N-ethyl-o(or p)-toluenesulphonamide

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:

16 Nov 2017 - 12 Jan 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))

Version / remarks:

22 July 2010

GLP compliance:

yes

Specific details on test material used for the study:

Identification: N-ethyl-o (or p) toluenesulphonamide (NETSA)
Appearance: Light yellow viscous liquid
Batch: 1703691015
Test item storage: At room temperature Keep containers tightly closed in a dry, cool and well-ventilated place
Stable under storage conditions until: 16 March 2019 (expiry date)

Additional information
Test Facility test item number: 201042/B
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: N-ethyl-o (or p) toluenesulphonamide
CAS number: 8047-99-2
Molecular structure: Not indicated
Molecular formula: C9H13NO2S
Molecular weight: 199.3
Volatile: Not indicated
Specific gravity / density: 1.2
Solubility in water: 0.47 g/l
Stability in water: Stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of NETSA tested was a light yellow viscous liquid with a purity of 100%. No correction was made for the purity/composition of the test item.
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts on an hourglass were added to the dark brown test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded.

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 pH was 7.8 on the day of testing.
- Pretreatment: The batch of sludge was used one day after collection. Therefore, 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids, i.e. 3.0 g/L of sludge. The amount of suspended solids in the final test mixture was 1.5 g/L.

Test type:

static

Water media type:

freshwater

Remarks:

water Tap water purified by reverse osmosis (Millipore Corp.,Bedford, Mass., USA)

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period.

Test temperature:

19 - 21°C

pH:

T 0hr control: 7.3 - 7.4
T 3hr control: 7.9 - 8.2

T 0hr test item: 7.3 - 7.4
T 3hr test item: 7.9 - 8.3

T 0hr 3,5 DCP: 7.4 - 7.6
T 3hr 3,5 DCP: 8.2 -8.4

Dissolved oxygen:

>60-70% of air saturation at test start

Nominal and measured concentrations:

Test substance
Nominal: 22, 46, 100, 220, 460 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Total fill volume: 500 mL (250 mL test solution (incl. synthetic medium) + 250 mL activated sludge in test medium)
- Aeration: yes (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 per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 4
- Sludge concentration (weight of dry solids per volume): 1.5 g/L sludge
- Nutrients provided for bacteria: synthetic medium acc. to OECD 209
- Nitrification inhibitor used : no

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel- Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation: 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.
- Synthetic medium:
16 g peptone (=sewage feed)
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake

TEST CONCENTRATIONS
- Combined limit / Range finding study
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; The combined limit/range-finding test showed 3%, 15% and 79% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/L, respectively. Therefore, the expected ELR50 was between a loading rate of 100 and 1000 mg/L. There was no oxygen uptake from abiotic processes.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

644 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CL: 551 - 775

Duration:

3 h

Dose descriptor:

other: ELR10

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CL: 73 - 145

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Inhibition of the Respiration Rate:
The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOELR and ELRx values. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg Ketjenflex 8 per litre. At higher loading rates the inhibitory effect of Ketjenflex 8 on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 14% inhibition at 220 mg/L to 66% at 1000 mg/L.

Acceptability of the test:
1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (26 mg oxygen per one gram of activated sludge in the final test).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (16%).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (7.2 mg/L in the final test).

Results with reference substance (positive control):

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.
EC50 = 7.15 mg/L ( 95% Cl: 5.05 - 9.30) (falls within the OECD reported range for total respiration, i.e. 2-25 mg/L)

Reported statistics and error estimates:

ECx and ELRx:
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 the test item, calculation of the ELRx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

NOELR determination
An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

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

Final Test

Treatment	Loading rate (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		39.39	26.26
T1	22	33.88	22.59	13.97
T2	46	35.14	23.43	10.78
T3	100	35.30	23.53	10.39
T4	220	33.88	22.59*	13.97
T5	460	23.65	15.77*	39.96
T6	1000	13.41	8.94*	65.95

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

In conclusion, NETSA was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOELR). The ELR10 and ELR50 were at a loading rate of 110 mg/L and 644 mg/L respectively.

Executive summary:

A study was performed to assess the effect of NETSA on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines.The final test was performed based on the result of a preceding combined limit/range-finding test. Micro-organisms in activated sludge were exposed to test concentrations of 22, 46, 100, 220, 460 and 1000 mg/l (5 replicates per loading rate) and an untreated control group (6 replicates) for 3 hours. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg NETSA per litre. At higher loading rates the inhibitory effect of NETSA on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 14% inhibition at 220 mg/L to 66% at 1000 mg/L. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.The ELR10 and ELR50 were determined to be at a loading rate of 110 mg/L (95% confidence interval: 73 - 145 mg/L) and 644 mg/L (95% confidence interval: 551 – 775 mg/L).

 

Description of key information

A study was performed to assess the effect of NETSA on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines.The final test was performed based on the result of a preceding combined limit/range-finding test. Micro-organisms in activated sludge were exposed to test concentrations of 22, 46, 100, 220, 460 and 1000 mg/l (5 replicates per loading rate) and an untreated control group (6 replicates) for 3 hours. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg NETSA per litre. At higher loading rates the inhibitory effect of NETSA on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 14% inhibition at 220 mg/L to 66% at 1000 mg/L. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.The ELR10 and ELR50 were determined to be at a loading rate of 110 mg/L (95% confidence interval: 73 - 145 mg/L) and 644 mg/L (95% confidence interval: 551 – 775 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

644 mg/L

EC10 or NOEC for microorganisms:

110 mg/L