# •

• TITLE PAGE Study Title: ACUTE TOXICITY OF DIAESUL BLACK ROT LIQUID TO SCENEDESMUS SUBSPICATUS ;**~** ·- -... -·- i. ·:,' •. I o:



### (OECD - Algae Growth Inhibition Test; including determination ofthe influence ofthe light absorption ofthe substance on the growth inhibition) Data Requirement:

OECD No. 201 Author; Mrs. 

### V. Wuthrich Study Completion Date:

November 18, 1993 

### Perfonning 

### Iahoratory: A C C UMWELTCHEMIE AG P.O. 

### Box CH -

### 4452 ltingen/BL Switzer1and Study Project No. : ACC PROJECT 333977 

### Page 1 

### of48



# RCC CONTAINS NO CBI

--------------------

GroupRCC 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 

### Natio

### nal 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 4e051II 

### 2

# •• RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID GOOD LABORATORY PRACTICE


### STATEMENT OF COMPLIANCE
PROJECT NUMBER: TESTARTICLE: 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 ofthe influence ofthe light absorption ofthe substance on the growth inhibition) This studywas conducted in compliance with Good Laboratory Practice Regulations and meets the requirements as listed underGLP 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 ofthe influence ofthe light absorption ofthe substance on the growth inhibition) Study procedures were periodically inspected, the protocol and this report were audited by the Quality Assurance Unit. The datesare given below. 

### Dates ofQAU Inspections I 

### Dates of Reports tothe 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 PROJECT333977 DIRESUL BLACK ROT LIQUID 

# CONTENTS Page TITLE PAGE
................................................................................................................... 

### 1 GLP COMPLIANCE STATEMENT
.................................................................................2 STATEMENTOF COMPLIANCE
...................................................................................3 PROJECT STAFF
..........................................................................................................4 QUALITYASSURANCE UNIT
........................................................................................

### 5 CONTENTS
.................................................................................................................6-9 GENERAL INFORMATION
.......................................................................................... 10 GOOD LABORATORY PRACTICE
.............................................................................. 

### 11 GUIDELINES
................................................................................................................ 11 SUMMARYOF PROTOCOLAMENDMENTS
.............................................................. 

### 11 SUMMARY
................................................................................................................... 12 

# -1 INTRODUCTION
................................................................................................. 13 2 MATERIALS AND METHODS
............................................................................. 13 2.1 DEFINITIONS
...................................................................................................... 13 2.2 TESTSYSTEM 

### 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 oftheTest
............................................................................... 19 2.5.4 Concentration ofTest Article
.................................................................... 19 2.5.5 Growth lnhibiton bythe 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 100OF 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 OFTHE TEST
...............................................................................22 3.6 RESULTS OF ANALYSES
.................................................................................22 

### 6RCC 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 PROJECT333977 DIRESUL BLACK ROT LIQUID 

# CONTENT CONT'D. -TABLES
Table 1A: Experimental part A (test .1- 3) Influence of DIRESUL BLACK ROT LIQUID on algae growth after 

### Page

72 hours ofexposure...............................................................................24 Table 1B: Experimental part B (test 3) Influence of DIRESUL BLACK ROT LIQUID on algae growth after 72 hours ofexposure...............................................................................27 Table 2A: Influence ofthe test substance on algae growth: growth rate 

### 1J (1/ day) and percentage inhibition of 1.1 in experimental partA.
.............................28 Table 2B: Influence ofthetest substance on algae growth: growth rate 

### 1J (1/ day) and percentage inhibition of 1.1 in experimental part B
..............................29 Table 3: pH ofthe algae suspension for all concentrations of DIRESUL BLACK ROT LIQUID ofexperimental partA.
........................................................30 Table 4: Influence of POTASSIUM DICHROMATE on algae growth after 72 hours ofexposure...............................................................................31 

### 8

# Fe 

# 4.

v;__ RCC PROJECT333977 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 ofScenedesmus subspicatus in experimental part A (test 1) incubated for 72 hours with differentconcentrations of DIRESUL BLACK RDT LIQUID
................................................................33 Figure 3: Growth curves ofScenedesmus subspicatus in experimental part A (test 2) incubated for 72 hours with different concentrations of DIRESUL BLACK RDT LIQUID................................................................ 34 Figure 4: Growth curves ofScenedesmus subspicatus in experimental part A (test 3) incubated for 72 hours with differentconcentrations of DIRESULBLACK RDT LIQUID
.................................................................35 Figure 5: Growth curves ofScenedesmus subspicatus in experimental part B incubated for 72 hours with different concentrations of DIRESUL BLACK RDT LIQUID
................................................................36 9RCC 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 ofthe influence ofthe light absorption ofthe substance onthe 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
November09, 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 10years. 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 furtherconsentRCC PROJECT 333977 DIRESUL BLACK ROT LIQUID 

# GOOD LABORATORY PRACTICE

The study will be performed in compliance with: "The OECD Principles ofGood 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 ofJuly 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 ofthe 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 

### ITest Apparatus/Medium Second Amendmentto Protocol: 1) TITLE 2) IntroductionRCC 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 ofthe test substance on the algae cells ordue to an indirect effect, a reduced algae growth by light absorption in coloured test solutions. The test included two experimental parts: Experimental partA: the algae grew in test medium with dissolved dyestuff in Erlenmeyerflasks. Experimental part B: the algae grew in separate containers (test tube) which were surrounded by the blackcoloured solutions ofthe 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 byseveral 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 PROJECT333977**~** 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 ofthe algae caused by real toxic effects ofthe dyestuffs or caused by an indirect effect, the light absorption bythe coloured test solution. The purpose ofthis modified test is to quantifythe algicidal effect ofthetested dyestuffversus the growth inhibition effect dueto reduced light intensities in the coloured test solutions. Exponentially growing cultures of the unicellular green algae Scenedesmus subspjcatus were exposed to various concentrations ofthe test substance in a modified test system. The inhibition ofgrowth in relation to control cultures was determined overa test period of72 hours.


# 2 MATERIALS AND METHODS 2.1 DEFINITIONS Cell density: The numberofcells/ml Growth:


### E,,Cso: The increase in cell density overthe test period. The concentration oftest substance which results in a 50% reduction in growth relative to the control.


## $

# l• NOEC (No Observed Effect Concentration): The concentration oftest substance which results in a 50 

### % reduction in growth rate. The highesttested concentration atwhich no significant inhibition of growth isobserved relative to the control. LOEC (Lowest Observed Effect Concentration): The lowesttested concentration atwhich 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 ofthe test article on the growth rate ofthe green alga Scenedesmys sybspicatys.

# l l 

### i RCC PROJECT333977 DIRESUL BLACK ROT LIQUID 

## 2.3 CHEMICALS 2.3.1 Test Article Data oftest 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 CompoundWater: stable for at least 48 hours Stable for at least 72 hours (determined by RCC underthe 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 ofthe 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 intest solution Stocksolution 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 Stocksolution 2 FeCI3 • 6H20 0.08 mg/1 NaEDTA · 2H20 0.1 mg/1 Stocksolution 3 H3B03 0.185 mg/1 MnCI2 •4H20 0.415 mg/1 Stocksolution 4 ZnCI2 3 x 10-3 mg/1 GoCI2 •6H20 1.5x 10-3 mg/1 Na2 MoO4 •2H20 7 x 10-3 mg/1 Stocksolution 5 CuCi2 • 2H20 10-s mg/1 Stocksolution 6 NaHC03 50 mg/1 The pH ofthe medium afterequilibrium 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 ofapproximately 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 ofthe test article in thetest medium withoutthe addition ofalgae 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 Erlenmeyerflasks 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 ofthe 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 untreatedcontrol. 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 over72 hours using the nominal concentrations 100, 1 0, 1and 0.1 mg/1 and an untreated control. The concentrations were prepared using the medium described in Directive 92169 EEC ofJuly 31, 1992. The test article was found to be stable using thistest 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, 25and 

### 12.5 mg/1. 

## i Main test 

### 2: 

## I 

# (Jl

# e The stock solution and dilutionswere 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 untreatedtest samples were incubated five fold. In experimental part B, DIRESUL BLACK ROT LIQUID containing treated and untreated test samples were incubated in duplicate. Referencetest: 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 ofGrowth Inhibition Numberofcells: 18

The number ofalgae after 24 hours was estimated by means of a microscope using a Neubauer chamberaccording tothe equation: Number

### ofcells counted 

### x 2.5 

### x10§ (dilution factor) (concentration factor) Area underthe growth curves (AUC): (N1 - No) (N1 

### + N2) - 2No area 

## = --------------- xt1 

### + ------------------------ x (t2 - t1)
... 

### 2 2 (Nn-1 

### + Nn) - 2No 

### + ---------------------------- 

### x(tn - tn-1) 

### 2 where is: No N1 N2 Nn 

## = = = = number ofcells/ml atthe start numberofcells/ml aftert1 

## =24 hours, number of cells/ml after

### t2

## =48 hours, numberofcells/ml aftertn. 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 

# IIRCC PROJECT 333977 DIRESUL BLACK ROT LIQUID Inhibition of Growth Rate 

### (lu): 

### IJJ 

## =------------ iJC where is: 

## = mean growth rate ofcontrol 

## = mean growth rate oftest concentration


### 2.5.2 pH 19

The pH was determined for all concentrations ofthetestarticle and ofthe control atthe 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 ofthe 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 ofTest Article 

# f Foranalytical purposes, the following samples were taken: Test 1: Duplicate 20 ml samples ofall concentrations and the control, atthe beginning and atthe end of the test. Test 2 and 3: Quadruplicate 20 ml samples ofall concentrations and the control, atthe beginning and atthe end ofthe test. All sampeswhich were not immediately analysed were stored at -20

# oc.
2.5.5 Growth lnhlblton bythe 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 100 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 

### 1A, 2A, 

### 1B and 28. In test 

### 1 and 2, {experimental part A was applied only) all test concentrations were statistically significant inhibited by thetestarticle. 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 matterwhetherthey 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 differenceswere observed between both experimental parts.

# ' RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID 21
The following results were obtained after 72 hours ofexposure (determined bythe area underthe 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

### +0.367x Log (dose) 95 % Confidence limits ofslope 0.365- 0.368 mg/1 NOEC- value 1.0 mg/1 LOEC -value between 1and 5 mg/1 linear regression ofgrowth inhibition versustest 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 after72 hours ofexposure 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 

### + 1.460x Log (dose) 1.455 - 1.466 mg/1 1-5mg/l about 5 mg/1



### y = 52.826x 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 forthe standard potassium dichromate are presented in table 4. They are archived underthe 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 limitsforthe slope were: 

### 2.085
- 2.120



## 3.4 pH OF ALGAE SUSPENSION The pH ofthe algae suspensions ofexperimental part A is presented in table 3. Atthe start ofthe tests, the pH ranged from 

### 8.1 to

### 9.5. At the end ofthe tests, the pH ranged from 

### 8.1 to 

### 8.9.



## ' 3.5 VALIDATION OFTHE TEST 

# l.l/; •. ------------------**,~···--.** '..'J ·r, The tests are regarded to be valid since the cell concentration in the control cultures increased by a factorof

### 25.4 (test 

### 1 ), 120 (test

### 2) and 

### 30.6 (test 

### 3) within three days in experimental part A and by 

### 50 (test3) in experimental part B.



## 3.6 RESULTS OF ANALYSES The results ofthe 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 

### 85.5o/o to 

### 107.7%ofthe 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 1and 5 mg/1 EbC 50: 1.84 mg/1 with the 95 % confidence limits of 1.37 - 2.33 mg/1 54.1 mg/1 with the 95 % confidence limits 41.7 - 74.7 mg/1 EIJC50:

Experimental part B NOEC: 1 -5 mg/1 about 5 mg/1 LOEC: EbC 50: 5.99 mg/1 with the 95%confidence limits of 5.58-6.44 mg/1 8.59 mg/1 with the 95% confidence limits 7.24- 10.9 mg/1 EiJC50: 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 uptoa concentration of50 mg/1.



### (1)
(2) 

# REFERENCES Cox, D.R.: Analysis of binary data. Methuen & Co. Ltd., 1977. Miller, R.G.: Simultaneous Statistical Inference SpringerVerlag, NewYork (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 after72 hours of exposure. Numberofcells are divided by 10'000. Area underthe curve (AUC) and response (Resp.) are divided by 10'000 and corrected or NO= 10'000. Significance (Sign) estimated with Dunnett-test: (* = 1

### %). Concentration Numberofcells 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**

## AUfilst

## 2l Number ofcells 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 Numberofcells 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 1B. Experimental part B (jest3) 1 Influence of DIRESUL BLACK ROT LIQUID on algae growth after72 hours of
,,\ exposure. Numberof cells are divided by 1 0'000. Area underthe curve (AUC) and 

## l response (Resp.) are divided by 10'000and corrected orNO= 10'000. Significance 

# I (Sign) estimated with Dunnett-test: (* = 1

### %). 

### l 

# J J ;·'i 

# ! 

# 1 Concentration Numberofcells 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 ofthetest substance on algae growth: growth rate 1.1 (1/day) and percentage inhibition of 1.1 in experimental part A TestArticle 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 28RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID Table 28: Influence ofthe 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



# •• 29RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID 30
Table 

### 3: pH ofthe algae suspension forall concentrations of DIRESUL BLACK ROT LIQUID of experimental partA.

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 after72 hours ofexposure. Numberofcells are divided by 10'000. Area underthe curve (AUC) and response (Resp.) are divided by 10'000 and corrected or NO= 10'000. Significaoce (Sign) estimated with Dunnett-test: (*

## =1

### %). Concentration Numberofcells 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 onthe growth of Scenedesmus sybspicatus after 72 hours ofexposure.
%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 Estillation + + + Part B.Observed values

,._ -- -x- - - ... Part B.Ln.reo of observed values 

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

### moll 32RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID 33 Figure 2: Growth curves ofScenedesmus 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.rsRCC PROJECT 333977 DIRESUL BLACK ROT LIQUID 34 Figure 3: Growth curves of Scenedesmus subspicatus in experimental part A (test 2) incubated for 72 hourswith 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 ofScenedesmus subspicatus in experimental partA (test 3) incubated for 72 hourswith 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 ofScenedesmus subspicatus in experimental 

### part B incubated for 72 hourswith 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 hoursRCC PROJECT 333977 DIRESUL BLACK ROT LIQUID



# t ATIACHMENT 1



# •• DETERMINATION OF THE CONCENTRATIONS OF DIRESUL BLACK ROT LIQUID IN TEST MEDIUM 37

# RCC UMWELTCHEMIE AG

PROJECT333977

DETERMINATION OFTHE CONCENTRATIONS OF DIRESUL BLACK ROT LIQUID IN TEST MEDIUM
ACUTE TOXICITYTO SCENEDESMUSSUBSPICATUS (OECD-Aigae Growth Inhibition Test; including determination ofthe influence ofthe lightabsorption 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 ProjectStaff
..........................................................................................................3 Archiving
..............................................................................................................3
INTRODUCTION
..................................................................................................4 2 MATERIALSAND METHODS
...............................................................................4 

### 2.1 TestArticle
............................................................................................................4 

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

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

### 2.3.1 Analysis ofSamples
.............................................................................................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
TestArticle Testing Facility

PROJECTSTAFF ResponsibleforAnalytics

ARCHIVING 3339n SANDOZAG Dr. R. Mislin Rothausstrasse 61 CH-4132 Muttenz Mr. E. Rudin Registration & Consulting Company Ltd. DIRESUL BLACK ROT LIQUID RCC UMWELTCHEMIEAG DepartmentofChemistry CH-4452 ltingen 

### 1Switzerland

K. Biedermann



# ..**~**

# .......if.._.f ..**~-4~~~** Date:

### Od~**~** 

## / --79'7'3 /ace

RCC, CH-4452 ltingen Raw data, protocol, report and copy of reportofthe present study will be archived for at leastten 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 oftest 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 least48 hours DIRESUL BLACK ROT LIQUID was found to be stable in the test medium overa period of72 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 oftest 2 and 3 were worked-up and quantified by spectrophotometry on the day ofsampling. Reserve samples were stored deepfrozen at-20

# oc until completion ofthe 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 afolded filter1
, aliquots were withdrawn from the 20 ml sample volume ofthe control, 1.0mg/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 ofthe 200 mg/1 stability test samples, and directly filled into 1em quartz cuvettes. The absorbance ofthese aqueous solutions was measured at580 nm versus bidistilled waterorversus 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 forcalibration ofthe spectrophotometer.

2.3.3 Spectrophotometric Determination (Example of Operating Conditions) Apparatus: Perkin-Elmer Lambda 15 UVNisspectrophotometer Wavelength: 580nm Quartz cuvette: 1em (pathlength)

1

Only samples oftest2and 3, collected after72hours, were filtered over afolded 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 latterwere 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 = Concentrationof 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 ofsample (20 ml)RCC PROJECT 

### 3339n DIRESUL BLACK ROT LIQUID 

# 3 RESULTS 7

The results obtained forthe concentrations of DIRESUL BLACK ROT LIQUID in the test medium oftests 1, 2and 

### 3are listed in Tables 2, 3and 4, respectively. The tabulated values represent rounded-off results obtained by calculations based on the exact rawdata. For this method of spectrophotometric analysis, the detection limit was calculated to be 1.0 mg DIRESUL BLACK ROT LIQUID per litertest medium. An example of acalibration curve is listed in Table 1.

# ' RCC PROJECT 333977 DIRESUL BLACK ROT LIQUID

Table 1: Example ofacalibration 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 PROJECT333977 DIRESUL BLACK ROT LIQUID

Table2: Results obtained forthe concentrations of DIRESUL BLACK ROT LIQUID in the test medium oftest 1. (Dates ofanalysis: 02/05-FEB-1993)
Nominal Time Actual Mean concentration of ln%of concentration concentration of samples Aand B nominal the testarticle (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 * :Stabilitytest (test medium without algae) 9



# 46RCC PROJECT333977 DIRESUL BLACK ROT LIQUID

Table 3: Results obtained forthe concentrations of DIRESUL BLACK ROT LIQUID in the test medium oftest2. (Dates ofanalysis: 22/25-FEB-1993)

Nominal Time Actual Meanconcentration of ln%of concentration concentration of samplesAand B nominal the testarticle (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 PROJECT333977 DIRESUL BLACK ROT LIQUID

Table 4: Results obtained forthe concentrations of DIRESUL BLACK ROT LIQUID in thetest medium oftest3. (Dates ofanalysis: 22/25-MAR-1993)
Nominal Time Actual Meanconcentration of ln%of concentration concentration of samples Aand B nominal the testarticle (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