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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-05 to 2017-01-06
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:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: 2015-04-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

OTHER SPECIFICS:

Molecular weight: 320
Volatile: 121 mmHg at 25°C
Specific gravity/density: 1.67 at 25°C
Solubility in water: Practically insoluble
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: 10, 100 and 1000 mg/L
Vehicle:
no
Remarks:
Not applicable
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of AsahiklinTM AC-2000 tested was a colourless liquid with a purity of 100.00%. No correction was made for the purity/composition of the test item.

The test item was volatile and 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 item 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 by pipetting the test item directly into the Milli-RO, and subsequently adding the used pipette tip to the resulting suspension, to ensure the quantitative addition of the amount of test item. Thereafter, 16 mLsynthetic 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 item and test organisms was ensured by applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation: 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 pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) 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:
not specified
Water media type:
other: Milli- RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test
pH:
The pH in all test vessels, before addition of sludge was between 7.2 and 7.4. After the 3 hour exposure period, the pH was between 7.1 and 8.5.
Nominal and measured concentrations:
10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass
- Aeration: 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.
- No. of vessels per concentration (replicates): The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- No. of vessels per control (replicates): A blank control (6 replicates) and a nitrification control (2 replicates)
- No. of vessels per vehicle control (replicates): Not applicable
- No. of vessels per abiotic control (replicates): an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates)
- Sludge concentration (weight of dry solids per volume): Suspended solids = 3.0 g/L of sludge
- Nutrients provided for bacteria: Synthetic medium (=sewage feed)
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU, Merck Schuchardt OHG)

TEST MEDIUM / WATER PARAMETERS
Medium: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot, The Netherlands) with the following composition:

CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

Synthetic medium (=sewage feed): Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4

OTHER TEST CONDITIONS:

Air supply: Clean, oil-free air

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: In a combined limit/range-finding test, loading rates of 10, 100 and 1000 mg/L were tested
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: ELR50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
Remarks on result:
not determinable
Remarks:
Test material was slightly toxic to waste water (activated sludge) bacteria at the loading rate of 1000 mg/L. Therefore, a NOELR could not be determined.
Details on results:
Inhibition of the respiration rate:

Detailed study results are presented in Table 3.

The combined limit/range-finding test showed 10, 6 and an average of 14% inhibition of the respiration rate at loading rates of 10, 100 and 1000 mg/L, respectively. Therefore, the ELR50 was above the highest loading rate tested (1000 mg/L). There was no oxygen uptake from abiotic processes and the result at the loading rate of 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was slightly lower than the total inhibition. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 37% inhibited. In the present test, AsahiklinTM AC-2000 was slightly toxic to waste water (activated sludge) bacteria at the loading rate of 1000 mg/L. Therefore, a NOELR could not be determined. As discussed with the sponsor, no further testing was performed to determine the NOELR.

Determination of effect parameters
Table 2 shows the effect parameters based on AsahiklinTM AC-2000 loading rates.

Experimental conditions
The pH values in the individual vessels are presented in Table 3. The pH in all test vessels, before addition of sludge was between 7.2and 7.4. After the 3 hour exposure period the pH was between 7.1 and 8.5. The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity. Details provided in Appendix 2 of the attached study report.

Table 2. Effect parameters

Parameter

AsahiklinTM AC-2000 Loading Rate

(mg/L)

NOELR

Not determined

ELR50

>1000

Table 3. Respiration rate/inhibition, pH Values

 

 

Replicate

 

Loading rate/

Concentration

(reference item)

(mg/L)

pH

Respiration rate

% Inhibition

respiration

rate

(mean value)

 

Start

 

End

 

(mg O2/L.h)

 

(mg O2/g.h)1

C1

0

7.4

8.0

39.10

26.07

 

C2

0

7.4

8.1

38.77

25.85

 

C3

0

7.4

8.0

40.49

26.99

 

C4

0

7.4

8.0

41.47

27.65

 

C5

0

7.4

8.0

41.32

27.55

 

C6

0

7.4

8.0

44.38

29.59

 

C Mean

 

 

 

40.92

27.28 (RTB)

 

SD

 

 

 

2.03

1.35

 

CV %

 

 

 

5

5

 

 

CN1

0

7.4

8.3

23.98

15.99

 

CN2

0

7.4

8.3

23.64

15.76

 

CN Mean

 

 

 

23.81

15.87 (RHB)

 

 

R1

1.0

7.4

8.1

35.79

23.86

12.54

R2

3.2

7.4

8.2

24.77

16.51

39.47

R3

10

7.4

8.1

20.29

13.53

50.42

R4

32

7.4

8.2

9.34

6.23

77.18

 

T1

10

7.2

8.2

36.66

24.44

10.41

T2

100

7.2

8.2

38.40

25.60

6.16

T3a

1000

7.2

8.2

35.63

23.75

12.93

T3b

1000

7.2

8.2

34.74

23.16

15.11

T3c

1000

7.2

8.2

35.39

23.59

13.52

T3 Mean

 

 

 

35.25

23.50 (RT:)*

13.85 (IT)

 

TN a

1000

7.2

8.5

23.31

15.54

2.10

TN b

1000

7.2

8.5

25.03

16.69

-5.12

TN c

1000

7.2

8.4

25.10

16.73

-5.42

TN Mean

 

 

 

24.48

16.32 (RH)

-2.81 (IH)

 

TA

1000

7.2

7.1

0#

0#

100

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

#: No respiration, therefore expressed as 0 mg O2/L.h

C: Blank control

CN: Nitrification control

R: Reference item, 3,5-dichlorophenol

T: Test item, AsahiklinTMAC-2000

TA: Abiotic control of AsahiklinTMAC-2000

TN: AsahiklinTMAC-2000 with N-allylthiourea

SD: Standard deviation

RTB:Total respiration blank

RHB: Heterotrophic respiration in the nitrification control

RT: Total respiration with AsahiklinTMAC-2000

RH: Heterotrophic respiration with AsahiklinTMAC-2000

IT: % inhibition of total respiration relative to RTB

IH: % inhibition of heterotrophic respiration relative to RHB

CV: Coefficient of variation

*  Statistically significantly different compared to control

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, AsahiklinTM AC-2000 was slightly toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L.

The ELR50 was above 1000 mg/L.
Executive summary:

The influence of AsahiklinTM AC-2000 on the respiration rate of activated sludge was investigated after a contact time of 3 hours in a guideline (OECD 209) study.

 

The test material was volatile and not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test material – Milli-RO water mixtures were prepared by pipetting the test material directly into the Milli-RO. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test material and test medium was ensured by applying continuous aeration and stirring during the 3- hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

 

In a combined limit/range-finding test, loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at the loading rate of 1000 mg/L, an abiotic control (1 replicate) and three replicates with a nitrification inhibitor were tested. Responses were compared to the blank and nitrification controls. The combined limit/range-finding test showed 10, 6 and an average of 14% inhibition of the respiration rate at loading rates of 10, 100 and 1000 mg/L, respectively.

 

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The test material was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L. Therefore, a NOELR could not be determined. As discussed with the sponsor, no further testing was performed to determine the NOELR.

 

The ELR50 was above 1000 mg/L.

Description of key information

1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L. Therefore, a NOELR could not be determined (As discussed with the sponsor, no further testing was performed to determine the NOELR).

 

The ELR50 was determined to be above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The influence of the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) on the respiration rate of activated sludge was investigated after a contact time of 3 hours in a Guideline (OECD 209) activated sludge respiration inhibition test (Charles River Laboratories Den Bosch BV., 2017d).

 

The test material was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L. Therefore, a NOELR could not be determined (As discussed with the sponsor, no further testing was performed to determine the NOELR).

 

The ELR50 was determined to be above 1000 mg/L.