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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:
June 16, 2010 to July 14, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)
Qualifier:
according to
Guideline:
other: ASTM E1191, and U.S. EPA FIFRA Subdivision E, Section 72-4
Principles of method if other than guideline:
salinity 8‰
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 concentrations of boron in test solutions were determined in samples collected seven days prior to initiation (day –7) and on study days 0, 7, 14, 21, and 28 (termination) of the definitive test. A single replicate sample was collected from the dilution-water control, and test-substance treatments; alternating replicates were sampled at each sampling event. The concentration of boron in the diluter stock solutions were determined in samples collected on the same days as the test solutions.

A 10-mL sample was collected from the dilution-water control, each test-substance treatment, and from the diluter stock solution. QC samples were prepared by fortifying water collected from the dilution-water control with boric acid at concentrations of 1.82 and 36.0 mg B/L. From each 10-mL sample, a 0.050-mL volume was removed from all but the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.020-mL volume was removed from each 10-mL sample of the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.025-mL volume of the diluted-diluter stock solution sample was further diluted with ABC reagent water to a volume of 10 mL. The standard curve range for these analyses was 0.000 to 0.800 mg B/L.

Test solutions

Vehicle:
no
Details on test solutions:
Diluter stock solutions were prepared at a target concentration of 6,140 mg B/L by diluting approximately 35.106 g (6.175 g as boron corrected for boron content in boric acid) of boric acid per liter of saltwater. Diluter stock solutions volumes of 4, 5, or 18 L were prepared in this manner on June 1, 2, 17, and 23 as well as July 2, 7 and 12, 2010. All diluter stock solutions were stored at room temperature and shielded from light.

Test organisms

Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
Mysid shrimp (Americamysis bahia) were obtained from in-house cultures that were maintained at ABC Laboratories, Inc. The initial parental mysids were obtained from Aquatic Bio Systems Inc. on October 29, 2008, and May 27, 2010. The organisms from Aquatic Bio Systems Inc. were received with documentation stating them to be Americamysis bahia. The adult mysids used to generate the juvenile test organisms were cultured in artificial saltwater with salinity of approximately 8‰ and at a temperature of approximately 25°C. The animals were fed brine shrimp nauplii (Artemia sp. <48 hours old). Periodic lots of feed were screened for contaminants. Records of feed suppliers and the results of the contaminant analyses are on file at ABC Laboratories, Inc. A record of mysid observations and juvenile production numbers during the culture period was maintained.

Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24-48 hours old) at least two times daily. The concentration of the nauplii in the food stock was determined by replicate counts of aliquots pipetted onto filter paper. The food stock concentration ranged from 280 to 920 brine shrimp/mL (mean ± standard deviation [SD]: 618 ± 157 brine shrimp/mL). The food stock was enriched the day of use with a mixture of Salt Creek HUFA Enrich, AlgaMac-3000, and AlgaMac-Enhance, all of which are commercially available components. All control- and test-substance treatments received, as near as reasonably possible, the same ration (i.e., volume) of food. The enrichment mixture was prepared by diluting 1 mL of Salt Creek HUFA Enrich and 1/8 teaspoon of an approximate 40:60 AlgaMac-3000:AlgaMac Enhance mixture in a household blender with 200 mL saltwater and then blended for approximately one minute. The enrichment mixture was then added to the food stock at the rate of 50 mL/L of food stock.

The body lengths of mysids (as measured by total midline body length) were measured to the nearest 0.1 mm with a dissecting microscope.



Study design

Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d

Test conditions

Test temperature:
24.1 to 25.6°C
pH:
7.68 to 8.11
Dissolved oxygen:
5.61 to 7.97 mg/L (74 to 102% saturation)
Salinity:
7.6 to 8.4 ‰
Nominal and measured concentrations:
The testing was performed at nominal concentrations of 0 (dilution-water control), 2.0, 4.0, 8.0, 16, and 32 mg B/L.
Mean Measured Concentrations as Total Boron: 1.39 (dilution water control), 3.46, 5.58, 9.63, 18.1, and 33.0 mg B/L
Mean Measured Concentrations as Added Boron: 0.0 (dilution water control), 2.00, 4.25, 8.16, 16.6, and 31.6 mg B/L
Details on test conditions:
The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marine Mix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater at a target salinity 8 ± 2‰. The laboratory freshwater consists of well water blended with well water that was demineralized by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO3/L. The dilution water was heated and aerated prior to its delivery into the diluter system. As the dilution water entered the diluter system, it passed through a particulate filter and an ultraviolet sterilizer.

During the definitive test, test solutions were delivered to three replicate test chambers and each replicate test chamber received approximately 0.67 L at each diluter cycle. The accuracy of the delivery of the test solution to the test chambers was verified by volumetric measurement before test initiation and adjusted such that the delivered volume was ±10% of the target volume.

Each test chamber used for the definitive test measured approximately 22 cm wide, 39 cm long, and 22 cm high. Each definitive-test chamber had a glass pane in the middle of the tank containing two holes near the bottom of the tank. This partition effectively cut the test chamber volume in half when the holes were stopped with silicone stoppers. The greatest depth of the test solution was approximately 12 cm, which yielded a maximum volume of approximately 10 L when one side was in use and approximately 20 L when both sides of the test chamber were in use. All test chambers were individually drained through the side of the chamber to a floor drain.

Mysids were maintained within retention baskets (constructed from nylon screening) to facilitate daily observations and enumeration. Because retention baskets were not completely isolated from one another, these retention baskets were not replicates. Only the test chamber containing the retention baskets was a true replicate.

Observations of mortality and sublethal responses were made daily for the duration of the testing period. Dead mysids were counted, differentiated by gender (if mature), and removed daily. Any missing mysids at the time of observation were noted as not found and were considered dead if not observed on following days; that mortality was subsequently reflected back to the time that the mysid was first observed missing. Any mysids that were inadvertently damaged or died as a result of becoming impinged onto the retention- or brood-basket mesh were removed from the initial number of mysids for those replicates and were not considered to be treatment-related mortality.

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
F0 Mysid Survival
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Male Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Female Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Female Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
F1 Mysid Survival
Remarks on result:
other: added boron
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The test acceptability criteria for this study were met. The test acceptability criteria for this study were met.
Conclusions:
The test acceptability criteria for this study were met. The water-quality characteristics remained within the tolerance limits set forth in the protocol. Survival of the dilution-water control F0 mysids was >90%. The percentage of dilution-water control F0-female mysids available to produce young that actually did produce a brood was 100%. The average total number of young produced per dilution-water control F0-female mysid was 16.0.
Based on mean total measured concentrations of boron during the 28-day exposure, the day-28 F0-mysid survival NOEC was 33.0 mg B/L, the highest boron concentration tested. The NOEC for F1-mysid survival on day 9 was 33.0 mg B/L. The 14-day F0-male mysid length was the most sensitive statistically significant endpoint and the NOEC for this parameter was 9.63 mg B/L. An EC10 could be computed for only one endpoint, mean total young per F0-female mysid, and was 16.0 mg B/L.
If the endpoints are stated based on added boron (total boron minus measured concentration natural boron in the control, Table 4), then the NOECadded born for the most sensitive endpoint was 8.16 mg Badded/L for the 14-day F0-female mysid length. The NOEC for all other endpoints, except for day-28 F0-female mysid length and mean total young per F0 female mysid, was 31.6 mg Badded/L. The NOEC for day-28 F0-female mysid length and mean total young per F0-female mysid was 16.6 mg Badded/L.