Dihexyl fumarate

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:

Experimental start date: 12 March 2018. Experimental completion date: 13 March 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 (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

Range-Finding Test
Test Item Preparation
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.

Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item Preparation
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.5 and was adjusted to pH 7.0 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item prior to the test item vessels being prepared as described in Section 3.4.1.1. Finally, two further control vessels were prepared.

Data from the inoculum control and reference item vessels was shared with similar concurrent studies.

The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 20 °C.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Test System and Supporting Information

A mixed population of activated sewage sludge micro-organisms was obtained on
12 March 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Test Water
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

Synthetic Sewage
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
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
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.

The pH of the synthetic sewage stock and the synthetic sewage stock used to feed the activated sewage sludge and used in the test was 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.3 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

duration

pH:

The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Nominal and measured concentrations:

10, 100, 1000 mg/L (nominal)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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 respiration due to nitrification

Details on results:

Range-Finding Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2. Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4. Observations made on the test preparations throughout the range-finding test are given in Table 5.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Results with reference substance (positive control):

Validation Criteria
The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.9 mg/L.
The specific respiration rate of the controls was 27.67 mg oxygen per gram dry weight of sludge per hour.
The coefficient of variation of oxygen uptake in the control vessels was 2.4%.

All validation criteria for the study were therefore satisfied.

 Inhibition of RespirationRate

 The following results were derived: 

	Dihexyl fumarate (EC 242-833-4)		3,5-Dichlorophenol
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>1000	-	2.1	-
EC20	>1000	-	3.0	-
EC50	>1000	-	8.9	7.0 - 11
EC80	>1000	-	27	-
NOEC	1000	-	-	-

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Table 1       pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	10	7.8	-
	100	7.7	-
	1000R1	7.6	-
	1000 R2	7.4	-
	1000 R3	7.4	-

-       = No adjustment required R   = Replicate

Table 2       Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	R1	5.75	65
	R2	5.68	64
	R3	6.38	72
	R4	6.01	68
Test Item	10	5.71	64
	100	5.78	65
	1000 R1	6.62	74
	1000 R2	5.62	63
	1000 R3	5.71	64
3,5-Dichlorophenol	3.2	6.72	76
	10	7.28	82
	32	7.68	86

R   = Replicate

Table 3       Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2Reading (mgO2/L)	Measurement Period (minutes)	Final O2Reading (mgO2/L)	O2 ConsumptionRates (mg O2/L/hour)	Inhibition(%)
Control	R1	3.5	2	2.1	42.00	-
	R2	4.2	3	2.2	40.00	-
	R3	5.5	5	2.0	42.00	-
	R4	4.2	3	2.1	42.00	-
Test Item	10	3.4	2	1.9	45.00	[8]
	100	4.7	3	2.2	50.00	[20]
	1000 R1	3.4	2	1.7	51.00	[23]
	1000 R2	5.6	4	2.3	49.50	[19]
	1000 R3	5.4	5	1.8	43.20	[4]
3,5- Dichlorophenol	3.2	5.1	6	1.9	32.00	23
	10	6.6	10	3.2	20.40	51
	32	7.6	10	6.6	6.00	86

R   = Replicate

-       = Not applicable

[  ] = Increase in respiration rate as compared to controls

Table 4       pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.7	7.4
	R2	7.6	7.5
	R3	7.5	7.5
	R4	7.4	7.5
Test Item	10	7.5	7.5
	100	7.4	7.5
	1000 R1	7.4	7.6
	1000 R2	7.7	7.4
	1000 R3	7.5	7.5
3,5-Dichlorophenol	3.2	7.5	7.8
	10	7.9	7.9
	32	7.5	7.7

R   = Replicate

Table 5       Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/brown dispersion, a layer of many oily globules of test item visible at the surface	Dark brown dispersion, a few oily globules of test item visible at the surface	Dark brown dispersion, a few oily globules of test item visible at the surface
	100	Pale yellow/brown dispersion, a layer of many oily globules of test item visible at the surface	Dark brown dispersion, a few oily globules of test item visible at the surface	Dark brown dispersion, a few oily globules of test item visible at the surface
	1000 R1	Pale yellow/brown dispersion, a layer of many oily globules of test item visible at the surface and oily globules of test item visible throughout	Dark brown dispersion, an oily layer of test item visible at the surface	Dark brown dispersion, an oily layer of test item visible at the surface
	1000 R2	Pale yellow/brown dispersion, a layer of many oily globules of test item visible at the surface and oily globules of test item visible throughout	Dark brown dispersion, an oily layer of test item visible at the surface	Dark brown dispersion, an oily layer of test item visible at the surface
	1000 R3	Pale yellow/brown dispersion, a layer of many oily globules of test item visible at the surface and oily globules of test item visible throughout	Dark brown dispersion, an oily layer of test item visible at the surface	Dark brown dispersion, an oily layer of test item visible at the surface
3,5- Dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

*Observations made prior to the addition of activated sewage sludge

R   = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

.