Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

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:
October 8, 2018 - April 16, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
yes
Remarks:
without any impact on the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MMHBB-002/18
- Expiration date of the lot/batch: 01-APR-2021
- Purity test date: 09-APR-2018
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas','s Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 7.4 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3.15 h
Remarks on exposure duration:
In the test the contact time was 3 hours and 15 minutes, due to a malfunctioning of the measuring device. The deviation of the contact time is relatively small and therefore this deviation has no impact on the outcome of the test.
Test temperature:
20 - 22°C
pH:
The pH in all test vessels, before addition of sludge was between 7.4 and 7.5. After the 3 hour and 15 minutes exposure period the pH was between 7.3 and 8.0.
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 10, 100 and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Aeration: 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 per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration: 1.5 g/L

TEST MEDIUM
- Synthetic medium (= 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 and 2.8 g K2HPO4 dissolved in Milli-RO water, made up to 1 litre and filtered (pH within 7.5 ± 0.5)
- Test medium: 16 mL synthetic medium made up to 50 mL with Milli-RO, mixed with 200 mL test item solution and then added with 250 mL activated sludge

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED:
After the 3-hour and 15 minutes 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 determination of oxygen consumption was performed with multiple oxygen sensors connected to a BlueBox, a multichannel measuring and controlling system.

OTHER PARAMETERS
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 temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels.

TEST CONCENTRATIONS:
- Range finding study: yes (combined limit/range-finding test)
- Test concentrations: 0 (control), 10, 100 and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3.15 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
Key result
Duration:
3.15 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
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 (7.2 mg/L).

Inhibition of the Respiration Rate

The combined limit/range-finding test showed no inhibition of the respiration rate at 10 and 100 mg/L. At 1000 mg/L an average of 5% inhibition of the respiration rate was observed. Therefore, the EC50 was above the highest concentration tested (1000 mg/L). No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg 1,3-Bis (4-hydroxy benzoyl) benzene per litre. Therefore, the NOEC was above the highest concentration tested (1000 mg/L). There was no significant oxygen uptake from abiotic processes.

Treatment

Loading rate (mg/L)

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h)¹

Control

 

38.49

25.66

 

T1

10

39.31

26.21

-2.13

T2

100

40.46

26.97

-5.11

T3a

 1000

35.79

23.86

7.02

T3b

 1000

35.32

23.55

8.24

T3c

1000

38.50

25.67

-0.02

Acceptability of the Test:

1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (25.66 mg oxygen per one gram of activated sludge, after a contact time of 3 hours). The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the combined limit/range-finding and final test (11%).

2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (7.2 mg/L in the combined limit/range-finding test) Since all criteria for acceptability of the test were met, this study was considered to be valid.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the test item was not toxic to waste water bacteria (activated sludge) at a concentration of 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.
Executive summary:

The toxicity of 1,3-Bis (4-hydroxy benzoyl) benzene to waste water bacteria (activated sludge) was investigated in a GLP-compliant study performed in accordance with OECD Guideline 209. In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg 1,3-Bis (4-hydroxy benzoyl) benzene per litre and therefore it was concluded that the NOEC is 1000 mg/L under the conditions of this test. The EC50 was above 1000 mg/L.

Description of key information

The toxicity of the test item to waste water bacteria (activated sludge) was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 209. Under the conditions of the test, the test item was not toxic to the bacteria at or below a concentration of 1000 mg/L (NOEC). The EC50 was higher than 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 toxicity of the test item to waste water bacteria (activated sludge) was investigated in a GLP-compliant study according to OECD 209. The study is considered as reliable (Klimisch 1) and is selected as the key study for this endpoint.