4-(cyclohexylamino)butane-1-sulfonic acid

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:

25 Apr 2019 - 26 Apr 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 (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTIONS
- Test item: A stock solution of 10 g/L was prepared by adding 5.0515 g test item to 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., USA). Magnetic stirring for 62 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 7.2) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations.
- Reference substance: A 1 g/L stock solution was prepared by dissolving 249.1 mg 3,5-dichlorophenol in Milli-RO water and making up to a total volume of 250 mL. The stock solution (pH 7.9) was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3 g/L, as used for the test). The pH was 7.5 on the day of testing. The batch of sludge was used one day after collection. Therefore, 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20-21°C

pH:

At start (before addition of sludge) (all test vessels): 7.5-7.6
After 3h (all test vessels): 7.4-8.2

Nominal and measured concentrations:

10, 100 and 1000 mg/L (nominal)

Details on test conditions:

A combined limit/range-finding test was performed. Based on the results, more testing was not needed.

TEST SYSTEM
- Test vessel: all-glass open bottles, 500 mL fill volume
- Aeration: continuous aeration with clean, oil-free air.
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels (1000 mg/L): 3
- No. of vessels (10 and 100 mg/L): 1 per concentration
- No. of vessels per control (replicates): 6
- No. of vessels per reference control (replicates): 1 replicate per concentration (4 concentrations used)
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)
- Synthetic medium: according to OECD 209

PERFORMANCE
The synthetic medium (=sewage feed, 16 mL) and an appropriate amount of the test item stock were mixed and made up to 250 mL with Milli-RO water. The pH was determined.
Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

Details on results:

- The combined limit/range-finding test showed 15%, no inhibition and an average of 7% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively (EC50: >1000 mg/L). See Table 1 in 'Any other information on results incl. tables' for details.
- The inhibition of the respiration rate of the sludge recorded at a concentration of 1000 mg/L was not statistically significant (NOEC: 1000 mg/L).
- There was no significant oxygen uptake from abiotic processes.

Results with reference substance (positive control):

The EC50 was in the accepted range of 2 to 25 mg/L for total respiration (3.8 mg/L).

Reported statistics and error estimates:

ECx:
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For 4-(cyclohexylamino)butane-1-sulfonic acid, no EC50-value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L).

NOEC determination:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Respiration rate and % inhibition

Concentration (mg/L)	Mean respiration rate (mg O2/g.h)	% inhibition of respiration rate
Control	27.16	-
10	23.08	15
100	32.80	-21
1000	25.15	7.4

 

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The substance was evaluated for its influence on the respiration rate of activated sludge in accordance with OECD 209. Based on the results of this study, the substance is not toxic to STP microorganisms (3h-NOEC: 1000 mg/L).

Executive summary:

The substance was evaluated for its influence on the respiration rate of activated sludge in accordance with OECD 209. In a combined limit/range-finding test, concentrations of 10, 100 and 1000 mg/L were tested. There was no significant oxygen uptake from abiotic processes and the study met the acceptability criteria. No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg/L (7%, mean of 3 replicates). Therefore it is concluded that based on the results of this study, the substance is not toxic to STP microorganisms (3h-NOEC: 1000 mg/L).

Description of key information

The substance was evaluated for its influence on the respiration rate of activated sludge in accordance with OECD 209. Based on the results of this study, the substance is not toxic to STP microorganisms (3h-NOEC: 1000 mg/L).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The substance was evaluated for its influence on the respiration rate of activated sludge in accordance with OECD 209. In a combined limit/range-finding test, concentrations of 10, 100 and 1000 mg/L were tested. There was no significant oxygen uptake from abiotic processes and the study met the acceptability criteria. No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg/L (7%, mean of 3 replicates). Therefore it is concluded that based on the results of this study, the substance is not toxic to STP microorganisms (3h-NOEC: 1000 mg/L).