FINAL REPORT

Study Title

ACTIVATED SLUDGE RESPIRATION INHIBITION TEST

(CARBON AND AMMONIUM OXIDATION)

WITH

Author

Test Facility

Laboratory Project Identification

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1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE ............................................................................................. 3

3. QUALITY ASSURANCE STATEMENT ............................................................................................ 4

4. SUMMARY ........................................................................................................................................ 5

5. INTRODUCTION .............................................................................................................................. 6

5.1. Preface ...................................................................................................................................... 6

5.2. Aim of the study ........................................................................................................................ 6

5.3. Guidelines ................................................................................................................................. 6

5.4. Storage and retention of records and materials ........................................................................ 6

5.5. Definitions.................................................................................................................................. 7

6. MATERIALS AND METHODS .......................................................................................................... 7

6.1. Test substance .......................................................................................................................... 7

6.1.1. Test substance information ............................................................................................. 7

6.1.2. Study specific test substance information ...................................................................... 8

6.1.3. Test substance – preparation of loading rates ............................................................... 8

6.2. Reference substance ................................................................................................................ 8

6.2.1. Reference substance ...................................................................................................... 8

6.2.2. Reference substance – preparation of stock and test concentrations ............................ 8

6.3. Combined limit/range-finding test ............................................................................................. 9

6.4. Test system ............................................................................................................................... 9

6.5. Test procedure and conditions ................................................................................................10

6.6. Electronic data capture ...........................................................................................................11

6.7. Interpretation ...........................................................................................................................11

6.7.1. Calculations ..................................................................................................................11

6.7.2. Acceptability of the test .................................................................................................12

6.8. List of deviations .....................................................................................................................12

6.8.1. List of protocol deviations .............................................................................................12

6.8.2. List of standard operating procedures deviations .........................................................12

7. RESULTS .......................................................................................................................................13

7.1. Combined limit/range-finding test ...........................................................................................13

8. CONCLUSION ................................................................................................................................13

TABLES 

Table 1 Experimental set-up........................................................................................................ 9

Table 2 Study results .................................................................................................................14

Table 3 EC50-value for 3,5-dichlorophenol .................................................................................15

FIGURES 

Figure 1 Influence of 3,5-dichlorophenol on the respiration rate of aerobic waste water 

(activated sludge) bacteria. ...........................................................................................15

APPENDICES 

APPENDIX 1 STUDY RESULTS ........................................................................................................14

APPENDIX 2 EC-VALUES ..................................................................................................................15

APPENDIX 3 STATISTICS .................................................................................................................16

Final Report


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4. SUMMARY 

The influence of on the respiration rate of activated sludge was investigated after a 

contact time of 3 hours. 

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In 

addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 

440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007). 

The batch of tested was a and not 

completely soluble in test medium at the loading rates initially prepared. Therefore, the test substance 

– Milli-RO water mixtures were magnetically stirred for a period of approximately 25h. Subsequently, 

synthetic sewage feed, sludge and Milli-RO water were added resulting in the required loading rates. 

Optimal contact between the test substance and test medium was ensured applying continuous 

aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded 

for 10 to 18 minutes. 

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest 

loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blankcontrols and a nitrification control of the sludge were included. Furthermore, an abiotic control and the 

highest concentration with a nitrification inhibitor were tested. 

The combined limit/range-finding test showed 15, 20 and an average of 30% inhibition of the 

respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. Thus, the EC50 was above the 

highest loading rate tested (1000 mg/l). 

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification 

inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total 

inhibition. 

The batch of activated sludge was checked for sensitivity by testing the reference substance 

3,5-dichlorophenol, which showed normal sensitivity. 

The study met the acceptability criteria prescribed by the protocol and was considered valid. 

In conclusion, was slightly toxic to waste water (activated sludge) bacteria. 

The EC50 was above the highest loading rate tested (1000 mg/l).

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5. INTRODUCTION 

5.1. Preface 

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Technical Coordinator

Study Plan Start : 8 December 2011

Completion : 8 December 2011

5.2. Aim of the study 

The purpose of the study was to evaluate the test substance for its ability to adversely affect aerobic 

microbial treatment plants, and if possible, to determine the EC50. 

5.3. Guidelines 

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 2: Effects on 

biotic systems, Guideline no. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and 

Ammonium Oxidation), adopted 22 July 2010. 

In addition, the procedures were designed to meet the test methods prescribed by the following 

guidelines: 

Council Regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of 

ecotoxicity, Publication No. L142, C 11:"Biodegradation: Activated sludge respiration inhibition 

test". 

 ISO Standard 8192, Water Quality - Test for inhibition of oxygen consumption by activated sludge 

for carbonaceous and ammonium oxidation (2007). 

5.4. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the archives 

for a period of at least 2 years after finalization of the report. After this period, the sponsor will be 

contacted to determine how the records and materials should be handled. will retain 

information concerning decisions made. 

Those specimens requiring refrigeration or freezing will be retained by for as long as the 

quality of the specimens permits evaluation but no longer than three months after finalization of the 

report. 

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will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Total respiration: heterotrophic respiration (carbon oxidation) and nitrification (ammonium oxidation) 

Respiration rate: the oxygen consumption of aerobic activated sludge or waste water microorganisms expressed generally as mg O2 per litre per hour. 

ATU: N-allylthiourea (nitrification inhibitor) 

EC50: the concentration of the test substance at which the respiration rate is 50% of the respiration 

rate of the controls under the conditions of the test. 

The No Observed Effect Concentration (NOEC) is the highest tested concentration at which no 

significant effect is observed relative to the controls. 

Activated sludge: the accumulated biological mass produced in the treatment of waste water by the 

growth of bacteria and other micro-organisms in the presence of dissolved oxygen. 

Suspended solids (SS): the solids removed from activated sludge by filtration and dried to a constant 

mass, expressed in grams per litre. 

6. MATERIALS AND METHODS 

6.1. Test substance 

6.1.1. Test substance information 

Identification 

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012 

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6.1.2. Study specific test substance information 

Hygroscopic No

Volatile No

Test substance handling Flush container with nitrogen after handling

Stability in water At least 24 hours

Solubility in water No

6.1.3. Test substance – preparation of loading rates 

The batch of tested was a 

The test substance was not completely soluble in test medium at the loading rates initially prepared. 

Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap 

water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 

2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 

approximately 25 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and Milli-RO 

water up to 500 ml were added resulting in the required loading rates (see also section 6.4). Optimal 

contact between the test substance and test organisms was ensured applying continuous aeration and 

stirring. 

6.2. Reference substance 

6.2.1. Reference substance 

Identification number RS92

Container B1

Identification 3,5-Dichlorophenol

Structure

OH

Cl Cl

Molecular formula Cl2C6H3OH

Molecular weight 163.00

CAS Number 591-35-5

Description Grey crystalline powder (determined at 

Batch 15402 DH

Purity 99.8%

Expiry date 20 March 2012

Storage conditions At room temperature in the dark

Stability under storage conditions Stable

Supplier Sigma-Aldrich Chemie GmbH, Steinheim, Germany 

Article number D70600

Certified Yes

Date of receipt 20 March 2008

6.2.2. Reference substance – preparation of stock and test concentrations 

The batch of activated sludge was checked for sensitivity by testing the reference substance 

3,5-dichlorophenol. 

A 3,5-dichlorophenol solution with a final concentration of 1 g/l was prepared. The pH as used for the 

test was 7.2. Aliquots of 25 ml were stored in a freezer until use. On the day of testing the reference 

substance solution was defrosted at room temperature and diluted to reach the test concentrations. 

Three concentrations were tested: 5.0, 12 and 30 mg/l. 

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6.3. Combined limit/range-finding test 

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest 

loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blankcontrol (6 replicates) was included. Furthermore, an abiotic control and the highest loading rate with a 

nitrification inhibitor were tested. 

Table 1 Experimental set-up

Type of bottle Test/reference 

substance

Synthetic 

medium

Made up to 

250 ml with 

Milli-RO 

water

Sludge

Blank-control (C) no 16 ml yes 250 ml

Nitrification control + ATU

(CN) no 16 ml yes 250 ml

Reference substance 

concentrations (R) yes 16 ml yes 250 ml

loading 

rates (T) yes 16 ml yes 250 ml

+ ATU

Highest loading rate (TN) yes 16 ml yes 250 ml

Abiotic control, 

Highest loading rate (TA) yes 16 ml yes

No sludge, made 

up to 500 ml with 

Milli-RO water

ATU: N-allylthiourea (Merck Schuchardt OHG, Hohenbrunn, Germany) 

6.4. Test system 

Test system Micro-organisms in activated sludge. 

Source Municipal sewage treatment plant: ' 

 receiving 

predominantly domestic sewage. 

Number of micro-organisms Number of micro-organisms was determined as the amount 

of Mixed Liquor Suspended Solids (MLSS) per litre test 

medium. 

Preparation of the sludge The sludge was coarsely sieved, washed and diluted with 

ISO-medium. A small amount of the sludge was weighed 

and dried overnight at ca. 105°C to determine the amount of 

suspended solids (3.0 g/l of sludge, as used for the test). 

The pH was 7.6 on the day of testing. The batch of sludge 

was used one day after collection; therefore 50 ml of 

synthetic sewage feed was added per litre of activated 

sludge at the end of the collection day. The sludge was kept 

aerated at test temperature until use. 

Medium Adjusted ISO medium, formulated using RO-water (tapwater purified by reverse osmosis; GEON 

Waterbehandeling, Berkel-Enschot, The Netherlands) with 

the following composition: 

CaCl2.2H2O 211.5 mg/l 

MgSO4.7H2O 88.8 mg/l 

NaHCO3

 

 46.7 mg/l 

KCl 4.2 mg/l 

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Rationale Recognized by international guidelines as the recommended 

test system. 

6.5. Test procedure and conditions 

Contact time 3 hours, during which aeration and stirring took place. 

Vessels All glass bottles/vessels. 

Milli-RO / Milli-Q water Tap-water purified by reverse osmosis (Milli-RO) and 

subsequently passed over activated carbon and ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, 

Mass., USA). 

Synthetic sewage feed 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

Dissolved in Milli-Q water, made up to 1 litre and filtered. 

The pH was within 7.5 ± 0.5. 

Inhibitor of nitrification A 2.32 g/l solution of N-allylthiourea (ATU, Merck 

Schuchardt OHG, Hohenbrunn, Germany) was prepared. 

2.5 ml of this solution was added to 500 ml final test medium 

(final ATU concentration: 11.6 mg/l).

Air supply Clean, oil-free air. 

Aeration The aeration was adjusted in such a way that the dissolved 

oxygen concentration at the start was above 60-70% 

saturation (60% of air saturation is > 5 mg/l at 20°C) and to 

maintain the sludge flocs in suspension.

Test set up See 6.3

Performance of the test The synthetic sewage feed (16 ml) and an adequate amount 

of the test substance loading were mixed and made up to 

250 ml with Milli-RO water in a 1 litre bottle. The pH was 

determined. Thereafter 250 ml activated sludge was added. 

This was the start of the test. The mixture was then aerated 

during the contact time, using a pipette as an aeration 

device. 

 After the 3-hour contact time, the oxygen consumption was 

recorded for a period of 10 to 18 minutes. During 

measurement, the sample was not aerated but continuously 

stirred on a magnetic stirrer. The pH and temperature was 

determined in the remaining part of the reaction mixture. 

This procedure was repeated for all test/reference substance 

concentrations/loading rates and controls. 

Oxygen recording Determination of oxygen was performed with oxygen probes 

connected to a BlueBox, a multichannel measuring and 

controlling system. 

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6.6. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following 

programme(s): 

REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, 

NJ, USA): Temperature.

BlueBox Software version 3.1.1.1 (GO-Systemelektronik GmbH, Germany): Oxygen.

6.7. Interpretation 

6.7.1. Calculations 

Calculation of oxygen uptakes 

The respiration rate (R) from each vessel, in mg O2/l.h, was calculated or interpolated from the linear 

part of the respiration curve, which was generally between 2 and 7 mg O2/l. 

Furthermore the respiration rate was also expressed as the amount of oxygen consumed per g dry 

weight of sludge per hour (Rs in mg O2/g.h). 

Rs = R / SS 

where SS is the concentration of suspended solids in the test mixture (1.5 g/l in the final text mixture). 

Calculation of the inhibition 

The inhibitory effect (percentage inhibition) at a loading rate was calculated as: 

% 1 x100%

mean Rc

Rt inhibition

in which Rc and Rt are respiration rates of controls and test/reference substance respectively 

(in mg O2/l.h). 

Interpretation of results

ECx 

For the reference substance the percentage inhibition was plotted against the logarithm of the 

concentrations and the EC50 was determined using linear regression analysis. 

For no EC50 could be calculated because the test substance proved to be nontoxic (EC50 > a loading rate of 1000 mg/l). 

NOEC estimation 

In this present test was slightly toxic to waste water (activated sludge) bacteria at 

all loading rates tested. Therefore, a NOEC could not be determined. 

For REACh registration (industrial chemicals) a NOEC is not required, relevant PNEC will be derived 

from EC50 value. 

Therefore, as discussed with the sponsor, no further testing was performed to determine the NOEC. 

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6.7.2. Acceptability of the test 

1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated 

sludge (dry weight of suspended solids) in an hour. 

 The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end 

of the definitive test. 

2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration 

(8.2 mg/l, see also APPENDIX 2).

Since all criteria for acceptability of the test were met, this study was considered to be valid. 

6.8. List of deviations 

6.8.1. List of protocol deviations 

There were no deviations from the protocol. 

6.8.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures were evaluated and filed in the study file. There 

were no deviations from standard operating procedures that affected the integrity of the study. 

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7. RESULTS 

7.1. Study results 

Detailed study results are presented in Table 3 of APPENDIX 1.

The combined limit/range-finding test showed 15, 20 and an average of 30% inhibition of the 

respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. 

The inhibition observed at a loading rate of 1000 mg/l was statistically significant (see APPENDIX 3).

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification 

inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total 

inhibition. 

7.2. Determination of effect parameters 

Table 2 shows the effect parameters based on loading rates. 

Table 2 Effect parameters for the inhibition of the respiration rate 

Parameter

Loading rate

(mg/l)

EC50 >1000

7.3. Experimental conditions 

The temperatures measured in the various vessels ranged between 19.3 and 20.8°C and were within 

the ranges prescribed by the protocol. The pH in all test series, before addition of sludge remained 

within the range of 7.5 ± 0.5. After the 3 hour exposure period the pH increased to a maximum of 8.5. 

8. CONCLUSION 

Under the conditions of this present test was slightly toxic to waste water 

(activated sludge) bacteria. 

The EC50 was above the highest loading rate tested (1000 mg/l).

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APPENDIX 1 STUDY RESULTS 

Table 3 Study results 

Flask

Conc. /

loading

Rate

(mg/l)

Temp.

(°C)

pH 

before 

addition 

of 

sludge

pH after 

3 h 

contact 

time

Oxygen 

conc. at 

the start 

(~ mg O2/l)

Respiration

rate

(mg O2/l h)

Respiration

Rate

(mg O2/g h)1

% 

Inhibition 

respiration 

relative to 

the control 

(mean 

value)

C 1 0 20.8 7.8 8.1 8.7 43 29

C 2 0 19.3 7.9 8.3 8.1 40 27

C 3 0 20.2 7.8 8.2 8.2 45 30

C 4 0 19.9 7.8 8.2 8.5 45 30

C 5 0 20.1 7.8 8.2 7.9 42 28

C 6 0 20.7 7.8 8.2 9.0 46 31

Mean 44 29

SD 2 2

CV

(%) 5 5

CN 0 20.7 7.8 8.5 8.8 26 17 40

R 1 5.0 20.1 7.8 8.4 8.8 26 17 40

R 2 12 20.7 7.8 8.3 8.7 19 13 56

R 3 30 20.6 7.8 8.4 8.9 8 5 82

T1 10 19.5 7.7 8.2 8.0 37 25 15

T2 100 19.9 7.7 8.1 8.1 35 23 20

T3a 1000 20.5 7.7 8.1 7.8 32 21 26

T3b 1000 19.8 7.7 8.1 7.8 29 19 33

T3c 1000 20.0 7.7 8.1 8.0 31 21 29

T3

mean

31 20 30

TA 1000 20.8 7.7 7.6 8.9 0 0 100

TN 1000 20.7 7.7 8.5 8.0 18 12 312

C: Blank Control 

CN: Nitrification control 

R: Reference substance, 3,5-dichlorophenol 

T: Test substance, 

CV: Coefficient of variation 

ATU N-allylthiourea 

1 The amount of suspended solids in the final test mixture was 1.5 g/l. 

2

 Relative to CN 

TA: Abiotic control of test substance. 

TN: Test substance with ATU. 

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APPENDIX 2 EC-VALUES 

Table 4 EC50-value for 3,5-dichlorophenol 

Parameter: Inhibition of respiration

Conc. X Y

(mg/l) Log conc. (mg/l) Inhibition (%)

5.0 0.699 40

12 1.079 56

30 1.477 82

Regression parameters

Slope: 54.0623

Intercept: 0.6708

Multiple R: 0.9924

n = number of observations: 3

Prediction of X values based on known Y values

Known Y 10Xreg 10X95%-10X95%+

Inhibition (%) Conc. (mg/l) Conc. (mg/l) Conc. (mg/l)

50 8.2 0.7 89.2

Regression line: Y= 54.06 X + 0.67

0

20

40

60

80

100

1 10 100

Inhibition (%)

Concentration (mg/l)

Figure 1 Influence of 3,5-dichlorophenol on the respiration rate of aerobic waste water 

(activated sludge) bacteria. 

 Dashed curves represent the 95 % confidence limits 

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APPENDIX 3 STATISTICS 

 Chi-Square Test for Normality

 ---------------------------------------------------------------------------- 

 Actual and Expected Frequencies 

 INTERVAL <-1.5 -1.5 to <-0.5 -0.5 to 0.5 >0.5 to 1.5 >1.5 

 ----- ------------- ----------- ----------- ----- 

 EXPECTED 0.6030 2.1780 3.4380 2.1780 0.6030 

 OBSERVED 1 2 2 4 0 

 ---------------------------------------------------------------------------- 

 Chi-Square = 3.0046 (p-value = 0.5571) 

 Critical Chi-Square = 13.277 (alpha = 0.01 , df = 4) 

 = 9.488 (alpha = 0.05 , df = 4) 

 ---------------------------------------------------------------------------- 

 Data PASS normality test (alpha = 0.01). Continue analysis. 

 F-Test for Equality of Two Variances 

 ---------------------------------------------------------------------------- 

 GROUP IDENTIFICATION VARIANCE F 

 ----- -------------- ------------ --------- 

 1 Controls 5.1000 

 2 1000 mg/l 2.3333 2.1857 

 ---------------------------------------------------------------------------- 

 (p-value = 0.6861) 

 Critical F = 199.2996 (P=0.01, 5, 2) 

 39.2982 (P=0.05, 5, 2) 

 Since F <= Critical F, FAIL TO REJECT Ho: Equal Variances (alpha = 0.01). 

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APPENDIX 3, continued FINAL TEST – STATISTICS 

 ANOVA Table

 ---------------------------------------------------------------------------- 

 SOURCE DF SS MS F 

 ---------------------------------------------------------------------------- 

 Between 1 329.3889 329.3889 76.4328 

 Within (Error) 7 30.1667 4.3095 

 ---------------------------------------------------------------------------- 

 Total 8 359.5556 

 ---------------------------------------------------------------------------- 

 (p-value = 0.0001) 

 Critical F = 12.2464 (alpha = 0.01, df = 1,7) 

 = 5.5914 (alpha = 0.05, df = 1,7) 

 Since F > Critical F REJECT Ho: All equal (alpha = 0.05) 

 2 Sample t-Test - TABLE 1 OF 2 Ho: Control=Treatment 

 ---------------------------------------------------------------------------- 

 TRANSFORMED MEAN CALCULATED IN SIG 

 GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

 ----- -------------------- ----------- ------------------ ------ --- 

 1 Controls 43.5000 43.5000 

 2 1000 mg/l 30.6667 30.6667 8.7426 * 

 ---------------------------------------------------------------------------- 

 Equal Var: t critical value = 2.3646 (2 Tailed, alpha = 0.05, df = 7) 

 (p-value = 0.0000) 

 

 2 Sample t-Test - TABLE 2 OF 2 Ho: Control=Treatment 

 ---------------------------------------------------------------------------- 

 Equal Variances: 

 ---------------------------------------------------------------------------- 

 NUM OF MIN SIG DIFF % OF DIFFERENCE 

 GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL 

 ----- -------------------- ------- ---------------- ------- ------------ 

 1 Controls 6 

 2 1000 mg/l 3 3.4711 8.0 12.8333 

 ---------------------------------------------------------------------------- 

Final Report