Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
MMBC
Appearance: brown solid
Storage conditions: in darkness at room temperature

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
yes
Details on test solutions:
REFERENCE SUBSTANCE
Identity 3,5-Dichlorophenol
The reference substance was prepared by dissolving 0.5 g of 3,5-dichlororphenol into 10 mL of 1N NaOH and diluting to approximately 30 mL with distilled water. A sufficient volume of 1N H2SO4 was then added under stirring to the point of incipient precipitation, and the final mixture diluted to one litre with distilled water.

PREPARATION
The test substance was dispersed into the test system using acetone as an auxiliary solvent (100 µL/L).

Test concentrations
Test substance: 99.6 mg A.I./L (in triplicate). Test concentrations for the definitive study were based on results from a preliminary study between 1 and 100 mg/L.

Reference substance: 3.2, 10 and 32 mg/L.

SYNTHETIC SEWAGE
A synthetic sewage was prepared by dissolving the following ingredients per litre of dilution water:
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

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms.
Source: The aeration stage of the Anglian Water (Godmanchester) sewage treatment plant treating predominantly domestic sewage.

Preparation
A sample of activated sewage sludge was collected from the sewage treatment plant on 7 October 1996. The sample was maintained at 20 ± 2 °C with continuous aeration and used within 24 hours of collection. The pH was 7.6 and the suspended solids level of the sample was 3.67 mg/L. Consequently, no adjustment of these characteristics was considered necessary.

Study design

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

Test conditions

Hardness:
Laboratory tap water, dechlorinated by passage through activated carbon and softened by reverse osmosis (hardness approximately 120 - 180 mg/L as CaCO3, pH 7.5 ± 0.5).
Test temperature:
20 °C
Dissolved oxygen:
The dissolved oxygen level was recorded for approximately 10 minutes, between approximately 6.5 and 2.5 mgO2/L.
Nominal and measured concentrations:
99.6 mg A.I./L
Details on test conditions:
At time "0", 16 mL of synthetic sewage was made up to 300 mL with water in a 1 litre glass beaker. An aliquot, 200 mL, of inoculum was added and the mixture aerated vigorously with compressed air via a narrow bore glass tube. This preparation was designated Control R1. Thereafter, at 15-minute intervals, further cultures were prepared following the same procedure but with the addition of appropriate amounts of test or reference substance to the vessels prior to adjusting the volume and adding the inoculum. Before the test substance cultures and immediately following them, solvent control cultures were tested. These were identical to the control cultures but for the addition of 50 µL of acetone (100 µL/L). The series was completed by a second Control (R2) culture. The cultures were then incubated for a maximum of 3 hours at 20 °C under normal laboratory lighting.

As each culture reached 3 hours contact time its pH was recorded and the rate of respiration measured using a Yellow Springs Dissolved Oxygen Meter (Model No. 57) and a magnetic stirrer. A 280 mL darkened glass BOD bottle was completely filled with the culture and sealed by the dissolved oxygen probe. Separate measuring bottles were used for control, test and reference cultures to eliminate the possibility of cross-contamination.

EVALUATION OF DATA
Percentage inhibition values for the test and reference cultures were calculated from the following equation:
% inhibition = 1 - [2Rs / (Rc1 + Rc2)] x 100
Where Rs = oxygen consumption rate for test or reference samples
Rc1 + Rc2 = oxygen consumption rates for Controls (or Solvent Controls, as appropriate) 1 and 2.

The EC50 value for the reference substance after 3 hours contact time was determined by plotting % inhibition against concentration and fitting a line by eye. The ECS0 value was derived by interpolation.

VALIDATION CRITERIA
(i) The respiration rates for the two Control, and Solvent Control, cultures R1 and R2 must be within 15 % of each other at 3 hours contact time.
(ii) The EC50 value of 3 ,5-dichlorophenol at 3 hours contact time must be within the accepted range of 5 - 30mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 99.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The oxygen consumption rates for the control, test and reference cultures after 3 hours contact time are given in Table 1, together with percentage inhibition values.

The EC50 value for 3 hours contact time, derived by interpolation, is given below:
Test substance: >99.6 mg A.I./L.

Variation in respiration rates of Controls 1 and 2: ± 5 %
Variation in respiration rates of Solvent Controls 1 and 2: ± 2 %.

The highest test concentration that could be prepared was 99.6 mg A.l. /L due to the limited solubility of the test substance in water. This concentration is also the maximum test concentration required under the EC guideline.
Results with reference substance (positive control):
The EC50 value for 3 hours contact time, derived by interpolation, is given below:
3,5-dichlorophenol: 7.5 mg/L.

Any other information on results incl. tables

Table 1 Oxygen Consumption Rates and Percent Inhibition for 3 h Contact Time

Culture

Concentration (mg/L)

O2 Consumption Rates (mgO2/L/min)

% Inhibition

Control

R1

-

0.567

-

R2

-

0.633

-

Solvent Control

R1

-

0.653

-

R2

-

0.627

-

Test

R1

99.6*

0.683

<7>

R2

99.6*

0.708

<11>

R3

99.6*

0.640

0

 

Reference

3.2

10

32

0.444

0.238

0.069

26

60

89

R1, R2 and R3 = Replicates 1, 2 and 3.

<increase>

*Concentrations given in mg A.I./L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (respiration inhibition) value for the test substance with activated sewage sludge was determined to be >99.6 mg A.l./L for the 3 hour contact time.
Executive summary:

The inhibitory effect of the test substance on the respiration of activated sewage sludge was investigated in accordance with standardised guidelines OECD 209 and EU Method C.11. The study was run as a limit test where cultures of activated sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance (three cultures containing 99.6 mg A.I/L). Aeration was interrupted after 3 hours and the rates of respiration measured electrochemically for each culture. Percentage inhibition of respiration was calculated for each culture after a 3 hour contact period by comparing oxygen depletion rates for the test substance with those for the negative control culture. A positive control was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. Under the conditions of the study, the EC50 value for the test substance was determined to be > 99.6 mg A.I/L for the 3 hour contact time.