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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-04/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
Following the special demand of authorities the test material was allowed to hydrolyse for 5 days in water prior to test .
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) for 115 hours. The prolonged pretreatment was performed following a special requirement by the European Chemicals Agency. The pH was measured and ranged between pH 6.2-7.2. The pH was adjusted to pH 7.2-7.4 with NaOH.
For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.
Test organisms (species):
sewage, domestic
Details on inoculum:
Test organism and synthetic sewage feed
Type : mixed population of aquatic microorganisms (activated sludge)
Origin : aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
Date of collection : 2015-03-30
Microbial inoculum : The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
Storage of sludge : aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
pH of the suspension before application : 7.7 (range finding test) 7.6 (main test)
Synthetic sewage feed : A synthetic waste water feed was prepared by dissolving the following amounts of substance per 1 litre of water.
16.0 g peptone
11.0 g meat extract
3.0 g urea
0.7 g NaCl
0.4 g CaCl2 x 2H2O
0.2 g MgSO4 x 7H2O
2.8 g K2HPO4
pH of the synthetic sewage feed : 7.5 ± 0.5
Test type:
other: static; 5 days pre-treatment of solution, lower concentration. level
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
20°C +/- 2 °C (including all test concentrations, controls and reference substance)
pH:
7 +/- 0.5 (including all test concentrations, controls and reference substance)
Dissolved oxygen:
recorded parameter
Salinity:
no data
Nominal and measured concentrations:
2.5, 5, 10, 20 and 40 mg/L (nominal)
Details on test conditions:
Test vessels: 300 mL glass Erlenmeyer flasks
Method of application: direct weighing
Concentration of the activated sludge : 800 mg/L suspended solids
Test temperature: 20 ± 2 °C
Stirring period of the test item before start of incubation time : 114 hours
Incubation time: 3 hours with permanent aeration
Control (6 replicates)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol, concentration (2.5, 5, 10, 20 and 40 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Result after aging of 7PPD at room temperature in water for a period of 5 days.
Validity criteria fulfilled:
yes
Remarks:
respiratory rates of the 2 controls were less than 30 % - The EC50 of the reference compound 3,5-Dichlorophenol should be in the range of 2 – 25 mg/L for total respiration. The EC50 of the reference compound was 14.954 mg/L; thus the test is valid.
Conclusions:
The test item showed 0 % respiration inhibition of activated sludge at the highest test item concentration of 40 mg/L. An EC50 and EC10 of >40 mg/L, and a NOEC of >= 40 mg/L were obtained in this test. The effect value relates to nominal concentration, since no analytical monitoring was performed.
Executive summary:

In order to test the toxicity to microorganism of 7PPD after aging in water for a period of 5 days, activated sludge from predominantly domestic sewage plant was exposed to the test solution of five nominal concentrations of the substance ( 2.5, 5, 10, 20 and 40 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours.

An EC50 and EC10 of >40 mg/L, and a NOEC of >= 40 mg/L were obtained.

The test item was added into Erlenmeyer flasks (incubation vessels) to about 400 mL deionised water and was stirred before testing (equilibration phase) for 114 hours.

Dissolved oxygen was recorded and respiration rates were claculated. The physico-chemical oxygen consumption has been determined at 40 mg/L test item concentration. As nearly no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations. 0 % respiration inhibition was observed at 40 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

The present study has been performed additionally to Currenta study 2014/0115/04 where no definite NOECs could be achieved.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09/2015 - 01/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) for 1 hour. The pH was measured and ranged between pH 6.0-6.4. The pH was adjusted to pH 7.2-7.4 with NaOH.
For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.
Test organisms (species):
sewage, domestic
Details on inoculum:
Test organism and synthetic sewage feed
Type: mixed population of aquatic microorganisms (activated sludge)
Origin: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
Date of collection: 2015-09-28
Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20 °C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
pH of the suspension before application: 7.8
Synthetic sewage feed: A synthetic waste water feed was prepared by dissolving the following amounts of substance per 1 litre of water.
16.0 g peptone
11.0 g meat extract
3.0 g urea
0.7 g NaCl
0.4 g CaCl2 x 2H2O
0.2 g MgSO4 x 7H2O
2.8 g K2HPO4
pH of the synthetic sewage feed: 7.5 ± 0.5
Test type:
other: static; freshly prepared solution
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
20°C +/- 2 °C (including all test concentrations, controls and reference substance)
pH:
7 +/- 0.5 (including all test concentrations, controls and reference substance)
Dissolved oxygen:
recorded parameter
Salinity:
no data
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L (nominal)
Details on test conditions:
Test vessels: 300 mL glass Erlenmeyer flasks
Method of application: direct weighing
Test concentration of the activated sludge: 800 mg/L suspended solids
Test temperature: 20 ± 2 °C
Stirring period of the test item before start of incubation time: 16 hours
Incubation time: 3 hours with permanent aeration
Control (6 replicates)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol, concentration (2.5, 5, 10, 20 and 40 mg/L)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
yes
Remarks:
respiratory rates of the controls were less than 30 % - The EC50 of the reference compound 3,5-Dichlorophenol should be in the range of 2 – 25 mg/L for total respiration. The EC50 of the reference compound was 12.612 mg/L; thus the test is valid.
Conclusions:
The test item showed 6.272 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L. An EC10 of > 100 mg/L and an EC50 of >100 mg/L were obtained in this test. The effect value relates to nominal concentration, since no analytical monitoring was performed.
Executive summary:

In order to test the toxicity to microorganism of the substance, activated sludge from a domestic sewage plant was exposed to the test solution of 100 mg/L, blank control solution and solution with positive reference substance for a period of 3 hours.

The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) for 1 hour.

Dissolved oxygen was recorded and respiration rates were claculated. No physico-chemical oxygen consumption was observed at the concentration of test substance. A respiration inhibition of 6.272 % was observed at 100 mg/L. An EC10 and an EC50 of > 100 mg/L were determined. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

Four tests with three different pre-treatments were permformed:
1. Short pre-treatment: 7PPD showed 6.272 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L with freshly prepared test solution. An EC10 of > 100 mg/L and an EC50 of >100 mg/L were obtained in this test. The effect value relates to nominal concentration, since no analytical monitoring was performed.
2. 17-hours-pretreatment: The test item showed 20.08 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L with a 17 h pretreated test solution. An EC10 of 96.7 mg/L and an EC50 of >1000 mg/l were obtained in this test.
3. Five-day-pre-treatment:
a) High concentration test: The test item showed 27.2 % respiration inhibition of activated sludge at the highest test item concentration of 400 mg/L. An EC50 of >400 mg/l was obtained in this test.
b) Low-concentration test: The test item showed 0 % respiration inhibition of activated sludge at the highest test item concentration of 40 mg/L. An EC50 and EC10 of >40 mg/L, and a NOEC of >= 40 mg/L were obtained in this test.
Finally, no significant effects were seen in any of these studies.

The stated key values for chemical safety assessment have to be read as >40 mg/L (EC10 and EC50), and >=40 mg/L (NOEC).

Key value for chemical safety assessment

EC50 for microorganisms:
40 mg/L
EC10 or NOEC for microorganisms:
40 mg/L

Additional information

1. Freshly prepared test solution:

In order to test the toxicity to microorganism of the substance, activated sludge from a domestic sewage plant was exposed to the test solution of 100 mg/L, blank control solution and solution with positive reference substance for a period of 3 hours.

The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) for 1 hour.

Dissolved oxygen was recorded and respiration rates were claculated. No physico-chemical oxygen consumption was observed at the concentration of test substance. A respiration inhibition of 6.272 % was observed at 100 mg/L. An EC10 and an EC50 of > 100 mg/L were determined.

2. Test solution with 17 hours pre-treatment in water:

In order to test the toxicity to microorganism of the substance, activated sludge from a domestic sewage plant was exposed to the test solution of five nominal concentrations of the substance (10, 32, 100, 320 and 1000 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours.

The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours.

Dissolved oxygen was recorded and respiration rates were claculated. No physico-chemical oxygen consumption was observed at the highest concentration of test substance. No respiration inhibition was observed at 100 mg/L; and 24.08 % inhibition at 1000 mg/L. An EC10 of 96.7 mg/L and an EC50 of > 1000 mg/L were determined.

3. Five day pre-treatment:

The test item was added into Erlenmeyer flasks (incubation vessels) to about 400 mL deionised water and was stirred before testing (equilibration phase) for 114 hours.

Currenta study 2014/0115/04:

In order to test the toxicity to microorganism of 7PPD after aging in water for a period of 5 days, activated sludge from a domestic sewage plant was exposed to the test solution of five nominal concentrations of the substance (25, 50, 100, 200 and 400 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours.

The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) for 115 hours.

Dissolved oxygen was recorded and respiration rates were claculated. The physico-chemical oxygen consumption has been determined at 400 mg/L test item concentration. As nearly no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations. 27.2 % respiration inhibition was observed at 400 mg/L.

As no clear NOEC could be derived from this study, another study (2014/0115/05) was performed covering the lower concentration level.

Currenta study 2014/0115/05:

To test the toxicity to microorganism of 7PPD after aging in water for a period of 5 days, activated sludge from predominantly domestic sewage plant was exposed to the test solution of five nominal concentrations of the substance (2.5, 5, 10, 20 and 40 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours.

The present study has been performed additionally to Currenta study 2014/0115/04 where no definite NOECs could be achieved.

An EC50 and EC10 of >40 mg/L, and a NOEC of >= 40 mg/L were obtained.

The afore-mentioned toxicity studies are classified as acceptable and satisfy the guideline requirements for the toxicity study to microoragnisms.

Other studies:

Two studies performed with p-hydroquinone, a secondary hydrolysis product of 7PPD, show that this substance is relatively toxic to organisms (NOEC 1 mg/L, IC50 71 mg/L).