Bis[(3,4-epoxycyclohexyl)methyl] adipate

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:

21 November 2017 to 14 February 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))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.

Vehicle:

no

Details on test solutions:

- Nominal amounts of test material were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 litres) 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 (Rinsed 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed three times with 10 mL of deionised 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:

no

Total exposure duration:

3 h

Test temperature:

19-20 °C

pH:

7.0-8.0 (adjusted)

Nominal and measured concentrations:

- Nominal concentrations: 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Material, size, headspace, fill volume: 500 mL
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 litre per minute.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 1 per concentration
- Sludge concentration: 3.0 g/L
- Nutrients provided for bacteria: Synthetic sewage consisted of 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 dissolved in a final volume of 1 litre of water with the aid of ultrasonication. The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range-finding, and definitive tests was between pH 7.2 and 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test material dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0 if necessary.
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED:
- Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge.
- 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.
- The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Range finding study test concentrations: 10, 100 and 1000 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 320, 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

RANGE-FINDING TEST
- 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 the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
- Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC value for the test material. Test concentrations of 100, 320 and 1000 mg/L were selected for the definitive test.

DEFINITIVE TEST
- The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 1. Oxygen consumption rates and percentage inhibition values for the control, test and reference materials after 3 hours contact time are given in Table 2.

INHIBITION OF RESPIRATION RATE
- The test material exhibited no statistically significant toxic effects at the test concentrations of 100 and 320 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentration of 1000 mg/L. Based on these results the NOEC for the test material after 3 hours exposure was 320 mg/L.
- 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.

Results with reference substance (positive control):

- For the reference material, 3,5-Dichlorophenol, the EC50 value was 7.8 mg/L (95 % confidence limits 6.1-10 mg/L) after 3 hours.

Reported statistics and error estimates:

- The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
- 95 % confidence limits were calculated for the reference material EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
- In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria

- The EC50 value (3-Hour contact time) for the reference material, 3,5-Dichlorophenol, was 7.8 mg/L.

- The specific respiration rate of the controls was 27.35 mg oxygen per gram dry weight of sludge per hour.

- The coefficient of variation of oxygen uptake in the control vessels was 9.3 %.

- All validation criteria for the study were therefore satisfied.

Table 1: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	R1	5.94	67
	R2	6.06	68
	R3	6.82	77
	R4	6.61	74
	R5	5.82	65
	R6	6.14	69
Test Material	100R1	5.85	66
	100R2	6.11	69
	100R3	6.02	68
	100R4	6.04	68
	100R5	5.97	67
	320R1	5.72	64
	320R2	5.82	65
	320R3	6.05	68
	320R4	5.71	64
	320R5	5.66	64

Table 1: Continued.

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Test Material	1000R1	5.73	64
	1000R2	5.61	63
	1000R3	5.59	63
	1000R4	5.64	63
	1000R5	5.69	64
3,5-Dichlorophenol	3.2	7.92	89
	10	8.36	94
	32	8.16	92

R = replicate

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	R1	5.5	5	2.2	39.60	-
	R2	4.8	4	2.1	40.50	-
	R3	4.8	4	2.4	36.00	-
	R4	4.6	4	2.0	39.00	-
	R5	4.5	3	2.2	46.00	-
	R6	4.8	4	1.8	45.00	-
Test Material	100R1	3.0	2	1.7	39.00	5
	100R2	4.8	4	2.0	42.00	[2]
	100R3	4.5	3	2.3	44.00	[7]
	100R4	4.9	4	2.1	42.00	[2]
	100R5	4.7	4	1.9	42.00	[2]
	320R1	4.3	3	2.1	44.00	[7]
	320R2	5.2	5	2.0	38.40	6
	320R3	5.0	5	1.8	38.40	6
	320R4	5.2	5	1.8	40.80	1
	320R5	5.0	4	2.2	42.00	[2]

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	Inhibition (%)
Test Material	1000R1	5.2	6	1.8	34.00	17
	1000R2	5.8	10	2.4	20.40	50
	1000R3	4.8	5	2.1	32.40	21
	1000R4	5.5	8	2.1	25.50	38
	1000R5	5.8	7	2.2	30.86	25
3,5-Dichlorophenol	3.2	5.3	6	2.3	30.00	27
	10	6.5	10	3.4	18.60	55
	32	7.8	10	7.0	4.80	88

R      = Replicate

[ ]    =Increase in respiration rate as compared to controls

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the No Observed Effect Concentration (NOEC) for the test material after 3 hours exposure was 320 mg/L.

Executive summary:

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions.

The Activated Sludge, Respiration Inhibition Test was performed to assess the effect of the test material on the respiration of activated sewage sludge.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 100, 320 and 1000 mg/L (five replicates) for a period of 3 hours at a measured temperature of between 19 and 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-Dichlorophenol.

The reference material gave a 3-Hour EC50 value of 7.8 mg/L, 95 % confidence limits 6.1 to 10 mg/L. All validation criteria for the study were therefore satisfied.

Under the conditions of this study, the No Observed Effect Concentration (NOEC) for the test material after 3 hours exposure was 320 mg/L.

Description of key information

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The Activated Sludge, Respiration Inhibition Test was performed to assess the effect of the test material on the respiration of activated sewage sludge.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 100, 320 and 1000 mg/L (five replicates) for a period of 3 hours at a measured temperature of between 19 and 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-Dichlorophenol.

The reference material gave a 3-Hour EC50 value of 7.8 mg/L, 95% confidence limits 6.1 to 10 mg/L. All validation criteria for the study were therefore satisfied.

Under the conditions of this study, the No Observed Effect Concentration (NOEC) for the test material after 3 hours exposure was 320 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

320 mg/L