AERO 5100 PROMOTER

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Apr - 08 May 1998

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was insufficiently soluble in water to allow the preparation of a suitable stock solution, so appropriate weights were added directly to test beakers. Dechlorinated tap water (284 mL) was added and the mixtures were treated with ultrasound for, at least, 10 minutes. Additions of synthetic sewage and microbial inoculum were then made as detailed in the schedule given under "Any other information on material and methods incl. tables" below.
- Controls: yes, test medium control

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Oakley sewage treatment works, a sewage plant treating predominantly domestic waste (obtained one day before testing)
- Method of cultivation: maintained under aerobic conditions until required
- Preparation of inoculum for exposure: On the day of collection, aliquots (25 mL) of the activated sludge were filtered through dried and pre-weighed filter papers which were then dried again, at least, at 105 °C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated and adjusted to 4 g/L by the removal of supernatant Synthetic sewage (50 mL/L) was added and the mixture aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/L by the addition of dechlorinated tap water. The pH of the sludge was also measured.
- Suspended solids: 1.6 g/L (determined on the day of collection and immediately before the start of the test)
- Synthetic sewage: Synthetic sewage feed for activated sludge was prepared by dissolving the following in one litre of ultrapure water (resistivity 18 Megohm/cm): peptone 16.0 g, meat extract 11.0 g, urea 3.0 g, sodium chloride 0.7 g, calcium chloride dihydrate 0.4 g, magnesium sulphate heptahydrate 0.2 g, di-potassium hydrogen phosphate 2.8 g

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

200 - 250 mg/L as CaCO3

Test temperature:

20.4 - 21.5 °C (test start)
20.6 - 21.2 °C (test end)

pH:

7.4 - 7.6 (test start)
7.9 - 8.3 (test end)

Nominal and measured concentrations:

control, 10, 50, 100, 500, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Aeration: Prepared mixtures were aerated using a Pasteur pipette connected to a laboratory supply of oil-free compressed air for 3 h.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water softened and treated by reverse osmosis and then purified to give a resistivity of 18 Megohm/cm. This water complies with the relevant standards (British Standard (BS) 2486, 1978; American Society for Testing and Materials (ASTM) D 1193-77) for classification as ultrapure water. The water used to prepare test mixtures was dechlorinated tap water.
- Intervals of water quality measurement: The pH and temperature of the samples were measured at the start and end of the test

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1, 10, and 100 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3,5-DCP)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

137.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CL: 117.4 - 159.0 mg/L

Results with reference substance (positive control):

EC50 (3 h) = 10.1 mg/L (95% CL: 8.2 - 12.5 mg/L)

Table 1: Results of definitive test

Test mixture	Respiration rate [mg O2/g/hour]	% inhibition
Control 1	39.5	-
AERO® 5100 Promoter
10	51.8	0
50	35.5	15
100	26.3	37
500	1.9	95
1000	1.0	98
3,5-DCP (mg/L)
3.0	34.9	17
10.0	20.8	50
32.0	8.0	81
Control 2	44.1	-

Results:

Sludge respiration rates in the test were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentrations (EC50) for 3,5-DCP was calculated by the Moving Average method to be 8.2 mg/L (95% confidence limits 6.7 - 9.9 mg/L) in the preliminary test, and 10.1 mg/L (95% confidence limits 8.2 - 12.5 mg/L) in the definitive test.

The specific respiration rate of control cultures established at the end of the two test series (31.9 and 44.1 mg O2/g/h) were 106% and 112% of the rates of those established at the start (30.0 and 39.5 mg02/g/h).

These results show that the tests were valid and that the samples of activated sludge employed were sensitive to inhibition.

Concentration related inhibition of respiration rates was seen at and above a test concentration of 50 mg/L. Respiration rates were inhibited by 95% and 98% at nominal AERO® 5100 Promoter concentrations of 500 and 1000 mg/L, respectively.

The EC50 of the test substance was calculated from the results of the definitive test using the values of inhibition observed at nominal levels of 50, 100, 500 and 1000 mg/L to be 137.5 mg/L (95% confidence limits 117.4 - 159.0 mg/L). The calculation of the EC50 of the test substance therefore excluded the result obtained at a nominal concentration of 10 mg/L (0% inhibition) as it could not be assumed that this was the no effect concentration. The EC20 and EC80 values were estimated from a graphical plot of the results to be 55 mg/L and 330 mg/L, respectively.

Description of key information

EC50 (3 h): 137.5 mg/L (nominal, OECD 209)

Additional information

One experimental study is available investigating the effects of the substance to activated sludge microorganisms (Cytec Industries, 1998). The study was performed according to OECD 209 (GLP) using activated sludge from a sewage treatment plant treating predominantly domestic sewage. Test substance was added to the test vessels directly to give nominal concentrations of 10, 50, 100, 500 and 1000 mg/L. After 3 h of incubation an inhibition of respiration was recorded resulting in an EC50 (3 h) of 137.5 mg/L (95% CI: 117.4 - 159.0 mg/L).