2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted on 4 April 1984

GLP compliance:

not specified

Analytical monitoring:

no

Test organisms (species):

activated sludge

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-8048 Zürich (3 September 2002;9.30 a.m.).
- Preparation of inoculum for exposure: the activated sludge was used immediately after sampling from the treatment plant without adaptation. 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

Nominal and measured concentrations:

125, 250, 500, 1000 and 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: flasks have been aerated by shaking (120 r.p.m.) for the whole test period of 3 hours.
- No. of vessels per concentration: one replicate sample (5 test concentrations) contained activated sludge + test medium + test substance.
- No. of vessels per blank control: two replicate samples contained activated sludge + test medium.
- No. of vessels per positive control:one replicate sample (3 test concentrations) contained activated sludge + test medium + 3,5-dichlorophenol as reference compound.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: synthetic sewage feed prepared with tap water. 16 ml of 100-fold sewage concentrate per 500 ml of final test mixture.
- Composition of tap water:
pH: 7.1 - 7.4
Conductivitv (20 °C): 510 - 585 µS/cm
CO3 - Hardness: 15.3 - 27.8 F
CO3 - Hardness after Hever: 15.2 - 26.9 F
Rest - Hardness: 2.4 - 4.1 F
Total Hardness: 17.7 - 31.9 F
KMnO4-consumption: 1.0 - 1.3 mg/l
E. Coli 44 °C: 0 per 100 ml
Colony count: none
Chloride: 7.5 - 12.0 mg/l
Sulfate: 17.5 - 21.0 mg/l
Phosohate 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
- Composition of the synthetic sewage feed:
Peptone 16 g
Meat extract 11 g
Urea 3 g
Sodium chloride 0.7 g
Calcium chloride dihvdrate 0.4 g
Maqnesium sulfate heptahvdrate 0.2 g
Dipotassium hvdrooen ohosohate 2.8 g
- pH: the initial pH-value was 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED
- Measuring apparatus: 300 ml flat bottom BOD-flask; polarographic oxygen electrode, connected to a potentiometric recorder.

VALIDITY
- The respiration rates of the two controls should be within 15 % of each other
- The EC50 of the reference compound should be in a range of 5 to 30 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 890 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The test substance showed significant toxic effects to the activated sludge (> 15 % respiration inhibition) at all test concentrations. The following percentages of respiration inhibition were observed: 2000 mg/l (51 %), 1000 mg/l (42 %), 500 mg/l (37 %), 250 mg/l (31 %), 125 mg/l (22 %).
Based on these data the EC20 and EC5 values of were calculated to be 91.2 and 1890 mg/|, respectively. The EC80 value was estimated to be > 2000 mg/l.
The variation between the two controls was ± 5.4 %. Therefore, the test was considered valid.

Results with reference substance (positive control):

The EC50 value of the reference substance 3,5-dichlorophenol was estimated to be 3.6 mg/l which was low but in the range of 5 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)	Vessel	O2 consumption rates (mg/O2/l/hr)	Respiration inhibition (%)
0	Blank	27.2	-
0	Blank	24.4	-
125	Test item	20.1	22.3
25	Test item	17.7	31.3
500	Test item	16.2	37.3
1000	Test item	15.0	42.1
2000	Test item	12.6	51.2
3	3,5-dichlorophenol	13.6	47.5
10	3,5-dichlorophenol	8.9	657
30	3,5-dichlorophenol	3.4	86.8

Conclusions:

EC50 (3h): 1989 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 were 125, 250, 500, 1000 and 2000 mg/l, respectively. These test concentrations were prepared by direct addition 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. The test substance showed significant toxic effects to the activated sludge (>15 % respiration inhibition) at all test concentrations. The following percentages of respiration inhibition were observed: 2000 mg/l (51 %), 1000 mg/l (42 %), 500 mg/l (37 %), 250 mg/l (31 %), 125 mg/l (22 %). Based on these data the EC20 and EC50 values were calculated to be 91.2 and 1890 mg/l, respectively. The EC80 value was estimated to be > 2000 mg/l.

Conclusion

EC50 (3h): 1989 mg/l (nominal)

Description of key information

Not harmful/toxic to mictoorganisms

Key value for chemical safety assessment

EC50 for microorganisms:

1 989 mg/L

Additional information

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 test substance showed significant toxic effects to the activated sludge (> 15 % respiration inhibition) at all tested concentrations. The following percentages of respiration inhibition were observed: 2000 mg/l (51 %), 1000 mg/l (42 %), 500 mg/l (37 %), 250 mg/l (31 %), 125 mg/l (22 %). Based on these data the EC20 and EC50 values were calculated to be 91.2 and 1890 mg/l, respectively. The EC80 value was estimated to be > 2000 mg/l.