Acute Toxicity (Dania rerio) 

DATA REQUIREMENT 

OECD 203 

92/69/EEC, Annex V, Part C1 

This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be used 

only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, 

publication or submission to other parties require the written permission of the Sponsor{s), with the exception of regulatory agencies 

acting within the limits of their administrative authority. 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 1 of47 
THIS PAGE IS INTENTIONALLY LEFT BLANK 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 2 of 47 
To be filled for USA EPA submission only: 

Sponsor 

Date 

Submitter 

This document is the property of the sPonsom (specified on page 1 ), use by other partles requires written pennlssion by the sponsors 

Page 3of47 
The following statement is for US EPA submissions only: 

The flagging criteria of 40 CFR 158.34 are not applicable for this study type 

This document is the property ofthe sponsors {specified on page 1), use by other parties requires written permission by the sponsors 

Page 4 of 47 
SIGNATURE PAGE 

This document Is the property of the sponsors (specified on page 1), use by other parties requires written pe~mlsslon by lh$ sponsors 

Page 5of47 
CONTRIBUTORS TO THE STUDY I SUPERVISORY LABORATORY PERSONNEL 

-

-

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 6 of47 
- - .... -- - -

T ...... .. .. "' "<" ... '"' ............... ' .. .. 

results to the Study Director and to Management. 

The final report reflects the raw data. 

This document is the property of the sponsors {specified on page 1), use by other parties requires written permission by the Sponsors 

Page 7 of47 
This document is the property of the sponsors (specified on page 1}, use by other parties requires written permission by the sponsors 

Page 8 of 47 
CONTENTS 

FLAGGING CRITERIA 

SIGNATURE PAGE 

CONTRIBUTORS TO THE STUDY I SUPERVISORY LABORATORY PERSONNEL 

STATEMENT OF THE QUALITY ASSURANCE UNIT 

CONTENTS 

LIST OF DEFINITIONS AND 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. MATERIALS AND METHODS 

3.1. TEST SUBSTANCE 

3.2. TEST ANIMALS 

3.3. HOUSING AND DIET 

3.3.1. Acclimatization 

3.3.2. Test water 

3.4. TEST GROUPS AND CONCENTRATIONS 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 9 of 47 
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 of test organisms 

3.8. OBSERVATIONS AND MEASUREMENTS 

3.8.1. Statistics 

3.8.2. Validity criteria 

4. RESULTS 

4.1. ANALYTICAL CONCENTRATION CONTROL 

4.2. MORTALITY AND 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) 

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 10 of 47 
LIST OF DEFINITIONS AND ABBREVIATIONS 

Lethali 

LCx 

LCo 

LCso 

NOEC 

Water-accommodated 

fraction (WAF): 

Loading rate: 

n.d. 

dissolved in dilution water that is calculated to result in x % lethality 

of the test batch of fish within a stated exposure period. 

maximum concentration causing no lethality within the period of the 

test; 

median lethal concentration; 

minimum concentration causing 100% lethality within the period of 

the test; 

No Observed Effect Concentration means the highest concentration 

of the test substance that does not have an adverse effect on the 

test organisms. 

An aqueous fraction containing the dissolved and/or suspended 

and/or emulsified fraction of a multi-component substance or a 

mixture. 

The mass to volume ratio of test substance to test medium (in mg/L) 

used to prepare the WAF test solution. ELx values are based on 

loading rate and calculated using the same statistical methods as 

for ECx. 

not 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 11 of47 
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 Guideline for 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 experiment the analytically determined concentrations were not 

in the required range. In a second experiment the samples taken at start 

of exposure were 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 

, This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 12 of 47 
Test Substance Preparation: The test solutions were prepared separately for each test 

vessel and each interval. Before the test substance was 

weighed it was 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 glass flask and made up with 10 L test 

water. The test solutions were stirred for about one day. Any 

undissolved test substance was not removed. To avoid 

potential losses of components by volatilization, the study 

was conducted in closed test vessels. 

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 of the total 

components of the test substance are soluble in water. 

However, the overall mean measured concentrations of the 

test substance were within 85 - 100 % of the mean initial 

measured concentrations, indicating that most of the soluble 

compounds remained in the solution over the exposure time. 

The concentration of the test substance was satisfactorily 

Since the test fish were exposed to the entire 

mass of the test substance for the duration of the test, the 

results should be evaluated based on the loading rate. 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 13 of47 
The following effect concentrations (mg/L) were obtained based on loading rate: 

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% of the initial concentrations throughout the test indicating that concentrations had 

been satisfactorily maintained. However, the test substance is a complex mixture and the 

analytically detected compounds only represent a fraction of the total substance. Since the 

test fish were exposed to the entire mass of the test substance for the duration of the test, the 

results should be evaluated based on the loading rate. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 14 of 47 
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 together with the raw data of this study. 

See also Section 3.4. 

2.3. TEST GUIDELINES 

The study was conducted according to the following test guidelines: 

OECD Guideline for Testing of Chemicals No. 203, July 1992 ("Fish Acute Toxicity Test") 

EEC directive 92/69, Annex V, 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) of the 

laboratories involved. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 15 of 47 
2.4. TIME SCHEDULE 

Test d . . .. 

The fish were exposed to the test substance for approxemately 96 hours. 

2.5. RETENTION OF RECORDS 

2.6. ANIMAL WELFARE 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 16 of 47 
3. MATERIALS AND METHODS 

3.1. 

The 

Name of test 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 over the exposure period 

was guaranteed by the manufacturer, and the manufacturer 

holds this responsibility. 

ADDITIONAL TEST SUBSTANCE INFORMATION 

Date of production: 

Physical state/ 

Appearance: 

Water solubility: 

Storage conditions: 

03 Apr 2007 

Liquid I dark brown 

Not applicable (mixture of multiple components) 

The test substance was stored at ambient temperature 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 17 of 47 
3.2. TEST ANIMALS 

Test species: Zebrafish (Dania rerio) 

Body weight (wet weight)*: 

Body length (from the top of the 

snout to the end of the caudal fin)*: 

0.44 g (0.39 - 0.48 g) 

3.4 em (3.3 - 3.5 em) 

Age of the animals: 

Hatching date: 

Arrival at the testing facility: 

Approx. 6 months 

17 Feb 2008 

28 May 2008 

*Measurements were performed with a representative sample of the fish batch. 

All animals used for the study were derived from the same batch of fish. 

The test fish were acclimatized to testing conditions prior to test initiation. During 

acclimatization, the general health status of the fish was observed for signs of sickness, 

injury, or other abnormalities. 

3.3. HOUSING AND DIET 

3.3.1. Acclimatization 

Duration of acclimatization to t""tinn 

;::14 days to test water, nh,olctne 

water and light regime: 

Photoperiod: 

Water quality: 

Total hardness: 

Acid capacity: 

Oxygen content: 

pH-value: 

Temperature: 

Diet: 

Withdrawal of feed: 

Medical treatment: 

Mortality during the last week 

before start of exposure: 

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 of exposure 

None during acclimatization 

<5% 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 18 of47 
3.3.2. Test water 

Water 

Total hardness: 

Conductivity: 

Ca content: 

Mg content: 

Acid capacity: 

pH-value: 

Temperature: 

Approx. 1 mmoi/L = 1 00 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 : 

One test vessel for the control group 

One test vessel per test 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 between 1 and 1 0 mg/L. 

This document is the property of the sponsors (specified on page 1}, use by other parties requires written permission by the sponsors 

Page 19 of 47 
3.5. TEST SUBSTANCE PREPARATIONS 

The nominal test concentrations are listed below. Fresh test solutions were prepared for each 

day of 

apprclxiir natelv minutes 

with a magnetic stir bar before use. The test substance is a mixture of several compounds 

with different water 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 

plastic weighing boat, which was inserted into a 10 L flask and made up with 10 L test water. 

The test solutions were stirred for about one day. Any undissolved test substance was not 

removed. To avoid potential losses of components by volatilization, the study was conducted 

in closed test vessels. 

Nominal Weighed amount of test substance (g) Volume Mean 

concentration of of test 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, 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 20 of47 
3.6. ANALYSES 

Detailed information on the method of the concentration control analyses is given in the 

Appendix. 

3.6.1. Stability 

The stability of the test substance as a solution in test water and under testing conditions was 

determined by concentration control analysis. 

3.6.2. Concentration control analyses 

Samples for analytical determination of the concentration were taken from new test solutions 

within 30 minutes before insertion of the 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 the test vessels 

from these concentrations were not stirred for the full duration due to a stir bar malfunction. 

Water samples were taken from the middle of the test vessel using a glass pipette and were 

transported to the analytical 

The transport to the Analytical Laboratory was done on the day of sampling. 

For the dates of analysis and storage of samples if applicable see Analytical report in the 

Appendix. 

The two samples from the end of the second interval were stored in the freezer. 

3.6.3. Water analysis 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 21 of 47 
3.7. EXPERIMENTAL PROCEDURE 

3.7.1. 

Due to 

semi static (daily renewal) system 

Test vessels: 

Volume of water: 

Animals I test vessel: 

Loading (g fish/L water): 

Test vessel/ 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 at the surface of the aquaria) 

22 'C 

None 

None 

3.7.2. Insertion oftest organisms 

The test organisms were introduced into the test vessels 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 after start of exposure. Fish were considered dead if there was no visible 

movement and no reaction after touching. Dead fish were removed from the test vessels. 

Daily observation of the appearance of the test solutions and dissolution behaviour of the test 

substance. 

Temperature, oxygen content and pH were measured in each test vessel within 1 hour after 

start of exposure and thereafter at start and end of each renewal interval. 

At the end of exposure the body weight and length of a representative sample of fish were 

determined. The representative sample consists of all surviving fish from the lowest 

concentration and control group. 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 22 of 47 
3.8.1. Statistics 

Them . nomina 

wasca . gp . . . . y ' : ~ 

possible the 95% confidence intervals were given as well. 

Symbols of the model of the dose response relationship: 

EC50 median lethal concentration (LCso) 

Kl Confidence interval 

e 

rations 

e. If 

In case that the data obtained were inadequate for the use of statistical methods for LCso 

calculation, an approximate LC50 was calculated. The geometric mean of the 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% of the saturation value 

Test concentrations within± 20% of nominal or mean of 

analytically determined concentrations 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 23 of 47 
4. RESULTS 

The analytically determined concentrations of the test 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 of the total components of the test substance are soluble in water. However, the 

overall mean measured concentrations of the test substance were within 85- 100 % of the 

mean initial measured concentrations, indicating that most of the soluble compounds 

remained in the solution over the exposure time. The concentration of the test substance was 

satisfactorily maintained during the test. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 24 of47 
' 

4.2. MORTALITY AND TOXIC SIGNS 

Toxic signs: 

nominal 

concentration number 1 hour 6 hours 24 hours 48 hours 72 hours 96 hours 

(mg/L) of fish 

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 of abreviations 

n no toxic signs observed 

D swimming at the bottom C swimming at the surface 

A apathy not observed I all animals dead 

Numbers in brackets ~ number of animals affected 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 25 of47 
(95% confidence limits) 

> 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 of exposure) 

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 data for calculation 

For calculation of effect cor1centn•tion' 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 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. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 27 of 47 
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 not vary by more the test. 

This document·is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 28 of47 
Dissolved oxygen content in mg/L during exposure (freshly prepared solution): 

- 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 of dissolved oxygen were maintained above 60% of the maximum 

saturation at the test temperature of 22°C (approx. 5.3 mg/L). 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 29 of47 
4.4. DISSOLUTION BEHAVIOUR 

in the 

rr;orniiiall conc;iiitrailiOri 

(mg/L) 1 hour 24 hours 48 hours 72 hours 96 hours 

Explanation of abreviations: 

0: clear test solution 

1: homogeneous dispersion of the test substance in water, turbidity 

2: strong turbidity caused by the test substance 

3: colouration caused by the test substance 

4: inhomogeneous dispersion of the test article, suspended particles 

5: precipitation, undissolved test substance at the bottom of the 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 the test substance 

analytically determined concentrations. 

the zebrafish (Dania rerio) 

and 2.52 mg/L based on the mean of 

The analytically determined concentrations of the test substance in the test solutions were 

within .±20% of the initial concentrations throughout the test indicating that concentrations had 

been satisfactorily maintained. However, the test substance is a complex mixture and the 

analytically detected compounds only represent a fraction of the total substance. Since the 

test fish were exposed to the entire mass of the test substance for the duration of the test, the 

results should be evaluated based on the loading rate. 

The incomplete stirring of the 2.2 and 5.0 mg/L test solutions during the first interval is not 

considered to have influenced the outcome of this study. The results in this study are 

consistent with all validity criteria and the test is valid according to the guidelines of this study. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 30 of47 
APPENDIX 

Statistical evaluation 

Analytical Report (Concentration Control Analysis) 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 31 of 47 
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 of surviving fish after 4-6 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 

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 32 of 47 
Number of surviving fish after 24 hours 

Concentration (mg/L) Number Number of surviving 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.: 

Mortality after 24 

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 of the sponsors {specified on page 1), use by other parties requires written permission by the sponsors 

Page 33 of 47 
Number of surviving fish after 48 hours 

Concentration (mg/L) Number Number of surviving 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 of the sponsors (specified on page 1}, use by other parties requires written permission by the sponsors 

Page 34 of47 
Number of surviving fish after 72 hours 

Concentration (mg/L) Number Number of surviving 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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 35 of 47 
Number of surviving fish after 96 hours 

Concentration (mg/L) Number Number of surviving 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 after 96 

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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 36 of47 
Number of surviving 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 of surviving fish after 4-6 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 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 37 of47 
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 after 24 hours 

2 3 

Concentration (mgll) 

x = observed mortality rate 

4 5 6 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 38 of 47 
Number of surviving fish after 48 hours 

Concentration (mg/L} Number Number of surviving 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 after 48 hours 

2 3 

Concentration {mg/L) 

x = observed mortality rate 

4 5 6 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 39 of47 
Number of surviving fish after 72 hours 

Concentration (mg/L) Number Number of surviving 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.: 

Mortality after 72 hours 

ProbabiV~~~--------

0.9 

0.8 

0.7 

0 2 3 

Concentration (mg!L) 

x = observed mortality rate 

4 5 6 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 40 of47 
Number of surviving fish after 96 hours 

Concentration (mg/L) Number Number of surviving fish 

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 after 96 hours 

2 3 

Concentration (mg/L) 

x =observed mortality rate 

4 5 6 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 41 of 47 
Test item 

Batch identification 

Date of production (test item) 

PSN 

Project no. 

Vehicle 

Sponsor 

Date of receipt of order 

Testing facility 

Study director 

Storage cond. test item 

Storage cond. vehicle 

Storage cond. samples 

Test period 

Storage of records 

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 UV detector and linked to an electronic data processing 

system. 

This document is the property of the sponsors {specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 42 of 47 
Reagents 

Sample preparation 

Test parameters 

Calibration 

Demineralized water (Milli-Q station) 

Acetonitrile (Sigma Aldrich) 

The delivered samples were Injected directly into the HPLCsystem. 

For calibration, adequate portions of the test item were weighed 

and dissolved resp. diluted with acetonitrile to depict a 

working range of about 0.05-50 mgll. 

After preparation, the calibration solutions were injected directly 

into the HPLC-system. 

Column: Purospher RP 18, 5pm, 125 x 3 mm (Merck) 

Mobile phase: A: Demineralized water 

B : Acetonitrile 

Gradient run· 

1 t(minl o 

I %(v/v)A I so 

I %(V/v B 40 

Flow rate: 

Injection volume: 

Temperature: 

15 

IO 

100 

0.66 ml/min 

20 ~I 

25°C 

20 

0 

100 

21 I 31 

60 I next inj. 

40 I 

This document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 43 of47 
*Note: estimated determination limit= 0.1 mgfl 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 44 of47 
Discussion of results All found values are mean values out of two injections and correspond 

maximal to about 62% with the expected concentrations. The reproducibility within the respective samples indicated that the gap between expected 

and found values is not caused by the analytical determination method. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 45 of 47 
• document is the property of the sponsors (specified on page 1 ), use by other parties requires written permission by the sponsors 

Page 46 of 47 
STATEMENT OF THE QUALITY ASSURANCE UNIT 

Inspection of Date of inspection 

Raw data: 

The final report reflects the raw data. 

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 47 of 47