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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
The synthetic medium (16 mL) made up to 50 mL with Milli-RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.

The medium temperature was recorded continuously in two temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
GLP compliance:
yes
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA), with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 10 minutes. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded unless otherwise requested by the Study Director.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.

Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Medium: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-
Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Rationale: Recognized by international guidelines as the recommended test system.
Test type:
not specified
Water media type:
other: Milli-RO water purified by reverse osmosis
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
18-21 °C
pH:
The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.4 and 8.3.
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: ELR50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
inhibition of total respiration
Validity criteria fulfilled:
yes
Conclusions:
No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Disperse Blue 359 per litre.
The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol and showed normal sensitivity.
The study met the acceptability criteria prescribed by the study plan and was considered valid.
Disperse Blue 359 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50was above 1000 mg/L
Executive summary:

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for approximately 10 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Disperse Blue 359 per litre.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Disperse Blue 359 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50was above 1000 mg/L

Description of key information

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for approximately 10 minutes. Subsequently. Synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Disperse Blue 359 per litre.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Disperse Blue 359 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50was above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for approximately 10 minutes. Subsequently. Synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Disperse Blue 359 per litre.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Disperse Blue 359 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50was above 1000 mg/L