Fatty acids, montan-wax, stearyl esters

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:

20 - 23 Aug 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Chemical analysis of test item concentrations in test media was not possible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, Annex 5 corrected 28 Jul 2011

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)

Version / remarks:

2000

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: 100 mg/L at test start and at test end
- Sampling method: At test start samples were taken from the test solution. At test end samples were taken from pooled samples.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 0.1 g test item and 1000 mL medium were magnetically stirred for 48 h to prepare the final concentration of 100 mg/L (nominal). Insoluble matter was not removed.
- Differential loading: No
- Controls: Test medium without test item.
- Evidence of undissolved material: Yes, the algae were exposed to a test solution with insoluble particles.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater algae
- Strain: ATCC 22662
- Source: American Type Culture Collection (30 Jun 1995)
- Age of inoculum: 3-d old pre-culture
- Method of cultivation: Algae were cultured in the test facility under sterile conditions, in OECD medium at 21 - 24 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.2 °C (mean)

pH:

0 h: 7.7
72 h: 7.9 - 8.0

Nominal and measured concentrations:

Control and 100 mg/L (nominal)
< LOD and < LOD (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilized 300 mL Erlenmeyer flask with gas-permeable Silicosen stoppers
- Initial cell density: 0.75 E+04 cells/mL (chlorophyll fluorescence value: 47, relative unit)
- Control end cell density (72 h): Chlorophyll fluorescence value: 3100 (mean), relative unit
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium according to testing guideline 201 (2006)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified water was used to prepare the OECD medium according to testing guideline 201
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: pH was measured at the start of exposure in a separate solution sampled from the preparation container. Temperature and light intensity were measured daily.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Alternative LED light with 90 µmol*m^-2*s^-1 (400 - 700 nm within ± 20% of nominal with ± 15% from the average light intensity)
- Conditions: Incubator with temperature and illumininationcontrol and continuous rotary shaking at approximately 100 rpm (LP-0.7LEDSS, Nippon Medical & Chemical Instruments Co., Ltd. Instrument No. SIN-SIN-008)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations (biomass): Spectrofluorometer (model RF-6000, Shimadzu, PF-005)
- Chlorophyll measurements: Every 24 h after the start of exposure. The insoluble matter in the test solution did not affect the chlorophyll fluorescence measurement.
- Observation of cell condition: System biological microscope (model BX41, Olympus Corporation), at the end of exposure

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, -2.5% growth inhibition after 3 d

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities: No
- Any stimulation of growth found in any treatment: Yes
- Any observations that might cause a difference between measured and nominal values: At the start of exposure, the test solution was a white suspension. At the end of exposure, the test solution was green due to algal growth.
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the result was within the stipulated range of 0.61 - 1.4 mg/L (mean ± 2 S.D., n = 32, fluorescent lights) to the background data in this test facility.
- ErC50 (72 h): 1.3 mg/L
- Other: Reference tests are performed periodically in the test facility. The most recent reference test was performed 21 - 24 May 2018.

Reported statistics and error estimates:

Statistical analysis of the growth rate resulted in a significant difference betweeen the treatment and control because the algal growth rate in the exposure level was higher than in the control. The statistically derived NOEC based on growth rate was ≥ solubility in medium.

VALIDITY CRITERIA

The study meets the validity criteria laid down by the guideline and is thus considered valid (Table 1).

 

Table 1. Validity criteria for OECD guideline 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The cell number in the control increased by a factor of 60 or more within 72 h.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation in the control was 12%.	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	The coefficient of variation of specific growth rates in replicate control cultures was 1.4%.	Yes

 

 

ANALYTICAL RESULTS

The measured test item concentrations at test start (0 h) and at test end (72 h) were below the limit of detection of 0.553 mg/L, reflecting the very low water solubility of the test item.

 

BIOLOGICAL RESULTS

Algal growth was statistically significantly higher in the 100 mg/L treatment level compared to the control. Microscopical observations showed that cells in the control and treatment group were normal. Thus, no adverse effects on algal growth rate were recorded up to the limit of test item solubility in the test medium, resulting in a NOErC (72 h) and ErC50 (72 h) for inhibition of algal growth rate of ≥ 100 mg/L and > 100 mg/L, respectively (nominal).

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

No effects up to the limit of water solubility (OECD 201, P. subcapitata)

Additional information

One experimental study is available, in which the toxicity of Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) to aquatic algae was assessed according to OECD guideline 201 and in compliance with GLP.

A static limit test was performed, in which the model alga Pseudokirchneriella subcapitata was exposed to a nominal limit loading rate of 100 mg/L for 72 h. Test item concentrations were analytically verified at the beginning (0 h) and end of the test (72 h) by HPLC.

The measured test item concentrations at test start (0 h) and at test end (72 h) were below the limit of detection of 0.553 mg/L, reflecting the very low water solubility of the test item.

After 72 h, algal growth was statistically significantly higher in the 100 mg/L limit concentration (mean growth inhibition rate = -5.2%) compared to the control. Microscopic observations showed that cells in the control and treatment group were normal. Thus, no adverse effects on algal growth rate were recorded up to the limit of test item solubility in the test medium, resulting in a NOELR (72 h) and EL50 (72 h) for inhibition of algal growth rate of ≥ 100 mg/L and > 100 mg/L (nominal), respectively. The study met the validity criteria of the guideline and the result is thus considered to be valid.