Pag e 1 o f 40 Study Title

Alga

, Inhibitio n Tes t with

ISOTA N L P RJ E 69140

Data Requirements / Test Guidelines

E U metho d C. 3 (2008)

OEC D T G 20 1 (2006)

Author:

Dr. Arnd Weyers

Stud y initiatio n date : 2009

- 10

- 01 Stud y completio n date : 2009

- 12

- 17

Star t o f Experimenta l Phase : 2009

- 10

- 06 En d o f Experimenta l Phase : 2009

- 12

- 17

Sponsor:

LANXES S Deutschlan d GmbH

Busines s Uni t Leather

5136 9 Leverkusen

Germany

Testing facilit y :

CURRENT A Gmb H & Co

. OHG

Service s Analytik

5136 8 Leverkusen

Germany

Laboratory Project Identification

Stud y No

. 2009/0010/15Pag e 2 o f 40

CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Thi s pag e i

s intentionall

y lef t blan k fo r th e purpos e o f sub m ittin g ad m inistrative

infor m atio n tha t i

s require d b

y regulation s pro m ulgate d b y variou s countries.Pag e 3 o f 40

CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

CONTENTS

Page

GL P DECLARATION 4

ARCHIVATIO N O F DOCUMENT S AN D TES T ARTICLES 4

QUALIT Y ASSURANC E STATEMENT 6

GL P CERTIFICATE 7

SUMMARY 8

INTRODUCTION 10

EXPERIMENTA L PROCEDURE 11

MATERIAL S AN D METHODS 13

Tes t item 13

Test species 14

Apparatus 14

Pre

- treatment of the test item 15

Exposur e conditions 15

Chemica l anal y s is 17

Expressio n o f biologica l results 20

Applie d SOP s an d methods 21

RESULTS 22

C ommen ts 37

Validity of the test 37

ANNE X 1 38

Nutrient medium of pre

- cultures and test cultures 38

ANNE X 2 39

Equation s fo r calculation 39Page 4 of 40

CURRENTA GmbH 

### & C o

. OHG

Services Analytik Study No.: 200910010115



### GLP DECLARATION

This study was conducted in compliance with the OECD principles of Good

Laboratory Practice (GLP, as revised in 1997) and with the Principles of Good

Laboratory Practice according to Annex I, German Chemical Law (Amendment of

Annex I, May 8, 200 1 ).

Date I Signature

Study Director 

## 'JoV'J

- / ? I t 

# 4 

# a

(Dr. Weyers I Netrtrahn)



### ARCHIVATION OF DOCUMENTS AND TEST ARTICLES

Study plans, raw data and study reports are archived for at least for 15 years at the

GLP archives of the test facility. No data will be discarded without the sponsor's

consent.

Test articles are archived at CURREN T A Services Analytik

.

The sponsor must ensure that information submitted about the test item (identity

testing or information about physical and chemical properties) is complete and that it

complies with the principles of Good Laboratory Practice.

The customer must also retain a sample of the test item used where sampling has

not been performed by CURREN T A Services Analytik

.Pag e 5 o f 40

CURRENT A Gmb H & Co

. OH G Stud y No. : 2009/0010/15

Service s Analytik

Thi s pag e i

s intentionall

y lef t blan k fo r th e purpos e o f sub m ittin g ad m inistrative

infor m atio n tha t i

s require d b

y regulation s pro m ulgate d b y variou s countries.P a g e 6 o f 4 0

CURRENTA GmbH & Co. OHG

SeNices Analytik Study No.: 2009/0010/15



### QUALITY ASSURANC E STATEMENT

This report was au d i ted by the Quality Assurance Unit CURRENTA Services

Analytik

, Quality Management at CURRENTA GmbH & Co. OHG and this statement

confirms that the final report reflects the raw dat a

.

The dates of Quality Assurance inspections and audits are given belo w

. During the

on

- site inspections procedures applicable to this type of short time study were

inspected

.

Audits Dates of QA Dates of

inspection report

study plan r

e v i e w 2 009

- 1 0

- 02 2009

- 10

- 02

process based inspection 2009

- 05

- 28 200 9

- 0 6

- 03

final report review 2009/0002/09

' 2 00 

### 9

- I 2

- 2 2

- ( (j (J 

### 9

- ; 'l

- 2..

Da t e I Signatu r e



### 10Vj

- 1 l

- Z.L

- 

# u?-

( &em baMg e " / Dr. Neupert)CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 7 o f 40 

### GLP CERTIFICATE

Pleas e note : Effectiv e Januar y 1 st

, 2008 the company name Bayer Industry Services

GmbH & Co. OHG was changed to CURRENTA GmbH & Co. OHGCURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 8 o f 40 

### SUMMARY

A stud y wa s performe d t o asses s th e advers e effect s o f ISOTA N L P RJ E 6914 0 on

th e yiel d (

= biomas s a t tim e t minu s initi al biomass ) an d th e growt h rate (= rate of

increas e i n cel l densit y wit h time ) o f t h e planktoni c freshwate r alga l species

Des m odes m u s subspicatu s (former name: Scenedes m u s subspicatu s ) ove r several

generations.

Th e stud y wa s conducte d i n accordan ce wit h Commissio n Regulatio n (EC)

N o 761/200 9 amendin g Regulatio n N o 440/2008

, Metho d C. 3 ‘Freshwate r Alga e and

Cyanobacteria

, Growt h inhibitio n test ’ (2009 ) whic h i s equivalen t t o OEC D Guideline

fo r Testin g o f Chemical s No

. 20 1 'A l ga

, Growt h Inhibitio n Test ' (2006).

Exponentiall y growin g alga l cell s wer e expo s e d fo r a perio d o f 7 2 hour s t o a rang e of

concentrations

, nominall y 0.32

, 1.0

, 3.2

, 1 0 an d 3 2 mg/ L o f ISOTA N L P RJ E 69140

dissolve d i n water.

Th e cel l densitie s wer e mea s ure d a t 2 4 hou r intervals

. Inhibitio n o f th e algal

populatio n wa s measure d a s reductio n i n growt h rat e (inde x r)

, relativ e t o control

culture s grow n unde r identica l conditions

. Gro w t h rate s wer e als o use d t o calculat e a

N o Observe d Effec t Concentratio n an d a Lowes t Observe d Effec t Concentration

accordin g t o William s Multipl e Sequentia l t

- Tes t Procedure

. Th e followin g values

were determined:

Results for drying residue / active ingredient [mg/L]:

E

r C 50* (0

- 48 h) : > 100 / > 100

E

r C 10* (0

- 48 h) : 1.6 / 1.4

NOEC [r] ( t

α 0.05 ) : 0.071 / 0.065

* Reductio n o f growt h rat e (E

r Cx

, NOE C [r] ) i s th e preferre d endpoin t accordin g to

OEC D 20 1 an d fo r regulator y purpose s i n t h e EU

. Result s relatin g t o yiel d (E y Cx,

NOE C [y] ) wer e calculate d t o fulfi l regulato ry requirement s i n som e countrie s (bu t not

i n th e EU ) an d ar e give n i n th e result s sectio n o f thi s report.

Th e E

r C 5 0 coul d no t b e calculate d wit h sufficien t p r ecision

. Fro m fig

. 3 a valu e o f ca.

100 0 mg/ L ca n b e read

, thu s th e E C 5 0 i s clearl y > 10 0 mg/L

Th e stud y dat a wer e analyse d o n a 4 8 h ba s is

. A s cel l number s increase d wel l above

16

- fol d ove r 4 8 h

, an d al l othe r validit y cri t eri a ar e met

, thi s i s a n optio n explicitly

covere d b y OEC D T G 20 1 (2006 ) an d E U metho d C. 3 (2009).CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 9 o f 40 SUMMARY

Th e result s ar e expresse d i n term s o f geometri c mea n measure d concentrations.

Effectiv e concentration s range d fro m 76.0

- 103.4 % o f nomina l value s a t 0 hours

, from

16.2

- 35.1 % o f nomina l value s a t 2 4 hours

, an d fro m 2.5

- 9

. 6 % o f nomina l value s at

4 8 hour s respectively

. Th e analytica l value s refe r t o th e dryin g residu e of

ISOTA N L P RJ E 6914 0 wit h a conten t o f 35.05 % (accor d in g t o dat a o f th e sponsor).

Thi s conten t wa s take n t o calculat e th e nomina l valu e o f ISOTA N L P RJ E 6914 0

.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 1 0 o f 40 

### INTRODUCTION

Thi s repor t contain s a descriptio n o f th e method s use d an d th e result s obtained

durin g a stud y t o investigat e advers e effect s o f ISOTA N L P RJ E 6914 0 o n th e yield

an d th e growt h rat e o f a populatio n o f th e planktoni c freshwate r alga l species

Des m odes m u s subspicatu s (former name: Scenedes m u s subspicatu s ). In the

present test, adverse effects were expres s e d a s thos e effectiv e concentration s which

reduce d yiel d (

= biomas s a t tim e t minu s initia l biomass ; inde x y ) and growth rate (=

rat e o f increas e i n cel l densit y wit h time ; inde x r ) by 10% ( E y C 10

, E

r C 10 ) an d 50%

( E y C 50

, E

r C 50 ) withi n a continuou s 7 2 hou r pe r io d o f exposure

. Th e stud y data

wer e analyse d o n a 4 8 h basis

, becaus e concentration s o f th e tes t ite m markedly

decrease d ove r tim e an d thu s th e 4 8 h valu e reflect s exposur e bette r tha n th e 7 2 h

values

. A s cel l number s increase d wel l abov e 16

- fol d ove r 4 8 h

, an d al l othe r validity

criteri a ar e met

, thi s i s a n optio n explicitl y covere d b y OEC D T G 20 1 (2006 ) an d EU

method C.3 (2009).

I n additio n t o th e E C 1 0 an d E C 5 0 values

, NOEC/LOE C wer e calculate d base d on

yield [y] and growth rate [r], respectively.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 11 o f 40 

### EXPERIMENTAL PROCEDURE

Th e stud y wa s conducte d i n accordan ce wit h Commissio n Regulatio n (EC)

N o 761/200 9 amendin g Regulatio n N o 440/2008

, Metho d C. 3 ‘Freshwate r Alga e and

Cyanobacteria

, Growt h inhibitio n test ’ (2009 ) whic h i s equivalen t t o OEC D Guideline

fo r Testin g o f Chemical s No

. 20 1 'A l ga

, Growt h Inhibitio n Test ' (2006).

Th e purpos e o f thi s metho d wa s t o det e rmin e thos e concentration s whic h cause d a

10 % an d a 50 % advers e effec t (

= E C 10

, E C 50) or, if conducted as a limit test, to

determin e th e advers e effect s a t a maximu m tes t concentratio n o f 10 0 mg/ L o r a t the

limi t o f wate r solubility

, respectively

. Eff e c t dat a ar e expresse d o n th e basi s o f yield

[ y ] and growth rate [r]

.

A range

- findin g (non

- GLP ) tes t precede d t h e mai n tes t an d provide d information

abou t th e rang e o f concentration s whic h wer e use d i n th e mai n test

. Th e following

nomina l concentration s o f th e tes t ite m wer e teste d i n th e range

- fi nding test: 1, 3.2,

10

, 3 2 an d 10 0 mg/L.

I n th e mai n test

, th e alga e wer e expose d t o th e tes t ite m adde d t o wate r a t a rang e of

concentration s fo r a perio d o f 7 2 hours

. Define d concentration s o f th e tes t ite m le d to

a certai n inhibitio n o f alga l yiel d an d gro w t h rat e a t th e en d o f th e 7 2 hou r study

period

. Cel l densitie s wer e recorde d a t 2 4 hou r intervals

. Th e 4 8 hou r E C 1 0 and

E C 5 0 (base d o n both

, yiel d [ y ] an d growt h rat e [r] )

, wer e calculate d b y probit

analysis.

Al l calculation s wer e carrie d ou t us i n g th e statistic s p r ogramm e ToxRatPro

Versio n 2.1 0 (release d 19.02.2009)

. Fo r th e calculation s al l alga e count s were

divide d b y a facto r o f 10000.

Durin g th e tes t a temperatur e rang e o f 2 1

- 24° C wa s maintaine d i n th e tes t vessels.

Th e maintenanc e o f tes t ite m concentration s wa s prove d b y analytica l measurements

an d th e p H wa s measure d a t th e beginnin g o f the test start and every 24 hours

thereafter

. I n orde r t o avoi d a n impairmen t o f th e tes t system

, additiona l replicates

wer e use d fo r qualitativ e examinatio n o f sample s (HPL C analysis ) an d pH

measuremen t afte r 0

, 2 4 an d 4 8 h

. P H mea s uremen t a t th e en d o f th e tes t were

performed using replicate I of the test.

I n orde r t o chec k whethe r o r no t significan t amounts of the test ite m ar e incorporated

int o th e alga l biomas s durin g the test period, test flasks a t th e tes t concentration

3 2 mg/ L w ithout algae were run in parallel to the geometric series of test.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 12 o f 40 EXPERIMENTA L PROCEDURE

Th e followin g validit y criteri a o f th e tes t wer e met:

Th e cel l densit y i n th e contro l culture s inc r ease d b y a facto r o f a t leas t 1 6 withi n 48

hours

. Th e tes t perio d wa s shortene d t o 4 8 hour s t o maintai n unlimited

, exponential

growth during the test.

Accordin g t o a n updat e o f OEC D T G 20 1 (2006)

, th e mea n coefficien t o f variatio n for

section

- by

- sectio n specifi c growt h rate s i n th e contro l culture s mus t no t excee d 35%.

Th e coefficien t o f variatio n o f averag e spe c ifi c growt h rate s durin g th e whol e test

perio d i n replicat e contro l c ultures must not exceed 7%.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 13 o f 40 

### MATERIALS AND METHODS

Test item

Test item (Product name): ISOTA N L P RJ E 69140

Dryin g residu e : 35.05%

Content of active

ingredien t : 31.94%

Parti e : RJ E 71120

Charge :

--

Expir y dat e : 2010

- 01

- 30

Appearanc e : liquid

Storage : room temperature

Data of active ingredient

Chemica l nam e : preparatio n o f hexame t hylenediisocyanate

bisulfite

- adduc t i n water

CA S numbe r : 38632

- 47

- 2 (N a salt)

Empirica l formul a : C 8 H 14 N 2 O 8 S 2

. 2Na

Molecula r weigh t : 37 6 g/mol

Stability of test concen

tration s durin g exposur e : Examine d b y chemica l analysi s (HPLC ) a t 0

, 24

an d 4 8 hours.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 14 o f 40 MATERIAL S AN D METHODS

Test species

Name : Des m odes m u s subspicatu s (formerly Scene

des m u s subspicatu s ) Strain No. 86.81 SAG

Source : Strain of the t es t specie s obtaine d fro m 'The

Collectio n o f Alga l Cultures ' of the Institute of Plant

Physiolog y a t th e Universit y o f Göttingen

(Germany).

Maintenance

an d Acclimatisatio n : Exponentially

- g rowin g stoc k culture s were

maintaine d i n th e tes t facilit y unde r constant

temperatur e condition s ( 2 3 +/

- 2°C ) a t a light

intensit y i n th e r ang e 6 0

– 12 0 μE

. x m - 2 x s - 1

(measure d i n th e rang e 40 0 t o 70 0 n m usin g a

spherica l quantu m flu x meter)

. Th e nutrient

mediu m (accordin g t o BRINGMAN N & KÜHN

(1977 ) wa s renewe d onc e a week

. Cel l density

measurement s wer e mad e usin g a microcell

counter

, Sysme x F300

, Digitana.

Preparatio n of

pre

- cultures : Pre

- cultures w er e se t u p thre e day s befor e the

star t o f a test

. The y wer e grow n unde r identical

exposur e condition s a s th e stoc k cultures

, except

from the use of a different nutrient medium

(anne x 1 )

.

Tes t culture s : Th e alga l inocu l a for a test were taken from an

exponentially

- growin g pre

- cultur e an d wer e mixed

wit h th e nutrien t mediu m (anne x 1 ) t o mak e u p to

a fina l cel l densit y o f abou t 500 0 cell s pe r millilitre

in the test medium.

Apparatus

– Analytica l balance

– PH

- meter

– Shakin g incubator

– Microcellcounter

– Variou s glas s materials : Erlenmeye r flasks, volumetric flasks, beakers,

pipettes etc.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 15 o f 40 MATERIAL S AN D METHODS

Pre

- treatment of the test item

A stoc k solutio n wa s prepare d t o giv e th e desire d serie s o f tes t concentrations

base d o n a dryin g residu e o f Isota n L P RJ E 6914 0 o f 35.05 % (accordin g to

dat a o f th e sponsor ) T o achiev e thi s 356

. 8 m g o f th e tes t ite m wer e adde d t o 1

litr e o f dilutio n water

. Th e p H wa s measure d t o b e p H 4.8.

Isota n L P RJ E 6914 0 hydrolysi s rapidl y i n water

, dependin g o n th e p H value.

T o minimis e hydrolysi s effect s th e p H of the concentrat e d nutrien t medium

wa s measure d t o b e p H 8. 0 an d wa s adjuste d t o p H 6.0.

T o produc e th e differen t tes t ite m con c entration s th e prepare d stoc k solution

wa s dilute d wit h dilutio n wate r t o a vo lume of 80 mL. Afterwards 10 mL of

nutrien t mediu m an d 1 0 m L o f alga l i noculu m wit h a cel l densit y o f 5000

cells/m L wer e adde d resultin g i n th e fina l nomina l concentrations

. Fo r each

test item concentration and contr o l 3 replicate s wer e prepared.

Exposure conditions

Tes t vessel s : 30 0 m L Erlenmeye r flask s wit h cotto n stoppers

Culturin g apparatu s : Ligh t chambe r i n whic h a tempera t ur e i n th e range

21° C t o 24° C wa s maintaine d a t +/

- 2°C

, and

continuou s unifor m illuminatio n wa s provide d in

th e spectra l rang e 40 0 t o 70 0 nm.

Ligh t intensit y : A t th e averag e o f th e tes t solutions

, a light

intensit y i n th e rang e 6 0 t o 12 0 μE

. x m - 2 x s - 1

, or

a n equivalen t rang e o f 400 0 t o 800 0 lx

, wa s used.

Ligh t intensit y wa s check ed before start of the

study.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 16 o f 40 MATERIAL S AN D METHODS

Cel l density

measurement s : Cel l densitie s wer e measure d i n a microcell

counte r (Sysme x F300

, Di g itana ) b y takin g small

aliquots from each test flask for measurements,

whic h wer e no t replaced.

Experimenta l desig n : 5 tes t concentration s plu s 1 control

3 replicate s pe r concentr a tion

, 3 replicate s per

control

Initia l cel l densit y i n th e tes t cultures

approximatel y 500 0 cell s pe r millilitre

Additionall y highes t tes t concentratio n without

algae

Test item

concentration/s : 0.32, 1.0, 3.2, 10 and 32 mg/L

Metho d o f administratio n : stoc k solution

Duratio n o f exposur e : 4 8 hours

Criteria of effects : The criteria of advers e effect s use d i n thi s study

wer e th e item

- induce d inhibitio n o f yiel d [ y ] and

growth rate [r]

, respectively

, o f th e algal

population.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 17 o f 40 MATERIAL S AN D METHODS

Chemical anal y s is

I n thi s chapte r th e method s use d t o determ i n e th e concentration s o f the test item are

described

. Th e quantificatio n o f t h e tes t ite m wa s performe d b y HPL C analysi s and

MS D

- detection

, accordin g t o SO P 0019 4 Versio n 1.

Materials and Methods

Test Item

Th e tes t ite m a s describe d i n th e biolog i ca l par t o f thi s stud y wa s als o use d to

prepar e th e spike d tes t wate r samples.

Analytical Procedure

Reagents and Solvents

Milliporewater / Ammoniumacetat / Methanol

Standard Solutions used for Calibration

29. 6 m g o f th e tes t ite m wer e diss o lve d i n Milliporewate r an d mad e u p with

Milliporewate r t o th e mar k i n a 10 0 m L volumetri c flas k t o prepar e a stock

solutio n o f 29 6 mg/ L respectivel y 103.74 8 mg/ L dryin g residu e o f 35.05%.

Define d volume s o f thi s stoc k solutio n wer e dilute d wit h Milliporewate r to

obtai n standar d solution s i n th e rang e o f 0.020 7 t o 4.15 0 mg/ L dryin g residue

of 35.05%.

Althoug h th e overal l metho d sensi t ivit y woul d hav e bee n better

, the

concentratio n o f th e lowes t use d calibr a tio n solutio n wa s employe d a s th e limit

o f quantification.

Thes e solution s wer e use d t o calibrat e th e HPLC

- system.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 18 o f 40 MATERIAL S AN D METHODS

Standard Solutions used for Verification of the Calibration and Blank Test

22. 7 m g o f th e tes t ite m wer e diss o lve d i n Milliporewate r an d mad e u p with

Milliporewate r t o th e mar k i n a 10 0 m L volumetri c flas k t o prepar e a

verificatio n solutio n wit h a nomina l c oncentratio n o f 22 7 mg/ L respectively

79.56 4 mg/ L dryin g residu e o f 35.05%

. Th e dail y verificatio n o f th e calibration

wa s performe d wit h thi s Standa r d Solutio n o f Verification

. Thi s solutio n was

dilute d wit h Milliporewate r t o obtai n a concentratio n o f 1.59 1 mg/ L drying

residue of 35.05%.

Th e mea n recover y rat e wa s calculated.

In addition, water without the test i t e m wa s analyse d (analytica l blank).

Analysis of Samples

Aliquot s o f th e sample s f r o m th e biologica l tes t wer e directl y analyse d by

HPL C an d MS

- detectio n (rang e o f th e i n jectio n volume : 1

- 5 0 μL

, dependin g on

th e expecte d concentration).

HPLC Conditions

HPLC system: e.g. Agilent T e chnologie s 110 0 syste m wit h MSD

Column: 2.1*150 mm

Stationar y phase : Inertsi l OD S

- 3; 3 μm (GL Sciences)

Eluen t A : 1.9 3 g Ammoniu m acetat e + 1 L Milliporewater

Eluen t B : 1.9 3 g Ammoniu m acetat e + 1 L Methanol

Isocratic: 95% A + 5% B (v/v)

Temperature : 4 0 °C

Flow rate: 0.2 mL/min

Detection: MSD / SIM Modus ( Positiv)

Recorde d mass : m/ z 249. 1 an d 331

Injectio n volume : 1

- 5 0 μLCURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 19 o f 40 y = peak area of test item in injected sample (counts)

x = concentration of test item in injected sample (mg/L)

a = 3988.43524

b

= 393430.65843

c = - 245.28965

(2) recovery % = 100 Calculation MATERIAL S AN D METHODS

Th e calibration

, verification

, b l an k an d sampl e solution s were

chromatographe d usin g th e condition s describe d i n th e paragrap h before.

Calculatio n wa s don e usin g a n exte r na l standardizatio n metho d an d a

calibratio n curve

. Th e calibratio n curv e wa s calculate d accordin g t o equation

(1).

(1) y = a

· x

2 + b

· x + c

where:

Th e correlatio n coefficien t R 2 was

R 2

= 0.99955

T o verif y th e calibratio n th e recover y rat e o f th e verificatio n solutio n was

calculate d accordin g t o equatio n (2).

mean value of assay

nomina l concentration

The recovery rate was 99.3% (number of determinations: 4).

Results

Th e analytica l result s ar e presente d i n detai l i n th e followin g tables

, together

wit h th e biologica l effec t data.

Sampling schedule:

Contro l : a t 0

, 2 4 an d 4 8 hours

Tes t concentration s : a t 0

, 2 4 an d 4 8 hoursCURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 2 0 o f 40 MATERIAL S AN D METHODS

Expression of biological results

• Cel l densit y measurements

, y i el d an d gro w t h rate s i n th e tes t and

contro l culture s wer e tabulate d accord i n g t o th e concentrati on of test item

and the time of measurement.

• Gro w t h curve s wer e plotte d fo r eac h tes t concentratio n an d contro l and

ar e presente d i n figure 1

.

• Th e percentag e inhibitio n of both, yield [b] and growth rate [r], was

calculate d fo r eac h tes t concentratio n usin g equation s [1] an d [3]

(anne x 2 )

.

• The gro w th rate [r] wa s calculate d fo r eac h tes t cultur e usin g equation

[2] i n anne x 2

.

• E C 5 0 an d E C 1 0 value s for both, yield [y] and growth rate [r], were

calculate d b y probi t analysis.

• NOE C an d LOE C o f both

, yiel d [y ] an d grow th rate [r], were determined

b y a multisamplin g compariso n (accor d in g t o William s Multipl e Sequential

t

- Test Procedure) (tabl e 9 an d 16)CURRENT A Gmb H & Co

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Service s Analytik Stud y No. : 2009/0010/15

Pag e 21 o f 40 MATERIAL S AN D METHODS

Applied SOPs and methods

SO P / Metho d Function

2030

- 7000806

- 0 1 D Alga l growt h inhibitio n test

0019 4 V. 1 HPL C analysis

Deviations : Deviatin g t o SOP 2030

- 7000806

- 01

bu t i n accordanc e wit h th e guidelin e th e cell

density at test star t wa s 500 0 cells/mL

an d fo r th e contro l 3 repli c ate s wer e tested

. The

spacing factor for the t es t concentration s wa s 3.2.

Deviatin g t o SO P 0019 4 V. 1 onl y on e single

analytica l determinatio n carrie d out.CURRENT A Gmb H & Co

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Service s Analytik Stud y No. : 2009/0010/15

Pag e 22 o f 40 

### RESULTS

Cell number in Desmodesmus subspicatus as Dependent on Concentration and Time

T ab

. 1 : Cel l numbe r i n Desmodesmu s subspicatu s a s dependen t o n concentratio n o f th e tes t ite m an d time;

Mean : arithmeti c mean ; Std.Dev. : standar d deviation ; n : numbe r o f replicates ; CV : coefficien t o f variation

(calculate d fro m InputRa w Data)

Treatm

. [mg/L] Control 0.071* 0.189* 0.467* 1.755* 8.413*

0 h 0.500 0.500 0.500 0.500 0.500 0.500

0.500 0.500 0.500 0.500 0.500 0.500

0.500 0.500 0.500 0.500 0.500 0.500

Mean: 0.500 0.500 0.500 0.500 0.500 0.500

Std.Dev.: 0.0000 0. 0000 0.0000 0.0000 0.0000 0.0000

n: 3 3 3 3 3 3

CV: 0.0 0.0 0.0 0.0 0.0 0.0

24 h 3.000 3.000 3.000 2.000 2.000 2.000

3.000 3.000 2.000 3.000 2.000 2.000

3.000 3.000 3.000 3.000 3.000 2.000

Mean: 3.000 3.000 2.667 2.667 2.333 2.000

Std.Dev.: 0.0000 0. 0000 0.5774 0.5774 0.5774 0.0000

n: 3 3 3 3 3 3

CV: 0.0 0.0 21.7 21.7 24.7 0.0

48 h 12.000 12.000 12.000 9.000 8.000 8.000

12.000 12.000 10.000 10.000 8.000 8.000

12.000 12.000 9.000 10.000 8.000 6.000

Mean: 12.000 12.000 10.333 9.667 8.000 7.333

Std.Dev.: 0.0000 0. 0000 1.5275 0.5774 0.0000 1.1547

n: 3 3 3 3 3 3

CV: 0.0 0.0 14.8 6.0 0.0 15.7

72 h 53.000 53.000 50.000 34.000 33.000 36.000

50.000 54.000 43.000 36.000 27.000 29.000

60.000 59.000 42.000 42.000 37.000 33.000

Mean: 54.333 55.333 45.000 37.333 32.333 32.667

Std.Dev.: 5.1316 3. 2146 4.3589 4.1633 5.0332 3.5119

n: 3 3 3 3 3 3

CV: 9.4 5.8 9.7 11.2 15.6 10.8

Comments : * Concentration s ar e give n a s geometri c mea n measure d values.

Nomina l value s rang e fro m 0.32

- 3 2 mg/ L wit h a spacin g facto r o f 3.2.CURRENT A Gmb H & Co

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Service s Analytik Stud y No. : 2009/0010/15

Pag e 23 o f 40 C e l l n u m b e r *1 0 ^ 4 RESULTS

C on t r o l

1.1 7 1

m g / L

1.2 8 9

m g / L

0

. 4 6 7 m g / L

1

. 7 5 5 m g / L

8

. 4 1 3 m g / L

1 0

. 0

1

. 0

0 2 4 4 8 7 2

Ti m e [ h ]

Fig. 1: Cell number in Desmodesmus subspicat u s as dependent on test item concentration and time.

Effects on Cell number

T ab

. 2 : Cel l numbe r (C ) an d it s inhibitio n relativ e t o contro l (%I ) a s compute d fro m th e ra w dat a fo r tes t intervals

selected.

Treatment 0

- 24 h 0

- 48 h

[mg/L] C % I C % I

Control 3.000 0.0 12.000 0.0

0.071 3.000 0.0 12.000 0.0

0.189 2.667 11.1 10.333 13.9

0.467 2.667 11.1 9.667 19.4

1.755 2.333 22.2 8.000 33.3

8.413 2.000 33.3 7.333 38.9CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 24 o f 40 RESULTS

Yield of Desmodesmus subspicatus cells as Dependent on Concentration and Time

T ab

. 3 : Yiel d o f Desmodesmu s subspicatu s cell s a s dependen t o n concentratio n o f th e tes t ite m an d time;

Mean : arithmeti c mean ; Std.Dev. : standar d deviation ; n : numbe r o f replicates ; CV : coefficien t o f variation

(calculate d fro m InputRa w Data)

Treatm. [mg/L] Control 0.071 0.189 0.467 1.755 8.413

0 h 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000

Mean: 0.000 0.000 0.000 0.000 0.000 0.000

Std.Dev.: 0.0000 0. 0000 0.0000 0.0000 0.0000 0.0000

n:

C V : 3 3 3 3 3 3

24 h 2.500 2.500 2.500 1.500 1.500 1.500

2.500 2.500 1.500 2.500 1.500 1.500

2.500 2.500 2.500 2.500 2.500 1.500

Mean: 2.500 2.500 2.167 2.167 1.833 1.500

Std.Dev.: 0.0000 0. 0000 0.5774 0.5774 0.5774 0.0000

n: 3 3 3 3 3 3

CV: 0.0 0.0 26.6 26.6 31.5 0.0

48 h 11.500 11.500 11.500 8.500 7.500 7.500

11.500 11.500 9.500 9.500 7.500 7.500

11.500 11.500 8.500 9.500 7.500 5.500

Mean: 11.500 11.500 9.833 9.167 7.500 6.833

Std.Dev.: 0.0000 0. 0000 1.5275 0.5774 0.0000 1.1547

n: 3 3 3 3 3 3

CV: 0.0 0.0 15.5 6.3 0.0 16.9CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 25 o f 40 RESULTS

Effects on Yield

T ab

. 4 : Yiel d (Y ) an d it s inhibitio n relativ e t o contro l (%I ) a s compute d fro m th e ra w dat a fo r tes t intervals

selected.

Treatment 0

- 24 h 0

- 48 h

[mg/L] Y % I Y % I

Control 2.500 0.0 11.500 0.0

0.071 2.500 0.0 11.500 0.0

0.189 2.167 13.3 9.833 14.5

0.467 2.167 13.3 9.167 20.3

1.755 1.833 26.7 7.500 34.8

8.413 1.500 40.0 6.833 40.6

Yield of Desmodesmus subspicatus

T ab

. 5 : %Inhibitio n cause d b y th e tes t ite m afte r 4 8 h.

Treatm.[mg/L] Mean Std. De v

. n % Decrease

Control 11.500 0.0000 3 0.0

0.071 11.500 0.0000 3 0.0

0.189 9.833 1.5275 3 14.5

0.467 9.167 0.5774 3 20.3

1.755 7.500 0.0000 3 34.8

8.413 6.833 1.1547 3 40.6CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 26 o f 40 % I n h i b i t i on RESULTS

Results of the probit anal y sis

T ab

. 6 : Result s o f th e probi t anal y sis : Selecte d effectiv e concentration s (ECx ) o f th e tes t ite m an d thei r 95%

- confidenc e limit s (accordin g t o F ieller` s theorem).

Parameter EC10 EC20 EC50

Valu e [mg/L] 0.110 0.581 14.125

lower 95%

- cl 0.000 0.006 3.686

upper 95%

- cl 0.502 1.925 44764.695

Computation of variances and con f idence limits was adjusted to metric data (Christensen & Nyholm

1984). Slope function after Litchfield and Wilcoxon: 44.335

(The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b); please note that small values refer to a steep concentration/response relation and large ones

t o a fla t relation.)

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

0

0

. 0 0 0 1 0

. 0 0 1 0

. 0 1 0

. 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0

C o n c e n t r a t i o n [ m g / L ] D a t a

F u n c t i o n

9 5 %

- C L

Fig. 2: Concentration

- effect curve showing the influence of the test item on yield of the introduced

Desmodesmus subspicatus as observed after 48 h.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 27 o f 40 RESULTS

Shapiro

- Wilk ́s Test on Normal Distribution

T ab

. 7 : Shapiro

- W ilk ́ s T es t o n Norma l Distribution ; Mean : arith m eti c mean ; n : sampl e size ; p(Shapiro W ilk ́ s W):

probabilit y o f th e W statistic

. I n cas e p(Shapiro W ilk ́ s W) i s greate r t ha n th e chose n significanc e level

, the

normalit y h y pothesis(Ho ) i s accepted.

Treatm

. [mg/L] Mean s n

Control 11.500 0.0000 3

0.071 11.500 0.0000 3

0.189 9.833 1.5275 3

0.467 9.167 0.5774 3

1.755 7.500 0.0000 3

8.413 6.833 1.1547 3

Resul t s:

Number of residues

= 7; Shapiro

- Wilk ́s W

= 0.992; p(W)

= 0.840; p(W) is greater than the selected

significance level of 0.050; therefore, treatment data do not sign ificantl y deviat e fro m normal

distribution.

Normality check was passed (p > 0.05).

Levene ́s test is chosen for variance homogeneity testing.

Levene ́s Test on Variance Homogeneity ( w ith Residuals)

T ab

. 8 : Levene ́ s T es t o n Varianc e Homogeneit y ( w it h Residuals) : Source : s ourc e o f variance ; SS : su m of

squares ; df : degree s o f freedom ; MSS : mea n su m o f squares ; F : tes t statistic : p : probability

Sourc e S S d f MS S F p(F)

Treatment 6.2222 5 1.2444 3.055 0.052

Residuals 4.8889 12 0.4074

Tota l 11.1111 17

Based on the pre

- selected significance level of 0.050, the Levene test indicates variance

homogeneity!

Variance homogeneity check was passed.

Normal distribution and variance homogeneity requirements are fulfilled.

A parametri c multipl e tes t i s advisable.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 28 o f 40 RESULTS

Williams Multiple Sequential t

- test Procedure

T ab

. 9 : Compariso n o f trea t ment s w it h "Control " b y th e t tes t procedur e afte r W illiams

. Significanc e w a s Alph a =

0.05

, one

- side d smaller ; Mean : arithmeti c mean ; n : sampl e size ; s : standar d deviation ; LhM : max.

likelihoo d mean ; %MDD : minimu m detec t abl e differenc e t o Contro l (i n per cent of Control); t: sample t; t*:

critica l t fo r Ho : μ 1

= μ 2

=

.. . = μk ; th e difference s ar e s i gnifican t i n cas e | t| > | t * | ( T h e residua l varianc e of

a n ANOV A w a s applied ; d f

= N

- k ; N : su m o f trea t men t replicate s n(i) ; k : numbe r o f treatments).

Treatm. [mg/L] Mean s df LhM % M DD t t* Sign.

Control 11.500 0.81650

0.071 11.500 0.81650 12 11.500

- 10.330 0.00

- 1.78

-

0.189 9.833 0.81650 12 9.833

- 10.858

- 2.50

- 1.87 +

0.467 9.167 0.81650 12 9.167

- 11.032

- 3.50

- 1.90 +

1.755 7.500 0.81650 12 7.500

- 11.119

- 6.00

- 1.92 +

8.413 6.833 0.81650 12 6.833

- 11.171

- 7.00

- 1.93 +

+: significant;

- : non

- significant

A NOE C o f 0.07 1 mg/ L i s suggeste d b y th e program.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 29 o f 40 RESULTS

Mean specific gro w th rate in Desmodesmus subspicatus as Dependent on

Concentration and Time

T ab

. 10 : Mea n specifi c gro w t h rat e i n Desmodesmu s subspicatu s a s dependen t o n concentratio n o f th e tes t item

an d time ; Mean : arithmeti c mean ; S t d.Dev. : standar d deviation ; n : numbe r o f replicates ; CV : coefficient

o f variatio n (calculate d fro m InputRa w Data)

Treatm. [mg/L] Control 0.071 0.189 0.467 1.755 8.413

0 h 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000

Mean: 0.000 0.000 0.000 0.000 0.000 0.000

Std.Dev.: 0.0000 0. 0000 0.0000 0.0000 0.0000 0.0000

n:

C V : 3 3 3 3 3 3

24 h 1.792 1.792 1.792 1.386 1.386 1.386

1.792 1.792 1.386 1.792 1.386 1.386

1.792 1.792 1.792 1.792 1.792 1.386

Mean: 1.792 1.792 1.657 1.657 1.521 1.386

Std.Dev.: 0.0000 0. 0000 0.2341 0.2341 0.2341 0.0000

n: 3 3 3 3 3 3

CV: 0.0 0.0 14.1 14.1 15.4 0.0

48 h 1.589 1.589 1.589 1.445 1.386 1.386

1.589 1.589 1.498 1.498 1.386 1.386

1.589 1.589 1.445 1.498 1.386 1.242

Mean: 1.589 1.589 1.511 1.480 1.386 1.338

Std.Dev.: 0.0000 0. 0000 0.0728 0.0304 0.0000 0.0830

n: 3 3 3 3 3 3

CV: 0.0 0.0 4.8 2.1 0.0 6.2CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 3 0 o f 40 RESULTS

Effects on Gro w th Rate

T ab

. 11 : Gro w

t h rat e (G ) an d it s inhibitio n relativ e t o con t ro l (%I ) a s compute d fro m th e ra w dat a fo r tes t intervals

selected.

Treatment 0

- 24 h 0

- 48 h

[mg/L] G % I G % I

Control 1.792 0.0 1.589 0.0

0.071 1.792 0.0 1.589 0.0

0.189 1.657 7.5 1.511 4.9

0.467 1.657 7.5 1.480 6.8

1.755 1.521 15.1 1.386 12.8

8.413 1.386 22.6 1.338 15.8

Gro w th Rate of Desmodesmus subspicatus

T ab

. 12 : %Inhibitio n cause d b y th e tes t ite m afte r 4 8 h.

Treatm.[mg/L] Mean Std

. De v

. n % Inhibition

Control 1.589 0.0000 3 0.0

0.071 1.589 0.0000 3 0.0

0.189 1.511 0.0728 3 4.9

0.467 1.480 0.0304 3 6.8

1.755 1.386 0.0000 3 12.8

8.413 1.338 0.0830 3 15.8CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 31 o f 40 % I nh i b i t i on RESULTS

Results of the probit anal y sis

T ab

. 13 : Result s o f th e probi t anal y sis : Selecte d effectiv e concentration s (ECx ) o f th e tes t ite m an d thei r 95%

-

an d 99%

- confidenc e limit s (accord i n g t o F ieller` s theorem).

Parameter EC10 EC20 EC50

Valu e [mg/L] 1.566 14.938 n.d.

lower 95%

- cl 0.149 4.658 n.d.

upper 95%

- cl 5.197 3946.777 n.d.

n.d.: not determined due to mathematical reasons or inappropriate data

Computation of variances and con f idence limits was adjusted to metric data (Christensen & Nyholm

1984). Slope function after Litchfield and Wilcoxon: 168.561

(The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b); please note that small values refer to a steep concentration/response relation and large ones

t o a fla t relation.)

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0 D a t a

F u n c t i o n

9 5 %

- C L

0

0

. 0 0 0 1 0

. 0 0 1 0

. 0 1 0

. 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C o n c e n t r a t i o n [ m g / L ]

Fig. 3: Concentration

- effect curve showing the influence of the test item on growth rate of the

introduced Desmodesmus subspicatus as observed after 48 h.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 32 o f 40 RESULTS

Shapiro

- Wilk ́s Test on Normal Distribution

T ab

. 14 : Shapiro

- W ilk ́ s T es t o n Norma l Distribution ; Mean : ari t hmeti c mean ; n : sampl e size ; p(Shapiro W ilk ́s

W): probabilit y o f th e W statistic

. I n cas e p(Shapiro W ilk ́ s W) i s greate r tha n th e c hose n significance

level

, th e normalit y h y po t hesis(Ho ) i s accepted.

Treatm

. [mg/L] Mean s n

Control 1.589 0.0000 3

0.071 1.589 0.0000 3

0.189 1.511 0.0728 3

0.467 1.480 0.0304 3

1.755 1.386 0.0000 3

8.413 1.338 0.0830 3

Resul t s:

Number of residues

= 8; Shapiro

- Wilk ́s W

= 0.991; p(W)

= 0.844; p(W) is greater than the selected

significance level of 0.050; therefore, treatment data do not sign ificantl y deviat e fro m normal

distribution.

Normality check was passed (p > 0.05).

Levene ́s test is chosen for variance homogeneity testing.

Levene ́s Test on Variance Homogeneity ( w ith Residuals)

T ab

. 15 : Levene ́ s T es t o n Varianc e Homogeneit y ( w it h Residuals) : Source : s ourc e o f variance ; SS : su m of

squares ; df : degree s o f freedom ; MSS : mea n su m o f squares ; F : tes t statistic : p : probability

Sourc e S S d f MS S F p(F)

Treatment 0.0001 5 0.0000 3.000 0.055

Residuals 0.0001 12 0.0000

Tota l 0.0001 17

Based on the pre

- selected significance level of 0.050, the Levene test indicates variance

homogeneity!CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 33 o f 40 RESULTS

Williams Multiple Sequential t

- test Procedure

T ab

. 16 : Compariso n o f trea t ment s w it h "Control " b y th e t tes t procedur e afte r W illiams

. Significanc e w a s Alpha

= 0.05

, one

- side d smaller ; Mean : arithmeti c mean ; n : sampl e size ; s : standar d deviation ; LhM : max.

likelihoo d mean ; %MDD : minimu m detec t abl e differenc e t o Contro l (i n per cent of Control); t: sample t; t*:

critica l t fo r Ho : μ 1

= μ 2

=

.. . = μk ; th e difference s ar e s i gnifican t i n cas e | t| > | t * | ( T h e residua l varianc e of

a n ANOV A w a s applied ; d f

= N

- k ; N : su m o f trea t men t replicate s n(i) ; k : numbe r o f treatments).

Treatm. [mg/L] Mean s df LhM % M DD t t* Sign.

Control 1.589 0.04676

0.071 1.589 0.04676 12 1.589

- 4.281 0.00

- 1.78

-

0.189 1.511 0.04676 12 1.511

- 4.500

- 2.05

- 1.87 +

0.467 1.480 0.04676 12 1.480

- 4.572

- 2.85

- 1.90 +

1.755 1.386 0.04676 12 1.386

- 4.608

- 5.31

- 1.92 +

8.413 1.338 0.04676 12 1.338

- 4.630

- 6.57

- 1.93 +

+: significant;

- : non

- significant

A NOE C o f 0.07 1 mg/ L i s suggeste d b y th e program.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 34 o f 40 RESULTS

Tabl e 17

, Anal y sis

Test item

concentration

[mg/L] HPL C values

[mg/L]

0 h 2 4 h 4 8 h

Control < 0.021

-- < 0.021

0.32 0.331 0.080 0.014

1.0 0.846 0.188 0.042

3.2 2.479 0.519 0.079

10 8.533 2.134 0.297

32 24.765 10.365 2.320

3 2 withou t algae 24.333 11.218 3.078

Comments : Th e tes t ite m concentration s 0.3 2 m g/ L an d 1. 0 m g/

L wer e calculated

(afte r 4 8 h

) base d o n th e m ea n recov ery rate of the test item

concentration s 3. 2 m g/L

, 1 0 m g/ L an d 3 2 m g/L.CURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 35 o f 40 RESULTS

Tabl e 18

, p H value s durin g th e study

Test item

concentration

[mg/L] p H value s a t 0 h

, 2 4 h

, 4 8 h

, an d 72h

0 h 2 4 h 4 8 h 7 2 h

Control 6.8 7.0 6.9 6.1

0.32 6.9 7.1 7.1 6.1

1.0 6.9 7.1 7.1 6.1

3.2 6.8 7.1 7.1 6.2

10 6.9 7.1 7.1 6.1

32 7.0 7.2 7.2 6.1

3 2 withou t algae 7.0 7.2 7.2 6.3

Comments : noneCURRENT A Gmb H & Co

. OHG

Service s Analytik Stud y No. : 2009/0010/15

Pag e 36 o f 40 RESULTS

Analysi s o f th e yiel d an d growt h rat e o f th e alga l populatio n withi n th e 4 8 h exposure

perio d (b y probi t analysi s an d William s Mul t ipl e Sequentia l t

- Tes t Procedure,

respectively ) gav e th e followin g results:

Results for drying residue / active ingredient [mg/L]:

95% confidence limits [mg/L]

dr y ing residue / active

i ng r ed i en t

E

r C 50 (0

- 48 h)

E

r C 1 0 (0

- 4 8 h) : > 100 / > 100

: 1. 6 / 1.4 n.d. / n.d

0.149

- 5.19 7 / 0.136

- 4.736

E y C 5 0 (0

- 4 8 h) : 14. 1 / 12.9 3.686

- 4476 5 / 3.359

- 40793

E y C 1 0 (0

- 4 8 h) : 0.1 1 / 0.10 0.0

- 0.502 / 0.0

- 0.457

NOEC [r] ( t

α 0.05 )

NOEC [y] ( t

α 0.05 ) : 0.071 / 0.065

: 0.071 / 0.065

n.d.: not determined due to mathematic a l reason s o r inappropriat e data

Th e E

r C 5 0 coul d no t b e calculate d wit h sufficien t p r ecision

. Fro m fig

. 3 a valu e o f ca.

100 0 mg/ L ca n b e read

, thu s th e E C 5 0 i s clearl y > 10 0 mg/L

Th e result s ar e expresse d i n term s o f geometri c mea n measure d concentrations.

Effectiv e concentration s range d fro m 76.0

- 103.4 % o f nomina l value s a t 0 hours

, from

16.2

- 35.1 % o f nomina l value s a t 2 4 hours

, an d fro m 2.5

- 9

. 6 % o f nomina l value s at

4 8 hour s respectively

. Th e analytica l value s refe r t o th e dryin g residu e of

ISOTA N L P RJ E 6914 0 wit h a conten t o f 35.05 % (accor d in g t o dat a o f th e sponsor).

Thi s conten t wa s take n t o calculat e th e nomina l valu e o f ISOTA N L P RJ E 6914 0

.

Th e growt h curve s ar e presente d i n figure 1

, th e concentratio n immobilisatio n curves

for Des m odes m u s subspicatu s ar e give n i n figure s 2 an d 3

.

Th e nutrien t mediu m fo r pre

- culture s an d tes t culture s ar e give n i n anne x 1

. The

equation s use d t o calculat e th e alga l yiel d an d growt h rate s a s wel l a s their

percentag e inhibitio n ar e presente d i n anne x 2

.

NOE C an d LOE C determine d accordin g t o William s Multipl e Sequentia l t

- Test

Procedur e ar e presente d i n table 9 an d 16

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Service s Analytik Stud y No. : 2009/0010/15

Pag e 37 o f 40 RESULTS

Comments

Th e stud y dat a wer e analyse d o n a 4 8 h basis

, becaus e concentration s o f th e test

ite m markedl y decrease d ove r tim e an d thu s th e 4 8 h valu e reflect s exposur e better

tha n th e 7 2 h values

. A s cel l number s inc r ease d wel l abov e 16

- fol d ove r 4 8 h

, an d all

other validity criteria are met, this is an optio n explicitl y covere d b y OEC D T G 201

(2006 ) an d E U metho d C. 3 (2009).

Validity of the test

Accordin g t o OEC D 201

, th e facto r o f th e biomas s paramete r

, measure d i n the

contro l betwee n 0 an d 4 8 h

, mus t b e a t leas t 16.

With the current test it was found to be 24.0. Th e tes t fulfill s thi s validit y criterion.

Evaluatio n o f th e section

- by

- sectio n growt h rates:

Arithmetic means of the contr o l replicate s fro m 0 h t o 4 8 h were : Replicat e 1 : 1.589;

Replicate 2: 1.589; Replicate 3: 1.589. [1/d].

Coefficient s o f variatio n i n contro l replicate s fro m 0 t o 4 8 h were:

Replicat e 1 : 18.0% ; Replicat e 2: 18.0%; Replicate 3: 18.0%.

Th e mea n o f th e repli c at e coefficient s o f variatio n i n th e section

- by

- sectio n growth

rate was: 18.0%.

Accordin g t o OEC D 201

, th e mea n coefficien t o f variation

, measure d i n th e control

from 0 to 48 h, must not be higher than 35%. Th e tes t fulfill s thi s validit y criterion.

Th e coefficien t o f variatio n o f th e mea n speci f i c growt h rat e replicate s i n th e control

betwee n 0 an d 4 8 h was : 0.0%.

Accordin g t o OEC D 201

, th e coefficien t o f v a riatio n o f th e mea n specifi c growt h rate,

measure d i n th e contro l fro m 0 t o 4 8 h

, m u s t no t excee d 7%

. Th e tes t fulfill s this

validit y criterion.CURRENT A Gmb H & Co

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Pag e 38 o f 40 

### ANNEX 1

Nutrient medium of pre

- cultures and test cultures

Nutrient Concentration

i n stoc k solution Fina l concentratio n i n the

solutio n o f th e pre

culture s an d tes t cultures

Stoc k solutio n 1 : macro

nutrients

NH 4 Cl 1. 5 g/L 1 5 mg/L

MgCl 2 x 6 H 2 O 1. 2 g/L 1 2 mg/L

CaCl 2 x 2 H 2 O 1. 8 g/L 1 8 mg/L

MgS O 4 x 7 H 2 O 1. 5 g/L 1 5 mg/L

KH 2 P O 4 0.1 6 g/L 1.6 mg/L

Stoc k solutio n 2 : Fe

- EDTA

FeCl 3 x 6 H 2 O

Na 2 EDT A x 2 H 2 O 8 0 mg/L

10 0 mg/L 8 0 μg/L

10 0 μg/L

Stock solution 3: trace elements

H 3 B O 3

MnCl 2 x 4 H 2 O

ZnCl 2

CoCl 2 x 6 H 2 O

CuCl 2 x 2 H 2 O

Na 2 MoO 4 x 2 H 2 O 18 5 mg/L

41 5 mg/L

3 mg/L

1.5 mg/L

0.0 1 mg/L

7 mg/L 18 5 μg/L

41 5 μg/L

3 μg/L

1. 5 μg/L

0.0 1 μg/L

7 μg/L

Soli d NaHC O 3 wa s adde d t o th e nutrien t medi a t o mak e u p a fina l concentratio n of

5 0 mg/ L i n th e solution s o f th e pre

- culture s an d tes t c ultures. Water hardness of the

fina l nutrien t mediu m wa s 1.3°dH

, correspondin g t o 22. 5 mg/ L CaC O 3

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Pag e 39 o f 40 

### ANNEX 2

Equations for calculation

Th e percentag e inhibitio n o f yiel d fo r eac h tes t concentratio n wa s calculated

accordin g to

Equatio n [1]

% I

y = ( Y

c - Y

T )

x 100 Y

c

Where

% I y wa s th e percentag e inhibitio n fo r yield,

Y C wa s th e mea n valu e fo r yiel d i n th e contro l group,

Y T was the value for yield for the treatment.

The growth rate [r]

Fo r eac h tes t concentratio n an d fo r th e contro l wa s calculate d accordin g to

Equatio n [2]

r = ln N 3

- ln N 0

t 3

Where

r was the growth rate,

l

n wa s th e natura l logarithm,

t 3 was the time of the last mea s urement at the end of the test,

N

o wa s th e nomina l initia l cel l density,

N 3 wa s th e measure d cel l densit y a t tim e t 3

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Service s Analytik Stud y No. : 2009/0010/15

Pag e 4 0 o f 40 ANNE X 2

Fro m th e mea n value s o f r th e percentag e inhibitio n o f growt h rat e fo r eac h test

concentratio n wa s calculate d accordin g to

Equatio n [3]

I r i = r c - r i

x 100

r c

Where

I r

i wa s th e percentag e inhibitio n (growt h rate) for test

concentratio n i,

r i

r c was the mean growth rate for test concentration i,

was the mean growth rate for the control.