Acute toxicity of benzoic acids to the crustacean Daphnia magna

Article abstract of DOI:10.1016/j.chemosphere.2004.11.003

The acute immobilization toxicity of benzoic acids substituted with hydroxyl and/or methoxyl groups on the aromatic ring was determined for the freshwater crustacean Daphnia magna under neutralized condition (initial pH: 7.45 ± 0.05). Toxicity, expressed as EC₅₀ value, varied depending largely on the number and position of phenolic hydroxyl groups. Especially, benzoic acids with ortho-substituted hydroxyl groups were more toxic than benzoic acids with meta- and/or para-substituted hydroxyl groups. Whereas the limited data indicated that methoxyl substitution had relatively small and variable effects on the toxicity. Of the tested compounds, 2,4,6- trihydroxybenzoic acid showed the highest toxicity with the 48 h EC₅₀ of 10 μmol l^-1. This was 700 times as toxic as the parent benzoic acid (48 h EC₅₀ = 7.0 mmol l^-1) and about two orders of magnitude higher than those previously reported for monohalogenated benzoic acid derivatives in Daphnia. Within the subgroups based on the number of hydroxyl groups (N_OH), the toxicity variations due to the position of hydroxyl groups appeared to be correlated with the logarithms of n-octanol/water partition coefficients (log P_ow). The toxicity of benzoic acids existing almost entirely as their ionized forms could be expressed as simple structure-toxicity relationships using these two descriptors (N_OH and log P_ow).

Full text of DOI:10.1016/j.chemosphere.2004.11.003

Chemosphere 59 (2005) 255–261
www.elsevier.com/locate/chemosphere
Acute toxicity of benzoic acids to the crustacean
Daphnia magna
*
Yasushi Kamaya , Yuki Fukaya, Kyoji Suzuki
Department of Environmental Science for Human Life, Faculty of Agriculture, Shizuoka University,
836Ohya, Shizuoka 422-8529, Japan
Received 5 March 2004; received in revised form 22 October 2004; accepted 3 November 2004
Abstract
The acute immobilization toxicity of benzoic acids substituted with hydroxyl and/or methoxyl groups on the aro-
matic ring was determined for the freshwater crustacean Daphnia magna under neutralized condition (initial pH:
7.45 0.05). Toxicity, expressed as EC50 value, varied depending largely on the number and position of phenolic hydro-
xyl groups. Especially, benzoic acids with ortho-substituted hydroxyl groups were more toxic than benzoic acids with
meta- and/or para-substituted hydroxyl groups. Whereas the limited data indicated that methoxyl substitution had rel-
atively small and variable effects on the toxicity. Of the tested compounds, 2,4,6-trihydroxybenzoic acid showed the
highest toxicity with the 48 h EC50 of 10 lmol lꢀ1. This was 700 times as toxic as the parent benzoic acid (48 h
EC50 = 7.0 mmol lꢀ1) and about two orders of magnitude higher than those previously reported for monohalogenated
benzoic acid derivatives in Daphnia. Within the subgroups based on the number of hydroxyl groups (NOH), the toxicity
variations due to the position of hydroxyl groups appeared to be correlated with the logarithms of n-octanol/water par-
tition coefficients (logPow). The toxicity of benzoic acids existing almost entirely as their ionized forms could be
expressed as simple structure–toxicity relationships using these two descriptors (NOH and logPow).
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Daphnia magna; Toxicity; Benzoic acids; Hydroxyl group; logPow
1. Introduction
of lignin, an important plant cell wall polymer (Chen
and Chang, 1985). Some of these naturally occurring
benzoic acid derivatives are shown to have various bio-
Benzoic acid derivatives having a general structure
C6–C1 are widely used as industrial chemicals, agro-
chemicals, pharmaceuticals and consumer products.
On the other hand, a number of benzoic acid derivatives
with variations including hydroxylation and methoxyla-
tion are known to be a group of secondary plant meta-
bolites and also aerobic microbial degradation products
´
´
logical activities (Tomas-Barberan, 2000).
Furthermore, significant environmental sources of
benzoic acids are the microbial metabolism of a variety
of natural and anthropogenic aromatic compounds,
since benzoic acid derivatives are very common struc-
tural units among the identified or estimated degrada-
tion products of aromatic compounds by aerobic
microorganisms (Smith, 1990; Habe and Omori, 2003).
However, toxic effects of stable degradation intermedi-
ates from aromatic compounds are not fully elucidated.
*
Corresponding author. Fax: +81 54 237 3028.
E-mail address: ahykama@agr.shizuoka.ac.jp (Y. Kamaya).
0045-6535/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.11.003

256
Y. Kamaya et al. / Chemosphere 59 (2005) 255–261
Numerous studies have been made on the antibacte-
(98), and 3,4,5-trihydroxybenzoic acid (98) from Nacalai
tesque, Kyoto, Japan; 2,4-dihydroxybenzoic acid (98),
2,6-dihydroxybenzoic acid (98), 3,5-dihydroxybenzoic
acid (98), 2,3,4-trihydroxybenzoic acid (95), 2,4,6-tri-
hydroxybenzoic acid (98), and 3-hydroxy-4-methoxy-
benzoic acid (98) from Tokyo Chemical Industries,
Tokyo, Japan; and 4-hydroxy-3,5-dimethoxybenzoic
acid (98) from Sigma, St Louis, MO, USA. 4-Hydroxy-
benzyl alcohol was prepared by the reduction of 4-
hydroxybenzaldehyde with NaBH4 in methanol at 0 ꢁC
in almost quantitative yield. Other reagents used for
Daphnia medium were of the highest purity available
and purchased from Wako.
rial, antifungal, and antiviral activities of natural and
synthetic phenolic compounds including various substi-
tuted benzoic acids (Friedman et al., 2003, and refer-
ences cited therein). However, studies on the effects of
benzoic acids, as well as other carboxylic acids, to aqua-
tic organisms have been limited (Fiorentino et al., 2003).
Toxicity data for some halogenated benzoic acids in the
bacteria, ciliate, daphnids, and fish have been reported
(Zhao et al., 1996; Muccini et al., 1999). Whereas the
toxicity of substituted phenols to Daphnia has been
investigated by many researchers (Kopperman et al.,
1974; LeBlanc, 1980; Devillers and Chambon, 1986;
Devillers, 1988; Kuhn et al., 1989; Jin et al., 1998; Abe
¨
et al., 2000).
Measured values of the logarithm of the n-octanol/
water partition coefficient (logPow) were obtained from
on-line interactive demo version of SRC physical prop-
erties database (Syracuse Research Corporation Web
site, http://esc.syrres.com). If a measured value was
unavailable, the logPow was estimated using the online
version of the CLOGP program (Daylight Chemical
Information Systems Web site, http://www.day-
light.com). The experimentally determined negative log-
arithms of the first dissociation constants (pKa) were
also obtained from the SRC database. If a measured
value was unavailable, the pKa value was estimated
using the SPARC on-line calculator (http://ibmlc2.chem.
uga.edu/sparc). Table 1 shows some physico-chemical
properties of benzoic acids and related compounds
tested in the present study.
The objective of this study was to establish data for
acute toxicity of benzoic acids substituted with hydroxyl
and/or methoxyl groups to the crustacean Daphnia mag-
na. Furthermore, the data were compared with those of
halobenzoic acids in the literature, and used to gain
some information regarding the structure–toxicity rela-
tionships underlying the toxicity of these compounds.
D. magna, an important freshwater invertebrate species
in aquatic food webs, has been used world-wide for
many years as a representative test species for ectoxico-
logical evaluation of industrial chemicals (OECD, 1984).
2. Materials and methods
To prepare the highest test solution, a test compound
was dissolved in the aerated test medium and the pH
was, where necessary, adjusted carefully with 1 mol lꢀ1
NaOH solution to 7.45 0.05, and then diluted to pre-
pare a series of test solutions. Test medium used was
‘‘moderately hard water’’ prepared from deionized and
2.1. Organism and culture conditions
Daphnia magna obtained from the National Institute
for Environmental Studies (NIES), Tsukuba, Japan was
used throughout this study. Neonatal daphnids were ob-
tained from continuous cultures in 1 liter glass beakers
at 21 0.3 ꢁC, in dechlorinated and conditioned tap
water (total hardness, ꢁ100 mg lꢀ1 as CaCO3; pH
7.5 0.1), 16 h light: 8 h dark photoperiod and a density
of below 20 per beaker. The medium was renewed three
times a weak and daphnids were fed daily with the green
alga Selenastrum capricornutum NIES-35 (3.0–3.5 ·
108 cells lꢀ1), cultured also in our laboratory.
distilled water (total hardness, ꢁ100 mg CaCO3 lꢀ1
;
USEPA, 1993), and after aeration the pH was adjusted
to 7.45 0.05 with 1 mol lꢀ1 HCl.
The solution pH decreased below 6 at concentrations
over 1 mmol lꢀ1 of benzoic acids when dissolved in the
test medium employed here, and daphnids exposed to
the solutions showed 100% immobility. Consequently,
the toxic effects of benzoic acids were not related to the
exposed concentrations alone, under the non-neutra-
lized condition. It was further noted that the pH adjust-
ment of some test solutions containing catechol and
hydroquinone type structures such as 2,3-, 2,5-, and
3,4-dihydroxybenzoic acids, and 2,3,4-, and 3,4,5-
trihydroxybenzoic acids resulted in slightly colored
preparations.
2.2. Chemicals and test solutions
Most chemicals (purity, minimum %) were used as
purchased from the following companies: benzoic acid
(99.5), 4-hydroxybenzoic acid (97), 2,3-dihydroxyben-
zoic acid (98), 2,5-dihydroxybenzoic acid (98), 4-hydro-
xy-3-methoxybenzoic acid (95), 4-methoxybenzoic acid
(98), 3,4-dimethoxybenzoic acid (97), 3,4,5-trimethoxy-
benzoic acid (98), methyl 4-hydroxybenzoate (99), and
4-hydroxybenzaldehyde (98), from Wako Pure Chemical
Industries, Osaka, Japan; 3-hydroxybenzoic acid (98), 2-
hydroxybenzoic acid (99.5), 3,4-dihydroxybenzoic acid
The concentrations of the resultant solutions were
checked before and after (48 h) exposure experiments
by the UV spectra (400–200 nm) using a UV–visible
spectrophotometer, UV mini 1240 (Shimadzu, Kyoto,
Japan). No significant spectral changes in most test solu-
tions were observed, indicating the stability of these

Y. Kamaya et al. / Chemosphere 59 (2005) 255–261
257
Table 1
Compounds tested in this study
Compound (common name)
Abbreviation CAS numbera MWb
logPow pKa
kmax (nm)c
Benzoic acid
BA
4-M
65-85-0
100-09-4
93-07-2
118-41-2
69-72-7
99-06-9
99-96-7
99-76-3
121-34-6
645-08-9
530-57-4
623-05-2
123-08-0
303-38-8
89-86-1
490-79-9
303-07-1
99-50-3
99-10-5
610-02-6
83-30-7
149-91-7
122.12 1.87
152.15 1.96
182.17 1.61
4.17
4.29
4.44
4.44d
2.99
4.07
224
246.5
4-Methoxybenzoic (p-anisic) acid
3,4-Dimethoxybenzoic (veratric) acid
3,4,5-Trimethoxybenzoic acid
3,4-DM
3,4,5-TM
2-H
250.5, 284.5
252.5, 277she
230, 295
223sh,e 286.5
212.2
1.26d
2-Hydroxybenzoic (salicylic) acid
3-Hydroxybenzoic acid
4-Hydroxybenzoic acid
138.12 2.26
138.12 1.50
138.121.58
3-H
4-H
4.54
452.5
Methyl 4-hydroxybenzoate [methyl paraben]
4-Hydroxy-3-methoxybenzoic (vanillic) acid
3-Hydroxy-4-methoxybenzoic (iso-vanillic) acid
152.15 1.96
168.15 1.43
168.15 1.36d
198.17 1.04
124.14 0.25
122.12 1.35
154.12 1.20
154.12 1.63
154.12 1.74
154.12 2.20
154.12 1.15
154.12 0.86
170.12 1.05
170.12 1.62d
170.120.70
(8.00)d 255.5, 294.5
VA
i-VA
4.52
251, 285
250, 286.5
261
4.50d
4.21
4-Hydroxy-3,5-dimethoxybenzoic (syringic) acid SA
4-Hydroxybenzyl alcohol
4-Hydroxybenzaldehyde
(9.82)
(7.67)
2.91
273
221.5, 286.5, 330.5
238, 306
2,3-Dihydroxybenzoic acid
2,3-DH
2,4-Dihydroxybenzoic (b-resorcylic) acid
2,5-Dihydroxybenzoic (gentisic) acid
2,6-Dihydroxybenzoic (c-resorcylic) acid
3,4-Dihydroxybenzoic (protocatechuic) acid
3,5-Dihydroxybenzoic (a-resorcylic) acid
2,3,4-Trihydroxybenzoic acid
2,4-DH
2,5-DH
2,6-DH
3,4-DH
3,5-DH
2,3,4-TH
2,4,6-TH
3,4,5-TH
3.11
2.95
247.5, 290.5
224sh,e 318.5
245, 305.5
250, 287.5
241, 295
1.05
4.26
4.04
3.13d
2.48d
255.5, 292
255, 289
2,4,6-Trihydroxybenzoic acid
3,4,5-Trihydroxybenzoic (gallic) acid
3.13
582.5
a
Chemical Abstract Service registry number.
Molecular weight.
b
c
UV absorbance in test solution.
Calculated value.
Shoulder peak.
d
e
compounds under the test conditions. However, 2,3,4-
and 3,4,5-trihydroxybenzoic acids showed an increase
of the absorbance and the peak broadening in UV re-
gion, with the darkening of their solution colors. At
pH > 7, 3,4,5-trihydroxybenzoic acid and its analogues
are readily oxidized by atmospheric oxygen, as shown
Probit analyses (USEPA, 1993), based on nominal
concentrations.
2.4. Analysis of structure–toxicity relationships
The relationship between the toxicity and the physi-
cochemical and/or structural descriptors was analyzed
by least-square regression using JMP 5.0 J software
(SAS Institute Inc., USA). Log (EC50, in mol lꢀ1) was
taken as the dependent variable, while the structural
and/or physicochemical descriptors were used as the
independent variables. The following descriptive infor-
mation was provided: n (number of observations used
in analysis), r2 (square of the correlation coefficient ad-
justed for degrees of freedom), s (the root of the mean
square for error), F (the Fisher statistic), p (the signifi-
cance level).
in a spectrometric study by Friedman and Jurgens
¨
(2000). In the present study no further investigation
was, however, made on the structural and toxicological
evaluation of products causing such observed changes.
2.3. Acute immobilization toxicity test
Neonates (<24 h old) from 2–3-week-old mothers
were placed in a 50 ml glass beaker containing 40 ml
of a test solution. All experiments for exposure and con-
trols without chemicals were made in four replicates and
performed at 21 0.3 ꢁC under 16 h light: 8 h dark pho-
toperiod. Immobility was used as the endpoint for deter-
mining acute toxicity; the daphnids showing no
movement within 15 s after gentle stirring were defined
to be immobile. After 24 and 48 h, the number of immo-
bile daphnids was recorded to determine the concentra-
tion able to achieve 50% immobilization and it was
indicated as EC50. The EC50 values were calculated by
3. Results and discussion
Acute immobilization toxicity of benzoic acid deriva-
tives and some related compounds for D. magna are
summarized in Table 2. In the present study, toxicity
of benzoic acid derivatives was determined at (initial)

258
Y. Kamaya et al. / Chemosphere 59 (2005) 255–261
Table 2
Acute toxicity data for tested compounds
Compound
Tested concentration 48 h EC50 (95% C.I.)a
24 h EC50 (95% C.I.)a
levels (mmol lꢀ1
)
(mmol lꢀ1
)
(mg lꢀ1
)
(mmol lꢀ1
)
(mg lꢀ1
2310
)
Benzoic acid
1.25–20
2–12
7.04 (6.25–7.85)
6.20 (5.60–6.70)
10.0 (9.03–11.0)
24.5 (22.1–27.2)
6.30 (5.60–6.90)
11.2 (8.90–13.6)
12.2 (10.3–14.1)
0.27 (0.23–0.31)
5.40 (4.8–6.0)
860
943
n.o.b
n.o.b
4-Methoxybenzoic acid
3,4-Dimethoxybenzoic acid
3,4,5-Trimethoxybenzoic acid
2-Hydroxybenzoic acid
3-Hydroxybenzoic acid
4-Hydroxybenzoic acid
Methyl 4-hydroxybenzoate
3.75–15
5–40
2–16
1820
5200
870
12.7 (11.6–14.0)
n.o.b
7.70 (6.50–8.80)
19.9 (16.8–22.9)
19.7 (17.8–21.9)
0.46 (0.41–0.59)
7.0 (5.5–8.8)
9.2(8.0–10.8)
n.o.b
1060
2750
2720
70.0
5–40
3.125–25
0.05–0.5
1550
1690
41.1
908
4-Hydroxy-3-methoxybenzoic acid 1–10
3-Hydroxy-4-methoxybenzoic acid 1.5–15
1180
1550
6.70 (6.10–7.40)
6.70 (6.20–7.20)
1130
1330
4-Hydroxy-3,5-dimethoxybenzoic
acid
1.25–20
4-Hydroxybenzyl alcohol
4-Hydroxybenzaldehyde
2,3-Dihydroxybenzoic acid
2,4-Dihydroxybenzoic acid
2,5-Dihydroxybenzoic acid
2,6-Dihydroxybenzoic acid
3,4-Dihydroxybenzoic acid
3,5-Dihydroxybenzoic acid
2,3,4-Trihydroxybenzoic acid
2,4,6-Trihydroxybenzoic acid
3,4,5-Trihydroxybenzoic acid
0.05–0.6
0.0625–1.0
0.25–8
0.24 (0.22–0.27)
0.404 (0.337–0.478)
2.9 (2.6–3.2)
29.8
49.3
447
0.60 (0.54–0.67)
0.908 (0.707–1.59)
5.40 (4.90–6.20)
1.22 (0.97–1.55)
n.o.b
74.5
111
832
0.25–8
0.125–2
0.785 (0.60–1.01)
0.386 (0.304–0.487)
0.0685 (0.0531–0.090)
2.4 (2.08–2.69)
120
188
59.5
10.6
370
0.0125–0.1
0.25–4
n.o.b
n.o.b
0.625–10
0.014–0.339
0.003–0.3
0.0625–1.0
4.0 (3.57–4.45)
616
5.86 (5.29–6.45)
0.123 (0.099–0.145)
0.0142 (0.0110–0.0179)
0.165 (0.150–0.181)
903
20.9
2.4
0.0686 (0.0548-0.0826)
0.0100 (0.0082–0.0122)
0.112 (0.094–0.133)
11.7
1.70
19.1
28.1
a
95% Confidence interval.
Not obtained.
b
pH of 7.45 0.05 greater than the pKa of acids (Table
1). Hence, the acids were almost entirely as their ionized
forms under the test conditions. The 48 h EC50 values
span over three orders of magnitude, ranging from
0.01 mmol lꢀ1 for 2,4,6-trihydroxybenzoic acid to
24.5 mmol lꢀ1 for 3,4,5-trimethoxybenzoic acid.
and 3,4,5-trihydroxybenzoic acids. Again, the number
of ortho-hydroxyl groups appeared to enhance the toxi-
city of substituted benzoic acids. The toxicity (48 h
EC50 = 10 lmol lꢀ1
) of 2,4,6-trihydroxybenzoic acid
with two ortho-hydroxyl groups was unexpectedly high-
er than those of 2,3,4- and 3,4,5-trihydroxybenzoic acids
containing a more reactive and biologically active cate-
cohol structure (Schweigert et al., 2001). This was 700
times as toxic as the parent benzoic acid. It should be
noted that 2,3,4-and 3,4,5-trihydroxybenzoic acids were
suggested to be air-oxidized under the test conditions as
described in materials and methods section. Therefore,
the observed toxicity may have been partly affected by
some oxidation products from the parent acid.
For simple monohydroxylated benzoic acid deriva-
tives, 2-hydroxybenzoic (salicylic) acid showed higher
toxicity (48 h EC50 = 6.3 mmol lꢀ1) than the other iso-
mers, 3- and 4-hydroxybenzoic acids (48 h EC50 = 11.2
and 12.2 mmol lꢀ1, respectively). But salicylic acid had
a similar toxicity to that of the parent benzoic acid
(48 h EC50 = 7.0 mmol lꢀ1). Compounds with additional
methoxyl groups, vanillic, iso-vanillic and syringic acids,
also had similar toxic levels to that of benzoic acid, but
the toxicites were higher than those of the corresponding
monohydroxylated benzoic acids.
Very recently, Friedman et al. (2003) reported the re-
sults of their extensive studies on the bactericidal activ-
ities of benzaldehyde and benzoic acid derivatives
substituted with hydroxyl and/or methoxyl groups.
They found that the carboxyl group was less active than
the aldehyde group and the compounds had the in-
creased activity with the increasing number of hydroxyl
groups in the molecule. Interestingly, 2,4,6-trihydroxy-
benzoic acid was inactive, while 3,4,5-trihydroxybenzoic
acid showed relatively higher activity among hydroxy-
benzoic acids tested. They showed that 2-OH and
The toxic effects of dihydroxybenzoic acids in
descending order were 2,6- > 2,5- > 2,4- > 2,3- > 3,4-
> 3,5-dihydroxybenzoic acids, suggesting the toxicity
enhancement by the ortho-hydroxyl substitution. All
dihydroxy derivatives showed higher toxicity than non-
hydroxylated and monohydroxylated benzoic acids.
As for trihydroxy derivatives, 2,4,6-trihydroxyben-
zoic acid was the most toxic isomer, followed by 2,3,4-

Y. Kamaya et al. / Chemosphere 59 (2005) 255–261
259
4-OH groups enhance the antimicrobial activities of
benzaldehyde but not those of benzoic acid. Further-
more, they found that compounds with mixed hydroxyl
and methoxyl groups exhibited variable results, i.e., in
some cases methoxyl groups enhanced activity and in
other cases they did not.
Muccini et al. (1999) studied the toxicities of benzoic
acid and its 14 halogenated derivatives for the ciliate
Tetrahymena pyriformis under neutralized and non-neu-
tralized conditions, and found that the ionized and un-
ionized forms of halobenzoic acids had different
contributions to toxicity. Zhao et al. (1996, 1998), study-
ing the acute toxicities of benzoic acid, 6 monohalogen-
ated benzoic acids, and three aminobenzoic acids to D.
magna at three different pH values (6.0, 7.8, and 9.0),
found that the toxicities decreased as the pH increased.
The 24 h EC50 values for monohalogenated benzoic
acids in Daphnia ranged from 0.78 mmol lꢀ1 for 3-
bromobenzoic acid to 7.08 mmol lꢀ1 for 4-fluorobenzoic
acid. The toxicity (48 h EC50) of 2,4,6-trihydroxybenzoic
acid was about two orders of magnitude higher than
those of monohalogenated benzoic acids. As for halo-
benzoic acids, compounds substituted at meta- and/or
para-positions are more toxic than those substituted at
ortho-positions. They explained the difference by the
general concept that the ortho-halogenated benzoic acids
having lower pKa values are more ionized, hence less
toxic than meta- and/or para-substituted ones. As for
the hydroxylated benzoic acids considered here, ortho-
hydroxylated benzoic acids also have lower pKa values,
but they showed higher toxicities than the meta- and/
or para- hydroxylated ones, indicating that the hydroxy-
benzoate derivatives behaved differently from the halo-
benzoates. Especially, polyhydroxybenzoates may have
higher affinity to daphnids than expected from their
low logPow and pKa values.
The esterification of 4-hydroxybenzoic acid to its
methyl ester (so-called methyl paraben) resulted in a sig-
nificant 45-fold increase in toxicity, giving the 48 h EC50
of 0.27 mmol lꢀ1 for methyl paraben. Methyl paraben is
used as an antimicrobial preservative in food, drug, and
cosmetics for over 50 years, and in animals it is known
to be practically non-toxic by both oral and pareteral
routs (Soni et al., 2002).
On the other hand, methylation of the phenolic hy-
droxyl group of 4-hydroxybenzoic acid to 4-methoxy-
benzoic acid resulted in 2-fold increase in toxicity.
These increase may be related to the increased hydro-
phobicity (logPow) of these derivatives.
However, the results with methoxybenzoic acids indi-
cated that the toxicity of benzoic acid decreased with
increasing methoxyl groups.
The effect of carboxyl group (COOH) was examined
by the comparative study with other p-hydroxylated C6–
C1 compounds such as 4-hydroxybenzaldehyde (C1 =
formyl, CHO), 4-hydroxybenzyl alcohol (C1 = hydroxy-
methyl, CH2OH), and 4-methylphenol (C1 = methyl,
CH3; p-cresol). The 48 h EC50 values of formyl and
hydroxymethyl substituted derivatives were experimen-
tally determined and a 48 h EC50 value of 0.071
mmol lꢀ1 for p-cresol was obtained from Kuhn et al.
As shown in Fig. 1A, there are not so significant
differences in relative magnitudes of toxicities among
benzoic, methoxybenzoic, monohydroxybenzoic, and
methoxylated monohydroxybenzoic acids. On the con-
trary, di- and tri-hydroxybenzoic acids appeared to have
higher toxicity than these compounds. These results sug-
gested the importance of the number of hydroxyl groups
in the toxicity. However, there were substantial varia-
tions depending on the position of hydroxyl groups.
Within the subgroups based on the number of hydroxyl
groups, the toxicity variations appeared to be well corre-
lated with the logPows, as shown in Fig. 1B. LogPow is a
hydrophobicity parameter determined for the unionized
form of a chemical. Probably, logPow functions here as a
relative indicator of partitioning of benzoate molecules.
Moreover, limited data in Fig. 1B showed that benzo-
ates substituted with both hydroxyl and methoxyl
groups (VA, i-VA, and SA) seemed to form a disparate
group from the other non-phenolic and mono-phenolic
benzoic acids.
¨
(1989). These data indicated that carboxyl group is con-
siderably less toxic than other C1 groups. The toxic
effects of C1 substituents in descending order were
CH3 > CH2OH > CHO > COOH. This also indicates
that oxidative transformation of aromatic methyl group
leading to carboxyl group, a common pathway in
aerobic microbial degradation of aromatic methyl group
(Smith, 1990), is the detoxification process for daphnids.
Similar toxic trend for such compounds has also been
reported for the bacteria, fish, and ciliate (Ribo and
Kaiser, 1983; Schultz et al., 1996), although respective
sensitivities to individual compounds are different.
The pH of test medium is an important factor espe-
cially to assess the ionizable compounds, because the un-
ionized form of a weak acid such as benzoic acid is
generally thought to be more toxic than the ionized
form. Wang and Lay (1989) reported that sodium 2-
hydroxybenzoate (24 h10 = 304 mg lꢀ1) was significantly
less toxic than the corresponding acid with the 24 h EC50
value of 230 mg lꢀ1 in Daphnia. The higher toxicity val-
ues for some hydroxybenzoic acids in the literature
Based on these observations, the relationship was
examined between the toxicity (log EC50), and structural
and physicochemical descriptors. Regression analysis
using both the number of hydroxyl groups (NOH) and
the hydrophobicity parameter (log Pow) revealed a good
relationship as follows:
(Kuhn et al., 1989; Fiorentino et al., 2003) than our
¨
present data may be ascribed to the differences in their
test conditions, especially the actual pH of exposure
solutions.

260
Y. Kamaya et al. / Chemosphere 59 (2005) 255–261
-1
-2
-3
-4
-5
-6
-1
B
A
3,4,5-TM
i-VA
4-H
3,4-DM
3-H
SA
2-H
-2
-3
-4
-5
-6
4-M
VA
3,5-DH
BA
2,3-DH
3,4-DH
2,4-DH
3,4,5-TH
2,5-DH
2,6-DH
2,3,4-TH
2,4,6-TH
0
1
2
3
0.5
1
1.5
log Pow
2
2.5
Number of
hydroxyl groups
Fig. 1. Plots of the 48 h log EC50 (in mol lꢀ1) versus the number of hydroxyl groups (A) and logPow (B) for benzoic acids substituted
with hydroxyl and/or methoxyl groups. For abbreviations of benzoic acids, see Table 1.
2
could be described by the use of these two descriptors,
NOH and logPow In contrast to the effects of OH
n ¼ 19; r2 ¼ 0:919; s ¼ 0:270; F ¼ 104;
log EC50 ¼ ꢀ0:321ðNOHÞ ꢀ 0:869ðlog PowÞ ꢀ 0:494;
groups, OMe groups had relatively smaller effects on
the whole toxicity.
p < 0:0001:
For only hydroxybenzoic acids the following rela-
tionship was obtained:
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Yasushi Kamaya, Ph.D., is an associate professor in the
Department of Environmental Science for Human Life at Shi-
zuoka University. His current research interests are microbial
transformation and biological effects of organic contaminants.
Schweigert, N., Zehnder, A.J.B., Eggen, R.I.L., 2001. Chemical
properties of catechols and their molecular modes of toxic