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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:

09 November 2017 to 21 November 2017

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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Due to the poor solubility of the test material, the test material was weighed directly into the test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the exit of the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf.
- Upon arrival in the test facility, the sludge was filtrated over a cotton cloth, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 57 mL (first experiment) and 47 mL (second experiment) nutrient solution / L sludge.
- On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 57 mL (first experiment) and 47 mL (second experiment) nutrient solution / L sludge. On the day of the experiment, the dry matter was determined once more.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.4 – 22.8 °C (first experiment (pre-test))
20.3 – 23.2 °C (second experiment (main test))

pH:

7.7 - 8.5

Nominal and measured concentrations:

- Pre-Test range finding study: 1, 10, 100, 1 000 mg/L (nominal)
- Main-Test concentrations: 46, 100, 220, 460, 1 000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (800 – 1 000 mL) were used as test vessels. They were cleaned before use.
- Type: closed.
- Material, size, headspace, fill volume: 500 mL
- Aeration: For safety reasons, the experiment was performed in a closed system without aeration. In order to ensure sufficient oxygen supply, a large headspace was provided and the mixtures were stirred vigorously.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 2 replicates before and 2 after measuring positive control and test material, respectively
- Sludge concentration (weight of dry solids per volume): Pre-test: 1.50 g/L, Main-test: 1.45 g/L.
- Nutrients provided for bacteria: Nutrient solution, 16 mL/vessel was provided. Composition of nutrient solution (synthetic sewage): Peptone 16.0 g, Meat Extract 11.0 g, Urea 3.0 g, NaCl 0.7 g, CaCl2*2H2O 0.4 g, MgSO4*7H2O 0.2 g, K2HPO4 2.8 g and Deionised water ad 1 000 mL.
- Test Vessel Preparation: In the control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution respectively the appropriate amount of test item into the respective test vessel, adding 16 mL nutrient solution and water to give 250 mL. Then, 250 mL inoculum was added in 5 minutes intervals and the mixtures were closed with sealed lids.
- Concentrations of test item and positive control were calculated using the concentration of the stock solution and the dilution factor.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

EFFECT PARAMETERS MEASURED:
- After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, poured in a 250 mL narrow-neck bottle and the oxygen consumption rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The following vessels were measured likewise in 5 minutes intervals. The respiration rate was measured over a period of only 5 minutes. In the guideline, a period of 5 to 10 minutes or until the oxygen concentration falls below 2 mg/L is recommended. As the linearity of all regression linear curves (r^2 of 0.99 or more) is give, the measurement period can be considered sufficiently long.

TEST CONCENTRATIONS
- Pre-Test range finding study: 1, 10, 100, 1 000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Strong significant inhibition was observed at the concentration 1 000 mg/L. Therefore, a second experiment (main test) experiment was performed with a highest concentration of 1 000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: inhibition of O2 consumption

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: inhibition of O2 consumption

Remarks on result:

other: 95 % Confidence Interval: 77 – 100 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: inhibition of O2 consumption

Remarks on result:

other: 95 % Confidence Interval: 110 – 140 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

240 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: inhibition of O2 consumption

Remarks on result:

other: 95 % Confidence Interval: 210 – 260 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

450 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: inhibition of O2 consumption

Remarks on result:

other: 95 % Confidence Interval: 400 – 500 mg/L

Details on results:

First Experiment (Pre-Test, Range Finder)
- Four concentrations were tested; an EC50 of 8.7 mg/L (95% confidence interval: 5.7 – 12 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.
- Strong significant inhibition was observed at the concentration 1000 mg/L. Therefore, a second experiment (main test) experiment was performed.

Second Experiment (Main Test)
- Significant inhibition of O2 consumption was observed at the following concentrations: 220 / 460 / 1 000 mg/L.

Results with reference substance (positive control):

A stock solution in deionised water containing 500 mg/L was freshly prepared for each experiment.
The calculated values for the positive control 3,5-Dichlorophenol were:
Pre-test: 3h-EC50 = 8.7 mg/L (95 % Confidence Interval = 5.7 – 12 mg/L)
Main-test: 3h-EC50 = 10 mg/L (95 % Confidence Interval = 5.0 – 16 mg/L)
All values lie within the recommended range of 2 – 25 mg/L.

Reported statistics and error estimates:

For determination of statistically significant difference between treatment and control, a NOEC will be determined by using a one-tailed significance test.
Clear inhibition was observed at 220 mg/L, whereas at 100 mg/L only slight inhibition was observed. For these treatments, it was tested whether the differences between treatment and blank control were significant.
The difference between the 100 mg/L treatment and the blank control can be considered as not significant as the calculated t-value lay below the tabulated t-value. The calculated t-value is greater than the tabulated t-value for the 220 mg/L treatment. Therefore, the concentration of 100 mg/L is stated as NOEC.
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

- Two experiments were performed.

- All validity criteria were met for both experiments. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response- equation. No observations were made which might cause doubts concerning the validity of the study outcome.

- In the second experiment (Main Test), for the test material the fits showed good statistical  correspondence of the data with the dose-response-equation. In the highest concentration (1000 mg/L) the test material showed 93 - 98 % inhibition. At 460 mg/L 75 – 89 % inhibition was observed. At 220 mg/L test material 33 – 48 % inhibition was reached. Inhibition values at 100 mg/L were not statistically significantly different from the control. Therefore, the NOEC can be stated at 100 mg/L.

O2-Consumption Inhibition Second Experiment (Main Test)

Vessel No.	Compound	Nominal Concentration in mg/L nominal	O2 consumption in mg/(L*min.)	O2 consumption in mg/(L*h)	Inhibition in %	pH
1	Blank Control	0	0.6285	37.712	0	7.9
2	Blank Control	0	0.7639	45.836	0	7.7
3	Positive Control	5	0.5126	30.756	21.0	7.8
4	Positive Control	10	0.3255	19.532	49.8	7.7
5	Positive Control	20	0.1287	7.720	80.2	7.7
6	Positive Control	40	0.810	4.859	87.5	7.8
7	Blank Control	0	0.6791	40.745	0	7.9
8	Blank Control	0	0.6737	40.423	0	7.9
9	Test Material	1000	0.0108	0.646	98.3	8.5
10	Test Material	1000	0.0203	1.219	96.9	8.5
11	Test Material	1000	0.0123	0.735	98.1	8.5
12	Test Material	1000	0.0395	2.371	93.9	8.3
13	Test Material	1000	0.0132	0.790	98.0	8.4
14	Test Material	460	0.1541	9.247	76.3	8.1
15	Test Material	460	0.0712	4.270	89.0	8.1
16	Test Material	460	0.1611	9.663	75.2	8.1
17	Test Material	460	0.1108	6.647	82.9	8.1
18	Test Material	460	0.1067	6.403	83.6	8.1
19	Test Material	220	0.4330	25.983	33.3	8.0
20	Test Material	220	0.3893	23.359	40.0	8.0
21	Test Material	220	0.3789	22.735	41.6	8.0
22	Test Material	220	0.3789	22.735	41.6	8.0
23	Test Material	220	0.3353	20.116	48.3	8.0
24	Test Material	100	0.6103	36.616	6.0	8.0
25	Test Material	100	0.7535	45.208	-16.1	8.0
26	Test Material	100	0.5658	33.947	12.8	7.9
27	Test Material	100	0.5663	33.978	12.8	7.9
28	Test Material	100	0.5627	33.764	13.3	7.9
29	Test Material	46	0.6447	38.684	0.7	7.9
30	Test Material	46	0.6416	38.495	1.2	8.0
31	Test Material	46	0.3375	38.250	1.8	7.9
32	Test Material	46	0.7125	42.750	-9.8	7.9
33	Test Material	46	0.5774	34.641	11.0	7.9
34	Blank Control	0	0.6094	36.563	0	7.9
35	Blank Control	0	0.5397	32.380	0	7.9

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 3-h NOEC of the test material was 100 mg/L, the 3-h EC50 was 240 mg/L (95 % confidence interval: 210 - 260 mg/L).

Executive summary:

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guidelines OECD 209 and EU Method C.11, under GLP conditions. Determination of the inhibition of the respiration of activated sludge when exposed to the test material was performed.

Two valid experiments were performed. The first experiment was performed as a range finder. The concentrations to be tested in the main test were based on the result of the range finder.

In the second experiment (main test) the test material was tested using 5 concentrations ranging from 1 000 to 46 mg/L nominal concentration. The dry matter of the inoculum was determined as 2.90 g suspended solids/L, giving a concentration of 1.45 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. For the positive control an EC50 of 10 mg/L (95 % confidence interval: 5.0 – 16 mg/L) was determined. The value lies within the recommended range of the OECD guideline. All validity criteria for both experiments were met.

Under the conditions of this study, the 3-h NOEC of the test material was 100 mg/L, the 3-h EC50 was 240 mg/L (95 % confidence interval: 210 - 260 mg/L).

Description of key information

Under the conditions of this study, the 3-h NOEC of the test material was 100 mg/L, the 3-h EC50 was 240 mg/L (95 % confidence interval: 210 - 260 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

240 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guidelines OECD 209 and EU Method C.11, under GLP conditions. Determination of the inhibition of the respiration of activated sludge when exposed to the test material was performed. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Two valid experiments were performed. The first experiment was performed as a range finder. The concentrations to be tested in the main test were based on the result of the range finder.

In the second experiment (main test) the test material was tested using 5 concentrations ranging from 1 000 to 46 mg/L nominal concentration. The dry matter of the inoculum was determined as 2.90 g suspended solids/L, giving a concentration of 1.45 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. For the positive control an EC50 of 10 mg/L (95 % confidence interval: 5.0 – 16 mg/L) was determined. The value lies within the recommended range of the OECD guideline. All validity criteria for both experiments were met.

Under the conditions of this study, the 3-h NOEC of the test material was 100 mg/L, the 3-h EC50 was 240 mg/L (95 % confidence interval: 210 - 260 mg/L).