Environment International 34 (2008) 1001–1005

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Environment International
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e n v i n t

Fate and effects of amphoteric surfactants in the aquatic environment

M. Teresa Garcia ⁎, Encarna Campos, Agustí Marsal, Isabel Ribosa
Surfactant Technology Department, IIQAB/CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Amphoteric surfactants form part of specialty surfactants available for formulators to improve or design new
Received 30 November 2007 formulations in response to environmental, toxicity, safety and performance demands. Nevertheless, limited
Accepted 12 March 2008 information on the ecological properties of amphoterics is available. In the present work, the aerobic and
Available online 5 May 2008
anaerobic biodegradability and the aquatic toxicity of different types of amphoteric surfactants (three alkyl
betaines, one alkylamido betaine and three alkyl imidazoline derivatives) were studied. The amphoteric
Keywords:
Amphoteric surfactants
surfactants tested were readily biodegradable under aerobic conditions (CO2 headspace test) and alkylamido
Betaines betaines and alkyl imidazoline derivatives were also easily biodegradable under anaerobic conditions (test
Imidazoline derivatives based on the ECETOC method). Toxicity to Photobacterium phosphoreum and Daphnia magna increased with the
Biodegradation fatty chain length of the surfactant. The EC50 toxicity values of the amphoterics tested were higher than 5 mg/L,
Aquatic toxicity and alkyl imidazoline derivatives, with EC50 values from 20 to N 200 mg/L, showed the lowest aquatic toxicity.
© 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Amphoterics are surfactants with ionic charge that can change
between anionic character, the isoelectric neutral stage and the
cationic character depending on the pH value (van Ginkel, 1995).
Amphoteric surfactants show stability against electrolytes, acids,
alkalis, and water hardness. They are compatible with anionic, cationic
and non-ionic surfactants (Porter, 1994). The amphoterics are
dermatologically mild surfactants because of their behaviour and
protein-like structure. They can form complexes with anionic
surfactants and are able to reduce their irritative properties with the
result that they are mainly used in cosmetics and toiletries or hand
dishwashing liquids as mild surfactants. Moreover, amphoterics are
important surfactants in some detergents, especially in light-duty
detergents and special wool care products (Domingo, 1995). Despite
the large variety of amphoterics, in practice only a small group of
products is used owing to limiting factors such as cost and complexity
of the reactions involved. Among the most important members of this
family are the alkyl and alkylamido betaines and the alkyl imidazoline
derivatives, whose production only in Western Europe is estimated at
33 and 6 ktons/year, respectively (Fraunhofer report, 2003). Never-
theless, ecological data concerning amphoterics are scarce. For
cocoamido betaine the EC50 value varies from 0.55 to 48 mg/L for
algae and for invertebrates and fish the range is between 1 and
100 mg/L (Madsen, 2001). As regards biodegradation, alkyl betaines
and alkylamido betaines have been demonstrated to be readily
biodegradable under aerobic conditions (Madsen, 2001).
⁎ Corresponding author. Tel.: +34 93 400 61 00; fax: +34 93 201 45 904.
E-mail address: mtgbet@iiqab.csic.es (M.T. Garcia).
The aim of this work was to obtain useful data for the selection of
technically efficient surfactants with a low impact on the aquatic
environment. To this end, the biodegradation of alkyl betaines,
alkylamido betaines and alkyl imidazoline derivatives under aerobic
and anaerobic conditions as well as their toxicity to two aquatic
organisms were investigated.
2. Materials and methods
2.1. Surfactants
The amphoteric surfactants tested were: three alkyl betaines (decyl dimethyl-,
dodecyl dimethyl- and tetradecyl dimethyl betaine; C10-Bet, C12-Bet and C14-Bet,
respectively), one alkylamido betaine (cocoamidopropyl dimethyl betaine; Amido-Bet)
and three alkyl imidazoline derivatives (decyl-, dodecyl- and tetradecyl monocarbox-
ymethylated imidazoline derivative; C10-Imi, C12-Imi and C14-Imi respectively). The
alkyl betaines and alkyl imidazoline derivatives were supplied by Albright and Wilson
Ltd. and the alkylamido betaine was supplied by Goldschmidt España S.A. The
surfactant structures and abbreviations used are given in Table 1.
2.2. Aerobic biodegradation test
The CO2 headspace test (ISO 14593, 1999) was used to assess the aerobic
biodegradability of the amphoteric surfactants. This method evaluates the ultimate
biodegradation under aerobic conditions (mineralization to carbon dioxide) of an
organic compound in aqueous medium by measuring the net increase in total inorganic
carbon over time with respect to a blank without the addition of the test substance. The
surfactants were tested at a concentration of 20 mgC/L. Samples were inoculated with
activated sludge (10 mg dry solids/L) collected from a municipal wastewater treatment
plant (Manresa, Barcelona) and then incubated in the dark at 22 ± 1 °C in 250 mL sealed
vessels (air headspace/1iquid volume ratio, 1:2). Sodium n-dodecyl sulphate was used as
the reference substance. Three replicates of the surfactants, blank and reference
substance were performed for each sampling day. The tests ran for 28 days. On each
sampling day, after injecting a sodium hydroxide solution to the vessels, shaking for 1 h
and allowing to settle, appropriate volumes were withdrawn by syringe from the liquid
phase of each vessel and kept in small beakers carefully filled to the brim and covered

0160-4120/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2008.03.010
1002 M.T. Garcia et al. / Environment International 34 (2008) 1001–1005
Table 1
Chemical structures and abbreviations of the amphoteric surfactants tested
Amphoteric Chemical structure
Alkyl betaines
Alkylamido betaines
Alkyl imidazoline derivatives
with a cap to prevent CO2 exchange with the air. The concentration of inorganic carbon
was determined in a carbon analyzer (Shimadzu TOC-5050). The biodegradation level
was expressed as a percentage of the theoretical amount of inorganic carbon based on
the initial amount of the test compound.
2.3. Anaerobic biodegradation test
A batch test system based on the method proposed by Birch et al. (1989) was
employed. This method evaluates the ultimate anaerobic biodegradation of a chemical
based on the production of biogas (methane and carbon dioxide) with respect to
unamended blanks. Sludge samples from the anaerobic digester of a municipal wastewater
treatment plant (Manresa, Barcelona) were used as inoculum. Total and volatile solids of
the anaerobic sludge samples were determined according to Standard Methods (APHA,
1998a,b) and the values obtained ranged from 45 to 50 g/L and from 47 to 55%, respectively.
After collection, sludge was washed with a mineral salt solution, as described in the
ECETOC-test (ECETOC,1988), to reduce the amount of inorganic carbon to a value ≤10 mg/L.
A final re-suspension step enabled adjustment of the dried solids concentration to 2.8–
3.4 g/L. C10-Bet, C14-Bet, C10-Imi and C14-Imi were tested at 50 and 100 mgC/L, C12-Bet and
C12-Imi were tested from 20 to 200 mgC/L and Amido-Bet was tested from 30 to 300 mgC/L.
Each surfactant concentration was tested in triplicate. Five replicates with anaerobic sludge
but without any added surfactant were performed to determine the endogenous biogas
production (control digesters). All samples were incubated in 250 mL pressure-resistant
glass bottles at 36 °C±1 and the gas/liquid volume ratio was 3:7. The bottles were fitted
with gas tight septa and aluminium crimp seals. After sealing the vessels and incubating
them for about 1 h excess gas was released to the atmosphere. Incubation proceeded in the
dark. The resulting pressure was measured with a digital manometer connected to a
syringe needle and the increase in headspace pressure in the closed bottles was used to
follow the mineralization process. The incubation time was 60 days. At the end of the test,
after allowing the sludge to settle, appropriate volumes were withdraw by syringe from the
clear supernatant of each vessel and kept in small beakers carefully filled to the brim and
covered with a cap to prevent CO2 exchange with the air. The dissolved amount of the
evolved carbon dioxide was determined as the concentration of inorganic carbon in the
clear supernatant using a carbon analyzer (Shimadzu TOC-5050).
2.4. Daphnia magna immobilisation test
D. magna, laboratory bred, not more than 24 h old were used in this test (OECD, 2004),
where the swimming incapabilility is the end point. The pH of the medium was 8.0 and the
Fig. 1. Aerobic biodegradation curves of alkyl and alkylamido betaines: (◊) C10-Bet,
(□) C12-Bet, (Δ) C14-Bet, (○) Amido-Bet.
R Abbreviation
R = decyl C10-Bet
R = dodecyl C12-Bet
R = tetradecyl C14-Bet
R = coco Amido-Bet
R = decyl C10-Imi
R = dodecyl C12-Imi
R = tetradecyl C14-Imi
total hardness was 250 mg/L (as CaCO3), with a Ca/Mg ratio of 4/1. Tests were performed in
the dark at 20 °C. Twenty daphnia, divided into four batches of five animals each, was used
at each test concentration. For each surfactant, ten concentrations in a geometric series
were tested in the concentration range first established in a preliminary test. The
percentage immobility at 48 h was plotted against concentration on logarithmic-
probability paper and a linear relationship was obtained. The Probit Method was employed
as the statistical procedure to determine the IC50 (the estimated concentration to
immobilise 50% of the daphnia after 48 h exposure) and the corresponding 95% confidence
interval (CI).
2.5. Photobacterium phosphoreum luminescence reduction test (MicrotoxR Test)
P. phosphoreum is a marine luminiscent bacterium naturally adapted to a saline
environment. These bacteria liberate energy in the form of visible light (maximum
intensity at 490 nm) as a consequence of a series of metabolic reactions. On exposure to
toxic chemicals, the light output is reduced and this reduction is proportional to the
toxicity of the sample. In the MicrotoxR bioassay (DIN, 1997) the concentration of a
chemical that causes a 50% reduction of the light emitted by the bacteria (EC50) is
estimated from a concentration–response curve by regression analysis. Toxicity data
were based on a 30 min exposure of bacteria to the surfactant solution at 15 °C.
3. Results and discussion
3.1. Aerobic biodegradability
As stated in the above section, the aerobic ultimate biodegradability of amphoteric
surfactants was evaluated by means of the CO2 headspace test. This test is included in
the European Regulation on biodegradability of detergent surfactants (EC, 2004) and is
the reference method for laboratory testing of ultimate biodegradability. A surfactant is
considered as readily biodegradable if the biodegradation level exceeds 60% within
28 days in this test.
The biodegradation curves obtained for the amphoteric surfactants tested are shown
in Fig. 1 (alkyl and alkylamido betaines) and Fig. 2 (imidazoline derivatives). A high
biodegradation level (N60%) was rapidly reached (b 7 days) by the alkylamido betaine
tested. On the other hand, the kinetics of biodegradation of alkyl betaines and imidazoline
derivatives showed a slower rate of mineralization for the former. At the end of the test, the
degree of biodegradation, calculated from the CO2 production, was higher than 60% for
Fig. 2. Aerobic biodegradation curves of imidazolinium derivatives: (◊) C10-Imi, (□) C12-Imi,
(Δ) C14-Imi.
 M.T. Garcia et al. / Environment International 34 (2008) 1001–1005
alkyl betaines and higher than 80% for the alkylamido betaine and imidazoline derivatives.
The percentages of surfactant removal attained under the stringent conditions of the
biodegradation test allows these surfactants to be classified as readily biodegradable and
therefore easily mineralized in aerobic aquatic environments.
3.2. Anaerobic biodegradability
The ultimate biodegradability of amphoteric surfactants under anaerobic condi-
tions was investigated by means of batch tests. Surfactant concentration in the tests
carried out ranged from 20 to 300 mgC/L, corresponding approximately from 10 to
150 mg surfactant/g dry sludge. Therefore, the typical values recommended in the
standard method for testing anaerobic biodegradability, 20–50 mgC/L, were included.
Moreover, higher surfactant concentrations were tested to assess the potential toxicity
of these compounds to methanogenic microorganisms. The ultimate biodegradation of
the amphoteric surfactants was evaluated by comparing the biogas production in the
digesters spiked with surfactant and in the control digesters. The evolution of the net
biogas production (i.e. subtracting the endogenous biogas generation) in the digesters
spiked with different surfactant concentrations is represented in Fig. 3 (alkyl and
alkylamido betaines) and Fig. 4 (imidazoline derivatives). The biogas production was
time dependent until a plateau was reached.
The alkyl betaines C10-Bet, C12-Bet and C14-Bet exhibited a similar pattern of biogas
production (Fig. 3a, b and c). Despite the occurrence of a slight stimulation of the
methanogenic activity at low alkyl betaine concentrations, a behaviour already reported
for other surfactants (Garcia et al., 2005, 2006), the biogas production in the spiked
digesters was lower than the endogenous biogas production when the surfactant
concentration was increased. Likewise, the biogas production decreased when
increasing the alkyl chain length of the surfactant. However, as negative effects on
the methanogenic activity were not observed at surfactant concentrations b 50 mg
surfactant/g dry sludge (5%), alkyl betaines are not expected to exhibit toxic effects on
the methanogenic microorganisms in the anaerobic digester of a wastewater treatment
plant.
The biogas production in the digesters spiked with cocoamido betaine (Fig. 3d) was
significantly higher than in the control digesters at the end of the test, and the level of
biogas production increased with the surfactant concentration. In the anaerobic
digesters spiked with the highest Amido-Bet concentration, 300 mgC/L (corresponding
to 150 mg surfactant/g dry sludge), an initial inhibition of the biogas production was
observed for about 4 weeks. After this adaptation phase, the methanogenic population
initiated biogas production. The increase in biogas production even at a surfactant/
Fig. 3. Evolution of the net biogas production (CH4 + CO2) in anaerobic digesters spiked with (a) C10-Bet, (b) C12-Bet, (c) C14-Bet and (d) Amido-Bet. Biogas production was calculated at
standard temperature and pressure. The standard deviation of experimental data ranged from 2 to 12%.
1003
biomass ratio as high as 150 mg surfactant/g dry sludge (15%) shows the low toxicity of
the alkylamido betaine to the methanogenic microorganisms. The extent of anaerobic
ultimate biotransformation of cocoamido betaine, calculated from the net biogas
production when the plateau was reached, exceeded 60% for all the concentrations
tested.
For the imidazoline derivatives, C10-Imi, C12-Imi and C14-Imi, net biogas production
in the anaerobic digesters increased with the surfactant concentration (Fig. 4). Adverse
effects on the methanogenic activity were not observed at a concentration as high as
100 mg surfactant/g dry sludge (10%). The percentage of anaerobic mineralization of the
imidazoline derivatives, calculated from the net biogas production when the plateau
was reached, exceeded 60% for all the concentrations tested.
The results obtained for the alkylamido betaine and the imidazoline derivatives in
this stringent test system indicate that these amphoteric surfactants will be readily
biodegradable under anaerobic conditions.
Data obtained here and the results of the anaerobic biodegradation of amine oxide
based surfactants reported in an earlier work (Garcia et al., 2007) suggest that under
anaerobic conditions the differences in biodegradability of alkyl betaines with respect
to alkylamido betaines and imidazoline derivatives can be attributed to the lack of a
potential site of enzymatic hydrolysis in the fatty alkyl chain of the alkyl betaines.
Alkylamido betaines and imidazoline derivatives can decompose into non-surface
active fragments by enzymatic hydrolysis of the amide bond, a process that does not
require the involvement of molecular oxygen.
3.3. Aquatic toxicity assessment
Acute toxicity tests on freshwater crustacea (D. magna) as well as on saltwater
bacteria (P. phosphoreum) were carried out to assess the aquatic toxicity of the
amphoteric surfactants. The results of both the D. magna 48 h immobilisation test and
the P. phosphoreum 30 min luminescence reduction test are given in Table 2.
The estimated concentration to immobilise 50% of the crustacea population after
48 h of exposure (EC50) ranged from 5 to more than 200 mg/L whereas the estimated
concentration to reduce 50% of light produced by the bacteria after 30 min of exposure
(EC50) ranged from 5 to 80 mg/L. In general, toxicity to daphnia and bacteria increased
(lower EC50 values) when extending the alkyl chain length of the homologue. The
homologues of alkyl imidazoline derivatives were less toxic than the alkyl betaine
homologues to the aquatic organisms tested. The cocoamido betaine toxicity to P.
phosphoreum resembled the toxicity of alkyl imidazoline derivatives. On the other hand,
although it was expected that the presence of an amide group in the alkyl chain would
1004 M.T. Garcia et al. / Environment International 34 (2008) 1001–1005

Table 3
Aquatic toxicity of some usual surfactants

Surfactant LC50 or EC50 Toxicity classification

(mg/L)
Anionic Linear alkyl benzene 4.1a Moderately toxicb, acute
surfactants sulphonates (mean value toxicity IIc (toxic to aquatic
C = 11.5–11.8) life)
Non-ionic C12–C15 (mean value C b 14)A, 0.63d Highly toxicb, acute toxicity
surfactants N 6–9 EO Ic (very toxic to aquatic life)
Cationic Alkyl trimethyl ammonium 0.1d Highly toxicb, acute toxicity
surfactants salts Ic (very toxic to aquatic life)
a
HERA-LAS, 2004.
b
Norwegian Foundation for Environmental Product Labelling, 2004.
c
US Fish and Wildlife Services, 1984.
d
OECD, 2001.

Table 4
Ecotoxicity hazard classification of the amphoterics tested based on the EC50 values on
Daphnia magna

Alkyl betaines (⁎) Moderately toxica

Alkylamido betaines (⁎)
Alkyl imidazoline derivatives (⁎)
Acute Toxicity IIb(toxic to aquatic life)
Moderately toxica
Acute Toxicity IIb (toxic to aquatic life)
Slightly toxica
Acute Toxicity IIIb (harmful to aquatic life)

(⁎) alkyl chain C12–C14.

a
US Fish and Wildlife Services, 1984.
b
OECD, 2001.

US Fish and Wildlife Services (1984). Based on the EC50 values from the short-term
crustacea test (Table 2), the aquatic toxicity classification of the amphoteric surfactants
studied is given in Table 4. As shown in this table, the amphoteric surfactants are less
toxic to aquatic organisms than common cationic and non-ionic surfactants (Table 3).

4. Conclusions

Alkyl betaines, alkylamido betaines and alkyl imidazoline deriva-

Fig. 4. Evolution of the net biogas production (CH4 + CO2) in anaerobic digesters spiked
with (a) C10-Imi, (b) C12-Imi and (c) C14-Imi. Biogas production was calculated at
standard temperature and pressure. The standard deviation of experimental data
ranged from 2 to 11%.
tives are readily mineralized under aerobic conditions. Alkylamido
betaines and alkyl imidazoline derivatives are readily biodegraded
under anaerobic conditions whereas alkyl betaines exhibit a negligible
biotransformation. Acute toxicity values of the amphoteric surfactants
studied are higher than 5 mg/L and alkyl imidazoline derivatives are
the compounds that are the least toxic to the aquatic organisms tested,
P. phosphoreum and D. magna. In the light of our results of
biodegradability and aquatic toxicity, alkyl imidazoline derivatives
show the lowest potential impact on the aquatic environment.
Acknowledgment

lead to a reduction in toxicity owing to the increase in the hydrophilic character of the The authors are grateful to Anna Lupon (EDAR Manresa, Barcelona)
molecule as was observed for amine oxide based surfactants (Garcia et al., 2007), the
for supplying the WWTP samples.
alkylamido betaine exhibited the same toxicity to D. magna than the alkyl betaine with
a similar alkyl chain length.
The acute aquatic toxicity values of some selected common anionic, non-ionic and References
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