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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2017 - 16 January 2018
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable.
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 dark brown test-bottles containing 200 mL Milli-RO water. The test item-water mixtures were then stirred for a short period, 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 of 10, 100 and 1000 mg/L in the range finder and 100, 180, 320, 560 and 1000 mg/L in the final test. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The source of the activated sludge was the municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. During the preparation of the sludge, the sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was then 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 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.
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
3 hours, during which aeration and stirring took place.
Post exposure observation period:
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.
Hardness:
Not stated
Test temperature:
20-22 °C during the test
pH:
The pH in all test vessels, before addition of sludge was between 7.3 and 8.9. After the 3-hour exposure period the pH was between 7.0 and 8.6.
Dissolved oxygen:
60-70% saturation
Salinity:
Not stated
Conductivity:
Not stated
Nominal and measured concentrations:
10, 100 and 1000 mg/L in the combined limit/range finder
100,180, 320, 560 and 1000 mg/L in the final test
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- 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.
- Combined limit/range finder test; The highest loading rate was tested in triplicate; lower loading rates consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.
Final test; Five replicates per test group and 6 replicates for the control were tested.
- Sludge concentration (weight of dry solids per volume): 3.0 g/L of sludge
- Medium used in preparation of the sludge: 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 (8.8 mg/L), NaHCO3 (46.7 mg/L) and KCl (4.2 mg/L).
Nutrients provided for bacteria: 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-RO water, made up to 1 litre and filtered.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).

MEASUREMENTS:
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. Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

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.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: ELR50
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: ELR10
Effect conc.:
440 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: ELR20
Effect conc.:
813 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Range-finder: The combined limit/range-finding test showed no inhibition at a loading rate of 10 and 100 mg/L and 31% inhibition of the respiration rate at a loading rate of 1000 mg/L. Therefore, the expected ELR50 was above a loading rate of 1000 mg/L.

Final test: The inhibitory effect of dilithium tetraborate on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from no inhibition at 100 mg/L to 26% at 1000 mg/L. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 320 mg Dilithium tetraborate per litre. For dilithium tetraborate, no ELR50-value could be calculated because effects were below 50% (ELR50 > 1000 mg/L).
Results with reference substance (positive control):
The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

Table 1: Range finder results

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

 

29.55

19.70

 

T1

10

32.84

21.89

-5.13

T2

100

30.96

20.64

-4.79

T3

1000

20.30

13.53 (RT)*

31.30 (IT)*

RT:   Total respiration with Dilithium tetraborate

IT:    % inhibition of total respiration relative to RC

* Statistically significantly different compared to control

Table 2          Final Test – Overview of the Results

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

 

34.90

23.26

 

T1

100

36.68

24.46

-5.13

T2

180

33.75

22.50

3.29

T3

320

33.19

22.13

4.88

T4

560

31.43

20.95*

9.92

T5

1000

25.90

17.26*

25.79

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

* Statistically significantly different compared to control

Table 3          Effect Parameters

Parameter

Dilithium tetraborate

Loading rate (mg/L)

NOELR

320

ELR10

440 (200 – 589 mg/L)

ELR20

813 (614 – 1211 mg/L)

ELR50

> 1000

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, dilithium tetraborate was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOELR). The ELR10 was at a loading rate of 440 mg/L (95% confidence interval: 200 - 589 mg/L). The ELR20 was at a loading rate of 813 mg/L (95% confidence interval: 614 - 1211 mg/L). The ELR50 was above a loading rate of 1000 mg/L.
Executive summary:

The objective of the study was to evaluate dilithium tetraborate for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR50and/or the no-observed effect loading rate (NOELR) using OECD guideline No. 209. The influence of dilithium tetraborate on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The inhibitory effect of dilithium tetraborate on aerobic waste water (activated sludge) bacteria (OECD TG 209) increased with increasing loading rate, ranging from no inhibition at 100 mg/L to 26% at 1000 mg/L. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 320 mg Dilithium tetraborate per litre.

It was therefore concluded that under the conditions of the test, dilithium tetraborate was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOELR). The ELR10 was at a loading rate of 440 mg/L (95% confidence interval: 200 - 589 mg/L), the ELR20 was at a loading rate of 813 mg/L (95% confidence interval: 614 - 1211 mg/L) and the ELR50 was above a loading rate of 1000 mg/L. The study met the acceptability criteria prescribed by the study plan and was considered valid.

Description of key information

The inhibitory effect of dilithium tetraborate on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from no inhibition at 100 mg/L to 26% at 1000 mg/L. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 320 mg dilithium tetraborate per litre.

It was therefore concluded that under the conditions of the test, dilithium tetraborate was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 320 mg/L (NOELR). The ELR10 was at a loading rate of 440 mg/L (95% confidence interval: 200 - 589 mg/L), the ELR20 was at a loading rate of 813 mg/L (95% confidence interval: 614 - 1211 mg/L) and the ELR50 was above a loading rate of 1000 mg/L.The study met the acceptability criteria prescribed by the study plan and was considered valid.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
440 mg/L

Additional information