effects of 46 dyes on population growth of freshwater green alga se-eksfrum capricornutum

8/2/2019 Effects of 46 Dyes on Population Growth of Freshwater Green Alga Se-Eksfrum Capricornutum

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Effects of 46 Dyes on Population Growth ofFreshwater Green Alga Se/eksfrum capricornutumBy Joseph C. Greene,Oregon State University, Corvallis, an d George L. B a u g h m a n , University of Georgia, Athens

qua tic toxicity s tudies are gener-A l ly con ducted w ith fish or Daph-n ia , but assessment of potential envi-ronmental impact often requires datafrom other organ isms. M uch of the lim-ited d ata available on d yes comes fromstudies with mosq uito larvae,' -3 Para-m ec iu m a ~ r e l i a , ~marine dinoflagel-late,5 duckw eed6 an d freshwater m i-croorganisms.' However, man y of thedyes used for these studies are com-er cially unim porta nt today."' Also,ost of these were motivatedinterest in phototoxicity-a mecha-that has recently been implicatedor dy es in wastewater.OIn the most com prehensive s tudy toe, Little and Chillingworth stud iede toxicity of 56 dyes to the green alga,capr icornu t u m g Theirincluded acid, basic, vat , dis-

d few dyes of recent commercializa-ova an d Solta examin ed the ef-30 dyes on the green a lgau s q u a dr i ca u d a a n d o n

toxicity of textilevaluationthe toxicity of 46 dyes. Toxicityperformed using theSelenastrumchronic toxicity test atmg/L of the active colorant in theAll except two of the dyes

strated toxicity.,, con cen tratio ns of 0.025 an dmg/L of colorant.

TERMSDyesGreen AlgaeSelenastrum capricornutumToxicitv

T u b i f e x t u b i f e x worms. IO A l thoughthei r s tudy included dyes from the di -rect, vat, acid an d fiber reactive classes,no structures were given. Burton et al .have sinc e exam ined the effect of twosolvent dyes on S. capricornutum."Thus, f iber react ive and metal l izeddyes are e ither absen t from previousstudies andfor they are represented bydyes of unstated colorant; i.e., molecu-lar structure.Interest in the to xicities of reactiveand/o r premetallized dyes is promptedby two primary considerations. First ,fiber reactiv e dyes are the o nly growtharea in dye manufacturing, but moresignificantly, the chemical structuresof their colorants lead to obvious ques-tions about their enviro nm ental behav-ior. This results from th e fact that onlyfiber reactive dyes h ave colorants thatbind to the substrate through formationof covalent bo nds an d also because, inmany, the reac t ive moie ty i s ach loro t r i az ine . T he l a t te r na tura l lyraises questions about potential phy-totoxicity due to structural similaritywith the important triazine herbicides.Sec ond , the pre senc e of metals, par-t i cu lar ly copper and chromium, indyes (i.e., premetallized dyes) causesconcern because of the wel l knowntoxicities of simp le metal salts. How-ever, t he d ye an d t ex t il e i ndus t r i eshave argued that the metals bound indye complexes a re much l ess tox icthan free metals-i.e. , ion ic metals.Thus there are regulatory implicationsvis-a-vis the us e of wa ter qu ality crite-ria, based on total metal conc entra tion,to establ ish effluent l imi tat ions formeta l s t ha t a re bound in dye com-plexes. Interestingly, the only reportedcase of metal toxicity from a texti le3ffluent wa s found to be due to zincthat wa s no t from a dye.12 Also, i tshould be n oted tha t de signation of aly e as fiber reactive or premetall izeds not mutual ly exclusive since someif the former are meta l complexes.In common usage, the term dye isrequently used to mean both a com-nercially form ulated prod uct that con-ains several compo nents such as de-

tergents, salts, oils, etc., as well as th eactual colorant molecu le(s) . A p re-metal l ized dye is one in which thecolorant is a metal complex , whe reasa reactive dye has a colorant tha t formsa covalent bon d to the substrate. Thes eterms will be used only in the mutu-ally exclusive sense here.Attempts to develop environmen-tally relevant data for dyes are con-f ron t ed w i t h d i f f i cu l t i e s t ha t a r eunparalleled elsewhere in the s tud y ofindus t r i a l chemica l s . Some of themajor problems are:Only about one half of all texti le

dyes in commerce have co loran tstructures that are both kno wn a ndavailable (in the pu blic dom ain), and* the fraction of struc tures av ailable iseven much l ess for the co loran t smost recently developed.Colorants are notorious ly d ifficult topurify and few are available exceptas comm ercial formulations.Although the majority of colorantsare ionic azo or anthraquinone com-pounds, colorants spa n a vast rangeof st ructures ( including many metalcomplexes), solubi l i ties , and reac-tivities.A dye often con tains several colorantmolecules.Informat ion on colorant s t ructureand usage is largely confined to thedye and texti le indus tries and is notfreely given o ut. .In the U.S., dyes cu rren tly comp riseabout 5000 commercial formulationsbased on approximate ly 1000colorants. Con sequent ly, it i s notsurprising that little data is availableconcerning the toxicity of dyes orcolorants to aquatic s pecies , espe -cially if f ish a nd was te t rea tmentmicroorganisms are excluded.Given that backgroun d, the stan-dard reference test alga S. capricornu-turn,was chosen to u se in toxicity testswith some com mercially available tex-tile dyes.Unicellular algae have been exten-sively used for toxicity tests and arevery sensi t ive. Their importance inoxygen evolution and as primary pro-

7996 or) Textile Chemistand Colorist 23


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ducers makes them suitable environ-me ntal indicator organisms. S. capri-cornutum is the green alga of choicebecause it has been demo nstrated to behighly sensitive to toxicants found inc o m p l e x c h e m i c a l m i x t u r e s a n d t ometals.13 Also, th e alga provides infor-mation on herbicidal activity of thechlorotriazine reactive dyes. Finally,t h e 9 6 - h o u r s t a t i c c h r o n i c t e s t i ss imple , rapid and cost effective.Two basic logistical cons idera tionsdetermined th e choice of dyes and thestudy co nditions. First , the large nu m-ber of dyes in com merce dictated thata manageable subse t be exam ined. Inorder to pro vide scientific credibility,t h e d y e s w e r e l i m i t e d t o t h o s e f o rwhich we could obtain s t ructures ofthe colorant molecules. Fulfillment ofthese criteria resulted in th e selectionan d testing of 46 commercial ly formu-lated textile d yes. The dec ision to usecomm ercial formulations was based onfour considerations:Purified single colorant com pou ndswere not available and purificationwas prohibitively expensive.Some of the fo rmulations were mix-tures of colorant compou nds.Data on the premetalized and fiberreactive dyes w ere needed.Components other than the colorantwere also of interest if they are toxic.A n ominal colorant concentra tionof 1mg/L was chosen as a compromisebetween the need for a concentrationas high or higher than is likely to befound in the environment , and a con-centration low en ough to minimize theeffect on th e alga of light screen ing bythe dye.

Materials and MethodsDye SelectionForty-six dyes (comprising 5 3 color-ants) were provided by the manufac-tu re r a s commerc ia l samples in theform (pow ders or liquids) usua lly dis-tributed to the textile industry. Theywere provided on the condi t ion thatcolorant s t ructures not be re la ted tocommercial names. Th us, Table I re-lates a num ber assigned to the commer-cial dye with the nu mb er of its respec-t ive coiorant s t ructure(s) is ted in theAppen dix. The C.I. numbers are notavailable.Except for two cations, the colorants(Append ix)are all anions, of wh ich 27are reac t ive ( 3 bromoacry la tes , 7chlorotriazines, 7 fluorotriazines and1 0 vinylsulfones). Eleven of th e reac-tive colorants are bifunctional, one istrifunctional and five are cop per com-plexes. Most of the colorants are azocompounds , 19 of which are metal (5copper , 2 cobal t and 12 c h r o m i u m )

complexes . Co loran t ch romo phoresi n c l u d e p h t h a l o c y a n i n e , f o r m a z a n ,anthraquinone, stilbene and oxazine.Each colorant comp rised at least 1 0%of the dye unless otherwise noted.Because of the im porta nce of relat-ing toxicity to compounds of knownstructure, the struc tures of a ll colorantsare given in the Ap pendix. However,it is important to note that the struc-tures are nominal s ince they do notreflect the com plex distr ibutio n of iso-mers that a lmost cer ta inly exis ts inmost dyes-especially if the colorantsare premetallized. For example, evensim ple complexes like Colorant 20 (seeAppe ndix) have more than 20 possiblei somers and many , if no t mos t , a reprobably present in an y sample of dyecontaining the colorant.Exam ination of Table I show s thatcolorants 2, 1 8 , 20, 44, 46, 47 and 49all appear in tw o dyes. Dyes 40 an d 25contain two and five co lorants, respec-tively. How ever, the dy es were differ-en t formulations.Two separate groups of commercialdye samples were obtained along withthe c oncentration of colorant but with -out structures. On receipt, dyes werenumbered and cor respond ing s tocksolutions were prepared. Immediatelyafter preparation, stock solutions wereshipp ed by ove rnight mail to OregonState University (OSU) wh ere tdxicityt e s ts w e r e i n i t i a t e d o n e a c h g r o u pw i t h i n 24 hours . S t ruc tu res o f thecolorants, which were obtained laterf r o m t h e m a n u f a c t u r e r , w e r e n o tknow n to OSU scientists until all tox-ic i ty resul ts w ere com pleted and re-sul ts analyzed.Toxicity TestingAlgal chronic toxicity tests were per-fo rmed accord ing to the method ofGreene et al.I4 Three replicates wereprepared for each dye a t a nominalconcentration of 1mg/L for the active

colorant. One mL of dye stock solution(nominal 50 mg/L of colorant) wasadded to 50 mL of algal assay med iumin 125-mL Er lenmeyer f l a sks . S.capricornutum in co nt inuous cul tureprovided the initial inoculu m (10,000algal cells/mL ). The algal c ells wereincubated in th e solutions for 96 hours.The di luent and negat ive controlwere algal assay mediu m (AAM).AMwas p repared by add ing 1 mL fromeach of five stock solutio ns to 900 mLof deionize d wa ter. After spikin g, thetotal volume was brought to 1 iter withdeionized water . The AAM pH wasadjusted to fall within the range of 7.0-7.5 m d stored in a refrigerator, in thedark, at 4C until u sed.P o p u l a t i o n g r o w t h o f S. capr i -cornutum was used to es tabl ish thepotential for toxicity or growth s t imu-lation. If the d ye inhib ited algal pop u-lation growth by 50% of that found inthe negative co ntrols, a defin itive testusing several dilutions of the dye wasperformed to allow for determinationof an EC5,, co nce ntra tion . Algal po pu -lations were mea sured, after 96 hoursof ex posu re, using an electro nic par-ticle counter fit ted with a mean cellv o l u m e c o m p u t e r . T h e T r i m m e dSpearman-Karber M etho d of Hamiltonet al., was us ed to calcu late EC50effectconcentrations a nd the associated 95%confidence limits.15Dun nett 's test wasperformed on al l resul ts that var iedf rom the con t ro l by &!O%. T h i smethod of statistical an alysis was usedto c o m p a r e t h e m e a n p o p u l a t i o ngrowth of controls to each of the testgroup means to determine statisticaldifferences.Quality AssuranceQuality control/quality assurance pro-cedures were adhered to during per-formance of this study. Performancecriteria were used to assess the qualityof the tests performed.

DyeNo.12345678910111213141516

Table 1. Dye No. and No. of Associated Colorant Structure(s) in AppendixColorants in D ye

3649383946273747,493443, 4334028412930

DyeNo.17181920212223242526272829303132

Colo rants in D ye26421815161819257

1 a,26,12,13,14251,52,5310832486,20

Compo nen t comprises 0.4%. bComponent comprises 4.0%.

DyeNo.3334353637383940414243444546

Colorants in Dye44,4531453592122,23, 434117501 120


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Positive control tests, using zinc,demonstra te thatf S. capricornutum waslaboratory exper iments , per-determ ine the toxicity ofECS0n the expec ted

A negat ive control , composed of, was performedeach test series to provide a m ea-e of the health 0f.S.capricornutum.g t he 96-hour static chronic tox-l cells in the co ntrol

l was also imp ortant because re-or stimulatory effects withinon the difference between then biomass of each exposed po pu-

T e m p e r a t u r e w a s c o n t i n u o u s l ythroughout the testing pe-thermom eters with record-and Discussion

confid ence limits , of algal biom-produced in control an d test

lation growth are also included.lation growth inhibition or posi-for pop ulation growth stimulation.

of that found in the3, 4, 23 ,24,25, 27 s t imula ted popu la t ionfrom 20.8-26.4%. Stat is t icals test dem on-

controls.11, 16,28 , 33 , 41, and

. Sta t is t ical analysis us ing thes test show ed that only dyes28 and 41 produced algal popula-were significantly differentl pop ulation . However,ition caused by dye 16 ,dered lo w toxicity. O n the other100 and 97.1% inhibitionwit h dyes 28 and 4 1, respec-

of dyes 28 and 41 wereive do se/resp onse test, wh ich

~

Table II. Average Yield (mglL dry weight S. apricornulum)an d 95% Confidence Limits for 96-HourAlgal Static Chronic Toxicity Tes tsDy eNo. Avg.Control 29.01 32.52 27.03 35.44 36.65 34.07 27.09 34.110 31.511 22.313 29.514 29.615 24.116 21.117 25.819 30.4

6 27.8a 32.a

12 28.8

18 28.720 28.821 29.822 32.323 36.4

Lower95 %C.L.26.124.622.330.434.929.623.721.725.130.029.920.224.527.122.414.423.920.222.024.026.433.1

24.8

18.3

Upper95%C.L.31.940.440.331 a38.438.531.934.040.433.124.337.531.434.325.727.933.233.540.635.635.539.6

38.1

38.2

PercentEffect+12.1-6.7+22.1~26.4+I73-4.1-3.8+13.1+17.6

-___-

+a.8-23.1-0.4+0.9+2.0-16.9-27.1-11.1-0.9+5.0-0.7~ 2 . 8+11.5+25.4

dium wi th 0.0,0.0001,0.001,0.01,0.1an d 1.0 mg/L of the colorants. Evalua-tion of the test re sult s, using Dunnettstest, indicated that the dyes caused apopulat ion growth that was s ta t is t i -cal ly less than that produced in thecontrols. A Trimm ed Spearman-Karberanaly sis of the test da ta resulted in anECS0 concentration of 0.025 mg/L fordye 28.j4 The low er and u pper 95%confid ence limits for the analysis were0.020 mg/L and 0.031 mg/L, respec-tively. Similarly , dye 41 resulted in anECS0 concentration of 0.247 mg/L forthe colorant with 95% confidence lim-i ts ranging k o a 0 .161 to 0.230 mg/L.Th e high toxicity of dyes 2 8 and 41 isentirely consistent with th e known tox-icity of basic; i.e., cation ic, dyes to bothS. capricornutum an d fish.gAlthough the cationic dyes are verytoxic, it is difficult to assess their be-havior in natural waters. Cationic dyescan be ex pected to strongly adsorb tos e d i m e n t s w h i c h w i l l r e d u c e t h e i rtoxic effects. How ever, since sorptio ncoefficients are not availa ble, the mag-nitud e of their impa ct on aquatic sys-tems canno t be estimated.Metal Complex ColorantsImportantly, none of the 19 metal com-plex colorants were toxic. A study withthe sa me alga includ ed five metal com-plex dyes (three cop per, one chromiumand one cobalt) and the only inhibi-tion reported (30 % after seven days at1 mg/L) was due to one of two cop-perized azo dyes.gAgain, the reasons for absence oftoxicity are not clear. As with the re-act ive dyes , b ioavai labi l i ty of such

DyeNo.24252627

Control2930313233343536373940414243444546

28

38

Avg.35.735.035.535.427.526.231.325.626.6

0.0128.2

19.826.826.8

28.826.8

27.530.6

0.7923.320.825.629.625.1

Lower95 %C.L.32.531.729.531.323.221.626.824.212.221.625.325.327.020.619.612.622.027.1

0.0019.8

18.418.9

0.65

20.8

Upper95% PercentC.L. Effect39.0 ,+23.338.4 +20.841.4 +22.439.5 c22.00.02 -100.031.8 -----30.8 -4.836.6 +2.535.8 +13.727.0 -7.027.4 -28.234.8 -3.434.8 -2.532.0 -2.529.7 0.035.8 +11.130.7 +4.833.0 -2.60.93 -97.127.0 -15.329.2 -24.329.3 -6.932.0 +7.429.6 -8.7

large molecules may be limiting sinc ethe molecular weig ht of the comp lexesis greater tha n 500.Other factors may be either a slowrate of release of bioavailable metalfrom the complexes (ligand exchangerate) or thermodynamic stability. ManyCr, and to a lesser extent Go, complexesare known to undergo slow er releaseof ionic m etal (ligand exchange) thanCu complexes. Th e release of Cu fromdyes was fo und by Hill et al.z6 o takeup to 1 2 hours while Cr release wasmuch lower.'^ It probably is signifi-cant that the Cu dyes in this study allhave phthalocyanine (dyes 2 ,8 a nd 44)or formazan (dyes 14 and 34) colorantsthat are less likely to undergo ligandexchange tha n the copperized azo di-rect dyes.In the absence of equilibrium con-stants, it is not possible to rule ou t thepossibility that the complexes are sim -ply so stable that th e alga cannot com -pete w ith the ligand.-This is probablythe case with the phthalocyanines an dperhaps for the other s tudied com-plexes.Little can be said with confidenceabout the Co complexes or about theCu azo com poun ds because of lack ofdata.For any of the d yes , absence of tox-icity in the 1 mg/L test is significantsince usual biological waste treatme ntis likely to result in conce ntrations t hatare much lower than 1 mg/l for anyindividual dye. Th is, in turn, suggeststhat many of the impo rtant colorantsused in reactive and/or m etallized dyesmay not lead to environmental toxic-ity at concentrations expected in re-


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ceiving w aters. However, since the pHof the receiving (o r test) water stronglyinfluences the equilibrium an d rate ofl igand exchange , toxic i ty may be astrong fun ction of pH.O'Neal and Boyter repor ted tha tmo st of the toxicity of Cu -dye con tain-ing texti le eff luents was due to freemetal that cou ld be separated from thecom plexes by ultrafiltration or ion ex-change.l8 Th is can only be observed ifthe therm odyna mics and kinetics favorthe complexes rather than free metal.Unfortunately, studie s of mill effluentshave c ontained l i t t le or no data on thenatur e of metallized dyes i n the waste-water.Reactive DyesNone of the reactive dyes exhibitedtoxicity. This is interesting since thisgroup has 23 colorants that contain thehalo t r iaz ine moie ty . O 'Kel ley andDeason have reported th at atrazine, achlorotriazine pesticide, was toxic to2 6 of 36 algal isolat es at the 0 .001 mglL level.zgRelevance of the test resultsto aquatic systems is based on the as-sumption that the reactive (unhydro-lyzed) forms were not hydrolyzed inthe test systems m ore rapidly than theyw o u l d b e i n t h e e n v i ro n m e n t . A l -though som e hydrolysis of the reactivegroups is expected, under the m ild testcondit ions, the rate must be low com-pared with that of dyeing operationsthat alw ays require high pH. A lso, ab-senc e of phytotoxicity could be d ue toinabi l i ty of the la rge colorant mol-ecules to cross cell membranes (i.e.,lack of bioavailability).Summary and C onclusions

s m ight be expected, the tw o basicdyes, numbers 28 and 41, were highlyoxic to the test alga S. capricornutum.he median ECS0effect conce ntrationsere 0.025 an d 0.247 mg1L activ e dyeolo rant, respectively.Data show the other dyes to be non-c to the test alga at concentrationsent. Results for the metallizedyes are consistent with th e view thatunder test condit ions, the m etals areor not av ailable to thea. The test results can be interpretedting that there w ou ld be littleon release of such

of the environ-st aw ait information on the effectsf pH o n the equilibria and kine tics ofange. Based on their known

dyes in genera l , is probablyy af fec ted by photochem ical

The data show no evidence of algaltoxicity caused by the reactive dyes;i.e., due to the chlorotriazine, fluoro-triazine, bromoacrylate and vinyl su l-fone reactive groups. In the a bsen ce ofother data, there is little reason to ex-pect chronic phytotoxicity from thesecom pou nds at levels that might be ex-pected in the environment.References1.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. 1 3, 1984,~ 1 3 6 6 .3. Pimprikar, G. D. et al., Southwestern Ento-mologist, Vol. 9, 19 84, p218.4. P rakash, C. J. and R. B. Misra,Biochemical andBiophysical 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. Ma rtin, D. F. and C. D. Norris, JournalofEnvi-ronmental ScienceandHealth, Vol. A23,19 88,

p765.7. Michaels, G. B. and D. L. Lewis, Environmen-tal ToxicologyandChemistry,Vol. 4,1985, p45.8. Tratnyek , P. G., M. S. Elovitz an d P. Colverson.Environmental Toxicology and Chemistry,Vol. 13, 1994, p27.9. Little, L. W. and M. A. Chillingworth, Dyesand the Environment-Reports on SelectedDyes and Their Effects, Vol. 11, Chapter 2,

American Dye Manufacturers Institute Inc.,September 1974.10. Dolezalova, L. a n d F. Solta, Vodni Hospo-darstvi. B, Vol. 35, 1985, p49.11.Burton, D. T., D. J. Fisher and R. L. Paulson,Chemosphere,Vol. 1 9,19 89, p1959.12. Wells, M. J. M., A. J. Rossano Jr. a n d E. C. Rob-erts, Archives of Environmental Contamina-tion an d Toxicolgy,Vol. 27, 199 4, p555.13. Miller, W. E. et al., Jourflal of EnvironmentalQuality, Vol. 4, 1985, p569.14. Greene, J. C. et al., Protocols for Short TermToxicity Screening of Hazardous Waste Sites,Report No. EPA 60013-88-029, U.S. Environ-mental Protection Agency, Corvallis, Ore. ,1988.15. Ha mil to n, M. A., R. C. Russo an d R. V.Thurston, Environmental Science Technology,Voi. 11,1983, p714.16. Hill, W. E., W. S. Perkins and G. S. Sandl in .-Textile Chemist an d Colorist,Vol. 25, 1993,p26.17. Hill, W. E., personal commu nication.18. O'Neal, W. G. and H. A. Boyter Jr., Proceed-ings, Water Environment Federat ion, 67thAnnual Conference an d Exposition, Chicago,1994, p567.19. O'Kelley, J .C. and T. R. Deason, Degradationof Pesticides by Algae. Report No. EPA-600/3-76-022, U.S. Environmental Protect ionAgency, Athens, Ga., 1976.Author's AddressGeorge L. Baughman, University ofGeorgia , Dawson Hal l , A thens , Ga.30602; telephon e 706-542-4883.

Appendix: Structures of Colorants in the Tested DyesColorant 1

pColorant 3 Three compoundswith X =OH and/or NHCH3CH3 CH3S04'

Colorant 4H& Colorant 5

2Na+, -03s'


5/8

Colorant 12

Colorant 13

Colorant 16Na+

0 YHo\ L z H 5H

Colorant 14

N=NNa+

Co lora nt 17 H3C.

H 3d

Colorant 150; Na+

0 NH

Colorant 18+Na+ SO


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Colorant 19

< Na+WO'H

Colorant 22

Colorant 20 - Colorant 21

3Na+0 2

\ NO2-03S

Colorant 25 CI

Na+ do2

FColorant 26

4Na+ ci

Na+


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Colorant 31 F

Colorant 33

I IH H

Colorant 35 H3c3; Colorant 36

37 CIColorant 38 A N

Na+ H \ k N A Y '-d / / H3C'CH2Na+so3'o3- - 'a+ 03s


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,Colorant 42 Na+ Colorant 43Na+

H HNa+ -03sNa+

H

Na+ Na+ Na+Colorant 47

Na+ Na+ Na+

Rings a, b, c, d contain one ofthe following substituents:-S03-Na+ (two total)-S02NH2

H H

NH2

Colorant 48 .Na+

-03S SO3 H

Colorant 49Rings a, b, c, d contain one ofthe following substituents:-S03 -Na+ (two total)

Colorant 50Rings a, b, c, d contain one ofthe following substituents:6 0 3 - Na+

Colorant 51 Colorant 52

2Na+

0so32N

09Nolorant 53

effects of 46 dyes on population growth of freshwater green alga se-eksfrum capricornutum

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