DR.U.NOACK-LABORATORIEN

Report Page 4 of 17 DOBA Fish (Zebrafish), Acute Toxicity Tes t, 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 Tes t, 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 D R .U.N OACK -L ABORATORIEN 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: LC 0 -, LC 100 -, NOEC-Values (0 - 96 h) 14

Table 5: LC 50 -Values with 95 % Confidence Intervals 14

Table 6: Water Parameters during the Definitive Study 16

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

Report Page 6 of 17 DOBA Fish (Zebrafish), Acute Toxicity Tes t, 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 Te sting of Chemicals No. 203 (1992) from 2006-
07-27 to 2006-07-31 at D R .U.N OACK -L ABORATORIEN , 31157 Sarstedt, Germany. A static limit test
with a saturated solution was conducted. T he preparation method has been chosen with regard to the test item properties and in accor dance 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 p H-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were deter mined to be within the acceptable limits. No test item analysis was carried out according to t he 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] LC 50 p = 95 %

24 > Saturated solution not applicable

48 > Saturated solution not applicable

72 > Saturated solution not applicable

96 > Saturated solution not applicable

LC 100 = > Saturated solution

Lowest test item concentration with 100 % mortality after 96 h

LC 0 = Saturated solution Highest test item concentration

with 0 % mortality after 96 h

NOEC (0-96 h) = No observed effect concentration Saturated solutionDR.U.NOACK-LABORATORIEN

Report Page 7 of 17 DOBA Fish (Zebrafish), Acute Toxicity Tes t, 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 temperatur e, protected from moisture and light

Retention At least 1 g has been retained.

Identification parameter Name, batch number, st ate, consistency, colour, melting point at test facility

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

Report Page 8 of 17 DOBA Fish (Zebrafish), Acute Toxicity Tes t, 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, a cc. 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 toxici ty 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, Osteicht hyes, 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 A QUARIUM AM A EGI , 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 t he 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 CaCO 3 /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 Tes t, 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, a cc. to OECD Series, No. 23 (2000)6. Food Stör perlets; S ERA G MB H, 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 t anks 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 introduc ed 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. FAZ 108572

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 wa s 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, W TW pH and water temperature: Multilab 340i, W TW Hardness: Spectrophotometer, CADAS 100 LPG 158, D R . L ANGE Rotating shaker: 3040, GFL DOC-analysator: Multi N/C 3000 A NALYTIK J ENA AG Thermohygrograph: L AMBRECHT , GöttingenDR.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. FAZ 108572

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 perfo rmed 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 we re observed. Measurements and
observations were carried out as described above.

EVALUATION The concentration leading to 0 and 100 % mortality (LC 0 and
LC 100 ) as well as the no effect concentration level (NOEC) after 96 h were determined directly from the test results.
LC 50 -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.

LC 0 The highest tested concentration of the test item with 0 % mortality.

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

LC 100 The lowest tested concentration of the test item with 100 % mortality.

VALIDITY CRITERIA - O 2 - 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 1DR.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. FAZ 108572

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. FAZ 108572



## 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 clear ly 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 0DR.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. FAZ 108572

Table 4: LC 0 -, LC 100 -, NOEC-Values (0 - 96 h)

LC 100 = > Saturated solution

Lowest test item concentration with 100 % mortality after 96 h

LC 0 = Saturated solution Highest test item concentration

with 0 % mortality after 96 h

NOEC (0-96 h) = No observed effect concentration Saturated solution

Table 5: LC 50 -Values with 95 % Confidence Intervals

Test Duration [h] LC 50 p = 95 %

24 > Saturated solution not applicable

48 > Saturated solution not applicable

72 > Saturated solution not applicable

96 > Saturated solution not applicableDR.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. FAZ 108572



## 5 Validity Criteria

The study met the validity criteria of the guideline:

• O 2 - 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 toxi city 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. FAZ 108572



## 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 CaCO 3 ]

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 2.5DR.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. FAZ 108572



## 9 GLP-Certificate of D R 

## .U.N OACK 

## -L ABORATORIENDR.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. FAZ 108572



## Robust Summary

Test Item DOBA
Remarks: none

Testing Facility D R .U.N OACK -L ABORATORIEN 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 CaCO 3 / 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 rep licate per concentration; 7 fish per replicate; gently aeration was gi ven; aquaria were covered by a glass plate; a natural photoper iod 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. FAZ 108572

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] LC 50 p = 95 %

24 > Saturated solution not applicable

48 > Saturated solution not applicable

72 > Saturated solution not applicable

96 > Saturated solution not applicable

LC 100 = > Saturated solution

Lowest test item concentration with 100 % mortality after 96 h

LC 0 = Saturated solution Highest test item concentration

with 0 % mortality after 96 h

NOEC (0-96 h) = No observed effect concentration Saturated solution