flux of 1L/min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- No. of vessels per abiotic control (replicates): n.a.
- Sludge concentration (weight of dry solids per volume): 7.2 g/L
- Nutrients provided for bacteria: Residual synthetic water prepared according to OECD 209
—Peptone 16 g
—Meat extract 11 g
—Urea 3 g
—NaCl 0,7 g
—CaCl2.2H2O 0,4 g
—MgSO4.7H2O 0,2 g
—K2HPO4 2,8 g
Diluted to 1000 mL with deionized water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated water
- Particulate matter: not specified

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: The incubation period begins when 200 mL of activated sludge is added at 15 minute intervals to each of the test vessels. All containers are aerated for a period of 3 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Oxygen measurements (mg/L) during the respiration inhibition test. Values were collected in 1 minute intervals for a period of 10 minutes (measured with an oxigen electrode cell).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

262 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Concentration of dissolved oxygen during the inhibition respiratory test where analyzed at 1 minute intervals.
Calculation of oxygen uptake rates are measured for each concentration and control.
Calculation of percentage of inhibition is also reported.
As a conclusion: the substance does not produce any respiratory inhibition in the activated sludges at the maximum concentration of tested substance corresponding to 100 mg/l.
CE50 (3h)>100mg/L

Results with reference substance (positive control):

Respiration rate of the test control are: 23.7 mgO2/L.h and 24.36 mgO2/L.h. The difference between both values is 2,7% which is inside the acceptation criteria (15%).
CE50-3hr of 3,5 Dichlorophenol is 12 mg/L, which is between the acceptation range of the OECD 209 guide (5mg/L - 30mg/L).

Table 1. Oxygen measurements (mg/L) during the respiration inhibition test:

conc (mg/L)	mg O2/L in the time interval (minutes
	0	1	2	3	4	5	6	7	8	9	10
control	7.35	6.90	6.46	5.99	5.56	5.08	4.65	4.19	3.73	3.28	2.84
10	7.28	6.82	6.36	5.94	5.45	5.01	4.57	4.11	3.64	3.20	2.75
32	7.26	6.86	6.42	6.02	5.60	5.19	4.77	4.38	3.95	3.54	3.12
100	7.00	6.67	6.36	6.06	5.73	5.40	5.09	4.77	4.46	4.14	3.84
320	7.22	7.04	6.85	6.65	6.47	6.27	6.08	5.88	5.79	5.51	5.31
1000	7.40	7.28	7.17	7.02	6.93	6.81	6.70	6.57	6.44	6.32	6.21
control	7.32	6.89	6.43	5.97	5.55	5.09	4.63	4.15	3.74	2.30	2.85

 

Table 2. Respiration rate (mg L-1 h-1) obtained for each of the concentrations and controls:

	control	10	32	100	320	1000	control
respiration rate (mg L-1 h-1)	27.06	27.18	24.84	18.96	11.43	7.14	26.82

 

 

Table 3. % Inhibition of respiration at 3h:

conc (mg/L)	% Inhibition of respiration
control	0
10	0
32	7.8
100	29.6
320	57.5
1000	73.5

 

 

Validity criteria fulfilled:

yes

Conclusions:

The test item Direct Black 155 RBB (batch: 12336-d) has a 3h EC50 value equal to 262 mg/L of based on active matter.

Executive summary:

The study of bacterial inhibition in activated sludge of the Direct Black 155 RBB dye 97.54% active matter (batch: 12336-d) has been carried out. For the determination of bacterial inhibition, OECD 209 (1984) and Royal Decree 363/1995 (BOE no. 133 of June 1995) have been followed. This method is used to evaluate the effect that the test substance has on microorganisms, measuring the respiration rate under certain conditions and at different concentrations. Activated sludge from an urban sewage treatment plant has been exposed to the dye Direct Black 155 RBB at test concentrations: 10, 32, 100, 320 and 1000 mg/L based on active material. The total duration of the test has been 3 hours. The result obtained in this test indicates that the dye Direct Black 155 RBB has an EC50 value (3h) equal to 262 mg/L based on active material after 3 hours of testing.

 

 

Description of key information

One study is available to determine the inhibition growth of micro-organisms of the substance. The study gives a value of EC50 of 262 mg/L, obtained by calculations based on experimental results.

Key value for chemical safety assessment

EC50 for microorganisms:

262 mg/L