Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

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:

06-February-2013-to-07-March-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with current Guideline and is GLP compliant.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

To prepare test vessels at 10 and 100 mg/L, the substance coating additives was weighed directly onto glass cover slips and these added to the appropriate test vessel (500 mL glass conical flask) to give the required test concentrations. The flasks had the appropriate amount of deionised water added (234 mL) and were ultrasonicated for ca. 10 min to aid dissolution. To prepare the test vessel at 1 mg/L, a 10 mg/L solution ofthe substance coating additives was prepared by adding the substance coating additives (10.10 mg) to deionised water (1 L) and ultrasonicating for ca. 10 min to aid dissolution. An aliquot (50 mL) from this 10 mg/L solution was added to the appropriate test vessel containing deionised water (184 mL).
The reference item, 3,5-DCP, was tested at nominal concentrations of: 5, 15 and 30 mg/L.
A 1 g/L reference item stock solution was prepared by adding 3,5-DCP (0.25019 g) to a volumetric flask (250 mL) and bringing to volume with deionised water. The solution was ultrasonicated for ca 10 min to aid dissolution. Aliquots were then added to the appropriate test vessel to give the required reference item test concentrations.
Prior to initiation of the test, both control vessels and reference item vessels were prepared by adding 16 mL of synthetic sewage feed and an appropriate volume of deionised water. An appropriate volume of the reference item was added to the reference item vessels and the weighed test item then added to the appropriate test item vessels. At time ‘0’ microbial inoculum (250 mL) was added to control vessel C1 to give a final volume of 500 mL. This process was repeated at 15 min intervals in the order outlined in the table in the 'test conditions' section below (i.e. from left to right, ending with C2).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: On arrival at the laboratory, approximately 400 mL of the clear supernatant was removed from each sample of sewage sludge and the sludge then aerated. The solid content of the sludge was determined by shaking the bottle of sludge to homogenise thoroughly. Duplicate sub-samples (5 mL) of the homogenised sludge were dried in an oven at approximately 105°C and the suspended solids content determined as 2.382 g/L. Ca. 600 mL of sludge was removed from each sample of sludge leaving 4 L remaining. Synthetic sewage feed was then added to the sludge at a rate of 50 mL/L of sludge and the sludge left aerating overnight. The solid content of the sludge was then re-determined and found to be 2.895 g/L, which was acceptable for use in the test.

Test type:

other: aerated

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

The dissolved oxygen concentration was recorded at 30 s intervals for a period of 10 min. The dissolved oxygen concentration was measured using a Yellow Springs Inc. Dissolved Oxygen meter (Model No. 5100) and BOD probe (Model No. 5010) with agitator paddle. The above processes were repeated for each vessel after the 3 hour contact time.

pH:

7.85 - 8.00

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flasks
- Type (delete if not applicable): open
- Fill volume: 500 mL
- Aeration: airflow rate = 1.0 L/min

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Charles River, Tranent
- Water bath temperature: 19.7-20.5°C (monitored throughout the test)

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of reference and control vessels was adjusted as necessary using 1 M HCL solution

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3,5-DCP). Aldrich (Batch No. MKBF6101V). Purity of 97%. EXP: January 2014

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: 3 h EC50 7.49 mg/L

Reported statistics and error estimates:

The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated, using the non-parametric Trimmed Spearman-Karber Method (Hamilton et al, 1977), as 7.49 mg/L, with 95 % confidence limits of 6.46 and 8.67 mg/L, under test conditions.

Dissolved Oxygen Measurements (mg O₂/L) at 3 h Contact Time

Time (min)	Dissolved Oxygen Measurements at 3 h Contact Time (mg O2/L)
	Control C1	Nominal Concentration of the substance coating additives (mg/L)					3,5-DCP (mg/L)			Control C2
		1	10	100 (I)	100 (II)	100 (III)	5	15	30
0.5	7.70	7.34	7.57	6.98	7.44	5.39	8.06	8.75	8.62	7.53
1	7.25	6.74	7.04	6.47	6.98	4.86	7.71	8.65	8.49	7.04
1.5	6.80	6.21	6.49	5.92	6.52	4.35	7.39	8.56	8.42	6.52
2	6.32	5.65	5.99	5.44	6.06	3.84	7.05	8.46	8.32	6.04
2.5	5.88	5.11	5.45	4.88	5.58	3.33	6.73	8.35	8.24	5.51
3	5.41	4.61	4.95	4.41	5.16	2.82	6.41	8.25	8.15	5.01
3.5	4.95	4.03	4.44	3.90	4.71	2.31	6.07	8.17	8.07	4.55
4	4.49	3.49	3.94	3.39	4.24	1.81	5.75	8.08	7.99	4.06
4.5	4.03	2.99	3.47	2.89	3.78	1.34	5.40	8.01	7.92	3.57
5	3.57	2.42	2.97	2.38	3.33	0.83	5.09	7.92	7.84	3.07
5.5	3.11	1.93	2.47	1.87	2.88	0.34	4.77	7.84	7.75	2.58
6	2.65	1.36	1.97	1.37	2.43	0.00	4.45	7.76	7.68	2.09
6.5	2.19	0.87	1.47	0.86	1.98	0.00	4.12	7.66	7.61	1.60
7	1.73	0.35	0.97	0.38	1.52	0.00	3.80	7.57	7.52	1.12
7.5	1.27	0.00	0.48	0.00	1.08	0.00	3.47	7.48	7.46	0.63
8	0.82	0.00	0.02	0.00	0.63	0.00	3.14	7.39	7.38	0.16
8.5	0.37	0.00	0.00	0.00	0.19	0.00	2.82	7.30	7.30	0.00
9	0.00	0.00	0.00	0.00	0.00	0.00	2.50	7.21	7.24	0.00
9.5	0.00	0.00	0.00	0.00	0.00	0.00	2.18	7.12	7.16	0.00
10	0.00	0.00	0.00	0.00	0.00	0.00	1.86	7.04	7.08	0.00

Note: When 2 consecutive readings of 0 mg O₂/L are recorded, further readings are not made and subsequent time points are allocated 0 mg O₂/L

Respiration Rates and Percentage Inhibition

Treatment	Concentration (mg/L)	Respiration Rate (mg O2/L/h)	% Inhibition
The substance coating additives (mg/L)	1	64.80	0.00
	10	60.93	0.00
	100 Rep I	61.35	0.00
	100 Rep II	54.60	4.53
	100 Rep III	61.60	0.00
Mean Respiration Rate for 100 mg/L		59.18	0.00
3, 5-DCP (mg/L)	5	39.00	31.81
	15	10.80	81.12
	30	9.73	82.99
Control 1	NA	55.32	NA
Control 2	NA	59.07	NA
Mean of Controls	NA	57.20	NA
Coefficient of Variation in Control Replicates (%)		4.64	NA

NA = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h EC50 and EC05 of the test substance for effects on activated sludge were both determined to be >100 mg/L (nominal).

Executive summary:

A study was conducted to determine the effects of the test substance, on the respiration rate of activated sludge microorganisms according to OECD guideline 209, in compliance with GLP. The test was conducted at the following nominal test concentrations: 1, 10 and 100 mg/L. Single vessels were tested for test concentrations of 1 and 10 mg/L, triplicate vessels were tested at 100 mg/L. A reference substance (3, 5 dichlorophenol) was tested as a positive control at the following nominal concentrations: 5, 15 and 30 mg/L. Duplicate control vessels were also tested. The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 30 sec intervals for up to a maximum period of 10 min. The respiration rate (mg O2/L/h) was calculated over the linear phase of oxygen consumption for each vessel (ca 7.0 – 2.0 mg O2/L). Where respiration rate was low, the calculation was conducted over the full linear range. The respiration inhibition (%) was compared to the mean control vessel respiration rate, for both the reference and test substances. The coefficient of variation of oxygen uptake rate in control replicates was 4.64%. No inhibition of respiration was noted at 1, 10 and 100 mg/L test concentrations. As there was no inhibition of respiration at any test concentration, the 3 h EC5/ELr5, EC20/ELr20 and EC90/ELr90 for the test substance to activated sludge respiration were each estimated as >100 mg/L (nominal). The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 7.49 mg/L, with 95 % confidence limits of 6.46 and 8.67 mg/L. The test met guideline validity criteria as the coefficient of variation in control replicates was less than 15%, the EC50 (3 h) for the reference substance was in the range 2 to 25 mg/L and the oxygen uptake in the controls was greater than 20 mg oxygen/g of activated sludge/h. Under the study conditions, the 3 h EC50 and EC05 of the test substance for effects on activated sludge were both determined to be >100 mg/L (nominal) (Knight, 2013).

Description of key information

The 3 h EC50 and EC05 values of the test substance for inhibition of respiration rate of the activated sludge were both determined to be >100 mg/L (nominal).

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

A study was conducted to determine the effects of the test substance, on the respiration rate of activated sludge microorganisms according to OECD guideline 209, in compliance with GLP. The test was conducted at the following nominal test concentrations: 1, 10 and 100 mg/L. Single vessels were tested for test concentrations of 1 and 10 mg/L, triplicate vessels were tested at 100 mg/L. A reference substance (3, 5 dichlorophenol) was tested as a positive control at the following nominal concentrations: 5, 15 and 30 mg/L. Duplicate control vessels were also tested. The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 30 sec intervals for up to a maximum period of 10 min. The respiration rate (mg O2/L/h) was calculated over the linear phase of oxygen consumption for each vessel (ca 7.0 – 2.0 mg O2/L). Where respiration rate was low, the calculation was conducted over the full linear range. The respiration inhibition (%) was compared to the mean control vessel respiration rate, for both the reference and test substances. The coefficient of variation of oxygen uptake rate in control replicates was 4.64%. No inhibition of respiration was noted at 1, 10 and 100 mg/L test concentrations. As there was no inhibition of respiration at any test concentration, the 3 h EC5/ELr5, EC20/ELr20 and EC90/ELr90 for the test substance to activated sludge respiration were each estimated as >100 mg/L (nominal). The 3 h EC50 (respiration inhibition) for 3,5-dichlorophenol to activated sludge was estimated as 7.49 mg/L, with 95 % confidence limits of 6.46 and 8.67 mg/L. The test met guideline validity criteria as the coefficient of variation in control replicates was less than 15%, the EC50 (3 h) for the reference substance was in the range 2 to 25 mg/L and the oxygen uptake in the controls was greater than 20 mg oxygen/g of activated sludge/h. Under the study conditions, the 3 h EC50 and EC05 of the test substance for effects on activated sludge were both determined to be >100 mg/L (nominal) (Knight, 2013).