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Testing surfactants for ultimate biodegradability
Chemosphere, Vol. 28, No. 8, pp. 1503-1523, 1994
Pergamon 0045-6535(94)E0073-3
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0045-6535/94 $7.00+0.00
TESTING SURFACTANTS FOR ULTIMATE BIODEGRADABILITY
J. Struijs° and J. Stoltenkamp
Laboratory for Ecotoxicology, National Institute of Public Health and Environmental Protection, P.O. BOX 1, 3720 BA Bilthoven, The Netherlands ° author to whom correspondence should be addressed (Received in Germany 3 September 1993; accepted 2 December 1993)
ABSTRACT The development of screening biodegradability tests is briefly reviewed. The "new chemicals" screening methods according to the OECD (1981) and the EC (1984) are based upon ultimate biodegradability or mineralization by making use of some analytical parameter which is not specific for the chemical. The methods are now known as "ready biodegradability" tests (RBTs). Screening tests that are specifically designed for surfactants have been developed by the OECD earlier. In the latter tests primary biodegradation is monitored by means of a specific analytical parameter. Recent harmonization and improvement of RBTs are evaluated in order to see if their applicability can be extended to screen surfactants for ultimate biodegradability. It is shown that conditions for biotransformation of a surfactant in one of the revised RBTs, i.e. the Closed Bottle Test, are similar or even more favourable than in the screening tests for surfactants. Testing ultimate biodegradability is proposed as a supplement to primary biodegradation of surfactants, at the level of both Screening and Confirmatory. Several revised RBTs were applied to evaluate the ultimate biodegradability of twelve surfactants. Results are in agreement with previous studies and correspond to validated structure biodegradability relationships for surfactants. It is concluded that a positive result in a RBT can safely be extrapolated to aerobic environments in regions where domestic waste water is processed by sewage treatment plants. The proposed test system exclusively employs standardized methods that are published by the OECD and EC. INTRODUCTION
Surface active substances applied in washing powders and other industrial and household products are high volume chemicals. Approximately 1,000,000 metric tons of linear
1503
1504 alkylbenzene sulphonates (LAS) and 470,000 metric tons of linear alcohol ethoxylates (LAE) are annually used in the United States, Western Europe and Japan (Richter and Knaut, 1988). Biodegradability criteria of surface active substances in detergents deviate from those in the regulation of other chemicals. The reasons for this are both historical and practical. However, recent developments in test methods and regulatory measures justify a new approach to evaluate surfactant biodegradability. Results from appropriate tests may be used to better predict the ultimate fate of surfactants in the environment, rather than to indicate that "elimination due to waste water treatment is acceptable". To achieve sufficient elimination during wastewater treatment, biodegradation is considered a process which should be fast enough to play a significant role. In detergent legislation, however, the term "biological degradability" is not further specified. It may refer toprimary
biodegradation, defined as the microbial alteration of a molecule sufficient to remove a characteristic property such as surface activity (foaming) or its response to an analytical procedure specific for the starting compound but not for its degradation products. For the two most important categories, the anionics and the nonionics, special reference is made to the European Community Directives 73/404 EEC and 73/405 EEC amended by 82/242 EEC and 82/243 EEC, specifying the test methods for determining biodegradability. In these standardized tests, biodegradation is quantified by means of a semi-specific analytical parameter, i.e. the disappearance of Methylene Blue Active Substances (MBAS) and Bismuth Active Substances (BIAS) response, respectively. This analytical approach determines essentially primary degradation by measuring the disappearance or removal of surfactant properties and not mineralization to CO2. However, no information from test data can be derived that is essential to evaluate the life-cycle of these compounds. Investigations reported by McEvoy and Giger (1986) and Brunner et al. (1988) revealed that persistent metabolites may have escaped our notice. Standardized biodegradability tests, specified by the new chemicals legislation, have been updated recently (OECD, 1993). Generally, methods that rely on mineralization have been adopted in the Annex to Commission Directive 92/69/EEC adapting to technical progress Council Directive 67/548/EEC, also known as the "notification directive". The purpose of this update was harmonization and improvement of the methods in order to enhance the applicability. In this study a comparison is made between the set of harmonized OECD/EC methods and the detergent OECD Screening Test. The latter has been designed to select "soft" surface
1505 active compounds so avoiding unnecessary expensive laboratory investigations. A selection of appropriate methods to screen surfactants is proposed which also accounts for ultimate
biodegradability in addition to primary degradation. Biodegradability" (RBTs)
OECD
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reduce uncertainty about effluent containing bioresistant
intermediates formed from the parent surfactant.
BIODEGRADABILITY TESTS SPECIFIED BY THE DETERGENT LEGISLATION
Elimination of the surface active properties during wastewater treatment has become a legislative requirement. Obviously, the first biodegradability test methods were designed so that elimination of surface active properties due to microbial transformation was the test criterion of choice. Those detergents that passed a biodegradability test, specified in the detergent laws, indeed did not foam during wastewater treatment and did not cause effluent toxicity problems. During the years of 1968-1970, an OECD group of experts worked on a common system of testing to be accepted internationally. As a result, a system was published (OECD, 1971) which consists of two stages, differing both in principle and in indications which can be drawn from them as to the biodegradability of anionic surface active substances:
The OECD Screening Test, a static flask test which is relatively quick and simple. This test must be considered as an "acceptance test" and not as an "rejection test". It selects "soft" anionic surfactants which do not have to be tested further as a high biodegradability is expected in sewage works.
The OECD Confirmatory Test, based on the simulation of the conditions existing in an activated sludge plant. This test should be used for any anionic surfactant which may not have passed the OECD Screening Test, in order to confirm or disprove the first results obtained. The results obtained at this stage are the only ones to be taken into consideration in the refusal or acceptance of products not accepted by the OECD Screening Test.
The degree of biodegradability is here defined as the percentage of MBAS eliminated under the conditions of the test. Thus only anionic surfactants which form a chloroform-
1506 soluble salt with methylene blue are detected. This analytical parameter responds to all synthetic anionic surface active agents used in detergents, but not to soap which is considered as an anionic surfactant derived from natural products. Since most detergent formulations contain a mixture of homologues and isomers, such a semi-specific analytical parameter proved very useful. Both tests are also appropriate for anionic surfactants contained in formulated products if use is made of well described extraction procedures (OECD, 1971). The stringency of the screening test is determined by the test duration, the pass level and the performance of the applied inoculum against two standards of biodegradability. The pass level is 80% degradation within 19 days. The alnot, nt of inoculum must be such that the "soft" standard Marion A, which is a commercial linear alkyl benzene sulphonate (LAS), is degraded to a value between) 90 and 95 per cent within 14 days. The disappearance of the "hard" standard, TBS (tetra propylene benzene sulphonate), should be less than 35% within the test duration. Usually 0.5 ml secondary eflluent per litre test solution will be adequate to cause the standards to follow the required degradation patterns. However, there are indications that microbial COlnmunities may have adapted to TBS, which probably has been present for several decades as a minor constituent of detergent products. European Community Directives 73/404 EEC and 73/405 EEC were published in 1973 in order to harmonize the regulations of the member states in this respect. The class of nonionic surfactants comprise numerous compounds with a wide structural diversity and usually lacking a common functional group. Obviously, the biological systems employed for testing anionics also suits the nonionics, however, the principal difficulty in establishing a test procedure tbr this group of chemicals was lying in the analytical methodology. After several ring tests, the OECD expert group decided to adopt the tetraiodobismuthate method according to Wickbold (1973). The procedure consists of preconcentration by sublation into ethyl acetate, precipitation of the nonionic agents by a bismuth containing reagent (Dragendorff reagent) and potentiometric titration of the bismuth content of the precipitate. In analogy to the anionics, analytical results are simply expressed as mg/L BiAS. Both the static test (OECD Screening Test) and the dynamic simulation test (OECD Confirmatory Test) are applicable to nonionic st, rfactants either as such or contained in formulated detergents. In the latter case, a prepurification procedure is necessary, consisting of an alcoholic extraction and ion-exchange because also anionic surfactants respond as BIAS. Although the Wickbold method refers specifically to water soluble ethoxylates, the OECD expert group considered that this was acceptable as nonionic
1507 surfactants predominantly consist of these compounds. The new test procedure (OECD, 1976) for anionic surfactants underwent minor changes with respect to 1971 and the analytical methods appropriate for the nonionics were added. Both for the anionics and the nonionics the "soft" and the "hard" standards are Marlon A and TBS, respectively.
BIODEGRADABILITY TESTS SPECIFIED BY NEW CHEMICALS LEGISLATION
For the very diverse class of "new chemicals" a simple screening is required in the framework of some sort of test hierarchy. A non-specific analytical parameter to follow the course of biodegradation is not only cost-effective but it also responds to any biodegradation residues or intermediates, indicating the extent of ultimate biodegradation of the tested compound. OECD experts modified and combined elements of the OECD Screening Test for detergents and other reported methods. This led to the acceptance of five tests which rely on a non-specific monitoring parameter: dissolved organic carbon (DOC) die away, oxygen uptake or carbon dioxide production. The five tests have in common with the OECD Screening Test that the test compound serves as the sole carbon source and that it is exposed to a relatively low amount of microbial biomass. A standardized test duration of 28 days gives the inoculum a limited opportunity to adapt to the test compound prior to the onset of biodegradation. Although standardization of the inoculum would improve the comparability of the methods, a mixed inoculum derived from a local source, f.i. a communal wastewater treatment plant (WWTP), is both practical and appropriate to ensure the presence of a wide variety of species in the tests. In view of the stringency, required in a screening test, preadaptation of the inoculum to the test compound is not allowed. Preliminary screening for rapid mineralization has become related to the term "ready biodegradability", which is an arbitrary classification of chemicals that have passed one of these static tests for ultimate biodegradability. The proposed tests are so stringent that it is assumed that positive chemicals will rapidly and completely biodegrade in a wide variety of natural aerobic environments (OECD, 1981). In 1984, these tests were adopted in Annex V (methods C.3 to C.7) of Directive 84/449/EEC, containing technical adaptation to the "notification directive" 67/548/EEC, Annex V (test guidelines for chemical substances).
In a correlation study on 44 compounds and several test methods, Gerike and Fischer (1979 and 1981) found a varying stringency of the RBT's, decreasing generally in the order
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1509 Closed Bottle Test, MITI(I), Modified OECD Test, Modified AFNOR test and the Modified Sturm Test. The lower stringency of the latter two tests compared to the Closed Bottle Test and the Modified OECD Screening Test is explained from the inoculum concentration which is several orders of magnitude higher (see Table 1). In spite of a relatively high biomass inoculum, the number of chemicals that are positive in the MITI(I) Test is rather low. This has been attributed to the prescribed culturing procedure which significantly reduces the metabolic diversity of the inoculum. Inoculum concentrations in the RBTs span more than 4 orders of magnitude, having an adverse effect on the mutual comparability of test results.
RBTs of 1981 generally offer a poor choice to select a method which is suitable for screening surfaetants, due to either the concentration of the test chemical or the applied amount of inoculum. All tests, with exception of the Closed Bottle Test, prescribe a relatively high concentration of the test substance being in the range of 10-100 mg/L. This is the consequence of the use of a non-specific analytical parameter. If the purpose is to screen the rather toxic surfactants for biodegradability, these tests should be considered inferior with respect to the OECD Screening Test, as the latter allows 5 mg/L. The test compound concentrations in the Closed Bottle Test and the OECD Screening Test are approximately equal. However, the prescribed inoculum concentration is ten times lower compared to the OECD Screening Test. As the latter test proved fall-safe, as far as primary degradation is concerned, the higher threshold for any biodegradation to occur in the Closed Bottle Test is not necessary and even at undesirable.
Harmonization of Ready Biodegradability Tests
A highly varying stringency in RBTs and differences in the mineral medium are not rational. Preparations to revise the existing guidelines 301 A to E (OECD, 1981) started in 1985 with the aim to harmonize the methods. Earlier attempts were made to adopt an additional test which is suitable for poorly soluble compounds. The Manometric Respirometry Test, proposed as a simplification of the MITI(1) test, appeared a reliable method in two international inter-laboratory exercises (Painter & King, 1985; CEC, 1985). Most of the weaknesses, mentioned in the foregoing, were assessed and discussed in a technical report on the statt, s of biodegradability testing by The European Chemical Industry
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1511 Ecology and Toxicology Centre (ECETOC, 1983), expressing the need for harmonization of the RBTs (Blok et al., 1985). Thirty-seven laboratories participated in 1988 in an OECD inter-laboratory test to investigate the validity of the proposed harmonization. Based on these results and after lengthy discussions as to the stringency of the new methods, the OECD published a new Guideline for Ready Biodegradability in 1993, containing six methods: DOC Die-away, the CO~ Evolution, Manometric Respirometry, the Modified OECD Screening Test, Closed Bottle Test and the MITI(1) test. The harmonization is reflected in the mineral medium which is similar in the first four tests, but also in the maximum permitted inoculum concentrations which span a range of only two orders of magnitude in all tests if the Modified OECD Screening Test is not taken into consideration. The °'10 days-window" was installed as an extra pass criterion for ready biodegradability stipt,lating that the time elapsed from 10% to 90% of the plateau in the biodegradation curve is no longer than 10 days, however, for the Closed Bottle Test 14 days. The concentration ratio of the test chemical and the inoculum is less favourable in the Modified OECD Screening test than in the other RBTs and therefore toxic effects restrict its applicability. The methods have now been adopted in the Annex to Commission Directive 92/69/EEC adapting to technical progress Council Directive 67/548/EEC. These methods, sunlmarized in Table 2, are referred to as "revised RBTs" in the following.
SCREENING SURFACTANTS FOR ULTIMATE BIODEGRADABILITY
Several revised RBTs seem appropriate to screen surfactants for ultimate biodegradability. At first sight the revised Closed Bottle Test is the method of first choice for the good soluble anionic and nonionic surfactants. The concentration of the test substance is as low as in the OECD Screening Test for detergents and generally will not cause inhibitory effects. If the highest inoculum concentration is applied, i.e. 5 ml secondary effluent/L, the test system would provide a better opportunity for any biodegradation to occur than the OECD Screening Test. Provided that the origin of the inoculum is similar for both tests, i.e. secondary effluent from the same WWTP, a surfactant that has passed the OECD Screening Test system, would certainly undergo complete primary degradation in the revised Closed Bottle Test. This is based upon two arguments. First, the amount of inoculum in the revised Closed Bottle Test is ten times higher than recommended in the OECD Screening Test. Second, the test duration in the Closed Bottle Test is 28 days compared to 19 days in the OECD Screening Test for
1512 detergents. Thus an observed negative result in the Closed Bottle, while criteria for sufficient primary biodegradation are met (___80% MBAS disappearance), can not be attributed to a lack of opportunity in the test system because the conditions are more favourable for biotransformation to occur.
Experimental results
Table 3 contains test results of twelve surfactants of varying ease of biodegradability. Most of these compounds were tested in the DOC Die Away, the Manometric Respirometry and the Closed Bottle Test. In the latter test the positive reference chemical (sodium acetate) displayed 70-90% ThOD in all runs after one week. All tests were conducted according to the revised OECD Guidelines (OECD, 1992). The amount of inoculum per litre medium was 5 mL secondary effluent in the Closed Bottle Test and 30 mg/L activated sludge (dry matter) in the other tests. The chemical concentrations were 2.5-4 mg/L in the Closed Bottle, 20 mg/L in the DOC Die-Away and 100 mg/L ThOD (approximately 50 mg test chemical/L) in the Manometric Respirometry.
LAS around Ct2 passed the DOC Die-Away and the Manometric Respirometry but not the Closed Bottle Test. Although in principle a substance may be classified as readily biodegradable if it is positive in one RBT but fails to pass another, it is worth to mention here that usually for readily biodegradable chemicals > 90% DOC removal is measured. For LAS only 74% was found which is in good agreement with previously reported results (Gerike, 1987). In a reply to a paper on the problem of ultimate biodegradability of LAS (Pitter & Fuka, 1979), Swisher (1981) has summarized the literature on this subject. He concluded that in the real world significantly higher DOC removals have been reported: averaging 80-90% in activated sludge experiments and up to 94% in river water. It also appeared that only 45-75% DOC removal was found from determinations in inoculated synthetic media, used in all kinds of static screening tests. This might be an indication that screening by means of a non-specific analytical parameter is rather sensitive for less degradable metabolites, as primary degradation of LAS in inoculated synthetic media is fast and usually exceeds 90%. This is confirmed in the present study and may be an explanation for the fact that in the Closed Bottle Test only 55 % was found, in contrast to sodium acetate for which 90% was measured in parallel runs.
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1514 The poorly degradable tetrapropylenebenzene sulphonate behaved as expected. Also the results from the anionic and nonionic surfactants with linear alkyl hydrophobic groups but without an aryl moiety, such as l-octane sulphonate, Na dodecyl sulphate and hexadecanol polyethoxylate (20 EO), are in agreement with validated generalizations relating surfactant structure and biodegradability (Swisher, 1987). These compounds consistently passed all three RBTs, apparently without the formation of stable intermediates as is reflected from DOC removals of 90% or higher. The branched sodium 3,9-diethyl, 6-tridecyl sulphate did not pass the oxygen consumption tests, probably because 17% of the carbon was refractory. Nonylphenol ethoxylate (10 EO) behaved erratically in the DOC Die-Away (55-70%) but did not pass the tests based on oxygen uptake as can be expected if only 70% DOC removal is measured. The branched iso-octylphenol ethoxylate (10 EO) with two quaternary carbon atoms did not pass any of the RBTs. In the DOC-Die-Away test 66% DOC reduction was measured after one week, however, this appeared to be a plateau as in the following three weeks no further DOC removal was found. This was confirmed by only 40% ThOD in the Manometric Respirometry. Tween 60 is not readily biodegradable although the results strongly suggest that primary biodegradation has occurred and was probably complete. The quaternary ammonium compounds (QACs) are not readily biodegradable as negative results were obtained in the Closed Bottle Test and Manometric Respirometry. However, either toxicity or low solubility (hexadecylpyridinium bromide) may be the reason rather than the inherent persistence of these compounds. In parallel runs the QACs were also tested in the presence of 200 mg/L ThOD sodium dodecyl sulphate (SDS), which is readily biodegradable. From the obtained respirograms a second wave with a height of 120 mg/L ThOD was observed for hexadecyltrimethylamnlonium chloride. This can only be attributed to complete mineralization (60% ThOD), which apparently was not inhibited by the toxic concentration of the QAC itself due to complex formation with SDS. In the absence of SDS the good soluble hexamethyltrimethylammonium chloride is toxic at the test concentration. From manometric respirometry experiments it was found that a concentration as low as 1 mg/L inhibited the mineralization of ghlcose/glutamic acid.
1515 PRIMARY AND ULTIMATE BIODEGRADABILITY IN A COMMON TEST SYSTEM FOR DETERGENTS
The primary biodegradation test at the screening stage proved predictive for wastewater treatment plants. Moreover, the sensitive semi-specific analytical procedure allows subdivision of positive results in categories, for example 80-85%, 85-90% and 90-95% MBAS/BiAS disappearance. Such data are an indication for the amount of less degradable constituents of which a surfactant mixture is composed. Although _>80% is a positive result, relatively low values (80-85 %), may coincide with a failure to pass the revised Closed Bottle Test. RBTs relying on oxygen consumption or carbon dioxide production have pass levels of 60% of the theoretical conversion to CO~_. An estimated 30% of the carbon of the tested compound is available for the growth of biomass, depending on the compound and the degrading organism. If in the test sample the proportion of recalcitrant surfactants is too high, it would not be available neither tbr assimilation nor for dissimilation (oxidation of substrate carbon to carbon dioxide). As a result a plateau below 60% of theoretical oxidation or carbon dioxide conversion may be obtained. It is not rare, however, that 90% of the theoretical oxygen consumption is obtained after 28 days for the positive reference chemical. Thus there may be a possibility that tbr samples containing 20-30% "hard" surfactants which remain unaltered during the 28 d incubation time, just 60% of the theoretical oxygen consumption is obtained after 28 days. Such samples would not pass the OECD Screening Test which has a pass level of 80% primary degradation already at day 19. Both a relatively high pass level for primary degradation after 19 days and testing ultimate biodegradation by means of a non-specific parameter is theretbre necessary. A positive result in one of the revised RBTs guarantees that a high level of primary degradation is due to ultimate biodegradation. Thus the set of revised RBTs should be considered as a supplement rather than a substitute for the OECD Screening Test for detergent surfactants. Application of a RBT constitutes a double screening to distinguish surfactants that are easily mineralizable from those being rapidly converted into stable intermediates. It should be emphasized that for cationic surfactants an international accepted standard method relying on semi-specific analysis is still lacking. Moreover, for those quaternary ammonium salts that are used as fabric softeners, specific analysis may not be applicable because of very low water solubility. Within the constraints as to solubility and toxicity, the DOC Die-Away, Carbon Dioxide Evolution, Manometric Respirometry and the Closed Bottle
1516
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DECISION
Figl. Evaluation of detergent surfactants in a common test system for both primary and ultimate biodegradability. Horizontal arrows indicate no further testing to be done; thin vertical arrows indicate which test(s) at the Confirmatory level are necessary if Screening proved inconclusive," thick vertical arrows mean that regulatory decisions have to be made.
1517 Test are considered applicable to these compounds.
A common test system for all surfactants, represented by Fig 1, includes screening and simulation as well as primary and ultimate biodegradation. If an anionic or nonionic passes both a primary and an ultimate biodegradability screening test, no further investigations are required. Thus any positive result at the screening stage is meant to indicate which simulation test is not necessary. These tests are still "acceptance tests" and will not have the power to reject chemicals. Testing mineralization in a laboratory simulation of a communal wastewater treatment plant is "confirmatory": If results of screening for primary and ultimate biodegradability are both negative, the confirmatory stage consists of testing both primary and ultimate biodegradation. The latter is tested in an activated sludge test or modifications of it, conform the current protocols (OECD, 1981; EC, 1884) and with a proposed pass-level of 70% DOC removal. If a surfactant is positive in the OECD Confirmatory Test for primary biodegradation, but fails to pass the mineralization analogue, the conclusion is justified that transformation products are persistent. A higher pass level in the OECD Confirmatory Test (_> 80% MBAS/BiAS elimination) is useful to ensure that stable ingredients in the detergent surfactant form only a minor part. If an anionic or nonionic surfactant has passed the OECD Screening Test but not any RBT, an activated sludge test based on DOC or COD removal of the test chemical should be conducted. Thus this test for ultimate biodegradability can reject a chemical if metabolites are not only stable in a screening mineralization test but also in a system which simulates a WWTP.
DISCUSSION
The predictive value of "ready biodegradability"
Surfactants represent one of the most prominent classes of chemicals not only present in raw sewage but also discharged with sewage sludge and to a minor extent with effluent. In view of the potential of widespread exposure, therefore, the life-cycle of surfactants should be carefully evaluated for relevant environmental conditions which may be considered the
1518 system sewage-WWTP-receiving surface water/soil (Fig 2). A positive result in a RBT has a high predictive value in this scenario. Although such a test system does neither provide any information on biodegradability under anaerobic conditions nor on the amount sorbed onto sewage sludge, a positive compound can safely be evaluated to the system sewage-WWTPreceiving surface water/soil. Only the mineralization rate in two component systems is required: the aeration tank of a WWTP and sludge amended aerobic soil. The first system is on the route to the receiving environment but largely prevents emission of the parent compound or its metabolites to surface water. The second system, sludge amended soil, is an "end station" in the life cycle of the surfactant where mineralization is fast with respect to the application rate of sludge to soil. Recent studies on LAS, which may be considered as a borderline case o.f "ready biodegradability", provide ample evidence for this assumption (Ward & Larson, 1989; Holt et al. 1989; Knaebel et al., 1990). Although mineralization largely determines the fate of a readily biodegradable chemical in the aeration tank of a WWTP, however, a significant portion will be adsorbed onto sewage sludge in the primary communal was~ watertreatmentFdant
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sedimentation tank. Then the surfactant may enter the terrestrial environment directly or after treatment in the anaerobic digester. Even if a surfactant is persistent in the digesting tank, mineralization will proceed once it is again exposed to aerobic conditions in sludge amended soil.
1519 The role of the WWTP is very critical in the chemical fate assessment of surfactants. Thus elimination of a readily biodegradable surfactant from the water phase during the activated sludge process is supposed to be complete, due to both sorption and mineralization at a rate of at least 3 hr -~ (Struijs et al., 1991). If elimination from the aqueous phase would be incomplete or if domestic waste water would be discharged without treatment, surfactants would enter the water compartment where it is subject to several simultaneous processes. In this scenario the surfactant may undergo degradation in the water phase, however it m.ay also be carried by suspended particles to the anaerobic sediment. This compartment should be considered a sink for surfactants or their metabolites and because anaerobic conditions prevail some surfactants may persist tbr a long time. Concentrations of LAS in the range of 0.2 to 69 mg/kg (dry basis) in the marine sediment in Tokyo bay have been reported by Kikuchi et al. (1986).
The problem of cationic surfactants
Although with 9 % only a minor share, these compounds may cause toxic effects to aquatic organisms. Fresh water concentrations in the range of 4 to 92 ppb (averaging 19 ppb) have been reported (Kappeler, 1982), indicating that biodegradation in a WWTP is insufficient. Analytical procedures such as DBAS (Disulphine Blue Active Substances) have been proposed for biodegradability testing (Knpfer & Waters, 1976), however, there is no standardized primary degradation test for this category. Only RBTs would cover these substances at the screening level. Most of the cationics are quaternary ammonium compounds and usually negative in a RBT. Testing mineralization in bench-scale activated sludge systems is hampered by several problems such as inhibitory effects, low solubility and the strong tendency to sorb onto sludge (sorption coefficients are typically higher than 10,000 L/kg). Test concentrations usually are at least 20 mg/L in order to allow DOC removal determinations from which mineralization of the test compound is evaluated. Unfortunately, either the QAC is water soluble but of the disinfectant type and thus toxic effects are expected at 20 mg/L or the QAC is of the fabric softener type and insoluble and not measurable as DOC. However, it is known that QACs form complexes with anionic surfactants (Boethling, 1984), showing completely different properties (toxicity and solubility). In the presence of a sufficient concentration of anionics, as normally is the case during wastewater treatment, toxic effects
1520 of QACs are generally lower and solubility is drastically increased. Commercially most important QACs, st,ch as distearyldimethylammonium chloride (DSDMAC) and ditallowdimethylammonium chloride (DTDMAC), have a very low solubility. Most published results are based on DOC removal of the anionic-cationic complex so allowing the measurement of DOC elimination of the QAC at concentrations up to 20 mg/L. Application of radiolabeled compounds at the confirmatory stage is required if such compounds are tested in the absence an excess neutralizing anionics. It has been reported that in laboratory scale activated sludge reactors, removal by sorption accounts for 5 to 15% of the total elimination of biodegradable QACs (Boethling, 1984). Emphasizing that those systems are not equipped with a primary sedimentation tank, such results are in good agreement with model predictions. For a readily biodegradable compound which is highly sorptive (K~, = 20,000 L/kg) a chemical fate model for waste water treatment plants (Struijs et al. 1991) would predict 12% removal by sorption. However, if primary sedimentation precedes the activated sludge process the predicted removal due to sorption would be up to 72 %, elimination by biodegradation 25 % and approximately 3 % would leave the installation via the effluent. If a QAC is persistent, the predicted elimination due to sorption would be approximately 90% so that 10% would enter the receiving surface water. Therefore, if a QAC is not readily biodegradable it is necessary to investigate the mineralization process in an activated sh,dge reactor, surface water and sludge amended soil.
Conclu.sions
The proposed test system exclusively employs standardized methods that have been published by the OECD and EC. Of these, the recently revised RBTs are screening tests and designed for classification of chemicals in view of notification purposes. As such they have no power to reject chemicals. Also in the current detergent legislation, a negative result of an OECD Screening Test can not be considered as conclusive. The here proposed double screening (both OECD Screening Test and an appropriate RBT) is not meant to reject surfactants but to limit further investigations as much as possible with a negligible risk of false positives. Although in the Closed Bottle Test ultimate biodegradation is measured as % ThOD, the test conditions provide better opportunity for primary biodegradation to occur than in the OECD Screening Test. The concentration of the tested compound is low in both systems (not higher
1521 than 5mg/L), but in the Closed Bottle Test the recommended concentration of biomass is 10 times higher and the test duration 9 days longer. In the DOC Die-Away, the Manometric Respirometry and the Carbon Evolution test, the concentration of the chemical is higher and may cause toxic effects. In spite of that, these tests should be taken into consideration as the concentration ratio compound/biomass is even more favourable than in the Closed Bottle and OECD Screening Test.
Screening for ultimate biodegradability by means of a RBT has the advantage of a high predictive value for environmental pathways through the aerobic environment. Numerous studies on LAS have demonstrated that both primary and t,ltimate biodegradation under a variety of laboratory and field conditions rapidly occurs (Larson, 1979; Larson & Payne, 1981; Ward and Larson 1989; Gerike, 1987 etc.). It has also been shown, though, that this is true provided that the environment is aerobic (Brunner et al. 1988). During anaerobic sludge treatment LAS seems rather stable. The predictive value of the RBT's enables the evaluation of a compound's life-cycle in densely populated industrial regions where treatment of wastewater is presumably 100%. RBTs cover all counpounds, also cationic surfactants, as non-specific analytical parameters are used to monitor the biodegradation process.
The dynamic test systems at the confirmatory stage, proposed in the common test system, need further research. Activated sludge tests and other related methodologies, based on the use of unspecific analysis (DOC or COD) which is indicative for ultimate biodegradation, have not been submitted yet to international ring exercises.
Standardized nnethods for anaerobic biodegradation are being submitted to international round-robin tests. In order to improve the test system for the evaluation of biodegradability of detergents, such methods, when published by the OECD, EC and ISO, should be included.
LITERATURE
Blok, J., de Morsier, A., Gerike, P., Reynolds, L., and Wellens, H. (1985). Harmonisation of ready biodegradability tests. Chemosphere 14, 1805-1820. Boethling, R.S. (1984). Environmental fate and toxicity in wastewater treatment of
1522 quaternary ammonium surfactants. Wat. Res., 18, 1061-1076. Brunner, P.H., S. Capri, A. Marcomini and W. Giger (1988). Occurrence and behaviour of linear alkylbenzenesulphonates, nonylphenol, nonylphenol mono- and nonylphenol diethoxylates in sewage and sewage sludge treatment. Wat. Res. 22, 1465-1472. CEC (1985) Degradation/Accumulation Sub-Group, Ring Test Programme 1983-1984, Assessment of biodegradability of chemicals in water by manometric respirometry, Final Report (H.A. Painter & E.F. King) ECETOC (1983). Technical Report No.8. Biodegradation testing: An assessment of the present status. EEC (1984), 84/449/EEC Directive, Annex Part C: Methods for the determination of ecotoxicity. EEC (1992), 92/69/EEC Directive, Annex Part C: Methods for the determination of ecotoxicity. Gerike, P. and W.K. Fischer (1979). A correlation study of biodegradability determinations with various chemicals in various tests. Ecotoxicol. Environ. Saf. 3, 159-173 Gerike, P. and W.K. Fischer (1981). A correlation study of biodegradability determinations with various chemicals in various tests. II Additional results and conclusions. Ecotoxicol. Environ. Saf. 5, 45-55 Gerike, P. (1987). Chp. 8 "Environmental Impact" In: Surfactants in consumer products (Ed. J. Falbe), Springer-Verlag, Berlin Heidelberg 1987. Holt, M.S., E. Matthijs and J. Waters (1989). The concentrations and fate of linear alkylbenzene sulphonate in sludge amended soils. Wat. Res. 23, 749-759.Kikuchi, M., A. Kappeler, T.U. (1982). Die aquatische Toxicit~t von DSDMAC. Tenside Detergents, 19, 169-176. Knaebel, D.B., T.W. Federle and J.R. Vestal (1990). Mineralization of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) in 11 contrasting soils. Environ. Toxicol. Chem. 9, 981-988. Kupfer, W. and J. Waters (1976). The determination of cationic surfactants in the presence of anionic surfactants in biodegradation test liquors. Analyt. Chim. Acta 85, 241-251. Larson, R.J. (1979). Estimation of biodegradation potential of xenobiotic organic chemicals. Appl. Environ. Microbiol. 38, 1153-1161. Larson, R.J. and A.G. Payne (1981). Fate of the benzene ring of linear alkylbenzene sulfonate in natural waters. Appl. Environ. Microbiol. 41,621-627.
1523
McEvoy, J. and W. Giger (1986). Determination of linear alkylbenzenesulfonates in sewage sludge by high-resolution gas chromatography/mass spectrometry. Environ. Sci. Technol. 20, 376-383. OECD (1971). Pollution by Detergents: Determination of the Biodegradability of Anionic Synthetic Surface Active Agents, OECD (Organization for Economic Cooperation and Development), Paris, 1971 OECD (1976). Environment Directorate. Proposed Method for the Determination of the Biodegradability of Surfactants used in Synthetic Detergents, OECD, Paris, 1976. OECD (1981) Guidelines for Testing Chemicals, Section III: Degradation and Accumulation, OECD, Paris, 1981 OECD (1993) OECD Guideline For Testing of Chemicals, "Ready Biodegradability" Painter, H.A. and E.F. King (1985). A respirometric method for the assessment of ready biodegradability: results of a ring test. Ecotoxicol. Environ. Saf. 9, 6-16. Pitter, P. and T. Fuka (1979). The problem of ultimate biodegradability of linear alkylbenzene sulphonates, Tenside Detergents 16, 298-302. Richter, H.J. and J. Knaut (1988). World prospects for surfactants. Proceedings, 2nd World Surfactant Congress, Paris, France, May 24-27, pp. 3-58. Struijs, J., J. Stoltenkamp and D. van de Meent (1991). A spreadsheet-based box model to predict the fate of xenobiotics in a municipal wastewater treatment plant. Wat. Res. 25, 891-900. Swisher, R.D. (1981). The problem of ultimate biodegradability of linear alkylbenzene sulfonates: an extension. Tenside Detergents 18, 57-63. Swisher, R.D. (1987). In: Surfactant Biodegradation, Marcel Dekker Inc.: New York, 1987. Ward, T.E. and R.J. Larson (1989), Biodegradation kinetics of linear alkylbenzene sulfonate in sludge-amended agricultural soils. Ecotoxicol. Environ. Saf. 17, 119-130. Wickbold, R. (1973). Analytical determination of small amounts of nonionic surfactants. Tenside Detergents I0, 179-182.
Pergamon 0045-6535(94)E0073-3
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0045-6535/94 $7.00+0.00
TESTING SURFACTANTS FOR ULTIMATE BIODEGRADABILITY
J. Struijs° and J. Stoltenkamp
Laboratory for Ecotoxicology, National Institute of Public Health and Environmental Protection, P.O. BOX 1, 3720 BA Bilthoven, The Netherlands ° author to whom correspondence should be addressed (Received in Germany 3 September 1993; accepted 2 December 1993)
ABSTRACT The development of screening biodegradability tests is briefly reviewed. The "new chemicals" screening methods according to the OECD (1981) and the EC (1984) are based upon ultimate biodegradability or mineralization by making use of some analytical parameter which is not specific for the chemical. The methods are now known as "ready biodegradability" tests (RBTs). Screening tests that are specifically designed for surfactants have been developed by the OECD earlier. In the latter tests primary biodegradation is monitored by means of a specific analytical parameter. Recent harmonization and improvement of RBTs are evaluated in order to see if their applicability can be extended to screen surfactants for ultimate biodegradability. It is shown that conditions for biotransformation of a surfactant in one of the revised RBTs, i.e. the Closed Bottle Test, are similar or even more favourable than in the screening tests for surfactants. Testing ultimate biodegradability is proposed as a supplement to primary biodegradation of surfactants, at the level of both Screening and Confirmatory. Several revised RBTs were applied to evaluate the ultimate biodegradability of twelve surfactants. Results are in agreement with previous studies and correspond to validated structure biodegradability relationships for surfactants. It is concluded that a positive result in a RBT can safely be extrapolated to aerobic environments in regions where domestic waste water is processed by sewage treatment plants. The proposed test system exclusively employs standardized methods that are published by the OECD and EC. INTRODUCTION
Surface active substances applied in washing powders and other industrial and household products are high volume chemicals. Approximately 1,000,000 metric tons of linear
1503
1504 alkylbenzene sulphonates (LAS) and 470,000 metric tons of linear alcohol ethoxylates (LAE) are annually used in the United States, Western Europe and Japan (Richter and Knaut, 1988). Biodegradability criteria of surface active substances in detergents deviate from those in the regulation of other chemicals. The reasons for this are both historical and practical. However, recent developments in test methods and regulatory measures justify a new approach to evaluate surfactant biodegradability. Results from appropriate tests may be used to better predict the ultimate fate of surfactants in the environment, rather than to indicate that "elimination due to waste water treatment is acceptable". To achieve sufficient elimination during wastewater treatment, biodegradation is considered a process which should be fast enough to play a significant role. In detergent legislation, however, the term "biological degradability" is not further specified. It may refer toprimary
biodegradation, defined as the microbial alteration of a molecule sufficient to remove a characteristic property such as surface activity (foaming) or its response to an analytical procedure specific for the starting compound but not for its degradation products. For the two most important categories, the anionics and the nonionics, special reference is made to the European Community Directives 73/404 EEC and 73/405 EEC amended by 82/242 EEC and 82/243 EEC, specifying the test methods for determining biodegradability. In these standardized tests, biodegradation is quantified by means of a semi-specific analytical parameter, i.e. the disappearance of Methylene Blue Active Substances (MBAS) and Bismuth Active Substances (BIAS) response, respectively. This analytical approach determines essentially primary degradation by measuring the disappearance or removal of surfactant properties and not mineralization to CO2. However, no information from test data can be derived that is essential to evaluate the life-cycle of these compounds. Investigations reported by McEvoy and Giger (1986) and Brunner et al. (1988) revealed that persistent metabolites may have escaped our notice. Standardized biodegradability tests, specified by the new chemicals legislation, have been updated recently (OECD, 1993). Generally, methods that rely on mineralization have been adopted in the Annex to Commission Directive 92/69/EEC adapting to technical progress Council Directive 67/548/EEC, also known as the "notification directive". The purpose of this update was harmonization and improvement of the methods in order to enhance the applicability. In this study a comparison is made between the set of harmonized OECD/EC methods and the detergent OECD Screening Test. The latter has been designed to select "soft" surface
1505 active compounds so avoiding unnecessary expensive laboratory investigations. A selection of appropriate methods to screen surfactants is proposed which also accounts for ultimate
biodegradability in addition to primary degradation. Biodegradability" (RBTs)
OECD
tests
for
"Ready
reduce uncertainty about effluent containing bioresistant
intermediates formed from the parent surfactant.
BIODEGRADABILITY TESTS SPECIFIED BY THE DETERGENT LEGISLATION
Elimination of the surface active properties during wastewater treatment has become a legislative requirement. Obviously, the first biodegradability test methods were designed so that elimination of surface active properties due to microbial transformation was the test criterion of choice. Those detergents that passed a biodegradability test, specified in the detergent laws, indeed did not foam during wastewater treatment and did not cause effluent toxicity problems. During the years of 1968-1970, an OECD group of experts worked on a common system of testing to be accepted internationally. As a result, a system was published (OECD, 1971) which consists of two stages, differing both in principle and in indications which can be drawn from them as to the biodegradability of anionic surface active substances:
The OECD Screening Test, a static flask test which is relatively quick and simple. This test must be considered as an "acceptance test" and not as an "rejection test". It selects "soft" anionic surfactants which do not have to be tested further as a high biodegradability is expected in sewage works.
The OECD Confirmatory Test, based on the simulation of the conditions existing in an activated sludge plant. This test should be used for any anionic surfactant which may not have passed the OECD Screening Test, in order to confirm or disprove the first results obtained. The results obtained at this stage are the only ones to be taken into consideration in the refusal or acceptance of products not accepted by the OECD Screening Test.
The degree of biodegradability is here defined as the percentage of MBAS eliminated under the conditions of the test. Thus only anionic surfactants which form a chloroform-
1506 soluble salt with methylene blue are detected. This analytical parameter responds to all synthetic anionic surface active agents used in detergents, but not to soap which is considered as an anionic surfactant derived from natural products. Since most detergent formulations contain a mixture of homologues and isomers, such a semi-specific analytical parameter proved very useful. Both tests are also appropriate for anionic surfactants contained in formulated products if use is made of well described extraction procedures (OECD, 1971). The stringency of the screening test is determined by the test duration, the pass level and the performance of the applied inoculum against two standards of biodegradability. The pass level is 80% degradation within 19 days. The alnot, nt of inoculum must be such that the "soft" standard Marion A, which is a commercial linear alkyl benzene sulphonate (LAS), is degraded to a value between) 90 and 95 per cent within 14 days. The disappearance of the "hard" standard, TBS (tetra propylene benzene sulphonate), should be less than 35% within the test duration. Usually 0.5 ml secondary eflluent per litre test solution will be adequate to cause the standards to follow the required degradation patterns. However, there are indications that microbial COlnmunities may have adapted to TBS, which probably has been present for several decades as a minor constituent of detergent products. European Community Directives 73/404 EEC and 73/405 EEC were published in 1973 in order to harmonize the regulations of the member states in this respect. The class of nonionic surfactants comprise numerous compounds with a wide structural diversity and usually lacking a common functional group. Obviously, the biological systems employed for testing anionics also suits the nonionics, however, the principal difficulty in establishing a test procedure tbr this group of chemicals was lying in the analytical methodology. After several ring tests, the OECD expert group decided to adopt the tetraiodobismuthate method according to Wickbold (1973). The procedure consists of preconcentration by sublation into ethyl acetate, precipitation of the nonionic agents by a bismuth containing reagent (Dragendorff reagent) and potentiometric titration of the bismuth content of the precipitate. In analogy to the anionics, analytical results are simply expressed as mg/L BiAS. Both the static test (OECD Screening Test) and the dynamic simulation test (OECD Confirmatory Test) are applicable to nonionic st, rfactants either as such or contained in formulated detergents. In the latter case, a prepurification procedure is necessary, consisting of an alcoholic extraction and ion-exchange because also anionic surfactants respond as BIAS. Although the Wickbold method refers specifically to water soluble ethoxylates, the OECD expert group considered that this was acceptable as nonionic
1507 surfactants predominantly consist of these compounds. The new test procedure (OECD, 1976) for anionic surfactants underwent minor changes with respect to 1971 and the analytical methods appropriate for the nonionics were added. Both for the anionics and the nonionics the "soft" and the "hard" standards are Marlon A and TBS, respectively.
BIODEGRADABILITY TESTS SPECIFIED BY NEW CHEMICALS LEGISLATION
For the very diverse class of "new chemicals" a simple screening is required in the framework of some sort of test hierarchy. A non-specific analytical parameter to follow the course of biodegradation is not only cost-effective but it also responds to any biodegradation residues or intermediates, indicating the extent of ultimate biodegradation of the tested compound. OECD experts modified and combined elements of the OECD Screening Test for detergents and other reported methods. This led to the acceptance of five tests which rely on a non-specific monitoring parameter: dissolved organic carbon (DOC) die away, oxygen uptake or carbon dioxide production. The five tests have in common with the OECD Screening Test that the test compound serves as the sole carbon source and that it is exposed to a relatively low amount of microbial biomass. A standardized test duration of 28 days gives the inoculum a limited opportunity to adapt to the test compound prior to the onset of biodegradation. Although standardization of the inoculum would improve the comparability of the methods, a mixed inoculum derived from a local source, f.i. a communal wastewater treatment plant (WWTP), is both practical and appropriate to ensure the presence of a wide variety of species in the tests. In view of the stringency, required in a screening test, preadaptation of the inoculum to the test compound is not allowed. Preliminary screening for rapid mineralization has become related to the term "ready biodegradability", which is an arbitrary classification of chemicals that have passed one of these static tests for ultimate biodegradability. The proposed tests are so stringent that it is assumed that positive chemicals will rapidly and completely biodegrade in a wide variety of natural aerobic environments (OECD, 1981). In 1984, these tests were adopted in Annex V (methods C.3 to C.7) of Directive 84/449/EEC, containing technical adaptation to the "notification directive" 67/548/EEC, Annex V (test guidelines for chemical substances).
In a correlation study on 44 compounds and several test methods, Gerike and Fischer (1979 and 1981) found a varying stringency of the RBT's, decreasing generally in the order
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1509 Closed Bottle Test, MITI(I), Modified OECD Test, Modified AFNOR test and the Modified Sturm Test. The lower stringency of the latter two tests compared to the Closed Bottle Test and the Modified OECD Screening Test is explained from the inoculum concentration which is several orders of magnitude higher (see Table 1). In spite of a relatively high biomass inoculum, the number of chemicals that are positive in the MITI(I) Test is rather low. This has been attributed to the prescribed culturing procedure which significantly reduces the metabolic diversity of the inoculum. Inoculum concentrations in the RBTs span more than 4 orders of magnitude, having an adverse effect on the mutual comparability of test results.
RBTs of 1981 generally offer a poor choice to select a method which is suitable for screening surfaetants, due to either the concentration of the test chemical or the applied amount of inoculum. All tests, with exception of the Closed Bottle Test, prescribe a relatively high concentration of the test substance being in the range of 10-100 mg/L. This is the consequence of the use of a non-specific analytical parameter. If the purpose is to screen the rather toxic surfactants for biodegradability, these tests should be considered inferior with respect to the OECD Screening Test, as the latter allows 5 mg/L. The test compound concentrations in the Closed Bottle Test and the OECD Screening Test are approximately equal. However, the prescribed inoculum concentration is ten times lower compared to the OECD Screening Test. As the latter test proved fall-safe, as far as primary degradation is concerned, the higher threshold for any biodegradation to occur in the Closed Bottle Test is not necessary and even at undesirable.
Harmonization of Ready Biodegradability Tests
A highly varying stringency in RBTs and differences in the mineral medium are not rational. Preparations to revise the existing guidelines 301 A to E (OECD, 1981) started in 1985 with the aim to harmonize the methods. Earlier attempts were made to adopt an additional test which is suitable for poorly soluble compounds. The Manometric Respirometry Test, proposed as a simplification of the MITI(1) test, appeared a reliable method in two international inter-laboratory exercises (Painter & King, 1985; CEC, 1985). Most of the weaknesses, mentioned in the foregoing, were assessed and discussed in a technical report on the statt, s of biodegradability testing by The European Chemical Industry
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1511 Ecology and Toxicology Centre (ECETOC, 1983), expressing the need for harmonization of the RBTs (Blok et al., 1985). Thirty-seven laboratories participated in 1988 in an OECD inter-laboratory test to investigate the validity of the proposed harmonization. Based on these results and after lengthy discussions as to the stringency of the new methods, the OECD published a new Guideline for Ready Biodegradability in 1993, containing six methods: DOC Die-away, the CO~ Evolution, Manometric Respirometry, the Modified OECD Screening Test, Closed Bottle Test and the MITI(1) test. The harmonization is reflected in the mineral medium which is similar in the first four tests, but also in the maximum permitted inoculum concentrations which span a range of only two orders of magnitude in all tests if the Modified OECD Screening Test is not taken into consideration. The °'10 days-window" was installed as an extra pass criterion for ready biodegradability stipt,lating that the time elapsed from 10% to 90% of the plateau in the biodegradation curve is no longer than 10 days, however, for the Closed Bottle Test 14 days. The concentration ratio of the test chemical and the inoculum is less favourable in the Modified OECD Screening test than in the other RBTs and therefore toxic effects restrict its applicability. The methods have now been adopted in the Annex to Commission Directive 92/69/EEC adapting to technical progress Council Directive 67/548/EEC. These methods, sunlmarized in Table 2, are referred to as "revised RBTs" in the following.
SCREENING SURFACTANTS FOR ULTIMATE BIODEGRADABILITY
Several revised RBTs seem appropriate to screen surfactants for ultimate biodegradability. At first sight the revised Closed Bottle Test is the method of first choice for the good soluble anionic and nonionic surfactants. The concentration of the test substance is as low as in the OECD Screening Test for detergents and generally will not cause inhibitory effects. If the highest inoculum concentration is applied, i.e. 5 ml secondary effluent/L, the test system would provide a better opportunity for any biodegradation to occur than the OECD Screening Test. Provided that the origin of the inoculum is similar for both tests, i.e. secondary effluent from the same WWTP, a surfactant that has passed the OECD Screening Test system, would certainly undergo complete primary degradation in the revised Closed Bottle Test. This is based upon two arguments. First, the amount of inoculum in the revised Closed Bottle Test is ten times higher than recommended in the OECD Screening Test. Second, the test duration in the Closed Bottle Test is 28 days compared to 19 days in the OECD Screening Test for
1512 detergents. Thus an observed negative result in the Closed Bottle, while criteria for sufficient primary biodegradation are met (___80% MBAS disappearance), can not be attributed to a lack of opportunity in the test system because the conditions are more favourable for biotransformation to occur.
Experimental results
Table 3 contains test results of twelve surfactants of varying ease of biodegradability. Most of these compounds were tested in the DOC Die Away, the Manometric Respirometry and the Closed Bottle Test. In the latter test the positive reference chemical (sodium acetate) displayed 70-90% ThOD in all runs after one week. All tests were conducted according to the revised OECD Guidelines (OECD, 1992). The amount of inoculum per litre medium was 5 mL secondary effluent in the Closed Bottle Test and 30 mg/L activated sludge (dry matter) in the other tests. The chemical concentrations were 2.5-4 mg/L in the Closed Bottle, 20 mg/L in the DOC Die-Away and 100 mg/L ThOD (approximately 50 mg test chemical/L) in the Manometric Respirometry.
LAS around Ct2 passed the DOC Die-Away and the Manometric Respirometry but not the Closed Bottle Test. Although in principle a substance may be classified as readily biodegradable if it is positive in one RBT but fails to pass another, it is worth to mention here that usually for readily biodegradable chemicals > 90% DOC removal is measured. For LAS only 74% was found which is in good agreement with previously reported results (Gerike, 1987). In a reply to a paper on the problem of ultimate biodegradability of LAS (Pitter & Fuka, 1979), Swisher (1981) has summarized the literature on this subject. He concluded that in the real world significantly higher DOC removals have been reported: averaging 80-90% in activated sludge experiments and up to 94% in river water. It also appeared that only 45-75% DOC removal was found from determinations in inoculated synthetic media, used in all kinds of static screening tests. This might be an indication that screening by means of a non-specific analytical parameter is rather sensitive for less degradable metabolites, as primary degradation of LAS in inoculated synthetic media is fast and usually exceeds 90%. This is confirmed in the present study and may be an explanation for the fact that in the Closed Bottle Test only 55 % was found, in contrast to sodium acetate for which 90% was measured in parallel runs.
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1514 The poorly degradable tetrapropylenebenzene sulphonate behaved as expected. Also the results from the anionic and nonionic surfactants with linear alkyl hydrophobic groups but without an aryl moiety, such as l-octane sulphonate, Na dodecyl sulphate and hexadecanol polyethoxylate (20 EO), are in agreement with validated generalizations relating surfactant structure and biodegradability (Swisher, 1987). These compounds consistently passed all three RBTs, apparently without the formation of stable intermediates as is reflected from DOC removals of 90% or higher. The branched sodium 3,9-diethyl, 6-tridecyl sulphate did not pass the oxygen consumption tests, probably because 17% of the carbon was refractory. Nonylphenol ethoxylate (10 EO) behaved erratically in the DOC Die-Away (55-70%) but did not pass the tests based on oxygen uptake as can be expected if only 70% DOC removal is measured. The branched iso-octylphenol ethoxylate (10 EO) with two quaternary carbon atoms did not pass any of the RBTs. In the DOC-Die-Away test 66% DOC reduction was measured after one week, however, this appeared to be a plateau as in the following three weeks no further DOC removal was found. This was confirmed by only 40% ThOD in the Manometric Respirometry. Tween 60 is not readily biodegradable although the results strongly suggest that primary biodegradation has occurred and was probably complete. The quaternary ammonium compounds (QACs) are not readily biodegradable as negative results were obtained in the Closed Bottle Test and Manometric Respirometry. However, either toxicity or low solubility (hexadecylpyridinium bromide) may be the reason rather than the inherent persistence of these compounds. In parallel runs the QACs were also tested in the presence of 200 mg/L ThOD sodium dodecyl sulphate (SDS), which is readily biodegradable. From the obtained respirograms a second wave with a height of 120 mg/L ThOD was observed for hexadecyltrimethylamnlonium chloride. This can only be attributed to complete mineralization (60% ThOD), which apparently was not inhibited by the toxic concentration of the QAC itself due to complex formation with SDS. In the absence of SDS the good soluble hexamethyltrimethylammonium chloride is toxic at the test concentration. From manometric respirometry experiments it was found that a concentration as low as 1 mg/L inhibited the mineralization of ghlcose/glutamic acid.
1515 PRIMARY AND ULTIMATE BIODEGRADABILITY IN A COMMON TEST SYSTEM FOR DETERGENTS
The primary biodegradation test at the screening stage proved predictive for wastewater treatment plants. Moreover, the sensitive semi-specific analytical procedure allows subdivision of positive results in categories, for example 80-85%, 85-90% and 90-95% MBAS/BiAS disappearance. Such data are an indication for the amount of less degradable constituents of which a surfactant mixture is composed. Although _>80% is a positive result, relatively low values (80-85 %), may coincide with a failure to pass the revised Closed Bottle Test. RBTs relying on oxygen consumption or carbon dioxide production have pass levels of 60% of the theoretical conversion to CO~_. An estimated 30% of the carbon of the tested compound is available for the growth of biomass, depending on the compound and the degrading organism. If in the test sample the proportion of recalcitrant surfactants is too high, it would not be available neither tbr assimilation nor for dissimilation (oxidation of substrate carbon to carbon dioxide). As a result a plateau below 60% of theoretical oxidation or carbon dioxide conversion may be obtained. It is not rare, however, that 90% of the theoretical oxygen consumption is obtained after 28 days for the positive reference chemical. Thus there may be a possibility that tbr samples containing 20-30% "hard" surfactants which remain unaltered during the 28 d incubation time, just 60% of the theoretical oxygen consumption is obtained after 28 days. Such samples would not pass the OECD Screening Test which has a pass level of 80% primary degradation already at day 19. Both a relatively high pass level for primary degradation after 19 days and testing ultimate biodegradation by means of a non-specific parameter is theretbre necessary. A positive result in one of the revised RBTs guarantees that a high level of primary degradation is due to ultimate biodegradation. Thus the set of revised RBTs should be considered as a supplement rather than a substitute for the OECD Screening Test for detergent surfactants. Application of a RBT constitutes a double screening to distinguish surfactants that are easily mineralizable from those being rapidly converted into stable intermediates. It should be emphasized that for cationic surfactants an international accepted standard method relying on semi-specific analysis is still lacking. Moreover, for those quaternary ammonium salts that are used as fabric softeners, specific analysis may not be applicable because of very low water solubility. Within the constraints as to solubility and toxicity, the DOC Die-Away, Carbon Dioxide Evolution, Manometric Respirometry and the Closed Bottle
1516
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DECISION
Figl. Evaluation of detergent surfactants in a common test system for both primary and ultimate biodegradability. Horizontal arrows indicate no further testing to be done; thin vertical arrows indicate which test(s) at the Confirmatory level are necessary if Screening proved inconclusive," thick vertical arrows mean that regulatory decisions have to be made.
1517 Test are considered applicable to these compounds.
A common test system for all surfactants, represented by Fig 1, includes screening and simulation as well as primary and ultimate biodegradation. If an anionic or nonionic passes both a primary and an ultimate biodegradability screening test, no further investigations are required. Thus any positive result at the screening stage is meant to indicate which simulation test is not necessary. These tests are still "acceptance tests" and will not have the power to reject chemicals. Testing mineralization in a laboratory simulation of a communal wastewater treatment plant is "confirmatory": If results of screening for primary and ultimate biodegradability are both negative, the confirmatory stage consists of testing both primary and ultimate biodegradation. The latter is tested in an activated sludge test or modifications of it, conform the current protocols (OECD, 1981; EC, 1884) and with a proposed pass-level of 70% DOC removal. If a surfactant is positive in the OECD Confirmatory Test for primary biodegradation, but fails to pass the mineralization analogue, the conclusion is justified that transformation products are persistent. A higher pass level in the OECD Confirmatory Test (_> 80% MBAS/BiAS elimination) is useful to ensure that stable ingredients in the detergent surfactant form only a minor part. If an anionic or nonionic surfactant has passed the OECD Screening Test but not any RBT, an activated sludge test based on DOC or COD removal of the test chemical should be conducted. Thus this test for ultimate biodegradability can reject a chemical if metabolites are not only stable in a screening mineralization test but also in a system which simulates a WWTP.
DISCUSSION
The predictive value of "ready biodegradability"
Surfactants represent one of the most prominent classes of chemicals not only present in raw sewage but also discharged with sewage sludge and to a minor extent with effluent. In view of the potential of widespread exposure, therefore, the life-cycle of surfactants should be carefully evaluated for relevant environmental conditions which may be considered the
1518 system sewage-WWTP-receiving surface water/soil (Fig 2). A positive result in a RBT has a high predictive value in this scenario. Although such a test system does neither provide any information on biodegradability under anaerobic conditions nor on the amount sorbed onto sewage sludge, a positive compound can safely be evaluated to the system sewage-WWTPreceiving surface water/soil. Only the mineralization rate in two component systems is required: the aeration tank of a WWTP and sludge amended aerobic soil. The first system is on the route to the receiving environment but largely prevents emission of the parent compound or its metabolites to surface water. The second system, sludge amended soil, is an "end station" in the life cycle of the surfactant where mineralization is fast with respect to the application rate of sludge to soil. Recent studies on LAS, which may be considered as a borderline case o.f "ready biodegradability", provide ample evidence for this assumption (Ward & Larson, 1989; Holt et al. 1989; Knaebel et al., 1990). Although mineralization largely determines the fate of a readily biodegradable chemical in the aeration tank of a WWTP, however, a significant portion will be adsorbed onto sewage sludge in the primary communal was~ watertreatmentFdant
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I Fig 2. Path and fate of readily biodegradable surfi~ctants or their metabolites in the ~ystem raw sewagewastewater treatment plant-receiving environment.
sedimentation tank. Then the surfactant may enter the terrestrial environment directly or after treatment in the anaerobic digester. Even if a surfactant is persistent in the digesting tank, mineralization will proceed once it is again exposed to aerobic conditions in sludge amended soil.
1519 The role of the WWTP is very critical in the chemical fate assessment of surfactants. Thus elimination of a readily biodegradable surfactant from the water phase during the activated sludge process is supposed to be complete, due to both sorption and mineralization at a rate of at least 3 hr -~ (Struijs et al., 1991). If elimination from the aqueous phase would be incomplete or if domestic waste water would be discharged without treatment, surfactants would enter the water compartment where it is subject to several simultaneous processes. In this scenario the surfactant may undergo degradation in the water phase, however it m.ay also be carried by suspended particles to the anaerobic sediment. This compartment should be considered a sink for surfactants or their metabolites and because anaerobic conditions prevail some surfactants may persist tbr a long time. Concentrations of LAS in the range of 0.2 to 69 mg/kg (dry basis) in the marine sediment in Tokyo bay have been reported by Kikuchi et al. (1986).
The problem of cationic surfactants
Although with 9 % only a minor share, these compounds may cause toxic effects to aquatic organisms. Fresh water concentrations in the range of 4 to 92 ppb (averaging 19 ppb) have been reported (Kappeler, 1982), indicating that biodegradation in a WWTP is insufficient. Analytical procedures such as DBAS (Disulphine Blue Active Substances) have been proposed for biodegradability testing (Knpfer & Waters, 1976), however, there is no standardized primary degradation test for this category. Only RBTs would cover these substances at the screening level. Most of the cationics are quaternary ammonium compounds and usually negative in a RBT. Testing mineralization in bench-scale activated sludge systems is hampered by several problems such as inhibitory effects, low solubility and the strong tendency to sorb onto sludge (sorption coefficients are typically higher than 10,000 L/kg). Test concentrations usually are at least 20 mg/L in order to allow DOC removal determinations from which mineralization of the test compound is evaluated. Unfortunately, either the QAC is water soluble but of the disinfectant type and thus toxic effects are expected at 20 mg/L or the QAC is of the fabric softener type and insoluble and not measurable as DOC. However, it is known that QACs form complexes with anionic surfactants (Boethling, 1984), showing completely different properties (toxicity and solubility). In the presence of a sufficient concentration of anionics, as normally is the case during wastewater treatment, toxic effects
1520 of QACs are generally lower and solubility is drastically increased. Commercially most important QACs, st,ch as distearyldimethylammonium chloride (DSDMAC) and ditallowdimethylammonium chloride (DTDMAC), have a very low solubility. Most published results are based on DOC removal of the anionic-cationic complex so allowing the measurement of DOC elimination of the QAC at concentrations up to 20 mg/L. Application of radiolabeled compounds at the confirmatory stage is required if such compounds are tested in the absence an excess neutralizing anionics. It has been reported that in laboratory scale activated sludge reactors, removal by sorption accounts for 5 to 15% of the total elimination of biodegradable QACs (Boethling, 1984). Emphasizing that those systems are not equipped with a primary sedimentation tank, such results are in good agreement with model predictions. For a readily biodegradable compound which is highly sorptive (K~, = 20,000 L/kg) a chemical fate model for waste water treatment plants (Struijs et al. 1991) would predict 12% removal by sorption. However, if primary sedimentation precedes the activated sludge process the predicted removal due to sorption would be up to 72 %, elimination by biodegradation 25 % and approximately 3 % would leave the installation via the effluent. If a QAC is persistent, the predicted elimination due to sorption would be approximately 90% so that 10% would enter the receiving surface water. Therefore, if a QAC is not readily biodegradable it is necessary to investigate the mineralization process in an activated sh,dge reactor, surface water and sludge amended soil.
Conclu.sions
The proposed test system exclusively employs standardized methods that have been published by the OECD and EC. Of these, the recently revised RBTs are screening tests and designed for classification of chemicals in view of notification purposes. As such they have no power to reject chemicals. Also in the current detergent legislation, a negative result of an OECD Screening Test can not be considered as conclusive. The here proposed double screening (both OECD Screening Test and an appropriate RBT) is not meant to reject surfactants but to limit further investigations as much as possible with a negligible risk of false positives. Although in the Closed Bottle Test ultimate biodegradation is measured as % ThOD, the test conditions provide better opportunity for primary biodegradation to occur than in the OECD Screening Test. The concentration of the tested compound is low in both systems (not higher
1521 than 5mg/L), but in the Closed Bottle Test the recommended concentration of biomass is 10 times higher and the test duration 9 days longer. In the DOC Die-Away, the Manometric Respirometry and the Carbon Evolution test, the concentration of the chemical is higher and may cause toxic effects. In spite of that, these tests should be taken into consideration as the concentration ratio compound/biomass is even more favourable than in the Closed Bottle and OECD Screening Test.
Screening for ultimate biodegradability by means of a RBT has the advantage of a high predictive value for environmental pathways through the aerobic environment. Numerous studies on LAS have demonstrated that both primary and t,ltimate biodegradation under a variety of laboratory and field conditions rapidly occurs (Larson, 1979; Larson & Payne, 1981; Ward and Larson 1989; Gerike, 1987 etc.). It has also been shown, though, that this is true provided that the environment is aerobic (Brunner et al. 1988). During anaerobic sludge treatment LAS seems rather stable. The predictive value of the RBT's enables the evaluation of a compound's life-cycle in densely populated industrial regions where treatment of wastewater is presumably 100%. RBTs cover all counpounds, also cationic surfactants, as non-specific analytical parameters are used to monitor the biodegradation process.
The dynamic test systems at the confirmatory stage, proposed in the common test system, need further research. Activated sludge tests and other related methodologies, based on the use of unspecific analysis (DOC or COD) which is indicative for ultimate biodegradation, have not been submitted yet to international ring exercises.
Standardized nnethods for anaerobic biodegradation are being submitted to international round-robin tests. In order to improve the test system for the evaluation of biodegradability of detergents, such methods, when published by the OECD, EC and ISO, should be included.
LITERATURE
Blok, J., de Morsier, A., Gerike, P., Reynolds, L., and Wellens, H. (1985). Harmonisation of ready biodegradability tests. Chemosphere 14, 1805-1820. Boethling, R.S. (1984). Environmental fate and toxicity in wastewater treatment of
1522 quaternary ammonium surfactants. Wat. Res., 18, 1061-1076. Brunner, P.H., S. Capri, A. Marcomini and W. Giger (1988). Occurrence and behaviour of linear alkylbenzenesulphonates, nonylphenol, nonylphenol mono- and nonylphenol diethoxylates in sewage and sewage sludge treatment. Wat. Res. 22, 1465-1472. CEC (1985) Degradation/Accumulation Sub-Group, Ring Test Programme 1983-1984, Assessment of biodegradability of chemicals in water by manometric respirometry, Final Report (H.A. Painter & E.F. King) ECETOC (1983). Technical Report No.8. Biodegradation testing: An assessment of the present status. EEC (1984), 84/449/EEC Directive, Annex Part C: Methods for the determination of ecotoxicity. EEC (1992), 92/69/EEC Directive, Annex Part C: Methods for the determination of ecotoxicity. Gerike, P. and W.K. Fischer (1979). A correlation study of biodegradability determinations with various chemicals in various tests. Ecotoxicol. Environ. Saf. 3, 159-173 Gerike, P. and W.K. Fischer (1981). A correlation study of biodegradability determinations with various chemicals in various tests. II Additional results and conclusions. Ecotoxicol. Environ. Saf. 5, 45-55 Gerike, P. (1987). Chp. 8 "Environmental Impact" In: Surfactants in consumer products (Ed. J. Falbe), Springer-Verlag, Berlin Heidelberg 1987. Holt, M.S., E. Matthijs and J. Waters (1989). The concentrations and fate of linear alkylbenzene sulphonate in sludge amended soils. Wat. Res. 23, 749-759.Kikuchi, M., A. Kappeler, T.U. (1982). Die aquatische Toxicit~t von DSDMAC. Tenside Detergents, 19, 169-176. Knaebel, D.B., T.W. Federle and J.R. Vestal (1990). Mineralization of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) in 11 contrasting soils. Environ. Toxicol. Chem. 9, 981-988. Kupfer, W. and J. Waters (1976). The determination of cationic surfactants in the presence of anionic surfactants in biodegradation test liquors. Analyt. Chim. Acta 85, 241-251. Larson, R.J. (1979). Estimation of biodegradation potential of xenobiotic organic chemicals. Appl. Environ. Microbiol. 38, 1153-1161. Larson, R.J. and A.G. Payne (1981). Fate of the benzene ring of linear alkylbenzene sulfonate in natural waters. Appl. Environ. Microbiol. 41,621-627.
1523
McEvoy, J. and W. Giger (1986). Determination of linear alkylbenzenesulfonates in sewage sludge by high-resolution gas chromatography/mass spectrometry. Environ. Sci. Technol. 20, 376-383. OECD (1971). Pollution by Detergents: Determination of the Biodegradability of Anionic Synthetic Surface Active Agents, OECD (Organization for Economic Cooperation and Development), Paris, 1971 OECD (1976). Environment Directorate. Proposed Method for the Determination of the Biodegradability of Surfactants used in Synthetic Detergents, OECD, Paris, 1976. OECD (1981) Guidelines for Testing Chemicals, Section III: Degradation and Accumulation, OECD, Paris, 1981 OECD (1993) OECD Guideline For Testing of Chemicals, "Ready Biodegradability" Painter, H.A. and E.F. King (1985). A respirometric method for the assessment of ready biodegradability: results of a ring test. Ecotoxicol. Environ. Saf. 9, 6-16. Pitter, P. and T. Fuka (1979). The problem of ultimate biodegradability of linear alkylbenzene sulphonates, Tenside Detergents 16, 298-302. Richter, H.J. and J. Knaut (1988). World prospects for surfactants. Proceedings, 2nd World Surfactant Congress, Paris, France, May 24-27, pp. 3-58. Struijs, J., J. Stoltenkamp and D. van de Meent (1991). A spreadsheet-based box model to predict the fate of xenobiotics in a municipal wastewater treatment plant. Wat. Res. 25, 891-900. Swisher, R.D. (1981). The problem of ultimate biodegradability of linear alkylbenzene sulfonates: an extension. Tenside Detergents 18, 57-63. Swisher, R.D. (1987). In: Surfactant Biodegradation, Marcel Dekker Inc.: New York, 1987. Ward, T.E. and R.J. Larson (1989), Biodegradation kinetics of linear alkylbenzene sulfonate in sludge-amended agricultural soils. Ecotoxicol. Environ. Saf. 17, 119-130. Wickbold, R. (1973). Analytical determination of small amounts of nonionic surfactants. Tenside Detergents I0, 179-182.