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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2011 - 24 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:
no
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
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
Test substance – preparation of concentrations
The batch of TDI-Urone tested was a clear colourless powder as described by the sponsor, with a purity of 97.5% active content (being a mixture of isomers). At NOTOX the test substance was observed to be a white powder. This minor difference in description of the appearance of the test substance was a result of individual objective observation and was discussed with the sponsor. The intended description of TDI-Urone was confirmed and the batch of TDI-Urone was used for the test.

A stock solution of 10 g/l was prepared by adding 10.0003 g test substance to 1000 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 5 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colorless 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:
Preparation of inoculum for exposure:
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 7.6 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:
Not applicable.
Hardness:
Not reported.
Test temperature:
20.1 - 21.1°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 8.5.
Dissolved oxygen:
The dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) -> 7.9-9.0 mg/L
Salinity:
Not applicable.
Nominal and measured concentrations:
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/l were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass bottles/vessels.
- Aeration: yes, clean, oil-free air. 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.
- No. of vessels per concentration (replicates): The highest concentration was tested in triplicate, lower concentrations consisted of one replicate.
- No. of vessels for blank control (replicates): 6
- Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested.

TEST MEDIUM / WATER PARAMETERS
- 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).
- Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

SYNTHETIC SEWAGE FEED:
- 16 g peptone
- 11 g meat extract
- 3 g urea
- 0.7 g NaCl
- 0.4 g CaCl 2 .2H 2 O
- 0.2 g MgSO 4 .7H 2 O
- 2.8 g K 2 HPO 4
- Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

INHIBITOR OF NITRIFICATION
- A 2.32 g/l solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/l).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate

TEST CONCENTRATIONS
- In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/l were tested.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Key result
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 TDI-Urone per litre (Two Sample t-Test : α=0.05 Toxstat).
Furthermore, there was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.
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 (8.2 mg/l).
Reported statistics and error estimates:
- Chi-Square Test for Normality
- F-Test for Equality of Two Variances
- ANOVA
- 2 Sample t-Test

Acceptability of the test

1. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.

2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (8.2 mg/l).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Table 1: Study results

 Flask  Conc. (mg/l)  Temp (°C)  pH before addition of sludge  pH after 3 h contact time  Oxygen conc. at the start (~ mg O2/l)  Respiration rate (mg O2/l h)  Respiration rate (mg O2/g h)¹  % Inhibition respiration relative to the control (mean value)
 C1  0  20.8  7.8  8.1  8.7  43  29  
 C2  0  19.3  7.9  8.3  8.1  40  27  
 C3  0  20.2  7.8  8.2  8.2  45  30  
 C4  0  19.9  7.8  8.2  8.5  45  30  
 C5  0  20.1  7.8  8.2  7.9  42 28   
 C6  0  20.7  7.8  8.2  9.0  46  31  
 Mean            44  29  
 SD            2  2  
 CV (%)            5  5  
                 
 CN (+ATU)  0  20.7  7.8  8.5  8.8  26  17  
                 
 R1  5.0  20.1  7.8  8.4  8.8  26  17  40
 R2  12  20.7  7.8  8.3  8.7  19  13  56
 R3 30   20.6  7.8  8.4  8.9  8  5  82
                 
 T1  10  20.9  7.8  8.1  8.2  46  31  -6
 T2  100  20.7  7.8  8.2  8.3  43  29  1
 T3a  1000  20.7 7.8   8.1  8.6  45  30  -3
T3b  1000   20.1  7.8  8.1 8.3   40  27  8
 T3c  1000  20.2  7.8  8.2  8.7  40  27  8
 T3 mean            42  28  4
                 
 TA (abiotic)  1000 21.1 7.9 7.6  8.4  0  0  100
 TN (+ATU)  1000  20.3  7.8  8.5  8.2  27  18  -4²

C: Blank Control

CN: Nitrification control

R: Reference substance, 3,5-dichlorophenol

T: Test substance, TDI-Urone

CV: Coefficient of variation

ATU N-allylthiourea

¹ The amount of suspended solids in the final test mixture was 1.5 g/l.

² Relative to CN

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test TDI-Urone was not toxic to waste water (activated sludge) bacteria at or below 1000 mg/l (NOEC).
The EC50 exceeded 1000 mg/l.
Executive summary:

The influence of TDI-Urone on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of TDI-Urone tested was a clear colourless powder as described by the sponsor, with a purity of 97.5% active content (being a mixture of isomers). The test started with a stock solution of 10 g/l. Magnetic stirring for 5 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colorless 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 concentrations 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.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg TDI-Urone per litre. Furthermore, there was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

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.

TDI-Urone was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/l (NOEC).

The EC50 exceeded 1000 mg/l.

Description of key information

TDI-Urone was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/l (NOEC).
The EC50 exceeded 1000 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The influence of TDI-Urone on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of TDI-Urone tested was a clear colourless powder as described by the sponsor, with a purity of 97.5% active content (being a mixture of isomers). The test started with a stock solution of 10 g/l. Magnetic stirring for 5 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colorless 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 concentrations 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.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg TDI-Urone per litre. Furthermore, there was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

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.

TDI-Urone was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/l (NOEC).

The EC50 exceeded 1000 mg/l.