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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
unavailable, the logPow was estimated using the online
version of the CLOGP program (Daylight Chemical
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
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