Encyclopedia of Color Science and Technology

2016 Edition
| Editors: Ming Ronnier Luo

Colorant, Environmental Aspects

Reference work entry
DOI: https://doi.org/10.1007/978-1-4419-8071-7_151



The environmental characteristics of colorants (dyes and pigments) encompass topics pertaining to the impact of such compounds on human health and the environment. Regarding human health, the concern is the potential for colorants to exhibit genotoxicity, namely, to cause adverse interactions between DNA and various compounds that produce a hereditable change in the cell or organism. Hereditable changes in humans include birth defects, carcinogenesis, teratogenesis, and other types of diseases. Mutations caused by molecular interactions with DNA are generally viewed as the first events in the onset of carcinogenesis. Consequently, screening methods have been developed to determine the mutagenic potential of colorants. Such tests include in vitro methods that use microorganisms (e.g., bacteria) or isolated tissues as substitutes for whole animal (in vivo) studies. The potential genotoxicity of dyes for various applications came to the forefront in the 1960s when it was found that azo dye manufacturing involving benzidine and 2-naphthylamine as precursors contributed to bladder cancers among plant workers. This outcome led to extensive testing of azo dyes and their aromatic amine precursors for mutagenic and/or carcinogenic potential. Regarding the environment, the key concern is the potential for colorants to harm aquatic life (plants or animals) or pollute drinking water. This topic became a matter of concern because as much as 15 % of the colorants produced can be lost during their manufacture and end-use application [1]. In the case of dyes, the principal source of losses is water-soluble colorants remaining in dyebaths following textile dyeing processes. Consequently, methods pertaining to the treatment of industrial wastewater prior to the release of effluents have been developed, along with pollution prevention methods, as key components of environmental stewardship.


The environmental properties of colorants are often determined by employing a battery of tests that are concerned principally with the potential for mutagenicity, carcinogenicity, and aquatic toxicity. An overview of progress in these areas is presented.


Some dyes are known to exhibit mutagenicity, and the most commonly used test for assessing mutagenicity potential is the reverse mutation assay employing specially engineered Salmonella bacteria. The standard Salmonella/mammalian microsome assay, often called the Ames test, is now the most widely used protocol as an initial screening test procedure for assessing the mutagenic potential of new experimental colorants. It was introduced in 1975 by Ames and co-workers [2], who observed that most mutagens were also carcinogens and that the extent of a compound’s mutation of DNA was related to its carcinogenic potential, due to the susceptibility of DNA to chemical mutagenesis. Nowadays, the correlation between mutagenic compounds in the Ames test and carcinogenicity seems to be >60 %.

The Ames test is an in vitro method that commonly uses one or more strains of Salmonella typhimurium. There are six strains of Salmonella typhimurium that are widely used in a mutagenicity test and are designed to detect different types of mutations involving colorants. TA98 and TA1538 are sensitive to frameshift mutagens, TA100 and TA1535 are used to detect base pair substitution mutations, and TA97 and TA1537 are used for base pair substitution and some frameshift mutations, which sometimes cannot be detected with the former strains. These strains cannot produce the amino acid histidine, an essential component for growth. Thus, the bacteria are unable to multiply unless a mutagen causes the proper type of reverse mutation in the histidine gene. Mutagenic activity can be measured quantitatively by simply counting the number of colonies present after incubating Salmonella bacteria with several doses of the test compound and other necessary test additives for a standard length of time. The change in the bacteria that permits growth is called a reverse mutation and the colonies that form are called revertant colonies. Generally, the test compound is considered mutagenic when the number of revertant colonies is more than twice that of the base count. Based on the number of the revertant colonies produced, the compound is characterized as a nonmutagen, a weak mutagen, or a strong mutagen. A solvent is added to facilitate adequate mixing, and an enzyme mixture may also be added to metabolize the test compound to enhance the sensitivity of the test, since bacteria do not have some of the enzymes present in mammalian tissues. The enzyme mixture is important because many compounds are mutagenic once they are metabolized in the liver and other tissues. An exogenous metabolic system, usually rat liver microsomal enzyme system, S9, is used in the Salmonella microsome assay.

Benzidine-based dyes such as CI Direct Black 38 (Fig. 1) are carcinogenic in several mammalian species. However, certain benzidine-based dyes (e.g., 1; CI Direct Red 28; Congo Red) are not mutagenic in the standard Salmonella microsome test. Consequently, the standard test was modified in 1982 to insure the liberation of the parent diamine (cf., 24; Fig. 2) and the maximum possible mutagenic activity in each of the benzidine-based azo dyes studied [3]. This is an important protocol for benzidine-based dyes and is known as the preincubation assay under reductive conditions, a test that employs hamster liver S9 which is believed to be richer in reductase enzymes.
Colorant, Environmental Aspects, Fig. 1

Molecular structure of CI Direct Black 38 (1)

Colorant, Environmental Aspects, Fig. 2

Reductase enzyme cleavage of CI Direct Red 28 liberating carcinogenic benzidine (4)

As a follow up to positive Ames/Prival outcomes, in vitro testing such as a gene mutation assay involving mammalian cells or the mouse lymphoma test, a chromosome aberration assay, has been recommended [4]. This approach complements the mutagenic evaluation of a compound providing a more accurate prediction of its mutagenic properties and carcinogenic potential.


Although a variety of synthetic colorants have been studied, the majority of our knowledge and major concerns in this area arises from the recognition of azo dye manufacturing involving benzidine and 2-naphthylamine as a source of bladder cancer in humans. This soon led to extensive evaluation of azo dyes and their precursors for carcinogenic potential. The volume of results from work in this area became so substantial and diverse that a concerted effort to digest it has been made [5]. A key goal was to determine (1) whether the production of tumors in animal studies was sufficient to designate a dye as a human carcinogen and (2) the reliability of the published literature. This inspired an assessment of published data on a group of 97 representative azo colorants associated with the dyestuffs industry, for the purpose of determining the scientific standing of the experimental work in this area and the potential human hazard associated with these compounds. This assessment contributed to a set of recommended guidelines for carcinogenicity testing that cover chemical purity of test compounds, number of animals tested and survival rates, study controls, dose levels, route of administration, duration of experiments, pathological considerations, number of species, and evidence of human carcinogenicity. Application of the guidelines to published data afforded six categories of animal carcinogens and noncarcinogens: (1) Class A – the colorant was tested in at least two species of animals, resulting in the generation of nonconflicting data, using a sufficient number of animals (25 per group) and having a sufficient number of survivors for about two-thirds their expected lifetime. Repeated injections and urinary bladder implantations should not be regarded as meaningful in the context of chemically induced neoplasia. For a positive response, a target organ was identified and the induced tumors diagnosed as malignant. (2) Class B – the colorant was tested according to Class A guidelines but meaningful data was generated for one species only. (3) Class C – testing of the colorant involved an insufficient number of test animals and/or too many premature deaths occurred for results to be conclusive. For a positive result or trend, an increase in the number of animals with malignant tumors was observed but a target organ not identified. (4) Class D – the colorant was tested in multiple animal assays, which individually did not permit a conclusion but taken together provide sufficient evidence for an opinion. (5) Class E1 – the colorant was tested in a method considered inappropriate for evaluation of chemical carcinogenicity, e.g., by bladder implantation or using too few animals. (6) Class E2 – there was insufficient data to permit a rational judgment regarding carcinogenicity.

Structure-activity relationships associated with azo dye carcinogenicity produced the groupings depicted in Figs. 3, 4, 5, 6, 7, and 8 [6]. Group 1 comprises carcinogenic azo dyes that are hydrophobic (lipophilic) colorants having a 4-aminophenylazo structure (5–8; Fig. 3) [7]. As illustrated in Fig. 4, the 4-amino group is susceptible to a series of metabolic steps leading to an electrophilic nitrenium ion (611) that can react with DNA. This illustration is important because it serves as a reminder that azo bond cleavage is not an absolute requirement for genotoxicity. Group 2 examples in Fig. 5 show that placement of the amino group ortho to the azo bond rather than para affords a nitrenium ion having an internal mechanism for deactivation (cf., 1215). Group 3 comprises azo dyes producing a carcinogenic aromatic amine upon cleavage of the azo bonds that are designated as carcinogenic, examples of which are shown in Fig. 6. In these examples, 2,4-dimethylaniline, 2,3,4-trimethylaniline, and benzidine are produced by cleavage of the azo bonds in 16–18. The properties of this dye grouping led to a ban on commercial products containing dyes derived from any of the family of 22 aromatic amines (now 24) known to be carcinogenic [8]. Similarly, dyes metabolized to benzidine are now classified by the IARC (International Agency for Research on Cancer) as belonging to Group 1 (carcinogenic to humans) [9, 10].
Colorant, Environmental Aspects, Fig. 3

Carcinogenic 4-aminophenylazo disperse dyes

Colorant, Environmental Aspects, Fig. 4

Metabolism of a 4-aminophenylazo dye leading to an electrophilic species

Colorant, Environmental Aspects, Fig. 5

Noncarcinogenic 2-aminoazo dyes (12–13) and metabolism leading to triazole (15) formation

Colorant, Environmental Aspects, Fig. 6

Azo dyes liberating a carcinogenic aromatic amine upon reductive cleavage

Colorant, Environmental Aspects, Fig. 7

Azo dyes liberating noncarcinogenic sulfonated amines upon reductive cleavage

Colorant, Environmental Aspects, Fig. 8

Examples of noncarcinogenic highly insoluble azo pigments

Group 4 azo dyes are metabolized to noncarcinogenic sulfonated aromatic amines and are regarded as safe to use (19–20). These structural types include FD&C Red 4 (19; Fig. 7), as well as FD&C Yellow 5 (Tartrazine, CI Food Yellow 4) and FD&C Yellow 6 (CI Food Yellow 3) [11]. Due to its benign nature, CI Food Black 2 was the first black dye used for ink-jet printing and remains the prototype for designing improved black dyes for this end-use area [12].

Insoluble organic pigments such as 21–22 characterize Group 5 colorants, which are noncarcinogenic (Fig. 8). They are not readily metabolized to their diamine precursors or absorbed into mammalian systems. The principal requirement for safety among organic pigments is the control of the concentration of unreacted (free) aromatic amine precursors in the commercial product [13].

Aquatic Toxicology

Aquatic toxicology is the study of the effects of chemicals such as colorants on aquatic organisms. The associated tests are used to measure the degree of response produced by exposure to various concentrations of dyes and pigments and may be conducted in the laboratory or field. Laboratory tests encompass four general methods: (1) static test (organisms are placed in a test chamber of static solution), (2) recirculation test (organisms are placed in a circulated test solution), (3) renewal test (organism is placed in a static test solution that is changed periodically), and (4) flow-through test (organisms are placed in a continuously flowing fresh test solution).

In the USA, the Clean Water Act (cf., Title 40 of the US Code of Federation Regulations 100–140, 400–470), which is administered by the Environmental Protection Agency (EPA), limits water pollution from industrial and public sources, stresses the importance of controlling toxic pollutants, and encourages investigations leading to waste-treatment technologies [13]. Similarly, the Office of Pollution Prevention and Toxics (OPPT) implements the Toxic Substances Control Act (TSCA) as part of its responsibility for evaluating and assessing the impact of new chemicals and chemicals with new uses to determine their potential risk to human health and the environment. Testing recommended includes acute and chronic toxicity for assessing environmental effects and bioconcentration, biodegradation, physicochemical properties, and transport/transformation studies for assessing environmental fate [14, 15]. Ecosystem-related toxicological testing includes acute and chronic toxicity tests employing species such as fish, Daphnia, earthworms, and green algae. The overall goal is to determine the potential for changes in the composition of plant or animal life, abnormal number of deaths of organisms (e.g., fish kills), reduction of reproductive success or the robustness of a species, alterations in the behavior of distribution of a species, and long-lasting or irreversible contamination of components of the physical environment (e.g., surface water). Specifically, acute toxicity tests are short-term tests designed to measure the effects of toxic agents on aquatic species during a short period of their life span. Acute toxicity tests evaluate effects on survival over a 24- to 96-h period. Chronic toxicity tests are designed to measure the effects of toxicants to aquatic species over a significant portion of the organism’s life cycle, typically one-tenth or more of the organism’s lifetime. Chronic studies evaluate the sublethal effects of toxicants on reproduction, growth, and behavior due to physiological and biochemical disruptions.

The most common static tests are performed with daphnids, mysid shrimp, amphipods, and fish (e.g., fathead minnow, zebrafish, and rainbow trout). Renewal tests are often used for life-cycle studies using Ceriodaphnia dubia (7 days) and Daphnia magna (21 days). Longer-term studies are usually performed with these tests. Flow-through tests are generally considered superior to static tests, as they are very efficient at retaining a higher-level of water quality, ensuring the health of the test organisms. Flow-through tests usually eliminate concerns related to ammonia buildup and dissolved oxygen usage as well as ensure that the toxicant concentration remains constant.

TL50 values (aqueous concentrations at which 50 % of the test organisms survive after a 96-h exposure) from a static bioassay using fathead minnow and a diverse group of commercial synthetic dyes were measured [16]. The most toxic dyes were Basic Violet 1 (Methyl Violet), with a TL50 of 0.047 mg/L, and Basic Green 4 (Malachite Green) at 0.12 mg/L, Disperse Blue 3 at 1 mg/L, Basic Yellow 71 at 3.2 mg/L, Basic Blue 3 at 4 mg/L, Acid Blue 113 at 4 mg/L, Basic Brown 4 at 5.6 mg/L, Mordant Black 11 at 6 mg/L, Acid Green 25 at 6.2 mg/L, and Acid Black 52 at 7 mg/L. These commercial products included 29 of the 47 dyes with TL50 >180 mg/L, many that were bisazo or trisazo dyes. Three dyes had TL50 values between 100 and 180 mg/L, and the remaining 14 had values <100 mg/L. These results are consistent with the frequent lack of correlation between dye structures leading to genotoxicity and those leading to aquatic toxicity. Whereas benzidine-based dyes such as CI Direct Black 38 (1) and Direct Blue 6 (18) are designated as carcinogenic, they did not exhibit acute toxicity when tested with aquatic organisms, along with their counterparts 23–24 – both of which are Cu complexes (Fig. 9). On the other hand, all of the cationic (basic) dyes and anthraquinone disperse and acid dyes (25–31; Fig. 10) were significantly, if not extremely, toxic in this assay. Interestingly, bisazo dye Acid Blue 113, unlike the bisazo direct dyes, and Cr-complexed dye Acid Black 52 and its unmetallized precursor, Mordant Black 11 (32–34; Fig. 11), were also significantly toxic. Water-insoluble vat dyes (35–37; Fig. 12) did not exhibit acute toxicity in this assay. In a study involving the sparingly water-soluble monoazo dye Disperse Red 1, aquatic toxicity was observed in a variety of animals, with Daphnia similis the most sensitive species in acute tests and Pseudokirchneriella subcapitata the most sensitive species in chronic tests [17]. Clearly, more studies are needed in this area in order to establish clear correlations between the molecular structures of colorants and their aquatic toxicity. While the picture is somewhat clearer regarding cationic dyes and vat dyes, it is much less so regarding sulfonated azo dyes.
Colorant, Environmental Aspects, Fig. 9

Examples of benzidine and benzidine congener-based dyes found negative in aquatic toxicity testing

Colorant, Environmental Aspects, Fig. 10

Examples of cationic, disperse, and acid dyes exhibiting aquatic toxicity

Colorant, Environmental Aspects, Fig. 11

Examples of bisazo and metal-complexed acid dyes exhibiting aquatic toxicity

Colorant, Environmental Aspects, Fig. 12

Water-insoluble vat dyes found negative in aquatic toxicity testing

Bioconcentration studies are performed to evaluate the potential for a chemical to accumulate in aquatic organisms that may subsequently be consumed by higher trophic-level organisms including humans. The extent to which a chemical is concentrated in tissue above the level in water is referred to as the bioconcentration factor (BCF). It is widely accepted that the octanol/water partition coefficient (e.g., Log Kow or Log P) can be used to estimate the potential for nonionizable organic chemicals to bioconcentrate in aquatic organisms.

With regard to water quality concerns, a variety of methods have been developed to treat wastewater prior to industrial discharges, in order to remove colorants that are clearly visible at very low levels (e.g., 1 ppm). The methods employed involve chemical and biological agents for reduction and oxidation, physical adsorption, membrane filtration, precipitation, and recycle and reuse. The latter methods are especially important since dyes are engineered to be stable compounds under end-use conditions and thus are completely decomposed with considerable difficulty. Also, care must be used when choosing a wastewater decoloration method. In several countries, effluents from biological treatment are decolorized using chlorine for simultaneous pathogen removal. It has been found, however, that the chlorination of wastewater containing certain disperse dyes can generate mutagenic compounds known as phenyl benzotriazoles (PBTAs; 38). These chlorinated compounds have been observed in drinking water. Similarly, the chlorination of commercial Disperse Red 1 (39; Fig. 13) produced higher mutagenicity than the untreated dye.
Colorant, Environmental Aspects, Fig. 13

Structures of compounds 38–39

Future Directions

Bearing in mind that organic colorants include naturally occurring and synthetic compounds and that the major concern generally lies with the latter types, there is renewed interest in exploring natural dye-based coloration because such dyes are widely regarded as biodegradable and of low inherent toxicity. In fact, various natural dyes are part of a normal diet (e.g., beta-carotene and lycopene, both of which are important antioxidants). Although few natural colorants have shown commercial viability (e.g., indigo, madder, logwood), they continue to find success in niche areas such as arts and crafts and enjoy exploration for potential use in mainstream products. It is worthwhile pointing out that the use of natural dyes as colorants for textiles often introduces the need for additives known as mordants, to help bond these dyes to fibers, since natural dyes have very low affinity for textiles. Mordants such as Cu and Cr are toxic to aquatic life and pose human health risks, making Al and Fe better choices. Regarding synthetic colorants, which encompass water- and/or solvent-soluble organic dyes and insoluble organic and inorganic pigments, they will continue to be the primary agents for adding color to substrates such as natural and synthetic fibers, paper, plastics, petroleum products, printing inks, hair, and food, drug, and cosmetic products. Due to their practically insoluble nature, pigments have very low bioavailability; hence much of the attention in the area of environmental concerns has fallen on synthetic dyes. Among the synthetic dyes, azo dyes lie at the forefront in this area, and the design of such dyes will continue to take into consideration the environmental properties of the required precursors and the metabolites formed in mammalian systems.



  1. 1.
    Zollinger, H.: Color Chemistry: Synthesis, Properties, and Applications of Organic Dyes and Pigments, “Analysis, Ecology, and Toxicology of Colorants”. Wiley-VCH, Switzerland (2003)Google Scholar
  2. 2.
    Maron, D.M., Ames, B.N.: Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173 (1983)CrossRefGoogle Scholar
  3. 3.
    Prival, M.J., Mitchell, V.D.: Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster. Mutat. Res. 97, 103 (1982)CrossRefGoogle Scholar
  4. 4.
    Hunger, K. (ed.): Industrial Dyes: Chemistry, Properties, and Applications, “Health and Safety Aspects”. Wiley-VCH, Germany (2003)Google Scholar
  5. 5.
    Longstaff, E.: An assessment and categorisation of the animal carcinogenicity data on selected dyestuffs and an extrapolation of those data to an evaluation of the relative carcinogenic risk to man. Dyes Pigments 4, 243–304 (1983)CrossRefGoogle Scholar
  6. 6.
    Gregory, P.: Azo dyes: structure-carcinogenicity relationships. Dyes Pigments 7, 45–56 (1986)CrossRefGoogle Scholar
  7. 7.
    Weisburger, E.K.: Cancer-causing chemicals. In: LaFond, R.E. (ed.) Cancer-The Outlaw Cell. American Chemical Society, Washington, DC (1978)Google Scholar
  8. 8.
    German ban on use of certain azo compounds in some consumer goods. Text. Chem. Color. 28(4), 11–13 (1996)Google Scholar
  9. 9.
    IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Aromatic Amines, Organic Dyes, and Related Exposures, vol. 99, 692 pp (2010)Google Scholar
  10. 10.
    Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Bouvard, V., Benbrahim-Tallaa, L., Cogliano, V.: Some aromatic amines, organic dyes, and related exposures. Lancet Oncol 9(4), 322–323 (2008)CrossRefGoogle Scholar
  11. 11.
    Marmion, D.M.: Handbook of U.S. Colorants: Foods, Drugs, Cosmetics, and Medical Devices. Wiley, New York (1991)Google Scholar
  12. 12.
    Carr, K.: Dyes for ink jet printing. In: Freeman, H.S., Peters, A.T. (eds.) Colorants for Non-textile Applications. Elsevier Science BV, Amsterdam (2000)Google Scholar
  13. 13.
    Code of Federal Regulations, Title 40, Protection of Environment, US EPA, amended (1972)Google Scholar
  14. 14.
    Fogle, H.C.: Regulatory toxicology: U.S. EPA/chemicals TSCA, In: M.J. Derelanko, M.A. Hollinger (eds.) Handbook of Toxicology, CRC Press, Boca Raton (1995)Google Scholar
  15. 15.
    Adams, W.J., Rowland, C.D.: Aquatic toxicology test methods. In: Hoffman, D.J., Rattner, B.A., Burton Jr., G.A., Cairns Jr., J. (eds.) Handbook of Ecotoxicology. Lewis Publishers (CRC Press), Boca Raton (2003)Google Scholar
  16. 16.
    Little, L.W., Lamb III, J.C.: Acute Toxicity of 46 Selected Dyes to the Fathead Minnow, Pimephales promelas. The University of North Carolina Wastewater Research Center, Department of Environmental Sciences and Engineering, Chapel Hill (1972)Google Scholar
  17. 17.
    Vacchi, F.I., Ribeiro, A.R., Umbuzeiro, G. A.: Ecotoxicity Evaluation of CI Disperse Red 1 Dye, SETAC North America 31st Annual Meeting. Portland, 7–11 Nov 2010Google Scholar

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Fiber and Polymer Science ProgramNorth Carolina State UniversityRaleighUSA