Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• (2) German Government (1962). Ordinance of the degradability of detergents in washing and cleaning agents. Bundesgesetzblatt, Pt.1 No 49: 698-706.

• (3) Painter HA and King EF (1978a). WRc porous-pot method for assessing biodegradability. Technical Report No 70, Water Research Centre, Medmenham, UK.

• (4) Painter HA and King EF (1978b). The effect of phosphate and temperature on growth of activated sludge and on biodegradation of surfactants. Wat. Res. 12: 909-915.

• (5) Eckenfelder, W.W (19) US EPA.

• (6) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability.

• (7) Chapter C.12 of this Annex, Biodegradation — Modified SCAS Test.

• (8) Chapter C.19 of this Annex, Estimation of the Adsorption Coefficient (KOC) on Soil and on Sewage Sludge Using High Performance Liquid Chromatography (HPLC).

• (9) Gerike P and Fischer WK (1979). A correlation study of biodegradability determinations with various chemicals in various tests. Ecotox. Env. Saf. 3:157-173.

• (10) Gerike P and Fischer WK (1981), as (9), II Additional results and conclusions. Ecotox. Env. Saf. 5: 45-55.

• (11) Painter HA and Bealing D (1989). Experience and data from the OECD activated sludge simulation test. pp. 113-138, In: Laboratory tests for simulation of water treatment processes. CEC Water Pollution Report 18. Eds. Jacobsen BN, Muntau H, Angeletti G.

• (12) ISO 11733 (1995; revised 2004). Evaluation of the elimination and biodegradability of organic substances in an aqueous medium — activated sludge simulation test.

• (13) Birch RR (1982). The biodegradability of alcohol ethoxylates. XIII Jornado Com. Espanol. Deterg.: 33-48.

• (14) Birch RR (1984). Biodegradation of noniomic surfactants. J.A.O.C.S. 61 (2): 340-343.

• (15) Gerike P, Fischer WK and Holtmann W (1980). Biodegradability determinations in trickling filter units compared with the OECD confirmatory test. Wat.Res. 14: 753-758.

• (16) Baumann U, Kuhn G and Benz M. (1998). Einfache Versuchsanordnung zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. Umweltchem. Ökotox. 10: 214-220.

• (17) Her Majesty’s Stationery Office (1982). Assessment of biodegradability. Methods for the examination of waters and associated materials. pp. 91-98 ISBN 0117516619.

• (18) ISO 14593 (1998). Water Quality — Evaluation in an aqueous medium of the ultimate biodegradability of organic compounds. Method by the analysis of inorganic carbon in sealed vessels.

Appendix 1

Figure 1

Equipment used for assessment of biodegradability

Husmann unit

Image omitted; see official source

• A.Storage vessel

• B.Dosing pump

• C.Aeration chamber (3l capacity)

• D.Settling vessel

• E.Air lift pump

• F.Collection vessel

• G.Aerator

• H.Air flow meter

Figure 2

Equipment used for assessment of biodegradability

Porous pot

Image omitted; see official source

• A.Storage vessel

• B.Dosing pump

• C.Porous aeration vessel

• D.Outer impermeable vessel

• E.Collection vessel

• F.Diffuser

• G.Air flow meter

Figure 3

Details of 3 litre porous pot aeration vessel

Image omitted; see official source

Appendix 2

Example of an elimination curve

Image omitted; see official source

Text of image

Polyethylene glycol 400 Test Concentration 20 mg/l DOC

Appendix 3

[INFORMATIVE]

COUPLING OF THE TEST UNITS

In order to try to equalise the microbial populations in sludges in a test unit, receiving sewage plus a test chemical, and in a control unit, receiving only sewage, a daily interchange of sludge was introduced (1). The procedure was called coupling and the method is known as coupled units. Coupling was initially performed using Husmann activated sludge units but it has also been done with Porous Pot units (2)(3). No significant differences in results were found as between non-coupled and coupled units, whether Husmann or Porous Pot so there is no advantage in expending the time and energy needed in coupling the units.

Sludge exchanges can give the appearance of quite a considerable removal, since some of the test chemical in transferred and the concentrations of test chemical in the test and control effluents become more nearly equal. Thus, correcting factors have to be used, which depend on the fraction exchanged and the mean hydraulic retention time. More details of the calculation have been published (1).

Calculate the corrected DOC or COD elimination degree using the general formula:

Image omitted; see official source

where

Dtc	=	corrected % DOC or COD elimination
---	---	---
Dt	=	determined % DOC or COD elimination
---	---	---
a	=	interchange fraction of the volume of the activated sludge units
---	---	---
r	=	mean hydraulic retention time (h)

If, for example, half of the volume of the aeration tank is exchanged (a = 0,5) and the mean hydraulic retention time is 6 h, the correction formula is:

Image omitted; see official source

LITERATURE:

• (1) Fischer W, Gerike P, Holtmann W (1975). Biodegradability Determinations via Unspecific Analyses (Chemical Oxygen Demand, DOC) in Coupled Units of the OECD Confirmatory Test. I The test. Wat. Res. 9: 1131-1135.

• (2) Painter HA, Bealing DJ (1989). Experience and Data from the OECD Activated Sludge Simulation Test. pp. 113-138. In: Laboratory Tests for Simulation of Water Treatment Processes CEC Water Pollution Report 18. Eds. Jacobsen BN, Muntau H, Angeletti G.

• (3) Painter HA, King EF (1978). Water Research Centre Porous Pot Method for Assessing Biodegradability. Technical Report TR70, Water Research Centre, Stevenage, UK.

Appendix 4

EVALUATION OF INHIBITION OF THE ACTIVATED SLUDGE

Process by test chemicals

• 1.A chemical (or a waste water) may not be degraded or removed in the simulation test and may even have an inhibitory effect on the sludge micro-organisms. Other chemicals are biodegraded at low concentrations but are inhibitory at higher concentration (hormesis). Inhibitory effects may have been revealed at an earlier stage or may be determined by applying a toxicity test, using an inoculum similar to or identical with that used in the simulation test (1). Such methods are inhibition of oxygen uptake (chapter C.11 of this Annex (2) and ISO 8192(3)) or inhibition of growth of sludge organisms (ISO 15522 (4)).

• 2.In the simulation test any inhibition will be manifest by the difference in dissolved organic carbon (DOC) or chemical oxygen demand COD between the effluent from the test vessel and that from the control being greater than the DOC added as test chemical. Expressed in another way, the percentage removal of DOC (and biochemical oxygen demand BOD, chemical oxygen demand COD, and/or NH⁺ 4) of the organic medium on treatment will be decreased by the presence of the test chemical. If this occurs, the test should be repeated reducing the concentration of the test chemical until a level is reached at which no inhibition occurs and perhaps further reducing the concentration until the test chemical is biodegraded. However, if the test chemical (or waste water) has adverse effects on the process at all concentrations tested, the indications are that the chemical is difficult, if not impossible, to treat biologically, but it may be worth repeating the test with activated sludge from a different source and/or subjecting the sludge to a more gradual acclimation.

• 3.Conversely, if the test chemical is bioeliminated at the first attempt in the simulation test, its concentration should be increased if it is required to be known whether the chemical could be inhibitory.

• 4.It should be remembered in trying to determine degrees of inhibition that the activated sludge population can change, so that with time the micro-organisms may develop a tolerance towards an inhibitory chemical.

• 5.Calculation of degree of inhibition:

• The overall percentage removals Ro, of BOD, DOC, COD etc., for the test and control units can be calculated from:

Image omitted; see official source

• where:

I	=	influent concentration of BOD, DOC, COD etc., for test or control vessels (mg/l)
---	---	---
E	=	respective effluent concentrations (mg/l).

• I and E must be corrected for the DOC due to the test chemical in the test units, otherwise the calculations of percentage inhibition will be incorrect.

• The degree of inhibition caused by the presence of the test chemical can be calculated from:

Image omitted; see official source

• where:

Rc	=	percentage removal in the control vessels
---	---	---
Rt	=	percentage removal in the test vessels

LITERATURE:

• (1) Reynolds L et al. (1987). Evaluation of the toxicity of substances to be assessed for biodegradability. Chemosphere 16: 2259.

• (2) Chapter C.11 of this Annex, Biodegradation — Activated Sludge Respiration Inhibition Test.

• (3) ISO 8192 (2007) Water quality — Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation.

• (4) ISO 15522 (1999) Water Quality — Determination of the inhibitory effect of water constituents on activated sludge microorganisms.

Appendix 5

Poorly water-soluble test chemicals — volatile chemicals

Poorly water-soluble chemicals

Few reports seem to have been published on subjecting poorly water-soluble and insoluble chemicals to tests simulating waste water treatment (1)(2)(3).

There is no single method of dispersal of the test chemical which is applicable to all insoluble chemicals. Two of the four types of method described in ISO 10634 (4) would seem to be suitable for attempting to disperse test chemicals for simulation testing; they are the use of emulsifying agents and/or of ultrasonic energy. The stability over at least 24h periods of the resulting dispersion should be established. Suitably stabilised dispersions, contained in a constantly stirred reservoir (paragraph 38), would then be dosed to the aeration tank separately from the domestic (or synthetic) sewage.

If the dispersions are stable, investigate how the test chemical can be determined in the dispersed form. It is unlikely that DOC will be suitable, so that a specific analytical method for the test chemical would have to be established which could be applied to effluents, effluent solids and activated sludge. The fate of the test chemical in the simulation of the activated sludge process would then be determined in liquid and solid phases. Thus, a ‘mass balance’ would be established to decide whether the test chemical had been biodegraded. However, this would indicate only primary biodegradation. Demonstration of ultimate biodegradation should be attempted by applying a respirometric test for ready biodegradability (chapter C.4 of this Annex (5) C, F or D) using as inoculum sludge exposed to the test chemical in the simulation test.

Volatile chemicals

The application of waste water treatment simulations to volatile chemicals is both debatable and problematic. As with poorly water-soluble test chemicals, very few reports seem to have been published describing simulation tests using volatile chemicals. A conventional type of complete-mixing apparatus is adapted by sealing the aeration and settling tanks, measuring and controlling the air flow using flow-meters and passing the exit gas through traps to collect volatile organic matter. In some cases, a vacuum pump is used to draw the exit gas through a ‘cold’ trap or a purge-trap containing Tenax and silica gel for gas-chromatographic analyses. The test chemical present in the trap can be determined analytically.

The test is carried out in two parts. The units are first operated without sludge but with the synthetic waste water plus test chemical being pumped into the aeration tank. Influent, effluent and exit gas samples are collected and analysed for the test chemical for a few days. From the data collected, the percentage (Rvs) of the test chemical stripped from the system may be calculated.

Then the normal biological test (with sludge) is performed under operating conditions identical to those in the stripping study. DOC or COD measurements are also made to check that the units are performing efficiently. Occasional analyses are made to determine the test chemical in the influent, effluent and exit gas in the first part of the test; after acclimation more frequent analyses are made. Again, from the data in the steady state, the percentage of removal of the test chemical from the liquid phase by all processes (RT) (physical and biological) may be calculated, as well as the proportion (RV) stripped from the system.

Calculation:

• (a) In the non-biological test, the percentage (RVP) of the test material stripped from the system may be calculated from: Image omitted; see official source where RVP = removal of test chemical by volatilisation (%), SVP = test chemical collected in trap expressed as equivalent concentration in liquid phase (mg/l), SIP = test chemical concentration in influent (mg/l).

• (b) In the biological test, the percentage (RV) of the test material stripped from the system may be calculated from: Image omitted; see official source where RV = removal of test chemical by volatilisation in biological test (%), SV = test chemical collected in trap in biological test, expressed as equivalent concentration in liquid influent (mg/l), SI = test chemical concentration in influent (mg/l).

• (c) In the biological test, the percentage (RT) of the test chemical removed by all processes is given by: Image omitted; see official source where SE = concentration of test chemical in the (liquid) effluent (mg/l).

• (d) Thus, the percentage (RBA) removed by biodegradation plus adsorption can be calculated from: Image omitted; see official source Separate tests should be carried out to determine whether the test chemical is adsorbed; if it is, then a further correction may be made.

• (e) A comparison between the proportion of test chemical stripped from the biological (Rv) and non-biological test (Rvp) systems indicates the overall effect that biological treatment has had on the emission of the test chemical into the atmosphere. Example: Benzene Sludge retention time = 4 days A synthetic sewage; retention time = 8 h. SIP = SI = 150 mg/l SVP = 150 mg/l (SEP = 0) SV = 22,5 mg/l SE = 50 μg/l Thus, RVP = 100, RV = 15 RT = 100 and RBA = 85. Benzene was assumed not to be adsorbed onto sludge.

LITERATURE:

• (1) Horn JA, Moyer JE, Hale JH (1970). Biological degradation of tertiary butyl alcohol. Proc. 25th Ind. Wastes Conference Purdue Univ.: 939-854.

• (2) Pitter P, Chudoba J (1990). Biodegradability of organic substances in the aquatic environment. CRC Press. Boston, USA.

• (3) Stover EL, Kincannon DF (1983). Biological treatability of specific organic compounds found in chemical industry waste waters. J. Wat. Pollut. Control Fed. 55: 97.

• (4) ISO 10634 (1995) Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

• (5) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability.

Appendix 6

Effects of sludge retention time (SRT) on treatability of chemicals

INTRODUCTION

• 1.The method described in the main text was designed to ascertain whether the chemicals tested (usually those known to be inherently, but not readily, biodegradable) can be biodegraded within the limits imposed in waste water treatment plants. The results are expressed in terms of percentage removal and percentage biodegradation. The conditions of operation of the activated sludge units and choice of influent allow rather wide variations in concentration of the test chemical in the effluent. Tests are carried out at only one nominal concentration of sludge solids or one nominal sludge retention time (SRT) and the sludge wastage regimes described can cause the value of SRT to vary considerably during the test, both from day to day and during a day.

• 2.In this variant (1)(2) the SRT is controlled within much narrower limits throughout each 24h period (just as happens on the large-scale) which results in a more constant concentration in effluents. Domestic sewage is recommended since it gives more consistent and higher percentage removals. Also, the effects of a number of SRT values are investigated and in a more detailed study the effects of a range of temperatures on effluent concentration may be determined.

• 3.There is no general agreement yet on which kinetic models operate when chemicals bio-degrade under conditions in waste water treatment. The Monod model of bacterial growth and substrate utilisation was chosen (1)(2) to be applied to the data collected, since the method was intended to be applied only to chemicals produced in high tonnages, resulting in concentrations in sewage of above 1 mg/l. The validity of the simplified model and the assumptions made was established using a series of alcohol ethoxylates having varying degrees of primary biodegradability (2)(3).

• Note: This variant method follows closely much of the text of this test method C.10-A and only those details which differ are given hereafter.

PRINCIPLE OF THE TEST

• 4.Activated sludge porous-pot units, designed to facilitate the (almost) continuous wastage of mixed liquor allowing very precise control of the sludge retention time (SRT, or θs), are operated in the non-coupled mode over a range of SRTs and, optionally, over a range of temperatures. The retention time is usually 2 to 10 days and the temperature between 5 and 20 °C. Sewage, preferably domestic, and a solution of the test chemical are dosed separately to the units at rates to give the required sewage retention time (3 to 6 hours) and the required concentration of test chemical in the influent. Control units receiving no test chemical are operated in parallel for comparative purposes.

• 5.Other types of apparatus can be used but great care should be exercised to ensure that good control of SRT is achieved. For example, when using plants, which incorporate a settler, allowance for loss of solids via the plant effluent may be necessary. Further, special precautions to avoid errors due to variation in the quantity of sludge in the settler should also be taken.

• 6.The units are operated at each selected set of conditions and, after equilibrium has been reached, the average steady state concentrations in the effluents of test chemical and, optionally, DOC are obtained over a period of about three weeks. Besides assessing the percentage removal of test chemical and, optionally, DOC, the relationship between plant-operating conditions and the concentration in the effluent is expressed in graphical form. From this tentative kinetic constants may be calculated and the conditions under which the test chemical can be treated may be predicted.

INFORMATION ON THE TEST CHEMICAL

• 7.Chapter C.10 A, paragraphs 12 and 13 apply.

PASS LEVELS

• 8.Chapter C.10 A, paragraphs 14 and 15 apply.

REFERENCE TEST CHEMICAL

• 9.Chapter C.10 A, paragraph 16 applies.

REPRODUCIBILITY OF TEST RESULTS

• 10.Chapter C.10 A, paragraphs 17 and 18 apply.

DESCRIPTION OF THE METHOD

Apparatus

• 11.A suitable unit is the modified porous pot system (Appendix 6.1). It consists of an inner vessel (or liner) constructed from porous polypropylene of 3,2 mm thickness and pore size of approximately 90 μm, the joint being butt-welded. (This makes a more robust unit than that described in paragraph 21 of this chapter, C.10 A). The liner is fitted into an impervious polyethylene outer vessel, which consists of two parts: a circular base in which holes are bored to accommodate two air lines and a sludge-wastage line, and an upper cylinder which screws on to the base and which has an outlet placed so as to give a known volume (3 l) in the porous pot vessel. One of the air lines is fitted with a diffuser stone and the other is open-ended and set at right-angles to the stone in the pot. This system produces the necessary turbulence to ensure that the contents of the pot are completely mixed, as well as providing concentrations of dissolved oxygen greater than 2 mg/l.

• 12.The appropriate number of units are maintained at controlled temperatures in the range of 5 to 20 °C (± 1 °C), either in water baths or in constant temperature rooms. Pumps are required to dose to the aeration vessels the solution of the test chemical and settled sewage at the required rates (0-1,0 ml/min and 0-25 ml/min, respectively) and a third pump to remove waste sludge from the aeration vessels. The necessary very low flow-rate of waste sludge is achieved by using a pump set at a higher rate and operated intermittently by the use of a timer-switch, e.g. operating for 10 seconds per min, pump delivery rate of 3ml/min yielding a wastage rate of 0,5 ml/min.

Filtration apparatus or centrifuge

• 13.Chapter C10 A, paragraph 23 applies.

Analytical equipment

• 14.Chapter C.10 A, paragraph 24 applies.

Water

• 15.Chapter C.10 A, paragraphs 25 and 26 apply.

Organic medium

• 16.Chapter C.10 A, paragraph 27 applies.

Synthetic sewage

• 17.Chapter C.10 A, paragraph 28 applies.

Domestic sewage

• 18.Chapter C.10 A, paragraph 29 applies.

Activated sludge

• 19.Chapter C10 A, paragraph 30 applies.

Stock solutions of test chemical

• 20.Chapter C.10 A, paragraphs 31 and 32 apply.

PROCEDURE

Preparation of the inoculum

• 21.Chapter C.10 A, paragraph 34 applies only — use activated sludge (about 2,5 g/l).

Number of test units

• 22.For a simple test, ie. to measure percentage removal, only a single SRT is required, but in order to acquire data necessary to calculate tentative kinetic constants 4 or 5 SRT values are required. Values between 2 and 10 days are usually chosen. Practically, it is convenient to perform a test at 4 or 5 SRTs simultaneously at one temperature; in extended studies the same SRT values, or perhaps a different range of values, are used at other temperatures within the range 5 to 20 °C. For primary biodegradation (the main use), only one unit per set of conditions is normally required. However, for ultimate biodegradability a control unit is required, for each set of conditions, which receives sewage but not test chemical. If the test chemical is thought to be present in the sewage used, it would be necessary to use control units when assessing primary biodegradation, and making the necessary correction in the calculations.

Dosage of organic medium and test chemical

• 23.Chapter C.10 A, paragraphs 36 to 39 apply, but note that the test chemical solution is dosed separately and that various sludge wastage rates are used. Also monitor and adjust, if necessary, to within ± 10 %, the flow-rates of influents, effluents and sludge wastage frequently, e.g. twice per day. If difficulties are encountered in the analytical methods when domestic sewage is used, carry out the test with synthetic sewage, but it must be assured that different media give comparable kinetic data.

Handling of activated sludge units

• 24.Chapter C.10 A, paragraphs 40 to 43 apply, but control SRT only by ‘constant’ wastage of sludge.

Sampling and analysis

• 25.Chapter C.10 A, paragraphs 44 to 50 apply, except that the concentration of the test chemical is to be determined and DOC determined optionally; COD should not be used.

DATA AND REPORTING

Treatment of results

• 26.Chapter C.10 A, paragraphs 52 to 54 apply.

Expression of test results

• 27.Chapter C.10 A, paragraphs 56 to 62 apply.

Calculation of kinetic constants

• 28.It is more realistic to quote the mean steady — state concentration of the test chemical in the effluent and to describe how this varies with plant-operating conditions than to quote percentage primary biodegradation. This can be done by consideration of equation (6) in Appendix 6.2, which can yield values for KS, μm and θSC, the critical sludge retention time.

• (Alternatively, approximate values of KS and μm may be obtained using a simple computer program to fit the theoretical curve calculated from equation 2 (Appendix 6.2) to the experimental values obtained. Although any given solution will not be unique, a reasonable approximation of KS and μm can be obtained.)

Variability of results

• 29.It is common experience that variable values of kinetic parameters for individual chemicals are obtained. It is thought that the conditions under which the sludge has been grown, as well as the conditions prevailing in the test used (as in paragraph 5 and in other tests), have a large effect on the resulting values. One aspect of this variability has been discussed by Grady et al (4), who have suggested that the terms ‘extant’ and ‘intrinsic’ should be applied to two extreme conditions representing the limits of physiological state a culture may attain during a kinetic experiment. If the state is not allowed to change during the test, the kinetic parameter values reflect the conditions in the environment from which the micro-organisms were taken; these values are called ‘extant’ or currently existing. At the other extreme, if conditions in the test are such as to permit the full development of the protein-synthesizing system allowing maximum possible growth rate, the kinetic parameters obtained are said to be ‘intrinsic’, and are dependent only on the nature of the substrate and the types of bacteria in the culture. As a guide, extant values will be obtained by keeping the ratio of concentration of substrate to competent micro-organisms (So/Xo) low, e.g. 0,025, and intrinsic values arise when the ratio is high e.g. at least 20. In both cases, So should equal or exceed the relevant value of Ks, the half-saturation constant.

• 30.Variability and other facets of biodegradation kinetics were discussed at a recent SETAC workshop (5). From such studies, reported and projected, a clearer view of kinetics operating in waste water treatment plants should be forth-coming to enable a better interpretation of existing data to be made, as well as to suggest more relevant designs for future Test Methods.

LITERATURE:

• (1) Birch RR (1982). The biodegradability of alcohol ethoxylates. XIII Jornado Com. Espanol Deterg.: 33-48.

• (2) Birch RR (1984). Biodegradation of nonionic surfactants. J.A.O.C.S., 61(2): 340-343.

• (3) Birch RR (1991). Prediction of the fate of detergent chemicals during sewage treatment. J. Chem. Tech. Biotechnol., 50: 411-422.

• (4) Grady CPL, Smets BF and Barbeau DS (1996). Variability in kinetic parameter estimates: A review of possible causes and a proposed terminology. Wat. Res., 30 (3): 742-748.

• (5) Biodegradation kinetics: Generation and use of data for regulatory decision making (1997). Workshop at Port Sunlight, UK. Eds. Hales SG, Feitjel T, King H, Fox K, Verstraete W. 4-6th Sept. 1996. SETAC- Europe, Brussels.

Appendix 6.1

Porous Pot with SRT Control

Image omitted; see official source

Appendix 6.2

Calculation of Kinetic Constants

• 1.By assuming Monod kinetics apply and considering a mass balance of active solids and substrate across the activated sludge system (1), the following steady state expressions can be obtained:

Image omitted; see official source	[1]

• or

Image omitted; see official source	[2]

• where:

S1	=	concentration of substrate in effluent, (mg/l)
---	---	---
KS	=	half-saturation constant, the concentration at which μ = μm/2 (mg/l)
---	---	---
μ	=	specific growth rate (d–1)
---	---	---
μm	=	maximum value of μm(d–1)
---	---	---
Kd	=	specific decay rate of active solids (d–1)
---	---	---
θS	=	sludge mean retention time, SRT (d)

• Examination of this equation leads to the following conclusions:

• (i) The effluent concentration is independent of that in the influent (S0); hence, the percentage biodegradation varies with the influent concentration, S0.

• (ii) The only plant-control parameter affecting S1 is the sludge retention time, θS.

• (iii) For a given concentration in the influent, S0, there will be a critical sludge retention time, such that: Image omitted; see official source [3] where: θSC = critical sludge retention time, below which the competent micro-organisms will be washed out of the plant.

• (iv) Since the other parameters in equation (2) are associated with growth kinetics, temperature is likely to affect the effluent substrate level and the critical sludge age, ie. the sludge retention time needed to obtain a certain degree of treatment would increase with decreasing temperature.

• 2.From a mass balance of solids in the porous pot system, and assuming that the solids concentration in the plant effluent, X2 is low compared with that in the aeration vessel, X1, the sludge retention time

Image omitted; see official source	[4]

• and

Image omitted; see official source

• where:

V	=	volume of the aeration vessel (l)
---	---	---
X1	=	concentration of solids in aeration vessel (mg/l)
---	---	---
X2	=	concentration of solids in effluent (mg/l)
---	---	---
Q0	=	flow rate of influent (l/d)
---	---	---
Q1	=	flow rate of waste sludge (l/d)

• Thus, it is possible to control the sludge retention time at any pre-selected value by the control of the waste sludge flow rate, Q1.

• Conclusions:

• 3.The main purpose of the test is thus to allow the effluent concentration, and hence the levels of test chemical in the receiving waters, to be predicted.

• 4.By plotting S1, vs. θS, the critical sludge retention time, θSC, can sometimes be readily evaluated, eg. curve 3 in Figure 1. When this is not possible, θSC may be calculated, together with approximate values of μm and KS, by plotting S1, vs. S1•θS.

• Rearrangement of equation (1) gives

Image omitted; see official source	[5]

• If Kd is small, then 1 + θs · Kd ~ 1 and [5] becomes:

Image omitted; see official source	[6]

• Thus, the plot should be a straight line (see Figure 2) of slope 1/μm and intercept KS/μm; also θS ~1/μm.

• Figure 1

Three temperatures; five SRTs Image omitted; see official source

Figure 2

Regression Line SRT · S1 vs S1 at T = 5 °C Image omitted; see official source

Glossary:

Effluent concentration: Curve:

Appendix 7

TEST AT LOW (μg/l) CONCENTRATION RANGE

• 1.Many chemicals are normally present in the aquatic environment, even in waste waters, at very low concentrations (μg/l). At such concentrations, they probably do not serve as primary substrates resulting in growth, but are more likely to degrade as non-growth, secondary substrates, concurrent with a variety of naturally occurring carbon chemicals. Consequently the degradations of such chemicals will not fit the model described in Appendic 6. There are many models which could be applied and, under the conditions prevailing in waste water treatment systems, more than one may be simultaneously operative. Far more research will be necessary to elucidate this problem.

• 2.Meanwhile the procedure given in the main text (chapter C.10 A) can be followed, but only for primary biodegradability, using suitably low concentrations (< 100 μg/l) and a validated analytical procedure. The percentage biodegradation may be calculated (see para. 54 of the Test Method) provided that abiotic processes (adsorption, volatility, etc.) are taken into account. An example is the study by Nyholm and his associates (1)(2) using a 4 h cycle in a fill and draw system. They reported pseudo first-order constants for 5 chemicals added in a synthetic sewage at 5 to 100 μg/l. (For ultimate biodegradability ¹⁴C-labelled test chemicals may be used. A description of this is beyond the scope of this Test Method since there are as yet no agreed procedures, though a proposed method for ISO 14592 (3) contains guidance on the use of ¹⁴C-labelled chemicals.

SCAS test

• 3.Later, a simpler two-stage test was proposed (4)(5)(6); the semi-continuous activated sludge (SCAS) method is followed by short-term kinetic tests on samples withdrawn from the SCAS units. The SCAS system is operated with known sludge wastage rates (unlike the original C.12 test method) and is fed a modified OECD synthetic sewage or domestic sewage. The synthetic sewage was modified (because of changing pH value and poor sludge settleability) by addition of phosphate as buffer, yeast extract, iron (III) chloride and trace element salts, and its COD was increased to about 750 mg/l by increasing the concentration of peptone and meat extract. The units were operated on a 24 h cycle: aeration for 23 h, wastage of sludge, settlement, withdrawal of supernatant (effluent) followed by addition of synthetic sewage plus test chemical, up to 100 μg/l, (i.e. at about the same concentration used in the short term test). Once per week 10 % of the total sludge was replaced by fresh sludge in order to maintain a balanced microbial population.

• 4.The concentrations of test chemical initially and at the end of aeration are measured and the test is continued until a constant removal of test chemical is attained; this takes from one week to several months.

Short-term test

• 5.A short test (e.g. 8 hours) is applied to determine the (pseudo) first order kinetic rate constant for the decay of the test chemical in activated sludge of known but different origins and histories. In particular, sludge samples are taken from the SCAS reactors — at the end of an aeration period when the concentration of organic substrate is low — during the course of an acclimatisation experiment (paragraphs 3, 4). Sludge may also be taken from a parallel SCAS unit not exposed to the test chemical, for comparison. Mixtures of sludge and the test chemical added at two or more concentrations in the range 1-50 μg/l are aerated, without the addition of synthetic sewage or other organic substrate. The test chemical remaining in solution is determined at regular intervals e.g. hourly depending on the degradability of the chemical, for a period not longer than 24h. Samples are centrifuged before appropriate analysis.

Calculations

• 6.Data from the SCAS units are used to calculate the percentage removal of test chemical (paragraph 54). Also, an average rate constant, K1, (normalised for concentration of suspended solids) can be calculated from:

Image omitted; see official source

• where:

t	=	aeration time (23h)
---	---	---
Ce	=	concentration at end of aeration period (μg/l)
---	---	---
Ci	=	concentration at beginning of aeration (μg/l)
---	---	---
SS	=	concentration of activated sludge solids (g/l)

• 7.In the short term test the log % concentration remaining is plotted against time and the slope of the initial part (10-50 % degradation) of the plot is equivalent to K1, the (pseudo) first order constant. The constant is normalised with respect to the concentration of sludge solids by dividing the slope by the concentration of sludge solids. The reported result must also include details of initial concentrations of the test chemical and suspended solids, sludge retention time, sludge loading and source, and details of pre-exposure (if any) to the test chemical.

Variability of results

• 8.Variability and other facets of biodegradation kinetics were discussed at a recent SETAC workshop (7). From such studies, reported and projected, a clearer view of kinetics operating in waste water treatment plants should be forth-coming to enable a better interpretation of existing data to be made, as well as to suggest more relevant designs for future Test Methods.

LITERATURE:

• (1) Nyholm N, Jacobsen BN, Pedersen BM, Poulsen O, Dambourg A and Schultz B (1992). Removal of micropollutants in laboratory activated sludge reactors. Biodegradability. Wat. Res. 26: 339-353.

• (2) Jacobsen BN, Nyholm N, Pedersen BM, Poulsen O, and Ostfeldt P (1993). Removal of organic micropollutants in laboratory activated sludge reactors under various operating conditions: Sorption. Wat. Res. 27: 1505-1510.

• (3) ISO 14592 (ISO/TC 147/SC5/WG4, N264) (1998). Water Quality — Evaluation of the aerobic biodegradability of organic compounds at low concentrations in water.

• (4) Nyholm N, Ingerslev F, Berg UT, Pedersen JP and Frimer-Larsen H (1996). Estimation of kinetic rate constants for biodegradation of chemicals in activated sludge waste water treatment plants using short-term batch experiments and μg/l range spiked concentrations Chemosphere 33 (5): 851-864.

• (5) Berg UT and Nyholm N (1996). Biodegradability simulation Studies in semi-continuous activated sludge reactors with low (μg/l range) and standard (ppm range) chemical concentrations. Chemosphere 33 (4): 711-735.

• (6) Danish Environmental Protection Agency. (1996). Activated sludge biodegradability simulation test. Environmental Project, No 337. Nyholm, N. Berg, UT. Ingerslev, F. Min. of Env. and Energy, Copenhagen.

• (7) Biodegradation kinetics: Generation and use of data for regulatory decision making (1997). Workshop at Port Sunlight, UK. Eds. Hales, SG. Feitjel, T. King, H. Fox, K. and Verstraete, W. 4-6th Sept. 1996. SETAC- Europe, Brussels.

C.10-B:   Biofilms

INTRODUCTION

• 1.Simulation tests are normally applied to chemicals which have failed a screening test for ready biodegradability (Chapter C.4 A to F of this Annex (9)), but have passed a test for inherent biodegradability. Exceptionally simulation tests are also applied to any chemical about which more information is required, especially high-tonnage chemicals, and normally the activated sludge test is applied (C.10 A). In some circumstances, however, specific information is required relating the behaviour of a chemical to methods of waste water treatment involving biofilms, namely, percolating or trickling filters, rotating biological contactors, fluidised beds. To meet this need various devices have been developed.

• 2.Gerike et al. (1) used large, pilot-scale trickling filters which they used in the coupled mode. These filters took up much space and required relatively large volumes of sewage or synthetic sewage. Truesdale et al. (2) described smaller filters (6 ft × 6 in. diameter) which were fed surfactant-free natural sewage but still required rather large volumes. As many as 14 weeks were required for the development of a ‘mature’ biofilm and an additional 4-8 weeks were needed after first introduction of the test surfactant before acclimatisation took place.

• 3.Baumann et al. (3) developed a much smaller filter which used polyester ‘fleece’ previously steeped in activated sludge as the inert medium supporting the biofilm. The test chemical was used as the sole source of carbon and biodegradability was assessed from measurements of dissolved organic carbon (DOC) in the influent and effluent, and from the amount of CO2 in the exit gas.

• 4.A quite different approach was made by Gloyna et al. (4) who invented the rotating tubular reactor. On the internal surface of the rotating tube a biofilm was grown on the known surface area by passage of influent introduced at the top end of the tube, inclined at a small angle to the horizontal. The reactor has been used to study the biodegradability of surfactants (5), as well as to investigate the optimal thickness of biofilm and diffusion through the film (6). These latter authors further developed the reactor, including modifying it to be able to determine CO2 in the exit gas.

• 5.The rotating tubular reactor has been adopted by the Standing Committee of Analysts (UK) as a standard method for assessing both the biodegradability of chemicals (7) and the treatability and toxicity of waste waters (8). The method described here has the advantages of simplicity, compactness, reproducibility and the need for relatively small volumes of organic medium.

PRINCIPLE OF THE TEST

• 6.Synthetic or domestic sewage, and the test chemical, in admixture or alone, are applied to the internal surface of a slowly rotating inclined tube. A layer of microorganisms, similar to those present on bio-filter media, is built up on the internal surface. The conditions of operation of the reactor are chosen to give adequate elimination of organic matter and, if required, oxidation of ammonium.

• 7.Effluent from the tube is collected and either settled and/or filtered before analysis for dissolved organic carbon (DOC) and/or the test chemical by a specific method. Control units receiving no test chemical are operated in parallel under the same conditions for comparative purposes. The difference between the concentrations of DOC in the effluent from the test and control units is assumed to be due to the test chemical and its organic metabolites. This difference is compared with the concentration of the added test chemical (as DOC) to calculate the elimination of the test chemical.

• 8.Biodegradation may normally be distinguished from bio-adsorption by careful examination of the elimination-time curve. Confirmation may usually be obtained by applying a test for ready biodegradation (oxygen uptake or carbon dioxide production) using an acclimated inoculum taken at the end of the test from the reactors receiving the test chemical.

INFORMATION ON THE TEST CHEMICAL

• 9.The purity, water solubility, volatile and adsorption characteristics of the test chemical should be known to enable correct interpretation of results to be made.

• 10.Normally, volatile and poorly soluble chemicals cannot be tested unless special precautions are taken (see Appendix 5 to chapter C.10 A). The chemical structure, or at least the empirical formula, should also be known in order to calculate theoretical values and/or to check measured values of parameters, e.g. theoretical oxygen demand (ThOD), DOC.

• 11.Information on the toxicity of the test chemical to micro-organisms (see Appendix 4 to chapter C.10 A) may be useful for selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values.

PASS LEVELS

• 12.Originally, the primary biodegradation of surfactants was required to reach 80 % or more before the chemical could be marketed. If the value of 80 % is not attained, this simulation (confirmatory) test may be applied and the surfactant may be marketed only if more than 90 % of the specific chemical is removed. With chemicals in general there is no question of a pass/fail level and the value of percentage removed can be used in proximate calculations of the probable environmental concentration to be used in hazard assessments posed by chemicals. In a number of studies of pure chemicals the percentage removal of DOC was found to be > 90 % in more than three-quarters, and > 80 % in over 90 %, of chemicals which showed any significant degree of biodegradability.

REFERENCE CHEMICALS

• 13.To ensure that the experimental procedure is being carried out correctly, it is useful occasionally to test reference chemicals whose behaviour is known. Such chemicals include adipic acid, 2-phenyl phenol, 1-naphthol, diphenic acid and 1-naphthoic acid.

REPRODUCIBILITY OF TEST RESULTS

• 14.The relative standard deviation within tests was found by a laboratory in the UK to be 3,5 % and between tests to be 5 % (7).

DESCRIPTION OF THE METHOD

Apparatus

Rotating tubular reactors

• 15.The apparatus (see figures 1 and 2 in the Appendix 8 consists of a bank of acrylic tubes each 30,5 cm long and 5 cm internal diameter, supported on rubber-rimmed wheels contained within a metal supporting frame. Each tube has an outside lip, approximately 0,5 cm deep, to retain it on the wheels, the internal surface is roughened with coarse wire wool and there is a 0,5 cm deep internal lip at the upper (feed) end to retain the liquid. The tubes are inclined at an angle of approximately one degree to the horizontal to achieve the required contact time when the test medium is applied to a clean tube. The rubber-tyred wheels are rotated using a slow, variable-speed motor. The temperature of the tubes is controlled by installation in a constant temperature room.

• 16.By enclosing each tube reactor inside a slightly larger, capped tube and ensuring that connections were gas-tight, exit CO2 gas could be collected in an alkaline solution for subsequent measurement (6).

• 17.A 24h supply, for each tube, of organic medium with added test chemical if applicable, is contained in a 20 l storage vessel (A)(see Figure 2). If required, the test chemical solution may be dosed separately. Near the bottom of each storage vessel there is an outlet which is connected by suitable tubing, e.g. silicone rubber, via a peristaltic pump (B) to a glass or acrylic delivery tube which enters 2-4 cm into the higher (feed) end of the inclined tube (C). Effluent is allowed to drip from the lower end of the inclined tube to be collected in another storage vessel (D). The effluent is settled or filtered before analysis.

Filtration apparatus-centrifuge

• 18.Device for filtration of samples with membranes filter of suitable porosity (nominal aperture diameter 0,45 μm) which adsorb organic chemicals or release organic carbon to a minimum degree. If filters are used which release organic carbon, wash them carefully with hot water to remove leachable organic carbon. Alternatively a centrifuge capable of achieving 40 000 m/sec² may be used.

• 19.Analytical equipment for determining:

• — DOC/total organic carbon (TOC), or chemical oxygen demand (COD);

• — specific chemical (HPLC, GC etc.) if required;

• — pH, temperature, acidity, alkalinity;

• — ammonium, nitrite, nitrate, if the tests are performed under nitrifying conditions.

Water

• 20.Tap water, containing less than 3 mg/l DOC.

• 21.Distilled or deionised water, containing less than 2 mg/l DOC.

Organic medium

• 22.Synthetic sewage, domestic sewage or a mixture of both may be used as the organic medium. It has been shown that the use of domestic sewage alone often gives increased percentage removed of DOC (in activated sludge units) and even allows the biodegradation of some chemicals, which are not biodegraded when OECD synthetic sewage is used. Thus, the use of domestic sewage is recommended. Measure the DOC (or COD) concentration in each new batch of organic medium. The acidity or alkalinity of the organic medium should be known. The medium may require the addition of a suitable buffer (sodium hydrogen carbonate or potassium hydrogen phosphate), if it is of low acidity or alkalinity, to maintain a pH of about 7,5 ± 0,5 in the reactor during the test. The amount of buffer, and when to add it, has to be decided in each individual case.

Synthetic sewage

• 23.Dissolve in each 1 litre of tap water: peptone, 160 mg; meat extract, 110 mg; urea, 30 mg; anhydrous dipotassium hydrogen phosphate, (K2HPO4), 28 mg; sodium chloride, (NaCl), 7 mg; calcium chloride dihydrate, (CaCl2.2H2O), 4 mg; magnesium sulphate heptahydrate, (MgSO4.7H2O), 2 mg. This OECD synthetic sewage is an example and gives a mean DOC concentration in the influent of about 100 mg/l. Alternatively, use other compositions, with about the same DOC concentrations, which are closer to real sewage. This synthetic sewage may be made up in distilled water in a concentrated form and stored at about 1 °C for up to one week. When needed, dilute with tap water. (This medium is unsatisfactory e.g. nitrogen concentration is very high, relatively low carbon content, but nothing better has been suggested, except to add more phosphate, as buffer, and extra peptone).

Domestic sewage

• 24.Use fresh settled sewage collected daily from a treatment works receiving predominantly domestic sewage. It should be collected from the overflow channel of the primary sedimentation tank, or from the feed to activated sludge plant, and be largely free from coarse particles. The sewage can be used after storage for several days at about 4 °C, if it is proved that the DOC (or COD) has not significantly decreased (i.e. by less than 20 %) during storage. In order to limit disturbances to the system, the DOC (or COD) of each new batch should be adjusted before use to an appropriate constant value, e.g. by dilution with tap water.

Lubricant

• 25.Glycerol or olive oil may be used for lubricating the peristaltic pump rollers: both are suitable for use on silicone-rubber tubing.

Stocks solutions of test chemical

• 26.For chemicals of adequate solubility prepare stock solutions at appropriate concentrations (e.g. 1 to 5 g/l) in deionised water or in the mineral portion of the synthetic sewage. For insoluble chemicals, see Appendix 5 in chapter C.10-A. This method is not suitable for volatile chemicals without modifications to the tubular reactors (paragraph 16). Determine the DOC and TOC of the stock solution and repeat the measurements for each new batch. If the difference between the DOC and TOC is greater than 20 %, check the water-solubility of the test chemical. Compare the DOC or the concentration of the test chemical measured by specific analysis of the stock solution with the nominal value to ascertain whether recovery is good enough (normally > 90 % can be expected). Ascertain, especially for dispersions, whether or not DOC can be used as an analytical parameter or if only an analytical technique specific for the test chemical can be used. Centrifugation of the samples is required for dispersions. For each new batch, measure the DOC, COD or the test chemical with specific analysis.

• 27.Determine the pH of the stock solution. Extreme values indicate that the addition of the chemical may have an influence on the pH of the activated sludge in the test system. In this case neutralise the stock solution to obtain a pH of 7 ± 0,5 with small amounts of inorganic acid or base, but avoid precipitation of the test chemical.

PROCEDURE

Preparation of organic medium for dosing

• 28.Ensure that all influent and effluent containers and tubing from influent vessels and to effluent vessels are thoroughly cleaned to remove microbial growths, initially and throughout the test.

• 29.Prepare the synthetic sewage (paragraph 23) freshly each day either from the solids or from the concentrated stock solution by appropriate dilution with tap water. Measure the required amount in a cylinder and add to a clean influent vessel. Also, where necessary, add the required amount of the stock solution of the test chemical or reference chemical to the synthetic sewage before dilution. If it is more convenient or necessary to avoid loss of the test chemical, prepare a separate diluted solution of the test chemical in a separate reservoir and deliver this to the inclined tubes via a different dosing pump.

• 30.Alternatively (and preferably), use settled domestic sewage (paragraph 24) collected freshly each day if possible.

Operation of rotating tubular reactors

• 31.Two identical tubular reactors are required for the assessment of one test chemical, and they are assembled in a constant temperature room normally at 22 ± 2 °C.

• 32.Adjust the peristaltic pumps to deliver 250 ± 25 ml/h of the organic medium (without test chemical) into the inclined tubes, which are rotated at 18 ± 2 rpm. Apply the lubricant (paragraph 25) to the pump tubes initially and periodically through the test to ensure proper functioning and to prolong the life of the tubing.

• 33.Adjust the angle of inclination of the tubes to the horizontal to produce a residence time of 125 ± 12,5 sec. for the feed in a clean tube. Estimate the retention time by adding a non-biological marker (e.g. NaCl, inert dye) to the feed: the time taken to reach peak concentration in the effluent is taken to be the mean retention time (when maximum film is present, the retention time can increase up to about 30 min.).

• 34.These rates, speeds and times have been found to give adequate removals (> 80 %) of DOC (or COD) and to produce nitrified effluents. The rate of flow should be changed if removal is insufficient or if the performance of a particular treatment plant is to be simulated. In the latter case, adjust the rate of dosing the organic medium until the performance of the reactor matches that of the treatment plant.

Inoculation

• 35.Airborne inoculation may be sufficient to start the growth of micro-organisms when synthetic sewage is used, but otherwise add 1 ml/l of settled sewage to the feed for 3 days.

Measurements

• 36.At regular intervals check that the dose-rates and rotating speeds are within the required limits. Also, measure the pH of the effluent, especially if nitrification is expected.

Sampling and analysis

• 37.The method, pattern and frequency of sampling are chosen to suit the purpose of the test. For example, take snap (or grab) samples of influent and effluent, or collect samples over a longer period e.g. 3-6 h. In the first period, without test chemical, take samples twice per week. Filter the samples through membranes or centrifuge at about 40 000 m/sec² for about 15 min (paragraph 18). It may be necessary to settle and/or coarse-filter the samples before membrane filtration. Determine DOC (or COD) at least in duplicate and if required BOD, ammonium and nitrite/nitrate.

• 38.All analyses should be performed as soon as possible after collection and preparation of the samples. If analyses have to be postponed, store the samples at about 4 °C in the dark in full, tightly stoppered bottles. If samples have to be stored for more than 48h, preserve them by deep-freezing, acidification or by addition of a suitable toxic chemical (e.g. 20 ml/l of a 10 g/l solution of mercury (II) chloride). Ensure that the preservation technique does not influence the results of analysis.

Running-in period

• 39.In this period, the surface biofilm grows to reach an optimal thickness, usually taking about 2 weeks and should not exceed 6 weeks. The removal (paragraph 44) of DOC (or COD) increases and reaches a plateau value. When the plateau has been reached at a similar value in both tubes, one is selected to be a control in the remainder of the test, during which their performance should remain consistent.

Introduction of test chemical

• 40.At this stage add the test chemical to the other reactor at the required concentration, usually 10-20 mg C/l. The control continues to receive the organic medium alone.

Acclimation period

• 41.Continue the twice weekly analyses for DOC (or COD) and, if primary biodegradability is to be assessed, also measure the concentration of the test chemical by specific analysis. Allow from one to six weeks (or longer under special conditions) after the test chemical has first been introduced for acclimation to occur. When the percentage removal (paragraphs 43-45) reaches a maximum value, obtain 12-15 valid values in the plateau phase over about 3 weeks for evaluation of the mean percentage removal. The test is considered completed if a sufficiently high degree of elimination is reached. Normally, do not exceed a test duration of more than 12 weeks after the first addition of the test chemical.

Sloughing of the film

• 42.The sudden removal of large quantities of excess film from the tubes, or sloughing, takes place at relatively regular intervals. To ensure that the comparability of results is unaffected, allow tests to cover at least two full cycles of growing and sloughing.

DATA AND REPORTING

Treatment of results

• 43.Calculate the percentage DOC (or COD) elimination of the test chemical for each timed assessment using the equation:

Image omitted; see official source

• where:

Dt	=	percentage elimination of DOC (or COD) at time t;
---	---	---
Cs	=	concentration of DOC (or COD) in the influent due to the test chemical, preferably estimated from the concentration in, and volume added, of the stock solution (mg/l);
---	---	---
E	=	measured DOC (or COD) in the test effluent at time t (mg/l);
---	---	---
Eo	=	measured DOC (or COD) in the control effluent at time t (mg/l).

• Repeat the calculation for the reference chemical, if tested.

Performance of the control reactor

• 44.The degree of DOC (or COD) elimination (DB) of the organic medium in the control reactors is helpful information in assessing the biodegradative activity of the biofilm during the test. Calculate the percentage elimination from the equation:

Image omitted; see official source

• where:

Cm = DOC (or COD) of the organic medium in the control influent (mg/l).

• 45.Calculate the removal (DST) of the test chemical, if measured, by a specific analytical method at each time assessment from the equation:

Image omitted; see official source

• where:

Si	=	measured or, preferably, estimated concentration of test chemical in the test influent (mg/l)
---	---	---
Se	=	measured test chemical concentration in the test effluent at time t (mg/l)

• If the analytical method gives a positive value in unamended sewage equivalent to Sc mg/l, calculate the percentage removal (DSC) from:

Image omitted; see official source

Expression of test results

• 46.Plot the percentage elimination Dt and DST (or DSC), if available, versus time (see Appendix 2 in chapter C.10- A). Take the mean (expressed to the nearest whole number) and standard deviation of the 12-15 values for DT (and for DST, if available) obtained in the plateau phase as the percentage removal of the test chemical. From the shape of the elimination curve, some conclusions may be drawn about the removal processes.

Adsorption

• 47.If a high DOC elimination of the test chemical is observed at the beginning of the test, the test chemical is probably eliminated by adsorption on to the biofilm. It may be possible to prove this by determining the adsorbed test chemical on solids sloughed from the film. It is not usual for the elimination of the DOC of adsorbable chemicals to remain high throughout the test; normally, there is an initial high degree of removal which gradually falls to an equilibrium value. If, however, the adsorbed test chemical was able to cause acclimation of the microbial population, the elimination of the test chemical DOC would subsequently increase and reach a high, plateau level.

Lag phase

• 48.As in static, screening tests many test chemicals require a lag phase before full biodegradation occurs. In the lag phase, acclimation (or adaptation) of the competent bacteria takes place with almost no removal of the test chemical; then the initial growth of these bacteria occurs. This phase ends and the degradation phase is arbitrarily taken to begin when about 10 % of the initial amount of test chemical is removed (after allowing for adsorption, if it occurs). The lag phase is often highly variable and poorly reproducible.

Plateau phase

• 49.The plateau phase of an elimination curve in a continuous test is defined as that phase in which the maximum degradation takes place. This phase should last at least 3 weeks and have about 12-15 measured valid values.

Mean degree of elimination of the test chemical

• 50.Calculate the mean value from the elimination values Dt (and Dst, if available) of the test chemical at the plateau phase. Rounded to the nearest whole number (1 %), it is the degree of elimination of the test chemical. It is also recommended to calculate the 95 % confidence interval of the mean value. In a similar way calculate the mean degree (DB) of elimination of the organic medium in the control vessel.

Indication of biodegradation

• 51.If the test chemical does not adsorb significantly on to the biofilm and the elimination curve has a typical shape of a biodegradation curve with lag, degradation and plateau phases (paragraphs 48, 49), the measured elimination can safely be attributed to biodegradation. If a high initial removal has taken place, the simulation test cannot differentiate between biological and abiotic elimination processes. In such cases, and in other cases where there is any doubt about biodegradation (e.g. if stripping takes place), analyse adsorbed test chemical on samples of the film or perform additional static (screening) tests for biodegradability based on parameters clearly indicating biological processes. Such tests are the oxygen uptake methods (Chapter C.4 of this Annex D, E and F) (9) or a test which measures CO2 production (Chapter C.4-C of this Annex or the Headspace method) (10); use as inoculum pre-exposed biofilm from the appropriate reactor.

• 52.If both the DOC removal and specific chemical removal have been measured, significant differences (the former being lower than the latter) between the percentages removed indicate the presence in the effluents of intermediate organic products, which may be more difficult to degrade; these should be investigated.

Validity of test results

• 53.Consider the test to be valid if the degree of DOC (or COD) elimination (DB) in the control units is > 80 % after 2 weeks operation and no unusual observations have been made.

• 54.If a readily biodegradable (reference) chemical has been tested, the degree of biodegradation should be > 90 % and the difference between duplicate values should not be greater than 5 %. If these two criteria are not met, review the experimental procedures and/or obtain domestic sewage from another source.

• 55.Similarly, differences between biodegradation values from duplicate units (if used) treating a test chemical should not differ by more than 5 %. If this criterion is not met but the removals are high, continue analysis for a further three weeks. If removal is low, investigate the inhibitory effects of the test chemical if not known and repeat the test at a lower concentration of test chemical, if that is feasible.

Test Report

• 56.The test report must include the following:

Test chemical:

• — identification data;

• — physical nature and, where relevant, physico-chemical properties.

Test conditions:

• — any modifications to test system, especially if insolubles or volatiles tested;

• — type of organic medium;

• — proportion and nature of industrial wastes in sewage, if used and if known;

• — method of inoculation;

• — test chemical stock solution — DOC (dissolved organic carbon) and TOC (total organic carbon) content; how prepared, if suspension; test concentration(s) used, reasons if outside range 10-20 mg/l DOC; method of addition; date first added; any changes in concentration;

• — mean hydraulic retention time (with no growth); rotational speed of tube; approximate angle of inclination, if possible;

• — details of sloughing; time and intensity;

• — test temperature and range;

• — analytical techniques employed.

Test results:

• — all measured data DOC, COD, specific analyses, pH, temperature, N chemicals, if relevant;

• — all calculated date of Dt (or Dtc), DB, Ds obtained in tabular form and elimination curves;

• — information on lag and plateau phases, test duration, the degree of elimination of the test chemical, of the reference chemical (if tested) and of the organic medium (in the control unit), together with statistical data and statements of biodegradability and validity of the test;

• — discussion of results.

LITERATURE:

• (1) Gerike P, Fischer W, Holtmann W (1980). Biodegradability determinations in trickling filter units compared with the OECD Confirmatory Test. Wat. Res. 14: 753-758.

• (2) Truesdale GA, Jones K, Vandyke KG (1959). Removal of synthetic detergents in sewage treatment processes: Trials of a new biologically attackable material.Wat. Waste Tr. J. 7: 441-444.

• (3) Baumann U, Kuhn G and Benz M. (1998) Einfache Versuchsanordnung zur Gewinnung gewässerökologisch relevanter Daten, UWSF — Z. Umweltchem. Ökotox. 10: 214-220.

• (4) Gloyna EF, Comstock RF, Renn CE (1952). Rotary tubes as experimental trickling filters. Sewage ind. Waste 24: 1355-1357.

• (5) Kumke GW, Renn CE (1966). LAS removal across an institutional trickling filter. JAOCS 43: 92-94.

• (6) Tomlinson TG, Snaddon DHM, (1966). Biological oxidation of sewage by films of micro-organisms. Int.J. Air Wat. Pollut. 10: 865-881.

• (7) Her Majesty’s Stationery Office (1982). Methods for the examination of waters and associated materials. Assessment of biodegradability, 1981, London.

• (8) Her Majesty’s Stationery Office (1984). Methods for the examination of waters and associated materials. Methods for assessing the treatability of chemicals and industrial waste waters and their toxicity to sewage treatment processes, 1982, London.

• (9) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability A-F.

• (10) ISO 14593 (1998). Water Quality-Evaluation in an aqueous medium of the ultimate biodegradability of organic substances. Method by analysis of released inorganic carbon in sealed vessels.

Appendix 8

Figure 1

Rotating tubes

Image omitted; see official source

Glossary:

Plan view:

View A/B:

Driven wheels:

Idling wheels:

Drive motor:

Reduction gear:

Internal flange:

Tilting mechanism:

Bevel gear drive:

Figure 2

Flow diagram

Image omitted; see official source

A : Feed Tank

B : Peristaltic Pump

C : Rotating Tube

D : Effluent Collection Vessel

DEFINITIONS:

Test chemical : Any substance or mixture tested using this Test Method.

Chemicals : It should be noted that the term ‘chemical’ is used broadly in the UNCED agreements and subsequent documents to include substances, products, mixtures, preparations, or any other terms that may be used in existing systems to denote coverage.

C.11.   ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST (CARBON AND AMMONIUM OXIDATION)

INTRODUCTION