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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:
11 Dec 2014 to 20 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
July, 2010
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
2007
GLP compliance:
yes
Specific details on test material used for the study:
- Name of the test material (as cited in study report): ZDEC
- Apearance: White to off-white powder
- Batch No.: 40101204.
- Purity/composition correction factor: No correction factor required
- Test substance storage: At room temperature
- Stable under storage until: 15 January 2016
- Solubility in water: 0.001 g/L at 20 °C
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24-27 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21.1 - 23.3 °C
pH:
- Test start: 7.4 - 7.6
- Test end: 7.4 - 8.1
Dissolved oxygen:
Adjusted to 60-70% saturation at the start of the test (60% of air saturation is > 5 mg/L at 20°C)
Nominal and measured concentrations:
- Nominal loading rates second full test: 0 (control), 2.0, 5.7, 16, 46, 130, 380 and 1070 mg/L (based on range-finding test)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass bottles (nominal volume 1L)
- Type: Open
- Test volume: 500 mL
- Aeration: Yes, continuous with clean oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Stirring: yes, continuously
- No. of vessels per concentration: 5
- No. of vessels per control: 6
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nitrification inhibitor used: None

TEST MEDIUM / WATER PARAMETERS
16 mL/test vessel of adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: 211.5 mg/L CaCl2.2H2O, 88.8 mg/L MgSO4.7H2O, 46.7 mg/L NaHCO3 and 4.2 mg/L KCl.

OTHER TEST CONDITIONS
- Adjustment of pH: No

PERFORMANCE OF THE TEST
The synthetic medium (16 mL) made up to 50 ml with Milli-RO and 200 ml test substance solution were mixed (total volume 250 mL) in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. After 3 hours incubation time the oxygen consumption was measured (see below). Furthermore, the pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls. The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

EFFECT PARAMETERS MEASURED: oxygen consumption
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test loading rates: 2.8
- Range finding study: Yes. This second definitive test is based on results of a preliminary test and a first definitive study. See 'Any other information on materials and methods incl. tables'.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
170 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.I.: 87 - 410 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.I.: 0.45 - 17 mg/L
Details on results:
No respiration inhibition was observed after 3 hours exposure up to a loading rate of 16 mg/L. At higher loading rates, significant inhibition of the respiration rate was observed, increasing in a dose dependent manner from 37% inhibition in the 46 mg/L loading rate up to 68% inhibition in the highest loading rate of 1070 mg/L. An overview of the results is provided in ‘Any other information on results incl. tables’.
Results with reference substance (positive control):
The 3-h EC50 value of the reference substance was determined to be 6.3 mg/L.
Reported statistics and error estimates:
Probit analysis for the determination of the effect values, Shapiro-Wilk Test for the determination of normality of distribution, Levene's Test on Variance Homogeneity (with residuals) for the determination of homogeneity of variance and a STUDENT-t test for Homogeneous Variances with Bonferroni-Holm Adjustment for the determination of NOEL.

Table: Overview of the results of the second definitive test

Loading rate (mg/L)

pH

Respiration rate

% Inhibition of respiration rate (mean value)

Start

End

range

(mg O2/L h)

mean

(mg O2/L h)

range

(mg O2/g h)(1)

mean

(mg O2/g h)(1)

Blank control

7.5 - 7.6

7.4 - 7.6

49 - 77

68

33 - 51

45

-

2

7.5

7.5 - 7.7

64 - 80

70

43 - 53

47

-4

5.7

7.5

7.6 - 7.7

50 - 69

60

33 - 46

40

11

16

7.5

7.6 - 7.7

54 - 74

62

36 - 49

41

8

46

7.4 - 7.5

7.6 - 7.8

35 - 48

43

23 - 32

28

37*

130

7.4 - 7.5

7.7 -7.8

27 - 34

31

18 - 23

20

55*

380

7.4

7.6

23 - 30

27

15 - 20

18

61*

1070

7.4

7.5 - 7.6#

19 - 24

22

13 - 16

14

68*

1) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

# pH of one vessel was not determined

ADDITIONAL EFFECT VALUES

- EL20: 18 mg/L ( 95% C.I.: 3.3 - 41 mg/L)

- NOEL: 16 mg/L

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'

Description of key information

The 3-h EC50 and 3-h EC10 values are 170 mg/L and 5.6 mg/L respectively in a respiration inhibition study with activated sludge of a predominantly domestic sewage.

Key value for chemical safety assessment

EC50 for microorganisms:
170 mg/L
EC10 or NOEC for microorganisms:
5.6 mg/L

Additional information

The toxicity of the substance to microorganisms was investigated in an acivated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (WIL Research Europe B.V., 2015). In this study activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 2.0, 5.7, 16, 46, 130, 380 and 1070 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring respiration rates and comparing these to an unexposed control. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a test substance loading rate of 16 mg/L. Inhibition of the respiration rate increased with the increasing loading rate ranging from 37% at 46 mg/L to 68% at 1070 mg/L. Based on these findings, the 3-h EC50 and 3-h EC10 values were determined at 170 mg/L and 5.6 mg/L, respectively (nominal).