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Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test animals were exposed to 10 - 14 concentrations of boric acid at 2 different water hardnesses.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
other: Bufo fowleri
Details on test organisms:
TEST ORGANISM
- Common name: Fowler's Toad
- Source: 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.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7.5 d
Hardness:
Hardness 50 : 57.4 mg/L
Hardness 200 : 207.5 mg/L
Test temperature:
23.7 °C
pH:
Hardness 50 : 7.6
Hardness 200 : 7.6
Dissolved oxygen:
Hardness 50 : 6.8 mg/L
Hardness 200 : 6.8 mg/L
Nominal and measured concentrations:
Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.1, 0.46, 0.97, 5, 9.4, 24, 48.7, 96, 189, 279 mg B/L
Measured concentrations for test with Hardness 200 : 0.05, 0.1, 0.45, 0.93, 4.5, 8.3, 22.3, 53.5, 93, 204, 308 mg B/L
Details on test conditions:
TEST SYSTEM
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 ml /hr for 300 ml test chambers. Syringe and peristaltic 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. 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 Gilson (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.
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.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
88.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Hardness 50; confidence limits: 25.8 - 302.7 mg/L
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
55.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Hardness 200; confidence limits: 38.4 - 79.6 mg/L
Reported statistics and error estimates:
For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. 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.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication that meets basic scientific principles. Non-standard design - only two exposures tested
Principles of method if other than guideline:
Eggs of amphibians were exposed to boron concentrations (0, 50, 100 mg B/L) under laboratory conditions.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Ambystoma jeffersonianum
Details on test organisms:
TEST ORGANISM
- Common name: Jefferson salamander
- Source: Egg masses were collected on April 2-3, 1997, from unirrigated ponds in the western portion of SGL 176 in Centre County, Pennsylvania. The masses were returned to the lab on ice and stored in pond water from their collection site. All eggs were in the blastula stage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
25 d
Hardness:
186.5 mg/L
Test temperature:
10 °C
pH:
6.5
Nominal and measured concentrations:
Nominal concentrations : 0, 50 and 100 mg B/L
Measured concentrations : 0, 49.50 and 100.24 mg B/L
The B concentrations changed little over the course of the experiments (<5%)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 2.4 L polyethylene containers
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): 5


OTHER TEST CONDITIONS
- Adjustment of pH: All solutions were adjusted to pH 6.5 with dilute H2SO4 or KOH to simulate the pH of irrigated ponds.
- Photoperiod: 12 h light : 12 h dark
- Light intensity: 40 W fluorescent overhead lights


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of dead, deformed, and hatched eggs was recorded weekly until the first evidence of hatching at witch point the containers were monitored every other day. The number of successfully hatchting larvae and the number of hatchlings with deformities were scored after hatching.
Duration:
25 d
Dose descriptor:
LOEC
Effect conc.:
<= 49.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
morphology
Remarks:
larval deformation
Remarks on result:
other: unbounded LOEC (7% effect at LOEC level)
Duration:
25 d
Dose descriptor:
NOEC
Effect conc.:
>= 100.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: hatching
Reported statistics and error estimates:
The proportion of eggs that successfully hatched and/or the proportion of deformed larvae were compared across levels in each experiment with one-way ANOVAs for each species, followed by Tukey multiple comparison tests if a significant effect was found. All proportional data were arcsine-transformed prior to analysis. Multiple comparisons were corrected with the sequential Bonferroni Type I error rate adjustment (Rice 1989). In this method, the p values from each of K comparisons are ranked in increasing order and compared to critical p values of 0.05/K, 0.05/K-1, 0.0K-2, and so on. If the lowest p value is lower than 0.05/K it is considered significant, and the next lowest p value is compared to 0.05/K-1. This continues until Pi >= 0.05/K-i when that and all subsequent p values are considered nonsignificant. All statistical tests utilized Minitab V.10 statistical software (Minitab 1994).
EC10 concentrations could not be calculated
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication that meets basic scientific principles. Non-standard design - only two exposures tested
Principles of method if other than guideline:
Eggs of amphibians were exposed to boron concentrations (0, 50, 100 mg B/L) under laboratory conditions.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Ambystoma maculatum
Details on test organisms:
TEST ORGANISM
- Common name: Spotted salamander
- Source: Egg masses were collected on April 2-3, 1997, from unirrigated ponds in the western portion of SGL 176 in Centre County, Pennsylvania. The masses were returned to the lab on ice and stored in pond water from their collection site. All eggs were in the blastula stage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
44 d
Hardness:
186.5 mg/L
Test temperature:
10 °C
pH:
6.5
Nominal and measured concentrations:
Nominal concentrations : 0, 50 and 100 mg B/L
Measured concentrations : 0, 49.50 and 100.24 mg B/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 2.4 L polyethylene containers
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): 5


OTHER TEST CONDITIONS
- Adjustment of pH: All solutions were adjusted to pH 6.5 with dilute H2SO4 or KOH to simulate the pH of irrigated ponds.
- Photoperiod: 12 h light : 12 h dark
- Light intensity: 40 W fluorescent overhead lights


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of dead, deformed, and hatched eggs was recorded weekly until the first evidence of hatching at witch point the containers were monitored every other day. The number of successfully hatchting larvae and the number of hatchlings with deformities were scored after hatching.
Duration:
44 d
Dose descriptor:
NOEC
Effect conc.:
24.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: larval deformation
Remarks on result:
other: Unbounded LOEC of 49.5 mg/L (17,8% at LOEC concentration); NOEC=LOEC/2 or 24.8 mg/L
Duration:
44 d
Dose descriptor:
LOEC
Effect conc.:
>= 49.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: larval deformation
Remarks on result:
other: 17.8 % effect at LOEC level
Duration:
44 d
Dose descriptor:
NOEC
Effect conc.:
>= 100.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: hatching
Reported statistics and error estimates:
The proportion of eggs that successfully hatched and/or the proportion of deformed larvae were compared across levels in each experiment with one-way ANOVAs for each species, followed by Tukey multiple comparison tests if a significant effect was found. All proportional data were arcsine-transformed prior to analysis. Multiple comparisons were corrected with the sequential Bonferroni Type I error rate adjustment (Rice 1989). In this method, the p values from each of K comparisons are ranked in increasing order and compared to critical p values of 0.05/K, 0.05/K-1, 0.0K-2, and so on. If the lowest p value is lower than 0.05/K it is considered significant, and the next lowest p value is compared to 0.05/K-1. This continues until Pi >= 0.05/K-i when that and all subsequent p values are considered nonsignificant. All statistical tests utilized Minitab V.10 statistical software (Minitab 1994).
EC10 concentrations could not be calculated
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test animals were exposed to 10 - 14 concentrations of boric acid at 2 different water hardnesses.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Rana pipiens
Details on test organisms:
TEST ORGANISM
- Common name: Leopard frog
- Source: 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.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7.5 d
Hardness:
Hardness 50 : 52.5 mg/L
Hardness 200 : 212.3 mg/L
Test temperature:
25 °C
pH:
Hardness 50 : 7.7
Hardness 200 : 7.7
Dissolved oxygen:
Hardness 50 : 7.7 mg/L
Hardness 200 : 7.7 mg/L
Nominal and measured concentrations:
Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.09, 0.39, 0.93, 4.5, 9.15, 32.5, 47.5, 84.5, 188, 298 mg B/L
Measured concentrations for test with Hardness 200 : 0.13, 0.53, 0.92, 4.6, 8.93, 25.2, 45.7, 86, 193, 288 mg B/L
Details on test conditions:
TEST SYSTEM
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 ml /hr for 300 ml test chambers. Syringe and peristaltic 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. 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 Gilson (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.
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.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
76.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks:
+ teratogenecity
Remarks on result:
other: Hardness 50; confidence limits: 60.8 - 97.0 mg/L
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
90.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks:
+ teratogenecity
Remarks on result:
other: Hardness 200; confidence limits: 70.6 - 115.9 mg/L
Reported statistics and error estimates:
For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. 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.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardnesses. Mortality was recorded and LC1 and LC50 calculated.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Rana pipiens
Details on test organisms:
TEST ORGANISM
- Common name: Leopard frog
- Source: 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.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7.5 d
Hardness:
Hardness 50 : 46.2 mg/L
Hardness 200 : 203 mg/L
Test temperature:
25.3 °C
pH:
Hardness 50 : 8.3
Hardness 200 : 8.4
Dissolved oxygen:
Hardness 50 : 7.7 mg/L
Hardness 200 : 7.8 mg/L
Nominal and measured concentrations:
Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.11, 0.51, 0.84, 4.57, 7.04, 9.6, 27.3, 56, 103, 205, 292 mg B/L
Measured concentrations for test with Hardness 200 : 0.05, 0.13, 0.42, 1, 4.98, 7.04, 10.5, 28.4, 55.2, 108, 244, 328 mg B/L
Details on test conditions:
TEST SYSTEM
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 ml /hr for 300 ml test chambers. Syringe and peristaltic 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. 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 Gilson (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.
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.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
27.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Hardness 50; confidence limits: 22.2 - 33.8 mg/L
Duration:
7.5 d
Dose descriptor:
LC10
Effect conc.:
21.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Hardness 200; confidence limits: 15.9 - 29.9 mg/L
Reported statistics and error estimates:
For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. 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.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication that meets basic scientific principles. Non-standard design - only two exposures tested
Principles of method if other than guideline:
Eggs of amphibians were exposed to boron concentrations (0, 50, 100 mg B/L) under laboratory conditions.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Rana sylvatica
Details on test organisms:
TEST ORGANISM
- Common name: Wood frog
- Source: Egg masses were collected on April 2-3, 1997, from unirrigated ponds in the western portion of SGL 176 in Centre County, Pennsylvania. The masses were returned to the lab on ice and stored in pond water from their collection site. All eggs were in the blastula stage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
23 d
Hardness:
186.5 mg/L
Test temperature:
10 °C
pH:
6.5
Nominal and measured concentrations:
Nominal concentrations : 0, 50 and 100 mg B/L
Measured concentrations : 0, 49.50 and 100.24 mg B/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 2.4 L polyethylene containers
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): 5


OTHER TEST CONDITIONS
- Adjustment of pH: All solutions were adjusted to pH 6.5 with dilute H2SO4 or KOH to simulate the pH of irrigated ponds.
- Photoperiod: 12 h light : 12 h dark
- Light intensity: 40 W fluorescent overhead lights


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of dead, deformed, and hatched eggs was recorded weekly until the first evidence of hatching at witch point the containers were monitored every other day. The number of successfully hatchting larvae and the number of hatchlings with deformities were scored after hatching.
Duration:
23 d
Dose descriptor:
LOEC
Effect conc.:
<= 49.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
morphology
Remarks:
larval deformation
Remarks on result:
other: 51% effect at LOEC level
Duration:
23 d
Dose descriptor:
NOEC
Effect conc.:
>= 100.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: hatching
Reported statistics and error estimates:
The proportion of eggs that successfully hatched and/or the proportion of deformed larvae were compared across levels in each experiment with one-way ANOVAs for each species, followed by Tukey multiple comparison tests if a significant effect was found. All proportional data were arcsine-transformed prior to analysis. Multiple comparisons were corrected with the sequential Bonferroni Type I error rate adjustment (Rice 1989). In this method, the p values from each of K comparisons are ranked in increasing order and compared to critical p values of 0.05/K, 0.05/K-1, 0.0K-2, and so on. If the lowest p value is lower than 0.05/K it is considered significant, and the next lowest p value is compared to 0.05/K-1. This continues until Pi >= 0.05/K-i when that and all subsequent p values are considered nonsignificant. All statistical tests utilized Minitab V.10 statistical software (Minitab 1994).
Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 8, 2010 till June 7, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Principles of method if other than guideline:
The potential reproductive toxicity of boric acid (BA) in the African clawed frog, Xenopus laevis, was evaluated following a modified Amphibian Growth and Reproduction Assay (AGRA) (U.S. Environmental Protection Agency (USEPA), Amphibian Growth and Reproduction Assay - Tier 2 (AGRA), Detailed Review Paper, 2004), now referred to as the Larval Amphibian Growth and Development Assay (LAGDA) (U.S. Environmental Protection Agency (USEPA), Weight of Evidence Guidance: Evaluating Results of EDSP Tier 1 Screening to Identify Candidate Chemicals for Tier 2 Testing - Draft for Public Comment, 2010)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test solution samples were collected for boron analysis at exposure phase initiation, weekly during the exposure phase, and at the in-life phase conclusion.

Immediately following collection and filtering, the samples were stored at 4°C ± 2° until shipped to ABC laboratories, Inc. Samples were shipped on ice weekly to the chemistry laboratory throughout the experimental period. ABC Laboratories, Inc. performed B analyses as soon as possible following sample receipt.

Water quality characteristics including pH, dissolved oxygen (DO), conductivity, hardness, alkalinity, ammonia, residual oxidants, pesticides, and metals were measured prior to beginning study. Each dechlorinated laboratory blank received by the analytical lab was analyzed for test substance boric acid (BA).
Vehicle:
no
Details on test solutions:
A target stock solution (3,563 mg BA/L) was used to prepare each test concentration.
Test organisms (species):
Xenopus laevis
Details on test organisms:
The test species used was the African Clawed Frog (X. laevis). Sexually mature X. laevis adults, originating from FEL colonies, were used exclusively throughout the study as the test system. The frogs were originally selected based on general productivity and health.

The adult frogs were fed Finfish Starter 50-15 pellets (Zeigler Brothers, Inc., Gardners, PA) ad libitum throughout the study.

The pre-exposure phase consisted of a 14-d acclimation period in which sexually mature male and female frogs were cultured in dechlorinated culture water and observed for signs of general health. Prior to exposure, eggs were flushed from females via super-ovulation and breeding.

Following 14 d of acclimation, female frogs were super-ovulated by injecting 500 IU of human chorionic gonadotropin (hCG) into the dorsal lymph sac s.c. Within 3-4 h post injection, oviposition generally occurred. Three of the super-ovulated females were paired with 3 males injected with approximately 200 IU hCG s.c. Each mating pair was placed in a polyethylene breeding chamber and allowed to breed overnight. In order to ensure that each female that was not mated fully discharged the majority of mature oocytes, the flanks at the anterior portion of the ovary were gently squeezed with posterior movement down the oviduct so as to strip the ovary of oocytes not yet released.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Hardness:
132 mg/L
Test temperature:
25 °C
pH:
7.5
Dissolved oxygen:
5.2 mg/L
Nominal and measured concentrations:
Nominal test concentrations: 0, 2.5, 5.0, 7.5, and 10.0 mg boron (B)/L as added boron.
The 30-d time weighted average measured exposure concentrations were <0.7 (control), 2.9, 5.2, 7.3, and 9.7 mg B/L
Expressed as added boron the 30-d average concentrations were: 0, 2.6, 4.9, 7.0, and 9.4 mg B/L
Details on test conditions:
A continuous-flow mini-diluter system with 10-L glass aquaria was used for the exposure system. Each aquarium or test chamber contained 4 L of test solution, which was renewed once every 2.7 hours (25 mL/min with 9 volume exchanges per day). A randomized design was used for the reproductive assay. This design was intended to randomize out the effects associated with the local environment (i.e., light and water) and possible trends associated with the diluter during testing. All frogs were randomly assigned to tanks prior to pre-exposure.

Eight adult female and eight adult male frogs were assigned to each of two replicates per treatment and maintained by sex, resulting in 16 female and male specimens exposed per treatment. Four frogs from each replicate aquarium (8 female and 8 male) were bred with unexposed frogs. Females were super-ovulated and baseline reproduction data were collected prior to in-life exposure. The primary endpoints measured were adult survival, growth (weight and snout-vent length [SVL]), necropsy data, reproductive fecundity, and development of progeny (F1) from the exposed frogs. Necropsy endpoints included gonad weight, gonado-somatic index (GSI), ovary profile (oocyte normalcy and stage distribution), and sperm count and dysmorphology. Assessment of reproductive fecundity involved evaluation of secondary sex characteristics and breeding success. Measurement of breeding success included number of eggs laid, fertilization, necrosis, and embryo-larval viability (survival and normalcy of development).

Laboratory prepared water, referred to as dechlorinated tap (DeCl2) water, was used as the dilution water and laboratory control for this study. Also, DeCl2 water was used as the analytical laboratory blank throughout the study. DeCl2 water was prepared by passing tap water through a 4 filter system; a multimedia filter to remove suspended solids in the feed water; a 10” pre-treatment filter (5 μm) to remove any additional solids; a 3.6 ft3 activated virgin carbon treatment filter to remove chlorine, ammonia, and higher molecular weight organics; and a 5 μm polishing filter to remove any carbon particles from the carbon treatment phase.
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element
Remarks:
boron
Basis for effect:
other: reproduction
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element
Remarks:
boron
Basis for effect:
other: length
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element
Remarks:
boron
Basis for effect:
behaviour
Details on results:
Concentrations were measured weekly and agreed reasonably well with nominal values. Control concentrations of boron were all below the minimum quantifiable level (0.7 mg B/L). The nominal concentrations were used, following the usual practice that a nominal concentration may be used if the measured values are reasonably close to the nominal.

The 30-d time weighted average exposure concentrations were <0.7 (control), 2.9, 5.2, 7.3, and 9.7 mg B/L which are equivalent to <4.0 (control), 16.4, 29.5, 41.7, and 55.4 mg BA/L. These were reasonably close to the nominal concentrations of 0, 2.5, 5.0, 7.5, and 10 mg B/L which are used in the table of Effect Concentrations

BA exposure to adult X. laevis did not alter any of the adult endpoints, or endpoints involving the embryo-larval viability of the F1 generation.
Reported statistics and error estimates:
All data from in-life portions of the study were tabulated in spreadsheets. Data were then summarized in the final report. Primary endpoints were analyzed using one-way ANOVA (parametric data sets) or Kruskal-Wallis (KW-) ANOVA on ranks (non-parametric data sets) accompanied by appropriate post-hoc comparison tests using SigmaPlot software (Systat Software, Inc., San Jose, CA).
Validity criteria fulfilled:
yes
Remarks:
Test validity was based on the following laboratory control criteria: • ≥ 80% survival in the controls over the course of the study; and • ≥ 70% induction of breeding in the controls as indicated by amplexus.
Conclusions:
Exposure to BA did not alter reproductive fecundity and F1 generation embryo larval development. Based on the results of the present study, the no observed adverse effects concentration (NOAEC) for reproductive fecundity and F1 embryo larval development was 10.0 mg B/L (nominal).
Executive summary:

Based on the results of this study, 30-d BA exposure did not induce reproductive or developmental (F1) toxicity at the concentrations selected. Thus, the NOAEC determined for reproduction and development (F1) was 10.0 mg B/L (or 9.7 mg B/L expressed as a 30-d time weighted average exposure concentration). A previous study reported effects at 8.8 mg B/L (nominal concentration). However the present study is considered more reliable because of multiple technical limitations of the historical study. These limitations suggest that the previous study be considered a preliminary or range-finding study. In fact it was used to set the exposure range of the present study. None of these endpoints were adversely affected at the highest exposure in the present study. Overall, the present study provides evidence that BA is not reproductively or developmentally toxic to adult X. laevis at ca. 10 mg B/L.

Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication that meets basic scientific principles. Non-standard design - only two exposures tested
Principles of method if other than guideline:
Eggs of amphibians were exposed to boron concentrations (0, 50, 100 mg B/L) under laboratory conditions.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Bufo americanus
Details on test organisms:
TEST ORGANISM
- Common name: American toad
- Source: Egg masses were collected on April 2-3, 1997, from unirrigated ponds in the western portion of SGL 176 in Centre County, Pennsylvania. The masses were returned to the lab on ice and stored in pond water from their collection site. All eggs were in the blastula stage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
23 d
Hardness:
186.5 mg/L
Test temperature:
10 °C
pH:
6.5
Nominal and measured concentrations:
Nominal concentrations : 0, 50 and 100 mg B/L
Measured concentrations : 0, 49.50 and 100.24 mg B/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 2.4 L polyethylene containers
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3


OTHER TEST CONDITIONS
- Adjustment of pH: All solutions were adjusted to pH 6.5 with dilute H2SO4 or KOH to simulate the pH of irrigated ponds.
- Photoperiod: 12 h light : 12 h dark
- Light intensity: 40 W fluorescent overhead lights


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of dead, deformed, and hatched eggs was recorded weekly until the first evidence of hatching at witch point the containers were monitored every other day. The number of successfully hatchting larvae and the number of hatchlings with deformities were scored after hatching.
Duration:
23 d
Dose descriptor:
LOEC
Effect conc.:
<= 49.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
other: hatching success
Remarks on result:
other: 35% effect at LOEC level
Reported statistics and error estimates:
The proportion of eggs that successfully hatched and/or the proportion of deformed larvae were compared across levels in each experiment with one-way ANOVAs for each species, followed by Tukey multiple comparison tests if a significant effect was found. All proportional data were arcsine-transformed prior to analysis. Multiple comparisons were corrected with the sequential Bonferroni Type I error rate adjustment (Rice 1989). In this method, the p values from each of K comparisons are ranked in increasing order and compared to critical p values of 0.05/K, 0.05/K-1, 0.0K-2, and so on. If the lowest p value is lower than 0.05/K it is considered significant, and the next lowest p value is compared to 0.05/K-1. This continues until Pi >= 0.05/K-i when that and all subsequent p values are considered nonsignificant. All statistical tests utilized Minitab V.10 statistical software (Minitab 1994).

Description of key information

30-d exposure to boric acid at concentrations of 0, 2.5, 5.0, 7.5, and 10.0 mg boron (B)/L as added boron did not induce reproductive or developmental (F1) toxicity in Xenopus laevis at the concentrations selected. Thus, the NOAEC determined for reproduction and development (F1) was 10.0 mg B/L (or 9.7 mg B/L expressed as a 30-d time weighted average exposure concentration.

The NOEC was ≥ 100.2 mg B/L for Rana sylvatica, exposed to concentrations of nominal 0, 50 and 100 mg B/L (disodium tetraborate, borax) for 23 days.

Bufo fowleri, 7.5 d, Boric acid, nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L, result: LC10 55.3 mg B/L

Rana pipens, 7.5 d, Borax, nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L, result: 21.8 mg B/L

Rana pipens, Boric acid, 7.5 d, nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L, result: LC10 76.8 mg B/L

Bufo americanus, Disodium tetraborate, 23 d, nominal concentrations : 0, 50 and 100 mg B/L, result: ≤ 49.5 mg B/L

Ambystoma maculatum, Disodium tetraborate, 44 d, nominal concentrations : 0, 50 and 100 mg B/L, result: 24.8 mg B/L (larval deformation), NOEC ≥ 100.2 mg B/L (hatching)

Ambystoma jeffersonianum, Disodium tetraborate, 25 d, nominal concentrations : 0, 50 and 100 mg B/L, result: NOEC ≥ 100.2 mg B/L (hatching)

Additional information