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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2016 to 04 October 2016
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
Version / remarks:
(version 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(version 2015)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for analytical measurement were taken at the beginning after a stirring period of approx. 5 minutes and at the end of the test.
Vehicle:
no
Details on test solutions:
SYNTHETIC NUTRIENT SOLUTION
- 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
- Deionised water: ad 1000 mL
The pH of the solution was 7.0 and therefore within the range of 7.5 ± 0.5. The nutrient solution was frozen immediately after preparation.

- Positive control: A stock solution in deionised water containing 500 mg/L was freshly prepared for the experiment.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf.
- Pre-treatment: Upon arrival in the test facility, the sludge was filtrated, 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 50 mL synthetic sewage feed /L.
- pH: 7.6
- Dry matter of sludge: 3.20 g suspended solids/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21.5 - 23.2 °C
pH:
7.4 - 7.7
Nominal and measured concentrations:
- Nominal concentrations: 0 (control)*, 3.2, 10, 32, 100 and 320 mg/L
- Measured concentrations (geometric mean): 0, 0.9, 2.3, 14.8, 25.5 and 55.1 mg/L (see 'Any other information on materials and methods incl. tables')

* In the blank solution, a substance was detected at the same retention time as the test item. The concentration was determined at 0.33 mg/L. However, as the shape of the peak in the test solution and the peak in the blank control is completely different, it was concluded that no test item was present in the blank control.
Details on test conditions:
TEST SYSTEM
- Test vessels: 2000 mL SCHOTT-flasks. They were cleaned before use.
- Measuring flasks: Narrow-neck glass bottles with flat bottoms (250 mL).
- Preparation of 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 50 mL/L sludge. On the day of the experiment, the dry matter was determined once more and determined at 1.60 g suspended solids/L.
- Replicates: 1 replicate (all concentrations positive control); 5 replicates (all concentrations test item);
- Blank control: 2 replicates before and 2 after measuring positive control and test item, respectively
- Water: tap water, see annex 2, page 13
- Feeding: nutrient solution, 16 mL/vessel

TEST PROCEDURE
In the blank 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 or the appropriate amount of test item into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minute intervals and the mixtures were closed with sealed lids. 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 respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes1. The following vessels were measured likewise in 5 minute intervals. The test item was added to the test vessels directly.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
72 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.:
7.5 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.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
A statistically significant effect was first seen at a test concentration of 2.3 mg/L (measured) at which 20.3% inhibition of respiration was noted. At the higher tested concentrations percentages inhibition of respiration plateaued at ca. 47.5%.
Results with reference substance (positive control):
3-h EC50 = 7.9 mg/L (95% C.L. 7.3 - 8.5 mg/L). The test was performed 29 September 2016.
Reported statistics and error estimates:
- EC50 and EC10: 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%.
- NOEC: The t-test was used for treatment 10 mg/L; whereas the WEIR test was used for treatment 3.2 mg/L.

Table: Oxygen consumption and percentages inhibition of respiration

Nominal test conc. (mg/L)

Measured test conc. (mg/L)

Oxygen consumption

Mean

% inhibition

mg/L

per min.

mg/L

per hour

%

Inhibition

0 (control)

0

0.6396

38.377

0

0

0.6505

39.028

0

0.6383

38.298

0

0.6205

37.230

0

0.7764

46.583

0

0.6407

38.445

0

3.2

0.9

0.6811

40.867

-3.0

-2.3

0.6530

39.182

1.2

0.6921

41.523

-4.7

0.6969

41.813

-5.4

0.6577

39.461

0.5

10

2.3

0.5187

31.119

21.5

20.3

0.5526

33.159

16.4

0.6208

37.250

6.1

0.5419

32.511

18.0

0.3995

23.970

39.6

32

14.8

0.2873

17.237

56.5

45.7

0.4200

25.200

36.5

0.3422

20.534

48.2

0.4026

24.156

39.1

0.3426

20.556

48.2

100

25.5

0.3813

22.875

42.3

45.4

0.3663

21.978

44.6

0.3116

18.695

52.9

0.3500

20.998

47.1

0.3960

23.759

40.1

320

55.1

0.3619

21.713

45.3

47.58

0.4063

24.377

38.5

0.2033

12.197

69.2

0.2986

17.915

54.8

0.4620

27.719

30.1

Note: The difference between treatment 3.2 mg/L and the blank control can be considered as not significant as

the calculated t-value lay below the significance limit and O2 consumption in the treatment was higher than in the

blank control. Therefore, the nominal concentration 3.2 mg/L resp. 0.9 mg/L measured concentration is stated as NOEC.

Validity criteria fulfilled:
yes
Conclusions:
The 3-h EC50, EC10 and NOEC values are 22.0, 2.1 and 0.9 mg/L, respectively in microorganisms in activated sludge.
Executive summary:

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (LAUS, 2017). In this study non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 3.2, 10, 32, 100 and 320 for 3 hours. As a certain degree of volatilisation could not be excluded, the test was performed in a closed system without aeration and the nominal test concentrations were analytically verified at the start and at the end of exposure. Within the test period of 3 hours, undissolved test item was dissolved, especially in the higher concentrated treatments and measured concentrations at the end of the test were clearly higher than at the beginning. Therefore, the geometric mean measured concentrations were calculated which were 0 (control)*, 0.9, 2.3, 14.8, 25.5 and 55.1 mg/L. Effect values are based on measured concentrations. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. A statistically significant effect was first seen at a test concentration of 2.3 mg/L (measured) at which 20.3% inhibition of respiration was noted. At the higher tested concentrations percentages inhibition of respiration plateaued  at ca. 47.5%. Based on these findings, the 3 -h EC50, EC10 and NOEC values were determined at 22.0, 2.1 and 0.9 mg/L, respectively.

* In the blank solution, a substance was detected at the same retention time as the test item. The concentration was determined at 0.33 mg/L. However, as the shape of the peak in the test solution and the peak in the blank control is completely different, it was concluded that no test item was present in the blank control.

Description of key information

The 3-h EC50, EC10 and NOEC values are 22.0, 2.1 and 0.9 mg/L, respectively in microorganisms in activated sludge. The EC10 value of 2.1 mg/L is used as key value for assessment.

Key value for chemical safety assessment

EC50 for microorganisms:
22 mg/L
EC10 or NOEC for microorganisms:
2.1 mg/L

Additional information