Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2016 - 01 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
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 (incl. certificate)
Remarks:
3 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: White crystalline solid
Storage conditions: At room temperature protected from light
Specific details on test material used for the study:
- Solubility: < 0.0001 g/l @ 19.5 - 20.5 °C (insoluble)
- Stability: not indicated

Sampling and analysis

Analytical monitoring:
no

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation was as much as possible performed under yellow light and/or low light conditions. 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. 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 applying continuous aeration and stirring.
- Controls: test medium without test item and treated in the same way as the test item solutions.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Waterschap Aa en Maas, 's Hertogenbosch, the Netherlands
- Method of cultivation: The source of test organisms is activated sludge (sampled near the exit of the aeration tank) freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage.
- Preparation of inoculum: 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 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g ss/L

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
17 - 21 °C during the test
pH:
Before addition of sludge: 7.7 -7.8
After 3 hours of exposure: 7.5 - 8.4 (includes conditions for reference item)
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass, 1 Litre, open bottles/vessels, fill volume: 500 mL
- Aeration: 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. Clean, oil-free air was used for air supply.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.5 g ss/L final test mixture
- Nutrients provided for bacteria: sewage feed (synthetic medium)
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: oxygen consumption after 3 hours contact time for a period of approx. 10 minutes.
Additional measurements: medium temperature was recorded continuously in a temperature control vessel. pH was determined at the start and at the end of the test for all test/reference item concentrations and controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Combined range finding/limit test concentrations: 10, 100 and 1000 mg/L + a control (6 replicates) and an abiotic control without sludge of 1000 mg/L test item (1 replicate) were tested
- Results used to determine the conditions for the definitive study: yes, the combined limit/range-finding test showed 30, 10 and 29% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively. No clear loading rate related response was observed. This effect might be caused by the fact that loading rates were tested resulting in comparable bioavailability of the test item for the bacterial population. The expected ELR50 was above loading rate of approximately 1000 mg/L. There was no oxygen uptake from abiotic processes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
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 on result:
other:
Remarks:
result was reported as 'ELR50', based on Loading Rates
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 on result:
other:
Remarks:
result was reported as 'NOELR', based on Loading Rates
Details on results:
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes
- Blank controls oxygen uptake rate: 43.94 mg O2/L*h
- Other: for BMS-587172-01 no ELR50-value could be calculated because the test item proved to be non-toxic (ELR50 > 1000 mg/L)
- Coefficient of variation of oxygen uptake rate in control replicates: 11%
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: ELR50: 12 mg/L (estimated value)
Reported statistics and error estimates:
-ELR50 determination: for the reference item ELR50 was calculated with the 2-param. normal CDF, a non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item. There was no significant fit, therefore the ELR50 was an estimated value. For the test item, no ELR50 could be obtained, because it proved to be non-toxic.
- NOELR determination: an effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the control revealed significant inhibition of the respiration rate (Dunnett´s multiple t-test procedure, α=0.05, one-sided, smaller). No significant inhibition of the respiration rate was observed at any of the tested loading rates. Therefore, the NOELR was considered to be the highest loading rate tested.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 Final test – Overview of the results

Treatment

Loading rate

(mg/L)

pH

Mean respiration rate

% Inhibition of the respiration

rate

(mean value)

Start

End

(mg O2/L h)

(mg O2/g h)¹

Control

 

7.8

7.6 - 8.2

43.94

29.29

 

T1

10

7.7 – 7.8

7.5 – 7.8

42.02

28.01

4.37

T2

32

7.7 – 7.8

7.6 – 7.8

46.16

30.77

-5.05

T3

100

7.7 – 7.8

7.5 – 7.7

46.37

30.91

-5.54

T4

320

7.7 – 7.8

7.7 – 7.8

40.89

27.26

6.95

T5

1000

7.7

7.8 – 7.8

45.48

30.32

-3.51

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

Table 2 Final test – Respiration rate and inhibition reference item

 

Concentration (mg/L)¹

2.0

4.0

8.0

16

Respiration rates (mg O2/L.h)

38.97

27.37

26.61

16.33

Respiration rates (mg O2/g.h)1

25.98

18.24

17.74

10.89

Inhibition %

2.24

31.36

33.25

59.04

1)

The amount of suspended solids in the final test mixture was 1.5 g/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1. Mean control oxygen uptake rate > 20 mg O2/g activated sludge in an hour (29 mg O2/g). 2. CV of oxygen uptake in controls < 30% (11%) 3. EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L for total respiration (estimated value: 12 mg/L).
Conclusions:
Under the conditions of this present test BMS-587172-01 was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 1000 mg/L (NOELR). The ELR50 was above a loading rate of 1000 mg/L.
Executive summary:

This study was performed to assess the influence of the test item on the respiration rate of activated sludge after a contact time of 3 hours. The study procedures were based on OECD guideline No. 209 and performed under GLP conditions.

Because the test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water, it was mixed with Milli-RO water before adding synthetic medium, sludge and the remaining Milli-RO water was added. During 3 hour exposure period, optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring. Oxygen consumption was recorded after 3 hours for approximately 10 minutes.

A combined range finding/limit test was performed with loading rates of 10 , 100 and 1000 mg/L. Based on the results from this test, a final test was conducted using loading rates of 10, 32, 100, 320 and 1000 mg/L. Five replicates per loading rate and six replicates per untreated control were tested.

No statistically significant inibition of respiration rate of the sludge was recorded at any of the tested loading rates in the final test.

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. In the final test, the temperature was below the range prescribed by the study plan (20 ± 2°C) during the exposure phase, i.e. 17-21°C. However, because the validity criteria for the controls and reference item were met, this deviation was considered to have no effect on the study.

The test item was shown to be not toxic to waste water bacteria (activated sludge) at or below a loading rate of 1000 mg/L (NOELR). ELR50 could not be determined (> 1000 mg/L).