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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 12, 2016 to September 15, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test Material Name: Stannous Octoate
Chemical Name: 2-Ethylhexanoic acid, Tin(2+) Salt (2:1)
Synonyms: Stannous Octoate S-26, Tin bis(2-ethylhexanoate)
Lot/Reference/Batch Number: A072E2N001
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 96.9% by gas chromatography with identification by nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy (Zhang, 2016).
Test Material Stability Under Storage Conditions: Stannous octoate was determined to be stable for 2 weeks at 55°C which is equivalent to 24 months under ambient storage conditions (Schoeb and Linehan, 2011).
Analytical monitoring:
yes
Details on sampling:
Analytical Measurements:
To confirm stannous octoate concentrations, the test solutions were analyzed for a dissociation product of the test material, 2-ethylhexanoic acid. Samples were collected at exposure initiation from the bulk test solutions and at termination from pooled replicate test solutions. The samples were analyzed by gas chromatography with electron impact ionization mass spectrometry (GC-EI-MS) detection in the multiple reaction ion monitoring (MRM) mode. All test solutions were analyzed within 24 hours of preparation; therefore, no stability assessment was required.
Vehicle:
yes
Remarks:
laboratory dilution water (LDW)
Details on test solutions:
Preparation of Test Solution:
Stannous octoate is expected to dissociate to 2-ethylhexanoic acid and tin ion (Sn2+) in LDW. Based on preliminary non-GLP work, 2-ethylhexanoic acid was expected to be soluble in LDW at the selected concentration for the definitive study. Observations of undissolved particulates during preliminary work and over the course of the definitive study were most likely due to the oxidation and precipitation of the tin ion as tin oxides.
The test solution was prepared via direct addition of the test material to LDW to create a bulk test solution. An aliquot containing 100 mg stannous octoate was added to a 1 L volumetric flask filled to volume with LDW. The flask was shaken and placed into a water bath sonicator for approximately 30 minutes. Following sonication, the 100 mg/L bulk mixture was slightly cloudy white with white particulates at the bottom and surface of the flask. Approximately 350 mL of the 100 mg/L bulk mixture was removed, avoiding visible white particulates, from the volumetric flask and was used as the 100 mg/L bulk test solution. Portions of this bulk solution were poured into individual test vessels. The bulk test solution was slightly cloudy white at test initiation.
Following 24 and 48 hours of exposure, the 100 mg/L test solutions were clear and colorless with fine white particulates at the bottom of the test vessels. The water control was clear and colorless at test initiation and over the course of the exposure period.
Test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solution was not required. The dispersal of the test material in the surrounding medium was considered to represent the most probable route of exposure in the environment.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test Organism:
The test organism was the freshwater daphnid, Daphnia magna, from in-house cultures initially obtained from Aquatic BioSystems, Inc. Fort Collins, Colorado. This species is widely accepted and recommended for toxicity testing by the test guidelines. Daphnid neonates less than 24 hours old were used to initiate the study.
Neonates were only used from daphnids which have had at least 3 broods. The day before neonates were needed for testing (<24 hours prior to test initiation), adults within the culture vessels were removed and transferred to new culture vessels. On the following day, neonates produced overnight in these culture vessels were removed and placed into a glass beaker containing LDW. These neonates (<24 hours old) were fed and held in an incubator until used for the study.

Typical culture conditions:
Test Species: Daphnia magna
Temperature: 20 ± 2°C
Photoperiod: 16-hour light/8-hour dark
Water: Lab dilution water
Culture Vessel: 2.9-L glass jars
Culture Vessel Cover: Plastic wrap
Culture Vessel Change-Out: At least 3 times weekly
Feed: Pseudokirchneriella subcapitata, a freshwater green alga (formerly known as Selenastrum capricornutum) and YTC (yeast, wheat grass, Zeigler Finfish starter #1) typically five times weekly
Test type:
static
Water media type:
other: laboratory dilution water (LDW)
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Daphnids were observed for immobility (inability to swim within approximately 15 seconds after gentle agitation of the test vessel) at approximately 24 and 48 hours of exposure (± 1 hour from exposure initiation). Any sublethal effects (abnormal behavior or appearance) were recorded.
Hardness:
Control Water (LDW) Hardness (mg/L CaCO3) on Day 0: 74
High Test Level (100 mg stannous octoate/L) Hardness (mg/L CaCO3) on Day 0: 76
Test temperature:
Temperature ranged from 19-20 °C.
pH:
pH ranged from 6.7-7.7
Dissolved oxygen:
Dissolved oxygen levels ranged from 8.6-8.7 mg/L (97-98 % air saturation)
Conductivity:
Control Water (LDW) Conductivity (μS/cm) on Day 0: 201
High Test Level (100 mg stannous octoate/L) Conductivity (μS/cm) on Day 0: 194
Nominal and measured concentrations:
Based on information from the range-finding test, a single nominal concentration of 100 mg stannous octoate/L, prepared as a single bulk solution and dispensed into four replicate exposure vessels, was used in the definitive study (limit test). Four replicate test vessels of LDW served as the water control.
Details on test conditions:
Culture and Test Water:
The water used for daphnid testing and culturing (referred to as laboratory dilution water or LDW) was Lake Huron water supplied to The Dow Chemical Company (TERC Laboratory) by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO2, carbon-filtered, and UV-irradiated. The water is typically monitored weekly for pH, alkalinity, hardness and conductivity. Periodically, the water is monitored for total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds. Results from the most recent analyses of the dilution water indicated that TOC was 1.9 mg/L, TSS was < 3 mg/L, and concentrations of contaminants were below levels of concern that may interfere with the results of this test.

Test Vessels:
The test vessels were 50-mL glass beakers each containing approximately 40 mL of control or test solution and covered to reduce evaporation. Each test vessel was uniquely labeled for identification purposes.

Definitive Test:
The definitive test was conducted under static conditions for approximately 48 hours from 12 to 14 April 2016. Four replicate test vessels, each containing approximately 40 mL of the appropriate test solution, were prepared for both the 100 mg/L test level and water control. Twenty daphnids (five daphnids per replicate) were impartially added to each test vessel within 30 minutes of completion of test solution preparations. The target test temperature was 20 ± 2 °C, and did not vary by > 1º C over the course of the exposure. The photoperiod was set at 16 hours light/8 hours dark per day. Daphnids were not fed during the exposure period.
Daphnids were observed for immobility (inability to swim within approximately 15 seconds after gentle agitation of the test vessel) at approximately 24 and 48 hours of exposure (± 1 hour from exposure initiation). Any sublethal effects (abnormal behavior or appearance) were recorded.
Dissolved oxygen, pH, and temperature were recorded for each bulk test solution at test initiation and from all test vessels (spent test solutions) at test termination. Water temperature was continuously monitored with a minimum/maximum thermometer placed in a representative vessel. Light intensity was measured at test initiation. Water hardness, alkalinity, and conductivity were measured from the control water and the 100 mg/L bulk solution at test initiation.

Selection of Test Concentration:
A non-GLP 48-hour range-finding test was conducted from 15 to 17 March 2016. Two replicates of five daphnids per test level were exposed to nominal concentrations 0 (water control), 1, 10 and 100 mg stannous octoate/L, over a 48-hour static exposure period. An aliquot containing 100 mg stannous octoate was added to a 1 L volumetric flask filled to volume with LDW, creating a 100 mg/L primary stock solution. The primary stock was then placed into a water bath sonicator for approximately 30 minutes. Following sonication, the solution appeared slightly cloudy white with white particulates visible at the bottom of the flask. Approximately 350 mL of the 100 mg/L primary stock was removed, avoiding visible white particulates, from the volumetric flask and was used as the 100 mg/L bulk test solution. The remaining bulk solutions were prepared as dilutions of the primary stock solution. Observations for immobility and sublethal effects were made at approximately 24 and 48 hours after initiation. Water quality (i.e., pH, D.O., temperature) measured at test initiation and termination remained within recommended ranges during the test. Following 48 hours of exposure, no immobility or sublethal effects were observed in any of the test levels, including the water control.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Biological Data:
No immobility or sublethal effects (change in daphnid behavior or appearance) were observed in the water control or the 100 mg stannous octoate/L test solution over the course of the exposure.
Reported statistics and error estimates:
Statistical Analysis:
Due to the absence of any immobility observed in the 48-hour exposure, the 24- and 48-hour EC50 values (the concentrations estimated to immobilize 50 % of the daphnids after 24 and 48 hours of exposure, respectively) were empirically determined to be greater than the highest (limit) concentration tested. Furthermore, since there was also an absence of sublethal effects (changes in daphnid behavior or appearance) observed in the test, the NOEC was empirically determined to be equal to the highest (limit) concentration tested.

Analytical Results:

Measured concentrations of the test level ranged from 95.9 to 97.4 % of the nominal concentration over the course of the exposure period. The resulting mean measured concentration was 96.7 mg stannous octoate/L. None of the analyses of the controls exhibited a stannous octoate concentration exceeding the lower limit of quantitation (LLQ) equivalent to 28.6 mg stannous octoate/L. Because the mean measured concentration was within ± 20 % of the nominal target concentration, results were based on the nominal (limit) concentration.

Test Conditions:

Dissolved oxygen levels ranged from 8.6-8.7 mg/L (97-98 % air saturation), temperature ranged from 19-20 °C, and pH ranged from 6.7-7.7. Light intensity ranged from 516-568 lux.

Water hardness was 74 mg CaCO3/L, alkalinity was 48 mg CaCO3/L, and conductivity was 201 μS/cm at test initiation in the water control. In the highest test (limit) concentration, water hardness was 76 mg CaCO3/L, alkalinity was 46 mg CaCO3/L, and conductivity was 194 μS/cm at test initiation. Therefore, presence of the test material (dissociation products) did not appear to affect water quality parameters at the concentration tested.

Validity criteria fulfilled:
not specified
Conclusions:
The acute toxicity values for Daphnia magna exposed to stannous octoate, over a 48-hour static exposure period and based on the nominal (limit) concentration, were as follows:
• 24- and 48-hour EC50 values: > 100 mg/L (limit concentration tested)
• The 48-hour NOEC: 100 mg/L (limit concentration tested)
Executive summary:

The purpose of this study was to assess the potential effects of stannous octoate to the freshwater cladoceran, Daphnia magna. The study was conducted as a limit test, with four replicate vessels of five daphnids per test level, exposed to nominal test concentrations of 0 (water control) and 100 mg stannous octoate/L, over a 48-hour static exposure period. Observations were made at 24 and 48 hours (±1 hour from exposure initiation) for daphnid immobility (inability to swim within approximately 15 seconds after gentle agitation of the test container) and any changes in behavior or appearance. Temperatures during the exposure period ranged from 19-20 °C. Dissolved oxygen levels ranged from 8.6-8.7 mg/L (97-98% air saturation). The pH ranged from 6.7-7.7 and the light intensity ranged from 516-568 lux.

Stannous octoate is expected to dissociate and oxidize in water, giving two moles of 2-ethylhexanoic acid and one mole of tin ion (Sn2+) for each mole of stannous octoate added. To confirm stannous octoate exposure concentrations, the test solutions were analyzed for the dissociation product, 2-ethylhexanoic acid at test initiation and exposure termination. Samples were analyzed by gas chromatography with electron impact ionization mass spectrometry (GC-EI-MS) detection operating in the multiple reaction ion monitoring (MRM) mode. None of the analyses of the water control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 28.6 mg stannous octoate/L. Measured concentrations of the limit exposures ranged from 95.9 to

97.4 % of the nominal concentration over the course of the exposure period. The resulting mean measured concentration was 96.7 mg stannous octoate/L. Because the mean measured concentration was within ± 20 % of the nominal target concentration, results were based on the nominal (limit) concentration.

The acute toxicity values for the daphnid (D. magna) exposed to stannous octoate over a 48-hour static exposure period and based on nominal concentrations were as follows:

• 24- and 48-hour EC50 : > 100 mg/L (limit concentration tested)

• 48-hour NOEC: 100 mg/L (limit concentration tested)

Description of key information

 In an acute, static, toxicity study with Daphnia magna,the 48-hour EC50 value for tin bis (2-ethylhexanoate) was > 100 mg/L based on nominal concentrations. 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

One static acute toxicity test withDaphnia magnaconducted according to OECD 202 under GLPwas available for tin bis (2-ethylhexanoate). This study was determined to be acceptable for use in risk assessment (Klimisch score = 1). The study was conducted as a limit test, with four replicate vessels of five daphnids per test level, exposed to nominal test concentrations of 0 (water control) and 100 mg/L tin bis (2-ethylhexanoate). Measured concentrations of the exposures ranged from 95.9 to 97.4% of the nominal concentration over the course of the exposure period. Observations were made at 24 and 48 hours for daphnid immobility and any changes in behavior or appearance. The 48-hour EC50 value was reported as > 100 mg/L based on nominal concentrations. The 48-hour NOEC value was reported as 100 mg/L based on nominal concentrations.