Journal of Cleaner Production 262 (2020) 121323

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Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Balancing chemical function with reduced environmental health

hazards: A joint probability approach to examine antimicrobial
product efficacy and mammalian toxicity
Rok Fink a, 1, Zhen Wang b, c, d, e, 1, Martina Oder a, Bryan W. Brooks b, e, *
a
University of Ljubljana. Faculty of Health Sciences, Zdravstvena pot 5, 1000, Jubljana, Slovenia
b
Department of Environmental Science, Institute of Biomedical Studies, Baylor University, Waco, TX, USA
c
Institute of Marine Sciences, Shantou University, Shantou, 515063, China
d
Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, China
e
Guangdong Key Laboratory of Environmental Pollution and Health, School of Environment, Jinan University, Guangzhou, 511443, China

a r t i c l e i n f o a b s t r a c t

Article history: The fourth principle of Green Chemistry, a critically important area of sustainability science, identifies
Received 11 August 2019 that substances should maintain intended functions while possessing limited intrinsic hazards to public
Received in revised form health and the environment. In the present study, we empirically determined efficacy of 20 cleaning
7 January 2020
products against E. coli attachment to surfaces. Subsequently, probabilistic assessments using novel
Accepted 23 March 2020
Available online 4 April 2020
chemical efficacy distributions (CEDs) were conducted. Results for most chemicals indicated bacterial
detachment with increasing concentration. Using a threshold concentration of 5th centiles, these
Handling editor: Jun Bi cleaning product ingredients were predicted to detach E. coli (Lowest Observable Effect Concentration) at
or below 0.7 (0.2, 2.2) mg L
1 for 5% of ingredients. We then employed chemical toxicity distributions
Keywords: (CTDs) to examine acute toxicity, based on currently available information, for common mammalian
Cleaning product ingredients models. Results demonstrate that probabilities of associated hazards for rat (oral exposure route), mouse
Antimicrobial efficacy (oral exposure route), rabbit (dermal exposure route), and rat (inhalation exposure route) were 43.2%,
Mammalian toxicity 36.7%, 38.6% and 75.5%, respectively. A novel joint probability distribution analysis approach was then
Chemical toxicity distributions
developed and applied to identify substances with various efficacy (CED) and hazard (CTD) character-
Thresholds of toxicological concern
istics. Our observations indicate that combining efficacy and toxicity information using joint probability
Joint probability distribution
curves may be useful for identifying classes of antimicrobial products, and other chemical classes, to
optimize efficacy while minimizing environment and health hazards.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction
Green chemistry presents critically important opportunities for
innovation of importance to sustainability (Anastas, 2009) and is
necessary to support the United Nations Sustainable Development
Goals (Anastas and Zimmerman, 2018; Brooks, 2018, 2019). In fact,
recent key question exercises have identified priority research
needs related to green and sustainable chemistry and engineering
to achieve more sustainable environmental quality (Van den Brink
* Corresponding author. Environmental Health Science Program, Department of
Environmental Science, Institute of Biomedical Studies, Baylor University, Waco, TX,
USA.
E-mail address: Bryan_Brooks@baylor.edu (B.W. Brooks).
1
R Fink and Z Wang contributed equally to this work.
et al., 2018; Fairbrother et al., 2019). To reduce hazards and risks to
public health and the environment, advances are being made in
green chemistry in which attributes of industrial chemicals are
being identified with improved safety profiles (e.g., lower toxicity,
increased biodegradability, decreased bioaccumulation)
(Voutchkova et al., 2010; Erythropel et al., 2018; Brooks, 2019).
Through diverse studies, including the Molecular Design Research
Network (MoDRN), sustainable molecular design guidelines are
being developed to identify attributes of chemicals with elevated or
reduced hazards profiles (Coish et al., 2018). This area directly ex-
tends from the fourth principle of Green Chemistry, which states
“Chemical products should be designed to preserve efficacy of
function while reducing toxicity” (Anastas and Warner, 1998).
However, research is specifically needed to ensure that unintended
consequences are avoided, including regretful substitutions of one

https://doi.org/10.1016/j.jclepro.2020.121323
0959-6526/© 2020 Elsevier Ltd. All rights reserved.
2 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323
chemical product for another that is not more sustainable and less
hazardous (Zimmerman and Anastas, 2015).
Efforts are also advancing to develop biologically derived or
inspired cleaning products (Marchant and Banat, 2012), ideally
with reduced hazards to public health and the environment. Such
advances are important because chemicals in cleaning products can
be absorbed dermally or through inhalation of household dust and
ingestion of chemical residues left on dishes and cutlery. In fact, we
recently performed novel comparative human health hazard as-
sessments of hundreds of cleaning products and formulary in-
gredients, which may support future product substitutions and
assessments (Wang et al., 2018, 2019). Cleaning products are
important interventions to manage interactions between bacteria
and material surfaces, and play important roles in food safety, the
pharmaceutical industry, medicine, households and in other facil-
ities and processes where hygiene is important (Fink et al., 2017b).
Because outbreaks are consistently reported, microorganisms pre-
sent critically important public health issues for which environ-
mental health professionals implement systems to reduce adverse
outcomes. Consequently, industries, public authorities and con-
sumers are concerned about health risks from bacteria and other
microorganisms (Faille et al., 2017), particularly associated with
compromised water quality and food safety, two of the most
commonly delivered programs by environmental health practi-
tioners (Gerding et al., 2019; Brooks et al., 2019).
For decades, chemical products have been employed to control
population growth of microorganisms. Thus, cleaning products are
often required within routine hygiene programs, from food prep-
aration and processing to consumer practices in residences.
Commonly used cleaning products in households and industrial
facilities include substances such as acids, bases, halogens, qua-
ternary ammonium compounds, amphoteric products, surfactants,
peroxides and others (Giaouris and Simo ~ es, 2018a). These chem-
icals commonly react with a number of cellular structures (e.g., cell
wall, membranes, enzymes, genetic material, cytoplasm) causing
destruction or reducing microbial reproduction (Simo ~ es, 2011; Srey
et al., 2013). Extensive research efforts examine inhibitory and
bacteriostatic activity of cleaning products, where bacterial cells in
liquid solution are exposed to various levels of chemicals and
product efficacy is subsequently identified (Kwak et al., 2017;
Pandya et al., 2017; Baptista et al., 2018). Such experimental
methods, however, do not include determination of cleaning
product efficacy for microorganisms that are attached to surfaces.
Surprisingly, little is known about the efficacy of cleaning
products on cells that are already attached to surfaces. In fact,
bacterial cells can be found as free flowing planktonic cells in bulk
solution, but commonly tend to adhere to surfaces (Flemming and
Wingender, 2010). Bacterial cells can attach to surfaces based on
gravitation, motility, diffusion or liquid shear forces, in which sur-
face interactions are based on van der Waals interactions, hydro-
phobic, acid-base and electrostatic interactions. Initial adhesion is a
reversible process where, due to Brownian motion and mild shear
forces, bacteria can be removed from surfaces (Simo ~es et al., 2010;
Khelissa et al., 2017). Subsequently, adhesion can become irre-
versible when cells starts anchoring appendages to surfaces and/or
producing exopolysaccharide substances to strengthen position.
Removal of irreversibly attached bacterial cells is difficult and re-
quires application of strong shear force or chemical agents (e.g.,
enzymes, detergents, surfactants, sanitizers, heat; Frank and
Chmielewski, 2001; Fink et al., 2017a). Joseph et al. (2001) previ-
ously demonstrated that 10 ppm of chlorine can kill 6 log bacterial
cells, but only kills 1 log when cells are attached to a surface.
Moreover, other researchers reported that total elimination of
adhered cells is difficult (Shi and Zhu, 2009; Fink et al., 2017a). The
traditional approach to control this problem has been to treat
material surfaces with classic and aggressive chemicals such as
chlorine, hydrogen peroxide or ozone that efficiently destroys and
remove cells from surfaces. Herein, selecting chemical products
with appropriate efficacy, but with limited environment and health
hazards and risks represents an important consideration for diverse
stakeholders. Through the Cleaning Product Ingredient Safety
Initiative (CPISI), we recently identified common ingredients in and
functional uses of cleaning products (DeLeo et al., 2018), which is
supporting novel environmental and human health hazard and risk
assessments. However, developing unique approaches to aid se-
lection of efficacious cleaning agents for surface attached bacteria
while balancing environment and health hazards is necessary.
In the present study, we present a novel joint probability dis-
tribution analysis (JPDA) approach to support balancing antimi-
crobial function with environmental health hazards. We initially
examined the efficacy of 20 selected cleaning product ingredients
to detach E. coli from a representative surface. Subsequently,
probabilistic hazard assessments (PHA) using chemical efficacy
distributions (CEDs) were conducted using newly generated effi-
cacy data here and corresponding mammalian toxicology data us-
ing chemical toxicity distributions (CTDs) from the publicly
available CPISI database (http://www.cleaninginstitute.org/CPISI/).
We also identified associated thresholds of toxicological concern
(TTCs), based on our analysis of these ingredients, which could be
extended to other classes or uses in the future. We then conducted
JPDA to identify potential relationships between cleaning product
efficacy and mammalian environmental hazards.
2. Methods
Here we studied 20 common cleaning products ingredients:
alcohol, benzotriazole, calcium chloride, Citrus aurantium dulcis
(Orange) peel oil, ethanolamine, magnesium hydroxide, magne-
sium sulfate, methyl alcohol, phenoxyisopropanol, Pinus palustris
oil, polyethylene glycol, Prunus amygdalus dulcis (sweet almond)
oil, sodium hypochlorite, sodium laureth sulfate, sodium lauroyl
sarcosinate, sodium metabisulfite, terpineol, titanium dioxide,
triethanolamine, and zinc chloride. All substances were purchased
from Sigma Aldrich (USA), except etheric oils of orange peel, pine
and almond were purchased from Favn (Slovenia).
2.1. Efficacy of cleaning products ingredients for E. coli detaching
Standard strains of E. coli ATCC 35218, obtained from the Czech
Collection of Microorganisms (Brno, Czech Republic), were used.
E. coli is a clinically important opportunistic pathogen, associated
with multiple infections. E. coli is thus often used as a model or-
ganism, an indicator of fecal contamination and is therefore
included in hygiene assessment (Belluco et al., 2016; Chien et al.,
2017; Oder et al., 2017).
Bacteria from the collection were transferred on nutrient agar
and incubated at 37  C 24h. After that, a single colony of strain was
transferred from nutrient agar to nutrient broth (Biolife, Italy) and
incubated in the same conditions. For adhesion testing the method
of Bohinc et al. (2014) was used with modifications as follows.
Overnight E. coli cultures were diluted in a 1:300 ratio with a fresh
nutrient medium. Clean and sterile glass 1 cm  1 cm (Isolab,
Germany) in size were transferred to asterile Petri dish and a pre-
pared suspension of E. coli bacteria was added. After this step,
samples were incubated for 1 h at 37  C to achieve initial bacterial
adhesion to glass. After the incubation period, the medium was
removed. Next, none or loosely attached cells were removed by
3 mL Phosphate-buffered saline (PBS) buffer (0.026 g KH2PO4,
0.047 g K2HPO4 in 1 L). Bacterial cells remaining on surface were
stained 0.1% crystal violet (Merck, Germany) suspension for 5 min.
 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323
Subsequently, dye was removed and the number of cells were
counted by light microscopy with an Olympus CX40 and CCD CMOS
camera. This approach allowed us to determine the number of cells
attached to the surface before cleaning products were applied.
Samples with attached E. coli were then exposed to each of the
cleaning products at five different concentrations (1, 2, 3, 4, and 4.3
log mg/L) for 10 min according to EN 13697 (ENAC, 2015). Next, the
cleaning product was removed, and the surface was washed with
3 mL of PBS buffer, stained with crystal violet and counted under
the microscope. Efficacy was determined as percentage of cells left
on the glass surface after exposure to cleaning product relative to
the control with no cleaning product. All experiments were per-
formed with five parallels and three repetitions.
The 5%, 10%, 20% and 50% effective concentrations (ECs) for each
chemical were calculated, respectively, from data distribution or
regression based on dose-response curves (treatment vs efficacy;
Solomon et al., 2000). For the observed non-linear relationships,
non-linear regression based on Hill’s model was used. For observed
linear relationships, a simple linear regression was employed
(GraphPad Prism™, version 5.00, San Diego, CA, USA). The no
observed effect concentration (NOEC) and/or lowest observed ef-
fect concentration (LOEC) for each experiment were also estimated
by comparing efficacy of treatments and control using one-way
analysis of variance (ANOVA) and corresponding post hoc Tukey’s
tests (R, version 3.5.1).
2.2. Probabilistic efficacy and hazard assessments
2.2.1. Chemical efficacy and toxicity distributions, and threshold
concentrations (TCs)
Probabilistic hazard assessment (PHA) using CEDs was con-
ducted using efficacy data (LOECs, NOECs, and EC10s/50s) for the
selected 20 cleaning product ingredients generated from the pre-
sent study. PHA was also performed for corresponding mammalian
hazard data (acute LD50s) and CTDs collected from the CPISI
database (http://www.cleaninginstitute.org/CPISI/). The CEDs and
CTDs generated using efficacy data and mammalian data, respec-
tively, were then compared using one-way analysis of covariance
(ANCOVA, SPSS, Chicago, IL, USA) for the differences of slopes and
intercepts of the both CEDs and CTDs, which were fitted with the
log-normal model. Visual comparisons of the extent of either
congruence or discrepancy of datasets were also employed.
CEDs and CTDs were generated followed previously reported
approaches (Wang et al., 2018). In brief, any outlier(s) detected by
the Grubb’s test (Grubbs, 1969) or Tietjen-Moore test (Tietjen and
Moore, 1972) was excluded for each dataset. A geometric mean
value was used when there were multiple data points available for
an ingredient. Datasets having a minimum of 5 data points (in-
gredients) were considered for CEDs and CTDs constructions.
Normality of residuals of each dataset (log-transformed) was
checked by the Shapiro-Francia test (Shapiro and Francia, 1972),
and the goodness of fit at the lower tails of each CED or CTD was
assessed by Anderson-Darling test (Anderson and Darling, 1954).
Efficacy and hazard data were then ranked in ascending order, and
percentiles were assigned from the Weibull formula:
i image analysis showed that sodium metabisulfite removed only a
Percentile ¼  100%
nþ1
where i is the rank of the datum in ascending order and n is the total
number of data points. The CEDs and CTDs were then constructed
(SigmaPlot, version 13.0, San Jose, CA, USA) and fitted by log-
normal regression model. For each CED or CTD, threshold concen-
tration (TC) values at 1st, 5th, 10th and 50th, 90th, 95th, and 99th
centiles and their 95% confidence intervals (95% CIs) were
3
determined from the parametric log-normal regression model,
multiplied by Monte Carlo simulation (resampled 5000 times).
These computations were accomplished with the model proced-
ures available in the Statistical Analysis System package (SAS,
version 9.4, Cary, NC, USA). Concurrent meta-analyses were also
conducted using similar substance for the pairwise datasets to
examine potential influences of chemical compositions for CEDs
and CTDs comparisons and the following JPD analyses.
2.2.2. Joint probability distribution analysis
JPDA was also conducted to further examine relationships be-
tween efficacy and mammalian toxicity, and joint probability
curves (JPCs) were developed following Solomon et al. (2000) with
minor adjustment. In the present study, a JPC was developed having
the percentile of chemicals toxicity being encountereed (p%) as x-
axis and probability of exceedance of concentration (TCp) affecting
efficacy of cleaning product ingredients as y-axis. In brief, the slope
(a) and intercept (b) parameters from the log-normal fitted CEDs or
CTDs were used to calculate the probability of concentrations (TCs)
associated with efficacy or toxicity (acute LD50s) in a specified
percentage of chemicals (p%):
Percent of exceedence ¼ 100%
NORMDISTðae  log10 xðpÞ
 Þ þ be Þ
(2)
where x(p) is the concentration affecting p% of chemicals from CEDs
or CTDs (e.g., TCs at 1st, 5th, 10th and 50th, 90th, 95th, and 99th
centiles), ae and be are the slope and intercept parameters for log-
normal fitted CEDs or CTDs, and NORMSDIST returns the standard
normal cumulative distribution function of a selected value.
3. Results and discussion
3.1. Efficacy of cleaning products ingredients for E. coli detachment
Results of our cleaning product ingredients efficacy experiments
showed that for most of the 20 chemicals examined E. coli
detaching potential was increased by concentration (Fig. 1). We
found for all chemicals at concertation of 1 log mg L
1 efficacy of
E. coli reduction was relatively small, but was increased by
increasing the concertation up to 4.3 log mg L
1. More detailed
analysis displayed large differences between ingredients. We found
that at the highest concentration (4.3 log mg L
1) tested the most
efficient was sodium hypochlorite, which resulted in total elimi-
nation of E. coli (100%), followed by ehanolamine (81%), sodium
laureth sulfate (74%), benzotriazole (72%) and titanium dioxide
(64%). For other tested chemicals, E. coli reduction efficacy was
below 50% relatively to control with no treatment. However, so-
dium metabisulfite and ethanol showed the lowest efficacy with
only 10% E. coli detachment at the highest concentration (4.3 log mg
L
1) examined (Fig. 1). Fig. 2 displays the number of E. coli cells on
the glass surface before exposure (Fig. 2a) to cleaning products and
after 10 min exposure to the highest concentration of sodium hy-
pochlorite (Fig. 2b) and sodium metabisulfite (Fig. 2c). Microscopic
(1) few bacterial cells in comparison to sodium hypochlorite, which
eradicated all cells from the surface.
3.2. Probabilistic efficacy and hazard assessments
Probabilistic hazard assessment using CTDs are useful to sup-
port hazard and risk assessment and comparative toxicology when
toxicity information for a chemical is not available, because these
4 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323

Fig. 1. Efficacy of cleaning products ingredients for E. coli detachment from a glass surface.

approaches inherently account for uncertainties and are data-
driven (Brain et al., 2006; Wang et al., 2018, 2019). The use of
CTDs allows hazard and risk assessors to generate a criterion
threshold value to protect a desired level of effect (e.g., 1%, 5% or
another centile of interest) for a particular endpoint for a particular
chemical class (Brain et al., 2006), as illustrated by our recent work
with common cleaning product ingredients (Wang et al., 2018).
Here we applied the CTD concept to toxicity data, and similarly
derived CEDs for newly derived empirical antimicrobial efficacy
information. Among all datasets used for CEDs and CTDs
 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323 5

Fig. 2. Number of E. coli cells on glass surface before and after exposure to cleaning products.

constructions, no outliers were identified; all data were thus used
in our analyses (see Table 1 for efficacy data and Table S1 for
mammalian toxicity data). With sufficient data points (5), CEDs
and CTDs were generated using efficacy data (LOECs, NOECs, and
EC10s/50s) and acute LD50s for rat (oral and inhalation), mouse
(oral), and rabbit (dermal), respectively (Fig. 3). All datasets passed
the Shapiro-Francia test (normality of residuals; p > 0.05) and
Anderson-Darling test (p > 0.05; Table S2). Each CED or CTD was
then fitted by a log-normal model; corresponding TCs and 95% CIs
estimated by Monte Carlo simulation are presented in Table 2.
Using TC5s from CEDs, for example, the cleaning product in-
gredients examined here would be expected to detach E. coli (LOEC)
at or below 0.7 (0.2, 2.2) mg/L for 5% of ingredients. Similarly, from
CTDs, these substances tended to be acutely toxic (lethal) at or
below 392 (192, 631), 22 (1.8, 89), 585 (163, 1166), and 913 (588,
1236) mg/kg bw/day for 5% of chemicals to rat under oral and
inhalation exposures, mouse under oral and rabbit under dermal
exposures, respectively. Comparatively, with more ingredients

Table 1
Summary of estimated ECs, NOECs and LOECs (if any) from dose-response relationships, and corresponding one-way ANOVA and Tukey’s post hoc test for E. coli exposed to
selected cleaning product ingredients.

Chemicals CAS Dose-response relationship ECs NOEC LOEC

Equation R2 EC5 EC10 EC20 EC50

Alcohol 64-17-5 NA NA NA NA NA NA 20000 NA

Benzotriazole 95-14-7 NA NA NA NA NA NA 1000 2000
Calcium Chloride 10043- NA NA NA NA NA NA 10000 20000
52-4
Citrus Aurantium Dulcis (Orange) 8008- NA NA NA NA NA NA 20000 NA
Peel Oil 57-9
Ethanolamine 141-43- y ¼ 100/((1 þ 10^(3.843- 0.818 17.4 (0.01, 37661) 187 (0.832, 711 (17.3, 6962 (1001, 100 1000
5 x)  0.608)) 42476) 29232) 48416)
Magnesium Hydroxide 1309- y ¼ 100/((1 þ 10^(4.48- 0.943 1.57 (0.013, 191) 19.0 (0.640, 561) 282 (39.4, 2026) 28611 (5477, 100 1000
42-8 x)  0.300)) 149449)
Magnesium Sulfate 7487- NA NA NA NA NA NA 100 1000
88-9
Methyl Alcohol 67-56-1 NA NA NA NA NA NA 10000 20000
Phenoxyisopropanol 770-35- NA NA NA NA NA NA NA 10
4
Pinus Palustris Oil 8002- NA NA NA NA NA NA 20000 NA
09-3
Polyethylene Glycol 25322- y ¼ 9.23xþ22.0 0.821 0.0145 (3.39E-31, 0.050 (2.19E-27, 0.610 (8.51E-20, 1083 (24.3, NA 10
68-3 2.94) 6.00) 26.0) 460257)
Prunus Amygdalus Dulcis (Sweet 8007- NA NA NA NA NA NA 20000 NA
Almond) Oil 69-0
Sodium Hypochlorite 7681- y ¼ 100/((1 þ 10^(2.531- 0.995 12.5 (3.75, 41.4) 28.8 (11.2, 74.6) 71.7 (35.5, 145) 340 (215, 537) NA 10
52-9 x)  0.891))
Sodium Laureth Sulfate 68891- y ¼ 100/((1 þ 10^(3.657- 0.967 33.8 (1.65, 692) 117 (11.3, 1218) 452 (87.2, 2341) 4538 (1872, 10 100
38-3 x)  0.601)) 11000)
Sodium Lauroyl Sarcosinate 137-16- NA NA NA NA NA NA 20000 NA
6
Sodium Metabisulfite 7681- NA NA NA NA NA NA 20000 NA
57-4
Terpineol 8000- y ¼ 100/((1 þ 10^(4.526- 0.906 0.133 (0.001, 153) 2.13 (0.222, 438) 96 (5.82, 1583) 33579 (3234, 10 100
41-7 x)  0.237)) 348691)
Titanium Dioxide 13463- y ¼ 9.94xþ33.4 0.985 0.0007 (3.64E-7, 0.003 (2.77E-6, 0.033 (0.0002, 69.5 (21.9, 153) NA 10
67-7 0.024) 0.06) 0.398)
Triethanolamine 102-71- NA NA NA NA NA NA 20000 NA
6
Zinc Chloride 7646- NA NA NA NA NA NA 20000 NA
85-7
6 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323

Fig. 3. Chemical toxicity distributions using (a) efficacy data (EC10/50s, NOECs and LOECs) generated from the current study and (b) acute LD50s for rat (oral and inhalation), mouse
(oral) and rabbit (dermal) using available data collated from the Cleaning Product Ingredient Safety Initiative database.

Table 2
Threshold concentrations (TCs; mg/kg bw/day) and their 95% confidence intervals (95% CIs) at 1st, 5th, 10th, 50th, 90th, 95th, and 99th percentiles for each chemical toxicity
distribution (CTD) considering all cleaning product ingredients of concern.
a
CTDs n y ¼ ax þ b TC1 (95% CI) TC5 (95% CI) TC10 (95% TC50 (95% TC90 (95% CI) TC95 (95% CI) TC99 (95% CI)
CI) CI)
a B R2
b
Efficacy LOEC (mg/L) 12 0.646 3.443 0.910 0.1 (0.01, 0.3) 0.7 (0.2, 2.2) 2.7 (0.8, 6.6) 256 (149, 24666 (10340, 90034 (31492, 1021373 (250088,
457) 89507) 440715) 8867332)
Efficacy NOEC c (mg/L) 16 0.595 3.014 0.764 0.3 (0.01, 2.) 3.7 (0.2, 16) 15 (1.4, 52) 2178 (866, 310132 (78065, 1264859 (243864, 17669786 (1979673,
4988) 2695337) 19535894) 750221673)
d
Efficacy EC10 (mg/L) 7 0.417 4.787 0.904 0.001 0.002 0.005 3.2 (0.7, 15) 2094 (244, 4701 (4571, 8976 (753, 1358313)
(0.00001, (0.00002, (0.0001, 328095) 497737)
0.014) 0.023) 0.04)
d
Efficacy EC50 (mg/L) 7 0.779 2.341 0.962 2.7 (0.5, 7.2) 20 (5.6, 41) 59 (21, 104) 2588 (1676, 114253 (54645, 334343 (137339, 2505773 (782528,
3576) 260881) 960768) 11261142)
Rat Oral Acute LD50 e (mg/ 20 54.3
141 0.809 162 (57, 297) 392 (192, 629 (365, 3328 (2781, 17613 (13336, 28247 (20101, 68515 (43029,
kg bw/day) 631) 948) 4481) 38755) 74491) 244768)
Rat Inhalation Acute LD50 f 7 0.713 2.400 0.879 2.4 (0.1, 16) 22 (1.8, 89) 71 (9.7, 228) 4426 (2187, 277481 (93467, 896878 (237949, 8099730 (1297518,
(mg/kg bw/day) 9492) 2443195) 13273962) 323875050)
Mouse Oral Acute LD50 5 1.559
1.222 0.976 197 (35, 510) 585 (163, 1046 (365, 8129 (5885, 63146 (35099, 112911 (55418, 335878 (127951,
(mg/kg bw/day) 1166) 1822) 11347) 149485) 353231) 1708911)
Rabbit Dermal Acute LD50 6 1.834
1.886 0.938 398 (217, 913 (588, 1420 (994, 6755 (5771, 32133 (24192, 49998 (35424, 114583 (71984,
(mg/kg bw/day) 605) 1236) 1818) 7784) 45405) 77045) 207656)

Note.
a
centile value ¼ NORMSDISTððae  log10 xðpÞÞ þ be Þ; where the NORMSDIST returns the standard normal cumulative distribution function of a selected value.
b
Lowest observed effect concentration (LOEC).
c
No observed effect concentration (NOEC).
d
10%/50% effective concentration (EC10/EC50).
f
Median lethal concentration (LC50).

included from diverse categories (n ¼ 314), a similar acute oral TC5
of 533 (506, 560) mg/kg bw/day was also identified for rats (over-
lapped 95% CIs of the both TC5s) (Wang et al., 2018).
In the current study, TC values and 95% CIs for a given endpoint
were estimated (Table 2). Our recent work, which examined many
more cleaning product ingredients than examined here, identified
novel TTCs for future hazard and screening level risk assessments
(Wang et al., 2018, 2019). It is also important to note that the linear
regression function for a particular distribution (e.g., y ¼ bx þ a;
Table 2) can be used in future efforts to estimate the probability of
finding a compound at or below a particular exposure threshold to
a common endpoint for a class of chemicals (Brain et al., 2006).
Additionally, CTDs can be useful to compare relative sensitivities
between models, toxicological or pharmacological endpoints, or
chemical classes among different distributions (Solomon et al.,
2000; Wang et al., 2018). In the present study, we also compared
CEDs generated using efficacy data to corresponding mammal
acute LD50 CTDs using all (Fig. 4: LOECs versus LD50s and
Figs. S1eS3 for NOECs/EC10s/EC50s versus LD50s) and similar
(Fig. S4) available data. Pairwise CEDs and CTDs between efficacy
data and mammalian acute LD50s visually diverged, which was
further supported by significant different slope and/intercept pa-
rameters (Table S3; except for efficacy NOECs and EC50s to inha-
lation acute LD50s using all and similar substances; respectively).
Corresponding TCs for both efficacy and acute LD50 CTDs were also
estimated and listed in Table S4.
In health hazard assessment, chemical (category) composition is
an important factor that can influence a probability distribution,
especially the lower end of CTDs where TC5s are normally derived,
and can also influence identification of potential candidates for
cleaning applications if higher efficacy with lower toxicity is
desired (as illustrated in Fig. 1). Considering category specific
 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323
Fig. 4. Comparison of chemical toxicity distributions (CTDs) using publicly available mammal acute LD50s and efficacy EC50s generated from the present study.
toxicity of cleaning product to the rat model (oral; Wang et al.,
2018), for example, chemicals examined here contained 17 sub-
stances from 9 categories and 3 chemicals of ungrouped sub-
stances. Nine categories included relative more toxic classes of
inorganic acids and salts (25%) and alkanolamines (10%) and ethers
(5%) with intermediate toxicity, whereas the least toxic categories
were aliphatic alcohols (15%) and glycerides (10%). Other categories
included alcohol ethoxysulfates (sodium laureth sulfate), aromatic
alcohols (phenoxyisopropanol), benzotriazoles (benzotriazole),
hydroxides (magnesium hydroxide). In addition, acute LD50s of the
20 selected chemicals (Fig. 2b) covered a wide range (7.0%e99.7%)
of CTD values when compared to our recent work (Wang et al.,
2018) examining acute LD50s of 314 cleaning product ingredients,
indicating the diverse toxicity of the selected chemicals considered
during the present study.
3.3. Joint probability distribution analysis
Balancing chemical use for protection of public health while
reducing hazards to humans and the environment has long rep-
resented an important consideration for environmental health
professionals. In the present study, the JPD concept (Solomon et al.,
2000) was employed to examine the probability of encountering
mammalian toxicity (CTDs) for a group of chemicals relative to
antimicrobial efficacy (CEDs) of removing attached E. coli (LOECs,
NOECs, EC10s/50s) for a proportion of these substances. In previous
7
applications for environmental risk assessment (Solomon et al.,
2000), the closer a JPC is to the coordinate axes, which represent
exposure and effects for a specific chemical, the lower the proba-
bility of risk. In the present study, we extended the JPD concept but
examined antimicrobial efficacy and effects. Here, differences
among the distance of the 4 JPCs (e.g., efficacy LOECs to mammalian
acute LD50s; Fig. 5a) indicated that rat (oral and inhalation), mouse
(oral) or rabbit (dermal) toxicity and efficacy of cleaning product
ingredients were markedly different. Based on a concentration at a
given 5th centile (i.e., TC5), the probability of the maximum haz-
ards for rat (oral), mouse (oral), rabbit (dermal), and rat (inhalation)
were 43.2%, 36.7%, 38.6% and 75.5% respectively (Table S4). This
hazard order (i.e., rat (inhalation) > rat (oral) > rabbit
(dermal) > mouse (oral)) was consistent with the results using ef-
ficacy data of NOECs, EC10s and EC50s compared to corresponding
mammalian acute LD50 CTDs (Fig. 5bed and Table S4).
Bacteria are becoming increasingly resistant to various types of
cleaning and disinfections products, which may result in increased
use of existing cleaning products or replacement with alternative
substances, but uncontrolled and inappropriate use of ingredients can
present environment and health risks. Because bacterial attachment
to surface can present important risks to public health, testing efficacy
of cleaning products ingredients should examine inhibitory and
bactericidal potential, and include efficacy for bacterial detaching
from surfaces as remains of dead cells (e.g., antimicrobial resistance
genes) on the surface could act as a source of cross-resistance (Verraes
8 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323

Fig. 5. Joint probability curves (JPCs) illustrating the probability of efficacy for E. coli surface detachment to cleaning product ingredients (a: LOECs, b: NOECs, c: EC10s and d: EC50s)
related to acute toxicity (LD50s) of the ingredients to rat (oral, inhalation), mouse (oral) and rabbit (dermal) models.

et al., 2013). Moreover, testing the potential of chemical compounds
only on planktonic cells, but not on attached cells to surfaces, may lead
to overestimation of antimicrobial activity because planktonic bac-
teria are more susceptive to chemicals (Giaouris and Simo ~ es, 2018).
Such information allows product efficacy assessments in worst case
scenarios when bacteria are attached to a surface and thus can start
forming a biofilm. However, identifying cleaning products that opti-
mize surface removal efficacy while lowering hazards represents a
timely opportunity to support product substitutions during alterna-
tives assessments.
In the present study, results of E. coli detaching potential
showed large efficacy variability among cleaning products ingre-
dient, ranging from 10% (sodium metabisulfite) to total eradication
(100%) for sodium hypochlorite at the same concentration of 4.3 log
mg L
1. One reason for this efficacy is oxidation by sodium hypo-
chlorite, which can target diverse bacteria and molds in short
contact times (Rossoni and Gaylarde, 2000). Though chlorine based
cleaning products are inexpensive and partly tolerate hard water,
these are relatively unstable and corrosive, and can further produce
disinfection byproducts (e.g. trihalomethanes, chloramines, halo-
acetic acids), which represent initially unintended tradeoffs asso-
ciated with protection of public health and the environment
(Giaouris and Simo ~ es, 2018a). In contrast, we found that sodium
metabisulfite was less efficient. Similar to observations in the pre-
sent study, Zhang et al. (2017) found no antibacterial activity of
sodium metabisulfite on S. aureus. Sodium metabisulfite is
commonly used in homebrewing and winemaking to sanitize
equipment. It is also used as a cleaning product for potable water
reverse osmosis membranes in desalination systems. Traditionally,
cleaning products are tested under strict protocols using standard
scenarios based on fats, proteins, starch and limescale added to
testing surface (e.g., ASTM, 2016), but less information is available
how efficacious substances are against microorganisms. Balancing
efficacy with lower hazards promises be economically acceptable,
have a broad spectrum of effectiveness, and have lower impacts on
the environment and human health.
3.4. Green chemistry and alternatives analyses
Historically, risk management of chemical substances primarily
has focused on reducing exposure, even though risk emerges from
the intersection of exposure and hazard (Brooks, 2019). Efforts
designing more sustainable and less hazardous chemicals, which
represent important principles of green chemistry (Anastas and
Warner, 1998), aim to reduce risk through design and use of less
toxic and more readily biodegradable substances (Erythropel et al.,
2018). For example, the American Chemical Society’s Green
Chemistry Institute (www.acs.org/content/acs/en/greenchemistry.
html) is an important resource to facilitate research and commu-
nication in sustainable and green chemistry and engineering.
Similarly, selecting alternative substances, or classes of sub-
stances, that are more sustainable and less hazardous for
 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323
substitutions in consumer and industrial products is advantaged
and increasingly expected by consumers (NRC, 2014). For example,
the U.S. Environmental Protection Agency’s Safer Choice program
(www.epa.gov/saferchoice) represents a non-regulatory effort to
advance sustainability. Through alternatives assessments by the
Design for the Environment program, companies submit informa-
tion related to their products, which are then evaluated for envi-
ronmental hazards using an alternatives assessment framework
(www.epa.gov/saferchoice/alternatives-assessment-criteria-
hazard-evaluation). If these products are identified with suffi-
ciently lower environment and health hazards, then a Safe Choice
label can be used by business to market their "greener" products.
Collectively, research and practice in green chemistry are fueling
innovation and advancing sustainability, particularly by incentivizing
protection of public health and the environment toward achieving the
United Nations Sustainable Development Goals (Anastas and
Zimmerman, 2018; Brooks, 2018). In the present study, we selected
group of chemicals in cleaning products for empirical microbiology
studies and examined their environmental health hazards. Fig. 6 il-
lustrates six JPCs of efficacy and toxicity, which may be useful in the
future to examine specific classes of substances or functional use
categories relative to environment or health toxicology information.
Whereas JPCs in the lower left corner of Fig. 6 includes chemicals with
low efficacy and toxicity, JPCs in the upper right corner chemicals with
high efficacy and toxicity. Chemicals associated with JPCs in the center
of Fig. 6 are characterized by decreased efficacy and toxicity; thus, the
green zone is interpreted as substances balancing function with
hazard, which directly links to the 4th principle of green chemistry
(Anastas and Warner,1998). Our novel observations indicate that JPCs
appear useful to examine relationships between probability of
chemical efficacy relative to mammalian toxicity, and thus to support
selection of less environmentally hazardous ingredients. Such ob-
servations are important because alternatives assessments are
increasingly employed during selection of other chemicals that are
more sustainable and less hazardous (NRC, 2014).
Fig. 6. Joint probability curves of chemical efficacy and toxicity may support future
examinations of specific classes of substances or functional use categories.
9
4. Conclusions
In the present study, we empirically identified antimicrobial effi-
cacy of 20 substances for surface adhered bacteria, a challenging
public health issue. Probabilistic distributions (i.e., CEDs and CTDs)
were subsequently constructed using these newly generated efficacy
data and likelihoods of encountering detachment thresholds for the
selected ingredients were determined. Mammalian toxicity CTDs
were then generated for mammalian species using acute LD50s for
oral, dermal or inhalation exposure routes. Because tools are needed
to balance product efficacy with reduced hazards, we developed a
novel PJPDA approach and compared probabilistic hazards of these
efficacy CEDs and mammalian toxicity CTDs; a general hazard trend
was identified: rat (inhalation) > rat (oral) > rabbit (dermal) > mouse
(oral). However, it is important to note that here we employed acute
toxicity from mammalian models, because we could not identify
sufficient sublethal and chronic toxicity for these specific 20 sub-
stances. We further did not examine traditional measures of antimi-
crobial efficacy or potential development of antimicrobial resistance
to these substances. Future studies are needed to expand the work
presented here with sublethal toxicity, data from standardized
studies of antimicrobial activity, and resistance development infor-
mation when it becomes available.
Important pursuits of green and sustainable chemistry include
identification of existing substances and sustainable molecular
design of new chemicals that are less hazardous to public health
and the environment yet maintain intended efficacy (Coish et al.,
2016; Erythropel et al., 2018). Research has often focused on al-
ternatives assessments of individual products to inform chemical
substitutions (NRC, 2014). Studies examining classes of substances
with desired functions has received relatively less attention
(Brooks, 2019). However, combining chemical efficacy and toxicity
information using JPCs may be useful for examining classes of
antimicrobial products, and other substances (Fig. 6). Moreover, use
of JPCs may support practitioners and decisions makers who aim to
balance chemical functions and hazards. For example, JPC appli-
cations could be particularly useful when examining specific clas-
ses of existing chemicals, but may also be valuable when new
classes of substances are designed or discovered with desirable
functional activities (Fig. 6). Future work using JPCs should thus
examine specific classes of substances (e.g., chemical categories,
product uses), including traditional chemicals or newly designed
ingredient candidates from renewable resources, to optimize
chemical selection while balancing product efficacy and environ-
ment and health hazards.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Rok Fink: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Investigation, Methodology, Project adminis-
tration, Resources, Supervision, Visualization, Writing - review &
editing. Zhen Wang: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology, Visualization, Writing - re-
view & editing. Martina Oder: Data curation, Formal analysis,
Investigation, Methodology, Resources, Supervision, Writing -
original draft. Bryan W. Brooks: Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology,
Project administration, Resources, Supervision, Visualization,
Writing - review & editing.
10 R. Fink et al. / Journal of Cleaner Production 262 (2020) 121323
Acknowledgements
RF, MO and BWB were supported by Slovenian Research Agency
under Slovenian-United States of America bilateral project “Less
hazardous cleaning agents potential for control the bacterial
adhesion capacity (BI-US/16-17-065)”. ZW and BWB were sup-
ported by an unrestrictive grant for the Cleaning Products In-
gredients Safety Initiative from the American Cleaning Institute,
and the U.S. National Science Foundation (CHE-1339637) with
additional support from the U.S. Environmental Protection Agency
to BWB through the Molecular Design Research Network (modrn.
yale.edu).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jclepro.2020.121323.
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