Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

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:

2 May - 26 July 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:

Adopted July, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt Baden-Württemberg, Karlsruhe, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item solutions were prepared individually by weighing the test item directly for each of the incubation vessels.
- Eluate: no
- Differential loading: no
- Controls: yes; blank control, reference item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No precipitation of the test substance was observable in any of the test vessels. The test substance was optically determined to be homogeneously dispersed in each test vessel.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal waste water treatment plant of Pforzheim (Germany), collected from the aeration tank, was used as the microbial inoculum for the test. This plant is predominantly treating domestic sewage. Activated sludge is kept under aerobic conditions in the period between sampling and inoculation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

Control: 7.7 - 7.9
Test item treatments: 7.8 - 7.9
Reference control: 7.6 - 7.7

Nominal and measured concentrations:

nominal: blank control, 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: volumetric flasks with 250 mL of medium per vessel
- Aeration: constant air supply
- No. of vessels per concentration (replicates): at least 2
- No. of vessels per blank control (replicates): at least 2
- Sludge concentration: 1.5 ± 0.3 g/L (suspended solids)
- Nutrients provided for bacteria: synthetic sewage feed
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: At the end of the three hour incubation period, aliquots of each test media preparation were transferred to the appropriate sample tube. The DO (dissolved oxygen) electrode to measure DO was sealed at the neck of the tube, ensuring air is completely excluded. The sample tube was stirred at a constant rate during DO measurements. DO was measured over a period of up to 10 minutes. DO content was measured using electronic oxygen probes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption was measured over a period of up to 10 minutes after termination of incubation. Respiration rates (mg O2/L/h) were determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: blank control, 10, 100 and 1000 mg test item / L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

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 heterotrophic 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 nitrification rate

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:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

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 nitrification rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation of the test substance was observable in any of the test vessels. The test substance was optically determined to be homogeneously dispersed in each test vessel.
- Blank controls oxygen uptake rate: Respiration rate was 32 mg/g/h.
- Coefficient of variation of oxygen uptake rate in control replicates: Coefficient of variation of the blank control respiration rate was 20 %.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
EC50 = 5.2 mg/L for total respiration (95% CI: 3.3 – 7.6)
EC50 = 13.2 mg/L for heterotrophic respiration (95% CI: 9.3 – 18.9)
EC50 ~ < 2 mg/L for nitrification respiration (95% CI: >0.1<2). Due to the high values obtained, the EC50 for nitrification respiration of 3,5-Dichlorophenol could not statistically be calculated, but was determined to not be lower than 0.1 mg/L.

Reported statistics and error estimates:

Calculation of Percentage of Inhibition:
The EC50 was estimated using an appropriate statistical method (e.g. Probit analysis) and, where appropriate, 95% confidence values given.
The EC50 of the reference inhibitor was estimated from a log-linear plot of concentration versus effect for both the range finding test and, where appropriate, the definitive test.

Table 1: Limit Test Mean Respiration Rate and Inhibition Data

Nominal Concentration mg/L	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate  (mg/L/h)	Nitrification Respiration Rate (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
Control	48.5	39.4	9.1	-	-	-
10	61.2	50.3	10.9	-26.3	-27.6	-20.3
100	83.1	36.6	46.5	-71.4	7.1	-412.2
1000	49.1	38.4	10.7	-1.3	2.5	-17.8

Table 2: Individual Values for Respiration Rates (Blank control and test item)

Nominal Concentration (mg/L)	Strathtox Run	Strathtox Tube Number	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate (mg/L/h)	Nitrification Respiration Rate (mg/L/h)
Control	1	1	51.4	53.9	-2.5
Control	1	5	41.8	40.2	1.8
Control	1	6	55.2	44.4	10.8
Control	2	1	45.2	35.2	10.0
Control	2	5	54.4	33.8	20.5
Control	2	6	45.5	30.2	15.3
Control	3	1	45.3	38.3	7.1
Control	3	6	49.1	39.3	9.8
10	1	2	61.0	56.0	5.1
10	2	3	61.4	44.6	16.8
100	1	3	48.6	45.0	3.6
100	2	3	117.5	28.2	89.3
1000	1	4	49.0	42.5	6.5
1000	2	4	50.5	32.2	18.3
1000	3	5	47.9	40.7	7.2

Table 3: Individual Values for Respiration Rates (Reference item)

Nominal Concentration (mg/L)	Strathtox Run	Strathtox Tube Number	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate (mg/L/h)	Nitrification Respiration Rate (mg/L/h)
0.1	3	2	57.8	49.5	8.2
2	3	3	28.5	34.2	-5.7
40	3	4	7.5	10.1	-2.6

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	32 mg/L	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	20%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The test item showed no inhibition to microorganisms up to the highest tested concentration (1000 mg/L).

Description of key information

EC50 (3h) > 1000 mg/L (respiration inhibition, OECD 209)

Additional information

In a key respiration inhibition study conducted according to OECD 209, the substance showed no inhibition to microorganisms up to the highest tested concentration (1000 mg/L).

Supporting information from the ready biodegradability study conducted according to the OECD guideline 301B (CO2 evolution), showed a biodegradation of 34 % within 14 days in the toxicity control. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration (biodegradation > 25 %).