Reactionmass of octan-2-yl prop-2-enoate and octan-3-yl prop-2-enoate and octan-4-yl prop-2-enoate

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:

June 20th, 2019

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M FMRD, S352546
- Expiration date: 2020.05.01
- Purity: 99.7%
- Physical state: Clear and colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (15-25°C).
- Stability under test conditions: Due to analytical issus, stability during the test is unknown.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
-Methods: Test substance directly weighed into the test vessel and distilled water added.
-Controls: blank (water only), abiotic, procedural control

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from aeration tank in the Shenyang North Sewage Treatment Plant, PRC
- Preparation of inoculum for exposure: Upon arrival at the laboratory, coarse particles and solids were removed by settling for 15 minutes. The decanted sludge was centrifuged for 5 minutes (2500 rpm) to obtain a sewage pellet and clear supernatant. The sludge pellet was washed by resuspension in dechlorinated tap water, and the above process was repeated. The washed, resuspended sludge was dried (10 mL suspended sludge, 5 replicates, at 105 C for 1 hour and 40 mins) to determine an initial suspended solids concentration and was further diluted to 3 g sludge solids/L. Sludge was fed with synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) and aerated one day prior to test.
- Preparation of inoculum for exposure: Sludge concentration was measured again on day of test and adjusted as needed to obtain 3 g sludge solids/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.4-21.6 °C

pH:

7.18-7.99

Nominal and measured concentrations:

Nominal: 0 (dilution water control), 10, 100, 300, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks, containing 500 mL volume
- Type: open during aeration
- Aeration: Yes, clean oil-free air, 0.5-1.0 mL/ min
- No. of vessels per concentration: The 10, 100, and 300 mg/L solutions were tested with 1 replicate and 1000 mg/L solution tested in triplicate and one abiotic control (1000 mg/L)
- No. of vessels per control (replicates): 4
- No. of vessels per abiotic control (replicates): 1
- No. of vessels per reference substance (replicates): 1 replicate at each of the five concentrations.
- Sludge concentration: 3 g/L
- Nutrients provided for bacteria: synthetic sewage per OECD 209 (peptone 16g, meat extract 11g, urea 3g, NaCl 0.7 g, CaCl2.2H2O 0.4 g, MgSO4.7H2O 0.2 g, K2HPO4 2.8g, (dissolved in 1 L of water and filtered).
- Test setup: For the exposure period, flasks received either pure test substance or reference substance stock solution. Distilled water was added to bring total volume to 234 mL. 16 mL synthetic sewage and 250 mL diluted sludge were then added and the flasks aerated until measurement. Solution pH was measured after aeration began.
- Nitrification inhibition used: None
- Biomass loading rate: 1.5 g sludge solids/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled tap-water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Details on termination of incubation: After the 3-hour exposure, a sample was transferred to a measuring cell. Using a HACH HQ40d oxygen probe with a sleeve adaptor, the probe was inserted into the flask and magnetic stirrer started. Dissolved oxygen concentration was continuously measured for 10 minutes.

EFFECT PARAMETERS MEASURED : Dissolved oxygen concentration. For each exposure, dissolved oxygen depletion and time interval were correlated and the slope of the regression line was calculated as the respiration rate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: variable
- Justification for using fewer concentrations than requested by guideline: test done as combined rangefinder/limit test

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Average percent inhibition at 1000 mg/L was 1.1%.

Details on results:

- Blank controls oxygen uptake rate: 20.7 mg/(g*hr), Details in Table 2
- Coefficient of variation of oxygen uptake rate in control replicates: 1.4%

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- EC50: 6.0 mg/L (95 CI: 3.9-9.2). The EC50 was within the 5 to 25 mg/L range considered acceptable for the test.

Reported statistics and error estimates:

For the study summary, a t-test was run on the difference in respiration rate in the 1000 mg/L concentrations and blank control, following report submission. No difference in mean rate was found (p=0.63).

Table 1. Dissolved oxygen concentration for total respiration during 10-minute measurement period after 3 hour contact time

Oxygen uptake	Test vessel No.	Nominal Concentration (mg/L)	1	2	3	4	5	6	7	8	9	10
Blank control (Total)	FBT1	/	8.61	8.10	7.59	7.08	6.57	6.06	5.55	5.04	4.53	4.02
	FBT2	/	7.76	7.24	6.72	6.20	5.68	5.16	4.65	4.14	3.63	3.12
	FBT3	/	7.24	6.72	6.20	5.68	5.16	4.64	4.12	3.61	3.10	2.59
	FBT4	/	6.20	5.67	5.14	4.61	4.08	3.55	3.03	2.51	1.99	1.47
Reference item (Total)	FRT1	0.5	7.77	7.27	6.77	6.27	5.77	5.28	4.79	4.30	3.82	3.34
	FRT2	1.3	6.85	6.40	5.98	5.55	5.11	4.68	4.24	3.82	3.37	2.93
	FRT3	3.5	7.63	7.19	6.78	6.37	5.98	5.60	5.22	4.85	4.49	4.13
	FRT4	9.4	7.52	7.30	7.08	6.86	6.64	6.42	6.20	5.97	5.74	5.51
	FRT5	25	9.42	9.39	9.33	9.27	9.21	9.14	9.08	9.02	8.96	8.90
Test item (Total)	FTT1	10	7.77	7.26	6.77	6.28	5.77	5.28	4.77	4.26	3.75	3.24
	FTT2	100	7.85	7.32	6.80	6.31	5.79	5.24	4.72	4.15	3.60	3.05
	FTT3	300	7.70	7.16	6.64	6.14	5.64	5.14	4.64	4.15	3.65	3.13
	FTT4-1	1000	6.88	6.36	5.84	5.32	4.81	4.29	3.77	3.24	2.72	2.18
	FTT4-2	1000	6.96	6.42	5.91	5.39	4.86	4.34	3.82	3.29	2.77	2.24
	FTT4-3	1000	7.20	6.71	6.19	5.67	5.16	4.67	4.19	3.72	3.25	2.79
	FA	1000	8.53	8.53	8.52	8.50	8.49	8.47	8.47	8.46	8.44	8.44

Table 2.  Oxygen uptake rate and percent inhibition of total respiration after 3 hour contact time

Oxygen uptake	Test vessel No.	Nominal Concentration (mg/L)	1-2	2-3	3-4	4-5	5-6	6-7	7-8	8-9	9-10	Average	R:mg/(L·h)	Rs: mg/(g·h)	% Inhibition
Blank control (Total)	FBT1	/	0.51	0.51	0.51	0.51	0.51	0.51	0.51	0.51	0.51	0.510	30.6	20.4	/
	FBT2	/	0.52	0.52	0.52	0.52	0.52	0.51	0.51	0.51	0.51	0.516	31.0	20.7	/
	FBT3	/	0.52	0.52	0.52	0.52	0.52	0.52	0.51	0.51	0.51	0.517	31.0	20.7	/
	FBT4	/	0.53	0.53	0.53	0.53	0.53	0.52	0.52	0.52	0.52	0.526	31.6	21.1	/
Reference item (Total)	FRT1	0.5	0.50	0.50	0.50	0.50	0.49	0.49	0.49	0.48	0.48	0.492	29.5	19.7	4.8%
	FRT2	1.3	0.45	0.42	0.43	0.44	0.43	0.44	0.42	0.45	0.44	0.436	26.2	17.5	15.5%
	FRT3	3.5	0.44	0.41	0.41	0.39	0.38	0.38	0.37	0.36	0.36	0.389	23.3	15.5	25.1%
	FRT4	9.4	0.22	0.22	0.22	0.22	0.22	0.22	0.23	0.23	0.23	0.223	13.4	8.9	57.0%
	FRT5	25	0.03	0.06	0.06	0.06	0.07	0.06	0.06	0.06	0.06	0.058	3.5	2.3	88.9%
Test item (Total)	FTT1	10	0.51	0.49	0.49	0.51	0.49	0.51	0.51	0.51	0.51	0.503	30.2	20.1	2.9%
	FTT2	100	0.53	0.52	0.49	0.52	0.55	0.52	0.57	0.55	0.55	0.533	32.0	21.3	-2.9%
	FTT3	300	0.54	0.52	0.50	0.50	0.50	0.50	0.49	0.50	0.52	0.508	30.5	20.3	1.9%
	FTT4-1	1000	0.52	0.52	0.52	0.51	0.52	0.52	0.53	0.52	0.54	0.522	31.3	20.9	-1.0%
	FTT4-2	1000	0.54	0.51	0.52	0.53	0.52	0.52	0.53	0.52	0.53	0.524	31.4	20.9	-1.0%
	FTT4-3	1000	0.49	0.52	0.52	0.51	0.49	0.48	0.47	0.47	0.46	0.490	29.4	19.6	5.3%
	FA	1000	0.00	0.01	0.02	0.01	0.02	0.00	0.01	0.02	0.00	0.010	/	/	/

Note: R: mg/(L·h), oxygen uptake rate; Rs: mg/(g·h), specific respiration rate

Average of I% was calculated based on average of Rs.

Validity criteria fulfilled:

yes

Remarks:

Control oxygen uptake rate > 20 mg O2/gram sludge/hour (20.7 mg O2/(h.g SS)); coeff. of variation <15% for control replicates (1.4%); ref. substance EC50 in range 5-25 mg/L (6.0 mg/L).

Conclusions:

3 hour EC50 >1000 mg/L in activated sludge for respiration inhibition (OECD 209)

Executive summary:

The toxicity of MTDID 44428 to activated sludge was assessed according to the OECD 209 guideline in a definitive test conducted at 10, 100, 300, and 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of MTDID 44428 to activated sludge is >1000 mg/L, as only 1.1% inhibition was observed at the highest concentration. There was no significant difference between the respiration rates of the blank control and the 1000 mg/L concentration. The study followed an international standard method and is GLP compliant. Therefore the study is deemed reliable without restrictions and can be utilized to calculate a PNEC for STP.

Description of key information

The toxicity of MTDID 44428 to activated sludge was assessed according to the OECD 209 guideline in a definitive test conducted at 10, 100, 300, and 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of MTDID 44428 to activated sludge is >1000 mg/L, as only 1.1% inhibition was observed at the highest concentration.There was no significant difference between the respiration rates of the blank control and the 1000 mg/L concentration.The study followed an international standard method and is GLP compliant.Therefore the study is deemed reliable without restrictions and can be utilized to calculate a PNEC for STP.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L