## FINAL REPORT

Study Title



## ACTIVATED SLUDGE RES PIRATION INHIBITION TEST

(CARBON AND AMMONIUM OXIDATION)

WITH

Author

Test Facility

Laboratory Project Identification

- Page 1 of 17

-- Page 2 -

1. CONTENTS

1. CONTENTS

...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE STATEMENT

............................................................................................ 4

4. SU MMARY

........................................................................................................................................ 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 EC 50 -value for 3,5-dichlorophenol

................................................................................. 15

F IGURES 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- Page 5 -

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 Co uncil 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 highestloading 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.

Th e 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. Th us , the EC 50 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 EC 50 was above the highest loading rate tested (1000 mg/l ).- Page 6 -

5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor (on behalf of the sponsor)

Test Facility

Study Director

Technical Coordinator

Study Plan Start : 8 December 201 1

C ompletion : 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 EC 50

.

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:

Co uncil 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.- Page 7 - 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 O 2 per litre per hour.

ATU: N-allylthiourea (nitrification inhibitor)

EC 50 : 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- Page 8 -

6.1.2. Study specific test substance information

Hygroscop ic 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

Identifi cation number RS92

Container B1

Identification 3,5

- Dichlorophenol

Structure

Molecular formula Cl 2 C 6 H 3 OH Molecular weight 163.00

CAS Number 591

- 35

- 5 Description Grey crystalline powder (determined at

Batch 1 5402 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.- Page 9 -

6.3. Combined limit/range-finding test

In a co mbined 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 consist ed ofone 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/ref erence
substance Synthetic

medium Made up to

250 ml with

Milli

- RO

water Sludge

Blank

- c ontrol (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

lo ading

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:

CaCl 2 .2H 2 O 211.5 mg/l

MgSO 4 .7H 2 O 88.8 mg/l

NaHCO 3 46.7 mg/l

KCl 4.2 mg/l- Page 10 -

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) andsubsequently 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 CaCl 2 .2H 2 O 0.2 g MgSO 4 .7H 2 O 2.8 g K 2 HPO 4 Dissolved in Milli-Q water, made up to 1 litre and filtered.

Th e 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 suspensio n.

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 wa s 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.- Page 11 -

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): Oxy gen.

6.7. Interpretation

6.7.1. Calculations

Calculation of oxygen uptakes

The respiration rate (R) from each vessel, in mg O 2 /l.h, was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O 2 /l.

Furthermore the respiration rate was also expressed as the amount of oxygen consumed per g dry weight of sludge per hour (R
s in mg O 2 /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:

in which Rc and Rt

are respiration rates of controls and test/reference substance respectively (in mg O 2 /l.h).

Interpretation of results

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

concentrations and the EC 50 was determined using linear regression analysis.

For no EC 50 could be calculated because the test substance proved to be nontoxic (EC 50 > 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 EC 50 value. Therefore, as discussed with the sponsor, no further testing was performed to determine the NOEC.- Page 12 -

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 EC 50 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.- Page 13 -

7. RESULTS

7.1. Study results

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

Th e 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

in hibition.

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)

EC 50 >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) bacter ia

. The EC 50 was above the highest loading rate tested (1000 mg/l ).- Page 14 -

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. a t
the start (~ mg O 2 /l) Respi

r ation

rate (mg O 2 /l h) Respi

r ation

Rate (mg O 2 /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 31 2

C: Blank Control

CN: Nitrification control R: Reference substance, 3,5-dichlorophenol T: Test substance,
CV: Coefficient of variation

A TU 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.- Page 15 -

APPENDIX 2 EC -VALUES

Table 4 EC 50 -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 10 Xreg 10 X95%

- 10 X95%+

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

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- Page 16 -

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).- Page 17 -

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

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