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A. N. Edginton et al.
filtered, irradiated well water at 18ꢀC under a light:dark cycle of 12:12
h. Adults were fed a rotation of beef or chicken liver and Frog Brittle
(Nasco, Fort Atkinson, WI) twice per week. Breeding was induced by
the injection of 600 and 800 IU of human chorionic gonadotrophin
(Sigma-Aldrich Canada, Oakville, ON, Canada) into the dorsal lymph
sac of males and females, respectively. Amplexus, egg laying, and
fertilization occurred within 12 h. Culture water used in all breeding
tanks, controls, and treatments conformed to the American Society of
Testing and Materials guideline for the performance of the Frog
Embryo Teratogenesis Assay–Xenopus (ASTM 1992). It comprised
625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaCl2, 60 mg
CaSO.42H2O, and 75 mg MgSO4/L of deionized water. The pH of the
culture water was adjusted to 7.2)7.4 using 1N HCl or 1N NaOH and
was 21ꢀC.
spiked with 0.8 lmol/L to 1.2 lmol/L 14C 2,4-D BEE. Twelve
experimental units represented 6 time points for the uptake phase (0.5,
1, 2, 4, 6, 8 h) with two repetitions. Fifty embryos, representing a
density of approximately 1.5 g/L, were added to each experimental
unit; 25 with an intact jelly coat, and 25 that had been dejellied. The
embryos were separated by placing one group in a small mesh basket
suspended about 1 cm from the bottom of the beaker. The other group
was placed on the bottom of the beaker. The group placed in the
basket was alternated for the two experiments. Two additional
experimental units contained 350 embryos at the same animal/water/
chemical ratio as in the uptake phase. At 8 h, these embryos were
moved to two 40-L aquaria each containing 20 L of clean culture
water. Sample time points for the excretion phase were 1, 2, 4, 8, 14,
and 22 h post-transfer for the first experiment, and 1, 2, 4, 8, 16, 40,
and 65 h post-transfer for the second experiment.
Two beakers containing no 14C 2,4-D BEE, one for dejellied em-
bryos and one containing embryos without a jelly coat, were included
in the experiment. At every sample time point, five embryos were
removed from each unit, placed on a small piece of tin foil, the excess
water drawn off, and subsequently weighed.
Test Substances and Analytical Chemistry
2,4-D acid (2,4-D) (99.5%; Lot # Moriss/1710) and the butoxyethyl
ester of 2,4-dichlorophenoxyacetic acid (2,4-D BEE) (99.5%; Lot #
1999C970153336) standards were supplied by Dow (Dow Agro-
Sciences, Indianapolis, IN). 14C-ring labeled 2,4-D was purchased
from Sigma (Sigma-Aldrich Canada, Oakville, ON, Canada). 14C
2,4-D had a formula weight of 221.0 g/mol, a specific activity of 2.9
mCi/mmol, and a radiochemical purity of 97.2%. Labeled 2,4-D BEE
was prepared by acid-catalyzed esterification from the reagents of
2-butoxyethanol (Sigma-Aldrich Canada) and 14C 2,4-D. First, the
following chemicals were added to a 50-ml round-bottom flask:
125 lCi of 14C 2,4-D dissolved in 6 ml of toluene + 2 ml 2-butoxy-
ethanol + 1 ml concentrated HCl. The flask plus an attached Dean-
Stark trap with a condenser was placed on a heating mantle with a
multivariate temperature control and gently boiled for 2 h. The tem-
perature was increased slightly for 30 min wherein 6 ml of additional
toluene, 1 ml of HCl, and 1 ml of 2-butoxyethanol was added to the
flask. After an additional 1 h of boiling, the flask was cooled. The
contents, containing unreacted alcohol, 14C 2,4-D, toluene, HCl, and
14C 2,4-D BEE, was loaded onto a C18 separatory column. The col-
umn was eluted with 3 · 10 ml of water, and 3 · 10 ml of CH3CN.
The CH3CN fraction contained 95% of the 14C 2,4-D BEE produced.
Using a rotary evaporator, the water and CH3CN fractions were
concentrated and resuspended in 1 ml acetonitrile. Eighty percent of
the radioactivity was recovered. 14C 2,4-D BEE was further purified
by high performance liquid chromatography (HPLC) using an Agilent
Series 1100 pump, autosampler, UV detector, and ChemStation
software. The system used an Ultracarb 5 ODS (C18) 4.6 mm · 25 cm
column and the following solvent system at a flow of 1 ml/min: 25%
CH3CN: 75% 0.1% formic acid in H2O for 10 min; 75% CH3CN: 25%
0.1% formic acid in H2O for the remainder of the 22-min run. The
radiolabel was quantified using an in-line EG&G Berthold Radioflow
LB508 detector (Berthold GmbH & Co., Bad Wildbad, Germany)
containing a 1000-ll flow cell and RadioStar software ver. 3.0.
Fraction collection and liquid scintillation counting (LSC) were used
when lower detection limits were needed. Retention time comparisons
with standards were used to verify 14C 2,4-D BEE and 14C 2,4-D
presence.
At each sample time point, two animal and two water samples were
taken, one for HPLC and one for LSC. The first animal sample was
prepared for analytical work using a modified method based on that of
Barron et al. (1990). Twenty embryos were homogenized using a
Polytronꢁ (Brinkmann Instruments, Mississauga, ON, Canada) tissue
homogenizer in 10 ml acetone/1% H3PO4. The homogenate was
shaken at 4ꢀC for 30 min, centrifuged at 6,000 g at 4ꢀC for 30 min,
and the supernatant was collected. After one additional 10-ml acetone/
1% H3PO4 extraction, the pooled supernatant was evaporated to near
dryness under vacuum at 45ꢀC using a rotary evaporator. The contents
were filtered through a 0.45-lm filter and samples were brought to
1 ml with deionized water (pH 3). The sample was loaded onto a C8
column under vacuum where 2 ml of methanol was loaded and the
elutant was collected. The sample was then brought to 1 ml under a
nitrogen stream. HPLC was used to separate 2,4-D BEE and its
metabolite, 2,4-D (the only metabolite observed). The mean recov-
ery € SEM for spiked embryo samples (with jelly coat) was 93.8
4.2% (n = 8) for 14C 2,4-D BEE and 94.5 € 4.3% (n = 6) for 14C 2,4-
D acid. No 2,4-D was observed in the 2,4-D BEE samples, demon-
strating that 2,4-D BEE was not hydrolyzed to 2,4-D during the
extraction procedure. For the second animal sample, total radioactive
residues in the embryos were determined by placing five embryos in a
7-ml glass vial containing 180 ll of tissue solubilizer (Hydamine
Hydroxideꢁ; ICN Biomedicals, Irvine, CA). Samples were placed in a
60ꢀC oven for 2–5 h until the tissue was digested. CytoScinct ES (ICN
Biomedicals) was added and the radioactivity was measured using a
Beckman LS6K-SC scintillation counter (Beckman Instruments Inc.,
Fullerton, CA). Limits of detection and quantification for radioactivity
in embryo samples were 0.24 gmol/g and 0.44 gmol/g, respectively.
To measure total radioactivity in the water, a 500-ll sample was
added to EcoLite (+) (ICN Biomedicals) and measured using LSC.
Water samples were directly injected into the HPLC.
Radiolabel Distribution Experiments
Whole-body autoradiography (WBARG), as described by Ullberg
et al. (1982), was performed to determine the distribution of radio-
active residues in embryos. About 80 embryos, at the blastula stages,
were water exposed to 3.6 gmol/ml 14C-2,4-D BEE in 2 · 100 ml of
culture water. The first exposure unit contained embryos with a jelly
coat inclusive of 3 clumps (10–20 embryos/clump) plus 20 single
embryos. The other exposure unit contained 40 embryos with their
jelly coats removed just prior to exposure. After a 4-h exposure,
embryos were removed from the experimental units by pipette and
rinsed for 5 s in de-ionized water. Embryos were embedded in a 5%
Experimental Protocol
Experiments began when embryos reached the blastula stages 8 to 9
(Gosner 1960). Minutes prior to exposure, half of the embryos were
gently swirled in a 2% w/v cysteine solution at pH 8.1 for about
4 minutes (ASTM 1992). This removed the jelly coat while leaving
the vitelline membrane and the developing embryo intact. Test units
consisted of 200-ml glass beakers containing 100 ml of culture water