[2-(1-ethoxyethoxy)ethyl]benzene

Toxicity to microorganisms

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:

19 January 2017 - 10 February 2017

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 (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2008 (amended by EC No. 2016/266)

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

Specific details on test material used for the study:

- Solubility in water: 453 mg/L at 24°C
- Stability in water: not indicated
- Volatility: 3.1 Pa

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition of test item to Milli-RO water (because the substance was not sufficiently soluble to allow for the preparation of a 10 g/L stock solution in water)
- Differential loading: 1-Litre test bottles were filled with 200 mL and the test item was transferred quantitatively into the Milli- RO water (tap water purified by reverse osmosis) with a positive displacement pipet. The initial loading rates of the test item – Milli - RO mixtures were 2.5 times the final loading rate. These mixtures were stirred briefly. Subsequently, synthetic medium, Milli-RO water and sludge were added resulting in the required loading rates, ranging from 100 to 1000 mg/L (spacing factor ca. 1.8)
- Controls: Test medium without test item and treated in the same way as the test item solutions
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

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. The pH was 7.2 on the day of testing.
- Pretreatment: 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: A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids, i.e. 3.0 g/L of sludge. The amount of suspended solids in the final test mixture was 1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

To ensure optimal contact between the test item and test medium, continuous aeration and stirring was applied during the exposure period.

Test temperature:

20-22°C

pH:

control: start: 7.2-7.5, end: 6.8-7.2
test item solutions: start: 7.2-7.5, end: 7.1-7.3
ref.item solutions: start: 7.5, end: 7.4-7.7

Dissolved oxygen:

>60-70% of air saturation at test start

Nominal and measured concentrations:

Nominal loading rates (definitive test): 100, 180, 320, 560 and 1000 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Total fill volume: 500 mL (250 mL test solution (incl. synthetic medium) + 250 mL activated sludge in test medium)
- Aeration: yes (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)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 4
- No. of vessels per abiotic control (replicates): 1 (only during RF test)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L sludge
- Nutrients provided for bacteria: synthetic medium acc. to OECD 209
- Nitrification inhibitor used : no

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water (tap water purified by reverse osmosis) was used to prepare the test medium (adjusted ISO acc. to OECD 209)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation: 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8
- Range finding study:
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; the combined limit/range-finding test showed -10%, -5% and 85% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively. Therefore the expected ELR50 was between a loading rate of 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

307 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% Confidence interval is: 267-352 mg/L

Duration:

3 h

Dose descriptor:

other: ELR10

Remarks:

(estimated value, based on extrapolation)

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% Confidence interval is: 39-81 mg/L

Details on results:

- Blank controls oxygen uptake rate: 31.45 mg O2/kg*h (n=6)
- Coefficient of variation of oxygen uptake rate in control replicates: 14%
- Other:
* There was no oxygen uptake from abiotic processes (RF test result).
* The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the guideline requirements.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 3.2 mg/L (falls within the OECD reported range for total respiration, i.e. 2-25 mg/L)
- Other: 95% C.I. = 2.5 - 4.0 mg/L

Reported statistics and error estimates:

* EC50 and ELRx
For the test item, calculation of ELRx values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.

* 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 (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Since Hyacinth body was toxic to waste water (activated sludge) bacteria at all loading rates tested, a NOELR could not be determined.

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

Results final test

The effects observed were slightly higher than what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of ELR_x values.

The substance was toxic to waste water (activated sludge) bacteria at all loading rates tested. Therefore, a NOELR could not be determined.

Final test - Overview of 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		47.17	31.45
T1	100	36.86	24.58	21.85*
T2	180	31.49	20.99	33.25*
T3	320	23.85	15.90	49.43*
T4	560	16.50	11.00	65.01*
T5	1000	5.95	3.97	87.39*

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

* Statistically significantly different compared to control

Validity criteria fulfilled:

yes

Remarks:

Control oxygen uptake rate exceeded 20 mg oxygen/g of activated sludge / hour (31 mg). The coefficient of variation of oxygen uptake in control replicates did not exceed 30% (10%). The EC50 of ref.control was in the range 2-25 mg/L (3.2 mg/L).

Conclusions:

The substance was toxic to waste water bacteria (activated sludge) at all loading rates tested. Therefore, a NOELR could not be determined.
The ELR10 was estimated to be 60 mg/L. The ELR50 was determined to be 307 mg/L.

Executive summary:

In a study performed according to OECD 209 and following the principles of GLP, the influence of the substance on the total respiration rate of activated sludge was investigated.

 

Micro-organisms in activated sludge (in test medium) were exposed to the substance for 3 hours, during which aeration and stirring took place, at nominal loading rates of 100, 180, 320, 560 and 1000 mg/L (5 replicates). In parallel, an untreated control (6 replicates) and a reference substance (3,5 -dichlorophenol, 4 replicates) were tested. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).

 

Under the conditions of the test the substance was toxic to waste water bacteria (activated sludge)at all loading rates tested. Therefore, a NOELR could not be determined. The ELR10 was estimated to be 60 mg/L. The ELR50 was determined to be 307 mg/L.

The reference substance showed inhibition in the accepted range of 2 to 25 mg/L for total respiration (actual EC50 is 3.2 mg/L).

Description of key information

In a study performed according to OECD 209 and following the principles of GLP, the influence of the substance on the total respiration rate of activated sludge was investigated.

 

Micro-organisms in activated sludge (in test medium) were exposed to the substance for 3 hours, during which aeration and stirring took place, at nominal loading rates of 100, 180, 320, 560 and 1000 mg/L (5 replicates). In parallel, an untreated control (6 replicates) and a reference substance (3,5-dichlorophenol, 4 replicates) were tested. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).

 

Under the conditions of the test the substance was toxic to waste water bacteria (activated sludge)at all loading rates tested. Therefore, a NOELR could not be determined. The ELR10 was estimated to be 60 mg/L. The ELR50 was determined to be 307 mg/L.

The reference substance showed inhibition in the accepted range of 2 to 25 mg/L for total respiration (actual EC50 is 3.2 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

307 mg/L

EC10 or NOEC for microorganisms:

60 mg/L