Appendix A.

Biomonitoring Report

Daphnia pulex & Pimephales promelas .

Prepared for Sasol by EE USA

Lab Report # Q-506-07

Sample ID: ALFOTERRA® 145-8S

October 31, 2007
NELAP Certificate No. : 0202 7

4W Biomonitoring Report

Daphnia pulex and Pimephales promela s

prepared for

SASOL - Alfoterra 145-85

Services Requested By: Mr. Geoffrey Russel l

D. pulex EE USA Project No. : Q-560-07

SURVIVAL LC50 2.89 mg/I P R

P. promelas EE USA Project No . : Q-5601-07

SURVIVAL LC50 1 .31 mg/I P R

Repo rt Date: October 31, 200 7

by

ENVIRONMENTAL ENTERPRISES USA, INC .

58485 PEARL ACRES ROAD, SUITE D

SLIDELL, LOUISIANA 7046 1

(800) 966-278 8

This report contains five pages plus six appendices, A - F. This

report must not be reproduced in part, only in whole . The results and

conclusions presented in this report apply only to the sample(s) tested.

/

1

Veronica McNew

PRluents Testing Supervisor

16A1 6 -7

DATE

LIU", U 'A

Mark A. O'Neil

QA/QC Supervisor

~~---

David L. Daniel

Laboratory Director

ll ilo7

DATE

DATE

D pulex Q-560-07

P. promelas Q-5601-0 7

1 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No : 0202 7

Daphnia pulex ACUTE STATIC-RENEWAL 48-HOUR DEFINITIVE TEST,

EPA-821-R-02-012 : SECTION 9, Method 2021 .

TEST OVERVIEW

A 48-hour static-renewal toxicity test was conducted by Environmental Enterprises USA, Inc (EE

USA) to determine toxicity of product (PR) Alfoterra 145-8S to Daphnia pulex neonates. Test organisms

were cultured at EE USA and less than 24.0-hours-old when this test was initiated. Reconstituted

laboratory-prepared water was used as the diluent and a laboratory performance control was evaluated .

Five replicates of each control solution and five PR concentrations were prepared initially and renewed

daily PR concentrations tested were 1, 2, 4, 8, and 16 mg/l . This test was initiated September 25, 2007,

at 1650 and completed September 27, 2007, at 1649 .

Methods, materials, and results are presented in this document. The test was conducted from

September 25 - 27, 2007, at the laboratory of EE USA. Those involved in pe rforming this test and in

preparation of this repo rt were Veronica McNew, Mark A . O'Neil, and David L. Daniel .

MATERIALS AND METHOD S

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of EFFluents and Receiving Waters to Freshwater and Marine Organisms .

Actual materials and methods are detailed below . This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2021 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental design .

D pulex was cultured and maintained in reconstituted moderately hard water at 20 +1°C . Neonates

were collected from approximately 300 adults selected from stock cultures and placed in a tempora ry

holding vessel . Test organisms were not fed .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, DP0710, with Potassium chloride (Sigma Chemical, Lot 096K0040). The

SRT test was initiated on September 26, 2007, with less than 24-hour old D. pulex neonates. Appendix F

contains D. pulex SRT control charts .

48-hr LC50 : 781 mg/I

Upper & Lower 95% Confidence Interval: 745 - 820 mg/I

On September 7, 2007, the PR sample was delivered to EE USA. Test chambers were labeled with

replicate identification and test chamber boards labeled with EE USA's project number. Six treatments,

five PR concentrations, and a laboratory performance control, were prepared daily (Appendix A, page 1) .

Each treatment was poured into a new acid-washed 0 .5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured .

At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix A, page 3) . Alkalinity and hardness were measured in the

performance control solution, September 25 and September 26 (Appendix A, page 1) .

D. pulex Q-560-07

P. promelas Q-5601-0 7

2 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No : 0202 7

On Day 0, the treatments were poured into one of two sets of test chambers, ten D. pulex neonates

were distributed randomly to each replicate, and then this test was placed in the environmental chamber .

On Day 1, the test was renewed : fresh treatment solutions were poured into the second set of test

chambers and surviving neonates were transferred by replicate from the old to the new solutions . Survival

was recorded daily (Appendix A, page 2) After 48 hours, the final survival data were recorded and this

test was terminated .

Summary of Experimental Condition s

Test Organisms : Less than 24 .0-hour-old Daphnia pulex neonate s

Dilution Water:

Temperature :

Photoperiod :

Test Chambers :

Test Solution Volume :

Aeration :

Test Solution Renewal :

Laboratory prepared Moderately- hard water

20+ 1°C

16 hours light; 8 hours dark

30 ml Disposable Plastic Cups

25 ml

No

Yes

RESULTS AND CONCLUSION

The 48-hour survival data were used to estimate the 48-hour LC50 : a point estimate of the

concentration expected to result in 50% mortality to exposed D. pulex neonates after 48 hours of

exposure . The 48-hour LC50 was 2 .89 mg/l PR, with a 95% confidence interval of 2 .42 to 3 .40% as

determined by Probit. Survival in the concurrent laboratory performance was 98.0%. Survival data

summary statistics are presented in Appendix C.

D. pulex Q-560-07

P promelas Q-5601-0 7

3of5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No . 02027

Pimephales promelas ACUTE STATIC-RENEWAL 96-HOUR DEFINITIVE TEST,

EPA-821-R-02-012 : SECTION 9, Method 2000 .

TEST OVERVIEW

A 96-hour static-renewal toxicity test was conducted by EE USA to determine toxicity of the PR

Alfoterra 145-8S to Pimephales promelas larvae. Test organisms were cultured at EE USA and three

days old when this test was initiated . Reconstituted laboratory-prepared water was used as the diluent and

a laboratory performance control was evaluated . Five replicates of control solution and five PR

concentrations were prepared initially and renewed at 48 hours . PR concentrations tested were 0 .5, 1, 2,

4, and 8 mg/I . This test was initiated September 25, 2007, at 1624 and completed September 29, 2007, at

1541

Methods, materials, and results are presented in this document . The test was conducted from

September 25 - 29, 2007, at the laboratory of EE USA . Those involved in performing this test and in

preparation of this report were Veronica McNew, Mark A. O'Neil, and David L . Daniel .

MATERIALS AND METHOD S

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms .

Actual materials and methods are detailed below . This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2000 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental desig n

P promelas was cultured and maintained in reconstituted moderately hard water at 25±1°C. Several

clutches from different females comprised the embryo pool from which the test organism population

hatched . Test organisms were fed Artemia nauplii less than 24-hours old twice daily .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, PP0710-48, with potassium chloride (Sigma Chemical, Lot 096K0040) .

The SRT test was initiated on September 26, 2007, with 2-day-old P. promelas larvae. Appendix F

contains P. promelas SRT control chart s

48-hr LC50 : 1450 mg/I

Upper & Lower 95% Confidence Interval: 1320 - 1600 mg/I

On September 7, 2007, the PR sample was delivered to EE USA . Test chambers were labeled with

test concentration, replicate, and EE USA' project number . Six treatments, five PR concentrations, and a

laboratory performance control, were prepared at initiation and upon renewal at 48 hours (Appendix B,

page 1) .

Each treatment was poured into a new acid-washed 0 .5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured .

At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix B, pages 4 & 4). Alkalinity and hardness were measured in the

performance control solution, September 25 and September 27 (Appendix B, page 1) .

D . pulex Q-560-07

P. promelas Q-5601-07

4 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No - 02027

On Day 0, the treatments were poured into their respective test chambers, ten P. promelas larvae were

distributed randomly to each, and then this test was placed in the environmental chamber . On Day 2, the

test was renewed : 90% of the treatment solution, excess food, and debris were poured or siphoned out of

each replicate . Aliquots of freshly prepared treatments were poured gently into each replicate as

appropriate. Surviving test organisms were disturbed as little as possible during renewal . Every 24 hours,

survival was recorded (Appendix B, pages 2 & 3). After 96 hours, the final survival data were recorded

and this test was terminated

Summary of Experimental Conditions

Test Organisms:

Dilution Water:

Temperature:

Photoperiod:

Test Chambers :

Test Solution Volume :

Aeration:

Test Solution Renewal :

3-day-old Pimephales promelas larvae

Laboratory prepared Moderately- hard water

20+ 1°C

16 hours light; 8 hours dark

Rectangular Pyrex Dish, 21cm x 11cm x 7cm

200 m l

No

Yes

RESULTS AND CONCLUSION

Total Volume = 1 .45 liters

The 96-hour survival data were also used to estimate the 96-hour LC50 : a point estimate of the

concentration expected to result in 50% mortality to exposed P. promelas neonates after 96 hours of

exposure . The 96-hour LC50 was 1 .31 mg/I PR, with a 95% confidence interval of 1 .23 to 1 .39% as

determined by Trimmed Spearman-Karber. Survival in the concurrent laboratory performance was

98 .0% . Survival data summa ry statistics are presented in Appendix C .

REFERENCES

EE USA. April 2007. Quality Assurance Plan . EE USA, Slidell, LA 70461 .

EE USA. April 2007. Standard Operating Procedures . EE USA, Slidell, LA 70461 .

2003 National Environmental Laboratory Accreditation Conference Standards , Chapter 5 Quality Systems .

EPA/600/R-04/003. Approved at Ninth NELAC Annual Meeting, June 5, 2003.

Tidepool Scientific Software . ToxCalc Version 5 .0 .

U S . Environmental Protection Agency . October 2002 . Methods for measuring the acute toxicity of

effluents and receiving waters to freshwater and marine organisms . EPA-821-R-02-012 . 5 "' Edition .

Office of Water (4303T) Washington, DC 20460 .

D pulex Q-560-07

P. promelas Q-5601-0 7

5 of 5
Environmental Enterprises USA, Inc.

APPENDIX A
9/25/07,2:59 P M

Environmental Enterprises USA, Inc .

Sasol - Alfoterra 145-8S

Geoff Russell (337) 494-5097

Preparation of Toxicant Simulated Effluent (EFF): Weigh 0.1627 g of Alfoterra 145-8S in a 25 ml

beaker. Add Alfoterra 145-8S to class A cylinder and rinse beaker with Moderately Hard Synthetic

Freshwater (MHSF). Add MHSF to cylinder for total volume of 3000 ml . The EFF is split into two

1500 ml aliquots One aliquot is the 16mg/1 EFF for testing, and the second aliquot is used to prepare

the 8 mg/l.

Preparation of 8 mg/1, 4 mg/I, 2 mg/I, and I mg/1 dilutions:

8 mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml of

this solution is the 8 mg/1 EFF

4 mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF; 1500 ml

of this solution is the 4 mg/1 EF F

2 mg 1l EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 2 mg/I EFF

I mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the I Ing/1 EFF

Data pages & Calculations by: /JA/QC Officer: 4

D. pulex = 5 Reps x 25 ml

= 125 ml

DHzO = Dilution Water = MHSF

LPC M

#

LP-c- M

#

Date 09/25 09/26

Alkalinity -?

Conductivity

Hardness

pH C%

TRC

LPC: Laboratory Performance Control, synthetic moderately hard water

Alkalinity. mg/1 as CaCO3 Conductivity: μS/cm Hardness: mg/1 as CaCO3

pH : su TRC : mg/I

Prep Date 09/25 09/26

Sample # 1 1

Initial

Comments: Feeding / Exposure Chambers - D. pulex - none / 30 ml medicine cups

P promelas - none / 16 oz . plastic cup s

Alfoterra 145-8S Q-560-07

I of 4
9/25/07,2:59 PM

Environmental Enterprises USA, Inc .

Cladoceran, Daphnia pulex

Acute Static Renewal 48 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 202 1

Sasol - Alfoterra 145-8S

Survival Data

Treatment mg/L PR

Time R; LPC R 1 R ; 2 R 4 R 16 Initia l Ei White E ppm E ppm E ppm E ppm E ppm

S

P! P Blue P Green P Yellow P ! Brown P Black

1i 10 6 10 11 10 16 10 21 10 26 10 09/25i0 7

O HR

2 ; 10 7 10 12 10 17 10 22 10 27 10

3fl:' c- 10 14 10 19 10 24 10 29 10

5 10 10 10 15 10 20 ; 10 25 10 30 ; 10

6 11 --1 16 21 2 6

24 09/26i0 2

HR 2 ; 7

C1'

12 17 22 2 7

3! 8 13 '• 18 '• ~~ 23 2 8

4 9 14 19 24 29

10 15 20 25 q 3 0

1! 6 11 21 2 2 6

48 o9/27/0 7

HR 2 7 12 2 7

3 8 13 F8! 23 2 8

~

4, 9 < 14

24 29 ; ~''

5! 10 ~ 15 20 25 30 !

()

n

1-7

C, S r

Counted by : QC/QA by:

Loaded ~y : Ii,~~

Test Organisms Age: hours old Test Organisms Source

Data Entry by:

Double Data Entry by: and/or

QC/QA by:

Alfoterra 145-8S

Cf /tS'4

Q-560-0 7

2 of 4
9/25/07,2:59 PM

Environmental Enterprises USA, Inc .

D. pulex Water Quality Data

LPC & All Treatments: Initial Temp. - 18.5 - 21.4 ; Initial DO - 9.2 < 4. 0

0 to 24 HR I Treatment mg/L PR

09/25-26/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Meter

DO I

~ ./ • V' 1~ V ' ~ ~D' \ '~ L✓

F - ~~ ~ >.~ ~~ : ~l ~• ~ ~~ ~

Temp I

lv~ ~

6

Cond. I

`3C-5 Zc

pH F

C

"

Initials Initials: Finals: •

Times Initial Time : l Final Time: y51

24 to 48 HR Treatment mg/L P R

09/26-27/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Mete r

DO I

C~ ~.

c

cL1

~

F

Temp I ___ ~• t~ l •? ~.~ ~%. ~ ~. s ~ ~ ~'~ ~ F

21 2c . z~ . 5 2~• ~ ~~- 2~> . I C~

Cond I , I

( L '

pH F ~/

C' ~~ S~ ~ f

y

G ~ cL~ > r0 ~L'• > ~~

Initials Initials: Finals :

Times Initial Time : Final Time : ,,~

DO, Dissolved Oxygen: mg/1 pH: SU Salinity : ppt Temp : °C

Comments

Alfoterra 145-8S Q-560-07

3 of 4
9/25/07,2 :59 PM

Environmental Enterprises USA, Inc .

Data Pages

2,- Company name & contact matches client file .

d Product matches client file

q/ Dilution series are correct:

Dilution series:

❑ Calculations on mixing page are correct. (sign mixing page)

Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

throughout data pages.

Format correct. ( spaces for all entries, page numeration, no split pages, etc.)

Initials Date

Chain-of-Custody

o Product on COC matches sample bottle .

a' Product on COC matches test data pages ,

d Lab # on COC matches sample bottle . B' Lab # on COC matches test data pages . ❑ Sample collection date : & Time: hrs.

expiration date : & Time: hrs. (Sample expired if >36

hrs) N/A

e' Sample volume is sufficient for test duration . (Sample volume in container(s)

checked against sample volume on mixing page )

Sample volume available: /c ~- mI

Sample volume needed: -trd~?

(Sample volume insufficient if sample volume available < sample volume needed)

Initials ZS" Date

Jugs & Labels

-6 Lab # on jug and labels matches test data pages .

El" Dilution water type is on jug . (i.e. 25 ppt, 20 ppt, MHSF, etc .)

~y Dilutions on jugs and labels match dilutions on test data pages .

a Jugs are color-coded . (see mixing page for appropriate color code sequence )

Initials Date

Raw Data QC/QA'd by ~~?~c i<' •'

~ , - ~ -

~~~~~% ~ G' l~l c"?

Alfoterra 145-8S Q-560-07

4 of 4
Environmental Enterprises USA, Inc.

APPENDIX B
9/25/07,2:59 PM

Environmental Enterprises USA, Inc .

Sasol - Alfoterra 145-8S

Geoff Russell (337) 494-509 7

Preparation of Toxicant Simulated Effluent (EFF): Weigh 0.1627 g of Alfoterra 145-8S in a 25 ml

beaker. Add Alfoterra 145-8S to class A cylinder and rinse beaker with Moderately Hard Synthetic

Freshwater (MHSF). Add MHSF to cylinder for total volume of 3000 ml . The EFF is split into two

1500 ml aliquots . One aliquot is the 16mg/1 EFF for testing, and the second aliquot is used to prepare

the 8 mg/l.

Preparation of 8 mg/l, 4 mg/l, 2 mg/I, 1 mg/l, and 0 .5 mg/I dilutions:

8 mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml of

this solution is the 8 mg/1 EFF

4 mg /I EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 4 mg/I EF F

2 mg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF; 1500 ml

of this solution is the 2 mg/i EFF

1 mg /I EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 1 mg/I EFF

0 .5 ma/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 0 .5 mg/1 EFF

Data pages & Calculations by:h~"rFc A/QC Officer :

P. promelas = 5 Reps x 200 ml

= 1000 ml

DH?O = Dilution Water = MHS F

LPC M

#

LPC M

#

Date 09/25 09/27

Alkalinity '7 (-p

Conductivity '?r- '7 ft-"s- C ; - f"";

Hardness lI c~ ll

H

TRC

~ vZ__

LPC: Laboratory Performance Control, synthetic moderately hard water

Alkalinity- mg/1 as CaCO3 Conductivity: μS/cm Hardness: mg/I as CaCO3

pH : su TRC : mg/ 1

Prep Date 09/25 09/27

Sample # { 1 j

Initia l

Comments: Feeding / Exposure Chambers • D. pulex - none / 30 ml medicine cups

P promelas - none / 16 oz . plastic cups

Alfoterra 145-8S Q-5601-07

1 of 6
9/25/07,2:59 PM

Environmental Enterprises USA, Inc .

Fathead Minnow, Pimepahles promelas

Acute Static Renewal 96 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 200 0

Sasol - Alfoterra L167-7S

Survival Data

Treatment mg/1L PR

Time R

E;

P!

LPC

White

R

E

P

0 . 5

pp m

Blue

R

E

P

I

ppm

Green

R ;

E

P

2

pp m

Yellow

R

E

P

4

pp m

Brown

R ; 8

E ppm

P! Black

Initial

S

O HR

1' 10 6 10 16 10 21 10 26 10 o9/zs/o7

2 ; 10 7 10 F; I 17 10 22 10 27 10

10 8 10 18 ; 10 23 ; 10 28 ; 10

4 ; 10 9 10 14 10 19 10 24 10 29 10

5 ; 10 10 10 15 10 20 10 25 10 30 10

24

1' , O 6 1~ 11 g 16 21 26 0

09/26/0 7

HR 2' ~ ~ 7 1 ~ 12 17 22 ~ 27

0

3' 8 !(U 13 ~ 1C 18 23 2

8

U

4 ; 9 I~ 14 19 24 i C) 2 9

~ 5 !

lJ

10 ID 15 20 ~p 25 C) 30

~

48

1! 6 11 16 21 26

09/27/0 7

HR 2; 7 12 17 ; 22 27

3!

! 1p 8 13 jp 18 23

"

28 t'

4! 9 14 19 24 ; ~ 29,

AL

5, 10

lb

15 20 25 30 C

m g/L

Sur

Counted by :

. QC/QA by : ,

Lo ded by: . .' 1

Test Organisms Age : _ 3 days old Test Organisms

Alfoterra 145-8S Q-5601-07

2 of 6
9/25/07,2:59 PM

Environmental Enterprises USA, Inc

P. promelas Survival Data Cont.

Treatment mg/1L PR

Time Ri LPC

E; Whit e

Pi

R 0 . 5

E PPm

P Blue

R I

E PPM

P Green

R I! 2

E PPm

P i Yellow

R 4

E PP M

P Brown

R 8

E ppP Black

Initial

s

72

1 ; f. 6 r~ 11, Q~ 16 21 26

09/28/0 7

HR 2! 7 /~

! I lJ

12

! ~

1 7 /+-

C~ '

22 27

3 8 18 23 28

4! 9 14 19 24 C) 29

Si D 10 15 20i 25 3 0

96

1 !! 6 11 16 21 26

09/29/07

HR 2 7 12 1 22 2 7

3! 8 13 , 18 23 2 8

14 ; 9 14

t

19 24 2 9

5 10 20 Cy 25 30

mg/L

Sur

Data Entryby.

e,

Double Data Entry by: and/or

QC/QA by

: - -- ~

Alfoterra 145-8S Q-5601-07

3 of 6
9/25/07,2 :59 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Dat a

LPC & All Treatments : Initial Temp. - 18.5 - 21.4; Initial DO - 9.2 < 4. 0

0 to 24 HR Treatment mg/L P R

09/25-26/07 LPC 0 .5 ppm I ppm 2 ppm 4 ppm 8 ppm Mete r

DO I ~J ( , {

F

x ~ Y

Temp I

'Ci L C':td 6•P, &'

.- 4

F

Cond. I

~~ ~

G~r•.~

, l~~ (~~ .l~ ~~ ~ , ~

pH F

•I z~ .

Initials Initials: Finals: &jZ

Times Initial Time :

iLot(i FinalTime :/,,,

48 HR Treatment mg/L P R

09/27/07 LPC 0 .5 ppm 1 ppm 2 ppm 4 ppm 8 ppm Meter

DO F

~ '~ ~ L•~ ~'t 5~

Temp F

~v ~.~•z ~'~ ;~. . •

pH F ~' L i?' ~ . C• ~. L% Cx Z

Initials Initials: Finals:

Times Initial Time: FinalTime :

DO, Dissolved Oxygen : mg/1 pH: SU Salinity : ppt Temp: °C

Comments :

Alfoterra 145-8S Q-5601-07

4 of6
9/25/07,2:59 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Data Cont.

48 to 72 HR Treatment mg/L P R

09/27-28/07 LPC 0 .5 ppm I ppm 2 ppm 4 ppm 8 ppm Mete r

DO I

,---

5

S-I

Temp I

F

7 ~ ~~ • ~l ~f% . ~1 .~~ ~, ~~ '

Cond. I

3&2 l ~~k) Zli

pH F

-e, . C,

Initials Initials: 4AAA Finals: rf

Times Initial Time : Final Time:

'- f ,

96 HR Treatment mg/L P R

09/29/07 LPC 0 .5 ppm 1 ppm 2 ppm 4 ppm 8 ppm Meter

DO F

Temp F

pH F C

L v ~~

Initials Initials: Finals :

Times 11 Initial Time- Final Time:

Alfoterra 145-8S Q-5601-0 7

5 of 6
9/25/07,2:59 PM

Environmental Enterprises USA, Inc

Data PagesData Pages

Company name & contact matches client file .

o Product matches client file . ❑-'Dilution series are correct:

Dilution series :

`-

❑ Calculations on mixing page are correct . (sign mixing page )

Q.--Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

throughout data pages.

4/ Format correct. ( spaces for all entries, page numeration, no split pages, etc .)

~, /Z- Initials Z-, -7 Date

Chain-of-Custody

tT Product on COC matches sample bottle .

d Product on COC matches test data pages .

Y Lab # on COC matches sample bottle . ❑/ Lab # on COC matches test data pages. ❑ Sample collection date : & Time:

expiration date: & Time:

h rs .

hrs. (Sample expired if >3 6

hrs N/A

Sample volume is sufficient for test duration . (Sample vol ume in container(s)

checked against sample volume on mixing page )

Sample volume available (Cf' ml

Sample volume needed- i t,2'7

o j~z5 /a'~

(Sample volume insufficient if sample volume available < sample volume needed)

Initials Date

Jugs & Labels

.a~ Lab # on jug and labels matches test data pages.

o' Dilution water type is on jug . (i.e . 25 ppt, 20 ppt, MHSF, etc . )

Z Dilutions on jugs and labels match dilutions on test data pages .

[o Jugs are color-coded . ( see mixing page for appropriate color code sequence )

/1 Initials 7 ZS G 7 Date

Raw Data QC/QA'd by :

Alfoterra 145-8S Q-560I-07

6of6
Environmental Enterprises USA, Inc.

APPENDIX C
Acute Toxicity Test-48 Hr Su rv ival

Start Date 9/24/2007 Test ID- dp56007 Sample ID: LAB-Lab Sample

End Date: 9/26/2007 Lab ID EE-Environmental Enterprise Sample Type . PRD-Product

Sample Date Protocol : EPAF 02-EPA Freshwater Test Species : DP-Daphnia pule x

Comments :

Conc-mg/L 1 2 3 4 5

PC-LP Control 0.9000 1 .0000 1 .0000 1 0000 1 .0000

1 0.9000 1 .0000 1 .0000 08000 0.9000

2 0 .6000 0.8000 0 .7000 0 .5000 0 7000

4 0 .4000 0 .4000 0 .3000 0 .5000 0.0000

8 0 .2000 0 .0000 0 .0000 0 .1000 0.2000

16 0 .0000 0 .0000 0 .0000 0 0000 0 0000

Transform : Untransformed Number Total

Conc-mg/L Mean CV% Mean Min Max CV°/a N Resp Numbe r

PC-LP Control 0 .9800 4 .563 0 .9800 0 .9000 1 .0000 4 .563 5 1 50

1 0 .9200 9 .094 0 .9200 0 .8000 1 .0000 9 .094 5 4 50

2 0 .6600 17 .275 0 .6600 0 .5000 0.8000 17 .275 5 17 50

4 0 .3200 60.111 0 .3200 0 .0000 0.5000 60 .111 5 34 50

8 0 .1000 100.000 0 .1000 00000 0.2000 100.000 5 45 5 0

16 0 .0000 0 .0000 0_0000 0.0000 5 50 5 0

Auxiliary Tests Statistic Critical Skew Kurt

Shapiro-Wilk's Test indicates normal distribution (p > 0 .01) 0.93767 0.888 -1 .0488 2 .01083

Bartlett's Test indicates equal variances (p = 0.12) 7.29394 13 .2767

Maximum Likelihood-Probi t

Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter

Slope 3.18189 0 .36483 2.46684 3 .89695 0 02 0 92839 7.81473 0 .82 0.46096 0.31428 3

Intercept 3.53326

TSCR 0 01 09

0 .21636 3.10919 3 .95733

. 9 0 . 01895 - 0 . 0 18 0 .05622 1 . 0

Point Probits mg/L 95% Fiducial Limits

EC01 2 674 0 .53684 0 .30506 0 90 .77645

EC05 3 .355 0 .87907 0 .57052 1 .17317 08

EC 10 3 718 1 .1434 079416 146631 07

EC15 3 964 1 .36532 0 .99069 1 70789

EC20 4 158 1 .57203 1 .17903 1 .93125 (U 06

EC25 4 326 1 .77413 1 .36667 2 .1495 a 05

EC40 4 747 2 .40626 1 .96308 2 .84351 (n

EC50 5 000 2 .89044 2 .41611 3 .39929 (U 0 4

EC60 5 253 3 .47204 2 .94305 4 .10601 0. 3

EC75 5 674 4 .70913 3 .98821 5 .75747

EC80 5 842 5 .31455 4 .46792 0 . 26.63046

EC85 6 036 6 .11916 5 .0816 784533 01

EC90 6 282 7 .3068 5 .94982 9 .73584 00

EC95 6 645 9.50397 7.4736 13.485 0 1 1 10 100

EC99 7 326 15.5627 11 3462 25 0997

Dose mg/L

G

Page 1 ToxCalc v5.0 .23 Reviewed by- /

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.
Environmental Enterprises USA, Inc.

APPENDIX D
Acute Toxicity Test-96 Hr Su rvival

Start Date 9/24/2007 Test ID: pp560107 Sample ID: LAB-Lab Sampl e

End Date 9/29/2007 Lab ID : EE-Environmental Enterprise Sample Type : PRD-Product

Sample Date Protocol- EPAF 02-EPA Freshwater Test Species- PP-Pimephales promela s

Comments -

Conc-mglL 1 2 3 4 5

PC-LP Control 0 .9000 1 .0000 1 .0000 1 0000 1 .0000

0 . 5 1 .0000 1 .0000 1 .0000 1 0000 1 .0000

1 0 .8000 1 .0000 0 .8000 1 .0000 0.8000

2 0 .0000 0 .0000 0 .0000 0 .0000 0.0000

4 0 .0000 0 .0000 0.0000 0.0000 0.0000

8 0 0000 0 .0000 0.0000 0.0000 0.0000

Transform: Untransformed Number Total

Conc-mglL Mean CV% Mean Min Max CV% N Resp Number

PC-LP Control 0 .9800 4.563 0.9800 0 .9000 1 .0000 4.563 5 1 5 0

05 1 .0000 0.000 1 .0000 1 .0000 1 .0000 0.000 5 0 50

1 0 .8800 12.448 0.8800 0 .8000 1 0000 12 .448 5 6 50

2 0 .0000 0.0000 0 .0000 0.0000 5 50 50

4 0 .0000 0.0000 0 .0000 0.0000 5 50 50

8 0 .0000 0.0000 0.0000 0.0000 5 50 50

Auxiliary Tests Statistic Critical Skew Kurt

Shapiro-Wilk's Test indicates normal distribution (p > 0 .01) 0.84333 0.835 0.46913 0.20879

Equality of variance cannot be confirmed

Trimmed Spearman-Karbe r

Trim Level EC50 95% CL

0 .0% 1 .3094 1 .2311 1 .3926

5 .0% 1 .3390 1 .2419 1 .4437

10.0% 1 .3537 1 .2154 1 .5076 1 0

20.0% 1 .3543 1 .3025 1 .4081 09

Auto-0.0% 1 .3094 1 .2311 1 .3926

0 8

0 . 7

06

c 0.5 0

N 0 4

W 0 3

0 .2

01

0 .0

-0 1

01 1 10

Dose mg/L

Page 1 ToxCalc v5.0 23 Reviewed by: ~~
Environmental Enterprises USA, Inc.

APPENDIX E
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APPENDIX F
D . pulex SRT, KCI mg/I

1400

1300

120 0

L°+ 1100 U

J

= 1000

0o

It 900

800

700

600

CV%=976

- -~---' -- - ---_- _

o~\oa\b~°o \~\ry~\°\~~~A\o^\~~°\o\~~~o\~o~ooo\o ~~\~~o \~~\h\°~~oo\o~P \o~o \^~\oo \ry6xo

Date of Test

+2 SD

+1 SD

Mean

-1 SD

-2 S D

Test # Test Date 48-hr LC50

mglL KCI

Cumulativ e

Mean LC50

-1 SD -2 SD +1 SD +2 SD Control

Survival

Toxicant Lot

#

DP0601 1/4/2006 1000 940 075K009 0

DP0602 2/24/2006 1120 1060 0000 975 1472 890 2944 1144 .8528 1229 7056 920 075K009 0

DP0603 3/22/2006 978 1032 6667 956 2381 879 8094 1109 0953 1185 5239 980 075K009 0

DP0604 4/12/2006 802 975-0000 843 8665 712 .7330 1106 1335 1237 .2670 98 .0 075K0090

DP0605 5/22/2006 958 971 6000 857 7808 743 9617 1085 4192 1199-2383 1000 075K00901

DP0606 6/15/2006 963 970 1667 868 3032 766 4397 1072 0301 1173 8936 1000 075K00901

DP0607 7/31/2006 854 953 5714 850 7385 747 9055 1056 4044 1159 2374 1000 075K00901

DP0608 8/10/2006 1090 970 6250 863 8985 757 1719 1077 3515 1184 .0781 100-0 075K00901

DP0610 9/28/2006 1010 975 0000 874 3074 773 6148 1075 6926 1176 3852 1000 075K00901

DP061 1 10/10/2006 1010 978 5000 882 9231 787 3462 1074 0769 1169 6538 100 .0 075K0090 1

DP0614 12/6/2006 949 975 8182 884 7108 793 6033 1066 9256 1158 0330 92-0 066K003 2

DP0701 1/31/2007 951 973 7500 886 5875 799 4251 1060 9125 1148 .0749 100 .0 066K003 2

DP0702 2/14/2007 1110 984 2308 892 6219 801 0130 1075 8397 1167 4486 100 0 066K0032

DP0703 3/28/2007 908 978 7857 888 4435 798 1012 1069 1280 1159 .4702 960 066K0032

DP0705 5/3/2007 897 973 3333 883 7528 794 1723 1062 9138 1152 4943 98 0 066K0032

DP0706 5/8/2007 983 973 9375 887 3608 800 7841 1060 5142 1147 0909 980 066K0032

DP0707 6/5/2007 831 965 5294 874 8162 784 1030 1056 2426 1146 9559 940 066K0032

DP0708 7/18/2007 926 963 3333 874 8367 786 3401 1051 8299 1140 3265 1000 096K0040

DP0709 8/29/2007 978 964 1053 878 0362 791 9672 1050 1743 1136.2433 960 096K0040

DP0710 9/26/2007 781 954 9500 861 7064 768 4628 1048 1936 1141 .4372 98 0 096K0040

November- EE cultures crashed before SRT was completed Purchased D pulex

DP0614 - ABS D pulex

DP0701 - ABS D pulex

DP0704 Control failure
P. promelasSRT, KCI mg/ I

CV%=29.2

160 0

1400

0 1200

W)

U

~ 1000

800

600

400

200

\R\o`5~°\0`o'~~\o°~~t\01\~6\0~^0`0°°\00~0\0~^°~\o`~~a\°6°~\0'1~°~\05~~\oR~°~\o ~o~o~~`~\oo ~~'°\°~~~\°~,~~\oA

Date of Test

+2 SD

+1 SD

Mean

-1 SD

-2 S D

Test # Test Date 48-hr LC5 0

mg/L KCI

Cumulative

Mean LC50 -1 SD -2 SD +1 SD +2 SD Control

Su rvival

Toxicant Lot

#

PP0601-48 1/4/2006 771 975 075K009 0

PP0602-48 2/14/2006 877 824 0000 749 .0467 674 .0934 898 .9533 973 9066 100 0 075K009 0

PP0603-48 3/16/2006 671 773 0000 669 .9854 566 9709 876 0146 979 .0291 97 5 075K009 0

PP0604-48 4/18/2006 975 823 5000 692 0631 560 6261 954 .9369 1086 3739 1000 075K0090 1

PP0605-48 5/26/2006 879 834 6000 718 .0976 601 5953 951 1024 1067 6047 1000 075K00901

PP0606-48 6/14/2006 720 815 5000 701 2761 587 0522 929 7239 1043 9478 100 0 075K00901

PP0607-48 7/26/2006 1490 911 8571 636 4203 360 9834 1187 2940 1462 7309 950 075K00901

PP0608-48 8/16/2006 689 884 0000 617.0998 350 1996 1150 9002 1417 .8004 1000 075K0090 1

PP0609-48 9/5/2006 645 857 4444 595 3795 333 3146 1119 5094 1381 5743 1000 075K0090 1

PP0610-48 10/10/2006 404 812 1000 526 .4283 240 7566 1097 7717 1383 4434 975 075K00901

PP0611-48 11/7/2006 787 809 8182 538 7006 267 5829 1080 9358 1352 .0534 1000 066K003 2

PP0612-48 12/14/2006 796 808 6667 550 1354 291 6041 1067 1979 1325 .7292 97 5 066K003 2

PP0701-48 1/4/2007 579 791 0000 535 4105 279 8210 1046 5895 1302 .1790 1000 066K003 2

PP0702-48 2/7/2007 958 802 9286 553 3429 303 7573 1052 5142 1302 0999 1000 066K003 2

PP0703-48 3/26/2007 907 809 8667 567 .8635 325 .8603 1051 8699 1293 8731 1000 066K0032

PP0704-48 4/4/2007 989 821 0625 583 0148 344 9670 1059 1102 1297 1580 100 .0 066K003 2

PP0705-48 5/2312007 909 826 2353 594 .7619 363 2885 1057 7087 1289 .1821 1000 0661<003 2

PP0706-48 6/12/2007 773 823 2778 598 3653 373 4528 1048 1903 1273 1027 1000 096K004 0

PP0707-48 7/26/2007 958 830 3684 609 6184 388 8683 1051 1185 1271 .8685 1000 096K004 0

PP0708-48 8/22/2007 1120 844 8500 620 4394 396 0287 1069 2606 1293 6713 1000 0961<004 0

PP0709-48 9/26/2007 1450 873 6667 618 1661 362 6656 1129 1672, 1384 6678 100 0 096K0040
Appendix B .

An Evaluation of the Chronic Toxicity of the ALFOTERRA® Samples to Freshwater Alga

Selenastrum capricornutum.

Prepared for Sasol by Pacific EcoRisk . October, 2007
~> PACIFIC E(ORIS K

Geoffrey Russell

Sasol North America, Inc .

2201 Old Spanish Trail

Westlake, LA 70669

Dear Mr. Russell :

November 5, 2007

I have enclosed two (2) copies of the report "Evaluation of the Chronic Toxicity of the Alfoterra®

Samples to the Freshwater Alga Selenastrum capricornutum" . The results of our testing are as

follows:

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra 145-8

The results of this test indicated that there were significant reductions in alg al growth at the

5,000 and 10,000 mg/L Alfoterra 145-8 (as WAF) concentrations. The algal growth NOEC was

2,500 mg/L; the IC25 was 5,111 mg/L and the IC50 was 9,378 mg/L .

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra L167- 4

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-4 (as WAF) concentrations. The algal growth NOEC was <625 mg/L;

the IC25 was 970 mg/L and the IC50 was 1,662 mg/L.

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra L167- 7

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-7 (as WAF) concentrations . The algal growth NOEC was <625 mg/L ;

the IC25 was 1,549 mg/L and the IC50 was 2,632 mg/L .

Chronic Toxicity of Alfoterra 145-8 S

The results of this test indicated that there were significant reductions in algal growth at the >2

mg/L Alfoterra 145-8S concentrations . The algal growth NOEC was 1 mg/L; the IC25 was 1. 2

mg/L and the IC50 was 1 .7 mg/L.

Chronic Toxicity of Alfoterra L167-4S

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-4S concentrations. The algal growth NOEC was <0 .5 mg/L; the IC25

was 0.25 mg/L and the ICSO was 0 .50 mg/L.

ORFORATF HEADQUARI ORS CON ORAL VALLEY SOUTHERN CALIFORNIA

_~>~ Cordelia R .~zJ 6a'0 PaciSe A-nue Or ?D 2792D7 Lker Avenur Su 100

. ai 6~ c CA ''dsil lur::con CA 9520- Carlsbad, CA 9201 0

flrane 70- LU ; o phone 2 0 9 952 113u phone 760 602 ?919

fas -(i- 211' ~91 ( fax 209 952 11311 fax 760 602 911 9

--pacrfirecm :sk co m

ENVIRONMENTAL CONSULTING & TESTING
Pacific EcoRisk Environmental Consulting and Testin g

Chronic Toxicity of Alfoterra L167-7S

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-7S concentrations. The algal growth NOEC was <1 mg/L ; the IC25

was 0.45 mg/L and the IC5o was 0 .89 mg/L.

If you have any questions regarding the performance and interpretation of these tests, please feel

free to contact me at (707) 207-7760.

Sincerely,

R. Scott Ogle, Ph.D.

CEO & Special Projects Director
An Evaluation of the Chronic Toxicity of the

Alfoterra® Samples to the Freshwater Alga

Selenastrunt capricornutunc

Prepared For:

Sasol North America, Inc .

2201 Old Spanish Trail

Westlake, LA 7066 9

Prepared By :

Pacific EcoRisk

2250 Cordelia Rd .

Fairfield, CA 94534

(707) 207-7760

October 2007

PACIFIC 1 (0 RIS K

ENVIRONMENTAL C ONSULTING & TESTING
Pacific EcoRisk Environmental Consulting and Testing

An Evaluation of the Chronic Toxicity of the Alfoterra' Samples

to the Freshwater Alga Selenastrum capricornutu m

Table of Contents

Page

] . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

2. ACUTE AND CHRONIC TOXICITY TEST PROCEDURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.1 Receipt and Handling of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 .2 Water Associated Fraction Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 .3 Algal Growth Toxicity Testing with Selenastrurn capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3 . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

3 .1 Chronic Toxicity of Alfoterra 145-8 to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3 .2 Chronic Toxicity of Alfoterra L167-4 to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

3 .3 Chronic Toxicity of Alfoterra L167-7 to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

3 .4 Chronic Toxicity of Alfoterra 145-8S to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

3 .5 Chronic Toxicity of Alfoterra L167-4S to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

3 .6 Chronic Toxicity of Alfoterra L167-7S to Selenastrum capricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. DATA QUALITY CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

5. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Appendices

Appendix A Test Data and Summary of Statistics for the Evaluation of Alfoterra®145-8 WAF

Toxicity to Selenastrum capricornutum

Appendix B Test Data and Summary of Statistics for the Evaluation of Alfoterra®L167-4 WAF

Toxicity to Selenastrum capricornutu m

Appendix C Test Data and Summary of Statistics for the Evaluation of Alfoterra ' L167-7 WAF

Toxicity to Selenastrum capricornutum

Appendix D Test Data and Summary of Statistics for the Evaluation of Alfoterra® 145-8S

Toxicity to Selenastrum capricornutum

Appendix E Test Data and Summary of Statistics for the Evaluation of Alfoterra°L167-4S

Toxicity to Selenastrufn capricornutum

Appendix F Test Data and Summary of Statistics for the Evaluation of Alfoterra"L167-7S

Toxicity to Selenastrum capricornutum
Pacific EcoRisk Environmental Consulting and Testin g

An Evaluation of the Chronic Toxicity of the Alfoterra® Samples to the

Freshwater Alga Selenastrum capricornutum

1 . INTRODUCTION

Sasol North America, Inc . (Sasol) has contracted Pacific EcoRisk (PER) to perform an

evaluation of the chronic toxicity of six new Alfoterra® products (designated 145-8, L167-4,

L167-7, 145-8S, L167-4S, and L167-7S) to algae . This evaluation consisted of performing the

US EPA 96-hr algal growth test with the green alga Selenastrum capricornutum (note - this

species was recently reclassified as Pseudokirchneriella subcapitata, but is still commonly

referred to as Selenastrum capricornutum). This report describes the performance and results of

these tests.

2. CHRONIC TOXICITY TEST PROCEDURE S

The method used in conducting these tests followed the guidelines established by the EPA

manual "Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving

Waters to Freshwater Organisms, Fourth Edition" (EPA/821/R-02-013).

2.1 Receipt and Handling of Sample s

Sasol collected and shipped samples of six Alfoterra products (designated 145-8, L167-4, L167-

7, 145-8S, L167-4S, and L167-75) to the PER laboratory in Fairfield, CA. Upon receipt, these

samples were stored at 4°C except when being used to prepare test solutions.

2.2 Preparation of Water-Accommodated Fractions (WAF )

Three of the Sasol products (145-8, L167-4, and L167-7) were known to be only sparingly

soluble in water; in order to test the potential toxicity of these compounds, water-accommodated

fractions (WAFs) were prepared so as to evaluate the toxicity of any constituent chemicals that

would partition into a water phase (and thus be able to cause toxicity to freshwater organisms).

The WAFs were prepared by adding 20 .0 gm of chemical product to 2 .0 L of reverse osmosis,

de-ionized (RO/DI) water (net concentration = 10 gm/L = 10,000 mg/L) in a 2-L+ glass beaker.

A Teflon-coated magnetic stir-bar was added to each beaker and the solutions mixed for -24 hrs

on magnetic stir plates with a -25% vortex. Upon stopping the mixing, the materials separated

out into 2 phases : water beneath and insoluble chemical product on top. Approximately 1800 mL

of WAF solution was drawn from the bottom water phase of each beaker, and comprised the

10,000 mg/L (as WAF) solution for each chemical product.

Page 1
Pacific EcoRisk Environmental Consulting and Testin g

2.3 Algal Growth Toxicity Testing with Selenastrum capricornutu m

The chronic algal toxicity test consists of a-96-hr bioassay in which the green alga Selenastrum

rapricornutum is exposed to a series of product dilutions and the effects on cellular reproduction

determined . The specific procedures used in this test are described below.

The Lab Control/diluent for these tests consisted of reverse-osmosis, de-ionized (RO/DI) water .

Aliquots of the Lab Control water and sample were spiked with nutrients and then filtered (using

sterile 0.45 pm filters) before use in these tests, as per EPA guidelines . The nutrient-amended

and filtered waters were then used to prepare test solutions at test treatment concentrations of:

• 625, 1250, 2500, 5000, and 10,000 mg/L for the WAF testing,

• 0.5, 1, 2, 4, and 8 mg/L for 145-8S, and L167-7S, and

• 0.25, 0.5, 1, 2, and 4 mg/L for L167-4S .

Routine water quality characteristics (pH, dissolved oxygen [D .O.], and conductivity) were

measured on these test solutions prior to their use in the test.

There were 4 replicates for each test treatment, each consisting of a 250-mL glass Erlenmeyer

flask containing 100 mL of test solution ; an additional replicate was established at each test

treatment for measurement of test solution water quality characteristics during the test and at test

termination. Each flask was inoculated to an initial cell density of 10,000 cells/mL of

Selenastrum from an ongoing laboratory culture that is maintained in log growth phase . These

flasks were loosely capped and randomly positioned within a temperature-controlled room at

25°C, under continuous cool-white fluorescent illumination . Each replicate flask was gently

shaken a minimum of 3 times daily.

After 96 (±2) hrs exposure, the algal cell density in each replicate flask was determined by

spectrophotometric analysis. The resulting cell density data were analyzed to evaluate any

impairment of algal growth caused by the leachate ; all statistical analyses (e .g., determination of

the NOEC, LOEC, and key IC point estimates) were made using the CETIS® statistical software

(Version 1 . 1 .2revL, TidePool Scientific, McKinleyville, CA).

Page 2
Pacific EcoRisk Environmental Consulting and Testin g

3 . RESULTS

3 .1 Chronic Toxicity of Alfoterra 145-8 (as WAF) to Selenastrum capricornutunt

The results of this test are summarized below in Table 1 . There were significant reductions in

algal growth at the 5,000 and 10,000 mg/L 145-8 (as WAF) concentrations . The algal growth

NOEC was 2,500 mg/L; the IC25 was 5,111 mg/L and the IC5 0 was 9,378 mg/L.

The test data and summary of statistical analyses for this test are presented in Appendix A .

Table 1 . Effects of Alfoterra 145-8 (as WAF) on Selenastrum capricornutum growth.

Test Treatment (mg/L [as WAF]) Mean Algal Cell Density (cells/mL x 106)

Lab Water Control 2 .94

625 2 .42

1250 2 .95

2500 2 .73

5000 2.23*

10,000 1 .36*

Summary of St a tistics

No Observable Effect Concentration (NOEC) = 2500 mg/L [as WAF]

IC25 = 5111 mg/L [as WAF]

IC5o = 9378 mg/L [as WAF]

* Significantly less than the Lab Water Control treatment at p<u .v~ .

Page 3
Pacific EcoRisk Environmental Consulting and Testing

3 .2 Chronic Toxicity of Alfoterra L167-4 to Selenastrunt capricornuturn

The results of this test are summarized below in Table 2 . There were significant reductions in

algal growth at each of the tested L167-4 ( as WAF) concentrations. The algal growth NOEC was

<625 mg/L ; the IC25 was 970 mg/L and the IC50 was 1,662 mg/L .

The test data and summary of statistical analyses for this test are presented in Appendix B .

Table 2. Effects of Alfoterra L167-4 (as WAF) on Selenastrum capricornutum growth .

Test Treatment (mg/L [as WAF]) Mean Algal Cell Density (cells/mL x 106 )

Lab Water Control 2 .94

625 2.60*

1250 1 .88*

2500 0.631 *

5000 0.000*

10,000 0 .000 *

Summary ot' Statistics

No Observable Effect Concentration (NOEC) _ <625 mg/L [as WAF]

IC25 = 970 mg/L [as WAF ]

IC5o = 1,662 mg/L [as WAF]

Significantly less than the Lab Water Control treatment at p<0 .05 .

Page 4
Pacific EcoRisk Environmental Consulting and Testing

3.3 Chronic Toxicity of Alfoterra L167-7 to Selenastruna capricornutu nc

The results of this test are summarized below in Table 3 . There were significant reductions in algal

growth at each of the tested L167-7 (as WAF) concentrations . The algal growth NOEC was <625

mg/L ; the IC25 was 1,549 mg/L and the ICSO was 2,632 mg/L .

The test data and summary of statistical analyses for this test are presented in Appendix C .

Table 3 . Effects of Alfoterra L167-7 (as WAF) on Selenastrum capricornutum growth .

Test Treatment (mg/L [as WAF]) Mean Algal Cell Density (cells/mL x 1 06)

Lab Water Control 2 .94

625 2 .23*

1250 2 .60*

2500 1 .55*

5000 0 .113*

10,000 0*

Summary of Statistics

No Observable Effect Concentration (NOEC) _ <625 mg/L [as WAF]

IC25 = 1,549 mg/L [as WAF]

IC5o = 2,632 mg/L [as WAF]

* Significantly less than the Lab Water Control treatment at p<0 .05 .

Page 5
Pacific EcoRisk Environmental Consulting and Testing

3 .4 Chronic Toxicity of Alfoterra 145-8S to Selenastrurn capricornutuna

The results of this test are summarized below in Table 4 . There were significant reductions in

algal growth at the >2 mg/L 145-8S concentrations . The algal growth NOEC was 1 mg/L; the

IC25 was 1 .2 mg/L and the IC5 0 was 1 .7 mg/L .

The test data and summary of statistical analyses for this test are presented in Appendix D .

Table 4 . Effects of Alfoterra 145-8S on Selenastrum capricornutum growth .

Test Treatment (mg/L) Mean Algal Cell Density (cells/mL x 106)

Lab Water Control 2 .58

0 .5 2 .42

1 2 .23

2 0.887*

4 0.652*

8 0.349*

Summary of Statistics

No Observable Effect Concentration (NOEC) = 1 mg/L

IC25 = 1 .22 mg/L

IC5o = 1 .70 mg/L

* Significantly less than the Lab Water Control treatment at p<U .U5 .

Page 6
Pacific EcoRisk Environmental Consulting and Testing

3.5 Chronic Toxicity of Alfoterra L167-4S to Selenastrum capricornutunt

The results of this test are summarized below in Table 5 . There were significant reductions in algal

growth at each of the tested L167-4S surfactant concentrations. The algal growth NOEC was <0 .5

mg/L: the IC25 was 0 .25 mg/L and the ICso was 0 .50 mg/L.

Note -- During the final 24 hrs of testing of this material, a`film' of some type was observed to have

developed on the surface of the 0 .25 mg/L test solutions. It was also observed that the visible algal

growth that is typical of the final 24 hrs of this test was not evident; this is reflected in the low algal

cell density that was measured at this test treatment . Upon consultation with Sasol staff, it was

hypothesized that the film may have been the result of microbial (or other) degradation of the

product, with subsequent inhibition of light penetration into the test replicate test solutions . As a

result of this artifactual interference, the results of this test treatment were not included in the

statistical analyses of these test results .

The test data and summary of statistical analyses for this test are presented in Appendix E .

Table 5 . Effects of Alfoterra L167-4S on Selenastrum capricornutum growth .

Test Treatment (mg/L) Mean Algal Cell Density (cells/mL x 10 )

Lab Water Control 2 .58

0 .25 0.398 a

0 .5 1 .29*

-____ 1 1 .11*

2

1 .09*

-- - 4 0 .086*

Numnlary ot ,~ tatlstlcs

No Observable Effect Concentration (NOEC) _ <0 .5 mg/L

IC25 = 0.25 mg/L

ICso = 0.50 mg/L

* Significantly less than the Lab Water c :ontroi treatment at p<v .u :) .

Page 7
Pacific EcoRisk Environmental Consulting and Testing

3 .6 Chronic Toxicity of Alfoterra L167-7S to Selenastrunt capricornutunc

The results of this test are summarized below in Table 6. There were significant reductions in algal

growth at each of the tested L167-7S surfactant concentrations. The algal growth NOEC was <1

mg/L ; the IC25 was 0 .45 mg/L and the IC50 was 0 .89 mg/L.

Note - During the final 24 hrs of testing of this material, a`film' of some type was observed to have

developed on the surface of the 0 .5 mg/L test solutions. It was also observed that the visible algal

growth that is typical of the final 24 hrs of this test was not evident ; this is reflected in the low algal

cell density that was measured at this test treatment . Upon consultation with Sasol staff, it was

hypothesized that the film may have been the result of microbial (or other) degradation of the

product, with subsequent inhibition of light penetration into the test replicate test solutions . As a

result of this artifactual interference, the results of this test treatment were not included in the

statistical analyses of these test results.

The test data and summary of statistical analyses for this test are presented in Appendix F .

Table 6 . Effects of Alfoterra L167-7S on Selenastrum capricornutum growth .

Test Treatment (mg/L) Mean Algal Cell Density (cells/mI. x 10)

Lab Water Control 2 .58

0 .5 0 .877

1 1 .02*

2 0.910*

4 1 .08 *

8 1 .53 *

Summary of Statistics

No Observable Effect Concentration (NOEC) _ <1 mg/L

IC25 = 0 .45 mg/L

IC5o = 0 .89 mg/L

* Significantly less than the Lab Water Control treatment at

Page 8
Pacific EcoRisk Environmental Consulting and Testing

4. DATA QUALITY CONTROL

Test Conditions - Test conditions (pH, D .O ., temperature, etc.) were all within acceptable limits

for all of the bioassays performed . All analyses were performed according to laboratory Standard

Operating Procedures.

Negative Control - The biological responses for the test organisms at the Lab Control treatments

were within acceptable limits.

5. SUMMARY AND CONCLUSION S

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra 145-8

The results of this test indicated that there were significant reductions in algal growth at the

5,000 and 10,000 mg/L Alfoterra 145-8 (as WAF) concentrations. The algal growth NOEC was

2,500 mg/L ; the IC25 was 5,111 mg/L and the IC50 was 9,378 mg/L .

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra L167- 4

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-4 (as WAF) concentrations . The algal growth NOEC was <625 mg/L ;

the IC25 was 970 mg/L and the IC50 was 1,662 mg/L.

Chronic Toxicity of the Water-Accommodated Fraction (WAF) of Alfoterra L167- 7

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-7 (as WAF) concentrations. The algal growth NOEC was <625 mg/L ;

the IC25 was 1,549 mg/L and the IC50 was 2,632 mg/L .

Chronic Toxicity of Alfoterra 145-8S

The results of this test indicated that there were significant reductions in algal growth at the >2

rngil, Alfoterra 145-8S concentrations. The algal growth NOEC was 1 mg/L; the IC25 was 1 .2

mg/L and the IC50 was 1 .7 mg/L.

Chronic Toxicity of Alfoterra L167-4S

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-4S concentrations. The algal growth NOEC was <0 .5 mg/L ; the IC25

was 0.25 mg/L and the IC50 was 0 .50 mg/L.

Chronic Toxicity of Alfoterra L167-7S

The results of this test indicated that there were significant reductions in algal growth at each of

the tested Alfoterra L167-7S concentrations. The algal growth NOEC was <1 mg/L; the IC25 was

0.45 mg/L and the IC5o was 0 .89 mg/L.

Page 9
Pacific EcoRisk Environmental Consulting and Testing

Appendix A

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra®145- 8

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Repo rt Date: 16 Oct-07 6,07 PM

Test Link : 17-9806-4476/25936

Selenastrum Growth Test Pacific EcoRis k

Test No : 10-5129-8000 Test Type : Cell Growth Duration: 95h

Start Date : 10 Oct-07 04 .25 PM Protocol : EPA/821/R-02-013 (2002) Species : Selenastrum capricornutu m

Ending Date , 14 Oct-07 03 00 PM Dil Water: Algal Culture Media Source: In-House Cultur e

Setup Date : 10 Oct-07 04,25 PM Brine: Not Applicabl e

Sample No: 01-9270-7133 Code : 12479 Client: SASOL

Sample Date : 05 Sep-07 12 00 PM Material : Effluent Project: Special Studie s

Receive Date: 19 Sep-07 12 00 PM Source: SASO L

Sample Age : 35d 4h (25 5° C) Station : ALFOTERRA L145-8

Comments : WAF

Comparison Summary

Analysis Endpoint NOEL LOEL ChV PMSD Method

09-3846-4913 Cell Density 2500 5000 3535 53 1065% Dunnett's Multiple Compariso n

Point Estimate Summa ry

Analysis Endpoint % Effect Conc-mg/L 95% LCL 95% UCL Method

04-9106-0921 Cell Density 1 75 96849 30 55822 429 889 Linear Interpolation

5 379 8425 152 7911 4399 1 8

10 2774 937 N/A 4753 198

15 3550 322 21407 6305 59 4

20 4325 707 2970 936 6815 25 7

25 5111 25 3506 .499 7318 82 6

40 7671 .153 6031 238 8907 19 2

50 9377 756 8377 871 10334 5 6

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD C V

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 9 74E+4 1 95E+5 6 .62 %

625 4 2 42E+6 2 29E+6 2 55E+6 5 36E+4 1 07E+5 4 43%

1250 4 2 95E+6 2 71 E+6 3.20E+6 1 04E+5 2 .07E+5 7 .03 %

2500 4 2 73E+6 2 56E+6 2 90E+6 8 77E+4 1 75E+5 6 42 %

5000 4 2 23E+6 1 98E+6 2 53E+6 1 36E+5 2 72E+5 1222%

10000 4 1 36E+6 1 25E+6 1 43E+6 3 92E+4 7 .83E+4 5 .74%

Cell Density Detai l

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4

0 Lab Water 2 89E+6 3 03E+6 3 15E+6 2 70E+6

625 2 55E+6 2 41 E+6 2 43E+6 2 29E+6

1250 2 88E+6 3 01 E+6 3 20E+6 2.71 E+6

2500 2 87E+6 2 .60E+6 2 90E+6 2 56E+6

5000 2 53E+6 1 .98E+6 2 01 E+6 2 37E+ 6

10000 1 43E+6 1 39E+6 1 25E+6 1 39E+6

000-034-101-2 CETIS"A9 v1 1 2revL Analyst Approval._______ c'
CETIS Analysis Detail

Comparisons : Page 1 of 1

Repo rt Date : 16 Oct-07 6 07 PM

Analysis : 09-3846-4913/2593 6

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Versio n

Cell Density Comparison 17-9806-4476 17-9806-4476 16 Oct-07 6 07 PM CETISv1 .1 2

Method Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMSD

Dunnett's Multiple Comparison C > T Untransformed 2500 5000 0 04 3535 53 1065%

Group Comparison s

Control vs Conc-mg/L Statistic Critical P-Value MSD Decision(O.05 )

Lab Water 625 400844 240711 00018 313316 Significant Effect

1250 -0 0615 240711 08508 313316 Non-Significant Effec t

2500 1 .63065 240711 0 1886 313316 Non-Significant Effec t

5000 550234 240711 00001 313316 Significant Effec t

10000 12 1233 240711 00000 313316 Significant Effec t

ANOVA Tabl e

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(O.05 )

Between 7 200E+12 1 440E+12 5 42 50 000000 Significant Effect

Error 6 099E+11 3 388E+10 1 8

Total 7 .8103E+12 1 .474E+12 2 3

ANOVA Assumptions

Attribute Test Statistic Critical P-Value Decision(0 .01 )

Variances Bartlett 466458 15 08627 0 .45817 Equal Variance s

Distribution Shapiro-Wilk W 0 96232 0 .48681 Normal Distributio n

Data Summa ry Original Data Transformed Dat a

Conc-mg/L Control Type Count Mean Minimum Maximum SD Mean Minimum Maximum S D

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 1 95E+ 5

625 4 2 42E+6 2 29E+6 2 55E+6 1 07E+ 5

125C 4 2 .95E+6 2 .71 E+6 3 20E+6 2 07E+ 5

250C 4 2 73E+6 2 56E+6 2 90E+6 1,75E+ 5

5000 4 2 .23E+6 1 98E+6 2 53E+6 2 .72E+ 5

10000 4 1 36E+6 1 .25E+6 143E+6 7 83C+ 4

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000-034-101 2 CETISTM v1 1 2revL Analyst, ~ Approval -N-c-
CETIS Analysis Detail

Linear Interpolation : Page 1 of 1

Report Date: 16 Oct-07 6-07 PM

Analysis : 04-9106-0921/2593 6

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Linear Interpolation 17-9806-4476 17-9806-4476 16 Oct-07 6-07 PM CETIS0 .1 .2

Linear Interpolation Option s

X Transform Y Transform Seed Resamples Exp 95% CL Method

Linear Linear 453528 280 Yes Two-Point Interpolatio n

Point Estimate s

% Effect Conc-mg/L 95% LCL 95% UCL

1 75 96849 30 55822 429 889

5 379 8425 152 7911 4399 1 8

10 2774.937 N/A 4753 198

15 3550 322 21407 6305 594

20 4325707 2970.936 6815.25 7

25 5111 25 3506 499 7318 826

40 7671 153 6031 238 8907 192

50 9377.756 8377 871 10334 56

Data Summary Calculated Variate

Conc-mg/ Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 94E+6 2 70E+6 3 .15E+6 3 98E+4 1 95E+5

625 4 2 42E+6 2 29E+6 2 55E+6 2 19E+4 1 .07E+ 5

1250 4 2 95E+6 2 71 E+6 3 20E+6 4 24E+4 2 07E+5

2500 4 2 73E+6 2 56E+6 2 90E+6 3 58E+4 1 75E+5

5000 4 2 23E+6 1 98E+6 2 53E+6 5 55E+4 2 72E+5

10000 4 1 36E+6 1 25E+6 1 43E+6 1 .60E+4 7.83E+4

Data Detail

Conc-mg/LControl Type Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 1 0

0 Lab Water 2 .89E+6 3 03E+6 3 15E+6 2 .70E+ 6

625 2 55E+6 2-41 E+6 2 43E+6 2 29E+ 6

1250 2 88E+6 3.01E+6 3 20E+6 2 71 E+ 6

2500 2 87E+6 2 60E+6 2 90E+6 2 56E+ 6

5000 2 .53E+6 1 98E+6 2.01 E+6 2 .37E+ 6

10000 1 43E+6 1 39E+6 1 25E+6 1 39E+6

Graphics

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000-034-101 2 CETIST" v1 1 2revL Analyst. /-V,./\ Approval ~ C'
Pacific EcoRisk. Environmental Consulting and Testi

Selenastrum capricornutum Cell Density Enumeration Data

Client SASOL Initial Count: 10,000 cells/mL

Test Material : ALFOTERRA145-8 Enumerating Scientist : &-19

Test Start Date : b (0(07 Start Time : (6 2 Project # : 1247 9

Test End Date : 6 ( t o

End Time: (~0-0 Test ID #: 25936

Treatment (m-/L Rep A Rep B Rep C Rep D Mean

Lab Water Control o ag 3,~~~ a• ~ 5 9 aA

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( cells/mL x 106)

% CV Date: Time : Signoff:

consistency with Test

Acceptability Criteria and/or

other issues of concern. ( © t "i ~b~ 0 ~p
Pactfic EcoRisk _ Environmental Consulting & Testing,

Selenastrum capricornutum

Client SASOL

Test Material ALFOTERRA 145-8

Algal Toxicity Test Water Quality Data

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Project # 12479 Control/Diluent 4th Ed .No EDTA

Test Treatment (mg/l') Temp (°C p D .O (mg/L) Conductivity

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Page 1 of I
Pacific EcoRisk Environmental Consulting and Testin g

Appendix B

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra® L 167-4

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Repo rt Date : 16 Oct-07 5 58 PM

Test Link : 14-8614-0101/25937

I Selenastrum Growth Test

Test No: 18-0350-5837

Start Date . 10 Oct-07 04 25 PM

Ending Date : 14 Oct-07 03 00 PM

Setup Date: 10 Oct-07 04 25 PM

Sample No : 01-9270-7133

Sample Date : 05 Sep-07 12 00 PM

Receive Date : 19 Sep-07 12 00 PM

Sample Age : 35d 4h (25 5°C)

Comments: WAF

Comparison Summa ry

Analysis Endpoint

04-9404-9358 Cell Density

Point Estimate Summary

Analysis Endpoint

09-3464-7837 Cell Density

Pacific EcoRisk~

Test Type : Cell Growt h

Protocol : EPAl821/R-02-013 (2002 )

Dil Water: Algal Culture Medi a

Brine: Not Applicabl e

Code : 1247 9

Material : Effluen t

Source : SASOL

Station: ALFOTERRA L167-4

NOEL LOEL ChV

< 625 625 N/A

Duration : 95 h

Species : Selenastrum capricornutum

Source : In-House Culture

Client: SASOL

Project: Special Studie s

PMSD

751 %

% Effect Conc-mg/L 95% LCL 95% UC L

1 54 15133 25 92772 158 517 9

5 270 7566 129 6386 792 589 5

10 541 5132 259 2772 846 081 1

15 713 549 448 1484 916 599 1

20 841 5738 627 2784 103719 4

25 969 5987 779 8463 1158 02 1

40 1368 839 1180 609 1543 .71 6

50 1662 343 1508 .298 1802 322

Method

Dunnett's Multiple Comparison

Method

Linear Interpolation

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD CV

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 9 74E+4 1 95E+5 6 .62 %

625 4 2 60E+6 2 50E+6 2 71 E+6 5 35E+4 1 07E+5 4 12%

1250 4 1 88E+6 1 80E+6 2 01 E+6 4 95E+4 9 89E+4 525%

2500 4 6 31 E+5 4 57E+5 7 44E+5 6 20E+4 1 24E+5 1964 %

5000 4 0 .00E+0 0 00E+0 0 00E+0 0 00E+0 0 00E+0 0 00 %

110000 4 0 00E+0 0 00E+0 0 00E+0 0 00E+0 0 .00E+0 000%

Cell Density Detail

Conc-mg/L Control Type Rep I Rep 2 Rep 3 Rep 4

0 Lab Water 2 89E+6 3 03E+6 3 15E+6 2 70E+ 6

625 2 71 E+6 2 67E+6 2 52E+6 2 50E+ 6

1250 2 01 E+6 1 91 E+6 1 82E+6 1 80E+ 6

2500 7 44E+5 6 86E+5 4 57E+5 6 38E+ 5

5000 0 00E+0 0 00E+0 0 00E+0 0 00E+0

10000 0 00E+0 0 00E+0 0 00E+0 0 00E+0

000-034-101-2 CETIST"" v1 1 2revL Analys/~_q_/\ Approval .--'<_
CETIS Analysis Detai

Comparisons : Page 1 of 1

Report Date : 16 Oct-07 5 58 PM

Analysis: 04-9404-9358/2593 7

Fselenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Comparison 14-8614-0101 14-8614-0101 16 Oct-07 5 58 PM CETISv1 .1 . 2

Method Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMS D

Dunnett's Multiple Comparison C > T Untransformed <625 625 NIA 7 51 %

Group Comparisons

Control vs Conc-mg/L Statistic Critical P-Value MSD Decision(O.05 )

Lab Water 625 351627 2 28739 0 0056 220851 Significant Effect

1250 10 9527 228739 00000 220851 Significant Effect

2500 23 9277 2 28739 0 0000 220851 Significant Effect

ANOVA Table

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(O.05)

Between 1 254E+13 4 18E+12 3 224 19 000000 Significant Effect

Error 2.237E+11 1 864E+10 1 2

Total 1.2763E+13 4 .199E+12 1 5

ANOVA Assumptions

Attribute Test Statistic Critical P-Value Decision(0 .01 )

Variances Bartlett 1 61412 11 .34487 065619 Equal Variance s

Distribution Shapiro-Wilk W 0 97274 0 88081 Normal Distributio n

Data Summary Original Data Transformed Dat a

Conc-mg/LControl Type Count Mean Minim um Maximum SD Mean Minimum Maximum S D

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 1 .95E+ 5

625 4 2 60E+6 2 50E+6 2 .71 E+6 1 07E+ 5

1250 4 1 88E+6 1 80E+6 2 01 E+6 989E+4

2500 4 6 31 E+5 4 .57E+5 7 .44E+5 1 24E+ 5

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000-034-101-2 CETIST"' v1 1 2revL Analystk'~ Approval~~!
CETIS Analysis Detai

Linear Interpolation : Page 1 of 1

Report Date : 16 Oct-07 5 .58 PM

Analysis : 09-3464-7837/2593 7

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Linear Interpolation 14-8614-0101 14-8614-0101 16 Oct-07 5 :58 PM CETISv1 .1 2

Linear Interpolation Option s

X Transform YTransfo rm Seed Resamples Exp 95% CL Metho d

Linear Linear 8144900 280 Yes Two-Point Interpolatio n

Point Estimates

% Effect Conc-mg/L 95% LCL 95% UCL

1 54 15133 25 92772 158 .517 9

5 270 7566 129 6386 792,589 5

10 541 5132 259 2772 846 081 1

15 713 549 448 .1484 916 5991

20 841.5738 627 .2784 1037 194

25 969 5987 779 .8463 1158 021

40 1368 839 1180 609 1543 716

50 1662 343 1508 298 1802 322

Data Summary Calculated Variate

Conc-mg/ Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 3 98E+4 1 95E+5

625 4 2 60E+6 2 50E+6 2 71 E+6 2 19E+4 1 07E+5

1250 4 1 88E+6 1,80E+6 2 01 E+6 2 02E+4 9 89E+4

2500 4 6.31 E+5 4 57E+5 7 44E+5 2 53E+4 1 24E+5

5000 4 0 00E+0 0 00E+0 0 00E+0 0 00E+0 0 00E+0

10000 4 0 DOE+0 0 00E+0 0 00E+0 0 00E+0 0 .00E+ 0

Data Detail

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 1 0

0 Lab Water 2 .89E+6 3 03E+6 3 15E+6 2 70E+ 6

625 2 71 E+6 2.67E+6 2 52E+6 2 50E+6

1250 2 01 E+6 1 .91E+6 1 82E+6 1 80E+ 6

2500 7 44E+5 6 86E+5 4 57E+5 6 38E+ 5

5000 0 00E+0 0 00E+0 0 00E+0 0 .00E+ 0

110000 0 00E+0 0 00E+0 0 00E+0 0 .00E+ 0

Graphics

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000-034-101-2 CETIST"' v1 1 2revL Analyst. Approval,
Pacific EcoRrck Environmental Consulting and Testin g

Client

Test Material

Selenastrum capricornutum Cell Density Enumeration Data

SASOL

A LFOTERRAL 167-4

Test Start Date . ~Q (0(0 7

Test End Datz~ : o p

L!If

Start Time : I k

End Time : I ~- p'(J

Initial Count: 10,000 cells/mL

Enumerating Scientist : (51-4)

Project #: 12479

Test ID #: 25937

Treatment (mg/L) Rep A Rep B Rep C Rep D Mean

Lab Water Control Z. ~d ~ 6 ~ _ O Z 9 ~_ I S Z Z-~0 9`f Z- g ~

625 6 -7 a, SIy 2- ~ o ;L 2~bo Z

1 25 0 2- .v(3 I .~o~ -7 S

!500 o-`t s

--

;ooo ~ 0 ~ CD -

10,000 o v ~ 0 a

Control Mean

This datasheet has been

Density % CV Date: Time : Signoff:

reviewed for completeness and (cells/mL x 10`)

consistency with Test

Acceptability Criteria and/or

other issues of concern. C3 /1 ~ ~ - ~ ( 0 (< <-( /b 7 p
PacLfic EcoRisk Environmental Consulting & Testin g

Selenastrum capricornutum Algal Toxicity Test Water Quality Dat a

Client

Test Material

SASOL Test ID # 25937 TCR #: (, e 1 6 1

Project # 12479 Control/Diluent 4th Ed.No EDTA

Test Treatment (mgiL) Temp (°C) pH D .O . (mg/L) Conductivity

(YS/CM) Sign-Off

Lab Water Control ~ . 4 9 (e Date ~„~ C Q+ro~

625 y• ~.~ S P a V

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I

Initial Test Conditions Alkalinity Hardness Light Intensity (ftc )

ALFOTERRA L16?-4

Pace I of I
Pacific EcoRisk Environmental Consulting and Testing

Appendix C

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra® L 167- 7

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Repo rt Date : 26 Oct-07 2 46 PM

Test Link : 10-3233-1941/25938

Selenastrum Growth Test Pacific EcoRis k

Test No . 13-6393-6192 Test Type : Cell Growth Duration : 95 h

Start Date : 10 Oct-07 04 25 PM Protocol : EPA/821 /R-02-013 (2 002) Species: Selenastrum capricornutu m

Ending Date : 14 Oct-07 03 00 PM Dil Water: Algal Culture Media Source : In-House Culture

Setup Date: 10 Oct-07 04 25 PM Brine: Not Applicable

Sample No : 01-9270-7133 Code : 12479 Client: SASOL

Sample Date : 05 Sep-07 12 00 PM Material : Effluent Project: Special Studie s

Receive Date : 19 Sep-07 12 00 PM Source : SASOL

Sample Age : 35d 4h (25 .5 °C) Station: ALFOTERRA L167-7

Comparison Summary

Analysis Endpoint NOEL LOEL ChV PMSD Method

02-1449-4219 Cell Density < 625 625 N/A 7 .53% Dunnett's Multiple Compariso n

Point Estimate Summary

Analysis Endpoint % Effect Conc-mg/L 95% LCL 95% UCL Method

11-8286-6834 Cell Density 1 34 82714 21 9084 58 46178 Linear Interpolation

5 174 1357 109 542 292 308 9

10 348 2714 219 084 584 617 8

15 522 407 328 626 1834 55 8

20 1337 13 53 81536 1665 268

25 1549 207 1197 .784 1858 247

40 2185 436 1965 798 2460 202

50 2632 473 2386 .688 2920 60 1

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD C V

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 9 74E+4 1 .95E+5 6 .62 %

625 4 2 23E+6 2 17E+6 2 28E+6 2 44E+4 4 88E+4 2 19%

1250 4 2 60E+6 2 33E+6 2 72E+6 9 22E+4 1 84E+5 7 10%

2500 4 1 55E+6 1 51 E+6 1 66E+6 3 65E+4 7 .29E+4 472%

5000 4 1 13E+5 2 40E+4 2 22E+5 4 66E+4 9 33E+4 82 90 %

10000 4 0 00E+0 0 00E+0 0 00E+0 0 .00E+0 0 00E+0 000%

Cell Density Detai l

Conc-mg/L Control Type Rep I Rep 2 Rep 3 Rep 4

0 Lab Water 2 89E+6 3 03E+6 3 15E+6 2 70E+6

625 2 17E+6 2 28E+6 2 22E+6 2 25E+6

1250 2 72E+6 2 71 E+6 2 33E+6 2 63E+6

2500 1 66E+6 1 52E+6 1 51 E+6 1 51 E+ 6

5000 2 40E+4 1 57E+5 2 22E+5 4 70E+4

10000 0 00E+0 0 00E+0 0 00E+0 0 00E+0

000-034-101-2 CETIST"" v1 1 2revL Analyst ~ Approval ~
CETIS Analysis Detail

Comparisons : Page 1 of 1

Report Date : 26 Oct-07 2 .46 PM

Analysis : 02-1449-4219/25938

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Comparison 10-3233-1941 10-3233-1941 26 Oct-07 11 28 AM CETISv1 1 2

Method Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMS D

Dunnett's Multiple Comparison C > T Untransformed <625 625 N/A 7 53%

Group Comparisons

Control vs Conc-mglL Statistic Critical P-Value MSD Decision(O.05 )

Lab Water 625 755746 2,35615 00000 221431 Significant Effect

1250 367631 235615 00039 221431 Significant Effect

2500 14 8409 2 .35615 0 .0000 221431 Significant Effect

5000 30 .1021 2 35615 00000 221431 Significant Effec t

ANOVA Tabl e

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(0 .05 )

Between 1 999E+13 4 998E+12 4 282 93 0 00000 Significant Effect

Error 2 65E+11 1 .766E+10 1 5

Total 2 0256E+13 5 015E+12 1 9

ANOVA Assumption s

A ttribute Test Statistic Critical P-Value Decision(0 .01 )

Variances Bartlett 659811 13 .27670 0 15871 Equal Variance s

Distribution Shapiro-Wilk W 0 .94343 027815 Normal Distribution

Data Summa ry Original Data Transformed Data

Conc-mg/L Control Type Count Mean Minimum Maximum SD Mean Minimum Maximum S D

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 I 95E+5

625 4 2 23E+6 2 17E+6 2 28E+6 4 88E+4

1250 4 2 60E+6 2 33E+6 2 72E+6 1 84E+5

2500 4 1 55E+6 1 51 E+6 1 .66E+6 7 .29E+4

5000 4 1 13E+5 2 40E+4 2 22E+5 9 33E+4

Graphics

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Conc-mg/L Rankit s

000-034-101-2 CETIST'1 v1 1 2revL Analyst-~ Approval ~ C-
CETIS Analysis Detail

Linear Interpolation : Page 1 of 1

Report Date: 26 Oct-07 2 46 PM

Analysis : 11-8286-6834/25938

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Linear Interpolation 10-3233-1941 10-3233-1941 26 Oct-07 1128 AM CETISv1 1 2

Linear Interpolation Option s

X Transform Y Transfo rm Seed Resamples Exp 95% CL Method

Linear Linear 7055475 280 Yes Two-Point Interpolatio n

Point Estimate s

% Effect Conc-mg/L 95% LCL 95% UCL

1 34 82714 21 .9084 58 4617 8

5 174 1357 109 542 292 308 9

10 348 2714 219 084 584 .617 8

15 522 407 328 626 1834 558

20 1337.13 53 .81536 1665.26 8

25 1549.207 1197 784 1858 247

40 2185.436 1965 798 2460 202

50 2632 473 2386 688 2920 60 1

Data Summary Calculated Variat e

Conc-mg/ Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 94E+6 2 70E+6 3 15E+6 3.98E+4 1 95E+5

625 4 2 23E+6 2 17E+6 2 28E+6 9 97E+3 4 88E+4

1250 4 2 60E+6 2 33E+6 2 72E+6 3 76E+4 1 84E+5

2500 4 1 55E+6 1 51 E+6 1 66E+6 1 49E+4 7 29E+4

5000 4 1 13E+5 2 40E+4 2 22E+5 1 90E+4 9 33E+4

10000 4 0 00E+0 0 00E+0 0 00E+0 0 00E+0 0 00E+0

Data Detail

Conc-mg/L Control Type Rep I Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 1 0

0 Lab Water 2 89E+6 3.03E+6 3 15E+6 2 .70E+ 6

625 2 17E+6 2 28E+6 2 22E+6 2 25E+ 6

1250 2 72E+6 2 71 E+6 2 .33E+6 2 .63E+ 6

2500 1 66E+6 1 .52E+6 1 51 E+6 1 51 E+6

5000 2 40E+4 1 57E+5 2 22E+5 4 70E+4

110000 0 00E+0 0 .00E+0 0.00E+0 0 .00E+0

Graphics

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Conc-mg/ L

000-034-101-2 CETIST" v1 12revL Analyst . . Approval~~
Pacific EcoRt s k

Client

Test MaterialEnvironmental ConsultinQ and Test i

Selenastrum capricornutum Cell Density Enumeration Data

SASOL

A LFOTERRA L 167- 7

Test Start bate : i ~36 i0 0 7

Test End Date . o(f 0 7

Start Time : b Z

End Time : I ~ fJ 0

Initial Count : 10,000 cells/mL

Enumerating Scientist : (,-, r)

Project # : 12479

Test ID #: 25938

Treatrr ent (mg/L) Rep A Rep B Rep C Rep D Mean

Lab Water Control

I

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10,000 o o

Control Mean

This datasheet has bee n

reviewed for completeness and

Density

(cells/mL x 10`)

% CV Date: Time : Signoff:

consistency with Test

Acceptability Criteria and/or r

other issues of concern . 2 . -7 ~ -Z~ (~

~
EcoRisk Environmental Consulting & T

Selenastrum capricornutum Algal Toxicity Test Water Quality Data

Client

Test Material

SASOL Test ID # 25938 TCR # (Q 12 k 5 (

Project # 12479 Control/Diluent . 4th Ed No EDT A

Test Treatment (mgiL) Temp CI pH D .O (mg/L) Conductivit y

(,U S/cm) Sign-Off

Lab Water Control 10;? Date

62~'5 '

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Initial Test Conditions Alkalinity Hardness Light Intensity (ftc)

SALFOTERRA L167-7

Paeelofl
Pacific EcoRisk Environmental Consulting and Testing

Appendix D

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra®145-8S

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Repo rt Date: 16 Oct-07 5 45 PM

Test Link : 01-4225-6361/25939

Selenastrum Growth Test Pacific EcoRis k

Test No : 13-0482-5674 Test Type : Cell Growth Duration: 96 h

Sta rt Date 10 Oct-07 05 1 5 PM Protocol : EPA/821/R-02-013 (2002) Species : Selenastrum capncornutu m

Ending Date : 14 Oct-07 05 00 PM Dil Water : Algal Culture Media Source: In-House Cultur e

Setup Date : 10 Oct-07 05 15 PM Brine: Not Applicabl e

Sample No: 01-9270-7133 Code : 12479 Client: SASOL

Sample Date : 05 Sep-07 12 00 PM Material : Effluent Project: Special Studies

Receive Date - 19 Sep-07 12 CO PM Source : SASOL

Sample Age : 35d 5h (25 5°C) Station: ALFOTERRA L145-8 S

Comparison Summary

Analysis Endpoint NOEL LOEL ChV PMSD Method

03-9589-8788 Cell Density 1 2 1 41421 15 01 % Dunnett's Multiple Compariso n

Point Estimate Summa ry

Analysis Endpoint % Effect Conc-mg/L 95% LCL 95% UCL Method

10-7402-8640 Cell Density 1 008210191 0005009478 0 816558 Linear Interpolatio n

5 04105096 002504739 09157482

10 0 7621586 008541107 1 20988 9

15 1 027917 02636756 1 18059 1

20 1 124003 09347823 1 28015 4

25 1 220089 1 066474 1 387922

40 1 508349 1 .345501 1 .807967

50 1 700522 1 508548 2 10053 5

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD CV

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 6 51 E+4 1 .30E+5 5 05%

05 4 2 42E+6 2 17E+6 2 86E+6 1 54E+5 3 07E+5 1268 %

1 4 2 23E+6 2 02E+6 2 35E+6 7 38E+4 1 48E+5 662%

2 4 8 87E+5 4 98E+5 1 26E+6 1 91 E+5 3 82E+5 4309%

4 4 6 52E+5 5 08E+5 7 89E+5 5 86E+4 1 17E+5 1797%

18 4 3 49E+5 2 18E+5 4 65E+5 6 .55E+4 1 31 E+5 37 .58 %

Cell Density Detai l

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4

0 Lab Water 2 60E+6 2 75E+6 2 45E+6 2 51 E+6

05 2 86E+6 2 17E+6 2 26E+6 2 39E+6

1 2 02E+6 2 30E+6 2 35E+6 2 25E+6

2 1 26E+6 1 17E+6 4 98E+5 6 23E+5

4 6 83E+5 7 89E+5 5 08E+5 6 26E+5

8 4 65E+5 2 18E+5 4 58E+5 2 54E+5

000-034-101-2 CETIST"^ v1 1 2revL Analy Approval . + ~
CETIS Analysis Detail

Comparisons : Page 1 of 1

Report Date : 16 Oct-07 5:45 PM

Analysis : 03-9589-8788/2593 9

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Comparison 01-4225-6361 01-4225-6361 16 Oct-07 5-45 PM CETISv1 .1 . 2

Method Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMS D

Dunnett's Multiple Comparison C > T Untransformed 1 2 100 1.41421 15.01 %

Group Comparisons

Control _ vs Conc-mg/L Statistic Critical P-Value MSD Decision(O.05 )

Lab Water 0 5 097672 240711 04322 386923 Non-Significant Effec t

' 2 17274 240711 00770 386923 Non-Significant Effec t

2 10 5184 240711 00000 386923 Significant Effect

4 11 9851 2 40711 0 0000 386923 Significant Effect

8 13 8685 240711 0 .0000 386923 Significant Effect

ANOVA Table

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(0 .05)

Between 1 984E+13 3 968E+12 5 76 79 000000 Significant Effect

Error 9 302E+11 5 168E+10 1 8

Total 2 0770E+13 4 02E+12 2 3

ANOVA Assumptions

Attribute Test Statistic Critical P-Value Decision(0 .01 )

Variances Bartlett 734334 15 08627 019633 Equal Variances

Distribution Shapiro-Wilk W 0 98341 0 .94947 Normal Distribution

Data Summary Original Data Transformed Data

Conc-mg/L Control Type Count Mean Minimum Maximum SD Mean Minimum Maximum SD

0 Lab Water 4 2 58E+6 2 .45E+6 2 75E+6 1 30E+ 5

0 5 4

1 4

2 42E+6 2 17E+6 2 .86E+6 3 07E+ 5

2 4

2 .23E+6 2 02E+6 2 35E+6 1 48E+ 5

4 4

8 87E+5 4 98E+5 1 .26E+6 3 .82E+ 5

8 4

6 52E+5 5 08E+5 7 89E+5 1 17E+ 5

3 49E+5 2 18E+5 4 .65E+5 1 31 E+ 5

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000-034-101 2 CETIST"' v1 1 2revL Analyst iL Approval 1

(_
CETIS Analysis Detail

Linear Interpolation : Page 1 of 1

Report Date: 16 Oct-07 5 45 PM

Analysis : 10-7402-8640/2593 9

Selenastrum Growth Test Pacific EcoRisk

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Linear Interpolation 01-4225-6361 01-4225-6361 16 Oct-07 5 .45 PM CETISv1 .1 . 2

Linear Interpolation Option s

X Transform Y Transform Seed Resamples Exp 95% CL Method

Linear Linear 7747401 280 Yes Two-Point Interpolation

Point Estimates

% Effect Conc-mglL 95% LCL 95% UCL

1 008210191 0 .005009478 081655 8

5 04105096 002504739 09157482

10 07621586 0 .08541107 1 .20988 9

15 1 027917 02636756 1 180591

20 1 124003 0 .9347823 1 280154

25 1 220089 1 066474 1 387922

40 1 508349 1 .345501 1 807967

50 1 700522 1 .508548 2 100535

Data Summa ry Calculated Variat e

Conc-mgl Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 2 66E+4 1 30E+5

0 .5 4 2 42E+6 2 17E+6 2 86E+6 6 27E+4 3 07E+5

1 4 2 23E+6 2 02E+6 2 35E+6 3 01 E+4 1 48E+5

2 4 8 87E+5 4 98E+5 1 26E+6 7 80E+4 3 82E+5

4 4 6 52E+5 5 08E+5 7 89E+5 2 39E+4 1 17E+5

8 4 3 49E+5 2 18E+5 4 65E+5 2 68E+4 1 .31 E+ 5

Data Detai l

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 1 0

0 Lab Water 2 60E+6 2 75E+6 2 45E+6 2 51 E+ 6

05 2 86E+6 2 17E+6 2 26E+6 2 39E+ 6

1 2 02E+6 2 30E+6 2 35E+6 2 25E+ 6

2 1 26E+6 1 17E+6 4 98E+5 6 23E+ 5

4 6 .83E+5 7 89E+5 5 08E+5 6 .26E+ 5

8 4 .65E+5 2.18E+5 4 58E+5 2 .54E+5

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000-034-101 2 CETISTM' v1 1 .2revL Analystl V Approval . --f-t;
Pacific EcoRlsk Environmental Consulting and T

Selenastrum capricornutum Cell Density Enumeration Data

Client

Test Materia

l Test Start Date d(, d

Test End Date. ( 0 tL-q 0

ALFOTERRA 145-8S

Start Time : 1 I ; (jEnd Time : J '7 QV

Initial Count : 10,000 eel I s/mL

Enumerating Scientist : t

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Project #: 12479

Test ID #: 25939

Trearr,ent (mg/L) Rep A Rep B Rep C Rep D Mea n

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This datasheet has been

Densit y

(cells/mL x 10`)

% CV Date: Time : Signoff:

rev iewed for completeness an d

consistency with Test

Acceptability Criteria and/o r

other issues of concern . t o

SASOL
Pacific EcoRisk Environmental Consulting & Test~

Selenastrum capricornutum Algal Toxicity Test Water Quality Data

Client SASOL Test ID # 25939 TCR # :

Test Material ALFOTERRA L145-8S Project # 12479 Control/Diluent. 4th Ed .No EDTA

Test Treatment (mg/L) Temp (°C) pH D .O (mg/L) Conductivit y

(}tS/cm) Sign-Off

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Pacific EcoRisk Environmental Consulting and Testin g

Appendix E

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra® L 167-4S

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Report Date : 30 Oct-07 11 51 AM

Test Link: 19-5672-1120/25940

Selenastrum Growth Test Pacific EcoRis k

Test No : 04-2440-8719 Test Type : Cell Growth Duration: 96 h

Start Date : 10 Oct-07 05 15 PM Protocol : EPA/821 /R-02-013 (2002) Species : Selenastrum capricornutu m

Ending Date . `4 Oct-07 05 00 PM Dil Water: Algal Culture Media Source : In-House Cultur e

Setup Date: "0 Oct-07 05 15 PM Brine: Not Applicabl e

Sample No : 01-9270-7133 Code : 12479 Client: SASOL

Sample Date : 05 Sep-07 12 00 PM Material : Effluent Project: Special Studies

Receive Date: 19 Sep-07 12 00 PM Source: SASOL

Sample Age : 35d 5h (25 5°C) Station: ALFOTERRA L167-4S

Comparison Summary

Analysis Endpoint NOEL LOEL ChV PMSD Method

09-3256-4663 Cell Density <0 5 0 5 N/A 1536% Dunnett's Multiple Comparison

Point Estimate Summa ry

Analysis

--

E ndpoint

--- - -

% Eff e

--- --------

ct Conc-mg/L 95% LCL 95% UCL Method

08-9626-4738 Cell Density 1 0009978711 0 00718025 001388845 Linear Interpolatio n

5 0 .04989355 0 03590125 0 .0694422 6

10 009978711 0 0718025 0 138884 5

15 0 1496807 0 1077038 02083268

20 0 1995742 0 143605 0277769

25 02494678 0 .1795063 0347211 3

40 0 3991484 0 28721 05555381

50 04989356 03590125 1 33888 6

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD C V

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 6 51 E+4 1 .30E+5 505%

0 25 4 3 98E+5 2 83E+5 4 76E+5 4 11E+4 8 21 E+4 2065%

05 4 1 29E+6 7 08E+5 1 58E+6 1 99E+5 3 .98E+5 30 91 %

1 4 1 11E+6 7 30E+5 1 39E+6 1 39E+5 2 .78E+5 25 12 %

2 4 1 09E+6 8 95E+5 1 30E+6 8 37E+4 1 67E+5 15 30 %

14 4 8 63E+4 1 70E+4 1 20E+5 2 34E+4 4 .69E+4 54 37 %

Cell Density Detai l

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4

0 Lab Water 2 60E+6 2 75E+6 2 45E+6 2 51 E+ 6

0 25 4 28E+5 4 76E+5 4 04E+5 2 83E+5

05 1 51 E+6 7 08E+5 1 35E+6 1 58E+6

1 1 39E+6 1 20E+6 1 10E+6 7 30E+5

2 1 12E+6 1 30E+6 1 06E+6 8 95E+5

4 1 .08E+5 1 20E+5 1 00E+5 1 70E+4

000-034-101 -2 CETIS111 v1 1 2revL Analyst tN/~ Approval~~
CETIS Analysis Detail

Comparisons : Page 1 of 1

Report Date : 30 Oct-07 11 48 AM

Analysis : 09-3256-4663/2594 0

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Li nk Control Link Date Analyzed Versio n

Cell Density Comparison 19-5672-1120 19-5672-1120 30 Oct-07 11 :47 AM CETISv1 1 2

Method __ Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMSD

Dunnett's Multiple Comparison C > T Untransformed <0 5 05 N/A 15 36°/o

Group Comparison s

Control vs Conc-mg/L Statistic Critical P-Value MSD Decision(O .05 )

Lab Water 05 768547 235615 00000 396015 Significant Effec t

1 8 75641 2 35615 0 .0000 396015 Significant Effec t

2 882632 235615 0 .0000 396015 Significant Effect

4 14 8251 235615 0 .0000 396015 Significant Effect

ANOVA Tabl e

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(0 .05 )

Between 1 265E+13 3 162E+12 4 55 97 000000 Significant Effec t

Error 8 475E+11 5 65E+10 1 5

Total 1 3496E+13 3 219E+12 1 9

ANOVA Assumption s

Attribute Test Statistic Critical P-Value Decision(0 .01 )

Variances Bartlett 10 08229 13 27670 003906 Equal Variance s

Distribution Shapiro-Wilk W 0 91063 0 .06552 Normal Distributio n

Data Summary Original Data Transformed Dat a

Conc-mg/L Control Type Count Mean Minimum Maximum SD Mean Minimum Maximum S D

0 Lab Water 4 2 58E+6 2 45E+6 2 .75E+6 1 30E+ 5

0 5 4

1 4

1 29E+6 7 .08E+5 1 58E+6 3 98E+5

2 4

1 11E+6 7 30E+5 1 39E+6 2 78E+5

4 4

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8 63E+4 1 70E+4 1 20E+5 4 69E+4

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000-034-101 -2 CETIST" v1 1 2revL Analyst- / -,A Approval IIIrA_
CETIS Analysis Detai

Linear Interpolation : Page 1 of 1

Repo rt Date : 30 Oct-07 11,48 AM

Analysis : 08-9626-4738/25940

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

CeII Density Linear Interpolation 19-5672-1120 19-5672-1120 30 Oct-07 11 48 AM CETISv1 1 2

Linear Interpolation Option s

X Transfor_m Y Transform Seed Resamples Exp 95% CL Method

Linear Linear 7055475 280 Yes Two-Point Interpolatio n

Point Estimate s

% Effect Conc-mg/L 95% LCL 95% UC L

1 0009978711 0 .00718025 001388845

5 0.04989355 003590125 00694422 6

10 009978711 00718025 0138884 5

15 0 1496807 0 .1077038 0.208326 8

20 0 1995742 0143605 0277769

25 02494678 01795063 0347211 3

40 03991484 028721 05555381

50 04989356 0 .3590125 1 338886

Data Summary Calculated Variat e

Conc-mg/ Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 2 66E+4 1 30E+5

0 5 4 1 29E+6 7 08E+5 1 58E+6 8 12E+4 3 98E+5

1 4 1 11E+6 7 30E+5 1 39E+6 5 67E+4 2 78E+5

2 4 109E+6 8 95E+5 1 30E+6 3 42E+4 1 .67E+ 5

4 4 863E+4 170E+4 1 20E+5 9 57E+3 4 69E+4

Data Detai l

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 1 0

0 Lab Water 2 .60E+6 2.75E+6 2 45E+6 2 .51 E+ 6

0 5 1 51 E+6 7 08E+5 1 35E+6 1 58E+ 6

1 1 .39E+6 1 .20E+6 110E+6 7 .30E+ 5

2 1 12E+6 1 30E+6 1 06E+6 8 95E+5

4 1 .08E+5 1 .20E+5 1 .00E+5 1 .70E+ 4

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000-034-101 2 CETIST"' v1 1 .2revL Analyst. AM1 Approval
Pacific EcoRisk Environmental Consulting and Testin g

Client

Test Material

Selenastrum capricornutum Cell Density Enumeration Data

Test Start Date In ( to 07

Test End Date : t o'?

SASOL

ALFOTERRA L1674S

Start Time : 1 7 ; ICEnd Time : ~ -1 Q'b

Treatment (mg/L) Rep A Rep B Rep C Rep D Mea n

Lab Water Control 2- (,0 (

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This datasheet has been

Density

(cells/mL x 106)

% CV Date: Time : Signoff:

rev iewed for completeness and

consistency with Test

Acceptability Criteria and/o r

other issues of concern. 5-7 $

f 0/ (Lt ~O -7 11 f

"

Initial Count: 10,000 cells/mL

Enumerating Scientist: (;L- % 7

Project # : 12479

Test ID #: 25940
Pacific EcoRisk Environmental Consulting & Testing

Selenastrum capricornutum Algal Toxicity Test Water Quality Data

Client SASOL

Test Material ALFOTERRA Ll /67-4S

Test ID # . 25940 TCR # -

Project # 12479 Control /Dtluent .

1~•7i5

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4th Ed .No EDTA

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o

~~
Pacific EcoRisk Environmental Consulting and Testing

Appendix F

Test Data and Summary of Statistics for the

Evaluation of the Toxicity of Alfoterra® L 167-7S

to Selenastrum capricornutum
CETIS Test Summary

Page 1 of 1

Report Date : 26 Oct-07 2 49 PM

Test Link : 01-9135-1216/2594 1

Selenastrum Growth Test Pacific EcoRis k

Test No: 02-8362-7350 Test Type : Cell Growth Duration : 96 h

Sta rt Date 10 Oct-07 05 1 5 PM Protocol : EPA/821/R-02-013 (20 02) Species : Selenastrum capricornutu m

Ending Date : 14 Oct-07 05 00 PM Dii Water: Algal Culture Media Source: In-House Cultur e

Setup Date : 10 Oct-07 05 15 PM Brine: Not Applicable

Sample No . 01-9270-7133 Code : 12479 Client: SASOL

Sample Date : 05 Sep-07 12 00 PM Material : Effluent Project : Special Studie s

Receive Date : 19 Sep-07 12 .00 PM Source: SASOL

Sample Age : 35d 5h (25 5°C) Station : ALFOTERRA L167-7 S

Comparison Summary

Analysis Endpoint NOEL LOEL ChV PMSD Method

05-7597-2072 Cell Density < 1 1 N/A 27 .64% Dunnett's Multiple Compariso n

Point Estimate Summa ry

Analysis Endpoint % Effe ct Conc-mg/L 95% LCL 95% UCL Method

08-8517-2287 Cell Density 1 001784701 001458114 00260409 Linear Interpolation

5 008923503 007290568 0 .130204 5

10 0 1784701 0 1458114 0 .26040 9

15 02677051 02187171 0 .390613 5

20 03569401 02916227 0 .52081 8

25 04461752 03645284 0651022 5

40 07138802 05832455 1 04163 6

50 0 .8923503 07290568 N/A

Cell Density Summa ry

Conc-mg/L Control Type Reps Mean Minimum Maximum SE SD C V

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 6 51E+4 1 30E+5 5 05 %

05 4 8 77E+5 7 72E+5 9 76E+5 5 41 E+4 1 08E+5 12 34%

1 4 1 02E+6 7 16E+5 1 64E+6 2 11 E+5 4 23E+5 4164%

2 4 9 10E+5 2 81 E+5 1 80E+6 3 19E+5 6 37E+5 70 07%

4 4 1 08E+6 8 75E+5 1 54E+6 1 55E+5 3 10E+5 28 65 %

8 4 1 53E+6 8 90E+5 1 91 E+6 2 33E+5 4 66E+5 3050 %

Cell Density Detail

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4

0 Lab Water 2 60E+6 2 75E+6 2 45E+6 2 51E+ 6

0 5 9 76E+5 9 65E+5 7 72E+5 7 96E+ 5

1 1 64E+6 8 40E+5 8 63E+5 7.16E+ 5

2 8 14E+5 1 80E+6 2 81 E+5 7 46E+ 5

4 9 45E+5 9 66E+5 1 54E+6 8 75E+ 5

8 1 47E+6 8 90E+5 1 84E+6 1 91 E+6

000-034-101 2 CETIST"' v1 1 2revL Analyst~ Approvall
CETIS Analysis Detail

Comparisons : Page 1 of 1

Report Date : 26 Oct-07 2-49 PM

Analysis : 05-7597-2072/2594 1

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Comparison 01-9135-1216 01-9135-1216 17 Oct-07 10-58 AM CETISv1 1 2

Method Alt H Data Transform Zeta NOEL LOEL Toxic Units ChV PMS D

Dunnett's Multiple Comparison C> T Untransformed <1 1 N/A 2764%

Group Comparisons

Control vs Conc-mg/L Statistic Critical P-Value MSD Decision(0 .05)

Lab Water 1 5 16672 235615 0 0002 712653 Significant Effect

2 551635

4 4 94438

8 347561

23561 5

2 3561 5

2 35615

0000 1

0 000 3

0 .0058

712653

712653

712653

Significant Effect

Significant Effec t

Significant Effec t

ANOVA Tabl e

Source Sum of Squares Mean Square DF F Statistic P-Value Decision(0 .05 )

Between 7 563E+12 1 891E+12 4 10 33 000032 Significant Effect

Error 2 745E+12 1 83E+11 1 5

Total 1 0307E+13 2 074E+12 1 9

ANOVA Assumption s

Attribute Test Statistic Critical P-Value Decision(0,01 )

Variances Bartle tt 5 50097 13 27670 023964 Equal Variance s

Distribution Shapiro-Wilk W 0 92772 0 13959 Normal Distributio n

Data Summary Original Data Transformed Dat a

Conc-mg/L Control Type Count Mean Minimum Maximum SD Mean Minimum Maximum SD

0 Lab Water 4 2 58E+6 2 45E+6 2 75E+6 1 30E+5

1 4 1 02E+6 7 16E+5 1 64E+6 4 23E+ 5

2 4 910E+5 2 .81E+5 1 80E+6 6 37E+ 5

4 4 1 08E+6 8 75E+5 1 54E+6 3 10E+5

8 4 1 53E+6 8 90E+5 1 91 E+6 4 66E+ 5

Graphics

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Conc-mg/L Rankits

000-034-101 2 CETIST" v1 1 2revL Analyst Approval
CETIS Analysis Detail

Linear Interpolation : Page 1 of 1

Report Date : 26 Oct-07 2 49 PM

Analysis : 08-8517-2287/2594 1

Selenastrum Growth Test Pacific EcoRis k

Endpoint Analysis Type Sample Link Control Link Date Analyzed Version

Cell Density Linear Interpolation 01-9135-1216 01-9135-1216 17 Oct-07 10 58 AM CETISv1 1 2

Linear Interpolation Option s

X Transform Y Transform Seed Resamples Exp 95% CL Method

Linear Linear 5334240 280 Yes Two-Point Interpolatio n

Point Estimate s

% Effect Conc-mg/L 95% LCL 95% UCL

1 001784701 001458114 00260409

5 008923503 007290568 0 130204 5

10 0 1784701 0 1458114 026040 9

15 02677051 02187171 0.390613 5

20 03569401 0 .2916227 052081 8

25 04461752 03645284 0.651022 5

40 07138802 05832455 1 041636

50 0 8923503 0 7290568 N/A

Data Summary Calculated Variate

Conc-mg/ Control Type Count Mean Minimum Maximum SE S D

0 Lab Water 4 2 58E+6 2 45E+6 2 .75E+6 2 66E+4 1 30E+5

1 4 1 02E+6 7 16E+5 1 64E+6 8 63E+4 4 23E+5

2 4 9 10E+5 2 81 E+5 1 80E+6 1 30E+5 6 37E+5

4 4 1 08E+6 8 75E+5 1 54E+6 6 33E+4 310E+5

8 4 1 53E+6 8 90E+5 1 .91 E+6 9 50E+4 4 66E+ 5

Data Detail

Conc-mg/L Control Type Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 Lab Water 2 60E+6 2 75E+6 2 45E+6 2 51 E+ 6

1 1 64E+6 8 40E+5 8 63E+5 7 16E+ 5

2 8 14E+5 1 .80E+6 2.81 E+5 7 46E+ 5

4 9 45E+5 9 66E+5 1 54E+6 8 75E+5

8 1 .47E+6 8.90E+5 1 .84E+6 1 .91 E+ 6

Graphics

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000-034-101-2 CETISTR9 v1 1 2revL Analyst _ t6 •A, Approval 7 C
EcoRisk Environmental Consulting and Testing

Selenastrum capricornutum Cell Density Enumeration Data

Cliert : SASOL Initial Count: 10,000 cells/mL

Test Material : ALFOTERRA L167-7S

Test Start Date : (0 ((0 D l

Test End Date . ~ ( Q

Start Time : -7' (

End Time : T. Q'O

Enumerating Scientist : QZ'

Project # : 12479

Test ID #: 25941

r---

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This datasheet has been

Densitv

( cells/mL x 106)

% CV Date: Time : Signoff:

reN iewed for completeness and

consistency with Test

Acceptabilit} Criteria and/o r

other issues of concern. -7
Pacific EcoRisk Environmental Consulting & Testing

Selenastrum capricornutum Algal Toxicity Test Water Quality Data

Client. SASOL

Test Material ALFOTERRA Ll67-7S

Test ID # 25941 TCR # :

Project # 12479 Control/Diluent . 4th Ed No EDTA

Test Treatment (mg/Lj Temp (°C)

---

H

I

p .O. (mg/L)

I

onductivit y

(μS/cm) ign-Off

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Appendix C.

Biomonitoring Report.

Daphnia pulex & Pimephales promelas.

Prepared for Sasol by EE USA

Lab Report # Q-6561-0 7

Sample ID: ALFOTERRA® L167-4S

October 31, 2007
NELAP Certificate No - 0202 7

40WBiomonitoring Repo rt

Daphnia pulex and Pimephales promelas

prepared for

SASOL - Alfoterra L167-4S

Services Requested By: Mr. Geoffrey Russel l

D. pulex EE USA Project No . : Q-561-07

SURVIVAL LC50 7 56 mg/I PR

P. promelas EE USA Project No. : Q-5611-07

SURVIVAL LC50 0 49 mg/1 P R

Report Date: October 31, 200 7

by

ENVIRONMENTAL ENTERPRISES USA, INC .

58485 PEARL ACRES ROAD, SUITE D

SLIDELL, LOUISIANA 7046 1

(800) 966-2788

This report contains five pages plus six appendices, A - F. This

report must not be reproduced in pa rt, only in whole . The results and

conclusions presented in this report apply only to the sample(s) tested .

!/~ ~- c r 3i n

Veronica McNew DATE

PRluents Testing Supervisor

Mark A O'Neil

QA/QC Supervisor

r

11/110-7

DATE

(r)David L Daniel

f Laboratory Director

l61 3l/-' 7

DATE

D. pulex Q-561-07

P. promelas Q-5611-0 7

1 of 5
ENVIRONMENTAL ENTERPRISES USA, INC . NELAP Certificate No . . 0202 7

Daphnia pulex ACUTE STATIC-RENEWAL 48-HOUR DEFINITIVE TEST,

EPA-821-R-02-012 : SECTION 9, Method 2021 .

TEST OVERVIEW

A 48-hour static-renewal toxicity test was conducted by Environmental Enterprises USA, Inc (EE

USA) to determine toxicity of product (PR) Alfoterra L167-4S to Daphnia pulex neonates . Test organisms

were cultured at EE USA and less than 24.0-hours-old when this test was initiated . Reconstituted

laboratory-prepared water was used as the diluent and a laboratory performance control was evaluated .

Five replicates of each control solution and five PR concentrations were prepared initially and renewed

daily PR concentrations tested were 1, 2, 4, 8, and 16 mg/l . This test was initiated September 25, 2007,

at 1707 and completed September 27, 2007, at 1655 .

Methods, materials, and results are presented in this document. The test was conducted from

September 25 - 27, 2007, at the laboratory of EE USA. Those involved in performing this test and in

preparation of this repo rt were Veronica McNew, Mark A . O'Neil, and David L . Daniel .

MATERIALS AND METHOD S

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms.

Actual materials and methods are detailed below. This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2021 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental design.

D pulex was cultured and maintained in reconstituted moderately hard water at 20±1°C . Neonates

were collected from approximately 300 adults selected from stock cultures and placed in a tempora ry

holding vessel . Test organisms were not fed .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, DP0710, with Potassium chloride (Sigma Chemical, Lot 096K0040). The

SRT test was initiated on September 26, 2007, with less than 24-hour old D. pulex neonates . Appendix F

contains D pulex SRT control charts .

48-hr LC50- 781 mg/1

Upper & Lower 95% Confidence Interval : 745 - 820 mg/I

On September 7, 2007, the PR sample was delivered to EE USA. Test chambers were labeled with

replicate identification and test chamber boards labeled with EE USA's project number. Six treatments,

five PR concentrations, and a laboratory performance control, were prepared daily (Appendix A, page 1) .

Each treatment was poured into a new acid-washed 0 .5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured .

At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix A, page 3). Alkalinity and hardness were measured in the

performance control solution, September 25 and September 26 (Appendix A, page 1) .

D pulex Q-561-07

P. promelas Q-5611-0 7

2 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No - 0202 7

On Day 0, the treatments were poured into one of two sets of test chambers, ten D. pulex neonates

were distributed randomly to each replicate, and then this test was placed in the environmental chamber

On Day 1, the test was renewed : fresh treatment solutions were poured into the second set of test

chambers and surviving neonates were transferred by replicate from the old to the new solutions . Survival

was recorded daily (Appendix A, page 2) . After 48 hours, the final survival data were recorded and this

test was terminated .

Summary of Experimental Condition s

Test Organisms : Less than 24 .0-hour-old Daphnia pulex neonates

Dilution Water:

Temperature :

Photoperiod:

Test Chambers :

Test Solution Volume :

Aeration:

Test Solution Renewal :

Laboratory prepared Moderately- hard water

20+ 1°C

16 hours light; 8 hours dark

30 ml Disposable Plastic Cups

25 ml

No

Yes

RESULTS AND CONCLUSION

The 48-hour survival data were used to estimate the 48-hour LC50 : a point estimate of the

concentration expected to result in 50% mortali ty to exposed D. pulex neonates after 48 hours of

exposure . The 48-hour LC50 was 7 56 mg/1 PR, with a 95% confidence interval of 6 .03 to 9.04% as

determined by Probit. Survival in the concurrent laborato ry performance was 92.0%. Survival data

summary statistics are presented in Appendix C .

D . pulex Q-561-07

P. promelas Q-5611-0 7

3 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No - 0202 7

Pimephales promelas ACUTE STATIC-RENEWAL 96-HOUR DEFINITIVE TEST,

EPA-821-R-02-012 : SECTION 9, Method 2000 .

TEST OVERVIEW

A 96-hour static-renewal toxicity test was conducted by EE USA to determine toxicity of the PR

Alfoterra L167-4S to Pimephales promelas larvae. Test organisms were cultured at EE USA and one day

old when this test was initiated . Reconstituted laboratory-prepared water was used as the diluent and a

laboratory performance control was evaluated . Five replicates of control solution and five PR

concentrations were prepared initially and renewed at 48 hours . PR concentrations tested were 0 .25, 0 .5,

1, 2, and 4 mg/I. This test was initiated September 25, 2007, at 1650 and completed September 29, 2007,

at 1438

Methods, materials, and results are presented in this document . The test was conducted from

September 25 - 29, 2007, at the laboratory of EE USA . Those involved in performing this test and in

preparation of this report were Veronica McNew, Mark A. O'Neil, and David L . Daniel .

MATERIALS AND METHOD S

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms .

Actual materials and methods are detailed below . This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2000 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental design

P promelas was cultured and maintained in reconstituted moderately hard water at 25±1°C . Several

clutches from different females comprised the emb ryo pool from which the test organism population

hatched . Test organisms were fed Artemia nauplii less than 24-hours old twice daily .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, PP0710-48, with potassium chloride (Sigma Chemical, Lot 096K0040)

The SRT test was initiated on September 26, 2007, with 2-day-old P. promelas larvae. Appendix F

contains P promelas SRT control charts .

48-hr LC50 . 1450 mg/I

Upper & Lower 95% Confidence Interval - 1320 - 1600 mg/I

On September 7, 2007, the PR sample was delivered to EE USA. Test chambers were labeled with

test concentration, replicate, and EE USA' project number. Six treatments, five PR concentrations, and a

laboratory performance control, were prepared at initiation and upon renewal at 48 hours (Appendix B,

page 1).

Each treatment was poured into a new acid-washed 0 5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured .

At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix B, pages 4 & 4). Alkalinity and hardness were measured in the

performance control solution, September 25 and September 27 (Appendix B, page 1) .

D . pulex Q-561-07

P. promelas Q-5611-07

4 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No : 02027

On Day 0, the treatments were poured into their respective test chambers, ten P. promelas larvae were

distributed randomly to each, and then this test was placed in the environmental chamber . On Day 2, the

test was renewed : 90% of the treatment solution, excess food, and debris were poured or siphoned out of

each replicate. Aliquots of freshly prepared treatments were poured gently into each replicate as

appropriate . Surviving test organisms were disturbed as little as possible during renewal . Every 24 hours,

survival was recorded (Appendix B, pages 2 & 3) . After 96 hours, the final survival data were recorded

and this test was terminated .

Summary of Experimental Condition s

Test Organisms:

Dilution Water:

Temperature:

Photoperiod:

Test Chambers :

Test Solution Volume :

Aeration:

Test Solution Renewal :

1-day-old Pimephales promelas larvae

Laboratory prepared Moderately- hard water

20+1°C

16 hours light ; 8 hours dark

Rectangular Pyrex Dish, 21cm x 11cm x 7cm

200 ml

No

Yes

RESULTS AND CONCLUSION

Total Volume = 1 .45 liters

The 96-hour survival data were also used to estimate the 96-hour LC50 : a point estimate of the

concentration expected to result in 50% mortality to exposed P. promelas neonates after 96 hours of

exposure . The 96-hour LC50 was 0 .49 mg/1 PR, with a 95% confidence interval of 0 .44 to 0.56% as

determined by Trimmed Spearman-Karber. Survival in the concurrent laborato ry performance was

96.0% . Survival data summa ry statistics are presented in Appendix C .

REFERENCES

EE USA. April 2007. Quality Assurance Plan . EE USA, Slidell, LA 70461 .

EE USA. April 2007. Standard Operating Procedures . EE USA, Slidell, LA 70461 .

2003 National Environmental Laboratory Accreditation Conference Standards , Chapter 5 Quality Systems .

EPA/600/R-04/003. Approved at Ninth NELAC Annual Meeting, June 5, 2003 .

Tidepool Scientific Software. ToxCalc Version 5.0.

U.S . Environmental Protection Agency . October 2002. Methods for measuring the acute toxicity of

effluents and receiving waters to freshwater and marine organisms . EPA-821-R-02-012 . 5`h Edition

Office of Water (4303T) Washington, DC 20460 .

D. pulex Q-561-07

P. promelas Q-5611-0 7

5 of 5
Environmental Enterprises USA, Inc.

APPENDIX A
9/25/07,3 :08 PM

Environmental Enterprises USA, Inc .

Sasol - Alfoterra L167-4S

Geoff Russell (337) 494-509 7

Preparation of Toxicant Simulated Effluent (EFF): Weigh 0.16000 g of Alfoterra L167-4S in a 25

ml beaker. Add Alfoterra L167-4S to class A cylinder and rinse beaker with Moderately Hard

Synthetic Freshwater (MHSF) . Add MHSF to cylinder for total volume of 3000 ml . The EFF is split

into two 1500 ml aliquots. One aliquot is the 16mg/1 EFF for testing, and the second aliquot is used to

prepare the 8 mg/1 .

Preparation of 8 mg/l, 4 mg/l, 2 mg/l, and 1 mg/1 dilutions :

8 mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml of

this solution is the 8 mg/1 EFF

4 m WI EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 4 mg/1 EFF

2 m 1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 2 mg/1 EF F

1 mg/I EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF; 1500 ml

of this solution is the 1 ~ng/1 EFF

Data pages & Calculations by: G~~~r.- i> v-QA/QC Officer: C)

D. pulex = 5 Reps x 25 ml

= 125 ml

DH2O = Dilution Water = MHSF

LPC M

#

LPC M

Date 09/25

Alkalinity `l [p lI //

Conductiv ity ' ' ,~ ~~

F

p,t5

Hardness ( CC' II ll

H

TRC HI

~- ~

LPC: Laboratory Performance Control, synthetic moderately hard water

Alkalinity : mg/1 as CaCO3 Conductivity : μS/cm Hardness : mg/1 as CaCO3

pH : su TRC : mg/1

Prep Date 09/25 09/2 6

Sample # 1 1

Initial ~ t'zComments: Feeding / Exposure Chambers : D. pulex - none / 30 ml medicine cups

P. promelas - none / 16 oz. plastic cups

Alfoterra L167-4S Q-561-07

1 of 4
9/25/07,3 :08 PM

Environmental Enterprises USA, Inc .

Cladoceran, Daphnia pulex

Acute Static Renewal 48 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 202 1

Sasol - Alfoterra L167-4S

Survival Da

T____ _Ir ntl

i l l~[lF1ll~:LLi ll1~fL 1 lt

Time Ri LPC

E ; White

Pi

R 1

E ppm

P Blue

R 2

E ppm

P Green

R 4

E ppm

P Yellow

R 8

E ppm

P Brown

R 16

E ppm

P Black

Initial

S

O HR

ii 10

!

6 10

!

11 10

!

16 10

!

21 10 26 10

!

09/25/07

2 ; 10 7 10 12 10 17 10 22 10 27 1 0

10 8 1 0 13 ; 10 18 10 3 10 28 10

~~

4~ 10 9 10 14 10 19 10 24 10 29 10

,~

5 ; 10 10 10 5 ; 10 20';10 25 10 30 10

24

6 ~~ 11 16 !(~ 21 26

09/26/07

HR 2! L7 7 (0 12 ;~~ 17 22 2 7

3 ! 8 13 18 23 28 (iO

~ i~U 4 9 ~ G)

14 t

9

f

~'i

24 2 9

5i l

.~V to

d

15 0 C) 2' 30 4

48

1 6 ! . ~ 11 16 21 26

09/27/0 7

HR 2; 7 12 ; 17 ; 22 ; ~ 27 ;

3! 13 18 23 28 !

4; 9 14 29

5! 10 IS 20! L 30

j_ s~~>~' ii7

Counted by : QC/QA by :

Loaded by :

Test Organisms Age- ~~ / hours old Test Organisms Source :

Data Entry by

Double Data En try b y : and/or

QC/QA by : - - -`~~

Alfoterra L167-4S Q-561-07

C3 ~Y,~.

.la•F- t

2 of 4
9/25/07,3 :08 PM

Environmental Enterprises USA, Inc .

D. pulex Water Quality Dat a

LPC & All Treatments: Initial Temp . - 18.5 - 21.4 ; Initial DO - 9 .2 < 4. 0

0 to 24 HR Treatment mg/L PR

09/25-26/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Mete r

DO I

F do

Temp I

~C( ~G' 2~ •Z _ q

~-};~

l~y

.

F

(

? (G Ij Cj jq 3 flr)

Cond. I

~ Z

Ck

~ 1 )

~ C

q a`~ l

` '

-~

pH F ~.~ t.% L0 ~ lJ ~,C) ~, .t✓ ~

Initials Initials : Finals:

Times Initial Time : ~~j Final Time:

24 to 48 HR Treatment mg/L PR

09/26-27/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Mete r

DO 1

7j

~3

F 3 ~7 U `y 9-S-_ S_`7(

I,- 1? ~~~ I?," `

Temp I 2L). ?C) '2 6 ~

~C), `Z ~C~. ~~•~ ~~

F

"2l'i . . • ~ ~~. ~ ~i. ~ 'T (k L~

Cond. I

[30 1 ,303 305- 130 1 302,

PH F

?

Initials Initials: 4 Finals:

Times Initial Time_ _ ) L Final Time :

DO, Dissolved Oxygen: mg/1 pH: SU Salinity: ppt Temp: °C

Comments :-

Alfoterra L167-4S Q-561-07

3 of 4
9/25/07,3 :08 PM

Environmental Enterprises USA, Inc .

Data Pages

a" Company name & contact matches client file .

0' Product matches client file .

d Dilution series are correct:

Dilution series:

❑ Calculations on mixing page are correct . (sign mixing page)

cv Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

throughout data pages .

L~ ' Format correct. (spaces for all entries, page numeration, no split pages, etc .)

1/1~.- Initials U9 g `l--- ? Date

Chain-of-Custody

d Product on COC matches sample bottle .

EJ Product on COC matches test data pages.

d Lab # on COC matches sample bottle .

L3, Lab # on COC matches test data pages.

❑ Sample collection date: & Time: hrs.

expiration date : & Time: hrs. (Sample expired if >36

hrs) N/A

a-," Sample volume is sufficient for test duration. (Sample volume in container(s)

checked against sample volume on mixing page )

Sample volume available : 1 ml

Sample volume needed . C . it 4* d

(Sample volume insufficient if sample volume available < sample volume needed)

Initials Ci z;

le"1 -1 Date 4- -

Jugs & Labels

Q-, Lab # on jug and labels matches test data pages .

a~ Dilution water type is on jug . (i.e . 25 ppt, 20 ppt, MHSF, etc. )

al Dilutions on jugs and labels match dilutions on test data pages.

Y Jugs are color-coded (see mixing page for appropriate color code sequence )

ff~,~ Initials 6 ~JZS~7 Date

Raw Data QC/QA'd by: Z ?

~ I k M

Alfoterra L167-4S Q-561-07

4 of 4
Environmental Enterprises USA, Inc.

APPENDIX B
9/25/07,3 :18 PM

Environmental Enterprises USA, Inc .

Sasol - Alfoterra L167-4 S

Geoff Russell (337) 494-509 7

Preparation of Toxicant Simulated Effluent (EFF): Weigh 0.16000 g of Alfoterra L 167-4S in a 25

ml beaker. Add Alfoterra L167-4S to class A cylinder and rinse beaker with Moderately Hard

Synthetic Freshwater (MHSF). Add MHSF to cylinder for total volume of 3000 ml . The EFF is split

into two 1500 ml aliquots . One aliquot is the 16mg/1 EFF for testing, and the second aliquot is used to

prepare the 8 mg/l. 8 mg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml

MHSF; 1500 ml of this solution is the 8 mg/I EFF

Preparation of 4 mg/1, 2 mg/l, I mg/1, 0 .5 mg/l, and 0.25 mg/l dilutions :

4 m 1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 4 mg/1 EFF

2 my,/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 m1 MHSF ; 1500 ml

of this solution is the 2 mg/1 EFF

1 mg /I EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 1 mg/1 EF F

0 .5 m WI EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 m1 MHSF ; 1500 ml

of this solution is the 0 .5 mg/1 EF F

0 .25 mg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the 0.35 mg/1 EFF

Data pages & Calculations by: c~$A/QC Officer:

P. promelas = 5 Reps x 200 ml

= 1000 m1

DHZQ = Dilution Water = MHSF

LPC M LPC M

Date 09/25 09/27

Alkalinity - 7 L 49

Conductivity ? l1l45

Hardness ~ " // //

H 91 () 611~

TRC llI ll /.%% sr

U ~G

LPC : Laboratory Performance Control, synthetic moderately hard wate r

Alkalinity: mg/I as CaCO3 Conductivity : μS/cm Hardness: mg/1 as CaCO3

pH : su TRC : mg/ 1

Prep Date 09/25 09/27

Sample # 1 1

initial 1A7 /-7

Alfoterra L167-4S Q-561I-07

I of 6
9/25/07,3:18 PM

Environmental Enterprises USA, Inc .

Fathead Minnow, Pimepahles promelas

Acute Static Renewal 96 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 2000

Sasol - Alfoterra L167-4S

Survival Data

Treatment mg/L PR

Time R;

E

P!

LPC

White

R

E

P

0.25

pp m

! Blue

R ;

E

P

0 . 5

pp m

Green

R

E

P

1

pp m

Yellow

R ;

E

P

2

pp m

Brown

R

E

P

4

pp m

Black

Initial

S

O HR

1! 10 6 10 10 16 10 21 10 26 10 o9/25io 7

2 ; 10 7 10 F 10 17 10 22 10 27 1 0

3 10 8 10 10 18 10 23 10 28 1 0

4 i 10 9 10 14 10 19 10 24 10 29 1 0

5 ; 10 10 10 15 10 20 10 25 ; 10 30 ; 1 0

24

1~ ~ 6 O 11 C) 16 21 26

09/26/07

HR 2; i o 7

ID 12 d 17 0

22 27

3; c) 8 1 3 18 0 3 C 2 88 C,

4! I© 9 t lU 14 0 19 0 24 C) 29

f`f~S 5! 10 15 Cj 20 25 Cl 3 0

48

1 ; 6 llj 16 21 26 i

09/27/0 7

HR

2

,i 7 42

17 22 2 7

3!! 8 13 • 18 ; C) 23 2 8

4! 9 14 C1

t~

19 24 t~ 2 9

5 ; 10 15 20,, 25 ; 3 0

mg/L

Sur

Counted by: QC/QA by

Loaded by

: ~

: C

~

~.•VJJ Test Organisms Age: days d Test Organisms Source :

Alfoterra L167-4S Q-5611-07

2 of 6
9/25/07,3 :18 P M

Environmental Enterprises USA, Inc .

P. promelas Survival Data Cont .

F

Treatment mg/L PR

Time Pi LPC R 0 .25 R 0 .5 R 1 R 2 R 4 Initial

E; White E ppm E ppm E ; ppm E ppm E ppm S

Pi P Blue P Green P Yellow P Brown P Blac k

72

1! q 6'( D 11 ~ 16 21 26 09i28/ 0 7

~ 2I 7 ID

12 10 17 0 22 2 7

3 ~8

~ O 13 lv 18 c~ 23 ~j 28 o

I (.0 4~ ( 4 9! rl4 5 19 24 c~ 29 O

5' C~ 10 15 °-T 20 C3 25 C) 3 0

96

i!~a a 6!~Q 11 16 21 26

09/29/0 7

HR 2i o 7 12 17i 22j 2 7

3

; C

J /~ 8 13 ~ 18 ; 23 ;i O 28 ;

4

; 10 9

14 19 24 C 29 ~. 4di

5 10 -7 15 20 25 30 ~

mg/L

Sur

Data Entry by :

Double Data Entry by: -and/or

QC/QA by:

Alfoterra L167-4S Q-5611-07

3 of 6
9/25/07,3 :18 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Data

LPC & All Treatments : Initial Temp. - 18.5 - 21.4; Initial DO - 9.2 < 4.0

0 to 24 HR Treatment mg/L PR

09/25-26/07 LPC 0.25 ppm 0.5 ppm I ppm 2 ppm 4 ppm Meter

DO I

F

Temp I ~i . cc i ~c ~~, ~ 2

F

Cond. I ~C~ ~ _ ~ ,~ • y~ ~ ~ ^ 6 ~

pH F

Initials Initials: Finals:

Times Initial Time : t 7'7 Final Time :

48 HR Treatment mg/L PR

09/27/07 LPC 0 .2 5

ppm

0 .5 ppm 1 ppm 2 ppm 4 ppm Meter

DO F

Temp F

2G. Ze! ~ 2c' ` ~" :

pH F

Initials Initials: Finals:

Times Initial Time : Final Time :

DO, Dissolved Oxygen : mg/1 pH : SU Salinity: ppt Temp: °C

Comments:

Alfoterra L 167-4S Q-561I-07

4 of 6
9/25/07,3 :18 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Data Cont.

48 to 72 HR Treatment mg/L PR

09/27-28/07 LPC 0 .25

ppm

0 .5 ppm 1 ppm 2 ppm 4 ppm Meter

DO I

~ 3 d•' D .~ ~•a ~ ^ `-s /

F V

Temp I

1 /

F C~

Cond. I

A

pH F

Initials i✓1 ~ Final

Times Time: Final Time : 09 oL "

96 HR Treatment mg/L P R

09/29/07 LPC 0 .25

ppm

0 .5 ppm 1 ppm 2 ppm 4 ppm Meter

DO F

`~ . ~

Temp F

pH F G :( y.

Initials Initials: Finals : ~rTimes Initial Time : PI) Final Time :

Alfoterra L 167-4S Q-5611-07

5 of 6
9/25/07,3 :18 PM

Environmental Enterprises USA, Inc .

Data Pages

al Company name & contact matches client file .

o' Product matches client file.

❑-- Dilution series are correct:

Dilution series:

❑ Calculations on mixing page are correct . (sign mixing page)

❑- Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

throughout data pages.

La' Format correct . (spaces for all entries, page numeration, no split pages, etc .)

Initials Date

Chain-of-Custody

g% Product on COC matches sample bottle.

❑-" Product on COC matches test data pages .

a,"' Lab # on COC matches sample bottle .

Y Lab # on COC matches test data pages .

❑ Sample collection date : & Time:

expiration date : & Time,

hrs), N/ A

~ Sample volume is sufficient for test duration.

checked against sample volume on mixing page)

Sample volume available- ~w ml

Sample volume needed : o (i, GU aa+<~

cs'l7~/b -1 r"_

hrs.

hrs . (Sample expired if>36

(Sample volume in container(s )

(Sample volume insufficient if sample volume available < sample volume needed)

Initials Date

Jugs & Labels

.,d Lab # on jug and labels matches test data pages.

e' Dilution water type is on jug . ( i .e 25 ppt, 20 ppt, MHSF, etc . )

o/ Dilutions on jugs and labels match dilutions on test data pages .

cd / Jugs are color-coded. ( see mixing page for appropriate color code sequence)

Initials ~JI 2 _ Date

_

Raw Data QC/QA'd by :

Alfoterra L167-4S Q-561I-07

6 of 6
Environmental Enterprises USA, Inc.

APPENDIX C
Acute Toxicity Test-48 Hr Surviva l

Start Date 9/24/2007 Test ID. dp56107 Sample ID: LAB-Lab Sample

End Date . 9/26/2007 Lab ID : EE-Environmental Enterprise Sample Type : PRD-Produc t

Sample Date . Protocol : EPAF 02-EPA Freshwater Test Species : DP-Daphnia pulex

Comments .

Conc-mg/L 1 2 3 4 5

PC-LP Control 0 .9000 0 .8000 1 .0000 1 .0000 0.9000

1 0 . 8000 1 .0000 0 .8000 0 .7000 1 .0000

2 0 .9000 0 .9000 0 .8000 0 .9000 0.8000

4 0 .8000 0 .6000 0 .7000 0 .8000 0.7000

8 0 .5000 0 .6000 0 .4000 0 .4000 0.4000

16 0 .1000 0 .0000 0 .0000 0 .1000 0.2000

Transform : Untransformed Number Total

Cone-mg/L Mean CV% Mean Min Max CV% N Resp Number

PC-LP Control 0 .9200 9 .094 0 .9200 0 .8000 1 .0000 9 .094 5 4 5 0

1 0 .8600 15.600 0 .8600 0 .7000 1 .0000 15 .600 5 7 5 0

2 0 .8600 6 .369 0 .8600 0 .8000 0.9000 6 .369 5 7 5 0

4 0 .7200 11 .620 0 .7200 0 .6000 0.8000 11 .620 5 14 5 0

8 0 .4600 19.444 0 .4600 0 .4000 0.6000 19.444 5 27 5 0

16 0 .0800 104.583 0 .0800 0 .0000 0.2000 104.583 5 46 5 0

Auxiliary Tests Statistic Critical Skew Kurt

Shapiro-Wilk's Test indicates normal distribution (p > 0 01) 0 .9468 0 .9 0 .13934 -0.8601

Bartlett's Test indicates equal variances (p = 0.70) 2.97269 15.0863

Maximum Likelihood-Probit

Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma [ter

Slope 3.70838 0.65824 2.41823 4.99852 0.08 1 .25902 7.81473 0.74 0.87854 0.26966 7

Intercept 1 74206 0.6247 0.51764 2.96647

TSCR 0. 1167 0. 02859 0. 06066 0. 17274 1 . 0

Point Probits mg/L 95% Fiducial Limits

EC01 2 .674 1 .78326 0 .73122 2 .78437 0 9

EC05 3 355 2.72261 1 .38637 3 .84634 0 8

EC10 3 718 3 .41156 1 .94424 45824 0 . 7

EC15 3 .964 3 .97239 2 .43768 5 .16734

EC20 4.158 4.48316 2 .91264 5 .69496 0 6

EC25 4.326 4 .9734 3 .3873 6 .20116 Q 0 5

EC40 4.747 6.45979 4 .89675 7 .77722 H

EC50 5 000 7.56025 6 .02741 9 .03757 ~ 0 4

EC60 5 .253 8.84817 7 .29599 10.6794 0 3

EC75 5 .674 11 .4926 9 .60062 14.7143

EC80 5.842 12.7493 10 .5743 16 9175 0 2

EC85 6 036 14.3887 11 .7651 200228 0 . 1

EC90 6.282 16.754 13 .373 24 9054 0 . 0

EC95 6.645 20.9936 16 .0449 34 .6819 0.1 1 10 100 EC99 7.326 32.0522 22 .3042 65 .3441 Dose mgl L

Page 1 ToxCalc v5.0 .23 Reviewed by: A*-D
Environmental Enterprises USA, Inc .

APPENDIX D
Acute Toxicity Test-96 Hr Surviva l

Start Date: 9/24/2007 Test ID: pp561107 Sample ID: LAB-Lab Sampl e

End Date 9/29/2007 Lab ID: EE-Environmental Enterprise Sample Type : PRD-Product

Sample Date- Protocol : EPAF 02-EPA Freshwater Test Species : PP-Pimephales promela s

Comments -

Conc-mg/L 1 2 3 4 5

PC-LP Control 0 .9000 1 .0000 1 .0000 1 .0000 0 .9000

0 25 1 .0000 1 .0000 1 .0000 0 .7000 0 .7000

0 .5 0.5000 0 .7000 0.4000 0 .4000 0 .5000

1 0.0000 0 .0000 0.0000 0 .0000 0 .0000

2 0 .0000 0 .0000 0.0000 0 .0000 00000

4 0.0000 0 .0000 0.0000 0 .0000 0 .0000

Transform : Untransformed Number Total

Conc-mg/L Mean CV% Mean Min Max CV% N Resp Number

PC-LP Control 0 .9600 5 .705 0.9600 0 .9000 1 .0000 5 .705 5 2 50

0 .25 0 .8800 18.672 0.8800 0 .7000 1 .0000 18.672 5 6 50

0 .5 0 .5000 24.495 0.5000 0 .4000 0 .7000 24.495 5 25 50

1 0 .0000 0.0000 0 .0000 0 .0000 5 50 50

2 0 .0000 0.0000 0 .0000 0 .0000 5 50 5 0

4 0 .0000 0.0000 0 .0000 0 .0000 5 50 5 0

Auxiliary Tests Statistic Critical Skew Kurt

Shapiro-Wilk's Test indicates normal distribution (p > 0 .01) 0.95749 0.835 -0.0407 -0.7411

Bartlett's Test indicates equal variances (p = 0.16) 3.67794 9.21034

Trimmed Spearman-Karber

Trim Level EC50 95% CL

0.0%

5.0%

10.0% 0.4950 0.4372 0.5605 10

20.0% 0.5002 0.4263 0.5869 09

Auto-8 .3% 0 .4941 0.4380 0.5574

0 . 8

0 .7

Q 0.6

CO 0 .5

~

0 .4

0 .3

02

01

0 .0--

0 .1

Dose mg/ L

Page 1 ToxCalc v5.0 .23

10

Pf

Reviewed by:- M&--O
Environmental Enterprises USA, Inc .

APPENDIX E
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Environmental Enterprises USA, Inc.

APPENDIX F
D. pulex SRT, KCI mg/I

1400

1300

120 0

L°e 1100 U

J

= 1000

00

900

800

700

600

CV%=976

_

---~

p p"ppo ^°P

o\~~~o`~° \~~ \'

6\

~~oo\~~\o\o~~°o\°~noo\°~~~o°\~°~°

Date of Test

+2 SD

+1 SD

Mean

-1 SD

-2 SD

Test # Test Date 48-hr LC5 0

mg/L KCI

Cumulativ e

Mean LC50 -1 SD -2 SD +1 SD +2 SD Control

Survival

Toxicant Lot

#

DP0601 1/4/2006 1000 94 .0 075K0090

DP0602 2/24/2006 1120 1060 .0000 975.1472 890 .2944 1144 .8528 1229 7056 92.0 075K0090

DP0603 3/22/2006 978 1032 6667 956.2381 879 .8094 1109 .0953 1185 .5239 98.0 075K0090

DP0604 4/12/2006 802 975 .0000 843 .8665 712 .7330 1106 .1335 1237 2670 98.0 0751(0090

DP0605 5/22/2006 958 971 6000 857 .7808 743 .9617 1085 .4192 1199 .2383 100 .0 0751C0090 1

DP0606 6/15/2006 963 970 1667 868 3032 766 .4397 1072 0301 1173 .8936 100 .0 0751(0090 1

DP0607 7/31/2006 854 953 5714 850 7385 747 .9055 1056 .4044 1159 .2374 100 .0 075K0090 1

DP0608 8/10/2006 1090 970 6250 863 .8985 757 1719 1077 .3515 1184.0781 1000 075K0090 1

DP0610 9/28/2006 1010 975 0000 874 .3074 773 6148 1075 .6926 1176 3852 100 .0 0751(0090 1

DP0611 10/10/2006 1010 978 5000 882 .9231 787 .3462 1074 0769 1169 .6538 100 .0 075K0090 1

DP0614 12/6/2006 949 975 8182 884 .7108 793.6033 1066 9256 1158 0330 92 .0 066K0032

DP0701 1/31/2007 951 973 7500 886 .5875 799 .4251 1060 9125 1148 0749 100 .0 066K0032

DP0702 2/14/2007 1110 984 2308 892 .6219 801 .0130 1075 8397 1167 .4486 100 .0 066K0032

DP0703 3/28/2007 908 978 7857 888 .4435 798 1012 1069 1280 1159 .4702 960 066K0032

DP0705 5/3/2007 897 973 3333 883 .7528 794 1723 1062.9138 1152.4943 98 .0 0661(003 2

DP0706 5/8/2007 983 973 9375 887 .3608 800.7841 1060 .5142 1147 .0909 980 066K0032

DP0707 6/5/2007 831 965 5294 874 .8162 784 .1030 1056 .2426 1146 .9559 94 .0 066K0032

DP0708 7/18/2007 926 963 3333 874 .8367 786 .3401 1051 .8299 1140 .3265 100 .0 096K0040

DP0709 8/29/2007 978 964 1053 878 .0362 791 .9672 1050 .1743 1136 .2433 96.0 096K0040

DP0710 9/26/2007 781 954 9500 861 7064 768 .4628 1048 .1936 1141 .4372 98.0 096K004 0

November- EE cultures crashed before SRT was completed Purchased D pulex

DP0614 - ABS D. pule x

DP0701 -ABS D. pulex

DP0704 - Control failure
P. promelas SRT, KCI mg/I

CV%=292

1600

1400

1200

V - --- --~_ -_- _ _ 100

0 80

0 600

400

20 0

^ \ ° ° \° N° ~ ~~ ~o ~ ' `~ \o c , \~~ ~ \° ~ \ ~~ ~\ `~ ~ ° ~ \° ° X06 °~ \° ~ `° \06 ° ° ~ \° ~ Xo

p

O ° \° \° 6 \ `~~ ° \\ ~6 ~ ~~ \°

0 0 0 0 0 0 0 0 0 '. N o 0 0 0 0 0 0

Date of Test

+2 SD

+1 SD

Mean

-1 SD

-2 SD

Test # Test Date 48-hr LC5 0

mg/L KCI

Cumulativ e

Mean LC50 -1 SD -2 SD +1 SD +2 SD Control

Survival

Toxicant Lo t

#

PP0601-48 1/4/2006 771 97 .5 075K0090

PP0602-48 2/14/2006 877 824 0000 749 0467 674 .0934 898 .9533 973 .9066 100 .0 075K0090

PP0603-48 3/16/2006 671 773 0000 669 .9854 566 .9709 876 .0146 979 .0291 97 .5 075K009 0

PP0604-48 4/18/2006 975 823 5000 692 .0631 560 .6261 954 .9369 1086 3739 100 .0 075K0090 1

PP0605-48 5126/2006 879 834 6000 718 0976 601 5953 951 1024 1067 .6047 100 0 075K0090 1

PP0606-48 6/14/2006 720 815 5000 701 .2761 587 0522 929.7239 1043 .9478 100 .0 075K0090 1

PP0607-48 7/26/2006 1490 911 8571 636 .4203 360 .9834 1187 .2940 1462 .7309 95 .0 075K0090 1

PP0608-48 8/16/2006 689 884 .0000 617 0998 350.1996 1150 9002 1417 .8004 100 .0 075K0090 1

PP0609-48 9/5/2006 645 857 .4444 595 3795 333 3146 1119 5094 1381 .5743 1000 075K00901

PP0610-48 10/10/2006 404 812 .1000 526 .4283 240 .7566 1097 7717 1383 .4434 97 .5 075K00901

PP061 1-48 11/7/2006 787 809 8182 538 .7006 267 .5829 1080 9358 1352 .0534 100 .0 066K0032

PP0612-48 12/14/2006 796 808 .6667 550 1354 291 .6041 1067 1979 1325 .7292 97 .5 066K003 2

PP0701-48 1/4/2007 579 791 .0000 535 .4105 279 .8210 1046 .5895 1302 .1790 100 .0 066K003 2

PP0702-48 2/7/2007 958 802 9286 553 3429 303 7573 1052 5142 1302 .0999 100 .0 066K003 2

PP0703-48 3/26/2007 907 809 8667 567 .8635 325 .8603 1051 8699 1293 .8731 100 .0 066K003 2

PP0704-48 4/4/2007 989 821 0625 583 .0148 344 9670 1059 .1102 1297 .1580 100 .0 066K003 2

PP0705-48 5/23/2007 909 826 .2353 594 .7619 363 .2885 1057 .7087 1289 .1821 1000 066K003 2

PP0706-48 6/12/2007 773 823 2778 598 .3653 373 4528 1048 1903 1273.1027 1000 0961(004 0

PP0707-48 7/26/2007 958 830 3684 609 .6184 388 .8683 1051 1185 1271 .8685 1000 0961(0040

PP0708-48 8/22/2007 1120 844 8500 620 .4394 396 .0287 1069 .2606 1293 .6713 1000 096K0040

PP0709-48 9/26/2007 1450 873 .6667 618 .1661 362 .6656 1129 .1672 1384 .6678 100 .0 096 1(0040
Appendix D.

Biomonitoring Report .

Daphnia pulex & Pimephales promelas .

Prepared for Sasol by EE USA

Lab Report # Q-562-07

Sample ID : ALFOTERRA® L167-7S

October 31, 2007
NELAP Certificate No : 0202 7

4W Biomonitoring Report

Daphnia pulex and Pimephales promelas

prepared for

SASOL - Alfoterra L167-75

Services Requested By: Mr. Geoffrey Russel l

D. pulex EE USA Project No. : Q-562-07

SURVIVAL LC50 15.8 mg/1 P R

P. promelas EE USA Project No. : Q-56,21-07

SURVIVAL LC50 1 40 mg/I P R

Report Date: October 31, 2007

by

ENVIRONMENTAL ENTERPRISES USA, INC .

58485 PEARL ACRES ROAD, SUITE D

SLIDELL, LOUISIANA 7046 1

(800) 966-2788

This report contains five pages plus six appendices, A - F. This

report must not be reproduced in part, only in whole . The results and

conclusions presented in this report apply only to the sample(s) tested .

Veronica McNe

l/. 2~

w

PRluents Testing Supervisor

111/

Mark A. O'Neil

QA/QC Supervisor

~ G'

(Ir~)David L . Daniel

Laboratory Director

po6t ~6 '7

DATE

DATE

~ e)6 a`7

DATE

D . pulex Q-562-07

P. promelas Q-5621-07

1 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No . . 0202 7

Daphnia pulex ACUTE STATIC-RENEWAL 48-HOUR DEFINITIVE TEST,

EPA-821-R-02-012: SECTION 9, Method 2021 .

TEST OVERVIEW

A 48-hour static-renewal toxicity test was conducted by Environmental Enterprises USA, Inc (EE

USA) to determine toxicity of product (PR) Alfoterra L167-7S to Daphnia pulex neonates. Test organisms

were cultured at EE USA and less than 24 .0-hours-old when this test was initiated . Reconstituted

laboratory-prepared water was used as the diluent and a laboratory performance control was evaluated .

Five replicates of each control solution and five PR concentrations were prepared initially and renewed

daily PR concentrations tested were 1 .9, 3 .8, 7.6, 15.3, and 30.5 mg/I. This test was initiated September

25, 2007, at 1720 and completed September 27, 2007, at 1701 .

Methods, materials, and results are presented in this document. The test was conducted from

September 25 - 27, 2007, at the laboratory of EE USA. Those involved in pe rforming this test and in

preparation of this repo rt were Veronica McNew, Mark A O'Neil, and David L . Daniel .

MATERIALS AND METHODS

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of EFFluents and Receiving Waters to Freshwater and Marine Organisms . Actual materials and methods are detailed below . This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2021 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental design

D pulex was cultured and maintained in reconstituted moderately hard water at 20 + 1°C. Neonates

were collected from approximately 300 adults selected from stock cultures and placed in a tempora ry

holding vessel . Test organisms were not fed .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, DP0710, with Potassium chloride (Sigma Chemical, Lot 096K0040) . The

SRT test was initiated on September 26, 2007, with less than 24-hour old D. pulex neonates . Appendix F

contains D pulex SRT control charts

48-hr LC50 : 781 mg/I

Upper & Lower 95% Confidence Interval : 745 - 820 mg/1

On September 7, 2007, the PR sample was delivered to EE USA. Test chambers were labeled with

replicate identification and test chamber boards labeled with EE USA's project number. Six treatments,

five PR concentrations, and a laborato ry performance control, were prepared daily (Appendix A, page 1) .

Each treatment was poured into a new acid-washed 0 .5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured . At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix A, page 3). Alkalinity and hardness were measured in the

performance control solution, September 25 and September 26 (Appendix A, page 1)

.

D. pulex Q-562-07

P. promelas Q-5621-0 7

2 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No : 0202 7

On Day 0, the treatments were poured into one of two sets of test chambers, ten D. pulex neonates

were distributed randomly to each replicate, and then this test was placed in the environmental chamber

On Day 1, the test was renewed : fresh treatment solutions were poured into the second set of test

chambers and surviving neonates were transferred by replicate from the old to the new solutions . Survival

was recorded daily (Appendix A, page 2) . After 48 hours, the final survival data were recorded and this

test was terminated .

Summary of Experimental Condition s

Test Organisms . Less than 24 .0-hour-old Daphnia pulex neonate s

Dilution Water:

Temperature :

Photoperiod:

Test Chambers :

Test Solution Volume :

Aeration:

Test Solution RenewalLaboratory prepared Moderately- hard water

20+ 1°C

16 hours light; 8 hours dark

30 ml Disposable Plastic Cups

25 ml

No

Yes

RESULTS AND CONCLUSION

The 48-hour survival data were used to estimate the 48-hour LC50 : a point estimate of the

concentration expected to result in 50% mortality to exposed D . pulex neonates after 48 hours of

exposure The 48-hour LC50 was 15 .8 mg/I PR, with a 95% confidence interval of 10.7 to 25.6% as

determined by Probit Survival in the concurrent laboratory performance was 94 .0% . Survival data

summary statistics are presented in Appendix C .

D. pulex Q-562-07

P. promelas Q-5621-0 7

3 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No . : 0202 7

Pimephales promelas ACUTE STATIC-RENEWAL 96-HOUR DEFINITIVE TEST,

EPA-821-R-02-012: SECTION 9, Method 2000 .

TEST OVERVIEW

A 96-hour static-renewal toxicity test was conducted by EE USA to determine toxicity of the PR

Alfoterra L167-7S to Plmephales promelas larvae . Test organisms were cultured at EE USA and one day

old when this test was initiated . Reconstituted laborato ry-prepared water was used as the diluent and a

laboratory performance control was evaluated Five replicates of control solution and five PR

concentrations were prepared initially and renewed at 48 hours. PR concentrations tested were 0 .25, 0.5,

1 . 2, and 4 mg/l. This test was initiated September 25, 2007, at 1656 and completed September 29, 2007,

at 1532

Methods, materials, and results are presented in this document . The test was conducted from

September 25 - 29, 2007, at the laboratory of EE USA . Those involved in performing this test and in

preparation of this report were Veronica McNew, Mark A . O'Neil, and David L Daniel

MATERIALS AND METHODS

Materials and methods for the work performed are stated in EPA-821-R-03-012 : Methods for

Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms .

Actual materials and methods are detailed below . This test was performed with strict adherence to the

requirements of EPA-821-R-03-012, Section 9, Method 2000 . Additionally, the recommendations and

suggestions made elsewhere in EPA-821-R-03-012 were incorporated whenever applicable to optimize

the experimental design

P. promelas was cultured and maintained in reconstituted moderately hard water at 25±1°C. Several

clutches from different females comprised the embryo pool from which the test organism population

hatched. Test organisms were fed Artemia nauplii less than 24-hours old twice daily .

Sensitivity of test organisms to a known toxicant was determined by performing an acute Standard

Reference Toxicant (SRT) test, PP0710-48, with potassium chloride (Sigma Chemical, Lot 096K0040) .

The SRT test was initiated on September 26, 2007, with 2-day-old P. promelas larvae. Appendix F

contains P promelas SRT control charts .

48-hr LC50 . 1450 mg/l

Upper & Lower 95% Confidence Interval- 1320 - 1600 mg/l

On September 7, 2007, the PR sample was delivered to EE USA . Test chambers were labeled with

test concentration, replicate, and EE USA' project number. Six treatments, five PR concentrations, and a

laboratory performance control, were prepared at initiation and upon renewal at 48 hours (Appendix B,

page 1 )

Each treatment was poured into a new acid-washed 0 .5-gallon plastic container and placed in an

environmental chamber to warm up to test temperature . After the test solutions reached test temperature,

initial water quality parameters (dissolved oxygen (DO), conductivity, and temperature) were measured .

At the end of each 24-hour exposure period, prior to renewal, the ending DO, temperature, and pH in each

treatment were recorded also (Appendix B, pages 4 & 4). Alkalinity and hardness were measured in the

performance control solution, September 25 and September 27 (Appendix B, page 1) .

D. pulex Q-562-07

P. promelas Q-5621-0 7

4 of 5
ENVIRONMENTAL ENTERPRISES USA, INC NELAP Certificate No. 02027

On Day 0, the treatments were poured into their respective test chambers, ten P. promelas larvae were

distributed randomly to each, and then this test was placed in the environmental chamber . On Day 2, the

test was renewed : 90% of the treatment solution, excess food, and debris were poured or siphoned out of

each replicate Aliquots of freshly prepared treatments were poured gently into each replicate as

appropriate. Surviving test organisms were disturbed as little as possible during renewal. Every 24 hours,

survival was recorded (Appendix B, pages 2 & 3). After 96 hours, the final survival data were recorded

and this test was terminated .

Summary of Experimental Condition s

Test Organisms.

Dilution Water:

Temperature.

Photoperiod .

Test Chambers .

Test Solution Volume .

Aeration :

Test Solution Renewal .

1-day-old Pimephales promelas larvae

Laboratory prepared Moderately- hard water

20+ 1°C

16 hours light; 8 hours dar k

Rectangular Pyrex Dish, 21cm x 11cm x 7cm

200 m l

No

Yes

RESULTS AND CONCLUSION

Total Volume = 1 .45 liters

The 96-hour survival data were also used to estimate the 96-hour LC50 : a point estimate of the

concentration expected to result in 50% mortality to exposed P. promelas neonates after 96 hours of

exposure . The 96-hour LC50 was 1 40 mg/1 PR, with a 95% confidence interval of 1 .21 to 1 .59% as

determined by Probit . Survival in the concurrent laboratory performance was 96.0% . Survival data

summary statistics are presented in Appendix C .

REFERENCES

EE USA. April 2007. Quality Assurance Plan EE USA, Slidell, LA 70461 .

EE USA. April 2007. Standard Operating Procedures. EE USA, Slidell, LA 70461 .

2003 National Environmental Laboratory Accreditation Conference Standards , Chapter 5 Quality Systems

EPA/6001R-04/003 . Approved at Ninth NELAC Annual Meeting, June 5, 2003 .

Tidepool Scientific Software ToxCalc Version 5 0 .

U S. Environmental Protection Agency . October 2002. Methods for measuring the acute toxicity of

effluents and receiving waters to freshwater and marine organisms. EPA-821-R-02-012 . 51h Edition

Office of Water (4303T). Washington, DC 20460

D pulex Q-562-07

P. promelas Q-5621-0 7

5 of 5
Environmental Enterprises USA, Inc.

APPENDIX A
9/25/07,3 :31 P M

Environmental Enterprises USA, Inc .

Sasol - Alfoterra L167-7S

Geoff Russell (337) 494-509 7

Preparation of ToxicantSimulated Effluent (EFF): Weigh 0.2966 g of Alfoterra L167-7S in a 25

ml beaker. Add Alfoterra L167-7S to class A cylinder and rinse beaker with Moderately Hard

Synthetic Freshwater (MHSF). Add MHSF to cylinder for total volume of 3000 ml. The EFF is split

into two 1500 ml aliquots. One aliquot is theO42mg/1 EFF for testing, and the second aliquot is used to

prepare the2~6 mg/1 . _-.- ';m°7

i6.3

(3.3 't,te A$ @0

Preparation of%6 mg/1,00 mg/1, W mg/1, and Ymg/1 dilutions:

CN-6 m 1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml of

~ this solution is the ~6 mg/1 EFF

i-5' s

'm 1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with. 1500 ml MHSF; 1500 ml

'1•t~ of this solution is the )'mg/I EFF

'1 4

g mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the mg/1 EFF

7.1i

mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is the ~g/1 EFF l

.

Data pages & Calculations by ~ QA/QC Officer: 119A-ujD pulex = 5 Reps x 25 ml

= 125 ml

DH2O = Dilution Water = MHSF

LPC M LPC M

Date 09/25 09/26

Alkalinity °1 g o

Conductivity 2zlq k5 36i A^

Hardness

H

TRC

LPC: Laboratory Performance Control, synthetic moderately hard water

Alkalinity: mg/1 as CaCO3 Conductivity : μS/cm Hardness: mg/1 as CaCO3

pH : su TRC : mg/ 1

Prep Date ~ 09/25 09/26

I Sample # ~ 1 1 I

Initial o'l , j,-

Conlments : Feeding / Exposure Chambers : D. pulex - none / 30 ml medicine cups

P. promelas - none / 16 oz. plastic cups

CcJ Ga .1 a-~ a , ps ccr a„

,a4-,2 1 .9, 15,3;

Alfoterra L167-7S

D AArj cbly"a~;,rh pm- 1 acs2

'30.5,~IL ~, . ►~~~Co ~ cXQ-562-07

I of 4
9/25/07,3 :31 PM

Environmental Enterprises USA, Inc .

Cladoceran, Daphnia pulex

Acute Static Renewal 48 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 202 1

Sasol - Alfoterra L167-7S

Survival Dat a

Treatment mg/1L PR

Time R;

E

P!

LPC

White

R

E

P

2

ppm

Blue

R ;

E

P

4

ppm

Green

R ;

E

P

8

ppm

Yellow

R ;

E

P

1 6

ppm

Brown

R

E

P

32 1

PP M

Black

1 Initial

0 HR

1! 10 6 10 16 10 21 10 26 10 o9/25io7

2i 10 7 10 F17 10 22 10 27 1 0

; 10 8 10 18 ; 10 23 10 28 1 0

4 10 9 10 14 10 19 10 24

E

29 1 0

_57_1 0 10 10 15 ; 10 20 ; 10 25 10 30 1 0

24

1

~

6

~

11 16 21 • 26

09/26/0 7

HR 2 ;

V

7 Q

l

12 ; 17 22 27 , ^

t-f~

3! 8 13 18 23 28

G 4i 9 14i 19i 24 1 o 29 i

5 10 15 20 25 30

1

!

6 16 21 26 09/27i0 7

HR

2 7

F

17 22 27

3! 8

~

-7 18 23! 28 !

r761 4 ; 9 14 , -7 19 24 29

5! IQ 10 15 20! 25! 30

Sur F`~ ~ z 17 c z L f 6-e' ~ CP

Counted by : QC/QA by:

Loaded by: W,

Test Organisms Age. hours old Test Organisms Source : CE t64

Data Entry by :

Double Data Entry by: _and/or

QC/QA by: - ~-------~~

Alfoterra L167-7S

2of4

Q-562-07
9/25/07,3 :31 PM

Environmental Enterprises USA, Inc .

D. pulex Water Quality Dat a

LPC & All Treatments: Initial Temp . - 18.5 - 21.4; Initial DO - 9 .2 < 4.0

0 to 24 HR Treatment mg/L PR

09/25-26/07 LPC 2 ppm 4 ppm 8 ppm 16 ppm 32 ppm Meter

DO I ~3•`~ - ~• c5 `~D

F

Temp I

>

F ~.~

~ ~ 1 < t l"I ~ ~"( .j t ► ~

Cond. I

pH F

R-N

. ~

Initials Initials . 777 Finals: 1ww

Times Initial Time : 1~~ Final Time: 6~9

24 to 48 HR Treatment mg/L PR

09/26-27/07 LPC 2 ppm 4 ppm 8 ppm 16 ppm 32 ppm Meter

DO I

S (P 8 g~~ S-~

F ~,S 2,5 S ~•Co ~65 ~~ S-7

Temp I

21-2 26 _9 2a u 21 .3 m s

F

ZD . 26 . 7I). 20- Z -W -

Cond. I 2D8 L3 ~~ I V ~ J(~ ) 302- ~

pH F

!

.~ ~ ~ ~ 2~ ~. ~ ~• ~ Q~

Initials Initials: 7-9 A Finals:

Times Initial Tim e : ~ Final Time :

DO, Dissolved Oxygen : mg/1 pH : SU Salinity: ppt Temp : °C

Alfoterra L 167-7S Q-562-0 7

3 of 4
9/25107,3 :31 PM

Environmental Enterprises USA, Inc .

Data Pages

E~ Company name & contact matches client file .

~ Product matches client file .

a~ Dilution series are correct:

Dilution series :

91 'A I U _

❑ Calculations on mixing page are correct. (sign mixing page)

d' Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

throughout data pages.

Format correct. (spaces for all entries, page numeration, no split pages, etc . )

~- Initials oq 2 5-16 -7 Date

Chain-of-Custody

Q-' Product on COC matches sample bottle .

w' Product on COC matches test data pages. W" Lab # on COC matches sample bottle . ©( Lab # on COC matches test data pages .

❑ Sample collection date: & Time

expiration date: & Time:

hrs .

hrs. (Sample expired if >3 6

hrs) N/A

❑ Sample volume is sufficient for test duration . (Sample volume in container(s)

checked against sample volume on mixing page)

Sample volume available: lc-b m l

Sample volume needed- o,

n p

q("'-

(Sample volume insufficient if sample volume available < sample vo lume needed)

Initials o 2S o'7 Date

Jugs & Labels

~' Lab # on jug and labels matches test data pages .

d Dilution water type is on jug . (i e. 25 ppt, 20 ppt, MHSF, etc . )

a" Dilutions on jugs and labels match dilutions on test data pages .

Y' Jugs are color-coded. (see mixing page for appropriate color code sequence )

1~ - Initials 0~j 40 - 7 Date

Raw Data QC/QA'd by: 15016- ?

Alfoterra L167-7S Q-562-07

4 of 4
Environmental Enterprises USA, Inc.

APPENDIX B
9/25/07,3 :35 PM

Environmental Enterprises USA, Inc .

Sasol - Alfoterra L167-7 S

Geoff Russell (337) 494-5097

Preparation of Toxicant Simulated Effluent (EFF): Weigh 0.2966 g of Alfoterra L167-7S in a 25

ml beaker. Add Alfoterra L167-7S to class A cylinder and rinse beaker with Moderately Hard

Synthetic Freshwater (MHSF). Add MHSF t9 cylinder for total volume of 3000 ml. The EFF is split

into two 1500 ml aliquots. One aliquot is the%1mg/1 EFF fertestirrg, and the second aliquot is used to

prepare the q,6 mg/l . tolb~VV

15-3 _1~ q

Preparation ot4mg/1,~1 mg/l,~mg/l~' mg/1, and~ mg/l dilutions :

{JXmg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml of

►5 .3 this solution is the mg/1 EFF

► • 3

Q mg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

-1-tv of this solution is the ~ mg/1 EF F

P-1-u'

Q f4mg/I EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

3~G of this solution is the I mg/1 EFF

~~

(DZ-mg/1 EFF : dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

t . R of this solution is the .2 mg/1 EFF

©i .9

x mg/1 EFF: dilute remaining 1500 ml of simulated EFF to 3000 ml with 1500 ml MHSF ; 1500 ml

of this solution is thdq g/1 EFF

oR

Data pages & Calculations by: Q ~>LA/QC Officer:

P. promelas = 5 Reps x 200 ml

= 1000 ml

DH2O = Diluticul Water = MHSF

LPC M

#

LPC M

Date 09/25 09/27

Alkalinity -t (p

Conductivity Q '36

Hardness ~ // 9~

pH ~f. o ~26 ~ b Q~

TRC HI

~/7~

LPC: Laboratory Performance Control, synthetic moderately hard water

Alkalinity : mg/I as CaCO3 Conductivity : μS/cm Hardness: mg/1 as CaCO3

pH : su TRC : mg/ 1

Prep Date 09/25 09/27

Sample # 1 1

Initial r h i,ti

Comments: Feeding / Exposure Chambers : D. pulex - none / 30 ml medicine cups

P. promelas - none / 16 oz. plastic cups

fj ~ .,Gd c j evE'if1 - erf.Tf ClCw ree~ 01_ ~~j A Ct'u fA.t ~e4 Cpl~tC'1.~R~lc~l+5 ta'ro-v

e

c2Qa5 O p

Alfo~ rra f 67-7S~~e o .~~s I ~ 3~,-r•rt, ~ r5 . ~ ~~(1 i R . +6%~/~ tr~ Q-5621-07

1 of 6
9/25/07,3 :35 PM

Environmental Enterprises USA, Inc .

Fathead Minnow, Pimepahles promelas

Acute Static Renewal 96 - Hour Definitive Test

EPA-821-R-02-012 : Section 9, Method 200 0

Sasol - Alfoterra L167-4S

Survival Data

Treatment mg/L PR

Time Ri

E i

P!

LPC

White

R

E

P

1

ppm

Blue

R

E

P

2

PP M

Green

R 4

E ppm

P Yellow

R

E

P

8

PP m

Brown

R

E

P

1 6

PP m

Black

Initial

S

0~ 1!;

10 6 ~ 10 11 10 16 10

!

21 10 26

!

10 09/25ro7

2 10 7 10 12 !

10 17 1 0

!

22 !

10 27

!

1 0

10 8 10 13 ; 10 18 10 23 10 28 ; 1 0

4 ; 10 9 10 14 10 19 10 24 10 29 1 0

5 ; 10 10 10 15 10 20 10 25 10 30 1 0

1 q 6 11 16 21 26 0

09/26/07

2 4

HR 2! ( p 7 (c) 12 17 -~ 22 rL 2 7

3, f D 813 18 ! f ~

~O

23 ~ 28 0

4!

, 9 14 C1 19 "7 24 29

(`f3~ 5! O 10 D 15 20 25 30 0

lj 6 16 21 ~ 26 O

09/27/0 7

4 8

HR 2 ; 7 r12 17 22 2 7

3i io g ~C~ 18 ; 23 ; 2g i

4! (rl 9 q 14 19 24 0 29 O

1

5;

;

10 15 20 25 o 30

mg/L

Sur

Counted by :

Loaded b

Test Organisms Age : _ I __ days old

byt

Test Organisms Sourc e

1

`0

ov,

Alfoterra L 167-7S

2 of 6

Q-5621-07
9/25/07,3 :35 PM

Environmental Enterprises USA, Inc .

P. promelas Survival Data Cont .

I

Treatment mg/L PR

Time R; LPC R I R 2 R 4 R 8 R 16 jj~ Initial

E; White E ppm E pp- E ppm E ppm E ppm

Pi P Blue sI P Green P Yellow P Brown P Blac k

72

11 16 Q 21 26

09/28/07

HR 2~ k O 7 I c~

l

12 3 17 C) 22 C) 27 Y-~

3! QO 8 ,(D 13 18 Q 23 28 V

4 ; ~fl 9 14 19i 24 C) 29i C)

S' 10 15

b" l

20 25 ~ 30 i n

96

6 11 ! © 16 21 26

O 09/29/07

HR 2 7 12 O 17 0 22 G 27 6

3 ; JO

l

8 ; O 13 18

6

23 C28 ;

4; 9 ; 14 19 24 29

5 10 15 20 25 30

Su/r ! U

Data Entry by: LLCC97

Double Data Entry by : 4~~& and/or

QC/QA by : - --

~~fY -

Alfoterra L167-7S Q-5621-07

3 of 6
9/25/07,3 :35 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Data

LPC & All Treatments: Initial Temp. - 18.5 - 21.4 ; Initial DO - 9.2 < 4. 0

0 to 24 HR Treatment mg/L P R

09/25-26/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Meter

DO I

CJ- CD _3 C7) U 0

F SS, fP S-7

Temp I D AA z ~b qF -Z(;'Y ` . i ZO .? 20 - 2a-7

a`Il~ra-'

Cond. I

pH

L

Initials Initials: Finals: ~7-K

Times Initial Time:

t b 2 Final. Time :

48 HR Treatment mg/L PR

09/27/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Mete r

DO F 'i , fp g .l ~ .l g+ S7

Temp F

~o .7 20 _ 20 _ ~ 26 _~ 26.~ &_~E

pH F

Initials Initials: Finals: 6J~,-7-4

Times Initial Time : FinalTime : pg~

DO, Dissolved Oxygen : mg/1 pH: SU Salinity: ppt Temp: °C

!/Z--

Alfoterra L167-7S Q-5621-07

4of6
9/25/07,3 :35 PM

Environmental Enterprises USA, Inc .

P. promelas Water Quality Data Cont.

48 to 72 HR Treatment mg/L PR

09/27-28/07 LPC 1 ppm 2 ppm 4 ppm 8 ppm 16 ppm Meter

DO I

3 /

F 8~ <~ "1

Temp I

/7 (4P

F CPo i a q q I~~ ge c>z~

Cond. I 3 c),3 .J D! 3 c)3 3 --- ~ 3 0.5r /19 .~

pH F 7,/ 0 0 (Pb O ~ ?0

Initials Initi als: J*G 1-).,4 Finals ~~-

Times Initial Time : / (,o 75- Final Time :0

96 HR Treatment mg/L PR

09/29/07 LPC I ppm 2 ppm 4 ppm 8 ppm 16 ppm Meter

DO F -

~ .5 8- s

Temp F

pH F

7. 0

Initials Initials : Finals:

Times _ Initial Time : Final Time:

/ v Jy

Alfoterra L167-7S Q-5621-07

5 of 6
9/25/07,3:35 PM

Environmental Enterprises USA, Inc .

Data Pages

t~ Company name & contact matches client file .

c~ Product matches client file .

Lj/ Dilution series are correct:

Dilution series :

-1_ 2- , 14 Y

❑ Calculations on mixing page are correct . (sign mixing page)

~d' Dates, dilutions, test method, # of replicates, replicate volume, product,

acceptance limits, data analysis endpoint, and test organisms are correct

,throughout data pages .

QT Format correct. (spaces for all entries, page numeration, no split pages, etc .)

Initials q Z 0Date

Chain-of-Custody

~ Product on COC matches sample bottle .

r~ Product on COC matches test data pages .

ur," Lab # on COC matches sample bottle .

Y" Lab # on COC matches test data pages.

❑ Sample collection date: & Time:

expiration date: & Time:

hr ) N/A

~ Sample volume is sufficient for test duration .

checked against sample volume on mixing page)

Sample volume available : I oh ml

Sample volume needed- D.~Fft+o)

031 aj0-7

w

hrs.

hrs . (Sample expired if >36

(Sample volume in container(s )

(Sample volume insufficient if sample volume available < sample volume needed)

Initials p 2 ki Date

Jugs & Labels

,rr Lab # on jug and labels matches test data pages .

q~ Dilution water type is on jug . (i.e . 25 ppt, 20 ppt, MHSF, etc . )

gi Dilutions on jugs and labels match dilutions on test data pages .

D/ Jugs are color-coded . (see mixing page for appropriate color code sequence)

Initials t ~ 6`~ Date

Raw Data QC/QA'd by:

Alfoterra L 167-7S Q-5621-07

6 of 6
Environmental Enterprises USA, Inc.

APPENDIX C
Acute Toxicity Test-48 Hr Survival

Start Date . 9/24/2007 Test ID: dp56207 Sample ID : LAB-Lab Sampl e

End Date . 9/26/2007 Lab ID: EE-Environmental Enterprise Sample Type : PRD-Product

Sample Date . Protocol : EPAF 02-EPA Freshwater Test Species : DP-Daphnia pule x

Comments :

Conc-mg/L 1 2 3 4 5

PC-LP Control 0.9000 0 .9000 0.9000 1 .0000 1 .0000

1 .9 0.8000 0.9000 0 .9000 0.9000 0.9000

3 .8 0 .6000 0.7000 0 .7000 0.7000 0.8000

7 .6 0 .9000 0.6000 0 .5000 0.5000 0.6000

15 .3 0 .5000 0.5000 0 .4000 0.4000 0 .5000

30.5 0 .3000 0.5000 0 .3000 0.4000 0 3000

Transform : Untransformed Number Total

Conc-mglL Mean CV% Mean Min Max CV% N Resp Number

PC-LP Control 0.9400 5 .827 0.9400 0 .9000 1 .0000 5 .827 5 3 50

1 .9 0.8800 5 .082 0.8800 0 .8000 0.9000 5.082 5 6 50

3.8 0.7000 10.102 0.7000 0 .6000 0.8000 10.102 5 15 50

7 .6 0.6200 26.503 0.6200 0.5000 0.9000 26.503 5 19 5 0

15 .3 0.4600 11 .907 0.4600 0.4000 0.5000 11 .907 5 27 5 0

30.5 0 .3600 24.845 0.3600 0.3000 0.5000 24.845 5 32 5 0

Auxiliary Tests Statistic Critical Skew Kurt

Shapiro-Wilk 's Test indicates normal distribution (p > 0 .01) 0 .90354 0 .9 1 .38261 3 .7233

Bartlett's Test indicates equal variances (p = 0.09) 9 .48417 15.0863

Maximum Likelihood-Probi t

Parameter Value SE 95% Fiducial Limits Control Chi-Sq Critical P-value Mu Sigma Iter

Slope 1 .30488 0.25384 0.80735 1 .80241 0.06 1 .49798 7.81473 0 .68 1 .19751 0.76635 4

Intercept 3.43739 0.27542 2.89758 3.9772

TSCR 0.05548 0. 03206 -0. 0073 0. 11831 1, 0

Point Probits mg/L 95% Fiducial Limits

ECO1 2 .674 0.25984 0.02336 0.7861 0 . 9

EC05 3 .355 0.86491 0.1596 1 .91935 0 . 8

EC 10 3 .718 1 .64206 0.44024 3.1192 0 7

EC15 3 .964 2.53068 0.86578 4.36447 y 0. 6 EC20 4.158 3.56888 1 .46912 5.7499

EC25 4.326 4.79307 2.28872 7.35977 a 0. 5

EC40 4.747 10.0776 6.39771 14.9872 ~ 0. 4

EC50 5.000 15.7583 10.6738 25.5733

EC60 5 .253 24.6413 16 .42 47.3251 0 . 3

EC75 5 .674 51 .8093 30.563 144.729 0 2

EC80 5 .842 69.5808 38.5211 228.977 0 1

EC85 6 .036 98.1261 50.1766 392.9 8

EC90 6 282 151 228 69 6054 779 25 4 0

0. 0

EC95 6 .01 1 100 10000 1000000 .645 287.11 112.323 2165. 5

EC99 7.326 955.684 272.645 14881 .3

Dose mg/L

Page 1 ToxCalcv5 .0 23 Reviewed by:
Environmental Enterprises USA, Inc .

APPENDIX D
Acute Toxicity Test-96 Hr Su rviva l

Start Date: 9/24/2007 Test ID: pp562107 Sample ID: LAB-Lab Sampl e

End Date 9/29/2007 Lab ID: EE-Environmental Enterprise Sample Type : PRD-Product

Sample Date- Protocol : EPAF 02-EPA Freshwater Test Species : PP-Pimephales promelas

Comments .

Conc-mg/L 1 2 3 4 5

PC-LP Control 0.9000 1 .0000 1 .0000 0 .9000 1 .0000

0 95 0.7000 0 .8000 1 .0000 0.7000 0.8000

1 .9 0.0000 0 .0000 0.2000 0.1000 0.7000

3 .8 0 .0000 0.0000 0.0000 0.1000 0.0000

7 .6 0 .0000 0.0000 0.0000 0.0000 0.0000

153 0 .0000 0.0000 0 .0000 0.0000 0.0000

Transform : Untransformed Number Total Conc-mg/L Mean CV% Mean Min Max CV% N Resn Number

LP Control 0.9600 5 .705 0.9600 0.9000 1 .0000 5.705 5 2 5 0

0.95 0 .8000 15.309 0 .8000 0.7000 1 .0000 15.309 5 10 50

1 9 0 .2000 145.774 0 .2000 0.0000 0 .7000 145.774 5 40 5 0

3 .8 0 .0200 223.607 0 .0200 0.0000 0 .1000 223.607 5 49 5 0

7 .6 0.0000 0 .0000 0 .0000 0 .0000 5 50 5 0

15.3 0.0000 0 .0000 0 .0000 0.0000 5 50 5 0

Auxi

Shapiro-Wilk's Test indicates non-normal distribution (p <= 0 .01) 0.80225 0.868 2.03272 6_46568 Bartlett's Test indicates unequal variances (p = 1 .86E-03) 14.955 11 .3449

Maximum Likelihood-Probi t

Parameter

Slope 5

Value SE 95% Fiducial Limits Control Chi-S Critical P-value Mu Sigma Iter .31138 0.79827 3.74677 6.87599 0.04 0 .83513 7.81473 0.84 0.14578 0.18827 3 Intercept 4.22569 0.20861 3.81681 4.63456

TSCR 003815 002702 -0 0148 0 091 1

Point Probits mg/L 95% Fiducial Limits

1 . 0

EC01 2 .674 0.51026 0.31266 0.67373 0' 9

EC05 3 .355 0.68564 0 .47156 0.85315 0 . 8

EC 10 3.718 0.8026 0 .58567 0 .96985

EC15 3.964 0 .89258 0 .67682 1 .05915 0 . 7

EC20 4.158 0 .97124 0 .75827 1 .13746 0. 6

EC25 4 .326 1 .04423 0.83485 1 .21077 a 0 5

EC40 4 .747 1 .25339 1 .05557 1 .42835 m

EC50 5 .000 1 .39889 1 .20572 1 .59051 0 4

EC60 5 .253 1 .56129 1 .3657 1 .78605 0 3

EC75 5 .674 1 .87402 1 .64673 2 .2094

EC80 5.842 2.01484 1 .76322 2 .41826 0'2

EC85 6.036 2 .19239 1 .9035 2 .69515 0 1

EC90 6.282 2 .43819 2 .08856 3 .09999

EC95 6 645 2 8541 2 3848

0 .0

EC99 7 .326 3.8351 3.0328 5

5 3.83305 0

.75656 .1 1 1 0 100

Dose mg/L

Page 1 ToxCalc v5.0.23 Reviewedby:
Environmental Enterprises USA, Inc .

APPENDIX E
~a

w

Cl)

w

~

>

O

0)

. . ...

cr

tih

y

~''•~-.~. ~~

0

4 4 a

U~

~ I U

sz rrJ

s. z

~

J

9- •j

~

-~-

-

l~

~.~

`

(II '

/1G

U)

wOoZ

U

-~

~

Z.) aU

-a

cu

va i

o aU CL . o E U

~

cl) J

LL

} -~- +~ .~ -~-

+

TO

EEE

F- F- 1- m 2

cu cu a 0 0 013

(DC

LO I

m~mm

~JI_-~.C~L

~w~~

~E

a) (u~a~UN0,

a)

F-~m0am~Q ) TcN

(L)

~

(3) (1)

F- E- F0 0 (L)

(a t0 ~ fII

~ ~ O~~0

mKN

~~U

~. ~

NE

F-

(3) ~05, ~Q)

Ln

=3

cr _

a) C~

a) EH

(1

)

C

6T~a ~ >NUNx

I 9

O0MXMO0NU0UN

4)ONU0.CUN~a)EX
Environmental Enterprises USA, Inc .

APPENDIX F
D. pulex SRT, KCI mg/l

CV% = 9 .76

1400

1300

120 0

110 0

U

J

Ir 1000

s

CO

a 900 _.~

800

700

600

°'?b 01\"\-b ~ o \, o \,

Date of Test

+2 SD

+1 SD

Mean

-1 SD

-2 SD

Test# Test Date 48-hr LC50

mg/L KCI

Cumulative

Mean LC50 _1 SD -2 SD +1 SD +2 SD Control

Survival

Toxicant Lot

#

DP0601 1/4/2006 1000 94 0 075K009 0 DP0602 2/24/2006 1120 1060 .0000 975 1472 890 .2944 1144 .8528 1229 .7056 920 075K0090 DP0603 3/22/2006 978 1032 6667 956 .2381 879 .8094 1109 0953 1185 .5239 98.0 075K0090

DP0604 4/12/2006 802 975 0000 843.8665 712 .7330 1106 .1335 1237 .2670 980 075K0090

DP0605 5/22/2006 958 971 .6000 857 .7808 743 .9617 1085 4192 1199 2383 100 .0 075K0090 1 DP0606 6115/2006 963 970 1667 868 .3032 766 4397 1072 0301 1173 8936 1000 075K0090 1 DP0607 7/31/2006 854 953 5714 850 7385 747 .9055 1056 4044 1159 .2374 1000 075K0090 1 DP0608 8/10/2006 1090 970 6250 863 .8985 757 .1719 1077 3515 1184 .0781 1000 075K0090 1 DP0610 9/28/2006 1010 975 0000 874 .3074 773 .6148 1075 6926 1176.3852 1000 075K0090 1 DP0611 10/10/2006 1010 978 5000 882 .9231 787 .3462 1074 .0769 1169 .6538 1000 075K0090 1 DP0614 12/6/2006 949 975 8182 884 7108 793 .6033 1066 .9256 1158 0330 920 066K0032 DP0701 1/31/2007 951 973 7500 886 .5875 799.4251 1060 9125 1148 0749 100 .0 066K003 2 DP0702 2/14/2007 1110 984 2308 892.6219 801 .0130 1075 .8397 1167 .4486 1000 066K003 2 DP0703 3/28/2007 908 978 7857 888 4435 798 .1012 1069 1280 1159 .4702 960 066K003 2 DP0705 5/3/2007 897 973 3333 883 7528 794 .1723 1062 9138 1152 4943 98 .0 066K003 2 DP0706 5/8/2007 983 973 9375 887 3608 800 7841 1060 5142 1147 .0909 980 066K0032 DP0707 6/5/2007 831 965 5294 874 .8162 784 1030 1056.2426 1146 .9559 940 066K003 2 DP0708 7/18/2007 926 963 3333 874 .8367 786 3401 1051 .8299 1140 .3265 100 .0 096K004 0 DP0709 8/29/2007 978 964 1053 878 .0362 791 .9672 1050 .1743 1136 2433 960 096K004 0 DP0710 9/26/2007 781 954 .9500 861 .7064 768 .4628 1048 .1936 1141 .4372 98 .0 096K0040

November- EE cultures crashed before SRT was completed Purchased D pulex

DP0614 - ABS D . pulex

DP0701 -ABS D pulex

DP0704 - Control failure
P . promelas SRT, KCI mg/ I

1600

1400

1200

N

U

~ 1000

x

C; 800

600

400

200

C1/% = 29 .2

~b

~p

`O

W,

bo tio~~`Oa~o`~0~060~~0~o6 o~ o~ ~~

6

~o6\o ax

o`

1~1

Date of Tes t

Test # Test Date 48-hr LC50

mg/L KCI

Cumulative

Mean LC50

-1 SD -2 SD +1 SD +2 SD

PP0601-48 1/4/2006 77 1

PP0602-48 2/14/2006 877 824 0000 749.0467 674 .0934 898 9533 973 906 6

PP0603-48 3116/2006 671 773 0000 669 9854 566 .9709 876 0146 979.0291

PP0604-48 4118/2006 975 823 5000 692 0631 560 .6261 954 9369 1086,3739

PP0605-48 5/26/2006 879 834 6000 718 .0976 601 .5953 951 1024 1067 .6047

PP0606-48 6/14/2006 720 815 5000 701 2761 587 0522 929 7239 1043 9478

PP0607-48 7/26/2006 1490 911 .8571 636 4203 360 .9834 1187 2940 1462.7309

PP0608-48 8/16/2006 689 884 .0000 617 0998 350 1996 1150 9002 1417.8004

PP0609-48 9/5/2006 645 857 4444 595 3795 333 3146 1119 5094 1381 .5743

PP0610-48 10/10/2006 404 812 1000 526 4283 240 7566 1097 7717 1383 .4434

PP0611-48 11/7/2006 787 809 8182 538 .7006 267 5829 1080 9358 1352 .0534

PP0612-48 12/14/2006 796 808 .6667 550 .1354 291 .6041 1067 1979 1325.7292

PP0701-48 1/4/2007 579 791 0000 535 .4105 279 .8210 1046 5895 1302.1790

PP0702-48 2/7/2007 958 802 .9286 553 .3429 303 7573 1052 5142 1302 0999

PP0703-48 3/26/2007 907 809 .8667 567 .8635 325 8603 1051 8699 1293 8731

PP0704-48 4/4/2007 989 821 0625 583 .0148 344 .9670 1059 1102 1297.1580

PP0705-48 5/23/2007 909 826 2353 594 .7619 363 2885 1057.7087 1289 1821 PP0706-48 6/12/2007 773 823 2778 598 .3653 373 4528 1048 .1903 1273 .1027

PP0707-48 7/26/2007 958 830 3684 609 6184 388 .8683 1051 1185 1271 .8685

PP0708-48 8/22/2007 1120 844 8500 620 .4394 396 .0287 1069 .2606 1293.671 3

PP0709-48 9/26/2007 1450 873 6667 618 1661 362 6656 1129 1672 1384.6678

+2 SD

+1 SD

Mean

-1 SD

-2 SD

Control Toxicant L

Survival #

97 5 075K0090

100.0 075K0090

97.5 075K009 0

100 .0 075K00901

100 .0 075K00901

100 .0 075K0090 1

1000 075K00901

1000 075K0090 1

975 075K00901

100.0 066K0032

97 .5 066K003 2

100 0 066K0032

1000 066K0032

100.0 066K0032

100 0 066K0032

1000 066K0032

100 0 096K0040

100 .0 096K0040

100.0 096KO040

100 .0 096K0040
Appendix E .

Biodegradation of Early Versions of ALFOTERRA® Propoxy Sulfate Surfactants

Primary Biodegradation of ALFOTERRA® Surfactants in the OECD 301A Tes t

ALFOTERRA® % MBAS Loss

Surfactant 1 6 9 10 13 15 17 22 28

12-3S 4.7 86.3 87.9 88 9 88.8 89 .1 95.2 -- 96 . 3

± Std. Dev. ± 1 .4 ± 0 .7 ± 0 .2 ± 1 .9 ± 0.1 ± 2.8 ± 0.3 -- ± 0. 4

123-2S 5.1 95 .8 97.4 97 6 97.2 98 .4 97.5 98.3 98 . 2

± Std. Dev . ± 0.5 ± 0 .2 ± 0 .2 ± 0 .3 ± 1 .5 ± 0.1 ± 0.2 ± 0.1 ± 0. 2

123-4S 10.1 876 86.9 868 88.6 91 .5 890 -- 91 . 1

± Std. Dev . ± 5.5 ± 2 .2 ± 0 .4 ± 0 3 ± 0.2 ± 0.6 ± 0.6 -- ± 0. 6

123-8S 5 .3 358 46.0 39.9 45.5 51 .6 45.3 -- 46 . 1

± Std. Dev ± 1 .7 ± 4 .2 ± 3 .2 ± 5 6 ± 0 .9 ± 2.5 ± 1 .3 -- ± 0. 3

145-2S 83 97 2 96 .7 97.6 97.8 98.6 -- -- 96 . 4

± Std . Dev ± 1 8 ± 0 2 ± 0 .1 ± 0 .1 ± 0 .3 ± 0 .2 -- -- ± 0. 1

145-4S 4 .7 86 .4 87 .9 88 .9 88.8 88.8 88.1 -- 88. 9

±Std.Dev . ±141 ±0.7 ±0.2 ±1 .9 ±0 .1 ±2 .5 ±2.5 -- ±2. 3

Ultimate Biodegradation of ALFOTERRA® Surfactants in the OECD 301A Tes t

ALFOTERRA® % DOC Loss

Surfactant 1 3 6 9 10 13 15 17 22 28

12-3S 13.8 1 7 5.8 13.8 24 .4 25.5 35.4 40 .0 45.7 46. 4

± Std . Dev. ± 7 .7 ± 2.4 ± 0.5 ± 2.2 ± 1 .2 ± 2 .5 ± 2 7 ± 0.1 ± 2.5 ± 4. 9

123-2S 84 20.8 29 .2 38.3 39 .6 43.0 47.3 53 7 56.7 52 . 1

±Std.Dev. ±11 .9 ±1 .6 ±0.1 ±1 .0 ±3.5 ±06 ±2 .8 ±21 ±1 .34 ±1 .4 1

123-4S 3 .9 1 4 8.8 16.1 17 .4 17.1 21 .5 28 .0 29.9 28. 7

±Std .Dev ±13 ±16 ±2.8 ±0 .4 ±1 .1 ±0 .2 ±2 .1 ±06 ±3 .3 ±2. 5

123-8S 0 0 1 3 0 10 .9 5 .7 4 .7 14 .0 151 10. 9

± Std . Dev ± 0 ± 0 ± 1 .8 ± 0 ± 4.2 ± 1 .3 ± 0 .8 ± 0.8 ± 2 .3 ± 1 . 6

145-2S 2 .4 21 6 288 37.8 42 .8 43.3 45.2 51 .5 546 54. 5

± Std . Dev ± 0 .2 ± 0 5 ± 0.5 ± 2 .2 ± 0.2 ± 0 .8 ± 2 .9 ± 0.8 ± 3 .1 ± 3. 5

145-4S 2 .7 2.9 133 18.2 24.8 26.3 28.1 34 .7 362 33. 9

±Std .Dev. ±0 .9 ±13 ±3.5 ±0 .8 ±0.1 ±2 .1 ±4 .2 ±0.6 ±1 .2 ±1 3

Acetate 88.9 97 4 96 5 98.5 96.5 99 .2 97.6 98 .3 96.5 100

± Std . Dev +9 .5 ± 0 .8 ± 0 5 ± 1 .5 ± 0 .3 ± 1 .3 ± 0 .9 ± 2.5 ± 1 .1 ± 0
~~H

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Appendix F .

Toxicity Reports on Earlier Versions of ALFOTERRA® Propoxy Sulfate Surfactants .

Daphnia magna and Pimephales promelas.

Prepared for Condea Vista Company by ENSR

Lab Report #8503-111-006 (015 thru 020)

Lab Report #8503-106-006 (001 thru 008)

August 1999
Consuiting • Engineering > Remediation

September 24, 1999

Allen M Nielsen, Ph .D

CONDEA Vista Company

PO Box 20013 5

Austin, TX 78720-0135

4303 West LaPorte Avenue

Fort Collins, CO 8052 1

(970) 416-0916

FAX (970) 493-8935

hftp .//www ensr co m

Subject: Definitive Acute Toxicity Tests of Three Materials on Daphnia magna

and Pimephales promelas

Dear Allen :

We have completed the definitive, static-renewal, acute toxicity tests with the three materials

sent to us by CONDEA Vista Company on August 10, 1999 . All three materials were tested

with both Daphnia magna and Pimephales promelas (fathead minnow) The following table

summarizes the test conditions for each species:

Test Conditions

Test Parameter

,

D~ • hnia ma rta

• ,

Pi

M

e 'faales romefas

Age <24 hours <24 hour s

Test Chambers 100-m1 glass beakers 500-ml glass beakers

Dilution Water Hard Moderately Har d

Volume per Replicate 70-80 ml 300 ml

Number of Replicates 4 4

Organisms per Replicate 5 1 0

Test Temperature 20±1 `C 20±1 ° C

Feeding None 0 1 ml brine shrimp @ 48 h ( 2

hours prior to renewal)

Test Duration 48 hours 96 hours

Endpoint Mortality Mortality

Test Concentrations Nominal Nominal

Test Guidance ASTM 1998 Standard Guide for Conducting Acute Toxicity Test s

on Test Materials with Fishes, Macro invertebrates, an d

Amphibians Method E729-96, pp 218-238 In 1998 Annual Boo k

of ASTM Standards, Vol . 11 05, Biological Effects an d

Environmental Fate ; Biotechnology, Pesticides. American Society

of Testing and Materials, West Conshohocken, P A

USEPA 1993. Methods for Measuring the Acute Toxicity o f

Effluents and Receiving Waters to Freshwater and Marin e

Organisms . Fourth Edition . EPA1600/4-90/027F .
~ :5 7

A Nielse n

24 September 1999

Page 2

Tests were renewed daily with test solutions prepared fresh each day . For the tests with D.

magna, renewal solutions on Day 1 were prepared with the original stock solution prepared

on Day 0 Day 0 stocks of 145-2PO-SO4 and 123-2PO-SO4 were also used to prepare the

fathead minnow test solutions each day . However, because of the larger volume of 12-3POS04 needed to renew the fathead minnow tests, a fresh stock solution of this material was

prepared dail y

A summary of the test results is presented in the following table More detailed information

on organism survival in each test is presented in the attached pages ,

LCso (95% CI) (mg/L)

Product

Daphnia magna 48-h Prrrrephales promelas 96-q

Alfoterra 12-3PO-SO4 8 88 (6 51-9 85) 10 99 (10 23-11 80 )

Alfoterra 123-2PO-SO4 2 06 (1 76-2.29) 4 65 (4..27-5 08 )

1 Alfoterra 145-2PO-SO4 2 .19 (1 .86-2.49) 2.31 (2.14-2.48)

Please give me a call if you have any questions about any of these results . We appreciate

the opportunity to conduct these studies for CONDEA Vista Company

Sincerely,

David A Pillard, Ph D

Senior Aquatic Ecologist/Toxicologist

attachments - Summary Reports

file: 8503-111-006
EIiF'.R Environmental Toxicology Services

SUMMARY REPORT - ACUTE TOXICITY TEST

SPONSOR : CONDEA Vista Company

TEST NUMBER: 8503-111-006-01 7

TEST SUBSTANCE: Alfoterra 12-3PO-SO4

ENSR SAMPLE NUMBER: 12569

TEST SPECIES : Daphnia magn a

TEST DATE(S) : 21-23 August 199 9

EXPERIMENTAL RESULTS

Nominal N4m4,e,'r,?E~pc~sed Number Surviving % Survlval @ 4 8

Concentration

m IL 2€ FioiifS 48 h.ou~s ;

hours

0 (Control) 20 20 20 100

5 20 20 20 10 0

10 20 20 5 25

12 .5 20 19 1 5

15 20 12 0 0

17.5 20 10 0 0

20 20 9 0 0

48=ho0r EGS ; m /L 85% Con#ide ace Inte rval m IL Metho d

8 .88 6 .51 - 9 .85 Probit
E1F'R

Environmental Toxicology Services

SUMMARY REPORT - ACUTE TOXICITY TEST

SPONSOR: CONDEA Vista Compan y

TEST NUMBER: 8503-111-006-020

TEST SUBSTANCE: Alfoterra 12-3PO-SO 4

ENSR SAMPLE NUMBER : 12569

TEST SPECIES: Pimephales promelas

TEST DATE(S): 27-31 August 1999

EXPERIMENTAL RESULTS

Nominal Nuf yil~er Number Surviving °!a ~~(rvi~at @

Concentratio n

m l,L

Exposed

24 houqs, 48 hburs 72 hours 96 hours

96 hours

0 (Control) 40 40 40 40 40 10 0

1 25 40 40 40 40 40 10 0

2 .5 40 40 40 40 40 10 0

5 40 40 40 40 40 10 0

10 40 40 35 33 30 75

125 40 36 25 23 18 45

15 40 9 4 3 1 2 .5

20 40 0 0 0 0 0

qS=hour LC m L : ;; ' 9'5% Confidence Intsrval;, ; tL : ' ~llethoi~

10 .99 10 .23 - 11 .80 Spearman-Karber
ER R

SPONSOR:

TEST NUMBER:

Environmental Toxicology Services

SUMMARY REPORT - ACUTE TOXICITY TES T

TEST SUBSTANCE:

ENSR SAMPLE NUMBER:

TEST SPECIES:

TEST DATE(S) -

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-116-006-01 5

Alfoterra 123-2PO-SO4

12570

Daphnia magna

21-23 August 1999

1Jon6 itlai Number Exposed Number Su rv iving % Surviva[ (M 4$

Concentration

m L 24 hours 48 hours

hours

0(Control) 20 20 20 10 0

1 20 20 20 10 0

2 20 20 11 5 5

3 20 17 1 5

4 20 6 0 0

5 20 9 0 0

48-hour LC im /L) 95% Confidence Interval (m!L . fulgthd -

2 .06 1 .76-2.29 Probft
EM'R

SPONSOR:

TEST NUMBER:

Environmental Toxicology Services

SUMMARY REPORT - ACUTE TOXICITY TEST

CONDEA Vista Company

8503-111-006-01 8

TEST SUBSTANCE:

ENSR SAMPLE NUMBER:

TEST SPECIES:

TEST DATE(S):

EXPERIMENTAL RESULTS

Alfoterra 123-2PO-SO4

12570

Pimephales promelas

27-31 August 199 9

Nominal Number Number Surviving % Survival @

Concentration

m L

Exposed

44liours; 48hours 72 hours 96 hours

96 hours

0 (Control) 40 40 40 40 40 10 0

1 25 40 40 40 40 40 10 0

25 40 40 40 40 40 10 0

5 40 28 23 23 20 50

75 40 0 0 0 0 0

12 .5 40 0 0 0 0 0

98-hour LG m/L , 95"Jo Confidence Inte rval m/L Method

4 65 4 .27-5.08 Spearman-Karber
EN'R

SPONSOR:

TEST NUMBER:

Environmental Toxicology Service s

SUMMARY REPORT - ACUTE TOXICITY TEST

TEST SUBSTANCE:

ENSR SAMPLE NUMBERTEST SPECIES:

TEST DATE(S):

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-116-006-01 6

Alfoterra 145-2PO-SO4

12571

Daphnia magna

21-23 August 1999

Nominal Number Exposed Number, Surv iving % Survival @ 4 8

Concentration

m L 24 haur5 48 hours

hours

0(Contro l ) 20 20 20 100

1 20 20 20 100

2 20 20 13 6 5

3 20 16 1 5

4 20 11 1 5

5 20 9 1 5

48-hour LC m LL 95%,Confidence:,lnterval ,; m L . Method

2 .19 1 .86-2.49 Probit
ER R

SPONSOR:

TEST NUMBER :

Environmental Toxicology Services

SUMMARY REPORT - ACUTE TOXICITY TEST

CONDEA Vista Company

8503-111-006-01 9

TEST SUBSTANCE:

ENSR SAMPLE NUMBER:

TEST SPECIES:

TEST DATE(S):

Alfoterra 145-2P0-SO4

12571

Pimephales promelas

30 August - 3 September 1999

EXPERIMENTAL RESULT S

NomiR;aF° .

t

Number Number Surviving %Survival- @

Conce n tV;a iori

m)L

Exposed

24,hGyC4 .,r~~ 48 hours 72 h,ours 96 hour's

96,hours

0 (Controi) 40 40 40 40 40 100

0 .5 40 40 40 40 40 100

1 40 39 39 39 39 97.5

2 40 40 39 36 35 87. 5

3 40 33 10 5 3 7 . 5

4 40 14 0 0 0 0

5 40 0 0 0 0 0

46-hour LC,( m .L' , 95°l Cqnfi'dence Inte~Method

2 31 2 .14-2.48 Spearman-Karber
~

Consulting • Engineering - Remediatio n

March 18, 1999

4303 West LaPorte Avenue

Fort Collins, CO 80521

(970) 416-091 6

FAX (970) 493-8935

http :1/www ensr corn

Allen M. Nielsen, Ph D

CONDEA Vista Company

PO Box 200135

Austin, TX 78720-013 5

Subject: Definitive Acute Toxicity Tests of Four Materials on Daphnia magna and

Pimephales promelas

Dear Allen:

We have completed the definitive, static-renewal, acute toxicity tests with the four materials

sent to us by CONDEA Vista Company on January 19, 1999 All four materials were tested

with both Daphnia magna and Pimephales promelas (fathead minnow) The following table

summarizes the test conditions for each species :

Test Conditions

Test Parameter Daphnia magna Pime hales romelas

Age <24 hours <24 hours

Test Chambers 100-m1 glass beakers 500-m1 glass beaker s

Dilution Water Hard Moderately Hard

Volume per Replicate 70-80 ml 300 ml

Number of Replicates 4 4

Organisms per Replicate 5 1 0

Test Temperature 20±1 °C 20±1 ° C

Feeding None 0 1 ml brine shrimp @ 48 h (2

hours prior to renewal )

Test Duration 48 hours 96 hour s

Endpoint Mortality Mortality

Test Concentrations Nominal Nominal

Test Guidance ASTM 1998 Standard Guide for Conducting Acute Toxicity Test s

on Test Materials with Fishes, Macro invertebrates, and

Amphibians Method E729-96, pp 218-238 In 1998 Annual Book

of ASTM Standards, Vol . 11 05, Biological Effects and

Environmental Fate ; Biotechnology ; Pesticides American Society

of Testing and Materials, West Conshokocken, P A

USEPA 1993 Methods for Measuring the Acute Toxicity o f

Effluents and Receiving Waters to Freshwater and Marin e

Organisms . Fourth Edition. EPA/600/4-90/027F.
EMOR

A Nielsen

18 March 1999

Page 2

Tests were renewed daily with test solutions prepared fresh each day Renewal solutions on

Day 1 were prepared with the original stock solution prepared on Day 0 New stock

solutions were prepared on Day 2 and used for renewal of the fathead minnow tests on Days

2 and 3

A summary of the test results is presented in the following table More detailed information

on organism survival in each test is presented in the attached pages

LC_,, (95% Cl) (rng/L)

Product

qaphnra magna Pime 'hales,promelas

Alfoterra 123-8PO-SO4 2 60 (2 12-3 20) 1 68 (1 .56-1 81 )

Alfoterra 145-4PO-SO4 2 34 (2 01-2 73) 1 62 (1 51-1 74 )

STEOL CS-370 11 40 (9 49-13 28) 437 (4 24-4 52 )

N 550 L. LAS 11 .93 (10.02-14 48) 3.91 (3 .62-4.23)

Please give me a call if you have any questions about any of these results We appreciate

the opportunity to conduct these studies for CONDEA Vista Compan y

Sincerely ,

David A Pillard, Ph D

Senior Aquatic EcologistlToxicologis t

attachments - Summary Reports

file: 8503-106-006
EPRIt ~csv~a~sae~taC 7axicaPag*y .$m ceea

SUMMARY REPORT - ACUTE TOXICITY TEST

SPONSOR : CONDEA Vista Company

TEST NUMBER: 8503-106-006-00 1

TEST SUBSTANCE. Alfoterra 123-8PO-SO 4

ENSR SAMPLE NUMBER. 11832

TEST SPECIES : Daphnia magn a

TEST DATE(S) : 2-4 Februa ry 1999

EXPERIMENTAL RESULTS

Nominal Number Exposed Number Surviving % Survival @4$

Concentratio n

(mg/L) 24 hours 48 hours

hQurs,

0 (Control) 20 20 20 10 0

0 156 20 20 20 10 0

D 312 20 20 20 10 0

0 625 20 20 20 10 0

1 25 20 20 17 8 5

25 20 19 15 7 5

5 .0 20 17 0 0

48-hour LC mg!L) 95% Confidence interval (mg/L) Methoe#, ;, .

2 60 212 - 3.20 Probit
EN!J g~~~ ~d &~ S~

SUMMARY REPORT - ACUTE TOXICITY TEST

SPONSOR: CONDEA Vista Compan y

TEST NUMBER- 8503-106-006-005

TEST SUBSTANCE: Alfoterra 123-8PO-SO4

ENSR SAMPLE NUMBER 11832

TEST SPECIES : Pimephales promelas

TEST DATE(S)- 2-6 February 1999

EXPERIMENTAL RESULTS

Nominal Number Number Surviving % S-urvivak~~

Concentratio n

(mg/L}°

Exposed

24 hours 48 hours 72 hours 96 hours

96 hour s

0 (Control) 40 40 40 40 40 100

0 156 40 40 40 40 40 100

0 312 40 40 39 39 39 97 5

0 625 40 40 40 40 39 97 5

125 40 40 38 38 38 9 5

25 40 5 1 1 1 2 5

5 .0 40 0 0 0 0 0

96-hour LC m/L a 95% Confidence Interval (mg/L) Method

1 68 1 56 - 1 .81 Spearman-Karbe r

3 LCs,s were calculated using initial nominal concentrations Nominal concentrations of

renewal solutions may have been slightly different because more or less volume was

needed These differences are very minor and have little or no impact on the LC,,

calculations
EAMR` 5isuvcowrceutw 7ax6col~ .$m,,ic"

SUMMARY REPORT - ACUTE TOXICITY TEST

SPONSOR: CONDEA Vista Compan y

TEST NUMBER: 8503-106-006-002

TEST SUBSTANCE : Alfoterra 145-4PO-SO4

ENSR SAMPLE NUMBER : 11833

TEST SPECIES : Daphnia magna

TEST DATE(S)- 9-11 Februa ry 1999

EXPERIMENTAL RESULTS

Nominal Number Exposed Number Surviving °!'Survival @ 4 8

Concentration

(m L 24 hours 48 hours

hours

0 (Control) 20 20 19 9 5

0 312 20 20 20 10 0

0 625 20 20 20 10 0

1 25 20 20 20 10 0

25 20 20 8 4 0

50 20 5 0 0

10 0 20 0 0 0

48-hour LC (mg/L) 95% Confi dencelnterval m/L Method

2 .34 2 .01 - 2 .73 Spearman-tCarber
ER'R~

-- - ~

SPONSOR:

TEST NUMBER5a~eo4e 7aze:~ smeez,;i,

SUMMARY REPORT - ACUTE TOXICITY TEST

CONDEA Vista Compan y

8503-106-006-006

TEST SUBSTANCE:

ENSR SAMPLE NUMBER:

TEST SPECI ES:

TEST DATE(S) :

EXPERIMENTAL RESULTS

Alfoterra 145-4PO-SO 4

11833

Pimephales promelas

9-13 February 1999

Nominal

C tr t

Number'

E

Number Surviving % Survival @

oncen a ion

(m g/L)

xposed

24 hours 4 8 hours 72 hours 96' hours

96 hours

0(Control) 40 40 40 40 40 10 0

0 312 40 40 40 40 40 10 0

0 625 40 40 40 40 40 10 0

1 25 40 40 40 39 35 87 5

2 5 40 11 0 0 0 0

50 40 0 0 0 0 0

10 .0 40 0 0 0 0 0

96-hour LC (mg/L)a 95% Confidenc e Interval (mg/L) Method

1 62 151-1.74 Spearman-Karber

a LC,,s were calculated using initial nominal concentrations Nominal concentrations of

renewal solutions may have been slightly different because more or less volume was

needed These differences are very minor and have little or no impact on the LC50

calculations
ENR

SPONSOR:

TEST NUMBER :

~~~~ ~,;rzc~

SUMMARY REPORT - ACUTE TOXICITY TES T

TEST SUBSTANCE:

ENSR SAMPLE NUMBERTEST SPECIES:

TEST DATE(S) :

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-106-006-003

STEOL CS-370

11834

Daphnia magna

2-4 February 199 9

Nomina l

Concentration

Number Exposed Number Sur•viving, % Survival @ 48

(mg/L) 24 hours 48 hours

hours

0 (Control) 20 20 20 100

312 20 20 20 10 0

625 20 20 18 9 0

125 20 18 9 45

18 75 20 18 3 1 5

250 20 14 0 0

48 hour LC (m L} 95% Confidence Intervaf m L} Metho d

11 40 9 .49 - 13 28 Probit
EI'4R

SPONSOR:

TEST NUMBER :

9,,6aasusreoata.a? 17axCc~ Sm,iced

SUMMARY REPORT - ACUTE TOXICITY TES T

TEST SUBSTANCE •

ENSR SAMPLE NUMBERTEST SPECIES:

TEST DATE(S) :

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-106-006-007

STEOL CS-370

11834

Pimephales promelas

2-6 February 1999

Nomina l

Concentration

Number

Ex os d

Number SurAving % Survival, @

(mg/L)

p e

24 hours 48 hours 72 hours 96 hours

96 hour s

0(Controi) 40 40 40 40 40 10 0

0156 40 40 40 39 38 9 5

0 312 40 40 40 40 38 9 5

0 625 40 10 3 2 1 2 5

125 40 0 0 0 0 0

25 40 0 0 0 0 0

96-hour LC (tn L)a 95% Confidence interval (rn /L Method

4 37 4 .24-4.52 Trimmed Spearman-Karber

a LC50s were calculated using initial nominal concentrations Nominal concentrations of

renewal solutions may have been slightly different because more or less volume was

needed These differences are ve ry minor and have little or no impact on the LC,,

calculations
EP&'R"

SPONSOR.

TEST NUMBER.

~ot(/flLOoINLP,ct~ 74YGf.4~ SQlu{CG a

SUMMARY REPORT - ACUTE TOXICITY TEST L

TEST SUBSTANCE:

ENSR SAMPLE NUMBER.

TEST SPECIES .

TEST DATE(S):

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-106-006-004

N 550 L LAS

11835

Daphnia magn a

9-11 Februa ry 1999

Nomina l

Concentration

Number Exposed Number Su rv iving %Survivai @ 48

(m /L 24 hours 48 hours

hours

0(Control) 20 20 20 100

1 56 20 20 20 100

312 20 19a 19 10 0

469 20 20 19 95

6 25 20 20 19 9 5

125 20 16 11 5 5

25 .0 20 0 0 0

~1- 01yanisnn was accidentally killed aurrng transfer at renewal, and was eliminated from analysi s

48-hour LC mg/L) 95% Corrfidenee interval (m /L Method

11 93 10 02 - 14 .48 Probit
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SPONSOR.

TEST NUMBER:

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SUMMARY REPORT - ACUTE TOXICITY TEST

TEST SUBSTANCE:

ENSR SAMPLE NUMBER'

TEST SPECIES :

TEST DATE(S).-

EXPERIMENTAL RESULTS

CONDEA Vista Company

8503-106-006-008

N 550 L LAS

11835

PimephaEes promela s

9-13 February 1999

Nomina l

Concentr ti

Numbe r

E

. ,

Number Survivin g . % Survival @

a o n

1g/L)

xposed

24 hours 48 hours, 72 h'ours 96'fiours

96 hours

0 (Control) 40 40 40 40 40 10 0

0 78 40 40 40 40 40 10 0

1 56 40 40 40 40 40 10 0

312 40 40 40 40 29 72 5

4 69 40 40 39 31 15 37 5

6 25 40 37 9 2 0 0

125 40 0 0 0 0 0

96-hour LC (mgtL)a 95% Confidence Interval (mg/L Method

391 3 62 - 4.23 Probit

a LC50s were calculated using initial nominal concentrations Nominal concentrations of

renewal solutions may have been slightly different because more or less volume was

needed These differences are very minor and have little or no impact on the LC,,

calculations