1/59 

REPORT

2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol 

Daphnia magna Reproduction Test 

according to the OECD Guideline No. 211 (2012) 

STUDY CODE: 

Study director: 

Test facility:

Sponsor:

 Project Number: 

 Order Number: 

Version: Final 

Study completion date: February 2014 

Head of the Branch 

.............................................

date 

This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is the 

property of the sponsor and may be used only for that purpose intended by the sponsor and as authorized under 

applicable law. Every other or additional use, exploitation, reproduction, publication or submission to other 

parties requires the written permission of the sponsor, except as otherwise required by applicable law (e.g., TSCA). 

The use of these studies/documents for regulatory, registration or other commercial purposes [including but not 

limited to Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH)], without the 

express written permission of the sponsor is unauthorized and strictly prohibited.
2/59 

CONTENTS:

LIST OF TABLES........................................................................................................................................3 

LIST OF FIGURES .....................................................................................................................................4 

LIST OF APPENDICES ..............................................................................................................................5 

DECLARATION OF STUDY DIRECTOR ...................................................................................................6 

PERFORMERS, AUTHORS .......................................................................................................................7 

STATEMENT OF THE QUALITY ASSURANCE UNIT ...............................................................................8 

ARCHIVING ................................................................................................................................................9 

DISTRIBUTION: ...................................................................................................................................... 10 

SUMMARY .............................................................................................................................................. 11 

1. AIM OF THE STUDY ........................................................................................................................... 13 

2. DATES ................................................................................................................................................. 13 

3. MATERIALS AND METHODS ............................................................................................................. 13 

3.1. Test item ........................................................................................................................................... 13 

3.2. Test organism ................................................................................................................................... 13 

3.2.1. Culturing of Daphnia magna ......................................................................................................... 14 

3.2.2. Culturing medium .......................................................................................................................... 14 

3.3. Test conditions ................................................................................................................................. 14 

3.3.1. Preliminary non-GLP tests ............................................................................................................ 15 

3.3.2. Definitive test ................................................................................................................................. 16 

3.3.3. Preparing concentrations of the test item ...................................................................................... 17 

3.4. Chemical analysis ............................................................................................................................ 18 

4. DEFINITIONS OF THE ENDPOINTS ................................................................................................. 18 

5. RESULTS ............................................................................................................................................ 18 

5.1. Preliminary non-GLP tests ............................................................................................................... 18 

5.2. Definitive test .................................................................................................................................... 19 

5.2.1. Endpoint values ............................................................................................................................. 20 

6. VALIDITY CRITERIA ........................................................................................................................... 22 

7. DEVIATIONS FROM THE STUDY PLAN ............................................................................................ 22 

8. REFERENCES .................................................................................................................................... 22 

9. STANDARD OPERATING PROCEDURES USED IN THE STUDY .................................................... 23 
3/59 

LIST OF TABLES 

Table 1. Results, first preliminary non-GLP test. ................................................................................... 24 

Table 2. Results, second preliminary non-GLP test. ............................................................................. 24 

Table 3. pH values and O2 concentration measured, definitive test ..................................................... 25 

Table 4. Conductivity measured, definitive test ..................................................................................... 26 

Table 5. The total hardness measured, definitive test .......................................................................... 26 

Table 6. Offspring per survivor per day in the control, definitive test .................................................... 27 

Table 7. Offspring per survivor per day in the WAF of a loading of 10 mg/L, definitive test ................. 27 

Table 8. Cumulative offspring per survivor in the control, definitive test ............................................... 28 

Table 9. Cumulative offspring per survivor in the WAF of a loading of 10 mg/L, definitive test ............ 28 

Table 10. Final body size of parent Daphnia magna [mm], definitive test ............................................ 29 

Table 11. Results, definitive test ........................................................................................................... 29 

Table 12. Endpoint values based on WAF of a loading of 10 mg/L, definitive test ............................... 30 
4/59 

LIST OF FIGURES 

Figure 1. Temperature, definitive test ................................................................................................... 31 

Figure 2. Mean cumulative offspring per survivor versus day of exposure, definitive test.................... 31 

Figure 3. Final body size of parent Daphnia magna, definitive test ...................................................... 32 
5/59 

LIST OF APPENDICES 

Appendix 1. Statistics for determination of endpoint values ................................................................. 33 

Appendix 2. Composition of the Elendt M7 medium ............................................................................. 38 

Appendix 3. Standard curves ................................................................................................................ 39 

Appendix 4. Copy of the Statement of GLP compliance ....................................................................... 42 

Appendix 5. Copy of the Study plan ...................................................................................................... 43 
6/59 

DECLARATION OF STUDY DIRECTOR 

The study ‘2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol Daphnia magna

Reproduction Test’ was carried out according to the order number 1089966059 dated September 17, 

2013 received from project 

number: 

The study coded was performed according to the OECD Guideline for Testing of Chemicals 

No. 211 (2012) [1], . The study was in compliance with the principles of Good Laboratory 

Practice, OECD 1997 [2]. The study was performed in compliance with the study plan. The preliminary 

tests were performed non-GLP. In the experimental part of the definitive test one deviation from the 

study plan occurred, concerning the variation of temperature as daily range (only on day 9), what did 

not have impact on the results. 

The study was performed by the 

Department of Ecotoxicology, which holds a Statement of GLP Compliance Registration Number 

 issued on by the Bureau for Chemical Substances, valid from 

 (Appendix 4) [3]. 

I hereby declare that the work was performed under my supervision and in accordance with the 

described procedures. It is assured that the reported results faithfully represent the raw data obtained 

during the experimental work. I declare that the report contains an authentic description of all results 

obtained and observations made during the study. I bear the responsibility for the technical conduct of 

the study as well as the interpretation, analysis, documentation and reporting of the results. 

Study director: 

 ...................................................

 date signature 
7/59 

PERFORMERS, AUTHORS 

Researchers performing the biological part: Date, signature 

.................................

...........................

...........................

........................

Head of the Department of Ecotoxicology:

...........................
8/59 

STATEMENT OF THE QUALITY ASSURANCE UNIT 

Study title: 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol, Daphnia 

magna, Reproduction Test 

Study code: 

Inspected test facility unit: Department of Ecotoxicology 

To fufill the requirements of GLP, the following inspections were conducted:

Types of 

inspections 

Number of the 

inspection 

protocol 

Date of the 

inspection 

Date of the 

inspection results 

submission to the 

study director 

Date of the 

inspection results 

submission to the 

Head of the test 

facility 

Study plan 08.01.2014 08.01.2014 08.01.2014 

Course of the study 

29.01.2014 

07.02.2014 

12.02.2014 

30.01.2014 

10.02.2014 

13.02.2014 

30.01.2014 

10.02.2014 

13.02.2014 

Draft report 24.02.2014 24.02.2014 24.02.2014 

Final report 28.02.2014 28.02.2014 28.02.2014 

The study was inspected according to the principles of GLP [2, 3], 

The person responsible for quality assurance programme confirms that the results presented in the 

final report precisely reflect the raw data obtained during the study, whereas all the methods, 

procedures and observations are described in an accurate and complete way. 

 Quality Assurance Unit:

...................................................................

 date 
9/59 

ARCHIVING 

The following documents will be archived in the Archives of Documentation and Test Samples in the 

 for 

10 years from study completion date 

- Copy of the order, 

- True copy of the study plan, 

- True copies of test sheets, results sheets, temperature records, descriptions and notes 

generated during the study, 

- Original documentation generated by the Quality Assurance Unit, 

- Copy of correspondence with sponsor, 

- True copy of the final report. 

After the archiving period, no raw data or material relating to the study will be discarded without the 

sponsor’s prior written consent. 

The archival sample of the test item will be stored in the Department of Test Material in the 

, until the expiry 

date 

The premises and conditions of archiving of the documentation and the archival standards are 

regulated according to the requirements of GLP and the quality system in the 

 
10/59 

DISTRIBUTION: 

Study plan: 

Original: 

True copy: 

Final report: 

Original: 

True copy: 

Raw data: 

Original: 

True copy: 

Electronic version 

of Final report: 
11/59 

SUMMARY 

In a semi-static reproduction test, young Daphnia magna (< 24 h old) were exposed to 2-(2Hbenzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol for 21 days. 

The test item was used in a single test item concentration as a limit test. Since the test item is of low 

solubility, the Water Accommodated Fraction (WAF) of the test item in test medium was used for 

exposure. The mixture of the test item in the test medium at 10 mg/L and the control (i.e. the test 

medium) were prepared. For each of them the sonication at 40°C temperature, prolonged mixing and 

then the filtration were made. The filtrates were used for exposure as the Water Accommodated 

Fraction, i.e. WAF of a loading of 10 mg/L and the control. 

The aim of a limit test was to demonstrate that the LOEC and NOEC values based on number of living 

offspring, immobilization and final body size (body length) of parent Daphnia magna are higher than 

the solubility limit of the test item in the test medium, or the WAF of a loading of 10 mg/L, respectively. 

The test was performed in glass beakers with a capacity of 150 mL. Each of them contained 100 mL. 

The content of each beaker was completely renewed three times a week. At each renewal the parent 

Daphnia magna were transferred from old to fresh solutions. The offspring produced in each test 

vessel was counted and removed daily. The observations on immobilization of parent Daphnia magna

were reported daily. The body size of each parent Daphnia magna was measured at exposure 

termination. 

No effects were observed on reproductive output, immobilization, final body size, intrinsic rate of 

population growth and age of first reproduction. 

The endpoint values were based on nominal concentration of the test item expressed as the WAF of a 

loading of test item. 

Material and methods 

Test item: 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol, 

Batch: 01100HJ0, Production date: 29 April 2010, Expiration date: 29 

April 2020, Purity: 99.66 area %. 

Test species: Daphnia magna Straus (< 24 h old at the exposure initiation); not first 

brood progeny; range of parent Daphnia magna age: 21 – 25 days; 

neonates collected from the laboratory culture cultivated at the 

 Laboratory of Aquatic Toxicology.

Test design: Semi-static system, test duration 21 days, the solutions exchanged three 

times per week, 10 replicates per treatment each with one parent 

daphnia held individually. 

Test concentrations: Control and the WAF of a loading of 10 mg/L (limit test). 

Test conditions: Temperature: 19.2 – 21.7 oC, pH of the control: 7.46 – 8.37 oxygen 
12/59 

concentration in the control: 8.0 – 9.4 mg/L, photoperiod: 16 h light : 8 h 

dark, mean light intensity: 1249 – 1348 lux, feeding: algal suspension 

mixture: Pseudokirchneriella subcapitata : Desmodesmus subspicatus 

(2:1, volume per volume) supplying ~ 0.2 mg organic carbon per parent 

Daphnia per day, no aeration. 

Statistics: Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance 

Homogeneity (with Residuals), Welch-t test for Inhomogeneous 

Variances, Student-t test for Homogeneous Variances. 

Endpoints: LOEC and NOEC. 

Conclusion: 

In the semi-static reproduction test on Daphnia magna, the endpoint values were determined on the 

basis of the WAF prepared from the loading of the test item. 

The results of the limit semi-static test demonstrated no effects of test item concentration i.e. WAF of a 

loading of 10 mg/L on reproductive output (the mean cumulative number of offspring per survivor), 

immobilization, final body size of parent Daphnia, intrinsic rate of population growth and age of first 

reproduction are not lower than the solubility limit of the test item in test medium. 

Since no immobilization or any other effects on parent Daphnia magna were observed, the 

conclusions of the estimations based on the number of living offspring produced per parent 

Daphnia alive at exposure termination (i.e. the mean cumulative number of offspring per survivor) 

are the same as the conclusions of the estimations based on the total number of living offspring 

produced per parent Daphnia introduced at exposure initiation (excluding parental accidental 

and/or inadvertent immobilization from the analysis). 

The endpoint values based on the number of living offspring produced per parent Daphnia alive 

at exposure termination, immobilization of parent Daphnia, the intrinsic rate of population 

growth, the final body size (body length) of parent Daphnia, age of first reproduction: 

LOEC >WAF of a loading of 10 mg/L, 

NOEC ≥ WAF of a loading of 10 mg/L, 

EC10/21 d >WAF of a loading of 10 mg/L, 

EC50/21 d >WAF of a loading of 10 mg/L. 
13/59 

1. AIM OF THE STUDY 

The primary aim of the study was to evaluate the toxic impact of the test item on the reproductive 

output and growth of Daphnia magna and to demonstrate that the lowest observed effect 

concentration (LOEC) and the no observed effect concentration (NOEC) values after 21 days of 

exposure based on reproductive output (i.e. the mean cumulative number of offspring per survivor), 

immobilization and final body size (body length) of parent Daphnia magna are higher than the 

solubility limit of the test item in the test medium, or the WAF of a loading of 10 mg/L, respectively. 

2. DATES 

The preliminary tests started: 18.11.2013 

The preliminary tests ended: 13.11.2013 

The study started: 15.01.2014 

The definitive test started: 22.01.2014 

The definitive test ended: 12.02.2014 

Draft report: 24.02.2014 

The study completion date: 28.02.2014 

3. MATERIALS AND METHODS 

3.1. Test item 

The test item 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol is a white solid1. The 

sample of the test item is labelled with Name: 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-

phenylethyl)phenol, Batch: 01100HJ0, Production date: 29 April 2010, Expiration date: 29 April 2020. 

The sample of the test item in amount of 50 g in a plastic container was provided by sponsor on 

October 02, 2013 with the material safety data sheet (MSDS), Specification of the test substance 

 and Analytical Report of Regulatory Purposes 

According to specification the solubility of test item in water is <0.04 mg/L at 20 °C and its purity is 

99.66 area %. 

The sample is stored according to the sponsor’s recommendations, at ambient temperature under dry 

conditions without exposure to light in a tightly sealed container [

Data relating to the identity, purity and stability of test item are the responsibility of the sponsor. 

3.2. Test organism 

The test organism Daphnia magna Straus originated from a standard culture maintained at the 

 

1

 The colour and the form were described on the basis of the information included in the specification of the test 

substance 
14/59 

. The sensitivity of the culture was monitored on regular basis with 

the reference substance potassium dichromate [SOP/W/72]. Young Daphnia magna (less than 24 h 

old), progeny of 21 – 25 days old parents, were used in the test. 

3.2.1. Culturing of Daphnia magna

Daphnia magna was cultured in glass beakers with a capacity of 150 mL (single parent per vessel) at 

room temperature 18 – 22o C with the light : dark cycle of 16 h : 8 h (electronic time programmer, 

KANLUX, Poland). The culture was maintained in Elendt M7 medium [SOP/W/63, SOP/W/67]. The 

Daphnia magna was fed with Pseudokirchneriella subcapitata algae suspension originating from 

culture in the Laboratory of Aquatic Toxicology [SOP/W/67, SOP/W/65]. Vitamins of group B and 

micronutrients necessary for proper growth were supplied with lyophilized Spirulina platensis

suspension. However, for three weeks before initiation of the definitive test each cultured Daphnia was 

fed with algal suspension mixture: Pseudokirchneriella subcapitata : Desmodesmus subspicatus 

originating from separate cultures in the Laboratory of Aquatic Toxicology 

Daphnia magna reproduce parthenogenetically, therefore in each beaker young Daphnia magna was 

produced. 

3.2.2. Culturing medium 

The Elendt M7 medium was used as the culturing medium for test organism and the diluent/solvent for 

the test item [1, 6]. Test medium was prepared on the basis of deionised water (SolPure 7 purification 

system [SOP/W/71]) by adding stock solutions of reagent-grade chemicals 

and aerated before use. 

The stock solutions were renewed on regular basis for Daphnia magna culturing and stored in a 

refrigerator. The composition of Elendt M7 medium and the concentration of each ingredient are 

presented in Appendix 2. 

3.3. Test conditions 

The definitive Daphnia magna reproduction test was performed according to the OECD Guideline for 

Testing of Chemicals No. 211 (2012) [1] and according to 

In order to find conditions of exposure for Daphnia magna in the definitive reproduction test the 

preliminary non-GLP acute immobilization test and preliminary non-GLP reproduction test were 

performed. 

The preliminary acute immobilization test was performed according to the OECD Guideline for Testing 

of Chemicals No. 202 (2004) [6] and according to The preliminary reproduction test was 

conducted according to general design features specified in the OECD Guideline No. 211. 
15/59 

3.3.1. Preliminary non-GLP tests 

The tests were performed using the Elendt M7 medium as a diluent necessary to prepare the test 

concentrations. Test vessels were glass beakers with a capacity of 150 mL containing at 100 mL of 

solution. Test vessels were covered with transparent plastic lids in order to minimize evaporation and 

to prevent accidental contamination. The preliminary tests were conducted under a daily cycle of 16 h 

light : 8 h dark (electronic time programmer, KANLUX, Poland); an artificial fluorescent light source 

was used. The temperature was continuously recorded using an electronic device with a sensor 

submerged in an additional test vessel with the test medium (thermologger HI 141, Hanna 

Instruments, USA) [

In the first preliminary test Daphnia magna was exposed for 48 h. The test was conducted in a static 

design. The test item concentration and the control were tested in four replicates. Five individuals of 

Daphnia magna were introduced in each replicate. In the test pH values and the dissolved oxygen 

concentration were measured at exposure initiation before division into replicates and at exposure 

termination of pooled replicates (pH-Oxi-meter inoLAB Level 3, WTW, Germany) ]. The test 

organisms were not fed during the exposure. 

In the second preliminary test Daphnia magna was exposed for 21 days. The test was conducted in a 

semi-static design. Each test item concentration and the control were tested in ten replicates with a 

single young Daphnia magna introduced at exposure initiation. During 21 days of exposure the test 

concentrations and the control were renewed on eight occasions. In the test the pH values and the 

dissolved oxygen concentration were measured in fresh solutions before splitting up into replicates (at 

exposure initiation and at each renewal) and in old solutions of pooled replicates (at each renewal and 

at exposure termination) (pH-Oxi-meter inoLAB Level 3, WTW, Germany) . At each 

renewal total volume in every test vessel was exchanged for fresh solutions. Solutions in test vessels 

were not aerated. The parent Daphnia magna was individually transferred to test vessels with fresh 

test solutions by Pasteur pipettes. At the same time mortality of parent Daphnia magna was recorded. 

The offspring produced by each parent Daphnia magna was removed, counted and recorded daily. On 

each day of the exposure every test vessel with test solution was supplied with algae suspension. Test 

organisms were fed with fresh suspension of Pseudokircheriella subcapitata acquired from standard 

mass laboratory culture in AAP medium concentrated by centrifugation and 

resuspended in deionized water . In order to provide 0.2 mg organic carbon per Daphnia

per day the sufficient amount of algae suspension was calculated based on a standard curve of the 

TOC content in algal feed. The standard curve shows the correlation of measured total organic carbon 

content vs. absorbance of algae suspension at 670 nm 

(Appendix 3). The test vessels were arranged at random and rearranged on each renewal. 

On day 21 every living parent Daphnia was measured (i.e. body length excluding the anal spine) by 

microscopic observations with accuracy of ±0.1 mm (using eyepiece micrometer disc, Microscope 

CX40, Olympus . 
16/59 

3.3.2. Definitive test 

The reproduction test on Daphnia magna was conducted in a semi-static design. The test was 

performed in Elendt M7 medium, the same as used in culture. Three renewals of test item 

concentration and the control per week were performed. Test vessels were glass beakers with a 

capacity of 150 mL containing at 100 mL of solution. The test item concentration and the control were 

tested in ten replicates with a single young Daphnia magna introduced at exposure initiation. The 

labelled test vessels were organised randomly and rearranged on each renewal. Test vessels were 

covered with transparent plastic lid in order to minimize evaporation and to prevent accidental 

contamination. 

The light : dark cycle of 16 h : 8 h was used with fluorescent light source application (electronic time 

programmer, KANLUX, Poland). The light intensity was measured at exposure initiation, twice during 

the exposure and at exposure termination (lux-meter with 2π receptor Sonopan L-50, Poland 

. 

The temperature of solutions was constantly measured in additional vessel and constantly recorded 

with a sensor submerged in an additional test vessel with 100 mL test medium (thermologger HI 141, 

Hanna Instruments, USA . The pH values, the dissolved oxygen concentration and the 

conductivity were measured in fresh solutions before splitting up into replicates (at exposure initiation 

and at each renewal) and in old solutions of pooled replicates (at each renewal and at exposure 

termination) Total hardness was measured at exposure initiation, on three renewals and 

at exposure termination in samples of the test item concentration and the control by complexometric 

titration with EDTANa2 (ethylenediaminetetraacetic acid disodium salt) (digital titration apparatus 

Solarus, Hirschmann Laborgeräte GmbH&Co., Germany 

At each renewal total volume in every test vessel was exchanged for fresh solutions. Solutions in test 

vessels were not aerated. Each parent Daphnia magna was individually transferred to test vessels 

with fresh test solutions by Pasteur pipettes. At the same time mortality of parent Daphnia magna was 

recorded. The offspring produced by each parent Daphnia magna was removed, counted and 

recorded daily. 

Test organisms were fed with algal suspension mixture: Pseudokirchneriella subcapitata : 

Desmodesmus subspicatus (2:1, volume per volume) acquired from standard mass laboratory cultures 

 The cultures of these algal species were concentrated by centrifugation , 

resuspended in Elendt M7 medium and combined. In order to provide 0.2 mg organic 

carbon per Daphnia per day the sufficient amount of algae mixture suspension was calculated based 

on chemical determinations of TOC (total organic carbon) ]. Once the content of TOC was 

determined, the algae mixture suspension of known TOC was stored in refrigerator and used during 

exposure. 

At exposure termination (on day 21) every living parent Daphnida was measured (i.e. body length 

excluding the anal spine) by microscopic observations with accuracy of ±0.1mm (using eyepiece 

micrometer disc, Microscope CX40, Olympus 
17/59 

3.3.3. Preparing concentrations of the test item 

The preliminary tests were conducted with the Water Accommodated Fraction of the test item in test 

medium used for exposure [4]. The preliminary tests were performed with a prolonged mixing to 

enable the highest solubility of the test item in the test conditions. 

In the first preliminary Daphnia magna acute immobilization static test the test item was mixed with 

test medium to obtain a mixture of 100 mg/L. The mixture was heterogeneous, particles were visible 

on the surface and bottom of flask therefore the mixture was sonicated for 15 min at 40°C temperature 

(ultrasonic bath, Sonic-10, Polsonic) . The mixture was heterogeneous with visible 

nondissolved particles suspended in the liquid phase, at the surface and at the bottom of flask. The 

mixture was mechanically shaken at 60 rpm at room temperature (mechanic shaker, JWElectronic) 

 After 24 hours of mixing, the mixture was visually unchanged; therefore, it was filtered 

through a conditioned nitrocellulose membrane filter (HAWG filter, pore size: 0.45 μm, Millipore) 

 The clear, colourless and homogeneous filtrate, i.e. the Water Accommodated Fraction 

(WAF) was used for exposure. 

In the second preliminary Daphnia magna reproduction semi-static test the test item was mixed with 

test medium to obtain a mixture of 10 mg/L. The mixture was heterogeneous, particles were visible on 

the surface and bottom of flask. Therefore the mixture was sonicated for 15 min at 40°C temperature 

(ultrasonic bath, Sonic-10, Polsonic) ]. The mixture was heterogeneous with visible 

nondissolved particles suspended in the liquid phase, at the surface and at the bottom of flask. The 

mixture was mechanically shaken at 60 rpm at room temperature (mechanic shaker, JWElectronic) 

. After 24 hours of mixing, the mixture was visually unchanged; therefore, it was filtered 

through a conditioned nitrocellulose membrane filter (HAWG filter, pore size: 0.45 μm, Millipore) 

 The filtrate, i.e. the Water Accommodated Fraction (WAF) was clear, colourless and 

homogeneous. In order to find if the test item impacts the reproduction of daphnids, the additional 

lower concentration of the test item was prepared by dilution of the WAF of a loading of 10 mg/L with 

the test medium for exposure within the second preliminary test. 

The definitive test was performed with a single test item concentration as limit test plus the control. 

Since the test item is of low solubility, the Water Accommodated Fraction of the test item in test 

medium was used for exposure [4]. The mixture of the test item in the test medium in concentration of 

10 mg/L and the control (i.e. test medium) were prepared Each of them was sonicated for 

15 min at 40°C temperature (ultrasonic bath, Sonic- 10, Polsonic and then mixed continuously by 

mechanic shaker at 60 rpm at room temperature for 24 h (mechanic shaker, JWElectronic) 

 Afterwards, the separation by a filtration through a conditioned nitrocellulose 

membrane filter was performed (HAWG filter, pore size: 0.45 μm, Millipore) . The filtrates 

were used for exposure as the Water Accommodated Fraction, i.e. WAF of a loading of 10 mg/L and 

the control. The filtrates, i.e. the Water Accommodated Fraction (WAF) were clear, colourless and 

homogeneous. 
18/59 

3.4. Chemical analysis 

As in previous acute toxicity test with Daphnia magna under GLP conditions ( project number 

) the test item concentrations were not chemically determined in the definitive 

reproduction test. In preliminary research, recoveries of the test item had been demonstrated to be 

clearly below a LoD of 1.0 mg/L, using a suitable HPLC technique. 

4. DEFINITIONS OF THE ENDPOINTS 

The effect of the test item on the reproductive output of Daphnia magna during 21 days was assessed 

on the basis of the mean cumulative number of offspring per survivor. The reproductive output was 

defined as the total number of living offspring produced per living parent Daphnia at the exposure 

termination. According to the OECD Guideline, the reproductive output should be supplemented with 

an additional response variable, i.e. the total number of living offspring produced at the exposure of 

the test per parent Daphnia at exposure initiation, excluding parental accidental and/or inadvertent 

immobilization from the analysis. 

The lowest observed effect concentration (LOEC) is the lowest test item concentration at which 

a statistically significant effect is observed when compared with the control. 

The no observed effect concentration (NOEC) is the highest test item concentration at which no 

statistically significant effect is observed when compared with the control. 

The ECx value is the concentration of the test substance that induces a x percent effect, e.g. reduction 

of reproductive output (i.e. the mean cumulative number of offspring per survivor), immobilization, final 

body size of parent Daphnia, intrinsic rate of population growth, age of first reproduction. 

Test organisms should be considered immobile if they show no ability to swim within 15 seconds after 

swirling the test vessel. The final body size (body length) of the parent Daphnia was determined by 

measuring their body length (excluding the anal spine measured at the exposure termination). 

The intrinsic rate of increase is a measure of population growth integrating reproductive output and 

age-specific mortality. 

5. RESULTS 

The effect of 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol on reproductive output 

(the mean cumulative number of offspring per survivor), immobilization, the final body size of Daphnia 

magna, intrinsic rate of population growth and age to the first reproduction after 21 days of exposure 

were assessed. 

5.1. Preliminary non-GLP tests 

In the first preliminary acute test observations of toxic effects on test organisms were conducted and 

recorded after 24 and 48 h of exposure. In the control and in the WAF of a loading of 100 mg/L neither 

immobilization nor other effects were observed during exposure (Table 1). The pH value measured at 
19/59 

exposure initiation was 7.41 in the control and 7.47 in the WAF of a loading of 100 mg/L. The pH at 

exposure termination was 7.44 in the control and 7.44 in the WAF of a loading of 100 mg/L. The 

temperature was in a range of 22.0 – 24.1oC. 

In the second preliminary test the mean number of live offspring per survivor parent Daphnia magna

and mean length of parent Daphnia magna measured at exposure termination were compared with 

the respective values in the control (Table 2). In fresh solutions measured pH values were in the range 

of 7.43 – 7.63 and the dissolved oxygen concentration was in the range of 8.0 – 9.0 mg/L. In old 

solutions measured pH values were in the range of 7.46 – 7.89 and the dissolved oxygen 

concentration was in the range of 8.0 – 9.4 mg/L. The temperature was in a range of 19.1 – 22.1oC. 

The mean light intensity was 1125.8 – 1275.5 lux. 

5.2. Definitive test 

The definitive test was performed in semi-static design with 21 days of exposure in a single test item 

concentration as a limit test. Since the test item is of very low solubility, the Water Accommodated 

Fraction of the test item in test medium was used for exposure. 

In fresh solutions of control measured pH values were in the range of 7.46 – 7.75 and the dissolved 

oxygen concentration was in the range of 8.1 – 8.8 mg/L. In old solutions of control measured pH 

values were in the range of 7.66 – 8.37 and the dissolved oxygen concentration was in the range of 

8.0 – 9.4 mg/L. In fresh solutions of WAF of a loading of 10 mg/L measured pH values were in the 

range of 7.44 – 7.79 and the dissolved oxygen concentration was in the range of 8.1 – 8.7 mg/L. In old 

solutions of WAF of a loading of 10 mg/L measured pH values were in the range of 7.69 – 8.43 and 

the dissolved oxygen concentration was in the range of 8.0 – 9.4 mg/L. The results are given in 

Table 3. The pH values in fresh solutions of WAF of a loading of 10 mg/L are similar to those in the 

control. 

The temperature was in a range of 19.2 – 21.7oC with a variation of 2.5oC (Figure 1). However, the 

daily range was with a variation of up to 2.0 oC with only one exception; on day 9 the daily range was 

2.1 oC. 

The mean light intensity was 1249 – 1348 lux. The conductivity was in the range of 574 – 612 μS/cm 

(Table 4). The total hardness determined in fresh and old solutions was in range of 198.83 – 

211.83 mg CaCO3/L (Table 5). 

The effects on reproduction were assessed based on the offspring produced by each parent Daphnia, 

removed from test vessels and counted daily. During the definitive test no aborted broods, dead 

progeny or ephippia were observed. Numbers and sizes of broods per parent Daphnia are presented 

in Tables 6 - 7. Age of parent Daphnia magna at first reproduction was similar in the WAF of a loading 

of 10 mg/L and the control (days 8 - 9). The cumulative number of living offspring (Tables 8 - 9) was 

used for statistical analysis. Based on the results, the cumulative offspring numbers in the WAF of a 

loading of 10 mg/L and the control were plotted against time of exposure (Figure 2). 
20/59 

The length of parent Daphnia at exposure termination representing the growth of Daphnia magna

during exposure is presented in Table 10. The mean final body size of each parent Daphnia magna

(i.e. mean length) in the control and the WAF of a loading of 10 mg/L is presented in Figure 3. 

No immobilization of parent Daphnia magna in the control and in the WAF of a loading of 10 mg/L was 

observed throughout the test (Table 11). The immobilization of parent Daphnia magna is defined as 

both the immobilization caused by the test item and also the accidental and/or inadvertent 

immobilization. 

In the control with individually held parent Daphnia magna, the coefficient of variation around the 

mean cumulative number of offspring per survivor was 7.0%, which is lower than 25%, compliant with 

recommendations of the OECD Guideline No. 211 (2012) [1]. 

The intrinsic rate of population growth integrates results of reproductive output and age-specific 

mortality. For growing populations it will be positive and for shrinking populations it will be negative [1]. 

The calculated intrinsic rates for the control and the WAF of a loading of 10 mg/L result that Daphnia 

magna populations would be growing under the chosen conditions. 

No effects were observed at the WAF of a loading of 10 mg/L on reproductive output, immobilization, 

final body size, intrinsic rate of population growth and age of first reproduction. 

5.2.1. Endpoint values 

The observed endpoints of Daphnia magna exposure to a single concentration of 2-(2H-benzotriazol2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol during 21 days were: reproductive output (the mean 

cumulative offspring per survivor), immobilization of parent Daphnia magna, final body size, intrinsic 

rate of population growth and age of first reproduction. The statistical analysis was performed with a 

commercial software ToxRat Professional 2.10 [5], [ , (Appendix 1). The results are 

presented in Table 12. 

The ECx values were not determined since in the limit test no effects were observed on any endpoint 

(reproductive output, immobilization of parent Daphnia magna, final body size, intrinsic rate of 

population growth and age of first reproduction). The limit test demonstrated that the EC10 values i.e. 

concentration of test item that results in 10% reduction in observed effect; reproductive output (the 

mean cumulative number of offspring per survivor), immobilization, final body size of parent Daphnia 

magna, intrinsic rate of population growth and age of first reproduction are higher than the test item 

concentration used i.e. WAF of a loading of 10 mg/L (limit test) (Table 12). 

Since no immobilization or any other effects on parent Daphnia magna were observed, the 

conclusions of the estimations based on the number of living offspring produced per parent 

Daphnia alive at exposure termination (the mean cumulative number of offspring per survivor) are 

the same as the conclusions of the estimations based on the total number of living offspring 

produced per parent Daphnia introduced at exposure initiation (excluding parental accidental 

and/or inadvertent immobilization from the analysis). 
21/59 

The reproductive output (the mean cumulative number of offspring per survivor) in the WAF of a 

loading of 10 mg/L was 5% higher than in the control. The statistical tests were performed with data on 

reproductive output after 21 days of exposure. Shapiro-Wilk’s Test on Normal Distribution confirmed 

normal distribution of data. Levene’s Test on Variance Homogeneity (with Residuals) indicated 

heterogeneity of the variances, Wetch-t test for Inhomogeneous Variances showed no significant 

difference between test item concentration and the control. Therefore, the lowest test item 

concentration causing reduction in reproduction output (LOEC) is higher than the test item 

concentration used, i.e. WAF of a loading of 10 mg/L, and the test item concentration not causing any 

effect on reproduction (NOEC) is higher than, or equal to, the WAF of a loading of 10 mg/L. 

No immobilization of parent Daphnia magna in the test item concentration and the control was 

observed. The lowest test item concentration causing immobilization of the parent Daphnia magna

(LOEC) is higher than the WAF of a loading of 10 mg/L, and the test item concentration not causing 

any effect on immobilization (NOEC) is higher than, or equal to, the WAF of a loading of 10 mg/L. 

The final body size of parent Daphnia in the WAF of a loading of 10 mg/L was 0.4% higher than in 

the control. The statistical tests were performed with data on length of parent Daphnia magna. 

Shapiro-Wilk’s Test on Normal Distribution confirmed normal distribution of data. Levene’s Test on 

Variance Homogeneity (with Residuals) indicated homogeneity of variances, Student-t test for 

Homogeneous Variances showed no significant difference between the test item concentration and 

the control. Therefore, the lowest test item concentration causing growth inhibition of parent Daphnia 

magna (LOEC) is higher than the WAF of a loading of 10 mg/L, and the test item concentration not 

causing any effect on growth of parent Daphnia magna (NOEC) is higher than, or equal to, the WAF of 

a loading of 10 mg/L. 

The intrinsic rate of population growth during 21 days was identical in the WAF of a loading of 

10 mg/L and the control. The statistical tests were performed with intrinsic rate data. Shapiro-Wilk’s 

Test on Normal Distribution confirmed normal distribution of data. Levene’s Test on Variance 

Homogeneity (with Residuals) indicated homogeneity of variances, Student-t test for Homogeneous 

Variances showed no significant difference between test item concentration and the control. 

Therefore, the lowest test item concentration causing reduction in intrinsic rate (LOEC) is higher than 

the WAF of a loading of 10 mg/L, and the test item concentration not causing any effect on intrinsic 

rate (NOEC) is higher than, or equal to, the WAF of a loading of 10 mg/L. 

The statistical tests were performed with the age of the first reproduction data. Levene’s Test on 

Variance Homogeneity (with Residuals) indicated homogeneity of variances, Student-t test for 

Homogeneous Variances showed no significant difference between test item concentration and the 

control. Therefore, the lowest test item concentration causing reduction in intrinsic rate (LOEC) is 

higher than the WAF of a loading of 10 mg/L, and the test item concentration not causing any effect on 

intrinsic rate (NOEC) is higher than, or equal to, the WAF of a loading of 10 mg/L. 
22/59 

6. VALIDITY CRITERIA 

In the definitive test, the following validity criteria specified in the OECD Guideline No. 211 were met: 

- the mortality of the parent animals (defined as immobilization together with accidental and/or 

inadvertent immobilization) in the control was 0%. Therefore, the criterion stating that it should 

not exceed 20% at the exposure termination was met. 

- the mean number of living offspring produced per parent animal surviving at the exposure 

termination (defined as the number of living offspring produced per parent Daphnia alive at 

exposure termination, i.e. the mean cumulative number of offspring per survivor) in the control 

was 135.2. Therefore, the criterion stating that it should be higher than or equal to 60, was met. 

7. DEVIATIONS FROM THE STUDY PLAN 

According to the OECD Guideline No. 211 [1] the recommended range of temperature is 18 - 22oC 

with variation not exceeding 2oC within these limits as daily range (Appendix 5). In the definitive test 

the temperature was in a range of 19.2 – 21.7oC with a variation of 2.5oC. However, the daily range 

was with a variation of up to 2oC with only one exception; on day 9 the daily range was 2.1oC, what did 

not have impact on the study results. All validity criteria were met. 

8. REFERENCES 

[1] OECD Guideline for the Testing of Chemicals No. 211 (Adopted October 2, 2012): ‘Daphnia 

magna Reproduction Test’. 

[2] Directive 2004/10/EC on the harmonisation of laws, regulations and administrative provisions 

relating to the application of the principles of good laboratory practice and the verification of 

their applications for tests on chemical substances (codified version), 

[3] Polish Act: Act of Parliament of February 25, 2011 on Chemical Substances and Mixtures 

(Dz.U. Nr 63, poz. 322). 

 Polish legislation: Regulation of Ministry of Health of 22nd May 2013 on Good Laboratory 

Practice and performing studies in compliance with Good Laboratory Practice (Dz.U. Nr 108, 

poz. 665). 

[4] OECD Series on Testing and Assessment No 23 “Guidance Document on Aquatic Toxicity 

Testing of Difficult Substances and Mixtures”, ENV/JM/MONO(2000)6, December 15, 2000. 

[5] ToxRat Professional 2.10 – Software for Statistical Evaluation of Biotests in Ecotoxicology, 

ToxRat Solutions GmbH, Alsdorf, Germany. 

[6] OECD Guideline for Testing of Chemicals No 202 (2004): ‘Daphnia sp., Acute Immobilization 

Test’. 
23/59 

9. STANDARD OPERATING PROCEDURES USED IN THE STUDY 

Proceeding with test item. 

Study coding. 

Analytical balance – instruction manual. 

Centrifuge- instructions manual. 

Microscope - manual instructions. 

Spectrophotometer – instructions manual. 

Preparation and labelling of chemical reagents. 

Daphnia, acute immobilization test. 

Daphnia magna, reproduction test. 

Documentation and archiving of test records and notes. 

inoLAB pH/Oxi Level 3 and Multi 3430 pH-oxi meter – instruction manual. 

NALGENE filtration system – instruction manual. 

Sonopan L-50 planar lux meter– instruction manual. 

Determination of total organic carbon by titration method. 

Solarus electronic titration apparatus – instruction manual. 

HI 141 temperature recorder– instruction manual. 

Mechanic shaker – instructions manual. 

Standardized Elendt M7 medium. 

Algae and cyanobacteria - laboratory cultivation. 

Daphnia magna - laboratory cultivation. 

ToxRat Professional – instruction manual. 

SolPure7 water deionization system – instruction manual. 

Reference tests. 

Absorbance and transmittance measurement for algae and cyanobacteria growth 

inhibition test. 

 Sonic-10 ultrasonic bath – instruction manual. 

Determination of hardness by titration method. 

Sample collection. 

 Method for determination of carbon in water. 

Receiving, distribution, storage, archiving and elimination of test item. 

Archiving of test documentation and samples. 

Control of study planning, experiment conduct, and final report preparation. 
24/59 

Table 1. Results, first preliminary non-GLP test. 

Concentration Number of 

Daphnia magna

Number of immobilized Daphnia magna Total of 

immobilized 

Daphnia 

magna [%] 

24 h 48 h 

Replicates 

A B C D A B C D 24 h 48 h 

Control 20 0 0 0 0 0 0 0 0 0 0 

WAF of a loading of 100 mg/L 20 0 0 0 0 0 0 0 0 0 0 

The exposure was on 19.11.2013 - 21.11.2013. 

Table 2. Results, second preliminary non-GLP test. 

Concentration 

Mean number of live 

offspring per parent 

Daphnia magna as 

compared with the 

control 

[%] 

Mean length of parent 

Daphnia magna as 

compared with the 

control 

[%] 

Total of immobilized 

parent Daphnia 

magna

[%] 

Control 100.0 100.0 0 

WAF of a loading of 

10 mg/L 104.5 100.3 0 

10-fold diluted WAF of a 

loading of 10 mg/L 101.1 100.7 0 

The exposure was on 22.11.2013 - 13.12.2013. 
25/59 

Table 3. pH values and O2 concentration measured, definitive test 

Sample 

pH values Dissolved oxygen 

concentration [mg/L] 

Control 

WAF of a 

loading of 

10 mg/L 

Control 

WAF of a 

loading of 

10 mg/L 

Fresh on day 0 7.53 7.53 8.3 8.2 

Old on day 2 8.34 8.43 9.4 9.4 

Fresh on day 2 7.52 7.54 8.8 8.7 

Old on day 5 8.37 8.28 8.5 8.6 

Fresh on day 5 7.62 7.60 8.1 8.1 

Old on day 7 7.84 7.70 8.0 8.0 

Fresh on day 7 7.54 7.55 8.3 8.1 

Old on day 9 8.04 7.96 8.0 8.0 

Fresh on day 9 7.75 7.78 8.4 8.4 

Old on day 12 7.71 7.69 8.3 8.3 

Fresh on day 12 7.47 7.44 8.2 8.4 

Old on day 14 7.87 7.75 8.1 8.3 

Fresh on day 14 7.59 7.59 8.2 8.2 

Old on day 16 7.66 7.75 8.2 8.1 

Fresh on day 16 7.46 7.47 8.3 8.7 

Old on day 19 8.26 8.28 8.2 8.6 

Fresh on day 19 7.74 7.79 8.2 8.4 

Old on day 21 7.84 7.87 8.1 8.2 

Fresh - measured in fresh samples before splitting up into replicates, 

Old - measured in old samples of pooled replicates. 
26/59 

Table 4. Conductivity measured, definitive test 

Sample 

Conductivity [μS/cm] 

Control WAF of a loading of 

10 mg/L 

Fresh on day 0 585 586 

Old on day 2 596 591 

Fresh on day 2 576 574 

Old on day 5 580 575 

Fresh on day 5 577 577 

Old on day 7 585 584 

Fresh on day 7 577 578 

Old on day 9 585 589 

Fresh on day 9 594 593 

Old on day 12 612 603 

Fresh on day 12 584 582 

Old on day 14 600 595 

Fresh on day 14 578 577 

Old on day 16 594 586 

Fresh on day 16 584 583 

Old on day 19 598 592 

Fresh on day 19 588 586 

Old on day 21 600 600 

Fresh - measured in fresh samples before splitting up into replicates, 

Old - measured in old samples of pooled replicates. 

Table 5. The total hardness measured, definitive test 

Sample 

Total hardness [mg CaCO3/L] 

Control WAF of a loading of 

10 mg/L 

Fresh on day 0 207.57 208.97 

Old on day 2 209.37 208.43 

Fresh on day 7 200.70 203.63 

Old on day 7 207.03 206.77 

Fresh on day 14 198.83 199.63 

Old on day 14 210.43 208.63 

Old on day 21 211.83 209.63 

Fresh - measured in fresh samples before splitting up into replicates, 

Old - measured in old samples of pooled replicates. 
27/59 

Table 6. Offspring per survivor per day in the control, definitive test 

mean – arithmetic mean 

SD – standard deviation 

Table 7. Offspring per survivor per day in the WAF of a loading of 10 mg/L, definitive test 

mean – arithmetic mean 

SD – standard deviation 
28/59 

Table 8. Cumulative offspring per survivor in the control, definitive test 

mean – arithmetic mean 

SD – standard deviation 

CV – coefficient of variation 

n.d. – not determined 

Table 9. Cumulative offspring per survivor in the WAF of a loading of 10 mg/L, definitive test 

mean – arithmetic mean 

SD – standard deviation 

CV – coefficient of variation 

n.d. – not determined
29/59 

Table 10. Final body size of parent Daphnia magna [mm], definitive test 

mean – arithmetic mean 

SD – standard deviation 

CV – coefficient of variation 

Table 11. Results, definitive test 

Concentration 

Mean number of live 

offspring per parent 

Daphnia magna as 

compared with the 

control 

[%] 

Mean length of parent 

Daphnia magna as 

compared with the 

control 

[%] 

Total of 

immobilized 

parent Daphnia 

magna

[%] 

Control 100.0 100.0 0 

WAF of a loading of 

10 mg/L 105.0 100.3 0 

The exposure was on 22.01.2014 – 12.02.2014 
30/59 

Table 12. Endpoint values based on WAF of a loading of 10 mg/L, definitive test 

Endpoint 

Mean 

cumulative 

offspring per 

survivor 

Mean 

cumulative 

number of 

offspring per 

survivor 

Immobilization 

of parent 

Daphnia 

magna

Final body size 

of parent 

Daphnia 

magna

Intrinsic rate r 

EC50 >10 >10 >10 >10 >10

EC20 >10 >10 >10 >10 >10

EC10 >10 >10 >10 >10 >10

LOEC >10 >10 >10 >10 >10

NOEC ≥10 ≥10 ≥10 ≥10 ≥10 
31/59 

Legend Ch1 Ch1 Lo Al Ch1 Hi Al

17.30

18.76

20.22

21.68

23.14

24.60

°C

Graph Time

2014-01-22

 10:30

2014-01-26

 02:43

2014-01-29

 18:57

2014-02-02

 11:10

2014-02-06

 03:23

2014-02-09

 19:37

2014-02-13

 11:50

Figure 1. Temperature, definitive test 

0

20

40

60

80

100

120

140

160

0 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Day of exposure

Mean cumulative offspring per survivor

Control

WAF of a loading of 10 mg/L

Figure 2. Mean cumulative offspring per survivor versus day of exposure, definitive test 
32/59 

3,58 3,59

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

Control WAF of a loading of 10 mg/L

Final body size of parent daphnids [mm]

Error bar = standard deviation 

Figure 3. Final body size of parent Daphnia magna, definitive test 
33/59 

Appendix 1. Statistics for determination of endpoint values 
34/59 
35/59 
36/59 
37/59 
38/59 

Appendix 2. Composition of the Elendt M7 medium 

Substance Concentration in the Elendt M7 medium 

[mg/L] 

Micronutrients

H3BO3 0.715 

MnCl2 · 4H2O 0.090 

LiCl 0.076 

RbCl 0.018 

SrCl2 · 6H2O 0.038 

NaBr 0.004 

Na2MoO4 · 2H2O 0.016 

CuCl2 · 2H2O 4.19· 10-3

ZnCl2 0.013 

CoCl2 · 6H2O 0.01 

KI 3.25 · 10-3

Na2SeO3 2.19 · 10-3

NH4VO3 5.75 · 10-4

Na2EDTA · 2H2O 0.625 

FeSO4 · 7H2O 0.249 

Macronutrients 

CaCl2 · 2H2O 293.8 

MgSO4 · 7H2O 123.3 

KCl 5.8 

NaHCO3 64.8 

Na2SiO3· 9H2O 10.0 

NaNO3 0.274 

KH2PO4 0.143 

K2HPO4 0.184 

Vitamins 

Thiamine hydrochloride 0.075 

Cyanocobalamine 0.001 

Biotine 7.5 · 10-4
39/59 

Appendix 3. Standard curves 

STANDARD CURVE

y = 1,0193x

R

2

 = 0,9986

0

50

100

150

200

250

300

350

400

450

0 50 100 150 200 250 300 350 400 450

Nominal total hardness [mgCaCO3/l]

Determined total hardness 

[mgCaCO3/l]

Figure A. Standard curve for analysis of total hardness by titration with EDTA 

Figure B. Standard curve for absorbance of algae versus TOC content determined by titration 

method, used in the preliminary test 

Content of total organic carbon (TOC) [mg C/L]

Absorbance at 670nm of 

50-fold diluted algae suspension 
40/59 

Figure C. Results of TOC analysis in algal suspension mixture, used in the definitive test 
41/59 

Figure D. Results of TOC analysis in algal suspension mixture, used in the definitive test
42/59 

Appendix 4. Copy of the Statement of GLP compliance 
43/59 

Appendix 5. Copy of the Study plan 
44/59 
45/59 
46/59 
47/59 
48/59 
49/59 
50/59 
51/59 
52/59 
53/59 
54/59 
55/59 
56/59 
57/59 
58/59 
59/59