Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 July 2012 - 16 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine

EC Number:

284-664-9

EC Name:

Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine

Cas Number:

84961-74-0

Molecular formula:

N/A

IUPAC Name:

Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1)

Test material form:

liquid: viscous

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Amounts of the test item (80, 140, 250, 450 and 800 mg) were separately dissolved in water with the aid of high shear mixing (approximately 7500 rpm, 5 mins) and the volume adjusted to 1 liter to give 80, 140, 250, 450 and 800 mg/L stock solutions. An aliquot (200 ml) of the 80 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sludge (250 mL) and water to a final volume of 500 mL, to give the required concentration of 32 mg/L. Similarly, aliqots (200 mL) of the 140, 250, 450 and 800 mg/L stock solutions were used to prepare the test concentrations of 56, 100, 180 and 320 mg/L. Each test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0 if necessary.
The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 18 July 2012 for the range-finding test and on 15 August 2012 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
The activated sewage sludge sample was maintained on contimuous aeration in the laboratory at a temperature of approximately 21°C overnight prior to use in the test. On the day of collection the activated sewage sludge (8.5 liters) was fed synthetic sewage sludge (425 mL). The pH of the sample on the day of the test was 7.7. The suspended solids concentration of the acitvated sewage sludge was determined to be equal to 3.0 g/L.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 ± 2°C

pH:

pH ranged from 7.3 to 7.6 at the start of the exposure period.
pH ranged from 7.6 to 8.2 at the end of the exposure period.

Dissolved oxygen:

The oygen concentrations were measured after 30 minutes contact time and ranged from 2.3 to 8.3 mg/L with no clear relation to test material concentration

Nominal and measured concentrations:

nominal concentrations:
0, 32, 56, 100, 180 and 320 mg test material/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1-L glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:material = glass, size = 1L, headspace = 0.5L, fill volume = 0.5 L
- Aeration: 0.5 - 1 liter per minute by oil-free compressed air via a narrow bore glass tube
- No. of vessels per concentration (replicates): 3 (reference material: 1 vessel per concentration)
- No. of vessels per control (replicates): 6
- Biomass loading rate: activated sludge concentration (suspendid solids) in test mixture was 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water containing 1.01 mg/L DOC.
- Synthetic sewage was prepared according to guidance
- Activated sewage sludge was obtained from a sewage treatment plant treating predominantly domestic sewage (3.0 g suspended solids/L)

OTHER TEST CONDITIONS
- Adjustment of pH: pH of test material stock solutions was measured and adjusted to pH 7.0 - 8.0
- Light: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference item and test material preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.
Oxygen consumption rate measurement:
As each vessel reached 3 hours contact time an aliquot was removed from the glass beaker and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.75
- Range finding study
- Test concentrations: 10, 100 and 1000 mg test material/L
- Results used to determine the conditions for the definitive study: No statistically significant toxic effects were shown at the test concentrations of 10 mg test material/L, however statistically significant toxic effects were shown at the test concentration of 100 and 1000 mg test material/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 190 - 260

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

360 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
- In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 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.
- The dissolved oxygen concentration after 30 minutes contact time in some vessels were lower than 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: For 3,5-dichlorophenol the following results were derived: 3-hour EC20 = 3.7 mg/L, 3-hour EC50 = 9.9 mg/L (95% CL 8.0 - 12 mg/L) and 3-hour EC80 =26 mg/L

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration, a line fitted uing the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
95% confidence limits were calculated for the EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
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).

Any other information on results incl. tables

Table 1. Observations on foam at the test preparations throughout the test period

Nominal concentration (mg test material/L)	0 hours*	30 minutes	3 hours
Control	No foam	No foam	No foam
32	Some foam on surface	No foam	No foam
56	Some foam on surface	No foam	No foam
100	Some foam on surface	No foam	No foam
180	Some foam on surface	No foam	No foam
320	Foam on surface	No foam	No foam
Reference	No foam	No foam	No foam

* Observations made prior to the addition of activated sewage sludge

Table 2 Oxygen consumption rates and percentage inhibition values after 3 hours contact time

Nominal concentration (mg /L)		Initial O2reading (mg O2/L)	Measurement period (minutes)	Final O2reading (mg O2/L)	O2consumption rates (mg O2/L/hour)	% inhibition
Control	R1	5.5	4	1.6	58.50	-
	R2	3.8	2	1.9	57.00	-
	R3	4.0	2	2.3	51.00	-
	R4	4.0	3	1.5	50.00	-
	R5	5.0	4	1.8	48.00	-
	R6	4.3	2	2.1	66.00	-
Test material	32 R1	3.8	2	2.1	51.00	7
	32 R2	2.9	1	2.0	54.00	2
	32 R3	5.8	5	2.0	45.60	17
	56 R1	2.8	1	2.0	48.00	13
	56 R2	5.2	5	1.9	39.60	28
	56 R3	3.8	3	1.9	38.00	31
	100 R1	2.7	1	1.8	54.00	2
	100 R2	3.4	2	2.0	42.00	24
	100 R3	2.8	1	2.0	48.00	13
	180 R1	3.8	3	2.1	34.00	38
	180 R2	3.9	4	1.8	31.50	43
	180 R3	4.3	4	2.3	30.00	46
	320 R1	6.0	10	3.3	16.20	71
	320 R2	5.2	10	2.7	15.00	73
	320 R3	3.7	5	2.4	15.60	72
3,5-dichlorophenol	3.2	3.1	2	1.5	48.00	13
	10	5.8	8	2.5	24.75	55
	32	7.3	10	5.8	9.00	84

 R1 -R6 = Replicated 1 to 6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour EC50 for Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with propanamine (1:1) on respiration of activated sewage sludge was 220 mg/L with 95% confidence limits of 190 - 260 mg/L.

Executive summary:

The toxicity of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with propanamine (1:1) to activated sludge was studied in a OECD 209 activated sludge respiration inhibition test. Following a preliminary range-finding test acitvated sludge was exposed to an aqueous solution of the test material (purity 97%) at concentrations of 32, 56, 100, 180 and 320 mg/L (3 replicates per concentration) for a period of 3 hours at a temperature of 20 ± 2°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5 -dichlorophenol.

The effect of the Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with propanamine (1:1) on the respiration of activated sewage sludge gave a 3-hour EC50 value of 220 mg/L, with 95% confidence limits of 190 - 260 mg/L; the 3-hour EC20 was 81 mg/L. The reference item, 3,5 -dichlorophenol gave a 3 -hour EC50 value of 9.9 mg/L, with 95% confidence limits 8.0 - 12 mg/L which shows the validity of the test.