Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

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:

18 July - 13 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

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)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: low solubility - therefore test item it had to be applied via inert and washed PE-foil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item applied as supplied
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: no
- Final preparation of a solid: no

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as supplied.

OTHER SPECIFICS: n/a

Analytical monitoring:

no

Details on sampling:

- Concentrations: 100 mg/L (limit test - determined after review of preliminary test results conducted at 10, 100 and 1000 mg/L)
- Sampling method: The different vessels must be 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. 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. For the controls the same procedure was made as with the test vessels, however without test or reference substance. All vessels were stirred and aerated throughout the contact time with room air to assure an oxygen saturation of 60-70% using a membrane pump. Before starting the test the oxygen saturation rate was measured.

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.

This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

- Sample storage conditions before analysis: No

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because of the high viscosity and the low water solubility of the test item it had to be applied via inert and washed PE-foil. To reach a test concentration of 100 mg/L an amount of 50.0 mg (+/- 5%) test item was added directly into the test vessels together with tap water and synthetic sewage feed and stirred for a few minutes without sludge to optimize the dilution of the test item. After this the sludge was added.
- Eluate: Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as test system. 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. 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. Synthetic sewage sludge feed was prepared as follows: per 1 L demineralised water - 16 g peptone; 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.
- Differential loading: n/a
- Controls: For the 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.): no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as test system (predominantly domestic waste).
- Method of cultivation: n/a
- Preparation of inoculum for exposure: The activated sludge was kept aerated until use at test temperature. Dry solid of the activated sludge was determined as 3.8 g/L (range finder test) and 3.2 g/L (limit test) by weight measurements before and after drying at 105°C (mean of triplicate measurements). 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. 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.
- Pretreatment: No
- Initial biomass concentration: 1.5 g/L dry solids

Test type:

static

Water media type:

other: Activated sewage sludge

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

O2 concentration measurements taken at 30 second intervals after the 180 minute incubation time had elapsed (6 measurements).

Hardness:

not determined

Test temperature:

21.3 - 21.9 ºC

pH:

7.4

Dissolved oxygen:

All vessels were stirred and aerated throughout the contact time with room air to assure an oxygen saturation of 60-70% using a membrane pump.

Salinity:

n/a

Conductivity:

not determined

Nominal and measured concentrations:

Nominal: 100 mg/L (main limit test)

Details on test conditions:

TEST SYSTEM
- Test vessel: Serum bottle and BOD bottles
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Serum bottle capacity = 600 mL (filled to 500 mL), BOD bottle capacity = 250-300 mL.
- Aeration: Yes, during 3 h incubation phase
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a
- No. of vessels per reference control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: not reported
- Nutrients provided for bacteria: Synthetic sewage feed
- Biomass loading rate: 1.5 g/L dry solids
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: n/a
- Particulate matter: n/a
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: 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. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test at 100 mg/L
- Justification for using fewer concentrations than requested by guideline: Test item found to be highly insoluble at higher concentrations, leading to unreliable and inconsistent results.
- Range finding study: Yes
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes - There was no inhibition observed in the test vessels with 10 and 100 mg/L test concentration. The test vessels with 1000 mg/L showed a very high variation of inhibition effects, which is probably caused by the glutinous appearance and the low water solubility of the test item. In accordance with the sponsor a limit test with 100 mg/L and five replicates was prepared afterwards.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
- Effect concentrations exceeding solubility of substance in test medium: Not reported
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: The blank controls oxygen uptake rate was higher than 20 mg oxygen per one gram of activated sludge per hour in both tests
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation (CV) of the blank respiration rates was less than 30% in both tests

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
- Other: n/a

Table 1       Oxygen concentration in the blank, test and reference vessels

Vessel	Concentration	Interval of measurements (secs)	Measurement after 180 mins
			O2 concentration (mg/L at time (secs)
1	Blank	30	7.14	6.77	6.39	6.01	5.62	5.22
2			7.19	6.89	6.57	6.26	5.92	5.58
3			7.09	6.77	6.41	6.05	5.70	5.37
7	Test Item (100 mg/L)		5.94	5.13	4.35	3.73	3.18	2.73
8			7.84	7.50	7.11	6.69	6.24	5.76
9			7.56	7.15	6.71	6.24	5.75	5.32
10			7.87	7.56	7.21	6.81	6.39	5.99
11			6.09	5.60	5.15	4.70	4.26	3.87
4	3,5-D (1 mg/L)		7.09	6.71	6.31	5.92	5.50	5.05
5	3,5-D (10 mg/L)		7.99	7.79	7.60	7.37	7.16	6.93
6	3,5-D (100 mg/L)		8.48	8.40	8.33	8.24	8.18	8.08
12	Blank		7.17	6.73	6.26	5.79	5.33	4.86
13			6.90	6.47	6.03	5.60	5.20	4.83
14			7.06	6.69	6.27	5.87	5.51	5.18

 

Table 2       Respiration of activated sludge after application of the test item

Concentration	O2 concentration in mg/L		Consumption rate (mg/L/h)	Consumption rate (mg/g sludge/h)	% Inhibition
	First measurement	Last measurement
Blank 1	7.14	5.22	46.1	30.7	-
Blank 2	7.19	5.58	38.6	25.8	-
Blank 3	7.09	5.37	41.3	27.5	-
Blank 4	7.17	4.86	55.4	37.0	-
Blank 5	6.90	4.83	49.7	33.1	-
Blank 6	7.06	5.18	45.1	30.1	-
Test item 1	5.94	2.73	77.0	51.4	-67
Test item 2	7.84	5.76	49.9	33.3	-8
Test item 3	7.56	5.32	53.8	35.8	-17
Test item 4	7.87	5.99	45.1	30.1	2
Test item 5	6.09	3.87	53.3	35.5	-16
3,5-D (1 mg/L)	7.09	5.05	49.0	32.6	-6
3,5-D (10 mg/L)	7.99	6.93	25.4	17.0	45
3,5-D (100 mg/L)	8.48	8.08	9.6	6.4	79

cov of blanks = 0.12 %

mean consumption rate of the blank controls = 30.7 mg/g sludge/h

mean consumption of the test vessels = 37.2 mg/g sludge/h

EC₅₀of reference item = 11.71 mg/L (95 % CL = 0.99 – 22.54 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

At a concentration of 100.0 mg/L, no inhibition of the total respiration rate was observed. The resulting EC50 and NOEC values were > 100 and 100 mg/L, respectivey.

Executive summary:

OECD 209 (2017) - The inhibitory effects of Amines, C12 -C14 -branched alkyl, dodecylbenzenesulfonates (1:1) on the respiration of activated sludge was assessed in an OECD 209 test.

 

The activated sludge (with sewage feed at a final concentration of 1.5 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

 

a) Blank control vessels (6) were prepared containing activated sludge, synthetic sewage feed and PE film (as a medium for ading the test item to the reactor vessels).

b) Test item flasks (5) were prepared containing activated sludge, synthetic sewage feed, PE film and test item. The test item was added directly to the sludge via the PE film to achieve a nominal concentration of 10 mg/L.

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed, PE film and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concertation’s of 1, 10 and 30 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured at 30 second intervals for 3 minutes. The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels.

 

At a test concentration of 100 mg/L, no inhibition of the total respiration rate was observed. The resulting EC₅₀ and NOEC values were concluded to be > 100 and 100 mg/L, respectively.

 

The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.

Description of key information

3 h EC50 = > 100 mg/L; 3 h NOEC = 100 mg/L; OECD 209; Brunswik-Titze, A. (2017)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

OECD 209 (2017) - The inhibitory effects of the test item on the respiration of activated sludge was assessed in an OECD 209 test. The activated sludge (with sewage feed at a final concentration of 1.5 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

a) Blank control vessels (6) were prepared containing activated sludge, synthetic sewage feed and PE film (as a medium for ading the test item to the reactor vessels).

b) Test item flasks (5) were prepared containing activated sludge, synthetic sewage feed, PE film and test item. The test item was added directly to the sludge via the PE film to achieve a nominal concentration of 10 mg/L.

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed, PE film and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concertation’s of 1, 10 and 30 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured at 30 second intervals for 3 minutes. The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels. At a test concentration of 100 mg/L, no inhibition of the total respiration rate was observed. The resulting EC₅₀and NOEC values were concluded to be > 100 and 100 mg/L, respectively. The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.