v -

SC77 4-02 Technical Bulletin 1 Shell Chemical Company

Ultimate biodegradation of an ~

alcohol ethoxylate and a nonylphenol ethoxylate under

real is t ic conditions

by L. Kravetz, H. Chung, K. F. Guin, W. T. Shebs

and L. S. Smith

Shell Development Company Houston, Texas

and H. Stupel

Shell Chemical Company Houston, Texas

Presented at the 1982 Annual Meeting of the Soap and Detergent Association, January 27-31 in Boca Raton, Florida

Abstract The biodegradabilities of the two largest volume nonionic surfactant types - a linear alcohol ethoxy- late and a branched nonylphenol ethoxylate - have been determined under realistic sewage treatment plant conditions. In order to provide detailed bio- degradation information, each of these surfactants was radiolabeled and fed continuously with sewage plant influent to closed biodegradation units set up at the site of a Houston domestic waste treatment plant. The results of this study indicate biodegrada- tion of the alcohol ethoxylate to carbon dioxide and water occurred more rapidly than for the nonyl- phenol ethoxylate. Considerably larger quantities of partially degraded intermediates were discharged in the effluent from the nonylphenol ethoxylate bio- degradation unit compared to the alcohol ethoxy- late unit. Radiotracer data suggest different biodeg- radation mechanisms for these two surfactant types.

Introduction Alcohol ethoxylates and alkylphenol ethoxylates are the two largest classes of nonionic surfactants in current use. Primary alcohol ethoxylates (AE), the fastest growing major surfactant,’ contain alkyl groups which are essentially linear while most commercial alkylphenol ethoxylates (APE) contain highly branched alkyl groups. In household deter- gent formulations, AE is the largest volume non- ionic. In the industrial sector, APE is the major surfactant .

The biodegradation of branched alkylbenzene sulfonates (ABS) and linear alkylbenzene sulfo- nates (LAS) has been studied extensively with LAS showing faster degradation than ABS. The branched alkyl chain of ABS is most likely responsible for its slower biodegradation. As shown in Figure 1, the chemical structures of the hydrophobes in com- mercial nonylphenol ethoxylates (NPE) and ABS

)

are similar in that each containsan aromatic portion and a branched alkyl chain. It is for this reason that most major detergent manufacturers, since the mid- 1960’s, have selected the more biodegradable alco- hol ethoxylates for formulation into such household products as laundry powders and liquids.

When a surfactant begins to biodegrade, it tends to lose its surface active properties. The loss of a measurable physical or chemical property of intact surfactant when exposed to microbial attack is termed primary biodegradation. Criteria used in many of the earlier surfactant biodegradation stud- ies generally were limited to foam height measure- ments, surface tension or capability to react with a chemical reagent specific to undegraded surfac- tant.*Jv4 While primary biodegradation tests may have been adequate in the past, more recent studies have focused on ultimate b i o d e g r a d a t i ~ n , ~ ~ ~ ~ ~ defined as microbial attack of an organic substrate to pro- duce carbon dioxide and water. As an example, the ultimate biodegradation of an alcohol ethoxylate having 13 carbon atoms in the alkyl chain and an average of nine ethylene oxide (EO) units in the polyoxyethylene (POE) chain can be shown as

~

__

Assuming no interferences from other organic mate- rials, the ultimate biodegradation of this nonionic may be followed by measuring loss of organic carbon, uptake of oxygen, evolution of carbon dioxide or production of water.

Prior laboratory studies comparing the biodegra- dation of AE (AE 25-9) and an octylphenol ethoxy- late (OPE-10) are summarized in Figure 2.899 They show much higher levels of ultimate biodegradation for the AE than for APE. However, in more realistic field tests,lo shown in Table 1, primary biodegrada- tion of AE and APE were found almost identical

Figure l/Structural similarities - NPE and ABS

O ( C H 2 C H 2 0 ) , H

CH C H f C H 2 - ~ H f C H 2 - C H I I I 3- I I

CH3-CH-CH2-CH-CH2-CH

C H 3 C H 3 C H 3 CH3( C H 3 / 2 C H 3

BRANCHED NONYLPHENOL ETHOXYLATE BRANCHED ALKYLBENZENE SULFONATE

3

Figure 2/Ultimate biodegradation by CO2 evolution shake flask test

100

80

z 0

D

K 0 W

8 40 m ae

- 60 a

a

-

20

0 0 8 16 24

DAYS 32

under summer conditions, while APE biodegraded much more slowly under winter conditions.

Ultimate biodegradation in the field had not been studied previously largely because such criteria as organic carbon removal and oxygen uptake would not differentiate between the surfactant substrate and the much higher background of other organic materials present in sewage treatment plants. The use of radiolabeled surfactants, found to be suc- cessful in small scale laboratory tests,8 would solve this problem but is unpractical because of the large

tage is that measurement of CO2 evolution, a highly important ultimate biodegradability criterion, is im- possible in field tests because COS evolved cannot be collected and measured quantitatively in open aeration units used in sewage treatment plants.

This paper discusses the results of a study designed to investigate the ultimate biodegradation of an AEand NPE under conditions modeling actual sewage treatment. quantities required in field tests. Another disadvan-

Table l/Prior studies - field tests

% BIODEGRADATION

PRIMARY ULTIMATE

SURFACTANT SUMMER WINTER

AE 25-9 > 90 89 NEVER TESTED

APE-9 80 20 NEVER TESTED

4

Materials and methods Scope of method To avoid the limitations of an ultimate biodegrada- tion test in the field, a powerful radiotracer approach combined with a slipstream influent of a domestic waste treatment plant was used. To accomplish this, radiolabeled surfactant substrates were fed to acti- vated sludge aeration units along with a slipstream of the plant influent. Activated sludge was obtained initially from the plant and placed in single-stage bench-scale aeration units installed in a portable laboratory trailer which was moved to the plant site. The use of a continuous slipstream assured that the aeration units received realistic concentrations of plant influent. The use of radiolabeled substrates permitted their accurate determination at various locations in the bench-scale units. An important criterion for the bench-scale units was the require- ment that they operate at least as efficiently as the plant activated sludge aeration units. To determine this, plant units were monitored as well as bench- scale units.

Choice of plant site An important criterion was the selection of a domes- tic waste treatment plant which operates efficiently and has cooperative personnel. The West Memorial Municipal Utility District plant in Harris County, Texas, near Houston was selected because it fulfilled these criteria and also provided essentially summer conditions throughout the year. This meant that bacterial populations would not be subjected to stresses imposed by drastic temperature changes such as are present in US. treatment plants in more

)

northern tiers. On this basis, a favorable biodegra- dation environment was provided the surfactants, particularly APE, biodegradation of which has been reported to be more sensitive to temperature changes.lo

Sewage plant description The slipstream surfactant biodegradation project was carried out at the West Memorial Municipal Utility District plant in Harris County, Texas. Official approval and a lease agreement was obtained from the Board of Directors of the West Memorial Utility District. Mainly domestic waste from three nearby residential communities (1208 sewer connections) is treated by activated sludge at this facility. The plant operates one circular and two rectangular extended aeration treaters with a total design outfall of 2.5 mgd. Typical of municipal sewage plants in the Houston, Texas area, primary clarification of the sewage influent is not employed. The treaters are equipped with facilities for demand sludge wasting and sludge dewatering by centrifugation. The chlori- nated effluents from the three treaters enter a common outfall for discharge into a small stream.

For the slipstream study, the circular treater (hereafter termed North Plant) was used as slip- stream influent and activated sludge source. Some of the operating and performance parameters of the North Plant are listed in Table 2. It should be noted that analysis of the surfactants in the West Memorial influent showed the presence of both AE and APE, indicating that plant activated sludge had been acclimated to these surfactant types.

~

__

Table 2/West Memorial Municipal Sewage Treatment Plant circular extended aeration treater (North Plant)

OPERATING AND PERFORMANCE PARAMETERS

CONTACT AND RE-AERATION TANK VOLUME CLARl FIE R VOLUME INFLUENT FLOW HYDRAULIC RESIDENCE TIME SLUDGE RESIDENCE TIME - MAINTAIN MLSS AT INFLUENT CTAS

ALCOHOL ETHOXYLATE ALKY LPHENOL ETHOXY LATE

INFLUENT MBAS TOTAL PLANT EFFLUENT BOD5 TOTAL PLANT EFFLUENT TSS TEMPERATURE, AERATION UNIT

175,000 45,000

350,000 12

4,000 2

1.7 0.3 3.6

6 14

25"

ga I gal gpd hr

mg/l mg/l mg/l mg/l mg/l mg/l

mg/l C

5

Radiolabeled substrates Radiolabeled substrates used in this study are shown in Figure 3. Their synthesis has been de- scribed in a separate paper.” AE 25-9 is a primary alcohol ethoxylate containing alkyl chain lengths having 12-15 carbon atoms with an average of 13.5 carbon atoms. Approximately 80% of the alkyl chains are linear with the remainder mostly methyl branched at the 2-alkyl position. NPE-9 is a branched nonylphenol ethoxylate. Both of these surfactants contain an average POE content of approximately 9 EO units/mole of alcohol or alkylphenol.

AE25-9 was labeled with tritium in thealkyl chain having a tritium distribution as follows:

1. 50% labeled at the l-carbon position. 2. 25% labeled at the 3-carbon position. 3. 25% generally labeled at carbon positions

NPE-9 was labeled with tritium in the 2,6aromatic positions of the alkylphenoxy chain.

Both AE 25-9 and NPE-9 were uniformly labeled with Carbon-14 in their respective POE chains.

The chemical and physical properties of the AE 25-9 and NPE-9 were found typical of these surfac- tants manufactured on a plant scale.

further down the chain.

Glucose, uniformly labeled with Carbon-14, was purchased from New England Nuclear Corporation and was used as a “soft” biodegradation standard.

Specific activities of the radiolabeled substrates were: ~ ~~~~~

1. For AE 25-9: 0.83 mCi/g 3H and 0.13 mCi/g 14C 2. For NPE-9: 0.15 mCi/g 3H and 0.10 mCi/g 14C. 3. For glucose: 0.21 mCi/g14C.

~

Closed bench-scale biotreater A schematic drawing of the closed continuous bench-scale biotreater system is shown in Figure 4. Four replicate bench units [A, B, C and D (control)] were color coded, installed in the portable trailer and deployed on-site at the West Memorial Munici- pal Sewage Treatment Plant. The units were seeded with fresh mixed liquor from a common sample obtained from the North Plant aeration basin. A slipstream of screened (less than 20 mesh) North Plant influent and the labeled or unlabeled sub- strates were continously fed to the bench aerators with excess influent returned to the North Plant wet well. Microorganisms were maintained in suspen- sion in the aerator by mild mechanical mixing. Aera- tion of the mixed liquor was accomplished by con-

~

Figure S/Substrate in Slipstream Study fed continously at rate - 5 mg/l

0 IAE25-91

R = CH3-CH-CH CH-CH CH- I 2-1 2-1

CH3 CH3 CH3

0-J

(UNIFORMLY LABELED WITH 14C)

0 I BLANK I

6

Figure 4/Closed continuous bench-scale biotreater

SLUDGE

,THERMOMETER

UNIT B

UNIT C - I IKD li l U I

MANOMETER

h CO2 ABSORBERS

S DOMESTIC INFLUENT SCREENED4 20 MESH

EFFLUENT ~ ~~~

REFRIGERATED AND PRESERVED

COMPOSITE INFLUENT REFRIGERATED AND

PRESERVED

tinuous sparging with bottled C02-free air. Mixed liquor overflowed by gravity to the external clarifier to settle the biosludge for recycling back to the aerator. The clarified effluent overflowed by gravity to a refrigerated receiver (5°C). Aqueous NaOH (1 N) was used in the COZ absorber train to trap the respired CO2, as Na~C03, in the off-gas during continuous biotreating.

To prevent biodegradation of substrate before biotreatment, the substrate delivery system was sterilized and continuously maintained under sterile conditions. Replicate delivery systems were set up to facilitate the step change between labeled and unlabeled substrate. By this procedure substrate dilution effects were avoided.

The on-site slipstream study was carried out from early September to the latter part of November, 1980. This period was chosen to avoid inclement

weather, and sudden changes in temperature which might have an adverse effect on microbial activity during wastewater treatment.

The bench biotreaters were operated in parallel at conditions simulating municipal plant operations as given in Table 3. Biotreating performance was based on steady-state operating conditions. Throughout the test period, bench units A, B and C were fed slipstream influent dosed with NPE-9, glucose and AE 25-9, respectively, at a concentra- tion of 5 mg/l. Unit D, control, was fed slipstream influent without substrate addition.

In order to assess the ultimate fate of the radio- labeled substrate with an acclimated activated sludge, the biodegradation test was divided into three phases:

7

Table 3/Bench units - operating parameters

SUBSTRATE FEED CONCENTRATION: 1.25 mg/ml

SUBSTRATE FEED RATE: 2 ml/hour

BIOTREATER VOLUME: 5 liters

INFLUENT FLOW RATE : 500 ml/hour

STEADY STATE SUBSTRATE CONC.:

HYDRAULIC RESIDENCE TIME: 8 hours

5 mg/l

1. 2.

3.

SLUDGE RESIDENCE TIME: MAINTAIN MLSS AT 4200 mg/l

SLUDGE RECYCLE RATE: 150 ml/hour

TEMPERATURE: 25°C

pH: 7-7.5

DISSOLVED OXYGEN: 2-5 mg/l

Acclimation to unlabeledsubstrate (14 days). Biodegradation test of radiolabeled substrate (1 4 days). Radiolabeled die-away with unlabeled sub- strate addition (12 days).

Unlabeled substrate addition during the die-away was made so that the disappearance of radioactivity in the effluent could be followed while permitting maintenance of acclimated biota.

The performance of the North Plant treater was monitored for comparison with the control bench unit, D.

Sampling procedures The bench units were sampled daily at 0900 hours for slipstream influent, biotreated effluent, MLSS and COZ absorber solutions. Plant effluent was sampled twice daily (0730 and 1830 hours) and Plant mixed liquor suspended solids (MLSS) on a less frequent basis. After completion of wastewater sampling of the bench units, the samples were shipped to Shell's research facility for work up and distributed for analysis. Influent and effluent sam-

ples were collected as 24-hour composites. MLSS was collected as grab samples.

The levels of radioactivity used during the course of this study were extremely low and posed no hazard. However, as a safety precaution all radioac- tive materials were confined to the portable trailer, shipped offsite to Shell's research facility in accor- dance with Department of Transportation regula- tions and disposed of in accordance with Texas Department of Health regulations.

Preservation methods BOD, was determined on samples collected under refrigeration (5" C) but otherwise unpreserved. Pres- ervation with formalin is not suitable for carbon determinations since the levels of formalin used are sufficiently high to interfere with these analyses. As a consequence, the slipstream influent, bench unit effluent and MLSS were preserved with 200 ppm HgCI, while on-site at the municipal plant. This level of HgCI, has been shown to have no interference with surfactant analyses. Wastewater samples for surfactant analyses were preserved additionally

8

with 1-5% formalin, which has been shown to be a more effective preservative than HgCl2.l2

Analytical methods Table 4 lists general, radiotracer and surfactant analyses performed on biodegradation samples.

Surface tension and foam height measurements of the treated effluent were made using the Soap and Detergent Association method.13

Total carbon (TC) and inorganic carbon (IC) analyses of the screened North Plant influent and filtered (0.45 micron) biotreated effluent were deter- mined using the Beckman Total Carbon Analyzer, Model 915. Prior to analysis, the influent was soni- cated so that the organic carbon particulates would be included in the assay. The total organic carbon

(TOC) values were obtained by differences between the TC and IC values.

Total suspended and volatile suspended solids (TSS/VSS) of the mixed liquor samples were soni- cated before sampling. TSSNSS measurements were made by a gravimetric pr0~edure. l~

Effluent NH3, NO3,-' and P04-3 were measured following standard procedures.14 ~

Sludge volume index (SVI) and interfacial settling velocity (ISV) measurements of the biosludges were made using a standard one liter graduate cylinder with one rpm peripheral stirrer.

Specific nonionic analyses which could differen- tiate between AE and APE were determined on influents using a procedure involving separation of nonionic surfactants from the environmental

__

_ _ ~

Table 4/Analyses - Slipstream Study

- GENERAL

ORGANIC CARBON CHEMICAL OXYGEN DEMAND BIOCHEMICAL OXYGEN DEMAND NITROGEN PHOSPHATE

TOTAL SUSPENDED SOLIDS FOAM HEIGHT SURFACE TENSION DISSOLVED 02 TOTAL CO2

RADIOTRACER

14c. 14c02t 3 ~ . 3 ~ ~ 0

SUR FACTANT

COBALT THIOCYANATE ACTIVE SUBSTANCE (CTAS) METHYLENE BLUE ACTIVE SUBSTANCE (MBAS) HYDROGEN BROMIDE - GAS CHROMATOGRAPHY (HBr-GC) SPECIFIC NONIONIC FOR AE AND APE

UNITS ANALYZED

SLIPSTREAM I NF LUENT SLIPSTREAM E F F L U E NT SLI PST R E AM SLUDGE C02 ABSORBERS PLANT EFFLUENT PLANT SLUDGE

NONIONIC ETHOXYLATES AN I ON I CS ALKYL 81 EO CHAINS

TOTAL ANALYSES REQUIRED: APPROXIMATELY 1600

9

samples followed by conversion of the surfactants to phenyl urethanes and determination by high performance liquid chromatography. This procedure has been developed by member companies of the Soap and Detergent Association and will be reported in a separate publication.

All other methods listed in Table 4 have been described previo~sly.~

Results and discussion Acclimation phase (days 1-14) As shown in Table 2, sewage plant influent con- tained AE and APE indicating the activated sludge units were acclimated to these surfactant types. Nevertheless, a 14-day acclimation period with 5 mg/l of unlabeled AE 25-9 and NPE-9 was initiated. The effluent cobalt thiocyanate active substance (CTAS) data in Table 5 show greater than 98% pri-

mary biodegradability of effluents from the NPE-9 (Unit A) and AE 25-9 (Unit C) test units throughout the entire test period. These results were obtained after substraction of residual CTAS found in the effluent control (Unit D). Lower levels of nonionic biodegradability found in the Glucose Unit, Control Unit and the North Plant are probably due to the presence in the influent of relatively low quantities of unidentified materials which respond to the CTAS procedure.

Foam height and surface tension data also showed essentially equivalent levels of primary biodegrada- tion (greater than 95%) for effluents from the AE 25-9 and NPE-9 units. On the basis of these primary biodegradation criteria, activated sludge units for AE 25-9 and NPE-9 were judged to be operating properly.

Table UPrimary biodegradation of AE 25-9 and NPE-9 - CTAS Method

1 98.2

5 100

7 99.0

9 99.6

15 96.8

21 99.6

37 99.8

70 100

8

GLUCOSE

88.3

96.8

90.0

90.0

80.0

92.1

77.1

94.1

C 5 mg L-’ AE 25-Qb)

99.6

100

100

100

98.4

98.8

100

98.8

D

CONTROL

89.2

95.2

90.6

90.6

89.2

90.0

66.7

83.5

WEST MEMORIAL NORTH PLANT

w’ E C )

75.0 66.7

92.1 95.3

92.3 89.2

91.2 90.0

73.3 78.3

89.3 92.1

85.4 78.2

76.7 86.0

a) BASIS CTAS ANALYSIS b) CORRECTED FOR CONTROL UNIT D c) GRAB SAMPLES

10

Radiolabeled feed phase (days 14-28) Effluent radiotracer results from the radiolabeled feed phase are plotted in Figures 5-8.

Figure 5 shows the formation of 3 H ~ 0 from the hydrophobes of AE 25-9 and NPE-9. As shown, AE 25-9 released approximately 90% of its tritium as 3H20 within one day. This level of 3H20 release remained essentially constant throughout the course of the radiolabeled feed phase. In contrast, release of tritium as tritiated water was considerably slower and less extensive for NPE-9, indicating its hydro- phobe was more resistant to ultimate biodegradation than AE 25-9. The relatively large variations in 3 H ~ formation from NPE-9 during this phase may be due to slight variable inhibiting effects from the influent which might have more of an effect on slower biodegrading substrates.

Figure 6 shows that evolution of CO, from the POE chain of AE 25-9 was only slightly greater than for NPE-9 during the radiolabeled feed phase.

The release of soluble 3H-labeled biodegradation intermediates from AE 25-9 and NPE-9 into effluent is plotted in Figure 7. These results indicate 35-50% of the hydrophobe of NPE-9 is discharged into the plant effluent while less than 5% of the hydrophobe of AE 25-9 appears in the effluent.

Figure 8 shows the release of soluble 14C-labeled biodegradation intermediates into effluent. As shown, 8-14% of the hydrophile of NPE-9 is dis-

)

charged to effluent while less than 2% of AE 25-9 is discharged.

The distribution of radioactivity to the ultimate biodegradation products COz and H20, to soluble 3H-and 14C-products and to 3H-and 14C-products found in cellular material obtained from activated sludge is listed in Table 6 for AE 25-9, NPE-9 and glucose. The data in this table indicate greater levels of ultimate biodegradability for the hydrophobe and the hydrophile of AE 25-9, compared to NPE-9, with greater discharge of metabolites from NPE-9 into effluent. Unexpectedly, larger quantities of 3H and 14C were found incorporated into cellular material from NPE-9 than from AE 25-9. It is interesting to note that 14C02 evolution from glucose was less than for AE 25-9 or NPE-9. However, more of the remain- ing 14C activity was found in cellular material for glucose than for AE 25-9 or NPE-9. The CO, results for glucose and AE 25-9 in these activated sludge tests are not in line with previously reported shake flask tests having more dilute bacterial i n o ~ u l a . ~ Dif- ferences in bacterial populations and types may account for these discrepancies.

~

__

Radiolabeled dieaway phase (days 28-40) Radiolabeled dieaway was determined while feed- ing the aeration units with unlabeled substrates. The percent conversion to radioactive metabolites was

Figure 5/Formation of 3H20 from AE 25-9 and NPE-9

2R

d I c)

0 I- z v)

w > z

0 a

8 w m 0 I L 0 a n > I

100

80

60

40

20

0

U

n 0 0 v o o " v U

0 AE 25-9

a NPE-9

n a 4 , I I I I I I I I I I I I

16 18 20 22 24 26 28 RUN DAY

-

11

Figure G/Evolution of I4CO2 from radiolabeled AE 25-9 and NPE-9

ae 6J E 2 0 + W 1

I n. 0 LT

> I U 0 z v) LT W > z 0 0

-

n

0

100

80

60

40

20

0

0 AE 25-9 a NPE-9

P

14 16 18 20 22 24 26 28

RUN DAY

~

Figure 7/Soluble 3H-labeled biodegradation products of AE 25-9 and NPE-9

100

80

60

40

20

0

0 AE 25-9

A NPE-9

14 16 18 20 22 24 26 28

RUN DAY

12

Figure 8/Soluble I4C-labeled biodegradation products of radiolabeled AE 25-9 and NPE-9

20

0 AE 25-9

NPE-9

W -1 m

16 2

12

8

4

0

L d 0 -

I

14 16 18 20 22 24 26 28 RUN DAY

) Table G/Distribution of radioactivity*

FOUND IN % CONVERSION CELLULAR

% CONVERSION TO . . . TO SOLUBLE.. . PRODUCTS, % HYDROPHOBE

AE 25-9 NPE-9

HYDROPHILE

25 } 3H20 3: } 3H PRODUCTS 5 33

AE 25-9 NPE-9 GLUCOSE

] 14C PRODUCTS 1

27 34 64

* A T COMPLETION OF RADIOLABELED FEED PHASE

13

calculated on the basis of radioactive material pres- ent at the end of the radiolabeled feed phase (day 28). Figure 9 shows 3Hz0 was released more rapidly from tritiated AE 25-9 metabolites than for tritiated NPE-9 metabolites. Also, more soluble tritiated metabolites are discharged into effluent from NPE-9 than from AE 25-9.

Figure 10 shows that during the dieaway l4COZ and 14C-metabolites to effluent are released at essentially equivalent rates for AE 25-9 and NPE-9.

Figure 11 shows l4CO, evolution and appearance in effluent of 14C metabolites during radioactive dieaway of glucose. Comparison with Figure 10 shows l4COZ evolution and appearance of 14C- metabolites to be slightly slower for glucose than for AE 25-9 and NPE-9.

Radioactivity balance As shown in Table 7, approximately 97% of tritium activity from AE 25-9 and NPE-9 was accounted for as 3H~0, soluble 3H-metabolites and 3H in the bio- mass accumulating during the course of the slip- stream study. Slightly lower levels of I4C activity from AE 25-9, NPE-9 and glucose were accounted for as I4CO2, soluble 14C-metabolites and 14C in the biomass. Slight losses of I4CO2 due to undetected leaks in the closed biodegradation units may be responsible for the lower 14C balance.

Mechanisms of AE 25-9 and NPE-9 biodegradation Table 8 lists the EO/hydrophobe ratio for NPE-9 from the start to the end of the radiolabeled feed phase. These results were obtained by calculating the 14W3H ratio in the effluent. As shown the EO/hydrophobe ratio is approximately 2.4 after approximately 8 hours of feeding radiolabeled NPE-9. This ratio is consistent with that found by other ~ o r k e r s ~ ~ ~ ~ ~ for NPE-9 using analytical tech- niques other than radiotracer. The rapid release of 3H20 by AE 25-9 and the rapid achievement of a steady state ratio of 2.4 for EO/hydrophobe from NPE-9, suggest the biodegradation mechanisms shown in Figure 12. Linear primary alcohol ethoxy- lates biodegrade by initial cleavage of hydrophobe from hydrophile followed by rapid &oxidation of the hydrophobe to COZ and HzO with slower oxida- tion of the hydrophile to COzand H20i7. Alkylphenol ethoxylates biodegrade by stepwise shortening of the POE chain to leave a relatively bioresistant

alkylphenol ethoxylate metabolite having approxi- mately two EO units in the POE chaini8.

Comparison of bench scale units with sewage treatment plant Biotreating performance of bench-scale units was compared with that of the North Plant. As shown in Tableg, measurement of such parameters as MLSS, BOD, COD, removal of nonionic and anionicsurfac- tants, effluent nitrate and phosphate levels, and sludge settling capabilities, indicate the bench- scale units performed essentially the same as the North Plant aeration unit except that the North Plant did not have good nitrification compared to that of the bench-scale units. This may have resulted from breakdown of the North Plant of several aeration pumps which led to low levels of dissolved oxygen (1 mg/l) in the plant aeration tank.

Summary Ultimate biodegradation has been recognized by the EPA as an important criterion for biodegradabil- ity. Recently, the EPA has included an ultimate bio- degradation test as part of their testing guidelines for premarket notification of new chemicals. The development of a powerful radiotracer approach has facilitated prior laboratory studies of the ulti- mate biodegradation of essentially linear AE and branched NPE. The results of these studiesshowed AE biodegraded faster and more extensively than NPE.

When combined with a slipstream influent of a domestic waste treatment plant, the use of a radio- tracer method overcomes the limitations previously imposed upon a field test for ultimate biodegrada- tion. Using this approach for AE and NPE, it has been shown that under realistic sewage treatment plant conditions AE biodegraded to COZ and HzO faster and more extensively than NPE. In addition, the AE formed fewer metabolites than the NPE.

For the AE, radiotracer analyses suggest cleavage of the POE chain from the alkyl chain as the initial biodegradation step with continuing biodegrada- tion of these fragments to COZ and HzO. For the NPE, radiotracer analyses indicate a relatively fast step- wise shortening of the POE chain to leave a biores- istant NPE metabolite having approximately 2 EO units.

14

Figure 9/Die-away of tritiated metabolites

I I I

0

20

40

60

I I I I I I

AE 25-9

I 0 0 SOLUBLE 3H-LABELED METABOLITE

28 30 32 34 36 38 40

RUN DAY

NPE-9 0

20 -

L

RUN DAY ___

15

Figure 1 O/Die-away of I4C-labeled metabolites

0

20

40

AE 25-9

0 SOLUBLE

0 1 4 ~ 0 ~

14C-LABELED METABOLITES

60 I I I I I I I I I I I I 28

0

20

40

60 28

30 32 34 36

RUN DAY

38 40

NPE-9 A 1

SOLUBLE 14C-LABELED METABOLITES

30 32 34 36

RUN DAY

38 40

16

Figure 1 1/Die-away of 14C-labeled glucose treated biosludge

20 -

40 -

60 -

0 14c02

SOLUBLE 14C-LABE ED ME ABOLITES

28 30 32 34 36 38 1

RUN DAY 3

Table 7/Radioactivity balance - Slipstream Study

ACT1 VI TY Dl STR I BUT ION AE 25-9 NPE-9 GLUCOSE - _ - 3~ 20 91.6 28.3

SOLUBLE 3H-METABOLITES 1.5 36.7 _ _ 3H IN BIOMASS 4.1 31.5 - - - -

TOTAL 97.2 96.5

65.4 54.0 42.8

SOLUBLE 14C-METABOLITES 1.3 8.5 1.4

52.0 14C IN BIOMASS 24.7 27.6

TOTAL 91.4 90.1 96.2

14c02

-

17

Table 8/Ratio EO/hydrophobe during biodegradation of NPE-9

TIME (DAYS)

0.08 (2 HOURS)

0.33 (8 HOURS)

1

18

24

28

RATIO, EO/HYDROPHOBE"

5.4

2.4

2.2

2.4

2.7

2.2

BASIS, 14W3H ACTIVITY IN EFFLUENT

Figure 12/Mechanisms

A€ 25-9

NPE-9 -

'CoA = COENZYME A

BIO R ESISTA N T

+ HOCH2CH20H or HOCH2COH fi'

+ C02 + H20

18

Table S/Comparison of bench-scale units with North Plant*

UNIT A UNIT B UNIT C UNIT D NORTH NPE-9 GLUCOSE AE25-9 BLANK PLANT

CTAS REMOVAL, % 97 91 97 85 83

TOC REMOVAL, % 92 93 94 93 93

SOLUBLE EFFLUENT BOD5, mg/l 1 1 1 1 1

SOLUBLE EFFLUENT COD, mg/l 13 13 12 12 14

EFFLUENT NH3, mg/l 3 2 3 4 7

EFFLUENT NO;-NITROGEN, mg/l 15 14 15 16 0.2 -3

EFFLUENT PO4, mg/l 33 34 32 32 30

MLVSS, mg/l 3000 3000 3000 3000 3000

"AVERAGES OVER 40 DAYS

Acknowledgement The authors are grateful to the Board of Directors and operating staff of the West Memorial Municipal Utility District of Harris County, Texas, for their per- mission to carry out this ultimate biodegradation study at their sewage treatment plant.

References Von Henning, D. H., Household and Personal Products Industry 74. 48 (1977).

2Frazee, C. D., Q; E. Osburn and R. 0. Crisler, J. Am. Oil Chem. SOC., 47, 808 (1964).

3Huddleston, R. L. and R. C. Allred, Ibid. 42, 983 (1965). 4Wickbold, R.. Vom Wasser 33, 229 (1966). 5St~rm, R. N., J. Am. Oil Chem. SOC. 50, 159 (1973). 6Kurata, N. and K. Koshida, Yukagaku 24, 879 (1975). 7Gledhill, W. E., Appl. Microbiol. 30, 922 (1975). 8Kravetz, L., J. Am. Oil Chem. SOC. 58, 58A (1981). gKravetz, L., H. Chung. J. C. Rapean, K. F. Guin and W. T. Shebs, "Ultimate Biodegradability of Detergent Range Alcohol Ethoxy- lates", Presented at the69th Annual Meeting of the American Oil Chemists Society, May 1978, St. Louis, Missouri.

'OMann. A. H. and V. W. Reid, J. Am. Oil Chem. SOC. 48, 794 (1971).

"Shebs, W. T. and L. S. Smith, "Synthesis of Two Nonionic Surfactants Labeled with Carbon-14 and Tritium", Presented at the 72nd Annual Meeting of the American Oil Chemists' Society, May 1981, New Orleans, Louisiana.

'*Shebs, W. T. and L. S. Smith, "A Novel Separation of Tritiated Water Formed By Biodegradation of Surfactant Samples and Its Application to a Sample Preservation Study", Presented at the 72nd Annual Meeting of the American Oil Chemists' Society, May 1981, New Orleans, Louisiana.

13Mausner, M. et. al., J. Am. Oil Chem. SOC. 46, 432 (1969). '?Standard Methods for the Examination of Water and Waste-

'5Rudling, L. and P. Solyom, Water Research 8, 115 (1974). 16SchoberI, P., E. Kunkel and K. Espeter, Tenside Deterg. 78, 64

"Patterson, S. C., C. C. Scott and K. B. E. Tucker, J. Am. Oil

'*Swisher, R. D., Surfactant Biodegradation, Marcel Dekker, Inc.,

water 13th Edition (1971).

(1 981 ).

Chem. SOC. 47,37 (1970).

N.Y. (1970).

Shell Chemical Company Sales Offices Warranty

Atlanta

Chicago

Cleveland

Houston

Los Angeles

West Orange

(404) 955-4600

(31 2) 887-5600

(216) 842-4000

(713) 526-4631

(71 4) 991 -9200

(201) 325-5200

320 Interstate North Parkway Atlanta, Georgia 30339 1415 West 22nd Street Oak Brook, Illinois 60521 7123 Pearl Rd. Middleburg Heights, Ohio 44130 2001 Kirby Dr. Houston, Texas 77019 51 1 N. Brookhurst Street Anaheim, California 92803 100 Executive Drive West Orange, New Jersey 07052

For international sales contact: Pecten Chemicals, Inc One Shell Plaza (713) 241-6161 Houston, Texas 77002

All products purchased from Shell are subject to terms and conditionsset out in thecontract, order acknowledge- ment and/or bill of lading. Shell warrants only that its product will meet thosespecifications designated as such herein or in other publications. All other information sup- plied by Shell isconsidered accurate but is furnished upon the expresscondition that the customer shall make its own assessment to determine the product's suitability for a particular purpose. No warranty is expressed or implied regarding such other information, the data upon which the same is based, or the results to be obtained from the use thereof; that any product shall be merchantable or fit for any particular purpose; or that the use of such other information or product will not infringe any patent.

October 1982 ~~ Printed in USA 1M