• 

• 

TITLE PAGE 

Study Title: 

ACUTE TOXICITY OF 

DIAESUL BLACK ROT LIQUID 

TO SCENEDESMUS SUBSPICATUS 

; ~ ·- -... -·-

i. ·:,' •. I o: 

(OECD - Algae Growth Inhibition Test; 

including determination of the influence of the light 

absorption of the substance on the growth inhibition) 

Data Requirement: 

OECD No. 201 

Author; 

Mrs. V. Wuthrich 

Study Completion Date: 

November 18, 1993 

Perfonning I ahoratory: 

A C C UMWELTCHEMIE AG 

P.O. Box 

CH - 4452 ltingen/BL 

Switzer1and 

Study Project No. : 

ACC PROJECT 333977 

Page 1 of 48 

RCC CONTAINS NO CBI --------------------

Group 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

EIDGENOSSISCHES DEPARTEMENT DES INNERN 

DtPARTEMENT FtD£RAL DE L'INT£RIEUR 

DIPARTIMENTO FEDERAL£ DELL'INTERNO 

GLP ComQliance Statement 

It is hereby certified that 

~ 8th - 12th, 19th am August 8th, 1991 

e the testinq facilities of 

-were i.n.spected by the Fe:EJ::al. Office of Public Health, the Federal Office of 

Fnvi.rcrmelt, Forests ani I.arrlscape am the Intercantalal Office for the 

Ccrltrol of Medicarrsnts with respect to the cxmpliance with the Swiss GU 

Q.ti.dellnes. 

'lhe inspectioo W!B perfODDed in agreenent with the OEI:D Glidelines for National GLP Inspecticns arx1 Auiits an:i cmprised the foil~ testing facilities: 

- RCC Research and Consultinq Calp!ny Ltd I~ and Geneva 

- RCC ~tchBDie JG It.ir9=n 

· e -RCC Aeqist.ratioo am Coosultilv; Carpmy Ltd (()lality Assurance OUt) Itingen 

- BRL Biological Research Laboratories Ltd (Toxicology) P1i1l.insdorf 

It \eS fOlD'Xi that the aforarentiooed testing facilities were operatinq in cxmpliance with the Swiss Principles of Goo:i Iaboratocy Practice (Gocxi I.aboratory 

Practice (GLP) in SWitzerl.W, Proce:iui:es and Principles, March 1986) at the 

tine they ~ inspected. 

Federal Departlnent of the Interior 

EeDle, lOth Septaltler 1991 

:UV 5000 4e051 I I 

2 
•• 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

PROJECT NUMBER: 

TEST ARTICLE: 

STUDY DIRECTOR: 

TITLE: 

333977 

DIRESUL BLACK ROT LIQUID 

Mrs. V. Wuthrich 

Acute Toxicity of DIRESUL BLACK ROT LIQUID 

to Scenedesmus sybspicatus 

(OECD -Algae Growth Inhibition Test; 

3 

including determination of the influence of the light 

absorption of the substance on the growth inhibition) 

This study was conducted in compliance with Good Laboratory Practice Regulations and meets 

the requirements as listed under GLP guidelines (page 11). 

Study Director: Mrs. V. Wuthrich 

.. \!..\\0 .. ~.~.!.\. ............ . Date: Dou~\:.u }9:,, A_C{C(~ 
r 

--

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

PROJECT STAFF 

STUDY DIRECTOR 

FOR THE MANAGEMENT 

4 

Mrs. V. Wuthrich 

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

Dr. A. Van Dijk 
I 

l 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

QUALITY ASSURANCE UNIT 

5 

R C C UMWEL TCHEMIE AG, CH-4452 ITINGEN I SWITZERLAND 

STATEMENT 

PROJECT NUMBER: 

TEST ARTICLE: 

STUDY DIRECTOR: 

; ft TITLE: 

333977 

DIRESUL BLACK ROT LIQUID 

Mrs. V. Wuthrich 

Acute Toxicity of DIRESUL BLACK ROT LIQUID 

to Scenedesmus sub5picatus 

(OECD -Algae Growth Inhibition Test; 

including determination of the influence of the light 

absorption of the substance on the growth inhibition) 

Study procedures were periodically inspected, the protocol and this report were audited by the 

Quality Assurance Unit. The dates are given below. 

Dates of QAU Inspections I Dates of Reports to the Study 

Audits Director and to Management 

30.10.1992 30.10.1992 

20.01.1993 20.01.1993 

•• 04.02.1993 04.02.1993 

18.03.1993 18.03.1993 

12./16.11.1993 16.11.1993 

Manager, Quality Assurance Unit Mrs. I. Wuthrich 

..................................................... ;M~J?·~ 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

CONTENTS Page 

TITLE PAGE ................................................................................................................... 1 

GLP COMPLIANCE STATEMENT ................................................................................. 2 

STATEMENT OF COMPLIANCE ................................................................................... 3 

PROJECT STAFF .......................................................................................................... 4 

QUALITY ASSURANCE UNIT ........................................................................................ 5 

CONTENTS ................................................................................................................. 6-9 

GENERAL INFORMATION .......................................................................................... 10 

GOOD LABORATORY PRACTICE .............................................................................. 11 

GUIDELINES ................................................................................................................ 11 

SUMMARY OF PROTOCOL AMENDMENTS .............................................................. 11 

SUMMARY ................................................................................................................... 12 -1 INTRODUCTION ................................................................................................. 13 

2 MATERIALS AND METHODS ............................................................................. 13 

2.1 DEFINITIONS ...................................................................................................... 13 

2.2 TEST SYSTEM I RATIONALE ............................................................................ 13 

2.3 CHEMICALS ....................................................................................................... 14 

2.3.1 Test Article ................................................................................................. 14 

2.3.2 Reference Compound ................................................................................ 14 

2.4 EXPERIMENTAL CONDITIONS ......................................................................... 15 

2.5 EVALUATIONS ................................................................................................... 18 

2.5.1 Determination of Growth Inhibition .......................................................... 18 

2.5.2 pH ............................................................................................................ 18 

2.5.3 Validation of the Test ............................................................................... 19 

2.5.4 Concentration of Test Article .................................................................... 19 

2.5.5 Growth lnhibiton by the standard compound Potassium Dichromate ....... 19 

3 RESULTS ............................................................................................................ 20 

3.1 RESULTS OF RANGE-FINDING TEST .............................................................. 20 

3.2 NOEC, LOEC, EbC 0, EbC 50 AND EbC 100 OF DIRESUL BLACK ROT 

LIQUID AFTER 72 HOURS OF EXPOSURE ................................................. 20-21 

3.3 NOEC, LOEC AND EbC 50 OF STANDARD POTASSIUM DICHROMATE 

AFTER 72 HOURS OF EXPOSURE ................................................................... 22 

3.4 pH OF ALGAE SUSPENSION ............................................................................ 22 

3.5 VALIDATION OF THE TEST ............................................................................... 22 

3.6 RESULTS OF ANALYSES ................................................................................. 22 

6 
RCC PROJECT 333977 

OIRESUL BLACK ROT LIQUID 

CONTENT CONT'D. Page 

4 CONCLUSION .................................•......................................................... 23 

5 REFERENCES ........................................................................................... 23 

TABLES 

(According to Contents Page 8) .....•.....................•................................ 24-31 

FIGURES 

(According to Contents Page 9) ............................................................ 32-36 

ATTACHMENT 1 

DETERMINATION OF THE CONCENTRATIONS OF DIRESUL 

BLACK ROT LIQUID IN TEST MEDIUM ............................................... 37-48 

7 
1-

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

CONTENT CONT'D. -TABLES 

Table 1 A: Experimental part A (test .1- 3) 

Influence of DIRESUL BLACK ROT LIQUID on algae growth after 

Page 

72 hours of exposure ............................................................................... 24 

Table 1 B: Experimental part B (test 3) 

Influence of DIRESUL BLACK ROT LIQUID on algae growth after 

72 hours of exposure ............................................................................... 27 

Table 2A: Influence of the test substance on algae growth: growth rate 1J (1/ day) 

and percentage inhibition of 1.1 in experimental part A. ............................. 28 

Table 2B: Influence of the test substance on algae growth: growth rate 1J (1/ day) 

and percentage inhibition of 1.1 in experimental part B .............................. 29 

Table 3: pH of the algae suspension for all concentrations of DIRESUL BLACK 

ROT LIQUID of experimental part A. ........................................................ 30 

Table 4: Influence of POTASSIUM DICHROMATE on algae growth after 

72 hours of exposure ............................................................................... 31 

8 
Fe 

4. v; __ 

RCC PROJECT 333977 

DIRESUL BLACK RDT LIQUID 

CONTENT CONT'D. - FIGURES 

Figure 1: Influence of DIRESUL BLACK RDT LIQUID on the growth of 

Page 

Scenedesmus subspicatus after 72 hours of exposure ............................ 32 

Figure 2: Growth curves of Scenedesmus subspicatus in experimental 

part A (test 1) incubated for 72 hours with different concentrations of 

DIRESUL BLACK RDT LIQUID ................................................................ 33 

Figure 3: Growth curves of Scenedesmus subspicatus in experimental 

part A (test 2) incubated for 72 hours with different concentrations of 

DIRESUL BLACK RDT LIQUID................................................................ 34 

Figure 4: Growth curves of Scenedesmus subspicatus in experimental 

part A (test 3) incubated for 72 hours with different concentrations of 

DIRESULBLACK RDT LIQUID ................................................................. 35 

Figure 5: Growth curves of Scenedesmus subspicatus in experimental 

part B incubated for 72 hours with different concentrations of 

DIRESU L BLACK RDT LIQUID ................................................................ 36 

9 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

GENERAL INFORMATION 

GENERAL 

Title: 

Sponsor: 

e Study Monitor: 

Testing Facility: 

RCC Project No.: 

Test Article: 

Test System: 

PROJECT STAFF 

Study Director: '. I.. Technical Coordinator: 

Analytical Chemistry: 

SCHEDULE 

Start of Experiments: 

Completion of Experiments: 

Study Completion Date: 

ARCHIVING 

Acute Toxicity of DIRESUL BLACK ROT LIQUID 

to Scenedesmus subspicatus 

(OECD -Algae Growth Inhibition Test; 

including determination of the influence of the light 

absorption of the substance on the growth inhibition) 

SANDOZ CHEMIKALIEN AG 

Dr. R. Mislin 

Rothausstrasse 61 

CH-4132 Muttenz 

Mr. E. ROdin 

Registration & Consulting Company Ltd. 

RCC 

UMWEL TCHEMIE AG 

CH-4452 ltingen/Switzerland 

333977 

DIRESUL BLACK ROT LIQUID 

Scenedesmus subspicatus 

Mrs. V. Wuthrich 

Mrs. H. Eckenstein 

K. Biedermann 

November 09, 1992 

March 25, 1993 

November 18, 1993/ zum 

10 

R C C, CH-4452 ltingen: Raw data, protocol, report and copy of report for at least 10 years. 

The test article reference sample (s) will be stored until expiration 

date. 

Thereafter, no raw data, protocol, report and copy of report will be withdrawn without the 

sponsor's further consent 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

GOOD LABORATORY PRACTICE 

The study will be performed in compliance with: 

"The OECD Principles of Good Laboratory Practice", Paris/France, 1981. 

"Good Laboratory Practice (GLP) in Switzerland, Procedures and Principles. March 1986". 

l 

I GUIDELINES ! .J. 

~~1 ' ~ 

l. Directive 92/69 EEC of July 31, 1992 (EEC Publication No. L 383, December 1992). 

11 

The test method is modified to differentiate between a reduced growth of algae due to real toxic 

effects of the test substance on the algal cells or due to an indirect effect, a reduced algal growth 

by light absorption in coloured test solutions. 

SUMMARY OF PROTOCOL AMENDMENTS 

First Amendment to Protocol: 

1) TITLE 

2) PREFACE I Schedule 

1 :e·· 3) Guidelines 

'~ '· 4) Introduction 

· 5) 2.3 Test Procedure I Test Apparatus /Medium 

Second Amendment to Protocol: 

1) TITLE 

2) Introduction 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

12 

t SUMMARY ,, ~ 

1 ,; 

! 

i 

l 

The influence of DIRESUL BLACK ROT LIQUID on the growth of the green algae Scenedesmus 

subspicatus was investigated. The study was performed according to the EEC Directive 92/69. 

The test method was modified to differentiate between a reduced growth of algae due to real 

toxic effects of the test substance on the algae cells or due to an indirect effect, a reduced algae 

growth by light absorption in coloured test solutions. 

The test included two experimental parts: 

Experimental part A: the algae grew in test medium with dissolved dyestuff in Erlenmeyer flasks. 

Experimental part B: the algae grew in separate containers (test tube) which were surrounded by 

the black coloured solutions of the same concentration as tested in part A 

Because of difficulties concerning the solubility of DIRESUL BLACK ROT LIQUID in different test 

media, several tests were performed. Finally, in experimental part A the following nominal 

concentration range was tested: 0.01 mg/1 to 200 mg/1. 

In experimental part B, the nominal concentration range of 0.01 mg/1 to 50 mg/1 was tested. 

The biological results of both experimental parts A and B were almost identical. This was shown 

by several calculated parameters, the NOEC, LOEC and the EC 50 values: 

In part A the NOEC was 1.0 mg/1, and in part B the NOEC was found to be between 1 and 5 

mg/1. The LOEC was found to be between 1 and 5 mg/1 in part A and it was about 5 mg/1 in part 

B. 

The percentage inhibition of algae biomass and the algae growth rate (J.J) after 72 hours of 

exposure were in the same order of magnitude. The EC 50 values calculated for both growth 

parameters, showed no significant differences. 

Experimental part A Experimental Part B 

EbC 50: 1.84 mg/1 5.99 mg/1 

95 % confidence limits: 1.37 · 2.33 mg/1 5.58 · 6.44 mg/1 

8.59 mg/1 

7.24 -10.9 mg/1 

E C 50: 

9S% confidence limits: 

54.1 mg/1 

41.7 · 74.7 mg/1 

Thus, the growth inhibition of Scenedesmus subspjcatus was in the same magnitude when the 

algae grew in test medium without test substance but under reduced light intensities by the filter 

effect (experimental part B) as in experimental part A, where the algae grew in test solutions 

without dissolved test substance. 

In conclusion, this modified algae test clearly demonstrated that the observed inhibition effect of 

the substance was caused only by the indirect effect, the light absorption in the coloured test 

solutions. Thus, a toxic effect of the test substance on the algae cells can be excluded up to a 

concentration of 50 mg/1. 
. r···· RCC PROJECT 333977 

~ DIRESUL BLACK ROT LIQUID 

13 

<f ;f 

·:1 

~~ 

1: 

;! 

1 c; 

1 INTRODUCTION 

The algae growth inhibition test when applied to dyestuffs cannot differentiate between a growth 

reduction of the algae caused by real toxic effects of the dyestuffs or caused by an indirect effect, 

the light absorption by the coloured test solution. 

The purpose of this modified test is to quantify the algicidal effect of the tested dyestuff versus the 

growth inhibition effect due to reduced light intensities in the coloured test solutions. Exponentially 

growing cultures of the unicellular green algae Scenedesmus subspjcatus were exposed to 

various concentrations of the test substance in a modified test system. The inhibition of growth in 

relation to control cultures was determined over a test period of 72 hours. 

2 MATERIALS AND METHODS 

2.1 DEFINITIONS 

Cell density: The number of cells/ml 

Growth: 

E,,Cso: 

The increase in cell density over the test period. 

The concentration of test substance which results in a 50% reduction in 

growth relative to the control. 

$ l• NOEC (No Observed 

Effect Concentration): 

The concentration of test substance which results in a 50 % reduction in 

growth rate. 

The highest tested concentration at which no significant inhibition of 

growth is observed relative to the control. 

LOEC (Lowest Observed 

Effect Concentration): The lowest tested concentration at which a significant inhibition of 

growth is observed relative to the control. 

2.2 TEST SYSTEM I RATIONALE 

The test was run with the OECD Guideline recommended species Scenedesmus subspicatus. 

The algae were grown in the laboratories off RCC under standardized conditions. 

This study should provide a rational basis to assess the influence of the test article on the growth 

rate of the green alga Scenedesmys sybspicatys. 
l 

l 

i 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

2.3 CHEMICALS 

2.3.1 Test Article 

Data of test article as delivered by the sponsor. 

Identity: 

Batch: 

Purity: 

Aggregate State: 

Solubility: 

DIRESUL BLACK ROT LIQUID 

1032812 

100% 

Black liquid 

Well soluble in water 

Appearance in the Test Medium: Black solution (determined by RCC) 

Stability: 

Stability in the Test Medium: 

Storage: 

Expiration Date: 

Safety Precautions: 

2.3.2 Reference Compound 

Water: stable for at least 48 hours 

Stable for at least 72 hours 

(determined by RCC under the present project number: 

see Attachment 1) 

At room temperature, in the dark 

March 31, 1993 

Routine hygienic procedures were sufficient to assure 

personnel health and safety. 

14 

As reference compound potassium dichromate of analytical grade was used (E. Merck, 

DarmstadVFederal Republic of Germany). It is periodically tested (two times per year) as check 

for the growth conditions and for the quality of the algae stem used for toxicity testing. 
• . 

L 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

2.4 EXPERIMENTAL CONDITIONS 

Test medium (according to the OECD and EEC Guidelines): 

Nutrient Final concentration in test solution 

Stock solution 1 

NH4CI 15 mg/1 

MgCI2 • 6H20 12 mg/1 

CaCI2 • 2H20 18 mg/1 

MgS04 • 7H20 15 mg/1 

KH2P04 1.6 mg/1 

Stock solution 2 

FeCI3 • 6H20 0.08 mg/1 

NaEDTA · 2H20 0.1 mg/1 

Stock solution 3 

H3B03 0.185 mg/1 

MnCI2 • 4H20 0.415 mg/1 

Stock solution 4 

ZnCI2 3 x 10-3 mg/1 

GoCI2 • 6H20 1.5 x 10-3 mg/1 

Na2 MoO 4 • 2H20 7 x 10-3 mg/1 

Stock solution 5 

CuCi2 • 2H20 10-s mg/1 

Stock solution 6 

NaHC03 50 mg/1 

The pH of the medium after equilibrium with air is about 8. 

15 

., 

I 

I 
• 

L 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

lncybatjon: 

16 

The experiments were started with a biomass of 1 0'000 cells per ml of nutrient solution. These 

cells were taken from a pre-culture which was set up 3 days prior to the test with the same 

conditions as in the test. 

In experimental part A toxic and indirect effects on algae growth were measured. For this 

purpose, the appropriate quantity of the pre-culture was diluted with test medium containing test 

article. Incubation was performed on a Lab shaker {120 strokes/min.) at 23 - 24 oc with 

continuous illumination with 8000 Lux. This illumination was achieved by 4 fluorescent tubes 

{Philips TL33, 20 W, 60 em) installed above the shaker at a distance of approximately 20 em from 

the incubation flasks. As incubation flasks, 50 ml Erlenmeyer flasks, stoppered with cotton-wool 

plugs, were used. 

In experimental part B indirect effects caused by light absorption were measured. For this 

purpose, solutions of the test article in the test medium without the addition of algae medium were 

prepared. Untreated test medium containing 1 0'000 algae cells/ml was filled into glass tubes 

stoppered with cotton plugs. The tubes were placed into the 250 ml Erlenmeyer flasks containing 

the test article concentrations corresponding to part A. The test conditions are indentical to those 

in experimental part A. 

The test was run for 72 hours. Samples of the incubated cultures were taken after about 24, 48, 

and 72 hours. 

Test Concentrations: 

Range-finding tests: 

Three range-finding tests were performed according to experimental part A. 

Range-finding test 1 : 

The test was performed over 72 hours using the nominal concentrations 1 00, 1 0, 1 and 0.1 mg/1 

and an untreated control. 

DIRESUL BLACK ROT LIQUID was found to be not soluble in the test medium which was 

prepared according to NEN 6506: Water Determination of Toxicity with Algae, {Dutch Standard) 

Netherlands Norrnalisatie lnstitut, Rijswijk (1979). 

Range-finding test 2: 

The test was prepared as described in range-finding test 1, but using Tween 80 as dispersant 

agent. 

Range-finding test 3: 

The test was performed over 72 hours using the nominal concentrations 100, 1 0, 1 and 0.1 mg/1 

and an untreated control. The concentrations were prepared using the medium described in 

Directive 92169 EEC of July 31, 1992. The test article was found to be stable using this test 

medium. 
r 

~. RCC PROJECT 333977 17 

] DIRESUL BLACK ROT LIQUID 

-~t ':] 

·r 

] 

,j 

j 

; 

~' 

Main tests: 

Three main tests were performed using the test medium described in the EEC Directive; test 1 

and 2 according. to experimental part A and test 3 according to experimental part A and B. 

Main test 1: 

100 mg DIRESUL BLACK ROT LIQUID were dissolved in test medium and filled to a volume of 

100 mi. From this stock solution, a series of sequential dilutions in test medium was prepared and 

the respective amount of algae added. The final nominal test concentrations were: 200, 100, 50, 

25 and 12.5 mg/1. 

i Main test 2: 

I ( Jl e The stock solution and dilutions were prepared as described in main test 1. The final nominal test 

! concentrations were: 15, 7.5, and 3.75 mg/1. 

:~ 

Main test 3: 

~ j 200 mg DIRESUL BLACK ROT LIQUID were dissolved in test medium and filled to a volume of 

! 200 mi. From this stock solution further dilutions in test medium were prepared and the respective 

i amount of algae added. The final nominal test concentrations were: 50, 5, 1, 0.5, 0.1 and 0.01 

mg/1. 

In experimental part A, DIRESUL BLACK ROT LIQUID containing test samples were incubated in 

triplicate and untreated test samples were incubated five fold. 

In experimental part B, DIRESUL BLACK ROT LIQUID containing treated and untreated test 

samples were incubated in duplicate. 

Reference test: 

As reference substance, potassium dichromate was used, tested January 1993 (run with RCC 

project 341201} according to experimental part A. 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

2.5 EVALUATIONS 

2.5.1 Determination of Growth Inhibition 

Number of cells: 

18 

The number of algae after 24 hours was estimated by means of a microscope using a Neubauer 

chamber according to the equation: 

Number of cells counted x 2.5 x1 0§ (dilution factor) 

(concentration factor) 

Area under the growth curves (AUC): 

(N1 - No) (N1 + N2) - 2No 

area = --------------- x t1 + ------------------------ x (t2 - t1) ... 

2 2 

(Nn-1 + Nn) - 2No 

+ ---------------------------- x (tn - tn-1) 

2 

where is: 

No 

N1 

N2 

Nn 

= 

= 

= 

= 

number of cells/ml at the start 

number of cells/ml after t1 = 24 hours, 

number of cells/ml after t2 = 48 hours, 

number of cells/ml after tn. 

Growth Inhibition (1}: 

area (control) -area (treated) 

= ----------------------------------------- X 1 00 

area (control) 

Calculations: 

The EbC 50 including confidence limits was estimated using the Logit-model (1). 

The NOEC and LOEC values were determined using the Dunnett-test (2). 

Growth Bate (u): 

In Nn -In No 

= ------------------- tn 

where is: 

tn = time (days) of ntn measurement 

\ 

I 

II. 

l

t.r! 'I 

II 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Inhibition of Growth Rate (lu): 

IJJ = ------------ iJC 

where is: 

= mean growth rate of control 

= mean growth rate of test concentration 

2.5.2 pH 

19 

The pH was determined for all concentrations of the test article and of the control at the beginning e and at the end of incubation. The pH of the controls should not deviate by more than 1.5 units 

during the test. 

2.5.3 Validation of the Test 

The cell concentration in the control cultures should have increased by a factor of at least 16 

within three days. 

2.5.4 Concentration of Test Article 

f For analytical purposes, the following samples were taken: 

Test 1: 

Duplicate 20 ml samples of all concentrations and the control, at the beginning and at the end of 

the test. 

Test 2 and 3: 

Quadruplicate 20 ml samples of all concentrations and the control, at the beginning and at the 

end of the test. 

All sampes which were not immediately analysed were stored at -20 oc. 

2.5.5 Growth lnhlblton by the standard compound Potassium Dichromate 

The influence of the reference compound Potassium Dichromate was tested under RCC Project 

No. 341201 in January 1993. 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

3 RESULTS 

3.1 RESULTS OF RANGE-FINDING TEST 

20 

Range-finding test 1 (performed using NEN test medium) indicated an EbC 50 in the range of 10 

and 100 mg/1. 

Range-finding test 2 (performed using Tween 80 and NEN test medium) indicated an EbC 50 in 

the range of 10 and 1 00 mg/1. 

Range-finding test 3 (performed using EEC recommended test medium) indicated an EbC 50 of 

>100 mg/1. 

3.2 NOEC, LOEC, EbC 0, EbC 50 AND EbC 100 OF DIRESUL BLACK 

ROT LIQUID AFTER 72 HOURS OF EXPOSURE 

The results are presented in tables 1 A, 2A, 1 B and 28. 

In test 1 and 2, {experimental part A was applied only) all test concentrations were statistically 

significant inhibited by the test article. 

In ~. (experimental part A and B was applied) no statistically significant influence was 

observed in part A even at the highest concentration of 50 mg where 72.7 % inhibition was 

observed. 

In part B, no statistically significant influence was observed up to 5 mg/1. 

Based on these results, the NOEC and the LOEC values are determined by computation of the 

observed results, no matter whether they were statistically significant or not. 

The biological results of both experimental parts A and B were almost identical. This is 

demonstrated by several calculated parameters, the NOEC/LOEC and the EC 50 values. 

Taking into account the variability of biological tests, the percentage inhibition of algal biomass 

and the algal growth rate ~ after 72 hours of exposure were in the same order of magnitude. 

Hence, no significant differences were observed between both experimental parts. 
' 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

21 

The following results were obtained after 72 hours of exposure (determined by the area under the 

growth curve): 

Experimental oart A 

EbC 50 1.84 mg/1 

95 % Confidence limit 1.37 - 2.33 mg/1 

Dose response Curve 

Logit = - 0.22 + 0.367 x Log (dose) 

95 % Confidence 

limits of slope 0.365- 0.368 mg/1 

NOEC- value 1.0 mg/1 

LOEC -value between 1 and 5 mg/1 

linear regression of growth 

inhibition versus test article 

concentration: 

Unear regression y = 26.693 x Log (dose) 

+ 31.758 (A= 0.782) 

EuC 0 0.065 mg/1 

EbC 50 4.8 mg/1 

EbC 100 360 mgil 

Results obtained after 72 hours of exposure by growth rate: 

95 % Confidence limit 

54.1 mg/1 

41.7-74.7 mg/1 

Exoerimental oart B 

5.99 mg/1 

5.58 - 6.44 mg/1 

-2.61 + 1.460 x Log (dose) 

1 .455 - 1.466 mg/1 

1-5mg/l 

about 5 mg/1 

y = 52.826 x Log (dose) 

6.542 (R = 0.998) 

0.75 mg/1 

6.65 mg/1 

58.8 mg/1 

8.59 mg/1 

7.24 - 1 0.9 mg/1 

Based on the results, the growth of algae was mainly inhibited by the light adsorption of the 

dyestuff DIRESUL BLACK ROT LIQUID. 
ACC PROJECT 333977 

DIAESUL BLACK ROT LIQUID 

3.3 NOEC, LOEC AND EbC 50 OF STANDARD POTASSIUM 

DICHROMATE AFTER 72 HOURS OF EXPOSURE 

The results for the standard potassium dichromate are presented in table 4. 

They are archived under the project-no.: 341201. 

22 

The EC 50 calculated by the Logit-Model was 0.312 mg/1 with the following confidence limits: 

95 o/o confidence limit: 0.299 - 0.326 mg/1 

The dose response curve was found to be: Logit = 2.45 + 2.103 x Log (dose). 

The 95 % confidence limits for the slope were: 2.085 - 2.120 

3.4 pH OF ALGAE SUSPENSION 

The pH of the algae suspensions of experimental part A is presented in table 3. 

At the start of the tests, the pH ranged from 8.1 to 9.5. 

At the end of the tests, the pH ranged from 8.1 to 8.9. 

' 3.5 VALIDATION OF THE TEST 

l.l/; 

,~···--. •. ------------------

'..'J 

·r, The tests are regarded to be valid since the cell concentration in the control cultures increased by 

a factor of 25.4 (test 1 ), 120 (test 2) and 30.6 (test 3) within three days in experimental part A and 

by 50 (test 3) in experimental part B. 

3.6 RESULTS OF ANALYSES 

The results of the analyses are reported in Attachment 1. 

DIRESUL BLACK ROT LIQUID was found to be stable under the test conditions. The 

concentration in all tests ranged from 85.5 o/o to 107.7% of the nominal concentration. 
'f' RCC PROJECT 333977 23 

] OIRESUL BLACK ROT LIQUID 

$ 

::1-. >t 

k 

Y.i ;I 

~ !< 

r. 

;( 

"t ,j 

4 CONCLUSION 

When DIRESUL BLACK ROT LIQUID was assessed for its algicidal effect on Scenedesmus 

_subspicatus over 72 hours, the following results were obtained: 

Experimental part A 

NOEC: 1 mg/1 

LOEC: between 1 and 5 mg/1 

EbC 50: 1.84 mg/1 with the 95 % confidence limits of 1.37 - 2.33 mg/1 

EIJC50: 54.1 mg/1 with the 95 % confidence limits 41.7 - 7 4. 7 mg/1 

Experimental part B 

NOEC: 1 -5 mg/1 

LOEC: about 5 mg/1 

EbC 50: 5.99 mg/1 with the 95% confidence limits of 5.58-6.44 mg/1 

EiJC50: 8.59 mg/1 with the 95% confidence limits 7.24- 10.9 mg/1 

In conclusion, this modified algae test showed that the observed growth inhibition effect of the 

test substance DIRESUL BLACK ROT LIQUID was caused only by the indirect effect, the light 

adsorption in the coloured test solutions. A toxic effect of DIRESUL BLACK ROT LIQUID can be 

excluded up to a concentration of 50 mg/1. 

(1) 

(2) 

REFERENCES 

Cox, D.R.: 

Analysis of binary data. 

Methuen & Co. Ltd., 1977. 

Miller, R.G.: 

Simultaneous Statistical Inference 

Springer Verlag, New York (1981}. 
r . 

. 

··.···.·:··1···· 

.-.' 

l 

~:r 

-.:! 

·-~ 

t 

! 

1 '1 

.J 

;~ 

: .. l 

·;·~-· 

' 

• ·-

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 1A: Experimental part A (test 1) 

24 

Influence of DIRESUL BLACK ROT LIQUID on algae growth after 72 hours of 

exposure. Number of cells are divided by 1 0'000. Area under the curve (AUC) and 

response (Resp.) are divided by 10'000 and corrected or NO= 10'000. Significance 

(Sign) estimated with Dunnett-test: (* = 1 %). 

Concentration Number of cells after AUC Resp. lnhib. Sign. 

(mg/1) 24h 48h 72h % 

Control 

3.1 6.9 21.1 433 

3.6 14.2 25.0 667 

1.7 4.7 21.1 347 

1.7 10.3 28.3 568 

3.9 6.7 31.7 575 

Mean 2.8 8.6 25.4 518 

Std. dev. 127 

12.5 

1.4 3.6 14.4 233 

1.9 5.8 7.2 211 

1.1 5.0 16.1 280 

Mean 1.5 4.8 12.6 241 277 53.4 * 

Std. dev. 35 

25.0 

0.6 4.4 4.4 113 

1.1 2.5 10.6 154 

1.7 3.1 7.8 149 

Mean 1.1 3.3 7.6 138 380 73.3 * 

Std. dev. 22 

50.0 

2.8 3.9 3.9 148 

0.6 6.1 4.4 154 

1.1 4.7 3.9 126 

Mean 1.5 4.9 4.1 142 376 72.5 * 

Std. dev. 15 

100.0 

0.6 1.7 0.0 -4 

0.6 3.3 5.0 94 

0.6 3.1 5.0 89 

Mean 0.6 2.7 3.3 59 459 88.6 • 

Std. dev. 55 

200.0 

1.7 3.1 2.2 82 

0.8 2.2 1.7 32 

0.6 0.8 2.2 0 

Mean 1.0 2.0 2.0 38 480 92.7 * 

Std. dev. 41 

.;j 

.t! 
RCC PROJECT 333977 25 

DIRESUL BLACK ROT LIQUID 

Table 1A: continued 

e~~rimfilotal12art A Ufilst 2l 

Number of cells after AUC Resp. lnhib. Sign. 

24h 48h 72h % 

Control 

9.2 23.3 125.0 2220 

5.3 28.6 155.6 2621 

4.4 18.3 111.1 1818 

3.3 13.1 91.7 1434 

9.2 27.8 116.7 2228 

Mean 6.3 22.2 120.0 2064 

Std. dev. 452 

3.75 

3.3 8.9 22.2 499 

'I ·::.-,.., 3.1 15.3 52.8 1015 

3.9 9.7 22.2 533 

Mean 3.4 11.3 32.4 682 1382 66.9 * 

Std. dev. 289 

7.5 

2.8 9.2 50.0 828 

5.0 14.1 19.4 631 

4.4 13.3 44.4 898 

Mean 4.1 12.2 37.9 786 1279 61.9 * 

Std. dev. 138 

15.0 

2.5 8.9 41.7 714 

3.1 8.1 47.2 775 

1.7 7.5 30.6 528 

Mean 2.4 8.2 39.8 672 1392 67.4 * 

Std. dev. 129 

• 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 1A: continued 

Experimental part A {lest 3) 

Concentration Number of cells after 

(mg/1) 24h 48h 72h 

Control 

2.5 7.2 16.7 

3.1 5.3 55.6 

1.7 6.1 22.2 

1.9 8.3 22.2 

0.8 3.9 36.1 

Mean 2.0 6.2 30.6 

Std. dev. 

0.01 

1.1 4.4 27.8 

2.2 7.8 47.2 

3.3 8.6 41.7 

Mean 2.2 6.9 38.9 

Std. dev. 

0.1 

0.8 3.6 16.7 

3.6 15.8 27.8 

1.1 5.0 63.9 

Mean 1.8 8.1 36.1 

Std. dev. 

0.5 

3.1 5.8 61.1 

2.5 10.0 77.8 

3.9 9.4 44.4 

Mean 3.2 8.4 61.1 

Std. dev. 

1.0 

3.3 7.8 25.0 

1.7 21.1 88.9 

2.8 13.1 19.4 

Mean 2.6 14.0 44.4 

Std. dev. 

5.0 

0.8 6.4 13.9 

2.2 8.9 19.4 

2.2 4.2 27.8 

Mean 1.7 6.5 20.4 

Std. dev. 

50.0 

1.1 5.0 11.1 

1.9 3.1 2.8 

0.8 5.8 0.0 

Mean 1.3 4.6 4.6 

Std. dev. 

n.s. : no statistically significant effect 

26 

AUC Resp. lnhib. Sign. 

% 

373 

809 

394 

451 

486 

503 

177 

406 

746 

726 

626 -122 -24.6 

191 

246 

739 

853 

613 -109 -21.9 n.s. 

323 

887 

1174 

792 

951 -447 -89.2 n.s. 

199 

506 

1554 

554 

872 -368 -73.4 n.s. 

591 

280 

439 

427 

382 121 24.0 n.s. 

89 

220 

94 

98 

137 365 72.7 n.s. 

71 
" l~\ 

''· 

RCC PROJECT 333977 27 

t Table 1 B. Experimental part B (jest 3) 

1 Influence of DIRESUL BLACK ROT LIQUID on algae growth after 72 hours of 

,, \ exposure. Number of cells are divided by 1 0'000. Area under the curve (AUC) and 

l response (Resp.) are divided by 10'000 and corrected or NO= 10'000. Significance I (Sign) estimated with Dunnett-test: (* = 1 %). 

l 

J 

J ;·'i 

! 

1 

Concentration Number of cells after 

(mg/1) 24h 48h 72h 

Control 

0.0 24.7 58.3 

0.6 31.1 41.7 

Mean 0.3 27.9 50.0 

Std. dev. 

0.01 

1.7 11.7 52.8 

0.3 13.6 30.6 

Mean 1.0 12.7 41.7 

Std. dev. 

0.1 

0.3 17.5 52.8 

0.3 13.3 52.8 

Mean 0.3 15.4 52.8 

Std. dev. 

0.5 

1.1 15.6 55.6 

0.8 19.7 27.8 

Mean 1.0 17.7 41.7 

Std. dev. 

1.0 

0.3 21.1 80.6 

0.6 20.0 30.6 

Mean 0.5 20.6 55.6 

Std. dev. 

5.0 

1.4 20.6 25.0 

0.8 16.9 27.8 

Mean 1.1 18.8 26.4 

Std. dev. 

50.0 

0.0 4.7 0.0 

0.3 2.2 0.0 

0.2 3.5 0.0 

n.s. : no statistically significant effect 

AUC Resp. lnhib. Sign. 

% 

1217 

1201 

1217 

22 

895 

641 

768 449 36.9 n.s. 

180 

1001 

900 

950 . 266 21.9 n.s. 

71 

1008 

766 

887 330 27.1 n.s. 

171 

1421 

802 

1111 106 8.7 n.s. 

438 

768 

698 

733 484 39.7 n.s. 

49 

53 

0 

26 1190 97.8 * 

37 
• 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 2A: Influence of the test substance on algae growth: growth rate 1.1 (1/day) and 

percentage inhibition of 1.1 in experimental part A 

Test Article Growth rate 1.1 (1/day) 

concentration 24 h 48h 72 h 

(mgll) 1.1 % J.J % _1.1 % 

I~st1; Control 1.03 --- 1.08 --- 1.08 --- 12.5 0.41 60.2 0.78 27.8 0.84 22.2 

25 0.10 90.3 0.60 44.4 0.68 37.0 

50 0.41 60.2 0.79 26.9 0.47 56.5 

100 -Q.51 -49.5 0.50 53.7 0.40 63.0 

200 0 100 0.35 67.6 0.23 78.7 

I~st2; Control 1.84 --- 1.55 --- 1.60 --- 3.75 1.22 33.7 1.21 21.9 1.16 27.5 

7.5 1.41 23.4 1.25 19.4 1.21 24.4 

15 0.88 52.2 1.05 32.3 1.23 23.1 

Test~; Control 0.69 --- 0.91 --- 1.14 --- 0.01 0.79 -14.5 0.97 -6.6 1.22 -7.0 

0.1 0.59 14.5 1.05 -15.4 1.20 -5.3 

0.5 1.16 -68.1 1.06 -16.5 1.37 -20.2 

1 0.96 -39.1 1.32 -45.1 1.26 -10.5 

5 0.53 23.2 0.94 -3.3 1.01 11.4 

50 0.26 62.3 0.76 16.5 0.51 55.3 

28 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 28: Influence of the test substance on algae growth: growth rate 1J (1/day) and 

percentage inhibition of 1J in experimental part B 

Test Article Growth rate 1J (1/day) 

concentration 24 h 48 h 72 h 

(mg/1} IJ % _j.l % IJ % 

T~sl J; Control -1.20 n.c. 1.66 -- 1.30 --- 0.01 0 n.c. 1.27 23.5 1.24 4.6 

0.1 -1.20 n.c. 1.37 17.5 1.32 -1.5 

0.5 0 n.c. 1.44 13.3 1.24 4.6 

1 -o.69 n.c. 1.51 9.0 1.34 -3.1 

5 0.10 n.c. 1.47 11.5 1.09 16.2 

50 -1.61 n.c. 0.63 62.1 0 100 

I n.c. : Not calculated 

•• 

29 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

30 

Table 3: pH of the algae suspension for all concentrations of DIRESUL BLACK ROT LIQUID of 

experimental part A. 

Concentration Test Time interval 

Article 

_{m_g/J)_ Start 72 hrs 

Test l: 0* 8.1 8.3 

12.5 8.2 8.2 

25 8.3 8.2 

50 8.5 8.2 

100 9.0 8.1 

200 9.5 8.2 

Temz: 0* 8.7 8.5 

3.75 8.6 8.3 

7.5 8.6 8.3 

15 8.6 8.2 

r~m ;3; 0* 8.5 8.6 

0.01 8.4 8.7 

0.1 8.3 8.9 

0.5 8.3 8.9 

1 8.3 8.8 

5 8.4 8.6 

50 8.8 8.2 

* Control 
----------------

r. 

. 

. 

CC PROJECT 333977 

~, DIRESUL BLACK ROT LIQUID 

31 

I 

I 

Table 4: Influence of POTASSIUM DICHROMATE on algae growth after 72 hours of exposure. 

Number of cells are divided by 1 0'000. Area under the curve (AUC) and response 

(Resp.) are divided by 10'000 and corrected or NO= 10'000. Significaoce (Sign) 

estimated with Dunnett-test: (* = 1 %). 

Concentration Number of cells after AUC Rasp. lnhib. Sign. 

(mg/1) 24h 48h 72h % 

Control 

4.2 13.6 116.1 1768 

3.9 16.9 66.7 1240 

3.3 22.2 47.2 1118 

3.3 10.6 25.0 574 

3.9 16.1 33.3 453 

Mean 3.7 15.9 57.8 1104 

Std. dev. 453 

0.25 

1.9 5.8 61.1 858 

2.5 6.7 27.8 494 

3.3 4.7 38.9 599 

Mean 2.6 5.7 42.6 650 453 41.1 

Std. dev. 187 

0.5 

4.2 1.9 22.2 353 

3.3 6.7 22.2 446 

3.6 4.2 11.1 260 

Mean 3.7 4.3 18.5 353 751 68.0 * 

Std. dev. 93 

1.0 

1.1 2.2 0.0 19 

1.7 2.2 5.6 101 

0.6 1.9 2.8 34 

Mean 1.1 2.1 2.8 51 1053 95.4 * 

Std. dev. 44 

2.0 

0.8 0.6 11.1 107 

0.8 0.3 0.0 -33 

0.3 0.3 5.6 22 

Mean 0.6 0.4 5.6 32 1072 97.1 * 

Std. dev. 71 

4.0 

1.1 0.0 0.0 -33 

1.1 0.6 2.8 14 

0.3 0.0 0.0 -52 

Mean 0.8 0.2 0.9 -23 1128 102.2 * 

Std. dev. 35 
• ; 

':_ 

RCC PROJECT 333977 

OIRESUL BLACK ROT LIQUID 

Figure 1: Influence of DIRESUL BLACK ROT LIQUID on the growth of Scenedesmus 

sybspicatus after 72 hours of exposure. 

% lnhllltlon 

100 . / 

80 

60 

40 

20 

---------------·-

" " " " 

+ 

" " " 

/ 

" " " 

" " " + 

/ " " 

" 

" " " 

" 

• 

/ " 

/ 

a / 

/ " I' a 

/ 

/ 

/ " 

" / " 

" 

/ 

/ " / / 

" " / " / 

" " 

X 

01jO~~~~mm~~~~~~~~~~~~~-rTTmmr-~~~~~~ 

w ~ ~ ~ ~ ~ 

a Part A.Observed values used for Loglt 

-----... Part Aaln.reg.of observed values 

a a 

"· • • • • ·,.. • • .. • ·• Part A, EC SO Est illation + + + Part B.Observed values ,._ ---x- - - ... Part B.Ln.reo of observed values 

------·-·-+ Part BoEC 50 Estimation .., .., .., Part BoObserve<l values used for Loglt 

moll 

32 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

33 

Figure 2: Growth curves of Scenedesmus subspjcatus in experimental part A (test 1) incubated 

for 72 hours with different concentrations of DIRESUL BLACK ROT LIQUID. 

tu1BER (F CELLS lx 10000) 

30 

25 

20 

15 

10 

5 

10 20 

•• ---·----• Control •· ••••• o- ••••• •0 12.5 mglt ._ ______ ..,. 25 mglt 

+-·-·--·-·-+ 50 mglt 

- - - - - )( 100 mg/l 

--··-+-···- 200 mg/1 

. . . 

. · . · 

30 40 50 60 

. . . . . . . . . 

.. .... 

~ 

. . . 

.... .. .. 

70 

ho.rs 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

34 

Figure 3: Growth curves of Scenedesmus subspicatus in experimental part A (test 2) incubated 

for 72 hours with different concentrations of DIRESUL BLACK ROT LIQUID. 

IU1BER CJ= CELLS tx 100001 

140 

120 

100 

80 

60 

40 

20 

0~--~~~~~~--~--~~---r--~--~~~~--~--~ 

60 70 

1-D..RS 

0 10 20 30 40 50 

•• ------·----• Control ••••••• o- ••••• ·0 3. 75 moll .,_ ______ ..,.7.5mgll 

------·-·-+ 15 mgll 
.. _., 

,,, 

•• 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

·-·~-----------------

35 

Figure 4: Growth curves of Scenedesmus subspicatus in experimental part A (test 3) incubated 

for 72 hours with different concentrations of DIRESUL BLACK ROT LIQUID. 

N..teER CF ffi.LS he 100001 

70 

60 

50 

40 

30 

20 

10 

0 

0 10 20 

• • • Control ••••••••••••• •0 0.01 moll 

.. - - --- - .. o. 1 moll 

-·-·-+-·-·-+ 0.5 moll 

-----•1mgll. _ ... _ .. __ 5 mgll. 

---vSOmgll 

I 

i 

i 

i 

/ 

I 

i 

i 

/ JC / ,/_,. 

I / .·A i , .· / . / :',' ,I , :, I/ .. ·, . . / I , :, '/ ,'/ / ,'/ /,, ,'/ 

r ,lf 

h ~~ ~ // / ... / , I / / . / . .;JI I A' /' 

/ I' A' ,•' , /.' / / i/:' ... .--...:-.,.. ~-~:.V 

., ..:.--·~=~-=-- ..... . ----· ~~-· __...., _____ ., 

-------

40 50 60 70 

hou's 
, •... 

.. 

·:.~ 

RCC PROJECT 333977 

OIRESUL BLACK ROT LIQUID 

36 

Figure 5: Growth curves of Scenedesmus subspicatus in experimental part B incubated for 

72 hours with different concentrations of DIRESUL BLACK ROT LIQUID. 

tU1BER (F CELLS be 100001 

60 

50 

20 

10 

10 20 

• • • Control ••••••• o- ••••• ·0 0. 01 mg/\ 

~ - - -- - - _. 0. 1 mg/\ ------·-·--t 0.5 mg/1 

-----)( 1 mg/\ 

---------- 5 mg/\ - --- - ... 50 mg/\ 

30 50 60 

I 

70 

hours 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

t ATIACHMENT 1 

•• 

DETERMINATION OF THE CONCENTRATIONS OF DIRESUL BLACK ROT LIQUID 

IN TEST MEDIUM 

37 
RCC 

UMWEL TCHEMIE AG 

PROJECT 333977 

DETERMINATION OF THE CONCENTRATIONS OF 

DIRESUL BLACK ROT LIQUID 

IN TEST MEDIUM 

ACUTE TOXICITY TO SCENEDESMUS SUBSPICATUS 

(OECD-Aigae Growth Inhibition Test; including determination of the influence of the 

light absorption of the substance on the growth inhibition) 

REPORT 

P.O.Box CH- 44521tingen 1 Switzerland 

Phone: 0611975 11 11 I Telex: 966 136 RCC CH 

Telefax: 0611971 52 66 
t 

• ' 

RCC PROJECT 3339n 

DIRESUL BLACK RDT LIQUID 

CONTENTS 

Page 

PREFACE 

General ................................................................................................................ 3 

Project Staff .......................................................................................................... 3 

Archiving .............................................................................................................. 3 

INTRODUCTION .................................................................................................. 4 

2 MATERIALS AND METHODS ............................................................................... 4 

2.1 Test Article ............................................................................................................ 4 

2.2 Sample Storage .................................................................................................... 4 

2.3 Analytical Procedure ............................................................................................. 5 

2.3.1 Analysis of Samples ............................................................................................. 5 

2.3.2 Standard Solutions ............................................................................................... 5 

2.3.3 Spectrophotometric Determination ........................................................................ 5 

2.3.4 Evaluation of Results ............................................................................................ 6 

3 RESULTS ............................................................................................................. 7 

TABLES ............................................................................................................... 8-11 

2 

3? 
RCC PROJECT 3339n 

DIRESUL BLACK ROT LIQUID 

PREFACE 

GENERAL 

RCC Project Number 

Sponsor 

Study Monitor 

Test Article 

Testing Facility 

PROJECT STAFF 

Responsible for Analytics 

ARCHIVING 

3339n 

SANDOZAG 

Dr. R. Mislin 

Rothausstrasse 61 

CH-4132 Muttenz 

Mr. E. Rudin 

Registration & Consulting Company Ltd. 

DIRESUL BLACK ROT LIQUID 

RCC 

UMWEL TCHEMIE AG 

Department of Chemistry 

CH-4452 ltingen 1 Switzerland 

K. Biedermann 

.. ~ ....... if.._.f .. ~-4~~~ Date: Od~ ~ / --79'7'3 /ace 

R C C, CH-4452 ltingen Raw data, protocol, report and copy of report of the 

present study will be archived for at least ten years. 

Test article reference sample(s) will be stored until the 

expiration date. 

3 

Thereafter, no raw data, protocol, report and copy of report will be withdrawn without the sponsor's further 

consent. 

~ ·"" L ··) 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

1 INTRODUCTION 

4 

This report describes the analytical method used and the results obtained for .the concentrations of 

DIRESUL BLACK ROT LIQUID in test medium. 

2 MATERIALS AND METHODS 

2.1 TEST ARTICLE 

Data of test article as delivered by the sponsor. 

Identity: 

Batch: 

Purity: 

Aggregate State: 

Solubility: 

Appearance in the 

Test Medium: 

Stability: 

Stability in the 

Test Medium: 

Storage: 

Expiration Date: 

Safety Precautions: 

DIRESUL BLACK ROT LIQUID 

1032812 

100% 

Black liquid 

Aqueous: very soluble 

Black solution (determined by RCC) 

Water: stable for at least 48 hours 

DIRESUL BLACK ROT LIQUID was found to be stable in the test medium 

over a period of 72 hours (determined by RCC) 

At room temperature, in the dark 

March 31, 1993 

Routine hygienic procedures were sufficient to assure 

personnel health and safety. 

2.2 SAMPLE STORAGE 

Samples of test 1 were stored in the refrigerator overnight prior to sample work-up and quantification by 

spectrophotometry. Samples of test 2 and 3 were worked-up and quantified by spectrophotometry on the 

day of sampling. Reserve samples were stored deepfrozen at -20 oc until completion of the final report. 
RCC PROJECT 3339n 

DIRESUL BLACK ROT LIQUID 

2.3 ANALYTICAL PROCEDURE 

2.3.1 Analysis of Samples 

5 

After shaking wen and filtration over a folded filter1

, aliquots were withdrawn from the 20 ml sample volume 

of the control, 1.0 mg/1, 3.75 mg/1, 5.0 mg/1, 12.5 mg/1, 15.0 mg/1, 50.0 mg/1 and 200 mg/1 samples, inclusive 

of the 200 mg/1 stability test samples, and directly filled into 1 em quartz cuvettes. The absorbance of these 

aqueous solutions was measured at 580 nm versus bidistilled water or versus algae test medium. 

2.3.2 Standard Solutions 

Stock solutions of the test article (see section 2.1) in bidistilled water or algae test medium with 

concentrations of 0.2 mg/ml and 1.0 mg/ml were freshly prepared on each day of analysis. Standard 

solutions in the concentration range from 1.0 J,lg/ml to 100 J.lg/ml were prepared by respective dilution of 

these stock solutions with bidistilled water or algae test medium. The absorbance of the latter standard 

solutions, as well as of the 0.2 mg/ml stock solution was measured at 580 nm versus bidlstilled water or 

algae test medium and used for calibration of the spectrophotometer. 

2.3.3 Spectrophotometric Determination 

(Example of Operating Conditions) 

Apparatus: Perkin-Elmer Lambda 15 UVNis spectrophotometer 

Wavelength: 580nm 

Quartz cuvette: 1 em (pathlength) 

1 Only samples of test 2 and 3, collected after 72 hours, were filtered over a folded filter. 
RCC PROJECT 3339n 

DIRESUL BLACK ROT LIQUID 

2.3.4 Evaluation of Results 

6 

Measured samples were quantified by absorbance with reference to the calibration curves. The latter were 

obtained by correlation of the absorbance of the analytical standards with their corresponding 

concentrations in IJg/ml. 

An example of a calibration curve is listed in table 1 . From this or a similar curve, the concentration Y of 

DIRESUL BLACK ROT LIQUID in IJQ/ml of a measured sample was calculated from equation 1. 

Y= a+b·X (1) 

where 

Y = IJg/ml DIRESUL BLACK ROT LIQUID of measured sample 

a - y-axis intercept 

b - slope 

X = absorbance of measured sample 

The concentrations of DIRESUL BLACK ROT LIQUID in test medium were calculated from equation 2. 

Y·V 

C= (2) 

s 

where 

C = Concentration of DIRESUL BLACK ROT LIQUID in test medium in mg/1 

Y = IJQ/ml of measured sample calculated by equation 1 

V = Final volume (20 ml) 

S - Volume of sample (20 ml) 
RCC PROJECT 3339n 

DIRESUL BLACK ROT LIQUID 

3 RESULTS 

7 

The results obtained for the concentrations of DIRESUL BLACK ROT LIQUID in the test medium of tests 1, 

2 and 3 are listed in Tables 2, 3 and 4, respectively. 

The tabulated values represent rounded-off results obtained by calculations based on the exact raw data. 

For this method of spectrophotometric analysis, the detection limit was calculated to be 1.0 mg DIRESUL 

BLACK ROT LIQUID per liter test medium. 

An example of a calibration curve is listed in Table 1. 
' 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 1: Example of a calibration curve of DIRESUL BLACK ROT LIQUID 

Standard Absorbance 

(J.lg/ml} (at 580 nm} 

5.0 0.039 

10 0.064 

20 0.129 

50 0.307 

100 0.617 

200 1.199 

Y = -1.54 + 167 · X (R2 = 1.00) 

where 

Y .. ).IQ/ml DIRESUL BLACK ROT LIQUID in measured solution 

X = absorbance of measured sample 

8 

. .- .. . ._, 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table2: Results obtained for the concentrations of DIRESUL BLACK ROT LIQUID 

in the test medium of test 1. 

(Dates of analysis: 02/05-FEB-1993) 

Nominal Time Actual Mean concentration of ln%of 

concentration concentration of samples A and B nominal 

the test article 

(mg/1) (hours) (mg/1) (mg/1) 

Control 0/A <5.0 --- --- Control 0/B <5.0 

Control 72/A <5.0 --- --- Control 72/8 <5.0 

12.5 0/A 10.35 10.69 85.5 

12.5 0/8 11.02 

12.5 72/A 11.12 11.20 89.6 

12.5 72/8 11.27 

50.0 0/A 44.83 45.08 90.2 

50.0 0/8 45.33 

50.0 72/A 45.29 45.37 90.7 

50.0 72/B 45.44 

200 0/A 185.8 185.5 92.7 

200 0/B 185.1 

200 72/A 181.5 181.6 90.8 

200 72/B 181.7 

200* 0/A 183.8 183.8 91.9 

200* 0/B 183.8 

200* 72/A 182.8 182.8 91.4 

200* 72/B 182.8 

NB : Samples in duplicate 

* : Stability test (test medium without algae) 

9 

46 
RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 3: Results obtained for the concentrations of DIRESUL BLACK ROT LIQUID 

in the test medium of test 2. 

(Dates of analysis: 22/25-FEB-1993) 

Nominal Time Actual Mean concentration of ln%of 

concentration concentration of samples A and B nominal 

the test article 

(mg/1) (hours) (mg/1) (mg/1) 

Control 0/A <1.0 --- --- Control 0/B <1.0 

Control 72/A <1.0 --- --- Control 72/B <1.0 

3.75 0/A 3.917 3.917 104.5 

3.75 0/B 3.917 

3.75 72/A 4.039 4.039 107.7 

3.75 72/B 4.039 

15.0 0/A 15.18 15.27 101.8 

15.0 0/B 15.36 

15.0 72/A 15.70 16.08 107.2 

15.0 72/B 16.45 

NB : Samples in duplicate 

10 

47 
' 

RCC PROJECT 333977 

DIRESUL BLACK ROT LIQUID 

Table 4: Results obtained for the concentrations of DIRESUL BLACK ROT LIQUID 

in the test medium of test 3. 

(Dates of analysis: 22/25-MAR-1993) 

Nominal Time Actual Mean concentration of ln%of 

concentration concentration of samples A and B nominal 

the test article 

(mg/1) (hours) (mg/1) (mg/1) 

Control 0/A <1.0 --- --- Control 0/B <1.0 

Control 72/A <1.0 --- --- Control 72/B <1.0 

1.0 0/A 0.9755 0.9755 97.6 

1.0 0/B 0.9755 

1.0 72/A 0.8824 0.8824 88.2 

1.0 72/B 0.8824 

5.0 0/A 4.973 4.973 99.5 

5.0 0/B 4.973 

5.0 72/A 4.921 4.921 98.4 

5.0 72/B 4.921 

50 0/A 50.03 50.12 100.2 

50 0/B 50.21 

50 72/A 52.65 52.92 105.9 

50 72/B 53.20 

NB : Samples in duplicate 

188 ON SNI\flN08 

11 

48