Acute Toxicity (Dania rerio) DATA REQUIREMENT OECD 203 92/69/EEC, AnnexV, Part C1

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The flagging criteria of40 CFR 158.34 are not applicable forthis study type

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### SIGNATURE PAGE

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### Page 5of47CONTRIBUTORS TO THE STUDY 

### I SUPERVISORY LABORATORY PERSONNEL



# -

-

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# - - .... 

## -- - - T

...... .. .. "' "<"
... '"'
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### results to the Study Director and to Management. The final report reflects the raw data.

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### Page 7of47This document is the property ofthe sponsors (specified on page 1}, use by other parties requires written permission by the sponsors Page 8of47CONTENTS

FLAGGING CRITERIA SIGNATURE PAGE CONTRIBUTORS TO THE STUDY 

### I SUPERVISORY LABORATORY PERSONNEL STATEMENT OF THE QUALITYASSURANCE UNIT
CONTENTS LIST OF DEFINITIONSAND ABBREVIATIONS 

### 1. SUMMARY 

### 2. INTRODUCTION 

### 2.1. OBJECTIVES 

### 2.2. SELECTION OF 

### 2.3. TEST GUIDELINES 

### 2.4. TIME SCHEDULE 

### 2.5. RETENTION OF RECORDS 

### 2.6. ANIMAL WELFARE 

### 3. MATERIALSAND METHODS 

### 3.1. TEST SUBSTANCE 

### 3.2. TESTANIMALS 

### 3.3. HOUSING AND DIET 

### 3.3.1. Acclimatization 

### 3.3.2. Test water 

### 3.4. TEST GROUPS AND CONCENTRATIONS

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### 3.5. TEST SUBSTANCE PREPARATIONS 3.6. ANALYSES 3.6.1. 3.6.2. 3.6.3. 3.7. 3.7.1. Test conditions 

### 3.7.2. Insertion oftest organisms 

### 3.8. OBSERVATIONS AND MEASUREMENTS 3.8.1. Statistics 

### 3.8.2. Validity criteria 

### 4. RESULTS 4.1. ANALYTICAL CONCENTRATION CONTROL 4.2. MORTALITYAND TOXIC SIGNS 4.3. WATER PARAMETER 4.3.1. Test temperature 

### 4.3.2. pH and dissolved oxygen 

### 4.4. DISSOLUTION BEHAVIOUR 5. CONCLUSION APPENDIX Statistical evaluation Analytical Report (Concentration Control Analysis)

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LCo LCso

NOEC
Water-accommodated fraction (WAF): Loading rate:
n.d. dissolved in dilution waterthat is calculated to result in x % lethality ofthetest batch offish within a stated exposure period. maximum concentration causing no lethality within the period ofthe test; median lethal concentration; minimum concentration causing 100% lethality within the period of the test; No Observed Effect Concentration meansthe highest concentration ofthe test substance that does not have an adverse effect on the test organisms. An aqueousfraction containing the dissolved and/or suspended and/or emulsified fraction ofa multi-component substance or a mixture. The masstovolume ratio oftest substanceto test medium (in mg/L) used to prepare the WAF test solution. ELxvalues are based on loading rate and calculated using the same statistical methods as for ECx. not

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### 1. SUMMARY Study

Location of raw data and final report: Test substance: Subject: Guidelines: Test concentrations: Time schedule:

Duration and system of exposure: Test species: Animal supplier: Fish weight: Fish length: Age: Physical conditions: Acute Toxicity Study with the OECD Guidelinefor Testing of Chemicals, No. 203 EEC Directive 92/69/EEC, Appendix V, Part C.1 0, 0.5, 1.0, 2.2, 5.0, 10 mg/L based on test substance mass

*In a first experimentthe analytically determined concentrations were not in the required range. In a second experiment the samples taken at start ofexposurewere not measured by the analytical laboratory. 

### 96 hours, static-renewal, 24 hour intervals

3.4 em (3.3- 3.5 em) Approx. 6 months Healthy, no visible abnormalities

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### Page 12 of47Test Substance Preparation: The test solutions were prepared separately for each test vessel and each interval. Before the test substance was weighed itwas melted, then homogenized by stirring the

Concentration Control Analysis: concentration were prepared individually as water accommodated fractions (according to guidance in OECD (2000) No.23. For each test concentration the melted test substance was weighed on a plastic weighing boat, which was inserted into a glassflask and made up with 10 Ltest water. The test solutions were stirred for about one day. Any undissolved test substance was not removed. To avoid potential losses ofcomponents by volatilization, the study was conducted in closed testvessels. Undissolved test substance was visible on the weighing boat in all concentrations. The overall mean concentrations deviated more than 20% from the nominal because only a fraction ofthe total components ofthe test substance are soluble in water. However, the overall mean measured concentrations ofthe test substance were within 85 - 100 % ofthe mean initial measured concentrations, indicating that most ofthe soluble compounds remained in the solution overthe exposure time. The concentration ofthe test substancewas satisfactorily

Since the test fish were exposed to the entire mass ofthe test substance for the duration ofthe test, the results should be evaluated based on the loading rate.

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### LCo LCso (95% r.niillclFm.'A NOEC > 

### 10 

### 5 10 10 10 2.1 2.1 2.1 The analytically determined concentrations of the test substance in the test solutions were within ±20% ofthe initial concentrations throughout the test indicating that concentrations had been satisfactorily maintained. However, thetest substance is a complex mixture and the analytically detected compounds only represent a fraction ofthetotal substance. Sincethe test fish were exposed to the entire mass ofthe test substance for the duration ofthe test, the results should be evaluated based on the loading rate.

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### 2. INTRODUCTION 2.1. 0

# . Theob substa

- • -•- .,- - -. •• - - -n,. - •• •- .- •• 

### 2.2. SELECTION OF CONCENTRATIONS The test concentrations of 0 lr.c,ntr·nl\ 

### basis of range finding tests

# ~ range finding test: LC50 

### between 1 and 10 mg/L. The raw data of the range finding test will be archived togetherwith the raw data ofthis study. See also Section 3.4. 2.3. TEST GUIDELINES The study was conducted according to the following test guidelines: OECD Guideline forTesting of Chemicals No. 203, July 1992 ("Fish Acute Toxicity Test") EEC directive 92/69, AnnexV, Part C1: "Acute Toxicity in Fish" This study also incorporates guidance in: OECD Series on Testing and Assessment No. 23 (2000) Guidance Document on Aquatic Toxicity Testing of Difficult The study was carried out in Procedures (SOPs) ofthe laboratories involved.

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### 2.4. TIME SCHEDULE Test d 

# . .

# ..

The fish were exposed to the test substancefor approxemately 96 hours. 

### 2.5. RETENTION OF RECORDS

2.6. ANIMAL WELFARE

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### 3. MATERIALSAND METHODS 3.1. The Name oftest substance: Test substance No.: Batch-Identification: CAS No.: Compostion:

Homogeneity: Storage stability: 

# -

-

Not homogeneous at visual inspection (for homogenization the test information from the The stability under storage conditions overthe exposure period was guaranteed by the manufacturer, and the manufacturer holds this responsibility. ADDITIONAL TEST SUBSTANCE INFORMATION Date of production: Physical state/ Appearance: Watersolubility: Storage conditions: 03 Apr2007 Liquid 

### I dark brown Not applicable (mixture of multiple components) The test substance was stored at ambient temperature

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### 3.2. TEST ANIMALS Test species: Zebrafish (Dania rerio)
Body weight (wet weight)*: Body length (from the top ofthe snoutto the end ofthe caudal fin)*: 0.44 g (0.39 - 0.48 g) 3.4 em (3.3 - 3.5 em) Age ofthe animals: Hatching date: Arrival atthetesting facility: Approx. 6 months 17 Feb 2008 28 May 2008 *Measurements were performed with a representative sample ofthe fish batch. All animals used forthe study were derived from the same batch offish. The test fish were acclimatized to testing conditions prior to test initiation. During acclimatization, the general health status 

### ofthe fish was observed for signs ofsickness, injury, orother abnormalities. 

### 3.3. HOUSING AND DIET 3.3.1. Acclimatization Duration ofacclimatization to t""tinn ;::14 daysto testwater, nh,olctne water and light regime: Photoperiod: Waterquality: Total hardness: Acid capacity: Oxygen content: pH-value: Temperature: Diet: Withdrawal offeed: Medical treatment: Mortality during the last week before start ofexposure: 16 hours light, 8 hours dark Non chlorinated charcoal filtered tap water (Frankenthal, Germany), mixed with deionized water. Approx. 1 mmoi/L 

### = 100 mg/L CaC03 Approx. 2.5 mmoi/L > 80% saturation Generally 7.5- 8.5 Approx. 23'C
During the last day before start ofexposure None during acclimatization <5% This document is the property ofthe sponsors (specified on page 1), use by other parties requireswritten permission by the sponsors Page 18 of47

### 3.3.2. Testwater Water Total hardness: Conductivity: Ca content: Mg content: Acid capacity: pH-value: Temperature: Approx. 1 mmoi/L 

### = 100 mg/L CaC03 Approx. 250**~S/cm** (at 25°C) Approx. 40 mg/L Approx. 5 mg/L Approx. 2.5 mmoi/L Generally 7.5- 8.5 Approx. 23°C


### 3.4. TEST GROUPS AND CONCENTRATIONS Test groups: Test concentrations: Reason for the selection of the test concentrations : Onetestvessel for the control group Onetestvessel pertest group 0, 0.5, 1.0, 2.2, 5.0, 10 mg/L based on test substance mass In a preliminary test using zebrafish the 96 hour LC50 was between1 and 10 mg/L.

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### 3.5. TEST SUBSTANCE PREPARATIONS The nominal test concentrations are listed below. Fresh test solutions were prepared for each day of apprclxiirnatelv minutes with a magnetic stir bar before use. Thetestsubstance is a mixture ofseveral compounds with differentwater solubility. Therefore, test solutions for each test concentration were prepared individually as water accommodated fractions (according to guidance in OECD (2000) No.23). For each test concentration the melted test substance was weighed on a plasticweighing boat, which was inserted into a 10 Lflask and made up with 10 Ltest water. The test solutions were stirred for about one day. Any undissolved test substance was not removed. To avoid potential losses ofcomponents by volatilization, the study was conducted in closed test vessels. Nominal Weighed amount oftest substance (g) Volume Mean concentration of oftest loading the test substance 1st interval 2nd interval 3'd interval 41 h interval water (L) rate (mg/L) 24 Aug 2008 25 Aug 2008 26Aug 2008 27Aug 2008 (mg/L) 0 10 0.5 0.0050 0.0050 0.0050 0.0050 10 0.5 1.0 0.0100 0.0100 0.0101 0.0100 10 1.0 2.2 0.0199 0.0220 0.0220 0.0220 10 2.1 5.0 0.0501 0.0501 0.0500 0.0501 10 5.0 10 0.1000 0.1001 0.1002* 10 10 *was not used in the test,

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### 3.6. ANALYSES

Detailed information on the method ofthe concentration control analyses is given in the Appendix.


### 3.6.1. Stability The stability ofthe test substance as a solution in testwater and undertesting conditions was determined by concentration control analysis.


### 3.6.2. Concentration control analyses Samples for analytical determination ofthe concentration were taken from newtest solutions within 30 minutes before insertion ofthe test organisms at the start of renewal intervals 1 and 4 and from 24 hour old solutions at the end of intervals 1 and 4. Additionally, samples from the second interval were taken from concentrations 2.2 and 5.0 mg/L only. These additional samples were analyzed because during the first interval solution preparation thetestvessels from these concentrationswere not stirred for the full duration due to a stir bar malfunction. Water samples weretaken from the middle ofthetest vessel using a glass pipette and were transported to the analytical The transport to the Analytical Laboratory was done on the day ofsampling. Forthe dates ofanalysis and storage ofsamples ifapplicable seeAnalytical report in the Appendix. The two samples from the end ofthe second interval were stored in the freezer.


### 3.6.3. Water analysis

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### 3.7. EXPERIMENTAL PROCEDURE 3.7.1. Due to semi static (daily renewal) system Testvessels: Volume ofwater: Animals 

### Itest vessel: Loading (g fish/Lwater): Testvessel/ concentration: Photoperiod: Light intensity: Temperature: Aeration: Feeding: Glass bottles covered with glass plugs (nominal volume: 10 L) 10 L 

### 7 0.31 1 16 hours light, 8 hours darkness Approx. 100 - 490 Lux (the light intensity is determined in regular intervals atthe surface ofthe aquaria) 22 

### 'C None None 3.7.2. Insertion oftest organisms The test organisms were introduced into thetestvessels according to a randomization plan prepared by using a statistical 

### 3.8. OBSERVATIONS AND MEASUREMENTS The fish were observed for mortality and toxic signs (changes appearance and abnormal behaviour in comparison to the control group) within 1 hour after start of exposure and 6, 24, 48, 72 and 96 hours afterstart ofexposure. Fish were considered dead ifthere was novisible movement and no reaction aftertouching. Dead fish were removed from the testvessels. Daily observation ofthe appearance ofthe test solutions and dissolution behaviour ofthe test substance. Temperature, oxygen content and pH were measured in each test vessel within 1 hourafter start ofexposure and thereafter at start and end of each renewal interval. Atthe end ofexposurethe body weight and length ofa representative sample offish were determined. The representative sample consists ofall surviving fish from the lowest concentration and control group.

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### 3.8.1. Statistics



# . Them nomina 

# . . . . . ' :**~** wasca gp y possible the 95% confidence intervals were given as well. Symbols ofthe model ofthe dose response relationship: EC50 median lethal concentration (LCso) Kl Confidence interval 

### e rations e. If

In case that the data obtained were inadequate forthe use ofstatistical methodsfor LCso calculation, an approximate LC50 was calculated. The geometric mean ofthe LCo and LC1oo was given as LC50 in case that no concentration between LC0 and LC10o was obtained
.


### 3.8.2. Validity criteria Validity criteria: 

# ...
No more than one mortality in the control group constant exposure conditions The pH should not vary by more than one unit Oxygen concentration::_ 60% ofthe saturation value Test concentrations within±20% ofnominal or mean of analytically determined concentrations

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### 4. RESULTS

The analytically determined concentrations ofthetest substance during exposure in the aquaria were: Nominal Analytically measured concentration Concentration Oh 24h (old) 24h (new) 48h (old) 72h (new) 96h (24h old) [mg/L] [mg/L] [mg/L] [mg/L] [mg/L] [mg/L] [mg/L] 0 < 0.1 < 0.1 

## - - < 0.1 < 0.1 0.5 0.27 0.25
- 

## - 0.29 0.30

1 0.45 0.42 

## - - 0.49 0.50 2.2 0.56 0.58 0.51* 0.64* 1.25 1.10

5 1.66 1.54 3.16* 3.05* 2.97 3.05 10 6.16 4.69 

## - - 

## - - Nominal Mean Initial Overall Mean Overall Mean Overall Mean Concentration Concentration Concentration Concentration [mg/L] [mg/L] [%Nominal] [%Initial] 0 0.5 0.28 0.28 56% 100%

1 0.47 0.47 47% 100% 5 2.60 2.57 10 6.16 5.43

- not determined *Additional analyses due to stir bar malfunction in interval 1. See section 3.6.2. for detailled results see analytical report The overall mean concentrations deviated more than 20% from the nominal because only a fraction ofthe total components ofthetest substance are soluble in water. However, the overall mean measured concentrations ofthetest substancewere within 85- 100 % ofthe mean initial measured concentrations, indicating that most ofthe soluble compounds remained in the solution overthe exposure time. The concentration ofthetest substancewas satisfactorily maintained during thetest.

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### 4.2. MORTALITYAND TOXIC SIGNS

Toxic signs: nominal concentration number 1 hour 6 hours 24 hours 48 hours 72 hours 96 hours (mg/L) offish 0 7 n n n n n n 0.5 7 n n n n n n 1.0 7 n n n n n n 2.2 7 n n n n n n 5.0 7 n n n T(3) A(3), T(1) A(3) 10 7 C(4), T(3) C(3), T(4) A(4), T(2)


## - - - Explanation ofabreviations n no toxic signs observed D swimming atthe bottom C swimming at the surface A apathy not observed Iall animals dead Numbers in brackets**~** numberofanimals affected

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### 10 > 

### 10 > 

### 10 10 10 10 NOEC 

### 5 

### 5 5 2.1 2.1 2.1
The following effect concentrations (mg/L) were obtained based on mean analytically determined concentrations: Observation time (hours after start ofexposure) 

### 1 hour 

### 6 hours 

### 24 hours 

### 48 hours 72 hours 

### 96 hours LCo 

### 5.43 5.43 2.57 0.77 0.77 0.77 LCso 

### > 

### 5.43 > 

### 5.43 6.19 2.62 2.62 2.52 (95% confidence limits)


## - 

## - - - - 

# - LC1oo 

### >

### 5.43 > 

### 5.43 > 

### 5.43 5.43 5.43 5.43 NOEC 

### 2.57 2.57 2.57 0.77 0.77 0.77

.. - 1nsuff1c1ent datafor calculation Forcalculation ofeffect cor1centn•tion'

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### 26 of

### 47

### 4.3. WATER PARAMETER

Nominal 

### 1 24 48 . 72 concentration [mg/L] hour hours hours hours 

### 0 22 22 22 22 

### 0.5 22 22 22 22 

### 1.0 22 22 22 22 2.2 22 22 22 22 

### 5.0 22 22 22 22 

### 10 22 22 

## - -

- not determmed Temperature in "C during exposure (old solution): Nominal 24 48 72 

### 96 concentration [mg/L] hours hours hours hours 

### 0 22 22 22 22 

### 0.5 22 22 22 22 

### 1.0 22 22 22 22 2.2 22 22 22 22 

### 5.0 22 22 22 22 

### 10 22 22 

# - - not determined The temperature was constant in a range of± 1"C.

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### 4.3.2. pH and dissolved oxygen pH val

- not determined pH values during exposure (old solution): Nominal 24 48 72 

### 96 concentration [mg/L] hours hours hours hours 0 8.2 8.2 8.1 8.1 0.5 8.1 8.2 8.1 8.0 1.0 8.2 8.1 8.0 8.1 2.2 8.2 8.0 8.0 8.1 5.0 8.2 8.0 8.0 8.1 10 8.2 8.0 

## - -

- not determined The pH did notvary by more the test.

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- not determined Dissolved oxygen content in mg/L during exposure (old solution): Nominal 24 48 

### 72 96 concentration [mg/L] hours hours hours hours 0 6.8 6.7 7.3 7.6 0.5 6.7 7.0 7.4 7.7 1.0 6.6 7.2 7.5 7.7 2.2 6.7 7.3 7.5 7.6 5.0 6.7 7.2 7.8 8.1 10 6.3 7.1 

## - -

- not determined The concentrations ofdissolved oxygen were maintained above 60% ofthe maximum saturation at the test temperature of22°C (approx. 5.3 mg/L).

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### 4.4. DISSOLUTION BEHAVIOUR in the 

### rr;orniiiall conc;iiitrailiOri (mg/L) 1hour 24 hours 48 hours 72 hours 96 hours

Explanation ofabreviations: 0: clear test solution 1: homogeneous dispersion ofthe test substance in water, turbidity 2: strong turbidity caused by the test substance 3: colouration caused by the test substance 4: inhomogeneous dispersion ofthe test article, suspended particles 5: precipitation, undissolved test substance at the bottom ofthe test vessel 6: undissolved test substance on the water surface 7: undissolved test substance on the weighing boat 8: undissolved test substance on the stir bar not determined 

### 5. CONCLUSION In conclusion the 96 hour LCso were 4.9 mg/L based on thetest substance analytically determined concentrations. the zebrafish (Dania rerio) and 2.52 mg/L based on the mean of The analytically determined concentrations ofthetest substance in the test solutions were within .±20% ofthe initial concentrations throughout thetest indicating that concentrations had been satisfactorily maintained. However, the test substance is a complex mixture and the analytically detected compounds only represent a fraction ofthe total substance. Since the test fish were exposed to the entire mass ofthetest substance forthe duration ofthe test, the results should be evaluated based on the loading rate. The incomplete stirring ofthe 2.2 and 5.0 mg/L test solutions during the first interval is not considered to have influenced the outcome ofthis study. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines ofthis study.

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### APPENDIX

Statistical evaluation
Analytical Report (Concentration Control Analysis)

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### Probitanalysis according to Finney based on loading rate



### 0.5

1 

### 2.1

5 10 7 7 7 7 

### 7 7 

### 7 7 7 7 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 

### <.0001 Lack of Fit No calculations possible

Number ofsurviving fish after4-6 hours Concentration (mg/L) Nu 

### 0.5

1 

### 2.1

5 10 7 7 7 7 7 7 7 7 7 7 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 

### <.0001 Lack of Fit No calculations possible

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### Page 32 of47

### Number ofsurviving fish after 24 hours Concentration (mg/L) Number Number ofsurviving fish



### 5 10 

### 7 7 7 6 EC50 95% Kl (lower) 95% Kl (upper)

11.296 Intercept Slope -21.24 8.760 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 Project No.: Mortalityafter24**Proba8~~~..----------** 0.40 0.38 0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14

0 2 3 4 5 6 7 8 9 10 11 Concentration (mgfl} x =observed mortality rate This document is the property ofthe sponsors {specified on page 1), use by other parties requireswritten permission bythe sponsors 

### Page 33 of47Number ofsurviving fish after48 hours Concentration (mg/L) Number Number ofsurviving fish

EC50

5.087 

### 5 10 

### 7 7 95% Kl (lower) 

### 4 0 95% Kl (upper)
Intercept Slope 

### -16.98 10.436 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 

### 1.0000**Probabi~i~~~--------** 0.9 0.8 0.7 0.6 0.5

0.1
0 2 3 4 5 6 7 8 9 10 11 Concentration (mg/L) x = observed mortality rate This document is the property ofthe sponsors (specified on page 1}, use by other parties requires written permission by the sponsors Page 34 of47Number ofsurviving fish after 72 hours Concentration (mg/L) Number Number ofsurviving fish



### 5 10 

### 7 7 4 0 EC50 95% Kl (lower) 95% Kl (upper)

5.087 Intercept Slope -16.98 10.436 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 

### Project No.:

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 Mortality

2 3 4 s s 7 a 9 10 11 Concentration (mg/L) x ""obse!Ved mortality rate This document is the property ofthe sponsors (specified on page 1), use by otherparties requireswritten permission by the sponsors Page 35 of47Number ofsurviving fish after 96 hours Concentration (mg/L) Number Number ofsurviving fish

EC50



### 4.910 5 10 7 7 95% Kl (lower) 

### 3 0 95% Kl (upper)
Intercept Slope 

### -15.81 9.932 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 Project No.: Mortality after96**ProbabW~~** .,---------- 0.9

0.4 0.3 0.2 0.1
0 2 3 4 5 6 7 8 9 10 11 Concentration (mg/L} x = observed mortality rate This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 36 of47Number ofsurviving fish after 1 hour
0.28 0.47 

### 0.77 2.57 5.43 

### 7 7 7 7 7 7 7 7 7 7 Goodness-of-Fit- Test (Pearson Chi-Square) p-value <.0001 Lack of Fit No calculations possible

Number ofsurviving fish after4-6 hours Concentration (mg/L) 0.28 0.47 0.77 2.57 5.43 

### 7 7 7 7 7 7 7 7 

### 7 7 Goodness-of-Fit- Test (Pearson Chi-Square) p-value <.0001 Lack of Fit No calculations possible

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### Number of surviving fish after 24 hours Concentration (mg/L) Number Number of surviving fish

2.57 5.43 

### 7 7 7 6 

### EC50 95% Kl (lower) 95% Kl (upper)

6.192 Intercept Slope -14.81 8.125 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 Project No.:

0.24 0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 Mortality after24 hours

2 3

Concentration (mgll) x

### = observed mortality rate 4 5 6

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### Page 38 of47Number ofsurviving fish after48 hours Concentration (mg/L} Number Number ofsurviving fish

2.57 

### 5.43 

### 7 7 4 0 EC50 95% Kl (lower) 95% Kl (upper)

2.618 Intercept Slope -9.306 9.668 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 Project No.:

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
0 Mortality after48 hours

2 3

Concentration {mg/L) x

### = observed mortality rate 4 5 6

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2.57 5.43 

### 7 7 4 0 EC50 95% Kl (lower) 95% Kl (upper)

2.618 Intercept Slope -9.306 9.668 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 

### Project No.: Mortality after 72 hours**ProbabiV~~~--------** 0.9 0.8 0.7

0 2 3 Concentration (mg!L) x

### = observed mortality rate 4 5 6

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2.57 5.43 

### 7 7 3 0 EC50 95% Kl (lower) 95% Kl (upper)

2.520 Intercept Slope -8.505 9.201 Goodness-of-Fit- Test (Pearson Chi-Square) p-value 1.0000 Project No.:

0.6 0.5 0.4 0.3 0.2 0.1
0 Mortality after96 hours

2 3

Concentration (mg/L) x =observed mortality rate 4 5 6

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Determination Method Apparatus Ambient Ambient


# -

Reversed phase HPLC with UV detection, gradient run modus and evaluation by the method of external standard. Automated HPLC system equipped with programmable autosampler and UVdetectorand linked to an electronic data processing system.

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Sample preparation

Test parameters

Calibration Demineralized water(Milli-Q station) Acetonitrile (SigmaAldrich)
The delivered samples were Injected directly intothe HPLCsystem. Forcalibration, adequate portions ofthe test itemwere weighed and dissolved resp. diluted with acetonitrileto depicta working range ofabout0.05-50mgll. After preparation, the calibration solutions were injected directly into the HPLC-system. Column: Purospher RP 18, 5pm, 125x 3 mm (Merck) Mobile phase: A: Demineralizedwater B :Acetonitrile Gradient run· 1t(minl 

### o I%(v/v)A 

### Iso I %(V/v B 40 Flow rate: Injectionvolume: Temperature: 15 

### IO

100 0.66 ml/min 20**~I** 25°C 20 0 100 21 I 31 60 

### I next inj. 40 I

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### Page 44 of47Discussion ofresults All found values are mean values out of two injections and correspond maximal to about62%with the expected concentrations. The reproducibility within the respective samples indicated that the gap between expected and found values is not caused bythe analytical determination method.

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Inspection of Date ofinspection Rawdata:

Thefinal report reflects the rawdata.

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