## Biodegradability**and Aq**  **uatic Toxicity of**  **AL FOTERRA**  **® Surfactants** 

**Research Report R&D**  **ENV 06**  **- 001 01**  **- 06** 

**Al len M. Nielsen**  **Geoff Russell**

**Jul y**  **2006**

Research & Development Lake Charles, Louisiana

## Interoffice Correspondence

To Victoria Stolarski From Allen M. Nielsen, Geoff Russell Date July 12, 2006 **Subject R&D ENV 0**  **6 -**  **00 1**  **01 -**  **06 Biodegradability and**  **Aquatic Toxicity of ALFOTERRA® Surfactants**  **Key Words**  : ALFOTERRA surfactants **,** biodegradability , aquatic toxicity This report docume nts the biodegradability and aquatic toxicity data that Sasol North America has generated on the ALFOTERRA® surfactants. These data will be used to support marketing efforts of the ALFOTERRA® product line into numerous applications in the US and overseas. The conclusions of these studies are as follows: 1. As the proportion of propylene oxide (PO) increases in an ALFOTERRA® surfactant, the rate and extent of primary and ultimate biodegradation decreases. It appears that four (4) moles of PO is the cut- off between acceptable (> 60% Theoretical CO2 production (ThCO2) or > 70% dissolved organic carbon (DOC) loss in 28 days) and unacceptable ultimate biodegradation in the OECD “ready” biodegradation tests. 2. None of the following had a significant change on the biodegradability of ALFOTERRA® surfactants: (1) Changes in the alkoxylation catalyst ( NOVEL® II or KOH), (2) Changes in the hydrophobe carbon chain length from C11 to C15, nor (3) insertions of up to 2 moles of ethylene oxide (EO) between the hydrophobe a nd the PO. 3. S ulfation decreases biodegradation of these surfactants. 4. High levels of primary biodegradation occur with ALFOTERRA® surfactants but very little, if any, ultimate biodegradation occurs during laboratory simulations of secondary wastewater tre atment. 5. Acute freshwater aquatic toxicities for ALFOTERRA® surfactants are similar to analogous commercially available alcohol ethoxylates (EO). Varying the hydrophobe chain length from C11 to C15 and the amount of PO from 2 to 8 moles do not significantl y affect aquatic toxicities. The ISOFOL® based - ALFOTERRA® surfactants appear to be slightly less toxic. **Future Work:** Future studies have been planned to address the following objectives : 1. To determine conclusively if sulfation of propoxylates impedes or decreases the biodegradation rate when compared to the analogous alkoxylates

. 2. To determine if biodegradation rate is affected by capping the propoxylate with ethylene oxide before sulfation.R&D ENV 06- 001 01 - 06

Page 3

3. To determine if sulfonation of alcohol propoxylates influence s biodegradation differently than su l fation. 4. To extend the above biodegradation work to hydrophobe chain length greater than C15. 5. To reconfirm the apparent poor ultimate biodegradation of ALFOTERRA® surfactants in simulated waste water treatment. Di stribution: D.T. Plummer, M.F. Cox, K.B. Youngblood, K. L. Matheson, K.K. Kinsley, M. Bose, J. Hackett, T.G. Grumbles, D. Penney, P. Filler, K. Sharp, F. Monterisi, E. Dallara, G. C assani, C. Schrö der , R. Cohen, D. Schär, Library, LivelinkR&D ENV 06- 001 01 - 06

Page 4 **INTRODUCTION** A LFOTERRA® surfactants have been developed by Sasol North America for many potential applica tions. ALFOTERRA® surfactants are based on Sasol’s non - linear alcohols (C11 - C18) such as ISALCHEM ®, ISOFOL®, TDA, LIAL® or SAFOL TM which are propoxylated ( 2 to 8 moles) and sulfated. They have been successfully used as the surfactant for clean - up of ground water contaminated with chlorinated hydrocarbons o r heavy oils. Numerous industrial and academic organizations have received samples of ALFOTERRA® surfactants for testing in a variety of additional applications. Most recently an opportunity has arisen for the possible use of ALFOTERRA® surfactants in Enhance d Oil Recovery (EOR)

. However, the use of ALFOTERRA® surfactants in the US will require submission of a US EPA Pre - Manufacturer Notification (PMN ) before commercial sales are allowed. Over the past several years some environmental fate and effects data has been generated by SNA which will be of value and interest as various applications of ALFOTERRA® surfactants are pursued. Specifically, multiple studies have addressed the effects of modifying the chemical structure of branched alcohol ethoxide or propoxide sulfates on the ultimate biodegradability including; (1) amount of propylene oxide ( PO ), (2) change s in the carbon chain length of the hydrophobe, (3) insertion of ethylene oxide (EO) between the hydrophobe and the PO, and (4) presence of a sulf( on )ate group

. Additional studies addressed the influence of the alkoxylation catalyst on the biodegradability of non - sulfated EO/PO alkoxylates

. 1,3 The detailed plan outlining the systematic examination of ALFOTERRA ® surfactants and the corresponding alkoxylates can be found in Research Project Plan AUS SBU 04 - 033

. 2 T he following report was written to documen t and summarize the available ALFOTERRA® surfactants’ environmental fate and effects data in a form that will be helpful in (1) evaluating the effects of selected changes to the chemical structure, (2) evaluating the effects of changing alkoxylation catal ysts and (3) supporting marketing efforts of the ALFOTERRA® product line. **OBJECTIVES**

 Determine the primary and ultimate biodegradability and aquatic toxicity profiles of ALFOTERRA® surfactants.  Estimate the biological removal of ALFOTERRA® surfactants in laboratory simulations of secondary wastewater treatment.  Evaluate the affect of hydrophobe chain length and propylene oxide (PO) quantity on biodegradability.  Examine the idea that insertion of ethylene oxide (EO) improves the biodegradability of ALFOT ERRA® surfactants.  Uncover any influence of the alkoxylation catalysts (KOH or NOVEL II) on the biodegradability of the ALFOTERRA® surfactants and the corresponding alkoxides.  Compare biodegradation data of an ALFOTERRA® surfactant and its alkoxylate analo gue to determine if sulfation has an effect.  Review freshwater aquatic toxicity data and correlate the results to structural changes in the ALFOTERRA® surfactant

. **CONCLUSIONS**

 As the proportion of propylene oxide (PO) increases in an ALFOTERRA® surfactan t, the rate and extent of primary and ultimate biodegradation decrease. It appears that four (4) moles of PO is the cut off between acceptable ( > 60 % Theoretical CO2R&D ENV 06- 001 01 - 06

Page 5 production ( ThCO2) or > 70% Dissolved Organic Carbon (DOC) loss in 28 days) and unacceptable ultimate biodegradation in the OECD “ready” biodegradation tests.

 None of the following had a significant effect on the biodegradation of ALFOTERRA® surfactants: (1) Changes in the alkoxylation catalyst ( NOVEL® II or KOH), (2) changes in the hydro phobe carbon chain length from C11 to C15 nor (3 ) insertion of up to 2 moles of EO prior to the propylene oxide.

 Surprisingly, it appears that sulfation decreases biodegradation of these surfactants.

 High levels of primary biodegradation occur with ALFO TERRA® surfactants but very little, if any, ultimate biodegradation occurs during laboratory simulations of secondary wastewater treatment.

 Acute freshwater aquatic toxicities for ALFOTERRA® surfactants are similar to analogous commercially available alco hol ethoxylates (AE). Varying the hydrophobe chain length from C11 to C15 and the moles of PO from 2 to 8 moles do not significantly affect aquatic toxicities. The I SFOL® alcohol based - ALFOTERRA® surfactants appear to slightly less toxic. **EXPERIMENTA L MA** 

**TERIA LS**  **AND METHODS Test Materials**  **AL FOTERRA** 

**® 123 -** **2S -**  Sample number for the propoxylate = V1110 - 170, M W of ISALCHEM ®123Alcohol = 194 mg/mmole, M W of PO = 123.5 mg/mmole, W ater = 327 ppm, Hydroxyl number = 189.0 mg KOH/g, PPG = 2.76 wt %, Free Alco hol = 10.19 wt %, NMR moles PO = 2.13, PO = 38.91 wt %, Average MW of propoxylate = 317.54 mg/mmole. Sample number for sulfate - V1086 - 30 - 2, Active = 26.4 wt %, Free oil = 0.86 wt %, Sodium Sulfate = 0.26%. KOH was the propoxylation catalyst. **AL FOTERR** **A®**

**123 -** **4S** - Sample number for propoxylate = V1110 - 174, MWof ISALCHEM ®123 Alcohol

– 194 mg/mmole, M W PO = 232.58 mg/ m mole, Water = 245 ppm, Hydroxyl number = 141.2 mg KOH/g, PPG = 4.78 wt %, Free alcohol = 1.49 wt %, NMR moles PO = 4.01, PO = 54.52 wt % , Average MWof propoxylate = 426.58 mg/mmoles. Sample number for sulfate - V1086 - 30 - 3, Active = 29.8 wt %, Free oil = 0.76 wt %, Sodium Sulfate = 0.32 wt %. KOH was the propoxylation catalyst. **AL FOTERRA** 

**® 123 -**  **8S -**  Sample number for propoxylate = V108 6 - 24 - 1, MWof ISALCHEM ®123 Alcohol = 194 mg/mmole, M W PO = 425.14 mg/mmole, W ater = 583 ppm, Hydroxyl number = 93.8mgKOH/g, PPG = 1.37%, Free alcohol = 0.27 wt %, NMR moles PO = 7.33, PO = 68.6y wt %, Average MW of propoxylate = 425.14 mg/mole, Sample nu mber for sulfate - V1086 - 25 - 1, Active = 28.8wt %, Free oil = 1.8 wt %, Sodium Sulfate = 0.28 wt %, sodium bicarbonate 0.13 wt %. KOH was the propoxylation catalyst. **AL FOTERRA** 

**® 145 -** **2S –**  Sample number for propoxylate = V1110 - 166, MWof ISALCHEM ® 145 Alcoh ol = 219 mg/ m mole, M W PO = 108.46 mg/mmole, Water = 481 ppm, Hydroxyl number = 183.8 mg KOH/g, PPG = 5.25 wt %, Free alcohol = 14.34 wt %, NMR moles PO = 1.87, PO = 33.12 wt %, Average M W of propoxylate = 327.46 mg/mmole, Sample number for sulfate - V 1 086 - 30 - 1, Active = 32.5 wt %, Free oil = 0.92 wt %, Sodium Sulfate = 0.30 wt %. KOH was the propoxylation catalyst.R&D ENV 06- 001 01 - 06

Page 6

**AL FOTERRA** 

**® 145 -** **4S –**  Sample number for propoxylate = V1086 - 22 - 1, M W of ISALCHEM ® 145 Alcohol

– 219 mg/mmole, M W PO = 207.6 mg/mmole, W ater = 245 ppm, Hydroxyl number = 130.4 mg KOH/g, Free Alcohol = 1.07 wt %, PPG = .99 wt %, NMR moles PO = 3.58, PO = 48.67 wt %, Average MWof propoxylate = 426.64 mg/mmole, Sample number for sulfate

– V1086 - 23 - 1, Active = 29.5 wt %, Free oil = 0.86 wt %, Sod ium Sulfate = 0.17 wt%. KOH was the propoxylation catalyst. **AL FOTERRA** 

**® I 12 -**  **3S** - Sample number for propoxylate = V1149 - 108 - 3, M W of ISOFOL® 12 Alcohol

– 186 mg/mmole, M W PO = 185.72 mg/mmole, Water = 581 ppm, Hydroxyl number = 183.4 mg KOH/g, Free Alcoh ol = 4.54 wt %, PPG = 13.27 wt %, NMR moles PO = 3.2, PO = 50.10 wt %, Average MWof propoxylate = 370.70, Samp mg/mmole, Sample number for sulfate

– V1086 - 30 - 5, Active = 30.1 wt %, Free oil = 1.72 wt %, Sodium Sulfate = 0.1 wt %. %. KOH was the propoxylation catalyst. **AL FOTERRA** 

**® S 23 -**  **3S**

– Sample number for propoxylate = V1086 - 151 - 1, M W of SAFOL TM 23 Alcohol = 193.9 mg/mmole, Hydroxyl number = 154.3 mg KOH/g, Free Alcohol = 0.6 wt %, PPG = 1.7 wt %, NMR moles PO = 2.93, Average M W of propoxylate = 363.6 mg/ mmoles, Sample number for sulfate

– V1086 - 151 - 1a also V1191 - 164 - 15, Active = 30.0 wt %, Fre e oil = 1.3 wt %, Sodium Sulfate = 0.20 wt %, Average MW of sulfate = 466.6 mg/mmole. KOMe was the catalyst. **AL FOTERRA** 

**® S 23 -**  **5S**

– Sample number for propoxylate = V1086 - 151 - 2, M W of SAFOL TM 23 Alcohol = 193.9 mg/mmole, Hydroxyl number = 122.5 mg KOH/g, Free Alcohol = 0.3 wt %, PPG = 4.3 wt %, NMR moles PO = 4.85, Average M W of propoxylate = 458.0 mg/mmole, Sample number for sulfate

– V1086 - 151 - 2a also V1191 - 164 - 16, Active = 31.6 wt %, Free oil = 2.3 wt %, Sodium Sulfate = 0.15 wt %, Average M W of sulfate = 561.0 mg/ mmoles. KOMe was the catalyst. **AL FOTERRA** 

**® S 23 -**  **8S**

– Sample number for propoxylate = V1086 - 151 - 3, M W of SAFOL TM 23 Alcohol = 193.9 mg/mmole, Hydroxyl number = 99.5 KOH/g, free Alcohol = 0.4 wt %, PPG = 4.5 wt %, NMR moles PO = 7.59, Average M W of proproxylate = 564.0 mg/mmole , Sample number for sulfate

– V10860151 - 3a and V1191 - 164 - 17, Active = 30.6 wt %, Free oil = 1.5 wt %, Sodium Sulfate = 0.13 wt %, Average M W of sulfate = 667.0 mg/mmole. KOMe was the catalyst. **AL FOTERRA** 

**® S 23 -** **4S –**  Sample nu mber for the sulfate = V1269 - 11 - 2, Active = 28.2 wt %, free oil = 0.4 wt %. Four mole propoxylate made from SAFOL TM 23 Alcohol using KOH as the catalyst. **AL FOTERRA** 

**® S 23 -** **4S** - Sample number for the sulfate = V1269 - 11 - 1, Active = 27.0 wt %, Free oil = 0.59 %. Four mole propoxylate made from SAFOL TM 23 Alcohol using the NOVEL® II catalyst. **AL FOTERRA** 

**® S 45 -** **2S**

– Sample number for the sulfate = V1269 - 12 - 1, Active = 24.6 wt %, Free oil = 0.63 wt %. Two mole propoxylate made from SAFOL TM 45 Alcohol using KOH as the catalyst. **AL FOTERRA** 

**® S 45 -** **2S**

– Sample number for the sulfate = V1269 - 12 - 2, Active = 24.6 wt %, Free oil = 0.6 3 wt %. Two mole propoxylate made from SAFOL TM 45 Alcohol using the NOVEL® II catalyst.R&D ENV 06- 001 01 - 06

Page 7 **AL FOTERRA** 

**® S 23 -** **4S**

– Sample number for the sulfate = V1269 - 8, Active = 29.2 wt %, Free oil = 0.35 wt %. Four mole propoxylate made from SAFOL TM 23 Alcohol using the NO VEL® II catalyst. **AL FOTERRA** 

**® S 23 -**  **0.5 EO -** **4S**

– Sample number for the sulfate = V1269 - 30, Active = 29.5 wt %, Free oil = 0.25 wt %. O.5 mole ethoxylate + four moles propoxylate made from SAFOL TM 23 Alcohol using the NOVEL® II catalyst. **AL FOTERRA** 

**® S 23 -**  **1.0 EO**  **- 4S**  - Sample number for the sulfate = V1269 - 31, Active = 29.0 wt %, Free oil = 0.33 wt %. One mole ethoxylate + four moles propoxylate made from SAFOL TM 23 Alcohol using the NOVEL® II catalyst. **AL FOTERRA** 

**® S 23 -** **2.0 EO -** **4S**

– Sample number for the sulfate = V1269 - 32, Active = 29.3 wt %, Free oil = 0.32 wt %. Two mole ethoxylate + four moles propoxylate made from SAFOL TM 23 Alcohol using the NOVEL® II catalyst. **AL FOTERRA** 

**® S 23 -** **2.0 EO -** **4S**

– Sample number for the sulfate = V1269 - 29, Active = 29.7 wt %, Free oil = 0.44 wt %. Two moles ethoxylate + four moles propoxylate made from SAFOL TM 23 Alcohol using KOH as the catalyst. **C12 derived LA S** 

– Sample number VO610 - 129 - 2, Active = 51.2 wt %, free oil = 1.25 wt %, Sodium Sulfate = 1.37 wt %. Aluminum Chloride alkyla tion of C12 olefin which is sulfonated and neutralized to a sodium salt. **JA PA**  **NESE ABS**  **AL KA**  **NE -**  **60**

– Sample number 4998 - 191, Active = 51.36%, free oil = 2.5 wt %, Sodium Sulfate = 0.86 wt %. Conventional polypropylene based alkylate which is highly branched in the C12 alkyl chain which is then sulfonated and neutralized to give the sodium salt. **NA LKYLENE® 550L derived LA** **S**

– Sample number

– V1086 - 20 - 1, Active = 51.84 wt%, oil = 1.26 wt %, Sodium Sulfate = 0.71 wt %. Average alkyl chain length = C11.69. **Bio test Sy** **stems The OECD 301A**  **: DOC die -** **aw ay test**  was done as described in the OECD Guideline for Testing of Chemicals adopted July 17,1992. This is a 28 - day aerobic biodegradation die - away test on 10 - 30 mg/L water soluble organic compounds (measured as dis solved organic carbon

– DOC) done in mineral salts medium inoculated with ≤ 30 mg/L total suspended solids (TSS) from natural sewage/soils or surface waters. Ultimate biodegradation is determined by measuring the DOC concentrations in the test flasks during the test. Primary biodegradation can also be measured by using some compound specific methodology such as methylene blue active substance (MBAS). The inoculum was derived fro m activated sludge obtained from the Anderson Mill Municipal W ater Reclamation Facility (Austin, TX) and the supernatant from a suspen sion of 100 g soil in 500 mL of the mineral salts medium. The test was done at a starting concentration of 20 mg DOC/L for the test materials. Toxicity tests were done with 20 mg DOC/L test surfactant and 20 mg DOC/L sodium acetate. The sampling and det ermination of DOC concentrations were done as described in Research Report 6951 - 01 - 22 - 92. Concentrated sulfuric acid (1 drop) was used to preserve each 30 mL DOC sample. The determination of MBAS was done as described in Sasol North America Analytical Me thod V1.025 - 85. The MBAS samples were preserved by the addition of 1% by volume formaldehyde. The details of this test are found in SNA Research Notebook V1160 pages 97 - 135 and SNA Research Notebook V1086 pages 73 - 91. The test started on 9/21/99 and was finished on 11/30/99.R&D ENV 06- 001 01 - 06

Page 8 **The OECD 302A**

**Inherent Biodegradability : Modified SCA** **S Test** was done as described in the OECD Guideline for Testing Chemicals adopted May 12, 1981. The inoculum was derived from activated sludge obtained from the Anderson Mill Mun icipal W ater Reclamation Facility (Austin, TX) . The test was done at a starting concentration of 20 mg DOC/L for the ALFOTERRA® surfactants and 20 mg active/L for the C12 LAS and ABS surfactants. The MBAS and DOC analyses were done as described in the OE CD 301A test. The test was started on 10/19/99 and was completed on 11/24/99. The details are in SNA research notebook V1160 pages 119 - 147 and SNA research notebook V1086 pages 71 - 97. **The OECD 301F Manometric Respirometry Test**  was done as described in the OECD Guideline for Testing Chemicals adopted July 17, 1992 with the following minor modifications: (1) reduction in test material concentration from 100 mg to 20 mg to reduce possible toxicity to the inoculum, (2) reduction in the pH from 7.4 to 7.0 to improve the efficiency of CO2 removal, and (3) reporting of % ThCO2 ( t heoretical maximum production of carbo n dioxide) instead of % ThO2 ( t heoretical maximum uptake of oxygen) to improve the accuracy of detecting ultimate biodegradation. The biodegra dation test was done on the ALFOTERRA® surfactants in quadruplicate using two 20 channel Coordinated Environmental Services (CES) Ltd (Cornwell, UK) aerobic electrolytic respirometer units following the OECD 301 F guideline with slight modifications (as no ted above). The workings of the CES respirometer units and the collection of the CO2 data are described in Research Report R&D ENV 05 - 006 01 - 05. The inoculum for these tests was derived from the recycled sludge from the City of Lake Charles, Louisiana Domestic Wa ste W ater Plant A. The sludge was aerated ~ 7 days prior to use. The inoculum in the test medium was adjusted to a final concentration of < 30 mg TSS/L. Sodium benzoate ( J.T. Baker

– 99% purity was used as the positive reference material). Inoculum toxicity tests with 20 mg sodium benzoate and 20 mg of the ALFOTERRA® surfactant were also completed. The details of this test are given in SNA research notebooks V1240 page 119 and V1255 page 69.

. **Definitive A cute A** **quatic Toxicity Tests on**  **Daphnia magna and Pimephales promelas** were done on several ALFOTERRA® surfactants and N ALKYENE ® 550L derived linear alkyl benzene sulfon ate ( LAS ) by ENSR ( Ft. Collins, CO). The methods used for doing these tests were detailed in US EPA Met hod EPA/600/4 - 90/02/027F

– 1993 and ASTM Method E729 - 96

– 1998. ENSR reports can be found in Appendix 1. **The OECD 301B CO2 Evolution ( Modified “**  **Sturm ”**  **Test) Biodegradation Test** was done as described in the OECD Guideline for Testing Chemicals a dopted July 17, 1992 on several propoxylates derived from SAFOL TM and ISALCHEM ® alcohols. These data were generated at contract laboratories in Europe at the request of Sasol Germany. These data are included in this report for comparative purposes only. **RESULTS AND DISCUSSION**  **Effect of PO on Primary and Ultimate B**  **iodegradation of ISA** **LCHEM ® ,**  **ISOFOL ® and**  **SA FOL**  **TM based -**  **AL FOTERRA**  **® Surfactants in OECD Screening Biodegradation Tests** The biodegradability of multiple ALFOTERRA® surfactants were tested using OECD 301A and 301F methods. Results showing the effect of PO on biodegradation of ALFOTERRA® surfactants are detailed in Table 1. Figure 1 shows the biodegradation trends as a function of moles of PO. The data in figure 1 was developed by averaging the biodegradation valuesR&D ENV 06- 001 01 - 06

Page 9 shown in Table 1 for the surfactants wit h the same number of moles of PO, irrespective of the hydrophobe. Complete documentation of these data is found in Tables 7, 8, 8A and Table 9. From these data it is clear that as the proportion of PO increases in the ALFOTERRA® surfactant, the rate and extent of primary and ultimate biodegradation decreases. **Table 1. Effect of PO on Primary**  **and Ultimate Biodegradation in OECD Screening Tests**

**AL FOTERRA** 

**® Surfactant OECD 301A**  **OECD 301F % M**  **BA S Loss**  **% DOC Loss % ThCO**  **2** Pass 10 - day Wi ndow 28 - Day

End Point Pass 10 -

day Wi ndow 28 - Day

End Point Pass 10 -

day Wi ndow 28 - Day

End Point ISALCHEM 123 - 2S Yes 98.2 No 52.1 -- -- ISALCHEM 145 - 2S Yes 96.4 No 54.5 -- -- ISOFOL I12 - 3S Yes 96.3 No 46.4 -- -- ISOFOL 123 - 4S Yes 91.1 N0 28.7 -- -- ISOFOL 145 - 4S Yes 88.9 No 3 3.9 -- -- ISOFOL 123 - 8S No 46.1 No 10.9 -- -- SAFOL TM S45 - 2.S a No 64.2 SAFOL TM S23 - 3S No 60.1 SAFOL TM S23 - 4S a No 55.5 SAFOL TM S23 - 5S No 42.4 SAFOL TM S23 - 8S No 43.3 a. Average of data from surfactants made from NII or KOH alkoxylatio n catalysts found in table 9. **Figure 1. Effect of PO on Primary**  **and Ultimate Biodegradation of ISA LCHEM® 123, 145,**  **and SA FOLTM 23, 45 A** **lcohol Based A LFOTERRA**  **® Surfactants in OECD Screening Tests** 

0 10 20 30 40 50 60 70 80 90 100

0 1 2 3 4 5 6 7 8 9 **Moles of PO B** **i o** **d e** **g r** **a d** **a t** **i o** **n** % MBAS Loss % DOC Loss % ThCO2

(a) Interpolated from 4 mole and 8 mole data (a)R&D ENV 06- 001 01 - 06

Page 10 Primary biodegradation was measured by methylene blue active substance (MBAS) analytical method which detects any alteration to or biological cleavage of the original surfactant. Ultimate biodegradation , or complete bre ak down of the molecule, was measured by the loss of total soluble organic carbon (DOC). It is also measured by the detection of CO2 produced by the biological activity (%ThCO2). Wh ile the high MBAS values suggests the rate and extent of primary biodegrad ation of ISALCHEM ® and ISOFOL® based - ALFOTERRA® surfactants with ≤ 4 moles PO is extensive , the ultimate biodegradation was not acceptable (< 70% DOC removal in 28 days in the OECD 301 D test). On the other hand, the SAFOL TM based - ALFOTERRA® surfactants with < 4 moles PO did show acceptable ultimate biodegradation (> 60% ThCO2 in 28 days in the OECD 301 F test). Table 8A contains data that confirms that ALFOTERRA® surfactants are not toxic to the inoculum

. It appears that if an ALFOTERRA® surfactant ha s ≥ 4 moles PO, it will not undergo “ready” biodegradability in the OECD Biodegradation screening tests. It also is apparent that changes in carbon chain length of the alcohol hydrophobe from C11 to C15 have very little, if any, effect on the rate or exte nt of biodegradation of ALFOTERRA® surfactants. These data are summarized in Table 1. The u se of KOH or NOVEL® II as the alkoxylat ion catalyst appears to have an insignificant effect on the biodegradation of the resulting ALFOTERRA® surfactant. As shown in table 2, the degradation end - points for surfactants made from either KOH or NOVEL II catalyst are not significantly different. **Table 2. Effect of PO Cataly** **st a**  **on Ultimate Biodegradation of SA** **FOL TM Based A**  **LFOTERRA ®**  **Surfactants AL**  **FOTERRA**

**® Surfactant (**  **SA FOL**  **TM) OECD 301F**  **Pass 10 -**  **Day Window 28**  **- Day**  **End Point Mean ± Std. Dev.**  S23 -4 NII S No 46.5 ND b S23 -4 NII S No 69.9 ± 10.3 S23 -4 KOH S No 50.0 ± 2.9 S23 - 2 EO -4 NII S No 59.2 ± 6.1 S23 - 2 EO -4 KOH S No 56.6 ± 8.8 S45 - 2 NII S No 62.3 ± 0.5 S45 - 2 KOH S No 66.1 ± 1.8

a NII = NOVEL® II catalyst, KOH = potassium hydroxide b ND = Not determined, only one value Insertion of up to 2 moles of EO before the 4 moles of PO in SAFOL TM based - ALFOTERRA® surfactants had very little, if any, effect on the rate or extent of ultimate biodegradation i n the OECD 301 F screening test as shown in Table 3 and documented in Table 9. All values are within the standard deviation of each other.R&D ENV 06- 001 01 - 06

Page 11 **Table 3. Effect of E**  **O Insertion on Ultimate Biodegradation of SA** **FOL TM Based A**  **LFOTERRA ®**  **Surfactants in OECD Screening Test AL**  **FOTERRA**

**® Surfactant (**  **SA FOL**  **TM) OECD 301F**  **Pass 10 -**  **Day Window 28**  **- Day**  **End Point Mean ± Std. Dev.**  S23 -4 S No 55.5 ± 12.6 a S23 - 0.5 EO -4S No 59.7 ± 5.6 S23 - 1.0 EO -4S No 60.8 ± 8.8 S23 - 2.0 EO -4S No 57.9 ± 7.5 a. Average of values from Table 9. Sulfation apparently does have a significant effect on the ultimate biodegradation of ALFOTERRA® surfactants. That is, the addition of the sulfate group significantly decreases the biodegradation that was observed for the parent alkoxylate. This effect was demonstrated by the data in Table 4 and by the documentation in Table 9 and 10. Although the sulfate and the alkoxylates were tested at different times and with slightly different methods, the significant dif ference in the %ThCO2 after 28 days leads to the conclusion that ultimate biodegradation was impeded by the sulfate group. That is, the sulfated 2 - mole EO / 4 - mole PO SAFOL based surfactant showed only 58% ThCO2 at the end of the 28 - day test while the parent alkoxylate showed 96%. **Table 4. Ap**  **parent Effect of Sulfation on Ultimate Biodegradation in OECD Screening Tests AL**  **FOTERRA**

**® (**  **SA FOL**  **TM) OECD 301F**  **OECD 301**  **B**

**Pass 10 -**  **Day Windo** **w 28**  **- Day**  **End Point %ThCO**  **2 Pass 10**  **- Day**  **Windo w**  **28 -**  **Day End**  **Point %ThCO 2**  S 23 - 2EO -4 PO - S No 57.9 **Al koxy**  **late (**  **SA FOL**  **TM)** S23 - 2EO -4PO Yes 95.7 S23 - 5EO -4PO Yes 96.1 S23 - 5EO - 5PO Yes 87.7 S23 - 5EO - 6PO Yes 86.1 This observation does not seem logical since sulfatase enzymes are widely distributed in nature. I t would be expected that the sulfate group should be easily and rapidly removed in biodegradation tests. (The high level of primary biodegradation noted above in Figure/Table 1 is consistent with this idea). However, MBAS reduced detection is not a specific indicator of sulfate removal but a measure of any change in the molecule structure. Th e decrease in biodegradation by the addition of the sulfate group has been reported in earlier work. A 1979 paper by Pitter showed that sulfation of nonylphenol ethoxylates deteriorates the bio degradation of these substances when compared to their alkoxylates. **Biological Removal of ISA**  **LCHEM ® and**  **ISOFOL ® based**  **- AL**  **FO TERRA**  **® Surfactants in Simulation of Secondary**  **Waste water Treatment (SCA** **S test)** ALFOTERRA® surfactants were evaluated by the SCAS test method which simulated the biological breakdown expected during processing at a water treatment facility. It is clear fro m the summary of SCAS data in Table 5 and more complete documentation in Tables 11,11A thatR&D ENV 06- 001 01 - 06

Page 12 all ALFOTERRA® surfactants will un dergo some primary biodegradation when in contact with the biomass in water treatment sludge . However, only those ALFOTERRA® surfa ctants with ≤4 moles of PO showed levels greater than 80% . This high level of primary biodegradation was probably due to the sulfatase enzymes activity present in the biomass. Only those ALFOTERRA® surfactants with ≤ 2 moles PO showed any (17 - 27%) ultimate b iodegradation as measured by loss of DOC. The ALFOTERRA® surfactants with more than 2 moles of PO did not undergo any measurable ultimate biodegradation at all . This result was similar to that observed for the propylene - based highly- branched ABS which wa s included as a negative control . In contrast, C12 LAS , added as a positive control, showed nearly complete ultimate biodegradation (97.5% DOC loss) in the SCAS test as expected

. The lack of ultimate biodegradation of the ALFOTERRA® surfactants was co n firmed by the loss of biomass ( measured by loss of TSS) in the SCAS units fed ALFOTERRA® surfactants as shown in Figure 2. Note that the loss of b iomass (TSS) in the uni ts fed ALFOTERRA® surfactants was even more rapid than that observed in the blank uni t which wa s fed only the background feed suggesting that the ALFOTERRA® surfactants were not a sufficient food source to support the biomass colony

. On the other hand , SCAS units fed the rapidly biodegraded C12 LAS showed an increase in biomass, as measured by TSS, over the same time period. **Table 5. Biological Removal of**  **ISA LCHEM**  **® -**  **based A LFOTERRA**  **® Surfactants in Laboratory Simulation of Secondary**  **Waste water Treatment (SCA** **S) AL**  **FOTERRA**

**® (**  **ISA LCHEM**  **®) %MBA** **S Loss %DOC Loss**  123 - 2S 99.8 17.2 145 - 2S 97.0 26.9 I12 - 3S (ISOFO**®** L) 88.4 0.0 145 -4S 82.8 0.0 123 - 8S 51.9 0.0 **Al ky**  **lbenzene Sulfonates** C12 LAS 99.8 97.5 ABS 37.7 0.0R&D ENV 06- 001 01 - 06

Page 13 **Figure 2.**

**Loss of TSS in SCA**

**S Units Fed ISA LCHEM**  **® or ISOFOL® Based A LFOTERRA®** 

**Surfactants**

0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750

0 1 2 3 4 5 6 7 8 9 10 11 12 **Day s** **T S** **S (** **m g** **/ L** **)** 2 Moles PO 3 Moles PO 4 Moles PO 8 Moles PO Blank

From the SCAS tests, it can be concluded that the I SALCHEM ®, and ISOFOL® based ALFOTERRA® s urfactants with ≤4 moles PO undergo high levels of p rimary b iodegradation. However, the ALFOTERRA® s urfactants , regardless of PO addition levels, undergo very little or , in some cases, no u ltimate b iodegradation during s imulation of s econdary waste water t reatment

. **Ac ute Freshwater A** **quatic Toxicity of**  **ISA LCHEM**  **® and ISOFOL** **® based A LFOTERRA**  **® Surfactants**  The results of acute aquatic toxicity tests for the ALFOTERRA® surfactants to invertebrates

– water fleas ( Daphnia magna) and fresh water fish - fathead mi nnows ( Pimephales promelas) are summarized in Table 6. The ENSR reports are found in Appendix one. **Table 6. Ac**  **ute A quatic Toxicity**  **of A LFOTERRA** 

**® Surfactants AL**  **FOTERRA**

**® Surfactant Daphnia magna**  **48 -**  **hr. LC 50**  **mg/L (95% CL) Pimephales promelas**  **96 -**  **hr. LC 50**  **m g/L (95% CL)**  123 - 2S 2.06 (1.76 - 2.29) 4.65 (4.27 - 5.08) 123 - 8S 2.65 (2.12 - 3.20) 1.68 (1.56 - 1.81) I12 - 3S 8.88 (6.51 - 9.85) 10.99 (10.23 - 11.80) 145 - 2S 2.19 (1.86 - 2.49) 2.31 (2.14 - 2.48) 145 -4S 2.34 (2.01 - 2.73) 1.62 (1.51 - 1.74) **Comparison Surfactant** N - 550L LAS 11.93 (10.02 - 14.48) 3.89 (3.55 -4.21)R&D ENV 06- 001 01 - 06

Page 14 Tests were done at ENSR, Ft. Collins, Colorado. Test numbers were 8503 - 111 - 006 - 15, 16, 17, 18, 19, 20 and 8503 - 106 - 006 - 001, 002, 003, 004, 005, 006, 007, 008. These results were reported in ENSR letters dated March 18, 1999, and September 24, 1999. The acute freshwater aquatic toxicity for the ALFOTERRA® surfactants are similar to those for analogous commercially available alcohol ethoxylates suggesting that the toxicity is acceptable

. Carbon chain length (C11 - C1 5) of the hydrophobe and moles of PO (2 to 8 moles) do not appear to be significant factors. The ISOFOL® based - ALFOTERRA® surfactant may be slightly less toxic than the I SALCHEM ® based s urfactants . **Future Work** There is a common percep tion that sulfat ed materials should be easily biodegraded by the plethora of sul fatase enzymes occurring naturally in soil and water. However, the above work suggests that the rate and extent of biodegradation of the sulfated ALFOTERRA® surfactants were significantly les s than that of their unsulfated counterparts. This theory has also been proposed in earlier work by Pitter and Fuka. 3 It is proposed that the conformational arrange ment of the propoxylate sulfate provide s a clue to the poor biodegradation observed

. Figure 3 shows a 3 - D space filling model of a linear ethoxylate sulfate, a branched ethoxylate sulfate, and a branched propoxylate sulfate. Note the difference in the packing around the sulfate head. It is believed that the bulkiness of the propoxylate group s urrounds the sulfate and thus, may impede the attack by the sulfatase enzymes. This impact in fact is greater than the effect observed for sulfated ethoxylates.



# Alcohol Ether Sulfate Types of Surfactants

C14 EO3 - sulfate (linear alcohol) 

### C14 EO3 - sulfate (C4 branch at the #2 carbon) 

### C14 PO3 - sulfate (C4 branch at the #2 carbon; Propylene oxide instead of ethylene oxide) **Figure 3. Space filling model for various alkoxy**  **late sulfat e.**  Continuing with this hypothesis, these models also explain why neither the choice of alkoxylation catalyst nor the insertion of EO showed an improvement to the biodegradation of the sulfated propoxylates. Neither condition changed the crowding around the sulfate group.R&D ENV 06- 001 01 - 06

Page 15 In a 1986 journal article, Gerike discusses biodegradability of propoxylates. 4 From his work it can be inferred that the biodegradability of alcohol propoxylates will improve with the addition of ethylene oxide after the PO ; if this h olds true then a similar affect should be seen with the sulfated version. Thus, comparing the propoxylate sulfate to a propoxylate sulfate with EO inserted between the two functional groups will be one of the objectives of the future work To continue to derive the structure/property relationship of alkoxylate sulfates the following objectives will be addressed in future work : 1. To determine conclusively if sulfation of propoxylates impedes or decreases the biodegradation rate when compared to the analogou s alkoxylates. 2. To determine if biodegradation rate is affected by capping the propoxylate with ethylene oxide before sulfation. 3. To determine if sulfonation of alcohol propoxylates influences biodegradation differently than su l fation. 4. To extend the above biodegradation work to hydrophobe chain length greater than C15. 5. To reconfirm the apparent poor ultimate biodegradation of ALFOTERRA® surfactants in simulated waste water treatment. **AC KNOWLEDGEMENTS**  The authors acknowledge Mark Bose for doing the DOC analysis; Rachel W ashburn for execution of the hand - shake MBAS analysis and Rebekah Cohen for excellent help with the data handling and execution of the electrolytic respirometer (OECD 301F) tests. **REFERENCES** 1. Rowe, L., Steiniger, D., Matheson, L. , and Russell , G

. 2004. Analytical and Performance Results for Commercially- Produced Biodac 2 - 32 and Lab - Produced NOVEL® II Biodac 2 - 32 Alkoxylates. SNA Research Report AUS ANL 03 - 004 01 - 03. 2. Russell, G. 2005. Gate B Document: Phase II ALFOTERRA® Surfacta nts: Improving the Biodegradability of ALFOTERRA® S urfactants, Alkyl Propoxylate Sulfates. 3. Schoenkaes, U., Grothe, R., and Mayer U., 2004. Low-foaming Surfactants Based on SAFOLTM 23 Alcohols, Project Marl TE 97/005 (TDO 153): Development of Biodegradable Alcohol EO - PO Derivatives. Technical Report FEA 5, April 15, 2004. 4. Pitter, P., and Fuka, T. Biodegradation of Non - sulfated and Sulfated Nonylphenol Ethoxylate Surfactants. Environment Protection Engineering (1979), 5 (1), 47 - 56 5. Gerike, P., Jasiak, W ., 1986. How Completely are Surfactants Biodegraded?. Tenside Detergents , 23 (1986) 6; pg 300 - 304.R&D ENV 06- 001 01 - 06

Page 16 **Table 7. Biodegradation of A** **LFOTERRA ® Surfactants in the OECD 301A**  **T est**  **Surfactant % MBA** **S Loss 1**  **6 9**  **10 13**  **15 17**  **22 28**  ALFOTERRA® 123 - 2S Std. Dev. 5.1 ± 0.5 95.8

± 0.2 97.4

± 0.2 97.6

± 0.3 97.2

± 1.5 98.4

± 0.1 97.5

± 0.2 98.3

± 0.1 98.2

± 0.2 ALFOTERRA® 123 -4S Std. Dev. 10.1 ± 5.5 87.6

± 2.2 86.9

± 0.4 86.8

± 0.3 88.6

± 0.2 91.5

± 0.6 89.0

± 0.6 --

-- 91.1 ± 0.6 ALFOTERRA® 123 - 8S Std. Dev. 5.3 ± 1.7 35 .8

± 4.2 46.0

± 3.2 39.9

± 5.6 45.5

± 0.9 51.6

± 2.5 45.3

± 1.3 --

-- 46.1 ± 0.3 ALFOTERRA® 145 - 2S Std. Dev. 8.3 ± 1.8 97.2

± 0.2 96.7

± 0.1 97.6

± 0.1 97.8

± 0.3 98.6

± 0.2 --

-- --

-- 96.4 ± 0.1 ALFOTERRA® 145 -4S Std. Dev. 4.7 ± 1.41 86.4

± 0.7 87.9

± 0.2 88.9

± 1.9 88.8

± 0.1 88.8

± 2.5 88.1

± 2.5 --

-- 88.9 ± 2.3 ALFOTERRA® I12 - 3S Std. Dev. 4.7 ± 1.4 86.3

± 0.7 87.9

± 0.2 88.9

± 1.9 88.8

± 0.1 89.1

± 2.8 95.2

± 0.3 --

-- 96.3 ± 0.4 C12 LAS Std. Dev. 0 ± 0 99.8 ± 0.3 98.9

± 0.4 98.7

± 0.6 99.7

± 0.1 100

± 0.1 98.4

± 0.9 99.0

± 0.2 99.0

± 0.3R&D ENV 06- 001 01 - 06

Page 17 **Table 8. Ultimate Biodegradation of A**  **LFOTERRA ® Surfactants in OECD 301A**  **T est**  **Surfactant % DOC Loss**  **1 3**  **6 9**  **10 13**  **15 17**  **22 28**  ALFOTERRA® 123 - 2S Std. Dev. 8.4 ± 11.9 20.8

± 1.6 29.2

± 0.1 38.3

± 1.0 39.6

± 3. 5 43.0

± 0.6 47.3

± 2.8 53.7

± 2.1 56.7

± 1.34 52.1

± 1.41 ALFOTERRA® 123 -4S Std. Dev. 3.9 ± 1.3 1.4

± 1.6 8.8

± 2.8 16.1

± 0.4 17.4

± 1.1 17.1

± 0.2 21.5

± 2.1 28.0

± 0.6 29.9

± 3.3 28.7

± 2.5 ALFOTERRA® 123 - 8S Std. Dev. 0 ± 0 0

± 0 1.3 ± 1.8 0

± 0 10.9 ± 4.2 5.7

± 1.3 4.7

± 0.8 14.0

± 0.8 15.1

± 2.3 10.9

± 1.6 ALFOTERRA® 145 - 2S Std. Dev. 2.4 ± 0.2 21.6

± 0.5 28.8

± 0.5 37.8

± 2.2 42.8

± 0.2 43.3

± 0.8 45.2

± 2.9 51.5

± 0.8 54.6

± 3.1 54.5

± 3.5 ALFOTERRA® 145 -4S Std. Dev. 2.7 ± 0.9 2.9

± 1.3 13.3

± 3. 5 18.2

± 0.8 24.8

± 0.1 26.3

± 2.1 28.1

± 4.2 34.7

± 0.6 36.2

± 1.2 33.9

± 1.3 ALFOTERRA® I12 - 3S Std. Dev. 13.8 ± 7.7 1.7

± 2.4 5.8

± 0.5 13.8

± 2.2 24.4

± 1.2 25.5

± 2.5 35.4

± 2.7 40.0

± 0.1 45.7

± 2.5 46.4

± 4.9 Acetate Std. Dev. 88.9 ± 9.5 97.4

± 0.8 96.5

± 0.5 98.5

± 1.5 96.5

± 0.3 99.2

± 1.3 97.6

± 0.9 98.3

± 2.5 96.5

± 1.1 100

± 0 C12 LAS Std. Dev. 13.4 ± 3.1 20.9

± 7.1 79.0

± 5.2 83.3

± 3.5 86.8

± 3.6 87.1

± 0.4 92.1

± 0.7 92.9

± 1.4 89.7

± 0.4 95.5

± 1.8R&D ENV 06- 001 01 - 06

Page 18 **Table 8A .**  **Non -** **Toxicity**

**of A LFOTERRA** 

**® Su rfactants**  **(A LFOTERRA** 

**® + Sodium A cetate)**  **AL FOTERRA** 

**® Surfactant (+ NaA** **cetate) 28**  **- Day**  **End Point % DOC Loss**  **Predicted Measured**  123 - 2S 75.4 79.3 123 -4S 62.6 66.3 123 - 8S 55.2 61.7 145 - 2S 73.3 80.3 145 -4S 64.4 69.5 I12 - 3S 71.3 73.5R&D ENV 06- 001 01 - 06 Page 19 **Table 9.**

**Biological Mineralization of A**  **LFOTERRA ® Surfactants in the OECD 301F Eletroly** **tic Respirometer Biodegradation Test**

**AL FOTERRA** 

**® Surfactants**  **%T hCO**  **2 1**  **2 3**  **4 6**  **8 10**  **12 14**  **16 18**  **20 22**  **24 26**  **28** SAFOL TM 45 - 2 (NII) S ± Std. Dev. 0.1 0.4 4.8 0.3 16.3 0.2 23.8 0.2 36.8 0.4 47.4 0.3 52.0 0.2 54.5 0.3 56.1 0.5 56.4 0.1 57.7 0.2 58.3 0.9 59.2 1.0 60.0 0.8 61.6 0.6 62.3 0.5 SAFOL TM 45 - 2 (KOH) S ± Std. Dev. 0 0 5.3 0.7 16.5 1.2 24.5 1.2 37.7 0.6 47.8 0.4 52.5 0 .4 55.5 0.6 57.3 0.7 58.8 0.3 60.5 0.3 61.2 0.6 62.5 0.8 63.8 1.3 65.3 1.6 66.1 1.8 SAFOL TM 23 - 3 (KOMe) S ± Std. Dev. 0 0 3.4 0.1 11.3 0.3 19.6 0.5 33.4 0.2 41.1 2.3 45.1 2.6 49.2 2.0 51.2 1.7 53.1 1.5 54.7 0.8 56.0 1.1 56.8 1.0 58.0 1.0 59.6 1.0 60.1 0.8 SAFOL TM 23 - 4 (NII) S 0 2.2 8.7 14.5 24.4 29.7 33.3 35.6 36.4 37.7 41.6 43.9 43.7 45.0 46.2 46.5 SAFOL TM 23 - 4 (KOH) S ± Std. Dev. 0.1 0.4 2.3 0.7 8.3 0.4 14.8 1.3 26.9 2.6 33.8 0.1 37.9 1.3 41.4 2.0 43.2 2.7 43.8 3.5 45.6 3.1 46.6 2.9 46.9 3.2 47.7 3.4 49.6 2.9 50.0 2.9 SAFOL TM 23 - 5 (KOMe) S 0.7 1.0 2.8 4.4 12.1 19.4 23.9 28.3 30.9 31.7 34.3 34.8 36.3 38.6 41.7 42.4 SAFOL TM 23 - 8 (KOMe) S ± Std. Dev. 0 0 1.3 0.7 4.1 0.3 8.3 0.1 20.8 2.0 26.8 2.2 30.3 2.4 33.7 2.6 35.6 2.4 36.6 2.7 38.2 2.4 39.2 2.5 40.3 3.0 4 1.3 3.1 42.9 3.2 43.3 3.3 SAFOL TM 23 - 4 (NII) S ± Std. Dev. 0.2 0.8 2.3 0.5 8.6 0.3 15.5 1.1 31.6 4.2 43.3 4.5 49.7 5.9 52.6 6.4 55.0 5.6 56.8 5.1 57.6 3.9 60.8 3.7 61.4 3.5 64.3 8.2 66.6 8.7 69.9 10.3 SAFOL TM 23 - 0.5EO 4 (NII) S ± Std. Dev. 0.6 1.2 2.9 1.4 9 .3 2.3 15.5 2.6 27.7 4.2 36.2 5.0 41.4 6.4 45.4 8.2 49.0 7.7 50.8 6.9 52.0 6.2 54.6 5.6 53.2 7.0 53.3 7.2 56.4 5.9 59.7 5.6 SAFOL TM 23 - 1EO - 4 (NII)S ± Std. Dev. 0.2 2.4 1.9 3.1 7.0 1.8 13.9 2.3 28.7 2.4 39.6 2.1 45.8 3.1 51.6 5.4 54.5 6.8 55.5 7.8 56.0 7.6 5 8.2 9.7 54.9 5.0 53.5 4.1 57.4 5.9 60.8 8.8 SAFOL TM 23 - 2EO - 4(NII)S ± Std. Dev. 0.1 1.2 1.2 0.6 7.6 0.6 14.0 0.9 27.0 1.7 36.2 1.2 42.9 1.3 46.3 0.6 49.5 3.0 53.5 8.1 54.1 7.9 54.3 6.9 53.5 2.5 53.5 1.3 56.9 3.8 59.2 6.1R&D ENV 06- 001 01 - 06 Page 20

**AL FOTERRA** 

**® Surfactants**  **%T hCO**  **2 1**  **2 3**  **4 6**  **8 10**  **12 14**  **16 18**  **20 22**  **24 26**  **28** SAFOL TM 23 - 2EO - 4 (KOH)S ± Std. Dev. 0.4 1.0 1.5 2.2 7.3 0.4 10.9 1.7 23.6 0.9 33.3 2.1 40.1 1.9 44.9 3.2 46.9 3.9 48.7 4.2 50.2 5.6 51.2 6.2 51.0 8.3 49.3 9.0 52.8 7.6 56.6 8.8 Sodium Benzoate ± Std. Dev. 2.6 2.7 39.2 2.3 50.9 3.6 62.6 4.9 78.1 5.9 83.9 5.4 89.6 4.8 91.0 4.3 93.2 4.0 95.4 3 .8 97.0 3.4 97.9 3.2 98.4 3.3 98.8 3.3 99.0 3.4 99.1 3.4R&D ENV 06- 001 01 - 06 Page 21 **Table 10. Comparative Data from OECD 301B CO**  **2 Evolution Test on EO/PO A** **lkoxides Al**  **koxide % ThCO**  **2 1**  **4 7**  **11 14**  **18 21**  **25 28**  SAFOL TM 23 - 2EO -4PO 4 a 0.3 18.3 49.7 76.6 86.9 93.2 95.2 97.0 95.7 SAFOL TM 23 - 5EO -4PO b 0.5 21.8 53.5 73.1 86.2 91.1 94.4 95.2 96.1 SAFOL TM 23 - 5EO - 5PO c 2.5 9.8 32.2 61.1 77.4 86.9 89.2 86.2 87.7 SAFOL TM 23 - 5EO - 6PO d 0.0 8.9 36.5 63.8 80.9 87.1 87.6 85.1 86.1

a BM G report number 451/f- 03 b BM G report number 638/e - 02 c BM G report number 451/a - 03 d BM G report number 451/b - 03 **Table 11. Removal of A** **LFOTERRA ® Surfactants in Semi Continuous A** **ctivated Sludge (SCA S) Test**  **Day s**  **% DOC Loss**

**1 2**  **3 4**  **9 11**  **14 15**  **16 22**  **Mean ± Std. Dev.**  **AL FOTERRA** 

**®** 123 - 2S 12.8 15.4 30.4 32.7 23.7 15.2 - 0.2 16.3 8.5 17.0 17.2 ± 9.8 123 -4S 0 0 0 0 0 0 0 0 0 0 0 ± 0 123 - 8S 0 0 0 0 0 0 0 0 0 0 0 ± 0 145 - 2S 20.4 26.9 39.5 37.7 37.1 29.9 24.1 22.9 13.3 17.9 26.9 ± 8.9 145 -4S 0 0 0 0 0 0 0 0 0 0 0 ± 0 I12 - 3S 0 0 0 0 0 0 0 0 0 0 0 ± 0 C12 LAS 98.2 93.7 94.3 -- -- -- -- 100 100 -- 97.5 ± 2.8 ABS 0 0 0 0 0 0 0 0 0 0 0 ± 0R&D ENV 06- 001 01 - 06

Page 22 **Table 11A .**  **SCA S Data**  **– MBA** **S Loss AL**  **FOTERRA**

**® Surfactant % MBA** **S Loss** 123 - 2S 99.8 123 -4S 87.3 123 - 8S 51.9 145 - 2S 97.0 145 -4S 82.8 I12 - 3S 88.4 C12 LAS 99.8 ABS 37.7 1. MBAS analysis done once on day 25 of SCAS TestR&D ENV 06- 001 01 - 06

Page 23 **Ap pendix** R&D ENV 06- 001 01 - 06 Page 24R&D ENV 06- 001 01 - 06 Page 25R&D ENV 06- 001 01 - 06 Page 26R&D ENV 06- 001 01 - 06 Page 27R&D ENV 06- 001 01 - 06 Page 28R&D ENV 06- 001 01 - 06 Page 29R&D ENV 06- 001 01 - 06 Page 30R&D ENV 06- 001 01 - 06 Page 31R&D ENV 06- 001 01 - 06 Page 32R&D ENV 06- 001 01 - 06 Page 33R&D ENV 06- 001 01 - 06 Page 34R&D ENV 06- 001 01 - 06 Page 35R&D ENV 06- 001 01 - 06 Page 36R&D ENV 06- 001 01 - 06 Page 37R&D ENV 06- 001 01 - 06 Page 38R&D ENV 06- 001 01 - 06 Page 39R&D ENV 06- 001 01 - 06 Page 40R&D ENV 06- 001 01 - 06 Page 41