# Biodegradability and Acute Aquatic

Toxicity of Alcohol Propoxy Sulfates or

Sasol’s ALFOTERRA ® Surfactants

Sasol Report Number R&D ENV 10

- 005 01

- 10

Sasol North America

May 2010

Work conducted by Geo ff Russell and Nomi hla Valashiya

- Mdleleni

Report edited by Marta Bourke

## 12/14/2011 Page 2 of 66



# Interoffice Correspondence

To Ricky Stackhouse

From Geoff Russell, Marta Bourke , Victoria Stolarski

Date May 30, 2010

**Subject :**  **PROJECT SUMMARY: Sasol Repo**  **rt R&D ENV 10**

**- 005 01** 

**- 10** 

**Biodegradability and Acute Aquatic Toxicity of Alcohol Propoxy Sulfate or**

**Sasol ’s**  **ALFOTERRA ®**  **Surfactant s.** 

**Key Words:**  ALFOTERRA ® , Alcohol propoxylates, Alcohol propoxy sulfates, Biodegradation,

Aquatic Toxicity, Oil recovery, Em ulsification

**Conclusions:** This report documents Sasol North America Inc.’s additional biodegradation and

aquatic toxicity studies on the new products, ALFOTERRA® alcohol propoxy sulfate surfactants

.

This work conf irms that the ALFOTERRA® alcohol propoxy sulfates, based on either C12

- C13,

C14

- C15, or C16

- C17 branched / linear primary alcohol, pose little risk to the environment. The

ALFOTERRA surfactants are not intended or expected to be released into the environment

when used as a surfactant for the enh anced recovery of petroleum oil

.

Using ALFOTERRA® 123

- 4S (ALFO 23

- 4S) surfactant as the model compound for the studies

containe d herein , the following conclusions can be made :

1) The rate and extent of the biodegradation of a lcohol propoxy sulfates , specifically

branched C12

- 13 alcohol 4

- mole propoxy sulfate (ALFO 23

- 4S) is notably less than that

of the standard detergent surfactant alcohol ethoxy sulfate

, or specifically

- linear

dodecyl 4

- mole ethoxy sulfate ( POLYSTEP B

- 43 0S from Stepan Company) as shown by

the remaining dissolved organic carbon in the ALFO 23

- 4S effluent from Porous Pot,

Die

- Away, and Electrolytic Respirometer testing.

2) Acute aquatic toxicities to fish of the neat material and the effluents from Porous Pot and

Die

- Away testing were determined using standard US EPA methods. The results

showed that both ALFO 23

- 4S and the control POLYSTEP B

- 430S had the same LC 50

values for the neat and effluent samples despite the significant re maining dissolved

organic car bon in the effluent of the ALFO 23

- 4S sample

.

3) Methylene Blue Active Substances (MBAS) analysis of both the neat and effluent

samples proved that ALFO 23

- 4S undergoes primary degradation and elimination of

surfactancy despite the significant remaining di ssolved organic carbon in the effluent of

the ALFO 23

- 4S sample af ter porous pot or 28

- day die

- away testing.

4) The acute aquatic toxicity to fish of the neat ALFO 23

- 4S is eliminated once the ALFO

23

- 4S passes through the porous pot or 28 die

- awa y test. Th e results of 96

- hour static

P. promelas (EPA

- 821

- R

- 02

- 013) showed for example that the LC50 of 14% dilution for

the ALFO 23

- 4S feed to the porous pot decreased to LC50 >100% dilution for the

effluent.

5) Using ALFOTERRA® surfactants for enhanced hydrocarbon recovery (EHR) will not

result in the discharge of any material into surface or ground water

. Any rinse aid

## 12/14/2011 Page 3 of 66

containing ALFOTERRA® surfactants, after processing through standard domestic or

industrial waste water treatment

, will pose no threat to the envir onment.

**Results Summary :**  Sasol designed and assembled a porous pot system in accordance with

the “ Fate, Transport and Transformation Test Guidelines OPPTS 835.3240 Simulation Test

— Aerobic Sewage Treatment: Activated Sludge Units Guideline” during the sp ring of 2009 to

further evaluate the biodegradation of branched alcohol propoxy sulfate surfactants

. The neat

surfactant, and the inf luent and effluent to the P orous P ot S ystem and additionally to the OECD

301

- A 28 D ay Die

- away Biodegradation Study was fu rther evaluated by 96

- hour static P.

promelas (EPA

- 821

- R

- 02

- 013) and Methylene Blue Active Substance. A ll s ample details,

experimental information, and discussion of the results are given in the following detailed report

and appendices

. A summary of the most relevant results is given here.

**Table I**

**- Porous Pot Data: ALFO 23** 

**- 4S compared to Benzoate Control** 

**DOC of Benzoate or ALFO 23-4S in Feed (mg/L)**

11.47 10.39

**Steady State established after foaming and zooglea issues**

**Blank Benzoate**  **ALFO 23-4S Blank**  **Blank Blank**  **Benzoate Benzoate**  **Benzoate Benz/Blk**  **ALFO 23-4S ALFO 23-4S**  **ALFO 23-4S Alfo/Blk** 

**Feed Feed**  **Feed Effluent**  **Effluent Mean**  **Effluent Effluent**  **Mean Mean Δ**  **Effluent Effluent**  **Mean Mean Δ** 

9-Jul 298.728 311.296 298.594

10-Jul 317.728 325.822 328.198 10.925 10.027 10.476 10.282 10.059 10.170 (0.306) 16.795 17.069 16.932 6.456

11-Jul 328.520 344.872 340.120 12.123 10.784 11.453 10.257 11.208 10.733 (0.721) 23.031 21.277 22.154 10.700

12-Jul 320.754 329.708 327.200 10.177 9.901 10.039 9.635 11.296 10.465 0.426 24.781 21.062 22.921 12.882

13-Jul 308.686 326.120 319.074 9.601 12.228 10.914 7.604 9.700 8.652 (2.262) 21.760 20.971 21.365 10.451

14-Jul 339.732 340.814 327.676 10.750 14.165 12.457 8.695 11.253 9.974 (2.483) 19.603 21.720 20.662 8.204

15-Jul 341.820 338.308 337.690 11.782 13.150 12.466 9.664 12.519 11.091 (1.375) 20.341 20.116 20.228 7.762

17-Jul 232.758 233.788 240.148 12.684 12.014 12.349 10.059 11.731 10.895 (1.454) 20.030 19.936 19.983 7.634

18-Jul 232.886 231.650 211.742 11.512 9.657 10.585 9.846 10.122 9.984 (0.601) 18.593 18.202 18.397 7.813

19-Jul 214.990 208.772 239.962 10.142 8.769 9.455 9.585 8.777 9.181 (0.274) 17.149 16.910 17.029 7.574

20-Jul 211.682 217.640 225.588 10.135 9.646 9.890 10.248 9.934 10.091 0.200 17.453 18.026 17.739 7.849

21-Jul 207.632 228.256 192.536 9.948 10.538 10.243 9.276 9.871 9.573 (0.670) 18.599 19.865 19.232 8.989

22-Jul 170.546 162.330 187.782 10.278 10.342 10.310 8.374 10.016 9.195 (1.115) 18.656 19.230 18.943 8.633

23-Jul 291.736 297.840 295.308 11.071 12.114 11.592 10.128 12.021 11.074 (0.518) 18.546 17.728 18.137 6.545

24-Jul 281.972 282.374 263.638 12.209 13.765 12.987 11.293 12.585 11.939 (1.048) 19.924 19.956 19.940 6.953

25-Jul 271.570 248.690 278.154 12.292 12.273 12.282 10.240 10.609 10.424 (1.858) 19.288 19.466 19.377 7.095

26-Jul 284.028 284.438 284.248 11.954 9.425 10.689 8.959 9.459 9.209 (1.481) 17.847 17.849 17.848 7.159

27-Jul 299.512 290.840 272.186 10.939 8.797 9.868 7.975 7.508 7.741 (2.127) 18.242 18.240 18.241 8.373

28-Jul 300.796 293.262 295.778 9.882 9.287 9.584 8.158 7.811 7.984 (1.600) 17.745 18.582 18.163 8.579

29-Jul 280.696 283.508 283.382 9.548 9.386 9.467 8.711 7.597 8.154 (1.313) 18.532 19.619 19.075 9.609

**Av erage**  **10.945 10.856**  **10.900 9.420**  **10.214 9.817**  **19.311 19.254**  **19.282**

**Std Dev 1.00**  **1.69 0.97**  **1.55 2.04**  **1.44**

**Mean Difference (Test-Blank Control)** -1.08 8.38

**Std Deviation (Test-Blank Control)** 0.81 1.61

**Number of Paired Observations** 19 19

**Calculated Student's T Value** 5.84 22.76

**Critical Student T Value** 1.73 1.73

**Percent Removal** 109.44% 19.33%



## The resulting porous pot data was analyzed as described in the OPPTS 835.3240 guidelines.

E ach sample, Blank, Benzoate, and ALFO 23

- 4S, was run in duplicate. T he benzoate control

showed on average 109% removal verses the blank feed. The ALFO 23

- 4S showed only 19%

removal verses the blank feed. Though the ALFO 23

- 4S does degrade somewhat when run

through the porous pot, a significant portion of the original d issolved organic carbon can be

seen in the effluent.

## 12/14/2011 Page 4 of 66

**Table II**

**- Porous Pot Data: ALFO 123** 

**- 4S compared to Linear C12 alcohol ethoxy sulfate** 

**(AES) , a standard commercial detergent surfactant** 

**DOC of AES or ALFO 23-4S in Feed (mg/L)**

16.91 16.04

**Steady state established after AES introduced as comparative sample**

**AES ALFO 23-4S**  **Blank Blank**  **Blank Blank**  **AES AES**  **AES AES**  **ALFO 23-4S ALFO 23-4S**  **ALFO 23-4S ALFO 23-4S** 

**Feed Feed**  **Feed Effluent**  **Effluent Mean**  **Effluent Effluent**  **Mean Mean Δ**  **Effluent Effluent**  **Mean Mean Δ** 

15-Aug 247.940 239.436 243.984

16-Aug 227.632 242.160 235.068 9.695 8.290 8.992 10.478 10.862 10.670 1.678 21.293 19.181 20.237 11.245

17-Aug 281.066 271.800 270.500 11.568 8.570 10.069 11.509 14.866 13.187 3.119 17.302 17.302 17.302 7.233

18-Aug 285.140 270.510 269.560 12.018 9.307 10.662 11.688 15.705 13.696 3.034 18.969 22.359 20.664 10.002

19-Aug 275.450 271.450 269.650 13.800 10.053 11.926 11.848 15.251 13.549 1.623 19.597 22.014 20.805 8.879

20-Aug 289.350 278.560 281.450 14.611 11.347 12.979 17.829 21.568 19.698 6.720 15.622 17.571 16.597 3.618

21-Aug 279.750 278.800 277.350 16.138 11.615 13.877 14.716 19.332 17.024 3.147 17.644 18.968 18.306 4.430

22-Aug 280.540 278.340 281.650 17.584 12.708 15.146 12.209 17.113 14.661 (0.485) 23.570 21.705 22.637 7.492

23-Aug 298.340 287.560 285.350 16.477 13.405 14.941 11.595 16.266 13.931 (1.010) 25.438 21.192 23.315 8.374

24-Aug 286.440 279.340 281.680 14.895 13.977 14.436 12.185 15.317 13.751 (0.685) 26.305 19.852 23.078 8.642

25-Aug 295.740 289.440 290.680 15.076 14.925 15.000 11.531 14.278 12.904 (2.096) 26.019 19.862 22.941 7.940

26-Aug 298.420 295.560 300.120 14.906 13.342 14.124 10.975 12.606 11.791 (2.333) 24.315 18.374 21.345 7.221

27-Aug 330.952 316.158 318.600 15.615 13.905 14.760 10.818 12.286 11.552 (3.208) 23.274 18.981 21.127 6.367

28-Aug 307.162 290.204 300.828 12.299 14.862 13.580 10.349 11.129 10.739 (2.841) 23.970 18.040 21.005 7.425

29-Aug 304.460 301.334 292.984 13.277 16.367 14.822 10.875 11.683 11.279 (3.543) 24.091 18.669 21.380 6.559

30-Aug 325.070 320.074 320.618 12.634 16.180 14.407 11.779 12.318 12.048 (2.358) 25.882 22.884 24.383 9.976

31-Aug 291.448 298.708 293.032 12.591 15.182 13.886 20.714 20.531 20.623 6.736 27.283 24.304 25.794 11.907

1-Sep 294.140 285.684 288.552 13.315 13.724 13.519 15.978 15.113 15.545 2.026 26.113 22.066 24.090 10.570

2-Sep 312.742 309.938 313.838 13.188 13.010 13.099 11.366 10.581 10.973 (2.126) 25.023 21.006 23.014 9.915

3-Sep 290.976 289.786 290.064 14.452 12.917 13.684 10.427 10.439 10.433 (3.252) 25.200 21.760 23.480 9.795

4-Sep 288.360 296.040 290.426 13.879 12.045 12.962 10.066 9.636 9.851 (3.112) 24.601 21.613 23.107 10.145

5-Sep 309.616 312.466 321.020 12.706 11.765 12.235 9.901 9.223 9.562 (2.674) 23.713 21.243 22.478 10.242

6-Sep 287.982 284.368 285.684 12.154 10.926 11.540 10.149 9.594 9.871 (1.669) 23.138 21.749 22.443 10.904

7-Sep 284.012 281.480 275.438 12.281 11.856 12.068 10.503 10.411 10.457 (1.611) 22.527 22.286 22.406 10.338

8-Sep 316.224 310.068 314.572 13.398 13.973 13.686 12.498 11.436 11.967 (1.719) 23.158 23.409 23.283 9.598

9-Sep 298.202 283.170 284.760 14.156 16.722 15.439 12.835 11.529 12.182 (3.257) 23.764 24.306 24.035 8.596

10-Sep 289.590 291.248 281.028 13.958 20.085 17.021 12.443 11.069 11.756 (5.265) 22.332 23.412 22.872 5.851

11-Sep 313.108 314.666 321.496 13.364 17.689 15.527 12.641 10.469 11.555 (3.972) 22.748 22.751 22.749 7.223

12-Sep 298.286 292.386 285.142 12.454 13.183 12.819 11.709 10.070 10.889 (1.929) 20.968 22.204 21.586 8.767

13-Sep 295.626 286.828 284.828 11.568 11.801 11.685 11.608 9.767 10.688 (0.997) 21.284 22.282 21.783 10.098

14-Sep 319.138 315.114 319.714 10.784 11.753 11.268 11.620 9.710 10.665 (0.604) 24.308 22.193 23.250 11.982

15-Sep 319.138 315.114 319.714 11.152 11.740 11.446 16.719 10.765 13.742 2.296 24.853 22.927 23.890 12.445

16-Sep 291.394 281.264 291.620 10.475 11.996 11.235 17.648 10.704 14.176 2.941 22.944 22.809 22.876 11.641

17-Sep 309.616 315.482 315.364 10.270 13.251 11.761 17.593 10.528 14.060 2.300 23.992 23.299 23.645 11.885

18-Sep 300.366 295.708 302.800 10.234 12.364 11.299 16.903 10.486 13.694 2.396 25.509 23.497 24.503 13.204

19-Sep 299.902 294.464 288.270 10.007 11.515 10.761 14.706 10.180 12.443 1.682 23.861 22.900 23.380 12.619

20-Sep 305.896 315.156 317.666 9.932 10.770 10.351 13.268 10.482 11.875 1.524 23.232 22.389 22.810 12.460

21-Sep 295.528 286.798 288.486 11.445 11.675 11.560 13.377 11.486 12.431 0.871 23.479 23.480 23.479 11.920

22-Sep 294.174 292.998 287.644 11.901 12.035 11.968 16.809 15.046 15.927 3.960 23.499 24.853 24.176 12.208

23-Sep 324.054 296.356 322.126 13.533 15.133 14.333 12.245 13.439 12.842 (1.491) 23.338 23.302 23.320 8.987

24-Sep 301.344 300.094 296.898 11.314 13.262 12.288 12.698 15.840 14.269 1.981 23.617 23.046 23.332 11.044

**Av erage**  **12.877 12.980**  **12.929 12.920**  **12.728 12.824**  **23.194 21.651**  **22.422**

**Std Dev 1.81**  **2.23 1.67**  **2.69 3.57**  **2.99 3.20**  **1.89 2.20** 

**Mean Difference (Test-Blank Control)** (0.105) 9.494

**Std Deviation (Test-Blank Control)** 2.90 2.32

**Number of Paired Observations** 40 40

**Calculated Student's T Value** (0.23) 25.87

**Critical Student T Value** 1.67 1.67

**Percent Removal** 100.62% 40.81%



## During the above time period the ALFO 23

- 4S showed 41% removal versus the blank feed.

However, the AES showed 100% removal versus the blank feed. Though similar surfactants,

the branching of the alcohol and alkoxylate portions of the ALFO 23

- 4S sig nif icantly inhibits

complete biodegradation within the evaluation period , unlike the all

- linear AES which quickly

and completely degrades.

## 12/14/2011 Page 5 of 66

**Graph I**

**- OECD 301** 

**-**

**A Die**

**- Away Data: ALFO 123** 

**- 4S compared to Linear C12 alcohol** 

**ethoxy sulfate (AES), a standard commercial detergent surfactant**



## OECD Die-A

**wa y DOC Loss** 

**0 20**  **40 60**  **80 100** 

**0 5**  **10 15**  **20 25**  **30**



### Days**% D** **O C** **L o** **s s** 

**AE S**  **ALFO Benz** 



## A 28

- d ay die

- away biodegradation study was run on the neat ALFO 23

- 4S and AES surfactants

shown above . A dd itionally, the die

- away test was run on the effluents on the porous pot to

show that the bi o degradation of the ALFO 23

- 4S, though slow, will continue (data in full report)

. Again the neat ALFO 23

- 4S shows only partial degradation in the 28 day period.

**Graph II**

**- Methyl Blue Active Substance**  **(MBAS) Data: ALFO 123** 

**- 4S compared to Linear** 

**C12 alcohol ethoxy sulfate (AES), a standard commercial deterg ent surfactant** 

**Porous Pot: MBAS Primary Biodegradation**

**0 5**  **10 15**  **20 25**  **30 35**  **40 45** 

**Blank AES**  **AL FO 23-4S** **M B** **A S** **( m** **g /** **L )**  **Influent**

**Effluent**



### Influent

Effluent

## 12/14/2011 Page 6 of 66

MBAS was run on the blank, AES, and ALFO 23

- 4S influent and effluent samples of the porous

pot study (PPS)

. The results show that significant primary biodegradation occurred fo r both

surfactants

. The MBAS percent reduction in activit y for the AES and ALFO 23

- 4S during the

PPS were 99% and 94% respectively.

Surfactan t cy (3mg/L) seen in the blank influent is due to the commercial laundry soap included

in the blank recipe.

**Table III** 

**- 96** 

**- hour static**  **P. promelas (EPA** 

**- 821** 

**- R** 

**- 02** 

**- 013)**  **of neat, influent and effluent**

**samples of ALFO 23**

**- 4S and AES** 

**Sample**

**Description Environmental** 

**Enterprise USA**

**Sample ID Survival** 

**NOEC % Survival** 

**LOEC % LC50 %** 

**(95% Confidence**

**Interval)**

PPS Blank Influent

Feed Q

- 642

- 09

- **1336**

**- 38** 

**- 1**  12% 25% 14.7 %

(12.4%

- 17.5%)

PPS AES Influent

Feed Q

- 643

- 09

- **1336**

**- 38** 

**- 2**  3.0% 6.0% 5.47%

(4.91%

- 6.10%)

PPS ALFO 23

- 4S

Influent Feed Q

- 644

- 09

- **1336**

**- 38** 

**- 3**  6.0% 12.0% 9.48%

(8.28%

- 10.9%)

AES Neat

(40 mg/L active) Q

- 645

- 09

- **1336**

**- 38** 

**- 4**  6.0%

(2.4 mg/L) 12.0%

(4.8 mg/L) 13.8% (5.52 mg/L)

(12.3%

- 15.5%)

ALFO 23

- 4S Neat

(40 mg/L active) Q

- 646

- 09

- **1336**

**- 38** 

**- 5**  3.0%

(1.2 mg/L) 6.0%

(2.4 mg/L) 7.76% (3.10 mg/L)

(6.64%

- 9.08%)

**PPS Blank**

**Effluent** Q

- 647

- 09

- **1336**

**- 38** 

**- 6**  **100% >100%**  **>100%**

**(NA )** 

**PPS A ES Effluent**  Q

- 648

- 09

- **1336**

**- 38** 

**- 7**  **100% >1**  **00% >100%** 

**(NA )** 

**PPS A LFO 23** 

**- 4S** 

**Effluent** Q

- 649

- 09

- **1336**

**- 38** 

**- 8**  **100% >100%**  **>100%**

**(NA )** 

ALFO 23

- 4S

Die

- Away

Influent

(50 mg/L) 0

- day Q

- 715

- 09

- **1336**

**- 58** 

**- 2**  6%

(3.0 mg/L) 12%

(6.0 mg/L) 8.8% (4.4 mg/L)

(8.0%

- 9.7%)

ALFO 23

- 4S

Die

- Away

Effluent

(50mg/L) 28

- day Q

- 716

- 09

- **1336**

**- 58** 

**- 1**  100% >100% >100%

NA

Data reported is the weight

- percent of the original test solution in the final dilution. For example, line 1 shows that in

order to achieve 50% survival rate, the PPS Blank Influent solution had to be diluted to 14 .7 % PPS Blank Influent and

85.3% water. The higher the value in the table the less effect the original solution had on the organisms. Values of

>100% indicate that all of the organisms survived in the solution as supplied to the test. No dilution was requi red.



## Acute aquatic toxicity testing on fat

- head minnows showed that the toxicity of the neat and

influent ALFO 23

- 4S is completely removed by processing through the porous pot system or 28

- day die

- away, despite the incomplete degradation seen. In fact, t he effluent toxicity of the ALFO

23

- 4S is equal to that of the AES which shows complete degradation in the laboratory tests.

It can be concluded from this data that ALFO 23

- 4S and similar branched alcohol propoxy

sulfates will not undergo complete bi odegr adation but will exhibit primary degradation which

renders the original molecule and resulting metabolites inert to aquatic life.

## 12/14/2011 Page 7 of 66 

# Biodegradability and Acute Aquatic Toxicity

of Alcohol Propoxy Sulfates or Sasol’s

ALFOTERRA

® Surfactants

Sasol Report Nu mber R&D ENV 10

- 005 01

- 10

Sasol North America

May 2010

Work conducted by Geoff Russell and Nomihla Valashiya

- Mdleleni

Report edited by Marta Bourke

## 12/14/2011 Page 8 of 66



# Ta **ble of Content s** 

TABLES

................................ ................................ ................................ ................................ ..... 9

TERMS

................................ ................................ ................................ ................................ ...... 9

INTRODUCTION

................................ ................................ ................................ ...................... 10

OBJECTIVES

................................ ................................ ................................ ........................... 10

CONCLUSIONS

................................ ................................ ................................ ........................ 11

RESULTS OF

................................ ................................ ................................ ........................... 11

EXPERIMENTS

................................ ................................ ................................ ........................ 11

TEST MATERIALS

................................ ................................ ................................ ................ 11

BIODEGRADATION STUDY

................................ ................................ ................................ . 13

Porous Pot System (PPS) Biodegradation Test Method

................................ .................... 13

Porous Pot System Daily Operations

................................ ................................ ................. 15

Porous Pot System Experimental Results

................................ ................................ ......... 18

PPS R esults Summary:

................................ ................................ ................................ ..... 22

OECD 301 A: DOC Die

- Away Experimental Results

................................ ............................. 22

MBAS Analysis

................................ ................................ ................................ .................. 24

Aquatic Acute 96hr Fathead Minnow Results

................................ ................................ .... 26

OECD 301F Biodegradability Electrolytic Respirometer Study

................................ .............. 29

OECD 301 F Electrolytic Respirometer Results:

................................ ................................ 32

OECD 301 F Electrolytic Respirometer Summary:

................................ ............................ 34

Discussion

................................ ................................ ................................ ................................ 35

Environmental Fate and Effects of ALFOTERRA ® 

## Surfactants

................................ .............. 35

Biodegradation Pathway of Anionic Surfactants Studied

................................ ....................... 36

REFERENCES

................................ ................................ ................................ ......................... 39

APPENDICES

................................ ................................ ................................ ........................... 40



# Ta **ble of Figures**

Figure 1: Porous Po t System (PPS) Assembly Test

................................ ................................ .. 14

Figure 2: Hydraulic Retention Time (HRT) Through Porous Pot System

................................ .. 15

Figure 3: Mixed Liquor T otal Suspended Solids (MLTSS) in Porous Pot Candle

...................... 16

Figure 4: Porous Pot Effluent TSS

................................ ................................ ........................... 17

Figure 5: Porous Pot Effluent pH Va lues

................................ ................................ .................. 17

Figure 6: Porous Pot DOC Feeds and Effluents

................................ ................................ ....... 18

Figure 7: Porous Pot Effluents DOC

................................ ................................ ......................... 19

Figure 8: Porous Pot Effluents

- Un

- acclimated and acclimated Response

.............................. 20

Figure 9: Return to Steady State (DOC)

................................ ................................ ................... 20

Figure 10: Porous Pot Effluent DOC: A Comparison of AES and ALFO 23

- 4S

........................ 21

Figure 11: Porous Pot Effluent: Increased HRT effect on DOC

................................ ............... 22

Figure 12: Die

- Away Porous Pot Effluent

................................ ................................ ................. 23

Figure 13: OECD 301A Die

- Away DOC Loss of neat ALFO 23

- 4S, AES, and Benzoate.

......... 24

Figure 14: Porous Pot MBAS Primary Biodegradation

................................ ............................. 25

Figure 15: ALFO 23

- 4S Comparison MBAS Porous Pot and Die Away

................................ .... 25

Figure 16: AES and ALFO 23

- 4S Neat Material Acute Toxicity Before Any Biodegradation

..... 27

Figure 17: Porous Pot Feed and Effluent: Aquatic Toxicity AES and ALFO 23

- 4S

................... 28

Figure 18: Acute Toxicity Test, Die

- Away Test ALFO 23

- 4S

................................ .................... 29

Figure 19: Biodegradation Study

- Alcohols (OECD 301F)

................................ ....................... 32

Figure 20: Biodegradation Study

- Alcohol 4 Mole Propoxylates (OECD 301F)

......................... 33

Figure 21: Biodegradation Study

- Alcohol 4 Propoxy Su lfate (OECD 301F)

........................... 34

Figure 22: Biodegradation profile of ALFO 23

- 4S Derivatives

................................ .................. 35

Figure 23: Alcohol Ether Sulfate Structure : AES and ALFO 23

- 4S

................................ .......... 36

## 12/14/2011 Page 9 of 66



# TA **BLE S** 

Table 1: Properties of the four mole propoxy alkoxylate derivatives (nonionic) of the six alcohols

tested

................................ ................................ ................................ ................................ ........ 12

Table 2: Properties of the anionic Surfactants based on the above propoxylates which are

comparable to the ALFOTERRA ® 

## Surfactant

................................ ................................ ............ 13

Table 3: Results of fa thead minnow 96hr acute toxicity test (see Appendix 23)

........................ 27



# TERMS

**TERM DEFINITION** 

(xxx

- xx

- x) Reference to internal laboratory sample numbers

AES Alcohol ethoxysulfate

ALFOTERRA

® Trade name given to the sodium salts o f branched

- alcohol propoxy

sulfate surfactants manufactured by Sasol Italy and imported by Sasol

North America

ALFO 23

- 4S ALFOTERRA ® 

## 123

- 4S Surfactant

DOC Demand Oxygen (mg/L)

HRT Hydraulic retention time (hours)

MBAS activity Methylene Blue Active Sub stances (mg/L)

MLTSS Mixed Liquor Total suspended solids (mg/L)

PEG Poly ethylene glycol

PPG Polypropylene glycol

PPS Porous Pot System test

TSS Total suspended solids (mg/L)

SDS linear alcohol sulfate dodecyl sulfate

SRT Sludge retention time (da ys)

## 12/14/2011 Page 10 of 66



# INTRODUCTION

Results presented in Research Report R&D ENV

- 07

- 024 01

- 07 “Biodegradability and Acute

Aquatic Toxicity of Alcohol Propoxy Sulfates” prompted Sasol to further evaluate the fate and

effects of alcohol propoxy sulfate surf actants or Sasol’ s ALFOTERRA ® 

## surfactants

. In this

second study, the ALFOTERRA ® 

## Surfactant s are evaluated following the “ Fate, Transport and

Transformation Test Guidelines OPPTS 835.3240 Simulation Test

—Aerobic Sewage

Treatment: Activated Sludge Units Guideline”. Specif ically, the simulated waste treatment

system referred to as the Porous Pot System (PPS) was requested.

Other studies were conducted to supplement the porous pot results and are also included in this

report. These additional studies are “OECD 301A: DOC Die

- Away” on the Porous Pot effluents

and the neat ALFOTERRA ® 

## Surfactant. Additionally, acute aquatic toxicity test by 96

- hour static

P. promelas (EPA

- 821

- R

- 02

- 013) was conducted on the effluents of the Porous Pot and Die

Away tests.

Finally, to furthe r understand the impact of the molecular structure on the biodegradation

response of the ALFOTERRA ® 

## Surfactant molecule, “OECD 301F: Electrolytic Respirometer

testing” (OECD, 1993) was conducted on alcohols, alcohol propoxylates, and alcohol propoxy

sulfat es with varying carbon chain structures.

. 

# OBJECTIVES

1. Set

- up and operate a PPS ev aluating a representative ALFOTERRA ® 

## Surfactant;

compare the ALFOTERRA ® 

## performance in the PPS against standards based on DOC

removal and MBAS analysis. Change PPS operating conditions throughout the study to

further enhance the understanding of PPS and fate of the ALFOTERRA ® 

## studied.

2. After reaching specific steady state in the PPS, determine the acute aquatic toxicity on

fathead minnow of PPS feeds, effluents and neat ALFOTERRA ®



## .

3. Conduct an OECD 301A; DOC Die

- aw ay on the PPS effluents and on the neat

ALFOTERRA

® 

## with two standards.

4. A t the end of the 28

- day die

- away , c onduct an acute aquatic minnow toxicity test on the

ALFOTERRA

® 

## die

- away water.

5. Conduct OECD 301F: Electrolytic R espirometer T est on a ) starting feed alcohols

, and b)

alcohol propoxylates, and c) the sodium sa lt of alcohol propoxy sulfates

.

## 12/14/2011 Page 11 of 66



# CONCLUSIONS

1. The rate and extent of the biodegradation of alcohol propoxy sulfates, specifically

branched C12

- 13 alcohol 4

- mole propoxy sulfate (ALFO 23

- 4S) is notably less than that

of the standard detergent surfactan t alcohol ethoxy sulfate or specifically linear dodecyl

4

- mole ethoxy sulfate (POLYSTEP B

- 430S from Stepan Company) as shown by the

remaining dissolved organic carbon in the ALFO 23

- 4S effluent from Porous Pot, Die

Away, and Electrolytic Respirometer tes ting.

2. Acute aquatic toxicities to fish of the neat material and the effluents from Porous Pot and

Die

- Away testing were determined using standard US EPA methods. The results

showed that both ALFO 23

- 4S and the control POLYSTEP B

- 430S had the same LC 50

val ues for the neat and effluent samples despite the significant remaining dissolved

organic carbon in the effluent of the ALFO 23

- 4S sample.

3. Methylene Blue Active Substances (MBAS) analysis of both the neat and effluent

samples proved that ALFO 23

- 4S under goes primary degradation and elimination of

surfactancy despite the significant remaining dissolved organic carbon in the effluent of

the ALFO 23

- 4S sample after porou s pot or 28

- day die

- away testing.

4. The acute aquatic toxicity to fish of the neat ALFO 23

- 4S is eliminated once the ALFO

23

- 4S passes through the porous pot or 28 die

- awa y test. The results of 96

- hour static

P. promelas (EPA

- 821

- R

- 02

- 013) showed for example that the LC50 of 14% dilution for

the ALFO 23

- 4S feed to the porous pot decreased to LC 50 >100% dilution for the

effluent.

5. Using ALFOTERRA® surfactants for enhanced hydrocarbon recovery (EHR) will not

result in the discharge of any material into surface or ground water. Any rinse aid

containing ALFOTERRA® surfactants, after processing thro ugh standard domestic or

industrial waste water treatment, will pose no threat to the environment

.



# RESUL **TS OF EXPERIMENT**  **S**

**TEST MATERIALS**

 ALFOTERRA ® 

## 123

- 4S (ref erred to as ALFO 23

- 4S)

- four mole propoxy sulfate b ased

on a C12

- 13 branched primary alcohol ISALCHEM ® 

## 123 . This is the product utilized in

all of the environmental fate and effect testing

. I t was chosen as the representative

struct ure as it is most likely the product w ith the highest lev el of concern of all

ALFOTERRA

® 

## materials listed in the PMN

- i.e

. high C12 content ( possible toxicity

issues) and highest branching c ontent (possible biodegradation issues).

 POLYSTEP B

- 430S (referred to as AES , Appendix 1 ) manufactu red by Stepan

- four

mole ethoxy sulfate based on a C12

- 14 linear primary alcohol

. This AES is a common

anionic surfactant found in a variety of household detergent products ; it is utilize d as the

comp arative anionic surfactant evaluated against ALFO 23

- 4S.

## 12/14/2011 Page 12 of 66

 ALFOL ® 

## 12, LIAL ® 

## 123, ALCHEM ® 

## 123, ISALCHEM ® 

## 123, SAFOL ® 

## 23 and TDA

alcohols which contain 12

- 13 carbons but vary in degrees of branching

. These alcohols

, their propoxyla te derivative s

, and the anionic sulfate ( sodium salt form s ) are used in a

systematic biodegradation exercise. This biodegradation test was conducted for the

purpose of shedding light on what aspect of the molecul e ’ s structure promotes a

n eg ative impact on the biodegradation prof ile.

**General Molecular Structure**  **: Al**  **cohol (branched**  **and/ or linear)**  **4**

**- Propoxy**  **late Sulfate**

**Surfactant**

**CH 3**  

## - **(CH 2**  

## )

**m** 

## - **CH**

**- (CH**  **2** 

## )

**n** 

## – **CH 3** 



## |

**CH 2**  

## – **O**

**- (CH**  **2** 

## – **CH**

**– O**  **)**

**y** 

## – **SO 3**  

### - 

## Na **+**



## |

**CH 3** 



## Table 1 list s the trade names and description s of the six al coho ls and propoxy derivatives

(nonionic) evaluated in this study. C arbon ch ain length

, degree of branching, molecular

we ights, percent propylene oxide by weight, and moles of propylene oxide are given for each of

the six products

. The red columns correspond to the alcohol and the red portions of the

compound above while the blue refers to the propoxylate portion.

**Table 1**  **: Properties of the four mole propoxy alkoxylate derivatives**  **(nonionic )**  **of the six alcohols** 

**tested**

**Base Al**  **cohol**

**(lab sample**

**number) alcohol** 

**carbon**

**chain a**  **lcohol**

**percent**

**branching MW** 

**propoxylate Percent by** 

**w eigh**  **t**

**PO (avg.)**  **moles PO**

**(avg.)**

ALFOL ® 

### 12

1314

- 159 C12 0% 414 54.8 3.91

ALCHEM ® 

### 123

1314

- 48

- 1 C12

- C13 5 % 41 4 52. 9 6 3.78

LIAL ® 

### 123

1314

- 46

- 1 C12

- C13 5 3 % 42 1 53. 6 0 3.89

SAFOL ® 

### 23

13 14

- 49

- 1 C12

- C13 50% 430 55.0 4.08

ISALCHEM ® 

### 123

1314

- 47

- 1 C12

- C13 94 % 4 25 54.1 2 3.96

TDA

1314

- 18

- 1 C13 100% 435 53.5 4.01



## Table 2 list s the anionic sodium salt derivatives of the six alcohol 4

- mole propoxylates described

in T able 1 as the ALFOTERRA ® 

## Surfactants. The ALFOTE RRA ® 

## Surfactant based on

ISALCHEM 123 alcohol ( ALFO 23

- 4S ) listed in bold is the primary product evaluated through out

this body of work

. The table includes the molecular weights, percent active of the anionic

sodium salt, percent active of the un

- sulfated prop o xylate and percent sodium sulfate. Wa ter

makes up the remaining ingredient of the ALFOTERRA ® 

## Surfactant commercial product. **alcohol (branched or linear)**

**sulfate propy**  **lene oxide**

**- avg.**  **4 moles**

## 12/14/2011 Page 13 of 66

**Table 2**  **: Properties of the**  **anionic Surfactants based on the**  **above p ropoxylates**  **which are**

**comparable to the AL**  **FOTERRA ®**  

### S **urfactant**

**An ionic**  **surfactant**

**Al cohol Base**  **(and lab**

**numbers) MW** 

**propoxylate**

**sulfate per**  **cent**

**anionic**

**active percent un** 

****

**sulfated**

**propoxylate percent** 

**Na 2**  

### SO **4**



### ALFOL ® 

### 12

131 1

- 197

- 1 516 29. 09 0.47 0.13

ALCHEM ® 

### 123

1311

- 74

- 4 51 6 27.20 0.48 0.10

LIA L ® 

### 123

1311

- 74

- 2 52 3 27.67 0.8 0.09

SAFOL ® 

### 23

1 311

- 74

- 5 532 27.42 0.47 0.11

**ISA LCHEM**  **®** 

### 123

**1311**

**- 74** 

**- 3**  **5 27**  **27.29 0.8**  **0.10**

TDA

1311

- 74

- 1 537 28.46 1.93 0.09



## Note: Ro **w h ighlighted bold** 

**- alcohol propoxy sulfate**  **is employed**  **in PPS**

**, Die** 

**- Aw**  **ay**

**studi es**  **and acute toxicity testing** 

**.**



# BIODEGRADATION STUDY

**Porous Pot Sy stem**  **(PPS) Biodegradation Test Method** 

The PPS (Figure 1) used in this study utilized the methods described in the US EPA OPPTS

Test Guideline 835.3220 “Porous Pot Test” and ASTM test E 1798

- 96 “Standard Test Method

for Assessing Treatability or Biodegradability, or Both, of Organic Chemicals in Porous Pots”

with a series of modifications designed to reduce the amount of feed utilized. The system

consisted of a series of s ix cylindrical glass containers approximately 17” x 2.75”. A constant

temperature of 25°C was maintained inside the glass cylinders by wrapping each cylinder with

tygon tubing held in place with duct

- tape. Porous high density polyethylene (HDPE) candles

were located on the interior of the glass containers. The candl es held an average of 250 mL of

mixed liquor activated sludge. A glass aeration line extended from the top of the glass container

to the bottom of the candle. House air was passed through AFP Flow Meters at 100

- 150

cc/min. Synth etic sewage feed was pumped into the units from three

- 1 gallon plastic jugs

placed in an ice chest. For replication, two feed lines/ jug delivered feed to two separate PPS by

means of six #QG metering pumps manufactured by F.M.I., Inc. (Oys ter Bay, NY). Each PPS

was connected to a separate 1 L side

- arm Erlenmeyer vacuum flask which captured effluent.

The source of the sludge for the candles was derived from the recycled sludge from the City of

Lake Charles, Louis iana Domestic Wa ste Wa ter Plant A and the SBR aeration basin from City

of Sulphur Regional Wa ste Wa ter Treatment Plant.

## 12/14/2011 Page 14 of 66

**Figure 1**  **: Porou s Pot**  **System (PPS) A**  **ssembly Test** 



## Synthetic sewage stock solution was made following the EPA Guideline 835.3220, except at 1⁄2

the concentration of each component. The recipe used for the sewage stock was 65 grams

glucose, 65 gram s beef extract, 65 grams nutrient broth, 65 grams di

- potassium hydrogen

phosphate and 25 grams of ammonia nitrate. The dry ingredients were added to 2 liters of DI

water and dissolved by stirring at 70°C. The synthetic sewage stock was re fri gerated to

maintain quality.

F or the various feeds delivered to the PPS

, the stock was diluted in DI water for a DOC

concentration of 250

- 300 mg/L on average. The basic recipe for synthetic f eed was 15 grams

synthetic sewage concentrate, 2 grams of a 1% stock solution of Tide Cold Powder (V1255

- 49

- 1), 10 grams of 1% stock solution of sodium sulfate. These concentrated ingredients (referred

to as the blank) were diluted to 3000 grams with tap water. The same recipe as the blank was

followed for the PPS test controls (sodium benzoate and AES) and study test material ( ALFO

23

- 4S ) except for the addition of 10

- 12 grams of 1% solution of benzoate, AES or ALFO 23

- 4 S

respectively

, bef ore dilution to 3000 grams . Synthetic sewage feeds we re made daily and

refrigerated fo r use the following day. Sewage f eeds in the ice chest were replaced daily with

the cooled

, refrigerated feed and c rushed ice packed around the one gallon feed jugs to

maintain the temperature ~ 4°C.

Tide Cold Powder was us ed because the simulated sewage recipe was de void of any

surfactants and the ALFO 23

- 4S test material is an anionic surfactant somewhat similar to the

surfactant contained in the Tide Cold Powder

. As th e sludge inoculate used in the PPS were

exposed/conditioned to commercial household surf actants

, it was deemed prudent to

consistently expose them to a common household detergent product in the synthetic sewage

recipe backgrou nd.

During the PPS study it was determined that a n antifoaming agent was necessary to control

foaming and lifting of the sludge from inside the porous candle s being fe d by either surf actant

. The antifoam used w a s J.T. Baker’s Antif o am B Silicone Emulsion ; 0. 3

- 0.6 mL was added daily

to all feeds including blank and benzoate

.

## 12/14/2011 Page 15 of 66



# Porous Pot Sy **stem Daily Operations** 

The six PPS were operated from April through early October 2009. Parameters that were

measured a lmost daily were hydraulic retention time (HRT), MLTSS concentrations in the PPS

candles, TSS concentrations in the effluents, pH of effluents and DOC concentration in the feed

and effluents. Hydraulic retention time (HRT) was maintained at 6 hours. The s ludge retention

time (SRT) was maintained between 5

- 10 days by wasting at least 1/ 10 th 

## to 1/5 th 

## of the mixed

liquor volume each day. The numbers in the following figures refer to the 6 pots run in parallel

with thre e feed material s (duplicate of each) at any time

.

In Figure 2, the out

- of

- range points are times when the feed line clogged and flow was reduced.

During the first part of July

, the fritted glass aerators were changed to a larger size as poor

mixing was observed inside the candle permitting DOC breakthrough. It was realized that the

larger glass aerators had displaced the working volume inside the candles from 250 mL to a

new volume of 215 mL. The flows were readjusted on the FMI pu mps to adjust HRT back to 6

hrs. To gather additional inf ormation on the impact of HRT on DOC concentration of the

effluents, the HRT was increased t o 12 hr s for the last 2

- weeks of the study.

In the following four tables, the legend numbers refer to each of the six candles of the PPS. The

specific composition within the candles will be addressed l ater. T he following four tables are

meant to show the consistency of retention time (HRT)

, initial mixed liquor to tal suspended

solids (MLTSS) and total suspended solids (TSS) of the PSS effluents throughout the duration

of the study despite the candle co ntents.

**Hy draulic Retention Time (HRT) Through Porous Pots** 

**0.0 2.0**  **4.0 6.0**  **8.0 10.0**  **12.0 14.0**  **16.0**

**6-A pr**  **26-Apr 16-May**  **5-Jun 25-Jun**  **15-Jul 4-Aug**  **24-Aug 13-Sep**  **3-Oct**

**Date H** **R T** **( h** **o u** **r s** **) 1**  **2 3** 

**4 5**  **6**



### Figure **2 : Hydraulic Retention Time (HRT) Through Porous Pot System** 



## Figure 2 shows the hydraulic retention time throughout the porous pot study. Wi th the exception

of the Z oogl ea l issue in early July and a secondary study in October, the HRT was maintained

consistently at 6 hrs.

## 12/14/2011 Page 16 of 66 **Mixed Liquor Total Suspended Solids (MLTSS) in Porous Pot Candle**

**Time period from April through October 2009**

**0 1,000**  **2,000 3,000**  **4,000 5,000**  **6,000 7,000**  **8,000 9,000**  **10,000**

**6-A pr**  **16-**

**Apr 26-** 

**Apr 6-** 

**May 16-** 

**May 26-** 

**May 5-Jun**  **15-**

**Jun 25-** 

**Jun 5-Jul**  **15-**

**Jul 25-** 

**Jul 4-A**  **ug 14-** 

**Aug 24-** 

**Aug 3-Sep**  **13-**

**Sep 23-** 

**Sep 3-Oct**  **13-**

**Oct**

**Date m** **g /** **L 1**  **2 3** 

**4 5**  **6**



### Figure **3 : Mixed Liquor Total Suspended Solids (MLTSS) in Porous Pot Candle** 



## Figure 3 depicts the variation in the concentration of the MLTSS during the study period. During

June, the MLTSS composition became enriched with bac teria species called Zooglea. This

species occurs in waste treatment plants and becomes a nuisance when its numbers bloom; the

bacteria produce a heavy slime layer on its surface resulting in a jelly like consistency in the

sludge. This jelly l ike consistency occurred in the sludge for all the candles and increased the

viscosity of the mixture. MLTSS concentrations were also increasing during this period

. The

aerators in the candles were not sufficiently large to accommodate this inc reased viscosity and

aeration /mixing was impaired . On July 4 th 

## , half of the volume of MLTSS was wasted to try and

control the problem. Increased wasting continued ( i.e. shortening SRT ) to try and control the

Zooglea.

## 12/14/2011 Page 17 of 66 **Porous Pot Effluent TSS**

**-10 10**  **30 50**  **70 90** 

**1-Jun 11-Jun**  **21-Jun 1-Jul**  **11-Jul 21-Jul**  **31-Jul 10-Aug**  **20-Aug 30-Aug**  **9-Sep 19-Sep** 

**Date T** **S S** **( m** **g /** **L )**  **1 2**  **3**

**4 5**  **6**

**0**



### Figure **4 : Porous Pot Effluent TSS** 



## Figure 4 depicts the TSS in the effluents during the trial ; effluent was disposed daily. The period

in June reflects what occurred in the candles during the Zooglea l bloom

- incre ased viscosity

and poor aeration/ mixing. Breakthrough of DOC into the effluent vessels allowed continued

biodegradation and increased microbial growth , thus, TSS in effluents was going through an

atypical increase. The porous pot has the capabil ity to block MLTSS from escaping into the

effluent. After correcting the problem in the candles, TSS in the effluents returned expected and

stabilized values fo r the duration of the test (July

- Sept) . TSS values oc curring below 0 mg/L

TSS are common on the occasions that fiber s on the filter pads break off ; it is then los t during

the filtering process

. **Figure 5. Porous Pot Effluent pH Values**

**5.00 5.50**  **6.00 6.50**  **7.00 7.50**  **8.00 8.50**  **9.00**

**1-Jun 21-Jun**  **11-Jul 31-Jul**  **20-Aug 9-Sep**  **29-Sep p** **H**

**1 2**  **3**

**4 5**  **6**



### Figure **5 : Porous Pot Effluent pH Values** 

## 12/14/2011 Page 18 of 66

Figure 5 depicts the pH values in the effluents during the trial ; t he starting pH of t he feeds

averaged 7.5. T he drop in the eff luent pH for units 3 and 6 were those units treating the ALFO

23

- 4S test material. This decrease began occurring after the concentration of the test material

was increased. T he drop in pH is indicative of partial /incomplete breakdown of t he parent

material into fatty acids. T o control/buffer the pH, 0.5 grams of sodium bicarbonate was added

each day to all the feeds. T he addition of t he buff er raised the pH slightly to 7.6. T his buffer

addition contr olled the effluent pH and was continued through the rest of the study period.

**Figure 6. Porous Pot DOC Feed and Effluents**

**0 50**  **100 150**  **200 250**  **300 350**  **400**

**5/7/2009 6/6/2009**  **7/6/2009 8/5/2009**  **9/4/2009 10/4/2009** **D O** **C m** **g /** **L** 1 Effluent

2 Effluent

3 Effluent

4 Effluent

5 Effluent

6 Effluent

Feed

Feed

Feed



### Figure **6 : Porous Pot DOC Feeds and Effluents** 



## Figure 6 depicts the PPS DOC values for the three feeds ( blank, benzoate, a nd sample ) in

blue s and the six effluents during the period of mid

- Ma y through early October when the study

ended. The effluent DOC values reflect a loss of steady state from the end of Ma y through early

June

- the period of the Zooglea l bloo m

. E ffluent DOCs were returning to a steady state in late

June but began to elevate when the HRT decreased due to volume displacement from the new

larger aerator/diffusers. Effluent values returned to steady state by early August when the feed

pumps were adjusted to correct the HRT back to 6 hrs. **In general**

**, during periods of stead**  **y**

**state (mid July through October)** 

**, DOC removal**  **rates for all units**  **wa s**  **above 90%**

**. This** 

**compares to the t**

**ypical removal efficiency seen in domestic waste treatment plants.**



# Porous Pot Sy **stem Experimental Results**

The primary objective of the PPS was to a ssess the fate of ALFO 23

- 4S after processing

through a simulated waste water treatment process. Additionally, there was interest in

developing a deeper understanding of the degradation mechanism of the alcohol propoxy

sulfate molecul es and how this degra dation compares to that of standard alcohol ethoxy

sulfate s

. As a result, the control materials, HRT rate, and bacteria condition s were

systematically varied throughout the study. In all s ituations, samples were run in duplicate

.

## 12/14/2011 Page 19 of 66

The next series of figures demonstrates the response of the effluent DOC values , especially

those for the ALFO 23

- 4S, at each specific test condition evaluated.

**Porous Pot Effluents DOC Pre Unacclimated A LFO 23-4S** 

**0 25**  **50 75**  **100 125**  **150**

**7-May 17-May**  **27-May 6-Jun**  **16-Jun 26-Jun**  **6-Jul 16-Jul**  **26-Jul**

**Date m** **g /** **L D** **O C**  **Effluent 1 Effluent 2**  **Effluent 3**

**Effluent 4 Effluent 5**  **Effluent 6**

feed increased to 15

mg/L active feed increased to 30 mg/L

active in #2 and #5



### Figure **7 : Porous Pot Effluents DOC** 



## Figure 7 shows the DOC values for the first three months of the PPS study. T he effluent from

the ALFO 23

- 4S candles are depicted in brown (effluent 3 and 6), the effluent from the benzoate

candles are depicted in green (e ffluen t 2 and 5) , and the effluent from the blank candles are in

blue (effluent 1 and 4).

The study was started in mid May with the addition of benzoate and ALFO 23

- 4S to blank

candle that had been acclimating since mid

- April. Shortly after this addition, the DOC values of

the ALFO 23

- 4S start ed to climb relative to the blank and benzoate samples. There was then a

period of upsets (f oaming, Z ooglia, aeration changes) that impacted the effluent quality as seen

by the erratic DOC values through out June and early July

. The system was brought under

control around July 5 and a steady state was re established by July 8. On Ju ly 9 th 

## t he

concentration s of benzoate and ALFO 23

- 4S were increased to 15 mg/L as active

. Shortly

af ter, an increase in the DOC of the ALFO 23

- 4S effluent w as again observed suggesting a

difference between the degradation rate of the ALFO23

- 4S and the benzoate

. The benzoate

DOC curve (green) i s identical to the blank (blue) under steady state conditions

.

To validate the increased DOC of the ALFO 2 3

- 4S, the benzoate was replaced with ALFO 23

- 4S in the feed of candles 2 and 5 (green on graph) on July 30 . Additionally, the ALFO 23

- 4S

concentration was raised in candles 2 and 5 to 30 mg/L as active or approximately 13 mg/L as

DOC. Immediately the DOC in the effluent of candles 2 and 5 increased to values equal to the

original ALFO 23

- 4S candles (3 and 6).

## 12/14/2011 Page 20 of 66 **Figure 8. Porous Pot DOC: A LFOTERRA** 

**Un-acclimated and A cclimated Response** 

**0 5**  **10 15**  **20 25**  **30**

**28-Jul 30-Jul**  **1-A ug**  **3-Aug 5-A**  **ug 7-A**  **ug 9-A**  **ug 11-Aug** 

**Date m** **g /** **L D** **O C**  **Blnk U-A**  **lfo A-Alfo** 

**Blnk U-A**  **lfo A-Alfo** 



### Figure **8 : Porous Pot Effluents** 

**- Un** 

**- acclimated and acclimated Response** 



## Figure 8 shows in more detail the change in effluent DOC values when ALFO 23

- 4S is added to

what was originally the benzoate candles (now labeled U

- ALFO for un

- acclimated )

. Most

interesting is that the original ALFO candles and the U

- ALFO candles have exactly the same

effluent DOC values , thus confirming the validit y of the response.

**Figure 9. Return to Steady State: Post Unacclimated A lfoterra Pre A**  **ES**

**0 5**  **10 15**  **20 25**  **30**

**7-A ug**  **9-A ug**  **11-Aug 13-Aug**  **15-Aug 17-Aug** **D O** **C m** **g /** **L**

**Benz Al**  **fo Blnk** 

**Benz Al**  **fo Blnk** 



### Figure **9 : Return to Steady State (DOC)** 

## 12/14/2011 Page 21 of 66

As a final confirmation of the DOC response to all samples (benzoate, blank, ALFO) the feed

conditions were chang ed such that the candles that were fed ALFO 23

- 4S from the beginning of

the study were now fed blank feed only (yellow and red above), The candles that were fed blank

feed from the beginning of the study were now fed benzoate (light blue, dark blue) , and t he

candles that were originally benzoate but had been switch ed to ALFO 23

- 4S f eed was kept with

the ALFO 23

- 4S (pink, purple) . Both the benzoate and the ALFO 23

- 4S feed had 30 mg/L as

active added during this period

. Figure 9 illustrates that the effluent DOC level s for the new

benzoate and new blank (formerly acclimated ALFO 23

- 4S) rapidly merged together within a

few days confirming that the PPS reacts rapidly to the feed changes and the response is real.

**Porous Pot DOC: A**

**Comparison of 1214E4S and A LFO 23-4S** 

**0 5**  **10 15**  **20 25**  **30**

**13-Aug 23-Aug**  **2-Sep 12-Sep**  **22-Sep**



### Date**D O** **C m** **g /** **L**

**AES Alfo**  **Blnk**

**AES Alfo**  **Blnk** 

### Feed changed from 30

to 40 mg.L as active



### Figure **10 : Porous Pot Effluent DOC: A**  **Comparison of A ES and A**  **LFO 23**

**- 4S** 



## Now that the PPS response for ALFO 23

- 4S ha d been established and verified, there was

interest in knowing how this response compared with t he common detergent surfactant a l cohol

ethoxy sulfate (AES)

. POLYSTEP B

- 430S (linear C12

- 14 alcohol based 4

- mole ethoxy sulfate

from Stepan Co. ) was added as the AES control replacing benzoate. The concentration for both

the AES and ALFO 23

- 4S was 40 mg/L as active or inf luent DOC values of 17 mg/L and 16

mg/L respectively. At the end of this period (Sept. 24 th 

## ), the effluent DOC values fo r AES (d ark

blue, l igh t blue) were approximately e qual to the blank (b rown, yellow) wh ile the ALFO 23

- 4S

(pink, purple) maintained its slightly higher effluent DOC response.

This difference in the effluent response of the ALFO 23

- 4S and the AES was not unexpected.

T here are multiple literature references, discussed in la ter sections of this report, that discuss

the how branching impedes the biodegradation response of molecules.

## 12/14/2011 Page 22 of 66 **Porous Pot: Increased Hydraulic Rentenion Time**

**(6hr-12hr)**

**0 5**  **10 15**  **20 25**  **30**

**19-Sep 24-Sep**  **29-Sep 4-Oct**  **9-Oct**



### Date**D O** **C m** **g /** **L AES**  **Alfo Blnk** 

**AES Alfo**  **Blnk**

**HRT from 6hr to 12hr**



### Figure **11 : Porous Pot Effluent: Increased HRT effect on DOC** 



## Finally, there was interest in seeing if the bi o degradation of the ALFO 23

- 4S would be increased

if the HRT was increased. Figure 11 illustrates the effluent DOC response when the treatment

time ( HRT ) was doubled from 6 to 12 hrs

. The FMI pum ps were turned down (reducing feed

rate) gradually each day starting on September 25 th 

## and completed on September 30 th



## . The

data shows that the effluent DOC for ALFO 23

- 4S (pink, purple) moves towards the 10 mg/L

baseline established for the DOC effluents of the AES (l ight blue, d ar k blue) and the blank

(br own , yellow) confirming that degradation of ALFO 23

- 4S continues with increased time

. The

PPS study ended on October 10 th



## .



# PPS Results Summary **:**

The results of the PPS study showed that even after processing ALFO 23

- 4S through a

simulated waste

- water treatment, a predominate amount of the original DOC remains in the

eff luent. This le d to two questions; 1) will the ALFO 23

- 4S continue to degrade after le aving a treatment facility and reach ing receiving waters and

, if so, what will the extent and timing of the

degradation be? And 2) i s the ALFO 23

- 4S sufficien tly broken down through waste water

treatment so t hat it no longer has the aquatic toxicity profile as the parent product? The

questions are addressed in the next two sections of this report.



# OECD 301 A: **DOC Die** 

**- Away Experimental Results** 

To determine the biodegradation rate of ALFO 23

- 4S DOC effluent, the effluent from the PPS

was subjected to a DOC Die

- away test. T his test was used to simulate degradation that could

be expected if the PPS effluent reached receiving waters.

Using OECD 301A: DOC Die

- Away

, experiments were conducted on the effluents from the PPS

system during the period of AES and ALFO 23

- 4S comparison. In conjunction, a DOC Die

-

## 12/14/2011 Page 23 of 66

Away test was conducted on benzoate and the neat or parent compound s fo r the AES and

ALFO 23

- 4S (before PPS treatment)

.

The effluents were collected from the PPS for two consecutive days on September 10 th 

## and 11 th



## . The effluent duplicates were combined and refrigerated on September 10 th 

## and then added to

the combined duplicates from September 11 th 

## . The pooled effluents for the blank, AES and

ALFO 23

- 4S were each added to 2

- liter Erlenmeyer flask s and placed on a bench top shaker fo r

mixing

.

To make the neat samples, o n the same day (Se ptember 11 th 

## ), mineral water solution was

added to six 2

- liter Erlenmeyer flask s . Using 1% stock solutions for benzoate, AES and ALFO

23

- 4S, additions were made to each flask for a concentration of 50 mg/L active for each test

compound ; each test material had a duplicate flask. The flasks were placed on the bench

- top

shaker and mix ed at 125 rpm.

Day

- 0 sampling for DOC occurred before the inoculate concentrate was added to each vessel.

The inoculate source was equal parts of aer ation sludge from City of Sulphur Regional W aste

Wa ter Treatment Plant and soil slurry supernatant

. T he inoculate concentrate was added to

each flask to a diluted target concentration of 25 mg/L TSS.



## Die-A **wa y Porous Pot Effluent** 

**0 5**  **10 15**  **20 25**  **30**

**0 5**  **10 15**  **20 25**  **30**

**Days D** **O C** **( m** **g /** **L )**  **PP Blank PP AES**  **PP A LFO** 



### Figure **12 : Die** 

**- Aw**  **ay Porous Pot Effluent**



## Results for the PPS effluent Die

- Away test can be seen in Figure 12. For all samples, t he DOC

continued to decline over the 28

- day period . However, the degradation of the ALFO 23

- 4S

(green) never reaches that of the blank (blue) or the AES (pink) in the time allotted

.

## 12/14/2011 Page 24 of 66 

## OECD Die-A

**wa y DOC Loss** 

**0 20**  **40 60**  **80 100** 

**0 5**  **10 15**  **20 25**  **30**



### Days**% D** **O C** **L o** **s s** 

**AE S**  **ALFO Benz** 



### Figure **13 : OECD 301A**  **Die**

**- Aw**  **ay DOC Loss of neat A**  **LFO 23**

**- 4S, A**  **ES, and Benzoate.**



## The die

- away test was conducted on the neat ALFO 23

- 4S, AES, and benzoate (not subjected

to porous pot ). In this cas e, the difference in the degradation rate of the ALFO 23

- 4S and the

AES is obvious. Benzoate and AES showed a rapid loss in DOC reaching 100% DOC loss by

day 17 w hereas the ALFO 23

- 4S had a modest DOC decline with 20% DOC loss at day 28.



# MBA **S An**  **al ysi**  **s**

In order to determine if p rimary degradation is occurring on the ALFO 23

- 4S during the porous

pot test and the die

- away test

, Methylene Blue Active Substances ( MBAS ) analysis was run on

the influents and effluents. Samples were preserved with formaldehyde for a final concentration

of 1% preservative.

## 12/14/2011 Page 25 of 66 **Porous Pot: MBAS Primary Biodegradation**

**0 5**  **10 15**  **20 25**  **30 35**  **40 45** 

**Blank AES**  **AL FO 23-4S** **M B** **A S** **( m** **g /** **L )**  **Influent**

**Effluent**



### Figure **14 : Porous Pot MBA**  **S Primary Biodegradation**



## Figure 14 shows the MBAS res ults for the inf luent and effluent for the porous pot study. The

small MBAS activity in the blank is attributed to the surfactant in the represent ative Tide Cold

Wa ter powder that was added to all three PPS feed , as discussed earlier

. A significant

decrease in surfactancy can be seen for both the AES and ALFO 23

- 4S after treatment through

the PPS . The green bar repres ents the PPS influent feed ( 40 mg/L as active fo r AES and ALFO

23

- 4S ) while the blue bar represents the respective effluent All samples were collected from

the PPS on September 11 th 

## . The MBAS percent reduction in activity for the AES and ALFO 23

- 4S during the PPS were 99% and 94% respec tively.

**Figure 15. Comparison Porous Pot and Die-away**

**0 10**  **20 30**  **40 50** 

**Porous Pot Die-away** **M B** **A S** **m g** **/ L**  **Influent Effluent** 



### Figure **15 : A**  **LFO 23** 

**- 4S**  **Comparison MBA S Porous Pot and Die A**  **way**



## Figure 15 compares the inf luent and effluent MBAS results for ALFO 23

- 4S through both porous

pot and die

- away degradation methods. In both cases, the inf luent contained approximately 40

mg/L as active ALFO 23

- 4S. The Porous pot effluent was generated after a 6hr HRT while the

## 12/14/2011 Page 26 of 66

die

- away effluent was taken after 28 days. The MBAS percent reduction in activity for ALFO 23

- 4S in the PPS was 94% and 90% in the 28

- day Die

- Away

.

**MBA S**  **Results Summary:** 

MBAS analysis of both the PPS and Die

- Away test methods confirm that the ALFO 23

- 4S

molecule undergoes significant primary biodegradation th ough most of the original DOC

remains

. The question that that remained unanswered was

- i s the residual ALFO 23

- 4S

material still aquatically toxic?



# Aq **uatic A cute 96hr Fathead Minnow Results** 

A 96

- hour static P. promelas (EPA

- 821

- R

- 02

- 013) test w as conducted by Environmental

Enterprises USA, Inc. (EE USA) during the PPS and Die

- Away study period . W ater samples of

the PPS synthetic sewage feeds (blank, AES + blank , and ALFO 23

- 4S + blank ), neat AES

, neat ALFO 23

- 4S (40 mg/L as active)

, and PPS effluents (blank, AES and ALFO 23

- 4S) were

sent for toxicity testing on September 9. Replicate effluents were combined and represented

two days (combined) to achieve the required volume needed for 5 replicates /concentration in

the fathead minnow test

. The toxicity endpoints for water samples represent ed in the PPS

study period is reported as the percent dilution of the test solution

, while AES and ALFO 23

- 4S

neat w ere compared at a “as is” concentration of 40 mg/L with toxicity endpoints reported mg/L.

On October 9 th 

## , a t the end of the 28

- day DOC Die

- A wa y for ALFO 23

- 4S

, the remaining solution

was put on ice and sen t for 96

- hour static minnow test. At this sa me date

, a fresh sample of

ALFO 23

- 4S at 50 mg/L was made and shipped on ice to EE USA to use as a control. The

results from the testing are given below in T able 3.

**Sample**

**Description Environmental** 

**Enterprise USA**

**Sample ID Survival** 

**NOEC**

**% Survival** 

**LOEC %**  **LC50 %** 

**( 95%**  **C onfidence Interval**  **)**

PPS Blank Influent

Feed Q

- 642

- 09

- **1336**

**-**

**38**

**- 1**  12% 25% 14.7 %

(12.4 %

- 17.5 % )

PPS AES Influent

Feed Q

- 643

- 09

- **1336**

**-**

**38**

**- 2**  3.0 % 6.0% 5.47%

(4.91%

- 6.10%)

PPS ALFO 23

- 4S

Influent Feed Q

- 644

- 09

- **1336**

**-**

**38**

**- 3**  6.0% 12.0% 9.48%

(8.28%

- 10.9%)

AES Neat

(40 mg /L active ) Q

- 645

- 09

- **1336**

**-**

**38**

**- 4**  6.0%

(2. 4

mg /L) 12.0%

(4.8 mg/L) 13.8% (5.52 mg /L)

(12.3%

- 15.5%)

ALFO 23

- 4S

(40 mg /L active) Q

- 646

- 09

- **1336**

**-**

**38**

**- 5**  3.0%

(1.2

mg /L) 6.0%

(2.4 mg /L) 7.76% (3.10 mg /L)

(6.64%

- 9.08%)

PPS Blank Effluent Q

- 647

- 09

- **1336**

**-**

**38**

**- 6**  100% >100% >100%

( NA )

PPS AES Effluent Q

- 648

- 09

- **1336**

**-**

**38**

**- 7**  100% >100% >100%

( NA )

PPS ALFO 23

- 4S

Effluent Q

- 649

- 09

- **1336**

**-**

**38**

**- 8**  100% >100% >100%

( NA )

ALFO 23

- 4S

Die

- Away Influent

(50 mg /L) 0

- d ay Q

- 715

- 09

- **1336**

**-**

**58**

**- 2**  6%

(3.0

mg /L) 12%

(6.0 mg /L) 8.8% (4.4 mg /L)

(8.0%

- 9.7%)

ALFO 23

- 4S

Die

- Away E ffluent

(50mg/L) 28

- day Q

- 716

- 09

- **1336**

**-**

**58**

**- 1**  100% >100% >100%

NA

## 12/14/2011 Page 27 of 66

**Table 3**  **: Results of fathead minnow 96hr acute toxicity test (see**  **Ap pendix**  **2 3**  **)**



## Note: numbers highlighted in bold are Sasol NA notebook number. Concentrations (mg/L) in red

were calculated as mg/L in the endpoint dilution value.

A quatic toxicity results from Table 3 ar e depicted in the following F igures

- 16, 17

, 18.

**AE S and A**  **LFO 23-4S**

**Ac ute Toxicity** 

**96 hr Fathead Minnow**

**0.0 2.0**  **4.0 6.0**  **8.0 10.0** 

**LC50 LOEC**  **NOEC**

**Toxicity Endpoint m** **g /** **L AES**  **AL FO 23-4S** 



### Figure **16 : A**  **ES and A L**  **FO 23** 

**- 4S**  **Neat Material Ac**  **ute Toxicity Before A**  **ny Biodegradation**



## Figure 16 is a comparison of aquatic toxicity for neat AES and ALFO 23

- 4S surfactants bef ore

any biological treatment . The toxicit i es for each endpoint are typical of anionic surfa ctants.

ALFO 23

- 4S has slightly higher end points than the AES suggestion that the neat AFLO 23

- 4S

is slightly more aquatically toxic than the neat AES. The LC50 for the neat, non

- biologically

treated ALFO23

- 4S was 5.7 mg/L.

## 12/14/2011 Page 28 of 66 **Figure 17. Porous Pot: A quatic Toxicity Test 96hr Minnow** 

**40mg/L AES and ALFO active**

**0 20**  **40 60**  **80 100**  **120**

**Blank Feed Blank Effluent**  **AES+Feed AES Effluent**  **ALFO+Feed ALFO Effluent** 

**Source P** **e r** **c e** **n t** **S o** **u r** **c e**  **LC50**

**LOEC**

**NOEC**



### Figure **17 : Porous Pot**  **Feed and Effluent**  **: A quatic Toxicity**  **A ES and A**  **LFO 23**

**- 4S** 



## Figure 17 compare s the toxicity results before and after the PPS. It shows that

, in all cases,

including the ALFO 23

- 4S, the aquatic toxicity is eliminated after a 6 hr retention time through

the porous pot. All three PPS effluents show no acute toxicity fo r the toxicity endpoints of L C50

, NOEC and LOEC

- all values being greater than 100% effluent

.

At the end of the 28

- day Die

- Away test on October 9 th 

## , the water remaining in the 2

- liter

Erlenmeyer shake flask for ALFO 23

- 4S was sent fo r toxicity testing. In addition, a sample of

neat ALFO 23

- 4S (50 mg/L as active )

, re flecting the Die

- Away concentration before bacterial

activity day

- 0

, was sent for testing

.

## 12/14/2011 Page 29 of 66 **Figure 18. OECD Die-A way**  **Test A LFOTERRA** 

**(50mg/active or 22mg/L DOC)**

**0 20**  **40 60**  **80 100**  **120**

**LC50 LOEC**  **NOEC**



### Toxicity **Endpoint P** **e r** **c e** **n t** **S o** **u r** **c e**  **AL FO Day 0**  **AL FO Day 28** 

**calculated LC50**

**= 4.4 mg/L**



### Figure **18 : A**  **cute Toxicity Test, Die**

**- Aw**  **ay Test A LFO 23** 

**- 4S** 



## Acute toxicity of ALFO 23

- 4S bef ore and after the 28

- day Die

- Away test was determined.

Figure 18 compares the toxicity from 96

- hr acute minnow of Die

- Away water at the end of th e 28

days (blue) with that at the beginning of the test (green). Tw o sets of water samples

, Day 0

(made identical to the ALFO 23

- 4S test water at the start containing 50 mg/L ALFO 23

- 4S ) and

the day 28, were evaluated

. The results are given as the calcula ted dilution percent of the two

water samples. The calculated concentration for the LC50 endpoint for the 0

- day sample was

12 % dilution or 4.4 mg/L

- similar to the toxicity observed from the neat in the PPS toxicity tests.

No toxicity remained in the ALFO 23

- 4S Die

- Away 28

- day water and toxicity endpoints for LC50,

LOEC and NOEC exceeded 100% effluent solution as provided

.

**Fat Head Minnow Results Summary:** 

Three test methods (Porous Pot, Die

- Away, Elect rolytic Respir o meter (from pr e vious report) )

ha ve shown th at ALFO 23

- 4S only loses ~30% of its original DOC values at the conclusion of

the test s suggesting alcohol propoxy sulfates do not biodegrade well. However, MBAS confirms

that at least primary degradation is occurring

. Mo re importantly, Acute Aquatic Testing on Fat

Head Minnows prove s that the ALFO 23

- 4S residual DOC in the effluent after standard

biodegradation treatment has lost the aquatic toxicity of the parent molecule.



# OECD 301F Biodegradability Electrolytic Respirometer Study

Though the above data suggests that with standard water treatment methods, ALFO 23

- 4S an d

similar molecules pose little

- to

- no threat the aquatic life, there is interest in understanding w hy

the residual DOC in ALFO 23

- 4S effluent after standard biological degradation test ing is so

much higher than for AES. To better understand the role of ch emical structure on the

## 12/14/2011 Page 30 of 66

biodegradation mechanism of alcohol propoxy sulfates, Electrolytic Respirometer (OECD 301F)

biodegradation s tudies were conducted on alcohol prop o xy sulfate raw materials with a known

structure. As detailed in Table 1 & 2, six alcohols in the C12

- C13 carbon range with varying

degrees of branching were studied. These six alcohols were then propxylated to a n average of

four moles of propy lene oxide using KOH catalyst and studied again. Finally, the six alcohol

propoxylates were sulfated using a thin

- film sulfa tion method to yield the sodium salt derivatives

and studied aga in

. Sodium benzoate was used as the standard in each test

.

The biodegradation tests were performed quadruplicate in using two 20 channel Coordinated

Environmental Services (CES) Ltd (Cornwell, UK) aerobic electrolytic respirometer u nits

following OECD 301F guideline s with slight modifications. The CES electrolytic respirometer

unit consists of 20 one

- liter vessels fitted with heads containing O 2 

## and CO 2 

## sensors. The

system measures the amount of CO 2 

## generated by means of a cuvette filled with 1% sodium

hydroxide and a conductivity probe that determines the change in conductivity as CO 2 

## is

absorbed from the headspace gases of the test vessel. A differential pressure sensor head is

designed to measure the amount of O 2 

## used by respiri ng microorganisms. It achieves this by

detecting the difference in pressure during the uptake of oxygen and generation of CO 2 

## and

then supplying an equal amount of O 2 

## back to the headspace using an electrolytic cell.

The electrolytic cell contains a cham ber filled with copper sulfate solution. Oxygen is generated

in this chamber by passing an electrical charge (initiated by the test vessel head pressure

change sensor) between electrodes. It is connected to the headspace of the sample vessel by

a vent. V alves are used to balance the pressure and return generated O 2 

## to the headspace of

the test vessel. The amount of CO 2 

## (mg) absorbed and O 2 

## (mg) generated is recorded by a

computer.

The quadruplicate samples were split between the two respirometer units; two replicates in Unit

1 and two replicates in Unit 2.

As stated above, slight deviations were made to some of the OECD 301F guidelines 8 

## to better

accommodate the poor water solubility of the alcohols and alcohol p ropoxylates evaluated in

this study. The inoculum for these tests was derived from the recycled sludge from the City of

Sulphur, LA Regional Waste Wa ter Treatment Plant; sludge was SBR aeration basin. The

sludge was allowed to settle and supernatant decan ted and refilled with mineral water to dilute

any background organic. This was repeated three times. The final decant sludge concentrate

was blended using a W ar ing Blender for two minutes on low. Total suspended solids (TSS)

w ere measured on the c oncentrate and the volume determined to add to the large pot of 45

liters of mineral water. The TSS in the medium is targeted to a final concentration of 25 mg/L

TSS to meet the OECD guideline specification of < 30 mg/L TSS. Sodium benzoate ( source:

J.T. Baker

– 99% purity) was used as the positive reference test material. From the 45 liter pot

of inoculum containing mineral water is 1kg is dispended into the 1

- liter test vessels.

The following describes the t echnique used for dispersing water insoluble alcohols and alcohol

propoxylates for biodegradation testing. Approximately 500 mg mound of silica gel (Aldrich,

Merck grade 9385) with a mesh size of 230

- 400 Ǻ was weighed onto a creased piece of

we ighing paper. A spatula was then used to tap the top of the mound and flatten it so that it

became a thick “plateau ” to prevent drop s of insoluble test material from rolling off of the silica

gel. Then, a 1 mL pipette was held vertically and individual drops of insoluble test material were

dispensed on to the silica gel until a weight close to 50

- 60 mg was reached. Special care was

taken to ensure that drops were not placed on top of each other so that none of the insoluble

solution soaked down to the weighing paper. The weight of the insoluble test material was then

## 12/14/2011 Page 31 of 66

recorded. The sodium benzoate powder (50

- 60mg) was added to the 5 00 mg silica gel in the

same manner. The weighing paper was then rolled into a cone and the silica gel/test material

was poured into a 125 mm x 15 mm test tube. The silica gel/test material was mixed using a

VW R Scientific Products, Mini Multiple Vortex er MV1, set at h ig hest speed (2500 rpm) for fifteen

minutes.

The 1

- liter test vessels containing 1 kg diluted inoculat e in the mineral medium were placed on a

magnetic stirrer and stirred prior to receiving the contents from the test tubes. The silica gel/test

material samples were then poured into the test vessels. A uniform dispersion of the test

material was immediately observed in all units and remained as such throughout the entire 28

- day test. The 1

- liter vessels were th en placed in the water bath (25 ± 1 °C) of the electrolytic

respirometer units. A higher temperature (25 instead of 22 ° C) was used for consistency with

the PPS temperature of 25°C ; also

, two of the alcohols (ALFOL ® 

## 12 and ALCHEM ® 

## ) are solids at

the guideline 22°C. The respirometer underwent a 60 minute initialization/equalization before

data collection began at every six hour interval

. Both CO 2 

## generation and O 2 

## uptake data were

recorded every six hours during the 28 day test. Each flask contained a stir bar positioned over

a magnetic stirrer to provide vigorous mixing. Blanks were also included in the study for validity

purposes. These blanks were prepared identically to the samples (i.e. test medium, inoculum,

and silica ge l ) but without the test compounds.

The procedure for the six anionic ALFOTERR A ® 

## Surf actants is achieved by adding 10 mL from

a 1% stock solution for each anionic surfactant into the 1

- liter vessel containing 990 g of

inoculated mineral water. Final con centration targeted is 100 mg/L as active. Since these are

anionic surfactants

, the silica gel dispersion technique did not apply.

The results for theoretical oxygen demand ( ThO2 ) is obtained by the COD wet chemistry HACH

method utilizing low range 0

- 1 50 mg/L COD vials and a DR/2000 spectrophotometer. The

results reported as T h O2 is determined by generating COD values on specific weight from each

test material, including benzoate. The mg O 2 

## / mg sample factor used was calculated by

dividing the COD mg/L value by the mg/L material used in the COD analysis

. The THO2 value

used for each material is the recorded weight in mg of sample multiplied by the mg O 2 

## / mg

added to each 1

- liter test vessel

.

## 12/14/2011 Page 32 of 66



# OECD 301 F Electr **o ly**  **tic Respirometer Results:**

**AL FOTERRA** 

**Hydrophobe Biodegradation Study**

**OECD 301F Theoretical Oxygen demand**

**0 20**  **40 60**  **80 100**  **120**

**0 5**  **10 15**  **20 25**  **30**

**Day s** **% T** **H O** **D Isalchem 23**  **Alchem 23**

**TDA Safol 23** 

**Lial 23 Alfol 12** 

**Benzoate**



### Figure **19 :**  **Biodegradation Study**

**- Alcohols**  **(OECD 301F)**



## The 28

- day biodegradation results for the six alcohols are shown in Figure 19. As expected, the

degradation rate decreases as t he amount of branching in the alcohol increases. This trend is

especially true for the first 10 days wh ere branching content appears to be the primary driver.

T he 100% linear ALFOL ® 

## 12 (d ar k green) gives the fastest and most complete biodegradation

wh il e the 100% branched TDA Alcohol ( red ) gives the slowest. The alcohols that are a mixture

of both linear and branched molecules fall in between. The only anomaly is the A LCHEM ®



## alcohol (brown) which is 100% linear and performs equal to the 100% l inear ALFOL ® 

## 12 at the

beginning of the study but does not reach the same 100% THOD after the 28 days. This can

be explained by the C13 content in the Alchem which must degrade slower than the 100% C12

content of the ALFOL ® 

## 12.

Also given in F igure 19 is a 3

- D model of the ALFOTERRA ® 

## Surfactant molecule . The circled

portion denotes the alcohol hydrophobe of the surfactant, which in the given case is

ISALCHEM ® 

## 123 alcohol . From the above data it can be concluded that the ISALCHEM ® 

## 123

alcohol porti on of the ALFOTERRA ® 

## molecule could i mpact the biodegradation in slightly

negative way due to its branching effect, especially when compared to the 100% linear C12 14

alcohol derived AES that was used in the earlier discussed studies as the control. How eve r, since all alcohols easily reached the 60% ThO2 threshold by day 28, the alcohol structure does

not completely explain the poor er biodegradation performance of the ALFO 23

- 4S.

## 12/14/2011 Page 33 of 66 **AL FOTERRA** 

**Hydrophobe 4PO Biodegradation Study**

**OECD 301F Theoretical Oxygen demand**

**0 20**  **40 60**  **80 100**  **120**

**0 5**  **10 15**  **20 25**  **30**

**Days %** **T H** **O D**  **Isalchem 23 Alchem 23** 

**TDA Safol 23** 

**Lial 23 Alfol 12** 

**Benzoate**



### Figure **20 :**  **Biodegradation Study**

**- Alcohol 4 Mole**  **Propoxylates (OECD 301F)**



## In order to determine the effect of the propoxylate portion on the biodegradation of the

ALFOTERRA

® 

## molecule , the six alcohol s were propoxylated using K OH catalyst to contain an

average 4 moles propylene oxide

. Figure 20 show s the resulting degradation curve after 28

days in the electrolytic respirometer. The most notable difference betw een the alcohol ( Figure

19) and alcohol propoxylate ( Figure 20) response curves is that the degradation of the

propoxylates within the first 10

- 15 days is generally slower than that of the alcohol. Secondly,

the branching in the alcohol back

- bone has less of an impact on the results. In fact, if not for the

slower start of the TDA alcohol and the lower final THOD of the ALFOL ® 

## 12 alcohol, all samples

would have performed statistically the same. Most surprising is that all samples biodegrade to

>80% i n 28 days. Ag ain, this leads one to conclude that the alcohol and propoxylate have only

a slight impact on the poor degradation of the ALFO 23

- 4S.

The ALFOTERRA ® 

## Surfactant molecule image in the figure now shows the alcohol propoxylate

portion of the molecule cir cled. Th e portion circled is a nonionic surfactant that has both a

hydrophobic (alcohol chain) tail and hydrophilic head group of a polyprop y lene glycol ( PPG )

chain. The PPG head group exten ds the hydrophobic property tha n imparts surfactancy. Thus,

the alcohol propoxylate has poor water solubility

.

## 12/14/2011 Page 34 of 66 **AL FOTERRA** 

**Hydrophobe 4PO Sulfate Biodegradation Study**

**OECD 301F Theoretical Oxygen demand**

**0 20**  **40 60**  **80 100**  **120**

**0 5**  **10 15**  **20 25**  **30**

**Day s** **% T** **H O** **D Isalchem 23**  **Al chem 23** 

**TDA Lial 23** 

**Safol 23 Al**  **fol 12**

**Benzoate**



### Figure **21 :**  **Biodegradation Study**

**- Alcohol**  **4 Prop**  **o xy**  **Sulfate (OECD 301F)**



## Finally, the sulfate (sodium salt) derivatives of the six alcohol propoxylates (4 mole s ) were

evaluated through electrolytic respirometer. The results are given in Figure 2 1 . Now

, the poor

biodegradation associated with the alcohol propoxy sulfate is seen ; the addition of the sulfate

head

- group or the interaction of the head

- group and the branched alcohol propoxylate

significantly de c reases the biodegradability of the molecule. This is somewhat surprising as the

sulfate molecules are extremely water soluble. In general, o ne would expect that water soluble

molecules are more bio

- available and would be much ea s ier for the microbes to degrade th a n

insoluble molecules such as the alcohol propoxylate

. However, this is not the case with this

molecule

.

Again, t here is some impact of branching on de gra dation. The molecule based on 100%

branched TDA alcohol ( red ) is the slowest to degrade while the 100% ALFOL ® 

## 12 alcohol (d ark

green) based product is the fastest. However, the branching affect is not so pronounced that

the bio degradation of the ALFO 2 3

- 4S could be much improved by going to a 100% linear

molecule.



# OECD 301 F Electroly **tic Respirometer Summary :** 

Though branching on the back

- bone of the alcohol hydrophobe and the addition of propylene

oxide does impact the biodegradation of alcohol propoxy sulfates when compared to the

industry standard linear alcohol ethoxy sulfates, it is the interaction of the sulfate head group

with the branched alcohol propo x ylate th at is resp onsible for the decrease in biodegradation.

To easily see the impact of each structural component for the model compound ALFO 23

- 4S,

the biodegradation curves for the ISALCHEM 123 alcohol, ISALCHEM 123

- 4PO and

ISALCHEM 123

- 4PO

- S (commercial name is ALFO TERRA ® 

## 1 23

- 4S) are given in Figure 22.

## 12/14/2011 Page 35 of 66 **Biodegradation Profile of Isalchem 123 Derivatives**

**OECD 301F Theoretical Oxygen Demand**

**0 20**  **40 60**  **80 100** 

**0 5**  **10 15**  **20 25**  **30**

**Day s** **% T** **H O** **D Isalchem123-4PO** 

**Isalchem 123-4POS**

**Isalchem 23 OH Al**  **cohol**

**4PO**

**4POS**



### Figure **22 : Biodegradation**  **p rofile of**  **AL FO 23** 

**- 4S**  **Derivatives**



# Discussion

**Environmental Fate and Effects of ALFOTERRA ®**  

# Surfactants

The results of the PPS studies reveal that DOC removal of ALFO 23

- 4S through the simulated

waste treatment plant is inadequate. At best there was a DOC loss of 60%. It was also shown

tha t the level of treatment reached a steady state and leveled off within a few days. The

standards (b enzoate and the model anionic AES surfactant ) showed excellent to complete

removal as compared to the blank. The poor performance in biodegradation of AL FO 23

- 4S is

also supported by the DOC Die

- Away and the electrolytic respirometer results.

In the PPS and DOC Die

- Away studies, more than 90% of MBAS activity of the ALFO 23

- 4S i s

los t . T his demonstration of high degree of primary degradation for the surfactant molecule is

also shown to have occurred in the model AES surfactant. The degree of primary

biodegradation for ALFO 23

- 4S and AES surfactants is si milar

.

Further evidence that primary degradation or breakdown of the parent molecule is occurring

come s from the aquatic toxicity testing

. The 96

- hour minnow acute toxicity LC50 of < 5 mg/L for

both the neat ALFO 23

- 4S and AES are typical concentratio ns for anionic surf actants.

However, the significantly improved acute aquatic toxicity in the effluents from the PPS and Die

Away at 28

- days indicates that the parent molecule for ALFO 23

- 4S has undergone significant

primary degradation so as to no longer be toxic

. I f the DOC that remains in both the PPS

effluent and Die

- Away of the ALFO 23

- 4S was intact parent compound, the resulting effluent

solution would have been toxic for at least the 100% solution w here the residual DOC is about

15 mg/L greater than the control samples

- clearly over the LC50 point

. However, the

remaining DOC is incapable of short te r m toxicity wh ich is demonstrated by the endpoints o f

LC50, NOEC and LOEC of greater than 100% effluents.

## 12/14/2011 Page 36 of 66



# Biodegradation Pathway **of Anionic Surfactants Studied**

**C14**

**br**

**- AES 3EO** 

**C4 branched # 2 carbon**  **ALFOTERRA ®**  **4PO**

**C4 branched # 2 carbon SO4** 

**PEG SO4**  **SO4**

**PEG PPG** 

**br**

**- C14**  **br**

**- C14** 

**AES 3EO**

**C14 linear alcohol**



### Figure **23: Alcohol Ether Sulfate Structure: A**  **ES and A LFO 23** 

**- 4S** 



## Figure 23 sh ows a 3

- D model fo r anionic surfactant molecules of a linear and branched

ethoxylated sulfate (AES) and a branched alcohol propoxy sulfate ( AL FOTERRA ® 

## Surfactant ) ;

each propoxylated with 4 moles of PO. Not e the reduced length and multiple appendages of

th e ALFOTERRA ® 

## molecule. The structure of the molecule w hen a branched alcohol chain an d

multiple propoxylate groups are present could act as a barrier to bacterial attack. Additionally,

note how the PPG branch and resulting sulfate posit ion protect the fourth sulfate oxygen (not

seen directly in this view) from direct exposure. In fact, almost all oxygen molecule s in the

ALFOTERRA molecule are protected by the bulky alcohol and /or p ropylene oxide carbon chain.

The experimental r esults confirm that the r ate of biodegradation fo r ALFO 23

- 4S is impacted by

the reduced linearity of the molecule ’ s structure f rom branching and PPG.

Surfactant molecules are highly complex and different degradation pathways are possible for

any given surfactant (Sharvelle, 2007 ). Complete removal may be difficult due to fo rmation of

by

- product s that are more slowly degraded compared to the parent compound. The recognized

and described metabolic pathways for biodegradation of alcohol ethoxylates and alcohol ethoxy

sulfates ( AES ) surfactant molecules is based on specific points of bacterial attack

.

Three routes have been identified whereby bacteria can initiate biodegradation of linear alcohol

ethoxylates (LAE) (the un

- sulfated form of AES). (W hite, 1993) The routes are (Figure 24):

 w/β

- oxida tion of the carbon chain of the alcohol hydrophobe in the intact surfactant

 exo

- cleavage of the ethylene glycol C2 units from the PEG moiety and/or

 cleavage at the ether bond which joins the PEG chain to the hydrophobic alkyl group

(central scission).

T he attack routes for AES are:

 omega oxidation on the carbon chain of the alcohol hydrophobe

 central fission separating the alcohol from the ethylene glycol ( E G ) chain

 ether cle avage along the E G chain and

 sulfate hydrolysis.

## 12/14/2011 Page 37 of 66

In previously published work Hales and W hite discuss the degradation of an alcohol ethoxy

sulfate

- linear dodecyl 3 mole ethoxy sulfate ( similar to linear dodecyl 4 mole ethoxy sulfate

used in this study )

. In their work, all four AES routes were identified in studies emplo ying four

bacteria species (Hales, 1986; Wh ite and Russell, 1998). In fact, they were able to identify

mono

- , di

- and triethyleneglycol sulfates as the metabolites of the ether cleavage of the three

ether bonds in the dodecyl triethox ysulfate

.

In 1982, Yoshimura (Yoshimura, 1982) showed the impact of branching in the alcohol

hydrophobe portion of AES. Yoshimura’s results showed that AES with a linear hydrophobe is

ready biodegradable, whereas a branched hydrophobe is much more resistant. The

degradation difficulty of the branched AES surfactant was attribute d to steric hindrance

preventing enzymic attack.

In this same study, Yoshimura also shows the impact of alkyoxylate structure on biodegradation

by comparing linear alcohol ethoxy sulf ate (AES) linear alcohol propoxyl sulfate (APS). The

r esults showed that the APS surfactant is more resistant to biodegradation than the AES. It was

suggested that this is likely due to the inclusion of a secondary carbon atom in the structure of

the propylene oxide grou p which shields the oxygen molecule from attack.

In general, it can be assumed that the AES is biodegraded through either of the following

pathways : (a) Enzymic hydrolysis of the sulfate group yielding sulfate and alcohol ethoxylate . T he latter is

further degraded through the biode gradation pathway of a primary alcohol ethoxylate by

wa y of central scission. Or

(b) En z ymes attack the terminal methyl group of the linear alcohol reducing the carbon chain

length as it moves forward. This is followed by β

- oxidation.

If both the AES and ALFO 23

- 4S used in this study follow route (a) then metabolites yielded can

be alcohol, PEG and /o r PPG chains respectively. It would then be expected that the AES and

ALFO 23

- 4S degradation would be more similar or that the ALFO 23

- 4S degradation would be

more similar to the alcohol propoxy sulfate from which i t was derived. This was not the case.

In a n extensive study comparing the biodegradation of low molecular weight polyethylene glycol

( PEG 300 ) with polypropylene glycol ( PPG 425 ) Zg ola (Zgola

- Grześkowiak, 2005) showed that

the biodegradation pathways of PEG and PPG are very different. PEG is biodegraded through

clipping of the PEG chain resulting in metabolites that were identified as short en ed PEG

etho x ymers . That is, the molecular weight of all PEG molecules is reduced consistently over

time throughout the degradation process. In contract, the biodegradation of PPG does not

involve chain scission at all. In fact, Zgola showed tha t while the homolog distribution pattern of

PPG propoxymers remains unchanged (average molecular weight do not decrease) during the

biodegradation test , the total quantity of molecules reduces over time. ; i.e. no metabolites that

are shorten ed version of the parent molecule are formed

. In other words, t he concentrations of

the individual homologs of PPG decrease instead of shifting to shorter or lower molecular weight

homolog s

. T he result is that the bacteria appear to be attacking the whole PPG molecule at one

time. Zg ola does not address how this mechanism is impacted with the addition of a sulfate

head group.

Finally, in a 2002 paper, Ellis identified enzymes that work to degrade branched alcohol sulfates

but not linear alcohol s ulfates, and vice verse. In this paper a novel a lkyl sul f atase enzyme was

isolated that is specific to a branched primary alkyl sulfate surfactant 2

- butyloctyl sulfate (Ellis,

2002)

. Th is identified novel sul f atase enzyme is produce d f ro m a s pecific strain of

## 12/14/2011 Page 38 of 66

Pseudomonas AE

- D bacteria grown on 2

- butyloctyl sulfate ; t his enzyme is not active on the

linear a lcohol sulfate dodecyl sulfate (SDS). In contrast

, Pseudomonas sp. Strain C12B, well

known for its ability to degrade linear SDS, was unable to grow on 2

- butyloc tyl sulfate

. Additionally, its alkylsulfatases which is responsible for initiating the degradation of SDS by

releasing the parent alcohol

, exhibited no hydrolytic activity towards 2

- butyloctyl sulfate.

Clearly small structural differences in otherwise similar molecules can drastically impact the

biodegradation rate, pathway and active bacterial species.

**ω**

**- /**  **β**

 

** Oxidation** **Central**

**Scission Ether** 

**Cleavage Sulfate** 

**Hydrolysis** 

### AE **S**

**APS No Cleavage** 

**Restricted**

**Sulfate**

**Hydrolysis ω** 

**- /**  **β**

 

** Oxidation** **Central**

**Scission Ether** 

**Cleavage Sulfate** 

**Hydrolysis** 

### AE **S**

**APS No Cleavage** 

**Restricted**

**Sulfate**

**Hydrolysis**



### Figure 2 **4 : A**  **nionic Linear**

**- AE**  **S and branched A PS (A**  **LFO 23**

**- 4S) Molecule Biodegradation** 

**Pathway**

**- Points of A**  **ttack**



## F igure 2 4 contains the 3

- D model s for AES and ALFO 23

- 4S anionic surfactant molecules. T he

arrows compar e points of attack as discussed in the literature references above

. For AES, all

points of attack can and do occur rapid ly ; h owe ver for ALFO 23

- 4S , the central scission of the

alcohol and PPG chain, steric hindrance preventing ether cleavage

, and restricted sulfate

hydrolysis all compromise the rate at which the molecule degrades.

Yet, in spite of the degradation barriers, the work detailed in this report prove s that primary

biodegradation of alcohol propoxy sulfate surfactants, and specifically ALFO 23

- 4S, does o ccur. Further, and more importantly, primary degradation results in the elimination of the aquatic

toxicity that is associated with the anionic surfactant parent . W ith responsible handling, and

treatment minimally through domestic or industrial waste wat er treatment facilities of any rinse

waters containing the alcohol propoxy sulfates, Sasol ’s ALFOTERRA ® 

## surfactants should be

harmless to the environment.

## 12/14/2011 Page 39 of 66



# REFERENCES

1. OECD.1993. OECD Guidelines for the Testing of Chemicals

– OECD 301A: DOC Die

Away and 301F: Manometric Respirometry Test. Organization of Economic Cooperation

and Development. Paris.

2. Sharvelle, S., Lattyak, R., Banks, M. K. (2007). Evaluation of biodegradability and

biodegradation kinetics for anionic, nonioni c, and amphoteric surfactants. W ater Air Soil

Pollut. 183, 177

- 186.

3. Wh ite G. F., (1993) Bacterial biodegradation of ethoxylated surfactants. Pestic Sci 37,

159

- 166.

4. Hales, S. G., W atson, G. K., Dodgson, K. S.,

5. Wh ite G. F. (1986). A comparative study of t he biodegradation of the surfactants sodium

dodecyltriethoxy sulfate by four detergent

- degrading bacteria. J. Gen. Microbiol 132,

953

- 961.

6. Wh ite, G. F., and Russell, N. J. (1988). Mechanisms of bacterial biodegradation of alkyl

sulfate and alkylpolyethoxy sulfate surfactant. Biodeterioration 7, 325

- 332.

7. Yoshimura, K., Masuda F., (1982). Biodegradation of sodium alkyl poly(oxyalkylene)

sulfates. JOACS 59, 328

- 332.

8. Zgola

- Grześkowiak, A., Grzeskowiak, T., Zembrzuska, J., Lukaszewski, Z., (2005).

Comparison of biodegradation of poly(ethylene glycol)s and poly(propylene glycol)s.

Chemosphere 64, 803

- 809.

9. Ellis, A. J., Hales, S. G., Ur

- Rehman, Naheed G. A., Wh ite, G. F., (2002). Novel

alkylsulfatases required for biodegradation of the branched primary alkyl sulfat e

surfactant 2

- butyloctyl sulfate. Appli. Environ. Microbiol. 68, 1, 31

- 36.

10. US EPA OPPTS 835.3220 “Porous Pot Test” Fate, Transport and Transformation Test

Guidelines (April 1992).

## 12/14/2011 Page 40 of 66



# AP **PENDICES**

Appendix 1: Stepan AES

................................ ................................ ................................ ........... 41

Appe ndix 2: Figure 2

- HRT

................................ ................................ ................................ ...... 42

Appendix 3: Figure 3

- MLTSS

................................ ................................ ................................ . 45

Appendix 4: Figure 4

- Porous Pot Effluent TSS

................................ ................................ ....... 48

Appendix 5: Figure 5

- Porous Pot Effluent pH Values

................................ ............................. 49

Appendix 6: Figure 6

- Porous Pot DOC Feed and Effluents

................................ .................... 51

Appendix 7: Figure 7

- PPS Effluent DOC Un

- acclimated ALFO 23

- 4S

................................ ..... 54

Appendix 8: Figure 8

- PPS f or Fragment Analysis Effluent

................................ ...................... 58

Appendix 9: Figure 9

- Return to Steady State: Post Un

- acclimated ALFO 23

- 4S

.................... 59

Appendix 10: Figure 10

- Porous Pot DOC: Comparison of 1214E4S and ALFO 23

- 4S

........... 60

Appendix 11: Figure 11

- PPS: Increased HRT

................................ ................................ ........ 61

Appendix 12: Figure 12

- Die

- Away Porous Pot Effluent

................................ .......................... 61

Appendix 13: Figure 13

- OECD Die

- Away DOC Loss %

................................ ......................... 61

Appendix 14: Figure 14

- Porous Pot: MBAS Primary Biodegradation

................................ ...... 62

Appendix 15: ALFO 23

- 4S Comparison Porous Pot and Die Away

................................ .......... 62

Appendix 16: Figure 16

- AES and ALFO 23

- 4S Acute Toxicity

................................ ............... 62

Appendix 17: Figure 17

- Porous Pot Aquatic Toxicity 96 Hour Minnow

................................ ... 62

Appendix 18: Figure 18

- OECD Die

- Away Test ALFO 23

- 4S

................................ ................... 62

Appendix 19: Figure 19: ALFOTERRA Hydrophobe Biodegradation Study

- OECD Demand

. 63

Appendix 20: Figure 20

- ALFOTERRA Hydrophobe 4PO Biodegrad ation Study

- OECD

....... 64

Appendix 21: Figure 21

- ALFOTERRA Hydrophobe 4PO Sulfate Biodegradation Study

- OECD

................................ ................................ ................................ ................................ ................. 65

Appendix 22: ALF OTERRA Hydrophobe Biodegradation Study, Comparison of Alcohol,

Nonionic, and Anionic

................................ ................................ ................................ ............... 66

Appendix 23: Aquatic Toxicity Reports from Environmental Enterprise USA Laboratory TSR No.

R&D ENV 10

- 00 4 01

- 10

................................ ................................ ................................ ............ 66

## 12/14/2011 Page 41 of 66

**Ap pendix**  **1 :**  **Stepan A ES** 



## POLYSTEP® B

**- 430S** 

Actives, % 28

Cloud Point 5oC (41oF)

Viscosity 53 cps @ 25oC (77oF)

Pour Point

- 2oC (28oF)

FDA Clearances 21 CF R 176.170, 176.180

Chemical Description SODIUM LAURYL ETHER SULFATE, 4 EO

Form at 25 ° C Liquid

## 12/14/2011 Page 42 of 66

**Ap pendix**  **2 :**  **Figure 2**

**- HRT** 

**1 2**  **3 4**  **5 6** 

6-A pr 5.98 6. 32 6. 08 5.9 2 6.16 6.19

7-A pr 5.89 5. 93 5. 91 5.9 8 5.88 5.96

8-A pr 5.90 5. 95 5. 96 5.9 4 5.93 5.94

9-A pr 5.97 6. 04 6. 02 5.9 7 6.03 5.94

10-A pr 5.94 6. 00 6. 00 5.9 4 5.95 6.01

11-A pr 5.91 5. 94 5. 92 5.8 5 6.05 5.93

12-A pr 5.83 6. 00 5. 98 5.9 2 5.92 5.90

13-A pr 5.90 6. 02 6. 00 5.9 8 5.92 6.05

14-A pr 5.91 5. 98 5. 96 5.9 5 5.94 5.99

15-A pr 6.22 5. 96 5. 95 5.9 0 5.93 5.98

16-A pr 5.90 6. 01 5. 97 5.9 0 5.96 5.97

17-A pr 5.90 5. 96 6. 14 6.0 7 6.13 6.14

18-A pr 5.90 5. 98 6. 09 5.9 2 6.00 5.99

19-A pr 5.97 5. 98 5. 90 5.9 2 6.01 6.09

20-A pr 5.93 6. 00 6. 01 6.0 1 6.07 6.03

21-A pr 5.95 6. 02 6. 04 5.9 0 6.03 6.05

22-A pr 5.95 6. 03 6. 02 6.1 5 6.06 6.06

23-A pr 5.99 6. 05 6. 00 5.9 3 6.14 6.04

24-A pr 6.01 5. 99 6. 01 5.8 9 5.80 6.07

25-A pr 6.01 5. 99 5. 98 5.9 5 5.99 5.96

26-A pr 6.00 5. 95 5. 96 5.9 6 6.10 6.04

27-A pr 5.98 5. 95 6. 00 5.9 4 6.06 6.11

28-A pr 5.89 6. 03 6. 07 5.9 8 5.99 6.02

29-A pr 5.83 5. 99 6. 02 5.9 5 5.92 6.07

30-A pr 5.88 5. 97 6. 11 6.0 0 5.92 6.11

1-Ma y 5.94 5. 97 5. 94 5.6 8 5.95 5.83

2-Ma y 5.90 6. 00 5. 99 5.8 9 5.93 6.01

3-Ma y 5.91 5. 88 5. 91 5.8 4 5.89 6.00

4-Ma y 5.87 5. 94 5. 94 5.9 1 5.96 6.05

5-Ma y 5.91 5. 94 5. 94 5.9 2 6.01 6.01

6-Ma y 5.90 5. 95 5. 95 5.9 0 5.99 5.99

7-Ma y 5.91 5. 96 5. 94 5.9 3 5.95 6.00

8-Ma y 5.91 5. 93 5. 92 5.9 3 5.94 5.95

9-Ma y 5.91 6. 00 5. 98 5.8 9 5.92 5.98

10-M ay 5.90 6. 03 5. 94 5.8 8 5.93 6.03

11-M ay 5.91 6. 04 6. 00 5.9 2 5.95 5.98

12-M ay 5.94 5. 99 5. 97 5.9 4 5.94 6.04

13-M ay 5.91 6. 06 5. 98 6.1 0 5.87 6.11

14-M ay 5.90 6. 00 5. 93 5.8 7 5.94 5.96

15-M ay 5.90 6. 00 5. 93 5.8 7 5.94 5.96

16-M ay 5.86 5. 89 5. 86 5.7 7 5.81 6.46

17-M ay 5.91 5. 97 5. 94 5.8 8 5.96 5.98

18-M ay 5.90 5. 97 5. 94 5.9 1 5.93 5.95

19-M ay 5.91 6. 02 6. 03 5.9 0 5.97 5.98

20-M ay 5.90 6. 01 5. 96 5.8 6 5.96 6.02

21-M ay 5.93 6. 04 5. 94 5.8 6 5.88 5.93

22-M ay 5.95 6. 03 6. 02 6.1 5 6.06 6.06

23-M ay 6.07 6. 14 6. 06 6.0 0 6.06 5.96

24-M ay 6.31 6. 15 5. 95 5.8 9 5.96 5.88

25-M ay 6.01 5. 90 5. 97 5.9 0 5.97 5.86

26-M ay 5.89 5. 93 5. 99 5.8 9 5.94 5.96

27-M ay 5.87 5. 93 5. 97 5.8 6 5.95 5.84

28-M ay 5.90 5. 91 5. 85 5.8 5 5.85 5.79

29-M ay 5.63 6. 04 5. 97 5.9 9 6.12 5.99

30-M ay 5.63 6. 04 5. 97 5.9 9 6.12 5.99

31-M ay 5.81 5. 90 5. 84 5.8 6 5.94 5.94

1-Jun 5.98 5. 89 5. 89 5.8 6 5.96 5.88

2-Jun 6.01 5. 92 5. 92 5.8 9 5.99 5.91

3-Jun 5.99 5. 93 5. 87 5.9 0 5.94 5.91

4-Jun 5.97 5. 71 5. 95 5.9 6 6.06 5.96 **Fi gur**  **e 2. Hydr** 

**au li**  **c Reten tio**  **n Ti** 

**me ( HR**  **T) T**  **hr oug**  **h P or**  **ou s P**  **ots (hr**  **s)**

## 12/14/2011 Page 43 of 66 **1 2**  **3 4**  **5 6** 

5-J un 6.05 6. 02 5. 96 5.8 8 5.95 5.89

6-J un 6.57 6. 03 5. 92 5.9 4 5.98 5.96

7-J un 5.75 6. 06 5. 93 5.9 4 5.94 6.01

8-J un 5.69 6. 06 6. 07 6.1 8 5.98 5.96

9-J un 5.98 6. 48 6. 12 6.1 0 6.18 6.10

10-Jun 5.76 5. 78 5. 90 5.9 1 5.94 5.88

11-Jun 5.83 6. 03 5. 92 5.9 4 5.95 5.92

12-Jun 5.79 5. 89 5. 86 5.8 8 5.95 5.94

13-Jun 5.87 5. 93 5. 87 5.8 8 6.08 5.95

14-Jun 5.96 6. 04 5. 89 5.9 9 6.02 6.07

15-Jun 5.92 5. 98 5. 99 5.9 0 5.99 6.04

16-Jun 5.79 5. 92 5. 87 5.9 4 5.92 5.91

17-Jun 5.89 5. 95 5. 89 5.9 4 5.98 6.03

18-Jun 5.84 5. 93 5. 88 5.9 7 5.96 6.06

19-Jun 5.85 5. 94 5. 89 5.9 6 5.78 5.87

20-Jun 5.87 5. 96 5. 88 6.0 2 6.06 5.89

21-Jun 5.85 5. 97 5. 89 6.0 8 5.87 5.94

22-Jun 5.99 6. 06 6. 04 5.8 5 6.02 6.01

23-Jun 6.01 6. 06 6. 04 5.9 0 5.94 6.02

24-Jun 5.97 6. 11 6. 03 5.8 9 6.03 6.02

25-Jun 5.84 5. 97 5. 91 5.7 7 5.93 5.97

26-Jun 6.15 5. 89 5. 85 5.8 3 5.96 5.99

27-Jun 6.08 5. 82 5. 84 5.8 6 5.91 5.88

28-Jun 5.86 5. 72 5. 89 5.8 7 6.10 5.94

29-Jun 6.00 6. 00 5. 90 6.0 8 5.72 5.93

30-Jun 5.83 6. 13 5. 90 6.0 4 5.76 5.96

1-J ul 5.29 4. 99 5. 12 5.2 6 4.99 5.14

2-J ul 5.14 5. 20 5. 21 5.7 4 5.08 5.23

3-J ul 5.14 5. 97 5. 40 5.0 5 5.05 5.26

4-J ul 5.20 5. 15 5. 42 5.3 0 5.21 5.69

5-J ul 4.88 4. 83 5. 06 4.8 0 4.89 5.02

6-J ul 4.94 4. 90 4. 85 5.0 9 4.90 5.09

7-J ul 4.92 4. 88 4. 83 4.8 4 4.89 4.91

8-J ul 5.01 4. 89 4. 86 4.8 8 4.95 4.93

9-J ul 5.10 4. 89 4. 82 4.8 0 4.91 4.94

10-Jul 4.97 4. 89 4. 86 4.8 8 4.93 4.89

11-Jul 4.90 4. 85 4. 77 4.7 6 4.82 4.73

12-Jul 5.01 8. 29 12. 59 4.9 3 5.20 4.96

13-Jul 5.00 4. 92 4. 85 4.9 5 4.89 4.79

14-Jul 4.97 4. 96 4. 84 4.8 9 4.92 4.79

15-Jul 5.07 5. 06 4. 99 17.3 0 5.11 5.06

16-Jul 5.13 5. 11 5. 26 5.0 1 5.16 5.05

17-Jul 5.14 5. 07 5. 25 5.4 4 5.10 5.27

18-Jul 5.14 4. 97 5. 22 5.2 8 5.22 5.27

19-Jul 5.13 5. 09 5. 20 5.2 7 5.14 5.24

20-Jul 5.12 5. 07 5. 24 5.4 1 7.07 5.25

21-Jul 5.11 5. 01 5. 23 5.0 1 5.11 5.23

22-Jul 5.56 5. 43 5. 75 5.4 9 5.62 5.88

23-Jul 5.90 5. 93 5. 91 5.9 4 6.17 5.95

24-Jul 5.86 5. 89 5. 92 5.9 3 5.96 5.88

25-Jul 5.96 6. 00 6. 04 6.0 4 6.29 6.00

26-Jul 6.15 6. 29 6. 32 6.2 4 6.55 6.31

27-Jul 5.99 5. 99 5. 99 5.9 8 5.95 5.97

28-Jul 5.92 5. 95 6. 11 6.0 8 5.91 6.16

29-Jul 5.92 5. 99 6. 01 6.0 3 5.94 5.97

30-Jul 6.09 6. 09 6. 03 6.0 9 6.01 6.12

31-Jul 5.96 6. 10 5. 98 5.9 9 5.93 6.02

1-A ug 5.93 5. 95 5. 93 5.9 5 5.88 5.96

2-A ug 6.25 5. 90 5. 98 5.9 7 5.91 5.94

3-A ug 6.22 5. 91 5. 96 5.9 5 5.89 5.96

4-A ug 5.81 5. 87 5. 94 5.9 7 5.90 5.95 **Fi gur**  **e 2. Hydr** 

**auli c Reten**  **tio n Ti** 

**me ( HR**  **T) Th**  **ro ug** 

**h P or**  **ou s P**  **ots (hr**  **s)**

## 12/14/2011 Page 44 of 66 **1 2**  **3 4**  **5 6** 

5-A ug 5.88 5. 87 5. 89 6.2 7 5.89 5.98

6-A ug 5.88 5. 88 5. 94 5.9 7 5.96 5.97

7-A ug 5.88 5. 88 5. 94 5.9 7 5.96 5.97

8-A ug 5.90 5. 87 5. 96 5.9 4 5.90 5.94

9-A ug 5.88 5. 93 6. 99 6.0 1 5.90 5.97

10-A ug 5.84 5. 90 6. 02 6.0 3 5.88 5.94

11-A ug 5.85 5. 92 5. 89 6.0 3 5.88 5.91

12-A ug 5.85 5. 88 5. 95 6.0 3 5.90 5.95

13-A ug 5.86 5. 92 5. 94 5.9 4 5.89 5.89

14-A ug 5.85 5. 85 5. 96 5.9 3 5.87 5.94

15-A ug 5.85 5. 89 5. 90 6.0 6 5.85 5.97

16-A ug 5.89 5. 98 6. 00 5.9 4 5.95 5.95

17-A ug 5.85 9. 92 5. 90 5.9 0 5.88 5.90

18-A ug 5.87 5. 92 6. 03 5.9 3 5.87 5.92

19-A ug 5.90 5. 92 5. 88 6.5 3 5.92 5.90

20-A ug 5.90 5. 94 6. 42 5.9 2 5.91 5.90

21-A ug 5.91 6. 13 5. 81 5.9 3 6.00 5.89

22-A ug 5.88 5. 91 5. 80 5.8 9 5.90 5.90

23-A ug 5.99 6. 02 6. 09 6.0 9 6.15 6.12

24-A ug 5.91 5. 87 6. 05 5.8 8 5.92 5.92

25-A ug 5.92 5. 90 5. 94 5.9 0 5.87 5.91

26-A ug 5.89 5. 87 5. 93 5.8 8 5.86 5.89

27-A ug 5.92 5. 91 5. 96 5.8 9 6.01 5.93

28-A ug 5.98 5. 95 6. 00 5.9 6 5.91 5.96

29-A ug 5.91 5. 98 5. 96 5.9 7 5.73 5.92

30-A ug 6.03 6. 03 6. 08 6.0 3 6.09 6.08

31-A ug 5.96 5. 91 5. 98 5.9 7 5.92 6.05

1-S ep 5.90 5. 89 5. 95 5.8 7 5.90 5.91

2-S ep 5.96 5. 88 5. 95 5.9 1 6.00 5.84

3-S ep 5.94 5. 85 5. 96 5.9 5 5.98 5.88

4-S ep 5.89 5. 86 5. 83 5.9 8 5.88 5.83

5-S ep 5.93 5. 81 5. 95 5.9 3 5.85 5.83

6-S ep 5.91 5. 87 5. 95 5.9 0 5.89 5.93

7-S ep 5.95 5. 89 5. 97 5.9 2 5.86 5.86

8-S ep 6.01 5. 93 5. 98 5.9 6 5.92 5.86

9-S ep 5.91 5. 94 6. 01 5.9 4 5.90 5.92

10-S ep 5.93 5. 90 6. 30 5.9 2 5.86 5.89

11-S ep 5.98 5. 98 5. 90 5.8 7 5.81 5.85

12-S ep 5.92 5. 88 5. 94 5.9 2 5.83 5.93

13-S ep 5.98 5. 88 5. 92 5.9 4 5.88 5.89

14-S ep 6.18 5. 91 5. 95 5.9 4 5.89 5.89

15-S ep 5.89 5. 89 5. 98 5.9 3 5.92 5.82

16-S ep 5.98 5. 86 5. 80 5.9 8 5.82 5.86

17-S ep 5.94 5. 87 5. 82 5.9 6 5.88 5.83

18-S ep 5.94 5. 93 5. 88 5.9 7 5.93 5.86

19-S ep 5.95 5. 92 5. 94 6.0 0 5.93 5.92

20-S ep 5.81 5. 83 5. 79 6.0 6 5.80 5.77

21-S ep 6.00 6. 01 6. 01 6.1 2 5.96 6.06

22-S ep 6.12 6. 12 6. 11 6.2 1 6.05 6.13

23-S ep 6.15 6. 15 6. 14 6.3 4 6.11 6.15

24-S ep 6.15 6. 15 6. 14 6.3 4 6.11 6.15

25-S ep 5.92 5. 92 5. 95 10.7 9 5.89 5.89

26-S ep 6.70 6. 50 6. 62 6.0 0 6.18 6.95

27-S ep 7.99 7. 58 7. 85 7.3 5 7.69 7.91

28-S ep 9.12 9. 04 8. 98 8.8 8 8.89 9.48

29-S ep 11 .15 10 .68 10. 43 10.8 8 10.48 10 .19

30-S ep 11 .68 12 .03 11. 55 11.8 0 11.64 11 .17

1-Oct 11 .78 11 .94 11. 51 11.9 3 11.60 12 .54

2-Oct 11 .59 12 .06 11. 53 11.9 0 12.17 11 .94

3-Oct

4-Oct 11 .66 12 .15 11. 58 12.0 9 11.61 11 .77

5-Oct 11 .60 11 .94 11. 59 11.6 7 11.84 12 .09

6-Oct 11 .46 11 .98 11. 43 12.0 1 11.57 11 .29

7-Oct 11 .61 11 .75 11. 35 11.7 3 11.62 11 .39 **Fi gur**  **e 2. Hydr** 

**auli c Reten**  **tio n**  **Ti**

**me (H**  **RT) T** 

**hr oug**  **h Po**  **ro us**  **P ots** 

**(hr s)** 

## 12/14/2011 Page 45 of 66

**Ap pendix**  **3 : Figure 3** 

**- MLTSS** 

**Po t**  **1 2**  **3 4**  **5 6** 

6-A pr 3, 720 3, 870 3,4 10 3,75 0 3,320 3, 440

7-A pr 4, 460 3, 850 3,7 40 3,92 0 3,670 3, 690

8-A pr 3, 880 4, 020 3,6 30 3,89 0 3,510 3, 760

9-A pr 3, 770 4, 130 3,6 40 3,79 0 3,390 3, 870

10-A pr 3, 990 4, 230 3,6 60 3,91 0 3,560 3, 990

11-A pr 3, 690 4, 240 3,9 60 4,07 0 3,590 3, 940

12-A pr 3, 800 4, 280 3,7 70 4,03 0 3,640 4, 150

13-A pr 3, 760 4, 230 3,9 10 4,03 0 3,600 3, 820

14-A pr 3, 580 4, 280 3,7 10 3,87 0 3,630 3, 660

15-A pr 3, 670 4, 360 3,6 70 3,93 0 3,470 3, 550

16-A pr 3, 620 4, 540 3,8 80 4,21 0 3,750 3, 750

17-A pr 3, 690 4, 580 4,0 60 4,22 0 3,850 4, 030

18-A pr 3, 700 4, 140 3,5 10 3,60 0 3,830 3, 620

19-A pr 3, 720 3, 980 3,7 00 3,42 0 3,630 4, 100

20-A pr 3, 680 3, 790 3,7 30 3,17 0 3,460 3, 860

21-A pr 3, 460 3, 430 3,7 00 3,36 0 3,670 3, 730

22-A pr 3, 400 3, 520 3,5 10 3,26 0 3,350 3, 500

23-A pr 3, 550 3, 680 3,2 90 3,30 0 3,440 3, 680

24-A pr 3, 530 3, 620 3,2 50 3,22 0 3,260 3, 420

25-A pr 3,400 4, 040 3, 440 3,3 60 3,23 0 3,900

26-A pr 3,370 3, 950 3, 460 3,1 90 2,89 0 3,510

27-A pr 3,280 3, 960 3, 390 3,1 80 2,75 0 3,180

28-A pr 3,380 3, 890 3, 380 3,1 20 2,75 0 3,150

29-A pr 3,410 3, 890 3, 320 2,9 90 2,71 0 2,650

30-A pr 3,530 4, 020 3, 310 2,8 20 2,72 0 2,680

1-Ma y 3,000 3, 290 2, 520 2,8 90 2,23 0 2,240

2-Ma y 3,130 3, 480 2, 930 3,0 60 2,85 0 2,390

3-Ma y 3,820 4, 030 3, 650 3,6 50 3,04 0 2,840

4-Ma y 4,280 4, 080 3, 680 3,8 10 2,98 0 3,150

5-Ma y 4,110 4, 070 3, 860 3,8 30 2,88 0 3,430

6-Ma y 4,270 4, 200 4, 030 3,9 80 2,72 0 3,690

7-Ma y 3,700 3, 560 3, 210 3,3 20 2,49 0 3,290

8-Ma y 3,790 3, 960 3, 330 3,3 80 2,69 0 3,420

9-Ma y 4,480 4, 090 3, 730 3,9 40 2,97 0 3,970

10-M ay 4,180 4, 190 3, 610 3,9 50 3,30 0 4,070

11-M ay 4,180 4, 520 3, 760 3,9 90 3,22 0 4,130

12-M ay 4,050 4, 460 3, 780 3,8 30 3,19 0 4,240

13-M ay 4,140 3, 800 4, 000 4,2 30 4,00 0 3,970

14-M ay 4,290 3, 980 4, 220 4,3 30 4,16 0 4,140

15-M ay 4,280 4, 240 4, 020 4,5 00 4,23 0 4,020

16-M ay 4,490 4, 418 4, 100 4,7 20 4,55 0 4,130

17-M ay 4,610 4, 690 4, 250 4,7 90 4,65 0 4,130

18-M ay 4,580 4, 540 4, 180 4,6 20 4,59 0 3,960

19-M ay 4,250 4, 270 4, 030 4,4 60 4,48 0 3,800

20-M ay 4,190 4, 270 4, 020 4,7 30 4,57 0 4,050

21-M ay 4,430 4, 270 4, 160 4,6 50 4,54 0 3,970

22-M ay 4,420 4, 310 4, 080 4,6 30 4,67 0 3,780

23-M ay 4,530 4, 180 4, 170 4,7 50 4,71 0 3,920

24-M ay 4,570 4, 380 4, 140 4,9 80 4,95 0 4,030

25-M ay 4,300 4, 370 3, 680 4,8 90 4,72 0 3,870

26-M ay 4,200 4, 250 3, 740 4,9 20 4,72 0 3,950

27-M ay 3,840 3, 920 3, 660 4,5 40 4,33 0 3,760

28-M ay 3, 230 3, 980 3,5 60 4,35 0 4,210 4, 990

29-M ay 3, 610 3, 390 3,3 70 3,95 0 3,930 3, 430

30-M ay 3, 540 3, 210 3,3 70 3,81 0 3,790 3, 460

31-M ay 3, 610 3, 510 3,3 00 3,92 0 3,770 3, 480

1-Jun 3, 300 3, 270 2,9 30 3,80 0 3,430 3, 070

2-Jun 3, 530 3, 450 3,1 00 3,93 0 3,550 3, 010

**Fi gu**  **re 3**  **. M ixed**  **L iq**  **uor T**  **ot al**  **Su**

**spen ded** 

**S olid**  **s (M LT**  **SS )** 

## 12/14/2011 Page 46 of 66 **Po t**  **1 2**  **3 4**  **5 6** 

3-Jun 3, 850 3, 390 3,2 10 4,24 0 3,810 3, 260

4-Jun 4, 140 3, 910 3,2 40 4,47 0 4,050 3, 520

5-Jun 4, 220 4, 220 3,6 10 4,67 0 4,460 3, 930

6-Jun 4, 320 4, 350 3,9 20 4,50 0 4,220 3, 700

7-Jun 4, 420 4, 390 4,3 30 4,55 0 4,380 3, 060

8-Jun 4, 370 4, 430 4,2 40 4,30 0 4,430 3, 280

9-Jun 4, 760 4, 270 4,4 90 4,37 0 4,580 3, 800

10-Jun 4, 880 4, 970 4,7 50 4,36 0 4,740 4, 140

11-Jun 4, 870 4, 950 4,7 10 4,41 0 5,030 4, 370

12-Jun 4, 890 5, 190 4,8 90 4,64 0 4,990 5, 330

13-Jun 4, 620 4, 880 5,0 10 4,39 0 4,880 4, 970

14-Jun 4, 390 4, 720 4,9 50 4,20 0 4,670 4, 650

15-Jun 3, 900 4, 320 4,9 50 4,16 0 4,640 4, 500

16-Jun 4, 290 4, 270 4,8 70 4,00 0 4,380 4, 230

17-Jun 4, 180 4, 140 4,8 50 4,01 0 4,260 4, 330

18-Jun 3, 830 3, 700 4,4 70 3,78 0 3,830 4, 160

19-Jun 3, 630 3, 580 4,1 00 3,73 0 3,770 4, 050

20-Jun 3, 580 4, 130 4,1 30 3,39 0 3,770 3, 900

21-Jun 3, 570 3, 000 4,1 00 3,27 0 3,890 3, 890

22-Jun 3, 040 2, 770 3,9 60 2,96 0 2,900 3, 680

23-Jun 2, 700 2, 550 3,5 60 2,79 0 4,350 3, 660

24-Jun 2, 640 2, 520 3,2 30 2,27 0 3,630 3, 800

25-Jun 2, 370 2, 480 3,3 10 2,40 0 3,810 3, 270

26-Jun 2, 500 1, 668 3,3 50 2,41 0 3,660 3, 510

27-Jun 2, 400 2, 410 3,2 10 2,62 0 3,790 3, 390

28-Jun 2, 440 2, 940 3,4 80 3,03 0 3,660 3, 530

29-Jun 2, 540 3, 360 3,2 70 3,29 0 3,360 3, 150

30-Jun 3, 190 3, 610 3,5 20 3,49 0 3,020 3, 440

1-Jul 3, 540 4, 340 4,0 10 3,49 0 3,200 3, 630

2-Jul 3, 780 4, 670 3,4 80 3,49 0 2,990 5, 780

3-Jul 4, 320 4, 630 3,2 40 3,68 0 2,920 3, 890

4-Jul 6, 370 5, 050 4,0 60 4,88 0 3,360 4, 160

5-Jul 5, 560 5, 450 4,7 70 5,10 0 4,640 4, 770

6-Jul 5, 030 5, 300 5,0 20 5,06 0 3,870 3, 880

7-Jul 3, 510 5, 240 6,3 20 4,84 0 3,750 4, 400

8-Jul 4, 710 4, 870 5,0 60 4,34 0 4,120 5, 050

9-Jul 5, 200 4, 930 4,7 60 5,35 0 4,680 5, 160

10-Jul 5, 240 4, 960 4,7 90 4,62 0 5,170 5, 340

11-Jul 6, 120 6, 250 5,3 80 7,02 0 5,660 5, 850

12-Jul 5, 650 4, 840 4,2 80 6,02 0 5,840 5, 470

13-Jul 5, 800 4, 370 4,8 00 6,63 0 6,110 5, 690

14-Jul 5, 040 5, 720 5,5 20 5,40 0 6,800 5, 750

15-Jul 6, 660 6, 790 6,3 00 4,41 0 6,420 6, 310

16-Jul 6, 380 6, 820 6,1 70 5,28 0 6,780 6, 010

17-Jul 5, 970 6, 400 6,0 80 6,03 0 6,750 6, 000

18-Jul 5, 510 5, 730 5,7 40 5,52 0 5,830 5, 530

19-Jul 5, 330 5, 590 5,2 40 5,31 0 5,530 5, 090

20-Jul 5, 310 5, 560 5,0 10 5,15 0 5,100 4, 950

21-Jul 5, 160 4, 380 4,9 60 5,39 0 5,000 4, 780

22-Jul 5, 320 5, 240 4,8 00 5,07 0 4,890 4, 620

23-Jul 4, 680 4, 610 4,3 50 3,64 0 4,510 4, 470

24-Jul 4, 610 4, 700 4,5 20 4,29 0 4,700 4, 280

25-Jul 3, 460 4, 260 4,7 60 4,25 0 4,060 4, 060

26-Jul 3, 460 4, 260 4,7 60 4,25 0 4,060 3, 960

27-Jul 4, 670 4, 170 4,5 10 4,92 0 4,650 4, 270

28-Jul 4, 600 4, 740 4,8 10 4,86 0 4,840 4, 400

29-Jul 4, 530 4, 440 4,4 00 4,54 0 4,700 4, 140

30-Jul 4, 210 4, 220 4,1 20 4,20 0 4,270 3, 840

31-Jul 4, 260 3, 880 3,8 90 4,10 0 4,120 3, 710

1-A ug 4, 320 3, 890 3,9 80 4,21 0 4,370 3, 880

2-A ug 4, 380 4, 200 4,1 80 4,10 0 4,230 3, 930

3-A ug 4, 230 4, 250 4,1 50 4,06 0 4,220 3, 990

4-A ug 4, 680 5, 020 4,8 20 4,79 0 4,910 4, 620

5-A ug 4, 430 4, 630 4,6 30 4,28 0 4,640 4, 290

**Fi gu**  **re 3. M**  **ixed L**  **iq uor**  **To tal**  **Su**

**spen ded** 

**So lid**  **s (M LT**  **SS )** 

## 12/14/2011 Page 47 of 66 **Po t**  **1 2**  **3 4**  **5 6** 

5-A ug 4, 430 4, 630 4,6 30 4,28 0 4,640 4, 290

6-A ug 4, 300 4, 740 4,4 60 4,35 0 4,360 4, 580

7-A ug 4, 120 4, 480 4,2 30 4,17 0 4,140 4, 350

8-A ug 4, 060 4, 340 3,2 30 4,01 0 3,970 4, 240

9-A ug 3, 620 3, 660 3,4 10 3,53 0 3,620 3, 730

10-A ug 4, 310 4, 140 3,7 10 4,09 0 3,900 4, 270

11-A ug 4, 100 3, 770 3,5 60 3,76 0 3,500 3, 950

12-A ug 3, 720 4, 040 3,5 20 3,89 0 3,470 3, 730

13-A ug 4, 230 4, 240 3,7 10 4,11 0 3,930 4, 040

14-A ug 4, 360 4, 350 3,9 60 4,12 0 4,090 4, 020

15-A ug 4, 790 4, 960 4,5 80 6,04 0 4,800 3, 360

16-A ug 4, 590 4, 160 4,7 80 4,65 0 4,650 4, 750

17-A ug 4, 530 4, 150 4,6 00 4,76 0 4,680 4, 040

18-A ug 4, 050 4, 350 4,3 40 4,21 0 4,010 4, 270

19-A ug 3, 860 4, 090 4,2 10 3,97 0 3,780 3, 800

20-A ug 3, 750 3, 890 4,1 00 3,86 0 3,690 3, 580

21-A ug 3, 700 3, 550 3,9 60 3,68 0 3,410 3, 080

22-A ug 3, 820 3, 300 3,9 20 3,56 0 3,420 3, 100

23-A ug 4, 090 3, 290 3,5 60 3,71 0 3,640 3, 510

24-A ug 4, 120 3, 070 3,2 80 3,31 0 3,710 3, 340

25-A ug 5, 230 4, 420 4,5 90 4,58 0 5,350 4, 550

26-A ug 4, 780 4, 340 4,5 00 4,58 0 4,910 4, 080

27-A ug 4, 870 4, 400 4,4 70 4,59 0 5,090 4, 140

28-A ug 4, 460 3, 090 5,3 00 4,12 0 4,650 3, 860

29-A ug 4, 240 3, 940 4,2 50 4,14 0 5,120 4, 170

30-A ug 4, 090 3, 510 4,1 30 4,01 0 4,240 3, 880

31-A ug 3, 770 3, 690 4,1 20 3,92 0 4,420 4, 040

1-S ep 3, 770 3, 420 3,8 90 4,06 0 4,590 3, 630

2-S ep 3, 900 3, 230 3,9 70 4,22 0 4,230 3, 470

3-S ep 4, 000 4, 710 3,9 70 4,28 0 4,200 3, 580

4-S ep 3, 760 3, 250 3,7 70 3,91 0 4,220 3, 220

5-S ep 4, 830 4, 670 4,9 60 5,24 0 4,980 4, 380

6-S ep 4, 940 4, 700 5,1 30 4,98 0 4,840 4, 560

7-S ep 4, 630 4, 440 4,9 50 4,88 0 5,120 4, 370

8-S ep 4, 400 4, 500 4,8 30 5,03 0 5,330 4, 530

9-S ep 4, 360 4, 780 4,8 40 5,09 0 5,160 3, 400

10-S ep 4, 300 4, 740 4,4 50 4,86 0 4,760 4, 680

11-S ep 4, 000 4, 560 4,1 30 4,67 0 4,590 4, 200

12-S ep 5, 770 6, 210 5,9 90 6,65 0 6,050 5, 860

13-S ep 4, 610 5, 300 5,2 30 5,26 0 4,810 4, 900

14-S ep 4, 680 5, 210 5,3 20 5,39 0 5,160 5, 550

15-S ep 4, 840 5, 270 5,2 60 5,33 0 5,110 5, 070

16-S ep 4, 460 5, 100 5,0 60 5,05 0 4,670 4, 650

17-S ep 4, 120 4, 650 4,6 90 4,40 0 4,170 4, 670

18-S ep 4, 440 4, 730 4,7 80 4,85 0 4,440 4, 900

19-S ep 5, 510 5, 780 6,0 50 5,91 0 5,640 6, 060

20-S ep 5, 420 5, 320 5,7 20 5,77 0 4,900 5, 830

21-S ep 4, 910 5, 160 5,5 20 5,28 0 5,180 6, 470

22-S ep 5, 880 5, 540 5,9 20 5,28 0 5,190 5, 850

23-S ep 4, 610 4, 620 4,7 40 4,35 0 4,430 5, 150

24-S ep 4, 600 4, 460 4,1 30 4,12 0 4,060 4, 530

25-S ep 4, 560 4, 300 4,3 20 3,41 0 4,640 4, 470

26-S ep 5, 760 5, 850 5,4 30 4,95 0 5,560 5, 850

27-S ep 5, 250 5, 550 5,2 00 4,26 0 5,150 5, 180

28-S ep 4, 600 4, 850 4,4 60 3,93 0 4,780 4, 580

29-S ep 4, 000 4, 130 3,8 30 3,11 0 3,880 3, 740

30-S ep 3, 800 3, 880 3,8 60 3,03 0 3,850 3, 740

1-Oct 3, 550 3, 960 3,7 90 3,49 0 4,070 3, 910

2-Oct 3, 600 3, 930 3,8 10 3,12 0 3,800 3, 640

3-Oct

4-Oct 4, 290 4, 700 4,5 40 3,76 0 4,660 4, 070

5-Oct 3, 730 4, 480 4,5 90 3,78 0 4,530 3, 990

6-Oct 3, 550 4, 640 4,3 50 2,81 0 5,330 3, 630

7-Oct 3, 650 4, 190 4,2 10 3,81 0 4,070 3, 520

**Fi gu**  **re 3. M**  **ixed L**  **iq uor**  **To tal**  **Su**

**spen ded** 

**So lid**  **s (M LT**  **SS )** 

## 12/14/2011 Page 48 of 66

**Ap pendix**  **4 : Figure 4** 

**- Porous Pot Effluent TSS** 

**PO T**  **1 2**  **3 4**  **5 6**  **POT 1**  **2 3**  **4 5**  **6**

2-Jun 24 22 32 18 18 38 26- Jul 1 0 -2 1 -1 -2

3-Jun 13 20 47 18 11 41 27- Jul 5 3 2 5 2 2

4-Jun 11 7 44 7 0 28 28- Jul 1 -3 -1 0 -2 -3

5-Jun 7 13 33 13 6 21 29- Jul 6 4 6 7 3 3

6-Jun 14 17 11 14 13 7 30- Jul 1 1 1 3 2 2

7-Jun 10 10 14 14 12 21 31- Jul 5 3 3 5 5 1

8-Jun 5 2 9 11 4 12 1-A ug -2 2 -4 0 2 0

9-Jun 9 2 10 18 9 12 2-A ug 0 4 0 3 2 1

10-Jun 6 7 7 19 11 9 3-A ug 2 6 2 2 5 2

11-Jun 9 5 5 11 0 9 4-A ug 0 4 3 3 3 -1

12-Jun 9 9 11 12 1 14 5-A ug -2 -1 0 -3 -1 -2 -6

13-Jun 8 13 9 17 3 15 6-A ug 4 6 5 4 5 0

14-Jun 13 22 17 33 9 14 7-A ug 1 3 1 3 3 -2

15-Jun 2 25 9 16 9 11 8-A ug -9 5 7 6 0 1

16-Jun 11 28 7 12 8 5 9-A ug 0 4 1 5 2 -1

17-Jun 19 31 6 14 16 6 10- Au g 2 3 2 7 2 0

18-Jun 7 31 5 30 18 7 11- Au g -1 0 -3 0 0 -5

19-Jun 21 30 7 17 25 9 12- Au g 3 1 2 2 2 1

20-Jun 29 36 14 32 27 18 13- Au g -2 -3 0 -3 -3 -2

21-Jun 39 42 26 46 38 25 14- Au g -1 -2 -5 1 -3 -3

22-Jun 34 14 11 19 19 9 15- Au g 2 1 3 4 2 5

23-Jun 37 20 13 19 12 9 16- Au g 4 5 9 4 4 2

24-Jun 48 23 22 34 19 15 17- Au g 5 -1 7 6 3 2

25-Jun 40 19 23 30 19 16 18- Au g 0 -2 5 2 1 0

26-Jun 33 25 24 36 38 18 19- Au g 1 -2 3 3 0 1

27-Jun 29 13 13 21 29 11 20- Au g 7 4 9 7 8 7

28-Jun 22 24 13 15 18 18 21- Au g -1 -3 0 0 -4 1

29-Jun 26 14 17 23 21 29 22- Au g 8 9 10 6 8 7

30-Jun 16 10 5 15 22 19 23- Au g

1-Jul 21 20 16 23 41 23 24- Au g 1 0 3 -1 -1 -1

2-Jul 16 10 13 12 34 16 25- Au g -2 -3 2 -2 -4 -2

3-Jul 16 13 13 11 31 24 26- Au g -1 -2 3 0 -4 -3

4-Jul 9 11 10 8 22 44 27- Au g -2 0 5 -3 -2 1

5-Jul 4 4 6 7 10 7 28- Au g -4 0 6 1 -3 0

6-Jul 1 1 0 0 10 0 29- Au g 7 4 15 0 3 4

7-Jul 0 -1 -2 3 3 5 30- Au g 1 4 12 8 2 2

8-Jul 4 4 1 3 6 1 31- Au g 2 1 4 2 -1 2

9-Jul 2 0 2 1 0 -4 1-S ep -1 0 9 1 1 3

10-Jul 0 1 3 2 3 2 2-S ep -3 -1 5 -2 -3 0

11-Jul 8 11 19 10 10 16 3-S ep 1 0 5 0 -2 -2

12-Jul -4 1 28 -2 -4 0 4-S ep -6 -5 -1 -1 -3 -1 3

13-Jul 10 11 18 12 8 12 5-S ep 3 0 5 4 0 0

14-Jul -2 -4 -2 -2 -4 -2 6-S ep -4 -8 -6 -4 -4 -2

15-Jul 5 2 2 9 1 1 7-S ep

16-Jul 2 2 5 4 3 4 8-S ep

17-Jul 2 1 4 2 3 4 9-S ep

18-Jul 1 1 4 -1 0 0 10- Se p 9 5 10 9 0 2

19-Jul 1 0 3 -1 -1 -2 11- Se p 2 -1 6 2 -2 -1

20-Jul 1 0 2 -2 0 -3 12- Se p -1 -4 6 -9 1 5

21-Jul 3 2 3 0 1 -1 13- Se p

22-Jul 1 -3 -3 -2 -2 -3 14- Se p -2 -3 2 -2 -4 -8

23-Jul 4 3 1 1 1 -3 15- Se p 6 2 7 4 0 1

24-Jul 2 -1 -6 1 -1 2 16- Se p

25-Jul 1 0 -2 1 -1 -2 17- Se p 2 -1 2 -1 -3 -6

26-Jul 1 0 -2 1 -1 -2 18- Se p -1 1 1 4 0 -2 **Fi gu**  **re 4 -**  **P or**  **ou s P**  **ot**

**Eff lu**  **ent TS**  **S**

## 12/14/2011 Page 49 of 66

**Ap pendix**  **5 : Figure 5** 

**- Porous Pot Effluent pH Values** 

**PO T**  **1 2**  **3 4**  **5 6** 

2-Jun 7.40 7. 30 7. 00 7.5 0 7.30 7.10

3-Jun 7.40 7. 30 7. 00 7.5 0 7.30 7.10

4-Jun 7.75 7. 43 7. 44 7.7 3 7.50 7.58

5-Jun 7.48 7. 49 7. 37 7.6 5 7.55 7.71

6-Jun 7.71 7. 45 7. 33 7.6 1 7.50 7.68

7-Jun 7.74 7. 66 7. 58 7.6 0 7.46 7.75

8-Jun 7.76 7. 71 7. 60 7.5 6 7.67 7.70

9-Jun 7.68 7. 71 7. 34 7.5 0 7.58 7.59

10-J un 7.59 7. 69 7. 38 7.4 4 7.34 7.57

11-J un 7.53 7. 67 7. 54 7.4 8 7.39 7.62

12-J un 7.60 7. 69 7. 35 7.4 7 7.39 7.64

13-J un 7.59 7. 64 7. 45 7.4 0 7.38 7.60

14-J un 7.56 7. 54 7. 43 7.4 2 7.29 7.59

15-J un 7.65 7. 65 7. 26 7.5 6 7.32 7.66

16-J un 7.70 7. 70 7. 30 7.5 3 7.52 7.68

17-J un 7.68 7. 64 7. 31 7.5 2 7.53 7.63

18-J un 7.58 7. 59 7. 29 7.5 1 7.52 7.62

19-J un 7.52 7. 55 7. 27 7.5 0 7.40 7.60

20-J un 7.53 7. 62 7. 29 7.4 8 7.25 7.61

21-J un 7.48 7. 60 7. 27 7.4 7 7.21 7.60

22-J un 7.49 7. 55 7. 29 7.4 7 7.48 7.61

23-J un 7.56 7. 55 7. 41 7.5 5 7.37 7.64

24-J un 7.64 7. 53 7. 48 7.6 0 7.42 7.68

25-J un 7.56 7. 41 7. 44 7.5 5 7.38 7.58

26-J un 7.57 7. 32 7. 43 7.5 2 7.47 7.46

27-J un 7.59 7. 29 7. 45 7.3 9 7.45 7.44

28-J un 7.55 7. 30 7. 48 7.5 3 7.37 7.48

29-J un 7.43 7. 22 7. 31 7.4 6 7.16 7.25

30-J un 7.45 7. 22 7. 33 7.6 0 7.27 7.53

1-Jul 7.28 7. 48 7. 45 7.3 1 7.21 7.67

2-Jul 7.43 7. 66 7. 62 7.4 8 7.35 7.66

3-Jul 7.39 7. 56 7. 54 7.4 4 7.20 7.67

4-Jul 7.40 7. 71 7. 57 7.5 0 7.27 7.78

5-Jul 7.39 7. 45 7. 07 7.5 0 7.30 7.02

6-Jul 7.35 7. 08 6. 68 7.3 5 7.37 6.64

7-Jul 7.25 7. 05 6. 60 7.2 9 7.29 6.70

8-Jul 7.42 7. 23 6. 56 7.5 5 7.32 6.85

9-Jul 7.21 7. 25 6. 60 7.4 9 7.26 6.85

10-J ul 7.19 7. 12 6. 62 7.4 1 7.21 6.71

11-J ul 7.25 7. 42 6. 47 7.5 0 7.24 6.60

12-J ul 7.27 7. 35 6. 18 7.3 1 7.22 6.46

13-J ul 7.27 6. 85 6. 12 7.3 1 7.20 6.42

14-J ul 7.32 7. 23 6. 01 7.2 6 7.23 6.13

15-J ul 7.43 7. 55 6. 66 7.5 6 7.35 6.69

16-J ul 7.54 7. 70 7. 05 7.2 1 7.38 7.12

17-J ul 7.60 7. 91 7. 11 7.3 7 7.36 7.12

18-J ul 7.48 7. 80 6. 57 7.2 4 7.15 6.58

19-J ul 7.39 7. 77 6. 30 7.1 0 7.09 6.43

20-J ul 7.52 7. 88 7. 08 7.3 4 7.24 7.09

21-J ul 7.50 7. 88 7. 26 7.5 4 7.31 7.38

22-J ul

23-J ul 7.52 7. 85 7. 29 7.5 7 7.49 7.46

24-J ul

25-J ul 7.62 7. 70 7. 51 7.3 9 7.28 7.59

26-J ul 7.54 7. 59 7. 55 7.4 2 7.32 7.56

27-J ul 7.54 7. 60 7. 63 7.4 6 7.41 7.60

28-J ul 7.54 7. 62 7. 63 7.4 6 7.47 7.55

29-J ul 7.58 7. 57 7. 64 7.4 3 7.40 7.54

**Fi gu**  **re 5. P**  **or ou**  **s P ot E**  **ff luen**  **t pH V**

**alu es** 

## 12/14/2011 Page 50 of 66 **PO T**  **1 2**  **3 4**  **5 6** 

30-Jul 7.61 7. 59 7. 66 7.4 6 7.45 7.53

31-Jul 7.59 7. 55 7. 58 7.4 6 7.41 7.44

1-A ug 7.55 7. 52 7. 63 7.5 1 7.42 7.49

2-A ug 7.50 7. 43 7. 59 7.4 7 7.38 7.47

3-A ug 7.61 7. 51 7. 63 7.5 5 7.41 7.51

4-A ug 7.56 7. 43 7. 56 7.5 0 7.36 7.46

5-A ug 7.50 7. 37 7. 50 7.5 3 7.39 7.39

6-A ug 7.43 7. 39 7. 40 7.5 0 7.43 7.31

7-A ug 7.40 7. 48 7. 43 7.5 7 7.45 7.33

8-A ug 7.32 7. 52 7. 39 7.5 0 7.42 7.32

9-A ug 7.27 7. 42 7. 38 7.3 9 7.37 7.33

10-Aug 7.27 7. 37 7. 45 7.4 6 7.32 7.35

11-Aug 7.32 7. 40 7. 48 7.5 4 7.36 7.43

12-Aug 7.33 7. 43 7. 42 7.5 4 7.41 7.40

13-Aug 7.37 7. 44 7. 45 7.4 7 7.45 7.34

14-Aug 7.37 7. 38 7. 44 7.5 4 7.46 7.35

15-Aug 7.41 7. 42 7. 36 7.5 6 7.43 7.39

16-Aug 7.50 7. 37 7. 35 7.5 5 7.37 7.37

17-Aug 7.53 7. 47 7. 32 7.4 8 7.40 7.35

18-Aug 7.55 7. 51 7. 35 7.4 4 7.43 7.40

19-Aug 7.41 7. 32 7. 29 7.2 9 7.38 7.28

20-Aug 7.37 7. 33 7. 36 7.3 0 7.43 7.23

21-Aug 7.39 7. 34 7. 44 7.4 2 7.57 7.36

22-Aug 7.51 7. 31 7. 45 7.3 9 7.62 7.40

23-Aug

24-Aug 7.61 7. 54 7. 54 7.6 1 7.67 7.57

25-Aug 7.57 7. 55 7. 50 7.6 1 7.63 7.50

26-Aug 7.70 7. 61 7. 42 7.7 0 7.51 7.53

28-Aug 7.52 7. 43 7. 35 7.6 9 7.51 7.52

29-Aug 7.46 7. 43 7. 23 7.6 4 7.40 7.41

30-Aug 7.51 7. 49 7. 21 7.7 0 7.34 7.32

31-Aug 7.45 7. 49 7. 35 7.6 5 7.34 7.35

1-S ep 7.62 7. 55 7. 48 7.6 5 7.44 7.37

2-S ep 7.69 7. 49 7. 52 7.6 9 7.46 7.33

3-S ep 7.74 7. 52 7. 69 7.7 4 7.53 7.52

4-S ep 7.75 7. 58 7. 76 7.7 9 7.59 7.60

5-S ep 7.72 7. 60 7. 78 7.7 9 7.54 7.64

6-S ep 7.72 7. 60 7. 78 7.7 9 7.54 7.64

7-S ep 7.69 7. 45 7. 44 7.7 1 7.41 7.74

8-S ep 7.60 7. 46 7. 32 7.7 4 7.37 7.60

9-S ep 7.62 7. 57 7. 18 7.7 3 7.32 7.51

10-Sep 7.67 7. 49 7. 10 7.7 5 7.30 7.49

11-Sep 7.70 7. 61 7. 21 7.7 4 7.34 7.56

12-Sep 7.72 7. 60 7. 35 7.7 8 7.35 7.69

13-Sep 7.63 7. 52 7. 39 7.7 4 7.33 7.78

14-Sep 7.57 7. 51 7. 34 7.7 0 7.36 7.83

15-Sep 7.55 7. 53 7. 24 7.6 5 7.34 7.82

16-Sep 7.68 7. 50 7. 26 7.7 1 7.32 7.77

17-Sep 7.70 7. 51 7. 29 7.7 1 7.33 7.76

18-Sep 7.84 7. 39 7. 25 7.7 1 7.35 7.77

19-Sep 7.62 7. 35 7. 27 7.6 8 7.29 7.69

20-Sep 7.58 7. 44 7. 34 7.6 7 7.26 7.62

21-Sep 7.88 7. 46 7. 34 7.6 5 7.35 7.69

22-Sep 7.67 7. 56 7. 41 7.4 5 7.34 7.65

23-Sep 7.76 7. 58 7. 37 7.4 0 7.33 7.59

24-Sep

25-Sep 7.66 7. 44 7. 29 7.2 6 7.26 7.35

26-Sep

27-Sep 7.51 7. 42 7. 34 7.3 8 7.44 7.23

28-Sep 7.61 7. 48 7. 36 7.5 1 7.55 7.26

29-Sep 7.65 7. 39 7. 35 7.5 4 7.55 7.20

30-Sep 7.64 7. 24 7. 33 7.5 6 7.54 7.30

1-Oct 7.64 7. 26 7. 33 7.6 4 7.51 7.37

2-Oct 7.63 7. 39 7. 32 7.6 7 7.49 7.30

3-Oct

4-Oct

5-Oct 7.60 7. 41 7. 36 7.7 5 7.49 7.31

6-Oct

7-Oct 7.60 7. 42 7. 36 7.7 8 7.47 7.39

**Fi gu**  **re 5. P**  **or ou**  **s P ot E**  **ff luen**  **t pH V**

**alu es** 

## 12/14/2011 Page 51 of 66

**Ap pendix**  **6 :**  **Figure 6**

**- Porous Pot DOC Feed and Effluents** 

**Fe ed**  **A Fe**  **ed B**  **Fe**

**ed C**  **1 2**  **3 4**  **5 6** 

12-M ay 310 .398 316. 130 316.7 72 17.19 5 11 .863 12 .431 12. 887 14.8 17 10.41 3

13-M ay 323 .046 321. 604 321.7 28 13.22 7 15 .342 13 .220 12. 990 16.0 77 12.14 2

14-M ay 313 .892 314. 746 321.6 10 14.68 4 17 .409 14 .468 13. 690 17.9 59 13.84 9

15-M ay 305 .876 327. 414 332.1 26 14.49 5 14 .161 18 .376 14. 024 15.2 09 17.92 5

16-M ay 318 .106 329. 830 331.6 40 13.71 1 12 .700 17 .998 13. 076 13.5 27 17.85 3

17-M ay 311 .960 327. 690 331.4 68 12.88 1 13 .083 18 .845 12. 278 12.4 77 19.94 4

18-M ay 318 .772 329. 720 330.0 44 12.90 6 12 .740 18 .765 13. 556 13.7 30 20.41 0

19-M ay 313 .296 323. 344 329.5 74 14.35 0 15 .506 20 .804 14. 350 14.1 81 19.87 6

20-M ay 272 .580 286. 502 284.8 56 14.86 1 14 .060 18 .592 14. 383 14.1 80 19.39 1

21-M ay 312 .214 320. 656 327.4 32 16.58 0 14 .957 21 .984 15. 965 17.3 24 21.71 2

22-M ay 321 .518 316. 058 310.7 22 18.51 9 16 .866 22 .555 16. 454 18.7 61 24.55 9

23-M ay 323 .230 323. 756 317.7 90 20.22 9 19 .077 23 .349 16. 571 17.0 46 24.23 1

24-M ay 308 .490 319. 932 315.6 58 21.05 2 20 .525 41 .704 17. 337 19.3 30 32.58 0

25-M ay 297 .152 309. 944 311.8 82 23.39 6 21 .497 75 .121 17. 636 19.9 86 34.10 3

26-M ay 290 .896 300. 350 305.6 54 24.67 7 25 .577 74 .135 18. 171 26.5 90 47.65 8

27-M ay 303 .948 320. 906 321.9 42 25.56 3 37 .302 48 .765 20. 633 31.9 11 39.95 1

28-M ay 310 .456 325. 792 324.3 02 27.79 5 38 .224 33 .350 25. 853 32.8 50 44.82 1

29-M ay 308 .198 329. 032 318.4 28 32.05 5 40 .734 53 .497 23. 635 39.6 29 51.59 1

30-M ay 296 .924 276. 622 268.1 86 38.55 6 36 .171 78 .989 21. 435 37.2 13 66.24 3

31-M ay 216 .942 296. 162 297.1 32 40.41 1 46 .503 109 .984 20. 625 52.8 42 86.84 5

1-Jun 296 .924 276. 622 268.1 86 11 0.84 7 39 .752 143 .003 21. 819 54.1 70 95.65 3

2-Jun 309 .826 312. 510 310.4 48 36.63 1 35 .625 122 .379 24. 765 31.5 62 60.39 4

3-Jun 314 .672 315. 498 303.9 30 35.40 8 34 .163 64 .634 23. 197 29.7 90 38.38 1

4-Jun 313 .684 306. 896 296.6 04 25.77 8 30 .849 45 .249 20. 403 25.6 52 34.20 4

5-Jun 314 .274 300. 292 299.7 38 24.41 4 26 .534 39 .658 18. 906 21.7 20 43.95 2

6-Jun 261 .456 257. 154 235.1 54 21.89 8 23 .696 43 .764 18. 753 23.7 38 40.28 7

7-Jun 204 .636 301. 458 278.5 46 21.51 2 21 .598 39 .313 18. 922 22.0 08 62.67 4

8-Jun 322 .322 335. 992 319.1 56 20.12 9 20 .363 36 .067 19. 547 19.2 82 37.60 1

9-Jun 336 .056 337. 702 330.4 60 18.25 7 16 .911 33 .803 18. 752 17.5 86 29.14 4

10-Jun 330 .406 334. 096 331.7 46 16.35 2 13 .618 26 .687 18. 165 16.2 79 30.17 3

11-Jun 310 .176 335. 696 313.5 58 19.10 5 14 .642 26 .226 17. 517 15.0 01 32.62 1

12-Jun 311 .394 322. 772 318.0 46 17.83 6 15 .879 27 .491 17. 122 13.9 82 31.40 9

13-Jun 289 .226 302. 326 289.0 92 16.74 5 17 .467 26 .470 16. 379 14.1 33 31.84 2

14-Jun 277 .004 304. 380 288.4 82 16.92 1 16 .429 24 .133 17. 134 13.4 17 30.07 2

15-Jun 323 .524 316. 720 311.2 28 16.83 0 16 .997 22 .378 16. 218 12.1 60 27.40 4

16-Jun 331 .302 328. 136 323.8 84 17.69 4 18 .709 24 .452 19. 130 14.6 97 27.25 2

17-Jun 333 .544 332. 582 320.9 76 19.06 6 19 .989 23 .082 18. 820 16.2 30 25.11 7

18-Jun 332 .956 335. 962 333.6 60 23.87 4 24 .348 24 .102 19. 231 23.8 53 24.86 1

19-Jun 329 .614 334. 470 331.1 58 26.43 3 29 .704 25 .941 19. 841 32.0 51 27.06 3

20-Jun 278 .082 291. 078 278.8 00 25.62 4 29 .934 24 .671 20. 212 27.4 98 23.93 4

21-Jun 286 .218 284. 252 280.4 66 29.82 0 31 .296 27 .129 28. 732 27.8 01 24.85 6

22-Jun 273 .466 276. 470 275.1 90 33.36 4 33 .462 30 .625 37. 706 35.4 63 27.31 5

23-Jun 276 .566 294. 430 279.0 50 34.68 3 32 .957 34 .865 40. 621 47.1 34 28.88 1

24-Jun 306 .758 311. 944 304.6 82 34.00 6 31 .203 36 .116 39. 870 48.7 25 28.02 4

25-Jun 293 .562 306. 332 297.5 78 35.44 0 30 .835 33 .907 41. 929 45.6 03 28.06 7

26-Jun 305 .384 306. 356 298.1 04 35.58 3 38 .343 32 .526 46. 820 38.5 63 27.10 5 **Fig ur**  **e 6. P or**  **ou s P**  **ot DO**  **C F**

**eed and**  **E fflu**  **ents (m**  **g/ l DOC)** 

## 12/14/2011 Page 52 of 66 **Fe ed**  **A Fe**  **ed B**  **Fe**

**ed C**  **1 2**  **3 4**  **5 6** 

27-Jun 277 .322 300. 150 282.7 06 36.63 8 45 .194 30 .356 41. 004 36.6 76 31.39 1

28-Jun 272 .306 293. 016 273.2 98 40.54 1 45 .753 29 .512 36. 005 38.0 39 36.94 5

29-Jun 272 .306 293. 016 273.2 98 54.94 7 40 .619 82 .448 34. 307 58.3 78 82.66 3

30-Jun 296 .130 297. 244 293.1 14 58.80 6 40 .602 89 .648 40. 328 53.5 57 59.33 4

1-Jul 307 .998 307. 116 310.2 64 39.93 3 37 .372 46 .079 46. 377 37.9 11 38.85 9

2-Jul 308 .760 316. 152 311.2 86 31.77 9 31 .256 42 .059 42. 875 53.2 76 33.02 1

3-Jul 283 .986 299. 444 277.8 36 28.97 0 30 .295 41 .395 41. 661 65.0 09 32.40 3

4-Jul 273 .722 284. 732 306.2 66 23.85 6 24 .307 29 .441 32. 022 38.5 71 30.05 1

5-Jul 272 .864 275. 804 271.3 66 14.30 9 11 .763 17 .811 14. 068 19.1 59 16.28 4

6-Jul 235 .468 239. 252 231.8 32 11.74 7 8.449 15 .297 11. 373 17.4 90 13.66 5

7-Jul 312 .272 311. 134 306.5 92 11.83 0 8.623 15 .178 13. 382 16.4 89 16.56 0

8-Jul 276 .180 312. 632 311.2 90 11.57 0 8.991 15 .406 13. 794 16.0 02 15.76 2

9-Jul 298 .728 311. 296 298.5 94 9.49 6 8.664 15 .919 11. 637 13.4 99 15.75 9

10-Jul 317 .728 325. 822 328.1 98 10.92 5 10 .282 16 .795 10. 027 10.0 59 17.06 9

11-Jul 328 .520 344. 872 340.1 20 12.12 3 10 .257 23 .031 10. 784 11.2 08 21.27 7

12-Jul 320 .754 329. 708 327.2 00 10.17 7 9.635 24 .781 9. 901 11.2 96 21.06 2

13-Jul 308 .686 326. 120 319.0 74 9.60 1 7.604 21 .760 12. 228 9.7 00 20.97 1

14-Jul 339 .732 340. 814 327.6 76 10.75 0 8.695 19 .603 14. 165 11.2 53 21.72 0

15-Jul 341 .820 338. 308 337.6 90 11.78 2 9.664 20 .341 13. 150 12.5 19 20.11 6

16-Jul

17-Jul 232 .758 233. 788 240.1 48 12.68 4 10 .059 20 .030 12. 014 11.7 31 19.93 6

18-Jul 232 .886 231. 650 211.7 42 11.51 2 9.846 18 .593 9. 657 10.1 22 18.20 2

19-Jul 214 .990 208. 772 239.9 62 10.14 2 9.585 17 .149 8. 769 8.7 77 16.91 0

20-Jul 211 .682 217. 640 225.5 88 10.13 5 10 .248 17 .453 9. 646 9.9 34 18.02 6

21-Jul 207 .632 228. 256 192.5 36 9.94 8 9.276 18 .599 10. 538 9.8 71 19.86 5

22-Jul 170 .546 162. 330 187.7 82 10.27 8 8.374 18 .656 10. 342 10.0 16 19.23 0

23-Jul 291 .736 297. 840 295.3 08 11.07 1 10 .128 18 .546 12. 114 12.0 21 17.72 8

24-Jul 281 .972 282. 374 263.6 38 12.20 9 11 .293 19 .924 13. 765 12.5 85 19.95 6

25-Jul 271 .570 248. 690 278.1 54 12.29 2 10 .240 19 .288 12. 273 10.6 09 19.46 6

26-Jul 284 .028 284. 438 284.2 48 11.95 4 8.959 17 .847 9. 425 9.4 59 17.84 9

27-Jul 299 .512 290. 840 272.1 86 10.93 9 7.975 18 .242 8. 797 7.5 08 18.24 0

28-Jul 300 .796 293. 262 295.7 78 9.88 2 8.158 17 .745 9. 287 7.8 11 18.58 2

29-Jul 280 .696 283. 508 283.3 82 9.54 8 8.711 18 .532 9. 386 7.5 97 19.61 9

30-Jul 284 .598 279. 092 271.9 36 9.37 7 8.547 18 .041 9. 578 7.7 21 18.56 7

31-Jul 301 .422 304. 646 308.0 92 10.39 3 9.521 19 .124 9. 134 7.8 09 18.23 8

1-A ug 231 .226 263. 306 256.8 08 9.07 8 16 .723 18 .275 9. 476 13.2 78 17.86 9

2-A ug 273 .140 249. 208 242.7 22 10.44 3 21 .702 19 .466 9. 916 19.5 07 17.99 4

3-A ug 251 .392 247. 012 248.2 48 10.18 6 19 .422 19 .289 9. 594 19.6 41 19.03 2

4-A ug 275 .774 281. 930 285.4 24 9.46 2 19 .051 19 .051 9. 397 19.2 72 18.11 4

5-A ug 276 .810 274. 422 265.7 08 8.54 4 18 .627 19 .138 8. 548 18.7 73 17.71 0

6-A ug 270 .762 271. 014 262.2 04 8.34 9 17 .032 17 .478 7. 592 17.7 35 16.69 9

7-A ug 274 .794 276. 876 274.8 50 8.61 2 16 .222 17 .900 8. 730 18.4 33 17.05 3

8-A ug 265 .740 277. 300 223.0 08 8.50 6 17 .371 21 .936 9. 676 18.4 21 17.22 2

9-A ug 270 .040 269. 878 264.6 78 8.63 3 18 .979 23 .501 10. 896 18.2 10 17.24 1

10-A ug 232 .574 247. 624 229.5 42 9.12 2 19 .029 22 .332 11. 119 21.1 36 18.45 2

11-A ug 276 .030 289. 588 263.9 90 9.64 7 18 .593 15 .954 11. 359 23.8 90 12.55 5

12-A ug 284 .890 258. 046 273.7 88 9.76 3 18 .736 11 .577 11. 251 22.7 79 9.15 0

13-A ug 269 .506 279. 070 262.1 04 10.86 9 18 .046 10 .947 12. 736 22.3 67 9.96 4

14-A ug 277 .934 276. 676 246.6 24 12.36 8 17 .517 10 .210 11. 895 21.0 79 10.96 1

15-A ug 247 .940 239. 436 243.9 84 11.79 7 16 .757 8. 796 11. 953 20.1 91 10.41 9

16-A ug 227 .632 242. 160 235.0 68 10.47 8 21 .293 8. 290 10. 862 19.1 81 9.69 5

17-A ug 281 .066 271. 800 270.5 00 11.50 9 17 .302 8. 570 14. 866 17.3 02 11.56 8

18-A ug 285 .140 270. 510 269.5 60 11.68 8 18 .969 9. 307 15. 705 22.3 59 12.01 8 **Fig ur**  **e 6. P or**  **ou s P**  **ot DO**  **C F**

**eed and**  **E fflu**  **ents (m**  **g/ l DOC)** 

## 12/14/2011 Page 53 of 66 **Feed A Feed B**  **Feed C 1**  **2 3**  **4 5**  **6**

19-Aug 275.450 271.450 269.650 11.848 19.597 10.053 15.251 22.014 13.800

20-Aug 289.350 278.560 281.450 17.829 15.622 11.347 21.568 17.571 14.611

21-Aug 279.750 278.800 277.350 14.716 17.644 11.615 19.332 18.968 16.138

22-Aug 280.540 278.340 281.650 12.209 23.570 12.708 17.113 21.705 17.584

23-Aug 298.340 287.560 285.350 11.595 25.438 13.405 16.266 21.192 16.477

24-Aug 286.440 279.340 281.680 12.185 26.305 13.977 15.317 19.852 14.895

25-Aug 295.740 289.440 290.680 11.531 26.019 14.925 14.278 19.862 15.076

26-Aug 298.420 295.560 300.120 10.975 24.315 13.342 12.606 18.374 14.906

27-Aug 330.952 316.158 318.600 10.818 23.274 13.905 12.286 18.981 15.615

28-Aug 307.162 290.204 300.828 10.349 23.970 14.862 11.129 18.040 12.299

29-Aug 304.460 301.334 292.984 10.875 24.091 16.367 11.683 18.669 13.277

30-Aug 325.070 320.074 320.618 11.779 25.882 16.180 12.318 22.884 12.634

31-Aug 291.448 298.708 293.032 20.714 27.283 15.182 20.531 24.304 12.591

1-Sep 294.140 285.684 288.552 15.978 26.113 13.724 15.113 22.066 13.315

2-Sep 312.742 309.938 313.838 11.366 25.023 13.010 10.581 21.006 13.188

3-Sep 290.976 289.786 290.064 10.427 25.200 12.917 10.439 21.760 14.452

4-Sep 288.360 296.040 290.426 10.066 24.601 12.045 9.636 21.613 13.879

5-Sep 309.616 312.466 321.020 9.901 23.713 11.765 9.223 21.243 12.706

6-Sep 287.982 284.368 285.684 10.149 23.138 10.926 9.594 21.749 12.154

7-Sep 284.012 281.480 275.438 10.503 22.527 11.856 10.411 22.286 12.281

8-Sep 316.224 310.068 314.572 12.498 23.158 13.973 11.436 23.409 13.398

9-Sep 298.202 283.170 284.760 12.835 23.764 16.722 11.529 24.306 14.156

10-Sep 289.590 291.248 281.028 12.443 22.332 20.085 11.069 23.412 13.958

11-Sep 313.108 314.666 321.496 12.641 22.748 17.689 10.469 22.751 13.364

12-Sep 298.286 292.386 285.142 11.709 20.968 13.183 10.070 22.204 12.454

13-Sep 295.626 286.828 284.828 11.608 21.284 11.801 9.767 22.282 11.568

14-Sep 319.138 315.114 319.714 11.620 24.308 11.753 9.710 22.193 10.784

15-Sep 319.138 315.114 319.714 16.719 24.853 11.740 10.765 22.927 11.152

16-Sep 291.394 281.264 291.620 17.648 22.944 11.996 10.704 22.809 10.475

17-Sep 309.616 315.482 315.364 17.593 23.992 13.251 10.528 23.299 10.270

18-Sep 300.366 295.708 302.800 16.903 25.509 12.364 10.486 23.497 10.234

19-Sep 299.902 294.464 288.270 14.706 23.861 11.515 10.180 22.900 10.007

20-Sep 305.896 315.156 317.666 13.268 23.232 10.770 10.482 22.389 9.932

21-Sep 295.528 286.798 288.486 13.377 23.479 11.675 11.486 23.480 11.445

22-Sep 294.174 292.998 287.644 16.809 23.499 12.035 15.046 24.853 11.901

23-Sep 324.054 296.356 322.126 12.245 23.338 15.133 13.439 23.302 13.533

24-Sep 301.344 300.094 296.898 12.698 23.617 13.262 15.840 23.046 11.314

25-Sep 300.364 292.060 297.084 11.130 22.490 11.974 15.955 22.102 11.065

26-Sep 317.504 321.496 321.742 10.436 22.485 11.243 16.001 21.810 10.419

27-Sep 290.010 302.172 291.054 10.373 22.744 10.329 15.872 20.982 11.089

28-Sep 306.476 296.318 288.270 10.404 23.165 10.408 14.406 21.075 11.006

29-Sep 281.454 265.706 259.256 10.932 23.759 10.758 13.608 20.633 10.953

30-Sep 309.146 315.066 326.780 10.891 22.285 10.801 13.099 20.355 11.242

1-Oct 281.634 299.206 305.734 10.495 22.540 10.844 14.001 19.488 10.390

2-Oct 309.044 304.350 305.666 11.365 26.022 11.399 12.684 20.468 10.976

3-Oct 299.736 282.914 293.654 12.626 20.104 10.331 11.082 19.193 11.403

4-Oct 281.762 263.418 266.922 12.244 20.534 10.092 11.092 19.931 12.310

5-Oct 300.662 297.672 298.714 12.810 19.083 8.635 10.097 19.076 12.698

6-Oct 294.066 283.294 279.308 13.882 19.841 9.215 10.321 18.704 13.530

7-Oct 280.762 283.152 278.744 12.498 20.916 8.630 10.060 19.672 13.849 **Figure 6. Porous Pot DOC Feed and Effluents (mg/l DOC)**

## 12/14/2011 Page 54 of 66

**Ap pendix**  **7 : Figure 7** 

**- PPS Effluent DOC Un** 

**- acclimated A**  **LFO 23**

**- 4S** 

**PO T**  **1 2**  **3 4**  **5 6** 

7-Ma y 13 .056 14 .259 17. 506 15.9 89 24.85 9 14 .375

8-Ma y 11 .390 12 .079 14. 995 13.0 05 14.41 9 10 .447

9-Ma y 11 .166 8. 076 13. 943 10.4 10 16.76 8 7.723

10-M ay 13 .851 9. 932 12. 975 13.7 80 16.50 4 8.973

11-M ay 14 .648 8. 654 12. 079 15.0 43 17.64 0 9.791

12-M ay 17 .195 11 .863 12. 431 12.8 87 14.81 7 10 .413

13-M ay 13 .227 15 .342 13. 220 12.9 90 16.07 7 12 .142

14-M ay 14 .684 17 .409 14. 468 13.6 90 17.95 9 13 .849

15-M ay 14 .495 14 .161 18. 376 14.0 24 15.20 9 17 .925

16-M ay 13 .711 12 .700 17. 998 13.0 76 13.52 7 17 .853

17-M ay 12 .881 13 .083 18. 845 12.2 78 12.47 7 19 .944

18-M ay 12 .906 12 .740 18. 765 13.5 56 13.73 0 20 .410

19-M ay 14 .350 15 .506 20. 804 14.3 50 14.18 1 19 .876

20-M ay 14 .861 14 .060 18. 592 14.3 83 14.18 0 19 .391

21-M ay 16 .580 14 .957 21. 984 15.9 65 17.32 4 21 .712

22-M ay 18 .519 16 .866 22. 555 16.4 54 18.76 1 24 .559

23-M ay 20 .229 19 .077 23. 349 16.5 71 17.04 6 24 .231

24-M ay 21 .052 20 .525 41. 704 17.3 37 19.33 0 32 .580

25-M ay 23 .396 21 .497 75. 121 17.6 36 19.98 6 34 .103

26-M ay 24 .677 25 .577 74. 135 18.1 71 26.59 0 47 .658

27-M ay 25 .563 37 .302 48. 765 20.6 33 31.91 1 39 .951

28-M ay 27 .795 38 .224 33. 350 25.8 53 32.85 0 44 .821

29-M ay 32 .055 40 .734 53. 497 23.6 35 39.62 9 51 .591

30-M ay 38 .556 36 .171 78. 989 21.4 35 37.21 3 66 .243

31-M ay 40 .411 46 .503 109. 984 20.6 25 52.84 2 86 .845

1-Jun 11 0.847 39 .752 143. 003 21.8 19 54.17 0 95 .653

2-Jun 36 .631 35 .625 122. 379 24.7 65 31.56 2 60 .394

3-Jun 35 .408 34 .163 64. 634 23.1 97 29.79 0 38 .381

4-Jun 25 .778 30 .849 45. 249 20.4 03 25.65 2 34 .204

5-Jun 24 .414 26 .534 39. 658 18.9 06 21.72 0 43 .952

6-Jun 21 .898 23 .696 43. 764 18.7 53 23.73 8 40 .287

7-Jun 21 .512 21 .598 39. 313 18.9 22 22.00 8 62 .674

8-Jun 20 .129 20 .363 36. 067 19.5 47 19.28 2 37 .601

9-Jun 18 .257 16 .911 33. 803 18.7 52 17.58 6 29 .144

10-Jun 16 .352 13 .618 26. 687 18.1 65 16.27 9 30 .173

11-Jun 19 .105 14 .642 26. 226 17.5 17 15.00 1 32 .621

12-Jun 17 .836 15 .879 27. 491 17.1 22 13.98 2 31 .409

13-Jun 16 .745 17 .467 26. 470 16.3 79 14.13 3 31 .842

14-Jun 16 .921 16 .429 24. 133 17.1 34 13.41 7 30 .072

15-Jun 16 .830 16 .997 22. 378 16.2 18 12.16 0 27 .404

16-Jun 17 .694 18 .709 24. 452 19.1 30 14.69 7 27 .252

17-Jun 19 .066 19 .989 23. 082 18.8 20 16.23 0 25 .117

18-Jun 23 .874 24 .348 24. 102 19.2 31 23.85 3 24 .861

19-Jun 26 .433 29 .704 25. 941 19.8 41 32.05 1 27 .063

20-Jun 25 .624 29 .934 24. 671 20.2 12 27.49 8 23 .934

21-Jun 29 .820 31 .296 27. 129 28.7 32 27.80 1 24 .856

22-Jun 33 .364 33 .462 30. 625 37.7 06 35.46 3 27 .315

23-Jun 34 .683 32 .957 34. 865 40.6 21 47.13 4 28 .881

24-Jun 34 .006 31 .203 36. 116 39.8 70 48.72 5 28 .024

25-Jun 35 .440 30 .835 33. 907 41.9 29 45.60 3 28 .067

26-Jun 35 .583 38 .343 32. 526 46.8 20 38.56 3 27 .105

27-Jun 36 .638 45 .194 30. 356 41.0 04 36.67 6 31 .391

28-Jun 40 .541 45 .753 29. 512 36.0 05 38.03 9 36 .945

29-Jun 54 .947 40 .619 82. 448 34.3 07 58.37 8 82 .663

30-Jun 58 .806 40 .602 89. 648 40.3 28 53.55 7 59 .334

1-Jul 39 .933 37 .372 46. 079 46.3 77 37.91 1 38 .859

2-Jul 31 .779 31 .256 42. 059 42.8 75 53.27 6 33 .021

3-Jul 28 .970 30 .295 41. 395 41.6 61 65.00 9 32 .403 **Fig ur**  **e 7. P or**  **ou s P**  **ot E**  **ff lue**  **nts D**  **OC Pre Una cclim**  **ated AL**  **FO 2**  **3-4 S**  **(m g/**  **l)**

## 12/14/2011 Page 55 of 66 **PO T**  **1 2**  **3 4**  **5 6** 

4-Jul 23 .856 24. 307 29.4 41 32.02 2 38 .571 30 .051

5-Jul 14 .309 11. 763 17.8 11 14.06 8 19 .159 16 .284

6-Jul 11 .747 8. 449 15.2 97 11.37 3 17 .490 13 .665

7-Jul 11 .830 8. 623 15.1 78 13.38 2 16 .489 16 .560

8-Jul 11 .570 8. 991 15.4 06 13.79 4 16 .002 15 .762

9-Jul 9. 496 8. 664 15.9 19 11.63 7 13 .499 15 .759

10-Jul 10 .925 10. 282 16.7 95 10.02 7 10 .059 17 .069

11-Jul 12 .123 10. 257 23.0 31 10.78 4 11 .208 21 .277

12-Jul 10 .177 9. 635 24.7 81 9.90 1 11 .296 21 .062

13-Jul 9. 601 7. 604 21.7 60 12.22 8 9.700 20 .971

14-Jul 10 .750 8. 695 19.6 03 14.16 5 11 .253 21 .720

15-Jul 11 .782 9. 664 20.3 41 13.15 0 12 .519 20 .116

16-Jul

17-Jul 12 .684 10. 059 20.0 30 12.01 4 11 .731 19 .936

18-Jul 11 .512 9. 846 18.5 93 9.65 7 10 .122 18 .202

19-Jul 10 .142 9. 585 17.1 49 8.76 9 8.777 16 .910

20-Jul 10 .135 10. 248 17.4 53 9.64 6 9.934 18 .026

21-Jul 9. 948 9. 276 18.5 99 10.53 8 9.871 19 .865

22-Jul 10 .278 8. 374 18.6 56 10.34 2 10 .016 19 .230

23-Jul 11 .071 10. 128 18.5 46 12.11 4 12 .021 17 .728

24-Jul 12 .209 11. 293 19.9 24 13.76 5 12 .585 19 .956

25-Jul 12 .292 10. 240 19.2 88 12.27 3 10 .609 19 .466

26-Jul 11 .954 8. 959 17.8 47 9.42 5 9.459 17 .849

27-Jul 10 .939 7. 975 18.2 42 8.79 7 7.508 18 .240

28-Jul 9. 882 8. 158 17.7 45 9.28 7 7.811 18 .582

29-Jul 9. 548 8. 711 18.5 32 9.38 6 7.597 19 .619

30-Jul 9. 377 8. 547 18.0 41 9.57 8 7.721 18 .567

31-Jul 10 .393 9. 521 19.1 24 9.13 4 7.809 18 .238

1-A ug 9. 078 16. 723 18.2 75 9.47 6 13 .278 17 .869

2-A ug 10 .443 21. 702 19.4 66 9.91 6 19 .507 17 .994

3-A ug 10 .186 19. 422 19.2 89 9.59 4 19 .641 19 .032

4-A ug 9. 462 19. 051 19.0 51 9.39 7 19 .272 18 .114

5-A ug 8. 544 18. 627 19.1 38 8.54 8 18 .773 17 .710

6-A ug 8. 349 17. 032 17.4 78 7.59 2 17 .735 16 .699

7-A ug 8. 612 16. 222 17.9 00 8.73 0 18 .433 17 .053

8-A ug 8. 506 17. 371 21.9 36 9.67 6 18 .421 17 .222

9-A ug 8. 633 18. 979 23.5 01 10.89 6 18 .210 17 .241

10-A ug 9. 122 19. 029 22.3 32 11.11 9 21 .136 18 .452

11-A ug 9. 647 18. 593 15.9 54 11.35 9 23 .890 12 .555

12-A ug 9. 763 18. 736 11.5 77 11.25 1 22 .779 9. 150

13-A ug 10 .869 18. 046 10.9 47 12.73 6 22 .367 9. 964

14-A ug 12 .368 17. 517 10.2 10 11.89 5 21 .079 10 .961

15-A ug 11 .797 16. 757 8.7 96 11.95 3 20 .191 10 .419

16-A ug 10 .478 21. 293 8.2 90 10.86 2 19 .181 9. 695

17-A ug 11 .509 17. 302 8.5 70 14.86 6 17 .302 11 .568

18-A ug 11 .688 18. 969 9.3 07 15.70 5 22 .359 12 .018

19-A ug 11 .848 19. 597 10.0 53 15.25 1 22 .014 13 .800

20-A ug 17 .829 15. 622 11.3 47 21.56 8 17 .571 14 .611

21-A ug 14 .716 17. 644 11.6 15 19.33 2 18 .968 16 .138

22-A ug 12 .209 23. 570 12.7 08 17.11 3 21 .705 17 .584

23-A ug 11 .595 25. 438 13.4 05 16.26 6 21 .192 16 .477

24-A ug 12 .185 26. 305 13.9 77 15.31 7 19 .852 14 .895

25-A ug 11 .531 26. 019 14.9 25 14.27 8 19 .862 15 .076

26-A ug 10 .975 24. 315 13.3 42 12.60 6 18 .374 14 .906 **Fig ur**  **e 7. P or**  **ou s P**  **ot E**  **ff lu**  **en ts**  **DO C**  **Pre Un**  **ac clim**  **ated**

**ALF O**  **23-4S**

**( mg/**  **l)**

## 12/14/2011 Page 56 of 66 **PO T**  **1 2**  **3 4**  **5 6** 

27-A ug 10 .818 23. 274 13.9 05 12.28 6 18 .981 15 .615

28-A ug 10 .349 23. 970 14.8 62 11.12 9 18 .040 12 .299

29-A ug 10 .875 24. 091 16.3 67 11.68 3 18 .669 13 .277

30-A ug 11 .779 25. 882 16.1 80 12.31 8 22 .884 12 .634

31-A ug 20 .714 27. 283 15.1 82 20.53 1 24 .304 12 .591

1-S ep 15 .978 26. 113 13.7 24 15.11 3 22 .066 13 .315

2-S ep 11 .366 25. 023 13.0 10 10.58 1 21 .006 13 .188

3-S ep 10 .427 25. 200 12.9 17 10.43 9 21 .760 14 .452

4-S ep 10 .066 24. 601 12.0 45 9.63 6 21 .613 13 .879

5-S ep 9. 901 23. 713 11.7 65 9.22 3 21 .243 12 .706

6-S ep 10 .149 23. 138 10.9 26 9.59 4 21 .749 12 .154

7-S ep 10 .503 22. 527 11.8 56 10.41 1 22 .286 12 .281

8-S ep 12 .498 23. 158 13.9 73 11.43 6 23 .409 13 .398

9-S ep 12 .835 23. 764 16.7 22 11.52 9 24 .306 14 .156

10-S ep 12 .443 22. 332 20.0 85 11.06 9 23 .412 13 .958

11-S ep 12 .641 22. 748 17.6 89 10.46 9 22 .751 13 .364

12-S ep 11 .709 20. 968 13.1 83 10.07 0 22 .204 12 .454

13-S ep 11 .608 21. 284 11.8 01 9.76 7 22 .282 11 .568

14-S ep 11 .620 24. 308 11.7 53 9.71 0 22 .193 10 .784

15-S ep 16 .719 24. 853 11.7 40 10.76 5 22 .927 11 .152

16-S ep 17 .648 22. 944 11.9 96 10.70 4 22 .809 10 .475

17-S ep 17 .593 23. 992 13.2 51 10.52 8 23 .299 10 .270

18-S ep 16 .903 25. 509 12.3 64 10.48 6 23 .497 10 .234

19-S ep 14 .706 23. 861 11.5 15 10.18 0 22 .900 10 .007

20-S ep 13 .268 23. 232 10.7 70 10.48 2 22 .389 9. 932

21-S ep 13 .377 23. 479 11.6 75 11.48 6 23 .480 11 .445

22-S ep 16 .809 23. 499 12.0 35 15.04 6 24 .853 11 .901

23-S ep 12 .245 23. 338 15.1 33 13.43 9 23 .302 13 .533

24-S ep 12 .698 23. 617 13.2 62 15.84 0 23 .046 11 .314

25-S ep 11 .130 22. 490 11.9 74 15.95 5 22 .102 11 .065

26-S ep 10 .436 22. 485 11.2 43 16.00 1 21 .810 10 .419

27-S ep 10 .373 22. 744 10.3 29 15.87 2 20 .982 11 .089

28-S ep 10 .404 23. 165 10.4 08 14.40 6 21 .075 11 .006

29-S ep 10 .932 23. 759 10.7 58 13.60 8 20 .633 10 .953

30-S ep 10 .891 22. 285 10.8 01 13.09 9 20 .355 11 .242

1-Oct 10 .495 22. 540 10.8 44 14.00 1 19 .488 10 .390

2-Oct 11 .365 26. 022 11.3 99 12.68 4 20 .468 10 .976

3-Oct 12 .626 20. 104 10.3 31 11.08 2 19 .193 11 .403

4-Oct 12 .244 20. 534 10.0 92 11.09 2 19 .931 12 .310

5-Oct 12 .810 19. 083 8.6 35 10.09 7 19 .076 12 .698

6-Oct 13 .882 19. 841 9.2 15 10.32 1 18 .704 13 .530

7-Oct 12 .498 20. 916 8.6 30 10.06 0 19 .672 13 .849

8-Oct

9-Oct

10-Oct

11-Oct

27-Jan 7. 838 24. 293 7.2 10 18.71 7 6.927 19 .858

28-Jan 7. 041 22. 283 6.7 89 18.12 9 7.411 17 .741

29-Jan 7. 092 21. 705 7.8 36 18.06 5 6.949 16 .841

30-Jan 10 .384 23. 765 7.7 04 20.32 8 10 .819 18 .908

31-Jan 10 .251 25. 144 7.1 03 19.60 7 10 .053 18 .549

1-Fe b 8. 195 22. 245 12.6 55 20.61 1 11 .038 18 .989

2-Fe b 10 .221 19. 207 5.5 64 16.98 1 8.556 15 .924 **Fi gu**  **re 7. P** 

**or ou**  **s P ot**  **E ff**  **lu en**  **ts DO**  **C P**

**re Unac**  **clim at**  **ed**

**ALF O**  **23-4S**

**( mg/**  **l)**

## 12/14/2011 Page 57 of 66 **PO T**  **1 2**  **3 4**  **5 6** 

3-Fe b 10 .358 18. 358 5.1 09 16.50 9 14 .327 14 .957

4-Fe b

- - - - - 15 .533

5-Fe b 12 .659 21. 732 9.7 62 20.25 1 28 .741 19 .345

6-Fe b 12 .166 21. 871 9.9 75 20.91 3 25 .082 19 .323

7-Fe b 11 .517 20. 609 9.9 82 21.59 8 22 .925 18 .706

8-Fe b 11 .990 19. 378 9.7 90 20.60 0 22 .808 17 .971

9-Fe b 12 .095 19. 527 10.1 09 19.72 1 21 .545 18 .237

10-F eb 10 .137 18. 812 9.5 86 19.78 4 21 .942 18 .208

11-F eb 9. 497 17. 350 8.8 65 19.10 6 21 .082 17 .630

12-F eb 9. 997 17. 536 9.1 53 19.34 1 21 .871 17 .226

13-F eb 10 .032 16. 785 8.3 74 18.51 7 21 .087 16 .087

14-F eb 10 .476 17. 272 8.2 70 20.28 2 24 .402 16 .910

15-F eb 11 .584 17. 668 8.4 07 20.10 3 24 .752 17 .514

16-F eb 11 .927 17. 335 8.7 90 19.78 4 24 .524 17 .137

17-F eb 10 .369 16. 625 7.5 78 18.73 3 24 .478 16 .751

18-F eb 9. 327 16. 554 8.0 15 19.23 5 25 .926 17 .089

19-F eb 8. 934 16. 010 8.1 14 18.96 4 25 .227 17 .168

20-F eb 7. 545 15. 258 7.7 47 17.63 6 21 .555 16 .359

21-F eb 7. 530 14. 440 7.1 86 17.53 1 20 .581 15 .640

22-F eb 7. 497 13. 977 7.4 69 16.37 8 20 .017 15 .460

23-F eb 9. 161 15. 515 11.8 41 17.48 1 20 .528 17 .275

24-F eb 8. 660 15. 284 9.2 24 17.86 2 19 .937 17 .141

25-F eb 9. 038 15. 848 9.1 30 19.52 4 19 .951 16 .359

26-F eb 8. 424 14. 550 8.7 73 20.42 1 17 .607 15 .653

27-F eb 9. 765 15. 294 9.4 59 20.58 3 17 .964 16 .959

28-F eb 7. 665 15. 685 11.3 15 19.86 4 17 .700 16 .761

1-Ma r 10 .946 17. 418 13.8 74 20.55 5 18 .541 18 .291

2-Ma r 10 .296 18. 553 13.8 16 20.33 4 18 .462 19 .059

3-Ma r 8. 601 17. 760 10.3 01 20.13 4 17 .334 16 .922

4-Ma r 9. 806 18. 244 10.6 68 22.05 6 17 .343 17 .279

5-Ma r 9. 758 17. 889 9.8 65 20.95 2 17 .186 17 .622

6-Ma r 9. 325 18. 858 9.1 08 19.87 6 15 .658 16 .091

7-Ma r 9. 454 21. 583 10.1 90 20.83 0 16 .501 17 .315

8-Ma r 8. 923 20. 579 10.8 87 20.85 5 17 .797 17 .471

9-Ma r 9. 719 20. 555 12.1 37 24.56 3 19 .187 18 .293

10-M ar 10 .355 20. 227 11.7 15 22.58 8 17 .553 16 .323

11-M ar 8. 056 20. 842 11.6 83 20.26 3 18 .006 16 .174

12-M ar 7. 939 21. 044 10.0 28 19.52 3 13 .407 15 .065

13-M ar 8. 215 20. 371 9.2 71 20.69 8 18 .992 14 .248

14-M ar 8. 206 20. 036 8.3 56 18.75 2 19 .452 13 .680

15-M ar 8. 368 19. 445 7.7 13 18.70 4 18 .478 13 .063

16-M ar 7. 378 19. 223 7.7 30 17.88 2 17 .791 12 .846

17-M ar 7. 465 18. 520 7.9 77 17.23 6 18 .189 12 .594

18-M ar 7. 618 18. 036 8.1 10 17.69 1 17 .503 12 .635

19-M ar 8. 326 17. 975 8.4 32 15.76 6 17 .404 12 .503

20-M ar 8. 933 16. 577 8.0 26 15.79 3 17 .617 12 .499

21-M ar 10 .063 16. 427 7.6 89 15.92 4 17 .235 12 .687

22-M ar 10 .129 15. 075 7.9 77 16.07 0 16 .588 12 .476

23-M ar 8. 997 15. 431 7.8 15 15.48 4 16 .694 12 .563

24-M ar 8. 445 15. 294 7.9 37 15.96 4 16 .825 13 .244

25-M ar 8. 544 15. 374 7.4 94 16.13 4 16 .928 13 .739

26-M ar 8. 237 14. 972 7.3 45 15.93 2 17 .945 13 .896 **Fig ur**  **e 7. P or**  **ou s P**  **ot E**  **ff**

**lue nts**  **DO C Pr** 

**e Unac clim**  **ated**

**AL FO**  **23-4S**

**( mg/**  **l)**

## 12/14/2011 Page 58 of 66

**Ap pendix**  **8 : Figure 8** 

**- PPS for Fragment A**  **nalysis Effluent**

**Bla nk (**  **1) Alfo**  **(2) Blan**  **k (3) Alfo**  **( 6)** 

27-J an 7.84 24 .29 7. 21 19 .86

28-J an 7.04 22 .28 6. 79 17 .74

29-J an 7.09 21 .71 7. 84 16 .84

30-J an 10.38 23 .77 7. 70 18 .91

31-J an 10.25 25 .14 7. 10 18 .55

1-Fe b 8.19 22 .25 12 .66 18 .99

2-Fe b 10.22 19 .21 5. 56 15 .92

3-Fe b 10.36 18 .36 5. 11 14 .96

5-Fe b 12.66 21 .73 9. 76 19 .35

6-Fe b 12.17 21 .87 9. 97 19 .32

7-Fe b 11.52 20 .61 9. 98 18 .71

8-Fe b 11.99 19 .38 9. 79 17 .97

9-Fe b 12.10 19 .53 10 .11 18 .24

10 -Fe b 10.14 18 .81 9. 59 18 .21

11 -Fe b 9.50 17 .35 8. 86 17 .63

12 -Fe b 10.00 17 .54 9. 15 17 .23

13 -Fe b 10.03 16 .79 8. 37 16 .09

14 -Fe b 10.48 17 .27 8. 27 16 .91

15 -Fe b 11.58 17 .67 8. 41 17 .51

16 -Fe b 11.93 17 .33 8. 79 17 .14

17 -Fe b 10.37 16 .63 7. 58 16 .75

18 -Fe b 9.33 16 .55 8. 02 17 .09

19 -Fe b 8.93 16 .01 8. 11 17 .17

20 -Fe b 7.55 15 .26 7. 75 16 .36

21 -Fe b 7.53 14 .44 7. 19 15 .64

22 -Fe b 7.50 13 .98 7. 47 15 .46

23 -Fe b 9.16 15 .52 11 .84 17 .27

24 -Fe b 8.66 15 .28 9. 22 17 .14

25 -Fe b 9.04 15 .85 9. 13 16 .36

26 -Fe b 8.42 14 .55 8. 77 15 .65

27 -Fe b 9.77 15 .29 9. 46 16 .96

28 -Fe b 7.67 15 .69 11 .31 16 .76

1-Ma r 10.95 17 .42 13 .87 18 .29

2-Ma r 10.30 18 .55 13 .82 19 .06

3-Ma r 8.60 17 .76 10 .30 16 .92

4-Ma r 9.81 18 .24 10 .67 17 .28

5-Ma r 9.76 17 .89 9. 86 17 .62

6-Ma r 9.32 18 .86 9. 11 16 .09

7-Ma r 9.45 21 .58 10 .19 17 .31

8-Ma r 8.92 20 .58 10 .89 17 .47

9-Ma r 9.72 20 .55 12 .14 18 .29

10 -Ma r 10.35 20 .23 11 .72 16 .32

11 -Ma r 8.06 20 .84 11 .68 16 .17

12 -Ma r 7.94 21 .04 10 .03 15 .06

13 -Ma r 8.21 20 .37 9. 27 14 .25

14 -Ma r 8.21 20 .04 8. 36 13 .68

15 -Ma r 8.37 19 .45 7. 71 13 .06

16 -Ma r 7.38 19 .22 7. 73 12 .85

17 -Ma r 7.46 18 .52 7. 98 12 .59

18 -Ma r 7.62 18 .04 8. 11 12 .64

19 -Ma r 8.33 17 .98 8. 43 12 .50

20 -Ma r 8.93 16 .58 8. 03 12 .50

21 -Ma r 10.06 16 .43 7. 69 12 .69

22 -Ma r 10.13 15 .08 7. 98 12 .48

23 -Ma r 9.00 15 .43 7. 81 12 .56

24 -Ma r 8.44 15 .29 7. 94 13 .24

25 -Ma r 8.54 15 .37 7. 49 13 .74

26 -Ma r 8.24 14 .97 7. 34 13 .90 **Po ro**  **us P**  **ot S tu**  **dy**

**f or**  **Fr agm**  **en t A**  **naly**

**sis E ff**  **luen t** 

## 12/14/2011 Page 59 of 66

**Ap pendix**  **9 : Figure 9** 

**- Return to Steady State: Post Un** 

**- acc**  **limated A LFO 23** 

**- 4S** 

**Po t**  **1 2**  **3 4**  **5 6** 

**Bl nk**  **U- Alf** 

**o A-**  **Alf**

**o Bln**  **k U-**  **Alf o**  **A- Al**  **fo**

30-Jul 9.38 8. 55 18. 04 9.5 8 7.72 18 .57

31-Jul 10 .39 9. 52 19. 12 9.1 3 7.81 18 .24

1-A ug 9.08 16 .72 18. 28 9.4 8 13.28 17 .87

2-A ug 10 .44 21 .70 19. 47 9.9 2 19.51 17 .99

3-A ug 10 .19 19 .42 19. 29 9.5 9 19.64 19 .03

4-A ug 9.46 19 .05 19. 05 9.4 0 19.27 18 .11

5-A ug 8.54 18 .63 19. 14 8.5 5 18.77 17 .71

6-A ug 8.35 17 .03 17. 48 7.5 9 17.73 16 .70

7-A ug 8.61 16 .22 17. 90 8.7 3 18.43 17 .05

8-A ug 8.51 17 .37 21. 94 9.6 8 18.42 17 .22

9-A ug 8.63 18 .98 23. 50 10.9 0 18.21 17 .24 **Fi gu**  **re 9**  **. Retu**

**rn to**  **St ea**  **dy**

**S ta**  **te: P**  **os t Un**  **acclim ate**  **d AL**  **FO 23-**  **4S**

**Pr e**  **AE S (** 

**mg /l DOC)** 

## 12/14/2011 Page 60 of 66

**Ap pendix**  **10 : Figure 10** 

**- Porous Pot DOC: Comparison of 1214E4S and A**  **LFO 23**

**- 4S** 

**Po t**  **1 2**  **3 4**  **5 6** 

**AE S**  **Alf**

**o Bln**  **k AES**  **Alfo Bln**  **k**

14-A ug 12 .37 17 .52 10. 21 11.8 9 21.08 10 .96

15-A ug 11 .80 16 .76 8. 80 11.9 5 20.19 10 .42

16-A ug 10 .48 21 .29 8. 29 10.8 6 19.18 9.69

17-A ug 11 .51 17 .30 8. 57 14.8 7 17.30 11 .57

18-A ug 11 .69 18 .97 9. 31 15.7 0 22.36 12 .02

19-A ug 11 .85 19 .60 10. 05 15.2 5 22.01 13 .80

20-A ug 17 .83 15 .62 11. 35 21.5 7 17.57 14 .61

21-A ug 14 .72 17 .64 11. 62 19.3 3 18.97 16 .14

22-A ug 12 .21 23 .57 12. 71 17.1 1 21.70 17 .58

23-A ug 11 .60 25 .44 13. 40 16.2 7 21.19 16 .48

24-A ug 12 .18 26 .30 13. 98 15.3 2 19.85 14 .90

25-A ug 11 .53 26 .02 14. 93 14.2 8 19.86 15 .08

26-A ug 10 .98 24 .32 13. 34 12.6 1 18.37 14 .91

27-A ug 10 .82 23 .27 13. 91 12.2 9 18.98 15 .62

28-A ug 10 .35 23 .97 14. 86 11.1 3 18.04 12 .30

29-A ug 10 .87 24 .09 16. 37 11.6 8 18.67 13 .28

30-A ug 11 .78 25 .88 16. 18 12.3 2 22.88 12 .63

31-A ug 20 .71 27 .28 15. 18 20.5 3 24.30 12 .59

1-S ep 15 .98 26 .11 13. 72 15.1 1 22.07 13 .31

2-S ep 11 .37 25 .02 13. 01 10.5 8 21.01 13 .19

3-S ep 10 .43 25 .20 12. 92 10.4 4 21.76 14 .45

4-S ep 10 .07 24 .60 12. 05 9.6 4 21.61 13 .88

5-S ep 9.90 23 .71 11. 77 9.2 2 21.24 12 .71

6-S ep 10 .15 23 .14 10. 93 9.5 9 21.75 12 .15

7-S ep 10 .50 22 .53 11. 86 10.4 1 22.29 12 .28

8-S ep 12 .50 23 .16 13. 97 11.4 4 23.41 13 .40

9-S ep 12 .84 23 .76 16. 72 11.5 3 24.31 14 .16

10-S ep 12 .44 22 .33 20. 08 11.0 7 23.41 13 .96

11-S ep 12 .64 22 .75 17. 69 10.4 7 22.75 13 .36

12-S ep 11 .71 20 .97 13. 18 10.0 7 22.20 12 .45

13-S ep 11 .61 21 .28 11. 80 9.7 7 22.28 11 .57

14-S ep 11 .62 24 .31 11. 75 9.7 1 22.19 10 .78

15-S ep 16 .72 24 .85 11. 74 10.7 6 22.93 11 .15

16-S ep 17 .65 22 .94 12. 00 10.7 0 22.81 10 .47

17-S ep 17 .59 23 .99 13. 25 10.5 3 23.30 10 .27

18-S ep 16 .90 25 .51 12. 36 10.4 9 23.50 10 .23

19-S ep 14 .71 23 .86 11. 51 10.1 8 22.90 10 .01

20-S ep 13 .27 23 .23 10. 77 10.4 8 22.39 9.93

21-S ep 13 .38 23 .48 11. 68 11.4 9 23.48 11 .44

22-S ep 16 .81 23 .50 12. 03 15.0 5 24.85 11 .90

23-S ep 12 .25 23 .34 15. 13 13.4 4 23.30 13 .53

24-S ep 12 .70 23 .62 13. 26 15.8 4 23.05 11 .31 **Fig ur**  **e 10. P**  **or ou**  **s P**

**ot DOC: A Co**  **mp ar** 

**iso n of**  **1214 E4**  **S an**  **d ALF**  **O 23**

**-4S**

**(m g/**  **l DOC)**

## 12/14/2011 Page 61 of 66

**Ap pendix**  **11 : Figure 11** 

**- PPS: Increased HRT** 

**Po t**  **1 2**  **3 4**  **5 6** 

**AE S**  **Alf**

**o Bln**  **k AES**  **Alfo Bln**  **k**

20-S ep 13 .27 23 .23 10. 77 10.4 8 22.39 9.93

21-S ep 13 .38 23 .48 11. 68 11.4 9 23.48 11 .44

22-S ep 16 .81 23 .50 12. 03 15.0 5 24.85 11 .90

23-S ep 12 .25 23 .34 15. 13 13.4 4 23.30 13 .53

24-S ep 12 .70 23 .62 13. 26 15.8 4 23.05 11 .31

25-S ep 11 .13 22 .49 11. 97 15.9 6 22.10 11 .07

26-S ep 10 .44 22 .48 11. 24 16.0 0 21.81 10 .42

27-S ep 10 .37 22 .74 10. 33 15.8 7 20.98 11 .09

28-S ep 10 .40 23 .17 10. 41 14.4 1 21.08 11 .01

29-S ep 10 .93 23 .76 10. 76 13.6 1 20.63 10 .95

30-S ep 10 .89 22 .29 10. 80 13.1 0 20.35 11 .24

1-Oct 10 .50 22 .54 10. 84 14.0 0 19.49 10 .39

2-Oct 11 .36 26 .02 11. 40 12.6 8 20.47 10 .98

3-Oct 12 .63 20 .10 10. 33 11.0 8 19.19 11 .40

4-Oct 12 .24 20 .53 10. 09 11.0 9 19.93 12 .31

5-Oct 12 .81 19 .08 8. 63 10.1 0 19.08 12 .70

6-Oct 13 .88 19 .84 9. 21 10.3 2 18.70 13 .53

7-Oct 12 .50 20 .92 8. 63 10.0 6 19.67 13 .85

8-Oct

9-Oct 12 .50 20 .92 8. 63 10.0 6 19.67 13 .85

10-Oct 12 .64 21 .18 8. 30 10.0 6 17.86 13 .49 **Fi gur**  **e 11 . P**  **or ous**  **P ot**  **: I nc**  **re ase**  **d Hy**  **dr au** 

**lic Ren ten**  **ion Tim**  **e**

**(6 Hr**  **t o 1**  **2 hr )**  **mg /l**  **DO C** 



### Ap **pendix 12**  **: Figure 12** 

**- Die** 

**- Aw**  **ay Porous Pot Effluent**

**Figu re**  **12. Die -A**  **way P or** 

**ou s P**  **ot Ef**  **fl uen**  **t (m**  **g/**

**l)**

**Da ys**  **0 1**  **4 7**  **12 17**  **24 28** 

PP B lank 16. 854 15.1 59 12 .412 5 11 .1985 9. 7205 7.8 225 7.48 25 6.954 5

PP A ES 11. 659 12.6 34 9.769 5 8. 5245 7. 7485 6.3 905 5.1 89 5.019 5

PP A LFO 23-4S 23. 473 22.2 32 21.46 1 20 .473 19. 2685 17 .359 16.5 91 16.43 6



### Ap **pendix 13**  **: Figure 13**

**- OECD Die** 

**- Aw**  **ay DOC Loss %**

**Figu re**  **13. OE CD Die-**  **Awa**

**y DOC L os** 

**s (% )** 

**Da ys**  **0 1**  **4 7**  **12 17**  **24 28** 

AE S 0 8. 26 54.2 9 60.68 93 .28 99 .49 98 .99 98 .9

AL FO 23-

4S 0 1. 62 9. 7 9.67 9.21 17 .87 19 .81 20 .5

Be nz 0 17. 75 95 .9 97.4 98.5 100 99 .63 99 .3

## 12/14/2011 Page 62 of 66

**Ap pendix**  **14 : Figure 14** 

**- Porous Pot: MBA**  **S Primary Biodegradation**

**In flue**  **nt Eff** 

**lu en**  **t**

Bl an k 1.2 0.1

AE S 44.0 0.1

AL FO 23-

4S 39.0 2.3

**In flue**  **nt Eff** 

**lu en**  **t**

Po rous P ot 39.0 2.3

Di e- aw ay 46.0 4.6 **Fig ure**  **14. P or**  **ou s P**  **ot : M**  **BA S**  **Pr im**  **ar**

**y**

**Bio de**  **gr ada**  **tion (m**  **g/l)**

**Fi gu**  **re 15. AL**  **FO 23**

**-4S Co**  **mp ar** 

**iso n**  **Po ro**  **us**

**P ot** 

**an d D**  **ie- away** 

**MB AS (**  **mg /l)** 



### Ap **pendix 15**  **: A LFO 23** 

**- 4S Comparison Porous Pot and Die A**  **way**

**Ap pendix**  **16 : Figure 16** 

**- AES and A**  **LFO 23**

**- 4S**  **Ac ute Toxicity** 

**Ap pendix**  **17 :**  **Figure 17**

**- Porous Pot A**  **quatic Toxicity 96 Hour Minnow**

**Ap pendix**  **18 : Figure 18** 

**- OECD Die** 

**- Aw**  **ay Test A LFO 23** 

**- 4S** 

**LC 50**  **LO EC**  **NOE C** 

AE S 5. 5 2.4 4.8

AL FO 23- 4S 3. 1 1.2 2.4

**LC 50**  **LO EC**  **NOE C** 

Bl an k Fe ed 14. 7 25.0 12.0

Bl an k Ef flue nt 100. 0 10 0.0 10 0.0

AE S + Fee d 5. 5 6.0 3.0

AE S Ef flu ent 100. 0 10 0.0 10 0.0

AL FO 23- 4S + Fee d 9. 5 12.0 6.0

AL FO 23- 4S E fflu ent 100. 0 10 0.0 10 0.0

**LC 50**  **LO EC**  **NOE C** 

AL FO 23- 4S Day 0 8. 8 12.0 6.0

AL FO 23- 4S Day 2 8 100. 0 10 0.0 10 0.0

**Acu te**  **T ox** 

**icit y 96 h**  **r Fath**  **ead**

**M in**  **no w (** 

**mg /l)**  **Fig ur**  **e 16. AE**  **S an**  **d AL**  **FO 23-4** 

**S**

**Fig ur**  **e 17**

**- Po**  **ro us** 

**P ot A**  **qu**

**atic To**  **xicity 96 h**  **r. Min**  **now**

**( mg**  **/l)**

**40 m g/l AES** 

**and AL**  **FO 2**  **3-4S**

**a ctive** 

**Fi gur**  **e 1 8. OE**  **CD D**  **ie -A**  **way T est**  **A LF**  **O 2 3-4S** 

**50 m g/**  **l activ**  **e or 22**  **mg/ l DOC** 

## 12/14/2011 Page 63 of 66

**Ap pendix**  **19 : Figure 19: A**  **LFOTERRA Hydrophobe Biodegradation Study** 

**- OECD Demand** 

**Isa lch**  **em**

**23 Al**  **chem**

**23 TD** 

**A Sa**  **fo l 23**  **Lia l 23**  **Alfo l**  **12 Es**  **ter**

0 0 0 0 0 0 0 0

1 10.30 13 .14 2. 34 12 .90 14.4 3 14.31 -0 .13

2 18.92 25 .66 9. 87 25 .39 23.7 5 27.47 0.27

3 23.13 37 .62 17 .48 33 .67 29.1 0 38.85 0.44

4 27.75 47 .88 22 .57 41 .31 35.0 1 49.49 0.61

5 33.45 56 .08 27 .13 48 .47 40.5 1 57.01 0.81

6 38.46 61 .58 31 .92 53 .53 45.2 1 61.79 0.96

7 43.08 65 .54 36 .19 58 .02 49.6 7 66.37 0.96

8 47.99 68 .90 40 .63 61 .31 54.0 2 70.68 0.93

9 53.21 71 .72 45 .31 63 .46 57.6 9 74.03 0.95

10 57.97 73 .63 49 .83 65 .43 60.7 3 76.40 1.00

11 61.95 75 .36 53 .71 67 .65 63.3 1 78.51 1.14

12 65.29 77 .04 56 .84 69 .77 65.4 8 80.38 1.27

13 68.15 78 .71 59 .40 71 .74 67.3 8 82.48 1.40

14 70.92 80 .82 61 .66 73 .60 69.1 3 84.78 1.50

15 73.54 82 .54 63 .56 75 .31 70.7 3 86.98 1.62

16 76.03 84 .41 65 .24 76 .86 72.5 0 89.28 1.74

17 78.28 86 .04 66 .67 78 .71 73.9 8 91.17 1.87

18 80.58 87 .28 68 .19 80 .61 75.6 8 92.68 1.97

19 82.75 88 .28 69 .72 82 .52 77.6 3 94.21 2.07

20 84.79 89 .14 71 .17 84 .10 79.4 3 95.38 2.19

21 86.78 89 .89 72 .44 85 .36 80.6 4 96.28 2.30

22 88.77 90 .77 73 .73 86 .75 81.6 3 97.14 2.44

23 90.58 91 .72 75 .22 88 .08 82.4 3 97.82 2.56

24 92.25 92 .52 76 .73 89 .33 83.1 3 98.49 2.68

25 93.84 93 .14 78 .26 90 .66 83.8 2 99.16 2.80

26 95.31 93 .62 79 .74 91 .55 84.5 2 99.84 2.94

27 96.74 94 .08 81 .26 92 .32 85.4 6 10 0.52 3.05

28 98.14 94 .48 82 .70 92 .97 86.4 6 10 1.26 3.18 **Fi gur**  **e 1 9. AL**  **FO TE**  **RRA Hy dr**  **op ho**  **be Bio**  **de gr** 

**adat ion**  **S tu**  **dy**

**OE CD 301F**  **The or** 

**etica l Oxyg**  **en dem**  **and**

## 12/14/2011 Page 64 of 66

**Ap pendix**  **20 : Figure 20** 

**- AL**  **FOTERRA Hydrophobe 4PO Biodegradation Study** 

**- OECD** 

**Day Isa**  **lch em** 

**23 Al**  **chem**

**23 TD** 

**A Sa**  **fo l 23**  **Lia l 23**  **Alfo l**  **12 Be**  **nz oat**  **e**

0 0 0 0 0 0 0 0

1 0.97 5.60 0. 28 4. 48 3.7 4 7.74 30.41

2 3.64 18 .64 1. 29 16 .24 11.9 3 18.98 57.22

3 9.64 22 .32 2. 81 21 .61 18.5 7 22.34 66.56

4 16.92 27 .55 3. 73 26 .46 24.7 2 26.49 71.08

5 22.86 32 .40 5. 95 31 .69 29.9 6 31.20 75.09

6 29.42 37 .16 11 .70 36 .57 35.1 0 35.36 78.74

7 35.81 41 .50 20 .36 40 .47 40.4 6 39.11 81.26

8 41.09 45 .68 31 .21 44 .46 45.2 6 42.74 83.09

9 45.94 49 .76 39 .65 48 .67 49.7 8 46.67 84.70

10 50.34 53 .41 46 .04 52 .66 53.7 4 51.72 86.08

11 54.80 56 .95 50 .94 56 .60 57.7 1 53.64 87.51

12 58.91 60 .29 55 .37 60 .29 61.4 2 57.09 88.76

13 62.74 63 .25 59 .24 63 .59 64.8 0 60.19 89.88

14 66.31 65 .88 62 .96 66 .56 67.8 9 62.87 90.86

15 69.79 68 .25 66 .27 69 .55 70.8 6 65.37 91.86

16 73.03 70 .50 68 .91 72 .45 73.6 4 67.73 92.90

17 75.95 72 .68 71 .40 75 .13 76.1 9 69.90 93.76

18 78.80 75 .02 74 .31 77 .78 78.7 2 72.09 94.74

19 81.52 77 .36 77 .24 80 .22 81.0 8 74.14 95.54

20 84.21 79 .69 79 .98 82 .71 83.4 7 76.30 96.47

21 86.83 82 .02 82 .32 85 .18 85.7 8 78.36 97.40

22 89.30 84 .15 84 .43 87 .26 88.0 2 80.14 98.02

23 92.08 86 .80 86 .79 89 .56 90.5 1 82.02 99.23

24 94.61 89 .21 89 .61 91 .41 92.6 7 83.45 10 0.06

25 97.12 91 .39 91 .79 93 .07 94.7 3 84.76 10 1.84

26 99.49 93 .47 93 .98 94 .55 96.6 4 85.94 10 2.25

27 10 1.92 95 .81 96 .29 95 .97 98.4 4 87.03 10 2.94

28 10 4.22 98 .51 98 .31 97 .44 100.2 1 88.25 10 4.18 **Fi gur**  **e 20 . AL**  **FOTE RRA Hyd**  **ro pho**  **be 4P**  **O Bi**  **ode gr**  **adat ion**  **St udy** 

**OE CD**  **3 01F**  **Theo re**  **tical Oxyg**  **en d em**  **and (%**  **)**

## 12/14/2011 Page 65 of 66

**Ap pendix**  **21 : Figure 21** 

**- AL**  **FOTERRA Hydrophobe 4PO Sulfate Biodegradation Study** 

**- OECD** 

**DAY Isa**  **lch em** 

**23 Al**  **chem**

**23 TD** 

**A Li**  **al 23 Sa**  **fo l 23**  **Alfo l**  **12 Be**  **nz oat**  **e**

0 0 0 0 0 0 0 0

1 1.25 7.86 0. 55 3. 54 4.9 9 7.44 26.91

2 4.08 13 .22 0. 54 9. 51 12.1 4 13.72 49.60

3 14.27 16 .76 4. 05 13 .56 15.3 3 17.18 60.60

4 17.71 19 .88 8. 41 17 .45 19.4 7 20.48 67.90

5 19.48 22 .42 11 .90 20 .79 22.8 9 22.84 73.19

6 20.99 25 .53 14 .09 23 .67 25.8 5 25.53 76.91

7 22.23 30 .40 15 .55 26 .55 29.3 6 30.10 79.54

8 24.20 34 .59 16 .73 28 .38 32.0 0 35.28 81.26

9 25.95 36 .97 17 .75 29 .76 33.7 4 37.88 82.50

10 27.33 38 .79 18 .60 31 .06 35.2 3 39.47 83.66

11 28.39 40 .01 19 .27 32 .03 36.2 9 40.72 84.57

12 29.21 40 .85 19 .82 32 .77 37.1 0 41.53 85.45

13 29.94 41 .53 20 .33 33 .32 37.8 8 42.20 86.25

14 30.61 42 .19 20 .86 33 .73 38.6 5 42.92 86.79

15 31.34 42 .97 21 .44 34 .61 39.4 9 43.84 87.82

16 32.08 43 .77 21 .96 35 .44 40.2 5 44.77 88.54

17 32.93 44 .60 22 .52 36 .20 41.1 3 45.60 89.33

18 33.62 45 .36 23 .01 37 .05 41.9 5 46.49 89.98

19 34.22 46 .04 23 .48 37 .83 42.7 3 47.71 90.58

20 34.74 46 .61 23 .90 38 .28 43.4 4 48.76 91.18

21 35.31 47 .23 24 .45 38 .82 44.1 6 49.72 91.78

22 35.71 47 .67 24 .86 39 .32 44.6 6 50.36 92.08

23 36.14 48 .11 25 .27 39 .78 45.1 5 51.05 92.50

24 36.64 48 .63 25 .80 40 .10 45.7 6 51.86 93.04

25 37.07 49 .09 26 .26 40 .61 46.2 9 52.59 93.44

26 37.45 49 .53 26 .66 41 .09 46.7 6 53.21 93.83

27 37.82 49 .93 26 .99 41 .34 47.1 6 54.13 94.26

28 38.28 50 .44 27 .42 41 .63 47.6 0 54.28 94.70 **Fi gu**  **re 21. A**  **LFOT ER**  **RA Hy** 

**dr oph**  **ob e 4P**  **O Su lf**  **at e Bio**  **deg rad**  **atio n S**  **tu dy** 

**OE CD 301F**  **The or** 

**etica l Oxyg**  **en dem**  **and**

## 12/14/2011 Page 66 of 66

**Ap pendix**  **22 : A**  **LFOTERRA Hydrophobe Biodegradation Study, Comparison of A**  **lcohol, Nonionic,**

**and A nionic** 

**Isa lche**  **m**

**23 Isal**  **ch em** 

**23 4P**  **O Isa**  **lch em** 

**23**

**4P O S** 

0 0 0 0

1 10.30 0.97 1. 25

2 18.92 3.64 4. 08

3 23.13 9.64 14 .27

4 27.75 16 .92 17 .71

5 33.45 22 .86 19 .48

6 38.46 29 .42 20 .99

7 43.08 35 .81 22 .23

8 47.99 41 .09 24 .20

9 53.21 45 .94 25 .95

10 57.97 50 .34 27 .33

11 61.95 54 .80 28 .39

12 65.29 58 .91 29 .21

13 68.15 62 .74 29 .94

14 70.92 66 .31 30 .61

15 73.54 69 .79 31 .34

16 76.03 73 .03 32 .08

17 78.28 75 .95 32 .93

18 80.58 78 .80 33 .62

19 82.75 81 .52 34 .22

20 84.79 84 .21 34 .74

21 86.78 86 .83 35 .31

22 88.77 89 .30 35 .71

23 90.58 92 .08 36 .14

24 92.25 94 .61 36 .64

25 93.84 97 .12 37 .07

26 95.31 99 .49 37 .45

27 96.74 10 1.92 37 .82

28 98.14 10 4.22 38 .28 **Fi gu**  **re 22.**  **AL FO**  **TE RR**  **A Hydr**  **op hob**  **e**

**Bio deg**  **rad atio**  **n S tu**  **dy**



### Ap **pendix 23**  **: Aq**  **uatic Toxicity Reports from Environmental Enterprise USA Laboratory**  **TSR No.**

**R&D ENV 10**

**- 004 01** 

**- 10** 



## Attached