Chlorination and bromination of 1,3-diphenylguanidine and 1,3-di-o-tolylguanidine: Kinetics, transformation products and toxicity assessment

Jou nal P e-p oo
Chlo ina ion and b omina ion o 1,3-diphenylguanidine and
1,3-di-o- olylguanidine: Kine ics, ans o ma ion p oduc s and oxici y
assessmen
Benigno J. Siei a, Rosa Mon es, A naud Tou e , Rosa io Rodil,
Ra ael Cela, He ´
e Galla d, Jos´
e Beni o Quin ana
PII: S0304-3894(19)31544-4
DOI: h ps://doi.o g/10.1016/j.jhazma .2019.121590
Re e ence: HAZMAT 121590
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Page 1
CHLORINATION AND BROMINATION OF 1,3-DIPHENYLGUANIDINE AND 1,3-DI-O-
TOLYLGUANIDINE: KINETICS, TRANSFORMATION PRODUCTS AND TOXICITY
ASSESSMENT
Benigno J. Siei a 1, Rosa Mon es 1, A naud Tou e 2, Rosa io Rodil 1, Ra ael Cela 1,
He é Galla d 2*, José Beni o Quin ana 1*
1 Depa men o Analy ical Chemis y, Nu i ion and Food Sciences, Ins i u e o Food
Analysis and Resea ch (IIAA), Uni e sidade de San iago de Compos ela, R/ Cons an ino
Candei a S/N, 15782 – San iago de Compos ela (Spain)
2 Ins i u e de Chimie des Milieux e des Ma é iaux de Poi ie s (IC2MP), École Na ionale
Supé ieu e d’Ingénieu s de Poi ie s (ENSIP), Uni e si é de Poi ie s, 1, ue Ma cel Do é,
TSA 41105, 86073 – Poi ie s (F ance)
* Co esponding au ho s:
He é Galla d: [email p o ec ed]
José Beni o Quin ana. [email p o ec ed]
G aphical abs ac
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Highligh s
 DPG and DTG eac apidly wi h chlo ine and b omine
 The pH dependance o he eac ion was modelled
 Se e al ans o ma ion p oduc s we e iden i ied by LC-QTOF
 Chlo o o m and dichlo oace oni ile a e also p oduced
 TPs a e mo e oxic han DPG and DTG
Abs ac
This wo ks in es iga es he chlo ina ion and b omina ion o wo ubbe and polyme
ela ed chemicals, which ha e eme ged as ele an wa e con aminan s, i.e. 1,3-di-o-
olylguanidine (DTG) and 1,3-diphenylguanidine (DPG). Kine ic cons an s a di e en
pH alues we e ob ained and modelled, aking in o accoun he pKa alues o DTG/DPG
and HClO, showing ha he maximum eac ion a e (kapp > 104 M-1 s-1) is ob ained a
pH alues 8.8 o DPG and 9.1 o DTG. B omina ion is also e y as , al hough unlike
chlo ina ion, de ia ion om he model was obse ed a neu al pH, which was
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a ibu ed o o ma ion o me as able ans o ma ion p oduc (TP). A o al o 35 TPs,
co esponding o halogena ion, hyd oxyla ion, o ma ion o monophenylguanidine
de i a i es and cycliza ion eac ions, we e en a i ely iden i ied. Fu he mo e i was
ound ha chlo o o m can be o med up o a 25% mola yield, while
dichlo oace oni ile was o med in o less han a 3% yield. Se e al eco oxicological
endpoin s we e p edic ed by quan i a i e s uc u e–ac i i y ela ionship models
(QSAR) o he TPs, some o which we e p edic ed o be mo e oxic han DPG/DTG.
Also a chlo ina ed solu ion in es iga ed by a Vib io Fishe i acu e oxici y es ,
con i med ha oxici y inc eases wi h chlo ina ion.
Keywo ds
Halogena ion; ans o ma ion p oduc s; high- esolu ion mass spec ome y (HRMS);
eco oxici y; disin ec ion by-p oduc s.
1. In oduc ion
Pola o ganic compounds, i pe sis en , sp ead along he wa e cycle, e en becoming a
human heal h p oblem i ha subs ances each d inking wa e s (He nández e al.
2015, Reem sma e al. 2016, Schulze e al. 2019). 1,3-Di-o- olylguanidine (DTG) and
1,3-diphenylguanidine (DPG) a e chemicals used as accele a o s in he ulcaniza ion
p ocesses o ubbe and o he polyme s manu ac u e, wi h a egis e ed p oduc ion in
Eu ope, acco ding o he REACH dossie s, in he 100-1000 ons (DTG) and 1000-10,000
ons (DPG) (ECHA 2018a, b). Howe e , so a , li le in o ma ion abou hei
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en i onmen al (pa icula ly wa e ) occu ence is a ailable. A i s s udy om Mon es e
al. (Mon es e al. 2017), in he ame o he p ojec PROMOTE(Me cal e e al. 2003),
epo ed DTG occu ing in en i onmen al wa e samples ac oss Eu ope by liquid
ch oma og aphy-high esolu ion mass spec ome y (LC-HRMS) sc eening. La e on,
wi hin he same p ojec , i has been shown ha DPG as well as DTG occu in di e en
wa e compa men s a he ng L-1 le el (Mon es e al. 2019, Schulze e al. 2019). Zahn
e al. ha e ecen ly shown ha , when conside ing na u al p ocesses, DPG (DTG was
no conside ed in ha s udy) pho olyzes and eac s wi h manganese oxide, bu does
no biodeg ade and is s able o hyd olysis (Zahn e al. 2019). Fu he mo e, his
compound has been iden i ied in d inking wa e in China a 0.7 mg L-1, mig a ing om
polye hylene pipes (Tang e al. 2015) and, mo e ecen ly, has also been ecen ly
iden i ied as being he majo chemical leaching om i e wea pa icles (Hübne e al.
2019, Zahn e al. 2019). Fu he mo e, he e is some li e a u e ha desc ibes DTG and
DPG oxici y and pha macological ac i i y in mice and a s (Ja amillo-lo anca and Es-
am 2015, Lamy e al. 2010).
Ye , he possible eac ion o bo h chemicals wi h chemical oxidan s used in d inking
wa e ea men plan s (DWTPs) and was ewa e ea men plan s (WWTPs) has no
been s udied so a . Se e al disin ec ion echniques and p ocesses a e employed in
DWTPs and WWTPs. Among hem, chlo ine is he oxidan used in he as majo i y o
DWTPs in Eu ope, and also in some WWTPs o a mino ex en (Beni ez e al. 2011,
Quin ana e al. 2014). Al hough chlo ine is e ec i e o inac i a e bac e ia, he
o ma ion o possible ha m ul ans o ma ion p oduc s (TPs), including well-known
disin ec ion byp oduc s (DBPs), as e.g. ihalome hanes, needs o be aken in o
accoun (Ace o e al. 2013, Pos igo and Richa dson 2014, Quin ana e al. 2014, Rodil e
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al. 2012). Such TPs (including DBPs) may in some cases be mo e (eco) oxic han he
p ecu so chemicals hemsel es (Pos igo and Richa dson 2014, Quin ana e al. 2014).
The e o e, hei iden i ica ion is necessa y in o de o ob ain a ele an in e p e a ion
o he eac ion.
Thus, he aim o his wo k was o pe o m a comp ehensi e s udy abou he
chlo ina ion o DTG and DPG in wa e . This includes a kine ic s udy and modelling,
iden i ica ion o TPs (including hose o med by b omina ion, since hypob omi e is
apidly o med om b omide in o solu ion du ing chlo ina ion (Beni ez e al. 2011)) by
LC-HRMS, quan i ica ion o he yield o known DBPs o med and p elimina y
(eco) oxicological assessmen .
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2. Ma e ials and me hods
2.1. Chemicals and s ock solu ions
DTG (99%) and DPG (97%) we e pu chased om Sigma-Ald ich (S einheim, Ge many)
and s ock solu ions we e p epa ed in ul a-pu e wa e . Ul a-pu e wa e was ob ained
di ec ly in he lab om a Milli-Q G adien A-10 sys em (Millipo e, Bed o d, MA, USA).
All solu ions and dilu ions necessa y o he expe imen s we e done in ul a-pu e
wa e un il desi ed concen a ion.
Sodium hypochlo i e (8-14% Cl2), ammonium chlo ide (> 99%) and po assium
phospha e dibasic ihyd a e (> 99%) we e ob ained om Sigma-Ald ich. Sodium
hiosul a e (99.5%) and po assium b omide we e om ACS Ac os O ganics (The mo
Fishe Scien i ic, Wal ham, MA, USA) and po assium di-hyd ogen phospha e (99.5%)
was om Pan eac (Ba celona, Spain). S anda d solu ions o chlo o o m and
haloace oni iles (HANs) we e p epa ed om EPA 551B Halogena ed Vola iles Mix
supplied om Supelco. The exac nominal ee chlo ine con en employed was
egula ly de e mined spec opho ome ically by measu ing he hypochlo i e anion
abso p ion a 292 nm (ɛ = 350 L-1 cm-1) (Johnson and Melbou ne 1996) o he s ock
solu ion (pH >10).
2.2. Real samples
Two samples we e used o s udy he ex en o he chlo ina ion eac ion wi h a eal
ma ix. A su ace wa e sample was collec ed om he Ri e Sa ela in San iago de
Compos ela (pH 6.8, Dissol ed O ganic Ca bon: 2.42 mg L-1, chlo ide: 7.98 mg L-1,
b omide: 0.043 mg L-1). A was ewa e e luen was collec ed om a WWTP comp ising
a p ima y and a seconda y con en ional sludge ea men (pH: 7.5, Dissol ed O ganic
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Ca bon: 14.1 mg L-1, chlo ide: 23.1 mg L-1, b omide: 0.075 mg L-1). Dissol ed o ganic
ca bon was measu ed wi h a Shimadzu 5000A TOC analyze (Duisbu g, Ge many),
while b omide and chlo ide we e de e mined wi h a Me ohm 850 P o essional Ion
Ch oma og aph (Zo ingen, Swi ze land).
2.3. Chlo ina ion expe imen s
Chlo ina ion o DTG and DPG we e pe o med indi idually in 100 mL ambe closed ials
a oom empe a u e. Also, expe imen s wi hou chlo ine we e p epa ed as a con ol.
Expe imen s o s udy chlo ina ion kine ics we e pe o med in a simila way, bu wi h
lowe compound concen a ions (1 µM), an excess o chlo ine (10 µM, 20 µM o 50
µM) and di e en pH o sample (5-12) being conside ed in 10 mM phospha e bu e
and NaOH o e y basic pHs. Aliquo s o 1 mL we e aken a di e en eac ion imes
and he eac ion s opped wi h 20 µL o 0.01 M sodium hiosul a e be o e esidual
concen a ion o guanidine was analysed by liquid ch oma og aphy-pho odiode a ay
de ec ion (LC-PDA). Ammonium chlo ide (1 mM) was used in some expe imen s as
“so ” quenching me hod as being selec i e o ee chlo ine and as o a oid any
Na2S2O3-induced back eac ion ha could in e e e wi h de e mina ion o a e
cons an (Dodd and Huang 2004). An expe imen was also pe o med wi hou s opping
he eac ion and aliquo s we e manually injec ed a di e en eac ion imes using a
Rheodyne al e in he LC-PDA sys em.
Expe imen s we e pe o med a oom empe a u e (22 ±1°C). The pH was measu ed
be o e and a e he expe imen , and a ia ion was less han 0.1 uni . F ee ac i e
chlo ine was analysed by DPD colo ime ic me hod (Clesce i e al. 1998) a he end o
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eac ion ime. Chlo ine consump ion was usually below 10% and pseudo- i s -o de
plo s we e always linea (see Figu e S1 o examples).
Addi ional expe imen s o he iden i ica ion o TPs (pe o med in iplica e) we e
ca ied ou wi h a simila p ocedu e. Fo hese expe imen s, ul apu e wa e adjus ed
a pH 7.0 was used, spiked wi h he compound a 10 µM, and ini ial chlo ine dose se
o 100 µM. TPs we e iden i ied a e educ ion by asco bic acid o eac ion imes o 30
s, 1 min, 2 min, 5 min, 10 min and 30 min.
DBPs o ma ion po en ials (chlo o o m and haloace oni iles) we e de e mined o a
eac ion ime o 2 days a pH 7.0 in ul apu e wa e wi h an ini ial concen a ion (o
ei he DPG o DTG) o 10 µM and mola chlo ine o guanidine a ios o 1, 10 and 100 in
headspace- ee condi ions.
2.4. B omina ion expe imen s
B omina ion expe imen s we e pe o med unde he same condi ions as chlo ina ion,
in o de o de e mine appa en a e cons an s and de ec TPs ha can be p oduced in
b omide con aining wa e s. B omine was gene a ed in he lab acco ding o he
p ocedu e desc ibed by Beni ez e al. (Beni ez e al. 2011). B ie ly, b omine was
p oduced om he eac ion be ween 9 mM HOCl and 10 mM po assium b omide. The
yield o his eac ion was ollowed spec opho ome ically (hypob omi e anion
maximum abso p ion wa eleng h a 329 nm wi h an ɛ = 332 M-1 cm-1 a pH abo e 11.5)
(Beni ez e al. 2011).
Kine ics o b omina ion we e s udied as o chlo ina ion o pH alues anging om 5
o 9 using di ec me hod in ba ch eac o wi h an excess o b omine (10 o 100 µM)
compa ed o 1 µM DPG o DTG solu ion (see Figu e S2 o examples o b omina ion o
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The alue o he a e cons an k was de e mined by a non-linea leas -squa e
eg ession o he expe imen al pH p o ile o he kapp alues using Sigma Plo 11.0
(Sys a So wa e Inc., San Jose, CA, USA).
The pH dependence o kapp is shown in Figu es 1a and 1b o DPG and DTG,
espec i ely. Fo bo h compounds, he pH p o ile exhibi s a maximum be ween pH 8
and pH 10. This maximum co esponds o he concomi an p esence o bo h HOCl and
neu al guanidine. The maximum pH alue is equal o he a e age alue o he pKa o
HOCl and guanidines i.e. 8.8 o DPG and 9.1 o DTG. As shown in Figu e 1, he model
i s well wi h he expe imen al da a conside ing only he eac ion o HOCl wi h neu al
guanidine. No imp o emen was ob ained by including he eac ion o ClO- wi h
neu al guanidine and he eac ion o HOCl wi h p o ona ed guanidine (see Tex S1 and
Table S5), which is in acco dance wi h he li e a u e (Debo de and on Gun en 2002).
The a e cons an s, k, o he eac ions be ween HOCl and DPG and DTG neu al species
de e mined om model i ing o he expe imen al alues a e 4.1 (±0.3) × 106 M-1 s-1
and 2.6 (±0.1) × 107 M-1 s-1 o DPG and DTG, espec i ely. The appa en a e cons an s
a neu al pH (~103 M-1 s-1) and in insic a e cons an s (k) o neu al species (~106 - 107
M-1 s-1) a e in he ange o a e cons an s o seconda y amines wi h chlo ine (Debo de
and on Gun en 2008). Howe e , N-chlo oamino compounds we e no de ec ed du ing
kine ic expe imen s and iden i ica ion o TPs would indica e ha ini ial eac i e si e is
he a oma ic ing. Lowe a e cons an o 19 M-1 s-1 was ob ained o e hyl guanidine a
pH 7.2 – 7.4 (Pa ison and Da ies 2001), which can be explained by he s onge basic
cha ac e o alkyl guanidines.
3.2. B omina ion kine ic s udy
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The appa en a e cons an s o b omina ion de e mined by using di ec and
compe i ion kine ics me hods a e lis ed in Table S3 and S4 o DPG and DTG,
espec i ely and a e plo ed e sus pH in Figu e 2. Examples o pseudo- i s -o de and
compe i ion kine ics plo s a e gi en in Figu e S2 and Figu e S3 o DPG. The appa en
a e cons an s ange om 76 o 2.89 x 105 M-1 s-1 o DPG and om 36 o 5.87 x 104 M-
1 s-1 o DTG. In con as o chlo ine, lowe a e cons an s we e de e mined o DTG,
which could be a ibu ed o s e ic e ec s be ween he bulky b omine a oms and he
me hyl g oups in DTG.
The expe imen al esul s i well wi h he p oposed model a pHs below 6 and abo e 9,
while a s ong de ia ion and e en disc epancies be ween a e cons an s de e mined
by he di ec kine ics me hod and he compe i ion kine ics me hod using BP as
e e ence compound in he pH 7 – 9 ange. Such de ia ions a e a ibu ed o a
me as able TP wi h oxidizing p ope ies which could no be iden i ied by LC-HRMS in
ha pH ange (see de ailed discussion in Tex S2 and Figu es S3-S6).
Excluding hose pH alues, and compa ed o chlo ina ion, maximum kapp alues a e
sligh ly shi ed o highe pH alues due o he highe pKa o HOB (pKa = 8.8).
Calcula ed in insic a e cons an s o he eac ion o HOB wi h neu al DPG and DTG
we e 8.3 (±0.4) x 106 and 5.5 (±0.7) x 106, espec i ely. While HOB eac s usually much
as e han HOCl wi h o ganic compounds (Heeb e al., 2014), he a e cons an o
HOB wi h DPG was only wice as high as he eac ion o HOCl wi h DPG and he a e
cons an o he eac ion o HOB wi h DTG was lowe han ha o HOCl. S e ic
hind ance (as men ioned), di e en eac i e si es and ype o eac ion (oxida ion s
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subs i u ion) migh explain his unexpec ed esul , which equi es u he
in es iga ion.
3.3. T ans o ma ion p oduc s
Iden i ica ion o TPs was pe o med o bo h DTP and DPG conside ing chlo ina ion
and b omina ion. Expe imen s we e ca ied ou as desc ibed in sec ions 2.3 and 2.4. All
samples we e analysed in he LC-QTOF equipmen as de ailed in 2.6. The p oposed
s uc u es o he TPs a e p esen ed in Figu es 3 and 4. Fu he de ails on o mulas,
mass e o s and sco es o he TPs, as well as indi idual s uc u es de i ed om he
in e p e a ion o MS/MS spec a a e p esen ed in Tables S6 and S7. TPs we e named
wi h he p ecu so compound abb e ia ion ollowed by he nominal mass o i s [M+H]+
ion. As i can be obse ed he empi ical o mula could be p oposed wi h a high deg ee
o ce ain y, wi h sco e alues highe han 95% and mass e o s lowe han 5 ppm,
excep o DTG-274, whose sco e was 79% and mass e o was 9.7 ppm due o i s low
in ensi y.
The p oposed s uc u es a e based on he in e p e a ion o he MS/MS spec a, which
a e p esen ed in o Figu es S7 and S8, o DPG and DTG TPs, espec i ely. Mo eo e ,
DPG-136 (i.e. monophenylguanidine) was unequi ocally iden i ied by pu chasing i s
au hen ic s anda d om Sigma-Ald ich.
The e a e ou main ypes o eac ions occu ing du ing chlo ina ion, i.e. ipso-
chlo ina ion o p oduce monoguanidine de i a i es, in oduc ion o chlo ine a oms
(b omine when samples a e b omina ed) in o an a oma ic ing, hyd oxyla ion and
in amolecula cycliza ion. Thus, DPG-136 was easily iden i ied by i s spec um (Figu e
S7), and because an au hen ic s anda d was a ailable, as men ioned. This TP u he
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eac s by halogena ion o he co esponding chlo ina ed o b omina ed de i a i es
(DPG-170 and DPG-214), easily iden i ied because hei MS/MS spec a is simila o
DPG-136 and exhibi he halogen iso opic pa e n (Figu e S7). DPG-119 is also
p oduced a long eac ion imes. Hyd oxyla ion o DPG p oduces DPG-228, while
halogena ion p oduces DPG-246 (chlo ina ion) and DPG-290 (b omina ion), all o hem
easily iden i ied by hei MS/MS spec a (Figu e S7).
A key TP is DPG-210, which a simila empi ical o mula han DPG i sel bu wi h one
u he double-bond equi alen (i.e. 2 a oms o H less), Table S6. I s MS/MS spec um
exhibi s i s he loss o ammonia o m/z 192.0671 and also he elimina ion o CH3N2 o
m/z 167.0720 om he p o ona ed molecula ion (Figu e S7e). This second ion would
no be possible unless a cycle is o med. Because o his, we hypo hesize he e ha
DPG-210 co esponds o he s uc u e shown in Figu e 3, by o ma ion o he 7-
membe ing cycle om DPG-228 (hyd oxyla ed DPG) by elimina ion o wa e . In ac he
maximum in ensi y o DPG-228 was obse ed a 0.5 min and hen i s in ensi y apidly
d ops, while DPG-210 maximum is eached a 1 min and hen d ops mo e slowly (see
Figu e S9a). The ac ha DPG-210 has also been obse ed as a pho olysis TP by Zahn
e al. (Zahn e al. 2019), al hough no s uc u e was p oposed in ha publica ion,
u he suppo s his hypo hesis. Once DPG-210 is o med, his molecule u he eac s
o yield a mono-hyd oxyla ed-TP (DPG-226), a hyd oxychlo o-TP (DPG-260) o a
dichlo o,hyd oxy-TP (DPG-294).
In he case o DTG, he eac ion was simila o DPG, as expec ed, bu a la ge numbe
o TPs could be iden i ied (23 s. 11 TPs). In gene al, he main di e ence in he TPs
p oduced is ha a g ea e deg ee o hyd oxyla ion and halogena ion is obse ed, e.g.
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DTG-308 (a dichlo ina ed de i a i e o DTG), which can be explained by he elec on-
dono e ec o he me hyl g oup on a oma ic ing. Al hough no MS/MS spec a (Figu e
S8) was ob ained o all TPs, due o he low in ensi y o some o hem, s uc u es
compiled in o Figu e 4 and Table S7 ( hose TPs wi hou MS/MS da a a e ma ked wi h a
* symbol in he Table) we e assigned on he basis o he MS/MS spec a (when
a ailable) and by analogy o DPG TPs.
As ega ds o he mos ele an TPs, Figu es S9b and 10b p esen he no malized
amoun o each TP and hei p ecu so s o a eac ion wi h 100 µM chlo ine and up o
30 min o con ac ime. No maliza ion was pe o med by using he signal o he
o iginal compound (DPG o DTG) as a su oga e o calcula e an app oxima e yield,
excep o DPG-136, DPG-119, DPG-170, DTG-150, DTG-166, DTG-184 and DTG-228,
whe e monophenyl-guanidine (DPG-136) was used ins ead, as being conside ed
s uc u ally close . In he case o DPG (Figu e S9), he mos in ense TP is DPG-136 wi h
a yield o ca. 5% a 0.5-2 min, d opping down o 3% a 30 min. The second mos
ele an TP is DPG-246 (monochlo o-DPG), wi h a yield o ca. 3% a 0.5-2 min, d opping
down o ca. 0.2% a 30 min. I is no ewo hy ha a 30 min, he e was no DPG
de ec able and he sum o all TPs in ensi ies would app oxima ely ep esen a 5%
yield. This could likely be a ibu ed o he o ma ion o ing-opening p oduc s like
chlo o o m wi h a ela i ely high yield (see 3.4) and he unce ain y o he semi-
quan i a i e app oach, due o he lack o au hen ic s anda ds o mos TPs.
In he case o DTG (Figu e S10), he mos in ense TP is DTG-254 (hyd oxyla ed cyclic
p oduc ) wi h a yield o ca. 30% a 0.5-2 min, ollowed by DTG-290 (hyd oxy,chlo o-
DTG) wi h a yield o ca. 15% a 0.5-2 min. These wo TPs a e also he mos ele an a
Jou nal P e-p oo
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30 min, ep esen ing an es ima ed yield o 4 and 8% espec i ely. Also, in he case o
DTG, he o al yield o TPs a 30 min is ca. 20%.
3.4. Disin ec ion by-p oduc s
Subsequen ly o TPs iden i ica ion, we in es iga ed he o ma ion po en ial o classical
DBPs (chlo o o m and HANs). Chlo o o m was measu ed as he only ihalome hane
ha can be o med wi hou b omide and ep esen ing he mos ele an g oup o
DBPs, while HANs is ano he impo an g oup o DBPs which can be p oduced om N-
con aining chemicals, as i is he case o DPG and DTG. Figu e S11 shows he mola
yields o CHCl3 and dichlo oace oni ile (DCAN), he only HAN de ec ed, p oduced om
he chlo ina ion o DPG and DTG a e 2 day eac ion ime. Simila yields we e
ob ained o bo h guanidines. Fo CHCl3, he yield o ca. 25% o a mola guanidine/Cl2
a io o 1:100 is simila o CHCl3 yields anging om 10 o 32% al eady desc ibed o
hyd oxyla ed and chlo ina ed a oma ic compounds (Galla d and on Gun en 2002).
This is consis en wi h he o ma ion o chlo o o /and hyd oxy DPG and DTG ha
u he eac wi h chlo ine leading o CHCl3 as end-p oduc a e ing clea age. Among
HANs, only DCAN was de ec ed a signi ican le els and wi h yields much lowe han
CHCl3. Fo a a io o 1:100, mola yields we e 4.4% and 2.3% o DPG and DTG,
espec i ely.
3.5. Reac ion in eal sample ma ices
The eac i i y o bo h guanidine compounds was es ed by spiking wo eal ma ices (a
su ace wa e and a was ewa e e luen ) wi h 1 µM (i.e., ca. 200 µg L-1) o ei he DPG
o DTG and 10 µM chlo ine and he eac ion kine ics ollowed o 20 min ( eac ion
quenched wi h asco bic acid) by LC-HRMS.
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Page 21
In he case o he su ace wa e , DPG and DTG eac ed apidly, being below 1% o hei
ini ial concen a ion a e 2 min (Figu e S12a-b). Con e sely, wi h he e luen
was ewa e (wi h a highe TOC), s ill an 87% o DPG and 73% o DTG emained a e 20
min (Figu e S12c-d). Indeed, o ma ion o TPs is easie o happen du ing d inking wa e
p oduc ion han by chlo ina ion o was ewa e (a e seconda y ea men ). E en so,
he chlo ina ed was ewa e was analysed o he TPs p e iously iden i ied in ul apu e
wa e and se e al o hem could be de ec ed. The amoun o b omide in hose samples
is e y low (<0.1 mg L-1, see 2.2), he e o e no b omina ed TPs we e de ec ed. When
excluding hose b omina ed TPs, 6 ou o 9 TPs whe e de ec ed o DPG (DPG-136,
DPG-228, DPG-210, DPG-226, DPG-260 and DPG-294) and 7 ou 16 whe e de ec ed o
DTG (DTG-150, DTG-256, DTG-290, DTG-239, DTG-254, DTG-270 and DTG-288).
3.6. (Eco) oxici y assessmen
To ob ain a p elimina y es ima ion o he eco oxicological implica ions o he
chlo ina ion eac ion, he US-EPA TEST so wa e was used in o de o p edic he
oxici y o he wo guanidines and hei TPs. This p edic ion was pe o med only o
Daphnia Magna LC50 (48 h), Te ahymena Py i o mis LC50 (48 h) and o al a LD50 (as a
p oxy o human oxici y), since he so wa e was unable o p oduce an es ima ion o
o he endpoin s. The esul s ob ained a e summa ized in Tables 1 and 2.
As i can be app ecia ed, o al a oxici y LD50 alues a e in he 602-805 mg Kg-1 bw o
he wo guanidines, which would classi y hem as Ca ego y 4 (i.e. he less oxic
ca ego y) acco ding o he ECHA Guidance (ECHA 2017). The TPs would also be
classi ied as Ca ego y 4. Hence human oxicological haza d is expec ed o be low.
Jou nal P e-p oo
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The p edic ed acu e aqua ic oxici y endpoin alues lie in he 5-28 mg L-1 and 3-6 mg L-
1 anges, o DPG and DTG espec i ely (Tables 1 and 2). Thus, hey would no be
classi ied as Ca ego y Acu e 1 ( he only acu e aqua ic oxici y ca ego y) acco ding o
he ECHA Guidance (ECHA 2017). As ega ds he TPs, he p edic ed aqua ic oxici y o
he monoguanidine TPs is lowe han he p ecu so guanidines o he c us acean
Daphnia Magna, while i could no be p edic ed o he ish T. py i o mis. On he o he
hand, pa icula ly TPs which a e halogena ed a e p edic ed o be mo e oxic. Thus,
DPG-294 and 14 TPs om DTG (see Table 1 and 2) would ha e a p edic ed oxici y
endpoin <1 mg L-1 and would hus be classi ied in he Ca ego y Acu e 1 o aqua ic
o ganisms. Ca e mus be aken wi h hese da a, since he hi d ophic le el (algae)
oxici y could no be p edic ed and alues ob ained o algae can likely esul in o mo e
eco oxici y. In ac DTG is classi ied in he REACH dossie as Aqua ic Acu e 1 (ECHA
2018a).
Fu he mo e, he acu e oxici y o DPG and DTG was assessed using he
bioluminescen Vib io ishe i es .
The EC50 and EC20 alues o Vib io ishe i es we e es ima ed om a se ies o
geome ical dilu ions wi h dilu ion ac o s om 1 o 256 and ini ial guanidine
concen a ion o 100 mg L-1. Figu e S13 shows wo dose- esponse cu es o DPG a e
an incuba ion ime o 30 min a 15°C. The ini ial EC20 o DPG was 40 ±2 mg L-1 and he
EC50 was es ima ed (as de ailed in Tex S3) o be 245 ±33 mg L-1 om ex apola ion o
he dose- esponse cu es. The oxici y o DTG was lowe and only an EC20 o 80 mg L-1
could be de e mined. These esul s con i m ha bo h guanidines ha e a low acu e
aqua ic oxici y. Due o solubili y limi a ions, he e ec o chlo ina ion on acu e oxici y
Jou nal P e-p oo
Page 23
was only es ed wi h a DPG solu ion o 40 mg L-1 co esponding o he EC20.
Chlo ina ion was pe o med wi h chlo ine doses o 40 and 400 mg Cl2 L-1 (i.e. mola
Cl2/DPG a io o 3 and 30). Toxici y es s we e conduc ed a e he absence o chlo ine
esidual was checked. Resul s in Figu e 5 shows ha he bioluminescence inhibi ion
s ongly inc eases om 14% be o e chlo ina ion o 45 and 99% o Cl2/DPG a ios o 3
and 30, espec i ely. Simila esul s we e gene ally obse ed in he li e a u e a e
chlo ina ion and we e assigned o mo e oxic halogena ed TPs (El Najja e al. 2013,
Tawk e al. 2015). E en hough he inc ease o oxici y could no be assigned o speci ic
TPs/DBPs, esul s o bioluminescen Vib io ishe i es we e in ag eemen wi h
p edic ed aqua ic oxici y endpoin s ob ained by QSAR.
4. Conclusions
DPG and DTG apidly eac wi h chlo ine and b omine a na u al wa e pH alues. This
eac ion leads o he o ma ion o se e al TPs ia ipso-halogena ion, hyd oxyla ion,
halogena ion and cycliza ion. Se e al o hese TPs a e p edic ed o be mo e oxic han
he o iginal guanidine compounds, which was con i med by measu ing he acu e
oxici y o a chlo ina ed mix u e by a Vib io Fishe i acu e oxici y assay. Mo eo e ,
chlo ina ion leads o he p oduc ion o he adi ional/ egula ed DBPs chlo o o m and,
o a mino ex en , dichlo oace oni ile, when he mola a io o chlo ine o DPG/DTG is
high.
Decla a ion o in e es s
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Page 24
☒ The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal
ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .
☐The au ho s decla e he ollowing inancial in e es s/pe sonal ela ionships which may be
conside ed as po en ial compe ing in e es s:
Acknowledgemen s
This wo k was suppo ed by he Wa e Challenges o a Changing Wo ld Join P og am
Ini ia i e (Wa e JPI) Pilo Call ( e . WATERJPI2013 – PROMOTE), unded by he Spanish
Minis y o Economy and Compe i i eness/Spanish Agencia Es a al de In es igación
( e s. JPIW2013-117 and CTM2017-84763-C3-2-R) and F ench O ice Na ional de l’Eau
e des Milieux Aqua iques ( e . PROMOTE). We also acknowledge he Galician Council
o Cul u e, Educa ion and Uni e si ies ( e . ED431C2017/36), Région Nou elle
Aqui aine and FEDER/EDRF unding.
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Page 31
pH
4 6 8 10 12
kapp (M-1 s-1)
1e+1
1e+2
1e+3
1e+4
1e+5
DPG expe imen al
DPG model
a)
pH
4 6 8 10 12
kapp (M-1 s-1)
1e+1
1e+2
1e+3
1e+4
1e+5
DTG expe imen al
DTG model
b)
Figu e 1. pH dependence o he expe imen al and modelled appa en a e cons an s o
chlo ina ion o (a) DPG and (b) DTG.
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Page 32
pH
4 6 8 10 12
kapp (M-1 s-1)
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
DPG model
pH
4 6 8 10 12
kapp (M-1 s-1)
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
DTG model
a)
b)
Figu e 2. pH dependence o he expe imen al (symbols) and modelled appa en a e
cons an s o b omina ion o (a) DPG and (b) DTG. Di ec kine ic me hod (ci cle) and
compe i i e kine ic me hod using 4-b omophenol (squa e) o 2,4,6- ib omophenol
( iangle) as e e ence compounds we e used o kapp de e mina ion. Only ull symbols
we e used o model calcula ion.
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Figu e 3. Schema ic ep esen a ion o DPG TPs
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Figu e 4. Schema ic ep esen a ion o DTG TPs
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Figu e 5. E olu ion o bioluminescence inhibi ion measu ed using Vib io ishe i
Mic o ox® es du ing DPG chlo ina ion. [DPG]o = 40 mg Cl2 L-1, mola DPG:Cl2 a ios o
1:3 and 1:30, chlo ina ion ime o 4 days a pH 7.0.
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Page 36
LIST OF TABLES
Table 1. QSAR P edic ed oxici y alues o DPG and i s TPs.
Daphnia magna
T. py i o mis
O al a
LC50 (48 h ) (mg/L)
IGC50 (48 h ) (mg/L)
LD50 (mg/kg)
DPG
5.09
28.5
805
DPG-119
16.4
np
493
DPG-136
30.6
np
500
DPG-170
11.5
np
455
DPG-210
5.29
11.4
908
DPG-214
1.14
9.53
1320
DPG-226
2.46
10.4
1143
DPG-228
5.24
21.1
2433
DPG-246
1.22
7.21
886
DPG-260
1.89
4.78
1241
DPG-290
1.14
9.53
1320
DPG-294
0.75
2.88
1019
np: no p edic ion possible
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Page 37
Table 2. QSAR P edic ed oxici y alues o DTG and i s TPs.
Daphnia magna
T. py i o mis
O al a
LC50 (48 h ) (mg/L)
IGC50 (48 h ) (mg/L)
LD50 (mg/kg)
DTG
2.92
5.53
602
DTG-150
27.5
np
498
DTG-166
23.5
np
993
DTG-184
22.7
np
429
DTG-228
5.00
np
519
DTG-238
3.14
6.35
553
DTG-254
1.68
8.26
1036
DTG-256
3.43
9.19
1259
DTG-270
2.16
6.52
1121
DTG-272
2.52
11.2
2352
DTG-274
0.70
2.29
1178
DTG-286
8.28
9.80
527
DTG-288
0.76
1.98
721
DTG-290
0.93
2.14
1014
DTG-306
0.95
2.06
1870
DTG-308
0.49
1.24
1109
DTG-318
0.39
4.35
1103
DTG-324
0.74
1.11
940
DTG-332
0.51
1.55
1627
DTG-334
0.87
3.21
927
DTG-340
0.31
1.11
1074
DTG-250
0.79
2.38
1485
DTG-396
0.19
1.46
563
DTG-412
0.28
1.21
823
DTG-426
0.03
0.55
384
np: no p edic ion possible
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Page1

SUPPORTINGINFORMATIONTO:

CHLORINATION AND BROMINATION OF 1,3‐DIPHENYLGUANIDINE AND 1,3‐DI‐O‐
TOLYLGUANIDINE: KINETICS, TRANSFORMATION PRODUCTS AND TOXICITY
ASSESSMENT
Benigno J. Siei a 1, Rosa Mon es 1, A naud Tou e  2, Rosa io Rodil1
, Ra ael Cela 1,
He éGalla d2*,JoséBeni oQuin ana1*
1Depa men o Analy icalChemis y,Nu i ionandFoodSciences,Ins i u eo Food
AnalysisandResea ch(IIAA),Uni e sidadedeSan iagodeCompos ela,R/Cons an ino
Candei aS/N,15782–San iagodeCompos ela(Spain)
2Ins i u deChimiedesMilieuxe desMa é iauxdePoi ie s(IC2MP)UMRCNRS7285,
ÉcoleNa ionaleSupé ieu ed’Ingénieu sdePoi ie s(ENSIP),Uni e si édePoi ie s,1,
ueMa celDo é,TSA41105,86073–Poi ie s(F ance)
*Co espondingau ho s:
He éGalla d:he e.galla d@uni ‐poi ie s. 
JoséBeni oQuin ana.[email p o ec ed]

Page2

TABLEOFCONTENTS:
CONTENT Page
Tex S1.Fullkine icmodel5
Tex S2.Fu he expe imen sin es iga ingguanidinesb omina ion 7
Tex S3.Desc ip iono Mic o ox® es inhibi ioncalcula ions. 9
TableS1.Expe imen allyob ainedkapp o DPGa  hedi e en pH aluesand
co espondinghal ‐li escalcula ed o 10µMCl2(i.e.0.71mgCl2L‐1).
10
TableS2.Expe imen allyob ainedkapp o DTGa  hedi e en pH aluesand
co espondinghal ‐li escalcula ed o 10µMCl2(i.e.0.71mgCl2L‐1).
11
TableS3.Expe imen allyob ainedkapp,kine icsme hods o b omina iono 
DPGa  hedi e en pH aluesandco espondinghal ‐li escalcula ed o 10
µMB 2(i.e.0.71mgB 2L‐1).BPandTBPa e4‐b omophenoland2,4,6‐
ib omophenol, espec i ely.Valueso k e a eappa en  a econs an so BP
andTBPcalcula ed omHeebe al.(2014).
12
TableS4.Expe imen allyob ainedkapp,kine icsme hods o b omina iono 
DTGa  hedi e en pH aluesandco espondinghal ‐li escalcula ed o 10
µM B 2(i.e.0.71mgB
2L
‐1). BP and TBP a e 4‐b omophenol and 2,4,6‐
ib omophenol, espec i ely.Valueso k e a eappa en  a econs an so BP
andTBPcalcula ed omHeebe al.(2014).
13
Table S5.Speci ic a econs an so halogena iono DPGandDTG
de e minedbykine icmodellingconside ingsimpleo  ullkine icmodel.
14
TableS6.Lis o chlo ina ionandb omina ionDPGTPs. 15
TableS7.Lis o chlo ina ionandb omina ionDTGTPs. 17
Figu eS1.Exampleso pseudo‐ is ‐o de kine icsplo sob aineddu ing he
chlo ina iono DPG(Phospha ebu e 10mM).(a)In luenceo pHand
quenchingme hods([DPG]01µM,[chlo ine]010µM).The eac ionwas
21
Page3

s oppedby hiosul a e( ullci cle),ammonium(openci cle)o manualdi ec 
injec ionwasused(opensqua e).Linea  eg essionsa eplo ed o 
educ ionby hiosul a e.(b)In luenceo chlo ineconcen a ions([DPG]01
µM,pH6.1).The eac ionwass oppedby hiosul a e.
Figu eS2.Exampleso pseudo‐ is o de kine icsplo sob ained o 
b omina iono DPG(Phospha ebu e 10mM).(a)In luenceo pH([DPG]01
µM).The eac ionwass oppedby hiosul a e.(b)In luenceo b omine
concen a ionandquenchingme hod([DPG]01µM,pH5.6).The eac ion
wass oppedby hiosul a e( ullci cle)o manualdi ec injec ionwasused
(openci cle).Linea  eg essionisplo ed o  educ ionby hiosul a e.
22
Figu e S3. De e mina ion o  appa en  second o de  a e cons an s o 
b omina ion o  DPG by using compe i ion kine ics me hod wi h (a) 4‐
b omophenol (BP) and (b) 2,4,6‐ ib omophenol (TBP) as e e ence
compound ([DPG]05µM,[BP]
0o [TBP]
05µM,[b omine]
00 o10µM,
phospha ebu e 10mM)
23
Figu eS4.De e mina iono appa en  a econs an o b omina iono TBP
(pH6.94,[TBP]01µM,[b omine]020µM,10mMphospha ebu e )
24
Figu eS5.Decayo oxidan  esponsedu ingb omina iono DPG(pH6.9,
[DPG]050µM,[b omine]05µM,phospha ebu e 10mM)
25
Figu e S6. UV/ isible spec a o  DPG solu ion be o e and a e  b omine
addi ion (pH 6.9, 10 mM phospha e bu e , [DPG]050µM,[B
2]0 50 µM,
educ ionbyanexcesso  hiosul a e)
25
Figu eS7.Ch oma og amsandMS/MSspec ao DPGandi sTPs. 26
Figu eS8.Ch oma og amsandMS/MSspec ao DTGandi sTPs. 38
Figu eS9.Plo summa izing he o ma iono TPs omDPG(10µMDPG+
100µMCl2)a di e en  eac ion imes:(a) esul sno malized o he ime
when heTP eachedi smaximum;(b) esul sno malizedbyassuming ha 
he esponseo  heTPswasequal oDPG,excep  o DPG‐136,DPG‐119and

Page10

Table S1. Expe imen ally ob ained kapp o DPGa  hedi e en pH aluesand
co espondinghal ‐li escalcula ed o 10µMCl2(i.e.0.71mgCl2L‐1).
pH kapp(M‐1s‐1) 1/2(s)
5.0 32 2203
5.6 73 950
5.9 170 408
6.0 194 357
6.1 230 301
6.4 580 120
6.5 670 104
6.7 1310 53
7.0 2400 29
7.5 3923 18
8.0 9760 7
8.4 11061 6
9.0 10776 6
9.5 4567 15
10.0 5999 12
11.0 1269 55
11.7 372 186



Page11

Table S2. Expe imen ally ob ained kapp o  DTG a  he di e en  pH alues and
co espondinghal ‐li escalcula ed o 10µMCl2(i.e.0.71mgCl2L‐1).
pH kapp(M‐1s‐1) 1/2(s)
4.9 25 2803
5.5 148 469
6.0 364 190
6.5 1764 39
7.0 5599 12
7.5 7941 9
8.0 11327 6
9.9 18297 4
11.0 8205 9


Page12

TableS3.Expe imen allyob ainedkapp,kine icsme hods o b omina iono DPGa  he
di e en pH aluesandco espondinghal ‐li escalcula ed o 10µMB 2(i.e.0.71mg
B 2L‐1).BPandTBPa e4‐b omophenoland2,4,6‐ ib omophenol, espec i ely.Values
o k e a eappa en  a econs an so BPandTBPcalcula ed omHeebe al.(2014).
pH Kine icme hod k e
(M‐1s‐1)
kapp
(M‐1s‐1)
½
(s)
5.00 di ec  ‐ 76 916.9
5.18 di ec  ‐ 84 827.0
5.50 di ec  ‐ 314 220.7
5.63 di ec  ‐ 195 354.7
5.74 di ec  ‐ 334 207.5
6.00 di ec  ‐ 228 304.0
6.83 di ec  ‐ 344 201.6
7.00 compe i ionwi hTBP 2112 311 222.9
7.08 di ec  ‐ 363 190.9
7.57 di ec  ‐ 460 150.7
8.00 di ec  ‐ 940 73.7
8.00 compe i ionwi hTBP 2842 4276 16.2
8.05 compe i ionwi hBP 26900 16829 4.1
8.50 di ec  ‐ 962 72.1
8.46 compe i ionwi hBP 50700 61934 1.1
8.95 compe i ionwi hBP 93300 158254 0.4
9.06 di ec  ‐ 2240 30.9
9.65 compe i ionwi hBP 439000 289338 0.2
10.00 compe i ionwi hBP 245910 246000 0.3
10.48 compe i ionwi hBP 93300 96164 0.7
11.03 compe i ionwi hBP 27700 37977 1.8
11.75 compe i ionwi hBP 5360 8245 8.4
Page13

TableS4.Expe imen allyob ainedkapp,kine icsme hods o b omina iono DTGa  he
di e en pH aluesandco espondinghal ‐li escalcula ed o 10µMB 2(i.e.0.71mg
B 2L‐1).BPandTBPa e4‐b omophenoland2,4,6‐ ib omophenol, espec i ely.Values
o k e a eappa en  a econs an so BPandTBPcalcula ed omHeebe al.(2014).
pHKine icme hod
k e 
(M‐1s‐1)
kapp
(M‐1s‐1)
½
(s)
5.22 di ec  ‐ 36 1918.4
5.55 di ec  ‐ 48 1438.6
6.04 di ec  ‐ 113 611.7
6.50 di ec  ‐ 277 250.3
6.85 di ec  ‐ 318 217.8
6.99 compe i ionwi hTBP 2112 127 545.8
7.50 di ec  ‐ 417 166.2
8.02 compe i ionwi hTBP 2842 797 86.9
9.15 compe i ionwi hBP 698000 47857 1.4
9.90 compe i ionwi hBP 294000 58707 1.2
10.55 compe i ionwi hBP 80300 42185 1.6
10.94 compe i ionwi hBP 33900 25430 2.7
11.80 compe i ionwi hBP 4780 8355 8.3

Page14

Table S5. Speci ic a e cons an s o  halogena ion o  DPG and DTG de e mined by
kine icmodellingconside ingsimpleo  ullkine icmodel.
 Fullmodel Simplemodel
k1k
2k
3R
2k R
2
Chlo ina ion DPG 4.1x106 1.4x10‐7 2.4x10‐6 0.898 4.1x106 0.898
DTG 2.4x107 2.5x10‐8 3.9x103 0.975 2.6x107 0.960
B omina ion DPG 8.3x106 2.2x10‐8 0 0.950 8.3x106 0.950
DTG 5.5x106 1.2x10‐6 0 0.997 5.5x106 0.997
Page15

TableS6.Lis o chlo ina ionandb omina ionDPGTPs.
Name Expe imen al
m/z
Molecula 
o mula
Theo e ical
m/z
E o 
(ppm)
E o 
(mDa) DBE Sco e
(%) S uc u e
DPG 212.1182 C13H13N3‐ ‐‐9‐

DPG‐119 119.0604 C7H6N2 119.0604 ‐0.21 ‐0.03 6 100.00

DPG‐136 136.0867 C7H9N3 136.0869 1.66 0.22 5 99.69

DPG‐170 170.0475 C7H8N3Cl 170.0480 2.67 0.45 5 98.90

DPG‐210 210.1025 C13H11N3 210.1026 0.35 0.07 10 99.97

DPG‐214 213.9968 C7H8N3B  213.9974 2.99 0.64 5 98.14

N
H
NH2
NH
B

Page16

DPG‐226 226.0975 C13H11N3O 226.0975 ‐0.05 ‐0.01 10 100.00

DPG‐228 228.1131 C13H13N3O 228.1131 0.17 0.04 9 99.99

DPG‐246 246.0795 C13H12N3Cl 246.0793 ‐1.01 ‐0.25 9 99.74

DPG‐260 260.0579 C13H10N3OCl 260.0585 2.38 0.62 10 98.51

DPG‐290 290.0287 C13H12N3B  290.0287 0.13 0.04 9 100.00

DPG‐294 294.0186 C13H9N3OCl2 294.0195 3.22 0.94 10 96.91

N
H
N
H
NH
B
Page17

TableS7.Lis o chlo ina ionandb omina ionDTGTPs.
Name Expe imen al
m/z
Molecula 
o mula
Theo e ical
m/z
E o 
(ppm)
E o 
(mDa) DBE Sco e(%) S uc u e
DTG 240.1495 C15H17N3‐‐9‐

DTG‐150 150.1026 C8H11N3 150.1026 ‐0.18 ‐0.03 5 100.00

DTG‐166 166.0971 C8H11N3O 166.0975 2.35 0.39 5 99.17

DTG‐184 184.0632 C8H10N3Cl 184.0636 2.19 0.40 5 99.17

DTG‐228 228.0131 C8H10N3B  228.0131 ‐0.06 ‐0.01 5 100.00

DTG‐238 238.1336 C15H15N3 238.1339 1.16 0.27 10 99.68
N
H
NH2
NH
B
HN
HN
HN
Page18

DTG‐254 254.1285 C15H15N3O 254.1288 1.14 0.29 10 99.66

DTG‐256 256.1436 C15H17N3O 256.1444 3.29 0.84 9 97.23

DTG‐270* 270.1238 C15H15N3O2 270.1237 ‐0.36 ‐0.10 10 99.96

DTG‐272 272.1391 C15H17N3O2 272.1394 0.93 0.25 9 99.75

DTG‐274* 274.1079 C15H16N3Cl 274.1106 9.71 2.65 9 79.46

DTG‐286* 286.1182 C15H15N3O3 286.1186 1.47 0.42 10 99.36

DTG‐288* 288.0895 C15H14N3OCl 288.0898 1.10 0.32 10 99.64

HN
HN
HN
OH
Cl
Page19

DTG‐290 290.1042 C15H16N3OCl 290.1055 4.38 1.27 9 95.41

DTG‐306* 306.0995 C15H16N3O2Cl 306.0931 2.89 0.88 9 97.39

DTG‐308 308.0706 C15H15N3Cl2 308.0716 3.19 0.98 9 96.81

DTG‐318 318.0600 C15H16N3B  318.0600 0.12 0.04 9 100.00

DTG‐324 324.0657 C15H15N3Cl2O 324.0665 2.46 0.79 9 97.98

DTG‐332* 332.0393 C15H14N3OB  332.0393 0.00 0.00 10 100.00

DTG‐334 334.0550 C15H16N3OB  334.0550 ‐0.15 ‐0.05 9 99.99




Figu


eS7.Ch o
m
m
a og ams andMS/M

Page26
Sspec ao DPGandi

sTPs.



Figu


eS7.Ch o
m
m
a og ams andMS/M

Page27
Sspec ao DPGandi sTPs.Con

inued.


Figu

eS7.Ch o
m
m
a og ams andMS/M

Page28

Sspec ao DPGandi sTPs.Con


inued.

Figu

eS7.Ch o
m
m
a og ams andMS/M

Page29

Sspec ao DPGandi sTPs.Con


inued.

Figu

eS7.Ch o
m
m
a og ams andMS/M

Page30

Sspec ao DPGandi sTPs.Con

inued.


Figu

eS7.Ch o
m
m
a og ams andMS/M

Page31

Sspec ao DPGandi sTPs.Con


inued.


Figu

eS7.Ch o
m
m
a og ams andMS/M

Page32


Sspec ao DPGandi sTPs.Con


inued.

Figu

eS7.Ch o
m
m
a og ams andMS/M

Page33

Sspec ao DPGandi sTPs.Con


inued.

Figu

eS7.Ch o
m
m
a og ams andMS/M

Page34

Sspec ao DPGandi sTPs.Con


inued.

Figu

eS7.Ch o
m
m
a og ams andMS/M

Page35

Sspec ao DPGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page42

Sspec ao DTGandi sTPs.Con


inued.


Figu

eS8.Ch o
m
m
a og ams andMS/M

Page43

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page44

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page45

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page46

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page47

Sspec ao DTGandi sTPs.Con


inued.


Figu

eS8.Ch o
m
m
a og ams andMS/M

Page48

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page49

Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page50


Sspec ao DTGandi sTPs.Con


inued.

Figu

eS8.Ch o
m
m
a og ams andMS/M

Page51

Sspec ao DTGandi sTPs.Con


inued.