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Study Title

Alga, Inhibition Test with 

ISOTAN LP RJE 69140

Data Requirements / Test Guidelines

EU method C.3 (2008) 

OECD TG 201 (2006)

Author:

Dr. Arnd Weyers

Study initiation date: 2009-10-01 Study completion date: 2009-12-17

Start of Experimental Phase: 2009-10-06 End of Experimental Phase: 2009-12-17

Sponsor:

LANXESS Deutschland GmbH 

Business Unit Leather

51369 Leverkusen 

Germany

Testing facility:

CURRENTA GmbH & Co. OHG

Services Analytik

51368 Leverkusen 

Germany

Laboratory Project Identification

Study No. 2009/0010/15
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CURRENTA GmbH & Co. OHG

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This page is intentionally left blank for the purpose of submitting administrative 

information that is required by regulations promulgated by various countries.
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CURRENTA GmbH & Co. OHG

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CONTENTS

Page

GLP DECLARATION 4

ARCHIVATION OF DOCUMENTS AND TEST ARTICLES 4

QUALITY ASSURANCE STATEMENT 6

GLP CERTIFICATE 7

SUMMARY 8

INTRODUCTION 10

EXPERIMENTAL PROCEDURE 11

MATERIALS AND METHODS 13

Test item 13

Test species 14

Apparatus 14

Pre-treatment of the test item 15

Exposure conditions 15

Chemical analysis 17

Expression of biological results 20

Applied SOPs and methods 21

RESULTS 22

Comments 37

Validity of the test 37

ANNEX 1 38

Nutrient medium of pre-cultures and test cultures 38

ANNEX 2 39

Equations for calculation 39
Page 4 of 40

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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, 2001).

Date I Signature

Study Director 'JoV'J-/ ?It 4a

(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 CURRENTA 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 CURRENTA Services Analytik.
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Services Analytik

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information that is required by regulations promulgated by various countries.
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CURRENTA GmbH & Co. OHG

SeNices Analytik

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QUALITY ASSURANC E STATEMENT

This report was audited 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 data.

The dates of Quality Assurance inspections and audits are given below. 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 review 2009-10-02 2009-10-02

process based inspection 2009-05-28 2009-06-03

final report review

2009/0002/09

'2009- I 2 - 22-

( (j(J 9- ;'l - 2..

Date I Signature

10Vj - 1 l - Z.L- u?-

(&embaMge"/Dr.Neupert)
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GLP CERTIFICATE

Please note: Effective January 1

st

, 2008 the company name Bayer Industry Services 

GmbH & Co. OHG was changed to CURRENTA GmbH & Co. OHG
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SUMMARY

A study was performed to assess the adverse effects of ISOTAN LP RJE 69140 on 

the yield (= biomass at time t minus initial biomass) and the growth rate (= rate of

increase in cell density with time) of the planktonic freshwater algal species 

Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several 

generations.

The study was conducted in accordance with Commission Regulation (EC) 

No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Algae and 

Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline 

for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of

concentrations, nominally 0.32, 1.0, 3.2, 10 and 32 mg/L of ISOTAN LP RJE 69140

dissolved in water.

The cell densities were measured at 24 hour intervals. Inhibition of the algal 

population was measured as reduction in growth rate (index r), relative to control 

cultures grown under identical conditions. Growth rates were also used to calculate a 

No Observed Effect Concentration and a Lowest Observed Effect Concentration 

according to Williams Multiple Sequential t-Test Procedure. The following values 

were determined:

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

ErC 50* (0-48 h) : > 100 / > 100 

ErC 10* (0-48 h) : 1.6 / 1.4

NOEC [r] (tα 0.05) : 0.071 / 0.065

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to

OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, 

NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not 

in the EU) and are given in the results section of this report.

The ErC 50 could not be calculated with sufficient precision. From fig. 3 a value of ca.

1000 mg/L can be read, thus the EC 50 is clearly > 100 mg/L

The study data were analysed on a 48 h basis. As cell numbers increased well above 

16-fold over 48 h, and all other validity criteria are met, this is an option explicitly 

covered by OECD TG 201 (2006) and EU method C.3 (2009).
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SUMMARY

The results are expressed in terms of geometric mean measured concentrations. 

Effective concentrations ranged from 76.0-103.4% of nominal values at 0 hours, from 

16.2-35.1% of nominal values at 24 hours, and from 2.5-9.6% of nominal values at

48 hours respectively. The analytical values refer to the drying residue of

ISOTAN LP RJE 69140 with a content of 35.05% (according to data of the sponsor). 

This content was taken to calculate the nominal value of ISOTAN LP RJE 69140.
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INTRODUCTION

This report contains a description of the methods used and the results obtained 

during a study to investigate adverse effects of ISOTAN LP RJE 69140 on the yield 

and the growth rate of a population of the planktonic freshwater algal species 

Desmodesmus subspicatus (former name: Scenedesmus subspicatus). In the 

present test, adverse effects were expressed as those effective concentrations which

reduced yield (= biomass at time t minus initial biomass; index y) and growth rate (=

rate of increase in cell density with time; index r) by 10% (EyC 10, ErC 10) and 50% 

(EyC 50, ErC 50) within a continuous 72 hour period of exposure. The study data 

were analysed on a 48 h basis, because concentrations of the test item markedly 

decreased over time and thus the 48 h value reflects exposure better than the 72 h 

values. As cell numbers increased well above 16-fold over 48 h, and all other validity 

criteria are met, this is an option explicitly covered by OECD TG 201 (2006) and EU 

method C.3 (2009).

In addition to the EC 10 and EC 50 values, NOEC/LOEC were calculated based on 

yield [y] and growth rate [r], respectively.
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EXPERIMENTAL PROCEDURE

The study was conducted in accordance with Commission Regulation (EC) 

No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Algae and 

Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline 

for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006).

The purpose of this method was to determine those concentrations which caused a 

10% and a 50% adverse effect (= EC 10, EC 50) or, if conducted as a limit test, to

determine the adverse effects at a maximum test concentration of 100 mg/L or at the 

limit of water solubility, respectively. Effect data are expressed on the basis of yield 

[y] and growth rate [r].

A range-finding (non-GLP) test preceded the main test and provided information 

about the range of concentrations which were used in the main test. The following 

nominal concentrations of the test item were tested in the range-finding test: 1, 3.2,

10, 32 and 100 mg/L.

In the main test, the algae were exposed to the test item added to water at a range of

concentrations for a period of 72 hours. Defined concentrations of the test item led to

a certain inhibition of algal yield and growth rate at the end of the 72 hour study 

period. Cell densities were recorded at 24 hour intervals. The 48 hour EC 10 and 

EC 50 (based on both, yield [y] and growth rate [r]), were calculated by probit 

analysis.

All calculations were carried out using the statistics programme ToxRatPro

Version 2.10 (released 19.02.2009). For the calculations all algae counts were 

divided by a factor of 10000.

During the test a temperature range of 21 - 24°C was maintained in the test vessels. 

The maintenance of test item concentrations was proved by analytical measurements

and the pH was measured at the beginning of the test start and every 24 hours 

thereafter. In order to avoid an impairment of the test system, additional replicates 

were used for qualitative examination of samples (HPLC analysis) and pH 

measurement after 0, 24 and 48 h. PH measurement at the end of the test were 

performed using replicate I of the test.

In order to check whether or not significant amounts of the test item are incorporated 

into the algal biomass during the test period, test flasks at the test concentration 

32 mg/L without algae were run in parallel to the geometric series of test.
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EXPERIMENTAL PROCEDURE

The following validity criteria of the test were met:

The cell density in the control cultures increased by a factor of at least 16 within 48 

hours. The test period was shortened to 48 hours to maintain unlimited, exponential 

growth during the test.

According to an update of OECD TG 201 (2006), the mean coefficient of variation for

section-by-section specific growth rates in the control cultures must not exceed 35%. 

The coefficient of variation of average specific growth rates during the whole test

period in replicate control cultures must not exceed 7%.
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MATERIALS AND METHODS

Test item

Test item (Product name): ISOTAN LP RJE 69140

Drying residue : 35.05%

Content of active

ingredient : 31.94%

Partie : RJE 71120

Charge : --

Expiry date : 2010-01-30

Appearance : liquid

Storage : room temperature

Data of active ingredient

Chemical name : preparation of hexamethylenediisocyanatebisulfite-adduct in water

CAS number : 38632-47-2 (Na salt)

Empirical formula : C8H14N2O8S2 . 2Na

Molecular weight : 376 g/mol

Stability of test concentrations during exposure : Examined by chemical analysis (HPLC) at 0, 24

and 48 hours.
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MATERIALS AND METHODS

Test species

Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG

Source : Strain of the test species obtained from 'The 

Collection of Algal Cultures' of the Institute of Plant 

Physiology at the University of Göttingen 

(Germany).

Maintenance

and Acclimatisation : Exponentially-growing stock cultures were

maintained in the test facility under constant 

temperature conditions (23 +/- 2°C) at a light 

intensity in the range 60 – 120 μE. x m

-2

x s

-1

(measured in the range 400 to 700 nm using a 

spherical quantum flux meter). The nutrient 

medium (according to BRINGMANN & KÜHN 

(1977) was renewed once a week. Cell density 

measurements were made using a microcellcounter, Sysmex F300, Digitana.

Preparation of

pre-cultures : Pre-cultures were set up three days before the

start of a test. They were grown under identical 

exposure conditions as the stock cultures, except 

from the use of a different nutrient medium 

(annex 1).

Test cultures : The algal inocula for a test were taken from an

exponentially-growing pre-culture and were mixed 

with the nutrient medium (annex 1) to make up to

a final cell density of about 5000 cells per millilitre 

in the test medium.

Apparatus

– Analytical balance

– PH-meter

– Shaking incubator

– Microcellcounter

– Various glass materials: Erlenmeyer flasks, volumetric flasks, beakers, 

pipettes etc.
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MATERIALS AND METHODS

Pre-treatment of the test item

A stock solution was prepared to give the desired series of test concentrations 

based on a drying residue of Isotan LP RJE 69140 of 35.05% (according to

data of the sponsor) To achieve this 356.8 mg of the test item were added to 1 

litre of dilution water. The pH was measured to be pH 4.8.

Isotan LP RJE 69140 hydrolysis rapidly in water, depending on the pH value. 

To minimise hydrolysis effects the pH of the concentrated nutrient medium 

was measured to be pH 8.0 and was adjusted to pH 6.0.

To produce the different test item concentrations the prepared stock solution 

was diluted with dilution water to a volume of 80 mL. Afterwards 10 mL of

nutrient medium and 10 mL of algal inoculum with a cell density of 5000 

cells/mL were added resulting in the final nominal concentrations. For each 

test item concentration and control 3 replicates were prepared.

Exposure conditions

Test vessels : 300 mL Erlenmeyer flasks with cotton stoppers 

Culturing apparatus : Light chamber in which a temperature in the range

21°C to 24°C was maintained at +/- 2°C, and

continuous uniform illumination was provided in 

the spectral range 400 to 700 nm.

Light intensity : At the average of the test solutions, a light

intensity in the range 60 to 120 μE. x m

-2

x s

-1

, or 

an equivalent range of 4000 to 8000 lx, was used. 

Light intensity was checked before start of the 

study.
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MATERIALS AND METHODS

Cell density

measurements : Cell densities were measured in a microcell

counter (Sysmex F300, Digitana) by taking small 

aliquots from each test flask for measurements, 

which were not replaced.

Experimental design : 5 test concentrations plus 1 control

3 replicates per concentration, 3 replicates per 

control

Initial cell density in the test cultures 

approximately 5000 cells per millilitre

Additionally highest test concentration without 

algae

Test item

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

Method of administration : stock solution

Duration of exposure : 48 hours

Criteria of effects : The criteria of adverse effects used in this study

were the item-induced inhibition of yield [y] and 

growth rate [r], respectively, of the algal 

population.
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MATERIALS AND METHODS

Chemical analysis

In this chapter the methods used to determine the concentrations of the test item are 

described. The quantification of the test item was performed by HPLC analysis and 

MSD-detection, according to SOP 00194 Version 1.

Materials and Methods

Test Item

The test item as described in the biological part of this study was also used to

prepare the spiked test water samples.

Analytical Procedure

Reagents and Solvents

Milliporewater / Ammoniumacetat / Methanol

Standard Solutions used for Calibration

29.6 mg of the test item were dissolved in Milliporewater and made up with 

Milliporewater to the mark in a 100 mL volumetric flask to prepare a stock

solution of 296 mg/L respectively 103.748 mg/L drying residue of 35.05%. 

Defined volumes of this stock solution were diluted with Milliporewater to

obtain standard solutions in the range of 0.0207 to 4.150 mg/L drying residue 

of 35.05%.

Although the overall method sensitivity would have been better, the 

concentration of the lowest used calibration solution was employed as the limit 

of quantification.

These solutions were used to calibrate the HPLC-system.
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MATERIALS AND METHODS

Standard Solutions used for Verification of the Calibration and Blank Test

22.7 mg of the test item were dissolved in Milliporewater and made up with 

Milliporewater to the mark in a 100 mL volumetric flask to prepare a 

verification solution with a nominal concentration of 227 mg/L respectively

79.564 mg/L drying residue of 35.05%. The daily verification of the calibration 

was performed with this Standard Solution of Verification. This solution was 

diluted with Milliporewater to obtain a concentration of 1.591 mg/L drying 

residue of 35.05%.

The mean recovery rate was calculated.

In addition, water without the test item was analysed (analytical blank).

Analysis of Samples

Aliquots of the samples from the biological test were directly analysed by 

HPLC and MS-detection (range of the injection volume: 1-50 μL, depending on 

the expected concentration).

HPLC Conditions

HPLC system: e.g. Agilent Technologies 1100 system with MSD 

Column: 2.1*150 mm

Stationary phase: Inertsil ODS-3; 3 μm (GL Sciences)

Eluent A: 1.93 g Ammonium acetate + 1 L Milliporewater 

Eluent B: 1.93 g Ammonium acetate + 1 L Methanol 

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

Temperature: 40 °C

Flow rate: 0.2 mL/min

Detection: MSD / SIM Modus (Positiv) 

Recorded mass: m/z 249.1 and 331

Injection volume: 1 - 50 μL
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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

MATERIALS AND METHODS

The calibration, verification, blank and sample solutions were 

chromatographed using the conditions described in the paragraph before.

Calculation was done using an external standardization method and a 

calibration curve. The calibration curve was calculated according to equation 

(1).

(1) y = a · x2 + b · x + c 

where:

The correlation coefficient R

2 was 

R

2

= 0.99955

To verify the calibration the recovery rate of the verification solution was 

calculated according to equation (2).

mean value of assay

nominal concentration

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

Results

The analytical results are presented in detail in the following tables, together 

with the biological effect data.

Sampling schedule:

Control : at 0, 24 and 48 hours

Test concentrations : at 0, 24 and 48 hours
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MATERIALS AND METHODS

Expression of biological results

• Cell density measurements, yield and growth rates in the test and 

control cultures were tabulated according to the concentration of test item 

and the time of measurement.

• Growth curves were plotted for each test concentration and control and 

are presented in figure 1.

• The percentage inhibition of both, yield [b] and growth rate [r], was 

calculated for each test concentration using equations [1] and [3] 

(annex 2).

• The growth rate [r] was calculated for each test culture using equation

[2] in annex 2.

• EC 50 and EC 10 values for both, yield [y] and growth rate [r], were 

calculated by probit analysis.

• NOEC and LOEC of both, yield [y] and growth rate [r], were determined 

by a multisampling comparison (according to Williams Multiple Sequential 

t-Test Procedure) (table 9 and 16)
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MATERIALS AND METHODS

Applied SOPs and methods

SOP / Method Function

2030-7000806-01 D Algal growth inhibition test

00194 V.1 HPLC analysis

Deviations: Deviating to SOP 2030-7000806-01

but in accordance with the guideline the cell 

density at test start was 5000 cells/mL 

and for the control 3 replicates were tested. The 

spacing factor for the test concentrations was 3.2.

Deviating to SOP 00194 V.1 only one single 

analytical determination carried out.
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RESULTS

Cell number in Desmodesmus subspicatus as Dependent on Concentration and Time

Tab. 1: Cell number in Desmodesmus subspicatus as dependent on concentration of the test item and time;

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation 

(calculated from InputRawData)

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: * Concentrations are given as geometric mean measured values. 

Nominal values range from 0.32-32 mg/L with a spacing factor of 3.2.
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Cell number *10^4

RESULTS

Control

1.1 71

mg/L

1.2 89

mg/L

0.467 mg/L

1.755 mg/L

8.413 mg/L

1 0.0

1.0

0 24 48 72

Tim e [h]

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

Effects on Cell number

Tab. 2: Cell number (C) and its inhibition relative to control (%I) as computed from the raw data for test 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 38.9
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RESULTS

Yield of Desmodesmus subspicatus cells as Dependent on Concentration and Time

Tab. 3: Yield of Desmodesmus subspicatus cells as dependent on concentration of the test item and time;

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation 

(calculated from InputRawData)

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:

CV:

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 16.9
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RESULTS

Effects on Yield

Tab. 4: Yield (Y) and its inhibition relative to control (%I) as computed from the raw data for test 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

Tab. 5: %Inhibition caused by the test item after 48 h.

Treatm.[mg/L] Mean Std. Dev. 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 40.6
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% Inhibition

RESULTS

Results of the probit analysis

Tab. 6: Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%-

confidence limits (according to Fieller`s theorem).

Parameter EC10 EC20 EC50

Value [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 confidence 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 

to a flat 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 10 0 0 1 0 0 0 0 1 0 0 0 0 01 0 0 0 0 0 0

C o n c e n tra tio n [m g /L ]

Data

 Fu n c tio n

 9 5 % -CL

Fig. 2: Concentration-effect curve showing the influence of the test item on yield of the introduced 

Desmodesmus subspicatus as observed after 48 h.
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RESULTS

Shapiro-Wilk ́s Test on Normal Distribution

Tab. 7: Shapiro-Wilk ́s Test on Normal Distribution; Mean: arithmetic mean; n: sample size; p(ShapiroWilk ́s W):

probability of the W statistic. In case p(ShapiroWilk ́s W) is greater than the chosen significance level, the 

normality hypothesis(Ho) is 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

Results:

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 significantly deviate from normal 

distribution.

Normality check was passed (p > 0.05).

Levene ́s test is chosen for variance homogeneity testing.

Levene ́s Test on Variance Homogeneity (with Residuals)

Tab. 8: Levene ́s Test on Variance Homogeneity (with Residuals): Source: source of variance; SS: sum of

squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability

Source SS df MSS F p(F)

Treatment 6.2222 5 1.2444 3.055 0.052

Residuals 4.8889 12 0.4074

Total 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 parametric multiple test is advisable.
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RESULTS

Williams Multiple Sequential t-test Procedure

Tab. 9: Comparison of treatments with "Control" by the t test procedure after Williams. Significance was Alpha =

0.05, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max.

likelihood mean; %MDD: minimum detectable difference to Control (in percent of Control); t: sample t; t*:

critical t for Ho: μ1 = μ2 = ... = μk; the differences are significant in case |t| > |t*| (The residual variance of

an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

Treatm. [mg/L] Mean s df LhM %MDD 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 NOEC of 0.071 mg/L is suggested by the program.
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RESULTS

Mean specific growth rate in Desmodesmus subspicatus as Dependent on

Concentration and Time

Tab. 10: Mean specific growth rate in Desmodesmus subspicatus as dependent on concentration of the test item 

and time; Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient 

of variation (calculated from InputRawData)

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:

CV:

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 6.2
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RESULTS

Effects on Growth Rate

Tab. 11: Growth rate (G) and its inhibition relative to control (%I) as computed from the raw data for test 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

Growth Rate of Desmodesmus subspicatus

Tab. 12: %Inhibition caused by the test item after 48 h.

Treatm.[mg/L] Mean Std. Dev. 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 15.8
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% Inhibition

RESULTS

Results of the probit analysis

Tab. 13: Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%-

and 99%-confidence limits (according to Fieller`s theorem).

Parameter EC10 EC20 EC50

Value [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 confidence 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 

to a flat relation.)

1 00

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

Data

Func tion

 9 5% -CL

0

0 .0 0 0 10 .0 0 1 0 .0 1 0 .1 1 1 0 1 00 1 00 0 1 0 0 0 01 0 00010 0 00100 0010 00 010 0 0 010 000 000 000 0000 0 0 0 0

C o n c e n tra tio 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.
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RESULTS

Shapiro-Wilk ́s Test on Normal Distribution

Tab. 14: Shapiro-Wilk ́s Test on Normal Distribution; Mean: arithmetic mean; n: sample size; p(ShapiroWilk ́s 

W): probability of the W statistic. In case p(ShapiroWilk ́s W) is greater than the chosen significance 

level, the normality hypothesis(Ho) is 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

Results:

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 significantly deviate from normal 

distribution.

Normality check was passed (p > 0.05).

Levene ́s test is chosen for variance homogeneity testing.

Levene ́s Test on Variance Homogeneity (with Residuals)

Tab. 15: Levene ́s Test on Variance Homogeneity (with Residuals): Source: source of variance; SS: sum of

squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability

Source SS df MSS F p(F)

Treatment 0.0001 5 0.0000 3.000 0.055

Residuals 0.0001 12 0.0000

Total 0.0001 17

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

homogeneity!
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RESULTS

Williams Multiple Sequential t-test Procedure

Tab. 16: Comparison of treatments with "Control" by the t test procedure after Williams. Significance was Alpha

= 0.05, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max.

likelihood mean; %MDD: minimum detectable difference to Control (in percent of Control); t: sample t; t*:

critical t for Ho: μ1 = μ2 = ... = μk; the differences are significant in case |t| > |t*| (The residual variance of

an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

Treatm. [mg/L] Mean s df LhM %MDD 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 NOEC of 0.071 mg/L is suggested by the program.
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RESULTS

Table 17, Analysis

Test item 

concentration

[mg/L]

HPLC values 

[mg/L]

0 h 24 h 48 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

32 without algae 24.333 11.218 3.078

Comments: The test item concentrations 0.32 mg/L and 1.0 mg/L were calculated 

(after 48 h) based on the mean recovery rate of the test item 

concentrations 3.2 mg/L, 10 mg/L and 32 mg/L.
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RESULTS

Table 18, pH values during the study

Test item 

concentration

[mg/L]

pH values at 0 h, 24 h, 48 h, and 72h

0 h 24 h 48 h 72 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

32 without algae 7.0 7.2 7.2 6.3

Comments: none
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RESULTS

Analysis of the yield and growth rate of the algal population within the 48 h exposure 

period (by probit analysis and Williams Multiple Sequential t-Test Procedure, 

respectively) gave the following results:

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

95% confidence limits [mg/L]

drying residue / active 

ingredient

ErC 50 (0-48 h)

ErC 10 (0-48 h)

: > 100 / > 100

: 1.6 / 1.4

n.d. / n.d

0.149-5.197 / 0.136-4.736

EyC 50 (0-48 h) : 14.1 / 12.9 3.686-44765 / 3.359-40793

EyC 10 (0-48 h) : 0.11 / 0.10 0.0-0.502 / 0.0-0.457

NOEC [r] (tα 0.05)

NOEC [y] (tα 0.05)

: 0.071 / 0.065

: 0.071 / 0.065

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

The ErC 50 could not be calculated with sufficient precision. From fig. 3 a value of ca.

1000 mg/L can be read, thus the EC 50 is clearly > 100 mg/L

The results are expressed in terms of geometric mean measured concentrations. 

Effective concentrations ranged from 76.0-103.4% of nominal values at 0 hours, from 

16.2-35.1% of nominal values at 24 hours, and from 2.5-9.6% of nominal values at

48 hours respectively. The analytical values refer to the drying residue of

ISOTAN LP RJE 69140 with a content of 35.05% (according to data of the sponsor). 

This content was taken to calculate the nominal value of ISOTAN LP RJE 69140.

The growth curves are presented in figure 1, the concentration immobilisation curves 

for Desmodesmus subspicatus are given in figures 2 and 3.

The nutrient medium for pre-cultures and test cultures are given in annex 1. The 

equations used to calculate the algal yield and growth rates as well as their 

percentage inhibition are presented in annex 2.

NOEC and LOEC determined according to Williams Multiple Sequential t-Test

Procedure are presented in table 9 and 16.
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RESULTS

Comments

The study data were analysed on a 48 h basis, because concentrations of the test

item markedly decreased over time and thus the 48 h value reflects exposure better 

than the 72 h values. As cell numbers increased well above 16-fold over 48 h, and all 

other validity criteria are met, this is an option explicitly covered by OECD TG 201 

(2006) and EU method C.3 (2009).

Validity of the test

According to OECD 201, the factor of the biomass parameter, measured in the 

control between 0 and 48 h, must be at least 16.

With the current test it was found to be 24.0. The test fulfills this validity criterion.

Evaluation of the section-by-section growth rates:

Arithmetic means of the control replicates from 0 h to 48 h were: Replicate 1: 1.589; 

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

Coefficients of variation in control replicates from 0 to 48 h were: 

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

The mean of the replicate coefficients of variation in the section-by-section growth 

rate was: 18.0%.

According to OECD 201, the mean coefficient of variation, measured in the control 

from 0 to 48 h, must not be higher than 35%. The test fulfills this validity criterion.

The coefficient of variation of the mean specific growth rate replicates in the control 

between 0 and 48 h was: 0.0%.

According to OECD 201, the coefficient of variation of the mean specific growth rate,

measured in the control from 0 to 48 h, must not exceed 7%. The test fulfills this 

validity criterion.
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ANNEX 1

Nutrient medium of pre-cultures and test cultures

Nutrient Concentration 

in stock solution

Final concentration in the 

solution of the precultures and test cultures

Stock solution 1: macronutrients

NH4Cl 1.5 g/L 15 mg/L

MgCl2 x 6 H2O 1.2 g/L 12 mg/L

CaCl2 x 2 H2O 1.8 g/L 18 mg/L

MgSO4 x 7 H2O 1.5 g/L 15 mg/L

KH2PO4 0.16 g/L 1.6 mg/L

Stock solution 2: Fe-EDTA

FeCl3 x 6 H2O

Na2EDTA x 2 H2O

80 mg/L

100 mg/L

80 μg/L

100 μg/L

Stock solution 3: trace elements

H3BO3

MnCl2 x 4 H2O

ZnCl2

CoCl2 x 6 H2O

CuCl2 x 2 H2O

Na2MoO4 x 2 H2O

185 mg/L

415 mg/L

3 mg/L

1.5 mg/L

0.01 mg/L

7 mg/L

185 μg/L

415 μg/L

3 μg/L

1.5 μg/L

0.01 μg/L

7 μg/L

Solid NaHCO3 was added to the nutrient media to make up a final concentration of

50 mg/L in the solutions of the pre-cultures and test cultures. Water hardness of the 

final nutrient medium was 1.3°dH, corresponding to 22.5 mg/L CaCO3.
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ANNEX 2

Equations for calculation

The percentage inhibition of yield for each test concentration was calculated 

according to

Equation [1]

% I y =

(Yc - YT)

x100 Yc

Where

% Iy was the percentage inhibition for yield,

YC was the mean value for yield in the control group, 

YT was the value for yield for the treatment.

The growth rate [r]

For each test concentration and for the control was calculated according to

Equation [2]

r = 

ln N3 -ln N0

t3

Where

r was the growth rate,

ln was the natural logarithm,

t3 was the time of the last measurement at the end of the test,

No was the nominal initial cell density,

N3 was the measured cell density at time t3.
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ANNEX 2

From the mean values of r the percentage inhibition of growth rate for each test

concentration was calculated according to

Equation [3]

Iri = r c - r i 

x 100

r c

Where

Iri was the percentage inhibition (growth rate) for test

concentration i,

ri

rc

was the mean growth rate for test concentration i,

was the mean growth rate for the control.