Dioctyl maleate, branched

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:

14 September 2017 to 20 October 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:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Bernel Ester DCM
Purity: 92.78%
Molecular Formula: C20H36O4
Molecular Weight: 340
Receipt date: 4 Sept 2017
Storage: Room temperature (15-30°C)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance was deemed of a sufficiently complex nature to permit addition via aqueous solution. Individually weighed quantities of test substance were added to the appropriate test vessels. The range-finder/limit test vessels, were prepared at concentrations of 1, 10, 100 and 1000 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated Sludge Inoculum
The activated sludge inoculum was collected from the sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material.

Preparation and Maintenance
The sludge was transported to the test facility in a closed container with an adequate headspace to prevent the sample becoming anoxic. On arrival at the test facility, the sludge was aerated with a compressed air supply delivered through a suitable distribution device. The suspended solids concentration was determined gravimetrically following homogenisation and adjusted using dechlorinated tap water to 3 g/L (± 0.3 g/L). The sludge was maintained at 20 ± 2°C with aeration. During storage it was maintained at 20 ± 2°C and fed with synthetic sewage concentrate at a rate of 50 mL/L.
The sludge for the range-finder/limit test was collected on 26 September 2017.

Synthetic Sewage
A synthetic sewage concentrate was used to feed the activated sludge during storage and to provide a uniform respiration substrate in the test cultures. Packets of pre-made synthetic sewage from Strathkelvin were added to water (1 packet per 250 mL of water). Batch number 2/05 was used throughout the study.

Inoculum
The suspended solids concentration was determined before the range-finder/limit test. The concentration was 3 g/L after adjustment with dechlorinated tap water and was within the nominal range of 3 ± 0.3 g/L. The pH of the sludge after adjustment was 7.22 and was within the acceptable range of 7.5 ± 0.5.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

190 min

Remarks on exposure duration:

180 minutes of aeration/incubation followed by 10 minutes in a sealed container with monitoring of change in dissolved oxygen.

Hardness:

Not specified

Test temperature:

The sludge was maintained at 20 ± 2°C with aeration for the incubation period.

pH:

7.05 to 7.64 (adjusted with NaOH for 2 of 3 testing runs)

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Range finder/Limit test: 1, 10, 100 and 1000 mg/L (nominal)

Details on test conditions:

Preparation of Test Vessels
Details of test vessel composition are presented in Appendix 2. Test vessels were ca 250 mL glass conical flasks (containing a total volume of 250 mL). Each vessel contained a synthetic sewage concentrate, dechlorinated tap water, test substance or reference substance and nitrification inhibitor, as appropriate. The reference substance and nitrification inhibitor were added to the test system in aqueous solution. At appropriate intervals, test samples were inoculated and aerated immediately. After addition of the test substance, inoculum and additional water, the nominal suspended solids concentration was 1.5 g/L. Each culture was aerated for ca 3 hours by bubbling air through the test system. The rate of aeration was sufficient to keep the test samples adequately mixed. Each vessel contained more than required test preparation for the number of samples assessed.

Nitrification Inhibitor
The nitrification inhibitor, N-allylthiourea (ATU), was added to appropriate test and reference vessels to give a final concentration of ca 11.6 mg/L ATU in the test system.

Incubation Conditions
The sludge was maintained at 20 ± 2°C with aeration for the incubation period.

Measurement of Respiration Rates
At the end of the 3-hour incubation period, a portion (20 mL) of each test preparation 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. Respiration rates (mg O2/L/h) were determined by measuring the gradient of the linear portion of the graph. Measurements at high (> 7.0 mg/L) or low (< 2.0 mg/L) DO levels were avoided, where possible. The Strathtox instrumentation generated both graphical data as well as calculated results.

Blank Control Vessels
Three blank controls were prepared to enable measurement in six vessels during the test series.

Measurement of Respiration Rates
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.
Respiration rates (mg O2/L/h) were determined by measuring the gradient of the linear portion of the graph. Measurements at high (> 7.0 mg/L) or low (< 2.0 mg/L) DO levels were avoided, where possible. The Strathtox instrumentation generated both graphical data as well as calculated results.

Incubation Conditions
The sludge was maintained at 20 ± 2°C with aeration for the incubation period.

Estimation of No-Effect and Effect Concentrations

Reference substance
Estimation of the EC50 for the reference substance was calculated from concentration versus effect for total and nitrification respiration for the range finder/limit test. The EC50 was based on a statistical analysis (linear interpolation) of concentration versus effect in total respiration and nitrification respiration.

Test substance
The NOEC for the test substance was based on both visual assessment of the data as well as from calculated data. Estimation of the EC50 for the test substance was derived using statistical analysis (linear interpolation) of concentration versus effect.

Validity Criteria
The following criteria were applied in order for the test to be considered valid:
The blank control respiration rate is ≥ 20 mg/L/h;
The coefficient of variation of the blank control respiration rates are ≤ 30%;
The EC50 values of the reference substance are as follows:
between 2 and 25 mg/L for total respiration
between 0.1 and 10 mg/L for nitrification respiration, where appropriate.

Reference substance (positive control):

yes

Remarks:

The validity of the test system was checked by inclusion of a series of preparations which contained 3,5-dichlorophenol (3,5-DCP), a standard respiration inhibitor reference. The concentrations used were 0.1, 2.0 and 40 mg/L.

Duration:

190 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

190 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

190 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

190 min

Dose descriptor:

other: NOAEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

190 min

Dose descriptor:

other: NOAEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

190 min

Dose descriptor:

other: NOAEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

Range-finder/Limit Test
The results of the range-finder/limit test are presented in Table 1 and Appendix 3.
Total, heterotrophic and nitrification respiration rates were similar across the controls and all treatment levels. No inhibition was calculated for any type of respiration at up to, and including, 1000 mg/L.
As there was < 10% inhibition observed in the test, the EC50 for total, heterotrophic and nitrification respiration could not be determined and is classed as > 1000 mg/L, which is the highest concentration level used in this test.
As there were slight enhancement effects noted (observed as negative inhibition), the endpoint for this is reported as No Observed Adverse Effect Concentration (NOEAC).

The NOAEC was therefore determined to be:
Total Respiration: 1000 mg/L
Heterotrophic Respiration 1000 mg/L
Nitrification Respiration: 1000 mg/L

Results with reference substance (positive control):

Reference substance (3,5-DCP) inhibition was calculated to be within the validity criteria:
Total Respiration: 2.5 mg/L
Nitrification Respiration: 0.9 mg/L

Reported statistics and error estimates:

The validity criteria (blank control respiration rate and the coefficient of variation between replicates) were also satisfied; the results of the limit test can therefore be considered to be valid.

Bernel Ester DCM Concentration (mg/L)

	Concentration (mg/L)	Concentration (mg/L)	Concentration (mg/L)
	Total Respiration	Heterotrophic Respiration	Nitrification Respiration
NOAEC	1,000	1,000	1,000
EC50	>1,000	>1,000	>1,000

Validity criteria fulfilled:

yes

Conclusions:

There were no statistically significant effects on respiration observed during the test. The EC50 for Bernel Ester DCM was greater than 1000 mg/L under the test conditions. The NOAEC value was determined to be 1000 mg/L, the highest concentration tested.

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/limit test, employing nominal Bernel Ester DCM concentrations of 1, 10, 100 and 1000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. The range-finder/limit test showed no statistically significant inhibition of total, nitrification or heterotrophic respiration at the highest concentration (1000 mg/L). As there were sufficient control replicates at the start and end of each exposure period and the highest concentration (1000 mg/L) was conducted in triplicate, as per the OECD 209 guideline, it was not therefore necessary to carry out any further testing. Determination of EC50 was not possible and is therefore reported to be greater than 1000 mg/L (the highest concentration of Bernel Ester DCM tested under these conditions). As there were slight enhancement effects noted (observed as negative inhibition), the endpoint for this is reported as No Observed Adverse Effect Concentration (NOEAC). The NOAEC for Bernel Ester DCM is therefore reported to be 1000 mg/L.

	Concentration (mg/L)	Concentration (mg/L)	Concentration (mg/L)
	Total Respiration	Heterotrophic Respiration	Nitrification Respiration
NOAEC	1,000	1,000	1,000
EC50	>1,000	>1,000	>1,000

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

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L