Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. Study on ammonium salt of hydrolysis product.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
The amount of suspended solids in the sludge was 3.3 g/L and therefore, outside the range of 4 g/L ± 10%. The respiration rate of the control and reference substance were at a level high enough to reliably evaluate the results.
Qualifier:
according to guideline
Guideline:
other: EEC directive 67/548, Part C:1988
Deviations:
yes
Remarks:
The amount of suspended solids in the sludge was 3.3 g/L and therefore, outside the range of 4 g/L ± 10%. The respiration rate of the control and reference substance were at a level high enough to reliably evaluate the results.
Qualifier:
according to guideline
Guideline:
other: ISO international Standard 8192:2007
Deviations:
yes
Remarks:
The amount of suspended solids in the sludge was 3.3 g/L and therefore, outside the range of 4 g/L ± 10%. The respiration rate of the control and reference substance were at a level high enough to reliably evaluate the results.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
ADONA
IUPAC Name:
ADONA
Details on test material:
- Name of test material (as cited in study report): MTDID 6675
- Substance type: single-constituent substance
- Physical state: clear colorless liquid
- Analytical purity: 29.9 ±0.1% in water
- Lot/batch no.: 140499-19/10
- Stability under test conditions: Stable in water at least 96 hours
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution of 16.72 g/L was prepared by adding 4.1797 g test substance to 250 mL of Milli-RO water (tap water purified by reverse osmosis, Millipore Corp., Bedford, Mass, USA). A concentration of 3340 mg/L, corresponding to 100 mL prepared stock solution in 500 mL final volume. This concentration of the total product equaled an active ingredient (a.i.) concentration of 1000 mg a.i./L. Optimal contact between the test substance and test medium was ensured by applying continuous aeration and stirring.
- Controls: test medium without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

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 (3.3 g/L of sludge, as used for the test). The sludge was collected one day before use. pH was 7.8 on day of sampling and 7.5 on 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 (*synthetic sewage feed: 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(H2O), 2.8 g K2HPO4 dissolved in 1 L RO water and filtered, final pH 7.1 (adjusted using NaOH)).

Study design

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

Test conditions

Hardness:
Not reported
Test temperature:
20 ± 2 °C as per the protocol
pH:
8.1 - 8.3
Nominal and measured concentrations:
Nominal concentration: 3340 mg/L total product, corresponding to 1000 mg/L active ingredient.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass bottles
- Type (delete if not applicable): Open during exposure.
- Material, size, headspace, fill volume: 100 mL test solution, 16 mL synthetic sewage, and 200 mL sludge suspension plus sufficient RO water to bring total volume to 500 mL in a 1L glass bottle.
- Aeration: Continuous aeration with pipette
- Nitrification assessed: No
- No. of vessels per concentration (replicates): 2
- No. of vessels per reference (replicates): 4
- No. of vessels per control (replicates): {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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to prepare media was tap water purified by reverse-osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q, Millipore Corp., Bedford, Mass., USA)
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Dissolved oxygen concentration. After a 3h contact time, a well mixed sample of the contents was poured into a 300 mL glass oxygen bottle and sealed with an oxygen electrode connected to a recorder, forcing the air out the vessel. oxygen consumption was measured and recorded for approximately 10 minutes. During analysis, sample was not aerated but continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test, one concentration
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol, 97% pure, Aldrich, Batch# 15809KI-392, Expiration: 24 June 2008

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: The 3h EC50 of ADONA for inhibition of respiration rate exceeded the nominal concentration of 3340 mg/L total product, which corresponds to 1000 mg/L active ingredient. 3h NOEC of ADONA = 1000 mg/L active ingredient.
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, Table 1
- Relevant effect levels: 3h EC50 = 5.5 mg/L The EC50 of 3,5-dichlorophenol was in the accepted range of 5 - 30 mg/L.
Reported statistics and error estimates:
No EC50 could be calculated for the test substance because it proved to be non-toxic (EC50 > 3340 mg/L total product (1000 mg/L active ingredient). 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 1, Study Results

Sample

Sample

Concentration

(mg/L)

Oxygen concentration at the start

(mg/L)

Respiration rate

(mg O2/L∙h)

% Inhibition

pH

Ref Control 1

0

8.7

30

 

8.2

Ref Control 2

0

7.9

37

 

8.2

Mean

 

 

33.5

 

 

 

 

 

 

 

 

Ref 1A

1.0

8.9

25

25

8.3

Ref 2A

3.2

9.1

20

40

8.4

Ref 3A

10

9.2

14

58

8.2

Ref 4A

32

9.0

7.2

79

8.2

 

 

 

 

 

 

Test Control 1

0

8.2

35

 

8.1

Test Control 2

0

8.4

32

 

8.3

Mean

 

 

33.5

 

 

 

 

 

 

 

 

Test 1B

1000C

(3340)

7.8

33

1.5

8.2

Test 2B

1000C

(3340)

7.9

32

4.5

8.2

A) Reference substance: 3-5,dichlorophenol

B) Test substance: ADONA

C) Concentration of active ingredient, in parentheses is corresponding nominal concentration to total product

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control respiration rate variability < 15%. Reference substance 3-h EC50 5.5 mg/L (within 5-30 mg/L).
Conclusions:
ADONA is not inhibitory to activated sludge respiration (3-h EC50 >1000 mg/L active ingredient).
Executive summary:

MeDONA is hydrolytically unstable. Aquatic toxicity testing on MeDONA would in practice be testing of the hydrolysis products. This test examined the ammonium salt of the hydrolysis product (ADONA).

The toxicity of ADONA to activated sludge organisms was assessed in an assay performed according to OECD TG209. No statistically significant inhibition was observed upon exposure to ADONA, with a 3h EC50 to sludge>1000 mg/L for the active ingredient (> 3340 mg/L total product). The 3h NOEC for ADONA is 1000 mg/L active ingredient (3340 mg/L total product).

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                          

3h EC50 >1000 mg/L active ingredient 95% C.I.: not applicable

3h NOEC = 1000 mg/L active ingredient Probit Slope: not applicable

Endpoint(s) Effected:  Respiration inhibition

Concentration corresponds to nominal 3340 mg/L total product