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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jan 2018 - 16 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Dose Verification:
The concentration of the active ingredient, 3 methyl sulfolane, in the test item was determined in each new test solution at 0 hours and in each old test solution at 72 hours by validated standard analytical methods. A blank consisting of only dilution water was also analyzed.
Vehicle:
no
Details on test solutions:
Test Item Preparation:
For the limit test, the stock solution of 200 mg/L concentration was prepared by dissolving 0.16 g of test substance into 800 mL of OECD medium and mixing for 15 minutes. An aliquot (600 mL) of the stock solution was then combined with 600 mL of algae to achieve the desired 100 mg/L concentration for testing. A positive control was prepared by combining 0.004 g of zinc chloride (Mfg: Acros, Lot: A0371487, Exp: 27 Feb 2022) with 200 mL of dilution water, to which was added 200 mL of algae, for a final concentration of 10 mg/L.

The definitive stock solution was prepared by dissolving 0.20 g of test item in 1000 mL of OECD medium and mixing for 15 minutes. This stock solution was diluted with dilution water to make five different test item solutions with five different concentrations for the definitive test. A test item control solution was made using only dilution water. A positive control solution was prepared using zinc chloride.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Experimental Organism
- Species: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), also known as Raphidocelis subcapitata
- Justification for Species: One of the freshwater alga species specified in the regulations
- Source: STILLMEADOW, Inc. cultures
- Age: Mass culture initiated 3-7 ± 1 days prior to dosing

Organism Husbandry
- Test Room:
- Range-Finder: Environmentally Controlled Chamber A
- Limit: Environmentally Controlled Chamber A
- Definitive: Algae Room
- Test Chambers: 250 mL sterile Erlenmeyer flasks with foam stoppers; filled with 100 mL of the appropriate solution
- Dissolved Oxygen Concentrations: Test containers received constant agitation on shaker table set at approximately 100 cycles/minute
- Test/Nutrient Medium: OECD media with a pH of 8.1 ±0.1.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Limit: 22-24ºC
Definitive: 22-25ºC
pH:
Limit: 8 - 7.9 (control)
Definitive: 8 - 8.6 (control)
Nominal and measured concentrations:
Limit test
Nominal: 100 mg/L
Measured geometric mean: 88.8 mg/L

Definitive test
Nominal: 6.25 mg/L, 12.5, 25, 50 and 100 mg/L.
Measured geometric mean: 1.4, 4.9, 18.6, 44.8 and 89.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks with foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250ml, filled with 100 mL of the appropriate solution
- Aeration: Test containers received constant agitation on shaker table set at approximately 100 cycles/minute
- Initial cells density:
Range-Finder: Initial cell density was 25,000 cells/mL
Limit: Initial cell density was 25,000 cells/mL
Definitive: Initial cell density was 50,000 cells/mL
- Control end cells density: Average 163.3 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6 and 3 for positive control

GROWTH MEDIUM
- Standard medium used: yes (OECD media)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: For the range-finding, limit and definitive test, the following measurements were made. The initial pH of each dosing solution and control solution were measured and recorded. The final pH of each treatment and control replicate was measured and recorded. Chamber temperature was measured at 0 hours and daily with a calibrated thermometer. Daily maximum and minimum temperatures were also recorded. Light intensity was measured daily in at least 5 locations in the test areas at the height of the test solution in the test chambers.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous lighting (4440-8880 lux ±15%)
- Actual Light Intensity: Range-finding: 4020-4920 lux
Limit: 4000-4880 lux
Definitive: 3860-5310 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: observation of inhibition were made for each test container by measuring the mean cell density with a hemocytometer.

TEST CONCENTRATIONS
- Range finding study: A preliminary range-finding test was conducted to determine the concentrations needed for the definitive test. The five concentrations used were 1 mg/L, 10 mg/L, 100 mg/L, 500 mg/L and 1000 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
zinc chloride
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 90 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 90 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Evaluation of Results for Limit Test
For the limit test, at 72 hours, the test concentration of 3-Methyl Sulfolane that resulted in EyC50 and ErC50 was determined to be > 88.8 mg/L. The yield and growth rate NOEC were <88.8 mg/L. The yield and growth rate LOEC were 88.8 mg/L.

For the definitive test:
the ErC10, at 72 hours, for 3-Methyl Sulfolane, was determined to be 11.2 mg/L. The ErC50 for 3-Methyl Sulfolane was determined to exceed the highest dose tested (90 mg/L). The growth rate NOEC for 3-Methyl Sulfolane was determined to 1.4 mg/L and the LOEC was 4.9 mg/L. For the definitive test, at 72 hours, the test concentration of 3-Methyl Sulfolane that resulted in EyC10 was determined to be 0.12 mg/L. The EyC50 was determined to exceed the highest dose tested (90 mg/L). At 72 hours, the cell density NOEC was determined to be 1.4 mg/L and the LOEC was 4.9 mg/L.

Test Validity
Per protocol, the test was considered valid because the 72-hour terminal control increased by a factor of at least 16 times. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 17.5% in the definitive test and 9.9% in the limit test. Additionally, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 3.5% (<7.0% per OECD 201). Although initial cell density exceeded 10,000 cells/mL (recommended by OECD 201), this had no adverse effect on the exponential growth throughout the incubation period without risk of nutrient depletion or on the integrity of the study.
Results with reference substance (positive control):
No growth occurred in the limit test or the definitive test.
Reported statistics and error estimates:
The mean cell counts of the test concentration containers were compared to the control counts to determine the concentration of the test item which resulted in a Median Effective Concentration (EC50) and the No Observed Effect Concentration (NOEC). The NOEC and EC50 were determined by a commercially available statistical program (ToxCalc Version 5.0. Probit analysis was used for ErCx and EyCx determination for the definitive test.
Validity criteria fulfilled:
yes
Remarks:
> 16-fold increase in controls, mean of coefficients of variation < 35%, specific coefficient of variation < 7%
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, 3-Methyl Sulfolane reduced growth rate and inhibited the yield of this fresh water algae species significantly at the 4.9 mg/L, 18.6 mg/L, 44.8 mg/L and 89.9 mg/L concentrations tested.

The EC50 for growth rate inhibition (72h-ERC50) exceeded 90 mg/L, the highest dose tested, based on analytically confirmed concentrations.
The EC50 for yield inhibition (72h-EYC50) exceeded 90 mg/L, the highest dose tested, based on analytically confirmed concentrations.
The 72h-NOEC for growth rate and yield inhibition based on statistical significance was 1.4 mg/L.

Executive summary:

A full OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata based on the results of a preceding range-finding test. Initially, a limit test was conducted using 100 mg/L concentration. To determine a No Observed Effect Concentration (NOEC), a definitive test was conducted. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg 3-Methyl Sulfolane per litre under static conditions. A positive control group consisted of three flasks treated with 10 mg/L zinc chloride. Dose verification was conducted by gas chromatography (GC) and performed during the study on new test solutions sampled at 0 and on old test solutions at 72 hours. In the definitive test, the dose verified concentrations were calculated using geometric means and were determined to be 1.4 mg/L, 4.9 mg/L, 18.6 mg/L, 44.8 mg/L and 89.9 mg/L for 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L and 100 mg/L, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of the present study 3-Methyl Sulfolane reduced growth rate and inhibited the yield of this fresh water algae species significantly at the 4.9 mg/L, 18.6 mg/L, 44.8 mg/L and 89.9 mg/L concentrations tested.

For the definitive test, the ErC10, at 72 hours, for 3 Methyl Sulfolane, was determined to be 11.2 mg/L. The ErC50 for 3 Methyl Sulfolane was determined to exceed the highest dose tested (> 90 mg/L) based on geometric mean concentration. The growth rate No Observed Effect Concentration (NOEC) for 3 Methyl Sulfolane was determined to 1.4 mg/L.

For the definitive test, at 72 hours, the test concentration of 3 Methyl Sulfolane that resulted in EyC50 was determined to exceed the highest dose tested (> 90 mg/L) based on geometric mean concentration. At 72 hours, the cell density NOEC was determined to be 1.4 mg/L. The EyC10 was determined to be 0.12 mg/L.

Description of key information

A full OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata based on the results of a preceding range-finding test. Initially, a limit test was conducted using 100 mg/L concentration. To determine a No Observed Effect Concentration (NOEC), a definitive test was conducted. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg 3-Methyl Sulfolane per litre under static conditions. A positive control group consisted of three flasks treated with 10 mg/L zinc chloride. Dose verification was conducted by gas chromatography (GC) and performed during the study on new test solutions sampled at 0 and on old test solutions at 72 hours. In the definitive test, the dose verified concentrations were calculated using geometric means and were determined to be 1.4 mg/L, 4.9 mg/L, 18.6 mg/L, 44.8 mg/L and 89.9 mg/L for 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L and 100 mg/L, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the conditions of the present study 3-Methyl Sulfolane reduced growth rate and inhibited the yield of this fresh water algae species significantly at the 4.9 mg/L, 18.6 mg/L, 44.8 mg/L and 89.9 mg/L concentrations tested.

For the definitive test, the ErC10, at 72 hours, for 3 Methyl Sulfolane, was determined to be 11.2 mg/L. The ErC50 for 3 Methyl Sulfolane was determined to exceed the highest dose tested (> 90 mg/L) based on geometric mean concentration. The growth rate No Observed Effect Concentration (NOEC) for 3 Methyl Sulfolane was determined to 1.4 mg/L.

For the definitive test, at 72 hours, the test concentration of 3 Methyl Sulfolane that resulted in EyC50 was determined to exceed the highest dose tested (> 90 mg/L) based on geometric mean concentration. At 72 hours, the cell density NOEC was determined to be 1.4 mg/L. The EyC10 was determined to be 0.12 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
90 mg/L
EC10 or NOEC for freshwater algae:
11.2 mg/L

Additional information

The ErC50 value was higher than the highest dose tested (nominally 100 mg/L).

Based on geomean measured concentrations this equals > 90 mg/L.