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Administrative data

field studies
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID

Data source

Referenceopen allclose all

Reference Type:
Reference Type:
Behavior of fluorescent whitening agents during sewage treatment.
Poiger T., Field J.A., Field T.M., Siegrist H., Giger W.
Bibliographic source:
Wat. Res. Vol. 32, N. 6 pp. 1939-1947. 1998

Materials and methods

Principles of method if other than guideline:
A field study was conducted at a full-scale mechanical-biological sewage treatment plant at Zuerich-Glatt, Switzerland. Samples of wastewater (raw sewage, primary and secondary effluent) and sludge (raw, activated and anaerobically- digested sludge) were collected during a 10-day period. The test substance concentrations in water samples were determined using solid-phase extraction with C18 disks and HPLC. The concentrations of the test substances in sludge were determined by supercritical fluid extraction and HPLC.
GLP compliance:
Type of measurement:
Mass flow of FWA .
Municipal sewage treatment plant.

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate
EC Number:
EC Name:
Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate
Cas Number:
Molecular formula:
disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate

Results and discussion

Any other information on results incl. tables


1.1 Extraction of FWA from Sludges

Liquid Extraction (LE)

Bases are believed to promote extraction by increasing the negative surface charge of the sludge matrix. With ion-pairing reagents, FWAs form strong lipophilic ion-pairs that partition more readily into organic solvents. The influence of increasing TBA concentration on the extraction efficiency was investigated using methanol as a solvent. A TBA concentration of 0.03 M was selected for subsequent experiments since no further increase in extraction efficiency was observed at higher concentrations. The use of TBA in methanol is less laborious as only dilution prior to HPLC analysis is necessary, rather than the evaporation and re-dissolution required when using basic extractants.

Supercritical Fluid Extraction (SFE)

FWA recovery in SFE, as well as in LE, largely depends on the type of sludge sample. Extraction of FWAs is less efficient from raw than from anaerobically digested sludge probably due to the much higher content of organic matter in raw sludge. Thus, the number ot extractions required tor quantitative recovery depends on the sludge type. Two consecutive SFE extractions were required to quantitatively extract FWAs from raw sludge samples while only a single extraction was required for digested sludge.

Results obtained by SFE and LE extraction procedures are in good agreement, the differences of FWA concentrations being well within the relative standard deviation (RSD) of each method. The higher RSD's obtained by SFE may, in part, be due to the smaller sample sizes (100 mg) used in SFE compared to 500 mg used in LE.

1.2 Enrichment ot FWA from Aqueous Samples

To determine the precision of the method, replicate sample extractions were performed using samples of raw sewage, primary effluent, secondary effluent and river water from the Zürich-Glatt sewage treatment plant and from Glatt river 1 km downstream of the plant. For the determination of FWA recovery, the samples were spiked with different amounts. Recovery rates were determined after an equilibration time of 15 h, to allow for partitioning of FWA between water and suspended particles.

Recovery rate was good for the test item: 84-91%.

Reproducibility of test substance determination and recovery from water samples

Matrix  Present
Raw sewage*
(10 ml)
23 6 25-100 86
Primary effluent*
(10 ml)
12 2 11-44 -
Secondary effluent*
(50 ml)
3 5 5-15 91
River water*
(200 ml)
0.4 3 1-5 84

*Samples trom Zürich-Glatt municipal sewage treatment plant and trom Glatt river 1 km downstream ot the plant.

**Relative standard deviation ot 4 replicate determinations.

***Spiked with 5-15 µl of standard solution and equilibrated tor 15 h.

****Average of six determinations.

1.3 Extraction of FWA from Lake Sediments

Lake sediments were extracted in the same way as sludge samples using LE with methanol containing 0.03 M TBA. The extraction of sediment samples was exhaustive, e.g. no FWA was found upon re-extraction and analysis of pre-extracted samples. In order to lower the detection limit compared to sludge analysis, the extracts were evaporated and the residue was redissolved in DMF:H2O (1:1). Assuming similar behaviour of FWA in sediment extracts as in elutes from Empore disks, evaporation of solvent and re-dissolution should not affect the recovery values. Assuming that recovery values will be less than 100%, the reported FWA levels in sediments from lake Biel should be treated as lower limit-values.

1.4 Chromatographic separation and detection

The combination of post-column irradiation and fluorescence detection provides the possibility to determine not only the total concentration of FWA in environmental matrices but also its isomeric composition. This is necessary to assess the environmental behaviour of FWAs, since different isomers behave differently. The sensitivity and selectivity of the method are adequate for the determination of FWAs in extracts of sewage wastewaters and sludges as well as in extracts of river water and lake sediment.


2.1 Octanol-Water partitioning

Hydrophobic interactions between natural organic matter and FWA were expected to be a significant contribution to the binding of FWA to suspended solids.

If more than one species contributes to the overall partitioning of a compound, as is the case with organic acids and bases, an octanol-water distribution ratio, Dow, is used instead of Kow. Hydrophobic organic acids may partition into octanol in their protonated or deprotonated form. The partition coefficients of the anions are usually more than 2 orders of magnitude smaller than of the neutral species. In addition, formation of ion-pairs of anions with cations such as Na+, K+, Mg2+, and Ca2+ has been found to promote partitioning into octanol[1 -2]. Thus, partitioning of organic acids (and bases) is strongly dependent on pH and ionic strength. Sulfonic acids usually have pKa values below 2[3]. Therefore, only anions have to be considered at pH 6-8. Ion-pair formation, however, might influence the partitioning behaviour of sulphonates, as was shown for linear alkyl benzene sulphonates (LAS)[4].

Octanol·water distribution ratios for all photoisomers of test substance were determined with aqueous phases containing different Ca2 + concentrations in large excess compared to FWA concentrations. Very low Dow value for test item was obtained in the absence of calcium ions in the aqueous phase, indicating that the sulphonated item is too hydrophilic to partition readily into octanol. The large increase of Dow in the presence of calcium ions indeed strongly supported the assumed ion·pair formation.

Octanol-water distribution ratios of test item isomers at different calcium concentrations ([CA2+]) in the aqueous phase.

Isomer [CA2+]/mM→ log Dow [lwater/loctanol]
0 0.1 1 10
(Z) -1.9 -0.6 0.2 0.6
(E) -1.1 0.0 0.9 1.6

Kow of (E)-isomers is greater than of (Z)-isomers. This is in agreement with the general finding, that hydrophobic molecules are more compatible with the aqueous phase, when their volume is smaller. (Z)·Stilbenes are more flexible than the corresponding (E)-stilbenes with its rigid conjugated π-system and may fold to yield smaller molecular volumes in aqueous solution.

Good agreement of experimental data and model calculation was found for both isomers of test item. The Kow of the FWA ion-pairs obtained by the model is more than 2 orders of magnitude higher than Kow for the native FWA. The two sulphonate moieties of the test substance are close together and it could even serve as a bidentate ligand. This might explain the relatively high conditional stability constant for the formation of ion-pairs obtained by the model calculation.

2.2 Sorption to River Sediment

Sorption equilibria were achieved within 20 h. Initially, both adsorption and desorption processes are fast, followed by a considerably slower step. In the desorption experiments, even a slight decrease in the amount of dissolved FWA was found after 6 h. This decrease was attributed to the fact that dry sediment was added to the river water in the beginning ol the experiment and that some swelling of the particles might have occurred.

In analogy to the Kow data, different distribution coefficients were obtained for different isomers. Distribution coefficient of (Z)-isomer was generally higher than of (E)-isomer.

Dow values for a Ca2+ concentration of 1 mM were used for the correlation in order to simulate the natural conditions as well as possible. However other cations such as Na+, K+, Mg2 +,and NH4+ may further increase Dow. The hydrophobic partitioning is not the only interaction between

FWA and natural suspended matter. Specific interactions and ion-exchange interactions between FWA and suspended solids may contribute strongly to the observed partitioning.

2.3 Photoisomerization and Partitioning in Sewage

While adsorbed FWAs are relatively stable, dissolved FWAs readily isomerize in the presence of light[5]. The constant ratio of (E)- and (Z)·isomers, the so-called photostationaly state, is achieved within a few minutes of exposure to direct sunlight. Because different isomers have different UV spectra, the isomer ratio is dependent on the spectrum of the irradiating light.

The spectrum of the light which actually reaches the dissolved FWA is altered by UV absorbing co-soIutes, as well as, in concentrated solutions by the FWA itself. Therefore, the isomer ratios are dependent also on the concentration of FWA (except for dilute solutions as sewage and river water samples) and the kind and concentration of co-solutes.

E/Z-ratios of FWA in the photostationary state after sunlight and UV Irradiation.

Isomer Isomer ratio after irradiation with
Sunlight* UV 254 nm**
(Z) 78% 75% 34%
(E) 22% 25% 66%

* Left column: aqueous sample was exposed to sunlight in a glass vial. Right column: isomer ratio in the aqueous phase of primary and secondary effluent samples taken after sunlight irradiation.

** Ratio of peaks for (E)-isomers ln HPLC chromatograms with and without post-column UV-irradiation.

Although the different isomers exhibit different partitioning characteristics, in a first step it is useful to treat the FWA as a sum of isomers and examine its average partitioning behaviour. This picture is then refined in a second step by looking at the isomeric compositions and the partitioning behaviour of individual isomers.

Isomerization of FWA in sewage was found to be very fast. The isomeric composition of the FWA tested varies between raw influent, and in primary and secondary effluent, respectively. This is not simply the result of different irradiation times.

A simple model was used to simulate the partitioning of FWA under the influence of sunlight. Irradiation yields and maintains a constant ratio of (E)- and (Z)-isomers in solution. The isomers adsorb to the solid phase with their individual distribution coefficients.

The isomer ratio of the test item is dependent on the amount of suspended particulate matter. ln solutions with low particle content, the ratio is dominated by the photochemically favoured (Z)-isomer. With increasing particle content, the more strongly adsorbing (E)-isomer is favoured and becomes the dominant species.

Very good agreement between model calculation and field data is obtained for primary and secondary effluent. For raw influent the model overestimates the fraction of the (Z)·isomer, most likely because the sample was not exposed to sunlight long enough to achieve photostationary conditions (one of the model assumptions). The same is also true for sludges, where the (E)-isomer is the absolutely dominant (>9O %) specie. Primary sludge is settled before isomerization can take place. Activated sludge is loaded with (Z)-isomer only during the day when isomerization takes place. During the night, this isomer is washed out due to their smaller affinity for suspended solids.

Comparison of measured and predicted isomer ratios.

Fraction of isomer [%]
measured predicted*
Anaerobically digested sludge 4.5 17
Activated sludge 9.5 18
Raw lnfluent 41 54
Primary effluent 60 63
Secondary effluent 70 72

* Model parameters: KEZ (Ratio (E) (Z) isomer in solution) = 3; KdZ = 2000 l/kg; KdE= 30000 l/kg.


3.1 Sewage In- and Effluents

Concentrations of test item in 24 h composite samples of primary and secondary sewage effluent from various treatment plants in the region of

Zürich, Switzerland are reported in Table. As these concentrations may vary considerably from day to day, the data presented should only be seen as first insight into the approximate range of concentrations at which FWA occur in wastewaters. Note that sand filtration does not reduce FWA concentrations significantly, because secondary effluent contains only little suspended solids.

FWA concentrations in wastewater*

Conc. in µg/l
Primary effluent
Zürich Glatt 11.4
Opfikon 9.8
Bülach 11.3
Niederglatt** 7
Secondary effluent
Zürich Glatt 2.6
Opfikon 2.8
Bülach 4.5
Niederglatt** 3.5
Tertiary effluent***
Opfikon 2.5

*24 h composite sample; **raw influent; ***sand filter

3.2 Sludge

The FWA concentrations in raw sludge were lower than those in anaerobically digested sludges, indicating that test item becomes relatively enriched in sludge due to solids reduction during anaerobic sludge digestion.

FWA concentrations in sewage sludges (mg/kg)

Sampling location Test item conc. Volatile
solids (%)
Anaerobically digested sludges
Opfikon 55 n.d.
Bauma 68 51
Nieclerglatt 55 46
Fällanden 75 46
Bassersdorf 69 54
Uster 57 46
Seuzach 65 44
Bülach 105 57
Zürich-Glat 96 46
Average 39 50


55-105 44-57
Raw sludges
Herisau 39 73
Zürich-Glat 50 70
Activated sludges
Zürich-Glat 38 n.d.

3.3 Diurnal and Daily Variations

The efficiency of removal during the activated sludge treatment increases.

When the FWA levels in primary effluent are high, the activated sludge is charged with FWA. Conversely, when FWA levels in primary effluent are low, FWA may be released from the sludge.

The levels of FWA in raw influent varied significantly during the day. The effluent levels, however, remained almost constant and were sometimes higher than the influent levels.

3.4 Elimination of FWA ,During Activated Sludge Treatment

Biological degradation of FWA would gradually reduce its concentration in activated sludge. The total concentration (sum of dissolved and adsorbed fraction) however, remained constant throughout the residence time of the wastewater in the activated sludge system, indicating that no detectable biochemical transformation processes occurred. To verify this result, samples of activated sludge were taken to the laboratory and aerated for another 48 h. Again no change in FWA concentrations occurred.

FWA concentrations, the fraction in the dissolved phase gradually decreased during the activated sludge treatment, and finally reached a value close to the one found in returned sludge. FWA is indeed removed from wastewater by adsorption to activated sludge. The differences in mass flows between primary and secondary effluent (e.g. the amount of FWA eliminated in the activated sludge treatment) may therefore be attributed to sorption processes and not to biodegradation.

3.5 Mass Flow

During primary clarification, approximately 69% of the test item in raw sewage was removed associated with primary sludge. Of the residual FWA in primary effluent, another 65% was removed during activated sludge treatment and secondary clarification. Residual mass in secondary effluent was 18 g/day (11%) of the corresponding influent level. As no biodegradation of FWA was observed during activated sludge treatment, test item removed during activated sludge treatment was quantitatively recovered in excess sludge.

Average FWA mass flows (g/day)

Raw influent 744
Primary effluent 230
Secondary effluent 80
Excess sludge 156
Raw sludge 664
Anaerobic sludge 635.0

Comparison of the mass flows associated with raw sludge (e.g. the sum of primary and excess sludge) and with anaerobically digested sludge yielded very good agreement (89% and 85%). Good agreement was obtained despite the fact that the anaerobically-digested sludges sampled in this study do not correlate with the influents and effluents sampled, because of the lang residence time of sludge in the digesters. This finding strongly indicates that FWA is also not biodegraded during anaerobic sludge treatment.

Isomerization in wastewater was found to be a fast process and photostationary conditions are achieved within a few minutes of exposure to sunlight. This raises the question, what impact sunlight has on the elimination of FWA during sewage treatment.

Sunlight will generally reduce the removal rates of FWA, because photoisomerization leads to isomers which are less sorptive than the parent isomers. A quantification of this reduction in removal rates, however, is more difficult since several factors are limiting the impact of sunlight:

- intense sunlight is only available during few hours a day

- sunlight only penetrates the top layer of the water in the settling tanks and in the activated sludge basin

- in the case of the test item isomerization leads to a significant fraction of the less sorptive (Z)-isomer.

As a result, the fractions of (Z)-test item in activated sludge and anaerobically digested sludge was far below the fractions calculated for photostationary conditions. A closer look should be given to test item: a rough estimate can be made by the content of the (Z)-isomer in sludge (4.5%) and its adsorption constant for sludge particles (2000 Ukg). The adsorption constant of (Z)-test item is very close to the adsorption constant of the isomer of another FWA tested and a similar elimination rate (50%) may therefore be assumed. This means that the amount of (Z)-test item released to the secondary clarifier is approximately equal to the amount in sludge. Thus the contribution of the (Z)-isomer (or the impact of sunlight) is in the range of only 10%.

3.6 Discharge of FWA to Surface Water and Farmland

Estimates of the fractions of FWA entering the Swiss STPs associated with raw sewage and leaving the STPs associated with anaerobically digested sewage sludge and sewage effluents can be made based on the levels of FWA in anaerobically-digested sludges and the elimination rates of FWA during sewage treatment. Since sludge is stored in the anaerobic digesters for several weeks, the FWA levels determined in anaerobically-digested sludge represent an average value of FWA removed from wastewater during this time period. Calculation of FWA discharge to surface water based on FWA levels in sludge and elimination rated as determined in the mass flow study are therefore more reliable than estimates based on effluent concentrations.

Estimation of the amounts in raw sewage, secondary effluents and anaerobically-digested sludge. Numbers in brackets indicate the fraction in each compartment relative to the consumption.

Annual consumptiona [t/y] 108 (= 100 %)
Elimination in STP [%] 89
Level in anaerobically
- digested sludge [mg/kg] 72.0
Amount in
- raw sewagedb [t/y] 21.0 (19%)
- secondary effluentec [t/y] 2.3 (2%)
- sludged 18.7 (17%)

a) Data from Ciba-Geigy AG.

b) Amount in sludge divided by elimination rate in STP

c) Amount in raw sewage multiplied by (100%-elimination rate)

d) Level in sludge multiplied by annua1 sludge production of 260000 t dry mass/y[6].

A considerable fraction (72 %) of the consumed test item is lost before the sewage treatment facility. This may be expected because FWAs are produced and added to detergents in order to adsorb to the textiles. Approximately 20% is bound to sewage sludge and another 2% is discharged to surface water.


1.5 % of the test item is finally found in river water.

Levels of FWA in river water samples.

River Sampling
Limmat  Gebenstort  67 ± 29
Rhein Rekingen  36 ± 8
Village-Neuf 372 ± 48
Aare  Brugg 94 ± 6
Rhone Chancy 63 ± 10
Glatt  Rheinsfelden 439 ± 24
Blank 12 ± 2
LOQ* 22

*2-wwek composite samples (November 29 to December 13, 1993) from automatic sampling stations of ttle NADUF-program. Samples were stored at 4°C, no preservatives were added.

**Limit o quantification (LOQ = Blank + 10 σ)

Mass flow and per capita flow of FWA calculated from data in the table above.

River Sampling
Hydraulic flow
Mass flow
Per capita mass flow
[mg/d person]
Limmat  Gebenstort  68 0.39 0.47
Rhein Rekingen  352 1.09 0.45
Village-Neuf 699 22.47 3.25**
Aare  Brugg 191 1.55 0.80
Rhone Chancy 248 1.35 0.95
Glatt  Rheinsfelden 6.3 0.24 0.70

*Data supplied by Landeshydrologie und -geologie, Bern, Switzerland.

**Chromatographic signals for test substance overlapped with signals from an unknown compound.

4.2 Sediments

The history of the application FWA as detergent ingredients and along, with it the history of FWA inputs into lake Biel is recorded in the concentration profile of FWA in the sediment core. Besides the nowadays mainly used the test substance and another FWA-2 (distyrylbiphenyls type[7]), another more hydrophobic FWA-6[8] is found in deeper sections of this core with a concentration maximum corresponding to the time period tram 1959-69. This FWA-6 has lost its significance as a detergent additive with the introduction of the test substance and the other FWA-2. This change in detergent formulation is recorded in the sediment core. The fact that the levels of the test substance and FWA-2 in the sediment are almost constant on a depth corresponding to a time period of more than 30 years strongly indicates their persistence under the environmental conditions of this sediment.


[1] West all J.C., Leuenberger C. and Schwarzenbach R.P., 1985. Influence of pH and ionic Strength on the Aqueous-Nonaqueous Distribution of Chlorinated Phenols. Environmental Science and Technology, 19, 193-198.

[2] Jafvert C. T., Westall J.C., Grieder E. and Schwarzenbach R.P., 1990. Distribution of Hydrophobic lonogenic Compounds between Octanol and Water: Organic Acids. Environmental Science and Technology, 24, 1795-1803.

[3] King JI.F., 1991, Acidity. In: The Chemistry of Sulfonic Acids, Esters and their Derivatives (eds. Patai :5. end Rappaport Z.), Wiley, 249-258.

[4] Brownawell B.J., Chen H., Zhang W. and West all J.C., 1991. Adsorption of Surfactants. In: Organic Substances and Sediments in Water (eds. Baker R. A.), Lewis Publishers, 127-147.

[5] Milligs B. and Holt l.A., 1974. Fluorescent Whitening Agents. I. Bis-4,4'-(4'-methoxy-6'-phenoxy-s-triazin2'-ylamino)stilbene-2,2'-disulphonic Acid: Its Photodecomposition in Solution and on Wool. Aust. J. Chem., 27, 195-203.

[6] Gujer N., 1989. Die Entwicklung des Klärschlammanfalles in der Schweiz. Mitteilungen der EAW.A.G, 28, 2-5.

[7] 4,4′-bis(2-sulfonatostyryl)biphenyl

[8] disodium 4,4'-bis[(4,6-dianilino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate

Applicant's summary and conclusion

Only adsorption to sewage sludge was found to be important for the elimination of FWA from wastewater. No evidence for biodegradation was found during (aerobic) activated sludge treatment and anaerobic digestion of sewage sludge.
Executive summary:

Study description

A field study was conducted at a full-scale mechanical-biological sewage treatment plant at Zuerich-Glatt, Switzerland. Samples of wastewater (raw sewage, primary and secondary effluent) and sludge (raw, activated and anaerobically- digested sludge) were collected during a 10-day period. The test substance concentrations in water samples were determined using solid-phase extraction with C18 disks and HPLC. The concentrations of the test substances in sludge were determined by supercritical fluid extraction and HPLC.

The method allowed the equally sensitive determination of the parent FWA as well as the isomers which are formed upon exposure to sunlight.

The mass flow of FWA was determined in a field study at the municipal sewage treatment plant Zürich-Glatt, Switzerland.


Analytical methods

The analytical methods based on reversed-phase high-performance liquid chromatography, post-column irradiation in combination with fluorescence detection provided an excellent tool for the sensitive and selective determination of the (highly fluorescent) parent compounds as well as the (non-fluorescent) isomers. FWAs were extracted from freezedried sewage sludges using either liquid extraction (LE) or supercritical fluid extraction (SFE).

Photoisomerization and partitioning

Isomerization of FWA in sewage was found to be very fast. The isomeric composition of the FWA tested varies between raw influent, and in primary and secondary effluent, respectively. A simple model was used to simulate the partitioning of FWA under the influence of sunlight. ln solutions with low particle content, the ratio is dominated by the photochemically favoured (Z)-isomer. With increasing particle content, the more strongly adsorbing (E)-isomer is favoured and becomes the dominant species. Very good agreement between model calculation and field data is obtained for primary and secondary effluent.

Primary sludge is settled before isomerization can take place.

Field determination

From field data the following conclusions concerning the behaviour and fate of FWA in a mechanical sewage treatment plant can be drawn:

(I) elimination of FWAs from wastewater occurs during both mechanical and biological treatment.

(II) overall removal rate of 98 % was observed.

(III) elimination is due to adsorption to primary and activated sewage sludge and the observed elimination, rate is consistent with the individual sorption behaviour of the FWA as investigated in laboratory experiments.

(IV) no evidence for biodegradation of FWA was found during the (aerobic) biological treatment of wastewater with activated sludge and during anaerobic-mesophilic digestion of raw sewage sludge.

(V) the FWA removed during wastewater treatment is thus quantitatively recovered in anaerobically digested, sewage sludge.