EPA-560/1-76-008
SENSITIVITY OF VERTEBRATE EMBRYOS TO BORON COMPOUNDS
April 1977
Final Report
Contract No. 68-01-3222
W.J. Birge, Principal Investigator
J.A. Black, Research Associate
Project Officer
Frank Kover
Environmental Protection Agency
Office of Toxic Substances
Washington, D.C. 20460
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Document is available to the public through the National Technical
Information Service, Springfield, Virginia 22151.
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NOTICE
This report has been reviewed by the Office of
Toxic Substances, EPA, and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute indorsement or
recommendation for use.
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TABLE OF CONTENTS
SUBJECT PAGE
LIST OF TABLES ii
LIST OF FIGURES AND PLATES iv
INTRODUCTION 1
EXPERIMENTAL METHODS 3
Selection and source of test animals 3
Test concentrations and exposure periods 3
Sample size and expression of data 4
Culture water for aquatic embryos . 4
Treatment of aquatic embryos 7
Treatment of chick embryos 11
Analytical procedures 11
Boron compounds 12
EXPERIMENTAL RESULTS 12
Bioassays with aquatic embryos 12
Rainbow trout 13
Channel catfish 15
Goldfish . 17
Amphibian embryos and larvae ... 18
Teratogenesis in aquatic embryos 19
Chick embryos 20
CONCLUSIONS 21
Environmental toxicity of boron 21
Comparative sensitivity of embryonic and posthatched stages ... 23
Comparative toxicity of borax and boric acid 23
Effects of water hardness on boron toxicity . . . « 24
Effects of other test parameters 24
Chick embryos . 25
SUMMARY 25
ACKNOWLEDGMENTS . 27
BIBLIOGRAPHY 64
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LIST OF TABLES
TABLE PAGE
1 Synthetic Culture Water 6
2 Performance of the Continuous Flow System 28
3A Analysis of Flow Rates for Brinkmann
Peristaltic Pump (Model 131900) 29
3B Analysis of Flow Rates for Gil son
Peristaltic Pump (Model HP8) 29
3C Analysis of Delivery Rates for Sage
Syringe Pump (Model 355) 29
4 Test Parameters Observed During Bioassays with
Boron Compounds 30
5 Exposure of Trout Embryos and Alevins (Salmo gairdneri)
to Boron (Boric Acid) in Water of 50 ppm CaCOg Hardness . . 31
6 Exposure of Trout Embryos and Alevins (Salmo gairdneri)
to Boron (Boric Acid) in Water of 200 ppm CaC03 Hardness . . 32
7 Exposure of Trout Embryos and Alevins (Salmo gairdneri)
to Boron (Borax) in Water of 50 ppm CaC03 Hardness 33
8 Exposure of Trout Embryos and Alevins (Salmo gairdneri)
to Boron (Borax) in Water of 200 ppm CaCOo Hardness .... 34
9 Exposure of Catfish Embryos and Fry (Ictalurus punctatus)
to Boron (Boric Acid) in Water of 50 ppm CaC03 Hardness . . 35
10 Exposure of Catfish Embryos and Fry (Ictalurus punctatus)
to Boron (Boric Acid) in Water of 200 ppm CaCQ3 Hardness . . 36
11 Exposure of Catfish Embryos and Fry (Ictalurus punctatus)
to Boron (Borax) in Water of 50 ppm CaC03 Hardness 37
12 Exposure of Catfish Embryos and Fry (Ictalurus punctatus)
to Boron (Borax) in Water of 200 ppm CaCO, Hardness .... 38
13 Exposure of Goldfish Embryos and Fry (Carassius auratus)
to Boron (Boric Acid) in Water of 50 ppm CaC03 Hardness . . 39
14 Exposure of Goldfish Embryos and Fry (Carassius auratus)
to Boron (Boric Acid) in Water of 200 ppm CaC03 Hardness . . 40
15 Exposure of Goldfish Embryos and Fry (Carassius auratus)
to Boron (Borax) in Water of 50 ppm CaC03 Hardness 41
16 Exposure of Goldfish Embryos and Fry (Carassius auratus)
to Boron (Borax) in Water of 200 ppm CaCO., Hardness .... 42 "
17 Exposure of Leopard Frog Embryos and Larvae (Rana pipi ens)
to Boron (Boric Acid) in Water of 50 ppm CaC03 Hardness . . 43
18 Exposure of Leopard Frog Embryos and Larvae (Rana pi piens)
to Boron (Boric Acid) in Water of 200 ppm CaC03 Hardness . . 44
ii
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TABLE PAGE
19 Exposure of Leopard Frog Embryos and Larvae (Rana jaipiens)
to Boron (Borax) in Water of 50 ppm CaC03 Hardness 45
20 Exposure of Leopard Frog Embryos and Larvae (Rana pi pi ens)
to Boron (Borax) in Water of 200 ppm CaC03 Hardness .... 46
21 Exposure of Fowler's Toad Embryos and Larvae (Bufg^ fowleri)
to Boron (Boric Acid) in Water of 50 ppm CaCOg Hardness . . 47
22 Exposure of Fowler's Toad Embryos and Larvae (Bufo fowleri)
to Boron (Boric Acid) in Water of 200 ppm CaCO^ Hardness . . 48
23 Survival Frequencies for Control
Populations of Aquatic Embryos 49
24 Log Probit Analysis of LC, Values in ppm
for Aquatic Embryos Exposed to Boron Compounds 50
25 Log Probit Analysis of LC5Q Values in ppm
for Aquatic Embryos Exposed to Boron Compounds 51
26 95% Confidence Limits for LC, Values in ppm
for Aquatic Embryos Exposed to Boron Compounds 52
27 95% Confidence Limits for LCj-Q Values in ppm
for Aquatic Embryos Exposea to Boron Compounds 53
28 Syringe Pump (Boron) and Peristaltic Pump (Culture Water)
Flow Rates with Resulting Dilution Ratios Obtained in
Treatment of Trout Embryos at 1 and 10 ppb Boron 54
29 Teratogenesis in Aquatic Embryos Surviving
Treatment with Boric Acid and Borax 55
30 Comparison of Sensitivity of Embryonic and
Posthatched Stages to Boron Compounds ... * 56
31 Toxicity of Boric Acid to Chick Embryos 57
32 Toxicity of Borax to Chick Embryos 58
33 Effect of Flow Rate on Metal Toxicity
to Catfish Embryos and Alevins . . . . -. 59
111
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LIST OF FIGURES AND PLATES
FIGURE PAGE
1 Continuous Flow Bioassay System 8
2 Bioassay Culture Assembly 9
3 Toxicity of Borax to Catfish Embryos 60
4 Toxicity of Boric Acid to Goldfish Embryos 61
5 Toxicity of Metals to Goldfish Embryos 62
PLATE
1 Boron-Induced Teratogenesis in Trout 63
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INTRODUCTION
Boron in natural ground and surface waters of the United States usually
does not exceed 1 mg/1, and generally is under 0.1 mg/1 (1,2). Out of 84
major basins and water supplies recently sampled, only 6 contained boron in
excess of 1 mg/1 (3). In streams and other water resources of the southeastern
U.S., Boyd and Walley (4) reported boron levels under 0.1 mg/1 for 86% of 199
samples taken. Considering 1546 samples of river and lake waters from various
parts of the United States, Kopp and Kroner (5, 6) reported a mean boron
concentration of 0.1 mg/1, with a maximum of 5.0 mg/1. However, in certain
western areas of the U.S., boron concentrations of 5-15 mg/1 have been
reported (1). Recent projections, based largely on dissipative commercial
uses of boron-containing compounds, forecast detectable increases in the boron
contamination of surface and ground waters. Based on 1972 production figures,
it has been estimated that 32,000 metric tons of boron contaminants enter the
U.S. environment yearly (7). In a report prepared for EPA by Versar, Inc.,
the principal sources include laundry products, certain agricultural chemicals
and fertilizers, mining and processing of borax, manufacture of glass and
ceramics, and coal combustion. Boric acid and borates (e...g_., borax) are the
major boron-containing pollutants affecting water resources (2, 7-9).
The most critical biological effect of boron so far reported concerns
certain plants. Though boron is an essential element in plant nutrition, the
growth of more sensitive plants (.§_.£., cherry, elm, grape, lemon, peach) is
inhibited by concentrations exceeding 0.3-1.0 ppm, depending on the species
(1, 2, 8, 10).
With respect to animals, bioassay experiments with several species of
adult fish have defined lethal dosages which are well above the boron levels
generally found in natural water resources (8). Working with the mosquito
fish (Gambusia affinis), Wall en, et al. (11) reported 96-hr TL, values of
. fu
3600 and 5600 mg/1 for sodium borate and boric acid, respectively. Using
the figure given for boric acid, this amounts to an approximate dosage of
980 ppm in boron equivalents. Turnbull, et_ al_. (12) determined a 24-hr TL^
of 15,000 mg/1 for bluegill treated with boron trifluoride administered at
20 C in Philadelphia tap water, and a 48-hr ID of 339 ppm boron has been
reported for 15-month old rainbow trout (2, 13). In addition, Sprague (2)
has reported safe limits of 30 ppm and 33 ppm for largemouth bass and bluegill,
respectively.
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Relatively few studies have been conducted on the toxic effects of boron
compounds upon developmental stages. However, boric acid has been found to
produce high frequencies of lethality and teratogenesis in embryos of several
vertebrate species. Takeuchi (15) has shown that limited exposure to a 1%
concentration of boric acid is highly toxic to early embryonic stages of
the toad (Bufo vulgaris formosus). When treatment was initiated at the yolk
plug stage and maintained for 24 hours, only 18% of the embryos survived to
reach the external gill stage. Anatomical abnormalities occurred among
survivors at a frequency of approximately 42%. The principal anomalies included
defects of the notochord, brain, and special sense organs.
Boric acid also has been found to induce mortality and anomalous development
in chick embryos (16-19). Landauer (18) treated White Leghorn chicken eggs
with 2.5 mg doses of boric acid injected into the yolk at 0-120 hours of
incubation. When treatment was initiated at the start of incubation, hatch-
ability was approximately 18% and 85% in experimental and control populations,
respectively. Treatment at 96 hours of incubation resulted in 19% hatchability
of experimental eggs, and approximately 76% of the embryos which survived
through 13 days of incubation were anomalous. In a later study, Landauer (19)
performed similar experiments with several strains of the domestic fowl. Using
the same dosage, he obtained 0.8%, 12.8%, 15.5%, and 19.6% hatchability for
Silver Gray Dorking, White Leghorn, Black Minorca, and White Minorca strains,
respectively. Control hatchability was 90% or better for all strains except
the Silver Gray Dorking (67%). Following the treatment of eggs with boric acid,
the frequency of anomalous chicks in surviving populations varied from 72% for
White Leghorns to 96% for the Silver Gray Dorking strain. Teratogenic defects
largely affected the skeletal and nervous systems, including rumplessness,
anomalous legs and feet, abnormal beaks, cleft palate, various degrees of
facial coloboma, reduced or defective eyes, and cyclopia (18, 19). Unfortu-
nately, Landauer did not base the dosage of boric acid on egg yolk volume.
However, if we assume a mean value of 18.5 grams for egg yolk, the 2.5 mg
dosage used would give an egg yolk concentration of 135 ppm boric acid, or
approximately 24 ppm boron. Laying hens were found to tolerate a daily ration
containing 0.5% boric acid (875 ppm boron equivalents), though egg production
ceased after 6 days of treatment. However, normal egg production resumed
within 2 weeks after treatment was discontinued (20).
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In addition, Kanakhia, et_ a]_. (21) recently noted that boric acid was
highly toxic to mammalian embryos. A concentration of 10 ppm boric acid
(1.75 ppm boron) prevented 51% of in vitro cultured mouse embryos from
reaching the blastocyst stage. By comparison, adult rats were unaffected
when maintained for 90 days on rations containing 525 ppm boron, administered
either as 3000 ppm boric acid or 4630 ppm borax, and 350 ppm dietary boron
produced no adverse effects over a two-year feeding period (2, 22).
Though the studies reviewed above are limited in scope, they provided
evidence that embryonic stages are more susceptible to boron contaminants than
are adult vertebrates. The principal objective of this study was to determine
more precisely the concentrations at which borax and boric acid produce
lethality and/or teratogenesis in representative piscine, amphibian, and avian
embryos and early posthatched stages.
EXPERIMENTAL METHODS
Selection and source of test animals. Species selected for study included
the domestic fowl (Gall us domesticus), leopard frog (Rana pi piens), Fowler's
toad (Bufo fowleri), rainbow trout (Salmo gairdneri), channel catfish (Ictalurus
punctatus). and the goldfish (Carassius auratus). This selection of animals
includes species representing poikilothermy and homeothermy, holoblastic and
meroblastic cleavage, two distinct patterns of gastrulajtion, and a number of
other important developmental variables (e...2.., egg type, yolk quantity,
hatching time) which may respond differentially to boron exposure (24).
Gravid fish and amphibians were obtained from State and Federal hatcheries
at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from
selected sites within Kentucky. Fertile chicken eggs (White Leghorn strain)
were obtained from the Poultry Science Department, University of Kentucky.
Test concentrations and exposure periods. Test animals were exposed to
10-14 concentrations of boric acid and borax. Depending on the sensitivity of
the animal species, tests were initiated at 50-300 ppm boron and continued at 2
to 10-fold dilutions untiHC^ and LC5Q values were determined. In all cases,
exposure levels of boric acid and borax were based on actual boron content
(boron equivalents), and verified by the chemical analysis of.culture water.
For aquatic species, boron treatment was initiated subsequent to ferti-
lization and maintained continuously through 4 days posthatching, giving
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exposure periods in days of 28, 9, and 7 for trout, catfish, and goldfish,
and 7.5 for amphibians. Chicken eggs were treated with boron compounds
immediately prior to the onset of incubation, and test responses were ascer-
tained upon completion of the hatching process. Mean hatching time was
21 days.
Sample size and expressjpn of data. Minimum sample size per test concen-
tration was set at 100 for the chick embryo and 125 for frog and fish embryos.
In the analyses of test results, embryos were classified as lethals, terato-
genic (anomalous) survivors, or normal survivors. Hatchability was based on
all embryos which lived to complete the hatching process. Normal survivors
were defined as animals free of the debilitating morphological defects which
characterized teratogenic survivors (e...g_., Plate 1). Test responses were
expressed as frequencies in experimental populations/frequencies in controls.
Survival of control populations is summarized in Table 23.
Log probit analysis was used to statistically determine LC, and LC,-,,
values (25). The probit regressions were performed with an IBM computer
(model 370-165), and were determined in each instance by using animal responses
(e..£., lethality) for the full range of test concentrations. When severely
truncated survival curves precluded use of the computer program for LC5Q
determinations, the Litchfield-Wilcoxon graphic method was used (35). Analysis
of variance and the t-test were used to determine statistical significance
of differences between boric acid and borax toxicity, Water hardness levels
and other test variables.
Culture water for aquatic embryos. We have given considerable attention
to the development of a synthetic culture water suitable for bioassays.
Synthetic water provides more stable test conditions than natural water,
as the latter is subject to substantial seasonal fluctuations in composition
(e..g_., total dissolved solids, hardness, pH). Also, problems with background
contaminants are minimized when prepared water is used for culture purposes.
However, it is essential to use a formulation for synthetic water which gives
chemical and physical characteristics similar to natural water.
The culture water described below has been used extensively during the
past 2 years,and is known to give toxicity responses with metals (e..£., Cd,
Cu, Hg, Zn) which are closely comparable to results obtained using natural
water of similar composition. Also, we have performed hatchability tests with
4 species of fish and 5 species of amphibians, comparing our synthetic culture
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water to natural water from Steele's Run, a local stream (limestone bed)
which supports a healthy aquatic ecosystem. Frequencies of hatchability
were statistically indistinguishable for the two water sources, averaging
92% for fish and 96% for amphibians (41).
The synthetic culture medium used for the boron bioassays was prepared
from distilled, double deionized water, having a conductivity of 0.25 umhos
or less. Routine monitoring was conducted for background contaminants,
including mercury, cadmium, lead, and other trace metals. Reagent grade
calcium, magnesium, sodium and potassium salts were added as given in Table 1.
This basic stock was prepared to give a water hardness level of 200 ppm
(as CaCOo) and a pH of 7.5-8.0. Different levels of hardness were achieved
by dilution of the basic culture water.
Our basic stock solution has essentially the same pH, conductivity,
and water hardness as given for Mount's test water (23), which consisted of a
mixture of natural spring water (emerging from limestone strata) and carbon-
filtered, deionized tap water. Also, the content of calcium, magnesium and
potassium are in close agreement. The concentrations of cations and anions
listed in Table 1 fall within ranges published for freshwater resources in
Arizona (26), Kentucky (27), and other areas of the U.S. (8). Total chloride
content, total dissolved solids, and the concentration of sodium plus potassium
are well under maximum levels of 170 mg/1, 400 mg/1, and 85 mg/1, observed
in 95% of U.S. waters found to support good, mixed fish' fauna (28). Also,
specific conductivity of 300 umhos falls within the range of 150-500 recommended
for fish propagation (8), and is typical of values recorded for freshwater
resources in Kentucky (27). In addition, osmolarity is well within the
maximum limit of 50 mOsm/Kg water suggested for freshwaters in the U.S. (!)
Total alkalinity exceeds the minimum recommended level of 20 mg/1 (as CaC03),
and closely approaches the range cited as optimum to support a diversified
aquatic life (1, 8). As maintained in the culture system described below,
(continuous aeration), the dissolved oxygen level is approximately 10 mg/1
at the culture temperature used for trout embryos (13° C). A minimum of 7 mg/1
has been recommended for trout and salmon spawning waters (1). The pH of our
culture medium also is well within the range found in natural waters. Bass
Becking, et_ al_. (29) observed a pH range of 6 to 8 for 85% of 347 freshwater
rivers and lakes surveyed, and pH values of 6.5-8.5 generally are found in
the more productive freshwater resources in the U.S. (1).
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TABLE 1
SYNTHETIC CULTURE WATER
DISSOLVED SALTS1 (mg/1 )
CaCl2
MgS04'7H20
NaHC03
KC1
150
150
100
5
CHEMICAL COMPOSITION (mg/1)
Ca
Mg
Na
K
Cl
HC03
so4
Total dissolved solids
54.2
14.8
27.4
2.6
98.2
72.6
58.5
328.3
PHYSICOCHEMICAL CHARACTERISTICS2
Hardness (as mg/1 CaCOg)
Total alkalinity (as mg/1 CaC03)
Conductivity (ymhos/cm)
Osmolarity (mOsm/Kg H20)
pH
200.0
82.0
300.0
12.0
7.9
'Prepared in distilled, deionized water (specific conductivity
of 0.2 ymhos).
^Measurements made at 22° C. Above figures represent mean
values for six measurements.
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Treatment of aquatic'embryos.- Boron bioassays were conducted using
the continuous flow system described in Figures 1 and 2. Fish and amphibian
eggs were cultured through 4 days posthatching in Pyrex chambers, through
which test water was perfused at prescribed flow rates. The toxicant was
administered to a mixing chamber situated ahead of each culture dish, using
graduated flow from a syringe pump. Synthetic culture water was delivered
to the mixing chamber by regulated flow from a peristaltic pump. Flow rates
from both syringe and peristaltic pumps were monitored by liquid flow meters.
Flow rate was set at 200 rol/hr for 300 ml test chambers. Syringe and peri-
staltic pumps were provided with calibrated, continuously variable speed
controls. The concentration of toxicant delivered to the mixing chamber was
regulated by adjusting the mixing ratio from the pumping units and/or by
varying the concentration of toxicant delivered from the syringe pump. For
example (Table 28), to obtain a treatment level of 1 ppb (ug/1), boron at
600 ug/1 was delivered from the syringe pump to the mixing chamber at 333 ul/hr,
and diluted 1:600 by culture water metered through the peristaltic pump at
200 ml/hr. Solutions from the two channels were mixed with a Teflon-coated
magnetic stirring bar, and delivered to the culture chamber under positive
pressure.
The system was operated using Brinkmann (model 131900) and Gil son (model
HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355). The
latter was fitted with a modified syringe holder, and operated using 10 ml
double-ground, glass syringes. Syringes were selected for equal stroke volume,
and peristaltic pump channels were fitted with tubing of matched diameters.
Initially, methylene blue dye was used to check the accuracy of a 1:1000
syringe/peristaltic pump mixing ratio, and to allow visual inspection of mixing
and flow patterns through the culture system. Results are summarized in
Table 2. Reproducibility of flow rates from syringe and peristaltic pumps is
treated in Table 3. The accuracy obtained in regulating boron toxicants is
shown in Tables 5-22. For test concentrations of 10 ppm or more, boron compounds
generally were added directly to the culture water in the peristaltic pump,^
reservoir, eliminating the need for the syringe pump channel. In such instances,
the toxicants remained stable at selected test concentrations for 24 hours
or more in polyethylene containers, and important test parameters of culture
water were not altered upon standing (.§_.£., pH, hardness).
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FIGURE 2
BIOASSAY CULTURE ASSEMBLY
2.1 Series of egg culture dishes (A) which received toxicant
and culture water from mixing chamber (B). Culture water
was supplied to mixing chambers by peristaltic pump (C).
Gilmont liquid flow meters (D) were used to measure flow rate.
2.2 Enlarged view of culture room showing culture dishes (A),
mixing chambers (B), peristaltic pumps (C), and flow
meters (D). Culture dishes exhausted water into plexiglass
drain troughs which were connected to waste reservoirs.
2.3 Syringe pump used to supply toxicant to mixing chamber.
Syringe pumps were mounted on outside of culture room wall
to avoid mechanical problems resulting from culture room
temperature and humidity. Note the modified holder which
supports six 10 ml syringes. A modified syringe mounting
bracket is shown inverted on the syringe drive carriage
(arrow).
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All aquatic bioassay cultures were maintained in walk-in environmental
rooms. Culture water was given continuous aeration (filtered air) in the
peristaltic pump reservoirs. For the 18 bioassays, pH ranged from 7.5-7.9
and 7.8-8.5 for boric acid and borax, respectively (Table 4). All cultures
were monitored at regular daily intervals for temperature, dissolved oxygen,
ammonia, water hardness and pH, using a YSI tele-thermometer with thermocouple,
YSI oxygen meter (model 51A), Orion ammonia and water hardness electrodes,
and a Corning digital pH meter (model 110, with expanded millivolt scale).
Flow rates from peristaltic and syringe pumps were monitored twice daily.
The concentration of ammonia was held below 0.1 ppm in all instances, and
other test parameters were maintained as given in Table 4.
Boron exposure levels were determined at regular daily intervals by
analysis of culture water. Cultures were maintained at temperatures of 13-14 C
for trout embryos, 24.7-29.4°C for catfish, and 24.8-27.0°C for goldfish,
giving average hatching periods of 24, 5, and 3 days, respectively (Table 4).
Hatching time averaged 3.5 days for Fowler's toad (24°C) and the leopard frog
(25°C). Test organisms were examined daily to gauge extent and frequency of
development, and to remove dead specimens. Control eggs were cultured simul-
taneously with experimentals and under identical conditions, except for omission
of the toxicant.
Particular attention was given to trout embryos, especially during the
"green stage". Harmful exposure to artificial light was" precluded (30, 31)
and cultures were maintained under semi-sterile conditions to minimize occur-
rences of soft egg disease and fungus. Prior to each use, culture rooms were
disinfected and irradiated with UV for 12 hours, and the rooms were maintained
under positive pressure.
In bioassays with boron toxicants, we maintained a flow rate of 200 ml/hr,
giving a turnover time of 1.5 hours. As shown in Table 33, we have not found
flow rate to constitute a substantial test variable in bioassays with certain
metallic toxicants (e_.£.» Cd), providing the turnover is sufficiently high
to prevent deterioration of culture water.
Trout eggs and sperm were collected for test purposes by artificial
spawning and milking, using methods of Leitritz (32). Fertilization was accom-
plished by mixing sperm and eggs for 15 minutes immediately prior to the onset
of boron exposure. For all other aquatic species, fertilized eggs were collected
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n
from natural spawn. Boron treatment was initiated immediately after spawning
for amphibian species and up to 2 hours post-spawning for goldfish and catfish.
Treatment of chick embryos. Chicken eggs were treated by yolk injection,
administering the selected boron compound in a 0.1 ml aliquot of sterile,
distilled water at a concentration calculated to dilute yolk volume to the
desired test level. The test solution was deposited in a "needle track"
extending through the diameter of the egg yolk.
Eggs were treated just prior to the start of incubation, and injections
were performed in a sterile transfer chamber, using a 1.0ml tuberculin syringe
equipped with a IV hypodermic needle. Prior to injection, the blunt end of
the egg was cleansed with an alcohol swab, and a small hole was drilled over
the air cell. The needle was inserted into the air cell of the egg and
extended through the diameter of the yolk. As the needle was withdrawn, the
test aliquot was deposited in the needle track, as previously described by
Birge and Just (33). This procedure provides more uniform dispersal of
toxicant than do injections which are directed into the central yolk mass.
Control eggs were injected in identical manner, except the toxicant was with-
held from the aliquot of distilled water. Hatching frequencies of control
eggs ranged from 78-89%. Fertility level, determined on independent samples
of uninjected eggs, varied from 82-91%. Control and experimental eggs were
incubated simultaneously for 21 days at 38°C and a relative humidity of 65%.
Analytical procedures. Quantitative determinations on boron were
accomplished using the curcumin method. Following the procedures given in
Standard Methods (34), this technique proved applicable for a concentration
range of 0.1-1.0 mg/1. Concentration and dilution of sample water were used
to extend the analytical range (34). Boron monitoring results obtained are
given in Tables 5-22. In addition, all lots of prepared culture water were
monitored for possible boron contamination prior to use for bioassay purposes.
In no instance was background boron contamination detected in the prepared
culture water, including that used for the maintenance of control animals.
To avoid possible contamination from borosilicate glassware (42), Vycor brand
evaporating dishes were used for analytical purposes.
Actual exposure levels below 0.1 ppm boron were not included in Tables
5-22, as analyses of standards prepared at 0.05 ppm or less were not fully
reproducible. However, in all such cases, boron levels delivered by syringe
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12
pumps to mixing chambers were well above the detection limit, and direct
analyses were conducted to confirm syringe pump boron concentrations. In
addition, flow rates from syringe pumps (boron) and peristaltic pumps (culture
water) were monitored at regular intervals, using both flow meters and direct
volumetric measurements, to determine actual boron dilution ratios obtained
in the mixing chambers. Dividing actual syringe pump boron concentrations by
boron dilution ratios, final boron levels delivered to the bioassay test
chambers usually were found to be within 3%,. and always within 5%, of selected
nominal exposure values. In Table 28, this alternative monitoring procedure
is shown for trout bioassays, which included boron exposure values of 0.01 ppm
(10 ug/1) and 0.001 ppm (1 ug/1). Though such measurements may be used to
confirm boron concentrations delivered to test chambers, they do not provide
direct measurements on the stability of exposure levels maintained within
the bioassay cultures. While it is unlikely that actual boron exposure values
could have exceeded the upper limit of variation (5%) shown for water supplied
to the cultures, possible losses of boron from culture water, through tissue
accumulation or other means, could have resulted in undetectable reductions in
exposure concentrations. However, as seen in Tables 5-22, a high degree of
culture stability was obtained for boron at concentrations as low as 0.1 ppm,
as determined by direct analyses of culture water.
Boron compounds. The boron compounds used for testing were obtained
from the Fisher Scientific Company. Both boric acid (FKBO-) and borax,
sodium tetraborate decahydrate (Na^B-O^lO FLO), were certified ACS grade.
All results were calculated and presented as ppm boron.
EXPERIMENTAL RESULTS
Bioassays with aquatic embryos. General water quality parameters maintained
in the aquatic bioassays are summarized in Table 4, and the resulting toxicity
data, together with analyses of actual boron exposure levels, are given in
Tables 5-22. In all instances, test responses are control adjusted, as noted
under Experimental Methods (i.e.., frequencies in experimental populations/.
frequencies in corresponding controls). Frequencies of survival and terato-
genesis for control populations of aquatic embryos are presented in Table 23.
Probit analyses of boron LC-| and LC5Q values are given in Tables 24 and 25.
Experimental results are based on continuous flow treatment from fertili-
zation through hatching and 4 days posthatching. Results given at 4 days
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13
posthatching include accumulative test responses recorded for the entire
exposure period. Frequencies of hatchability are based on all animals which
lived to complete the hatching process, including teratogenic survivors.
Percentages of teratogenic animals observed in hatched populations are given
parenthetically in Tables 5-22. Their frequencies include only animals found
to bear gross, debilitating anomalies, as determined by microscopic and cine-
matographic observations. Animals free of such defects were treated as normal
survivors. For convenience, results are presented using nominal concentrations
for exposure levels and water hardness. Actual boron exposure values are given
in Tables 5-22 for bioassays with boric acid and borax administered in soft
and hard water (50 and 200 ppm CaCO^). Measurements on water hardness are
shown in Table 4.
Rainbow trout. Developmental stages of the trout were exposed for 28 days
to boron concentrations ranging from 1 ppb to 200 ppm. Average hatching time
was 24 days at 13-14°C, and the results are summarized in Tables 5-8. From
1 ppm to 200 ppm boron, percent hatchability of trout eggs generally was
inversely proportional to exposure level. At 1 ppm boron, hatchability varied
from 94-99%. Borax at or below 0.5 ppm boron did not reduce hatching frequency,
compared to control populations. Treatment with low concentrations of boric
acid gave more variable results, with 5-8% embryonic lethality occurring at
boron concentrations as low as 0.01 ppm (Tables 5, 6). High concentrations of
boron were required to produce substantial levels of embryonic mortality. At
200 ppm, complete lethality was observed with borax, and hatching frequencies
were reduced to 55% and 65% for boric acid in soft and hard water, respectively.
Using 50 ppm boron, egg hatchability varied from 77-82% for boric acid to
60-80% for borax.
Boric acid produced the highest frequencies of teratogenic survivors,
particularly when used at a water hardness level of 200 ppm CaCOo. In the latter
case, 51% of those animals which survived 100 ppm boron were grossly defective
(Plate 1), and frequencies dropped to 26%, 11%, and 5% at 1.0, 0.01, and 0.001
ppm boron, respectively. It is especially noteworthy that a high incidence
of teratogenesis was observed over a broad range of exposure levels, varying
from 200 ppm to 1.0 ppm boron (Table 5). Results were much more variable and
less pronounced when boric acid was administered in soft water (50 ppm CaCO.,),
with 27%, 1%, 21%, and 5% of survivors bearing anomalies at exposure levels of
200, 50, 1.0, and 0.01 ppm boron (Table 5).
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14
Exposure of trout eggs to borax consistently produced 2% to 3% teratogenic
survivors among populations exposed to boron concentrations of 0.001 to 10.0 ppm.
Frequencies of anomalous survivors exceeding 10% occurred only at 25 ppm boron
or more, and water hardness did not appear to constitute a significant variable.
To best determine the effects of boron treatment on survival of test
organisms, frequencies of lethality and teratogenesis were combined and the
resulting data are expressed in Tables 5-8 (.§_.£., percent dead or teratogenic).
Survival frequencies for animals found to be free of gross teratogenic anomalies
also are given (percent normal survival). Though classified as normal, such
animals were not screened for possible physiological, genetic or behavioral
impairments. Using the combined data, whether expressed as percent normal
survival or as percent dead or teratogenic, results were somewhat less propor-
tional to boron exposure level than was percent egg hatchability. This was
more evident in studies with boric acid, in which teratogenesis was substan-
tially less dependent on boron concentration than was embryonic mortality
(Tables 5, 6).
Probit analyses of the combined data for boric acid at 4 days posthatching
gave LC5Q and LC, values of 100 ppm and 0.1 ppm boron in soft water, and 79 ppm
and 0.001 ppm boron in hard water (Tables 24, 25). With borax, LC^g and LC^
values at 4 days posthatching were 27 ppm and 0.07 ppm boron in soft water and
54 and 0.07 ppm in hard water. These values reflect the sum total of three
test responses, including embryonic mortality, embryonic teratogenesis, and
posthatched mortality. At boron concentrations ranging from LC, values to
50 ppm, embryonic mortality and teratogenesis were the predominant responses.
This is evident in the values for percent normal survival which showed little
or no decline from hatching to 4 days posthatching, except at extremely high
boron concentrations (Tables 5-8).
Boric acid produced higher rates of embryonic mortality and teratogenesis
when administered in hard water (Tables 24, 25). However, water hardness was
not an appreciable factor concerning the embryopathic effects of borax. This
is especially evident from a comparison of borax LC^ values (Table 24), as
well as results obtained at exposure levels of 0.001 to 10.0 ppm boron (Tables
7, 8). Concerning the 4 trout experiments (Table 24), the order of increasing
boron toxicity to embryonic stages included boric acid in soft water (LC, = 0.1),
borax in hard water or soft water (LC, = 0*07 ), and boric acid in hard water
(LC] = 0.001).
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15
The best indication of boron sensitivity of the posthatched trout alevins
is given by comparing survival at hatching and 4 days posthatching for exposure
levels of 50-200 ppm (Tables 5-8). Results indicate that, in contrast to
embryonic stages, trout alevins are more susceptible to borax and that toxicity
for both boric acid and borax is slightly higher in soft water. For example,
borax at 50, 100, and 200 ppm boron gave posthatched survival values of 22%,
0%, and 0% in soft water and 73%, 0%, and 0% in hard water (Tables 7, 8).
It is of interest that the survival of posthatched stages was not substantially
affected by boron until exposure levels exceeded 25-50 ppm and 100 ppm in bio-
assays with borax and boric acid, respectively. However, except for boric acid
administered in hard water, survival of posthatched stages dropped sharply
once these levels were exceeded.
In all four trout bioassays, boron exposure levels spanned a dilution range
of 200,000 without achieving the full range of test responses (e...g_., 0% to
100% normal survival). The test responses most characteristic of boron toxicity
to embryonic and early juvenile stages of the rainbow trout may be summarized
as follows: generally, high frequencies of both embryonic and postembryonlc
mortality were recorded only at boron concentrations of 50 ppm or more.
Embryonic mortality and teratogenesis were the principal boron-induced responses
at 50 ppm or less. Using borax, embryonic mortality and teratogenesis decreased
progressively with concentration to 3-4% at 1 ppm boron, and 2-3% teratogenic
impairment occurred over a broad, near-threshold range of 0.5-0.001 ppm.
Though results with boric acid were more variable, trout embryos suffered
mortality and teratogenesis at frequencies of 10% or more at boron levels as
low as 0.01 ppm, and 4-7% of test animals were affected by 0.001 ppm at 4 days
posthatching. While water hardness did not exert a profound influence on
boron bioassays with trout, hard water generally increased embryonic lethality
and teratogenesis, and soft water increased boron toxicity to posthatched alevins.
The fact that high concentrations (25-200 ppm) were required to consistently
produce substantial impairment of test populations .indicates that boron compounds
are not highly toxic to trout embryos and alevins. However, compared to trace
metals such as cadmium and mercury (24), borax and boric acid are unusual"in
that they exert low level embryopathic effects on trout over a wide span of
exposure levels (e..£., 0.001-1.0 ppm).
Channel catfish. Developmental and early posthatched stages of the catfish
were treated continuously for 9 days with boron compounds. Average hatching
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16
time was 5 days at 25-29°C. Boron exposure levels ranged from 0.01-300 ppm
for boric acid bioassays and 0.05-300 ppm in tests with borax. Both toxicants
were used in soft and hard water (50 and 200 ppm CaCO~). For all catfish
bioassays, percent egg hatchability and percent normal survival at hatching
and 4 days posthatching gave good inverse correlations with boron concentration.
For example, correlation coefficients for boron exposure level and percent
normal survival at hatching were -0.95, -0.93, -0.95, and -0.98 for catfish
embryos treated with boric acid at a water hardness of 50 ppm CaC03, boric acid
at 200 ppm CaCO.,, borax at 50 ppm Ca(XLs and borax at 200 ppm CaCOg, respectively
(Tables 9-12). In all four tests, 300 ppm boron produced lethality or terato-
genesis at hatching in 100% of the experimental populations, and normal survival
at 4 days posthatching was only 0-2% at 200 ppm boron. Using boric acid in
soft water, frequencies of normal survival at 4 days posthatching increased
to 56%, 75%, 95%, and 99% at concentrations of 150, 10, 1.0, and 0.5 ppm boron.
Normal survival was 100% at and below 0.1 ppm. Treating with boric acid in
hard water, normal survival values at 4 days posthatching were 9%, 65%, 86%,
and 98% at 75, 10, 1.0, and 0.01 ppm boron, respectively.
Exposure to borax in soft water gave normal survival frequencies at 4 days
posthatching of 65%, 70%, 94%, and 98% at 150, 50, 10, and 5 ppm. Normal
survival was 100% at and below 1.0 ppm boron. In hard water, continuous expo-
sure to borax for the 9-day treatment period resulted in normal survival for
38%, 71%, 90%, and 96% of test populations at 100, 25, 10, and 1.0 ppm boron.
At 0.5 ppm or less, survival was not impaired, except for a 1% deflection at
0.1 ppm.
At 4 days posthatching, LC, and LCrQ values for boric acid were 0.5 ppm
and 155 ppm boron in soft water, and 0.2 ppm and 22 ppm in hard water (Tables
24, 25). By comparison, LC-, and LC5Q values for borax were 5.5 ppm and 155 ppm
boron in soft water, and 1.7 and 71 ppm in hard water. Using LC-, values, the
order of increasing toxicity was borax in soft water, borax in hard water,
boric acid in soft water, and boric acid in hard water. The survival curve
for borax in hard water at hatching is illustrated in Figure 3. Boric acid
was moderately more toxic than borax to both catfish embryos and early fry,
and both compounds produced greater impairment of test populations when admin-
istered in hard water (Tables 9-12). Similar to results with trout, embryonic
mortality and teratogenesis increased in hard water, with boric acid producing
higher frequencies than borax. However, differences were observed with post-
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17
hatched stages. Catfish fry were somewhat more sensitive to boron in hard
water, as seen by comparing differences in frequencies of normal survival at
hatching and 4 days posthatching for high exposure levels (Tables 9-12). As in
trout bioassays, boron concentrations of 50 ppm or more were required to produce
high levels of posthatched mortality. However, catfish fry were somewhat more
susceptible than were trout alevins to lower concentrations of boron. For
example, in tests with boric acid in hard water, appreciable levels of post-
hatched mortality occurred at all concentrations down to 1.0 ppm boron (Table 10).
The boron dilution ranges required to give test responses varying from
0-100% were approximately 300 for borax in soft water, 600 for borax in hard
water, 3000 for boric acid in soft water, and 30,000 for boric acid in hard
water. It is apparent that the effective dilution range increases with the
order of toxicity. As the boron level required to produce 100% mortality was
approximately the same in the 4 different bioassays, this increase largely
reflects extensions at the LC-, end of the exposure range, correlating principally
with observed embryopathic effects of boron (e..c[., mortality, teratogenesis).
Considering posthatched LC-j values, catfish stages collectively were less
sensitive to boron than trout stages by approximate factors of 200, 80, 25,
and 5 for boric acid in hard water, borax in soft water, borax in hard water,
and boric acid in soft water. Differential sensitivity was greatest under
conditions which proved most toxic to both species (j_.e_., boric acid in hard
water).
Goldfish. Developmental stages of the goldfish were treated continuously
with boron compounds for 7 days. Average hatching time was 3 days at 25-27°C.
As seen in Tables 13-16, hatchability and normal survival gave good inverse
correlations with boron concentration. Boron at 200 ppm produced complete
lethality in all tests. Treating with boric acid in soft water, frequencies
of normal survival at 4 days posthatching were 4%, 52%, 94%, and 98% for boron
concentrations of 100, 50, 10, and 1.0 ppm (Table 13). With hard water, normal
survival averaged 35%, 67%, 85%, and 98% for the same exposure levels (Table 14).
Borax at 50 ppm hardness gave normal posthatched survival at frequencies of
4%, 78%, 91%, and 97% for 100, 50, 10, and 1.0 ppm boron. Survival frequencies
for the same exposure levels were 0%, 77%, 93%, and 95% when hard water was
used.
Posthatched LC, and LC5Q values for boric acid, given in ppm boron, were
0.6 and 46 for soft water, and 0.2 and 75 for hard water. For borax, they
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18
were 1.4 and 65 in soft water and 0.9 and 59 in hard water. The order of
toxicity for LC, values was the same as given for the catfish. However,
differences among the 4 bioassays were not as pronounced, as LC, values were
restricted to a narrow range of 0.2-1.4 ppm boron. The LC5Q values at 4 days
posthatching also fell within a narrow range of 46-75 ppm boron, indicating
that water hardness and boron source were less significant than for other
piscine species.
As for the trout, substantial levels of posthatched mortality were found
only at the higher boron concentrations, and posthatched stages were somewhat
more susceptible to boric acid in soft water. Unlike the trout and catfish,
appreciable frequencies of teratogenesis occurred only at high exposure levels.
Frequencies exceeding 10% were observed only at or above 100 ppm boron.
In all instances, normal posthatched survival was 92% or more at and below
7.5 ppm boron, and there was a broad near-threshold range extending to 0.05
ppm boron at which low levels of embryonic mortality and/or teratogenesis
consistently were observed. This is illustrated in Figure 4 for boric acid
in soft water, and the boron survival curve may be compared to those for
cadmium and zinc obtained with the same test system (Figure 5).
The effective dilution ranges were approximately 4000 and 6000 for borax
and boric acid, respectively. Using posthatched LC, values, goldfish were
less sensitive than trout stages by approximate factors of 200, 20, 13, and
6 for boric acid in hard water, borax in soft water, borax in hard water, and
t
boric acid in soft water. As for catfish and trout, the greatest differential
in sensitivity occurred under conditions found most toxic to trout and goldfish
(j_.e.., boric acid in hard water).
Amphibian embryos and larvae. Developmental stages of the leopard frog
and Fowler's toad were given continuous treatment for 7.5 days. Average hatching
time was 3.5 days at 24-25°C. Leopard frog embryos suffered 100% lethality
or teratogenesis when treated with borax and boric acid at exposure levels of
200 ppm and 300 ppm boron, respectively (Tables 17-20). Using boric acid in
soft water, normal posthatched survival for the leopard frog increased to 87%,
93%, and 99% at boron levels of 100, 10, and 1.0 ppm. In hard water, normal
survival values for the same exposure levels were 88%, 97%, and 100%. Borax in
soft water gave normal posthatched survival frequencies of 2%, 39%, 93%, and
99% at concentrations of 100, 50, 10, and 1.0 ppm boron. When borax was
administered in hard water, normal survival averaged 16%, 52%, 90%, and 99%
for the same exposure levels.
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19
Posthatched boron LC, and LCj-Q values for the leopard frog treated with
boric acid were 13 and 130 ppm for soft water, and 22 and 135 ppm for hard water.
In bioassays with borax, they were 5 and 47 for soft water, and 3 and 54 for
hard water. As reflected by these values, borax was distinctly more toxic than
boric acid, and water hardness was not a significant factor (Tables 26, 27).
In bioassays with embryos and larvae of Fowler's toad, exposure to boric
acid produced 100% lethality or teratogenesis at 200 ppm boron (Tables 21, 22).
When treatment was maintained in soft water, normal posthatched survival
frequencies were 73%, 98%, and 99% at 100, 50, and 10 ppm boron. Using hard
water, survival values were 65%, 72%, 93%, and 98% for exposure levels of 100,
50, 10, and 1.0 ppm. Posthatched LC, and LCj-Q values were 25 and 145 ppm
when soft water was used, and 5 and 123 ppm for hard water (Tables 24, 25).
Amphibian larvae were surprisingly tolerant to boron compounds, as
substantial frequencies of mortality seldom occurred between hatching and 4
days posthatching (Tables 17-22). Also, compared to the fish bioassays. hatch-
ability of amphibian eggs was far less affected by boron treatment. In 4 of 6
tests with amphibian eggs, hatchability ranged from 87-91% at 200 ppm boron.
Appreciable reductions in hatchability occurred only with borax treatment of
leopard frog eggs at exposure levels of 100-300 ppm boron (Tables 19, 20).
Though amphibian embryos and larvae survived boron treatment considerably better
than piscine developmental stages, teratogenic impairment was substantially
greater at high boron concentrations, frequently involving 90-100% of surviving
t
populations. However, frequencies of teratogenesis dropped sharply between
200 and 50 ppm boron, always reaching 10% or less by 25 ppm, and remaining at
1-3% at lower concentrations. As a consequence, there was an exceptionally
broad concentration range, which started at 10-100 ppm boron and extended
through 0.05 ppm, where normal survival approached or exceeded 90% but remained
below 100%. This unique trend is obvious from data presented in Tables 17-22.
When frequencies for mortality and teratogenesis were combined, amphibian stages
did not appear significantly more tolerant to LCrQ concentrations of boron
than did piscine species (Tables 25, 27). However, they were appreciably
more tolerant to LC-, concentrations of boric acid, particularly in soft water
at hatching (p < 0.0001) and 4 days posthatching (p < 0.03).
Teratogenesis in aquatic embryos. All control and experimental populations
were screened for frequencies of teratogenesis by microscopic and cinemato-
graphic observations. Representative cultures were maintained under general
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20
observation for 2 weeks following withdrawal of boron treatment. Embryonic
anomalies selected for study were grouped in 6 general categories, as given
in Table 29.
Dwarfed body involved morphological irregularities and disproportionate
size reductions in the trunk and tail. The body generally was significantly
reduced in size compared to the head, and linear compressions in the trunk
often were evident, especially between the head and dorsal fin. A moderate
case is shown in Plate 1, figure 4. Defects of the cranium largely included
reduced or truncated upper jaw (Plate 1, figure 5), immobile, amphiarthrotic
lower jaw, or cephalic twinning (duplicitas anterior). The latter condition
varied from the occurrence of partial, secondary heads (Plate 1, figure 3) to
complete twinning of the head and anterior trunk (Plate 1, figure 2). Amphi-
arthrotic jaws usually were fixed in the open position (Plate 1, figures 1
and 5).
Defects of the vertebral column were far more frequent than other anomalies,
constituting the single, most reliable indicator of boron teratogenicity. This
was the only teratogenic response found to occur over the full range of aquatic
bioassays (Table 29). The more common patterns included acute lordosis,
scoliosis, kyphosis, and extreme, rigid coiling of the vertebral column (Plate 1,
figures 1, 3, 4, and 5, respectively). In the order named above, frequencies
for these different vertebral defects in trout embryos were 17%, 19%, 14%, and
30% for boric acid in hard water, and 36%, 12%, 22%, and 14% for boric acid
in soft water, amounting to 80% and 84% of the total disabilities.
Defective fins were treated as gross anomalies only when absent or reduced
sufficiently to impair locomotion. The most common impairment of the nervous
system included absent or defective eyes. Retinal coloboma, though not classified
as a gross anomaly, frequently occurred in trout alevins which suffered acute
defects of the skull or vertebral column (Plate 1, figures 1, 5, and 6).
Other boron-induced abnormalities included deformed or edematous yolk sacs
in fish embryos, and acute abdominal edema in amphibian larvae. Retarded yolk
sac resorption was classified as a teratogenic response only when complicated
by morphological defects. Frequencies for the different categories of terato-
genesis are summarized for the 18 aquatic bioassays in Table 29.
Chicj< embryos. Chicken eggs were injected with doses of boric acid and
borax calculated to dilute egg yolk volume to boron concentrations of 0.01-50
ppm. Treatment was initiated immediately prior to onset of incubation, and
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21
all frequencies of hatchability were control adjusted as given under Experi-
mental Methods. Boric acid treatment gave hatchability values of 0%, 54%,
and 92% at boron exposure levels of 50, 1.0, and 0.01 ppm (Table 31). The
frequency of teratogenic chicks surviving treatment ranged from 4% at 0.50 ppm
boron to 10% at 10 ppm. Treatment with borax gave hatching frequencies of
0%, 49%, and 95% at 50, 1.0, and 0.01 ppm boron (Table 32). However, frequencies
of teratogenesis were higher with borax, ranging from 42% at 25 ppm to 2-3%
at 0.10-0.25 ppm,.
The patterns of teratogenic development produced by boric acid and borax
were comparable. Nearly 91% of the observed embryonic anomalies involved
defective feet and/or toes, frequently accompanied with motor imbalance.
Defects of the gut, coelom and ventral body wall comprised 5% of boron-induced
defects, including abdominal cysts, hernias, eventrations, and unresorbed yolk
sacs. The remaining anomalies (4%) involved absent eyes, occipital encephaloceles,
defective beaks and facial coloboma. More bizarre conditions (je-ji., rumplessness)
were not observed among surviving populations, as grossly defective embryos
failed to complete the hatching process.
Combining teratogenesis and lethality, the LCgQ values for boric acid and
borax were approximately 1.0 ppm and 0.5 ppm boron, respectively. The LC-, values
for these compounds appeared close to 0.01 ppm boron. No teratogenic devel-
opment was observed at this exposure level, and hatchability was 92-95% of
that observed for controls. ,
CONCLUSIONS
Environmental toxicity of boron. Depending on water hardness and the
boron compound administered, the ranges of LC, values at 4 days posthatching
were 0.1-0.001 ppm for trout, 0.2-1.4 ppm for the goldfish, and 0.2-5.5 ppm
for the catfish. The LC5Q values ranged from 27-100 ppm for the trout, 46-75 ppm
for the goldfish, and 22-155 ppm for the catfish. Amphibian species were more
tolerant to boron, particularly at low concentrations, with LC, and LC5Q values
at 4 days posthatching of 3-25 ppm and 47-145 ppm, respectively.
Due to the somewhat variable responses obtained at low concentrations and
the unusual shape of the boron survival curve, 95% confidence limits for LC,
values could not always be determined by the probit program. However, the
probit LC.J values were in reasonable agreement with test responses given in
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22
Tables 5-22. For the 18 bioassays, using control adjusted data, measurable
impairment of test populations was always observed at and above LC, concen-
trations, both at hatching and 4 days posthatching. In addition, the 36
independently derived LC, values correlated with actual concentrations which
produced mortality and/or teratogenesis at frequencies of 1-7% above control
levels.
Generally, concentrations at 100-300 ppm boron produced complete lethality
for all species, and differences in sensitivity were reflected primarily at
lower exposure levels. Consequently, variations in boron sensitivity among
the 5 species were greater for LC, values, and significantly less for LC5Q
levels. On the basis of LC, values, the order of decreasing species sensitivity
was rainbow trout, goldfish, channel catfish, leopard frog, and Fowler's toad.
Comparing these results to findings reported in the literature, as
reviewed in the Introduction above (p. 1), it would appear that boron compounds
are much more toxic to developmental and early posthatched stages than to adult
fish. For example, Turnbull, ert a]_. 02) gave a 24-hr TL of 15,000 ppm boron
tri fluoride for the bluegill, and Wall en, et^aj.. (11), working with Gambusia,
reported 48-hr and 96-hr LC5Q values of 8200 and 3600 ppm borax and 10,500 and
5600 ppm boric acid. Though animal species and test conditions vary, values
reported by Wallen3 et aj_. are considerably above those found for goldfish,
catfish and amphibian embryos, where LC5Q values reported at hatching were
based on exposure periods of 3-5 days (Table 25). While boron LC, values for
piscine embryos range down to 0.001 ppm, an exposure level of 10 ppm borax is
required to produce a chronic, irritant response in adult fish (36). Also,
boron induces detectable frequencies of embryonic mortality and teratogenesis
at concentrations significantly below those at which certain, more toxic
elements (.e.cj.., As, Hg, Pb, Zn) initiate avoidance and other behavioral responses
in adult fish (37-39).
Such considerations emphasize the need to evaluate tolerances of sensitive
life cycle stages in establishing protective guidelines for aquatic ecosystems.
Depending on water hardness and the particular compound, boron levels of 0.001-
0.1 ppm could reduce reproductive potential of more sensitive piscine species
(e_.o_., trout), and concentrations exceeding 0.2 ppm likely would impair survival
of developmental stages for other species, at least under conditons providing
continuous exposure from fertilization through 4 days posthatching. As noted
in the Introduction (p. 1), boron concentrations in this range are not uncommon
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23
in U.S. freshwater resources (1-6). As water hardness, pH, total alkalinity,
conductivity, and other physical and chemical parameters most likely to affect
boron toxicity were well within ranges found for the majority of U.S. fresh-
waters (pp. 5,6; Table 4), we would expect these results to apply to most
freshwater ecosystems.' As maintained in these experiments, the exposure period
varied from 7 days for goldfish to 28 days for trout, and developmental
impairments may prove more extreme with longer exposure.
Comparative sensitivity of embryonic and posthatched stages. Results given
at 4 days posthatching included a summation of 3 independent responses, embryonic
mortality, teratogenesis and posthatched mortality. All three effects were
observed at substantial frequencies at high boron concentrations (e..£., 50-300
ppm). However, embryonic mortality and teratogenesis were the predominant
responses at lower exposure levels. Accordingly, results clearly indicated
that fish and amphibian embryos are considerably more sensitive to boron
compounds than early posthatched stages, at least under conditions where exposure
is continuous from fertilization through 4 days posthatching. This trend is
obvious from data given in Table 30, where mortality and teratogenesis were
expressed per day of boron treatment. Frequencies of test responses per day
were higher for embryonic stages in all but one of the 16 bioassays. The sole
exception occurred when trout stages were treated with borax in soft water.
Averaging results for all aquatic species, test responses per day of boron
exposure were significantly greater for embryonic stages (p < 0.001).
Comparative toxicity of borax and boric acid. Basing exposure levels on
actual boron concentration, no substantial consistent difference was observed
in the order of toxicity of the two boron compounds. Combining data for all
aquatic species, borax and boric acid toxicity could not be shown to differ
statistically at 4 days posthatching for either water hardness level. However,
certain consistent interactions were observed. Borax always was more toxic to
amphibian embryos and larvae at both LC-j and IC exposure levels. Piscine
developmental stages were more sensitive to boric acid at LC, concentrations,
due to higher frequencies of teratogenesis. This was particularly true for
bioassays using hard water, where analysis of variance showed boric acid to
be significantly more toxic than borax to fish embryos (p < 0.025). The only
exception was for trout bioassays conducted in soft water, where the two com-
pounds produced similar effects.
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24
At intermediate and higher boron exposure levels, the catfish remained
more sensitive to boric acid when administered in hard water, but no signi-
ficant difference was observed between the two compounds when they were used
in soft water. This is reflected in the LC5Q values given in Table 27.
However, trout and goldfish embryos and posthatched stages, like amphibians,
generally were more sensitive to bora* than boric acid at higher exposure
levels. The only exception was for posthatched goldfish LC-0 values determined
in soft water. Combining data for all aquatic species except the catfish,
borax proved more toxic at the IC level in both hard (p < 0.02) and soft
water (p < 0.001). This is consistent with bioassays on adult fish reported
by Wallen, et_ al_. (11), in which borax was somewhat more toxic than boric
acid to adult fish.
Effects of water hardness on boron toxicity. When LC, and IC data were
_____ i ,j(j
averaged for all species, hardness level did not exert a statistically signi-
ficant effect on toxicity of either borax or boric acid, though certain trends
were discernible. Boron toxicity to embryonic stages generally was greater
in hard water, especially for boric acid. This was most pronounced at low
exposure levels, and is reflected in the LC, values (Table 24). Though less
evident, particularly for borax, this effect also occurred in most bioassays
at the LC5Q level.
Though boron-induced embryonic mortality and teratogenesis were generally
favored by hard water, there was some indication that posthatched stages were
more susceptible to boron administered at the 50 ppm hardness level. This
effect was observed for trout, goldfish, and leopard frog stages treated with
boric acid, where percent normal survival declined more sharply from hatching
to 4 days posthatching in soft water (Tables 13, 14, and 25). However, water
hardness did not appear to constitute a significant variable when posthatched
stages were treated with borax. The fact that water hardness does not appreciably
affect boron toxicity to early fry is consistent with findings by Led ere and
Devlaminck (40) who reported 6-hr minimum lethal dosages of boric acid for
adult minnows at 18,000-19,000 mg/1 in distilled water and 19,000-19,500 mg/1
in hard water.
Effects of_ other test parameters. Though efforts were not undertaken
to evaluate the effects of temperature, dissolved oxygen and pH on boron
toxicity, certain observations are noteworthy. In the 18 aquatic bioassays,
temperature was varied from 13-29°C, dissolved oxygen ranged from 6.4-10.3 ppm,
-------
25
and pH fluctuated from 7.5-8.5 (Table 4). Though moderate effects on boron
toxicity could not have been deduced from the data, no substantial correlations
could be established between test responses and the observed variations in
temperature, oxygen, and pH.
Chick embryos. The approximate boron LC-, and LC5Q values for chick embryos
were 0.01 and 1.0 ppm for boric acid, and 0.01 and 0.5 ppm for borax. The
concentration ranges between LC, and LCrn values were considerably less than
for fish and amphibian embryos, and toxicity was moderately greater with
borax, primarily because of higher frequencies of embryonic teratogenesis
(Tables 31, 32). The embryonic tolerance to boron is far below that of laying
hens, which can survive a daily ration containing approximately 875 ppm boron
(20). Results indicate that trace levels of boron in avian eggs may substan-
tially reduce hatchability.
SUMMARY
Developmental stages of fish and amphibian embryos were treated from
fertilization through 4 days posthatching with boric acid and borax, using
a continuous flow bioassay system. Exposure levels were based on actual
boron concentrations and each toxicant was administered at water hardness
levels of 50 and 200 ppm CaCOg. Test responses analyzed included embryonic
mortality, embryonic teratogenesis, and posthatched (larval) mortality. Only
gross debilitating anomalies were tabulated for frequencies of teratogenesis.
Developmental stages of the rainbow trout (Salmo gairdneri) were treated
continuously for 28 days, with hatching occurring at 24 days (13-14°C).
Combining all test responses, boric acid LC1 and LC50 values, expressed in
ppm boron at 4 days posthatching, were 0.1 and 100 in soft water, and 0.001
and 79 in hard water. With borax, these values were 0.07 and 27 in soft water,
and 0.07 and 54 in hard water.
Embryonic and posthatched stages of the channel catfish (Ictalurus
punctatus) were exposed continuously for 9 days, with hatching occurring on
day 5 (25-29°C). The LC, and LC50 values for boric acid (ppm boron) were 0.5
and 155 in soft water, and 0.2 and 22 in hard water. By comparison, these
values for borax were 5.5 and 155, and 1.7 and 71.
Embryos and early fry of the goldfish (Carassius auratus) were treated
continuously for 7 days, with hatching occurring on day 3 (25-27°C). Boron
LC.J and LC^Q values for boric acid in ppm were 0.6 and 46 in soft water, and
-------
26
0.2 and 75 in hard water. For borax, they were 1.4 and 65 in soft water, and
0.9 and 59 in hard water.
Developmental stages of the leopard frog (Rana pipi ens) received continuous
treatment for 7.5 days, with hatching occurring at 3.5 days (24-25°C). Post-
hatched LC, and IC values for boric acid were 13 and 130 ppm boron in soft
water, and 22 and 135 ppm in hard water. In bioassays with borax, these
values were 5 and 47 ppm, and 3 and 54 ppm.
Embryos and larvae of Fowler's toad (Bufp fowleri) were given continuous
exposure for 7.5 days, with hatching occurring by 3.5 days (24-25°C). The LCn
and LCj-Q values for boric acid were 25 and 145 ppm boron in soft water, and
5 and 123 ppm in hard water, taken at 4 days posthatching.
Generally, boron concentrations of 100-300 ppm produced complete lethality
for all species, and differences in sensitivity were observed primarily at the
LC, end of the exposure range, which extended down to 0.001 ppm boron for
trout stages. Though marked differences between boric acid and borax toxicity
or soft and hard water were not observed, certain distinct trends were evident.
Boric acid usually was more toxic at LC, concentrations, and borax normally
was slightly to moderately more toxic at intermediate (LC^) and high boron
concentrations. Though embryonic mortality and teratogenesis were greater in
hard water, mortality of early fish fry and amphibian larvae was higher for
most species in soft water. The latter was most evident at exposure levels of
50 ppm boron or more. While all three test responses were appreciable at
higher exposure levels, embryonic mortality and teratogenesis were predominant
at lower boron concentrations.
In the bioassays conducted with the 5 aquatic species, temperature ranged
from 13-29°C, dissolved oxygen varied from 6.4-10.3 ppm, and pH fluctuated
from 7.5-8.5. No substantial correlations were observed between these para-
meters and boron toxicity. Other characteristics of the prepared culture water,
which was formulated to approximate the ionic composition of Mount's spring
water (23), included mean values of 328 mg/1 total dissolved solids, total
alkalinity of 82 mg/1 CaCOg, conductivity of 300 umhos/cm, and an osmolarity
of 12 mOsm/Kg. The total chloride content was S8 mg/1 and sodium plus
potassium was 30 mg/1. As the principal chemical and physical parameters most
likely to affect boron toxicity were within ranges characteristic of U.S.
freshwaters, test results should be applicable to most freshwater ecosystems.
-------
27
Results clearly show boron compounds to be substantially more toxic to
embryos and larvae than to adult fish and amphibians, emphasizing the need
to evaluate tolerances of sensitive developmental stages in establishing
protective guidelines for aquatic ecosystems. Depending on water hardness
and the particular compound, boron levels of 0,001 to 0.1 ppm could reduce
reproductive potential of more sensitive piscine species (e>g_., trout),
and concentrations exceeding 0.2 ppm likely would impair survival of develop-
mental stages for other fish species, at least under conditions providing
continuous exposure from fertilization through 4 days posthatching.
Tolerances for amphibian stages were higher than for fish, with 3.0 ppm
boron representing the limiting LC, value (borax, hard water) for an exposure
period of 7.5 days.
Chick embryos also were found to be much more sensitive than adult fowl
to boron compounds, with an LC, value of 0.01 ppm boron for both boric acid
and borax, and LC5Q values of 1.0 ppm and 0.5 ppm for boric acid and borax,
respectively. Treatment of chick embryos was initiated by egg yolk injection
just prior to the onset of incubation, and exposure values were calculated
to give ppm boron for a mean egg yolk mass of 18.4 i 0.1 gm.
ACKNOWLEDGMENTS
The bioassay system used for boron measurements was developed under
support from the National Science Foundation (RANN), including grant numbers
GI 43623 and AEN 74-08768-A01. We are also grateful for technical assistance
provided by Jarvis E. Hudson, Manu C. Parekh, Barbara A. Ramey, and Albert G.
Westerman.
-------
28
TABLE 2
PERFORMANCE OF THE CONTINUOUS FLOW SYSTEM
Reproducebility of 1:1000 Mixing Ratio for an Aqueous Solution of Methylene
Blue Dye (1000 yg/ml) Delivered at 0.3 ml/hr by a Sage Syringe Pump to Culture ,
Chambers Receiving 300 ml/hr of Culture Water Provided by Gil son Peristaltic Pump'
CULTURE ACTUAL DYE CONCENTRATION2»3>4
CHAMBER NO. dig/ml)
1 1.00 ±0.03
2 0.98 ±0.03
3 0.99 ±0.03
4 ' 1.00 ±0.01
5 0.99 ±0.03
6 0.96 ±0.02
The pumping rates stated above were set to give a final, theoretical dye concentra-
tion of 1 yg/ml culture water. Dye concentrations were determined spectrophoto-
metrically (Gary model 17 recording spectrophotometer).
2
Each concentration given as the mean value ± standard error for 20 measurements
taken at regular intervals over a seven day period of continuous operation.
A single Sage syringe pump (model 355) was used to operate all six syringes, and
one multichannel Gil son peristaltic pump (model HP8) provided 6 separate supply
lines for culture water.
4
Sections of tygon tubing used for the different peristaltic pump channels were
selected for matching diameters. Six double ground, 10 cc syringes were mounted
on the syringe pump, using a modified syringe holder.
-------
29
TABLE 3A. ANALYSIS OF FLOW RATES FOR
BRINKMANNPERISTALTIC PUMP (MODEL 131900}1
r1ow Rate
(ml/hr)
50
100
150
300
1
49.8*0.3
99.4*0.4
150.8*2.4
299.9*2.0
2
49.5*0.2
97.4*0.5
146.6*1.0
294.6*2.7
Channel
3
49.2*0.1
98.6*0.3
150.2*0.8
296.1*2.0
Number^
4
50.0*0.6
101.5*0.4
150.9*0.9
304.6*2.0
5
50.8*0.2
101.6*0.4
149.1*1.2
302.2*2.1
6
50.1*0.2
99.7*0.3
151 .4*1.2
297.7*2.3
Tygon tubing - 1/16" ID, 1/8" OD.
Mean * standard error; each value based on 20 repl icatesover a 7 day
period.
TABLE 3B. ANALYSIS OF FLOW RATES FOR
GILSON PERISTALTIC PUMP (MODEL HP8}1
:1ow Rate
(ml/hr)
50
100
175
275
1
48.7*0.4
96.7*0.4
170.4*0.6
267.3*0.8
2
50.4±0.7
100.5*0.5
173.6*0.6
273.9*2.0
Channel
3
50.8*0.6
99.9*0.5
177.2*0.5
278.5*0.9
Number^
4
48.7*0.4
100.1*0.5
173.8*0.6
274.1*0.6
5
48.3*0.0
96.5*0.4 '
172.4*0.4
268.5*0.7
6
48.3*0.0
96.5*0.4
169.4*0.5
265.5*9.7
Tygon tubing - 1/16" ID, 1/8" OD.
/
Mean * standard error; each value basad on 20 replicates over a 7 day
oeriod.
TABLE 3C. ANALYSIS OF DELIVERY RATES FOR
SAGE SYRINGE PUMP (MODEL 355)
:1ow Rate
/hr X 10
10.002
30.002
30. OO3
60. OO3
Uj
10
31
30
64
Syri nae
1
.07*0
.40*0
.14*0
.00*1
.24 9
.45 31
.57 30
.24 57
2
.ao*o.
.80*0.
.09*0.
.11*2.
17 10
48 32
67 32
20 60
3
.00*0.
.00*0.
.05*0.
.38*1.
Numbe
16 10.
37 31 .
76 31.
31 63.
r^
4
21*0.21
50*0.48
13*0.82
79*1.24
5
9.53*0
31.50*0
30.50*0
61.67*2
.13
.50
.58
.16'
6
10.42*0.1
32.60*0.3
30.14*0.6
64.00*1.1
Mean * standard error; each value based on 20 reol i cates over a 7 dayperiod
10 ml glass syri nge (single ground).
10 ml plastic disposable syrinae.
-------
30
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PLATE 1
BORON- INDUCED TERATOGENESIS IN TROUT
Photographs 1-6 show congenital deformities frequently observed
in trout alevins which survived treatment with boron compounds. Photo-
graphs were taken 2 weeks subsequent to hatching at a magnification of
1QX. The anomalies most frequently encountered included various acute
defects of the vertebral column, amphiarthrotic (immobile) jaw
articulation, absent or reduced fins, anomalous or absent eyes,
dwarfed or irregular trunk morphology, Siamese twinning, and retarded ^
yolk sac resorption.
1. Boron-induced skeletal anomalies including an acute,
lordotic spine, attenuated tail fin, and a defective,
immobile lower jaw (arrow).
2. Twinning of head and trunk, with a single tail. A
single yolk sac possesses paired yolk stalks, one
serving each partial twin.
3. Acute scoliosis of the vertebral column and cephalic J|
twinning. Secondary head (arrow) is only partially
formed.
4. Kyphosis of the vertebral column with irregular, ^
dwarfed trunk structure.
5. Anomalous, inflexible curvature of spinal column; irregular,
truncate upper jaw, and immobile lower jaw. As shown in
all other figures, yolk sac resorption is retarded. Also, mt
as seen in Figures 1 and 6, incomplete closure of the " J1
choroid fissure has resulted in coloboma of the retina.
This condition is discernible as a vertical cleft at the
mid-inferior border of the eye.
6. Irregular, fixed curvature of the vertebral column similar
to that shown in Figure 5.
*
I
I
I
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BIBLIOGRAPHY
1. National Technical Advisory Committee on Water Quality Criteria, U.S.
Dept. Interior. 1968. 234 pp.
2. Sprague, R.W. 1972. The Ecological Significance of Boron. U.S. Borax
Research Corporation, Anaheim, Calif. 58 pp.
3. Proposed Criteria for Water Quality. 1973. U.S. Environmental Protection
Agency, vol. 1. 425 pp.
4. Boyd, C.E. and W.W. Walley. 1972. Studies of the biogeochemistry of boron.
Am. Mid. Nat., 88(1): 1-14.
5. NAS-NAE Committee on Water Quality Criteria. 1973. Water Quality
Criteria 1972. U.S. Govt. Print. Off., Washington, D.C. 593 pp.
6. Kopp, J.F. and R.C. Kroner. 1970. Trace Elements in Waters of the United
States, Oct. 1, 1962-Sept. 30, 1967. U.S. Dept. Interior, Fed. Wat.
Pollut. Control Admin., Division of Pollution Surveillance, Cincinnati,
Ohio,
7. Preliminary Investigation of Effects on the Environment of Boron, Indium,
Nickel, Selenium, Tin, Vanadium and Their Compounds. 1974. Versar,
Inc., (prepared for Office of Hazardous Materials Control of EPA),
vol. 1, Boron. 110 pp.
8. McKee, J.E. and H.W. Wolf. 1963. Water Quality Criteria. The Resource
Agency of California (2nd ed.). State Water Quality Control Board,
Publ. #3A. 548 pp.
9, Waggott, A. 1969. An investigation of the potential problem of increasing
boron concentrations in rivers and water courses. Wat. Res., 3: 749-765.
10. Diagnosis and Improvement of Saline and Alkali Soils. 1954. U.S. Salinity
Laboratory, U.S. Dept. Agric. Handbook #60.
11. Wallen, I.E., W.C. Greer and R. Lasater. 1957. Toxicity to Gamfausia affinis
of certain pure chemicals in turbid waters. Sew. Ind. Waste, 29: 695-711.
12. Turnbull, H., J.G. DeMann and R.F. Weston. 1954. Symposium on waste
disposal in the petroleum industry. Ind. Eng. Chem., 46: 324-333.
13. Alabaster, J.S. 1957. The toxicity of certain weed killers to trout.
Proc. Brit. Weed Contr. Conf., 3: 807.
14. Lawrence, J.M. 1958. Methods for Controlling Aquatic Weeds in Fish Ponds.
Alabama Polytechnic Institute Agr. Exp. Sta. Progr. Rep. Ser. #69.
15. Takeuchi, T. 1958. Effects of boric acid on the development of the eggs
of the toad, Bufo vulgaris formosus. Sci., Rep. Tohoku Univ. Ser.4,
Biol. 24(1): 33-43.
64
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65
16. Hermanni, H.H. 1972. Boric acid produced malformations of the posterior
extremities of chicken embryos and their histogenesis. Wilhelm Roux'
Archiv., 171: 200-222.
17. Goldi, M. and H. Stierholz. 1964. Histologic features of the development
of the rump!ess chick embryo induced with boric acid. J. Am. Osteo.
Assoc., 63(9): 879-880.
18. Landauer, W. 1952. Malformations of chicken embryos produced by boric
acid and the probable role of riboflavin in their origins. J. Exptl.
Zoo!., 120(3): 469-507.
19. . 1953. Genetic and environmental factors in the teratogenic effects
of boric acid on chicken embryos. Genetics, 38: 216-228.
20. Hove, E., C.A. Elvehjem and E.B. Hart. 1939. Boron in animal nutrition.
Amer. J. Physiol., 127: 689-701.
21. Kanakhia, A.P., B.U. Leonov and V.A. Shaternikov. 1970. Organism Sreda,
Konf. Gig Kafedr. (ed. by Cherkinskii, S.N. Pervui Mosk Med. Insti.),
6th (1): 102-104. Moscow.
22. Weir, R.J., Jr.., and R.S. Fisher. 1972. Toxicologic studies on borax
and boric acid. Toxicol. Appl. Pharmacol., 23(3): 351-364.
23. Mount, D.I. 1968. Chronic toxicity of copper to fathead minnows (Pimephales
promelas. Rafinesque). Water Res., 2: 215-223.
24. Birge, W.J., A.G. Westerman and O.W. Roberts. 1974. Lethal and teratogenic
effects of metallic pollutants on vertebrate embryos. _In_ 2nd Annual
NSF-RANN Trace Contaminants Conference, Asilomar, Calif., 2: 316-320.
25. Daum, R.J. 1969. A- revison of two computer programs for probit analysis.
Bull. Entomol. Soc. Am., 16: 10-15.
26. Dutt, G.R. and T.W. McCreary. 1970. The quality of Arizona's domestic,
agricultural, and industrial waters. Univ. Arizona Ag. Exper. Station,
Report #256. 83 pp.
27. Water Resources Data for Kentucky, Part 2. 1970. U.S. Dept. Interior
Geol. Survey. 173 pp.
28. Hart, W.B., P. Doudoroff, and J. Greenbank. 1945. Evaluation of Toxicity
of Industrial Wastes, Chemicals and Other Substances to Freshwater
Fishes. Water Control Lab., Atlantic Refining Co., Philadelphia.
29. Bass Becking, L.G.M., I.R. Kaplan, and D. Moore. 1960. Limits of the
natural environment in terms of pH and oxidation-reduction potentials.
J. Geol., 68: 243-284.
30. MacCrimmon, H.R. and W.H. Kwain. 1969. Influence of light on early
development and meristic characters in the rainbow trout (Salmo gairdneri
Richardson). Can. J. Zoo!., 47: 631-637.
-------
66
31. Eisler, R. 1957. Some effects of artificial light on salmon eggs and
larvae. Trans. Am. Fish. Soc., 87: 151-162.
32. Leitritz, E. 1972. Trout and Salmon Culture. State of California,
Dept. Fish and Game, Fish. Bull. #107.
33. Birge, W.J. and J.J. Just. 1975. Sensitivity of Vertebrate Embryos to
Heavy Metals as a Criterion of Water Quality, Phase II. U.S. Dept.
Interior, Res. Rep. #84. 36 pp.
34. Standard Methods for the Examination of Water and Wastewater. 1971.
Amer. Pub. Health Assoc.. Amer. Wat. Works Assoc., and Wat. Pollut.
Control Fed. (joint eds., M.J.Taras, A.E. Greenbert, R.D. Hoak and
M.C. Rand), Washington, D.C. (13th ed.). 874 pp.
35. Litchfield, J.F., Jr., and F. Wilcoxon. 1949. A simplfied method of
evaluating dose-effect experiments. J. Pharmacol. and Exp. Ther.,
96: 99-113.
36. Hiatt, R.W., J.J. Naughton, and D.C. Matthews. 1953. Relation of chemical
structure to irritant responses in marine fish. Nature, 172: 904.
37. Weir, P.A. and C.H. Hine. 1970. Effects of various metals on behavior in
conditioned goldfish. Arch. Environ. Health, 20: 45-51.
38. Waller, W.T. and J. Cairns, Jr. 1972. The use of fish movement patterns
to monitor zinc in water. Wat. Res., 6: 257-269.
39. Sparks, R.E., J. Cairns, Jr., and A.G. Heath. 1972. The use of bluegill
breathing rates to detect zinc. Wat. Res., 6: 895-911.
40. LeClerc, E. and F. Devlaminck. 1955. Fish toxicity tests and water
quality. Bull, de Beige. Condument Eaux., 28: 11.
41. Birge, W.J., A.G. Westerman, and J.A. Black. 1975. Sensitivity of Vertebrate
Embryos to Heavy Metals as a Criterion of Water Quality, Phase III.
U.S. Dept. Interior, Res. Rep. #91. 27 pp.
42. Green, G.H., C. Blincoe, and H.G. Weeth. 1976. Boron contamination from
borosilicate glass. J. Agric. Food Chem., 24(6): 1245-1246.
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TECHNICAL REPORT DATA
(Please read Ins&ucnons on the reverse before completing)
\. REPORT NO. 2.
EPA- 560/ 1-76-008
I. TITLE AND SU8TITLS
Sensitivity of Vertebrate Embryos to Boron Compounds
. AUTHOR(S)
Wesley J. Birge, Jeffrey A. Black
PERFORMING ORGANIZATION NAME AND ADDRESS
Thomas Hunt Morgan School of Biological Sciences
University of Kentucky
Lexington, Kentucky 40506
I. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
3. RECIPIENT'S ACCE5SIOf*NO.
5. REPORT DATE
April 1977
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-3222
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
i. SUPPLEMENTARY NOTES
. ABSTRACT
Developmental stages of rainbow trout, channel catfish, goldfish, leopard frog,
and Fowler's toad were treated in a continuous flow system with boric acid and borax
concentrations ranging from 0.001-300 ppm boron. Exposure was initiated subsequent
to fertilization and maintained through 4 days posthatching, using water with
hardness levels of 50 and 200 ppm CaC03. Expressed in ppm boron at 4 days post-
hatching, LC] values for trout, catfish, and goldfish were 0.1, 0.5, and 0.6 for
boric acid in soft water, 0.001, 0.2, and 0.2 for boric acid in hard water, 0.07,
5.5, and 1.4 for borax in soft water, and 0.07, 1.7, and 0.9 for borax in hard
water. LCso values in ppm for trout, catfish, and goldfish were 100, 155, and 46
for boric acid in soft water, 79, 22, and 75 for boric acid i,n hard water, 27,
155, and 65 for borax in soft water, and 54, 71, and 59 for borax in hard water.
The LCso values for amphibian embryos and larvae ranged from 47 for borax in soft
water to 145 for boric acid in soft water. By comparison, boron LCso values for
chick embryos, treated by yolk injection, were 1.0 and 0.5 ppm for boric acid and
borax, respectively.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Bioassay
Boron
Embryonic mortality
Teratogenesis
JlsrmaUTION STATEMENT "" ' "
Release unlimited
b.lDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (This page)
Unclassified
c. COSATI Field/Group
21. NO. OF PAGES
22. PRICE
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