Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jun 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 9580
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 99%
- Expiration date of the lot/batch: 31 Dec 2007
- Stability under test conditions: Not completely soluble in test medium at a loading rate of 100 mg/L. Reacts slowly with oxygen, e.g. in the presence of moisture, resulting in acid fluorides, carboxylic acids and HF. Exposure to air was kept at a minimum during the preparation procedures, since the test substance was indicated to be volatile.
- Storage condition of test material: At room temperature in the dark under nitrogen

Sampling and analysis

Analytical monitoring:
no

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because of low solubility, MV 31 was added to reverse osmosis-purified water at a loading rate of 2000 mg/L, with 24 hours' stirring in closed 1-L test bottles. For the test, the loading rate brought to 1000 mg/L with activated sludge, sewage feed, and RO water.

- Controls: blanks, 3,5-Dichlorophenol as reference substance
- Chemical name of vehicle: No vehicle
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None noted

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Sludge was collected from municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.The sludge was coarsely sieved, washed and diluted with ISO medium. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (4.1 g/L of sludge, as used for the test). The sludge was collected one day before use. The pH was 7.5 on day of sampling and adjusted from 5.8 to 7.2 on the day of testing.

ISO medium (in reverse osmosis [RO]-purified tap water)
211.5 mg/L CaCl2∙2(H2O)
88.8 mg/L MgSO4∙7(H2O)
46.7 mg/L NaHCO3
4.2 mg/L KCl

ISO medium was used only for sludge preparation, not during the test.

- Method of cultivation: Because the sludge was used one day after collection, 50 mL of synthetic sewage feed was added to each litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
Not reported
Test temperature:
18.2 - 20.5°C
pH:
7.5 - 8.2
Nominal and measured concentrations:
Nominal concentration 1000 mg/L. No analytical monitoring.
Details on test conditions:
TEST SYSTEM
- Test vessels: all glass
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 1L, with 250 mL test solution, 16 mL synthetic sewage, and 200 mL sludge suspension plus sufficient RO water to bring total volume to 500 mL.
- Aeration: Continuous aeration with pipette
- No. of vessels per concentration (replicates): 2
- No. of vessels per reference (replicates): 4
- No. of vessels per control (replicates): 4 total (2 for test substance, 2 for reference substance). One analysed at the start of the test and one analysed at the end of the test
- Biomass loading rate: 1.64 g/L Mixed Liquor Sludge Solids


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges
- Standard medium used: Synthetic sewage
- Detailed composition: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCI, 0.4 g CaCI2·2(H2O), 0.2 g MgSO4·7(H20), 2.8 g K2HPO4 dissolved in 1 L RO water, filtered and the pH adjusted to 7.0

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Dissolved oxygen concentration

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Single concentration tested
- Justification for using less concentrations than requested by guideline: Limit test, MV 31 not soluble in water.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No reduction in respiration rate was recorded at nominal 1000 mg/L
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: yes. However, report does not mention whether undissolved material was present in sample bottle after stirring.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 7.7 mg/L which is within the required range of 5 - 30 mg/L
Reported statistics and error estimates:
No EC50 could be calculated for MV 31 because it proved to be non-toxic (No effects seen at highest nominal concentration tested, 1000 mg/L). For the reference substance the percentage of inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.

Any other information on results incl. tables

Table, Oxygen concentration, oxygen consumption, percentage inhibition and pH for Negative controls (C), 3,5-dichlorophenol (R), and MV 31 (T)

Replicate

Concentration of substance (mg/L)

Initial oxygen conc. (mg/L)

Oxygen consumption rate (mg O2/L/h)

Respiration inhibition (%)

pH

C1

0

7.4

46

8.0

C2

0

8.3

43

8.0

Mean C1+C2

44.5

R1

1.0

7.8

42

5.6

8.1

R2

3.2

8.6

30

33

8.3

R3

10

8.7

20

55

8.2

R4

32

9.0

9

80

8.2

C3

0

7.3

48

7.8

C4

0

7.7

44

7.7

Mean C3+C4

46

T1

1000

5.7

52

-13

7.5

T2

1000

5.7

54

-16

7.6

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
ontrol respiration rate variability < 15%. Reference substance 3-h EC50 7.7 mg/L (within 5-30 mg/L)
Conclusions:
MV 31 is not inhibitory to activated sludge respiration (3-h EC50 >1000 mg/L)
Executive summary:

The toxicity of MV 31 to activated sludge organisms was assessed in an assay performed according to OECD TG209. The test solution was made by stirring MV 31 for 24 hours in a closed vessel at a loading rate of 2000 mg/L, with subsequence dilution to 1000 mg/L for the test. No inhibition was observed upon exposure to MV 31 at the loading rate. The 3-hour EC50 to sludge is therefore >1000 mg/L.

The study was conducted in accord with GLP criteria and satisfies the guideline requirements for test method OECD209, activated sludge respiration inhibition test. It is classified as reliable without restrictions and is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.

Results Synopsis  

Test Type: Static                          

EC50:  >1000 mg/L                       95% C.I.:  not applicable

NOEC:  1000 mg/L                        Probit Slope:  not applicable

Endpoint(s) Effected:  Respiration inhibition