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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 2017 to September 2017
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))
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:
- Appearance: Pink liquid
- Test item storage: At room temperature protected from light
- Purity/Composition correction factor: No correction factor required
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, aliquots corresponding to the required loading rate were pipetted directly into the dark brown test bottles containing 400 mL Milli-RO water. Since the test item was known to be volatile the pipette tip was immersed below the surface of the Milli-RO when transferring the test item. The test item – Milli-RO water mixtures were magnetically stirred briefly to mix (3-10 minutes) and subsequently, 32 mL synthetic medium made up to 100 mL with Milli-RO water and 500 mL sludge were added resulting in the required loading rates.
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 7.9 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 h
Remarks on exposure duration:
Additional measurements were performed after 30 minutes of incubation: since the test item is volatile, a reduced contact time was included in the test, as recommended by the test guideline.
Test temperature:
18 - 21°C
pH:
7.9 - 8.3 (after the 3-hour contact time)
Nominal and measured concentrations:
Nominal concentrations (definitive test): 0 (control), 100, 180, 320, 560 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): 5
- No. of vessels per control (replicates): 6
- 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: 32 mL synthetic medium made up to 100 mL with Milli-RO, mixed with 400 mL test item solution and then added with 500 mL activated sludge

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED:
After a contact time of 30 minutes, approximately half of the sample was transferred to a beaker for the recording of oxygen consumption for a period of approximately 10 minutes. The remaining half of the sample was aerated until a contact time of 3 hours was reached. 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 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 part of the reaction mixture remaining after measurement. 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
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
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 h
Dose descriptor:
other: EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
other: NOELR
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
other: EL50
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 measured EC50 of 3,5-dichlorophenol in the final test was within the accepted range of 2 to 25 mg/L for total respiration (8.3 and 5.8 mg/L in the 30 minute and 3 hour contact time, respectively).
Reported statistics and error estimates:
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 blank control revealed significant inhibition of the respiration rate.
the Multiple Sequentially-rejective U-test After Bonferroni-Holm Procedure for the 30 minute contact time and the Multiple Sequentially-rejective t-test After Bonferroni-Holm Procedure for 3 hour contact time, both with α=0.05, one-sided, smaller were performed.
Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Inhibition of the Respiration Rate

A summarized overview of the study results is presented in the table below. No significant inhibition of the respiration rate was observed at any of the tested contact times and loading rates. Therefore, the NOELR was considered to be the highest loading rate tested (1000 mg/L). No ELR50-value could be calculated because the test item proved to be non-toxic (ELR50 > 1000 mg/L).

30-min contact time

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

 

50.51

33.67

 

T1

100

45.61

30.41

9.69

T2

180

45.43

30.29

10.05

T3

320

47.30

31.54

6.34

T4

560

49.81

33.21

1.38

T5

1000

50.28

33.52

0.45

3-hour contact time

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

 

42.78

28.52

 

T1

100

35.90

23.93

16.08

T2

180

35.35

23.57

17.35

T3

320

38.46

25.64

10.08

T4

560

40.49

27.00

5.34

T5

1000

41.61

27.74

2.73

Acceptability of the Test

1. The mean control oxygen uptake rate in the final test exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (33.67 and 28.52 mg oxygen per one gram of activated sludge after a contact time of 30 minute and 3 hours, respectively in the final test). The coefficient of variation of oxygen uptake in control replicates of the final test did not exceed 30% at the end of the both the 30 minute and 3 hour contact time (10% and 18%, respectively).

2. The measured EC50 of 3,5-dichlorophenol in the final test was within the accepted range of 2 to 25 mg/L for total respiration (8.3 and 5.8 mg/L in the 30 minute and 3 hour contact time, respectively.

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 or below a loading rate of 1000 mg/L (NOELR). The ELR50 was higher than 1000 mg/L.
Executive summary:

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. 109. Under the conditions of the test, the test item was not toxic to the bacteria at or below a loading rate of 1000 mg/L (NOELR). The ELR50 was higher than 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. 109. Under the conditions of the test, the test item was not toxic to waste water bacteria at or below a loading rate of 1000 mg/L (NOELR). The ELR50 was higher than 1000 mg/L.

Key value for chemical safety assessment

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, with minor deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.