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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 2 to 5 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ASTM E 1440-91
Deviations:
yes
Remarks:
(Test duration extended to measure reproductive endpoints)
Principles of method if other than guideline:
The test protocol that follows is based on American Society of Testing and Materials E 1440-91 with modifications from the research publication “A 2-d Life Cycle Test with the Rotifer Brachionus calyciflorus” by Snell and Moffat (Env Toxicol Chem 11: 1249-1257 (1992)).
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sigma Aldrich Boric Acid, lot 079K0115 and lot 010m0008, 99.9% purity

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of boron in test solutions was measured in composite test solution samples collected at 0 and 72 hours of the definitive test. Control and boric acid-fortified samples were also prepared for analysis at each sample period. Ten-milliliter samples were collected from the control and test substance treatment at 0 and 72 hours. Samples were diluted with reagent water to provide final sample concentrations within the analytical standard concentrations range. QC samples were prepared by fortifying laboratory saltwater with boric acid at concentrations of 5.05 and 116 mg B/L. All samples were directly analyzed using an ICP-MS system.

Test solutions

Vehicle:
no
Details on test solutions:
A 0.572 mg boric acid/mL primary stock solution was prepared at test initiation by weighing 0.5727 g of boric acid, adjusting for purity (99.9%) and diluting to a volume of 1,000 mL with dilution water.

Test organisms

Test organisms (species):
other: Brachionus calyciflorus (Rotifera)
Details on test organisms:
Brachionus calyciflorus cysts were obtained from Aquatic Eco-Systems, Apopka, Florida. Rotifer cysts were placed into Petri dishes with approximately 10 mL of warm blended freshwater. The Petri dishes were placed in an environmental chamber set at 25°C under full light for 16-20 hours. Cultures were monitored to ensure collection of larval rotifers hatched within approximately two hours of each other. Rotifers were fed an algal suspension of Nannochloropsis sp. during the test, prepared along with test concentrations at study initiation. The larvae were considered acceptable with no signs of abnormal appearance or behavior, high mortality or excessive delay in hatching. Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.

Rotifers were fed an algal suspension prepared for the control and each test substance treatment. The suspension contained approximately 3 x 106 Nannochloropsis cells/mL.

Six rotifers were added to test chambers for the dilution water control and each test substance treatment. Rotifers were impartially added to a set of labeled containers with each container representing one treatment. One individual was added to each labeled container starting with the control then proceeding from the low to high test substance treatments. The individuals within each container were then transferred from each container into the corresponding test chamber using a pipet. Observations of live and dead rotifers, and rotifers with eggs were made after 72 hours.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
150 mg CaCO3/L
Test temperature:
24.6 - 24.9
pH:
7.6 - 8.2
Dissolved oxygen:
7.0 - 7.5 mg/L (89 - 94% saturation)
Nominal and measured concentrations:
Nominal Test Concentrations: 0 (control), 6.5, 13, 25, 50, and 100 mg B/L
Mean Measured Concentrations as Total Boron: 0.775 (dilution-water control), 7.13, 14.2, 25.4, 51.7, and 108 mg B/L
Mean Measured Concentrations as Added Boron: 0 (dilution-water control), 6.35, 13.4, 24.6, 51.0, and 107 mg B/L
Details on test conditions:
Brachionus calyciflorus cysts were obtained from Aquatic Eco-Systems, Apopka, Florida. Rotifer cysts were placed into Petri dishes with approximately 10 mL of warm blended freshwater. The Petri dishes were placed in an environmental chamber set at 25°C under full light for 16-20 hours. Cultures were monitored to ensure collection of larval rotifers hatched within approximately two hours of each other. Rotifers were fed an algal suspension of Nannochloropsis sp. during the test, prepared along with test concentrations at study initiation. The larvae were considered acceptable with no signs of abnormal appearance or behavior, high mortality or excessive delay in hatching. Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.

The definitive and range-finding tests were conducted in 10-mL glass test tubes measuring 100 mm in height by 15 mm in diameter. The tubes contained 8 mL of control or test substance solution and were capped tightly with screw caps. The test tubes were placed in a rotating plastic bottle revolving at approximately 2 rpm. Tests were performed without light to prevent additional algal growth.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
24.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
24.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
other: reproduction
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
other: reproduction
Remarks on result:
other: added boron
Details on results:
The nominal concentrations were used in the effect concentrations table, following the usual practice that a nominal concentration may be used if the measured values are reasonably close to the nominal.
Observations of live and dead rotifers, and rotifers with eggs were made after 72 hours. After 72 hours, the mean number of surviving rotifers was 21.2, 14.2, 21.4, 20.2, 7.2, and 5.0 live rotifers in the 0 (control), 6.5, 13, 25, 50, and 100 mg B/L treatments, respectively. One dead rotifer was observed in the 6.5, 13 and 50 mg B/L test substance treatments and four dead rotifers were observed in the 25 and 100 mg B/L treatments. Rotifers with eggs were observed in all replicates of all test substance treatments except replicates A and B of the 6.5 mg B/L treatment. After 72 hours, the mean number of rotifer reproduction was 15.2, 8.2, 15.4, 17.3, 2.0, and 0 rotifers in the 0 (control), 6.5, 13, 25, 50, and 100 mg B/L treatments, respectively. Based on mean measured total boron concentrations, the EC50 for total survival was estimated to be 48.3 mg B/L. Based on mean measured total boron concentrations, the EC50 for reproduction was estimated to be 50.0 mg B/L. The slope of the 72-hour concentration-response could not be calculated. The nominal NOEC and LOEC for total survival and reproduction was 25 and 50 mg B/L, respectively.
Reported statistics and error estimates:
All statistical analyses were performed using SAS software (SAS version 9.1 for Windows). Inferences of statistical significance were based upon a p = 0.05 unless otherwise noted.
The no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) for mortality/immobility data were determined by using a one-tailed Dunnett’s test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The nominal NOEC and LOEC for both survival and reproduction were 25 and 50 mg B/L, respectively.Based on mean measured total boron concentrations, the 72-hour EC50 for survival and reproduction were estimated to be 48.3 mg B/L and 50.0 mg B/L, respectivly (95% confidence limits could not be determined). The slope of the 72-hour concentration-response could not be calculated.
Based on the added measured concentrations the NOEC and LOEC for survival and reproduction were 24.6 and 51.0 mg B/L, respectively.