3-butoxypropylamine

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:

22 August 2017

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: BOPA
- CAS Number: 16499-88-0
- Molecular weight: 131 g/mol
- Chemical Name: 3-Butoxypropylamine
- Source and lot/batch No.of test material: PFW160573
- Expiration date of the lot/batch: 26 September 2018
- Purity: 99.39%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable

OTHER SPECIFICS:
- Physical state/Appearance: clear colorless liquid

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 item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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 dissolved directly in water.
A nominal amount of test item (2500 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.
The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 to pH 8.0 if necessary.

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 25 minutes. The pH of this stock solution was measured to be pH 6.2 and was adjusted to pH 7.1 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.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: mixed population of activated sewage sludge micro-organisms was obtained on 21 August 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 (4.5 liters) was fed synthetic sewage (225 mL). The pH of the sample on the day of the test was 7.6 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 3 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Pretreatment: No
- Initial biomass concentration: 3.0 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

20”C

pH:

prior to adjustment: 7.5 - 11.6
after adjustment: 7.5 - 7.1

Dissolved oxygen:

5.4 - 8.3 mg O2/L or 61 - 93% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Range-Finding Test
Nominal test concentrations of 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks of 500 mL
- Type (delete if not applicable): closed (see below for aeration)
- Material, size, headspace, fill volume: 200 mL
- Aeration: aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute.
- No. of vessels per concentration (replicates): one flask each for 10 and 100 mg/L and three for 1000 mg/L
- No. of vessels per control (replicates): one per concentration (3.2, 10 and 32 mg/L)
- No. of vessels per vehicle control (replicates): 4
- No. of vessels per abiotic control (replicates): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 3h
- Light intensity: normal laboratory lighting

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 100 - < 1 000 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 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 item after 3 hours contact time are given in Table 2.
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.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 1.6% and the specific respiration rate of the controls was 28.65 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied .

Inhibition of Respiration Rate
The following results were derived:
FAT 93588/A TE 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 - 1.4 -
EC20 <1000 - 2.1 -
EC50 >1000 6.8 5.2-8.8
EC80 >1000 - 22 -
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.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.2 to 8.8 mg/L.

Reported statistics and error estimates:

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).

Table 1. 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)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.22	70
	R2	5.65	63
	R3	6.36	71
	R4	6.17	69
Test Item	10	6.04	68
	100	5.43	61
	1000 R1	5.96	67
	1000 R2	6.24	70
	1000 R3	6.26	70
3,5-dichlorophenol	3.2	7.14	80
	10	7.81	88
	32	8.28	93

R₁– R₄= Replicates 1 to 4

Table 2. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding 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	5.1	4	2.2	43.50	-
	R2	5.0	4	2.2	42.00	-
	R3	6.1	5	2.5	43.20	-
	R4	5.8	5	2.2	43.20	-
Test Item	10	5.1	5	1.5	43.20	[1]
	100	4.9	4	2.0	43.50	[1]
	1000 R1	5.7	6	1.9	38.00	12
	1000 R2	6.2	6	2.3	39.00	9
	1000 R3	6.2	6	2.4	38.00	12
3,5-dichlorophenol	3.2	6.1	7	2.6	30.00	30
	10	7.3	10	4.5	16.80	61
	32	8.3	10	7.5	4.80	89

R₁– R₄= Replicates 1 to 4

[Increase in respiration rate as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms resulted in a 3-hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was less than 1000 mg/L.

Description of key information

Under the conditions of a guideline study (Best, 2017), the effect of the test item on the respiration of activated sewage sludge micro-organisms resulted in a 3-hour EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was less than 1000 mg/L but above 100 mg/L. Results of this study are reliable without restrictions.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L