Reactive Orange 143

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:

27 January 2012 to 27 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in water: Stability for at least 6 hours at room temperature
Solubility in water: More than 80 g/L

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of FAT 40854/A tested was a reddish-brown powder with a purity of 46.2 % (4 main constituents). Preparation started with a stock solution of 10 g/l, prepared by dissolving 5.0059 g test substance in 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Volumes of the clear dark red stock solution corresponding to the test concentration were then added to the test media. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was coarsely sieved, washed and diluted with ISO-medium. 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 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

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 and temperature were determined in the remaining part of the reaction mixture.

Test temperature:

Between 18.8 and 21.7 °C

pH:

The pH in all test series, before addition of sludge remained within the range of 7.5 ± 0.5.
After the 3 hour exposure period the pH increased to a maximum of 8.4.

Dissolved oxygen:

The oxygen concentration at the start of the measurements was at least 7.1 mg O2/L.

(Mean) respiration rates:
Controls: 42 mg O2/L h
10 mg/L: 51 mg O2/L h
100 mg/L: 43 mg O2/L h
1000 mg/L: 44 mg O2/L h

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: All glass
- Aeration: During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 10 (1); 100 (1) and 1000 mg/L (3)
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.5 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The respiration rate from each vessel, in mg O2/L/hr, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.

TEST CONCENTRATIONS
- Test concentrations in a combined limit/range-finding test: 10, 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg FAT 40854/A per litre (Two Sample t-Test : α=0.05 Toxstat). There was no significant oxygen uptake from abiotic processes and no heterotrophic inhibition was observed at 1000 mg/l (tested with a nitrification inhibitor).

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (7.6 mg/L).

Reported statistics and error estimates:

ECx
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis. For FAT 40854/A no EC50 could be calculated because the test substance proved to be non-toxic (EC50 >1000 mg/L).


NOEC estimation
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

Validity criteria fulfilled:

yes

Conclusions:

NOEC of FAT 40854/A to activated sludge bacteria was at or below a 1000 mg/L and the EC50 was >1000 mg/L.

Executive summary:

In a GLP-compliant study, the influence of FAT 40854/A on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures were based on the OECD guideline 209 and EU method C11. The batch of FAT 40854/A tested was a reddish-brown powder with a purity of 46.2 % (4 main constituents). Preparation started with a stock solution of 10 g/l. Volumes of the clear dark red stock solution corresponding to the test concentration were then added to the test media. Optimal contact between the test substance and test medium was ensured 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 concentration of 10, 100 and 1000 mg/l were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested. In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg FAT 40854/A per litre. There was no significant oxygen uptake from abiotic processes and no heterotrophic inhibition was observed at 1000 mg/l (tested with a nitrification inhibitor). The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. FAT 40854/A was not toxic to wastewater (activated sludge) bacteria at a concentration of 1000 mg/l (NOEC). The EC50 exceeded 1000 mg/l.

Description of key information

The NOEC and EC50 of test substance to activated sludge bacteria was identified to 1000 and >1000mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In a GLP-compliant study, the influence of FAT 40854/A on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures were based on the OECD guideline 209 and EU method C11. In a combined limit / range-finding test concentration of 10, 100 and 1000 mg/l were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested. In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg FAT 40854/A per litre. There was no significant oxygen uptake from abiotic processes and no heterotrophic inhibition was observed at 1000 mg/l (tested with a nitrification inhibitor). The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. FAT 40854/A was not toxic to wastewater (activated sludge) bacteria at a concentration of 1000 mg/l (NOEC). The EC50 exceeded 1000 mg/l.