Triboron trimethyl hexaoxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 203

Qualifier:

according to guideline

Guideline:

other: ASTM E729-96

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The concentration of boron in test solutions was measured in samples collected at 0 and 96 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 and 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 10.1 and 232 mg B/L. All samples were directly analyzed using an ICP-MS system.

Details on test solutions:

A 1.14 mg boric acid/mL primary stock solution (equivalent to 200 mg B/L) was prepared at test initiation by diluting 20.5200 g of boric acid into an 18,000 mL volume of dilution water.

Test organisms (species):

other: Litopenaeus vannemei

Details on test organisms:

Marine shrimp (Litopenaeus vannemei) were received from Shrimp Improvement Systems (Plantation Key, Florida) on June 4, 2010 and maintained at ABC Laboratories, Inc. The initial supplier provided the species identification. The shrimp were at growth stage PL-8 (post larval) upon receipt. The shrimp were maintained in artificial saltwater at a temperature of approximately 23 to 28°C. The shrimp were gradually acclimated from an initial approximate 35‰ saltwater salinity to a target saltwater salinity of 20 ± 2‰ over a period of 11 days. The animals were fed shrimp food and brine shrimp nauplii (Artemia sp. <48 hours old) during the holding period. Periodic lots of feed were screened for contaminants and records of feed suppliers and the results of the contaminant analyses are on file at ABC Laboratories, Inc. A record of shrimp observations and during the culture period was maintained. No mortalities were recorded during the holding period.
Shrimp used for the definitive test were collected on July 9, 2010. The shrimp were at PL-44 (post larval) stage. Shrimp were fed ad libitum newly hatched brine shrimp (Artemia sp.; approximately 24-48 hours old) nauplii with at least two feedings daily. All treatments and controls received approximately the same ration (i.e., volume) of food. The control shrimp were measured at termination and ranged from 14 to 19 mm in length (mean and standard deviation = 17 ± 1.5 mm) and from 0.0357 to 0.1013 g in blotted wet weight (mean and standard deviation = 0.0767 ± 0.0194g).

During the test shrimp were offered brine shrimp ad libitum daily to reduce predation.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.8 to 25.1°C

pH:

7.4 to 8.0

Dissolved oxygen:

5.7 to 7.4 mg/L (76 to 99% saturation)

Salinity:

19.0 to 19.9‰

Nominal and measured concentrations:

Nominal Concentrations: 0 (control), 13, 25, 50, 100, and 200 mg B/L
Mean Measured Concentrations as added Boron: 0 (dilution-water control), 14.0, 27.2, 55.8, 103, and 215 mg B/L
Mean Measured Concentrations as Total Boron: 2.23 (dilution-water control), 16.2, 29.5, 58.0, 105, and 217 mg B/L

Details on test conditions:

The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marinemix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater at a target salinity 20 ± 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.

The test chambers utilized during the range-finding test were 1-L glass beakers containing approximately 800 mL of control or test solution. In the definitive test, the test chambers were 10-L glass jars containing approximately 8 L of control or test solution. Animal size during the definitive test required larger test chambers to avoid biological loading problems. The test chambers in both tests were covered with plastic Petri dishes. The test chambers were maintained at 21 to 25°C and within a range of 2°C in a temperature-controlled water bath. Fluorescent lighting was maintained on a 16-hour light and 8-hour dark photoperiod with 30-minute simulated dawn and dusk periods. The measured light intensity at the termination of the definitive test ranged from 658 to 710 lux as measured with a LI-COR Model LI-189 light meter equipped with a photometric sensor.

Ten shrimp were added to test chambers for the dilution water control and each test substance treatment. Shrimp 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 released from each container into the corresponding test chamber. Observations were made daily (± 1 hour of test initiation) for mortality and sublethal effects. The initial loading rate was 0.0959 grams of shrimp per liter of test solution which meets the loading rate criterion.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

130 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

boron

Basis for effect:

mortality

Remarks on result:

other: added boron (110 - 154) (Salinity: 19.0 to 19.9‰)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

103 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

boron

Basis for effect:

mortality

Remarks on result:

other: added boron (Salinity: 19.0 to 19.9‰)

Details on results:

After 96 hours of exposure, mortality was 0, 0, 0, 0, 20, and 100% in the 0 (dilution-water control), 13, 25, 50, 100, and 200 mg B/L nominal treatments. Based on mean measured total boron concentrations, the 24- and 48-hour LC50 was estimated to be >217 mg B/L, the highest concentration tested, as determined by the trimmed Spearman-Karber method. The 72-hour LC10 and LC50 values, based on mean measured total boron concentrations, were estimated to be 106 mg B/L (95% confidence limits of 50.5 and 136 mg B/L) and 163 mg B/L (95% confidence limits of 124 and 221 mg B/L), respectively, as determined by the probit method. The 96-hour LC50, based on mean measured total boron concentrations, was estimated to be 132 mg B/L with 95% confidence limits of 112 and 156 mg B/L as determined by the untrimmed Spearman-Karber method. The slope of the 96-hour concentration-response could not be calculated. There were no sublethal effects noted in the control or test substance treatments during the definitive test. The 96-hour NOEC and LOEC values were 105 and 217 mg B/L, respectively, based on total measured boron concentrations.

Reported statistics and error estimates:

All statistical analyses were performed with SAS software. Estimates of LC50 values and their 95% confidence limits were calculated using the probit method and Spearman-Karber method (trimmed or untrimmed). When the P value for Goodness of Fit was >0.05 and there was no other evidence of questionable convergence, the probit method was selected for reporting. When this criterion was not achieved, the Spearman-Karber method was selected for reporting. The NOEC was determined by using a Fisher’s exact test. A Hochberg adjustment was used to control the experiment wise error rate for the Fisher’s test at the same alpha level.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured total boron concentrations, the 24- and 48-hour LC50 was estimated to be >217 mg B/L, the highest concentration tested. The 72-hour LC10 and LC50 values, based on mean measured total boron concentrations, were estimated to be 106 mg B/L (95% confidence limits of 50.5 and 136mgB/L) and 163 mg B/L (95% confidence limits of 124 and 221 mg B/L), respectively. The 96-hour LC50, based on mean measured total boron concentrations, was estimated to be 132 mg B/L with 95% confidence limits of 112 and 156 mg B/L. The slope of the 96-hour concentration-response could not be calculated. There were no sublethal effects noted in the control or test substance treatments during the definitive test. The 96-hour NOEC and LOEC values were 105 and 217 mg B/L, respectively, based on nominal concentrations. The 96-hour NOEC and LOEC values were 103 and 215 mg B/L, respectively, based on added concentrations.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, limited documentation, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Semi-static acute toxicity test with Mytilus

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Sampling method: density reading on refractometer

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Narragansett Bay seawater was obtained via the in-house sand filtration system and was re-filtered through a 10 µm Aqua Pure filter (Cuno, Inc., Connecticut).

Test organisms (species):

other aquatic mollusc: Mytilus edulis

Details on test organisms:

TEST ORGANISM
- Common name: blue mussel
- Source: Narragansett Bay in early January of 1993 using a scallop dredge.
- Length at study initiation (length definition, mean, range and SD): 5-7 cm


ACCLIMATION
- cultured at United States Environmental Protection Agency - Atlantic Ecology Division Laboratory, Narragansett, Rhode Island (USEPA, formerly USEPA-ERLN) and placed in a 760 L flow-through seawater holding tank
- Acclimation conditions (same as test or not): Unfiltered seawater was pumped into the tank and was maintained at 3°C, ambient field temperature. After being held in the tank for 1 week, approximately 200 mussels were taken out of the flow-through tank and placed in a flow-through acclimation table. The water temperature was raised every 3 d by 2°C until the mussels were acclimated to 15°C.
- Acclimation period: After the mussels had been maintained in 15°C water tor 3 d, they were removed and placed in the dosing system
- Type and amount of food: unicellular algae, T-Iso, a strain of lsochrysis galbana

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

15 ± 0.5°C

pH:

7.5 ± 0.2 (0% Methanol), 7.7 ± 0.1 (1%), 7.6 ± 0.05 (2%), 7.8 ± 0.1 (3%), 8.1 ± 0.1 (5%), 8.2 ± 0.01 (10%)

Dissolved oxygen:

7.3 ± 0.2 (0% Methanol), 7.0 ± 0.6 (1%), 7.3 ± 0.2 (2%), 7.3 ± 0.4 (3%), 7.3 ± 0.01 (5%), 7.3 ± 0.01 (10%)

Salinity:

29 - 31 g/kg

Nominal and measured concentrations:

0%, 1%, 2%, 3 %, 5 % and 10% methanol/seawater (v/v) (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1-L glass beakers
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): The test system was contained in a temperature controlled water bath and consisted of a diluter, algae pump, solvent pump, 6 carboys containing the test solutions for the six treatments, 1 carboy containing algae solution, eighteen 1-L glass beakers, and a timer that controlled the pumps (Nelson 1990). Every hour, 750 mL of test solution (pre-mixed) and 150 mL of algae were transferred to each respective chamber of the diluter. In each diluter chamber, the test solution was split three ways, delivering 250 mL of test solution and 50 mL of algae solution to each of three replicate beakers per treatment. This entire process occurred over a period of 3 min. Over a 24-h period, the total volume of each beaker was replaced six times. The solutions were prepared daily; the temperature of the water bath was maintained at 15 ± 0.5°C.
- No. of organisms per vessel: 6
- No. of vessels per concentration (replicates): 3

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

15 900 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

7 960 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

In summary, an LC50 of 15900 mg/L of methanol, equivalent to 2% (v/v), was determined. Sublethal effects (e.g. narcosis) were observed for exposure concentrations of 2% and 3% methanol. Data from the literature suggest that some sublethal effects may be reversed if methanol is removed from the system. In marine ecosystems, exposure to extremely high concentrations (> 5%) for 12 hours or more is lethal to Mytilus edulis. Mussels exposed to 3% methanol survived for greater than 72 hours, and those exposed to 1% exhibited no adverse effects during the entire 96 hr exposure period.

Reported statistics and error estimates:

Two statistical methods were used to determine an LC5O for the binomial distribution. With the Trimmed Spearman-Karber method (TSK) (Hammilton et al. 1977), an α value of 0.05 is generally chosen as the amount of mortality data to be trimmed from the ends of the tolerance distribution. In this experiment, all mortality fell within 95% of the central mortality distribution and the data were not trimmed. In applying the TSK method, the
mortality data from each respective treatment is pooled, thus variability between replicates is not considered. In contrast, the Inhibition Concentration Percentage method (ICP) (Norberg-King 1988) accounts for the variability between replicates when determining an LC-50.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

not specified

Remarks:

The study is a publication with no certain GLP status

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

CULTURING
- Culturing: single clone Daphnia magna (clone K6) cultures were held in 1 L glass recipients filled with aerated and biologically filtered tap water, each containing 20 - 25 individuals.
- Conditions: the temperature in the temperature and light controlled chamber (Type WT150'/+5DU-WB, Weiss Technik, Liedekerke, Belgium) was maintained at 20 ± 1 °C with a photoperiod of 14 h light/10 h dark throughout culturing and exposure experiments.
- Water renewal: three times weekly
- Feeding: the daphnids were fed a mixture of P. subcapitata and Chlamydomonas reinhardtii in a 3/1 ratio (4 x 10E5 cells/ml)

- Age of daphnids at test initiation: < 24 h

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

initial water hardness: 250 mg CaCO3

Test temperature:

20 ± 1 °C

pH:

initial pH: 7.8-8.2

Nominal and measured concentrations:

- Nominal concentrations (linear 1/2 dilution series): 1 - 32 g/L

Details on test conditions:

TEST SYSTEM
- Test vessels: glass recipients
- Test volume: 50 ml
- No of animals/replicate: 10
- No of replicates/concentration: 3
- Test medium: reconsituted water
- Test media renewal: every 24 h

TEST MEDIUM
- Medium: reconstituted water

TEST CONDITIONS
- Feeding: daphnids were fed the same algae mixture as during culturing
- Photoperiod: 14 h light/ 10 h dark

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

18 260 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: ASTM E729-95 Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1020 (Gammarid Acute Toxicity Test)

GLP compliance:

no

Remarks:

results obtained from a peer-reviewed publication

Analytical monitoring:

yes

Details on sampling:

Each treatment was sampled at beginning and end of test

Vehicle:

no

Test organisms (species):

other aquatic crustacea: Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Common name: Hyalella, scud, amphipod
- Source: In-house culture
- Age at study initiation (mean and range, SD): 7 to 14 d
- Feeding during test: none
- Food type:
- Acclimation: 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

103 mg/L

Test temperature:

21.0 °C

pH:

8.4

Dissolved oxygen:

> 6.0 mg/L

Nominal and measured concentrations:

Nominal: Control, 25, 50, 100, 200, 400 mg-B/L
Measured: <0.027, 27, 53, 100, 205, 395 mg-B/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml beaker
- Type: open
- Material, size, headspace, fill volume: glass, 40 ml
- Aeration: none
- Renewal rate of test solution: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RMHRW (reconstituted moderately hard reconstituted water, per Smith et al. 1997. Modified to make pH = 8.5 (increased amount of NaHCO3 to 398 mg/L rather than 96 mg/L like standard RMHRW)
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine: 0 nominal
- Ca/mg ratio: 5.40 (mass:mass)
- Conductivity:
- Salinity:
- Culture medium different from test medium: Cultured in RMHRW, acclimated 3-4 days in test water
- Intervals of water quality measurement: boron, pH, conductivity, alkalinity, hardness at beginning and end of test, temperature and D.O. daily

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 L: 8 D

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality (daily)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: (95% CI 41-101 mg-B/L)

Reported statistics and error estimates:

LC50 calculated using trimmed Spearman-Karber method

Data

Concentration (mg B/L)	# dead (out of 20)	Percent
<0.027 (control)	2	10.0%
27	5	25.0%
53	9	45.0%
100	13	65.0%
205	15	75.0%
395	20	100.0%

Description of key information

TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found folllowing release to the environment. Therefore, an assessment of ecotoxicological potential was conducted taking account of the effects of the hydrolysis breakdown products of TMBX upon the different trophic levels.

The 96 hour toxicity of methanol to daphnia spp. was established in a semi-static system (Dom et al 2012), the EC50 was determined to be 18260 mg/L (nominal) based on mobility.

The acute aquatic toxicity of borates to freshwater invertebrates was examined across a variety of species and water conditions by Soucek et al 2010. The most sensitive species was Hyalella azteca under test conditions of 103mg/L hardness and pH8.4. The calculated LC50 (geometric mean) at 96hrs was 64 mg/L (95% CI 41-101 mg/L) expressed as elemental Boron.

The 96 hour toxicity of methanol to blue mussels (Mytilus edulis) was established in a semi-static system (Helmstetter et al, 1996), the EC50 was determined to be 15900 mg/L (nominal) based on mortality and the NOEC established at 7960 mg/L.

The acute (96hr) aquatic toxicity to marine invertebrates (Marine shrimp; Litopenaeus vannemei) was determined in a static system (Bergfield et al 2011). The 96-hour LC50, based on mean measured total boron concentrations, was estimated to be 132 mg B/L with 95% confidence limits of 112 and 156 mg B/L; The 96-hour NOEC and LOEC values were 105 and 217 mg B/L, respectively, based on total measured boron concentrations.

Based on the above results, the LC50 values for boron were used to calculate equivalent values for TMBX:

Freshwater 96 hr L(E)C50 = 64 x (173.5/32.4) = 342.7 mg/L

Seawater 96 hr L(E)C50 = 132 x (173.5/32.4) = 706.9 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

342.7 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

706.9 mg/L