Dihexyl ether

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

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Details on sampling:

At the end of the 3-hour incubation period, a portion (20 mL) of each test preparation (see Treatment of Test Substance Vessels) was transferred to an appropriate sample tube containing a PTFE stirrer. For those samples requiring amendment with ATU, this was undertaken at least 10 minutes prior to oxygen consumption measurement.
The DO (dissolved oxygen) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.110 minutes prior to measurement of heterotrophic respiration, 1.15 mL of nitrification inhibitor (2.32 g/L ATU) was added to the remaining vessel volume of 230 mL portion of the culture to give a final concentration of ca 11.6 mg/L ATU.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances) :
The test substance was insufficiently soluble to permit addition via aqueous solution.
Individually weighed quantities of test substance were added to the appropriate test vessels. For the test, the vessels were prepared at concentrations of 1, 10, 100 and 1000 mg/L. Each vessel was used as an individual replicate for respiration assessments.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Burley Menston sewage treatment works (Yorkshire, UK)
- Method of cultivation: at 20 ± 2°C with aerationand fed with synthetic sewage concentrate at a rate of 50 mL/L
- Preparation of inoculum for exposure: The pH of the sludge was 7.00 following adjustment from 5.90 and was adjusted by addition of 2M NaOH to be within the acceptable range of 7.5 ± 0.5.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

7.03-7.37

Nominal and measured concentrations:

Nominal concentrations of 1, 10, 100 and 1000 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks (containing a total volume of 250 mL)
- Aeration: yes
- No. of vessels per concentration 1-100 mg/l (replicates): 2
- No. of vessels per concentration 1000 mg/l (replicates): 3
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Pre-made synthetic sewage were added to water.

OTHER TEST CONDITIONS
- Adjustment of pH: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10E+01
- Justification for using fewer concentrations than requested by guideline:
A range-finder test, employing nominal Dihexyl ether concentrations of 0, 1.0, 10, 100 and 1000 mg/L, was undertaken and showed no inhibition of heterotrophic, nitrification or total respiration for any
concentrations of the test substance.

Reference substance (positive control):

yes

Remarks:

Single preparations were tested containing the reference substance (3,5-DCP) at nominal concentrations of 0.1, 2.0 and 40 mg/L.

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

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 heterotrophic respiration

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 nitrification rate

Results with reference substance (positive control):

Reference substance (3,5-DCP) inhibition (EC50) was observed to be within the validity criteria.
Total Respiration: 9.11 mg/L
Nitrification Respiration: 1.78 mg/L

Nominal Concentration mg/L	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate (mg/L/h)	Nitrification Respiration Rate (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
Control	47.1	29.6	17.5	not applicable	not applicable	not applicable
	47.0	30.5	16.5
	53.7	32.1	21.6
	55.7	33.6	22.1
	51.4	29.0	22.4
	53.5	31.0	22.5
1	53.0	33.9	19.1	-11.5	-14.2	-7.2
	61.6	36.8	24.8
10	46.4	29.7	16.7	2.5	-0.1	6.5
	53.8	32.3	21.5
100	61.8	38.0	23.8	-25.1	-24.8	-25.5
	66.8	39.3	27.5
1000	43.4	30.4	13.0	5.8	-7.6	26.3
	47.7	34.6	13.1
	54.1	35.0	19.1

Validity criteria fulfilled:

yes

Conclusions:

No inhibition of heterotrophic, nitrification or total respiration for any concentrations of the test substance was observed in this test system. This effect was confirmed to be statistically nonsignificant when compared to the control.

Executive summary:

The aim of this study was to determine the no observable effect concentration (NOEC) and estimate the effect concentration for an x% effect (ECx) for activated sludge exposed to the test substance, where possible. The study was undertaken according to OECD Guideline 209, Activated Sludge, Respiration and Inhibition Test (Carbon and Ammonium Oxidation), adopted July, 2010.

The activated sludge inoculum was collected from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K.), which has a predominantly domestic catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material. A range-finder test, employing nominal Dihexyl ether concentrations of 0, 1.0, 10, 100 and 1000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive dose response test. The test showed no inhibition of heterotrophic, nitrification or total respiration for any concentrations of the test substance. This effect was confirmed to be statistically nonsignificant (Bonferroni Adj t Test, α=5%, p values = 0.7901, 1.0000 and 1.0000 for heterotrophic, nitrification and total respiration, respectively) when compared to the control.

As there was < 0%, < 30% and < 6% inhibition of heterotrophic, nitrification and total respiration, respectively, the EC50 could not be determined statistically and is therefore classed as ≥1000 mg/L, which is the highest concentration used in this test.

The NOEC (No Observed Effect Concentration), based on statistical analysis, was determined to be:

Total Respiration: ≥1000 mg/L

Heterotrophic Respiration: ≥1000 mg/L

Nitrification Respiration: ≥1000 mg/L

The validity criteria applied to this study type were met and therefore the data are considered valid.

Description of key information

The effect of Dihexyl ether on the respiration rate of activated sludge (Carbon and Ammonium Oxidation) was assessed in a valid experimental GLP-study according to OECD guideline 209.

The test showed no inhibition of heterotrophic, nitrification or total respiration for any concentrations of the test substance, resulting in a NOEC ≥ 1000 mg/l. As there was < 0%, < 30% and < 6% inhibition of heterotrophic, nitrification and total respiration, respectively, the EC50 could not be determined statistically and is therefore classed as >1000 mg/L, which was the highest concentration used in the test.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Supporting these findings, no inhibition was observed for dihexyl ether in the available biodegradation studyy according to ‘Manometric Respirometry Test' (OECD Guideline 301F). The degradation process was followed by the oxygen uptake in the test solutions using three different test item concentrations (corresponding to 25 mg ThOD/L, 50 mg ThOD/L,and 100 mg ThOD/L) and an additional toxicity control was included. Neither the test concentrations, nor the toxicity control showed any signed of inhibition during the study period. The toxicity control showed 81% degradation within 28 days, therefore the test item is not considered to be inhibitory according to the OECD 301F test guideline.