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 

## DANIO RERIO MITIGATED WITH HUMIC ACID

Authors 

### M. Kean Technical Support 

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

### 26/7/2010 Document number 

### 2.418.625 Study number 

### T09066 EFB
Client 

### Akzo Nobel Surfactants America 

# REPORT Technolo gy & P O B o x 9300 T + 31 26 366 26 7 3 Engineering B.V. 6800 S B Arnh e m F + 31 26 366 5820

g g E nv i r o nm e n tal The Nethe r la n ds w ww. a kz o n obe l. co m /te Chemistry Department



## DUOMEEN 2 HT

M

. K ean I. G arttener, J. van Dam, B. Helming

A kzo Nobel S ur f actants America T 09066 EFB 

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## ABSTRACT
The acute toxicity of Duomeen 2 HT to the fresh water fish Danio Rerio (Zebra Fish) mitigated with humic acid 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 for mortality of 50 mg/L. This data was used to estimate the concentration range for the definitive test.
A definitive test with nominal test concentrations in the range of 10 to 104 mg/L including a control was carried out. The results indicate that the test substance had 100% lethal effect at 104 mg/L. 1 further mortality was observed at 58.3 mg/L. At the other test concentrations of 10.0, 18.0 and 32.4 mg/L, no effects were observed.
Based on the test results a maximum concentration causing no lethal or physical effects (0-96h) or NOEC and the lowest tested concentration causing lethal or physical effects (0-96h) or LOEC can be derived of 32.4mg/L and 58.3 mg/L respectively. Based on the available data an LC 50 

### was calculated of 74.7 mg/L. The concentration causing 100% mortality or LC 100 within the period of the test was 104 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. Duomeen 2 HT

Duomeen 2 HT 2.418.625 T 09066 EFB 

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## ACUTE TOXICITY OF DUOMEEN 2HT TO DANIO RERIO (ZEBRA FISH) 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 di rector 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. 

## DUOMEEN 2HT

Akzo Nobel S ur f actants America 2 81 Fields Lane B rewste r N ew Y or k 10 5 09 U SA M. G yimesi D

. Hillib o ld

AkzoNobel Technology and Engineering Environmental C hemistry departmen t V elperweg 7 6 P. O . Box 9300 6800 S B Arnhem Th e N et h er l an d s M

. K ean J.van Dam, I. G arttener, B.Helming H . van D aa l en R. Borgmann- S trahsen

AkzoNobel Technology & Engineering Arnhem

AkzoNobel Technology & Engineering Arnhem 2.418.625 T 09066 EFB 

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## GLP COMPLIANCE STATEMENT
The study was conducted in compliance with the following Good Laboratory Practice regulations:

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:

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

The range finding study.

Analytical method development, optimization, and stab ility testing. Study director

Management R. Borgmann-Strahsen R. Borgmann- S trahsen 2.418.625 T 09066 EFB


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### 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 20.5.2010 Study Plan 20.5.2010 20.5.2010 Test (Calibration Curve) 20.5.2010 9.7.2010 Report 10.7.2010

Quality Assurance Unit H. van Daalen H . van D aa l en AkzoNobel Technology and Engineering Arnhem. 2.418.625 T 09066 EFB 

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## CONTENTS

INTRODUCTION 8 MATERIALS 10 METHODS 11 RESULTS 14 QUALITY CRITERIA 16 AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 17 CONCLUSION 17 REFERENCES 18 ANNEX 1 23 ANNEX 2 33 2.418.625 T 09066 EFB 

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## INTRODUCTION
The purpose of this study was to determine the acute lethal toxicity of Duomeen 2 HT to the freshwater fish Danio Rerio (Zebra Fish) under continuous exposure during 96 hours in a semi static test with 20 mg/L humic acid dissolved in 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 for risk assessment in combination with test results from a standard fish test (where a semi static approach must be used to maintain exposure ) 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:

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

The control replicates were considered to be a representative sample of the test media as used in all test replicates. DOC analysis was conducted as NPOC on this replicate only.

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 concentrat ions at the start of the test and directly after refreshment.

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

Danio Rerio ( Zebra fish) were used for testing. This species is 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 this fish species was permitted for this type of test. 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. Duomeen 2 HT 2.418.625 T 09066 EFB 

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### 7 fish per replicate were used instead of 10 per replicate recommended in the guideline. Due to some variation in length and weight of the test batch from the supplier. 7 fish per replicate were selected instead of 10 to allow compliance with the length and weight criteria of the guideline. The nature of the test chemical is such that steep dose response curves are observed. For this reason it was decided that use of 7 fish would have almost no impact on the calculated endpoint. Body mass of the fish however is known to have more of an impact on the endpoint. Larger fish often being less susceptible. The study director therefore chose to minimize fish size variation by excluding larger animals from the test.

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. 2.418.625 T 09066 EFB 

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## 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 if 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 Standard Operation Procedure K 10 (8.10)

Test vessels As test vessels, 5 L aquaria containing 3 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. Duomeen 2H T 2.418.625 T 09066 EFB 

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### Test animals The test was performed with juvenile Zebra fish, 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 2.6 cm with a minimum of 2.3 and a maximum of 2.8 cm long. The average weight was 0.216 g. The mean biomass loading was 0.5 g/L. Batch 16032010 B was used for testing. Excessively small or large fish from this batch were deliberately not used for testing in order to meet the length and weight variation criteria of the guideline.


## 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. 14 fish in 2 replicates of 7 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. 2.418.625 T 09066 EFB 

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### Preparation of the stock solution Due to the low solubility of the test chemical large volumes of stock were required. 3 stocks at approximately 104 mg/L were made by weighing approximately 0.52g 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 were followed:
The test substance was ultra sonicated in 100 mL of DSW for approximately 8 minutes. Care was taken to avoid excessive heating of the test chemical. 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 under agitation. The beaker used for preparation was filled and sonicated for a second and third time for one minute to remove test substance residue on the glass wear. All liquid was transferred to the stock solution flask in the normal manner. Visible reaction with the dissolved humic acid was observed forming a finely dispersed precipitate. The pH of the stock solutions was then reduced from 8.8 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.
The following final nominal test concentrations were prepared: 10.0, 18.0, 32.4, 58.3, 104 mg/L. A control containing DSW and 20 mg/L humic acid was also included in the test.
Preparation of the test medium Standard laboratory Dutch Standard Water (DSW) was used as media. 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. Test medium with the above mentioned specification was prepared by an automatic dosing system for the acclimatization of the fish batch to be used for testing and for the definitive 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 water-saturated and purified by an activated charcoal and cotton filter. 2.418.625 T 09066 EFB 

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### Chemical analyses Chemical analysis was conducted in all concentrations 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 are specified in Annex 1. 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. DOC was measured as NPOC in the control at the beginning of the test.
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. DOC was measured using a Shimadzu NPOC analyzer following the standard laboratory operating procedures for this apparatus. Details on standard curves, and control standards are recorded in the study journal. The sample was filtered through a 0.45μm filter prior to analysis. 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 no effects the lowest concentration causing effects and the concentration causing 100% mortality were derived directly from the test observations. 2.418.625 T 09066 EFB 

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## RESULTS
Range finding test The test concentrations were estimated based on a 50 mg/L NOEC with zebra fish from preliminary testing. No lethality was observed at 50 mg/L. Behavioral effects were however observed at 1 mg/L.

Definitive test The results of the test on survival and sub-lethal effects are summarized in Table 2 on page 19. 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 100% lethal effect at 104 mg/L after 48 hours. 1 mortality and 1 fish with reduced activity were observed at 58 mg/L after 96 hours. See figure 1 on page 20 for the dose response curve taken from Toxcalc 5.0.23 software.
Based on the observed effects a maximum concentration causing no effects (NOEC) and the lowest tested concentration causing visible effects (LOEC) can be derived as 32.4 mg/L and 58.3 mg/L respectively.

Based on the available data a 96h LC 50 

### was calculated of 74.7 mg/L using the Trimmed Spearman Karber method and Toxcalc 5.0.23 software.
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 on page 21

Other observations Interaction of the test substance with the humic acid was clearly seen as expected in all test replicates during the test period. A brown sediment was formed in all concentrations except the control.

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 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 were used for endpoint calculations. 2.418.625 T 09066 EFB 

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### A full description of the analytical method and results table is provided in Annex 1. A summary of the observed measurements as a percentage of the nominal concentration is reported in table 1 below: All components of the test substance were analyzed. Table 1 :Measured concentrations as a percentage of the nominal Test Samples T=0 % of nominal T= 48h % of nominal C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 Control


### -- -- -- -- -- -- 10 mg/L 

### 102.6 104.0 101.4 18 mg/L 

### 94.2 95.5 95.7 32.4 mg/L 

### 94.5 95.0 94.8 94.9 93.0 95.3 58.3 mg/L 

### 101.2 103.8 105.6 104 mg/L 

### 94.9 99.1 98.5 Stock* 

### 95.2 97.1 95.9 98.3 97.8 98.5
Oxygen, pH, Temperature, Hardness and DOC Measurement Oxygen concentrations varied at the most from 8.9 at the start to 7.9 mg/L at the 48 hour refreshment. The concentration with the highest variation in oxygen concentrations was the 32.4 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 .
The pH did not vary more than 0.2 pH units in any replicate and is therefore considered acceptable according to the guideline. The results of the pH measurements are summarized in Table 5 .
The temperature in the control test vessels during the test period ranged from 20.4 to 21.1oC. 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 in line with the relevant guideline.
Water hardness was measured at the start of the test as 74 mg/L of CaCO 3 

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

### ) stated in the guideline. The total hardness (a sum of Mg and Ca) was measured as 13.1 odH (German degrees of hardness). C 16, C 16 C 16, C 18 C 18, C 18 C 16, C 16 C 16, C 18 C 18, C 18 2.418.625 T 09066 EFB 

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### DOC was measured as NPOC using a Shimadzu Total organic Carbon Analyzer (TOC-V CPH) apparatus at the start of the test in the control only. The test media used was identical for the whole test. The control is therefore considered representative of all test replicates prior to addition of the test chemical. All NPOC measurements were analyzed at least in triplicate and an average is reported. A calibration and standard samples were analyzed in the same run as test sample to ensure system accuracy. The result of the measurement is displayed in Table 6. Previous studies (8.7) with surface active substances have shown that due to interaction with the test substance NPOC measurements in the test concentrations do not produce meaningful results hence the measurement of the control only.



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

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

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

The measured concentrations of test substance at the start of the test did not differ more than 20% of the nominal concentrations.

Deviations in the test temperature was not greater than ± 2 °C.

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

The length of the fish used did not exceed 3.0 cm (OECD Criteria)

The average load of fish in the test aquaria did not exceed 0.5 g/L. 2.418.625 T 09066 EFB 

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## AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN Amendments to the study plan

The analytical method was optimized

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

The Batch/Lot number was amended after receiving the analysis certificate from the sponsor. Deviations from the study plan

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 were present as stated in the study plan.

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

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.

The acquisition parameters in the analytical method were further refined and a rinsing step in the method was added. Described method differs therefore from that stated in the study plan.

7 fish per replicate were tested instead of 10 fish per replicate as stated in the study plan due to variation in fish size in the test batch. Due to the steep effect curve 100 % effect was observed from one concentration to the next and due to the lack of partial responses the use of 7 test animals per replicate instead of 10 has not affected the accuracy of the calculated endpoints.


## CONCLUSION In conclusion all quality criteria were met and the test substance concentrations were analytically confirmed with a validated analytical method. Deviations from the guideline were documented and justified. This study can be considered an accurate representation of the effects of the test substance to Danio Rerio in the presence of 20 mg/L of humic acid. 2.418.625 T 09066 EFB 

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## 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, 2 03: 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. 8.7 Akzo Nobel, Surfactants America Acute Toxicity of Berol R-648 To Danio Rerio In The Presence of 20 mg/L Humic Acid, T09023OFA Kean 2009. Akzo Nobel, Surfactants America Acute Toxicity of Berol R-648 2.418.625 T 09066 EFB 

# SANITIZED2.418.625 T09066 EFBTable 2:TABLES AND FIGURESOBSERVED SURVIVAL AND SUB-LETHAL EFFECTSTestConcentration(mg/L)No.Exposed0 hours 4 hours 24 hours 48 hours 72 hours 96 hoursNo.deadeffectsNo.deadeffectsNo. dead effectsNo.dead effectsNo. dead effectsNo. dead effects0 Control + HA I 7AN AN AN AN AN AN0 Control + HA II 7AN AN AN AN AN AN10 I 7AN AN AN AN AN AN10II 7AN AN AN AN AN AN18 I 7AN AN AN AN AN AN18II 7AN AN AN AN AN AN32.4I 7AN AN AN AN AN AN32.4II 7AN AN AN AN AN AN58.3 I7ANANANANAN16AN58.3 II7ANANANANAN1C6AN104 I7ANAN43 *104 II7ANAN31* 3 C3Observed Effects: AN = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; R = lying on bottom*= Euthanized for ethical reasons.C= reduced activity2.418.625T09066EFBSANITIZED2.418.625 T09066 EFB



### Figure 1: Acute Fish Test-96 Hr Survival Start: 17/5 Test ID: T 09066 Sample ID: End:21/5 Lab ID: Sample Type: Sample Date: Protocol: OPPTS 850.1085 Test Species: BR-Brachydanio rerio Comments: Toxicity Duomeen 2HT to Brachdanio Rerio Mitigated with 20mg/L Humic A

Conc-mg/L 1 2 control 1,0000 1,0000 10 1,0000 1,0000 18 1,0000 1,0000 32,4 1,0000 1,0000 58,3 0,8571 1,0000 104 0,0000 0,0000

Trimmed Spearman-Karber Trim Level EC50 95% CL 0,0% 74,690 68,929 80,933 5,0% 76,005 68,014 84,934 10,0% 76,152 72,714 79,753 20,0% 76,152 72,714 79,753 Auto-0.0% 74,690 68,929 80,933

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

1 10 100 1000 R e s p o n s e

Dose mg/L y Duomeen 2HT t 2.418.625 T 09066 EFB



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

0.22 2.7 0.24 2. 5 0.21 2. 8 0.21 2. 5 0.22 2.4 0.23 2.6 0.19 2. 5 0.20 2. 5 0.26 2.7 0.18 2.3
Average length was 2.6 cm and measured average biomass 0.5 g The measured fish are a representative sample and not all fish have been measured. 

### Table 4: Oxygen measurements (mg/L)
Dilution (mg/L) 0 hours 48 hours New 48 Hours

Old 96 hours

0 Control 8. 9 8. 9 8. 5 8. 8 0 Control 8. 9 9.0 8.4 8. 5 10. I 8. 9 9.1 8. 5 8. 8 10.II 8. 9 9.1 8.2 8.3 18.I 8. 9 9.1 8.4 8.4 18. II 8. 9 9.0 8.3 8.7 32.4I 8. 9 9.0 7. 9 8.1 32.4II 8. 9 9.0 8.6 8.4 58.3 I 8. 8 9.0 8.6 8.7 58.3 II 8. 8 9.0 8. 5 8.6 104 I 8.7 - - - 104 II 8.7 - - - 

### - = Not measured (no surviving fish) 2.418.625 T 09066 EFB 

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### Table 5: pH-measurements
Dilution (mg/L) 0 hours 48 hours New 48 Hours

Old 96 hours

0 Control 8.2 8.3 8.3 8.1 0 Control 8.2 8.3 8.3 8.1 10. I 8.2 8.4 8.2 8.1 10.II 8.2 8.4 8.2 8.1 18.I 8.2 8.4 8.2 8.1 18. II 8.2 8.4 8.2 8.1 32.4I 8.2 8.4 8.2 8.1 32.4II 8.2 8.4 8.2 8.1 58.3 I 8.2 8.4 8.3 8.1 58.3 II 8.2 8.4 8.2 8.1 104 I 8.2 - - 

### - 104 II 8.2 - - 

### - - = Not measured (no surviving fish) Table 6: NPOC m 

### easurements

Vial Sample NPOC measurement mg/L 9 T09066 EFB Control + 20 mg/L HA 6.6



### *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 water sample hence may also be referred to as DOC 2.418.625 T 09066 EFB


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## ANNEX 1
Analytical procedure for the quantification of Duomeen 2 HT in aqueous samples using an LCMS/MS system

Introduction A method is described to determine the concentration of the test substance in aqueous samples (i.e. Dutch Standard Water).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 components 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 three components were analyzed in the test media. Results showed that quantification based on each of the three components met the required accuracy and repeatability criteria. Regardless of which component was analyzed almost identical recoveries were observed. Software used for integrations was mass hunter Quantitative analysis version 13.01.

N R

R NH 2

Figure I: Structure of Duomeen 2 HT R= carbon chain (R= mainly C16 - C18) (C16 C16 and C18 C18 also occur)



### Analytical procedure The following conditions were used to determination of the test compound at the start of the test. 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 f Duomeen 2 HT

f Duomeen 2 HT C16 - C18) (C16 C16 C18 C18 2.418.625 T 09066 EFB 

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### 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

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 high amounts of salts 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. 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 40 C16, C16 C16, C18 C18, C18 2.418.625 T 09066 EFB 

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### 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 204 and 390 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 theconcentration range 0 – 500 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 before analysis. Further dilutions if necessary were done in leaching solution.

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 area peak Sample ion concentrat Sample  



### Method validation
Linearity The analytical method was found to be linear over the concentration range of 0 – 500μg/L with the system settings described above for all three components (paragraph: Analytical procedure). Figure II is example of calibration curve 2.418.625 T 09066 EFB


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C16,C16 - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs

Concentration (g/L) -50 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 5 5 4 x10

0 1 2 3 4 5 6 7 8 9 y = 181.0213 * x + 596.9105 R^2 = 0.99973679



### Figure II: calibration curve component C16,C16 (17-05-2010)
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%.
Table V repeatability lowest and highest standard concentration Sample response C16,C16 response C16,C18 response C18,C18 Sample response C16,C16 response C16,C18 response C18,C18 10 μg/L 1 287.2 586.7 578.6 500 μg/L 1 11152.5 23286.0 23886.5 10 μg/L 2 284.0 597.3 578.0 500 μg/L 2 11286.6 23511.1 24461.9 10 μg/L 3 280.0 590.5 577.4 500 μg/L 3 10855.5 22532.8 23229.9 10 μg/L 4 269.3 584.4 562.2 500 μg/L 4 11193.4 23583.0 24652.1 10 μg/L 5 274.4 597.0 563.3 500 μg/L 5 11379.2 23845.7 24505.0 10 μg/L 6 264.8 578.5 613.8 500 μg/L 6 10978.8 22851.7 23875.9 mean 276.6 589.1 578.9 mean 11141.0 23268.4 24101.9 stdev 8.7 7.3 18.7 stdev 194.2 491.2 539.0 CV (%) 3.1 1.2 3.2 CV (%) 1.7 2.1 2.2 SD = standard deviation CV = coefficient of variation * Testing of the repeatability of the method took place during study T09066 OFA 

### C16,C16

p C 16,C1 6 p C 16,C1 8 p C 18,C1 8 p C 16,C1 6 p C 16,C1 8 p C 18,C1 8 2.418.625 T 09066 EFB 

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### 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 4.0 - 4.5 min and 5.2 - 5.4 min.) The lowest concentration standard i.e. 10 μg/L on 20-05-2010 had the lowest S/N ratio of 222, 191 and 88 for the components C18,C18 C18,C16 and C16,C16 respectively. The LOD and LOQ were 0.14 and 0.45 μg/L for C18,C18, the LOD and LOQ were 0.15 and 0.52 μg/L for C18,C16 and the LOD and LOQ were 0.34 and 1.14 μg/L for C16,C16.
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 C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 calibration st. 17-05-2010 27447.9 58380.5 72432.1 -- -- --

control st. 17-05-2010 29034.0 61775.1 73057.0 105.8 105.8 100.9 calibration st. 20-05-2010 3414.8 6711.9 7534.0 -- -- --

control st. 20-05-2010 3308.8 6779.4 7125.1 96.9 101.0 94.6 

### C18,C18 C18,C16 C16,C16 r r C18,C18, r C18,C16 r C16,C16.

C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 2.418.625 T 09066 EFB 

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### Example chromatogram

3 x10

0 0.25 0.5 0.75

1 1.25 1.5 1.75

2 + TIC MRM (** -> **) 17-05-2010 sample4.d

1 1 2 2 3 3

Counts vs. Acquisition Time (min) 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 

### Figure IV: calibration standard 10 μg/L

3 x10

0 0.25 0.5 0.75

1 1.25 1.5 1.75

2 + MRM (579.3 -> 562.3) 17-05-2010 sample4.d

1 1 2 2 3 3

Counts vs. Acquisition Time (min) 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 

### Figure V: component C18,C18 of 10 μg/L calibration standard

3 x10

0 0.25 0.5 0.75

1 1.25 1.5 1.75

2 + MRM (551.3 -> 534.3) 17-05-2010 sample4.d

1 1 2 2 3 3

Counts vs. Acquisition Time (min) 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 

### Figure VI: component C16,C18 of 10 μg/L calibration standard C18,C18

C16,C18 2.418.625 T 09066 EFB 

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3 x10

0 0.25 0.5 0.75

1 1.25 1.5 1.75

2 + MRM (523.3 -> 506.3) 17-05-2010 sample4.d

1 1 2 2 3 3

Counts vs. Acquisition Time (min) 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 

### Figure VII: component C16,C16 of 10 μg/L calibration standard C16,C16 2.418.625 T 09066 EFB 

# SANITIZED2.418.625 T09066 EFBTable VII: all analyzed samples during the testSample nameAcq. Date-TimeDil.ResponseC16,16Calc. Conc.(μg/L) C16,C16Final Conc.(μg/L) C16,C16Response C16,18Calc. Conc.(μg/L) C16,C18Final Conc.(μg/L) C16,C18Response C18,18Calc. Conc.(μg/L) C18,C18Final Conc.(μg/L) C18,C18blanco 17-05-10 15:46 1 187.0 0.0 0.0 624.2 0.0 0.0 864.5 0.0 0.00 μg/L 17-05-10 16:03 1 181.5 0.0 0.0 496.4 0.0 0.0 545.3 0.0 0.010 μg/L 17-05-10 16:18 1 2715.8 11.7 11.7 6000.4 9.5 9.5 7779.0 8.4 8.425 μg/L 17-05-10 16:31 1 5177.2 25.3 25.3 11858.0 25.4 25.4 16376.1 28.1 28.175 μg/L 17-05-10 16:44 1 13994.9 74.0 74.0 30448.9 75.7 75.7 36132.9 73.4 73.4150 μg/L 17-05-10 16:58 1 27447.9 148.3 148.3 58380.5 151.4 151.4 72432.1 156.6 156.6300 μg/L 17-05-10 17:11 1 55963.8 305.9 305.9 116900.7 309.8 309.8 138914.8 309.1 309.1500 μg/L 17-05-10 17:24 1 90579.8 497.1 497.1 184750.0 493.6 493.6 218983.3 492.7 492.7blanco 17-05-10 19:51 1 154.1 0.0 0.0 399.2 0.0 0.0 477.7 0.0 0.0control T=0 17-05-10 20:05 2 84.7 0.0 0.0 160.0 0.0 0.0 158.5 0.0 0.010 mg/L T=0 17-05-10 20:18 50 37731.3 205.1 10256.9 79267.3 207.9 10396.2 92532.9 202.7 10135.918 mg/L T=0 17-05-10 20:31 50 62003.9 339.2 16961.3 129401.7 343.7 17184.4 154422.0 344.6 17231.932.4 mg/L T=0 17-05-10 20:45 100 56019.3 306.2 30616.5 116121.6 307.7 30772.6 138037.6 307.1 30706.758.3 mg/L T=0 17-05-10 20:58 250 43334.4 236.1 59022.8 91913.9 242.2 60542.8 111495.2 246.2 61550.4104 mg/L T=0 17-05-10 21:11 250 72075.2 394.9 98715.4 154792.1 412.4 103111.5 182815.2 409.7 102437.0Stock T=0 17-05-10 21:25 250 72295.9 396.1 99020.1 151684.7 404.0 101007.8 178187.8 399.1 99784.2blanco 17-05-10 21:38 1 310.5 0.0 0.0 812.5 0.0 0.0 886.6 0.0 0.0control st. 150 μg/L 17-05-10 21:51 1 29034.0 157.1 157.1 61775.1 160.6 160.6 73057.0 158.1 158.10 μg/L 20-05-10 12:03 1 15.5 0.0 0.0 43.2 0.0 0.0 47.2 0.0 0.010 μg/L 20-05-10 12:16 1 317.9 0.8 0.8 704.2 1.9 1.9 771.0 0.0 0.025 μg/L 20-05-10 12:30 1 754.2 22.4 22.4 1628.1 25.1 25.1 1754.8 23.4 23.475 μg/L 20-05-10 12:43 1 2102.7 89.2 89.2 4202.4 89.9 89.9 4595.1 92.4 92.4150 μg/L 20-05-10 12:56 1 3411.0 154.0 154.0 6937.7 158.8 158.8 7534.0 163.7 163.7300 μg/L 20-05-10 13:10 1 6454.1 304.7 304.7 12913.9 309.2 309.2 13704.6 313.4 313.4500 μg/L 20-05-10 13:23 1 10277.2 494.0 494.0 20086.0 489.7 489.7 20797.7 485.5 485.5blanco 20-05-10 13:36 1 11.5 0.0 0.0 40.1 0.0 0.0 39.8 0.0 0.0control st. 150 μg/L 20-05-10 15:49 1 3414.8 154.1 154.1 6711.9 153.1 153.1 6912.5 148.6 148.6blanco 20-05-10 16:02 1 8.0 0.0 0.0 16.5 0.0 0.0 19.0 0.0 0.0control T=48h new 20-05-10 16:16 2 3.0 0.0 0.0 10.6 0.0 0.0 10.6 0.0 0.0pC16,16C16,C16)C16,C16C16,18)C16,C18C16,C18e C18,18C18,C18C18,C182.418.625T09066EFBSANITIZED2.418.625 T09066 EFBSample nameAcq. Date-TimeDil.ResponseC16,16Calc. Conc.(μg/L) C16,C16Final Conc.(μg/L) C16,C16Response C16,18Calc. Conc.(μg/L) C16,C18Final Conc.(μg/L) C16,C18Response C18,18Calc. Conc.(μg/L) C18,C18Final Conc.(μg/L) C18,C1832.4 mg/L T=48h new 20-05-10 16:29 100 6509.0 307.4 30739.1 12604.0 301.4 30141.7 13507.9 308.6 30863.8Stock T=48h new 20-05-10 16:42 250 8616.0 411.7 102935.4 16673.4 403.8 100961.9 17660.5 409.4 102351.4control st. 150 μg/L 20-05-10 16:55 1 3308.8 148.9 148.9 6779.4 154.8 154.8 7125.1 153.8 153.8pC16,16C16,C16C16,C16C16,18C16,C18C16,C18C18,18C18,C18C18,C182.418.625T09066EFBSANITIZED32 

### Tables VIII & IX Results summarized in mg/L and as a percentage of the nominal concentrations.

Test Samples T=0 % of nominal T= 48h % of nominal C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 Control



### -- -- -- -- -- -- 10 mg/L 

### 102.6 104.0 101.4 18 mg/L 

### 94.2 95.5 95.7 32.4 mg/L 

### 94.5 95.0 94.8 94.9 93.0 95.3 58.3 mg/L 

### 101.2 103.8 105.6 104 mg/L 

### 94.9 99.1 98.5 Stock* 

### 95.2 97.1 95.9 98.3 97.8 98.5
*= Stock concentration was 104 mg/L at T=0 and 103.9 mg/L at T=48h 

### Test Samples T=0 (mg/L) T=48h (mg/L) C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 Control 

### < LOD < LOD < LOD < LOD < LOD < LOD 10 mg/L 

### 10.3 10.4 10.1 18 mg/L 

### 17.0 17.2 17.2 32.4 mg/L 

### 30.6 30.8 30.7 30.7 30.1 30.9 58.3 mg/L 

### 59.0 60.5 61.6 104 mg/L 

### 98.7 103.1 102.4 Stock* 

### 99.0 101.0 99.8 102.9 101.0 102.4 C 16, C 16 C16,C18 C18,C18 C 16, C 16 C 16, C 18 C 18, C 18

C 16, C 16 C16,C18 C18,C18 C 16, C 16 C 16, C 18 C 18, C 18 

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## ANNEX 2 

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