Study initiation 

Data Requirements I Test Guidelines ~· ·---

Author: 

Study completion 

End of Experimental 

Sponsor: 

Testing facility: 

Laboratory Project Identification 

Study No. 

Page 1 of 22 
Page 2 of 22 
ARCHIVATION OF DOCUMENTS AND TEST ARTICLES 

QUALITY ASSURANCE STATEMENT 

GLP CERTIFICATE 

SUMMARY 

INTRODUCTION 

EXPERIMENTAL PROCEDURE 

Test item 

Test organisms 

Pre-treatment of the inoculum 

Pre-treatment of the test Item 

Pre-treatment of the reference compound 

Pre-treatment of the toxicity control 

Exposure conditions 

Chemical analysis 

Applied SOPs and methods 

RESULTS 

Comments 

DEGRADATION CURVES 

Page 3 of 22 

4 

6 

7 

8 

9 

10 

11 

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12 

14 

14 

14 

14 

15 

15 

16 

21 

22 
Date I Signature 

Study Director 

ARCHIVATION OF DOCUMENTS AND TEST ARTICLES 

Study plans, raw data and study reports are archived at the GLP archives of the test 

facility. 

Test articles are archived 

The sponsor must ensure that information submitted about the test item (identity testing 

or information about physical and chemical properties) is complete and that it complies 

with the principles of Good Laboratory Practice. 

The customer of the test item used where sampling has not 

been performed 

Page 4 of 22 

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Page 5 of 22 
The dates of Quality Assurance inspections and audits are given below. During the on- site inspections procedures applicable to this type of short time study were inspected. 

Audits 

study plan review 

process based inspection 

final report review 

Date: 

Dates ofQA 

inspection 

Dates of 

report 

·- .~ .---- -· 

Quality Assurance Unit: 

Page 6 of 22 
Please note: 

GLP-Bnchelnlgu.ng/St•t•ment of GLP Compllanc• 

(gemla/accordlng to 11tb Ab». 1 Chemlkallenguetz} 

Elne GlP~ zur 0beTWachUng det EJr\halll.mg cMr AUtUmtlll or C'Oflfonntly Mth GUO IIC'COI'dl11-' 10 GLP·Grundfltuo gernU Chemikallengnetz hrw. RlcNinie r:Mmlhlkn~ :.d Olmttw IU3201CEC 1t: 8&1320iEGwurde durchg.tiltwt In: 

Pr!ifungen nach Katogorien Areas of EJq>ertise (;end~. OlemYW·GLP Nt. 5.3o«CO gt,ifanct) ltce«dkki ChemllwV.Q,. ,..., 5.31'0f.CO ~) Kategorie 1 category 1 

Prfifungen zur Sestimmung der ph~lltalltch- physlcal-dlemlcal tMii'lg ehetnischen Eigensdlaft•l und Geheltsbesllmnungen 

Kategono 4 eategory -4 

Okoloxikologlsche Prllfungen zur Bestlmmung der environmental toxicity tluclles on aqooUc ond Auswlr1c:unoe" auf aqualische und letrestriseh.e t.,..trlal ot;anlsma 

Organ! ~men ""'-5 catogo<y 5 PrQfungen zum Verhalten 1m Boden, IM Wasser und In studies on behavtour In water, sol and air; dar Luh: PrOfungen n.~r Bloakkumuletion und zur bloacc;umulatlon Melabolisrerung 

KArl_., 8 colegoryl 

Analytrsthe PrUfungen an bio{ogischen Uaterlalien analytlcal and dlnlcal chemlstty tesllng 

Datum der lnspektion Date of Inspection 

~ 

. . . 

. 

-·---~· 

Die geoannte Pt\'tfek'lrtchtung beftndM Sleh 1m natr GLP-Oberwlld\unoSwrlahren und -Mtd IIIQIIIImiB,iy •uf GLP Complianl.:a PfOUtllnlftMI .,u;S Is lnspect.d on I ,.g~ar Einhal'tung derGLP·GtuMsllze Oberwecht. bests. 

Auf der GtuncLJge des ~'fidl~ Md hivm¥\. B;,.w.ll.KttiwiMp«::onNpartlteanbetonflrrned,thattia be.stlllgt. da» In dleser PrOfelnrk::htunu die Dben tettfaciJtyllebleto~lhe•foremenlionedstuc:lesin 

genann.ten PrQfungen unter Einr'oltung· der GLP· toft'C)IIanoo wtlb tho Prtndpktaot GLP. Grundsltl' du~.rotvt Mrden kOnnen. 

Page 7 of 22 
Inherent 

operating 

Regulation (EC) No 

in a mineral medium was inoculated with 40 % 

sludge of cipal sewage treatment plants (STP) and 20 % 

sludge of an industrial STP and incubated under aerobic conditions. During this period, 

degradation was followed by continuous automated BOD determinations. 

Therefore, 

86 % degradation after 7 days 

1 00 % degradation after 14 days 

100% degradation after 21 days 

1 00% degradation after 27* days 

considered to be inherently biodegradable. 

The reference compound sodium benzoate showed 68 % degradation after 7 days and 

67% degradation after 14 days. 

* By mistake, programming of the automated BOD-meter (OxiTop bottles) ended one 

day earlier than foreseen in the study plan (i.e. on day 27). The test duration is within 

the limits given in the guideline (14-28d) and, as 100% biodegradation were observed, 

has no influence on the overall study result. There is indication of biodegradation, and 

clearly above the pass level for the test item of 70 %. 

Page 8 of 22 
y 

edified MITI 

~ (based on standard 

CD Guideline _.-ancf the Council Regulation (EC) No 

~method ._"Manometric Respirometry Test"-· with fresh inocula 

~nt sources. 

A measured volume of inoculated mineral medium, containing a known concentration of 

26.2 mg active ingredient (a.i.)/L of the test item was stirred in a closed flask at a 

constant temperature (25 ± 2 oc) for up to 28 days. The consumption of oxygen (BOD) 

was determined by measuring the drop in pressure in the automated OxiTop respirometer flask. Evolved carbon dioxide was absorbed in sodium hydroxide. The 

amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run 

in parallel) was expressed as a percentage of chemical oxygen demand (COD). 

Page 9 of 22 
p

(

) 

p

a

e

 to check the operation of the procedures. 

A toxicity control (test item and reference compound mixed) was run in parallel, to 

ensure that the chosen concentration of the test item was not inhibitory to microorganisms. 

Degradation was followed by the determination of oxygen uptake and measurements 

were taken at frequent intervals to allow the identification of the beginning and end of biodegradation. 

The test lasted 28 days, but programming of the automated BOD-meter (OxiTop bottles) ended one day earlier than foreseen in the study plan (i.e. on day 27). 

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate, for the reference compound in duplicate. 

The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH of the contents of the flasks was measured. 

A correction of the oxygen consumed by nitrification was not necessary, because of the determination of the chemical oxygen demand of the test item. 

Start of the incubation period : 

End of the incubation period : 

Page 10 of 22 
Drying residue 

Content of active 

ingredient 

Partie 

Charge 

Expiry date 

Appearance 

Storage 

Data of active ingredient 

Chemical name 

CAS number 

Empirical formula 

Structural formula 

Molecular weight 

Stability of test concentrations during exposure : 

35.05% 

31.94% 

liquid 

room temperature 

376.3 g/mol 

Page 11 of 22 
Origin 

Date of collections 

Concentration of 

inoculum (dry weight) 

Pre-treatment of the inoculum 

2 x 40% activated sludge of an aeration tank of two 

different wastewater treating predominant~ 

domestic water authority ... 

sewage 

treatment plant treating predominantly sewage of 

industrial origi . 

---....,_. -- ~ ]._ . 

100 mg/L suspended solids (ss) for all treatments 

with test item and toxicity control and 30 mg/L (ss) in 

the reference compound, respectively 

-

t~e three sludges were ~ixed proportionally ¥Is described before ¥ 

. 

- the sludge was washed twice by adding mineral medium and centrifuging for 1 0 min at 2000 rpm and 20 oc and decanting off the supernatant 

- then the sludge was first dissolved in mineral medium and afterwards stored 

at room temperature under continuous shaking with aeration until use for 

two ~ays 

.; :~ \f• 

' 1.076 g of the wet sludge were d~ied in order W calculate; the amount of wet 

sludge to achieve a concentration of activated sludge of·5 g/1:. (ss) 

Page 12 of 22 
1. Mineral salt solution 

2. Magnesium sulphate 

solution 

3. Calcium chloride 

solution 

4. Iron (Ill) chloride 

solution 

Potassium phosphate, monobasic (KH2P04) 8.5 g 

di-Potassium hydrogen phosphate (K2HP04) 21.75 g 

di-Sodium hydrogen phosphate dodecahydrate 

(Na2HP04 · 12 H20) 44.6 g 

Ammonium chloride (NH4CI) 1.7 g 

The components were dissolved in deionised water and 

filled up to 1 litre. 

The pH of the solution was adjusted to 7 .4. 

Magnesium sulphate heptahydrate 

(MgS04 · 7 H20) 22.5g 

The substance was dissolved in deionised water and filled 

up to 1 litre. 

Calcium chloride dihydrate 

(CaCI2 · 2 H20) 36.4 g 

The substance was dissolved in deionised water and filled 

up to 1 litre. 

Iron (Ill) chloride hexahydrate 

(FeCia · 6 H20) 0.25g 

The substance was dissolved in deionised water and filled 

up to 1 litre. 

To prepare the mineral medium 3 ml each of the mineral salt solution, the magnesium 

sulphate-, calcium chloride- and the iron (Ill) chloride solution are mixed and made up to 

1000 ml with deionised water. 

Page 13 of 22 
Afterwards the flask content was made up to 250 ml with mineral medium 

containing inoculum to give a test concentration of 26.2 mg test item (a.i.)/L. 

Pre-treatment of the reference compound 

Reference compound name : sodium h""''"7"'"'td 

Purity : 100.1% 

Batch-no. : -

25 mg of the reference compound were weighed out on aluminium foil. Both were 

added into the test flasks, which had already been filled with 200 ml of mineral 

medium. Afterwards the flasks were made up to 250 ml with mineral medium 

containing inoculum to give a test concentration of 1 00 mg reference compound/L. 

Pre-treatment of the toxicity control 

20.5 mg of the test item and 7.5 mg of the reference compound were weighed 

out on piece of plastic I aluminium foil, respectively. Both were added to the test 

flasks, which had already been filled with 200 ml of mineral medium. Afterwards 

the flask content was made up to 250 ml with mineral medium containing inoculum to give a test concentration of 26.2 mg/L test item and 30 mg/L reference compound. 

Exposure conditions 

Test volume 

Test apparatus 

Mixing 

Incubation time 

Incubation temperature 

250ml 

OxiTop System (WTW) 

1 magnetic stirrer per test vessel 

28 days 

25 ± 2 oc 

Page 14 of 22 
COD 

Method 

Determination of Chemical Oxygen Demand of 

solids and substances which are not watersoluble (COD). 

Calibration Standard 

Test apparatus 

Potassium hydrogen phthalate 

Titrator I Methrohm 7301759 

Applied SOPs and methods 

SOP I Method 

00090 Version 1 

2011-0619601-070 

Deviations 

Function 

Manom. Respirometry Test 

Determination of Chemical Oxygen 

Demand of solids and substances 

In deviation to the SOP 00090 Version 1, in all treatments 

with test item the test was conducted with 100 mg/L (dry 

weight) sludge inoculum of three origins (see test organisms) 

and 26.2 mgll test item. The mineral medium used is that of 

the "Modified MITI Test (II)" (OECD 302C) and is described 

in the report. All resulting deviations are documented in the 

raw data and in the report. 

Page 15 of 22 
flask no. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

pH-value 

7.2 

7.3 

7.3 

8.3 

8.5 

6.4 

6.5 

6.4 

6.7 

Page 16 of 22 
mean compound control 

flask no. 6 7 8 1 2 3 4 5 9 

code* 

a1 a2 a3 b1 b2 b3 bm r1 r2 t1 time (d) 

0 0 0 0 0 0 0 0.0 0 0 0 

1 -5.5 9.7 -8.3 -2.8 -1.4 -5.5 0.0 26.3 19.4 -4.1 

2 -1.4 19.4 -5.5 2.8 4.1 1.4 2.8 44.2 45.6 -1.4 

3 5.5 35.9 -1.4 6.9 6.9 5.5 6.4 101 105 4.1 

4 22.1 48.4 6.9 9.7 12.4 9.7 10.6 115 119 15.2 

5 27.7 58.1 18.0 15.2 15.2 15.2 15.2 119 126 20.7 

6 34.6 66.4 24.9 18.0 18.0 18.0 18.0 126 131 26.3 

7 37.3 73.3 29.0 22.1 20.7 20.7 21.2 135 135 30.4 

8 42.9 78.8 34.6 24.9 23.5 23.5 24.0 136 140 34.6 

9 48.4 85.7 38.7 30.4 27.7 27.7 28.6 138 144 38.7 

10 56.7 89.9 42.9 31.8 29.0 29.0 29.9 138 147 44.2 

11 62.2 92.6 45.6 33.2 30.4 30.4 31.3 139 148 49.8 

12 65.0 96.8 49.8 37.3 33.2 34.6 35.0 140 151 55.3 

13 69.1 99.5 55.3 40.1 34.6 35.9 36.9 146 152 56.7 

14 71.9 101 59.5 38.7 35.9 35.9 36.8 145 153 58.1 

15 71 .9 102 63.6 41.5 35.9 37.3 38.2 146 153 59.5 

16 74.7 104 66.4 42.9 37.3 40.1 40.1 147 156 62.2 

17 70.5 101 65.0 40.1 33.2 35.9 36.4 150 152 59.5 

18 74.7 106 67.7 45.6 37.3 41.5 41.5 148 156 62.2 

19 78.8 108 69.1 45.6 40.1 42.9 42.9 149 158 65.0 

20 76.0 105 69.1 45.6 37.3 41.5 41.5 148 156 65.0 

21 77.4 106 70.5 48.4 38.7 44.2 43.8 151 158 66.4 

22 78.8 106 70.5 49.8 38.7 45.6 44.7 151 158 66.4 

23 81.6 108 73.3 49.8 40.1 45.6 45.2 152 160 66.4 

24 83.0 109 73.3 51.2 41.5 47.0 46.6 153 160 69.1 

25 83.0 111 73.3 52.5 41.5 48.4 47.5 151 159 67.7 

26 85.7 111 74.7 53.9 42.9 49.8 48.9 152 160 69.1 

27 88.5 111 74.7 53.9 42.9 49.8 48.9 152 160 69.1 

Comments: *Abbreviations according to test guideline. 

Page 17 of 22 
yg

(analytically determined) 

corrected BOD 

test item (a-bm) 

mg 02/L) time (d) a1 a2 a3 

0 0.0 0.0 0.0 

1 0.0 9.7 0.0 

2 0.0 16.6 0.0 

3 0.0 29.5 0.0 

4 11.5 37.8 0.0 

5 12.5 42.9 2.8 

6 16.6 48.4 6.9 

7 16.1 52.1 7.8 

8 18.9 54.8 10.6 

9 19.8 57.1 10.1 

10 26.8 60.0 13.0 

11 30.9 61.3 14.3 

12 30.0 61.8 14.8 

13 32.2 62.6 18.4 

14 35.1 64.2 22.7 

15 33.7 63.8 25.4 

16 34.6 63.9 26.3 

17 34.1 64.6 28.6 

18 33.2 64.5 26.2 

19 35.9 65.1 26.2 

20 34.5 63.5 27.6 

21 33.6 62.2 26.7 

22 34.1 61.3 25.8 

23 36.4 62.8 28.1 

24 36.4 62.4 26.7 

25 35.5 63.5 25.8 

26 36.8 62.1 25.8 

27 39.6 68.7 32.4 

Comments: none 

g 02/mg 

% degradation 

a1 a2 a3 

0 0 0 

0 71 0 

0 100 0 

0 100 0 

84 100 0 

91 100 20 

100 100 50 

100 100 57 

100 100 77 

100 100 74 

100 100 94 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

100 100 100 

Page 18 of 22 

% 

degradation 

mean 

0 

24 

33 

33 

61 

71 

84 

86 

93 

91 

98 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 
e

ght 144.1 g/mol 

Theoretical oxygen demand 1.665 mg 0/mg 

corrected BOD 

reference 

compound % degradation % degradation 

(r-bm) mean 

(mg 02/L) time (d) r1 r2 r1 r2 

0 0.0 0.0 0 0 0 

1 26.3 19.4 16 12 14 

2 41.4 42.8 25 26 25 

3 94.6 98.6 57 59 58 

4 104.4 108.4 63 65 64 

5 103.8 110.8 62 67 64 

6 108.0 113.0 65 68 66 

7 113.8 113.8 68 68 68 

8 112.0 116.0 67 70 68 

9 109.4 115.4 66 69 68 

10 108.1 117.1 65 70 68 

11 107.7 116.7 65 70 67 

12 105.0 116.0 63 70 66 

13 109.1 115.1 66 69 67 

14 108.2 116.2 65 70 67 

15 107.8 114.8 65 69 67 

16 106.9 115.9 64 70 67 

17 113.6 115.6 68 69 69 

18 106.5 114.5 64 69 66 

19 106.1 115.1 64 69 66 

20 106.5 114.5 64 69 66 

21 107.2 114.2 64 69 67 

22 106.3 113.3 64 68 66 

23 106.8 114.8 64 69 67 

24 106.4 113.4 64 68 66 

25 103.5 111 .5 62 67 65 

26 103.1 111.1 62 67 64 

27 103.1 111.1 62 67 64 

Comments: none 

Page 19 of22 
corrected BOD 

toxicity control (t-bm) % degradation 

(mg Oz/L) 

time (d) t1 t1 

0 0.0 

1 0.0 

2 0.0 

3 0.0 

4 4.6 

5 5.5 

6 8.3 

7 9.2 

8 10.6 

9 10.1 

10 14.3 

11 18.5 

12 20.3 

13 19.8 

14 21.3 

15 21.3 

16 22.1 

17 23.1 

18 20.7 

19 22.1 

20 23.5 

21 22.6 

22 21.7 

23 21.2 

24 22.5 

25 20.2 

26 20.2 

27 20.2 

0 

0 

0 

0 

7 

9 

13 

15 

17 

16 

22 

29 

32 

31 

33 

33 

35 

36 

33 

35 

37 

36 

34 

33 

35 

32 

32 

32 

Comments: The used concentrations of the test item did not show toxic effects to 

bacteria. 

Page 20 of 22 
s considered to be "Inherently 

The degradation curves are given in figure 1. 

Comments 

The validity criteria of the test method were met: 

The percent degradation of the reference compound sodium benzoate reached the level 

of<!: 40% after 7 days and<!: 65% after 14 days. 

No toxicity of the test item was observed in the toxicity control. 

* By mistake, programming of the automated BOD-meter (OxiTop bottles) ended one 

day earlier than foreseen in the study plan (i.e. on day 27). The test duration is within 

the limits given in the guideline (14-28d) and, as 100% biodegradation were observed, 

has no influence on the overall study result. There is indication of biodegradation, and 

clearly above the pass level for the test item of 70 %. 

Page 21 of 22 
:fl. 

II 

:s 

c" 

0 ~ "' ., Q 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Tlme,[t]"d 

Fig. 1 Degradation curves 

compound sodium n.on..,.,,,..,,1to 

toxicity control and reference 

Page 22 of22