Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Remarks:
Respiration and nitrification inhibition
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
2007
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution of the substance in water

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Incubation: 3 hours

Test solutions

Vehicle:
no
Details on test solutions:
Two stock solutions with the test item were prepared in pure water with the following concentrations: 5g/L and 50g/L. Stock solution one with a concentration of 5g/L was used for the preparation of test concentrations 10, 32 and 100mg test item/L. Stock solution two was used for the preparation of test concentrations 320 and 1000mg/L. Appropriate volumes of stock solution were added to each test flask and pure water was added.
In parallel to the study with the test item, the reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4, and 16 mg/L (five replicates each test concentration) under otherwise identical test conditions. A stock solution of 3,5-dichlorophenol was prepared accord-ing to the OECD Guideline No. 209: 0.5 g of 3,5-dichloro-phenol was dissolved in 500 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The pH was adjusted with Sodium Hydroxide to 7.2 and therefore in the range of 7 to 8.
In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions. A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L N-allylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to about 3.0 g/L on dry weight basis. This level gives a concentration of 1.2 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.7 and therefore no adjustment necessary.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
For the measurement of the respiration rate a sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes.

Test conditions

Test temperature:
20°C ± 2°C during pre-incubation (3 hours) and evaluation period
pH:
7.0 - 8.0
Dissolved oxygen:
4.5 - 8.7
Nominal and measured concentrations:
Nominal 10, 32, 100, 320 and 1000 mg of the test item
1, 4 and 16 mg 3,5-Dichlorophenol/L and
Six inoculum controls
Details on test conditions:
Surrounding Type: Controlled environment room
Temperature: 20°C ± 2°C during pre-incubation (3 hours) and evaluation period
Aeration: Compressed air (1.017 litre per minute)
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
187.8 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
not determinable
Duration:
3 h
Dose descriptor:
EC50
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
not determinable
Duration:
3 h
Dose descriptor:
NOEC
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
not determinable
Details on results:
Inhibition of Respiration Rate (Total Respiration):
Total respiration:
In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited at the two lower test concentrations and only slightly inhibited for the other test concentrations. The inhibition rates were -13.1% at a test concentration of 10 mg/L and -18.8% at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 9.1%, and 10.1%, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition rate of 16.1%. Concentrations exceeding 1000 mg/L nominal were not tested.

3-hour EC10, EC20 and 3-hour EC50 based on Total Respiration:
The 3-hour EC10 and for total respiration were established to be 187.8 mg/L (95% confidence limits (cl): lower cl: 32.3 mg/L, higher cl: 1091.6 mg/L). The 3-hour EC50 The EC50 was established to be > 1000mg/L. The NOEC was determined to be at a test item concentration of 320 mg/L.

Inhibition of Respiration Rate (Heterotrophic Respiration without Nitrification):
In comparison to the inoculum controls the respiration was slightly inhibited for all test concentrations. The inhibition rates were 26.3%, 22.4% and 16.1% at test item concentrations of 10 mg/L, 32 mg/L and 100 mg/L, respectively. At the concentrations of 320 mg/L and 1000 mg/L the inhibition of the respiration rate was 22.0% and 23.9%.

3-hour EC10, EC20 and 3-hour EC50 based on Heterotrophic Respiration without Nitrification:
The 3-hour EC50, EC20 and EC10 for heterotrophic respiration could not be established. The NOEC could not be established.

Inhibition of Respiration Rate based on Nitrification:
In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were not inhibited at the test item concentrations.


3-hour EC10, EC20 and 3-hour EC50 based on Oxygen Uptake due to Nitrification:
For the oxygen uptake due to nitrification the 3-hour EC50 was established to be 1889.5mg/L (lower cl: n.d., higher cl: n.d.); the NOEC was determined to be >= 1000 mg/L.


Results with reference substance (positive control):
Inhibition of Respiration Rate of Total Respiration:
The inhibition of the activated sludge treated with the reference item was in the range of 31% to 84% for the test concentrations from 1 until 16 mg/L.
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Total Respiration:
The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 0.3 0.8 3.8
lower 95%-cl 0.1 0.3 1.2
upper 95%-cl 0.9 2.0 12.2
n.d.: not determined due to mathematical reasons

Inhibition of Respiration Rate of Heterotrophic Respiration without Nitrification:
The inhibition of the activated sludge treated with the reference item was in the range of 1% to 74% for the test concentrations from 1 until 16 mg/L.
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Heterotrophic Respiration:
The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 2.8 4.2 9.0
lower 95%-cl 1.8 2.7 5.3
upper 95%-cl 4.5 6.5 15.2
n.d.: not determined due to mathematical reasons

Inhibition of Respiration Rate of Oxygen Uptake due to Nitrification:
The inhibition of the activated sludge treated with the reference item was in the range of 83% to 103% for the test concentrations from 1 until 16 mg/L.
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Oxygen Uptake due to Nitrification: Based on measured inhibition rates, the 3-hour EC10, EC20 and EC50 and their 95% confidence limits were determined. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 0.001 0.003 0.043
lower 95%-cl 0.0 0.0 0.0
upper 95%-cl 42202.5 62554,3 n.d.
n.d.: not determined due to mathematical reasons
Reported statistics and error estimates:
For normal distribution of the data, the Shapiro-Wilk`s Test was used, the variance homogeneity was tested by the Leven`s Test. Subsequently, the Williams Multiple Sequential t-test Procedure (one-sided smaller) with Levenberg-Marquardt optimization was used to determine the 3-hour ECx and their 95 %-confidence limits and LOEC and NOEC values, if possible.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Any other information on results incl. tables

Influence of the test item and the Reference Item 3,5-Dichlorophenol on Total Oxygen Consumption of Activated Sludge

Flask No. Treatment Concen-tration Oxygen consumption [mean]1 Coefficient of Variation [mean]1 Inhibition [mean]1 pH-values [mean]1 Oxygen concentration [mean][mg O2/L]1
    [mg/L] [mg O2/L h] % % start* end* start* end*
1, 2, 18, 19, 45,46  control  --- 39.0 14.4 --- 7.0 7.7 4.5 7.3
3-7 3.5-DCP  1 26.8 --- 31.4 7.0 8.0 5.8 7.6
8-12 3.5-DCP  4 22.4 --- 42.5 7.0 8.0 4.0 8.0
13-17 3.5-DCP  16 6.1 --- 84.3 7.0 8.0 7.7 8.7
20-24 Test item 10 44.1 --- -13.1 7.0 8.0 4.9 6.9
25-29 Test item 32 46.3 --- -18.8 7.0 8.0 6.2 7.3
30-34 Test item 100 35.4 --- 9.1 7.0 7.0 6.1 6.3
35-39 Test item 320 35.1 --- 10.1 7.0 7.0 6.4 7.0
40-44 Test item 1000 32.7 --- 16.1 7.0 7.0 5.9 6.7
1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:  3,5-Dichlorophenol
Test item:  Sanolin Lave Blue A VP 5453
CV:  standard deviation / mean * 100

Influence of the test item and the Reference Item 3,5-Dichlorophenol on Heterotrophic Oxygen Consumption of Activated Sludge, with addition of ATU

Flask No. Treatment Concen-tration Oxygen consumption [mean]1 Coefficient of Variation [mean]1 Inhibition [mean]1 pH-values [mean]1 Oxygen concentration [mean][mg O2/L]1
    [mg/L] [mg O2/L h] % % start* end* start* end*
47, 48, 64, 65, 91, 92  control  --- 24.6 12.2 --- 7.0 8.0 5.9 8.4
49-53 3.5-DCP  1 24.4 --- 1.0 7.0 8.0 2.2 8.1
54-58 3.5-DCP  4 20.0 --- 18.5 7.0 8.0 4.2 8.4
59-63 3.5-DCP  16 6.5 --- 73.7 7.0 8.0 6.7 9.1
66-70 Test item 10 18.1 --- 26.3 7.0 8.0 5.5 8.6
71-75 Test item 32 19.1 --- 22.4 7.0 8.0 5.6 8.1
76-80 Test item 100 20.6 --- 16.1 7.0 8.0 6.2 8.1
81-85 Test item 320 19.2 --- 22.0 7.0 8.0 5.9 7.9
86-90 Test item 1000 18.7 --- 23.9 7.0 8.0 6.2 8.4
1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:  3,5-Dichlorophenol
Test item:  Sanolin Lave Blue A VP 5453
CV:  standard deviation / mean * 100

Influence of the test item and the Reference Item 3,5-Dichlorophenol on Oxygen Consumption based on Nitrification of Activated Sludge

Treatment Concentration [mg/L] Oxygen consumption [mean]1[mg O2/L h] Coefficient of Variation [mean]1%   Inhibition [mean]1% 
         
control  --- 14.4 5.0 ---
3.5-DCP  1 2.4 --- 83.3
3.5-DCP  4 2.4 --- 83.3
3.5-DCP  16 -0.4 --- 102.5
Test item 10 26.0 --- -80.6
Test item 32 27.3 --- -89.3
Test item 100 14.8 --- -2.8
Test item 320 15.9 --- -10.3
Test item 1000 14.0 --- 2.6
1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:  3,5-Dichlorophenol
Test item:  Sanolin Lave Blue A VP 5453
CV:  standard deviation / mean * 100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item had only slightly effects on the respiration (total respiration and nitrification respiration) of activated sludge microorganisms up to a concentration of 32 mg/L; low effects were observed for heterotrophic respiration up to test item concentrations of 320 mg/L. A test item concentration of 1000 mg/L caused moderate effects on total respiration, heterotrophic respiration and nitrification respiration.
The NOEC was determined to be at 320 mg /L for total respiration, could not be established for heterotrophic respiration and was determined to be ≥ 1000 mg/L for nitrification respiration.
The 3-hour EC50 could not be established for total respiration, could not be established for heterotrophic respiration) and was found to be 1889.5 mg/L (lower cl: n.d., higher cl: n.d., nitrification respiration).
Executive summary:

Title:

Sanolin Lave Blue A VP 5453: Toxicity to Activated Sludge in a Respiration Inhibition Test

 

 

Guidelines/Recommendations:

This study was designed to comply with the following methods:

-     Commission Regulation (EC) No 440/2008, Method C.11: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Union No.L 142/559-563, dated

-     OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted

-     DIN EN ISO 8192: Wasserbeschaffenheit – Bestimmung des Sauerstoffverbrauchs von Belebtschlamm nach Kohlenstoff- und Ammonium-Oxidation (ISO 8192:2007)

 

 

Purpose:

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item Sanolin Lave Blue A VP 5453 on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of several controls. Additionally the oxygen uptake by nitrification was evaluated in presence of an inhibitor, e.g. N-allythiourea (ATU).

The test method of application and the test system are recommended by the test guidelines.

 

 

Test Concentrations:

Nominal 10, 32, 100, 320 and 1000 mg Sanolin Lave Blue A VP 5453/L;
1, 4 and 16 mg 3,5-Dichlorophenol/L and
Six inoculum controls

 

 

Results:

 

Toxicity of Test Item:

Two stock solutions with the test item Sanolin Lave Blue A VP 5453 were prepared in pure water, with the following concentrations: 5g/L and 50g/L. Stock solution one with a concentration of 5g/L was used for the preparation of test concentrations 10, 32 and 100mg test item/L. Stock solution two was used for the preparation of test concentrations 320 and 1000mg/L. Appropriate volumes of stock solution were added to each test flask and pure water was added.

Total respiration:

In comparison to the inoculum controls the total respiration rate of the activated sludge was not inhibited at the two lower test concentrations and only slightly inhibited for the other test concentrations. The inhibition rates were -13.1% at a test concentration of 10 mg/L and -18.8% at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 9.1%, and 10.1%, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition rate of 16.1%. Concentrations exceeding 1000 mg/L nominal were not tested. The 3-hour EC10and for total respiration were established to be 187.8 mg/L (95% confidence limits (cl): lower cl: 32.3 mg/L, higher cl: 1091.6 mg/L). The EC50was established to be > 1000mg/L. The NOEC was determined to be at a test item concentration of 320 mg/L. 

Respiration without nitrification (Heterotrophic Respiration):

In comparison to the inoculum controls the respiration was slightly inhibited for all test concentrations. The inhibition rates were 26.3%, 22.4% and 16.1% at test item concentrations of 10 mg/L, 32 mg/L and 100 mg/L, respectively. At the concentrations of 320 mg/L and 1000 mg/L the inhibition of the respiration rate was 22.0% and 23.9%. The 3-hour EC20and EC50for heterotrophic respiration could not be established. The NOEC could not be determined.

Respiration based on nitrification:

In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were not inhibited at any of the tested concentrations (inhibition below 3%). For the oxygen uptake due to nitrification the 3-hour EC50was established to be 1889.5 mg/L (lower cl: n.d., higher cl: n.d.); the NOEC was determined to be higher than a test item concentration of 1000 mg/L.

Toxicity of 3,5-Dichlorophenol:

The positive control 3,5-Dichlorophenol was tested in the same way as the test item.The 3-hour EC50for total respiration was found to be 3.8 mg/Lthus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The EC20was 0.8 mg/L, the EC10was 0.3 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50was 0.043 mg/L. The 3-hour EC50for heterotrophic respiration was found to be 9.0 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used. The 3-hour EC50of 0.043 for the oxygen uptake due to nitrification indicates that the activated sludge used is even more sensitive to the reference substance than required by the guideline, therefore providing a more conservative endpoint and an even more robust test system.

This study is classidied acceptable and satisfies the guideline requirements for toxicity to microorganisms studies.