DR.U.NOACK-LABORATORIEN 

Report Page 4 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Personnel Involved

Study Director: Dirk Scheerbaum 

 (Biologist) 

Deputy: Silke Fiebig 

 (Engineer, Biotechnologist) 

Technical Staff: Christian Rebl 

 

Quality Assurance Unit: Gudrun Möhrmann-Kalabokidis 

 (Biologist) 

Deputy: Ute Kutzner 

 (Technical Assistant) 

Head of Dr. Udo Noack 

Testing Facility: (Biologist) 
DR.U.NOACK-LABORATORIEN 

Report Page 5 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Table of Content 

 Page 

 Title Page 1 

 Statement of GLP Compliance 2 

 Statement of Quality Assurance Unit 3 

 Personnel Involved 4 

 Table of Content 5 

 List of Tables 5 

1 Summary 6

2 Characterisation Data of the Test Item 7

3 Method 8

4 Results 13

4.1 Definitive Test 13

5 Validity Criteria 15

6 Conclusions 15

7 Literature / References 15

8 Physicochemical Data 16

8.1 Water Quality Measurements 16

8.2 DOC Analysis of the Test Media for Detection of the Test Item 16

9 GLP-Certificate of DR.U.NOACK-LABORATORIEN 17

List of Tables 

 Page 

Table 1: LC-Values with 95 % Confidence Intervals 6

Table 2: Observations in the Test Vessels 13

Table 3: Cumulative Mortality [%] in the Test Vessels 13

Table 4: LC0-, LC100-, NOEC-Values (0 - 96 h) 14

Table 5: LC50-Values with 95 % Confidence Intervals 14

Table 6: Water Parameters during the Definitive Study 16

Table 7: DOC Content of the Test Media 16
DR.U.NOACK-LABORATORIEN 

Report Page 6 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

1 Summary 

The acute toxicity of the test item DOBA (batch no.: BES06024) to fish (zebrafish) was 

determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2006-

07-27 to 2006-07-31 at DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany. A static limit test 

with a saturated solution was conducted. The preparation method has been chosen with regard 

to the test item properties and in accordance with the relevant OECD guidance document 

(2000). Duration of the test was 96 h. 7 test organisms were exposed to the saturated solution 

and the control. Water quality parameters pH-value, temperature and oxygen-saturation 

measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits. No 

test item analysis was carried out according to the sponsor. The acute toxicity of the test item 

DOBA is summarised in the following table: 

 Table 1: LC-Values with 95 % Confidence Intervals 

 

Test duration [h] LC50 p = 95 % 

24 > Saturated solution not applicable 

48 > Saturated solution not applicable 

72 > Saturated solution not applicable 

96 > Saturated solution not applicable 

LC100 = > Saturated solution 

Lowest test item concentration 

with 100 % mortality after 96 h 

LC0 = Saturated solution 

Highest test item concentration 

with 0 % mortality after 96 h 

NOEC (0-96 h) = 

No observed effect concentration 

Saturated solution 
DR.U.NOACK-LABORATORIEN 

Report Page 7 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

2 Characterisation Data of the Test Item 

TEST ITEM DOBA 

Batch Number BES06024 

Purity > 98.1 % 

Appearance Colourless solid 

Water solubility < 1 g/L 

Stability Not specified 

Density 0.55 g/L (20 °C) 

Melting point 143 – 145 °C 

 

Expiry date 2007-03-06 

Recommended storage Room temperature (20 °C) 

Storage at test facility Room temperature, protected from moisture and light 

Retention At least 1 g has been retained. 

Identification parameter Name, batch number, state, consistency, colour, melting point 

at test facility 

The test item and the information concerning the test item were provided by the sponsor. 
DR.U.NOACK-LABORATORIEN 

Report Page 8 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

#

) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test 

conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

3 Method 

TEST GUIDELINE OECD-Guideline for Testing of Chemicals No. 203 (1992) 

 

TYPE AND PURPOSE Determination of the acute toxicity of DOBA to zebrafish with the 

saturated solution#

 under static conditions. 

The saturated solution was tested as limit concentration.

TEST SYSTEM Danio rerio (zebrafish) 

 Gnathostoma, Pisces, Osteichthyes, Teleostei, Clupeiformes, 

Cyprinidae 

Reason for the selection According to the guideline Danio rerio is suitable for this kind 

of the test system of study. 

 

Origin AQUARIUM AM AEGI, Volgersweg 6, D-30175 Hannover 

 All fish used in the test originated from the same delivery of the 

supplier. 

Holding Holding was performed at the testing facility at 23 ± 2 °C and 

diffuse light (0.1-10 μmol photons · m-2 · s-1, natural photoperiod). 

The water was changed at least once per week. The dissolved 

oxygen concentration was more than 80 % of the air saturation 

value. 

 Zebrafish with at least 12 days of acclimatisation and mortality 

 < 5 % within these days before the study starts were used in the 

test. No disease treatments were administered throughout holding 

and testing. 

Water Tap water of local origin was used for holding and testing. The 

water was filtered on activated charcoal and aerated for at least 

24 h to remove chlorine. 

 Nominal water parameters: 

 Total hardness: 10 - 250 mg CaCO3/L 

 pH-value: 6.0 - 8.5 

 The water is analysed biannual acc. to German tap water 

regulation. 

Feeding Food was provided 3 times per week. The amount of food was 

4 % of the fish body weight per feeding day. The test fish were 

not fed 24 h before the test started. 
DR.U.NOACK-LABORATORIEN 

Report Page 9 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

#

) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test 

conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

Food Stör perlets; SERA GMBH, D-52518 Heinsberg 

Test Item DOBA

Preparation of the saturated A suspension (100 mg/L test item was weighed out) was 

solution prepared with dilution water, shaken with 20 rpm for 24 h and 

filtrated (0.45 μm). 

Limit concentration Saturated solution# (100 mg/L) 

CONTROL 7 fish in dilution water (without test item) were tested under the 

same test conditions as the test replicates. 

REFERENCE ITEM No reference item is recommended for this test according to the 

guideline. 

TEST METHOD A static limit test was performed with one application at the test 

initiation. 

Test duration 96 h 

Test vessel Glass-aquaria loosely covered by glass tops were used. 

Test volume About 3 L per vessel 

Aeration Gentle aeration was provided. 

Dilution water Same as for holding. 

Number of test fish 7 zebrafish were used per test concentration and control. 

Loading, length of fish Fish density in the tanks was less than 1 g fish per litre test 

solution. 

 Average body length at the test start: 2.53 cm 

 Average body weight at the test start: 0.2 g 

Replicates One replicate per test concentration and control. 

Introduction of fish Fish were introduced randomly to individual replicates. 
DR.U.NOACK-LABORATORIEN 

Report Page 10 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Feeding of test fish The fish were not fed during the test. 

Water temperature 23 ± 2 °C 

Dissolved oxygen 

concentration Not less than 60 % of air saturation value. 

Light intensity 0.1 - 10 μmol photons · m-2 · s-1

Photoperiod A natural photoperiod occurred during the test. 

KIND AND FREQUENCY OF

MEASUREMENT AND

OBSERVATIONS 

Water quality pH-value, temperature and oxygen saturation were measured 

measurements in all vessels at the beginning of the test and every 24 h. 

 Total hardness of the water was determined at the beginning out 

of the test control. During the test the room temperature was 

recorded continuously with a thermohygrograph. 

Observation intervals Observations were made after 24, 48, 72 and 96 h. 

Criteria of effects Fish were considered dead if there was no visible movement (e.g. 

gill covers movement) and if touching of the caudal peduncle 

produced no reaction. Records were kept of visible abnormalities 

(e.g. loss of equilibrium, swimming behaviour, respiratory 

function, pigmentation, etc.). 

Measurement of fish size At test start body weight and total length of a representative 

number of 7 test fish was determined. These fish were not used 

for testing. 

Equipment Oxygen: Oxygen meter Oxi 197 - S, WTW 

 pH and water temperature: Multilab 340i, WTW 

 Hardness: Spectrophotometer, CADAS 100 LPG 158, DR. LANGE

 Rotating shaker: 3040, GFL 

 DOC-analysator: Multi N/C 3000 ANALYTIK JENA AG 

 Thermohygrograph: LAMBRECHT, Göttingen 
DR.U.NOACK-LABORATORIEN 

Report Page 11 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

DETERMINATION OF The limit test concentration and control was analytically 

THE TEST ITEM verified via analysis of organic carbon (DOC, according to DIN 

EN 1484) at test start. 

COURSE OF THE STUDY The definitive study was performed as a limit test with the 

saturated solution. 

 One control with dilution water (without test item) was tested 

under the same conditions as the test group. In the tested 

concentration and control 7 fish were tested. The test fish were 

not fed during the test. After 24, 48, 72 and 96 h mortality and 

behaviour of the test fish were observed. Measurements and 

observations were carried out as described above. 

EVALUATION The concentration leading to 0 and 100 % mortality (LC0 and 

LC100) as well as the no effect concentration level (NOEC) after 

96 h were determined directly from the test results. 

 LC50-values have not to be calculated in a limit test. 

NOEC (No observed The highest tested concentration level of a test item with no 

effect concentration) significant effect when compared to control. 

LC0 The highest tested concentration of the test item with 0 % 

mortality. 

LC50 The concentration of the test item with a mortality of 50 %. 

LC100 The lowest tested concentration of the test item with 100 % 

mortality. 

VALIDITY CRITERIA - O2 - saturation ≥ 60 % 

- Mortality in the control ≤ 1/7 fish 

 

DATES 

Study initiation 2006-06-19 

Experimental starting 2006-07-26 

Experimental completion 2006-07-31 

Study completion Please refer to page 1 
DR.U.NOACK-LABORATORIEN 

Report Page 12 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

DEVIATIONS FROM DOC analysis only at the beginning of the test was carried out 

THE GUIDELINE according to the sponsor’s request. Therefore no information on 

stability and recovery of the test item under test conditions was 

given.

DEVIATIONS FROM None 

THE STUDY PLAN 

ARCHIVING The following will be retained in the archive of the testing facility 

for the period as specified in the operative national GLP 

regulations: 

• all raw data 

• study plan 

• final report 

• all records performed by the quality assurance programme 

 including master schedules 

• sample of the test item 

 Additionally, microfilms will be retained in a safe-deposit by 

Volksbank Sarstedt, D-31157 Sarstedt. 
DR.U.NOACK-LABORATORIEN 

Report Page 13 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

4 Results 

4.1 Definitive Test 

Symptoms of toxicity during the test are given in table 2. The cumulative mortality at each time 

of observation is shown in table 3. 

The test item solution was clearly dissolved throughout exposure. 

The saturated solution of the test item caused no mortality or non-lethal effects. Therefore, the 

NOEC was laid down as > saturated solution. 

Table 2: Observations in the Test Vessels 

 (n=7) 

 Test Duration [h] 

 Effect*)

24 48 72 96 

Saturated solution (1) 7/7 7/7 7/7 7/7 

Control (1) 7/7 7/7 7/7 7/7 

*) The number in brackets correspond to the following observation: 

 (1) = Normal behaviour 

 

Table 3: Cumulative Mortality [%] in the Test Vessels 

Test Duration [h] 

24 48 72 96 

Saturated solution 0 0 0 0 

Control 0 0 0 0 
DR.U.NOACK-LABORATORIEN 

Report Page 14 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Table 4: LC0-, LC100-, NOEC-Values (0 - 96 h) 

 

LC100 = > Saturated solution 

Lowest test item concentration 

with 100 % mortality after 96 h 

LC0 = Saturated solution 

Highest test item concentration 

with 0 % mortality after 96 h 

NOEC (0-96 h) = 

No observed effect concentration 

Saturated solution 

Table 5: LC50-Values with 95 % Confidence Intervals 

Test Duration 

[h] LC50 p = 95 % 

24 > Saturated solution not applicable 

48 > Saturated solution not applicable 

72 > Saturated solution not applicable 

96 > Saturated solution not applicable 
DR.U.NOACK-LABORATORIEN 

Report Page 15 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

5 Validity Criteria 

The study met the validity criteria of the guideline: 

• O2 - saturation was ≥ 60 % 

• Mortality in the control was 0 % 

6 Conclusions 

In this study DOBA did not cause any effects to zebrafish after 96 h when tested with the 

saturated solution. The NOEC (0 – 96 h) is laid down as the saturated solution. 

7 Literature / References 

(1) OECD Guideline 203 for Testing of Chemicals: Fish, Acute Toxicity Test. Adopted July 17, 

1992

(2) OECD (2000): Guidance document on aquatic toxicity testing of difficult substances and 

mixtures. OECD series on testing and assessment no. 23

(3) DIN Richtlinie EN 1484 (1997): Anleitung zur Bestimmung des gesamten organischen 

Kohlenstoffs (TOC) und des gelösten organischen Kohlenstoffs (DOC)
DR.U.NOACK-LABORATORIEN 

Report Page 16 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

8 Physicochemical Data 

8.1 Water Quality Measurements 

Table 6: Water Parameters during the Definitive Study 

 

Day OxygenSaturation pH-Value Temperature Total Hardness 

[%] [°C] [mg/L as CaCO3] 

Saturated solution 100 7.46 22.3 

0 

Control 100 7.53 22.4 87 

Saturated solution 100 7.65 22.6 

1 

Control 98 7.67 22.6 

Saturated solution 100 7.54 22.9 

2 

Control 100 7.51 22.9 

Saturated solution 98 7.53 23.2 

3 

Control 95 7.44 23.2 

Saturated solution 94 7.55 23.4 

4 

Control 96 7.39 23.3 

8.2 DOC Analysis of the Test Media for Detection of the Test Item 

Table 7: DOC Content of the Test Media at Test Start

 [mg/L] 

 Parallel determination Mean 

Saturated 

solution 4.8 4.8 4.8 

Control 2.5 2.6 2.5 
DR.U.NOACK-LABORATORIEN 

Report Page 17 of 17 

DOBA 

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

9 GLP-Certificate of DR.U.NOACK-LABORATORIEN 
DR.U.NOACK-LABORATORIEN 

Robust Summary Page 1 of 2 

DOBA

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Robust Summary 

Test Item DOBA

Remarks: none 

Testing Facility DR.U.NOACK-LABORATORIEN

Käthe-Paulus-Str. 1, D-31157 Sarstedt 

 Tel. +49(0)5066 70670, email: info@noack-lab.de 

Author Dirk Scheerbaum 

Report issued 2006-08-11 

Method 

Method/Guideline followed: OECD-Guideline No. 203 for Testing of Chemicals (1992) 

Type: Static 

GLP: Yes [X] No [ ] 

Year: 2006 

Test system: Zebrafish (Danio rerio) 

Exposure period: 96 h 

Analytical monitoring: DOC 

Test Conditions 

Test fish: Zebrafish (Danio rerio), length: 2.53 cm, weight: 0.2 g, loading: 

ca. 0.47 g fish / L test solution 

Dilution water: Tap water of local origin was used for holding and testing. 

 The water was filtered on activated charcoal and aerated for at 

least 24 h to remove chlorine. 

Water Hardness: 87 mg CaCO3 / L 

Stock solution, 

Dispersion treatment: The stock solution was shaken with 20 rpm for 24 h (rotating 

 shaker) and filtrated (0.45 μm) 

Limit concentration: Saturated solution 

Allowed water 

temperature range: 23 ± 2 °C 

Stability of the test media: Stable in water. 

Exposure vessel type: Glass aquaria; one replicate per concentration; 7 fish per 

replicate; gently aeration was given; aquaria were covered by a 

glass plate; a natural photoperiod occurred during the test.
DR.U.NOACK-LABORATORIEN 

Robust Summary Page 2 of 2 

DOBA

Fish (Zebrafish), Acute Toxicity Test, Static, 96 h Project-No. 060601CL 

acc. to OECD 203 Study-No. FAZ108572 

Table 1: Water parameters during test exposure

 Control Saturated solution 

Dissolved oxygen 

range [%] 95 - 100 94 - 100 

pH-value range 7.39 - 7.67 7.46 - 7.65 

Temperature range [°C] 22.4 - 23.3 22.3 - 23.4 

DOC [mg/L] 2.5 4.8 

Results

 Table 2: Element values: LC with 95 % Confidence Intervals 

 

Test duration [h] LC50 p = 95 % 

24 > Saturated solution not applicable 

48 > Saturated solution not applicable 

72 > Saturated solution not applicable 

96 > Saturated solution not applicable 

LC100 = > Saturated solution 

Lowest test item concentration 

with 100 % mortality after 96 h 

LC0 = Saturated solution 

Highest test item concentration 

with 0 % mortality after 96 h 

NOEC (0-96 h) = 

No observed effect concentration 

Saturated solution