Boric acid

Toxicity to other aquatic organisms

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid

EC Number:

233-139-2

EC Name:

Boric acid

Cas Number:

10043-35-3

Molecular formula:

H3BO3

IUPAC Name:

boric acid

Details on test material:

Test article boric acid (CAS No. 10043-35-3; Lot No. 9H03), obtained from the Sponsor, was > 99% pure, as confirmed by certificate of analysis.

Sampling and analysis

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).

Test solutions

Vehicle:

no

Details on test solutions:

A target stock solution (3,563 mg BA/L) was used to prepare each test concentration.

Test organisms

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.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Test conditions

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.

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Effect concentrations 3

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

Effect concentrations 4

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).

Applicant's summary and conclusion

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.