Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

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:

November 15th, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study described was conducted in accordance with SN EN 45001.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted on 4 April 1984

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Final test concentrations were prepared with test medium by dilution of stock solutions of the test substance.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Inoculum: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned.
- Source: ARA Werdhölzli, CH-80 48 Zürich(15 November 2000; 9.30 a.m.)
- Pretreatment: the activated sludge was used immediately after sampling from the treatment plant without adaptation. lf the sludge was used on the following day, 50 ml of the synthetic sewage concentratewere added per liter of activated sludge and the sludge aerated overnight.
- Preparation of inoculum for exposure: prior to the test, the sludge was washed twice with tap water.
- Initial biomass concentration: 1.6 g/l dry matter in the final mixture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

22 ± 0.5 °C

pH:

Whitin an acceptable range.

Nominal and measured concentrations:

62.5, 125, 250, 500, 1000, 2000 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 ml flasks containing a total volume of test suspension of 500 ml
- Aeration: vessels were aerated by shaking (120 r.p.m.).
- No. of vessels per concentration: one replicate sample (5 test concentrations) contained activated sludge + test medium + test substance.
- No. of vessels per control: two replicate samples contained activated sludge + test medium.
- No. of vessels per reference compound: one replicate sample (3 test concentrations) contained activated sludge + test medium + 3,S-dichlorophenol as reference compound.
- Abiotic control: contained test substance + test medium, but no sludge

TEST MEDIUM / WATER PARAMETERS
- Test medium: synthetic sewage feed prepared with tap water; 16 ml of 100-fold sewage concentrate per 500 ml of final test mixture.
- pH: 7.1 - 7.4
- Conductivitv (20 "C): 510 - 585 µS/cm
- CO3 Hardness: 15.3 - 27.8 °F
- CO3 Hardness after Heyer: 15.2 - 26.9 °F
- Rest Hardness: 2.4 - 4.1 °F
- Total Hardness: 17.7 - 31.9 °F
- KMnO4-consumotion: 1.0 - 1.3 mg/l
- E. Coli (at 44 °C): 0 % ml
- Colony count: 0 % per ml at 20 "C; 0- 5 % per ml at 30 °C.
- Chloride: 7.5 - 12.0 mg/l
- Sulfate: 17.5 - 21.0 mg/l
- Phosphate as PO4-P: <0.002 - 0.038 mg/l
- Ammonia as NH4-N: <0.01 mg/l
- Nitrite as NO2-N: <0.002 mg/l
- Nitrate as NO3-N: 7.0 - 12.0 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to pH 7.5 ± 0.5 with NaOH or H2SO4, if necessary.

EFFECT PARAMETERS MEASURED
Measuring apparatus: 300 ml flat bottom BOD flask; polarographic oxygen electrode, connected to a potentiometric recorder.
The median effective concentration (EC50 value) of the test substance is the calculated or interpolated inhibition value at which the respiration rate is 50 % of that shown by the blank control.

SAMPLING
The first sample (blank control B,) was analyzed 3 h after starting the test followed by the analysis of the test suspensions (T.) at 10-15 min intervals. Finally, the second blank control B, was analyzed.

Reference substance (positive control):

yes

Remarks:

3,S-dichlorophenol

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

The variation between the two controls was ± 0 %

Results with reference substance (positive control):

The EC50 value of the reference substance 3,S-dichlorophenol was estimated to be 5.0 mg/l thus confirming suitability of activated sludge and test conditions.

Activated sludge respiration rates after 3 h of exposure to the test material and calculation of the inhibition.

Nominal concentration (mg/l)	O2 consumption rates (mg O2/l/h	Respiration inhibition (%)
Control 0	53.7	-
Control 0	53.7	-
Test item 62.5	52.9	1.6
Test item 125	53.4	0.6
Test item 250	53.8	-0.2
Test item 500	52.6	2.1
Test item 1000	50.5	6.0
Test item 2000	52.7	1.8
3,S-dichlorophenol 3	33.2	38.3
3,S-dichlorophenol 10	18.1	66.3
3,S-dichlorophenol 30	5.8	89.3

Validity criteria fulfilled:

yes

Remarks:

the two control respiration rates were within 15 % of each other and EC50 (3h) of 3,5-dichlorophenol has been recorded in the accepted range 5 -30 mg/l

Conclusions:

IC50 (3h) > 2000 mg/l (nominal)
IC10 (3h) > 2000 mg/l (nominal)

Executive summary:

The inhibitory effect of test item on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions. The inhibitory effect may include the effect on respiration and nitrification. The exposure time of the test substance on the activated sludge was 3 hours.

The nominal concentrations of test item were 62.5, 125,250,500, 1000 and 2000 mg/l, respectively. These test concentrations were prepared by corresponding dilution of stock solutions of the test substance.

No chemical analysis of test concentrations was conducted. For the calculation of the effective concentration leading to 20, 50, and 80 % respiration inhibition (EC20, EC50, EC80) the nominal content of the test substance was used, assuming the test compound to be stable in water over 3 h.

At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

Conclusion

IC50 (3h) > 2000 mg/l (nominal)

IC10 (3h) > 2000 mg/l (nominal)

Description of key information

IC10 (3h) > 2000 mg/l (nominal)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

2 000 mg/L

Additional information

The inhibitory effect of test item on the oxygen consumption of activated sludge microorganisms was assayed in two separated studies.

In the key study of municipal sewage from treatment plant was investigated under aerobic static conditions, over a period of 3 hours. The nominal concentrations of test item were 62.5, 125,250,500, 1000 and 2000 mg/l, respectively. The test substance showed no significant toxic effects to the activated sludge and the percentage of respiration inhibition was determined to be 1.6, 0.6, -0.2, 2.1, 6 and 1.8, respectively; the respiration rates were in the same range as determined for the two blank controls, thus the EC10 resulted to be higher than 2000 mg/l (nominal).

The supporting experiment was conducted at 10 and 100 mg/l. During the test, it was noted that aeration caused the test solutions to foam and it was deduced that some of the test article was lost via foam. Under these conditions, the respiration rate of the sludge was slightly inhibited at 3 hours (18.9 %) at the highest concentration of 100 mg/l.

The percentage of inhibition in the second experiment results to be not consistent with the outcomes of the key study: 18.9 % of inhibition was recorded at 100 mg/l, while in the main test the inhibition was in the range of -0.2 - 6.0 % at concentrations between 125 and 2000 mg/l. Based on the available information, the foam observed in the second experiment may be responsible for the outcomes. This supposition can be considered as reliable if it is taken into consideration the consistency of the results obtained in the key study and the fact that 4 higher concentrations were assayed without significant impact (inhibition of less than 10 % of population).