Limnology (2017) 18:233–241
DOI 10.1007/s10201-016-0508-5

RESEARCH PAPER

A simple method for preserving environmental DNA in water
samples at ambient temperature by addition of cationic surfactant
Hiroki Yamanaka1 • Toshifumi Minamoto2 • Junichi Matsuura3 • Sho Sakurai4 •Satsuki Tsuji4 • Hiromu Motozawa4 • Masamichi Hongo4 • Yuki Sogo4 •Naoki Kakimi4 • Iori Teramura1 • Masaki Sugita1 • Miki Baba1 • Akihiro Kondo5

Received: 31 August 2016 / Accepted: 31 October 2016 / Published online: 19 November 2016Ó The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Environmental DNA (eDNA) analysis is a
powerful tool within ecology for the study of the distribution or abundance of aquatic species, although the simplification of water sampling is required for enabling lightand fast field sampling to expand further application of
eDNA analysis. Here, certain candidate chemicals
belonging to the group of cationic surfactants were examined for their effectiveness as preservatives for eDNA
water samples by simply adding the chemicals to water
samples to suppress the degradation of eDNA. The quaternary ammonium compound benzalkonium chloride(BAC) at a final concentration of 0.01% was effective toretain 92% of eDNA derived from the bluegill sunfish
Lepomis macrochirus in an 8-h incubation test at ambient
temperature, which assumed a transportation of water
samples in 1-day field sampling during the daytime.
Meanwhile, eDNA in water samples without BAC retained
only 14% of the initial eDNA. Moreover, an additional

long-term incubation test (up to 10 days) revealed BACtreated samples retained *70 and 50% of bluegill DNA
compared to the initial amount after 1- and 10-day incubation at ambient temperature, respectively. Meanwhile,
eDNA in naı̈ve samples reduced to 20% after 1-day incubation and reached undetectable levels after 10 days. Up tonow, many eDNA studies have adopted on-site filtration
followed by filter fixation, which requires many pieces ofequipment. Addition of BAC can protect eDNA in water
samples with less effort and equipment resulting in an
increase of measurement accuracy of the eDNA quantityand detection probability of rare species by preventing thedisappearance of rare sequences in water samples.
Keywords eDNA  Degradation  Less equipment
requirements  Macroorganism  Preservatives

Introduction
Handling Editor: Hideyuki Doi.
& Hiroki Yamanaka
yamanaka@rins.ryukoku.ac.jp
1

Faculty of Science and Technology, Ryukoku University, 1-5
Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan

2

Graduate School of Human Development and Environment,
Kobe University, 3-11 Tsurukabuto, Nada-ku,
Kobe 657-8501, Japan

3

Settsu Oil Mill Inc., 1-5-10 Chikkoshinmachi, Nishi-ku,
Sakai, Osaka 592-8331, Japan

4

Graduate School of Science and Technology, Ryukoku
University, 1-5 Yokotani, Seta Oe-cho, Otsu,
Shiga 520-2194, Japan

5

Hiyoshi Corporation, 908 Kitanosho-cho, Omihachiman,
Shiga 523-0806, JapanThe biological monitoring of aquatic vertebrates by environmental DNA (eDNA) analysis is currently experiencing rapid
expansion in multiple disciplines, such as rare species conservation (Rees et al. 2014; Fukumoto et al. 2015) and invasive species management (Ficetola et al. 2008; Jerde et al.
2011; Takahara et al. 2013). In most cases, the species-specificdetection of eDNA shed by a target species into surrounding
water is commonly applied to reveal the macroorganism distribution (Goldberg et al. 2011; Keskin 2014; Yamanaka andMinamoto 2016) and in some cases to estimate the biomass orabundance (Takahara et al. 2012; Klymus et al. 2015;
Lacoursière-Roussel et al. 2016). Recently, a metabarcoding
technique has been implemented for eDNA analysis using
universal primers and a next-generation sequencer to simultaneously determine the species composition (Thomsen et al.

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2012; Kelly et al. 2014; Miya et al. 2015), thereby enabling an
extensive high-throughput analysis of faunas from multiple
target sites. Therefore, the establishment of a simple sampling
method is required to leverage the potential of eDNA analysis
by collecting samples from a number of sampling sites, whichcannot be covered by other conventional monitoring methods,
such as direct capture of organisms.
Improved preservation of the initial state of an eDNA
sample at the time of sampling would increase the reliability of analysis results in terms of the number of species
detected by metabarcoding or the biomass estimated by thequantification of eDNA from a target species. This isbecause the eDNA sample in its initial state is most likely
the best reflector of the fauna at the time of sampling.
eDNA is suggested to degrade rapidly in water, with therate varying among species (Thomsen et al. 2012; Maruyama et al. 2014; Pilliod et al. 2014). Temperature-dependent variation has also been reported, with decreased
degradation rates at lower temperatures (Eichmiller et al.
2016). This problem hampers the comparison of the eDNA
concentration among sampling sites or among species
because of the time lag of sampling between sites or the
species-specific degradation rate of eDNA. Consequently,
eDNA degradation in water after sampling might reduce
the quality of eDNA analysis.
In previous studies, various eDNA sample preservation
techniques, such as cooling the water samples in a cool box(Takahara et al. 2012; Eichmiller et al. 2014; LacoursièreRoussel et al. 2016), freezing the water samples (Takahara
et al. 2015) and on-ice preservation/ethanol fixation/immersion in lysis buffers of obtained filter samples (Goldberg
et al. 2011; Takahara et al. 2012; Renshaw et al. 2015;
Minamoto et al. 2016), have been followed to decelerate thedegradation rate of eDNA. However, on-site filtration
requires a sophisticated system setup, and cooling or freezing of water and filter samples requires a cool box or
portable freezer, resulting in increased equipment requirements for field sampling and an increased contamination
risk. The requirement of numerous pieces of equipment
hinders the expansion of the sampling area by requiring
multiple survey teams or citizen scientists. In addition,
cooling the water sample is insufficient to avoid sample
degradation as eDNA degrades even at 4–5 °C (Pilliod et al.
2014; Eichmiller et al. 2016), whereas freezing the water
samples might inhibit polymerase chain reactions (PCRs)(Takahara et al. 2015). Therefore, developing a novel simple
method of preserving water samples for eDNA analysis
using less equipment and a simpler on-site procedure isessential. Ideally, developing a preservative that is efficienteven at ambient temperatures and merely requires its addition to water samples would be the optimal solution.
We examined certain chemical agents, categorised as
cationic surfactants, for their potential as preservatives.

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These chemicals, commonly used as antiseptic drugs inmedical and sanitary fields, were used in the present study tosuppress eDNA degradation by microorganisms. The efficiency of each preservative was determined in terms of its
ability to decelerate/halt the reduction of the eDNA concentration depending on the incubation duration of sample
water at ambient temperature after sampling.

Materials and methods
Preservatives tested
Three cationic surfactants were selected as candidate
preservatives. Cationic surfactants are adsorbed on thenegatively charged bacterial cell membrane because of
their positive surface electric charge. This produces anantiseptic effect, resulting in the disruption of normal cellular function (Ziani et al. 2011). The three candidates
varied in their number of benzene rings.
Benzalkonium chloride (BAC)
Benzalkonium chloride (BAC), also known as alkyldimethylbenzylammonium chloride, is the cationic surfactant mostfrequently used in ophthalmic solutions as a preservative(Fig. 1a). In the present study, we used 10% Takeclean A-ST
(Settsu Oil Mill Inc., Sakai, Japan) as the BAC source.

Fig. 1 Chemical structures of candidate cationic surfactants examined in the present study: a benzalkonium chloride (BAC), R = –C8H17-C18H37, b benzethonium chloride (BEC) and c didecyldimethylammonium chloride (DDAC)

Limnology (2017) 18:233–241

Benzethonium chloride (BEC)
BEC, also known as hyamine, is a synthetic quaternaryammonium salt (Fig. 1b). It is used as an ingredient inmedicaments, deodorants and mouthwashes and also to
disinfect equipment in food processing and pharmaceuticalindustries, during surgery and as a preservative. In thepresent study, we used 10% hyamine solution (Daiichi
Sankyo Espha Co., Ltd., Tokyo, Japan) as the BEC source.
Didecyldimethylammonium chloride (DDAC)
DDAC, a quaternary ammonium compound, is antiseptic
and is used in many biocidal applications as a disinfectant(Fig. 1c). In the present study, we used 7% Catiogen
DDM-PG (DKS Co., Ltd., Kyoto, Japan) as the DDAC
source.
Experiment 1: short-term test using BAC
Treatments
Ex1-On-ice-Initial No preservative was added to thewater samples. Samples were immediately filtered on site,
and filter samples were transported to the laboratory on ice.
They were incubated on ice for 6 h (3.0 ± 0.1 °C) until
filtering of Ex1-BAC0.01 samples was complete.
Ex1-BAC0.01 BAC at a final concentration of 0.01% wasadded to samples on site immediately after water sampling.
The samples were transported to the laboratory and filteredafter incubation at ambient temperature (25.3 ± 0.1 °C)
for 6 h after completing the filtration of the Ex1-On-iceInitial samples.
An incubation time of 6 h was set by assuming a 1-day
field trip to collect water samples during the daytime. Fivereplicates of water samples in each treatment were sampled. Each treatment contained a negative control sample
comprising 500-ml ultrapure water sourced from the laboratory. The negative control samples were analysed alongwith the experimental samples to assess unintended contamination during the transportation, incubation, filtrationand extraction steps. In all of the following experiments,
the negative control samples were included and analysed in
the same manner.
Water sampling
Water samples were obtained at 15:40 on 18 September
2015 from the Wani River (35.154509 N, 135.922484 E), a
tributary of Lake Biwa, Japan, located at the southwestern
shore of the north basin of the lake. The fish fauna of thisriver include many migratory fish species including ayu

235sweetfish Plecoglossus altivelis (Plecoglossidae, Osmeriformes), swimming up to the river from Lake Biwa. Eachwater sample was collected directly from the water surface
by submerging a plastic container (volume, *10 l; madeof polyethylene) and divided into two containers (3 l each;made of polyethylene). One container was assigned to Ex1On-ice-Initial and the other to Ex1-BAC0.01, and 3-ml of
10% BAC at a final concentration of 0.01% was added to
the latter container. Using a beaker with a handle, 500 ml
water from each container was dispensed into each of thefive different plastic bags (DP16-TN1000; Yanagi,
Nagoya, Japan; made of polyethylene), and the bags were
subsequently sealed. The five bags of Ex1-On-ice-Initialwere immediately filtered on site according to the method
described in the next paragraph. Filter samples were stored
on ice in a cool box. The five bags of Ex1-BAC0.01 were
subjected to 6-h incubation at ambient temperature in a
cardboard box and transported to a laboratory during the
incubation. Using the hand sensors HI 98128 pHep 5, HI
98312 DiST 6 and HI 98312 DiST 6 (HANNA Instruments,
Woonsocket, RI, USA), the pH, electrical conductivity(EC) and temperature of the water at the sampling site were
measured to be 7.89, 0.26 mS/cm and 25.0 °C, respectively. The temperature conditions in the cool box used for
the Ex1-On-ice-Initial filter samples and in the cardboard
box for Ex1-BAC0.01 were measured using temperatureloggers (UA-001-64; Onset Computer Corp., Bourne, MA,
USA). All plastic containers used for water sampling weresterilised before use using 10% bleach solution. Thisdecontamination procedure was adopted at water samplingin all of the following experiments.
Filtration of water samples
The five Ex1-On-ice-Initial water samples were filtered onsite immediately after sampling using GF/F glass fiber
filters (diameter = 47 mm, nominal pore size = 0.7 lm;
GE Healthcare), filter holders (PP-47, ADVANTEC,Tokyo, Japan) and an aspirator pump (DP0105-X1-0001,
Nitto Kohki, Tokyo, Japan) powered by a 12-V car battery.
Each sample bag containing water was connected to thefilter holder and filtered. The five Ex1-BAC0.01 water
samples were subjected to incubation at ambient temperature under darkness in a cardboard box for 6 h, following
which they were filtered in the same manner as the Ex1On-ice-Initial samples. In all treatments, 500 ml water fromeach bag was filtered. For each sample, the filter disc wasremoved from the filter holder, folded inward, wrapped inaluminium foil and placed in a plastic bag using forceps.
All filters including Ex1-On-ice-Initial and Ex1-BAC0.01
filter samples were stored in a freezer at -20 °C immediately after filtration for the Ex1-BAC0.01 samples until thesubsequent DNA extraction. All equipment used for

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filtration, including filter holders and forceps, were sterilised before use using 10% bleach solution.
DNA extraction
Filter samples were subjected to DNA extraction following
the method suggested by Miya et al. (2015). Briefly, eachfilter was rolled into a cylindrical shape using sterile forceps and placed into a spin column (EZ-10 SpinColumn &Collection Tube; Bio Basic Inc., Ontario, Canada). The
silica membranes of the columns were removed before use.
The columns were centrifuged at 60009g for 1 min andexcess water on the filter recovered as the filtrate wasdiscarded. Two hundred microlitres ultrapure water, 100 ll
buffer AL and 10 ll proteinase K were dispensed onto thefilter, and the spin columns were then incubated at 56 °C
for 15 min. The buffer AL and proteinase K from a DNA
extraction kit (DNeasy Blood and Tissue Kit; Qiagen,Hilden, Germany) were used for this step. The column wasthen centrifuged at 60009g for 1 min, and the filtrate wasstored in a microtube. TE buffer (pH 8.0, 200 ll) was
added onto the filter and centrifuged at 60009g for 1 min
to recover the remaining DNA on the filter after 1-min
incubation at room temperature. The first filtrate was stored
in a microtube, whereas the second filtrate comprising TE
buffer was mixed in a 2-ml collection tube. After adding
200 ll buffer AL and 600 ll ethanol to the 2-ml collection
tube, the mixture of *1210 ll was subjected to DNA
purification using a DNeasy spin column, according to the
manufacturer’s instructions. During the final elution step,
DNA trapped on the silica membrane of the spin column
was eluted with 100 ll buffer AE.

Limnology (2017) 18:233–241

DNA of target sequences. In all real-time PCR assays, negative
controls in triplicate were adopted to assess the occurrence of
unintended cross contamination using ultrapure water insteadof the DNA template. The thermal conditions for PCR were
2 min at 50 °C and 10 min at 95 °C, followed by 55 cycles of
15 s at 95 °C and 60 s at 60 °C. The R2 values of the standard
curve for all qPCR experiments exceeded 0.98.
Experiment 2: test using three cationic surfactants
Treatments
Ex2-Initial No preservative was added to the water
sample. Samples were immediately filtered on site andstored for 8 h in a portable -18 °C freezer (MR040F;
Sawafuji Electric Co., Ltd., Ota, Japan) powered by a 12-V
car battery until the other samples had been filtered. In thepresent experiment, on-site filtration followed by filter
sample preservation in a portable freezer was adopted toprepare the initial sample as we found considerable
reduction of eDNA in Ex1-On-ice-Initial samples, eventhough they were filtered on site and transported to thelaboratory on ice (see ‘‘Results’’).
Ex2-Naı̈ve No preservative was added to water samples.
Samples were transported from the sampling site to thelaboratory and filtered after incubation at ambient temperature (22.5 ± 0.2 °C) for 8 h after the filtration of Ex2Initial samples.
Ex2-BAC0.1, 0.01, 0.001 BAC at final concentrations of
0.1, 0.01 and 0.001% was added on site immediately after
water sampling. Samples were transported to the laboratory
in the same manner as Ex2-Naı̈ve and filtered.

Real-time polymerase chain reaction (PCR) assay
The DNA of the ayu in each sample was quantified using
StepOnePlusÒ Real-Time PCR System (Life Technologies,
Foster City, CA, USA) to determine DNA degradation. The
sequences of the forward and reverse primers and a probe forthe cytochrome b gene of ayu were as follows: Paa-CyB-Forward, 50 -CCTAGTCTCCCTGGCTTTATTCTCT-30 ; PaaCyB-Reverse, 50 -GTAGAATGGCGTAGGCGAAAA-30 and
Paa-CyB-Probe, 50 -FAM-ACTTCACGGCAGCCAACCCC
C-TAMRA-30 developed by Yamanaka and Minamoto (2016).
PCR was conducted in triplicate in a 15-ll reaction mixture
comprising 900 nM of each of the primers and 125 nM of theprobe at final concentrations in 19 Gene Expression MasterMix (Life Technologies) with 1 ll template DNA. Quantification standards adjusted to the copy numbers of 3 9 101–
3 9 104 copies per reaction were adopted for all real-time PCR
assays. The quantification standards originated from a dilution
series of a plasmid, which included an artificially synthesised

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Ex2-BEC0.1, 0.01, 0.001 BEC at final concentrations of
0.1, 0.01 and 0.001% was immediately added after water
sampling on site. Samples were transported in the samemanner as Ex2-Naı̈ve and filtered.
Ex2-DDAC0.07, 0.007, 0.0007 DDAC at final concentrations of 0.07, 0.007 and 0.0007% was added on site
immediately after water sampling. Samples were transported and filtered in the same manner as Ex2-Naı̈ve.
Ex2-SH0.1, 0.01, 0.001 Sodium hypochlorite (SH) atfinal concentrations of 0.1, 0.01 and 0.001% was added onsite immediately after water sampling. Samples weretransported and filtered in the same manner as Ex2-Naı̈ve.
SH is an oxidising agent and an ingredient in bleach, which
can act as a powerful antiseptic chemical in a different way
from BAC, BEC and DDAC. SH degrades DNA molecules
and therefore has been used for decontaminating DNA in aLimnology (2017) 18:233–241

237

number of eDNA studies. In the present experiment, SH
was used as a control.
An incubation time of 8 h was set by carrying out a
1-day field trip to collect water samples during daytime.
Water samples in each treatment were sampled in triplicate. Each treatment contained a negative control water
sample comprising 500-ml ultrapure water sourced fromthe laboratory. Negative control samples were analysed
along with the other experimental samples to assess unintended contamination during the transportation, incubation,
filtration and extraction steps.

CA-30 , Bluegill_CytB_R 50 -ACGTCCCGGCAGATGTG
T-30 and Bluegill_CytB_probe 50 -FAM-CGACATCGCAACTGCCTTCTCTTCAGT-BHQ-30 , as suggested by Takahara et al. (2013). Moreover, the volume of DNA template
per PCR reaction was changed to 2 ll in a 15-ll PCR
mixture. The R2 values of the standard curve for all qPCR
experiments exceeded 0.98.

Water sampling

Ex3-Naı̈ve-0d, -1d, -3d, -5d, -10d No preservative wasadded to water samples. Ex3-Naı̈ve-0d samples wereimmediately filtered on site and frozen in a portable freezer
at -18 °C (MR040F) and transported to the laboratory.
Ex3-Naı̈ve-1d, -3d, -5d and -10d samples were transported
to the laboratory without filtration and filtered after incubation at ambient temperature (21.4 ± 1.2 °C) for 1, 3, 5
and 10 days after the filtration of Ex3-Naı̈ve-0d initialsamples.

Water samples were obtained from a pond (TsukinowaOike pond, Otsu, Japan; 34.981480 N, 135.945857 E) at
11:15 on 15 October 2015. Each water sample wasobtained directly from the water surface by submerging aplastic container (volume, *30 l; made of polyethylene).
Using a beaker with a handle, 500 ml of water was dispensed into each of the 42 plastic bags (DP16-TN1000),
and the bags were subsequently sealed. The 500-ml water
samples were randomly assigned to the Ex2-Initial, Naı̈ve,
BAC, BEC, DDAC and SC treatments. Ex2-Initial samples
were immediately filtered on site using the same method as
in Experiment 1. Five per cent BAC, 5% BEC, 3.5%
DDAC and 5% SH were added to all bags assigned to Ex2BAC, BEC, DDAC and SC, respectively, and adjusting to
the three levels of final concentrations described in theprevious paragraph. All bags except those of Ex2-Initialwere subjected to 8-h incubation at ambient temperature ina cardboard box and were subsequently transported to thelaboratory. Water at the sampling site was measured tohave a pH of 8.57, EC of 0.08 mS/cm and temperature of
21.3 °C using the same hand sensors as in Experiment 1.
The temperature condition in the cardboard box for incubation was measured using a temperature logger (UA-00164). Equipment for water sampling was sterilised in thesame manner as in Experiment 1.
Three Ex2-Initial water samples were filtered on site,whereas the remaining samples in triplicate, respectively,
were filtered after 8-h incubation using the method
described in Experiment 1. All filter samples were stored ina freezer at -20 °C until the subsequent DNA extraction.
All equipment used for filtration, including filter holders
and forceps, was sterilised before use using 10% bleachsolution. DNA was extracted from filter samples using themethod described in Experiment 1. The PCR assay was
also conducted using the method described in Experiment
1; however, the target species was changed to bluegill
sunfish Lepomis macrochirus (Centrarchidae, Perciformes)
inhabiting the pond. The primer and probe sequences were
Bluegill_CytB_F
50 -GCCTAGCAACCCAGATTTTAA

Experiment 3: long-term test using BAC
Treatments

Ex3-BAC0.01-0d, -1d, -3d, -5d, -10d BAC at a final
concentration of 0.01% was added on site immediately
after water sampling. Ex3-BAC0.01-0d samples wereimmediately filtered on site and frozen in a portable freezer-18 °C (MR040F). Ex3-BAC0.01-1d, -3d and -5d, -10d
samples were transported to the laboratory and analysed in
the same manner as the corresponding Ex3-Naı̈ve samples.
The incubation duration, i.e., up to 10 days, was set tocover the whole degradation process of naı̈ve samples toreach an undetectable level to illustrate the long-term
effectiveness of BAC. Water samples in each treatment
were sampled in triplicate. Each treatment contained a
negative control water sample comprising 500 ml ultrapure water sourced from the laboratory. Negative control
samples were analysed along with the other experimental
samples to assess unintended contamination duringtransportation, incubation, filtration and extraction steps.
Water sampling
Water samples were obtained from the same site as inExperiment 2 at 9:05 on 9 December 2015. Water samples
were obtained directly from the water surface by submerging a plastic container (volume, *20 l; made ofpolyethylene). Using a beaker with a handle, 500 ml water
was decanted into each of the 30 plastic bags (DP16TN1000), and the bags were subsequently sealed. Each bag
was randomly assigned to each control. Ten percent BAC
was added at a final concentration of 0.01% to all bags
assigned to Ex3-BAC0.01. All bags assigned to Ex3Naı̈ve-0d and Ex3-BAC0.01-0d were immediately filtered

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Limnology (2017) 18:233–241

on site using the same method as in Experiment 1. All of
the remaining bags were subjected to incubation up to
10 days at ambient temperature in a cardboard box. The
samples were transferred to a laboratory during the incubation. Water quality variables at the sampling site were
measured at a pH of 7.33, EC of 0.08 mS/cm and watertemperature of 11.1 °C using the same hand sensors as inExperiment 1. The temperature condition in the cardboard
box for incubation was measured using a temperaturelogger (UA-001-64). Equipment for water sampling wassterilised in the same manner as in Experiment 1.

ambient temperature. Ex1-On-ice-Initial samples contained
less DNA than those in Ex1-BAC0.01 samples (P \ 0.001;
65,772.7 ± 23,616 and 456,326.4 ± 43,240.6 copies/
500 ml, respectively; Fig. 2). The latter contained a 6.94
times higher concentration of ayu DNA. Ex1-On-ice-Initialsamples were filtered on site, and filter samples weretransported on ice, although the DNA concentration was
lower than that of Ex1-BAC0.01 samples, which wereincubated at ambient temperature for 6 h before filtration.

Filtration of water samples

eDNA degradation was confirmed in Experiment 1, even in
the cases where the water samples were filtered on site, andthe filter samples were transported to the laboratory on ice.
Therefore, in Experiment 2, initial samples were filtered onsite, and the filter samples were transported to the laboratory in a frozen state using a portable freezer. Each of thethree candidate preservatives was used at three different
concentrations for treatments. The DNA concentrations inmost of the treatments using addition of preservatives otherthan SH were higher than those of Ex2-Naı̈ve samples after
8-h incubation at ambient temperature (P \ 0.05 in all
combinations; Fig. 3). A significant difference was notfound only in the combination of Ex2-Naı̈ve and Ex2BEC0.1 (P = 0.104). In comparison with Ex2-Initialsamples, significant reductions in the DNA concentrationin all treatments with preservatives other than SH were notobserved, regardless of their concentrations (P C 0.05 in

Water samples assigned to Ex3-Naı̈ve-0d and Ex3BAC0.01-0d were filtered on site, whereas the remaining
samples were filtered after a series of incubation times (1,3, 5 and 10 days) using the same method as in Experiment
1. All filter samples were stored in a freezer at -20 °C
until the subsequent DNA extraction. All equipment used
for filtration, including filter holders and forceps, wassterilised before use with 10% bleach solution.
DNA was extracted from filter samples using the method
in Experiment 1. The PCR assay on bluegill was conductedfollowing the method used in Experiment 2. The R2 values ofthe standard curve for all qPCR experiments exceeded 0.98.
Statistical analysis

Experiment 2

DNA concentrations were calculated as DNA copies per
filtered water volume (500 ml) based on real-time PCR
results and subjected to statistical analyses. All statisticalanalyses were performed using R version 3.0.2 software (RCore Team 2013). The average DNA concentration wascompared among treatments using a t test or one-way analysis of variance (ANOVA) followed by a post hoc test. In
Experiment 3, the lm function of R was used to determine thedifference in the concentration of eDNA (log-transformed
before analysis) between naı̈ve and BAC-added controls
using the presence/absence of BAC and the incubation days
as explanatory variables. In the present analysis, the average
eDNA concentration in triplicate samples in each treatment
was used; however, replicates without positive result were
excluded from the calculation of the averages. Alpha, or thesignificance level, was set to 0.05 in all analyses.

Results
Experiment 1
Experiment 1 determined the deceleration effect of BAC
on ayu DNA degradation within 6 h of incubation at

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Fig. 2 DNA concentrations of the ayu sweetfish Plecoglossus
altivelis determined in Experiment 1. Ex1-BAC0.01 was treated withbenzalkonium chloride (BAC). See ‘‘Materials and methods’’ fortreatment details. Asterisk indicates a significant difference between
the groups. Error bars show the standard deviation of each group(n = 5, each)Limnology (2017) 18:233–241

239

all combinations; Fig. 3). Although the differences were
not significant, slight reductions in the DNA concentrations
were observed in all treatments with cationic surfactant
preservatives (Fig. 3). In Ex2-BEC0.1, the average DNA
concentration in the control was determined to be 56% ofthat of Ex2-Initial. Significant differences in DNA concentrations were detected between Ex2-Initial and Ex2Naı̈ve samples (P \ 0.001), namely 6409.6 ± 1067.4 and926.9 ± 344.1 copies/500 ml, respectively. In controls
using the same preservative, the retention rates of DNA
were not different among treatments, regardless ofpreservative concentrations (P C 0.05 in all combinations).
DNA was not detected at all in any treatments with SH
addition.
Experiment 3
In Experiment 3, only BAC was used as a preservative at
0.01% concentration because of the resulting relatively
high retention rate of DNA in Experiment 2 (*92.1%),
although the difference was not significant among threepreservatives. No significant difference in the DNA concentrations between Ex3-Naı̈ve-0d and Ex3-BAC0.01-0d
was observed (P [ 0.05). The linear model (lm) analysis
revealed significant effects of incubation days (P \ 0.001)
on the concentration of eDNA (Fig. 4). Although thepresence/absence of BAC did not have a significant effect(P [ 0.05), it affected the eDNA concentration through aninteraction effect with incubation days (P \ 0.001). An
ANOVA test revealed that the DNA concentrations in all

Fig. 3 DNA concentrations of the bluegill sunfish Lepomis macrochirus determined in Experiment 2 using various preservatives anddifferent concentrations after 8-h incuation at room temperature: BAC
benzalkonium chloride, BEC benzethonium chloride, DDAC didecyldimethylammonium chloride, SH sodium hypochlorite. See ‘‘Materials and methods’’ for treatment details. Asterisk indicates asignificant difference from the Ex2-Initial. Error bars show astandard deviation of each group (n = 3, each)Fig. 4 Time-dependent decrement of DNA concentrations of the
bluegill sunfish Lepomis macrochirus in benzalkonium chloride(BAC)-treated and naı̈ve water samples determined in Experiment
3. DNA concentrations are shown as their common logarithm. See‘‘Materials and methods’’ for treatment details. Error bars show thestandard deviation of each group (n = 3, each)water samples without a preservative (Ex3-Naı̈ve-1d, -3d, 5d, -10d) were different from those in Ex3-Naı̈ve-0d
samples regardless of the incubation days (P \ 0.05 in all
combinations; Fig. 4). The reduction rates were 83.6, 97.0,
99.3 and 100%, in Ex3-Naı̈ve-1d, -3d, -5d and -10d,
respectively (Fig. 4). On the other hand, no significant
reduction in DNA concentrations in BAC controls (Ex3BAC0.01-1d, -3d, -5d, -10d) was observed in comparison
with those in Ex3-Naı̈ve-0d samples (P [ 0.05 in all
combinations) even after 10-day incubation (Ex3BAC0.01-10d). Although the reductions in DNA concentrations in the BAC controls were not significant, their
reduction rates were calculated as 30.3, 45.3, 48.5 and52.3% in Ex3-BAC0.01-1d, -3d, -5d and -10d, respectively(Fig. 4).
In all experiments, no occurrence of DNA cross contamination among samples during filtration, transportationand the subsequent molecular analysis was observed, as
indicated by negative results for all of the negative controls, including PCR-negative controls.

Discussion
The present study suggested that water samples for eDNA
analyses can be preserved for a considerable duration atambient temperature under the effect of the preservatives
described here. On average, BAC appeared to be the mosteffective; however, all the remaining preservatives

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belonging to the group of cationic surfactants were also
effective in halting or significantly decelerating thedegradation rate of DNA in water samples, as confirmed by
the results of Experiment 2. In Experiment 3, which
focused on BAC, a significant reduction in DNA concentration was not observed in treatments with BAC addition
at 0.01% concentration even after 10-day incubation atambient temperature (*21 °C), and the retention rate of
DNA was determined to be *50%. This result isremarkable in comparison with the failure of DNA detection in a control without BAC after 10-day incubation.
Even after 1-day incubation, approximately 80% of DNA
was degraded in the control without BAC. Based on theseresults, the effectiveness of BAC as an eDNA preservative
can be considered exceptional. Using these cationic surfactants as eDNA preservatives, the degradation rate of
DNA in a sample water could be reduced to *10 and 50%
within 8-h and 10-day incubations, respectively, at an
ambient temperature. Cationic surfactants are adsorbed
onto the bacterial surface because of their positively
charged cell membrane, following which the normal
function of the bacterial cells is considerably disturbed(Ziani et al. 2011). Moreover, it may influence DNase to
reduce its degrading effect on DNA, thereby contributing
to DNA preservation. The preservation methods of eDNA
water samples shown in the present study can contribute toincreasing the accuracy of eDNA quantification and to
reducing false-negative results in case of detecting rare
species or invasive species by reducing the probability ofthe disappearance of rare sequences in a water sample
during transportation. In the present study, we focused on a
freshwater habitat, although we also confirmed the effectiveness of BAC as a preservative for eDNA in saltwater bytargeting blackhead seabream Acanthopagrus schlegelii(Bleeker, 1854; Sparidae, Perciformes). Twenty-four-hour
incubation of sample water with BAC at ambient temperature did not produce a significant difference in the DNA
concentration from the initial value (Minamoto et al.,unpublished). The effectiveness of the eDNA preservatives
is remarkable, although further detailed examinations are
merited, e.g., on their effect as a PCR inhibitor, as there
was a slight reduction of the eDNA concentration insamples to which a higher concentration of BEC was added
in Experiment 3 (Fig. 3). In addition, future studies should
clarify whether there are biases in the effectiveness of thesepreservatives among target species or among different
biomasses.
In Experiment 1, a considerable reduction in the DNA
concentration in the initial sample (Ex1-On-ice-Initial)
without preservatives was observed, probably because of
the DNA degradation during transportation, even when thewater sample was filtered on site and filter samples weretransported to the laboratory on ice. Moreover, in our

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Limnology (2017) 18:233–241

preliminary experiment in which a water sample wastransported to the laboratory in a cool box within 7.5 h andfiltered, the resulting DNA concentration in the sample wasconsiderably lower than that of a 3-day incubated sample
that was treated with 0.01% BAC at ambient temperature(Yamanaka et al. unpublished). These results suggest thatthe DNA concentrations measured in previous studies,
which were affected by the method of sample transportation used, such as water transportation on ice (e.g. Takahara et al. 2012; Eichmiller et al. 2014; LacoursièreRoussel et al. 2016) or on-site filtration and transportationof filter samples on ice (Takahara et al. 2012), might notreflect the actual DNA concentration at the sampling time
because of DNA degradation during sample transportation.
Pilliod et al. (2014) noted a 38% reduction in the eDNA
concentration of the Idaho giant salamander Dicamptodon
aterrimus in 2 days, which was incubated at 4 °C, although
the detection rate appeared to be unaffected according to
the PCR assay. This may cause difficulties in the eDNA
quantification in studies where the concentration is used toestimate the biomass or abundance of the target species,
particularly when water samples are collected from multiple sites because of the difference in the transportation
time of water/filter samples among the sites. In Experiment
3, on-site filtration followed by filter preservation in a
portable freezer at -18 °C was adopted to prepare theinitial sample (Ex3-Naı̈ve-0d). This more careful sample
treatment resulted in the statistical equality of the average
DNA concentrations between the initial sample and the
BAC-treated sample, which was also filtered on site andpreserved in the same freezer (Ex3-BAC0.01-0d). Taken
together, it appears that the DNA concentration measured
by the following two methods will provide the best estimate of the actual initial DNA concentration in the fieldwater: (1) on-site filtration followed by filter preservation
under a freezing temperature (or followed by preservation
with ethanol or other preservatives instead, e.g. Goldberg
et al. 2011, Renshaw et al. 2015) or (2) on-site addition of apreservative. In any case, only halting DNA degradation
using the appropriate methods is required to obtain the best
estimate of the DNA concentration as degradation begins
immediately after water sampling. Further, comparing thetwo methods based on the amount of equipment required
for field work, the method using a preservative is simpler.
In the present study, we focused on the DNA concentration of a specific species and confirmed the effectivenessof cationic surfactants as eDNA preservatives. We did nottest the effectiveness to preserve eDNA samples formetabarcoding analysis, although the method we present
here is likely to be effective for that as well because thedeactivation of microorganisms will result in whole eDNA
preservation in the water sample. However, further studies
verifying the use of these preservatives in metabarcoding

Limnology (2017) 18:233–241

studies and in various water qualities are required. The
adoption of eDNA analysis reduces the considerable
amount of effort required in field work, thereby enabling
biological monitoring at a very large scale. Similar to the
Ocean Sampling Day (https://www.microb3.eu/myosd)
initiative, the future involvement of citizen scientists inbiological monitoring programs is inevitable to facilitate
nation-wide, continent-wide or ocean-wide surveys. The
statistical equivalence of the eDNA concentration between
the initial sample and 8-h incubated samples in Experiment
2 suggests that usage of these preservatives can assure thestrict measurement of the eDNA concentration without an
on-site filtration protocol in daily field surveys. The simplification of eDNA sampling would accelerate theexpansion of further applications of eDNA analysis invarious studies.
Acknowledgements This work was supported by a Grant-in-Aid forthe new technology development project of small and medium
enterprises of Shiga Prefecture and partly supported by the Ryukoku
University Technology Fund to HY. JM is an employee of Settsu Oil
Mill, Inc., and AK is an employee of Hiyoshi Corp.
Open Access This article is distributed under the terms of theCreative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide alink to the Creative Commons license, and indicate if changes were
made.

References
Core Team R (2013) R: A language and environment for statisticalcomputing. R Foundation for Statistical Computing, Vienna
Eichmiller J, Bajer PG, Sorensen PW (2014) The relationshipbetween the distribution of common carp and their environmental DNA in a small lake. PLoS One 9:e112611. doi:10.1371/
journal.pone.0112611
Eichmiller J, Best SE, Sorensen PW (2016) Effects of temperatureand trophic state on degradation of environmental DNA in lakewater. Envir Sci Tech 50:1859–1867. doi:10.1021/acs.est.
5b05672
Ficetola GF, Miaud C, Pompanon F, Taberlet P (2008) Species
detection using environmental DNA from water samples. Biol
Lett 4:423–425. doi:10.1098/rsbl.2008.0118
Fukumoto S, Ushimaru A, Minamoto T (2015) A basin-scale
application of environmental DNA assessment for rare endemic
species and closely related exotic species in rivers: a case studyof giant salamanders in Japan. J Appl Ecol 52:358–365. doi:10.
1111/1365-2664.12392
Goldberg CS, Pilliod DS, Arkle RS, Waits LP (2011) Molecular
detection of vertebrates in stream water: a demonstration using
Rocky Mountain tailed frogs and Idaho giant salamanders. PLoS
One 6:e22746
Jerde CL, Mahon AL, Chadderton WL, Lodge DM (2011) ‘‘Sightunseen’’ detection of rare aquatic species using environmental
DNA. Conserv Lett 4:150–157. doi:10.1111/j.1755-263X.2010.
00158.x

241
Kelly RP, Port JA, Yamahara KM, Crowder LB (2014) Using
environmental DNA to census marine fishes in a large mesocosm.
PLoS One 9:e86175. doi:10.1371/journal.pone.0086175
Keskin E (2014) Detection of invasive freshwater fish species using
environmental DNA survey. Biochem Systemat Ecol 56:68–74.
doi:10.1371/journal.pone.0022746
Klymus KE, Richter CA, Chapman DC, Paukert C (2015) Quantification of eDNA shedding rates from invasive bighead carp
Hypophthalmichthys nobilis and silver carp Hypophthalmichthys
molitrix. Biol Conserv 183:77–84. doi:10.1016/j.biocon.2014.11.
020
Lacoursière-Roussel A, Côté G, Leclerc V, Bernatchez L (2016)
Quantifying relative fish abundance with eDNA: a promising
tool for fisheries management. J Appl Ecol 53:1148–1157.
doi:10.1111/1365-2664.12598
Maruyama A, Nakamura K, Yamanaka H, Kondoh M, Minamoto T(2014) The release rate of environmental DNA from juvenile andadult fish. PLoS One 9:e114639. doi:10.1371/journal.pone.
0114639
Minamoto T, Naka T, Moji K, Maruyama A (2016) Techniques forthe practical collection of environmental DNA: filter selection,
preservation, and extraction. Limnol 17:23–32. doi:10.1007/
s10201-015-0457-4
Miya M, Sato Y, Fukunaga T, Sado T, Poulsen JY, Sato K, Minamoto
T, Yamamoto S, Yamanaka H, Araki H, Kondoh M, Iwasaki W(2015) MiFish, a set of universal PCR primers for metabarcoding
environmental DNA from fishes: detection of more than 230
subtropical marine species. R Soc Open Sci 2:150088. doi:10.
1098/rsos.150088
Pilliod DS, Goldberg CS, Arkle RS, Waits LP (2014) Factors
influencing detection of eDNA from a stream-dwelling amphibian. Mol Ecol Resour 14:109–116. doi:10.1139/cjfas-2013-0047
Rees HC, Maddison BC, Middleditch DJ, Patmore JRM, Gough KC,
Crispo E (2014) The detection of aquatic animal species using
environmental DNA-a review of eDNA as a survey tool in ecology.
J Appl Ecol 51:1450–1459. doi:10.1111/1365-2664.12306
Renshaw MA, Olds BP, Jerde CL, McVeigh MM, Lodge DM (2015)
The room temperature preservation of filtered environmental
DNA samples and assimilation into a phenol–chloroform–
isoamyl alcohol DNA extraction. Mol Ecol Resour
15:168–176. doi:10.1111/1755-0998.12281
Takahara T, Minamoto T, Yamanaka H, Doi H, Kawabata Z (2012)
Estimation of fish biomass using environmental DNA. PLoS One
7:e35868. doi:10.1371/journal.pone.0035868
Takahara T, Minamoto T, Doi H (2013) Using environmental DNA toestimate the distribution of an invasive fish species in ponds.
PLoS One 8:e56584. doi:10.1371/journal.pone.0056584
Takahara T, Minamoto T, Doi H (2015) Effects of sample processing
on the detection rate of environmental DNA from the Common
carp (Cyprinus carpio). Biol Conserv 183:64–69. doi:10.1016/j.
biocon.2014.11.014
Thomsen PF, Kielgast J, Iversen LL, Møller PR, Rasmussen M,
Willerslev E (2012) Detection of a diverse marine fish faunausing environmental DNA from seawater samples. PLoS One
7:e41732. doi:10.1371/journal.pone.0041732
Yamanaka H, Minamoto T (2016) The use of environmental DNA offishes as an efficient method of determining habitat connectivity.
Ecol Indic 62:147–153. doi:10.1016/j.ecolind.2015.11.022
Ziani K, Chang Y, McLandsborough L, McClements DJ (2011)
Influence of surfactant charge on antimicrobial efficacy of
surfactant-stabilized thyme oil nanoemulsions. J Agric Food
Chem 59:6247–6255. doi:10.1021/jf200450m

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