Ecotoxicology and Environmental Safety 84 (2012) 274–281

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Ecotoxicology and Environmental Safety

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Biodegradability, toxicity and mutagenicity of detergents: Integratedexperimental evaluations

Roberta Pedrazzani a, Elisabetta Ceretti b, Ilaria Zerbini b, Rosario Casale c, Eleonora Gozio d,Giorgio Bertanza e, Umberto Gelatti b, Francesco Donato b, Donatella Feretti b,n

a Department of Mechanical and Industrial Engineering, University of Brescia, 38 Via Branze, 25123 Brescia, Italyb Department of Experimental and Applied Medicine Institute of Hygiene, Epidemiology and Public Health, University of Brescia, 11 Viale Europa, 25123 Brescia, Italyc Laboratory of Sanitary and Environmental Engineering, University of Salerno, Ponte Don Melillo, 84084 Fisciano, Italyd ARPA, Regional Environmental Agency of Brescia, 20 Via Cantore, 25128 Brescia, Italye Department of Civil Engineering, Environment, Land and Architecture, University of Brescia, 43 Via Branze, 25123 Brescia, Italy

a r t i c l e i n f o

Article history:

Received 21 December 2011

Received in revised form

16 July 2012

Accepted 24 July 2012Available online 14 August 2012

Keywords:

Detergents

Ready biodegradability

Ames test

Comet test

Allium cepa test

Toxicity tests

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.07.023

esponding author. Fax: þ39 030 3717688.

ail addresses: roberta.pedrazzani@ing.unibs.it

med.unibs.it (E. Ceretti), zerbini@med.unibs.

unisa.it (R. Casale), e.gozio@arpalombardia.i

bertanza@ing.unibs.it (G. Bertanza), gelatti@m

med.unibs.it (F. Donato), feretti@med.unibs.

a b s t r a c t

The widespread use of detergents has raised concern with regard to the environmental pollution caused

by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex

mixtures of different substances, in which synergistic effects may occur, we aimed to assess the

mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready

biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio

fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet

assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All

detergents presented ready biodegradability. EC50 values varied for all products, according to the

marker organism used, but were always higher than the more stringent value considered for aquatic

toxicity assessment (V. fischeri 10–60 mg/L; D. magna 25–300 mg/L; A. cepa 250–2000 mg/L). None of

the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an

increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one

commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in

human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such

as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic

features.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

Detergents are complex mixtures of surfactants, builders,bleaching agents, enzymes, bleaching agent activators, fillers andother minor additives, such as dispersing agents, fabric softeningclay, dye-transfer inhibiting ingredients, optical brighteners andperfumes (Ho Tan Tai, 2000; Pettersson et al., 2000; Stjerndahl andHolmberg, 2005; Yangxin et al., 2008).

Due to their huge consumption, more stringent environmentalregulations and increasing awareness of the potential toxicity ofdetergents to humans and ecosystems have resulted in efforts todevelop new classes of surfactants, builders and other constituents

ll rights reserved.

(R. Pedrazzani),

it (I. Zerbini),

t (E. Gozio),

ed.unibs.it (U. Gelatti),

it (D. Feretti).

with a lower impact in terms of persistence and toxicity and betterwashing performance, as well as economic convenience. Besidesconventional surfactants, often deriving from biological molecules,increasing attention has been also given to the so-called biosur-factants (Banat et al., 2000; Gunjikar et al., 2006; Stjerndahl andHolmberg, 2005; Yakimchuk et al., 2004; Yangxin et al., 2008). Inparallel with the development and optimization of new sub-stances, the evaluation of possible risks for consumers, and forhuman health and the environment in general, has become acrucial topic in science and policy, in accordance with recentEuropean Regulations (EC 648/2004, 2004; EC 907/2006, 2006; EC1336/2008, 2008 and EC 1272/2008, 2008).

The behaviour of a substance in the environment can bepredicted according to its physical and chemical properties; more-over, biodegradability in water, soil and sediments provides impor-tant information for assessing its final fate (Glathe and Schermer,2003; Sanderson et al., 2006; Gomez et al., 2007; Sharvelle et al.,2007). Several standard tests have been proposed and discussed inthe scientific literature for evaluating biodegradability – ready,

R. Pedrazzani et al. / Ecotoxicology and Environmental Safety 84 (2012) 274–281 275

inherent, ultimate – (OECD, 1993, 2006; Blok and Struys, 1996;Pagga, 1997; Reuschenbach et al., 2003; Guhl and Steber, 2006; O’Malley, 2006; Boethling and Lynch, 2007; Stasinakis et al., 2008).Suitable tests should be established, based on usage, and hence onthe main environmental compartments where a chemical can befound. In the case of detergents, for instance, aquatic environmentshould be considered, together with wastewater treatment plants.Although all conditions should be applied, aerobiosis is absolutelythe most significant, since metabolic pathways are energeticallyfavoured by molecular oxygen and degradation rates are usuallyhigher. Nowadays, OECD 301 A–F ready biodegradability tests are avaluable means for monitoring the extent of biodegradation ofchemicals, taking into account abiotic processes such as adsorptionand hydrolysis. At present, as far as detergents are concerned, theinadequacy of these protocols for technical surfactant evaluation,based on their particular chemical nature, has been underlined(Richterich and Steber, 2001). In addition, some prescriptions, suchas the 10-d window criterion (see Section 2.1), have been modifiedby legislative documents (Regulation EC 648/2004, 2004).

The aim of this research was to apply different characteriza-tion tests on final detergent formulations: according to the above-mentioned regulations, and the Ecolabel assignment criteria, purechemicals or mixtures of structurally similar chemicals must beconsidered separately and a global evaluation must be carried outby means of specific calculations (e.g., for the assessment of CDV,Critical Dilution Volume, the limits of which in ecotoxicity impactassessment have recently been highlighted, together with alter-native methods, such as USEtox (Van Hoof et al., 2011). Fulldetails are available for several categories and also for specificcompounds (Takamatsu et al., 1996; Jensen, 1999; Scott andJones, 2000; Stjerndahl and Holmberg, 2005; Belanger et al.,2006; Mohan et al., 2006; Sharvelle et al., 2007; Sibila et al.,2008). A more accurate prediction of the eco-compatibility of adetergent, however, should not take only biodegradability intoaccount, because synergistic factors can amplify toxicity, andbiodegradation metabolites themselves can also become signifi-cant (Pettersson et al., 2000). Furthermore, specific aspects suchas mutagenesis and alteration of endocrine systems, becomemore and more relevant, based on scientific knowledge.

Single components of detergents may exhibit toxic and muta-genic activity to different organisms, even at low concentrations.Quaternary ammonium compounds induce micronuclei in mam-malian and plant cells (Ferk et al., 2007). Among the others,Garcıa et al., 2001, showed that the estimated concentration ofamino-oxide-based surfactants yielding 50% immobilisation ofDaphnia magna (IC50) ranges from 6 to 45 mg/L, whereas theestimated concentration resulting in a 50% reduction of bacterialuminescence EC50 ranges from 0.1 to 11 mg/L; non-ionic surfac-tants and their degradation products are toxic to marine andfresh-water organisms (Ying, 2006) and have endocrine effects onfish (Jobling and Sumpter, 1993; Purdom et al., 1994; De Weertet al., 2011); sodium citrate induces chromosomal aberrations inAllium cepa (Turkoglu, 2007); sodium perborate and polycarbox-ylates induce point mutations in bacteria (Kaplan et al., 2004;Seiler, 1989); corrosion suppressants, such as benzotriazole, aswell as other additives, are toxic to water organisms, such asbacteria, invertebrates (crustaceans) and fish used as standardaquatic laboratory test organisms (Pillard et al., 2001); thecomponents of some perfumes, e.g., cinnamaldehyde, react withDNA (Stammati et al., 1999), and limonene produces tumours inmale rats by a non-DNA-reactive mechanism which is notrelevant to humans (IARC, 1999). Very few studies that haveconsidered the toxic effects of final detergent formulates or, morespecifically, taken the effects on algae into account, showing theability of these mixtures to interfere with cellular permeability,affect cell mobility, and inhibit some specific functions, such as

photosynthesis or biosynthesis and biodegradation pathways(Aizdaicher and Markina, 2006; Azizullah et al., 2011). Acutetoxicity in D. magna was also studied (Pettersson et al., 2000) butno information on their mutagenicity is available.

A more accurate assessment of the mutagenicity of a detergentshould take into account possible interactions between singlechemicals, which might play a crucial role in such complexmixtures. Therefore, it may be useful to test raw detergents ratherthan single components. Furthermore, some properties, such asmutagenicity and the ability to influence endocrine systemactivity (‘‘endocrine disruption’’), have raised more and moreconcern in recent years (WHO, 2002,, 2004).

This paper reports the results of a battery of biological testsperformed on various pre-manufacture (not yet available on themarket) and commercial detergents for laundry, dishwashers andwashing up.

2. Materials and methods

Ready biodegradability, toxicity and genotoxicity of some detergents in their

complete formulation were examined. Three pre-manufacture detergents pending

ecolabel certification (A, B and C, for laundry, dishwashers and washing-up,

respectively), two ecolabelled commercial detergents (E and F, for dishwashers

and laundry, respectively) and a conventional one (D, for dishwashers) were

studied.

2.1. Detergents tested

Six detergent formulations, either pre-manufacture or already commercially

available, were examined. Their usage, surfactant contents and other constituents,

and COD (chemical oxygen demand) concentration are listed in Table 1.

Detailed manufacturer’s information on surfactant content was used for new

detergents, and consumer information appearing on the packaging or website

allowed a rough evaluation for commercial products.

2.2. Ready biodegradability test

Detergents underwent a ready biodegradability test according to OECD 301 F

(Manometric Respirometry Test) (OECD, 1993).

The biodegradation test described by OECD 301 F was carried out using the

respirometric BOD OxiTop method (APHA, 1998). Activated sludge taken from a

municipal waste water treatment plant was used as inoculum. Abiotic sterile control

was obtained by dosing NaClO. Glucose and sodium glutamate (1:1 w/w ratio) were

added as reference organic compounds. Since OECD 301 F consists of a respirometric

test, the ‘‘pass level’’ for ready biodegradability corresponds to a 60% decrease in initial

ThOD (theoretical oxygen demand) or initial COD, in cases where the test material was

insufficiently defined and ThOD could not be calculated. Measurement of COD

(colorimetric method 5220-D) (APHA, 1998) was carried out before and at the end

of testing. Raw samples were filtered through 0.45 mm cellulose acetate membranes

(model 25CS045AN, Advantec MFS Inc.).

2.3. Toxicity tests

EC50 (24 h) tests were conducted with Cladocer crustacean D. magna Straus for

each detergent studied, according to official Italian standards (APAT–IRSA/CNR,

2003); liquors deriving from the OECD 301 F test underwent the assay for direct

evaluation of effluent toxicity after 24 and 48 h (APAT–IRSA/CNR, 2003). In EC50

evaluation, preliminary screening tests were carried out on detergent formulation

concentrations from 0.1 to 100 g/L, as prescribed, whereas subsequent tests were

carried out on dilutions down to a concentration of 1 mg/L. Daphnids (neonates

less than 24 h old, obtained by hatching ephippia—Ecotox LDS Srl) were introduced

into 50 mL glass vessels (5 and 10 organisms/vessel for preliminary and final tests,

respectively), together with samples diluted in a standard mineral solution

(pH¼7.5–8.5; alkalinity¼110–120 mg CaCO3/L; hardness¼140–160 mg/L CaCO3/

L; 10 mg KCl/L, 192 mg NaHCO3/L, 53 mg MgSO4/L, 183 mg CaSO4 �2H2O). Bioas-

says were conducted under static conditions, measuring dissolved oxygen and pH

in each sample at both the start and end of testing. Dosage calculation was based

on mass/volume ratio; in the event of liquid formulations, density data were used.

EC50 and 25% confidence limits were calculated as prescribed by the standard

method. In the toxicity evaluation of the liquors after biodegradability tests,

crustaceans were introduced directly into the samples. All the experiments were

performed in triplicate. Test conditions (temperature: 2071 1C—kept in a refri-

gerated thermostat, Ecotox LDS Srl, mod. ECO 96/CRS.A; darkness/irradiation-

Table 1Summary of detergents studied.

Code Detergent Detergent use Ecological

definition

Composition COD

(g/L)

A New pre-

manufacture

Laundry Yes Potassium cocoate, sodium carbonate, C9-11 fatty alcohol

ethoxylates, C10 fatty alcohol ethoxylates, sodium gluconate,

sodium chloride, sodium lauryl sulphate,

alkyliminodipropionate, 2-phenoxyethanol

450

B New pre-

manufacture

Dishwashers Yes Glutamic acid derivative, sodium citrate, sodium metasilicate,

polyaspartate, sodium hydroxide, alkyliminodipropionate, C10

fatty alcohol ethoxylates, C9-11 fatty alcohol ethoxylates

200

C New pre-

manufacture

Washing-up Yes Sodium lauryl sulphate, alkylamidopropylbetaine, sodium

chloride, C9-11 fatty alcohol ethoxylates, C10 fatty alcohol

ethoxylates, sodium iminodisuccinate, 2-phenoxyethanol,

alkyliminodipropionate, citric acid

270

D Commercial Dishwashers No PEG-30-PEG-40, PEG-130-PEG-150, sodium polyacrylate,

tetraacetylethylenediamine (TAED), tetrasodium etidronate,

fatty alcohol alkoxylate, benzotriazole, limonene

350

E Commercial Dishwashers Yes 2-propenoic acid, telomer with sodium hydrogen sulfite,

sodium polyacrylate-sulfonate, PEG-75,

tetraacetylethylenediamine (TAED)

450

F Commercial Laundry Yes C12-18 alkyl sulphate, tetraacetylethylenediamine (TAED),

sodium salt of 2-propenoic acid, 2,5-furandione polymer, C12-14

fatty alcohols ethoxylates, C12-18 fatty alcohols ethoxylate, alkyl

polyglycosides, 4,40-bis(triazinylamino)stilbene-2,20-disulfonic

acid, tetrasodium editronate, benzyl salicylate, hexyl cinnamal

250

R. Pedrazzani et al. / Ecotoxicology and Environmental Safety 84 (2012) 274–281276

provided by cool white fluorescent lamps, 300 lux-duration: 8 and 16 h, respec-

tively) and procedures (dissolved oxygen measurement, carried out using a WTW

340i oxymeter and immobile organism evaluation) were as reported by standard

methods.

Vibrio fischeri (strain NRRL-B-11177) was also used to evaluate toxicity (after

15 min) of raw products, in accordance with method 8030 (APAT–IRSA/CNR,

2003). The luminometer (Lumistox 300) and dehydrated bacteria were supplied

by Hach–Lange GmbH. Detergents were diluted in a 2% NaCl aqueous solution, the

pH of which was adjusted to 7.070.2. Temperature was kept at 15 1C throughout

the test, as prescribed. Detergent concentration in the preliminary tests ranged

from 50 to 5000 mg/L. All the experiments were performed in triplicate; EC50 and

95% confidence limits were calculated as well. Assessment of toxicity test

reliability was confirmed by means of a Z-score test (ISO, 2005) performed at

certified laboratories, under the supervision and control of the Italian National

Environmental Agency (ARPA).

2.4. Mutagenicity tests

Three different mutagenicity tests were carried out on the detergents in order

to assess their behaviour in terms of ability to induce genetic damage in target

cells of different organisms (bacteria, plant cells and human leukocytes).

2.4.1. Salmonella/microsome test

The samples diluted in distilled water underwent the Salmonella/microsome

test (Ames test) at increasing doses, with Salmonella typhimurium TA98 and TA100

strains (as recommended in the Standard Methods for the Examination of Water

and Wastewater, APHA, 1998), with and without metabolic activation (S9 mix 4%)

to highlight the presence of indirect and direct mutagenic substances. A range of

doses from 5 to 1000 mg/plate for each detergent was applied. This reversion test

allows the detection of point mutation: the TA98 strain detects frame-shift

mutagens and the TA100 strain responds to base-pair substitution mutations

(Maron and Ames, 1983). For positive control, 2-nitrofluorene for TA98 without S9

(10 mg/plate), sodium azide for TA100 without S9 (10 mg/plate) and 2-aminofluor-

ene for both strains with S9 mix (20 mg/plate) were used, respectively. Distilled

water was used as a negative control. All the experiments were conducted in

duplicate and performed twice. The results were expressed as mutagenicity ratio,

dividing the average revertants/plate by the spontaneous mutation rate. The

results were considered positive if two consecutive dose levels or the highest non-

toxic dose level produced a response at least twice that of the solvent control and

at least two of these consecutive doses showed a dose–response relationship

(APHA, 1998).

2.4.2. Allium cepa test

In a preliminary toxicity assay, equal-sized young onion bulbs were exposed

for 96 h in the dark to different concentrations (from 10 to 2000 mg/L) of each

detergent. Root length was used to calculate the EC50 value of the six detergents

and identify the three concentrations to undergo the A. cepa genotoxicity assay,

the highest of which corresponded to the EC50 value found (Fiskesjo, 1993, 1985).

Other macroscopic parameters (turgescence, consistency, colour change, root tip

shape) were used as toxicity indexes (Fiskesjo, 1993, 1985).

The A. cepa micronucleus test was performed using six equal-sized young

bulbs per sample (Ma et al., 1995). After 72-h pre-germination in mineral water,

the bulbs were exposed to three concentrations of each detergent for 24 h. They

were then replaced in mineral water for 44 h of recovery time, fixed in acetic acid

and ethanol (1:3) for 24 h and lastly stored in 70% ethanol. Distilled water was

used as negative control and maleic hydrazide (1 mg/L, 6-h exposure) was used as

positive control. Five roots of each sample were considered for microscopic

analysis: 5000 cells (1000 cells/slide) were scored for mitotic index (as a measure

of cellular division and hence sample toxicity) and 10,000 cells (2000 cells/slide)

were scored for micronucleus frequency. The results were recorded as number of

micronuclei per 100 cells and the data were analysed using the w2 and Dunnett’s

tests, respectively.

2.4.3. Single cell gel electrophoresis (SCGE) assay

The SCGE, or comet assay, was performed according to the method proposed

by Singh et al. (1988). Human leukocytes were treated with different doses of each

detergent (range 1–2000 mg/L) at 37 1C for 1 h. Negative (distilled water) and

positive (ethyl methanesulfonate, 2 mM) controls were performed. After treat-

ment, the assay was performed only on samples with viability 470%, according to

the procedure proposed by Tice et al. (2000). After slide preparation and cell lysis,

DNA was subjected to 20-min unwinding and 20-min electrophoresis (pH413,

0.8 V/cm and 300 mA). The slides stained with ethidium bromide were examined

under a fluorescence microscope (Olympus CX 41RF) equipped with a BP 515–

560 nm excitation filter and an LP 580 nm barrier filter. Fifty randomly-selected

cells per slide (two slides per sample) were analysed. The extent of DNA migration

was evaluated by both ‘‘visual score’’ (based on visual classification of DNA

damage) and the comet parameter ‘‘tail intensity’’ (percentage of DNA migration

in the tail) detected using an automatic imaging system (Komet 5, Kinetic Imaging

Ltd). Significance of the effect of each dose against the negative control was

determined using Dunnett’s test.

3. Results

3.1. Ready biodegradability test

Detergent A reached a final removal percentage of about 80%,much higher than the ‘‘pass level’’ of 60%, even when consideringa slight decrease due to possible nitrification (a reaction mediatedby autotrophic aerobic bacteria, which contribute to oxygenconsumption without parallel organic carbon degradation)(Fig. 1). For detergent B, the dissolved oxygen consumptioncorresponded to 55% of initially measured COD. Furthermore,

Fig. 1. OECD 301 F. Manometric respirometry test for ready biodegradability assessment: removal (%) of initial COD.

Table 2Summary of toxicity test results: EC50 values obtained in V. fischeri, D. magna and

A. cepa toxicity tests (upper and lower 95% confidence limits for V. fischeri and

D. magna tests are reported in brackets).

Detergent V. fischeri D. magna A. cepa

EC50 (mg/L) EC50 (mg/L) EC50 (mg/L)

A 20 (18; 22) 50 (45; 56) 2000

B 45 (39; 52) 300 (244; 369) 1500

C 10 (9; 12) 70 (62; 79) 1500

D 60 (54; 67) 300 (250; 360) 250

E 40 (34; 47) 100 (87; 115) 500

F 15 (13; 18) 25 (21; 30) 750

Time exposure: 15 min for V. fischeri; 24 h for D. magna; 96 h for A. cepa.

R. Pedrazzani et al. / Ecotoxicology and Environmental Safety 84 (2012) 274–281 277

since the nitrogen content of this detergent is significant (4%compared to COD), taking the contribution of nitrification intoaccount, the results (55% consumed oxygen compared to initialCOD) appeared even less positive.

Test 301 F, carried out on detergent C, exhibited a dissolvedoxygen consumption of about 80% of initial measured COD,showing the positive behaviour of this preparation. Furthermore,these values should not be decreased due to the nitrificationprocess, since nitrogen content is not significant (o1% comparedto COD), hence the related possible oxygen consumption can beneglected.

The degradation percentage for commercially available pro-ducts was high, around 70% and 80%, in the case of the conven-tional dishwasher (D) and ecolabelled laundry detergents (F),respectively, whereas it was quite low for sample E, ecolabelleddishwasher detergent (51%). In general, commercial productsrequired a longer acclimatization period compared to pre-manufacture ones.

3.2. Toxicity tests

Table 2 shows the EC50 values obtained in the V. fischeri,

D. magna and A. cepa toxicity tests. Bacteria luminescence wasinhibited to a similar extent in all cases (range 1–60 mg/L),although higher dosages were observed with dishwasher deter-gents (B, D, E). In D. magna the EC50 range was broader(25–300 mg/L): detergents A and F exhibited the highest toxicity.Likewise, D. magna immobilization was less relevant for dish-washer detergents (100–300 mg/L). The other detergents gavesimilar results, whereas formulations C and E had a lower impactcompared to the pre-manufacture one (B). Generally speaking,EC50 in A. cepa roots was higher than observed in V. fischeri andD. magna (250–2000 mg/L), suggesting a lower toxicity towards

this organism. At the end of the 301 F modified OECD screeningtest, all the liquors underwent a toxicity test on D. magna. This isrecommended by EC 648/2004 as an additional test for assessingthe toxicity of biodegradation test liquors. Organism immobiliza-tion did not occur in any cases, after 24- and 48-h contact,suggesting the absence of toxic by-products (at least at significantconcentrations), due to the biodegradation process.

3.3. Mutagenicity assays

3.3.1. Salmonella/microsome test

None of the detergents analysed exhibited mutagenicity in thebacterial test on TA98 or TA100 strains with or without metabolicactivation at the tested doses: from 5 to 2000 mg/plate for newformulations, A, B and C, and 1000 mg/plate for conventional ones,D, E and F (Table 3). One of the new detergents (sample C) showedtoxicity on TA98 strain without metabolic activation at almost alldoses tested, whereas two of the commercially available products(samples D and F) showed only light toxicity at the highest testeddose (1000 mg/plate) on the same strain. These effects vanishedwhen S9 was added, due probably to the detoxifying property ofthe enzymatic mixture.

3.3.2. Allium cepa test

In the toxicity test, the commercial products (samples D, E and F)showed a greater cytotoxic effect on A. cepa roots compared to thenew formulations (samples A, B and C) (Table 2).

All three new formulations (detergents A, B and C) and two ofthe three commercial ones (samples D and E) gave negativeresults in the A. cepa micronucleus test, showing no significantdifferences in micronucleus frequency compared to thenegative control (Table 4). The third commercial product, how-ever, the ecolabelled laundry detergent F, induced a micronucleusfrequency significantly higher than in the negative control(3.071.1% versus 1.170.9%, po0.01 in accordance withDunnett’s test) but only at the concentration of 0.50 g/L. A higherconcentration of the same detergent (0.75 g/L) did not induce asignificant genotoxic effect, but it did induce a mitotic indexsignificantly lower than in the negative control (10.1% versus13.1%, po0.05 in accordance with w2 test), showing that it wasalready toxic, which probably prevented genotoxic activity.

3.3.3. Single cell gel electrophoresis assay (comet assay)

Five detergents showed toxic effects in human leukocytesevaluated with the viability test, so they were tested for geno-toxicity at different concentration ranges. The assay was per-formed only on sample doses having viability 470%, according to

Table 3Ames test results expressed as mutagenicity ratio.

Sample dose

(mg/plate)

Sample A Sample B Sample C Sample D Sample E Sample F

�S9 þS9 �S9 þS9 �S9 þS9 �S9 þS9 �S9 þS9 �S9 þS9

TA985 0.7 1.2 0.6 1.6 tox 1.1 1.1 1.2 0.8 1.5 0.6 1.4

10 1.0 1.1 0.5 1.4 tox 1.3 1.1 1.4 1.1 1.7 0.9 1.4

25 0.9 1.4 0.7 1.2 0.7 1.1 0.8 1.2 0.7 1.5 1.1 1.9

50 0.9 1.3 0.8 1.3 0.6 1.4 1.0 1.6 0.7 1.5 1.0 1.3

100 0.9 1.4 0.7 1.1 tox 1.2 0.9 1.5 1.2 1.4 0.8 1.6

500 1.1 1.2 0.8 1.2 tox 1.1 1.0 1.6 1.0 1.3 1.1 1.3

1000 0.7 1.4 0.9 1.2 tox 1.0 tox 1.3 0.7 1.5 tox 1.5

2000 0.6 1.4 0.6 1.4 tox 1.0 – – – – – –

TA1005 0.9 0.9 1.0 0.8 0.9 0.8 1.1 0.9 1.2 1.0 1.2 0.9

10 1.0 0.8 0.9 0.8 1.0 1.2 0.9 1.0 1.2 1.0 1.2 0.8

25 1.2 1.1 1.1 0.8 0.9 0.8 1.1 0.9 1.2 1.0 1.2 1.0

50 1.1 0.9 1.1 1.0 0.9 1.0 1.1 1.1 1.3 1.0 1.1 0.9

100 1.0 1.1 1.0 1.0 1.1 0.9 1.3 1.1 1.2 1.0 1.2 1.0

500 1.1 0.9 1.0 0.8 0.8 1.0 1.2 1.1 1.4 1.0 1.3 1.1

1000 1.1 0.9 1.1 0.9 1.0 1.0 1.0 1.0 1.3 1.1 0.9 0.8

2000 0.8 0.9 1.0 0.9 0.8 0.9 – – – – – –

tox¼toxic for bacteria; –¼not tested; negative control (revertants/plate): 29.374.9 (TA98�S9), 20.375.0 (TA98þS9), 75.2710.4 (TA100�S9, samples A-B-C), 82.376.6

(TA100þS9, samples A-B-C), 69.277.7 (TA100�S9, samples D-E-F), 87.076.9 (TA100þS9, samples D-E-F)

positive control (revertants/plate): 41000 (TA987S9 and TA1007S9)

Table 4A. cepa micronuclei test results.

Sample dose Sample A Sample B Sample C Sample D Sample E Sample F

(g/L) MI (%) MCN (%) MI (%) MCN (%) MI (%) MCN (%) MI (%) MCN (%) MI (%) MCN (%) MI (%) MCN (%)

0.05 – – – – – – 11.5 0.570.6 – – – –

0.1 – – – – – – 11.7 1.170.7 15.5 0.870.1 – –

0.25 – – – – – – 9.7a 0.370.4 15.4 1.671.2 14.3 0.470.5

0.5 12.7 0.370.4 10.8 0.970.5 10.9 0.870.4 – – 10.7a 1.270.3 13.3 3.071.1n

0.75 – – – – – – – – – – 10.1a 2.172.7

1.0 11.1 0.170.2 11.0 0.070.0 7.2b 1.271.2 –– – – – – –

1.5 tox tox 11.0 0.970.7 tox tox – – – – – –

2.0 tox tox – – – – – – – – – –

Negative control 14.2 1.070.5 10.7 0.670.4 11.9 0.871.0 13.2 0.871.0 14.2 1.472.0 13.1 1.170.9

MI¼mitotic index; MCN¼micronuclei frequency.n Statistically significant vs. negative control according to Dunnett’s test (po0.01). Positive control: maleic hydrazide (10 mg/L): 9.0% MI; 4.071.8% MCN.a Statistically significant vs. negative control according to w2 test (po0.05).b Statistically significant vs. negative control according to w2 test (po0.01).

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the procedure proposed by Tice et al. (2000). Only one product(E) showed no toxic effects at any tested doses.

All the new formulations (samples A, B and C) showedgenotoxic effects on human leukocytes (Fig. 2). Sample A, testedfrom 20 to 140 mg/L because it showed toxic effects over 160 mg/L, induced a significant increase in DNA damage, starting fromlow doses (40 mg/L). Sample C, tested from 10 to 200 mg/L andshowing toxicity from 300 mg/L, induced a significant increase inDNA migration at medium doses (from 160 mg/L). Sample B,tested from 45 to 2000 mg/L and showing toxicity only at the highdose of 2500 mg/L, induced a significant genotoxic effect only atvery high doses (720 mg/L). Furthermore, the increase in DNAmigration induced by samples A and B was much higher than thatinduced by sample C. With regard to the commercial products(Fig. 2), sample D showed toxic effects at very low doses, from100 mg/L, so it was tested from 0.5 to 50 mg/L. It was also theonly detergent in this group to determine a significant increase inDNA migration at low doses, from 1 mg/L. Sample F showed toxiceffects only, from 250 mg/L, and was tested from 1 to 100 mg/L,inducing no increase in DNA damage. Sample E did not show toxic

or genotoxic activity on human leukocytes at any of the testeddoses, from 10 to 1000 mg/L.

4. Discussion

The impact of complex mixtures like detergents on the aquaticecosystem and human health has been a matter of debate for manydecades, because of their widespread production and use: in the EUalone, soap and cleansing agents accounted for 11% of chemicalindustry sales in 2007 (CEFIC, 2009). Data on the ingredients ofdetergents and experimental evidence for their toxicity and geno-toxicity are well known (Ferk et al., 2007; Garcıa et al., 2001; IARC,1999; Kaplan et al., 2004; Pillard et al., 2001; Purdom et al., 1994;Stammati et al., 1999; Ying, 2006). Since detergents are complexmixtures, their overall effect may not simply be the sum of theeffects of the single components, due to interactions among them.Despite this, few studies have considered the toxic features of thefinal formulations, mainly taking into account the effects on algaeand daphnia (Aizdaicher and Markina, 2006; Azizullah et al., 2011;

Fig. 2. Comet assay. DNA damage induced by the three new formulations (left graph) and the three commercialised products (right graph) and detected by the comet assay

as visual score, according to doses (semilogarithmic scale). The doses given in the figure had viability 470%; the last dose reported for each detergent (except for sample E)

was the highest non-toxic dose. Negative control: visual score¼118.0075.35. Positive control: visual score = 314.67713.00. n po0.05, nn po0.01 and nnn po0.001 using

Dunnett’s test (reference group: negative control).

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Pettersson et al., 2000), and very little information is available ontheir mutagenicity, an important feature as regards both theenvironment, because of their possible dispersion and accumulationin surface waters, and health.

Ready biodegradability in aquatic aerobic conditions wasassessed by applying OECD 301 F test. Degradation analysisshowed positive results for A, C, D and F, whereas B and E werecharacterized by less effective biodegradation. Despite the finaldegradation percentage, it is worth noting, however, that thecommercial detergents required a longer acclimatization periodcompared to the ecological pre-manufacture formulations.Although many authors have pointed out several critical aspectsin the execution and interpretation of the results of OECD readybiodegradability tests (Boethling and Lynch, 2007; O’ Malley,2006; Richterich and Steber, 2001), especially in the case ofsurfactants, these findings suggest that testing mixtures (in thiscase end products) could provide useful information, which isotherwise lacking when assessing environmental impact based onsingle component behaviours. For instance, it is worth noting thatall components of pre-manufacture formulations are declared as‘‘readily biodegradable’’ by the manufacturers, based on OECDtests, whereas when final formulations were tested, in one casethe product did not reach the ‘‘pass level’’ (sample E). This aspectshould be taken into account, especially when applying thecalculation for Ecological Label, based on chemical and toxicolo-gical data for each mixture component, thus neglecting possiblesynergistic effects (both positive and negative).

Toxicity studies showed extremely variable EC50 values accord-ing to the marker organism used. EC50 values in V. fischeri were verylow (10–60 mg/L), showing predictable high toxicity toward pro-karyotic cells. Despite confidentiality constraints about new pro-ducts and qualitative information about commercial ones, it ispossible draw a rough overall toxicity estimation: detergent Acomposition correlates well with D. magna toxicity tests (based onthe content of C9–11 fatty alcohol ethoxylates, C10 fatty alcoholethoxylates and potassium cocoate). In the case of product C, whoseEC50 is moderately higher (70 mg/L), but still below 100 mg/L, arough correlation may well be due to the contribution of C9–11 fattyalcohol ethoxylate and C10 fatty alcohol ethoxylate. Their amount islower than in the case of product A, however, thus explaining thebetter behaviour (Lundahl and Cabridenc, 1978). Product F is anecolabelled dishwasher detergent, but unfortunately its compositionis only qualitative. It should be pointed out, however, that theingredients sodium percarbonate and proteases have extremely lowEC50 values (4.9 and 0.1–13.0 mg/L, in the case of different enzymes,respectively) (HERA, 2011; IUCLID, 2011). Furthermore, its alcoholethoxylates are composed of C12–18 and C12–14 mixtures: theincrease in hydrophobicity (i.e., longer alkyl chains and shorter EOchains, higher Kow) is correlated with the increase in toxicity (Boeijeet al., 2006; Morrall et al., 2003).

The reference organisms (crustaceans), according to EC 1272/2008, are always characterized by EC50 higher than 10 mg/L,which is the most stringest value considered for aquatic toxicityassessment. Lower EC50 values (C and F) for D. magna could beascribed to the amount (quantified in the case of C) of fattyalcohol ethoxylates; data provided by manufacturers are similarto those presented in the literature, although it is difficult toassess the toxicity of such mixtures, which can be composed of upto 100 isomers (Belanger et al., 2006; Morrall et al., 2003). Thesecompounds, however, are characterized by a high ready biode-gradability (Battersby et al., 2001; Wind et al., 2006), whichmeans their concentration in wastewater treatment plant efflu-ents and in aquatic environments is extremely low. In effect, theirremoval by means of activated sludge treatment has beenestimated at more than 99% (Battersby et al., 2001; Wind et al.,2006). Nevertheless, it is worth noting that an appreciablefraction of these molecules can be adsorbed onto bioflocs (VanCompernolle et al., 2006), thus affecting bioavailability itself andglobal mass balances based on degradation processes. In the caseof product F, other ingredients such as percarbonate and pro-teases may contribute to toxicity to D. magna. Moreover, based onthe amounts of surfactants in tested detergents, it was seen, as ageneral trend, that the higher the concentration of surfactants, thelower the EC50 values (data not shown), as already stated byPettersson et al. (2000).

With regard to genotoxicity tests, none of the detergentsstudied induced point mutations in bacteria. However, onecommercial ecolabelled product (F) induced an increase in micro-nucleus frequency in A. cepa root cells. All the pre-manufactureproducts (A, B and C) and one commercial detergent (D), whichgave negative results in Ames and A. cepa tests, induced DNAdamage in human leukocytes. Although this is potentially repar-able primary damage, this mutagenicity is of concern because ofthe wide diffusion of mutagenic compounds in the environment.Severity of DNA damage caused by mutagenic compoundsdepends on both the intensity and duration of exposure and theefficiency of the DNA repair system activated by the exposureitself.

Overall, these results suggest that biodegradability estimationbased only on properties of each component can be flawed (e.g.,for detergent B) and therefore needs to be confirmed byfurther assays. Again, the results of each assay (mutagenicity,toxicity and biodegradability) are not well explained by referringto their composition, there being in general, for instance, hightoxicity in the case of a large amount of surfactants and highmutagenicity in the case of huge concentrations of cocoate. Theaim of this work, however, was to assess the mutagenicity andpotential environmental impact of end products, by directlytesting products used by consumers and dissipated in theenvironment.

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5. Conclusion

The studied detergents were shown not to be completely freeof toxic and genotoxic effects. They are complex mixtures ofsubstances which may contribute to deterioration in wastewaterquality, hence increasing the presence of compounds with muta-genic properties or their precursors (Feretti et al., 2012).

It is worth underlining that, as stated in the literature, biode-gradation products should also be monitored, since they exhibitdifferent toxicity characteristics (in terms of dose and targets)compared to parent compounds. In addition, liquors from washingprocesses should also be tested, because cleaning conditions canaffect degradation processes and the formation of by-products. Inthis case, in order to estimate actual environmental loads, specificstudies should be based on dosage and consumption data.

Nevertheless, the detergent formulations themselves, whichare well known, may contain substances (even at trace concen-trations) released into wastewater, and hence into the aquaticenvironment, including surface and groundwater, which is ulti-mately intended for human consumption, thus constituting apossible safety hazard for. Wastewater disinfection as well canplay an important role in DBP formation (Monarca et al., 2000).

Many authors focus on QSAR (quantitative structure-activityrelationship) studies applied to specific categories of detergentingredients; toxic and genotoxic effects are dependent on theconcentration of the detergent. Besides, potential synergistic andadditive effects may be present due to the large number of toxicand allergenic molecules in these mixtures. For this reason, it isessential to promote mutagenicity studies on raw detergents, notonly on single components, as in the intention of EC 1272/2008.

In conclusion, a more accurate evaluation of the potentialimpact of complex mixtures such as detergents requires a batteryof tests to describe degradation, as well as toxicological andmutagenic features. This work confirms that mutagenicity testscarried out on different organisms can be a valuable means forstudying the mutagenic activity of complex mixtures introducedinto the environment.

Acknowledgment

The authors would like to thank Dr. Silvia Palladini of Bensos(Villanuova sul Clisi, Brescia, Italy) for her collaboration and forsupplying the new detergents tested in this work.

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