Science of the Total Environment 613–614 (2018) 1220–1227

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Science of the Total Environment

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

Short-term organic carbon migration from polymeric materials in contact

with chlorinated drinking water
Guannan Mao a,b, Yingying Wang a,⁎, Frederik Hammes b,⁎
a
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of Environmental
Science and Engineering, Nankai University, Tianjin 300071, China
b
Department of Environmental Microbiology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf 8600, Switzerland

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Established migration and growth po-

tential assays in chlorinated water
• Test materials consumed significant
amounts of chlorine during the migra-
tion assay.
• Chlorine hardly affected TOC and AOC
migration from materials.
• Migration and biofilm formation poten-
tial of materials remained after migra-
tion.
• Distinct migration behavior and biofilm
development observed on different
materials

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

Article history: Polymeric materials are widely used in drinking water distribution systems. These materials could release organ-
Received 18 July 2017 ic carbon that supports bacterial growth. To date, the available migration assays for polymeric materials have not
Received in revised form 14 September 2017 included the potential influence of chlorination on organic carbon migration behavior. Hence, we established a
Accepted 16 September 2017
migration and growth potential protocol specifically for analysis of carbon migration from materials in contact
Available online xxxx
with chlorinated drinking water. Four different materials were tested, including ethylene propylene
Editor: D. Barcelo dienemethylene (EPDM), poly-ethylene (PEX b and PEX c) and poly-butylene (PB). Chlorine consumption
rates decreased gradually over time for EPDM, PEXc and PB. In contrast, no free chlorine was detected for PEXb
Keywords: at any time during the 7 migration cycles. Total organic carbon (TOC) and assimilable organic carbon (AOC)
Polymeric materials was evaluated in both chlorinated and non-chlorinated migrations. TOC concentrations for EPDM and PEXb in
Chlorinated chlorinated migrations were significantly higher than non-chlorinated migrations. The AOC results showed pro-
Drinking water nounced differences among tested materials. AOC concentrations from chlorinated migration waters of EPDM
Migration and PB were higher compared to non-chlorinated migrations, whereas the opposite trend was observed for
Biofilm
PEXb and PEXc. There was also a considerable difference between tested materials with regards to bacterial
growth potential. The results revealed that the materials exposed to chlorine-influenced migration still exhibited
a strong biofilm formation potential. The overall results suggested that the choice in material would make a con-
siderable difference in chlorine consumption and carbon migration behavior in drinking water distribution
systems.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding authors.
E-mail addresses: wangyy@nankai.edu.cn (Y. Wang), Frederik.hammes@eawag.ch (F. Hammes).

https://doi.org/10.1016/j.scitotenv.2017.09.166
0048-9697/© 2017 Elsevier B.V. All rights reserved.
 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227
1. Introduction
Water utilities aim to produce and distribute drinking water with
high chemical and biological stability. Synthetic polymeric materials
are commonly used in distribution systems and particularly in building
plumbing systems. These include materials such as cross-linked poly-
ethylene (PEX), polyvinyl chloride (PVC) and ethylene propylene
dienemethylene (EPDM) that are used for pipes, sealing rings and flex-
ible connection hoses. Such materials are used for both cold and hot
water installations due to their versatility, light weight and low cost
(Al-Malack and Sheikheldin, 2001; Bucheli-Witschel et al., 2012). One
critical concern facing all these materials is the migration of organic car-
bon contaminants such as additives, stabilizers, and antioxidants (Stern
and Lagos, 2008). A certain fraction of these migrated contaminants
comprises biodegradable organic carbon that can be used by bacteria
as source of energy. Bacterial consumption of such carbon would result
in biologically unstable water with increasing planktonic and sessile
(biofilm) bacterial concentrations compared with stainless steel and
copper pipes (Van der Kooij et al., 2005; Zhu et al., 2014). Excessive bio-
film development could lead to the establishment and proliferation of
opportunistic pathogens and other biofouling problems (Feazel et al.,
2009). Hence, there is a clear need for polymeric materials in contact
with potable water to undergo strict quality control. Ideally such mate-
rial quality controls should consist of migration potential assays and bi-
ological growth potential assays in case of application of drinking water
installations (Bucheli-Witschel et al., 2012).
Migration assays in the European Union (EU) are performed ac-
cording to established EU standards (EN 12873-1, 2014), which
focus on TOC migration but not on the biological availability of the
migrated carbon. In addition, a number of different growth potential
tests are available (Koetzsch et al., 2016). Wen et al. (2015) devel-
oped a fast and comprehensive material testing assay called
“BioMig”, which is a combination of a high-temperature migration
potential (MP) assay and an ambient temperature biofilm formation
potential (BFP) assay. The MP assay includes determination of the
total organic carbon (TOC) and assimilable organic carbon (AOC).
The BFP assay describes biofilm formation potential, comprising mi-
crobial growth in the planktonic phase and the cells attached to the
surface of the material (Bucheli-Witschel et al., 2012; Wen et al.,
2015; Koetzsch et al., 2016).
Migration could be affected by several important factors, including
temperature, surface-to-volume ratio, water quality, stagnation time
and pipe brand. For example, higher temperatures increase migration
rates while higher surface-to-volume ratios enhance the migration of
organic carbon (Bucheli-Witschel et al., 2012; Inkinen et al., 2014;
Wen et al., 2015; Proctor et al., 2016). Some previous studies demon-
strated that different pipe brands made from the same polymeric mate-
rial formulation may still differ considerably in carbon release (Kelley
et al., 2015; Connell et al., 2016). However, very little work has been car-
ried out so far on the impact of chlorine on the migration behavior of
materials. In many countries, drinking water is distributed with residual
disinfectants (i.e. chlorine, chloramine) to ensure biological stability
(Gillespie et al., 2014; Proctor and Hammes, 2015). Free chlorine is a
strong oxidizing agent and has been used to inactivate microbial con-
taminants in drinking water, leading to the decay of chlorine that has
been extensively observed (Hallam et al., 2002; Ngwenya et al., 2013;
Wang et al., 2014; Zheng et al., 2015). Previous research indicated that
chlorinated water could change the rate of substrate migration from
polymeric pipes (Whelton and Nguyen, 2013) and reduce the materials'
service life due to changed surface and bulk properties with long-term
chlorinated water exposure (Dear and Mason, 2006; Whelton et al.,
2011). Despite this evidence, standardized material assay for migration
and biofilm growth potential never considers the impact of chlorinated
water on the materials (EN 12873-1, 2014; Wen et al., 2015; Koetzsch
et al., 2016). In this work, we have focused on organic carbon migration
in the presence of chlorinated water and subsequent biofilm formation
1221
on the surface of materials after chlorination, with emphasis on short-
term migration and growth potential assays.
Here we developed a chlorinated water BioMig assay to determine
the effect of chlorination on the migration behavior of materials and
the bioavailability of the migrated organic carbon. Specific objectives
of this study were to (i) analyze chlorine consumption for different
polymeric materials in contact with chlorinated water, (ii) characterize
chlorine-influenced migration of organic carbon from polymeric mate-
rials, and (iii) compare whether chlorine-influenced migration impact-
ed subsequent biofilm formation.
2. Materials and methods
2.1. Materials
The following polymer materials were tested: poly-ethylene silane
cross-linked (PEXb), poly-ethylene electron beam cross-linked (PEXc),
poly-butylene (PB), and ethylene propylene dienemethylene contain-
ing 2% of plasticizer (EPDM). All the materials have drinking water ap-
plication approval: PEXb, PEXc and PB are used for drinking water
pipes and EPDM is used in drinking water distribution systems as
sealing material.
2.2. Migration and growth potential assays
The new material assay was based on the BioMig assay described by
Wen et al. (2015). In short, this assay combines a high-temperature mi-
gration potential (MP) assay and an ambient temperature biofilm for-
mation potential (BFP) assay. The MP assay run for seven sequential
24-hour (Mig1–Mig7) cycles at 60 °C and Mig1, Mig3 and Mig7 are
used to determine the total (TOC) and assimilable (AOC) organic car-
bon. The BFP assay measures planktonic and biofilm batch growth in
the presence of the material (Wen et al., 2015; Koetzsch et al., 2016).
Bottled water (Evian, France) without any disinfectant was chosen as
test water. A surface-to-volume ratio of 1 cm−1 was used in all assays,
and a minimal medium solution containing sufficient phosphate, nitro-
gen, and iron was added in order to ensure the only growth-limiting fac-
tor is carbon (Wen et al., 2015). The BFP assays were performed for
14 days at 30 °C with shaking at 120 rpm. After 2 weeks of incubation,
both planktonic cells and biofilm cells (detached by sonication) were
quantified with flow cytometry (FCM) and the remaining dissolved or-
ganic carbon (DOC) was analyzed. The assay was adapted to test the in-
fluence of free chlorine (below).
2.2.1. Chlorine-influenced MP assay
For the chlorine-influenced migration, hypochlorite (final
concentration = 1.8 mg/L) was added to the water at the start of
every migration cycle (M1–M7). Negative controls contained all com-
ponents except for the test materials (Fig. 1A). After each migration
cycle the residual chlorine was measured and the water was refilled.
As in the normal BioMig assay, Mig1, Mig3 and Mig7 were used to deter-
mine the total (TOC) and assimilable (AOC) organic carbon.
2.2.2. BFP assay
For a comparison of the influence of a pre-migration with and with-
out chlorine on biofilm formation potential, the test materials following
7-day migration (above) were transferred to the BFP assay. Biofilm for-
mation on new materials without any prior migration was used as a
control (Fig. 1B).
2.3. Rate of chlorine decay
Material samples with a surface area of 100 cm2 were placed into
sealed jars with 100 mL sterile-filtered bottled water (Evian, France).
Chlorine (hypochlorite) was added to a final concentration of 1.8 mg/L
free chlorine. All experiments were carried out at 60 °C (sealed and in
1222 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227

Fig. 1. Outline of a strategy for testing polymeric materials in contact with chlorinated water for (A) migration potential and (B) biofilm formation potential (BFP), adapted from Wen et al.
(2015). TOC = total organic carbon; AOC = assimilable organic carbon.

the dark). The remaining free chlorine was measured after the desired μL/min. All data were processed using the BD Accuri CFlow® software
contact time (i.e. 0.5, 1, 2, 4, 8 and 24 h) within 24 h. Linear fitting as described previously (Prest et al., 2013).
was used to fit all data. The rate of chlorine consumption was calculated
by the following equation: d[C] / dt = −k. Where [C] the chlorine con-
2.7. Statistical analysis
centration, t is the time, and k is the rate constant. All experiments in-
cluding negative control (excluded material) were performed in
Mean and standard deviation values were calculated for all remain-
triplicate.
ing free chlorine concentrations, TOC, AOC and BFP results. t-tests with
an error alpha value of 0.05 were performed in SPSS statistical software
2.4. TOC and AOC analysis
or Excel.
All glassware used for TOC and AOC measurement were muffled at
500 °C for 6 h to ensure they were carbon-free. Quenching (i.e. sodium 3. Results and discussion
thiosulfate (5 μL, 10% w/v) per 100 mL or 1.5 mL 50 mM sodium nitrite
per 100 mL) was applied prior to TOC and AOC measurements in order 3.1. Chlorine consumption by different polymeric materials
to avoid chlorine interfering with results. Aliquots of the migration
water with quenching were transferred into carbon-free TOC vials. All synthetic polymeric materials reacted with chlorine during the
TOC content was analyzed through a total carbon analyser (TOC- migration experiments, resulting in considerable chlorine decay within
VCPH, Shimadzu, Japan). The method described by Hammes and Egli 24 h. The free chlorine in the negative controls decayed approximately
(2005) was used to determine AOC concentration. The influence of 19.2% during each migration cycle due to reaction with the water matrix
quenching solutions on TOC and AOC measurement was monitored and the elevated incubation temperatures (Fig. 2). Every material con-
(Fig. S1). sumed significantly more chlorine than the negative control (p b 0.01,
n = 14). Every material showed large initial chlorine consumption,
2.5. Chlorine measurements ranging from 1 μg/cm2 to 1.3 μg/cm2 (concentrations to surface area
unit conversions based on S/V ratio which was 1 cm−1) on the first con-
The chlorine concentrations were measured by the N,N-diethyl-p- tact with chlorinated water (Mig1). This suggests that chlorine rapidly
phenylenediamine (DPD) colorimetric method using a benchtop spec-
trophotometer (Hach-Lange DR3900, Germany) after every 24-hour
migration. After reading blank, the samples are added to chlorine re-
agent cuvette (Hach-Lange LCK 310, Germany) and followed by 1 min
incubation prior to measurement. The solution turns red and the color
intensity is proportional to the chlorine concentration. The range of
the chlorine reagent kit is 0.05 to 2 mg/L. Measurements done with
this method have an accuracy of ±3%.

2.6. Fluorescent staining and flow cytometry (FCM) analysis

FCM analysis and fluorescent staining were carried out based on the
methods described previously (Hammes et al., 2008; Prest et al., 2013).
Briefly, for total cells concentration (TCC) measurements, 1 mL of the
sample (preheated 3 min and diluted if necessary) were stained with
10 μL SYBR® Green I (100× diluted; Invitrogen), mixed and incubated
in the dark for 10 min at 37 °C. FCM analysis was performed on a BD
Accuri C6® flow cytometer (BD Accuri cytometers, Belgium) equipped
with a 50 mW laser emitting at a fixed wavelength of 488 nm. Bacterial
Fig. 2. Remaining free chlorine concentrations in the seven migrations for EPDM PEXc,
signals were selected using electronic gating on the density plots of PEXb and PB materials. Each migration was added the same chlorine concentrations
green fluorescence (FL1; 533 nm) and red fluorescence (FL3; dashed dotted line, and performed at 60 °C for 24 h. Error bars indicate standard
N670 nm). Measurements were performed at a pre-set flow rate of 66 deviation of duplicate measurements.
 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227
reacted with a variety of compounds in the bulk water and the materials
surface.
Over 7 days of chlorinated-influenced migration, the remaining
chlorine progressively increased for all materials except for PEXb mate-
rial (Fig. 2). In the case of EPDM, no free chlorine was detected after mi-
grations 1 and 2, but free chlorine concentrations gradually increased
from migration 3 onwards. After seven migration cycles, the free chlo-
rine concentrations in the EPDM samples were approximately 0.4
μg/cm2 (Fig. 2). In contrast, the PEXc and PB samples contained measur-
able chlorine concentrations after the first migration (0.3 and 0.1
μg/cm2 respectively), and chlorine consumption decreased with subse-
quent migration cycles. Chlorine residuals in the PEXc and PB experi-
ments were 1.0 and 0.5 μg/cm2 respectively after seven sequential 24-
hour migration cycles (Fig. 2). This is in agreement with previous stud-
ies that chlorine demand decreased with subsequent flushes for HDPE
and cPVC (Heim and Dietrich, 2007) as well as PE, uPVC and PPR
(Zhang et al., 2014). Interestingly, the PEXb experiment never had de-
tectable concentrations of free chlorine at any time during the seven mi-
gration cycles (Fig. 2). Such substantial differences in chlorine
consumption between PEXb and PEXc are likely due to their different
additives and manufacturing methods (Whelton and Nguyen, 2013).
Specifically, PEXc is manufactured without crosslinking regents, while
PEXb is created using silane crosslinking with hot water for several
hours (Peacock, 2001). The differences in chlorine consumption could
also be due to variability across brands between the same material
(Kelley et al., 2015; Connell et al., 2016). Although it is unclear whether
surface reactions or the migrated contaminants from the materials con-
sumed the chlorine, a previous study showed no effect of chlorine on
the hydrophobicity and roughness of the HDPE surface (Mathieu et al.,
2016).
Due to the considerable difference in chlorine consumption among
the different materials, the rates of chlorine consumption at 60 °C
from different materials was investigated in further detail (Fig. 3). In
the negative control, free chlorine decayed approximately 23.3% during
24 h, consistent with migration results above (Fig. 2). The trends in chlo-
rine consumption were fitted to linear regressions (R2 ≥ 0.95 for all test
materials) and the calculated rates of chlorine consumption for the four
tested materials distinguished two groups. In the “fast” group (EPDM
and PEXb), about 95% of free chlorine was lost within 8 h, with a
decay rate of − 0.21 μg/(cm2·h). This rate was about 4 times faster
than the slow group (PEXc and PB), which was approximately −0.05
μg/(cm2·h). A small amount of free chlorine (0.33 and 0.15 μg/cm2 re-
spectively) remained in the PEXc and PB experiments after 24 h. Free
Fig. 3. Consumption rate of free chlorine by the four tested materials, measured at 60 °C in
24 h. The two different groups of chlorine consumption rates were observed and the slope
of the linear fitted data during 8 h for the faster group (dashed line) was −0.2 μg/(cm2·h)
and for the slower group during 24 h (dashed dotted line) was −0.05 μg/(cm2·h).
1223
chlorine was not detected after 8 h in the EPDM and PEXb experiments.
Chlorine is a relatively unstable chemical that easily reacts with (in)or-
ganic compounds in the bulk water or leached from pipes, thus causing
its decay in the distribution system (Abd El-Shafy and Grunwald, 2000).
A free chlorine residual of 0.3 mg/L should be maintained in building
plumbing (WHO, 1997). It was noted that water often stagnated in
building plumbing more than 8 h, resulting in free chlorine concentra-
tions lower than the required standard (Cook et al., 2014). Compared
with other kinds of pipe materials, the new polymeric materials con-
sumed less chlorine (Table 1). The polymeric pipes exhibited relatively
low reactivity with free chlorine, suggesting a lower free chlorine dos-
age would be required to maintain chlorine residuals compared with
copper or cast iron materials (Tamminen et al., 2008).
3.2. Migration of TOC and AOC
Overall, the amount of TOC (ΣMig) migrated from materials into the
water phase was higher in the presence of chlorine than absence (Fig. 4
and Table 2). TOC in the migration water from the non-chlorinated neg-
ative control and chlorinated negative controls had no significant differ-
ence (p = 0.35, n = 9) (Fig. S3). AOC concentrations followed the same
pattern (p = 0.37, n = 18) and remained between 0.03 and 0.06 mg/L
(Fig. S3). In order to calculate noise-corrected TOC and AOC results,
the corresponding values of the negative controls were subtracted
from the values of four testing materials.
The four materials showed clearly different TOC migration behavior.
PB and PEXb released more TOC (ΣMig = 2.7–3.1 μg/cm2 (measured
TOC concentration normalized to material surface area, based on the
S/V ratio of 1 cm−1)) than EPDM and PEXc (ΣMig = 1.1–1.3 μg/cm2)
with or without chlorinated water. TOC concentrations were highest
after the first migration (ranging from 56% to 68% of ΣMig) and de-
creased exponentially with increasing migration cycles under both
chlorinated and non-chlorinated conditions (for details see Table 2),
which were consistent with previous studies (Bucheli-Witschel et al.,
2012; Wen et al., 2015; Proctor et al., 2016). For the first migration
cycle (Mig1), the difference in TOC migration in the presence and ab-
sence of chlorine was smaller than that in Mig3 and Mig7. With increas-
ing migration steps, the difference between chlorinated and non-
chlorinated migrations from all materials exhibited an increase in TOC
migration. For example, the difference between chlorinated and non-
chlorinated TOC migration for EPDM increased from 8% (Mig1) to 40%
(Mig7), and other materials showed similar trends (PEXc from 3% to
34%, PEXb from 1% to 25%, PB from 0.5% to 12%). It should be noted
that TOC migration from polymeric materials was the least in Mig7.
Hence the influence of chlorine on TOC migrations was limited. Only
EPDM and PEXb showed significant differences (p b 0.05, n = 3,
ΣMig) between chlorinated and non-chlorinated migrations. Although
EPDM and PEXb consumed the largest chlorine concentrations after mi-
grations 1 and 2, that exact mechanism for TOC release was not eluci-
dated. Previous reports showed comparable results, where TOC
migration from PEX (Kelley et al., 2014) and HDPE (Heim and
Table 1
Free chlorine decay rate constant of different materials.
Material Chlorine decay Material References
constant (h−1) conditions
Coppera 1.20 Used but cleaned Zheng et al. (2015)
Cast irona 0.67 New Hallam et al. (2002)
Galvanized irona 0.90 Used but cleaned Zheng et al. (2015)
PVCa 0.09 New Hallam et al. (2002)
Polymericb EPDM 0.19 New This study
PEXb 0.20
PEXc 0.05
PB 0.06
a
At room temperature conditions.
b
At elevated temperature conditions.
1224 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227
Fig. 4. Total organic carbon (TOC) and assimilable organic carbon (AOC) concentrations in chlorinated migration assays for four materials. Migration were performed in the absence of
chlorine (TOC, black columns; AOC, hatched columns) or in the presence of 1.8 mg/L free Cl2 (TOC, gray columns; AOC, grid columns). Three of seven sequential migrations (Mig1,
Mig3 and Mig7) were measured. ΣMig was represented as the sum of Mig1, 3 and 7.
Dietrich, 2007) pipes was not influenced by chlorination. These findings
only investigate one material and short-term chlorinated-influence mi-
grations. However, large-scale water distribution systems may be com-
posed of a variety of different materials and long stagnation, interaction
of different materials on TOC and chlorine decay requires further
research.
To facilitate the TOC and AOC data comparison, a conversion factor 1
× 107 cells/μg-C was used to calculate AOC concentrations from growth
potential data (Wen et al., 2015). The AOC concentrations demonstrated
clear differences among the testing materials. For EPDM and PB in con-
tact with chlorine, AOC concentrations followed the same pattern as
TOC results. ΣAOC values were 0.67 μg/cm2 and 0.61 μg/cm2 under chlo-
rinated conditions respectively, which was higher than that under non-
chlorinated conditions (0.48 μg/cm2 and 0.36 μg/cm2 respectively)
(Table 2). This may be due to AOC formation from the oxidation of com-
plex TOC molecules with chlorine (Ramseier et al., 2011). In contrast,
PEXb and PEXc released more AOC (0.41 μg/cm2 and 0.47 μg/cm2) in
the absence of chlorine than in the presence of chlorine (0.28 μg/cm2
and 0.32 μg/cm2) (Fig. 4 and Table 2). This may be due to chlorine in-
creasing the toxic effects of released compounds (aromatic compo-
nent) from PEX (Kelley et al., 2014) and producing disinfectant
byproducts such as trihalomethane (Heim and Dietrich, 2007),
both of which negatively influence AOC assays. Inhibitory effects of
leached toxicity have been reported from previous studies (Wen
et al., 2015; Proctor et al., 2016). It cannot be ignored that difference
pipe brands within the same material could also influence the
T(A)OC migration. The proportion of the bioavailable fraction from
migrated carbon varied dramatically (ranging from 9% to 57%)
(Table 2). With this wide range, there is not sufficient evidence
that chlorination could be linked to efficient AOC conversion (i.e.
the percentage of AOC from the total TOC).
3.3. Relationship of cumulative chlorine decay and TOC migration
The cumulative chlorine decay and ΔTOC migration (subtracting
non-chlorinated TOC from corresponding chlorinated TOC) were calcu-
lated for all materials (Fig. 5). The relationship between cumulative
chlorine decay with ΔTOC migration showed a significantly difference
among testing materials. It should be noted that TOC was determined
only in migrations 1, 3 and 7, whereas chlorine concentration was mea-
sured at every migration. While such a cumulative comparison underes-
timates the total TOC migration values, it still reveals their relationship
to chlorine decay.
Comparison of the cumulative ΔTOC exhibited an effect of chlorine
decay on TOC migration from materials (Fig. 5). PEXb showed the
most total chlorine decay (9.4 μg/cm2) as well as the most ΔTOC in-
crease (0.35 μg/cm2) after seven migrations among all materials.
EPDM followed the same pattern (total chlorine decay 8.9 μg/cm2 and
ΔTOC 0.22 μg/cm2). For PB and PEXc, ΔTOC was similar at 0.09 μg/cm2
and 0.11 μg/cm2 respectively. However, total chlorine decay for PB
was 2 times more than for PEXc. This result showed that differences in
chlorine decay did necessarily translate in differences in TOC migration,
suggesting that no direct relationship exist between chlorine decay and
TOC migration. It suggests that chlorination does not change the
physico-chemical characteristics of polymeric material surfaces in
short-term (Mathieu et al., 2016), which can influence the amount of
TOC released.
3.4. Growth potential after migration
The BFP assay was carried out for all the tested materials after the
seven sequential migrations with or without chlorine, together with
 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227 1225

Table 2
TOC and AOC concentrations at Mig1, Mig3, Mig7 and ΣMig (=sum of Mig1, Mig3, Mig7) under (non-)chlorinated conditions for different materials, as well as the percentage of AOC from
the total TOC under (non-)chlorinated conditions.

Material Mig1 Mig3 Mig7 ΣMig Efficiency

Non-chlorinated Chlorinated Non-chlorinated Chlorinated Non-chlorinated Chlorinated Non-chlorinated Chlorinated Non-chlorinated Chlorinated
(μg/cm2) (μg/cm2) (μg/cm2) (μg/cm2) (μg/cm2) (μg/cm2) (μg/cm2) (μg/cm2) (%) (%)

TOC
EPDM 0.61 0.66 0.22 0.29 0.14 0.23 0.97 1.18
PEXc 0.71 0.73 0.27 0.29 0.16 0.24 1.14 1.26
PEXb 1.87 1.89 0.51 0.72 0.36 0.48 2.74 3.09
PB 1.69 1.69 0.65 0.68 0.51 0.57 2.85 2.94

AOC
EPDM 0.31 0.39 0.09 0.14 0.08 0.14 0.48 0.67 49 57
PEXc 0.38 0.29 0.06 0.02 0.03 0.01 0.47 0.32 41 25
PEXb 0.30 0.16 0.05 0.06 0.06 0.06 0.41 0.28 15 9
PB 0.29 0.51 0.04 0.07 0.03 0.03 0.36 0.61 13 21

new materials without any prior migration (Fig. 6). Growth in the
planktonic phases (pBFP) and biofilm phases (sBFP), as well as the re-
maining DOC (rDOC) in the bulk water was analyzed. The correspond-
ing conversion to organic carbon from BFP data was calculated using
the calibration factor of 1 × 107 cells/μg-C (Wen et al., 2015).
As expected, the new materials without any prior migration sup-
ported the highest bacterial growth among all materials (Fig. 6). How-
ever, the differences between new materials and materials with prior
migration were notably small, ranging from 13% to 45%. This is interest-
ing because most of these pre-migrated materials showed particularly
little TOC migration after seven migration cycles (Fig. 4 and Table 2).
These results indicate that migration behavior from materials will per-
sist for a long period and that migrated biodegradable organic sub-
stances are favorable for biofilm to grow. Bucheli-Witschel et al.
(2012) also showed that PEXb continued to release organic carbon dur-
ing 14 migration cycles at 60 °C, with values remaining stable between
Mig7 and Mig14. Previous studies demonstrated polymeric pipe mate-
rials could release organic substances and support both short-term
(14 days) (Wen et al., 2015) and long-term (over 80 days) high biofilm
growth (Lehtola et al., 2004; Waines et al., 2011). The results of this
study are consistent with previous studies. Here the highest growth
(Σ BFP of 1.60–2.37 × 108 cells/cm2) was supported by EPDM whereas
the results for PEXb, PEXc and PB (Σ BFP of 6.28 × 106–4.05 × 107 cells/-
cm2) exhibited a lower potential for planktonic and sessile biofilm for-
mation, indicating that different production properties and additives
(e.g. antioxidant, plasticizer and stabilizer) could lead to variations in
BFP results. The biofilm fraction contributed to a high proportion of
the total growth. For instance, the biofilm fraction ranged between 70
and 91% of the total growth for PEXb, PEXc and PB, and 50% for EPDM.
This indicates variation in material characteristics have a direct and pro-
found influence on fraction of BFP on the surface (Waines et al., 2011).
For example, high porosity (NRC, 2006) and intrinsic hydrophobicity
(Mathieu et al., 2016) is beneficial to biofilm development. Others
have also found distinct biofilm communities among different types of
materials in contact with drinking water (Jang et al., 2011; Proctor
et al., 2016). The results demonstrated that not only new polymeric ma-
terials but also aged materials should be strictly monitored for biofilm
formation.
Different pipe conditions (e.g. service age, disinfectant) and material
composition will influence biofilm formation on pipe surfaces (Wang
et al., 2014; Prest et al., 2016). Little information is available on BFP
after chlorine-influenced migration. Such a situation is frequently en-
countered in the food industry and/or bottled water production,
where rinsing of disinfected surfaces is often done with drinking
water. Even more so, the plumbing of new buildings are typically sub-
jected to pressure tests with drinking water which then stagnates
until commissioning of the property, which can be days to weeks
later. The results showed that materials subjected to chlorine-
influenced migration exhibited a higher biofilm formation potential
than those subjected to chlorine-free migration. However, the differ-
ences are small and no significant difference was found among all

Fig. 5. Relationship between cumulative chlorine decay and cumulative Δ TOC.
Cumulative Δ TOC is the summing of subtracting non-chlorinated TOC from chlorinated
TOC over the entire migration (triangle, only migration 1; square, summing of 3
migrations; circle, summing of 7 migrations). Cumulative chlorine decay was sampled
from summing of corresponding migration.
Fig. 6. Biofilm formation potential assay (BFP) for four materials. The biological
information (pBFP, planktonic biofilm formation potential and sBFP, sessile biofilm
formation potential) was converted to intuitive organic carbon. 14 days total organic
carbon was the sum of pBFP, sBFP and remaining dissolved organic carbon (rDOC).
Three conditions for each material (i.e. migration without chlorine, migration with
chlorine, new without migration) were compared.
1226 G. Mao et al. / Science of the Total Environment 613–614 (2018) 1220–1227
testing materials (EPDM, p = 0.07, n = 3; PB, p = 0.15, n = 3; PEXb, p =
0.30, n = 3) except for PEXc (p b 0.05, n = 3). The concentration of
rDOC in the bulk water, indicator of non-consumed organic carbon,
showed a similar trend. Materials that were exposed to chlorination
may have an increase in organic carbon release, and will in turn enhance
biofilm formation potential. However, it has limited influence compared
with other factors, such as different temperatures, various types of pipes
and stagnation (Bucheli-Witschel et al., 2012; Mathieu et al., 2016;
Proctor et al., 2016). Our data suggested that chlorine-influenced migra-
tion did not significantly affect organic carbon release from materials
and biofilm formation on the surface of materials compared to
chlorine-free migration. Since polymeric pipes are relatively unreactive
with chlorine, chlorinated water would not considerably change the
physical integrity inside the polymeric materials in short-term. Hence
chlorination has limited effect on the subsequent biofilm formation.
4. Practical implications
The findings of the present work have important practical implica-
tions. The application of chlorine remains one of the frequently used
strategies to ensure the disinfection of drinking water distribution sys-
tems and biological stability. New polymeric materials in building
plumbing systems release organic carbon but also contribute signifi-
cantly to the chlorine decay. Such chlorine decay, particularly during
stagnation, will favor unwanted bacterial growth which has impacts
on drinking water aesthetics, hygienic quality and unmask underlying
odors (Lund et al., 2011). However, previously available migration as-
says have not included potential influences of chlorinated water on or-
ganic carbon migration behavior. Here, on the basis of extending an
existing migration and growth potential method, we have developed a
chlorinated water BioMig assay. The results showed that the choice in
plumbing material can make a considerable difference in chlorine con-
sumption and migration behavior. Furthermore, the data showed that
different kinds of materials released different amounts organic carbon
as well as different abilities of chlorine consumption, highlighting the
complexities when choosing materials used in building plumbing.
5. Conclusions
• A chlorine-influenced migration and growth potential assay was de-
veloped. The method yielded essential information on the assessment
of migration behavior and biological impact of materials in chlorinat-
ed water.
• For EPDM, PEXc and PB, residual chlorine concentrations gradually in-
creased with elapse time from the second migration cycle onward.
However, free chlorine was not detected in water exposed to PEXb
at any time during the test run. The calculated rates of chlorine con-
sumption at 60 °C decreased in the following order EPDM ≈ PEXb
N PEXc ≈ PB.
• The amount of TOC migration from EPDM and PEXb was significantly
higher under chlorinated conditions comparing to non-chlorinated
conditions. The AOC results showed no significant effect of chlorine
on AOC leached from all four polymeric materials tested.
• Materials with 7-days (non-)chlorinated migration still supported a
substantial amount of biofilm formation. Different materials support-
ed biofilm growth to various degrees; EPDM showed more growth po-
tential than PEXb, PEXc and PB. Chlorinated migration did not
significantly enhance the capacity of organic carbon migration and
biofilm formation of materials.
Acknowledgement
The authors are grateful to the financial support from Chinese Na-
tional Key Basic Research Development Program (2015CB459000), Chi-
nese National Science Foundation (31322012, 31670498) and for
technical support from Jürg Sigrist, Stefan Kötzsch, Franziska Rölli and
Romina Sigrist.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2017.09.166.
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