Marine Pollution Bulletin 157 (2020) 111320

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Marine Pollution Bulletin

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Ecological risk assessment of an antifouling biocide triphenyl T

(octadecylamine) boron in the Seto Inland Sea, Japan
⁎
Toshimitsu Onduka , Kumiko Kono, Mana Ito, Nobuyuki Ohkubo, Takeshi Hano, Katsutoshi Ito,
Kazuhiko Mochida
National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-
0452, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, we derived the predicted no-effect concentrations (PNEC) for triphenyl (octadecylamine) boron
Algal growth inhibition test (TPB-18) and investigated the occurrence of triphenylboranes (TPBs), including TPB-18, for ecological risk as-
Crustacean reproduction test sessment in the Seto Inland Sea, Japan. We tested algal growth inhibition, crustacean immobilization, and re-
Early-life stage toxicity test productive toxicity and performed toxicity tests in fish to assess acute and chronic toxicity and generate the
Species sensitivity distribution
PNEC for TPB-18. The minimum toxicity value was 0.30 μg/L, as determined by the 72-h no-observed-effect
concentration (NOEC) for the alga Chaetoceros gracilis. The 5th-percentile of hazardous concentration (HC5),
derived from NOECs using the species sensitivity distributions approach, was 0.059 μg/L, which indicated the
PNEC of 0.0059 μg/L. In comparison, the highest concentration in seawater sampled from the Seto Inland Sea
was 0.00034 μg/L, suggesting that the ecological risks posed by TPB-18 are currently low.

1. Introduction
Restrictions on the use of organotin antifoulants have led to the use
of novel antifouling compounds on ship hulls and fishing nets. Among
these biocides, Polycarbamate (PC) is most commonly used, accounting
for > 78% of total emissions this decade, followed by boron-based
biocides like triphenylborane, with a couple of modifying groups, such
as pyridine and octadecylamine, which contributed 22% of the total
emissions, as reported by the Japanese PRTR (Pollutant Release and
Transfer Register, PRTR Emissions, 2019). This article focuses on the
ecological toxicity of the triphenyl (octadecylamine) boron (TPB-18)
that has been widely used mainly on fishing nets over the past decade.
The toxicity of boron-based compounds, particularly TPB-18, is
poorly described relative to other biocides. In a previous study, we
demonstrated the primary risks associated with pyridine triphenyl
borane (PTPB, Mochida et al., 2012), which has only been used on ship
hulls in Japan. TPB-18 toxicity has been reported in a few marine or-
ganisms—for instance the luminous bacteria Aliivibrio fischeri, the brine
shrimp Artemia salina (Tsuboi and Okamura, 2012), the oyster Cras-
sostrea gigas, and the sea urchin Hemicentrotus pulcherrimus (Tsunemasa
et al., 2013). However, data on TPB-18 toxicity on marine organisms
remains limited.
The principal aim of this study was to assess the acute and chronic
toxicity values of TPB-18 for marine organisms representing three
trophic levels (algae, crustacea, and fish), and to estimate the predicted
no-effect concentration (PNEC) for ecological risk assessment using
conventional and probabilistic approaches based on previously re-
ported data and current data in this study. We assessed the ecological
risk from the derived PNEC values and environmental concentration
data for triphenylboranes (TPBs) from seawater in the vicinity of fishing
nets, fishing ports, harbors, river mouths, and shipping routes in the
Seto Inland Sea, Japan. Owing to the difficulty in discriminating TPB-18
from other TPBs, we were not able to test for this biocide specifically.
2. Materials and methods
2.1. Organisms
The test organisms used in this study included algae: Tetraselmis
tetrathele, Chaetoceros gracilis, and Skeletonema marinoi-dohrnii complex
(NIES-324); crustacea: Tigriopus japonicus and Japanese blue crabs
(Portunus trituberculatus); and fish: the Arasaki strain of the mummichog
(Fundulus heteroclitus, Shimizu, 1997) and red sea bream (Pagrus major).
Nauplii of T. japonicus (< 24 h after hatching) and hatched zoea of

⁎
Corresponding author at: National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi,
Hiroshima 739-0452, Japan.
E-mail address: onduka@fra.affrc.go.jp (T. Onduka).

https://doi.org/10.1016/j.marpolbul.2020.111320
Received 8 April 2020; Received in revised form 25 May 2020; Accepted 26 May 2020
Available online 10 June 2020
0025-326X/ © 2020 Elsevier Ltd. All rights reserved.
T. Onduka, et al.
Japanese blue crabs were used in crustacea immobilization and/or re-
production tests. Juvenile red sea bream (total length, 47 ± 5.3 mm;
body weight, 1450 ± 165 mg, average ± standard error) and
mummichog larvae (total length, 6.4 ± 0.1 mm; body weight,
2 ± 0 mg) were used in the acute toxicity tests. Mummichog embryos
were used for early-life stage (ELS) tests. Detailed information on
sources of test organisms and their culture and acclimation conditions
are provided in the Supplementary material (SM1).
2.2. Chemicals
TPB-18 (molecular weight, 510.6; purity, 94.7%) was obtained from
Nitto Seimo, Co., Ltd. (Tokyo, Japan). Stock solutions of TPB-18 were
prepared by dilution with dimethyl sulfoxide (DMSO, plant cell culture
tested, Sigma-Aldrich St. Louis, MO, USA) or DMSO containing 40%
polyoxyethylene hydrogenated castor oil (HCO-40, Nikko Chemicals,
Tokyo, Japan).
2.3. Dilutions
For algal and crustacean toxicity tests, the test solutions were ob-
tained by dilution of the stock solution and the DMSO (for the solvent
control, SC) 10,000 times with modified SWM3 medium (hereafter
SWM3, Imai, 2012) for algae, 10,000 times with seawater filtered
through sand, activated carbon, and a glass fiber filter (GF/C,
Whatman, Maidstone, UK; hereafter “filtered seawater”) for T. japo-
nicus, and 1000 times with filtered seawater for P. trituberculatus (Table
S1). The acute and chronic fish toxicity tests were conducted in a flow-
through system, with pretest solutions obtained by diluting the stock
solutions between 1000 and 16,000 times. Details of preparation of test
solutions for the fish toxicity tests are provided in the Supplementary
material (SM2). DMSO and HCO-40 concentrations are shown in Table
S2.
2.4. Toxicity tests
Toxicity tests were carried out following the test guidelines of the
Organization for Economic Cooperation and Development (OECD,
1992a,b, 2006) with modifications for marine organisms. Algal growth
inhibition tests, crustacean immobilization tests, and the crustacean
reproduction test were performed following Onduka et al. (2010, 2013)
and Kikkawa et al., 2010. Organisms were tested in a test solution of
SWM3 medium, in filtered seawater (as described above), or in sea-
water filtered only through sand. The details of these test methods are
provided in the Supplementary material (SM3). Water quality para-
meters for the TPB-18 tests are shown in Table S3.
2.5. Environmental seawater sample collection
Water samples (400 mL) were collected with a stainless-steel
bucket, from the top of the water column from 35 stations located in the
central (St. 1–18, 22, 23) and eastern part (St. A – O) of the Seto Inland
Sea (Fig. 1). Sampling was conducted only once per year at each site
between June and December of 2017 and 2018.
2.6. Chemical analysis
To determine the actual toxicant concentrations of TPB-18 in the
toxicity tests, test solutions were sampled 0 and 72, 0 and 24, and 24
and 96 h after the start of the algal growth inhibition, crustacean im-
mobilization, and fish acute toxicity tests, respectively. For the crusta-
cean reproduction test, water was sampled once a week, before and
after the renewal of the test solution. In the ELS tests, water was sam-
pled every 10 days. Pyridine was added to each test solution im-
mediately after collection (final concentration: 1% (v/v)) to stabilize
TPB-18. TPB-18 was extracted from the water samples with Sep-Pak
2
Marine Pollution Bulletin 157 (2020) 111320
C18 Plus short cartridges (WAT020515, Nihon Waters K.K., Tokyo,
Japan) following the manufacturer's instructions, and eluted from car-
tridges with acetonitrile containing 1% pyridine for further analysis.
TPB-18 concentrations in the water samples were analyzed fol-
lowing methods specified by the Chemical Evaluation and Research
Institute, Japan (2015). This method measures TPBs derived from TPB-
18 and other TPB complexes. To stabilize TPB-18 in the seawater, 0.2 M
NaOH (special grade; Wako Pure Chemical Industries, Ltd., Osaka,
Japan) was added to 400 mL of each sample immediately after collec-
tion (pH: approximately 12.8). These solutions were loaded onto
Presep®-C Agri (Short) cartridges (291-26851, Wako Pure Chemical
Industries, Ltd.), conditioned with 10 mL of acetonitrile and distilled
water was added sequentially. TPBs were eluted from the cartridges
with 5 mL of acetonitrile, following which the elute was condensed to
1 mL prior to further analysis.
TPBs concentrations were assessed by liquid chromatography (LC)
(LC-30AD Prominence system, Shimazu, Kyoto, Japan), following the
method of Zhou et al. (2007). Details on the chemical analysis of TPB-
18 using LC-MS, are shown in the Supplementary material (SM4).
The TPB-18 detection limit of test solution used for the toxicity tests
(0.02 μg/L) was calculated as ten times the signal to noise ratio. We
used geometric mean values or time-weighted means (for the crusta-
cean reproduction test) of the TPB-18 concentrations to estimate the
median effect concentration (EC50), median lethal concentration (LC50),
the lowest observed effect concentrations (LOECs), and the no observed
effect concentration (NOECs). When TPB-18 was not detected in the test
solution, its concentration was determined as half of the detection limit
(0.01 μg/L), following the OECD guidelines (OECD, 2000). For water
samples, the recovery rate in seawater analysis and the quantification
limit of TPB-18 were determined as follows: seawater for the recovery
test was spiked with a specified amount of the TPB-18 (0.09 ng/L) and
pretreated as for the seawater analysis. The recovery test was per-
formed seven times, and the standard deviation of the TPB-18 con-
centration and average recovery rate were determined. Method quan-
tification limit (MQL) for TPB-18 was determined as ten times the
standard deviation (0.12 ng/L) of the limit, and the average recovery
rate and its standard deviation were 100% ± 13% and 0.012 ng/L.
2.7. Data analysis
Estimation of the 72-h EC50, 24-h LC50, and 96-h LC50 values, and
the multiple comparison tests were carried out with R (R Development
Core Team, 2019) software, primarily with the drc, coin (Cochran-Ar-
mitage test), and multcomp (Dunnett's test) packages (Ritz et al., 2015;
Hothorn et al., 2006, 2008). For the crustacean reproduction test, the
survival, number of larvae developing to copepodid stage, sex ratio,
number of brood individuals, and number of nauplii in the group were
analyzed using the Cochran-Armitage test for the linear trends of the
proportions (Agresti, 2002). Time to development, time until produc-
tion of the first clutch, number of clutches, and the number of nauplii
per individual and clutch were analyzed by Dunnett's test. Differences
between groups were considered significant at p < 0.05. The max-
imum acceptable toxicant concentration (MATC) was calculated as the
geometric mean of LOEC and NOEC (Rand, 1995). The acute-to-chronic
ratio (ACR) was calculated as the ratio between the acute toxicity value
and the chronic toxicity value. In case of inability to estimate chronic
toxicities directly, they were directly estimated using the ACR.
Species sensitivity distributions (SSDs) were estimated using a
Bayesian approach (Mochida et al., 2012; Hayashi and Kashiwagi,
2010, 2011). The SSD analysis for TPB-18 was performed using acute
(EC50 and LC50) and chronic toxicity data obtained in this study. The
72-h NOEC for algae, or the geometric mean of LOEC and NOEC for
crustacea and fish, were used as measures of chronic toxicity. For
species where two toxicity values were available, the geometric mean
was used for the SSD analysis (Table 1). SSD analyses were conducted in
WinBUGS (Lunn et al., 2000) and R, using a Bayesian model and
T. Onduka, et al.
Fig. 1. Map showing the sampling stations (black circles) in the central (St. 1–18, 22, 23) and eastern (St. A–O) parts of the Seto Inland Sea. The maps were processed
based on the information available from the Kokudo Chiri-in (Geographical Survey Institute), Japan. https://fgd.gsi.go.jp/download/mapGis.php.
assuming a normal distribution. Independently and identically dis-
tributed (i.i.d.) random variables were drawn as follows:
Model:Xij ~i. i. d. N (θ, σ 2 )
where θ is the mean and σ2 is the variance of the normal distribution.
Details of the estimation are provided in the Supplementary material
(SM5). The conventional approach, as per the guidelines of the Che-
mical Substances Control Law, Japan (http://www.meti.go.jp/policy/
chemical_management/english/cscl/about.html), OECD (2011), and
EU (2003), calculates PNEC as the lowest value from the chronic toxi-
city values obtained for three different trophic levels, divided by an
uncertainty factor of 10.
3. Results
3.1. Toxicity
3.1.1. Algae
The measured TPB-18 concentrations averaged 38% ± 2% of the
nominal concentrations obtained from the algal growth inhibition tests
(Tables 1, S4). The 72-h EC50 of TPB-18 for C. gracilis, the S. marinoi-
dohrnii complex, and T. tetrathele from both experiments averaged 1.6,
2.5, and 23.0 μg/L, respectively. The results of these tests (repeated
twice) were reproducible. The algal growth inhibition tests using dia-
toms (C. gracilis and the S. marinoi-dohrnii complex) satisfied the per-
formance criteria of the OECD test guidelines (OECD, 2006). For T.
tetrathele, the 72-h EC50 of TPB-18 should be used for reference only,
since the recorded growth rate in the control was lower than 0.92/day
in the test period.
3
Marine Pollution Bulletin 157 (2020) 111320
3.1.2. Crustacea
The measured TPB-18 concentrations averaged 40% ± 3% of the
nominal concentrations obtained from the crustacean immobilization
tests (Tables 1, S5). In these tests, the immobilization in the SC group
was ≤10%. The 24-h LC50 of TPB-18 for Japanese blue crabs and T.
japonicus were about 150 and 22 μg/L respectively, and the results of
these tests (repeated twice) were reproducible.
In the crustacean reproduction test, the measured concentrations of
TPB-18 were 15% ± 1% of the nominal concentrations (Table 2).
Survival and the number of larvae developing to copepodid stage sig-
nificantly decreased as the TPB-18 concentration increased (p < 0.05
in the 40 μg/L exposure groups). Additionally, the sex ratio (at 7-d)
significantly increased, while the size of brood per group significantly
decreased in response to the increase in TPB-18 (in the 20 and 40 μg/L
exposure groups). In contrast, no effect of TPB-18 was observed in any
of the experimental groups on time of development to the copepodid
stage, sex ratio at 21-d, or time to produce the first clutch. For assess-
ment of time of development to the copepodid stage, time to produce
first clutch, number of clutches, number of nauplii per organism, and
number of nauplii per clutch, all TPB-18 exposure groups were com-
pared with the SC, since these variable were same in the control and SC
groups. Our results showed that the number of clutches was sig-
nificantly lower in the 40 μg/L exposure group than in the SC group,
while the number of nauplii per organism was significantly lower in the
5, 20, and 40 μg/L exposure groups compared to the SC group. The
number of nauplii per clutch was also significantly lower in the 5 and
20 μg/L exposure groups than in the SC group (Table 2). However, the
5 μg/L exposure group had a higher number of brood individuals than
the other groups (including the control and SC groups). Therefore, the
T. Onduka, et al. Marine Pollution Bulletin 157 (2020) 111320

Table 1
Toxicity values of triphenyl (octadecylamine) boron toward various marine organisms, such as algae, crustacea, and teleosts, used for species sensitivity distribution
analysis.
Taxon Species Endpoints Toxicity value Geometric mean

Algae
Chaetoceros gracilis 72-h EC50 1.9 (1.6–2.3) μg/La 1.6 μg/La
1.3 (1.1–1.5) μg/La
72-h NOEC 0.30 μg/La 0.30 μg/La
Skeletonema marinoi-dohrnii complex 72-h EC50 2.5 (1.9–3.5) μg/La 2.5 μg/La
2.5 (2.0–3.3) μg/La,c
72-h NOEC 0.56 μg/La 0.54 μg/La
0.52 μg/La
Tetraselmis tetrathele 72-h EC50 21 (18–26) μg/La 23 μg/La
26 (21−32) μg/La
72-h NOEC 2.0 μg/La 2.1 μg/La
2.3 μg/La
Invertebrate
Crustacean Artemia salina 48-h LC50 40 (15–87) μg/Lb,d 40 μg/Lb,d
Portunus trituberculatus 24-h EC50 190 (170–210) μg/La 150 μg/La
120 (110−130) μg/La
Tigriopus japonicus 24-h EC50 22 (19–25) μg/La 22 μg/La
22 (6.6–51) μg/La
21-d LOEC 1.6 μg/La
21-d NOEC 2.8 μg/La
MATC 2.1 μg/La
Bivalve Crassostrea gigas 24-h LC50 10 (9.5–12) μg/Lb 10 μg/Lb,e
Echinoderm Hemicentrotus pulcherrimus 48-h LC50 73 (68–79) μg/Lb 73 μg/Lb,e
Vertebrate
Teleost fish Mummichog 96-h LC50 980 (960–990) μg/La 980 μg/La
(Fundulus heteroclitus) 50-d LOEC 120 μg/La
50-d NOEC 48 μg/La
MATC 76 μg/La
Red sea bream 96-h LC50 1000 (970–1200) μg/La 920 μg/La
(Pagrus major) 840 (830–850) μg/La

The 95% confidence limits are given in parentheses. MATC, maximum acceptable toxicant concentration.
a
The toxicity value was based on measured concentration.
b
The toxicity value was based on the nominal concentration.
c
The same values were obtained from the 1st and 2nd toxicity tests.
d
The data were reported by Tsuboi and Okamura (2012).
e
The data were reported by Tsunemasa et al. (2013).

lower number of nauplii per clutch in the 5 μg/L exposure group
compared to the SC group (Table 2) could be explained by increased
competition for food.
Our results indicated that the sex ratio at 7-d, size of brood, number
of nauplii per organism, and number of nauplii per clutch were the most
sensitive endpoints. Based on the measured TPB-18 concentrations,
LOEC and NOEC were estimated to be 2.8 and 1.6 μg/L (5.5 and
3.1 nM), respectively.
3.1.3. Fish
The measured TPB-18 concentrations averaged 68% ± 17% of the
nominal concentrations obtained from the fish acute toxicity tests
(Tables 1, S6). Survival in the SC group was 100%. In both trials, the
96-h LC50 of TPB-18 for the red sea bream and the mummichog were
920 and 980 μg/L, respectively. The results of these tests (repeated
twice) were reproducible.
In the ELS tests, the measured TPB-18 concentrations were
20% ± 5% of the nominal concentrations (Table 3). Since there were
no significant differences between the control and SC groups, all TPB-
18 exposure groups were compared to the SC group at all endpoints.
Further, although there were no obvious effects on hatchability and the
time to hatch in any of the test groups, the 30-d survival in the 400 μg/L
exposure group was significantly lower than in the SC group, and
reached zero after 40 d. Survival in the other TPB-18 exposure groups
was not significantly different from the SC group, but tended to de-
crease as TPB-18-concentration increased, especially at 30-d. Further,
TPB-18 toxicity had no significant effect on the growth of mummichogs,
except in the 50 μg/L exposure group, where body weight was sig-
nificantly lower (p < 0.05) than in the SC group. Since survival in this
group was better than in the other groups (including the control and
SC), higher organism density may have increased competition for nu-
trients, thereby inhibiting growth. However, the ELS test for TPB-18 did
not show morphological deformities in any of the experimental groups.
Thus, survival was the most sensitive endpoint, and based on the
measured TPB-18 concentrations, LOEC and NOEC were estimated as
120 and 28 μg/L (235 and 94 nM) respectively.
3.2. SSD and PNEC
The SSD analysis for TPB-18 was performed using acute (EC50 and
LC50) and chronic toxicity data obtained in this study. The values used
included ten acute toxicity values (for three algae, five crustacean, and
two fish species) and five chronic toxicity values (for three algae, one
crustacean and one fish species). These values were common log-
transformed prior to SSD derivation using the Bayesian statistical model
(Fig. 2). Further, the Shapiro-Wilks test confirmed the normality of the
log-transformed SSD data for the acute and chronic toxicity values
(p = 0.4884 and p = 0.343, respectively). The median of the 5% ha-
zardous concentration (HC5) with 90% confidence intervals for the
acute and chronic toxicity values was 0.62 (0.040–3.0) and 0.059
(0.00029–0.49) μg/L, respectively. To be on the safer side while esti-
mating risk, the HC5 for the chronic toxicity values was used for cal-
culating PNEC. Deviance information criterion and the medians of θ
and σ (the normal distribution mean and variance), with their 90%
confidence intervals for the acute and chronic toxicity data, are de-
scribed in Table S7. Using the conventional method, we obtained a
PNEC of 0.030 μg/L by dividing the lowest chronic toxicity value
(0.30 μg/L, for the marine alga, Chaetoceros calcitrans, Table 1) by an

4
 Table 2
Effect of long-term exposure to various concentrations of triphenyl (octadecylamine) boron (TPB-18) on the survival, number of developments, development time, sex ratio, number of brood individuals, time to
production of the first clutch, number of clutches, and fecundity of Tigriopus japonicus.
Group Conc. (μg/L) Survival (%) Number of Development Sex ratio Number of Time to Number of Fecundity (number of nauplii)
T. Onduka, et al.

developments time (N to C, (male/all brood production clutches

(N to C)b day)b %) individuals of the first
clutch
Nominal Actual Day 7 Day 21 Day Day (days) Whole /Individual /Clutch
7 21 group
Change 1 Change 2 Change 3 Time-
weighted
0h 48 h 0h 48 h 0h 48 h meana

Control 0 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 100 90 20 5.3 ± 0.1 58 56 8 13.5 ± 0.5 2.6 ± 0.3 271 34 ± 4 13 ± 2
Solvent cont. 0 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 100 90 20 5.0 ± 0.1 44 44 10 12.7 ± 0.4 3.6 ± 0.2 487 49 ± 5 13 ± 1
TPB-18 2.5 1.41 < 0.02 1.83 0.10 1.61 0.04 0.43 100 85 20 4.8 ± 0.1 47 47 9 13.3 ± 0.2 3.4 ± 0.2 487 54 ± 9 15 ± 1
5 2.90 < 0.02 2.82 0.15 3.82 < 0.02 0.69 100 80 20 5.0 ± 0.3 22 19 14 13.5 ± 0.3 2.7 ± 0.3 368 26 ± 5* 9 ± 1*
10 5.98 < 0.02 6.09 0.33 8.59 0.10 1.60 95 95 19 5.3 ± 0.2 50 50 9 13.3 ± 0.6 3.4 ± 0.2 459 51 ± 6 15 ± 1
20 11.75 < 0.02 11.32 0.54 17.69 0.08 2.81 100 70 20 5.1 ± 0.2 74* 64 5* 13.2 ± 0.7 2.8 ± 0.6 112 22 ± 5* 7 ± 1*
40 24.52 < 0.02 19.53 2.18 35.75 0.06 5.55 90* 10* 18v 5.7 ± 0.4 67* 0 5* 11.6 ± 0.4 1.8 ± 0.2* 94 19 ± 5* 10 ± 2

a
The actual concentration was calculated using half the detection limit (0.01 μg/L) when the test chemical was not detected.
b
Development time, from nauplius to copepodid. Data are expressed as means ± standard error.
* Indicates significant differences from the values of solvent control at p < 0.05.

5
Table 3
Effect of long-term exposure to various concentrations of triphenyl (octadecylamine) boron (TPB-18) on the hatching time, hatchability, survival, and growth of the mummichog (Fundulus heteroclitus).
Group Conc. (μg/L) No. of used Hatch time Hatchability Survival (%) Growth (50 d)

Nominal Actual

Number of sampling Geometric mean

1 2 3 4 5 Embryosa Hatch (d) (%) (30 d) (40 d) (50 d) TL (mm) BW (mg)

Control 0 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 60 16 ± 0.43 87 ± 6.0 100 91 ± 6.8 80 ± 7.6 22 ± 0.43 150 ± 9.7
Solvent Cont. 0 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 60 16 ± 0.52 88 ± 4.7 100 94 ± 1.9 78 ± 5.8 22 ± 0.38 140 ± 7.7
TPB-18 25 8.8 1.3 1.3 2.3 1.6 2.2 ± 1.5 60 16 ± 0.52 93 ± 2.7 95 ± 3.4 89 ± 4.6 82 ± 6.2 22 ± 0.49 130 ± 8.8
50 21 2.3 2.5 7.1 3.7 5.0 ± 3.4 60 14 ± 0.43 98 ± 1.7 93 ± 5.1 91 ± 4.5 85 ± 7.5 21 ± 0.29 110 ± 4.6*
100 33 32 20 25 34 28 ± 2.7 60 18 ± 2.5 95 ± 3.2 91 ± 3.5 86 ± 4.7 75 ± 8.9 21 ± 0.41 120 ± 7.7
200 74 77 32 53 26 48 ± 11 60 20 ± 1.2 82 ± 5.0 89 ± 6.4 72 ± 9.2 66 ± 8.4 21 ± 0.50 130 ± 8.7
400 160 94 140 92 ND 120 ± 15 60 17 ± 1.2 90 ± 3.3 7.1 ± 5.1* 0 0 ND ND

ND, not determined (no fish survived for 40 d in the 400 μg/L group); d, days.
a
15 × 4 subgroups for 60. Data are expressed as means ± standard error.
⁎
Indicates significant differences from the solvent control at p < 0.05.
Marine Pollution Bulletin 157 (2020) 111320
T. Onduka, et al. Marine Pollution Bulletin 157 (2020) 111320

Table 4
Concentrations of triphenylboranes (as TPB-18) in the surface seawater samples
from the Seto Inland Sea, Japan.
Sampling station Conc. (ng/L)

St. No. Year

2017 2018

1 n.d. n.d.
2 0.23 n.d.
3 0.21 n.d.
4 0.25 n.d.
5 n.d. n.d.
6 n.d. n.d.
7 n.d. n.d.
8 n.d. n.d.
9 n.d. n.d.
10 n.d. n.d.
11 n.d. n.d.
12 n.d. n.d.
13 n.d. n.d.
14 0.32 n.d.
15 n.d. n.d.
16 n.d. n.d.
17 n.d. n.d.
18 n.d. n.d.
Fig. 2. Species sensitivity distribution to triphenyl (octadecylamine) borane
22 n.d. n.d.
derived from the model using acute (black lines) and chronic (red lines) toxicity
23 n.d. n.d.
values. Solid and dashed lines indicate the median and 90% confidence inter- A n.d. n.d.
vals, respectively. The 90% confidence interval of the 5th percentile of ha- B 0.34 n.d.
zardous concentration (HC5) is also indicated with horizontal solid lines. C n.d. n.d.
Symbols are toxicity values for algae (circles), crustacea (triangles), and fish D n.d. n.d.
(crosses). Blue solid symbols are toxicity values based on nominal concentra- E n.d. n.d.
tions, and the other symbols are toxicity values based on measured con- F n.d. n.d.
centrations. (For interpretation of the references to colour in this figure legend, G n.d. n.d.
H n.d. n.d.
the reader is referred to the web version of this article.)
I n.d. n.d.
J n.d. n.d.
uncertainty factor of 10. Using the probabilistic method, we obtained a K n.d. n.d.
L n.d. n.d.
PNEC of 0.0059 μg/L by dividing the HC5 (0.059 μg/L, obtained from
M n.d. n.d.
the chronic toxicity values of the text taxa) by an uncertainty factor of N n.d. n.d.
10. O n.d. n.d.

3.3. Distribution and levels of TPBs in Seto Inland Sea n.d., not detected.

TPB concentrations in surface seawater were determined in the
central (St. 1–18, 22, 23) and eastern regions (St. A – O) of the Seto
Inland Sea (Table 4). In the central region, TPBs in surface seawater
were detected at St. 3 (0.21 ng/L) and St. 4 (0.25 ng/L) in June, and St.
2 (0.23 ng/L) and St. 14 (0.32 ng/L) in December 2017. In the eastern
part of the Seto Inland Sea, TPBs were only detected in surface seawater
at St. B (0.34 ng/L) in September 2017. Further, in 2018, TPBs were not
detected in surface seawater sampled at any of the stations.
4. Discussion
In the present study, we calculated the PNECs using conventional
and probabilistic techniques, and identified both, acute and chronic
toxicity, of TPB-18 to marine organisms such as algae, crustacea, and
fish under laboratory conditions. We compared the toxicity of TPB-18
with the other commonly used antifouling biocides and compared
PNECs with observed values of TPBs in the Seto Inland Sea.
4.1. Acute and chronic toxicities
The measured TPB-18 concentrations were 15–68% of the nominal
concentrations because TPB-18 has photolytic properties (Tsuboi,
2013). Thus, toxicity values were estimated based on the measured
concentrations in the present study. The toxicity of TPB-18 to marine
organisms relative to other biocides depends on the biocide and the
responses of the organisms. Cu2O is the most commonly used biocidal
compound in Japan (Okamura and Mieno, 2006). However, informa-
tion on Cu2O toxicity, as opposed to other copper sources, such as
CuSO4, is scarce (Burridge and Zitko, 2002) and unavailable for any of
the organisms tested in the present study. Therefore, we compared TPB-
18 toxicity to values for copper from CuSO4 or CuCl2. We also compared
TPB-18 toxicity values to those of PTPB, another boron-based biocide
and Copper pyrithione [CuPT, bis-(hydroxy-2(H)-pyridine thionate-
O,S)-copper], the second most common biocide among antifouling
paints registered in the Japanese market (Okamura and Mieno, 2006).
We observed higher toxicity of TPB-18 to Skeletonema sp. (72-h
EC50: 2.5 μg/L, 4.9 nM; 72-h NOEC: 0.54 μg/L, 1.1 nM) and T. japonicus
(24-h EC50: 22 μg/L, 43 nM;) than has been reported for copper: the
acute toxicity values for copper (calculated using CuSO4) on the marine
algae Skeletonema costatum and the crustacea T. japonicus (adult) were
reported as 4600 and 9100–18,600 nM (96-h LC50, Kwok et al., 2008;
Bao et al., 2011), respectively; and the chronic toxicity values for
copper (calculated using CuCl2) on the marine algae Skeletonema cost-
atum was reported as 56 nM (72-h NOEC, Van Sprang et al., 2008). Our
results indicated that the toxicity of TPB-18 to the marine fish species,
red sea bream (96-h LC50: 920 μg/L, 1800 nM) and mummichog (96-h
LC50: 980 μg/L, 1900 nM), were similar to that of copper. The acute
toxicity values of copper (using CuSO4) to red sea bream and mum-
michog were reported to be 1300 nM (96-h LC50, Mochida et al., 2012)
and 110 to > 6100 nM (96-h LC50, Grosell et al., 2007), respectively. In
addition, the TPB-18 toxicity to Skeletonema sp., T. japonicus, Japanese
blue crabs (24-h EC50: 150 μg/L, 290 nM), red sea bream, and

6
T. Onduka, et al.
mummichog were similar to that of PTPB (Mochida et al., 2012), i.e.,
72-h EC50: 2.2 μg/L, 6.8 nM; 24-h EC50: 6.6 μg/L, 21 nM; and 24-h EC50:
32 μg/L, 100 nM, 96-h LC50: 290–400 μg/L, 900–1200 nM, 96-h LC50:
370–470 μg/L, 1100–1500 nM respectively. Thus, TPB-18 showed si-
milar toxicity for marine algae, crustacea, and fish compared to PTPB,
another boron-based biocide (Mochida et al., 2012). Further, the TPB-
18 toxicity to Skeletonema sp., T. japonicus, and Japanese blue crabs (24-
h EC50: 150 μg/L, 290 nM) was similar to that of CuPT (Onduka et al.,
2010; Mochida et al., 2008), i.e., 72-h EC50: 1.5 μg/L, 5.4 nM; 24-h
EC50: 23 μg/L, 83 nM; and 24-h EC50: 32 μg/L, 100 nM, respectively.
Interestingly, the TPB-18 toxicity observed for mummichog in the
present study (96-h LC50: 980 μg/L, 1900 nM) was approximately 100
times less than that of CuPT (96-h LC50; 2.9–8.4 μg/L, 9.2–27 nM,
Mochida et al., 2006). Our toxicity results also show that algae, espe-
cially diatoms (C. gracilis), are more sensitive to TPB-18 than crustacea
and fish, as observed with other antifouling biocides, including PTPB
and CuPT (Mochida et al., 2012; Onduka et al., 2010). However, results
for the alga T. tetrathele should be used for reference only, since the
recorded growth rate in the control was lower than 0.92/day during the
test period. This was slower than the rate recommended by the OECD
test guideline (OECD, 2006), which stipulates “the biomass in the
control cultures should have increased exponentially by a factor of at
least 16” for adequate performance of the test. In future studies on T.
tetrathele, it may be necessary to extend the test period to meet this
performance criterion.
To the best of our knowledge, this is the first study to report the
chronic toxicity values of TPB-18 to T. japonicus and mummichog, for T.
japonicus, and the Japanese blue crab; the MATC for TPB-18 was 2.1 μg/
L (4.1 nM), while ACR was 10 (=22/2.1 μg/L) for T. japonicus.
According to Kenaga (1982), the chronic toxicity of organic chemicals
can be calculated from acute toxicity values by assuming an ACR of
≤15. Moreover, analysis of daphnia ecotoxicity datasets estimated the
median (10th, 90th percentile) ACR as 10 (2.6, 92.3) (Hayashi and
Kashiwagi, 2015). Thus, our estimated chronic toxicity value for T.
japonicus appears to be reasonable. Following the approach of Kenaga
(1982), assuming ACR ≤ 15, the chronic toxicity value was estimated
to be 15 μg/L for the Japanese blue crab. For mummichog and the red
sea bream, the MATC for TPB-18 was 76 μg/L (149 nM), while the ACR
was 13 (=980/76 μg/L) for mummichog. Assuming ACR ≤ 15
(Kenaga, 1982), the estimated chronic toxicity value for the red sea
bream was 71 μg/L. In the crustacean reproduction test, the sex ratio
was 0% at 21 d in the 40 μg/L exposure group. However, the survival
rate was only 10% (two individuals) at 21 d. Meanwhile, the sex ratio
was 67% at 7 d in the group, and no concentration-dependent changes
were observed in the sex ratio at 21 d. Thus, these results were not
enough data to indicate TPB-18 has anti-androgenic or estrogenic ef-
fects.
4.2. Environmental risk assessment
Toxicity values are needed for a sufficiently large and ecologically
diverse sample of species to obtain a reliable PNEC estimate. In this
study, acute and chronic toxicity value-based SSD analyses were carried
out using ecotoxicity data for ten and five species, respectively. In ad-
dition, both, the acute and chronic toxicity values, were obtained from
species belonging to three trophic levels, following recommended
practice (Chemical Substances Control Law, Japan; OECD, 2011; EU,
2003). Using a probabilistic approach, an SSD is obtained by assuming
a log-logistic distribution of species sensitivities, such as acute and
chronic toxicity values. The number of different species required for an
SSD varies among national guidelines: e.g., ≥4 in The Netherlands, ≥5
in Denmark, Australia, and the OECD, and ≥8 in the US (RIVM, 2001;
ANZECC, 2000; Stephen et al., 1985). TenBrook et al. (2009) pointed
out that toxicity data on ≥5 species were essential for SSD analysis in
cases where the toxicity data distribution was assumed to be log-
normal. In addition, Okkerman et al. (1991) mentioned that
7
Marine Pollution Bulletin 157 (2020) 111320
considering cost performance, ecotoxicity data on ≥5 species was en-
ough for an SSD analysis. Thus, the HC5 value obtained in the present
SSD analysis, based on ten species, conformed to the prescribed
guidelines.
In implementing the probabilistic approach, we obtained the PNECs
by using the 5th percentile of the SSD, called the HC5 (Kooijman, 1987;
Van Straalen and Denneman, 1989; Posthuma et al., 2002). For reg-
ulatory purposes, this factor was determined by a panel of experts by
taking into account the number of toxicity values and the composition
of the species used for the SSD analysis (RIVM, 2001; EFSA, 2013). HC5
is seldom used per se as a PNEC. While some guidelines recommend
using an uncertainty factor of 1–5 to obtain PNEC from HC5 values
(RIVM, 2001; Stephen et al., 1985; EFSA, 2013), other guidelines re-
commend an uncertainty factor of 10 (RIVM, 2001). In the present
study, we used 10 as the uncertainty factor, as this is the most con-
servative value in the range of recommended values.
The PNEC values obtained by the conventional and probabilistic
approaches in the present study (0.030 μg/L and 0.0059 μg/L, respec-
tively) were higher than the concentration values estimated for sea-
water samples from the Seto Inland Sea. The concentration of TPBs in
the surface seawater was close to the MQL, < 0.12–0.32 and <
0.12–0.34 ng/L in the central and eastern part of the Seto Inland Sea,
respectively. During 2017 and 2018, TPBs were not detected con-
tinuously at any of the sampling stations. Since photolysis limits the
half-life of TPBs to 16–67 h (Tsuboi, 2013), the presence of TPBs in the
surface seawater implied a continuous input from TPB sources in the
vicinity of the sampling stations. This was confirmed by the close vi-
cinity of harbors and shipping routes to the sampling stations. These
results suggest that the TPBs detected in the surface seawater in the
Seto Inland Sea were derived from TPBs used as antifouling biocides on
ship hulls. The maximum values of TPB found in this study exceeded
those reported in a previous study in Hiroshima Bay (also in the Seto
Inland Sea) including marinas, where concentrations ranged within
4.8–21 pg/L (Mochida et al., 2012). The contamination level of TPB
found in this study was similar to those reported in a previous study in
coastal areas, including river mouths and ports of Japan, where con-
centrations ranged from < 0.023–0.37 ng/L (Ministry of the
Environment Government of Japan, 2019). The monitoring of the
concentration of TPBs in sediment is a future task because monitoring
of sediment was not performed in the present study. In this study, we
determined seven acute and five chronic toxicity values of marine or-
ganisms and performed the SSD analysis using ten acute and five
chronic toxicity values. Although we only tested a small number of
species relative to the total number of species occurring in the study
region, we did include representatives from three trophic levels, fol-
lowing recommended practice (Chemical Substances Control Law,
Japan; OECD, 2011; EU, 2003). Moreover, while seawater sampling
was carried out only once per year per sampling location, sampling was
conducted throughout the Seto Inland Sea in locations where TPB-18
emissions were expected to be high. Sampling in this study, thus was
geographically and temporally representative with respect to species
composition. We are therefore confident that we have followed best
practices to estimate the ecological risk posed by TPB-18 in the Seto
Inland Sea.
5. Conclusions
In this study, we used a conventional and a probabilistic approach
to derive the PNEC of TPB-18 for marine organisms. The highest con-
centration of TPBs in the Seto Inland Sea was 0.34 ng/L (Table 4).
Considering the worst-case scenario regarding the ecological impact of
TPB-18, in which all of the detected triphenylborane originated from
TPB-18, the PNEC values derived from both the conventional and
probabilistic approaches were higher than the environmental con-
centrations observed in this study (Table 4) and reported in a previous
study (Mochida et al., 2012, Ministry of the Environment Government
T. Onduka, et al.
of Japan, 2019). Based on our results we propose that it is thus likely
that ecological impact of TPB-18 contamination in the Seto Inland Sea
is currently low.
CRediT authorship contribution statement
Toshimitsu Onduka:Conceptualization, Methodology,
Validation, Formal analysis, Investigation, Resources, Data cura-
tion, Writing - original draft, Visualization, Project
administration.Kumiko Kono:Investigation, Writing - review &
editing.Mana Ito:Investigation, Writing - review & Kikkawa, T., Takaku, H., Itoh, Y., 2010. Shiodamarimijinnko no hannsyoku sogai si-
editing.Nobuyuki Ohkubo:Investigation, Writing - review &
editing.Takeshi Hano:Formal analysis, Investigation, Resources,
Writing - review & editing.Katsutoshi Ito:Investigation, Writing -
review & editing.Kazuhiko Mochida:Conceptualization,
Methodology, Software, Formal analysis, Investigation, Writing -
review & editing, Visualization, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We extend our gratitude to Nitto Seimo, Co., Ltd. for kindly pro-
viding TPB-18; Dr. Yutaka Okumura (Tohoku National Fisheries
Research Institute) for kindly providing test algae; Ms. Miki Shoda
(National Research Institute of Fisheries and Environment of Inland
Sea) for her kind assistance; and to the crews of the Shirahuji-maru and
Kotaka-maru, the research vessels of the National Research Institute of
Fisheries and Environment of Inland Sea; the Japan Fisheries Research
and Education Agency, for field sampling support. We would like to
thank Editage (www.editage.com) for English language editing. This
work was supported in part by a grant-in-aid from NITTO SEIMO, Co.,
Ltd., Japan.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.marpolbul.2020.111320.
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