sec-butylamine

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:

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The synthetic sewage feed was prepared to contain the following constituents per liter:
- 16 g peptone
- 11 g meat extract
- 3 g urea
- 0.7 g sodium chloride
- 0.4 g calcium chloride dihydrate CaCl2, 2H2O
- 0.2 g magnesium sulfate heptahydrate MgSO4, 7H2O
- 2.8 g di-potassium hydrogen phosphate K2HPO4
The final volume was filled up with ultra-pure water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The inoculum was obtained on 10 April 2017 from the activated sludge of the biological wastewater treatment plant from Mourenx (France, 64) which handles predominantly domestic sewage.
On arrival at the laboratory, the activated sludge (around 10L) was centrifuged (1000g for 10 minutes) and the solid sewage inoculum pellet was re-suspended in 10L deionised water. 500 mL synthetic sewage feed was then added. Sewage sludge was maintained under aerated conditions for a maximum of 3 days and fed daily with synthetic sewage feed.
In order to obtain the required sludge suspended solids concentration of 3 g/L in the stock solution to be used in the experiments, the solid content of the 10-L sludge solution was determined by removing a sub-sample of homogenised sludge and drying in an oven at approximately 105°C. The suspended solids concentration was subsequently adjusted with the source water until the recommended guideline value of 3 g/L ± 10% is achieved.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21.2 - 22°C (min-max)

pH:

The pH of the test item stock solution was adjusted before dilutions. pH laid between 7 and 8.

Nominal and measured concentrations:

Nominal: control ; 6.5 ; 32.5 ; 97.7 ; 312.5 ; 1000 mg/L
Measured: none (not requested by the guideline).

Details on test conditions:

RANGE-FINDING TEST: PREPARATION OF TEST SOLUTIONS
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. Tests solutions were prepared from dilutions of a stock solution: a known volume of test item (6.93 mL) was poured into a volumetric flask, the volume was then made up to 1000 mL with dilution water. The solution was kept under high speed stirring at room temperature during a few minutes. Aliquots of the stock solution were added to activated sewage sludge, synthetic sewage and water to achieve final concentrations, each with two replicates. Upon addition of the microbial inoculum, the pH was measured in order to identify if there was any variation in pH caused by the addition of the test item. The pH of the test item stock solution was adjusted before dilutions.
The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum: a stock solution at 1 g/L was prepared by dissolving the reference item (101.6 mg) directly in previously heated water to aid dissolution, the volume was then made up to 100 mL with ultra-pure water. Aliquots of the stock solution were added to activated sewage sludge, synthetic sewage and water to achieve final concentrations of 3.2, 10 and 32 mg/L, each with a single replicate.

At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum were added to the first blank control vessels Fba (final suspended solid of nominally 1.5 g/L/dw). Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with test vessels in which 16 mL of synthetic sewage were made up to 250 mL with the appropriate amount of reference or test item diluted in ultra-pure water. As for the control, 250 mL of activated sludge inoculum were then added. Fifteen minutes after the preparation of the last treatment, the last blank control vessel Fbb was prepared at last. The fifteen minutes interval was done so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel.

DEFINITIVE TEST: PREPARATION OF TEST SOLUTIONS
The definitive test was conducted using a range of concentrations deduced from the preliminary range-finding test: activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 9.5, 32.5, 97.7, 312.5 and 1000.0 mg/L. In order to obtain both NOEC and EC50, four controls and five treatment concentrations in a geometric series with 2 replicates were prepared. Tests solutions were prepared from dilutions of a stock solution prepared as described above. The same inoculum as for the range-finding test was used.
The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm again the suitability of the inoculum (test solutions were prepared with a single replicate from the same stock solution as the range-finding test).
The test protocol was identical to the one described for the range-finding test, except that more test item concentrations were tested. Upon addition of the microbial inoculum the pH was measured in order to identify if there was any variation in pH caused by the addition of the test item. The pH was not adjusted.

INCUBATION AND OBSERVATIONS
The addition of the inoculum was considered as the start of the 3-hours incubation time period. All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L. The temperature was monitored throughout the test period.

Reference substance (positive control):

yes

Remarks:

1,3-dichlorophenol

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.:

313 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

It was not possible to derive a reliable 3h-EC10. However, a reliable 3h-NOEC was derived for respiration inhibition. NOEC = 313 mg/L.

Results with reference substance (positive control):

The 3h-EC50 for 3,5-dichlorophenol (13.6 mg/L) was between 2 and 25 mg/L for total respiration.

Reported statistics and error estimates:

Results were processed as described in the guideline. Toxrat 3.2 was used to derive both ECx and NOEC/LOEC where possible.

Table 1: mean total respiration inhibition per tested concentration.

 

Test item nominal concentration (mg/L)	Inhibition of total respiration (%)
9.5	-8.6
32.5	-11.3
97.7	-9.1
312.5	-4.8
100.0	33.0

 

Validity criteria fulfilled:

yes

Conclusions:

3h-NOEC was 313 mg/L for respiration inhibition.

Executive summary:

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010). Total respiration inhibition was assessed in this study. The test was conducted at the following nominal concentrations:9.5, 32.5, 97.7, 312.5 and 1000.0 mg/L

A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 3.2, 10.0 and 32.0 mg/L.

The prepared vessels were aerated for 3h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time and expressed as mg O₂/L/h for the linear part of the graphs (then converted in mg O₂/g of activated sludge dry weight of suspended solids/h). 

 

The definitive test met the validity criteria of the guideline detailed as follows:

        § The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the test: it              was determined to be 25.7%.

§ The 3h-EC50 for 3,5-dichlorophenol (13.6 mg/L) was between 2 and 25 mg/L for total respiration.

§ The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: the mean total respiration rate was 44.8 mg O₂/g of activated sludge (dry weight of suspended solids)/h.

  

3h-NOEC was 313 mg/L and 3h-EC50 was higher than the highest concentration tested (i.e., 1000 mg/L).

Description of key information

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010). Total respiration inhibition was assessed in this study. The test was conducted at the following nominal concentrations:9.5, 32.5, 97.7, 312.5 and 1000.0 mg/L

A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 3.2, 10.0 and 32.0 mg/L.

The prepared vessels were aerated for 3h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time and expressed as mg O₂/L/h for the linear part of the graphs (then converted in mg O₂/g of activated sludge dry weight of suspended solids/h). 

 

The definitive test met the validity criteria of the guideline detailed as follows:

       § The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the test: it             was determined to be 25.7%.

§ The 3h-EC50 for 3,5-dichlorophenol (13.6 mg/L) was between 2 and 25 mg/L for total respiration.

§ The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: the mean total respiration rate was 44.8 mg O₂/g of activated sludge (dry weight of suspended solids)/h.

  

3h-NOEC was 313 mg/L and 3h-EC50 was higher than the highest concentration tested (i.e., 1000 mg/L).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

313 mg/L