butanedioic acid, 2-octenyl

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This was a well conducted study compliant with the OECD guideline as well as good laboratory practices

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

To confirm octenylsuccinic acid concentrations and associated stability over the course of the exposure, the collected samples were analyzed by high performance liquid chromatography/mass spectrometry (HPLC/MS). All test solutions were analyzed within 24 hours of preparation; therefore, no stability assessment was required.

The bulk exposure solutions (LDW control 63, 125, 250, 500 and 1000 mg/L solutions) were sampled for analytical confirmation of test material. At test initiation (0 hours), single aliquots (~ 5 mL) of LDW control, 125, 250, and 500 mg/L bulk exposure solutions were collected using a glass pipette and transferred to 20 mL vials. At test initiation, four aliquots (~ 5 mL) of 63 and 1000 mg/L bulk exposure solutions were collected from the top, middle, and bottom of their container to evaluate homogeneity. At 48 hours and at exposure termination (96 hours), single aliquots (~ 5 mL) were collected from all test vessels using an Eppendorf pipette and transferred to 20 mL glass vials.

Samples were diluted to an acceptable analysis range with acetonitrile. These samples were analyzed by HPLC/MS-MS. The concentration of Octenylsuccinic acid were quantified by using external standard calibration, and reported concentrations were not corrected for purity of the test material.

Vehicle:

no

Details on test solutions:

At test initiation, a primary stock solution was prepared at a volume of 40 L with a target nominal concentration of 1000 mg octenylsuccinic acid/L (equivalent to the nominal high test concentration). An aliquot containing 40 g of octenylsuccinic acid (no adjustment for purity) was added directly to a glass carboy containing 40 L of LDW. The solution was stirred on a magnetic stir plate utilizing a Teflon® lined magnetic stir bar (~3.5 hours). While stirring, the pH of the stock solution was adjusted utilizing 5N sodium hydroxide (initial pH = 3.4; final pH = 8.0). Prior to pH adjustment, the stock solution appeared slightly cloudy/white. Following pH adjustment, the stock solution was clear and colorless.

The test solutions were prepared as dilutions of the 1000 mg/L stock solution. The solutions were stirred with a glass stirring rod. The remaining 1000 mg/L primary stock solution was decanted to a volume of ~15 L and was used as the highest nominal test concentration. LDW without the addition of any test material was utilized as the water control. All test solutions were clear and colorless.

Test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solutions 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):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test organism was the freshwater fish, the rainbow trout (Oncorhynchus mykiss). This species is widely accepted and recommended for toxicity testing by the test guidelines. The fish were obtained as juveniles from Troutlodge Inc., Sumner, Washington, and assigned a lot number (RBT 030116). Upon arrival, the juveniles were visually inspected and put into a holding tank under conditions similar to the test (i.e., test temperature and dilution water) for a minimum of 14 days. The fish were observed daily for mortality to evaluate the health of the population. The mortality did not exceed 5% (0.3% actual) of the population in the 48-hour period prior to test initiation. Feed was withheld for a minimum of 24 hours prior to test initiation (~33 hours actual).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

Water control = 64 mg CaCO3/L
Highest test concentration (704 mg/L, measuredl) = 64 mg CaCO3/L

Test temperature:

Temperatures ranged from 13-14 °C.

pH:

pH ranged from 7.0-7.9.

Dissolved oxygen:

Dissolved oxygen levels ranged from 6.7-9.0 mg/L (65-87% air saturation).

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations = 0 (water control), 63, 125, 250, 500, 1000 mg/L
Measured concentrations = < lowest limit quantified (water control), 57.2, 93.9, 174, 326, 704 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 17 L glass cylindrical jars (30.5 X 30.5 cm)
- Type (delete if not applicable): loosley covered
- Material, size, headspace, fill volume: 15L
- Aeration: No
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.3 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 analysis of the dilution water indicated that TOC was 0.95 mg/L, TSS was < 3 mg/L, and concentrations of contaminants were below levels that may have interfered with the results of this test.

OTHER TEST CONDITIONS
- Adjustment of pH: the 1000 mg/L stock solutin was pH adjusted utilizing 5N sodium hydroxide (initial pH = 3.4; final pH = 8.0).
- Photoperiod: 16 hrs light/8 hrs dark
- Light intensity: 536-654

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The fish were observed at approximately 24, 48, 72 and 96 hours of exposure (±1 hour from exposure initiation) for mortality (no response to touching of the caudal peduncle and no opercula movement) and sublethal effects (behavioral and/or gross pathological findings). Dead fish were recorded and removed when observed. Sublethal effects were also recorded when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0 (water control), 63, 125, 250, 500, and 1000 mg octenylsuccinic acid/L
- Range finding study
- Test concentrations: 0 (water control), 10, 25 and 100 % of a 1000 mg octenylsuccinic acid/L saturated solution. Test solutions were prepared from a 1000 mg octenylsuccinic acid/L saturated solution (equal to the high test concentration). An aliquot containing 30,000 mg (30 g) octenylsuccinic acid (no adjustment for purity) was added to a glass carboy containing 30 L of LDW. The mixture was stirred on a magnetic stir plate and was subsequently filtered (0.45 μm) creating a saturated solution. The mixture was slightly cloudy with foam/bubbles on the surface prior to filtration. After filtration, the resulting saturated solution was pH-adjusted with 1N sodium hydroxide and 1N hydrochloric acid (initial pH = 3.2; final pH = 7.6). Prior to pH adjustment, the saturated solution was clear and colorless with foam/bubbles at the surface. Following pH adjustment, the solution was clear and colorless. The remaining test solutions were prepared as dilutions of the saturated solution.
- Results used to determine the conditions for the definitive study: Following 96 hours of exposure, one mortality (1 of 5) was observed in the 100 % test level. The remaining test organisms (4 of 5) at this test level appeared normal over the course of the 96-hour exposure. No mortality or sublethal effects were observed in the remaining test levels, including the water control, during the 96-hour static exposure. Based on information from the range-finding test, one replicate at a single nominal exposure concentration of 1000 mg octenylsuccinic acid/L (limit test), was used in a first attempt at the definitive study. One additional replicate of lab dilution water (LDW) served as the water control. Following 48 hours of exposure, mortality was observed in 60% (6 of 10) of the test organisms at the limit concentration (1000 mg/L nominal). Based on the excessive mortality, the limit test was considered invalid and was terminated following the 48-hour observations. Thus, a second attempt at the definitive study was conducted with nominal test concentrations of 0 (water control), 63, 125, 250, 500 and 1000 mg octenylsuccinic acid/L..

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 704 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 704 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 704 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

559 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

326 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks:

and sublethal effects

Details on results:

The mean fish wet (blotted dry) weight was 0.572 g (range: 0.478 to 0.669 g) and the mean total fish length was 4.3 cm (range: 4.0 to 4.5 cm). The length of the largest fish was no more than twice that of the shortest fish. The biological loading rate, based on the test solution volume at any one time (instantaneous loading rate) and calculated as [(mean fish weight of 0.572 g × 7 fish per vessel)/15 L of solution per vessel], was 0.3 g fish/L of test solution.

The fish were observed for mortality and sublethal effects at approximately 24, 48, 72, and 96 hours of exposure. Sublethal effects such as partial loss of equilibrium, complete loss of equilibrium, lethargy, swimming at the surface, melanosis, and immobility were observed at the 704 mg octenylsuccinic acid/L test level. These sublethal effects were observed following 24 hours of exposure and were apparent throughout the remainder of the exposure period. Mortality was observed in 43 (3 of 7) and 71 % (5 of 7) of the fish at the high test level following 72 and 96 hours of exposure, respectively. No fish mortality or sublethal effects were observed at the remaining test levels or in the water control over the course of the exposure period. Since mortality and sublethal effects were only observed at the highest exposure concentration (704 mg/L), which is expected to
represent the solubility limit of the test material under the conditions tested, it is possible that the observed toxicity could include physical effects of undissolved test material.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

An appropriate U.S. EPA computer program was used to calculate the 24-, 48-, 72-, and 96-hour LC50 values (the concentration estimated to result in 50 % mortality of the test population within the respective exposure periods) and corresponding 95 % confidence intervals, when possible. Two statistical methods were available: Probit Program, Version 1.5, and Trimmed Spearman-Karber (TSK) Program, Version 1.5. The order of preference used to select the method for reporting LC50 values was Probit analysis followed by trimmed Spearman-Karber. However, the appropriateness of a given method was determined by the concentration-response data, e.g., the number of concentrations resulting in mortality between 0 and 100 %. The NOEC was empirically determined based on biological interpretation of the data, and/or the highest concentration tested exhibiting no significant fish mortality or sublethal effects as determined by a Fisher’s Exact Test.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity values for Oncorhynchus mykiss exposed to octenylsuccinic acid, over a 96-hour static exposure period and based on mean measured concentrations, were as follows:
• The 24-, 48-, and 72-hour LC50 values: >704 mg/L (highest concentration tested)
• The 96-hour LC50 value: mg/L (95% confidence interval = non calculable)
• The 96-hour NOEC: 326 mg/L (highest concentration exhibiting no mortality or sublethal effects.)
Based on these results and the observed solubility behavior under the conditions tested, the test material can be regarded as not acutely toxic to Oncorhynchus mykiss at its solubility limit.
(~ 704 mg/L).

Executive summary:

The purpose of this study was to assess the potential effects of octenylsuccinic acid to the freshwater fish, the rainbow trout, Oncorhynchus mykiss. The study was performed under static exposure conditions for 96 hours with target nominal concentrations of 0 (water control), 63, 125, 250, 500, and 1000 mg octenylsuccinic acid/L. Observations for mortality and sublethal effects were made at approximately 24, 48, 72, and 96 hours (±1 hour from exposure initiation). Temperatures during the exposure period ranged from 13 -14 °C. Dissolved oxygen levels ranged from 6.7-9.0 mg/L (65-87% air saturation). The pH ranged from 7.0-7.9 and the light intensity ranged from 536-654 lux.

Test solutions were analyzed at test initiation and termination by high performance liquid chromatography/mass spectrometry (HPLC/MS). None of the analyses of the water control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 4.16 mg octenylsuccinic acid/L. Mean measured concentrations were <LLQ, 57.2, 93.9, 174, 326 and 704 mg octenylsuccinic acid/L. These analytical results indicate that the test material was stable in the exposure medium over the 96-hour exposure period, and that the test material was not fully soluble at the 1,000 mg/L nominal concentration. The data collected were used to calculate the 24-, 48-, 72-, and 96-hour LC50 values (the concentrations estimated to result in 50% mortality of the test population after 24, 48, 72, and 96 hours of exposure, respectively) and a 96-hour no-observed-effect concentration (NOEC).

The acute toxicity values for rainbow trout (O. mykiss) exposed to octenylsuccinic acid over a 96-hour static exposure period and based on mean measured concentrations were as follows:

• 24-, 48-, and 72-hour LC50 : >704 mg/L (highest concentration tested)

• 96-hour LC50 : 559 mg/L

• 96-hour NOEC: 326 mg/L (Based on the highest concentration exhibiting no mortality or sublethal effects)

Based on these results and the observed solubility behavior under the conditions tested, the test material can be regarded as not acutely toxic to Oncorhynchus mykiss at its solubility limit (~ 704 mg/L).

Description of key information

A single study (Reliability 1) of the short-term toxicity of octenylsuccinic acid to fish is available. The GLP compliant study was performed according to the OECD 203 testing guideline. The reported 96-hour LC50 value for mortality was 559 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

559 mg/L

Additional information

In a single acute toxicity study with the freshwater fishOncorhynchus mykiss, organisms were exposed to nominal concentrations of 0, 63, 125, 250, 500, and 1000 mg octenylsuccinic acid/L for 96 hours under static conditions. Test solutions were analyzed for octenylsuccinic acid concentrations by high performance liquid chromatography/mass spectrometry at test initiation and termination. The resulting 96-hour LC50 value for mortality based on mean measured concentrations was 559 mg/L, with a no-observed-effect concentration (NOEC) of 326 mg/L.