1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 05 - March 19, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study compliant with TG guideline and conducted under GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

not required

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: All amounts of test substance and all test substance concentrations listed in the present report are expressed as amount of water- and minor impurity-free test substance per volume of aqueous solution, suspension or medium, unless otherwise stated. This was accounted for during preparation of the test solutions by applying a factor of 1.16 to all test substance concentrations as follows: Test substance concentration administered = 1.16 times the test substance received at the testing laboratory. A stock solution of 0.5 g/L was prepared by adding 579.9 mg test substance to 1000 mL of tap water purified by reverse osmosis. Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. Preparation was as much as possible performed under yellow light and/or low light conditions.
Synthetic sewage feed (16 mL), activated sludge (200 mL) and an adequate amount of the test substance stock were mixed and made up to 500 mL with tap-water (purified by reverse osmosis) in a 1 litre bottle.
- Controls: Two controls without test substance were tested in each test series, one at the start and one at the end.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: The sludge was coarsely sieved, washed and diluted with adjusted ISO-medium (see Table 1). A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.8 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic 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.
- Pretreatment: none
- Initial biomass concentration: 3.8 g/L. The number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS)
per litre test medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

during exposure, aeration and stirring took place

Test conditions

Hardness:

not indicated

Test temperature:

18-20 °C

pH:

controls: 8.4 (controls); 8.2-8.5 (treated)

Dissolved oxygen:

controls: 7.8-8.0 mg/O2/L (controls); 6.6-8.8 mg/O2/L (treated)

Salinity:

not applicable (freshwater)

Nominal and measured concentrations:

Nominal: 0, 32, 56, 100, 180, 300 mg/l (expressed as water and minor impurity-free test substance)

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass, approximately 300 mL oxygen bottles and 1 litre test bottles
- Type (delete if not applicable): open
- Aeration: aerated with clean, oil-free air using a pipette as an aeration device
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.5 g/l

TEST MEDIUM / WATER PARAMETERS
Tap-water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges

- Intervals of water quality measurement: pH, temperature and oxygen concentration were measured after the 3-hour contact time

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration inhibition after 3-hour contact time

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.8

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol; 0, 5.0, 12, 30 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water- and minor impurity-free test material

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL: 120-160 mg/L

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water- and minor impurity-free test material

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL: 41-57 mg/L

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

62 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water- and minor impurity-free test material

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL: 53-72 mg/L

Effect concentrations 4

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

290 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water- and minor impurity-free test material

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL: 250-340 mg/L

Details on results:

No inhibition of respiration rate of the sludge was recorded at the lowest concentration (32 mg/L*). Thereafter, the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 16% inhibition at 56 mg/L* to 81% inhibition at the highest concentration (300 mg/L*). See Table 1.
____________________________________________________________________________________________________ 
* Expressed as water- and minor impurity-free test substance

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect level: EC50 = 7.4 mg/L (accepted range: 5-30 mg/L)

Reported statistics and error estimates:

The EC-values were estimated using linear regression (regression line: Y= 87.11 X - 135.79, Y=inhibition (%) and X= concentration (mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Criteria of OECD Guideline 209: The difference between the controls was within 15% of each other. The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/l.