butanedioic acid, 2-octenyl

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were collected from the bulk test solutions at test initiation and from pooled replicate samples at termination. Blank replicates were analyze separately. To assess analytical method precision and solution homogeneity, three additional samples were collected on day 0 from the highest and l west concentration bulk test solutions. To confirm octenylsuccinic acid concentrations, the collected samples were analyzed using high performance liquid chromatography/mass spectrometry (HPLC/MS).

The bulk exposure solutions (AAP medium control 9.5, 31, 987, 313 and 1000 mg/L solutions) were sampled for analytical confirmation of test material. At test initiation (0 hours), single aliquots (~ 5 mL) of AAP medium control, 31, 98, and 313 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 9.5 and 1000 mg/L bulk exposure solutions were collected from the top, middle, and bottom of their container to evaluate homogeneity.

The contents of algal test vessels were pooled at exposure termination (72 hours) to provide one composite sample at each exposure concentration and sampling point for analytical confirmation. Aliquots (2 mL) were collected using an Eppendorf pipette and transferred to 20 mL glass vials. Pooled samples were vortex-mixed. Single aliquots (5 mL) were collected from the single counting blank test vessel (containing test material but not inoculated with algae) 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:

A primary stock solution was prepared at the highest test concentration of 1000 mg octenylsuccinic acid/L. Octenylsuccinic acid (actual weight: 2.00 g) was added to 2 L of algal assay procedure medium(AAP) to achieve 1000 mg octenylsuccinic acid /L. The flask was covered, and the solution was shaken vigorously until the solution appeared homogenous. The stock solution was then pH adjusted utilizing 1N sodium hydroxide (initial pH = 3.1; final pH = 7.2). Prior to pH adjustment, the solution was slightly cloudy/white. Following pH adjustment, the stock solution was clear and colorless. Remaining test solutions were prepared as dilutions of the primary stock solution.

All dilutions made from the primary stock (1000 mg/L) were shaken vigorously until the solutions appeared homogenous and were clear and colorless. 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 (AAP) was considered to represent the most probable route of exposure in the environment.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata from in-house cultures was initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 060215A) in June 2015. This species is widely accepted and recommended for toxicity testing by the test guidelines. Stock cultures of this organism were maintained axenically by periodic transfer into sterile algal assay procedure (AAP) medium. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2 ºC.

The algal inoculum for the test was prepared from a 3-day old stock culture. A Coulter Multisizer 3 (Beckman Coulter, Brea, California) was used to determine the cell density of the stock culture. This evaluation determined that a 0.424 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 10,000 cells/mL.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not measured

Test temperature:

Temperature during the exposure period was 24 ºC

pH:

The pH ranged from 7.0 to 7.2 at test initiation. At test termination the pH ranged from 7.1 to 7.4 and 6.7 to 7.1 in replicates with and without algae, respectively.

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations = 0 (AAP medium control), 9.5, 31, 98, 313, 1000 mg/L
Measured concentrations = < lowest limit quantified (AAP medium control), 8.59, 28.2, 78.7, 243, 874 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL borosilicate Erlenmeyer flasks
- Type (delete if not applicable): plugged with foam stoppers
- Material, size, headspace, fill volume: contained 50 mL test medium
- Aeration: No
- Initial cells density:
- Control end cells density: 3,366,000 at 72h
- No. of organisms per vessel: initial cell density of 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes (algal assay procedure medium-AAP)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algae was cultured and tested in freshwater algal nutrient medium (i.e., AAP medium), prepared with sterile deionized water and reagent grade chemicals. The water source for the deionized water system was municipal water produced by the City of Midland Water Treatment. The base water used to prepare the medium was passed through a series of activated carbon, (two) deionization polymer (US Filter Mixed Bed, Type 1), and a final filtration unit, prior to collection and autoclaving in clean glass containers. Prior to treatment, the base water used to prepare the medium is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The 1000 mg/L stock solution was pH adjusted utilizing 1N sodium hydroxide (initial pH = 3.1; final pH = 7.2).
- Photoperiod: 24h continuous light
- Light intensity and quality: 4480-5450 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea, California) fitted with a 100 μm aperture tube. Total cell counts were determined at approximately 24, 48, and 72 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. Two cell count readings were made per replicate and averaged. The readings for the blank replicates were used to correct for background in daily calculations. The adjusted cell counts were converted to cells x 10,000/mL (cell density) for statistical analysis and reporting. In addition, at test termination morphological observations were done on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus BH Microscope (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0 (AAP medium control), 9.5, 31, 98, 313, and 1000 mg octenylsuccinic acid/L
- Range finding study
- Test concentrations: 0 (AAP control), 1, 10, and 100 mg octenylsuccinic acid/L. Test solutions were prepared similarly as in definitive test. A primary stock solution was prepared at the highest test concentration of 100 mg octenylsuccinic acid/L. Remaining bulk test solutions were prepared as dilutions of the primary stock. All bulk test solutions were pH adjusted prior apportioning into individual test vessels. Two replicate flasks per treatment level (6 for control) were inoculated with a predetermined aliquot of algal inoculum to achieve 10,000 cells/mL. An uninoculated replicate (counting blank) was prepared at each treatment level and control.
- Results used to determine the conditions for the definitive study: Cell density was not inhibited for any of the treatment levels tested following 72 hours of exposure. Based on these results, the EC50 for cell yield was estimated at > 100 mg/L. Based on information from the range-finding test, the following nominal test concentrations were set for the definitive study: 0 (AAP control), 9.5, 31, 98, 313, and 1000 mg octenylsuccinic acid/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

375 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Remarks on result:

other: 95% CL = 211-665 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.59 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 874 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

78.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All cell yield data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Mean yields at 72 hours were 335.6, 327.7, 247.1, 284.3, 200.8, and 104.0 (x 10,000) cells/mL for the control, 8.59, 28.2, 78.7, 243 and 874 mg octenylsuccinic acid/L test levels, respectively. Between 0 and 72 hours, the mean inhibition response relative to the control ranged from 2 to 69% inhibition of yield. At 72 hours, yields in the 28.2, 243, and 874 mg octenylsuccinic acid/L test levels were significantly different from the control. The 78.7 mg octenylsuccinic acid/L test level was not significantly different from the control at 72 hours. However, using the most conservative approach, the 72-hour NOEC for yield is reported as 8.59 mg octenylsuccinic acid/L. The calculated EyC50 (95% confidence intervals) was 375 (211-665) mg octenylsuccinic acid.

All growth rate data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Results are presented in Table 6 (individual data are in Appendix F). Growth curves are presented in Figure 2. Mean specific growth rates between 0 and 72 hours were 1.939, 1.929, 1.833, 1.884, 1.751, and 1.550 (day-1) for the control, 8.59, 28.2, 78.7, 243 and 874 mg octenylsuccinic acid/L test levels, respectively. From 0 to 72 hours, mean inhibition response relative to the control ranged from 1 to 20% of the mean specific growth rate. Mean specific growth rates for 243 and 874 mg octenylsuccinic acid/L at 72 hours were significantly different from the control. Thus, the 0-72 hour NOEC was 78.7 mg octenylsuccinic acid/L. The calculated ErC50 was > 874 (95 % confidence interval not determined) mg octenylsuccinic acid.

Microscopic evaluation of cells at each test concentration and the control at test termination revealed no abnormal observations in the control, 8.59, 28.2, 78.7, 243 mg octenylsuccinic acid/L test concentrations. Misshapen algal cells were observed in the 874 mg octenylsuccinic acid/L test concentration.

The following acceptance criteria set forth in the OECD 201 guideline (2006) were met:
1) The cell density in the control cultures increased exponentially by a factor of at least 16 within 72 hours. The 72-hour mean cell density increased by approximately 337-fold, thereby exceeding the criteria for minimal increase in cell density.
2) The coefficient of variation average for specific growth rates throughout the exposure (days 0 – 3) in replicate control cultures was 1.5%, which is below the limit of 7% set by the guideline.
3) The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) must be 35% or less. The growth rate (per day) for each control replicate was calculated for each time period. A mean, standard deviation, and coefficient of variation was calculated for each replicate based on each daily growth rate. The mean coefficient of variation for the control replicates was 7.1%, which was below the maximum criterion.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

For each endpoint, mean values were calculated for each exposure concentration level and control level. Percent inhibition values were calculated based on differences in mean exposure concentrations compared to control values.

The data (untransformed, square root transformed for number of cells, and the log of average specific growth rate) were tested for normality using the Shapiro-Wilk’s Test (α = 0.01) and for homogeneity of variance using the Levene’s Test (α = 0.01). If the data were normal and homogeneous, a one-tailed Dunnett’s means comparison procedure was used to determine no-observed-effect concentrations (NOECs) (α = 0.05). If the assumptions of normality and/or homogeneity were not met and a nonparametric analysis was required, the ranks of the values were determined and then the above analysis was performed on the ranks. The 72-hour EyC50 (the concentration which reduces yield by 50% relative to the control) and the 72-hour ErC50 value (the concentration which reduces the average specific growth rate by 50% relative to the control) were calculated. Two sigmoid-shaped nonlinear models were used for calculation of EC50 values and 95% confidence intervals. One model used to describe the response to increasing concentrations was the four-parameter logistic model (percent inhibition versus concentration). The second model used was the cumulative normal model (response compared to log concentration). The model that best fit the experimental data was used to report study endpoints. All statistical analyses were conducted in SAS version 9.2 (SAS Institute, Cary, North Carolina). GraphPad Prism (version 6.07 for Windows, GraphPad Software) was used to draw the growth curves.

All cell yield data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). All growth rate data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01).

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity values for Pseudokirchneriella subcapitata exposed to octenylsuccinic acid over a 72-hour static exposure period and based on mean measured concentrations were as follows:

Cell yield:
72-hour EyC50 : 375 mg/L
72-hour NOEC : 8.59 mg/L

Growth rate:
72-hour ErC50 : > 874 mg/L (highest concentration tested)
72-hour NOEC : 78.7 mg/L

Executive summary:

The purpose of this study was to assess the potential effects of octenylsuccinic acid to the freshwater green alga,Pseudokirchneriella subcapitata. The study was performed for 72 hours with target nominal concentrations of 0 (algal assay procedure medium (AAP)), 9.5, 31, 98, 313, and 1000 mg octenylsuccinic acid/L. Cell counts were determined at approximately 24, 48 and 72 hours (±1 hour from exposure initiation). Temperatures during the exposure period were maintained at 24 °C. The pH ranged from 6.7-7.4 and the light intensity ranged from 4480-5450 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 media control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 4.16 mg octenylsuccinic acid/L. Mean measured concentrations were < LLQ, 8.59, 28.2, 78.7, 243 and 874 mg octenylsuccinic acid/L. The data collected were used to determine EC50(the concentration causing 50% inhibition) values for 72-hour cell yield and 0-72-hour average specific growth rate. The no-observable-effect concentrations (NOEC) were determined for each endpoint based on the highest exposure concentration for which growth parameters were not significantly different from the control.

 

The acute toxicity values forPseudokirchneriella subcapitataexposed to octenylsuccinic acid over a 72-hour exposure period and based on mean measured concentrations were as follows:

Cell yield:

72-hour EyC50: 375 mg/L

72-hour NOEC = 8.59 mg/L

Growth inhibition:

72-hour ErC50: >874 mg/L (highest concentration tested)

72-hour NOEC : 78.7 mg/L

Description of key information

A single study (Reliability 1) of the toxicity of octenylsuccinic acid to freshwater algae is available. The GLP compliant study was performed according to the OECD 201 testing guideline. The reported 72-hour EC50 values for growth rate inhibition and biomass yield were >874 mg/L and 375 mg/L, respectively. The corresponding NOEC values were 78.7 and 8.59 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

874 mg/L

EC10 or NOEC for freshwater algae:

78.7 mg/L

Additional information

In a single toxicity study with the freshwater alga Pseudokirchneriella subcapitata, organisms were exposed to nominal concentrations of 0, 9.5, 31, 98, 313, and 1000 mg octenylsuccinic acid/L for 72 hours. Test solutions were analyzed for octenylsuccinic acid concentrations by high performance liquid chromatography/mass spectrometry at test initiation and termination. The resulting 72-hour EC50 values for growth rate inhibition and biomass yield based on mean measured concentrations were greater than 874 mg/L (highest concentration tested) and 375 mg/L, respectively. The 72-hour no-observed-effect concentrations (NOEC) based on the highest measured exposure concentrations for which growth parameters were not significantly different from the control were 78.7 mg/L and 8.59 mg/L, respectively, for growth rate inhibition and biomass yield.