87
Effects of 46 Dyes on Population Growth of
Freshwater Green Alga Se/eksfrum capricornutum
By Joseph C. Greene, Oregon State University, Corvallis, and George L. Baughman, University of Georgia, Athens
A
nia,
quatic toxicity studies are gener-
ally conducted with fish or Daph-
but assessment of potential envi-
ronmental impact often requires data
from other organisms. Much of the lim-
ited data available on dyes comes from
studies with mosquito larvae,'-3 Para-
mecium a ~ r e l i aa, marine
~ dinoflagel-
late,5 duckweed6 and freshwater mi-
croorganisms.' However, many of the
dyes used for these studies are com-
mercially unimportant today."' Also,
most of these were motivated
by interest in phototoxicity-a mecha-
nism that has recently been implicated
for dyes in wastewater.O
In the most comprehensive study to
date, Little and Chillingworth studied
the toxicity of 56 dyes to the green alga,
Selenastrum capricorn u t u m g Their
study included acid, basic, vat, dis-
perse and direct dyes. It included no
fiber reactive dyes: only four metal
complex-Le., premetallized-dyes
and few dyes of recent commercializa-
tion. In a more recent study,
Dolezalova and Solta examined the ef-
fect of 30 dyes on the green alga
Scenedesmus quadricauda and on
ABSTRACT
Questions about the toxicity of textile
mill discharges to plant life in
receiving waters led to an evaluation
of the toxicity of 46 dyes. Toxicity
assessment was performed using the
standardized 96-hour Selenastrum
capricornutum chronic toxicity test at
1 mg/L of the active colorant in the
dyes. All except two of the dyes
examined have anionic colorants,
many of which are reactive andlor
metal complexes. Only the two
cationic dyes demonstrated toxicity.
The two cationic dyes resulted in
EC,, concentrations of 0.025 and
0.247 mg/L of colorant.
KEY TERMS
Dyes
Green Algae
Selenastrum capricornutum
Toxicitv
April 7996 or)
Tubifex tubifex worms.IO Although
their study included dyes from the di-
rect, vat, acid and fiber reactive classes,
no structures were given. Burton et al.
have since examined the effect of two
solvent dyes on S. capricornutum."
Thus, fiber reactive and metallized
dyes are either absent from previous
studies andfor they are represented by
dyes of unstated colorant; i.e., molecu-
lar structure.
Interest in the toxicities of reactive
and/or premetallized dyes is prompted
by two primary considerations. First,
fiber reactive dyes are the only growth
area in dye manufacturing, but more
significantly, the chemical structures
of their colorants lead to obvious ques-
tions about their environmental behav-
ior. This results from the fact that only
fiber reactive dyes have colorants that
bind to the substrate through formation
of covalent bonds and also because, in
many, the reactive moiety is a
chlorotriazine. The latter naturally
raises questions about potential phy-
totoxicity due to structural similarity
with the important triazine herbicides.
Second, the presence of metals, par-
ticularly copper and chromium, in
dyes (i.e., premetallized dyes) causes
concern because of the well known
toxicities of simple metal salts. How-
ever, the dye and textile industries
have argued that the metals bound in
dye complexes are much less toxic
than free metals-i.e., ionic metals.
Thus there are regulatory implications
vis-a-vis the use of water quality crite-
ria, based on total metal concentration,
to establish effluent limitations for
metals that are bound in dye com-
plexes. Interestingly, the only reported
case of metal toxicity from a textile
3ffluent was found to be due to zinc
that was not from a dye.12 Also, it
should be noted that designation of a
lye as fiber reactive or premetallized
s not mutually exclusive since some
if the former are metal complexes.
In common usage, the term dye is
requently used to mean both a com-
nercially formulated product that con-
ains several components such as de-
tergents, salts, oils, etc., as well as the
actual colorant molecule(s). A pre-
metallized dye is one in which the
colorant is a metal complex, whereas
a reactive dye has a colorant that forms
a covalent bond to the substrate. These
terms will be used only in the mutu-
ally exclusive sense here.
Attempts to develop environmen-
tally relevant data for dyes are con-
fronted with difficulties that are
unparalleled elsewhere in the study of
industrial chemicals. Some of the
major problems are:
Only about one half of all textile
dyes in commerce have colorant
structures that are both known and
*
available (in the public domain), and
the fraction of structures available is
even much less for the colorants
most recently developed.
Colorants are notoriously difficult to
purify and few are available except
as commercial formulations.
Although the majority of colorants
are ionic azo or anthraquinone com-
pounds, colorants span a vast range
of structures (including many metal
complexes), solubilities, and reac-
tivities.
A dye often contains several colorant
molecules.
Information on colorant structure
and usage is largely confined to the
dye and textile industries and is not
freely given out. .
In the U.S., dyes currently comprise
about 5000 commercial formulations
based on approximately 1000
colorants. Consequently, it is not
surprising that little data is available
concerning the toxicity of dyes or
colorants to aquatic species, espe-
cially if fish and waste treatment
microorganisms are excluded.
Given that background, the stan-
dard reference test alga S. capricornu-
turn, was chosen to use in toxicity tests
with some commercially available tex-
tile dyes.
Unicellular algae have been exten-
sively used for toxicity tests and are
very sensitive. Their importance in
oxygen evolution and as primary pro-
Textile Chemist and Colorist 23
 I
ducers makes them suitable environ- complexes. Colorant chromophores colorant. One mL of dye stock solution
mental indicator organisms. S. capri- include phthalocyanine, formazan, (nominal 50 mg/L of colorant) was
cornutum is the green alga of choice anthraquinone, stilbene and oxazine. added to 50 mL of algal assay medium
because it has been demonstrated to be Each colorant comprised at least 10% in 125-mL Erlenmeyer flasks. S.
highly sensitive to toxicants found in of the dye unless otherwise noted. capricornutum in continuous culture
complex chemical mixtures and to Because of the importance of relat- provided the initial inoculum (10,000
metals.13Also, the alga provides infor- ing toxicity to compounds of known algal cells/mL). The algal cells were
mation on herbicidal activity of the structure, the structures of all colorants incubated in the solutions for 96 hours.
chlorotriazine reactive dyes. Finally, are given in the Appendix. However, The diluent and negative control
the 96-hour static chronic test is it is important to note that the struc- were algal assay medium (AAM). AAM
simple, rapid and cost effective. tures are nominal since they do not was prepared by adding 1 mL from
Two basic logistical considerations reflect the complex distribution of iso- each of five stock solutions to 900 mL
determined the choice of dyes and the mers that almost certainly exists in of deionized water. After spiking, the
study conditions. First, the large num- most dyes-especially if the colorants total volume was brought to 1liter with
ber of dyes in commerce dictated that are premetallized. For example, even deionized water. The AAM pH was
a manageable subset be examined. In simple complexes like Colorant 20 (see adjusted to fall within the range of 7.0-
order to provide scientific credibility, Appendix) have more than 20 possible 7.5 m d stored in a refrigerator, in the
the dyes were limited to those for isomers and many, if not most, are dark, at 4C until used.
which we could obtain structures of probably present in any sample of dye Population growth of S. capri-
the colorant molecules. Fulfillment of containing the colorant. cornutum was used to establish the
these criteria resulted in the selection Examination of Table I shows that potential for toxicity or growth stimu-
and testing of 46 commercially formu- colorants 2, 18, 20, 44, 46, 47 and 49 lation. If the dye inhibited algal popu-
lated textile dyes. The decision to use all appear in two dyes. Dyes 40 and 25 lation growth by 50% of that found in
commercial formulations was based on contain two and five colorants, respec- the negative controls, a definitive test
four considerations: tively. However, the dyes were differ- using several dilutions of the dye was
Purified single colorant compounds ent formulations. performed to allow for determination
were not available and purification Two separate groups of commercial of an EC5,, concentration. Algal popu-
was prohibitively expensive. dye samples were obtained along with lations were measured, after 96 hours
Some of the formulations were mix- the concentration of colorant but with- of exposure, using an electronic par-
tures of colorant compounds. out structures. On receipt, dyes were ticle counter fitted with a mean cell
Data on the premetalized and fiber numbered and corresponding stock volume computer. The Trimmed
reactive dyes were needed. solutions were prepared. Immediately Spearman-Karber Method of Hamilton
Components other than the colorant after preparation, stock solutions were et al., was used to calculate EC50effect
were also of interest if they are toxic. shipped by overnight mail to Oregon concentrations and the associated 95%
A nominal colorant concentration State University (OSU) where tdxicity confidence limits.15Dunnett's test was
of 1mg/L was chosen as a compromise tests were initiated on each group performed on all results that varied
between the need for a concentration within 24 hours. Structures of the from the control by &!O%. This
as high or higher than is likely to be colorants, which were obtained later method of statistical analysis was used
found in the environment, and a con- from the manufacturer, were not to compare the mean population
centration low enough to minimize the known to OSU scientists until all tox- growth of controls to each of the test
effect on the alga of light screening by icity results were completed and re- group means to determine statistical
the dye. sults analyzed. differences.
Materials and Methods Toxicity Testing Quality Assurance
Algal chronic toxicity tests were per- Quality control/quality assurance pro-
Dye Selection formed according to the method of cedures were adhered to during per-
Forty-six dyes (comprising 53 color- Greene et al.I4 Three replicates were formance of this study. Performance
ants) were provided by the manufac- prepared for each dye at a nominal criteria were used to assess the quality
turer as commercial samples in the concentration of 1mg/L for the active of the tests performed.
form (powders or liquids) usually dis-
tributed to the textile industry. They
were provided on the condition that Table 1. Dye No. and No. of Associated Colorant Structure(s) in Appendix
colorant structures not be related to Dye Dye Dye
commercial names. Thus, Table I re- No. Colorants in Dye No. Colorants in Dye No. Colorants in Dye
lates a number assigned to the commer- 1 36 17 26 33 44,45
cial dye with the number of its respec- 2 49 18 42 34 31
tive coiorant structure(s) listed in the 3 38 19 18 35 4
Appendix. The C.I. numbers are not 4 39 20 1516 36 5
5 46 21 18 37 35
available. 6 27 22 19 38 9
Except for two cations, the colorants 7 37 23 25 39 21
(Appendix)are all anions, of which 27 8 47,49 24 7 40 22,23,24
are reactive ( 3 bromoacrylates, 7 9 34 25 1 a,26,12,13,14 41 3
10 43,44 26 2 42 41
chlorotriazines, 7 fluorotriazines and 11 33 27 51,52,53 43 17
10 vinylsulfones). Eleven of the reac- 12 40 28 10 44 50
tive colorants are bifunctional, one is 13 28 29 8 45 11
trifunctional and five are copper com- 14 41 30 32 46 20
15 29 31 48
plexes. Most of the colorants are azo 16 30 32 6,20
compounds, 19 of which are metal (5
copper, 2 cobalt and 12 chromium) Component comprises 0.4%. bComponent comprises 4.0%.

24 Textile Chemist and Colorist QJ) Vol. 28, No. 4

 ~

Positive control tests, using zinc, Table II. Average Yield (mglL dry weight S.capricornulum)and 95% Confidence Limits for 96-Hour
were performed to demonstrate that Algal Static Chronic Toxicity Tests
the sensitivity of S. capricornutum was Lower Lower
Upper Upper
within established norms. The con-
Dye 95% 95% Percent Dye 95% 95% Percent
trolled laboratory experiments, per- No. Avg. C.L. C.L. Effect No. Avg. C.L. C.L. Effect
formed to determine the toxicity of
zinc to the test alga, resulted in ECS0 Control 29.0 26.1 31.9 -___- 24 35.7 32.5 39.0 ,+23.3
1 32.5 24.6 40.4 +12.1 25 35.0 31.7 38.4 +20.8
concentrations within the expected 2 27.0 22.3 31.a -6.7 26 35.5 29.5 41.4 +22.4
range. 3 35.4 30.4 40.3 +22.1 27 35.4 31.3 39.5 c22.0
A negative control, composed of 4 36.6 34.9 38.4 ~26.4 28 0.01 0.00 0.02 -100.0
algal assay medium, was performed 5 34.0 29.6 38.5 +I73 Control 27.5 23.2 31.8 -----
6 27.8 23.7 31.9 -4.1 29 26.2 21.6 30.8 -4.8
with each test series to provide a mea- 7 27.0 21.7 34.0 -3.8 30 28.2 19.8 36.6 +2.5
sure of the health 0f.S.capricornutum. a 32.a 25.1 40.4 +13.1 31 31.3 26.8 35.8 +13.7
During the 96-hour static chronic tox- 9 34.1 30.0 38.1 +17.6 32 25.6 24.2 27.0 -7.0
10 31.5 29.9 33.1 +a.8 33 19.8 12.2 27.4 -28.2
icity test, the algal cells in the control 11 22.3 20.2 24.3 -23.1 34 26.6 18.4 34.8 -3.4
flasks must produce a population of 12 28.8 24.5 37.5 -0.4 35 26.8 21.6 32.0 -2.5
->1,000,000 cells/mL to achieve the 13 29.5 27.1 31.4 +0.9 36 26.8 18.9 34.8 -2.5
acceptability criterion. The negative 14 29.6 24.8 34.3 +2.0 37 27.5 25.3 29.7 0.0
15 24.1 22.4 25.7 -16.9 38 30.6 25.3 35.8 +11.1
control was also important because re- 16 21.1 14.4 27.9 -27.1 39 28.8 27.0 30.7 +4.8
sults were used in the calculation of 17 25.8 18.3 33.2 -11.1 40 26.8 20.6 33.0 -2.6
all toxic or stimulatory effects within 18 28.7 23.9 33.5 -0.9 41 0.79 0.65 0.93 -97.1
each test. Effect calculations were 19 30.4 20.2 40.6 +5.0 42 23.3 19.6 27.0 -15.3
based upon the difference between the 20 28.8 22.0 35.6 -0.7 43 20.8 12.6 29.2 -24.3
21 29.8 24.0 35.5 ~2.8 44 25.6 22.0 29.3 -6.9
mean biomass of each exposed popu- 22 32.3 26.4 38.2 +11.5 45 29.6 27.1 32.0 +7.4
lation and the mean biomass of the 23 36.4 33.1 39.6 +25.4 46 25.1 20.8 29.6 -8.7
negative control.
Temperature was continuously
monitored throughout the testing pe- dium with 0.0,0.0001,0.001,0.01,0.1 large molecules may be limiting since
riod using thermometers with record- and 1.0 mg/L of the colorants. Evalua- the molecular weight of the complexes
ing charts. tion of the test results, using Dunnett’s is greater than 500.
test, indicated that the dyes caused a Other factors may be either a slow
Results and Discussion population growth that was statisti- rate of release of bioavailable metal
Table I1 lists the average yield, and cally less than that produced in the from the complexes (ligand exchange
95% confidence limits, of algal biom- controls. A Trimmed Spearman-Karber rate) or thermodynamic stability. Many
ass (mg/L) produced in control and test analysis of the test data resulted in an Cr, and to a lesser extent Go, complexes
cultures. Data for the percent effect ECS0 concentration of 0.025 mg/L for are known to undergo slower release
each dye demonstrated upon algal dye 28.j4 The lower and upper 95% of ionic metal (ligand exchange) than
population growth are also included. confidence limits for the analysis were Cu complexes. The release of Cu from
Percent effect is either negative for 0.020 mg/L and 0.031 mg/L, respec- dyes was found by Hill et al.z6to take
population growth inhibition or posi- tively. Similarly, dye 41 resulted in an up to 1 2 hours while Cr release was
tive for population growth stimulation. ECS0 concentration of 0.247 mg/L for much lower.'^ It probably is signifi-
the colorant with 95% confidence lim- cant that the Cu dyes in this study all
Basic Dyes its ranging k o a 0.161 to 0.230 mg/L. have phthalocyanine (dyes 2,8 and 44)
Seven basic textile dyes stimulated The high toxicity of dyes 28 and 41 is or formazan (dyes 14 and 34) colorants
growth by 220% of that found in the entirely consistent with the known tox- that are less likely to undergo ligand
control cultures. Dyes 3, 4, 23, 24, 25, icity of basic; i.e., cationic, dyes to both exchange than the copperized azo di-
26 and 27 stimulated population S. capricornutum and fish.g rect dyes.
growth from 20.8-26.4%. Statistical Although the cationic dyes are very In the absence of equilibrium con-
analysis using Dunnett’s test demon- toxic, it is difficult to assess their be- stants, it is not possible to rule out the
strated, however, that the enhanced havior in natural waters. Cationic dyes possibility that the complexes are sim-
yields were not statistically different can be expected to strongly adsorb to ply so stable that the alga cannot com-
from the controls. sediments which will reduce their pete with the ligand.-This is probably
Also,,dyes 11, 16, 28, 33, 41, and toxic effects. However, since sorption the case with the phthalocyanines and
43 inhibited population growth by coefficients are not available, the mag- perhaps for the other studied com-
>20%. Statistical analysis using the nitude of their impact on aquatic sys- plexes.
Dunnett’s test showed that only dyes tems cannot be estimated. Little can be said with confidence
16, 28 and 41 produced algal popula- about the Co complexes or about the
tions that were significantly different Metal Complex Colorants Cu azo compounds because of lack of
from the control population. However, Importantly, none of the 19 metal com- data.
the 27.1% inhibition caused by dye 16, plex colorants were toxic. A study with For any of the dyes, absence of tox-
is considered low toxicity. On the other the same alga included five metal com- icity in the 1 mg/L test is significant
hand, the 100 and 97.1% inhibition plex dyes (three copper, one chromium since usual biological waste treatment
observed with dyes 28 and 41, respec- and one cobalt) and the only inhibi- is likely to result in concentrations that
tively, was significant and required tion reported (30% after seven days at are much lower than 1 mg/l for any
further testing. 1 mg/L) was due to one of two cop- individual dye. This, in turn, suggests
Toxicities of dyes 28 and 41 were perized azo dyes.g that many of the important colorants
Further defined by the performance of Again, the reasons for absence of used in reactive and/or metallized dyes
3 definitive dose/response test, which toxicity are not clear. As with the re- may not lead to environmental toxic-
2onsisted of spiking algal assay me- active dyes, bioavailability of such ity at concentrations expected in re-

April 7996 (xx) Textile Chemist and Colorist 25


ceiving waters. However, since the pH
of the receiving (or test) water strongly
influences the equilibrium and rate of
ligand exchange, toxicity may be a
strong function of pH.
O'Neal and Boyter reported that
most of the toxicity of Cu-dye contain-
ing textile effluents was due to free
metal that could be separated from the
complexes by ultrafiltration or ion ex-
change.l8 This can only be observed if
the thermodynamics and kinetics favor
the complexes rather than free metal.
Unfortunately, studies of mill effluents
have contained little or no data on the
nature of metallized dyes in the waste-
water.
Reactive Dyes
None of the reactive dyes exhibited
toxicity. This is interesting since this
group has 23 colorants that contain the
halotriazine moiety. O'Kelley and
Deason have reported that atrazine, a
chlorotriazine pesticide, was toxic to
26 of 36 algal isolates at the 0.001 mgl
L level.zgRelevance of the test results
to aquatic systems is based on the as-
sumption that the reactive (unhydro-
lyzed) forms were not hydrolyzed in
the test systems more rapidly than they
would be in the environment. Al-
though some hydrolysis of the reactive
groups is expected, under the mild test
conditions, the rate must be low com-
pared with that of dyeing operations
that always require high pH. Also, ab-
sence of phytotoxicity could be due to
inability of the large colorant mol-
ecules to cross cell membranes (i.e.,
lack of bioavailability).
Summary and Conclusions
As might be expected, the two basic
dyes, numbers 28 and 41, were highly
toxic to the test alga S. capricornutum.
The median ECS0effect concentrations
were 0.025 and 0.247 mg1L active dye
colorant, respectively.
Data show the other dyes to be non-
toxic to the test alga at concentrations
well above levels expected in the en-
vironment. Results for the metallized
dyes are consistent with the view that
under test conditions, the metals are
either not toxic or not available to the
alga. The test results can be interpreted
as suggesting that there would be little
chronic toxicity on release of such
compounds to natural waters at pHs
similar to the test condition. A more
definitive assessment of the environ-
mental toxicity of metallized dyes
must await information on the effects
of pH on the equilibria and kinetics of
ligand exchange. Based on their known
stability, the longer term fate of the
anionic dyes in general, is probably
strongly affected by photochemical
processes.
26 Textile Chemist and Colorist
The data show no evidence of algal
toxicity caused by the reactive dyes;
i.e., due to the chlorotriazine, fluoro-
triazine, bromoacrylate and vinyl sul-
fone reactive groups. In the absence of
other data, there is little reason to ex-
pect chronic phytotoxicity from these
compounds at levels that might be ex-
pected in the environment.
References
1. Barbosa. P. and T. M. Peters, Journal of Medi-
cal Entemology., Vol. 7, 1970, p693.
2. Carpenter, T. L., N. C. Respico and J.R. Heitz,
Environmental Entomology, Vol. 13, 1984,
~1366.
3. Pimprikar, G. D. et al., Southwestern Ento-
mologist, Vol. 9, 1984, p218.
4. Prakash, C. J. and R. B. Misra, Biochemical and
Biophysical Research Communication, Vol.
139,1986, p79.
5. Martin, B. B. and D. F. Martin, Journal of En-
vironmental Science and Health, Vol. A23,
1988, p757.
6. Martin, D. F. and C. D. Norris, JournalofEnvi-
ronmental ScienceandHealth, Vol. A23,1988,
p765.
7. Michaels, G. B. and D. L. Lewis, Environmen-
tal ToxicologyandChemistry,Vol. 4,1985, p45.
8. Tratnyek, P. G., M. S. Elovitz and P. Colverson.
Environmental Toxicology and Chemistry,
Vol. 13, 1994, p27.
9. Little, L. W. and M. A. Chillingworth, Dyes
and the Environment-Reports on Selected
Dyes and Their Effects, Vol. 11, Chapter 2,
Appendix: Structures of Colorants in the Tested Dyes
Colorant 1
Colorant 3
Three compoundswith X =OH and/or NHCH3
CH3 CH3S04'
Colorant 4
H&
, -03s'
American Dye Manufacturers Institute Inc.,
September 1974.
10. Dolezalova, L. and F. Solta, Vodni Hospo-
darstvi. B, Vol. 35, 1985, p49.
11. Burton, D. T., D. J. Fisher and R. L. Paulson,
Chemosphere, Vol. 19,1989, p1959.
12. Wells, M. J. M., A. J. Rossano Jr. and E. C. Rob-
erts, Archives of Environmental Contamina-
tion and Toxicolgy, Vol. 27, 1994, p555.
13. Miller, W. E. et al., Jourflal of Environmental
Quality, Vol. 4, 1985, p569.
14. Greene, J. C. et al., Protocols for Short Term
Toxicity Screening of Hazardous Waste Sites,
Report No. EPA 60013-88-029, U.S. Environ-
mental Protection Agency, Corvallis, Ore.,
1988.
15. Hamilton, M. A., R. C. Russo and R. V.
Thurston, Environmental Science Technology,
Voi. 11,1983, p714.
16. Hill, W. E., W. S. Perkins and G. S. Sandlin.
-Textile Chemist and Colorist, Vol. 25, 1993,
p26.
17. Hill, W. E., personal communication.
18. O'Neal, W. G. and H. A. Boyter Jr., Proceed-
ings, Water Environment Federation, 67th
Annual Conference and Exposition, Chicago,
1994, p567.
19. O'Kelley, J . C. and T. R. Deason, Degradation
of Pesticides by Algae. Report No. EPA-600/
3-76-022, U.S. Environmental Protection
Agency, Athens, Ga., 1976.
Author's Address
George L. Baughman, University of
Georgia, Dawson Hall, Athens, Ga.
30602; telephone 706-542-4883.
p
Colorant 5
2Na+
Vol. 28, No. 4
 Colorant 12

Colorant 14
Colorant 15

0; Na+
Colorant 13

0 NH

N=N

Na+

Colorant 16

Na+
Colorant 17 H3C.
+ Colorant 18

0 YH

o\ NI L z H 5
H
H3d
Na+ SO;

April 7996 co3 Textile Chemist and Colorist 27

J
Colorant 19 Colorant 20 -
Colorant 21

< Na+ 3Na+

WO' H \ NO2
02

-03S

Colorant 22

Na+ do2

F
Colorant 25 CI
Colorant 26

4Na+ ci

Na+

28 Textile Chemist and Colorist Vol. 28, No. 4

 Colorant 31 F

I I
H H

Colorant 33

Colorant 35 3;
,
H3c
Colorant 36

Colorant 37 CI
Colorant 38 A N

8-
Na+
Na+

' ,
&p
d
so3- -
/

''
H \ k N A Y
/ H3C'CH2

so3'
'
Na+
03s

April 1996 Textile Chemist and Colorist 29

,
Colorant 42 Na+ Colorant 43
Na+

Na+
Na+ -03s
H H H

Na+ Na+ Na+ Na+ Na+ Na+

Colorant 47
Rings a, b, c, d contain one of
the following substituents:
Colorant 48 .
-S03-Na+ (two total)
Na+
-S02NH2
H H -03S
SO3 H

NH2

Colorant 49
Rings a, b, c, d contain one of Colorant 50
the following substituents:
Rings a, b, c, d contain one of
-S03-Na+ (two total) the following substituents:
6 0 3 - Na+

Colorant 51
Colorant 52 Colorant 53 09N

2Na+

0
O2N
so3

30 Textile Chemist and Colorist a Vol. 28, No. 4