SPONSOR 

STUDY TITLE 

Acute Toxicity Screen of Tinuvin 900 to 

Scenedesmus subspicatus 

AUTHORS 

STUDY COMPLETION DATE 

September 28, 1993 

PERFORMING LABORATORY 

' PROJECT ID 

Final Report

Page 1 of 67 

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

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

Every other or additional use, exploitation, reproduction, publication or submission to other parties requires the written 

permission of the sponsor, except as otherwise required by applicable law (e.g., TSCA). The use of these studies/

documents for regulatory, registration or other commercial purposes [including but not limited to Registration, 

Evaluation, Authorization and Restriction of Chemical Substances (REACH)], without the express written permission of 

the sponsor is unauthorized and strictly prohibited.
STUDY NONCOMPUANCE STATEMENT 

Study Noncompliance Statement for Final Report entitled "Acute 

Toxicity Screen of Tinuvin 900 to Scenedesmus subspicatus" for

In accordance with intent that aU aquatic toxicity tests conducted by our 

facility follow good laboratory practices, study director for the above test 

herein confirms that the study was conducted in the spirit of Good Laboratory Practice 

standards, and the final report received a Limited review by Quality Assurance Unit. 

However, the in-life portion of the study was not audited by Quality 

Assurance Unit. The data are intended for the study sponsor's internal use only and are not 

suitable for any regulatory submission. No analytical confirmation of test concentrations was 

done nor was the stability of the test substance under test conditions investigated. 

AU original raw data were provided to with the final report and a copy was 

retained at

^'^B-?3 

Date 

 Study Director 
OUALITY ASSURANCE STATEMENT 

Quality Assurance Unit reviewed Study "Acute Toxicity Screen of 

Tinuvin 900 to Scenedesmus subspicatus," for . 

The following audit was conducted on this study. 

Date Reported to Date Reported to 

Date of Inspection Phase Inspected Study Director Management 

09-21-93 Fmal Report and 09-21-93 09-22-93 

Raw Data 

The undersigned conducted the final report audit. The audit indicates that the report is an 

accurate reflection of the study as it was conducted by

Date 

Quality Assurance Officer n 
SIGNATURE PAGE 

Submitted by: 

Prepared by: 

Approved by: 

:nab 

^-;iS'f5 

Date 

9~ajy) 

Date 

yyy/9j 

Date 

^A?A. '1 

Date 
TABLE OF CONTENTS 

Page 

SUMMARY 6 

METHODS AND MATERIALS 7 

I. Test Species 7 

n. Test Substance 7 

m. Static Test Procedure 8 

IV. Statistical Analysis 8 

RESULTS 9 

REFERENCES 10 

APPENDICES - RAW DATA 11 

PROJECT PERSONNEL 12 

APPENDIX I - BIOLOGICAL RAW DATA 13 

APPENDIX n - STATISTICAL ANALYSIS DATA 28 

A. Student r-test Data 29 

B. ANOVA and Dunnett's Multiple Means Test Data 39 

APPENDIX m - CORRESPONDENCE AND PROTOCOL 53 
SUMMARY 

Subject: "Acute Toxicity Screen of Tinuvin 900 to Scenedesmus subspicatus,"

Final Report

Sponsor:

Testing Facility:

Location of the Original Raw Data and Final Report: 

Test Substance: Tinuvin 900 

Nominal Test Concentrations: Control (0.0), Vehicle Blank (DMF), 1.0, 10, and 100 mg/L 

Dilution Media: 7.4 (pH) 

Experimental Test Dates: Initiation — September 3, 1993 

Termination — September 6, 1993 

Length of Study: 72 hours 

Results: 72-hour EC50 = > 100 mg/L (95% confidence limits could not be determined) 

72-hour no-observed effect concentration was 100 mg/L 

Test Species: Scenedesmus subspicatus 

Source of Organisms: Department of Botany, Culture Collection of Algae, University of Texas 

at Austin 

Age of Culture at Study Initiation: 3 days old 
METHODS AND MATERIALS 

The study was performed following the procedures outlined in as 

approved by on February 25, 1993, and the study 

director on March 10, 1993. 

I. Test Species 

The parent stock of Scenedesmus subspicatus (supplier batch #UTEX-2594,

culture 93-T-Sc) used in the definitive test was obtained from the 

Department of Botany, Culture Collection of Algae, University of Texas at 

Austin, Austin, Texas, on August 26, 1993. The parent stock of algae was 

received growing on an agar slant contained in a 50-mL culture tube. The algae 

culture was identified as Scenedesmus subspicatus on the culture tube label. 

Scrapings were taken from the slant and placed in 250-mL Erlenmeyer flasks 

containing 100 mL of sterile algae nutrient media. These flasks were incubated 

in an environmental chamber at 24 + 2°C to facihtate algae growth. 

Periodically, new Scenedesmus subspicatus cultures were initiated using a lot of 

the parent stock or cloned from an existing culture derived from the parent stock 

in 100 mL of sterile culture medium. Cultures of Scenedesmus subspicatus at 

ABC Laboratories were maintained under the same environmental test conditions 

as for the definitive test. The algal culture used for this toxicity test was 3 days 

old at test initiation. 

n . Test Substance 

The Tinuvin 900 test substance (batch #EN 306296.12) was received from

 February 10, 1993 as a white powder. The compound was assigned 

 reference #TS-6313 and was stored at room temperature. 

Purity of the test substance was reported to be > 99 % and the test concentrations 

were prepared based on total product of Tinuvin 900. 

For definitive testing, 1.0, 10, and 100 mg/L working standards were prepared 

by quantitatively transferring 0.0005, 0.0050, and 0.0500 g, respectively, of the 

test substance into 500-mL volumetric flasks, and each was brought to volume 

with sterile nutrient media. After each working standard was bought to volume, 

0.050 mL of DMF was injected into each. Each working standard was then 

sonicated for 30 minutes and then magnetically stirred for 30 minutes. The 

vehicle blank was prepared by injecting a 0.010-mL aliquot of DMF into each 

vehicle blank replicate containing 100 mL of algal nutrient media. After 

preparation of test solutions, the 1.0 mg/L solution was clear, the 10 mg/L 

solution was clear with a precipitate, and the 100 mg/L solution was cloudy with 

a precipitate. The sterile algal nutrient medium was prepared as described in the 

protocol. 
m. Static Test Procedure 

The static Scenedesmus subspicatus toxicity screen was conducted in 250-mL 

Erlenmeyer flasks containing 100 mL of algae nutrient media or test solution. 

Each test level and the controls were prepared in triplicate and received 1.0 mL 

of algal inoculum containing approximately 1.0 x 10* cells/mL, resulting in 

approximately 1.0 x 10'^ cells/mL for each flask. Initial cell counts of the 

control and vehicle blank replicates resulted in an actual mean cell count of 9.3 

X 10^ and 8.5 x 10^ ceUs/mL, respectively. The water quality parameters of 

temperature and pH were measured in each test solution at test initiation. The 

temperature for all test solutions was 23°C and the pH ranged from 7.4 to 7.6. 

After preparation, the test vessels were positioned randomly, using a computergenerated random table, and incubated for 72 hours at 24 + 2°C under 

continuous cool-white fluorescent lighting and constant rotary agitation. Light 

intensity was maintained at 800 + 10% ft-c (approximately 8600 lux) and the 

agitation rate was 100 rpm. Temperature, light intensity, and oscillation rate were 

monitored throughout the study. 

Algal cell counts were conducted at 0 hour in the control and vehicle blank 

replicates and in all test chambers after 72 hours. The algal cell counts were accomplished using a hemacytometer and an Olympus Model BH-2 microscope. 

The hemacytometer has two chambers each with nine squares, 1 mm on a side. 

The average number of cells per 1 mm^ was designated as Q. The center square 

was subdivided into twenty-five 0.20-mm squares. The average number of cells 

per 0.04 mm^ was designated as R. The cell density (d) for a given suspension 

of algal cells could be calculated from either of the following equations: 

A. d(cells/mL) = IO'* x Q (average number of cells/1 mm^) 

B. d(cells/mL) = 10* x V4R (average number of cells/0.04 mm^) 

In general, equation B was only used with very dense cell populations such as 

those encountered with a 4- to 7-day-oId algal culture typically used to prepare 

an algal test inoculum. Algal cell counts during the definitive study used equation 

A. When the average number of ceUs per I-mm square was less than 11 algal 

cells, all nine 1-mm squares were counted and divided by 9 to obtain the average 

number of cells per 1-mm square (Q) or the replicate mean value. For aU 

suspensions with at least 11 ceUs per 1-mm square, the four comer 1-mm squares 

could be counted and averaged to obtain Q (repUcate mean value). The cells per 

milliUters for each repUcate were calculated and recorded. 

IV. Statistical Analysis 

The SAS program (1) prints out the raw data, plots, charts; computes the mean 

number of ceUs by treatment and hour; and tests for significant differences from 

the control and vehicle blank mean at each timepoint. 
A Student f-test was conducted to determine if the control and vehicle blank ceU 

count values were significantly different {p < 0.05) from each other. The test 

indicated that there was not a significant difference after 72 hours. The control 

and vehicle blank values were then pooled for aU further statistical analyses. 

A one-way analysis of variance (using PROC GLM in SAS) was conducted for 

each timepoint with a Dunnett's comparison to the pooled controls. A one-tailed 

Dunnett's test was conducted at the 0.05 level of significance with the alternate 

hypothesis being that the mean number of ceUs has been reduced in comparison 

to the pooled controls mean. Prior to the Dunnett's test, a Levene's test (2) was 

conducted to test for homogeneity of variance over treatments at each hour. If the 

p value from the Levene's test is greater than 0.01, indicating insignificant 

heterogeneity, then the analysis is performed on the ceU counts divided by 10"*. 

If the p value is less than 0.01 for some hour(s), then the cell counts for each 

repUcate are transformed using the square root of the cell count, the 

recommended transformation for count data (3). 

RESULTS 

A 72-hour screen with Scenedesmus subspicatus exposed to Tinuvin 900 was conducted at test 

concentrations of 1.0, 10, and 100 mg/L along with a control and vehicle blank (DMF). Test 

concentrations were based on the solubiUty of Tinuvin 900 in algae nutrient media. Logarithmic 

phase growth was confirmed at 72 hours with a mean count of 6.9 x 10^ and 

6.8 X 10^ ceUs/mL in the control and vehicle blank repUcates, respectively, that were 74- and 

80-fold increases, respectively, from the initial 9.3 x 10^ and 8.5 x 10^ ceUs/mL. After 

72 hours, algal ceU counts for the I.0-, 10-, and 100-mg/L test levels were 104, 94, and 94% 

of the pooled control population, respectively. A precipitate was observed in the 10 and 100 

mg/L test repUcates at 72 hours. 

A 72-hour EC50 for Scenedesmus subspicatus exposed to Tinuvin 900 was estimated to be 

>10 mg/L since 50% inhibition was not observed in any of the test concentrations. The 

72-hour no-observed effect concentration was 100 mg/L. 
REFERENCES 

(1) PC DOS SAS/STAT Release 6.04 Copyright 1985, 1987, 1988 by SAS Institute Inc., 

Cary, North CaroUna, 27512-8000 USA. 

(2) MilUken, G.A. and D.E. Johnson. Analysis of Messy Data, 1984, Vol. 1, p. 22. 

(3) Zar, J.H. Biostatistical Analysis, 1984, 2nd edition, p. 241. 

ABC LABS #40858-10 
APPENDICES — RAW DATA 

NOTE: Some of the records that appear in this raw data appendix have been provided as 

exact copies of original records on fUe at . This has been done 

by necessity for certain data that are used commonly in several studies at

 Such records may include analytical standard solution preparation 

sheets, organism culture logs, and hard blended water chemical screen results. 

The foUowing symbols may appear on the raw data pages included in this report. 

C Calculation error R Recording enor 

E Entry error F Form change 

S SpelUng enor T Transcription enor 

 PgOn I I 
PROJECT PERSONNEL 

Name Title 

 Biologist n/Study Director 

 Biological Technician n 

Biologist n 

 Biologist I 

pgOO I 2 
APPENDIX I - BIOLOGICAL RAW DATA 

> 

 pgOO I 3 
Test Material: 

Study Director: _ 

Study 

Day: O'^ovr" 

Concentration 

(Units: la^ L ) 

CONTROL A 

B 

CW C 

> /I 1 1 _/ ^ 

^Jhfi^k 3 

QW c 

A 

/ A B 

Clr,f C 

A 

10 ^ 1 ^ cir y C 

A 

|0t> B 

CM.P c 

A 

B 

c 

A 

B 

c 

Remarks: Counts w 

Hemacytometer 

di 

Reviewed By:

ALGA 

y.:na^J 

Time/Date/ 

Total 

(9Q) 

iO 

^ 

U 

10 

i l 

7 

ith Olympus 

'y-2t-^^ ^ 

LCELL 

or^ 7£) 

Cell C 

'l.D.: HIn 

Q l 

BH-2 Mic 

e^r 

P; 

COUNT, 

0 

ounts 

5^pm/^' 

dividual 

cells/rr 

Q2 

roscope 

' SollKTlOT-

' Pfe^ipU 

S (Q SO 

Protoc( 

Study 

--5'^3/5^ 

Q Coun 

iL/IO"^ 

Q3 

 Mat 

l i clear- . 

O.K SiH 

UARES] 

Dl No.:

No.:

^ [ 

ts 

0 4 

• 

erial #16: 

fK-f9-3-fJ 

9'3'^3 

Calculat 

)ate/I.D.: SL 

Replicate 

Mean 

(cells/mL 

/104) 

I.II 

I.OO 

O.G?7 

I.II 

afai 

o.7e 

3-384) and 

; 

Date: ^ 

ons 

8-^5/404 

Cone. 

Mean 

(cells/mL 

/lO^) 

0.^3 

0.06 

•-»• . 

P^^^ l 

pg 0 0 \ ^ 
Test Material: 

Study Director: _ 

Study 

Day: ~77--h'>^»r 

Concentration 

(Units: rv^^- ) 

CONTROL A 

B 

C 

1 f% t t t - l/r. _ . 

0- 5l6A g 

c 

A 

1.0 B 

c 

A 

10 B 

^ C 

A 

//)0 . B 

f C 

A 

B 

C 

A 

B 

C 

Remarks: Counts w 

Hemacytometer Pr 

/7y eicH 9-t!,'9i 

Reviewed Bv: 

ALGA 

i n U'J 

Time/Datey 

Total 

(9Q) 

ith Olympus 

PrecifUAK S 

LCELL 

>Y^ 9<9( 

Cell C 

'l.D.: ^ 

in 

BH-2 Mic 

•"ir' 

COUNT. 

3 

ounts 

-'^^^'./^ 

dividual 

cells/n 

roscope 

S (OSO 

Protoc( 

Study 

-f^^hc 

f 

QCoun 

iL/IO"^ 

 Mat 

UARES] 

Dl No.:.

Mo.: 

/M [ 

ts 

erial #16. 

Calculat 

)ate/I.D.: ^ 

Replicate 

Mean 

(cells/mL 

/104) 

^g.GO 

7/. 7.6 

(p0.5^ 

no.00 

b5iD0 

(Q^60 

b&.ns 

bn.n5 

7f?.75 

(oD.OO 

b6i26 

n0iZ6 

6JI,Q.S 

nitoo 

bioo 

3-384) and 

"*> 

Date: *? 

ons 

M3/^C ^ 

Cone. 

Mean 

(cells/mL 

/10^) 

^ ^ 

68 

nx 

bS 

b<h 

' M 7 

pg 0 0 1 5 
Tes t M< 

Study C 

Concen. 

te&k' 

Co/jfrJ 

\}.6lQl0k 

ItO 

10 

iod 

a 

" YSI Mo 

• pH E 

C 

Remarks: C 

Reviewed 

aterial: 

)irecto 

'6LI4 s ; 

0 

Temp." 

»c 

4^ 

9 1 

Si-i 

r?^ 

^--h 

}^c 

del 64 A 

fCornina 

] 

^ £ SCM 

bv: 

r: 

'ifrri 

- Hour ( 

DO" 

mg/L 

«¥? 

' ^ 

— 

^ thern 

RC Olssc 

140 ph 

?-?'93 

T. 

F T ^ 

Sbserv 

PH° 

7.Y 

zV 

7Yr? 

7(ff 

7. ^ 

lomet 

)lved ( 

l/mVr 

nuvifN ^ 

5 

ation 

flrzp 

ft 

fth 

f 

*- . 

er 

)xygen Meter, 

neter, ma 

pH pen, W 

?t /%f ^ 

oo 

Temp." 

»C 

mat 

terial con 

odel 

r J C-^ 

- Hour ( 

DO' 

mg/L 

erial cor 

trol *17 

*i faft / 

Wi 

^bserv 

pH" 

trol n 

14-17 

^i < 

ATER QUA 

Protocol N 

Study #: _ 

ation 

1905-485 

5 and Fisher 1 

ff/ukzSfyy ..rj-H 

LLITY 

0 . :

Temp." 

"C 

3-620-28 

f'1'9 

- Hour 0 

DO' 

mg/L 

7 Electro 

> 

bserv 

pH° 

de 

ation 

Temp." 

"C 

Hour O 

DO' 

mg/L 

Date:_ 

bserv 

pH' 

7-

ation 

9-^-5 

C3-
1 

Test Ma 

1 Study D 

Date 

9-.=^'9^ 

"1-^-93 

I'^-iJ 

q-<a-^i 

^ LI-CC 

#J^0 

^ Merc 

"" Oscill 

Mate 

Reviewe 

/) 

terial: 

irector:

Light^ 

Reading 

(LUX) 

eeS3 

^"/^o 

^IZ S 

8(o3^ 

R Model Ll-ie 

%A^ ) and Ll 

-3MV 

jry thermome 

ation rate tak( 

rial#1900-00( 

d by:

iLGAE - ENVIRONM 

Ti-nUfUiXi ^h O 

Footcandles 

(LUX X 0.0929) 

7 9 ^ 

7^^? 

?-// 

^ oZ 

JaBT Quantum/Radio 

-COR Model Photon 

ter. 

3n directly from gat 

ENTAL C\ 

Protocol 

Study Nc 

Temp** 

°C 

SS 

ZS 

zr 

Z5 

meter/Phc 

netric Sen 

ige on En\ 

1AMBER 

No.

).

Oscillation*^ 

Rate 

(RPMs) 

/oo 

t o o 

/ CO 

/ o o 

>tometer (

sor (Serial »?\ 

/ironmental QX 

Date: 

Observations 

Si-tl 

3 ^ 

STV 

5 3 V 

Material 

1-4391-) 

lamber

h M S 

pgOOI I 
r ' ••• •• -

1 

Tes t Materi a 

ga^h 

fcrpt No. : EMJ 

^ 

Ct'ii>lw M n •

 Stud y Di» 

Balance : [ 

[ 

Balanc e chec l 

D.OIOO 

"~TCTiii"S"i; i 

0»iOOO 

TcTass S wi 

Weighed By : 

Gros s 

Weight 

<g) 

OMb^ 

O.om^D 

0.050D 

Remarks: ^W« i 

Prepare d wit ^ 

each contain ] 

control(s) : 

MSidii-JJ3.(ii . 

^B)c\ \ ^ZAiaicoAUe 

Form Preparec 

Checked By : 

Stud y Direct* 

^'REPflROTIOl 

D ' 

I : hAiu^iiii 

iPU2i(^^3. . 

 »

-ector : 

3'^rtorio i 

DOther : 

< wit h Cla< 

g + 

•77- ^ 

9 • . 

,. ) 

Tar e 

Weight 

(g) 

O.OCX>0 

OiOOO O 

COOO O 

ii>AiCkifiiH')- 3 

1 —ic^pkJ-

" 9 —LQ.Q_ . 

(describe ) 

F-.m±i:3i-. 

fjffoka^ ha J t 

i By :

> r:

s* OF TEST ( 

Veliminar j 

900 

Purity : 

iS R 300 S, 

iS S weight 

OiOOO ( 

TTiFi ) 

O'OOC 

<Tare) 

- f1 - — 

Net 

Weight 

(g) 

0.0005 

O'OOSO 

0-050(^ 

•^5 (Dio/ujlwi, 

.t___ repli c 

._nil o f _^ : 

J/^^LL-I^IL L 

lOS/nL bmP( 

:ONCENTRflTIOr 

.' n Def J 

>^°i %

Z

 Materi e 

. s : 

0 g = • 

.^.-__ 9 = 

Adjuste d ^ 

Wt. Corre d 

f o r Puri t 

<g) 

— 

nneliarw'.^k'i^rn.if 

•at e __^5^__« 

kLjkkko'^1 

.iid.injJiar^2i^ . 

On 

io^ 

'itAnHp^s ^ -i^jtit 

— . — 

.—. 

*B (Wf 

niti s 

l l «1S 

O 

—7F1 

0 

(f^i 

^ 

l e t 

ed 

y 

lUJt. $t* 

fil te : 

% ^ 

BiqjL f 

IlLbt 

ihiiiJifjK 

M l/iiZc 

>ight/volum e 

'e i^cretA. 

)05-475 

02_0O 5 

n i l w€. T 

^01^? 5 

n a l wf. ) 

Date : ?' ' 

Dilutio n 

Volume 

(1) 

O.Sb 

0.50 

0.60 

1 

i t ^essels/ c 

o-% als o pre i 

\r^reftj>^Jjj:^t. 

t3S3saXuiL. 

lOOrrtC etlyt- «• 

> eocJi. s-ctt 9-:JDate : __ f 

Date : ^ 

Date : _^ z 

») 

/ 

1 

1-^3 

(3 

Fina l 

Concentratio n 

(mg/l ) 

I.O® 

, o ^ 

mo® 

u clauJv w W a preilpi'Uk 

roncent ra t i on . 

3ared were 

dAi^n^QLfi'sLea^_nfik<uLlt 

.«J/rt. StJi 9-3-93 

?.? 

:3:li 

:/_ij_ 

/5rfj. 

PgOO I 8 
OBSERVATIONS AND/OR REMARKS FORM 

Test Material: ^iniuMini %0 Protocol No. '

Study Director: Study #:

fi'fi//^ 

'/» 

^'i/fak J $oycaHorZ> o f /^ / Sp/uA^OAJia-l B'^O o, 

%rfr7/)ijajtj SoAjiM-fiVAj o f /« / so/u^o/js ^^ H'''^/>>yi 

Af-/tr prf^''^^^^'^ ^^e /£>ync/l, SJU/'OA J '-^os c/&tK^ iht. 

Q) BrMiff*/ Si>/Ji^^f>f /naj^ridl ^ l02hS' 3 

NOTE: Individual eptt^ies mustbe elated, and initialed 

Reviewed by:. 

Study Director: 

Date: f-^ n 

Date: f/J-^-/ 

PiOfiil R 
OBSERVATIONS AND/OR REMARKS FORM 

Test Material: VARIOUS Protocol No. VARIOUS 

Study Director: VARIOUS Study #: VARIOUS 

09/02/93: ^ 

A visible solubility test was performed on Tinuvin 900 using 

the solvents^^cetonerDMF (Dimethylformamide), ancrfethanol. 

Three stock solutions were prepared by weighingn .OOOOg of 

Tinuvin 900 in 3 separate lOmL volumetric flasks. Each was 

brought to volume of lOmL with Acetone, DMF, and Ethanol 

respectively. All three volumetrics w/ 100 mg/mL solutions 

were sonicatecrin excess of 1/2 an hour. In the volumetrics 

containing Tinuvin 900 and Acetone, and Tinuvin 900 and Ethanol 

a large amount of the compound was not in solution. The volumetric 

containing Tinuvin 900 and DMF seem to dissolve a little better 

than the other solvents, but there was still a significant amount 

of compound left undissolved. 

Next a 50mg/mL solution of Tinuvin 900 was prepared by weighing 

0.5000g of test material in a lOmL volumetric and bringing it to 

volume with DMF. All compound dissolved, but when 0.1 OmL of the 

50mg/mL solution was injected in 500ml Dl water an excess of white 

precipitate was evident. Even after sonicating this lOmg/L solution 

the white precipitate was abundant. 

tS) f-vj-l i l 73/0/^ 3 * © BrAtiSt^TS S-i«,o -Ton.t^^o r 

/fljii^tji X atiifoi a lOjQ^-Hk^ 

NOTE: Individual entries must be dated and initialed. 

Reviewed by: Date: 9-/3-9^ " 

Study Director:. Date:_^i^3iM. 

 Pg002 0 
1 

Test 

Stuc 

1 Cult 

Mea 

• 

(a) 

• 

(b) 

Prep 

Revi 

Ma 

iy Di 

ure I 

A 

a 

(D, 

% 

CG 

(i>-

n R^ 

/ 

v 

Ha 

sp 

Ur 

01 

He 

ared 

ewei 

terial: 

rector: 

.ot No.: 

Igal cells/n 

stock 

jlls/mL = F 

R(.2 mm ! 

^ -

^ -

^ -

^ — 

^alue = — 

Final volun 

of test 

inoculum (n 

(Washed sti 

ceUd 

[ icelh 

— X 

— 

indbook of 

onsored bv 

jiversity Pr 

ympus BHsmacytome 

By:

[JBy:

T.' 

93 

^L 

cul 

^ 1 

squ 

-^ 

ne\ 

^L)] 

oak 

'ens 

ilml 

It 

X ] 

Ph 

' t h 

BSS 

2 V 

ter 

AL 

C 

ir»u-v 

- T 

n v\ 

turc 

/ 4 ) 

are 

-

-

-

-

X 

cub 

ity 

I) 

9,06 

[0« 

yco 

e P 

• P 

i/iicr 

.Q)B 

GAI 

] D 

n r i 

' S c 

/ash 

i 

< IC 

s)"" 

( FU 

Cell 

[(c t 

ture) 

1 

10 

X 

logi 

hyc< 

p. : 

osc 

U H 

. I N 

efin 

^< 

ed 

|6(al 

w l 

I De 

db/i 

X 

1. 

cal 

3log 

302 

ope 

9-3 

OCU 

itive 

DO 

Test) 

nsity 

'nL) j 

(volur 

pe 

\ 

X 

0 

Meth 

ical! 

-303 

•93 

LU 

^^^ 

— 

X 

ne 

r t 

— 

od 

3o( 

^ 

M PREPARA 

D Prelimi 

Protocol Nc 

Study No.: 

Alga 

Ce 

0 

^5 

/OH 

90 

8 ^ 

Mean Q Va 

( Test 

Solution 

[volume (mL) 

of inoculum\ 

est vessel 

(ml) j 

— 

s, 1973, edi 

:iety of Ame 

/laterial #16: 

TION 

nary 

>.:

1 cells/m 

Test in 

lls/mL = 

(1 mm s 

lue = 9< 

\ 

volur 

) _ stoc 

a 

ino 

Alok'-

_ uAi 

a 1 

ted Jane 

rica, Inc 

3-384) a 

Date: _ 

Date: _ 

\/sci-<f^(n. 

L in prepared 

oculum 

Q X 10*'-' 

quares) "" 

/09 

/?'; 

/03 . 

//3 

^ 

ne of washed 

k culture to 

dd to test 

culum {mL) 

D./itlioAJ "-f f^oiheJ efti 

^ o f /ueed St/t^t meofj 

loKc >^«f *« > >'"' 'i'i-^ i 

t R. Stein 

Cambridge 

nd 

4-3-^7 

1-SA-n 

pgOOZ I 
RANDOM GROUP ASSIGNMENT 1 PAGE 1 

TEST MATERIAL 

STUDY NUMBER 

TEST GROUP 

DATE 

VARIOUS 

09-01-1993 

n 

1 

2 

3 

4 

5 

6 

7 

a 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

27 

Group # 1 

ID 

40858 10 C 

40857 0. 10 B 

CONTROL A 

40857 0.10 C 

40858 1.0 B 

40857 1.0 C 

40858 10 A 

40857 VBA 

40857 VBC 

40858 VBB 

CONTROL C 

40857 1.0 B 

40858 100 A 

40858 1.0 C 

40857 10 B 

40857 VBB 

40857 10 A 

40857 0.10 A 

CONTROL B 

40858 100 C 

40858 VBA 

40857 10 C 

40858 1.0 A 

40858 10 B 

40858 100 B 

40857 1.0 A 

40858 VBC 

DATA ENTERED BYs

REVIEWED BY:

STUDY DIRECTOR: ^'/6-93 

pg0O^ 2 
COMPOUND RECEIPT 

Jcci3 

Compound / i nu^ i rj '^OO 

SUPPLIED INFORMATION 

Date Received: 0'^/jc>/^. 5 inggfld In By: K- h f ^ Ref # TS^^B^ S 

Storage 

Remarks 

: K^^ ^ "l^ryj p Total Weight: JO'O ^ 

Physical Description: tc^H C Z^^jj^ g 

SUPPLIER PROVIDED INFORMATION 

Phone: V<'-^//4');gVg 8 

Batch/Lot No. E^ ^ 30C>Z%>.I ^ CAS # AJA< ^ Purity: >99 ^ 

Amount Declared: " ^ "^^ ^ Expiration Date: ^'^/'^ ^ 

Storage Instructions / ^ ^ 

Other 0<^ /ai^^ / • "7 ^ i:i'^^ 3 

"ItS^ it an •xact copy of 

The original document" 

By date 9-B-n 

I pg0 0 23 
"snamysote liSfiigho erfT 

„,. sisb-.. 
Test Material:. 

Study Director: 

Date Prepared/ 

V liters of 

(1 mLA.) of the 

autoclaved

f withgrw JM H 

pH adjustment 

then stored in a 

Macronutrlent £ 

water 

Micronutrient Si 

O F SCH 1-3-'}^ 

Reviewed by: 

Study Director:

ALGAE NUTRIENT SOLUTIOI 

mriou ^ 

\JaKiniJ< 

nitlals ^'3^?/4t>y 

sterile nutrient media were prep 

nutrient solutions listed below. 

 reagent water. The solution v 

_ using a Corning 140 pH mete 

the solution was passed througl 

1 refrigerator. 

>tock Solutions: Each prepared 

NaNO, 

NaHCOj 

MgS04«7H20 

MgClj-eHjO 

CaCla^aHjO 

K,HP04 

took Solutions: Prepared In IOC 

MnCl2»4H20 

NajEDTA^aHjO 

HaBO, 

FeCI,«6HjO 

Na2Mo04»2H,0 

ZnClj 

CoClj-eHjO 

CuCl3»2H,0 

yi PREPARATION 

Protocol No.: "Janou 

Studv #: \lat\ox\ 

>ared with nutrient aliquots 

The solution was prepared v 

i/as pH adjusted to 7.5 ±0. 1 

r, LD. No.1714-175. A 

\\ 0.45-/mi Millipore* filters ar 

in 1000 mL of sterile reagent 

Target 

Weight 

25.500 g 

15.000 g 

14.700 g 

12.164 g 

4.410 g 

1.044 g 

K) mL of sterile reagent wate 

Target j f ^ 

Weight [[ | ^ 

415.4 mg § j 1 

300 mg | 5 

185.5 mg *= g

159.8 mg J »

7.3 mg g S

3.3 mg £ 5

1.4 mg r

12.0/ig J 

is 

Date: f-^r- ^ 

Date 9-/S- n 

4 

vith 

fter 

Id 

t 

\ 

\ 

PgOO 2 4 
J 

< , 

e 
V, 

Lot #/^er]esi ^ 

Date Initiated: 

Date 

9z\y 3 

^'5-^ 3 

"i^r^^ 

q-io-^^ 

q-w-iS 

9~/S'^3 

S9-^-n 

I 

(3)/ah M^ / ^'/

?3-T-^c 

?-: 

Obs. ID 

^ 

,$UJ 

4 

5-t// 

S o ^ 

i% 

<>l[^ 

"fhi i 

Thi»< 

Sl

-^^. W 

Culture R 

Scenedesmut 

Initiate 

)l-S3 

Lot# 

T-Sc 

Tr^'^ 

7,yc 

T-Sc 

Ts'Sc 

Tj^-sc. 

T-S L 

isanexacto 

H^- o 

ecord for 

? subspicatus 

Jd from: ^^-i 4^ '^i-Ss^iH-H 

Comments 

/rt/^O/oJec^ 

iyi/.fafJ 

I /7.V(V3vl-<»c/ 

f C rm/A/aJe c/ 

hrnn^'if J lyf^ir J 

//^I'Vii^Vr/ 

/>//;« Ac/

)pyof 

Bant" 

f-/5''?3 

6'i'$iv?-/o-yjj 

i Prepared by:_ 

Reviewed by:. 

Date:_£i2MLi__ 

Date: ^"^- ^ "^-^ 

pg0 02 5 
y 
OBSERVATIONS AND/OR REMARKS FORM 

Test Material: VARIOUS Protocol No. VARIOUS 

Study Director: VARIOUS Study #: VARIOUS 

8/31/93 

^ 

On August 26, 1993 a slant labeled UTEX 1648 Selenastrum 

capricornutum !ot# 93-1648-4), a test tube labeled UTEX LB2308 

Skeletonema costatum lot# 93-LB2308-4) containing a live culture in 

suspension, a slant labeled UTEX 2594 Scenedesmus subspicatus lot# 

93-2594-4), a slant labeled UTEX 1444 Anabaena flos-aquae lot# 93-

1444-4), and a slant labeled UTEX 661 Navicula pelliculosa lot# 93-661 

4) were received. Slants and suspension were stored at room temperature. 

8/31/93 ' - ^ : 

Scrapings of the live cultures were taken and asceptically placed in sterile 

250 mL E-flasks, each containing 100 ml of their respective algal nutrient 

media. The two Skeletonema costatum cultures were placed in an environmental chamber at 20°C ±2°C with a 16-hr. light/8-hr. dark photo period. 

The two Anabaena flos-aquae cultures were placed in an environmental 

chamber at 25°C ±1'*C with continuous lighting. Two Selenastrum 

capricornutum and two Scenedesmus subspicatus cultures were placed in an 

environmental chamber at 24°C ±2°C with continuous lighting and agitation. 

Preparation of all plugs was done asceptically under a U.V, hood. 

By 

"This is an exact copy of 

The original document" 

.date-±2±2^ 

NOTE: Individual entries must be dated and initialed. 

Reviewed by:

Study Director:

Date:__iilii3 _ 

Date: A// h 

pg 0 0 2 b 

Pi 0 0^ 1 

( , 

APPENDIX n - STATISTICAL ANALYSIS DATA 

A. Student Mest Data 

B. ANOVA and Dunnett's Multiple Means Test Data 

 pgfl0 2 8 
A. Student Mest Data 
PRINTOUT OF RAW DATA 

FOR CONTROLS 

OUR 

72 

72 

72 

72 

72 

72 

LEVEL 

CON 

CON 

CON 

VEH 

VEH 

VEH 

REP 

A 

B 

C 

A 

B 

C 

CELL_104 NO_CELLS 

N = 6 

ANALYSIS BY; 

REVIEWED BY; 

DATE: 08SEP93 

DATE: ^-13 -'73 

pg00 3 0 
DISTRIBUTION OF CELL COUNTS FOR EACH HOUR 

LOOK FOR UNUSUAL VALUES WHICH MAY BE INCORRECT DATA 

HOUR=72 

FREQUENCY OF CELL 104 

SYMBOL LEVEL 

C CON 

SYMBOL LEVEL 

V VEH 

ANALYSIS BY: 

REVIEWED BY: 

DATE: 08SEP93 

DATE: (/-/3- fJ 

pg0 0 3 l 
MEAN CELL COUNTS FOR CONTROLS BY HOUR 

MEAN OF CELL_104 BY LEVEL GROUPED BY HOUR 

HOUR LEVEL CELL_104 

FREQ MEAN 

72 CON 

VEH 

*********************************** 3 69.41667 

********************************** 3 68.16667 

10 20 30 40 50 60 70 

CELL 104 MEAN 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: J 

pg 0 0 3 2 
DESCRIPTIVE STATISTICS (N, MIN, MAX, MEAN, STANDARD DEVIATION, AND CV) 

FOR NUMBER OF CELLS/ML DIVIDED BY 10**4 

LEVEL 

CON 

VEH 

NO_REPS 

3 

3 

MIN_C104 

68 

65 

HOUR=72 -

MAX_C104 

71.75 

70.00 

AVR_C104 

69.4167 

68.1667 

STD_C104 

2.03613 

2.75379 

CV_C104 

2.93320 

4.03978 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: ^-/i'93 

pg0 03 3 
DESCRIPTIVE STATISTICS (N, MIN, MAX, MEAN, STANDARD DEVIATION, AND CV) 

FOR NUMBER OF CELLS/ML 

LEVEL 

CON 

VEH 

NO_REPS 

3 

3 

MIN_CELL 

680000 

650000 

HOUR=72 -

MAX_CELL 

717500 

700000 

_—^^^^^^^———^ 

AVR_CELL 

694166.67 

681666.67 

STD_CELL 

20361.32 

27537.85 

CV_CELL 

2.93320 

4.03978 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9'/?"?^ 

PgO0 3 4 
REPEATED MEAStJRES ANALYSIS OF VARIANCE COMPARING CONTROLS 

AVERAGED OVER ALL TIME POINTS FROM 72 HOURS 

IF PR>F FOR SOURCE=LEVEL (BOTTOM LINE) IS LESS THAN .05 

AND IF DIFFERENCE IS BIOLOGICALLY MEANINGFUL 

THEN USE VEHICLE OR CARRIER AS CONTROL 

General Linear Models Procedure 

Class Level Information 

Class 

LEVEL 

REP 

HOUR 

Levels 

2 

3 

1 

Values 

CON VEH 

A B C 

72 

Number of observations in data set = 6 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9-J^-93 

^ pg0 0 3 5 
REPEATED MEASURES ANALYSIS OF VARIANCE COMPARING CONTROLS 

AVERAGED OVER ALL TIME POINTS FROM 72 HOURS 

IF PR>F FOR SOURCE=LEVEL (BOTTOM LINE) IS LESS THAN .05 

AND IF DIFFERENCE IS BIOLOGICALLY MEANINGFUL 

THEN USE VEHICLE OR CARRIER AS CONTROL 

ANALYSIS BY: 

REVIEWED BY: 

DATE; 08SEP93 

DATE: 9'13-93 

pg00 3b 
REPEATED MEAStTRES ANALYSIS OF VARIANCE COMPARING CONTROLS 

AVERAGED OVER ALL TIME POINTS FROM 72 HOURS 

IF PR>F FOR SOURCE=LEVEL (BOTTOM LINE) IS LESS THAN .05 

AND IF DIFFERENCE IS BIOLOGICALLY MEANINGFUL 

THEN USE VEHICLE OR CARRIER AS CONTROL 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9'/3'93 

pg00 3:i 
9 

STUDENT T-TEST COMPARING CONTROLS 

AT EACH TIME POINT STARTING AT 72 HOURS 

IF PR0B>|T | FO R EQUA L VARIANCES IS LESS THAN .05 

AND IF DIFFERENCE IS BIOLOGICALLY MEANINGFUL 

THEN USE VEHICLE OR CARRIER AS CONTROL 

ANALYSIS BY: 

REVIEWED BY: 

DATE: 08SEP93 

DATE: 9-13-^3 

pg0fl3 8 
B. ANOVA and Dunnett's Multiple Means Test Data 

pg 0 0 3 9 
ANALYSI

S BY: 

REVIEWED BY:

DATE: 08SEP93 

'T3 

TgOfl4 0 
ANALYSI

S BY: 

REVIEWED BY: 

DATE: 08SEP93 

r/3-fJ 

TgOOlt I 
ANALYSI

S BY: 

REVIEWED BY: 

DATE: 08SEP93 

 9'/3-'?3 

 pg004 2 
ANALYSI

S BY: DATE: 08SEP93 

REVIEWED BY: DATE: ^'/3-9^ 

 Pi004 3 
ANALYSI

S BY: DATE: 08SEP93 

REVIEWED BY: DATE

: 9-/3-'^3 

 Pg0044^ 
ANALYSI

S BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9'l3-93 

Pg00 4 5 
ANALYSI

S BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9-/3-9J : Pg0fl4b 
8 

CONT_MSG CONT_LEV 

POOLED CONTROL USED FOR COMPARISONS TO CONTROL WITH DUNNETT'S TEST POOLED 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: '?'/3-93 

^ Pg0 0 4 1 
RESULTS OF LEVENE'S TEST FOR HOMOGENEITY OF VARIANCE 

ACROSS TREATMENTS FOR EACH HOUR 

IF P IS LESS THAN .01 THEN THE ANALYSIS IS 

PERFORMED ON THE TRANSFORMED VALUE (SQUARE ROOT TRANSFORMATION) 

OBS HOUR NUM_DF DEN_DF F P 

1 72 3 11 1.79670 0.20594 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: 9-/3-93 

pg0 04 8 
10 

ANOVA AND DUNNETT'S MULTIPLE MEANS COMPARISON 

ON SQR_CELL = SQRT(NO. OF CELLS/ML + 0.5) 

OR NUMBER OF CELLS/ML DIVIDED BY 10**4 

DEPENDING ON RESULTS OF LEVENE'S TEST 

FOR THAT HOUR 

HOUR=72 

General Linear Models Procedure 

Class Level Information 

Class Levels Values 

TREATMNT 4 DUNNETT/CONTROL LEVEL-1 LEVEL-2 LEVEL-3 

Number of observations in by group = 15 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE; 9-/3-93 

Pg0 0 4 9 
ANOVA AND DUNNETT'S MULTIPLE MEANS COMPARISON 

ON SQR_CELL = SQRT(NO. OF CELLS/ML +0.5) 

OR NUMBER OF CELLS/ML DIVIDED BY 10**4 

DEPENDING ON RESULTS OF LEVENE'S TEST 

FOR THAT HOUR 

11 

HOUR=72 

ANALYSIS BY:

REVIEWED BY:

DATE: 08SEP93 

DATE: 9'/3-9 j 

pgOOS O 
12 

ANOVA AND DUNNETT'S MULTIPLE MEANS COMPARISON 

ON SQR_CELL = SQRT(NO. OF CELLS/ML + 0.5) 

OR NUMBER OF CELLS/ML DIVIDED BY 10**4 

DEPENDING ON RESULTS OF LEVENE'S TEST 

FOR THAT HOUR 

HOUR=72 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: fVJ-fJ 

 PgOOS i 
13 

ANOVA AND DUNNETT'S MULTIPLE MEANS COMPARISON 

ON SQR_CELL = SQRT(NO. OF CELLS/ML +0.5) 

OR NUMBER OF CELLS/ML DIVIDED BY 10**4 

DEPENDING ON RESULTS OF LEVENE'S TEST 

FOR THAT HOUR 

HOUR=72 

ANALYSIS BY: DATE: 08SEP93 

REVIEWED BY: DATE: 4-/3-93 

 Pg0 0 5 2 
ADDENDUM TO ANALYTICAL BIO-CHEMISTRY'S 

REPORT NO.

The following corrections were identified for the above cited study: 

Page 9, RESULTS. Second Paragraph, First Sentence 

Replaced " > 10 mg/L" with " > 100 mg/L". This correction reflects 

the actual results of the study. 

We are issuing this addendum to report to provide the corrected page for the above 

cited final report and does not change any reported results. This addendum will be attached 

to the final report for the above cited study. 

Team Leader, Aquatic Toxicology 
A Student ^test was conducted to determine if the control and vehicle blank cell 

count values were significantly different {p < 0.05) from each other. The test 

indicated that there was not a significant difference after 72 hours. The control 

and vehicle blank values were then pooled for all further statistical analyses. 

A one-way analysis of variance (using PROC GLM in SAS) was conducted for 

each timepoint with a Dunnett's comparison to the pooled controls. A one-tailed 

Dunnett's test was conducted at the 0.05 level of significance with the alternate 

hypothesis being that the mean number of cells has been reduced in comparison 

to the pooled controls mean. Prior to the Dunnett's test, a Levene's test (2) was 

conducted to test for homogeneity of variance over treatments at each hour. If the 

p value from the Levene's test is greater than 0.01, indicating insignificant 

heterogeneity, then the analysis is performed on the ceU counts divided by IO'*. 

If the p value is less than 0.01 for some hour(s), then the cell counts for each 

replicate are transformed using the square root of the cell count, the 

recommended transformation for count data (3). 

RESULTS 

A 72-hour screen with Scenedesmus subspicatus exposed to Tinuvin 900 was conducted at test 

concentrations of 1.0, 10, and 100 mg/L along with a control and vehicle blank (DMF). Test 

concentrations were based on the solubility of Tinuvin 900 in algae nutrient media. Logarithmic 

phase growth was confirmed at 72 hours with a mean count of 6.9 x 10^ and 

6.8 X 10^ cells/mL in the control and vehicle blank replicates, respectively, that were 74- and 

80-fold increases, respectively, from the initial 9.3 x 10^ and 8.5 X 10^ cells/mL. After 

72 hours, algal cell counts for the 1.0-, 10-, and 100-mg/L test levels were 104, 94, and 94% 

of the pooled control population, respectively. A precipitate was observed in the 10 and 

100 mg/L test replicates at 72 hours. 

A 72-hour EC50 for Scenedesmus subspicatus exposed to Tinuvin 900 was estimated to be 

>100 mg/L since 50% inhibition was not observed in any of the test concentrations. The 

72-hour no-observed effect concentration was 100 mg/L.