Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

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:

29 June 2017 - 07 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines (OECD 209) and in accordance with GLP. All relevant guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

Three replicates per test concentration and six replicates for the blank control performed, in conjunction with the recommendation of the German Federal Environment Agency.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Remarks:

Test item was added directly

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item was added directly to the test vessels (at concentrations: 9.54, 30.5, 97.65, 312.5 and 1000 mg/L) numbered 7 - 21 and 31 - 45 with a test volume of 500 mL. To avoid the foaming of the test item, 200 µL of the antifoam agent "Silicone Antifoam" was added into each vessel. The antifoam agent was shown to have no inhibition effects on the activated sludge following a limit test. The pH of the test solution was measured before addition of inoculum. A stock solution of 2.32 g/L of nitrification inhibitor N-allylthiourea (ATU) was prepared in chlorine-free tap water. An amount of 2.5 mL of this stock solution was added to the vessels 25 - 48. Preparation of reference item: A weight of 49.8 mg of 3,5-dichlorophenol were dissolved in deionised water and filled up to 50 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels 4, 5 and 6 in order to obtain final concentrations of 1, 10 and 30 mg/L. Three additional reference vessels (28, 29 and 30) with the concentrations of 1, 10 and 30 mg/L were prepared the same way as described but with 2.5 mL ATU. At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 mL with chlorine-free tap water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 mL with 1.5 g/L dry solids.
- Eluate: N/A
- Differential loading: N/A
- Controls: For the blank controls the same procedure was made as with the test vessels, however without test or reference substance.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: N/A
- Name and location of sewage treatment plant where inoculum was collected: Collection from municipal wastewater treatment plant Staufener Bucht. The treatment plant has a capacity of 140 000 inhabitant equivalents and constitutes predominantly domestic wastewater. Sampling date of activated sludge was 5 December 2017.
- Method of cultivation: N/A
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Pretreatment: The activated sludge was kept aerated until use at test temperature.
- Initial biomass concentration: Dry solid of the activated sludge was determined as 4.7 g/L by weight measurements before and after drying at 105°C (mean of triplicate measurements).

Test type:

static

Water media type:

other: Activated sewage sludge

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

The different vessels were started separately with an interval of exactly 15 min in order to be able to measure the respiration rates one by one while making sure that the aeration time is 3 h in every vessel.

Post exposure observation period:

After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.

Hardness:

N/A

Test temperature:

20.7 - 22°C

pH:

(Test item) 7.3-7.4
(Reference item) 7.3
(Blank) 7.3

Dissolved oxygen:

Oxygen saturation of 60-70%

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal:
9.54, 30.5, 97.65, 312.5 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Serum bottles - Schott, Mainz, Germany
- Type (delete if not applicable): open / closed
- Size: 600ml, fill volume: 500ml
- Aeration: Membrane pump connected with glass pipes via PolyEthylen (PE)-tubes
- No. of vessels per concentration (replicates): Three replicates per test concentration
- No. of vessels per control (replicates): Six replicates for blank control; three replicates for reference control
- No. of vessels per vehicle control (replicates): N/A
- No. of vessels per abiotic control (replicates): N/A
- Sludge concentration (weight of dry solids per volume): A final concentration of 1.5 g/L dry solids in the test
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: N/A
- Nitrification inhibitor used: N-allylthiourea
- Biomass loading rate: N/A
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Chlorine-free tap water
- Particulate matter: N/A
OTHER TEST CONDITIONS
- Adjustment of pH: pH adjustment not necessary for reference and test item; pH adjusted in activated sludge
- Photoperiod: N/A
- Light intensity: N/A
- Details on termination of incubation: After 3 h the aeration of the vessel was shut off and the respiration rate was measured
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Inhibiting effects of the test time on total and heterotrophic respiration of activated sludge
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: N/A
- Range finding study: An initial range finder test was performed with the test concentrations 10, 100 and 1000 mg/L. After detecting that the test item has strong foaming properties the range finder test was repeated with the concentrations 10, 100 and 1000 mg/L but with an additional antifoam agent. 200 µL of Silicone Antifoam was added in every test vessel.
- Test concentrations: 9.54, 30.5, 97.65, 312.5 and 1000 mg/L
- Results used to determine the conditions for the definitive study: An EC50 test was realized with six replicate test vessels in the untreated control and three test vessels in the treatments with 9.54 - 1000 mg/L test item (Factor 3.2). For observing the heterotrophic and nitrification consumption rate the same set of reaction mixtures were prepared but with an additional 2.5 mL of ATU.

Reference substance (positive control):

yes

Remarks:

49.8 mg of 3,5-dichlorophenol dissolved in deionised water (Sigma-Aldrich, Lot MKBS0425V); final concentrations of 1, 10 and 30 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

169.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

78.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

30.5 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.:

>= 9.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

63.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

16.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

97.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 30.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: N/A
- Effect concentrations exceeding solubility of substance in test medium: N/A
- Adsorption (e.g. of test material to the walls of the test container): N/A
- Blank controls oxygen uptake rate: The total respiration rates of the six blank control were within 27.0 and 38.1 per one gram activated sludge.
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation for blank controls was 17%.
The coefficient of variation for nitrification controls was > 120 %.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of the reference item was within the acceptable range of 2 to 25 mg/l for the total respiration (presently 5.4 mg/L), of 5 to 40 mg/L for the heterotrophic respiration (presently 8.4 mg/L) and 0.1 to 10 mg/L for the nitrification respiration (presently 2.5 mg/L)
- Other: N/A

Reported statistics and error estimates:

The EC50, LOEC and NOEC calculations was performed with the ToxRat software (ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH) by probit analysis using linear maximum likelihood regression. The EC50 is calculated from the slope of the curve.
Inhibition of the total respiration rate of activated sludge with confidence interval:
EC50 = 169.2 mg/L (95%-CL: 92.620 - 308.571 mg/L)
EC20 = 101.9 mg/L (95%-CL: 61.667 - 168.110 mg/L)
EC10 = 78.2 mg/L (95%-CL: 45.890 - 133.181 mg/L)
LOEC = 30.5 mg/L
NOEC ≥ 9.54 mg/L
Inhibition of the heterotrophic respiration rate of activated sludge with confidence intervals:
EC50 = 63.1 mg/L (95%-CL: 7.279 - 531.344 mg/L)
EC20 = 25.9 mg/L (95%-CL: 4.388 - 152.198 mg/L)
EC10 = 16.242 mg/L (95%-CL: 2.474 - 106.614 mg/L)
LOEC = 97.65 mg/L
NOEC ≥ 30.5 mg/L

Results from the definitive test and the calculated effect concentrations are found in the tables below.

Table 1. Total respiration rate of activated sludge after application of test item and reference item solutions

No.	Vessel	Consumption rate [mg/(L*h)]	Consumption rate [mg/(g sludge*h)]	Inhibition (%)
Test item vessels
1	Blank 1	52.8	35.2	-
2	Blank 2	53.5	35.7	-
3	Blank 3	57.1	38.1	-
22	Blank 4	40.6	27.0	-
23	Blank 5	40.8	27.2	-
24	Blank 6	38.2	25.4	-
7	Test 9.54 mg/L 1	45.1	30.1	4
8	Test 9.54 mg/L 2	42.0	28.0	11
9	Test 9.54 mg/L 3	42.0	28.0	11
10	Test 30.5 mg/L 1	38.4	25.6	19
11	Test 30.5 mg/L 2	38.2	25.4	19
12	Test 30.5 mg/L 3	33.6	22.4	29
13	Test 97.65 mg/L 1	33.8	22.6	28
14	Test 97.65 mg/L 2	33.1	22.1	30
15	Test 97.65 mg/L 3	41.5	27.7	12
16	Test 312.5 mg/L 1	6.7	4.5	86
17	Test 312.5 mg/L 2	7.9	5.3	83
18	Test 312.5 mg/L 3	4.3	2.9	91
19	Test 1000 mg/L 1	4.8	3.2	90
20	Test 1000 mg/L 2	4.1	2.7	91
21	Test 1000 mg/L 3	3.6	2.4	92
Reference item vessels
1	Blank 1	52.8	35.2	-
2	Blank 2	53.5	35.7	-
3	Blank 3	57.1	38.1	-
22	Blank 4	40.6	27.0	-
23	Blank 5	40.8	27.2	-
24	Blank 6	38.2	25.4	-
4	3,5-D 1 mg/L	42.0	28.0	11
5	3,5-D 10 mg/L	15.4	10.2	67
6	3,5-D 30 mg/L	4.3	2.9	91

Table 2. Heterotrophic respiration rate of activated sludge after application of the test item reference item solutions and ATU

No.	Vessel	Consumption rate [mg/(L*h)]	Consumption rate [mg/(g sludge*h)]	Inhibition (%)
Test item vessels
25	Blank 1	19.0	12.6	-
26	Blank 2	35.5	23.7	-
27	Blank 3	20.6	13.8	-
46	Blank 4	51.4	34.2	-
47	Blank 5	38.2	25.4	-
48	Blank 6	28.6	19.0	-
31	Test 9.54 mg/L 1	50.2	33.4	-56
32	Test 9.54 mg/L 2	38.2	25.4	-19
33	Test 9.54 mg/L 3	59.0	39.4	-83
34	Test 30.5 mg/L 1	25.0	16.6	22
35	Test 30.5 mg/L 2	25.0	16.6	22
36	Test 30.5 mg/L 3	18.5	12.3	43
37	Test 97.65 mg/L 1	14.2	9.4	56
38	Test 97.65 mg/L 2	10.6	7.0	67
39	Test 97.65 mg/L 3	11.5	7.7	64
40	Test 312.5 mg/L 1	6.5	4.3	80
41	Test 312.5 mg/L 2	6.7	4.5	79
42	Test 312.5 mg/L 3	5.5	3.7	83
43	Test 1000 mg/L 1	6.7	4.5	79
44	Test 1000 mg/L 2	7.4	5.0	77
45	Test 1000 mg/L 3	6.5	4.3	80
Reference item vessels
25	Blank 1	19.0	12.6	-
26	Blank 2	35.5	23.7	-
27	Blank 3	20.6	13.8	-
46	Blank 4	51.4	34.2	-
47	Blank 5	38.2	25.4	-
48	Blank 6	28.6	19.0	-
28	3,5-D 1 mg/L	29.3	19.5	9
29	3,5-D 10 mg/L	14.4	9.6	55
30	3,5-D 30 mg/L	7.4	5.0	77

Table 3. Results for the inhibition of the total respiration rate, and heterotrophic respiration rate of activated sludge

Effect	Inhibition of activated sludge (mg/L)	95% Confidence Limit (mg/L
Total respiration rate
EC50	169.2	92.620 – 308.571
EC20	101.9	61.667 - 168.110
EC10	78.2	45.890 – 133.181
LOEC	30.5	-
NOEC	≥9.54	-
Heterotrophic respiration rate
EC50	63.1	7.279 – 531.344
EC20	25.9	4.388 – 152.198
EC10	16.24	2.474 – 106.614
LOEC	97.65	-
NOEC	≥30.5	-

Validity criteria fulfilled:

yes

Conclusions:

The inhibiting effect of the test item Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts on the total and heterotrophic respiration of activated sludge was observed in this test, conducted with five concentrations (9.54, 30.5, 97.65, 312.5 and 1000.0 mg/L).
The test met all guideline validity criteria, and there was one deviation from OECD 209 whereby three replicates for each test concentration were prepared (as according to the Question-and-Answer Document by the German Federal Environment Agency on the new OECD Guideline 209 "Activated Sludge, Respiration Inhibition Test (Carbon and ammonium Oxidation)" (Version: 02.03.2012)) instead of five.
The EC50 for inhibition of the total respiration rate of activated sludge was 169.2 mg/L (95%-CL: 92.620 - 308.571 mg/L).
The EC50 for inhibition of the heterotrophic respiration rate of activated sludge was 63.1 mg/L (95%-CL: 7.279 - 531.344 mg/L).
The results for inhibition of the nitrification respiration rate of activated sludge were considered inappropriate for analysis, thus it was not possible to determine if there are any effects on nitrification caused by the test substance.

Executive summary:

An OECD 209 Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) (22 July 2010) was conducted with Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts to assess the inhibition effect of different concentrations of test item to micro-organisms of activated sludge by measuring the total and heterotrophic respiration rate after adding a standard amount of synthetic sewage feed and a standard amount of a nitrification inhibitor.

An initial range finding study was performed with test concentrations of 10, 100 and 1000 mg/L. The test item displayed strong foaming properties and so the range finding study was repeated with the same concentrations, and an additional antifoam agent (Silicone Antifoam).

Six blank controls were used, as well as three replicate reference controls and three replicate test item vessels per test concentration.

For observing the heterotrophic and nitrification consumption rate the same set of reaction mixtures were prepared but with an additional 2.5 mL of ATU. The test concentrations used were 9.54, 30.5, 97.65, 312.5 and 1000 mg/L, at an exposure duration of 3 h.

The results for the inhibition of the total respiration rate of activated sludge are:

EC₅₀= 169.2 mg/L (95%-CL: 92.620 – 308.571 mg/L)

EC₂₀= 101.9 mg/L (95%-CL: 61.667 – 168.110 mg/L)

EC₁₀= 78.2 mg/L (95%-CL: 45.890 – 133.181 mg/L)

LOEC = 30.5 mg/L

NOEC≥9.54 mg/L

The results for the inhibition of the heterotrophic respiration rate of activated sludge are:

EC₅₀= 63.1 mg/L (95%-CL: 7.279 – 531.344 mg/L)

EC₂₀= 25.9 mg/L (95%-CL: 4.388 – 152.198 mg/L)

EC₁₀= 16.242 mg/L (95%-CL: 2.474 – 106.614 mg/L)

LOEC = 97.65 mg/L

NOEC ≥ 30.5 mg/L

Due to substance properties, nitrification inhibition was also analysed.  However, the results generated from the test are considered inappropriate for further analysis.  In the controls, the coefficient of variation is large and no monotonic response was generated. Thus the data produced and any effects seen are either down to natural variation or testing limitations.  No meaningful analysis can be conducted, and it is not possible to determine if there are any effects on nitrification caused by the test substance.

Description of key information

OECD 209; OECD 209; Brunswik-Titze (2017)

The results for the inhibition of thetotal respiration rateof activated sludge are:

EC₅₀= 169.2 mg/L (95%-CL: 92.620 – 308.571 mg/L)

EC₂₀= 101.9 mg/L (95%-CL: 61.667 – 168.110 mg/L)

EC₁₀= 78.2 mg/L (95%-CL: 45.890 – 133.181 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:

169.2 mg/L

EC10 or NOEC for microorganisms:

78.2 mg/L

Additional information

An OECD 209 Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) (22 July 2010) was conducted with Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts to assess the inhibition effect of different concentrations of test item to micro-organisms of activated sludge by measuring the total and heterotrophic respiration rate after adding a standard amount of synthetic sewage feed and a standard amount of a nitrification inhibitor.

An initial range finding study was performed with test concentrations of 10, 100 and 1000 mg/L. The test item displayed strong foaming properties and so the range finding study was repeated with the same concentrations, and an additional antifoam agent (Silicone Antifoam).

Six blank controls were used, as well as three replicate reference controls and three replicate test item vessels per test concentration.

For observing the heterotrophic and nitrification consumption rate the same set of reaction mixtures were prepared but with an additional 2.5 mL of ATU. The test concentrations used were 9.54, 30.5, 97.65, 312.5 and 1000 mg/L, at an exposure duration of 3 h.

The results for the inhibition of the total respiration rate of activated sludge are:

EC₅₀= 169.2 mg/L (95%-CL: 92.620 – 308.571 mg/L)

EC₂₀= 101.9 mg/L (95%-CL: 61.667 – 168.110 mg/L)

EC₁₀= 78.2 mg/L (95%-CL: 45.890 – 133.181 mg/L)

LOEC = 30.5 mg/L

NOEC≥9.54 mg/L

The results for the inhibition of the heterotrophic respiration rate of activated sludge are:

EC₅₀= 63.1 mg/L (95%-CL: 7.279 – 531.344 mg/L)

EC₂₀= 25.9 mg/L (95%-CL: 4.388 – 152.198 mg/L)

EC₁₀= 16.242 mg/L (95%-CL: 2.474 – 106.614 mg/L)

LOEC = 97.65 mg/L

NOEC ≥ 30.5 mg/L

Due to substance properties, nitrification inhibition was also analysed.  However, the results generated from the test are considered inappropriate for further analysis.  In the controls, the coefficient of variation is large and no monotonic response was generated. Thus the data produced and any effects seen are either down to natural variation or testing limitations.  No meaningful analysis can be conducted, and it is not possible to determine if there are any effects on nitrification caused by the test substance.