Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized

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:

02-08-2017 to 19-10-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017; signature: November 2017

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Range Finder concentrations: 5, 50, 500 mg ; Definitive Test concentrations: 9.8, 30.6, 97.7, 312.6, 1000 mg. The respective concentrations of test item were each separately dispersed in approximately 200 mL deionised reverse osmosis water and subjected to ultrasonication for approximately 20 minutes. All test vessels were shielded from light during mixing. Synthetic sewage (16 mL), activated sludge (250 mL) and water were added to a final volume of 500 ml to give the required concentrations. Range Finder: 10, 100, and 1000 mg/L ; Definitive Test: 9.8, 30.6, 97.7, 312.6 and 1000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): In the range finding test: At concentrations of 10 and 100 mg/L, no undissolved test item was visible although there was a Dark /brown dispersion with a few oily globules of test item visible on the surface. At concentrations of 1000 mg/L, the test item was Dark /brown dispersion with oily slick and small oily globules of test item visible on surface. In the definitive test: at concentration 9.8 to mg/L 30.6 nominal: there was no undissolved test item. At 100 to 312.6 mg/L there was a dark brown dispersion with small oily globules of test item visible on surface. At 1000 mg/L there was brown dispersion with an oily slick and small oily globules of test item visible on surface

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: aerobic activated sludge from sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage (dates provided in the full study report).
- Preparation of inoculum for exposure: 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 sludge (14 litres) was fed synthetic sewage sludge (700 mL). The pH of the sample on the day of the test was 7.8.
- Initial biomass concentration: Determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 3.0 g/L prior to use.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test
start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was done in the Range Finder, when necessary. It was not necessary in the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

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 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 approximately 7.0 mg O2/L and 2.0 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 the low oxygen consumption, the rate was determined over an approximate 10 minute period.

Test temperature:

20 ± 2 °C

pH:

0 hours: control: 7.4 - 8.0 and test groups: 7.2 - 7.6 and reference item: 7.2 - 7.4
3 hours: control: 7.3 - 7.6 and test groups: 7.2 - 7.6 and reference item: 7.5 - 7.8

Dissolved oxygen:

30 minutes: control: 5.83 - 7.21 mgO2/L and test groups: 5.94 – 7.63 mgO2/L and reference item: 7.53 - 8.69 mgO2/L (all dissolved oxygen concentrations were above 60% saturation).

Nominal and measured concentrations:

Range-finder: nominal: 0 (control) 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and four for control).
Definitive test: nominal: 0 (control) 9.8, 30.6, 97.7, 312.6 and 1000 mg/L (in three replicates; six for control)
Reference item was completed: nominal: 3.2, 10, 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass conical BOD flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six (negative) and three (reference item)
- Nitrification inhibitor used (delete if not applicable): Not applicable.
- Biomass loading rate: See table.

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 Dissolved Organic Carbon (DOC). Synthetic sewage was subsequently prepared.
- Culture medium different from test medium: Not applicable.
- Intervals of water quality measurement: pH and temperature were determined in all test media and controls; prior to and at the end of the 3-hour incubation period. Dissolved oxygen values were determined in all vessels 30 minutes after incubation had initiated.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item dispersions was measured and adjusted to between pH 7.0 and 8.0, if necessary.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive study: 0 (control) 9.8, 30.6, 97.7, 312.6 and 1000 mg/L
- Range finding study: 0, (control) 10, 100, 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

214 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

82.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

44.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

30.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

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

Validity Criteria:
1. The mean specific respiration rate of the blank controls was 25.91 mg oxygen per gram dry weight of sludge per hour (i.e. was > 20 mg oxygen per gram dry weight of sludge per hour).
2. The coefficient of variation of the oxygen uptake rate in the control replicates was 5.95% (i.e. < 30%).
3. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 5.5 (C.I. 4.0 – 7.5) mg/L. This was within the expected range: 2 to 25 mg/L.
Therefore, the validity criteria was met.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: total respiration: actual 5.5 (C.I. 4.0 – 7.5) mg/L. This was within the expected range: 2 to 25 mg/L. Full information is provided in the full study report.

Reported statistics and error estimates:

95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon.
Analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control 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 analysis were performed using the SAS computer software package.

Table 1: Range-finding test: Control, reference substance and test substance oxygen concentration, oxygen consumption rate and percentage inhibition of the respiration rate.

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.1	6	1.9	42.0	-
	R2	5.5	5	2.1	40.8	-
	R3	6.1	6	2.0	41.0	-
	R4	5.6	5	2.0	43.2	-
Test Item	10	5.2	5	1.7	42.0	[1]
	100	5.4	6	2.1	33.0	21
	1000 R1	7.4	10	5.2	13.2	68
	1000 R2	7.7	10	5.7	12.0	71
	1000 R3	7.6	10	5.3	13.8	67
3,5-Dichlorophenol	3.2	6.8	10	2.4	26.4	37
	10	7.5	10	4.8	16.2	61
	32	7.8	9	7.5	2.0	95

[ ] = Increase in respiration rate as compared to controls

 

Table 2: Definitive test: Control, reference substance and test substance oxygen concentration, oxygen consumption rate and percentage inhibition of the respiration rate

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	4.4	4	1.9	37.50	-
	R2	5.7	6	2.0	37.00	-
	R3	5.4	5	1.8	43.20	-
	R4	4.7	4	2.2	37.50	-
	R5	5.2	5	1.9	39.60	-
	R6	5.7	5	2.5	38.40	-
Test Item	9.8R1	4.5	4	2.0	37.50	4
	9.8R2	4.8	5	1.9	34.80	10
	9.8R3	5.3	5	2.3	36.00	7
	30.6R1	4.7	5	1.7	36.00	7
	30.6R2	4.4	4	2.1	34.50	11
	30.6R3	4.4	4	2.0	36.00	7
Test Item	97.7R1	5.7	7	2.3	29.14	25
	97.7R2	5.7	8	2.0	27.75	29
	97.7R3	5.8	8	1.7	30.75	21
	312.6R1	6.4	10	3.6	16.80	57
	312.6R2	6.5	10	3.7	16.80	57
	312.6R3	6.0	10	3.0	18.00	54
	1000R1	6.7	10	4.3	14.40	63
	1000R2	7.4	10	4.2	19.20	51
	1000R3	7.2	10	5.0	13.20	66
3,5-Dichlorophenol	3.2	6.8	10	2.9	23.40	40
	10	7.4	10	4.8	15.60	60
	32	8.2	10	7.5	4.20	89

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3-hour EC50 for total inhibition was 214 mg/L. The EC20 was 82.5 mg/L, the EC10 was 44.6 mg/L. The NOEC was 30.6 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration, a definitive test was conducted examining total respiration only was carried out under static conditions. The test item concentrations were 9.8, 30.6, 97.7, 312.6, 1000 mg/L (three replicates per test concentration) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was 5.95% and the specific respiration rate of the controls was 25.91 mg oxygen per gram dry weight of sludge per hour. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/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. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 5.5 (C.I. 4.0 – 7.5) mg/L. All validity criteria were considered to have been satisfied. Under the conditions of the study, the 3-hour EC50 for total inhibition was 214 mg/L. The EC20 was 82.5 mg/L, the EC10 was 44.6 mg/L. The NOEC was 30.6 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Description of key information

EC50 = 214 mg/L (nominal), 20 °C, OECD TG 209, 2018

EC10 = 44.6 mg/L (nominal), 20°C, OECD TG 209, 2018

NOEC = 30.6 mg/L (nominal), 20 °C, OECD TG 209, 2018

Key value for chemical safety assessment

EC50 for microorganisms:

214 mg/L

EC10 or NOEC for microorganisms:

30.6 mg/L

Additional information

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration, a definitive test was conducted examining total respiration only was carried out under static conditions. The test item concentrations were 9.8, 30.6, 97.7, 312.6, 1000 mg/L (three replicates per test concentration) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was 5.95% and the specific respiration rate of the controls was 25.91 mg oxygen per gram dry weight of sludge per hour. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/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. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 5.5 (C.I. 4.0 – 7.5) mg/L. All validity criteria were considered to have been satisfied. Under the conditions of the study, the 3-hour EC50 for total inhibition was 214 mg/L. The EC20 was 82.5 mg/L, the EC10 was 44.6 mg/L. The NOEC was 30.6 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.