TS-CKHI02

Attachment #10

Environment Effects Data on the New Chemical Substance 

Acute Fish Toxicity Test 
LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta 

Phone: +36 88 545 300 

Fax: +36 88 545 301

FINAL REPORT

ACUTE TOXICITY

TEST WITH

 Polymer

ON ZEBRAFISH

(Brachydanio rerio)

Date of Final Report: 04 April 2011 STUDY CODE: 10/339-009H 



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GENERAL INFORMATION

STUDY TITLE : Acute toxicity test with Polymer

on Zebrafish (Brachydanio rerio) 

TEST ITEM : 

SPONSOR :

TEST FACILITY : LAB Research Ltd.

 Address: H-8200 Veszprém, Szabadságpuszta 

 Hungary 

 Phone: +36 88 545-300 

Fax: +36 88 545-301 

STUDY DIRECTOR : István Ágh M.Sc. 

 Phone: 36 88 545 344 

 e-mail: istvan.agh@labresearch.hu 

QUALITY ASSURANCE : Ramóna Heiderné Grób B.Sc. 

TECHNICAL STAFF : Ecotoxicological staff

START OF EXPERIMENT : 21 February 2011 

END OF EXPERIMENT : 25 February 2011 

BASIS OF STUDY:

– OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", 

adopted July 17, 1992 

– EPA Health Effects Test Guidelines, OPPTS 850.1075 

– Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test 

methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACH), Annex Part C, C.1 (published in the Official Journal of 

the European Union L 142 of 31 May 2008) 
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CONTENTS 

Page

1. SUMMARY 7

2. INTRODUCTION 8

3. MATERIALS AND METHODS 8

3.1. TEST ITEM AND CONTROLS AND VEHICLE 8

3.2. EXPERIMENTAL ANIMALS 9

3.3. ENVIRONMENTAL TEST CONDITIONS 10

3.4. DESCRIPTION OF THE TEST PROCEDURE 10

3.5. OBSERVATIONS 11

3.6. ANIMAL WELFARE 11

3.7. PERMISSION OF THE IACUC 11

3.8. STATISTICAL EVALUATION 11

3.9. ANALYTICAL MEASUREMENTS 11

3.10.ARCHIVES 12

3.11.VALIDITY CRITERIA OF THE STUDY 12

3.12.DISTRIBUTION OF THE FINAL REPORT 12

3.13.REFERENCES 13

4. RESULTS 14

4.1. VALIDITY 14

4.2. MORTALITY DATA 14

4.3. SUB-LETHAL EFFECTS 14

4.4. BODY WEIGHT 14

5. CONCLUSION 15

APPENDICES 16 

Data of Test conditions 1 of Appendix 1 

Body Length of Test Fish 1 of Appendix 2 

Copy of the GLP Certificate 1 of Appendix 3 
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1. SUMMARY

The acute toxicity of Polymer was assessed with acute fish toxicity 

test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours. 

Toxic effects were not observed during the preliminary concentration range-finding test 

therefore a limit test was performed in the main experiment using only one test 

concentration at the limit of solubility of the test item in the test medium (100 % v/v 

saturated solution) and one control group. Because the test item is extremely poorly 

soluble in water or in any practicable organic solvent, and in addition no analytical 

measurement is possible for the determination of stability in the test medium over the 

test duration, the test item solution used in the test was prepared by a method which 

ensured that the test was carried out at the limit of solubility of the test item (limit test 

concentration) during the whole test period. 

One aquarium was used in test group and one in the control group. Both aquaria 

comprised seven fish and twenty litre test solution.

The number of mortalities and any sub-lethal effect were determined 3, 6, 24, 48, 72 

and 96 hours after the start of exposure. 

All validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on zebrafish (Brachydanio 

rerio) the observed endpoints for the effect of Polymer were the 

followings: 

The 24h, 48h, 72h and 96h LC50 value: > 100 % v/v saturated solution

The 96h NOEC: 100 % v/v saturated solution

The 96h LOEC: > 100 % v/v saturated solution

The 96h LC100 value: > 100 % v/v saturated solution

Based on the results of this study, the test item Polymer had no

toxic effect at aquatic saturation on fish; the LC50 results and the LOEC are 

higher than the solubility level of the test item in the test medium.
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2. INTRODUCTION

The objective of this study was to determine the median lethal concentration (LC50

value) of the test item in acute toxicity test on zebrafish (Brachydanio rerio). 

Fish were exposed to aqueous test media containing the test item at one concentration 

at the limit of solubility to demonstrate that the test item has no influence on test 

animals up to at least this test concentration level. 

The test method of application and the test species zebrafish (Brachydanio rerio) are 

recommended by the test guidelines. 

3. MATERIALS AND METHODS 

3.1. TEST ITEM AND CONTROLS AND VEHICLE

3.1.1. Test Item

Name:

Chemical name:

Lot Number: 

Purity: 98.6 % 

Description: white / waxy solid

Expiry date: 23 August 2012 

Storage condition: Room temperature (15-30°C); humidity (40-70%);

in the dark 

Safety Precautions: Routine safety precautions (gloves, goggles, face mask, 

lab coat) for unknown materials will be applied to assure 

personnel health and safety 

Manufacturer: see Sponsor 

The test item of a suitable chemical purity was supplied by the Sponsor. All 

precautions required in the handling and disposal of the test item were outlined by the 

Sponsor. These documents are part of the raw data. Identification of the test item was 

performed in the Central Dispensary of LAB Research Ltd. on the basis of the 

information provided by the Sponsor. 
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3.1.2. Dilution and Preparation of Testing Solutions 

Because the test item is extremely poorly soluble in water or in any practicable organic 

solvent, the test was carried out in a saturated solution of the test item in water (limit 

test concentration) during the whole test period in a recirculation system. Due to the 

extremely poorly water solubility, no analytical measurement was possible for the 

determination of concentration or stability in the test medium.

The method is based on the elution of the test substance with the test medium from a 

column, which is charged with an inert support material (e.g. glass beads or quartz 

sand) mixed with an excess of the test substance. For this method aquarium water is 

used as eluent.

Small particles of test substance (with a relatively high surface area) were mixed with 

support material (glass beads were used in this test). This mixture was poured into the 

column and then filled with aquarium water. The column was connected to a recirculating pump. The flow through the column was started approximately 10 hours 

before the introduction of the fish (to achieve the constant saturated solution) and run 

over the test period. The flow rate of the water pumped through the column filled with 

test item allowed a theoretical recirculation of the whole tank volume approximately 

every 12 minutes, so 10 hour represented a recirculation of approximately 50 times.

3.1.3. Untreated Control 

The dilution water (circulated and filtered tap water) was used without of addition of 

the test item.

3.2. EXPERIMENTAL ANIMALS

Species: Zebrafish (Brachydanio rerio)

Source: “Black Molly” Fish Farm, Veszprém, Hungary

Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient 

species for acute fish toxicity test.

Number of animals: seven fish per test group 

Body length of animals: 2.7 – 3.2 cm

Food and Feeding: The fish were not fed during the test 

Acclimatisation: >12 days 

Animal health: Fish were bred in a well-known fish farm, under diseaseand parasite-controlled conditions. Fish were observed 

during the acclimatisation and test periods, the fish were 

healthy.

There was no mortality of the population for seven days 

before the test.
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3.3. ENVIRONMENTAL TEST CONDITIONS

The water temperature, oxygen concentration and pH were measured at the start of the 

test and daily thereafter. 

The test temperature was between 22.0 and 22.3 °C. 

The dissolved oxygen concentration was in the range of 91 – 93 % of the air saturation 

value at the temperature used. 

The pH was in the range of 8.08 – 8.20. 

The hardness of the dilution water was determined as 249 mg/L (as CaCO3). 

The light-dark cycle during the test was 16 hours light and 8 hours darkness. 

The test conditions above are detailed in Appendix 1. 

3.4. DESCRIPTION OF THE TEST PROCEDURE 

The test duration was 96 hours. One aquarium was used for test group and for the 

control group respectively. Both aquaria comprised 7 fish and 20 L test solution. The 

animals were not fed during the test. The loading of the test aquaria was less than 1.0 g 

fish/L test solution at the start of the experiment. 

The choice of the test concentrations was done on the basis of the results of the 

preliminary range-finding test. 

3.4.1. Preliminary Range-Finding Test 

Three fish were exposed to a single test concentration at aquatic saturation and one 

control in order to demonstrate that the test item had no influence on fish up to at least 

this test concentration (100 % v/v saturated solution). The test solution was prepared 

using method as described in section 3.1.2..

No mortality or any toxic effect was detected in the treated concentration (100 % v/v 

saturated solution) and in the control groups during the 96 h exposure period. 

3.4.2. Concentration Levels Investigated in the Main Test 

Because toxicity was not observed during the preliminary test, only one test 

concentration at aquatic saturation and one untreated control were tested in a limit test. 
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3.5. OBSERVATIONS

The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 

and 96 hours. Mortality and any sub-lethal effect were determined in each observation 

period. 

The test conditions (pH, temperature, oxygen saturation) were examined daily during 

the test.

The body weights of the fish were recorded before the introduction of fish. The body 

weight of 7 fish per aquarium was registered. The loading of the aquaria was 

calculated on the basis of these body weights (g fish/litre testing liquid).

3.6. ANIMAL WELFARE

After the experiment, all fish were humanely killed. Euthanasia for all animals was 

applied by an overdose of an aqueous solution of 200 mg/L Tricaine 

Methanesulphonate (MS 222; CAS No.: 886-86-2). This technique is considered to 

provide a humane euthanasia by direct depression of the brain and vital centers. 

3.7. PERMISSION OF THE IACUC

The conduct of the study was permitted by Institutional Animal Care and Use 

Committee (IACUC) of LAB Research Ltd.

Date of IACUC approval: 02 February 2011 

3.8. STATISTICAL EVALUATION

No statistical analysis was performed because of the lack of toxic effects.

The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

3.9. ANALYTICAL MEASUREMENTS

Due to the extremely low aquatic solubility of the test substance, analytical 

determination of its concentration in the test medium was not possible. A system was 

used to assure that a saturated solution was used throughout the study by recirculation 

of the aquatic media through a column containing the test item. To demonstrate that 

the test solution was saturated during the whole exposure period, visual assessment of 

the test substance-support material mixture was undertaken at the beginning and end 

of the test. These observations showed that test item was present throughout the study 

in the recirculation system at ~50 recirculations per 10 hours over a high surface area 

of test item; this is considered sufficient evidence that the solution was saturated

during the study. 
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3.10. ARCHIVES

The study documents: 

- study plan and amendment, 

- all raw data,

- sample of test item,

- study report, and any amendments, 

- correspondence 

are stored according to the Hungarian GLP and to the LAB Research Ltd. SOP-s in the 

archives of LAB Research Ltd. 8200 Veszprém, Szabadságpuszta, Hungary. 

After the retention time has elapsed all the archived materials listed above will be 

returned to the Sponsor or retained for a further period if agreed by a contract. 

Otherwise the materials will be discarded. 

3.11. VALIDITY CRITERIA OF THE STUDY

- Constant conditions should be maintained in this procedure. 

- The mortality in the controls should not exceed 10 % at the end of the test (or one 

fish if less than ten are used). In case of limit test, no mortality should be occurred. 

- The dissolved oxygen concentration (throughout the test) has to be at least

60 % of air saturation value. 

3.12. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound 

 1x copy, unbound 

 1x PDF file

Archive: 1x original, bound 
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3.13. REFERENCES

1. OECD Principles of Good Laboratory Practice, adopted by Council on 26th

November 1997 [C(97)186/Final], Environment Directorate, Organisation for 

Economic Co-operation and Development, Paris 1998. 

2. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30) EÜM-FVM joint 

decree of the Minister of Health and the Minister of Agriculture and Regional 

Development which corresponds to the OECD GLP, 1997

3. Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test 

methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACH), Annex Part C, C.1 (published in the Official Journal of the 

European Union L 142 of 31 May 2008) 

4. EPA Guideline 712-C-96-118: OPPTS 850.1075, "Fish Acute Toxicity Test, 

Freshwater and Marine" April 1996 

5. OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute 

Toxicity Test", adopted July 17, 1992. 

6. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series 

on Testing and Assessment No. 23, Paris September 2000. 

7. Guidelines for studies on the new chemical substance as required by the Law 

Concerning the Evaluation of Chemical Substances and Regulation of their 

Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the 

reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and 

KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance 

of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, 

Labour and Welfare (MHLW) and Ministry of the Environment (MOE)
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4. RESULTS

4.1. VALIDITY

The validity criteria were within acceptable limits and therefore the study can be 

considered as valid. Evidence that the test item was present at saturation during the 

study is considered to be sufficient.

4.2. MORTALITY DATA

Cumulative mortality data from the exposure of zebrafish to the test material during 

the definitive test are given below in Table 1. 

There were no mortalities in 7 fish in the control group or in 7 fish exposed to a test 

concentration of 100 % v/v saturated solution. 

Table 1: Cumulative mortality data in the definitive test

Test Group

Mortality rate of treated fish (Dead fish/Treated fish)

3h 6h 24h 48h 72h 96h

Control 0/7 0/7 0/7 0/7 0/7 0/7

100 % v/v saturated solution 0/7 0/7 0/7 0/7 0/7 0/7

4.3. SUB-LETHAL EFFECTS

No sub-lethal effects were observed in the control and test group for a period of 

96 hours. 

4.4. BODY WEIGHT

The body weight of 7 fish was weighed at the start of the test. The measured and 

calculated data are listed below in Table 2. 

Table 2: Measured and calculated data of bodyweight

Test Group Measured weight 

of 7 fish (g)

Calculated mean 

weight of 1 fish (g)

Loading of testing 

aquarium

(g fish/L testing liquid)

Control 4.05 0.58 0.20

100 % v/v saturated 

solution 4.09 0.58 0.20

There was no considerable difference observed concerning body weights between 

the groups. 
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5. CONCLUSION

The acute toxicity of Polymer was assessed with acute fish toxicity 

test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours. 

One concentration at the solubility level of the test item in the test medium (100 % v/v 

saturated solution) plus an untreated control was tested in a limit test. 

All validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on zebrafish (Brachydanio 

rerio) the observed endpoints for the effect of Polymer were the 

followings: 

The 24h, 48h, 72h and 96h LC50 value: > 100 % v/v saturated solution

The 96h NOEC: 100 % v/v saturated solution

The 96h LOEC: > 100 % v/v saturated solution

The 96h LC100 value: > 100 % v/v saturated solution

Based on the results of this study, the test item Polymer had no

toxic effect at aquatic saturation on fish; the LC50 results and the LOEC are 

higher than the solubility level of the test item in the test medium.
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APPENDICES
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APPENDIX 1

DATA OF TEST CONDITIONS

Table 3: Temperature (°C)

Test Group Measuring 

0h 24h 48h 72h 96h

Control 22.2 22.2 22.0 22.3 22.0

100 % v/v saturated solution 22.2 22.1 22.1 22.3 22.0

Table 4: Dissolved oxygen concentration (% of the air saturation value) 

Test Group

Measuring 

0h 24h 48h 72h 96h

Control 92 92 93 92 93

100 % v/v saturated solution 91 92 92 92 92

Table 5: pH 

Test Group Measuring 

0h 24h 48h 72h 96h

Control 8.12 8.10 8.14 8.10 8.16

100 % v/v saturated solution 8.17 8.08 8.12 8.09 8.20
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APPENDIX 2 

BODY LENGTH OF TEST FISH 

Table 6: Body length of test fish at the end of the test 

Test Group Body length [cm]

1 2 3 4 5 6 7 

Control 2.8 3.0 3.0 2.9 3.0 3.2 2.9

100 % v/v saturated solution 2.9 3.0 3.2 3.1 3.1 2.7 2.8

Remark: At the end of the test, body length of test fish were measured in order to check their 

compliance with the size range recommended for the species by the test guideline [Commission 

Regulation (EC) No 440/2008; Annex Part C, C.1]
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APPENDIX 3 

COPY OF THE GLP CERTIFICATE