[No public or meaningful name is available]

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:

December 2021

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))

Deviations:

yes

Remarks:

The temperature in the main test was in a range of 19.8 – 24.3°C instead of 20 ± 2 °C. This deviation was stated as uncritical, as normal respiration activity of the control could be observed.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the main test was in a range of 19.8 – 24.3°C instead of 20 ± 2 °C. This deviation was stated as uncritical, as normal respiration activity of the control could be observed.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 0921SA107S

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5°C)
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Stable

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activation basin of the sewage treatment plant in 67482 Edenkoben, In den Seewiesen, Germany
- Preparation of inoculum for exposure: Upon arrival in the test facility, the sludge was filtrated, washed with tap water three times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

0.90 mmol/L

Test temperature:

19.8-24.3°C

pH:

7.2-7.7

Dissolved oxygen:

Not reported

Salinity:

N/A

Conductivity:

233 µS/cm at 25°C

Nominal and measured concentrations:

Nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: SCHOTT flasks
- Type: closed
- Material, size, headspace, fill volume: 2000 mL
- No. of vessels per concentration (replicates): 1 replicate/concentration (first experiment, lower concentrations); 5 replicates/concentration (first experiment, highest concentration); 5 replicates/concentration (second experiment)
- No. of vessels per blank control (replicates): 2 replicates before and 2 after measuring positive control and test item, respectively
- No. of vessels per positive control (replicates): 1 replicate/concentration (all experiments)
- Sludge concentration (weight of dry solids per volume): 1.54-1.58 g suspended solids/L
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See 'Any other information on maerials and methods incl. tables'
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption
TEST CONCENTRATIONS
- Range finding study : 1, 10, 1000 mg/L
- Test concentrations: 32, 100, 320, 1000, 3200 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS no. 591-35-5)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 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.:

680 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% confidence interval: 520-840 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% confidence interval: 1600-2600 mg/L

Details on results:

See 'Any other information on results incl. tables'

Results with reference substance (positive control):

See 'Any other information on results incl. tables'

Reported statistics and error estimates:

See 'Any other information on results incl. tables'

O2 consumption, inhibition in the first experiment (pre-test)

The values of the O2 consumption (which is a measure for the viability of the bacteria) of control, test and positive control vessels and the calculated inhibition are presented in the following table. The measured pH at the end of the test is also stated.

Vessel No.	Content	Concentration (mg/L)	O2 consumption (mg/(L*min))	O2 consumption (mg/(L*h))	Inhibition (%)	pH
1	Blank control	0	0.6744	40.467	0	7.8
2	Blank control	0	0.6755	40.530	0	7.7
3	Positive control	5	0.4776	28.654	28.6	7.7
4	Positive control	10	0.2874	17.244	57.0	7.6
5	Positive control	20	0.1485	8.909	77.8	7.6
6	Positive control	40	0.0741	4.448	88.9	7.6
7	Blank control	0	0.6131	36.786	0	7.8
8	Blank control	0	0.6978	41.871	0	7.8
9	Test item	1000	0.4872	29.229	27.2	7.2
10	Test item	1000	0.5259	31.555	21.4	7.2
11	Test item	1000	0.5108	30.646	23.7	7.1
12	Test item	1000	0.5368	32.210	19.8	7.1
13	Test item	1000	0.5231	31.385	21.8	7.1
14	Test item	100	0.6831	40.987	-2.1	7.6
15	Test item	10	0.6102	36.610	8.8	7.8
16	Test item	1	0.6621	39.728	1.0	7.8
17	Blank control	0	0.6789	40.737	0	7.8
18	Blank control	0	0.6747	40.481	0	7.9

 

 

O2 consumption, inhibition in the second experiment (main test)

The values of the O2 consumption (which is a measure for the viability of the bacteria) of control, test and positive control vessels and the calculated inhibition are presented in the following table. The measured pH at the end of the test is also stated.

Vessel No.	Content	Concentration (mg/L)	O2 consumption (mg/(L*min))	O2 consumption (mg/(L*h))	Inhibition (%)	pH
1	Blank control	0	0.7998	47.989	0	7.8
2	Blank control	0	0.8061	48.367	0	7.8
3	Positive control	5	0.6592	39.551	20.4	7.7
4	Positive control	10	0.3833	22.995	53.7	7.7
5	Positive control	20	0.1900	11.403	77.0	7.7
6	Positive control	40	0.0766	4.594	90.8	7.8
7	Blank control	0	0.7327	43.965	0	7.8
8	Blank control	0	0.8134	48.805	0	7.8
9	Test item	3200	0.1519	9.116	81.7	6.5
10	Test item	3200	0.1755	10.532	78.8	6.5
11	Test item	3200	0.2523	15.139	69.5	6.6
12	Test item	3200	0.1818	10.909	78.0	6.5
13	Test item	3200	0.1818	10.909	78.0	6.5
14	Test item	1000	0.7390	44.342	10.7	7.3
15	Test item	1000	0.6566	39.397	20.7	7.3
16	Test item	1000	0.7379	44.247	10.9	7.4
17	Test item	1000	0.7188	13.128	13.2	7.3
18	Test item	1000	0.7554	45.323	8.8	7.4
19	Test item	320	0.8194	49.167	1.0	7.7
20	Test item	320	0.8586	51.514	-3.7	7.7
21	Test item	320	0.7640	45.839	7.7	7.7
22	Test item	320	0.8407	50.440	-1.5	7.6
23	Test item	320	0.8080	48.481	2.4	7.7
24	Test item	100	0.8843	53.058	-6.8	7.7
25	Test item	100	0.8806	52.837	-6.4	7.8
26	Test item	100	0.9257	55.544	-11.8	7.7
27	Test item	100	0.8794	52.765	-6.2	7.8
28	Test item	100	0.9133	54.799	-10.3	7.8
29	Test item	32	0.9387	56.319	-13.4	7.8
30	Test item	32	0.9364	56.185	-13.1	7.8
31	Test item	32	0.9405	56.429	-13.6	7.7
32	Test item	32	0.9381	56.285	-13.3	7.8
33	Test item	32	0.9216	55.296	-11.3	7.8
34	Blank control	0	0.9162	54.973	0	7.7
35	Blank control	0	0.8998	53.988	0	7.7

 

For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.

Because no inhibition was observed in the three lowest concentrated treatments, the two lowest concentrated treatments were not used for evaluation to receive a linear regression.

Statistical Determination of the NOEC

For the treatments with the test item concentration 320 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used.

In order to select a suitable test for significance, it was checked whether equality of variance was given.

With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with degree of freedom: n1 + n2 – 2, level of significance 95 %).

The difference between treatment 320 mg/L and the blank control can be considered as not significant as the calculated t-value lay below the tabulated t-value. Therefore, the concentration 320 mg/L is stated as NOEC.

EC10, EC50

For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauss-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.

Validity

The calculated values for the positive control 3,5-dichlorophenol are presented in the following table:

Date of experiment	3-h EC50	95% Confidence interval
7 Dec. 2021	8.8 mg/L	6.0-12 mg/L
15 Dec 2021	10 mg/L	7.2-13 mg/L

 

All values lie within the recommended range of 2 – 25 mg/L.

The coefficient of variation of oxygen uptake rate in blank control replicates should not be more than 30% at the end of the test.

Date of experiment	Mean	Standard deviation	Coefficient of variation
7 Dec. 2021	40.145 mg/(L*h)	1.731 mg/(L*h)	4.3%
15 Dec 2021	49.681 mg/(L*h)	4.113 mg/(L*h)	8.3%

 

All values lie below the recommended limit of 30%.

The blank controls oxygen uptake rate should not be less than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour.

Date of experiment	Oxygen Uptake Rate in mg/h per gram activated sludge
7 Dec. 2021	26.07 mg oxygen per gram sludge in 1 hour
15 Dec 2021	31.44 mg oxygen per gram sludge in 1 hour

 

All values meet the required limit.

Validity criteria fulfilled:

yes

Conclusions:

Two experiments were performed. In the first experiment (pre-test) the test item was tested using four concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, five replicates were used, for the other treatments, one replicate each. Because significant inhibition was observed, an additional experiment was performed under the same test conditions.

In the second experiment (main test) the test item was tested using five concentrations ranging from 3200 to 32 mg/L nominal concentration. Since the inhibition values from the main test allow a graphical determination of the EC10 and EC50 values, all biological results are based on the results of the main test.

The following results were determined:
3h NOEC = 320 mg/L
3h EC10 = 680 mg/L (Confidence interval: 520 - 840 mg/L)
3h EC50 = 2000 mg/L (Confidence interval: 1600 - 2600 mg/L)

Executive summary:

Two experiments were performed. In the first experiment (pre-test) the test item was tested using four concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, five replicates were used, for the other treatments, one replicate each. Because significant inhibition was observed, an additional experiment was performed under the same test conditions.

In the second experiment (main test) the test item was tested using five concentrations ranging from 3200 to 32 mg/L nominal concentration. Since the inhibition values from the main test allow a graphical determination of the EC10 and EC50 values, all biological results are based on the results of the main test.

The following results were determined:

3h NOEC = 320 mg/L

3h EC10 = 680 mg/L (Confidence interval: 520 - 840 mg/L)

3h EC50 = 2000 mg/L (Confidence interval: 1600 - 2600 mg/L)

All validity criteria were met. No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed to discern between inhibition of nitrificators and inhibition of total population. No observations were made which might cause doubts concerning the validity of the study outcome.

Description of key information

Two experiments were performed. In the first experiment (pre-test) the test item was tested using four concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, five replicates were used, for the other treatments, one replicate each. Because significant inhibition was observed, an additional experiment was performed under the same test conditions.

In the second experiment (main test) the test item was tested using five concentrations ranging from 3200 to 32 mg/L nominal concentration. Since the inhibition values from the main test allow a graphical determination of the EC10 and EC50 values, all biological results are based on the results of the main test.

The following results were determined:

3h NOEC = 320 mg/L

3h EC10 = 680 mg/L (Confidence interval: 520 - 840 mg/L)

3h EC50 = 2000 mg/L (Confidence interval: 1600 - 2600 mg/L)

All validity criteria were met. No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed to discern between inhibition of nitrificators and inhibition of total population. No observations were made which might cause doubts concerning the validity of the study outcome.

Key value for chemical safety assessment

EC50 for microorganisms:

2 000 mg/L

EC10 or NOEC for microorganisms:

680 mg/L