Technology & 

Engineering B.V. 

PO Box 9300 

6800 SB Arnhem 

The Netherlands

T +31 26 366 2673 

F +31 26 366 5820 

Environmental www.akzonobel.com/te 

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

PO Box 9300 T +31 26 366 2673

Engineering B.V. g g 6800 SB Arnhem F +31 26 366 5820

Environmental The Netherlands www.akzonobel.com/te

DUOMEEN 2 HT 

AkzoNobel Surfactants America 

M. Kean

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

Technology &

Chemistry Department 

T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

2

 ABSTRACT 

The acute toxicity of Duomeen 2 HT to the fresh water fish Danio 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 (Danio 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 LC50 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 

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

3

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 director 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 

Surfactants America 

281 Fields Lane

Brewster

New York

10509

USA

M.Gyimesi

D.Hillibold

AkzoNobel Technology and Engineering 

Environmental Chemistry department

Velperweg 76 

P.O. Box 9300

6800 SB Arnhem

The Netherlands

M.Kean 

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

H. van Daalen

R. Borgmann-Strahsen 

AkzoNobel Technology & Engineering Arnhem

AkzoNobel Technology & Engineering Arnhem 

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

4

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.Kean 

R. Borgmann-Strahsen 

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

5

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

6

QUALITY ASSURANCE STATEMENT 

This report was audited by the Quality Assurance Unit contracted by AkzoNobel Technology and 

Engineering Arnhem. It is considered to be an accurate presentation of the methods and procedures 

applied in the course of the study and an accurate reproduction of the data recorded. 

Listed below are the dates of inspection of this study by the Quality Assurance Unit and the dates on 

which its findings were reported to Study Director and Management. 

Dates of inspection Phase of study Dates of QA reports 

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. 

H. van Daalen

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

7

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 OFA

SANITIZED
2.419.597 T09066 OFA 

8

 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. 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 (LC50),

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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

9

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 2HT

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

10 

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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

11 

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 CaCO3 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 NaHCO3, 20 mg of KHCO3, 200 mg 

of CaCl22H2O and 180 mg of MgSO47H2O. 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 OFA

SANITIZED
2.419.597 T09066 OFA 

12 

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 CaCO3 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 LC50 was calculated with the computer program TOXCALCTM 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 OFA

SANITIZED
2.419.597 T09066 OFA 

13 

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 LC50 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 OFA

13

SANITIZED
2.419.597 T09066 OFA 

14 

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

C16,C16 C16,C16 C16,C16 C16,C16

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

15 

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 

C16,C16 C16,C16 

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

16 

Water hardness was measured at the start of the test at 74.3 mg/L of CaC03. This is acceptable based 

on a maximum hardness of 180mg/L (as CaC03) 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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

17 

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 OFA

17

SANITIZED
2.419.597 T09066 OFA 

18 

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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

19 

REFERENCES

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

Humic Acid April 1996 

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

Freshwater and Marine April 1996 

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

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

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

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

estimating medial lethal concentrations in toxicity bioassays’. Env.Sci. & Technol. 11, 714-719. 

Correction, 12 (1978)417. 

8.6 C.E. Stephan, 1977: ‘Methods for calculating a LC50’. 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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

20 

Table 2: TABLES AND FIGURES 

OBSERVED SURVIVAL AND SUB-LETHAL EFFECTS Test Concentration(mg/L) 

No.

Exposed 

17/5/2010 17/5/2010 18/5/2010 15/5/2010 20/5/2010 21/5/2010

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

No. 

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

Control I 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN Control II 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN 4.7 7 7 AN 7 AN 7AN 7C 4 3C 34.7 7 7 AN 7 AN 7 AN 2 3C 2 * 8.5 7 7 AN 7 AN 4 3 38.5 7 7 AN 7 AN 1C* 1* 515.4 7 7 AN 7 AN 715.4 7 7 AN 7 AN 727.7 7 7 AN 1A 5C 627.7 7 7 AN 7C 750.0 7 7AN 5 2N*50.0 7 7AN 6 1N*

19/5/2010 19/5/2010 20/5/2010 21/5/2010 22/5/2010 23/5/2010

Control I 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN Control II 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN 1.45 I 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN 1.45 II 7 7 AN 7 AN 7 AN 7 AN 7 AN 7 AN 2.61 I 7 7AN 7 AN 7AN 7 AN 1 6AN 1 5C2.61 II 7 7AN 7 AN 7AN 7 AN 7AN 7CObserved Effects: AN = appearing normal; E = erratic swimming; A = surfacing; N = loss of equilibrium; R = lying on bottom *= Euthanized for ethical reasons. 2.419.597 

C= reduced activity

T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

21 

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 OFA

21

SANITIZED
2.419.597 T09066 OFA 

22 

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

Additional 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 OFA

22

SANITIZED
2.419.597 T09066 OFA 

23 

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

Additional 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 OFA

23

SANITIZED
2.419.597 T09066 OFA 

24 

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

NH2

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 OFA

24

SANITIZED
2.419.597 T09066 OFA 

25 

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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

26 

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 MgCl2 * 6H2O in a 

methanol: 2-propanol 50:50 (v/v) solution. From this stock solution dilutions were made in the 

concentration 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 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

27 

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

Sample peak area constant

Sample concentration  

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 55

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 T09066 OFA

C16,C16 

SANITIZED
2.419.597 T09066 OFA 

28 

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

C16,C16

p

C16,C16

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

29 

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 T09066 OFA

SANITIZED

C16,C16 C16,C16 
2.419.597 T09066 OFA 

30 

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 T09066 OFA

C18,C18 

t C16,C18 

C16,C16 

SANITIZED
2.419.597 T09066 OFA 

31 

Table VIII: Raw data of analyzed samples during the test 

Sample name Acq. Date-Time Dil. Response C16,16 Calc. Conc. (μg/L) C16,C16 Final Conc. (μg/L) C16,C16 Response C16,18 Calc. Conc. (μg/L) C16,C18 Final Conc. (μg/L) C16,C18 Response C18,18 Calc. Conc. (μg/L) C18,C18 Final Conc. (μg/L) C18,C18 

blanco 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 8.4 25 μg/L 17-5-2010 16:31 1 5177.2 25.3 25.3 11858 25.4 25.4 16376.1 28.1 28.1 75 μg/L 17-5-2010 16:44 1 13994.9 74 74 30448.9 75.7 75.7 36132.9 73.4 73.4 150 μg/L 17-5-2010 16:58 1 27447.9 148.3 148.3 58380.5 151.4 151.4 72432.1 156.6 156.6 300 μg/L 17-5-2010 17:11 1 55963.8 305.9 305.9 116900.7 309.8 309.8 138914.8 309.1 309.1 500 μg/L 17-05-10 17:24 1 90579.8 497.1 497.1 184750 493.6 493.6 218983.3 492.7 492.7 blanco 17-05-10 17:38 1 154.1 0 0 399.2 0 0 477.7 0 0Control T=0 17-05-10 17:51 2 96.7 0.0 0.0 274.5 0.0 0.0 318.3 0.0 0.0 4.7 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 3300.8 8.5 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 6292.6 15.4 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 13411.2 27.7 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 26199.1 50 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 48305.7 Stock T=0 17-05-10 19:11 250 72270.3 395.9 98984.7 148366.1 395.0 98761.1 185621.9 416.2 104046.0 blanco 17-05-10 19:24 1 198.3 0.0 0.0 533.7 0.0 0.0 662.1 0.0 0.0 control st. 150 μg/L 17-05-10 19:38 1 27967.1 151.2 151.2 59662.6 154.8 154.8 73236.2 158.5 158.5 blanco 20-05-10 11:36 1 113.7 0.0 0.0 313.6 0.0 0.0 296.8 0.0 0.0 blanco 20-05-10 11:50 1 17.4 0.0 0.0 45.1 0.0 0.0 54.4 0.0 0.0 0 μg/L 20-05-10 12:03 1 15.5 0.0 0.0 43.2 0.0 0.0 47.2 0.0 0.0 10 μg/L 20-05-10 12:16 1 317.9 1.2 1.2 704.2 5.0 5.0 771.0 0.0 0.0 25 μg/L 20-05-10 12:30 1 754.2 22.8 22.8 1628.1 28.1 28.1 1754.8 20.3 20.3 75 μg/L 20-05-10 12:43 1 2073.0 88.0 88.0 4202.4 92.3 92.3 4595.1 89.8 89.8 150 μg/L 20-05-10 12:56 1 3411.0 154.2 154.2 6937.7 160.5 160.5 7534.0 161.8 161.8 300 μg/L 20-05-10 13:10 1 6454.1 304.8 304.8 12913.9 309.6 309.6 13704.6 313.0 313.0 500 μg/L 20-05-10 13:23 1 10277.2 494.0 494.0 20086.0 488.5 488.5 20797.7 486.7 486.7 blanco 20-05-10 13:36 1 11.5 0.0 0.0 40.1 0.0 0.0 39.8 0.0 0.0 

p

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

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

32 

Sample name Acq. Date-Time Dil. Response C16,16 Calc. Conc. (μg/L) C16,C16 Final Conc. (μg/L) C16,C16 Response C16,18 Calc. Conc. (μg/L) C16,C18 Final Conc. (μg/L) C16,C18 Response C18,18 Calc. Conc. (μg/L) C18,C18 Final Conc. (μg/L) C18,C18 

Control 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 0.0 Control 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 0.0 1.45 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 1290.6 2.61 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 3088.1 4.7 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 2761.3 4.7 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 7644.2 8.5 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 8950.3 Stock 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 95167.0 blanco 20-05-10 15:36 1 23.0 0.0 0.0 56.9 0.0 0.0 66.8 0.0 0.0 control st. 150 μg/L 20-05-10 15:49 1 3414.8 154.4 154.4 6711.9 154.9 154.9 6912.5 146.6 146.6 blanco 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 5.0 5.0 632.7 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 84701.2

p

 C16,16 C16,C16 ) C16,C16 C16,18 C16,C18 C16,C18 C18,18 C18,C18 ) C18,C18

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

33 

Sample name Acq. Date-Time Dil. Response C16,16 Calc. Conc. (μg/L) C16,C16 Final Conc. (μg/L) C16,C16 Response C16,18 Calc. Conc. (μg/L) C16,C18 Final Conc. (μg/L) C16,C18 Response C18,18 Calc. Conc. (μg/L) C18,C18 Final Conc. (μg/L) C18,C18 

blanco 

21-05-10 16:24 1 28.3 0.0 0.0 66.2 0.0 0.0 69.3 0.0 0.0

control 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 9.0 9.0 560.6 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 9.5

p

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

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

34 

Sample name Acq. Date-Time Dil. Response

 C16,16 

Calc. Conc. 

(μg/L)

C16,C16 

Final Conc. 

(μg/L)

C16,C16 

Response 

C16,18 

Calc. Conc. 

(μg/L)

C16,C18 

Final Conc. 

(μg/L)

C16,C18 

Response 

C18,18 

Calc. Conc. 

(μg/L)

C18,C18 

Final Conc.

(μg/L)

C18,C18

500 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 23875.9 508.4 508.4

 C16,16

C16,C16 C16,C16

C16,18 

C16,C18 C16,C18

C18,18 

C18,C18 C18,C18

2.419.597 T09066 OFA

SANITIZED
2.419.597 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 --

C16,C16 

(g)

C16,C18 C18,C18 C16,C16

( g

C16,C18 C18,C18

C16,C16 C16,C16 C16,C16 C16,C16

C16,C16 C16,C16 C16,C16 C16,C16

2.419.597 T09066 OFA

SANITIZED
2.419.597 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 

( )

C16,C16 C16,C16 

(g)

C16,C16 C16,C16 

)

2.419.597 T09066 OFA

SANITIZED
2.419.597 T09066 OFA 

37 

ANNEX 2 

2.419.597 T09066 OFA

SANITIZED