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 

## ACUTE TOXICITY OF DUOMEEN 2 HT TO 

# DANIO RERIO

Authors 

### M. Kean
Technical Support 

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

Date 

### 26/07/2010
Document number 

### 2.419.597
Study number 

### T09066 OFA

Client 

### AkzoNobel Surfactants America 

# REPORT 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 nvir o nm e n ta l The Nethe r la n ds w ww. a kz o n obe l. co m /te



## DUOMEEN 2 HT

A kzoNobel S ur f actants America M

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

Chemistry Department



### T 09066 O F A 

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# ABSTRACT

The acute toxicity of Duomeen 2 HT to the fresh water fish D anio Rerio (Zebra Fish) was tested under semi static conditions for 96 hours in accordance with EPA guideline OPPTS 850.1075 (8.2). The test was performed in compliance with Good Laboratory Practice (GLP).

Duomeen 2 HT is poorly soluble in water. A preliminary test for Zebra fish (D anio rerio ) gave a NOEC value for mortality of 10 mg/L and 100% mortality was observed at 50 mg/L. This data was used to estimate the concentration range for the definitive test. During the definitive test the test organisms proved significantly more sensitive than in the range finding test.

A definitive test with nominal test concentrations in the range of 1.45, 2.61, 4.7, 8.5, 15.4 27.7 and 50.0 mg/L including a control was carried out. The results indicate that the test substance had 100% lethal effect from 4.7 mg/L upwards. Two mortalities in one replicate were observed at 2.61 mg/L. At 1.45 mg/L no effect was observed.

Based on the test results a maximum concentration causing no lethal or physical effects (0-96h) and the lowest tested concentration causing lethal or physical effects (0-96h) can be derived of 1.45 mg/L and 2.61 mg/L respectively. Based on the available data an LC 50 

### was calculated of 3.22 mg/L. The lowest concentration causing 100% mortality within the period of the test was 4.7 mg/L.

The results of the chemical analyses show that the time weighted averages of measured test substance concentrations at the start and end of the test and before and after solution replacement differed less than 20% from the nominal test concentrations with the exception of the lowest test concentration which had a slightly lower recovery. The study director considers that there is enough evidence to justify basing the effect data on the nominal concentrations of the test chemical as received. Corrections have not been made for active ingredient. Duomeen 2 HT

Duomeen 2 HT T09066 O F A 2.419.597 

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# ACUTE TOXICITY OF DUOMEEN 2HT TO DANIO RERIO 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 17/5/2010 Experimental completion date 23/5/2010



# 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 281 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 76 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 2.419.597 T 09066 O F A 

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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 Dr Lange test kits. GLP is not claimed for these measurements.

Optimization of analytical method.

Stability study in test media

The Range finding study.

Study director M.Kean

Management R. Borgmann-Strahsen M

. K ean

R. Borgmann- S trahsen 2.419.597 T09066 O F A 

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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 Test (Calibration curve) 28.6.2010 Report & Journal 29.6.2010

Quality Assurance Unit H. van Daalen AkzoNobel Technology and Engineering Arnhem.

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# CONTENTS

INTRODUCTION 8
MATERIALS 9
METHODS 10
RESULTS 13
QUALITY CRITERIA 17
AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN 17
CONCLUSION 18
REFERENCES 19
ANNEX 1 24
ANNEX 2 37 2.419.597 T09066 O F A 

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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 D anio Rerio (Zebra Fish) under continuous exposure during 96 hours in a semi static test. Preliminary stability studies demonstrated that there was potential for the concentration of the test substance to fall below 80% of the nominal values during the test period. A semi static testing method was therefore considered the most appropriate testing method to maintain exposure as far as was possible according to OPPTS 850.1075 (8.2).

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 850.1075 Fish Acute Toxicity Freshwater and Marine. (EPA712–C–96–118 April 1996) With the following deviation from the guideline:

Due to the test animals displaying a higher sensitivity than expected based on the range finding test in the first 48 hours of testing, 2 additional concentrations were tested to ensure all desired endpoints could be determined and excessive animal use with a repeat test be avoided. A total of 7 concentrations were therefore tested. The same fish batch and identical test procedures and conditions were used. There were no other deviations to the guideline.

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. Duomeen 2 HT 2.419.597 T 09066 O F A 

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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.

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 laboratory Standard Operation Procedures.

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 2 H T 2.419.597 T 09066 O F A


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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 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 2.5 cm with a minimum of 2.3 and a maximum of 2.7 cm long. The average weight was 0.233 g. The mean biomass loading was 0.54 g/L. Batch 10032010 A was used for testing. Excessively small or large fish from this batch were not used for testing in order to meet the length 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. 2.419.597 T 09066 O F A 

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### Preparation of the stock solution Due to the low solubility of the test chemical large volumes of stock were required. 2 stocks at approximately 100 mg/L were made by weighing approximately 0.5 g 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 150 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 under agitation. The beaker used for preparation was filled and sonicated for a second and third time for one minute to remove residue on the glass wear. All liquid was transferred to the stock solution flask in the normal manner. The pH of the stock solutions was then reduced from 8.8 to 8.2 with HCL of the test media. The volume of the stock was then made up to 5 litres with DSW to reach the desired concentration. An opaque solution without visible particles was obtained

Preparation of the test solutions DSW medium were made 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 initially prepared: 4.7 8.5 15.4 27.7 and 50mg/L on 17/5/2010. 2 additional concentrations of (1.45 and 2.61 mg/L) were started on 19/5/2010 together with the refreshment of the original concentrations using identical procedures by amendment of the study plan. The additional concentrations were closed 48 hours later than the original concentrations to give 96 h exposure data for all test concentrations. A control containing DSW was also included in the test the control was extended for 48 hours as a control for the additional concentrations.

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 74.3 mg/L. Total hardness measured as a sum of calcium and magnesium carbonate or odH is approximately 13 odH. Test medium with the above mentioned specification was used for the acclimatization of the fish batch and for testing. 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. 2.419.597 T09066 O F A


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### Chemical analyses Chemical analysis was conducted at the start, the end, the stock and in the old and new concentrations before and after refreshment 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, 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.

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. 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 NOEC, LOEC and LC100 were derived directly from the test observations. 2.419.597 T09066 O F A 

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# RESULTS

Preliminary test The definitive test concentrations were estimated based on a 10 mg/L NOEC with zebra fish from preliminary testing. No lethality was observed at 10 mg/L. 100% mortality was observed at 50mg/L. This test was conducted with the same fish species, but a different batch to that used in the definitive test.

Definitive test The results of the test on survival and sub-lethal effects are summarized in detail in Table 2 on page 20. 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 50, 27.7, 15.4, 8.5 and 4.7 mg/L. Due to these results differing considerably from the non GLP preliminary test, 2 additional concentrations of 2.61 and 1.45 mg/L were added to the test by means of an amendment. 2 mortalities were observed in one replicate at 2.61 mg/L and all surviving fish in both replicates displayed reduced activity. At 1.45 mg/L no effects were observed.

Based on the observed effects a maximum concentration causing no effects (NOEC 96h) and the lowest tested concentration causing visible effects (LOEC 96 h) can be derived as 1.45 mg/L and 2.61 mg/L respectively. A NOEC can also be determined statistically using a Dunnetts test but the minor effects observed at 2.61 mg/L are not considered significant by this test and the non lethal effects are not taken into account. For this reason the NOEC was based on the observed effects. The NOEC was therefore 1.45 mg/L.

Based on the available data a 96h LC 50 

### was calculated of 3.22 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. 2.419.597 T09066 O F A

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### Observations When prepared the test solutions were slightly turbid with no particles. During the test in the higher concentrations slight precipitation was observed at 48 hours. This can be attributed to the poor solubility of the test substance and indicates that the substance is not indefinitely stable at the tested concentrations, hence the use of a semi static test. Considering the solutions were refreshed at 48 hours and the chemical analysis demonstrated sufficient average recovery this is not considered to have negatively influenced the test results and can be considered an inherent characteristic of the test chemical. No physical effects on the test animals were observed. The stability of this test chemical in solution and poor solubility should be taken into consideration during risk assessment.

Chemical analyses All test solutions were analyzed at the start and at the end of the test and both before and after refreshment of the solutions at 48 hours. All test concentrations were observed to be within ± 21% of the expected nominal concentrations based on time weighted means, with the exception of the lowest concentration that fell below this. Stock analysis demonstrated that in theory the correct amount of test substance was added to all replicates including the lowest concentration. At the lowest concentration greater loss as a percentage of the nominal concentration was observed. This is likely to be due to binding of the test chemical to glass surfaces or the fish themselves which is characteristic of these types of substances. The study director considers that there is sufficient evidence to justify the use of nominal concentrations for the calculation of endpoints as the exposure was maintained as far as was possible by replacement of the test solutions. Additionally the calculated LC50 occurred at a concentration where >80% analytical recovery was observed. A description of the analytical method and results tables is provided in Annex 1. 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 old % of nominal T=48h new % of nominal T=96h old % of nominal C16,C16 C16,C16 C16,C16 C16,C16 Control



### -- -- -- -- 1.45 

### 91.5 40.9 111.3 43.1 2.61 

### 122.2 58.1 111.2 56.6 4.7 

### 81.5 77.0 188.2* 83.5 8.5 

### 89.7 104.4 -- -- 15.4 

### 92.1 -- -- -- 27.7 

### 95.4 -- -- -- 50 

### 94.5 -- -- -- Stock 

### 99.0 -- 97.8 -- * outlier -- Not measured in replicates where no surviving fish were present C 16, C 16 C 16, C 16 C 16, C 16 C 16, C 16 2.419.597 T09066 O F A 

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### Table 1A Time weighted mean values as a percentage of the nominal concentrations.

Test Samples time weighted mean 0-48h (%) time weighted mean 48-96h (%) C16,C16 C16,C16 Control



### -- -- 1.45 

### 66.2 77.2 2.61 

### 90.1 83.9 4.7 

### 79.2 *135.8 8.5 

### 97.0
-- 15.4 

### 92.1**

-- 27.7 

### 95.4**

-- 50 

### 94.5**

-- ** values only based on T=0 measurements ---- Not measured in replicates where no surviving fish were present.



### Discussion Chemical analysis Only the C16 –C16 component was used for quantification during the test as the other components did not meet the quality criteria for storage. The test substance can be accurately quantified based on any of its components. See Annex 1.

The anomalous result highlighted with * at 48 h new is most likely an outlier. Based on the stock concentration, the measurement of the old solution and the amount of the stock that was added to this replicate such a high result is not possible and is considered invalid. The average based on this measurement highlighted in red should also be interpreted with care. Conclusions can still be drawn on the 0 – 48 and the 96 hour measurements for this concentration. The result is also not of great significance for the LC50 calculation as all fish died at this concentration.

Oxygen, pH, Temperature, Hardness Measurement Oxygen concentrations varied at the most from 9.0 at the start to 8.1 mg/L at the end of the test. The concentration with the highest variation in oxygen concentrations was the second control 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 0.1 pH units in any replicate and is therefore are 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. C16 –C16 component C 16, C 16 C 16, C 16 2.419.597 T 09066 O F A 

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### Water hardness was measured at the start of the test at 74.3 mg/L of CaC0 3 

### . This is acceptable based on a maximum hardness of 180mg/L (as CaC0 3 

### ) stated in the guideline. The total hardness (a sum of Mg and Ca) was measured as 13.1 odH (German degrees of hardness). 2.419.597 T 09066 O F A 

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# 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)

Variation in the test temperature was no 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.

The following quality criterion was not met:

The average measured concentrations of test substance did differ more than 20% of the nominal concentrations in the lowest test concentration. Evidence suggests that the correct amount of test substance was added to the replicate based on the stock analysis. Due to the remainder of the chemical analysis measuring around 80% or greater the study director considers that there is enough evidence to justify calculating the endpoints using nominal values. 2.419.597 T09066 O F A

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# AMENDMENTS TO AND DEVIATIONS FROM THE STUDY PLAN

Due to the higher sensitivity of the fish in the definitive test additional concentrations were added to the study plan by amendment to ensure all endpoints could be determined.

Analytical calibration procedures and settings were optimized and settings therefore differ slightly from that of the study plan. Minor typing errors regarding the quality criteria and an incorrect address were also amended.

The Batch/Lot number was amended after receiving the analysis certificate. 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.

An additional wash step to reduce salt precipitation was included in the analytical method to remove potential contamination in the LCMSMS apparatus. Settings differ slightly from those in the study plan. Definitive settings are reported in Annex 1.



# CONCLUSION
In conclusion all critical quality criteria were met and the test substance concentrations were analytically confirmed with a validated analytical method. Deviations from the guideline and study plan were documented and justified. After excluding the one analytical anomaly measured stability of the test substance at 4.7 mg/L can still be demonstrated by surrounding measurements and is not of influence to the LC 50 calculation.

The initial proposed testing concentrations were not sufficient to determine the required endpoints due to an unexpected difference in sensitivity between range finding and definitive fish batches. This was rectified by the addition of two additional concentrations by study plan amendment following identical procedures and using the same fish batch under identical test conditions. Additional concentrations were tested in preference to a complete test repeat for ethical reasons. The study director considers the data generated to be an accurate representation of the effects of the test substance to D.Rerio. 2.419.597 T 09066 O F A 

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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.

8.8 Akzo Nobel Surfactants America Toxicity of Duomeen 2HT to Cyprinus carpio mitigated with humic acid T09066 EFC Kean 2010

8.9 Akzo Nobel Surfactants America Toxicity of Duomeen 2HT to Brachdanio Rerio mitigated with humic acid T09066 EFB Kean 2010 Akzo Nobel, Surfactants America Acute Toxicity of Berol R-648

Akzo Nobel Surfactants America Toxicity of Duomeen 2HT

Akzo Nobel Surfactants America Toxicity of Duomeen 2HT 2.419.597 T 09066 O F A 

# SANITIZED2.419.597 T09066 OFA20Table 2:TABLES AND FIGURESOBSERVED SURVIVAL AND SUB-LETHAL EFFECTSTestConcentration(mg/L)No.Exposed17/5/2010 17/5/2010 18/5/2010 15/5/2010 20/5/2010 21/5/20100 hours 4 hours 24 hours 48 hours 72 hours 96 hoursNo.deadeffectsNo.deadeffects No. dead effectsNo.dead effectsNo.deadeffectsNo.deadeffectsControl I 77 AN 7 AN 7 AN 7 AN 7 AN 7 ANControl II 77 AN 7 AN 7 AN 7 AN 7 AN 7 AN4.7 77 AN 7 AN 7AN 7C43C 34.7 77 AN 7 AN 7 AN23C 2 *8.5 77 AN 7 AN4338.5 77 AN 7 AN 1C* 1*515.4 77 AN 7 AN715.4 77 AN 7 AN727.777 AN1A 5C627.777 AN7C750.077AN52N*50.077AN61N*19/5/2010 19/5/2010 20/5/2010 21/5/2010 22/5/2010 23/5/2010Control I77 AN7 AN7 AN7 AN7 AN7 ANControl II77 AN7 AN7 AN7 AN7 AN7 AN1.45 I77 AN7 AN7 AN7 AN7 AN7 AN1.45 II77 AN7 AN7 AN7 AN7 AN7 AN2.61 I77AN7 AN7AN7 AN16AN15C2.61 II77AN7 AN7AN7 AN7AN7CObserved Effects: AN = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; R = lying on bottom*= Euthanized for ethical reasons.C= reduced activity2.419.597T09066OFASANITIZED2.419.597 T09066 OFA

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### Figure 1: LC 50 Calculation

Acute Fish Test-96 Hr Survival Start Date: 17-mei Test ID: T09066 Sample ID: End Date: 23-mei Lab ID: Sample Type: Sample Date: Protocol: EPA 850,1075 Test Species: brachdanio Rerio Comments: OFA 96 hours

Conc-mg/L 1 2 0 1,0000 1,0000 1,45 1,0000 1,0000 2,61 0,7143 1,0000 4,7 0,0000 0,0000 8,5 0,0000 0,0000 15,4 0,0000 0,0000 27,7 0,0000 0,0000

50 0,0000 0,0000

Trimmed Spearman-Karber Trim Level EC50 95% CL 0,0% 3,2202 2,8849 3,5946 5,0% 3,2806 2,8822 3,7340 10,0% 3,3218 2,8019 3,9382 20,0% 3,3349 3,0943 3,5942 Auto-0.0% 3,2202 2,8849 3,5946

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 R e s p o n s e

Dose mg/L 2.419.597 T09066 O F A

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### Table 3: Biomass and weight measurements of 10 fish

Mass Grams Length CM 0.3 2.7 0.25 2. 5 0.11 2.6 0.25 2.7 0.20 2.3 0.25 2.6 0.24 2. 5
0.25 2. 5
0.25 2. 5 0.23 2.4

Average length was 2.78cm and measured average biomass loading was 0.5 g/liter The measured fish are a representative sample and not all fish have been measured.


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

Conc mg/L 0 hours 48 hours

new 48 hours old 96 hours old I II I II I II I II
Control 9.0 9.0 9.0 9.0 - 8. 5 8. 8 8.1
4.7 9.0 9.0 9.0 9.0 8.4 8.7 - 8. 9
8.5 8.9 8.9 - - 8. 5
15.4 8.9 8.9 27.7 8.9 8.9 50 8.9 8.9 A dditional concentrations
1.45 9.0 9.0 9.4 9.3 8.7 8.4 8. 9 8. 9

2.61 9.0 9.0 9.3 9.4 8. 5 8.3 8.3 8.7
- = Not measured Note Replicates were aerated 2.419.597 T09066 O F A

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### Table 5: pH-measurements

Conc mg/L 0 hours 48 hours

new 48 hours old 96 hours old I II I I II II I II
Control 8.2 8.2 8.3 8.3 - 8.2 8.1 8.1
4.7 8.2 8.2 8.3 8.3 8.2 8.2 8.1 -
8.5 8.2 8.2 - - 8.3
15.4 8.2 8.2 27.7 8.2 8.2 50 8.2 8.2 A dditional concentrations
1.45 8.3 8.3 8.1 8.1 8.1 8.1 8.1 8.1
2.61 8.3 8.3 8.1 8.1 8.0 8.0 8.1 8.1 

### - = Not measured 2.419.597 T09066 O F A

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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) 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. Previous analysis on this chemical with tests T09066 EFB and T09066 EFC reference 9.0 &9.1 has shown that quantification can be conducted on any of the components and almost identical recovery was observed. During this study however samples were stored. Only C16-C16 component met the required quality criteria for storage. The C16-C16 component only was therefore used for quantification of test substance.

N R

R NH 2

Figure I: Structure of Duomeen 2 HT R= carbon chain (R= mainly C16 - C18) (C16-C16& C18C18 are also present) 

### f Duomeen 2 HT

f Duomeen 2 HT 

### C16-C16 e C16-C16

y C16 - C18) (C16-C16& C18C18 2.419.597 T09066 O F A

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### Analytical procedure The following conditions were used for 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
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 40 C16, C16 C16, C18 C18, C18 2.419.597 T 09066 O F A 

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### 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



### Additional procedure 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.

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. 2.419.597 T 09066 O F A 

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### 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 – 500 μg/L with the system setting described above for all three components (paragraph: Analytical procedure). Figure II is example of calibration curve

Figure II: calibration curve component C16,C16 (17-05-2010) 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



### C16,C16 2.419.597 T 09066 O F A

C16,C16 

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### 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 Sample response C16,C16 10 μg/L 1 287.2 500 μg/L 1 11152.5 10 μg/L 2 284.0 500 μg/L 2 11286.6 10 μg/L 3 280.0 500 μg/L 3 10855.5 10 μg/L 4 269.3 500 μg/L 4 11193.4 10 μg/L 5 274.4 500 μg/L 5 11379.2 10 μg/L 6 264.8 500 μg/L 6 10978.8


### mean 276.6 mean 11141.0 stdev 8.7 stdev 194.2 CV (%) 3.1 CV (%) 1.7 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 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 88 for the components C16,C16. The LOD and LOQ were 0.34 and 1.14 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. C16,C16. p C 16,C1 6 p C 16,C1 6 2.419.597 T 09066 O F A 

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### Table VI: Control standards Sample Respons (counts) % of nominal C16,C16 C16,C16 

### calibration st. 17-05-2010 27447.9

-- 

### control st. 17-05-2010 27967.1 101.9 

### calibration st. 20-05-2010 3411.0

-- 

### control st. 20-05-2010 3414.8 100.1 

### calibration st. 21-05-2010 3582.9 -- 

### control st. 21-05-2010 3439.0 96.0 

### calibration st. 25-05-2010 3367.7 -- 

### control st. 25-05-2010 3569.7 106.0 Control standard = 150μg/l 

### Some samples were stored during the test, therefore stability samples (stored under the same conditions as the test samples) were analyzed to determine stability during storage. Stability samples 1 - 3 were taken and stored during the test. Stability samples 4 – 6 were prepared at the day of analysis. The results of table VII indicating that the storage conditions were only stable for compound C16,C16 not for C16,C18 and C18,C18. C16,C16 was therefore considered representative of the test substance in this study. This component only is reported. Results of the other two compounds can be found in the raw data (table VIII) for additional information.

Table VII: Stability storage conditions Sample Concentration (mg/L) % of nominal C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 

### Stability sample 1 2.59 1.97 1.30 99.1 75.4 49.8 

### Stability sample 2 2.60 2.00 1.26 99.4 76.7 48.3 

### Stability sample 3 2.53 1.90 1.19 96.8 72.9 45.7 

### Stability sample 4 2.84 2.78 2.65 108.6 106.6 101.6 

### Stability sample 5 3.16 3.12 3.13 121.1 119.4 119.8 

### Stability sample 6 2.97 2.99 3.02 113.8 114.7 115.7 

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

C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 2.419.597 T 09066 O F A 

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### Example chromatogram

Figure IV: calibration standard 10 μg/L

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

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

Figure VII: component C16,C16 of 10 μg/L calibration standard 2.419.597 T 09066 O F A



### C18,C18

t C16,C18

C16,C16 

# SANITIZED2.419.597 T09066 OFA31Table VIII: Raw data of 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 17-5-2010 15:46 1 187 0 0 624.2 0 0 864.5 0 00 μg/L 17-5-2010 16:03 1 181.5 0 0 496.4 0 0 545.3 0 010 μg/L 17-5-2010 16:18 1 2715.8 11.7 11.7 6000.4 9.5 9.5 7779 8.4 8.425 μg/L 17-5-2010 16:31 1 5177.2 25.3 25.3 11858 25.4 25.4 16376.1 28.1 28.175 μg/L 17-5-2010 16:44 1 13994.9 74 74 30448.9 75.7 75.7 36132.9 73.4 73.4150 μg/L 17-5-2010 16:58 1 27447.9 148.3 148.3 58380.5 151.4 151.4 72432.1 156.6 156.6300 μg/L 17-5-2010 17:11 1 55963.8 305.9 305.9 116900.7 309.8 309.8 138914.8 309.1 309.1500 μg/L17-05-10 17:241 90579.8 497.1 497.1 184750 493.6 493.6 218983.3 492.7 492.7blanco17-05-10 17:381 154.1 0 0 399.2 0 0 477.7 0 0Control T=0 17-05-10 17:51 2 96.7 0.00.0 274.5 0.0 0.0 318.3 0.0 0.04.7 mg/L T=0 17-05-10 18:04 20 35248.1 191.4 3828.4 74762.2 195.7 3914.5 76101.6 165.0 3300.88.5 mg/L T=0 17-05-10 18:18 20 69612.6 381.3 7625.1 136514.3 362.9 7259.0 141334.7 314.6 6292.615.4 mg/L T=0 17-05-10 18:31 50 51945.3 283.7 14183.0 106829.3 282.6 14128.1 121099.2 268.2 13411.227.7 mg/L T=0 17-05-10 18:44 100 48433.4 264.3 26425.9 100259.7 264.8 26477.2 118380.7 262.0 26199.150 mg/L T=0 17-05-10 18:58 200 43375.4 236.3 47263.5 92029.0 242.5 48496.5 109457.4 241.5 48305.7Stock T=0 17-05-10 19:11 250 72270.3 395.9 98984.7 148366.1 395.0 98761.1 185621.9 416.2 104046.0blanco 17-05-10 19:24 1 198.3 0.0 0.0 533.7 0.0 0.0 662.1 0.0 0.0control st. 150 μg/L 17-05-10 19:38 1 27967.1 151.2 151.2 59662.6 154.8 154.8 73236.2 158.5 158.5blanco 20-05-10 11:36 1 113.7 0.0 0.0 313.6 0.0 0.0 296.8 0.0 0.0blanco 20-05-10 11:50 1 17.4 0.0 0.0 45.1 0.0 0.0 54.4 0.0 0.00 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 1.21.2 704.2 5.0 5.0 771.0 0.0 0.025 μg/L 20-05-10 12:30 1 754.2 22.8 22.8 1628.1 28.1 28.1 1754.8 20.3 20.375 μg/L 20-05-10 12:43 1 2073.0 88.0 88.0 4202.4 92.3 92.3 4595.1 89.8 89.8150 μg/L 20-05-10 12:56 1 3411.0 154.2 154.2 6937.7 160.5 160.5 7534.0 161.8 161.8300 μg/L 20-05-10 13:10 1 6454.1 304.8 304.8 12913.9 309.6 309.6 13704.6 313.0 313.0500 μg/L 20-05-10 13:23 1 10277.2 494.0 494.0 20086.0 488.5 488.5 20797.7 486.7 486.7blanco 20-05-10 13:36 1 11.5 0.0 0.0 40.1 0.0 0.0 39.8 0.0 0.0pC16,16C16,C16C16,C16C16,18C16,C18C16,C18C18,18C18,C18C18,C182.419.597T09066OFASANITIZED2.419.597 T09066 OFA32Sample 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,C18Control T=48h old 20-05-10 13:49 2 8.0 0.0 0.0 22.4 0.0 0.0 26.4 0.0 0.0Control T=48h new 20-05-10 14:03 2 7.8 0.0 0.0 20.5 0.0 0.0 23.0 0.0 0.01.45 mg/L T=0 20-05-10 14:16 4 6997.6 331.7 1326.8 13370.8 321.0 1283.9 14100.7 322.7 1290.62.61 mg/L T=0 20-05-10 14:29 8 8350.1 398.6 3189.1 16295.3 393.9 3151.3 16687.1 386.0 3088.14.7 mg/L T=48h old 20-05-10 14:43 20 3951.4 181.0 3619.3 6896.8 159.5 3189.9 6564.3 138.1 2761.34.7 mg/L T=48h new 20-05-10 14:56 20 9230.8 442.2 8844.4 17220.7 417.0 8339.7 16532.1 382.2 7644.28.5 mg/L T=48h old 20-05-10 15:09 20 9258.7 443.6 8872.0 18439.7 447.4 8947.8 19198.2 447.5 8950.3Stock T=48h new 20-05-10 15:22 250 8206.9 391.6 97888.4 15670.4 378.3 94580.6 16469.0 380.7 95167.0blanco 20-05-10 15:36 1 23.0 0.0 0.0 56.9 0.0 0.0 66.8 0.0 0.0control st. 150 μg/L 20-05-10 15:49 1 3414.8 154.4 154.4 6711.9 154.9 154.9 6912.5 146.6 146.6blanco 21-05-10 12:13 1 7.0 0.0 0.0 27.7 0.0 0.0 27.3 0.0 0.0blanco 21-05-10 12:30 1 2.0 0.0 0.0 2.0 0.0 0.00 μg/L 21-05-10 12:47 1 4.0 0.0 0.0 2.0 0.0 0.010 μg/L 21-05-10 13:04 1 304.9 5.05.0 632.7 6.8 6.8 680.3 4.7 4.725 μg/L 21-05-10 13:18 1 751.1 24.9 24.9 1556.9 27.2 27.2 1720.7 26.2 26.275 μg/L 21-05-10 13:31 1 1956.9 78.5 78.5 4083.3 83.1 83.1 4500.4 83.5 83.5150 μg/L 21-05-10 13:44 1 3582.9 150.8 150.8 7530.5 159.3 159.3 8023.0 156.2 156.2300 μg/L 21-05-10 13:58 1 7017.9 303.7 303.7 14121.2 305.0 305.0 15200.1 304.3 304.3500 μg/L 21-05-10 14:11 1 11366.6 497.1 497.1 22620.2 492.9 492.9 24407.8 494.3 494.3blanco 21-05-10 14:24 1 13.4 0.0 0.0 35.4 0.0 0.0 37.0 0.0 0.0Control T=96h old 21-05-10 14:38 2 3.0 0.0 0.0 9.0 0.0 0.0 9.9 0.0 0.0Control T=96h new 21-05-10 14:51 2 3.0 0.0 0.0 3.0 0.0 0.01.45 mg/L T=48h old 21-05-10 15:04 4 3526.3 148.3 593.3 7202.7 152.0 608.2 7996.5 155.7 622.71.45 mg/L T=48h new 21-05-10 15:18 4 9258.7 403.4 1613.4 18439.7 400.5 1602.0 19198.2 386.8 1547.32.61 mg/L T=48h old 21-05-10 15:31 8 4453.4 189.6 1516.5 8773.8 186.8 1494.3 9474.6 186.2 1489.42.61 mg/L T=48h new 21-05-10 15:44 8 8350.1 362.9 2903.4 16295.3 353.1 2824.7 16687.1 335.0 2680.04.7 mg/L T=96h old 21-05-10 15:58 20 4603.7 196.3 3925.1 8561.1 182.1 3641.7 8930.9 175.0 3499.1Stock T=48h new 21-05-10 16:11 250 8428.1 366.4 91600.2 16472.1 357.0 89250.6 16871.5 338.8 84701.2pC16,16C16,C16) C16,C16C16,18C16,C18C16,C18C18,18C18,C18) C18,C182.419.597T09066OFASANITIZED2.419.597 T09066 OFA33Sample 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,C18blanco21-05-10 16:24 1 28.3 0.0 0.0 66.2 0.0 0.0 69.3 0.0 0.0control st. 150 μg/L 21-05-10 16:38 1 3439.0 144.4 144.4 6942.2 146.3 146.3 7278.2 140.9 140.9blanco 25-05-10 13:40 1 1.0 0.0 0.0 23.0 0.0 0.0blanco 25-05-10 13:53 1 17.1 0.0 0.0 34.9 0.0 0.0 42.6 0.0 0.00 μg/L 25-05-10 14:06 1 13.9 0.0 0.0 27.0 0.0 0.0 33.2 0.0 0.010 μg/L 25-05-10 14:20 1 242.4 9.09.0 560.6 9.1 9.1 556.4 8.2 8.225 μg/L 25-05-10 14:33 1 615.3 26.3 26.3 1334.5 26.3 26.3 1368.3 25.6 25.675 μg/L 25-05-10 14:46 1 1668.2 75.1 75.1 3539.8 75.4 75.4 3621.1 74.0 74.0150 μg/L 25-05-10 14:59 1 3367.7 153.9 153.9 7260.5 158.2 158.2 7520.0 157.6 157.6300 μg/L 25-05-10 15:13 1 6444.0 296.6 296.6 13601.1 299.4 299.4 14119.1 299.1 299.1500 μg/L 25-05-10 15:26 1 10845.8 500.8 500.8 22513.5 497.8 497.8 23408.0 498.4 498.4blanco 25-05-10 15:39 1 26.2 0.0 0.0 76.1 0.0 0.0 81.9 0.0 0.0Control T=144h old 25-05-10 15:52 2 21.1 0.0 0.0 35.1 0.0 0.0 30.9 0.0 0.01.45 mg/L T=96h old 25-05-10 16:06 4 3365.3 153.8 615.3 9623.0 210.8 843.3 11441.5 241.7 966.92.61 mg/L T=96h old 25-05-10 16:19 8 3965.0 181.6 1453.0 8720.0 190.7 1525.8 9030.9 190.0 1520.1stabiliteits monster 1 25-05-10 16:32 8 7018.2 323.3 2586.0 11203.5 246.0 1968.1 7744.0 162.4 1299.2stabiliteits monster 2 25-05-10 16:45 8 7043.1 324.4 2595.2 11393.0 250.2 2001.8 7513.5 157.5 1259.7stabiliteits monster 3 25-05-10 16:59 8 6855.3 315.7 2525.6 10838.6 237.9 1903.1 7124.5 149.1 1192.9stabiliteits monster 4 25-05-10 17:12 8 7690.4 354.4 2835.4 15773.8 347.8 2782.0 15621.9 331.4 2651.1stabiliteits monster 5 25-05-10 17:25 8 8567.6 395.1 3161.0 17646.2 389.4 3115.5 18388.0 390.7 3125.7stabiliteits monster 6 25-05-10 17:38 8 8056.9 371.4 2971.4 16957.3 374.1 2992.8 17769.7 377.5 3019.6control st. 150 μg/L 25-05-10 17:51 1 3569.7 163.3 163.3 7708.2 168.2 168.2 7871.4 165.1 165.110 μg/L 1 25-05-10 18:05 1 287.2 11.0 11.0 586.7 9.7 9.7 578.6 8.7 8.710 μg/L 2 25-05-10 18:18 1 284.0 10.9 10.9 597.3 9.9 9.9 578.0 8.7 8.710 μg/L 3 25-05-10 18:31 1 280.0 10.7 10.7 590.5 9.7 9.7 577.4 8.7 8.710 μg/L 4 25-05-10 18:44 1 269.3 10.2 10.2 584.4 9.6 9.6 562.2 8.4 8.410 μg/L 5 25-05-10 18:58 1 274.4 10.4 10.4 597.0 9.9 9.9 563.3 8.4 8.410 μg/L 6 25-05-10 19:11 1 264.8 10.0 10.0 578.5 9.5 9.5 613.8 9.5 9.5pC16,16C16,C16C16,C16C16,18C16,C18C16,C18C18,18C18,C18C18,C182.419.597T09066OFASANITIZED2.419.597 T09066 OFA34Sample nameAcq. Date-TimeDil.ResponseC16,16Calc. Conc.(μg/L)C16,C16Final Conc.(μg/L)C16,C16ResponseC16,18Calc. Conc.(μg/L)C16,C18Final Conc.(μg/L)C16,C18ResponseC18,18Calc. Conc.(μg/L)C18,C18Final Conc.(μg/L)C18,C18500 μg/L 1 25-05-10 19:24 1 11152.5 515.0 515.0 23286.0 515.0 515.0 23886.5 508.7 508.7500 μg/L 2 25-05-10 19:37 1 11286.6 521.2 521.2 23511.1 520.0 520.0 24461.9 521.0 521.0500 μg/L 3 25-05-10 19:51 1 10855.5 501.2 501.2 22532.8 498.2 498.2 23229.9 494.6 494.6500 μg/L 4 25-05-10 20:04 1 11193.4 516.9 516.9 23583.0 521.6 521.6 24652.1 525.1 525.1500 μg/L 5 25-05-10 20:17 1 11379.2 525.5 525.5 23845.7 527.4 527.4 24505.0 521.9 521.9500 μg/L 6 25-05-10 20:30 1 10978.8 507.0 507.0 22851.7 505.3 505.3 23875.9 508.4 508.4C16,16C16,C16C16,C16C16,18C16,C18C16,C18C18,18C18,C18C18,C182.419.597T09066OFASANITIZED2.419.597 T09066 OFA

35 

### Tables IX a-e Measured concentrations and time weighted means as mg/L and as percentages of the nominal concentrations. Table IXa
Test Samples T=0 (mg/L) T=48h old (mg/L) C16,C16 C16,C18 C18,C18 C16,C16 C16,C18 C18,C18 Control 

### < LOD < LOD < LOD < LOD < LOD < LOD 1.45 mg/L 

### 1.3 1.3 1.3 0.6 0.6 0.6 2.61 mg/L 

### 3.2 3.2 3.1 1.5 1.5 1.5 4.7 mg/L 

### 3.8 3.9 3.3 3.6 3.2 2.8 8.5 mg/L 

### 7.6 7.3 6.3 8.9 8.9 9.0 15.4 mg/L 

### 14.2 14.1 13.4 -- -- -- 27.7 mg/L 

### 26.4 26.5 26.2 -- -- -- 50 mg/L 

### 47.3 48.5 48.3 -- -- -- Stock 

### 99.0 98.8 104.0 -- -- --

Table IXb & Table IXc

Test Samples T=0 % of nominal T=48h old % of nominal T=48h new % of nominal T=96h old % of nominal C16,C16 C16,C16 C16,C16 C16,C16 Control



### -- -- -- -- 1.45 

### 91.5 40.9 111.3 43.1 2.61 

### 122.2 58.1 111.2 56.6 4.7 

### 81.5 77.0 188.2* 83.5 8.5 

### 89.7 104.4 -- -- 15.4 

### 92.1 -- -- -- 27.7 

### 95.4 -- -- -- 50 

### 94.5 -- -- -- Stock 

### 99.0 -- 97.8 -- * outlier Test Samples T=0 (mg/L) T=48h old (mg/L) T=48h New (mg/L) T=96h old (mg/L) C16,C16 C16,C16 C16,C16 C16,C16 Control 

### < LOD < LOD < LOD < LOD 1.45 mg/L 

### 1.3 0.6 1.6 0.6 2.61 mg/L 

### 3.2 1.5 2.9 1.5 4.7 mg/L 

### 3.8 3.6 8.8* 3.9 8.5 mg/L 

### 7.6 8.9 -- -- 15.4 mg/L 

### 14.2

-- -- -- 27.7 mg/L 

### 26.4

-- -- -- 50 mg/L 

### 47.3

-- -- -- Stock 

### 99.0 -- 97.9 -- C 16, C 16 

### (g) C 16, C 18 C 18, C 18 C 16, C 16 

### ( g C 16, C 18 C 18, C 18

C 16, C 16 C 16, C 16 C 16, C 16 C 16, C 16

C 16, C 16 C 16, C 16 C 16, C 16 C 16, C 16 2.419.597 T 09066 O F A


# SANITIZED2.419.597 T09066 OFA

36 

### Table IXd

Test Samples time weighted mean 0-48h (%) time weighted mean 48-96h (%) C16,C16 C16,C16 Control



### -- -- 1.45 

### 66.2 77.2 2.61 

### 90.1 83.9 4.7 

### 79.2 135.8 8.5 

### 97.0

-- 15.4 

### 92.1**

-- 27.7 

### 95.4**

-- 50 

### 94.5**

-- ** values only based on T=0 measurements Table IXe

Test Samples time weighted mean 0-48h (mg/L) time weighted mean 48-96h (mg/L) C16,C16 C16,C16 Control



### -- -- 1.45 

### 1.0 1.1 2.61 

### 2.4 2.2 4.7 

### 3.7 6.4 8.5 

### 8.2

-- 15.4 

### 14.2**

-- 27.7 

### 26.4**

-- 50 

### 47.3**

-- ** values only based on T=0 measurements ( ) C 16, C 16 C 16, C 16



### (g) C 16, C 16 C 16, C 16 

### ) 2.419.597 T 09066 O F A



# SANITIZED2.419.597 T09066 OFA

37 

# ANNEX 2 2.419.597 T 09066 O F A


# SANITIZED