Technology &

Engineering B.V. PO Box 9300 6800 SB Arnhem The Netherlands T +31 26 366 2673 F +31 26 366 5820 www.akzonobel.com/te Environmental Chemistry Department



# CONFIDENTIAL

Title 

## TOXICITY OF DUOMEEN 2 HT TO 

## CYPRINUS CARPIO MITIGATED

WITH HUMIC ACID

Authors 

### M. Kean

Technical Support 

### I. Garttener, J. van Dam, B.Helming

Date 

### 28/07/2010

Document number 

### 2.419.605

Study number 

### T09066 EFC

Client 

### AkzoNobel Surfactants America



# REPORT2.419.605 T09066 EFC

2



## ABSTRACT

The acute toxicity of Duomeen 2 HT to the fresh water fish Cyprinus carpio (Common Carp) was tested under semi static conditions for 96 hours in accordance with EPA guideline OPPTS 850.1085 (8.1). The test was performed in compliance with Good Laboratory Practice (GLP).

Duomeen 2 HT is poorly soluble in water. A preliminary test (mitigated with humic acid) for zebra fish ( Danio rerio

) gave a NOEC value of 50 mg/L. This data was used to estimate the concentration range for the definitive test. An additional range finding test with Common Carp was not conducted for ethical

reasons.

A definitive test with nominal test concentrations in the range of 14.3 to 150.0 mg/L including a control was carried out. The results show that the test substance had 100% lethal effect at 83.3 and 150 mg/L, but no lethal or sub-lethal effects were observed at the other test concentrations of 14.3, 25.7 and 46.3 mg/L. Based on the test results a maximum concentration causing no mortality (0-96h) (NOEC) and the lowest tested concentration causing an effect (0-96h) (LOEC) can be derived of 46.3 mg/L and 83.3 mg/L respectively. Based on the available data an LC 50 

### was calculated of 62.1 mg/L. The minimum concentration causing 100% mortality (LC 100) within the period of the test was 83.3 mg/L.

The results of the chemical analyses show that the measured test substance concentrations at the start of the test and the new solutions after replacement differed less than 20% from the nominal test

concentrations. The effect data are based on the nominal concentrations of the test chemical as received. Corrections have not been made for active ingredient.2.419.605 T09066 EFC

3



## ACUTE TOXICITY OF DUOMEEN 2HT TO CYPRINUS CARPIO (COMMON CARP) STUDY ORGANIZATION

Sponsor Akzo Nobel Surfactants America 281 Fields Lane Brewster New York 10509 USA Study monitor M.Gyimesi Study Contact Person D.Hillibold

STUDY ORGANISATION Location AkzoNobel Technology and Engineering Environmental Chemistry department Velperweg 76 P.O. Box 9300 6800 SB Arnhem The Netherlands Study director M.Kean Others J.van Dam, I, Garttener,B.Helming Quality Assurance Unit H. van Daalen Management R. Borgmann-Strahsen Experimental starting date 26/4/2010 Experimental completion date 30/4/2010



## ARCHIVING AND STORAGE

The project file including the final report, amendments to the final report, the study plan, amendments to the study plan, records of quality assurance inspections, all letters, memos and notes and raw data pertaining to the study will be retained in the archives of AkzoNobel Technology & Engineering Arnhem for a period of ten years. Other records including master schedule sheet, laboratory notebooks, logbooks, records of the maintenance and calibration of equipment, summary of training, curricula vitae and job descriptions of the personnel involved in the study, records related to location and storage of the test substance will also be kept in the AkzoNobel Technology & Engineering Arnhem archives for a period of ten years. Test material will be stored deep frozen under the sample code T 09066 for ten years or only as long as the quality of the test substance permits evaluation.2.419.605 T09066 EFC

4



## GLP COMPLIANCE STATEMENT

The study was conducted in compliance with the following Good Laboratory Practice regulations:

x The study was performed in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM (98)17, 21 January 1998, including all supplements published up to the starting date of the study

.

The following aspects of the study were not conducted under GLP:

x Measurement of water hardness was conducted using standard test kits. GLP is not claimed for

these measurements.

x The range finding study.

x Analytical method development, optimization, and stability testing.

Study director M.Kean

Management R. Borgmann-Strahsen2.419.605 T09066 EFC

52.419.605 T09066 EFC

6



### QUALITY ASSURANCE STATEMENT

This report was audited by the Quality Assurance Unit contracted by AkzoNobel Technology and Engineering Arnhem. It is considered to be an accurate presentation of the methods and procedures applied in the course of the study and an accurate reproduction of the data recorded. Listed below are the dates of inspection of this study by the Quality Assurance Unit and the dates on which its findings were reported to Study Director and Management.

Dates of inspection Phase of study Dates of QA reports 11.4.2010 Draft Study Plan 11.4.2010 28.4.2010 Study Plan 28.4.2010 Study (Analysis + Stock) 1.7.2010 Report 1.7.2010

Quality Assurance Unit

H. van Daalen2.419.605 T09066 EFC

7



## CONTENTS

INTRODUCTION 8

MATERIALS 10

METHODS 11

RESULTS 14

QUALITY CRITERIA 16

AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 17

REFERENCES 18

ANNEX 1 23

ANNEX 2 312.419.605 T09066 EFC

8



## INTRODUCTION

The purpose of this study was to determine the acute lethal toxicity of Duomeen 2 HT to the

freshwater fish Cyprinus carpio (Common Carp) under continuous exposure during 96 hours in a semi static test with 20mg/L humic acid added to the test water. A semi static testing method was considered the most appropriate testing method as the results generated in this report are intended to be used by the American EPA in combination with test results from a standard fish test (where a semi static approach was also used) according to OPPTS 850.1075 (8.2). This allows therefore a more meaningful comparison between the two tests.

Acute toxicity is the discernible adverse effect induced in an organism within a short time of exposure

to a substance.

The acute toxicity of a test substance is normally expressed as the median lethal concentration (LC 50 

### ), which is the concentration in water which kills 50% of a test batch of fish within a period of continuous exposure. Lethality of the fish is defined as the lack of opercular movement. Sub-lethal effects such as deviations in the behavior or the appearance of the fish were recorded if

observed.

Test guidelines, modifications and deviations The test was carried out in accordance with EPA Ecological Effects Test Guidelines OPPTS 850.1085 Fish Acute Toxicity Mitigated by Humic Acid. (EPA712–C–96–117 April 1996) With the following deviations from the guideline:

x No additional control without Humic acid (HA) was tested for ethical reasons.

x DOC (as NPOC) measurements were not conducted in all replicates with test substance as this is known to deliver non representative results with surfactants due to strong interactions with the test chemical. The control replicates were considered to be a representative sample of the test media as used in all test replicates. DOC analysis was conducted on this replicate only.

x Although specific chemical analysis is not given in the guideline as a strict requirement, chemical analysis was conducted at all concentrations to demonstrate correct addition of the test chemical

and confirm the nominal concentrations at the start of the test and after refreshment.

x European water is slightly harder than the guideline recommendations. Due to the test animals being bred and reared in hard water this was not lowered for the test to avoid influencing the health

of the animals.2.419.605 T09066 EFC

9



### x Common Carp ( Cyprinus carpio) used for testing are not strictly specified in guideline OPPTS 850.1085. This is however an approved species listed in the OPPTS 850.1075 guideline. During previous contact with the EPA use of fish species from this guideline was permitted. The same logic has been applied to this study therefore. OPPTS 850.1075 and OECD 201 (8.2 &8.3) were consulted for additional information if not present in the main test guideline.

x Feeding was stopped 24 hours prior to the start of the test according to OECD 203 criteria. Not 48 hours as stated in the test guideline.2.419.605 T09066 EFC

10



## MATERIALS

Test substance

The test substance (project sample code T 09066) was received on 22-12-2009 Data on the handling, stability, composition, purity or other characteristics of the test substance supplied by the sponsor were accepted and used without further check.

Test substance name Duomeen 2HT

Batch/Lot no. 2268-45-1 Analysis Certificate See Annex 2. Purity >90% Appearance White waxy solid Water solubility Insoluble in cold water Stability Will not biodegrade during the test period. Will interact strongly with Humic acid. Storage until required In original container in the dark

Chemicals All reagents used were of reagent grade quality. Humic Acid supplier was Sigma Aldrich CAT 1,675-2,

Lot16308-118.

De-ionized water The de-ionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not more than 2.0 mg/L and a conductivity of less than 5 μS/cm. This water was produced from tap water in a water purification system according to laboratory Standard Operation Procedures.

Test vessels As test vessels, 5 L aquaria containing 4 L of test solution were used. Aquaria were covered with glass plates and gently aerated during the test.

Test room and light regime The test was carried out in a temperature controlled room, the light regime was 16 hours of ambient light per day, provided by fluorescent tubes.2.419.605 T09066 EFC

11



### Test animals The test was performed with juvenile Common Carp which were obtained from Wageningen University department of animal sciences (Zodiac). These fish were accepted according to the quality control criteria of the guideline after a quarantine period of at least 14 days. The fish in stock were fed one to three times per day with commercially available dry, deep-frozen food, or fresh nauplii. The feeding was stopped 24 hours before the test was started, and the animals were not fed during the test. A representative number of test fish were measured and weighed. The average length of a sample of 10 fish randomly taken from the stock aquarium was 3.3 cm with a minimum of 3.0 and a maximum of 3.8 cm long. Average weight was 0.28 g. The mean biomass loading was 0.7 g/L.



## METHODS

Test principle and procedures The test was performed as a semi static test which means the test media was replaced during 96 hours, once, half way through the test. 20 fish in 2 replicates of 10 were used per test concentration and per control.

Under otherwise identical test conditions, the fish were exposed to the chosen concentrations of the test substance as described below and mortality and sub-lethal effects were recorded at least within the first six hours and then at approximately 24, 48, 72 and 96 hours. The fish were considered dead when a lack of opercular movement was observed and touching of the caudal peduncle produced no reaction. Dead fish were removed from the test vessels directly after being observed, which was checked at least twice each day. In addition to death, sub-lethal effects such as erratic swimming, loss of reflex, increased excitability etc were recorded. Fish that were convulsing or showing other severe forms of distress not considered transient in nature and likely to become more severe before the exposure is terminated, were sacrificed for humane reasons. These fish were treated as having died in

the test.

The fish were randomly placed in the test vessels which were positioned in the test room in a random manner. During the test the vessels were covered with glass plates. The test solutions present in the test vessels were aerated with water-saturated air purified by an active coal filter and a cotton filter during testing.2.419.605 T09066 EFC

12



### Preparation of the stock solution Due to the low solubility of the test chemical large volumes of stock were required. 4 stocks at approximately 150 mg/L were made by weighing approximately 0.750g of the test substance on an analytical balance and dispersing this in 5 litres of test media. For all stock solutions including those prepared for the solution refreshment the following preparation procedure was followed:

The test substance was ultra sonicated in 150 mL of DSW for approximately 10 minutes. Care was taken to avoid excessive heating of the test chemical. After every minute of sonication the stock was allowed to cool for around 30 seconds. When test substance particles were no longer visible, the power on the ultra sonic mixer was reduced to approximately 50 % also to prevent overheating. The

stock solution formed was milk white in colour. This was then added to 4.5 litres of test media containing 20 mg/L of humic acid whilst stirring. Visible reaction with the dissolved humic acid was observed forming a finely dispersed precipitate. The pH of the stock solutions was then reduced from 8.9 to 8.2 with HCl in line with the normal pH of the test media. The volume of the stock was then made up to 5 litres to reach the desired concentration.

Preparation of the test solutions The stock was used as the highest concentration. Dilutions of the stock solution in freshly prepared DSW test medium containing 20 mg/L humic acid were used to prepare the desired concentration range in the aquaria prior to addition of the test animals. All preparation was conducted whilst agitating the media with magnetic stirrers.

Due to the interaction of the humic acid and test substance an additional stock without humic acid at the same concentration was prepared to investigate any potential effects on analytical recovery. The following final nominal test concentrations were prepared: 14.3, 25.7 46.3 83.3 150.0 mg/L. A control containing DSW and 20 mg/L humic acid was also included in the test.

Preparation of the test medium As test medium, the standard laboratory Dutch Standard Water (DSW) media was used. The pH of DSW is approximately 8.0 ± 0.2 and the hardness of DSW measured as CaCO 3 

### is approximately 80 mg/L. Total hardness measured as a sum of calcium and magnesium carbonate or odH is approximately 12 odH or (degrees of German Hardness). Test medium with the above mentioned specification was used for keeping the fish prior to testing and also for use during the test. The final composition used contained: 100 mg of NaHCO 3 

### , 20 mg of KHCO 3 

### , 200 mg of CaCl 2 

###  2H 2 

### O and 180 mg of MgSO 4 

###  7H 2 

### O. The test medium was aerated before being used in the test. The air was watersaturated and purified by an activated charcoal and cotton filter.2.419.605 T09066 EFC

13



### Chemical analyses Chemical analysis was conducted at the start of the test and in the stock and in one concentration after solution replacement, to quantify the concentration of the test substance in the test solutions. The analytical method used was an LC MS/MS method. Details of the method details of this method are specified in Annex 2.

Dissolved oxygen, pH, temperature, hardness TSS, and NPOC measurements in the test solutions The dissolved oxygen and pH were measured at the beginning and the end of the test in all concentrations, and in the control vessels. Temperature was measured continuously throughout the test in water in the vicinity of the test. Hardness was measured at the beginning of the test. The total suspended solids (TSS) were not measured due to synthetic not natural water being used. No TSS are therefore present. NPOC was measured in the control at the beginning of the test. All samples for NPOC measurement were filtered prior to analysis. NPOC can therefore be considered representative of DOC (dissolved organic carbon) for the analyzed sample.

Apparatus used The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter, the pH was measured using a microcomputer pH-meter, the temperature was measured with a temperature sensor linked to a data recorder and the hardness was measured using a Dr Lange

LCK 362 CaCO 3 

### test kit and a Dr Lange LCK 327 total hardness test kit. NPOC was measured using a NPOC analyzer following the standard laboratory operating procedures for this apparatus. Details on standard curves, and control standards are recorded in the study journal. Dr Lange test kits were considered of sufficient accuracy for determination of water parameters and were not internally

validated. Hardness measurements are therefore not included in the GLP claim.

Evaluation of data If possible the LC 50 

### was calculated with the computer program TOXCALC TM 

### version 5.023, using the trimmed Spearman-Karber method (8.5). The concentrations causing zero and 100% immobility were derived directly from the test observations.2.419.605 T09066 EFC

14



## RESULTS

Range finding test

No preliminary test with Common Carp was conducted in order to reduce fish usage. The test range was estimated based on a 50 mg/L NOEC with zebra fish.

Definitive test

The results of the test on survival and sub-lethal effects are summarized in Table 2. All results are calculated based on the test substance as received and are therefore not corrected for active ingredient content. The results show that the test substance had lethal effect at 83.3 and 150 mg/L, but no lethal or sub-lethal effects were observed at the other test concentrations of 14.3, 25.7 and 46.3 mg/L respectively.100% lethal effects at 150 mg/L were observed after 24 hours. 100% lethal effects at 83.3 mg/L were also observed after 48 hours. Animals displaying effects between these two time points were euthanized for ethical reasons to minimise suffering. These fish were recorded as having died due to test substance effects. See table 2 below for full record of results / observations. See figure 1 for the dose response curve taken from Toxcalc 5.0.23 software. Based on the test results a maximum concentration causing no effect (NOEC) (0-96h) and the lowest tested concentration causing an effect (LOEC) (0-96h) can be derived of 46.3 mg/L and 83.3 mg/L respectively. Based on the available data an LC 50 

### was calculated of 62.1 mg/L. The minimum concentration causing 100% mortality(LC 100) within the period of the test was also 83.3 mg/L. Due to no partial responses being observed the LOEC and the LC100 are represented by the same value 83.3 mg/L in this test, and should therefore be interpreted with care.

The length and weight of 10 fish was determined. Fish weight was recorded as wet weight in grams. Fish length was recorded in centimetres. The results of the length and mass determinations are presented in Table 3.2.419.605 T09066 EFC

15



### Chemical analyses All test solutions were analyzed at the start of the test and the stock and the middle concentration were analysed after refreshment at 48 hours. All test concentrations were observed to be within ± 20% of the expected nominal concentrations. The stock at 48 hours did fall slightly below 80 % of the expected nominal concentrations but it can be seen from the measurement of the middle

concentration that the concentration was correct in the test. The inevitable decrease of the

concentration of the test substance with time due to interaction with humic acid in the test media is acceptable according to the Guideline and nominal concentrations may be used for endpoint concentrations. The reaction observed in the stock solution with humic acid did not negatively influence the recovery of the test chemical. This was demonstrated by the analysis of a control stock at 150 mg/L without humic acid.

A full description of the analytical method and results table is provided in Annex 2. A summary of the observed measurements as a percentage of the nominal concentration is reported in table 1 below:

Table 1 Test Samples T=0 % of nominal T=48h % of nominal

Control 

### <LOD - 14.3 mg/L 

### 87.4 - 25.7 mg/L 

### 84.2 - 46.3 mg/L 

### 83.9 84.2 83.3 mg/L 

### 88.0 - 150 mg/L 

### 85.3 - Stock 150 mg/L + 20 mg/L

H.A. 

### 85.3 76.5

Control Stock 150 mg/L 

### 79.9 -

Oxygen, pH, Temperature, Hardness and NPOC Measurement Oxygen concentrations varied at the most from 8.4 at the start to 7.3 mg/L at the end of the test. The concentration with the highest variation in oxygen concentrations was the 14.3 mg/L replicate. The Oxygen concentrations did however not fall below 60% saturation and are therefore considered acceptable according to OECD guidelines. (Specific saturation criteria not listed in OPPTS) (8.3). The results of the oxygen measurements are summarized in Table 4.

Control pH concentrations varied during the test from 8.2 at the start to 7.9 at the end of the test. The concentration with the highest variation in pH was the 14.3 mg/L replicate. The pH did however not vary more than 1.0 pH units and is therefore considered acceptable according to the guideline. The results of the pH measurements are summarized in Table 5.2.419.605 T09066 EFC

16



### The temperature in the control test vessels during the test period ranged from 20.55. to 22.15oC. Temperature variation therefore met criteria in the study plan and the EPA guideline. The temperature in the test room was however set 1oC lower than the study plan value inline with the relevant guideline.

Water hardness was measured at the start of the test at 82 mg/L of CaCO 3 

### . This is acceptable based on a maximum hardness of 180mg/L (as CaCO 3 

### ) stated in the guideline. The total hardness (a sum of Mg and Ca) was measured as 13.4 odH (German degrees of hardness).

NPOC was measured at the start of the test in the control only. NPOC measurements were not used as a method of indirect chemical analysis as specific analysis was used. NPOC analysis was used to quantify the amount humic acid as NPOC in the test media only. The test media used was identical for the whole test. The control is considered representative of all test replicates. All NPOC measurements were analyzed at least in triplicate and an average is reported. Standard samples were analyzed in the same run as test samples to ensure system accuracy. The results of the NPOC measurements are

summarized in Table 6.



## QUALITY CRITERIA

The following quality criteria have been met in the present study:

x Mortality in the control did not exceed 10 % at the end of the test.

x The dissolved oxygen concentration was >60% of the air saturation value throughout the test. (OECD criteria)

x The average measured concentrations of test substance did not differ more than 20% of the

nominal concentration at the start of the test..

x Deviations from the test temperature should be no greater than ± 2 °C.

x Deviation in pH throughout the test did not exceed pH 1.5 units (OECD Criteria)

x The length of the fish used did not exceed 4 cm (OECD Criteria)

The following quality criterion has not been met in the present study:

x The average load of fish in the test aquaria was < 0.5 g/L. The average biomass loading during the test was 0.7 g/L. The advised loading of EPA guideline OPPTS 850.1085 followed for this test was therefore exceeded. The loading was however still below that recommended in the OPPTS 850.1075 and OECD 203 fish test guidelines of <0.8g/L and <1.0g/L respectively. This deviation is therefore not considered to have significantly affected the reliability of the study.2.419.605 T09066 EFC

17



## AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN

Amendments to the study plan

x The analytical method was optimized.

x Minor typing errors were corrected and further refinements to the analytical procedure were added.

x The lot number in the study plan was amended.

Deviations from the study plan

x Temperature was measured continuously next to the test in water. The temperature was not measured at the beginning and end of the test in replicates where living fish are present as stated in the study plan.

x Temperature was not also measured with a min / max digital thermometer as stated in the study plan. It was measured with a temperature sensor and data recorder.

x Temperature in the test room was not set at 23 oC as stated in the study plan but at 22oC as recommended by the guideline.

x The acquisition parameters in the analytical method were further refined and differ therefore slightly from the values stated in the study plan.



## CONCLUSION

In conclusion all critical quality criteria were met with the exception of the loading. Test substance concentrations were confirmed with a validated analytical method. Deviations from the guideline and amendments from the study plan were not critical to the validity of the study and were documented and justified. This study can be considered an accurate representation of the effects of the test

substance to Cyprinus carpio in the presence of 20 mg/L of humic acid.2.419.605 T09066 EFC

18



## REFERENCES

8.1 EPA Ecological Effects Test Guidelines OPPTS 850.1085 Fish Acute Toxicity Mitigated by

Humic Acid April 1996

8.2 EPA Ecological Effects Test Guidelines OPPTS 850.1075 Fish Acute Toxicity Test,

Freshwater and Marine April 1996

8.3 OECD Guidelines for testing of chemicals, 203: Fish, acute toxicity test (7/1992).

8.4 EEC Directive 79/831, Annex V, part C: Methods for the determination of ecotoxicity, part

C.1. Acute toxicity to fish (update 11/1989).

8.5 M.A. Hamilton, P.C. Russo and R.V. Thurston, 1977: ‘Trimmed Spearman-Kärber method for

estimating medial lethal concentrations in toxicity bioassays’. Env.Sci. & Technol. 11 , 714-719. Correction, 12 (1978)417.

8.6 C.E. Stephan, 1977: ‘Methods for calculating a LC 50 

### ’. In: Aquatic toxicology and hazard

evaluation, F.L. Mayer and J.L. Hamelink (Eds.), 65-84.2.419.605 T09066 EFC

19



### Table 2 OBSERVED SURVIVAL AND SUB-LETHAL EFFECTS 

## TABLES AND FIGURES

Concentration (mg/L) No.

Exposed 0 hours 4 hours 24 hours 48 hours 72 hours 96 hours

No. dead effects No.

dead effects No. dead effects No.dead effects No. dead effects No. dead effects

0 Control + HA I 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

0 Control + HA II 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

14.3 I 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

14.3II 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

25.7 I 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

25.7II 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

46.3I 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

46.3II 10 

### 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN

83.3 I 10 

### 10 AN 10 AN A 4 2A 4R 6*

83.3 II 10 

### 10 AN 10 AN A 7 1A 2R 3*

150.0 I 10 

### 10 AN A 10

150.0 II 10 

### 10 AN A 10

Observed Effects: AN = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; R = lying on bottom 

### *= Euthanized for ethical reasons.2.419.605 T09066 EFC

20 

### Figure 1:

Ac ute Fis h Te s t-9 6 Hr S urviva l Start D ate26/4 Tes t ID : T09066 E FC S a m p le ID : D u o m e e n 2 H T End D ate30/4 Lab ID : T& E E n viro n m e n ta l C h e m is try D E P T S am ple Type: Sam ple D ate: Protocol: EP A OP PTS 850,1085 Tes t S pecies : Co m m on C arp C om m ents : T09066 E FC Conc -m g/L 1 2 control 1,0000 1,0000 14,3 1,0000 1,0000 25,7 1,0000 1,0000 46,3 1,0000 1,0000 83,3 0,0000 0,0000 150 0,0000 0,0000

Not Fis he r 's 1 -Ta ile d Num ber Total Conc -m g/L Mean N-Mean Resp Resp Total N Exact P Critical Resp Number control 1,0000 1,0000 0 20 20 2 0 20 14,3 1,0000 1,0000 0 20 20 2 1,0000 0,050 0 0 20 25,7 1,0000 1,0000 0 20 20 2 1,0000 0,050 0 0 20 46,3 1,0000 1,0000 0 20 20 2 1,0000 0,050 0 0 20 83,3 0,0000 0,0000 20 0 20 2 20 20 150 0,0000 0,0000 20 0 20 2 20 20

Hypothe s is Te s t (1 -ta il, 0 .0 5 ) NOEC LOEC ChV TU Fis her's Exact Tes t 46,3 83,3 62,1031

Tr im m e d S pe a r m a n Ka rbe r Gra phic a l M e tho d Trim Le vel EC50

0,0% 62,103

62,103

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

1 1 0 100 1000 R e s p o n s e

D ose m g/L



### Table 3: Biomass and weight measurements of 10 fish Mass (g) Length (cm)

0.3 3.2

0.21 3.4

0.34 3.3

0.24 3.1

0.30 3.8

0.31 3.2

0.35 3.4

0.20 3.2

0.23 3.4

0.35 3.0

Average length was 3.3 cm and measured average biomass 0.28 g The measured fish are a representative sample and not all fish have been measured.2.419.605 T09066 EFC

21 

### Table 4: Oxygen measurements (mg/L)

Dilution (mg/L) 0 hours 48 hours

old 48hours

new 96 hours

0 Control 8.4 8.2 8.8 7.9

0 Control 8.4 8.1 8.9 8.4

14.3 I 8.3 8.2 8.8 8.1

14.3II 8.4 7.6 8.8 7.3

25.7I 8.3 7.6 8.7 8.0

25.7II 8.3 7.7 8.7 8.1

46.3I 8.3 7.2 8.6 8.0

46.3II 8. 3 7.4 8.6 8.2

83.3 I 8.2 7.0

-

83.3 II 8.2 7.0

-

150 I 8.3

-

150 II 8.3

- 

### - = Not measured (no surviving fish)

Table 5: pH-measurements

Dilution (mg/L) 0 hours 48 hours

old 48hours

new 96 hours

0 Control 8.2 8.0 8.1 7.9

0 Control 8.2 8.0 8.1 7.9

14.3 I 8.1 8.1 8.1 7.9

14.3II 8.2 8.0 8.1 7.8

25.7I 8.2 8.2 8.2 8.0

25.7II 8.2 8.1 8.2 7.9

46.3I 8.2 8.0 8.1 7.9

46.3II 8.2 8.0 8.1 7.9

83.3 I 8.2 8.0

-

83.3 II 8.2 8.0

-

150 I 8.2

-

150 II 8.2

- 

### - = Not measured (no surviving fish)2.419.605 T09066 EFC

22 

### Table 6: NPOC m 

### easurements

Vial Sample NPOC

measurement mg/L 1 100mg/L standard calibration Criteria Met 5,10,50 & 100 2 Wash 0.0 3 10 mg/L control standard 9.9

4 Wash 0.0

5 T09066 EFC Control Sample t =0 8.3

6 Wash 0.0

7 10 mg/L control standard 9.7

8 Wash 0.0



### *All results are averages of at least three analytical measurements per sample *Calibration curves and control standards were all found to be in range according to SOP K7. *Raw data is archived in study journal * Non purgeable organic carbon was measured in a filtered sample hence may also be referred to as dissolved organic carbon or DOC2.419.605 T09066 EFC

23 

## ANNEX 1

Analytical procedure for the quantification of Duomeen 2 HT in aqueous samples using an LC-MS/MS system

Introduction A method is described to determine the concentration of the test substance in aqueous samples (i.e. Dutch Standard Water) at the start, middle and end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). The test substance is a mixture of a component with multiple carbon chain lengths (see figure I). Samples were quantified using a calibration curve. Although methods were detailed in the study plan the full method with all apparatus settings are reported here. All 3 components of the test chemical were measured. However only the C16-C16 component met the quality criteria for a valid analytical run. All reported analysis is therefore based on the C16-C16 component. The remaining measurements for other components have been archived in the raw data.

N R

R NH 2

Figure I: Structure of Duomeen 2 HT R= carbon chain (R= mainly C16 - C18) (C16-C16 &C18-C18 are also present)2.419.605 T09066 EFC

24 

### Analytical procedure The following conditions were used to determine the test compound at the start of the test and after 48 hours in the middle concentration.. The conditions for HPLC are given in table I,

for MS in table II and III.

Table I: Parameters for HPLC (Agilent Technologies 1200 series) Column: Zorbax SB-C18 2.1x30 mm 3.5μm Column temp.: 50 °C Flow: 0.5 ml/min

Mobile phase: A= HPLC water (with TFA, ammonia and Acetic acid)

B= Methanol:2-propanole (with TFA, ammonia and Acetic acid)

Gradient mobile phase: 0 min. 50 % A 50 % B

3 min. 0 % A 100 % B 6 min. 0 % A 100 % B post time 3 min. (50% A 50% B)

Injection volume: 10 μl

Table II: Parameters for MS (Agilent Technologies G 6410B, triple quad) Ion source: ElectroSpray Polarity: Positive mode Detector mode: Multiple Reaction Mode Delta EMV 300 V

Source parameters: Gas temperature: 350 °C Gas flow: 12 l/min Nebulizer: 60 psi Capillary voltage: 2500 V

Table III: MS acquisition parameters

Compound Precursor

Ion MS 1

RES Product

Ion MS 2

RES Dwell

time (Ms) Frag.

(V) Collision

Energy (V) C16, C16 523.3 Unit 506.3 Unit 100 225 35 C16, C18 551.3 Unit 534.3 Unit 100 180 35 C18, C18 579.3 Unit 562.3 Unit 100 185 402.419.605 T09066 EFC

25 

### Table IV: Time segment

Time segment Time

(min.) Scan type Ion Polarity Diverter

Valve Delta EMV

(V) Stored

1 0 MRM Positive To waste 0 Yes

2 4 MRM Positive To MS 300 Yes

3 5.6 MRM Positive To waste 0 Yes



### Due to the high amount of salt in the samples, MS was rinsed for 1.5 minutes with 2- propanol: HPLC water 30:70 (v/v) at a flow of 2 ml/min. between each injection to remove the salts which precipitated in the MS.

Preparation Mobile phase:

Mobile phase A 1 liter of HPLC water was added to a 1000 ml measuring cylinder, approximately 3⁄4 of this volume was added to a glass 1 liter bottle. Subsequently 0.65 ml of trifluoroacetic acid, 0.75 ml ammonium hydroxide solution (25% v/v), 1.15 ml acetic acid and the remaining HPLC water in the measuring cylinder was added. Content of the bottle was mixed thoroughly.

Mobile phase B 500 ml Methanol was added to a 1 liter glass bottle, subsequently 0.65 ml of trifluoroacetic acid, 0.75 ml ammonium hydroxide solution (25% v/v), 1.15 ml acetic acid and 500 ml 2- propanol was added. Content of the bottle was mixed thoroughly.

Sample preparation For the standards a stock solution of the test substance was made in leaching solution at a concentration of 316 mg/L. This Leaching solution contains 100 g/L MgCl 2 

### * 6H 2 

### O in a methanol: 2-propanol 50:50 (v/v) solution. From this stock solution dilutions were made in the concentration range 0 – 1000 μg/L for the calibration standards (n  5). Control standards from the middle range of the calibration series were analyzed at a minimum rate of one per ten samples and after each test series.

Aqueous samples Aqueous samples were diluted 50:50 (v/v) with leaching solution and filtered over a 0.45 μm Pall GHP membrane acrodisc syringe filter to remove the suspended solids and or algae before analysis. Further dilutions if necessary were done in leaching solution.2.419.605 T09066 EFC

26 

### Calculation of concentrations Quantification was done by measurement of peak areas. The concentrations of the test substance in the samples were calculated from the relation between concentration of standards (Cs) and peak area (PAs) obtained with linear regression analysis:

PAs = Cs * Slope + constant

slope constant areapeak Sample ion concentrat Sample 



### Method validation

Linearity The analytical method was found to be linear over the concentration range of 0 – 1000 μg/L with the system setting described above (paragraph: Analytical procedure).

Figure II: calibration curve (26-04-2010)

Repeatability Six aliquots of the lowest and highest standard concentration prepared from a single homogenous sample were analyzed. Mean values, standard deviations and variation of coefficients are given in Table below. Repeatability of the analytical method was concluded to be acceptable, hence the coefficient of variation was below the required 20%.2.419.605 T09066 EFC

27 

### Table V repeatability lowest and highest standard concentration Sample Response (counts) of

component C16,C16 Sample Response (counts) of component C16,C16 50 μg/L # 1 2845.649 1000 μg/L 1 54776.6449 50 μg/L # 2 2736.808123 1000 μg/L 2 53311.43133 50 μg/L # 3 2655.992116 1000 μg/L 3 55521.46646 50 μg/L # 4 2718.315 1000 μg/L 4 54896.95085 50 μg/L # 5 2691.384548 1000 μg/L 5 55059.35437 50 μg/L # 6 2690.0405 1000 μg/L 6 53791.73852

mean 2723.0 mean 54559.6 stdev 66.1 stdev 834.7 CV (%) 2.4 CV (%) 1.5 SD = standard deviation CV = coefficient of variation



### LOD/LOQ The Limit of Detection (LOD) was calculated using the signal to noise ratio (S/N). The LOD is calculated as 3 * S/N whereas the Limit of Quantification (LOQ) is calculated as 10 * S/N. S/N was determined with the software of the LC-MS/MS (i.e. Mass Hunter) based on peak height. (signal definition; peak height, noise definition; peak to peak, noise region; retention time 4.1 - 4.3 min.) The lowest concentration standard i.e. 50 μg/L on 26-04-2010 (start test) had a S/N ratio of 569.7 The LOD and LOQ were 0.26 and 0.88 μg/L, respectively.

Accuracy/Precision The control standards analyzed during sample series were all within the specified quality criteria i.e. remaining within 10% of the expected values based on the calibration curve standard, see table VI.

Table VI: Control standards Sample Response (counts) % of nominal

calibration st. 26-04-2010 23585 -- control st. 26-04-2010 21240 90.1

calibration st. 28-04-2010 19437 -- control st. 28-04-2010 18253 93.9 Control Standard =150μg/L2.419.605 T09066 EFC

28 

### Example chromatogram

Figure IV: calibration standard 50 μg/L2.419.605 T09066 EFC

29 

### Raw data Table VII: all analyzed samples during the test

Sample name Acq. Date-Time Dil. Response Calc. Conc. (μg/L) Final Conc.

(μg/L)

blanco 26-04-10 16:43 1 210.0 1.9 1.9 blanco 26-04-10 16:53 1 87.8 0.4 0.4 0 μg/L 26-04-10 17:03 1 59.7 0.0 0.0 50 μg/L 26-04-10 17:12 1 4331.8 54.1 54.1 150 μg/L 26-04-10 17:22 1 12553.0 158.1 158.1 300 μg/L 26-04-10 17:32 1 23585.0 297.7 297.7 500 μg/L 26-04-10 17:42 1 37765.3 477.1 477.1 750 μg/L 26-04-10 17:52 1 56751.8 717.3 717.3 1000 μg/L 26-04-10 18:01 1 81868.4 1035.1 1035.1

blanco 26-04-10 18:11 1 52.7 0.0 0.0 Control T=0 26-04-10 18:21 1 50.0 0.0 0.0 14.3 mg/L T=0 26-04-10 18:31 50 19815.8 250.0 12498.4 25.7 mg/L T=0 26-04-10 18:41 100 17168.4 216.5 21647.1 46.3 mg/L T=0 26-04-10 18:50 100 30753.5 388.4 38836.0 83.3 mg/L T=0 26-04-10 19:00 250 23236.5 293.2 73312.1 150 mg/L T=0 26-04-10 19:10 250 40507.7 511.8 127944.0 Stock I + 20 mg/L H.A. 26-04-10 19:20 250 40487.8 511.5 127881.0 Stock no H.A. 26-04-10 19:30 250 37949.8 479.4 119852.9 control st. 300 μg/L 26-04-10 19:39 1 21240.0 268.0 268.0 blanco 28-04-10 15:19 1 988.0 7.1 7.1 blanco 28-04-10 15:29 1 109.1 0.0 0.0 0 μg/L 28-04-10 15:39 1 25.8 0.0 0.0 50 μg/L 28-04-10 15:49 1 3464.0 48.0 48.0 150 μg/L 28-04-10 15:58 1 10230.7 160.0 160.0 300 μg/L 28-04-10 16:08 1 19436.8 312.3 312.3 500 μg/L 28-04-10 16:18 1 29666.3 481.6 481.6 750 μg/L 28-04-10 16:28 1 46556.6 761.1 761.1 1000 μg/L 28-04-10 16:38 1 60745.3 995.8 995.8 blanco 28-04-10 16:47 1 54.9 0.0 0.0 46.3 mg/L T=48h 28-04-10 16:57 100 24126.2 389.9 38992.2 Stock T=48h 28-04-10 17:07 250 28289.7 458.8 114702.3 control st. 300 μg/L 28-04-10 17:17 1 18253.0 292.7 292.7 50 μg/L # 1 28-04-10 17:27 1 2845.6 37.8 37.8 50 μg/L # 2 28-04-10 17:36 1 2736.8 36.0 36.0 50 μg/L # 3 28-04-10 17:46 1 2656.0 34.7 34.7 50 μg/L # 4 28-04-10 17:56 1 2718.3 35.7 35.7 50 μg/L # 5 28-04-10 18:06 1 2691.4 35.3 35.3 50 μg/L # 6 28-04-10 18:15 1 2690.0 35.2 35.2 1000 μg/L 1 28-04-10 18:25 1 54776.6 897.1 897.1 1000 μg/L 2 28-04-10 18:35 1 53311.4 872.8 872.8 1000 μg/L 3 28-04-10 18:45 1 55521.5 909.4 909.4 1000 μg/L 4 28-04-10 18:55 1 54897.0 899.0 899.0 1000 μg/L 5 28-04-10 19:04 1 55059.4 901.7 901.7 1000 μg/L 6 28-04-10 19:14 1 53791.7 880.8 880.82.419.605 T09066 EFC

30 Table VIII recoveries as a % of the nominal value 

### Test Samples T=0 % of nominal T=48h % of nominal

Control 

### -- 14.3 mg/L 

### 87.4 25.7 mg/L 

### 84.2 46.3 mg/L 

### 83.9 84.2 83.3 mg/L 

### 88.0 150 mg/L 

### 85.3 Stock + 20 mg/L H.A. 

### 85.3 76.5 Nominal stock concentration was 150 mg/L2.419.605 T09066 EFC

31 

## ANNEX 2