Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 December 2010 - 13 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and EC guidelines and in compliance with GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
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)
Version / remarks:
(2007)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
EC Number:
619-508-4
Cas Number:
381209-09-2
Molecular formula:
C13 H24 O2
IUPAC Name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The batch of CAT-Acid tested was a clear orange liquid with big orange crystals and a purity of 99.7%. Since CAT-Acid was hardly soluble in water test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Before handling of the test substance (weighing or pipetting), the test substance was melted at 50°C.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Received from the Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.

- Preparation of the sludge: 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, 3.7 and 3.6 g/l of sludge, as used in experiment 1, 2 and 3, respectively). The pH was 7.9, 7.7 and 7.5 on the day of testing in experiment 1, 2 and 3, respectively. 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
aeration and stirring took plcae during the test duration
Post exposure observation period:
no

Test conditions

Hardness:
not reported
Test temperature:
18.5 - 20.8°C
pH:
7.2 - 7.8
Dissolved oxygen:
not reported
Details on test conditions:
Three experiments were performed during this project.

In the first experiment 1-litre test bottles were filled with 200 ml of test substance mixtures with initial loading rates of 25 mg/l, 250 mg/l and 2500 mg/l. These mixtures were stirred in closed dark brown glass bottles for 24½-25 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and
Milli-RO water up to 500 ml were added resulting in loading rates of 10, 100 and 1000 mg/l, respectively.

Based on the results of the first experiment (see section 7.1) a second and third experiment were performed with loading rates from 10 to 1000 mg/l, increasing with a factor 3.2. Preparation of the test vessels was similar to the method as applied for the first experiment, except that the test substance stirring period was 24-27 hours.

The first and second experiment were performed without the addition of a nitrification inhibitor, whereas N-allylthiourea (ATU), a nitrification inhibitor was added to all bottles of the third experiment; except for the bottle of the blank-control. This blank-control was used to evaluate the acceptability criterion.

In all experiments optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

TEST SYSTEM
- Test vessel: All glass, approximately 300 ml oxygen bottles and 1-litre test bottles.
- Aeration: yes (The aeration was adjusted in such a way that the dissolved oxygen concentration at the start is above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.)
- Air supply: Clean, oil-free air
- No. of organisms per vessel: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates):
--- blank controls: 6 replicates without nitrification inhibitor, 1 replicate with nitrification inhibitor
--- nitrification controls: 5 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium

OTHER TEST CONDITIONS
- Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Inhibition of respiration rates (nitrification potential)

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 10, 100 and 1000 mg/L (nominal, loading rate)
- Results used to determine the conditions for the definitive study: although no significatnt inhibition of respiration rate was observed up to 100 mg/L, in the final test the range 10 to 1000 mg/L was tested in order to allow for determination of NOEC and EC10,20,50
Final test:
- Test concentrations: 10, 32, 100, 320 and 1000 mg/L (nominal, loading rate)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rates (RT and RH)
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rates (RT and RH)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rates (RT and RH)
Details on results:
Range finding test:
No biological significant inhibition (<10%) of respiration rate of the sludge was recorded at or below a loading rate of 100 mg CAT-Acid per litre. At a loading rate of 1000 mg/l the inhibition of the respiration rate was between 19 and 30%. Furthermore, the nitrification potential of the sludge was
40% and no oxygen consumption was observed in the abiotic-control at 1000 mg/l.

The amount of suspended solid in the final text mixture of experiment 1 was 1.48 g/L.

Final tests:
In the experiment without a nitrification inhibitor (no. 2) and the experiment with a nitrification inhibitor (no. 3) the overall results were comparable. In both experiments no statistically significant effects on respiration rate were found at or below a loading rate of 320 mg/l compared to the control (Bonferroni t-Test: a=0.05).
Thus, the no observed effect concentration (NOEC) for CAT-Acid equalled a loading rate of 320 mg/l and the lowest observed effect concentration (LOEC) equalled a loading rate of 1000 mg/l.
The EC50-values for the inhibition of the respiration rates, RT and RH were above the highest concentration tested (> a loading rate of 1000 mg/l).

The amount of suspended solid in the final text mixture of experiment 2 was 1.85 g/L.
The amount of suspended solid in the final text mixture of experiment 3 was 1.82 g/L.
Results with reference substance (positive control):
EC50 for heterotrophic respiration: 37.6 mg/L
EC50 for total respiration: 7.2-9.8 mg/L
The validity criteria as described in OECD 209 (2010) were met.
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 CAT-Acid no EC50 could be calculated (EC50 > a loading rate of 1000 mg/l) and based on the results it was also not possible to determine a reliable EC10 and EC20.

NOEC estimation
The NOEC was based on statistical analysis of the data. For the control and the test concentrations the respiration rates were tested for normality and for homogeneity of variance. An effect was considered to be significant if statistical analysis of the data revealed a significant effect for the test concentrations compared with those obtained in the control (Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).

Any other information on results incl. tables

The test was accepted for the following reasons:

1. The blank-controls oxygen uptake rate was not below 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 (RT) and 5 to 40 mg/l for heterotrophic respiration (RH). (RT was 9.8 and 7.2 mg/l for experiment 1 and 2, respectively. RH was 38 mg/l for experiment 3.

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, CAT-Acid was not toxic to waste water (activated sludge) bacteria up to and including a loading rate of 320 mg/l prepared by direct addition of CAT-Acid to the test vessels (=overall NOEC).
The overall EC50 of CAT-Acid exceeded the highest concentration tested, being a loading rate of 1000 mg/l.