Alcohols, C13-15-branched and linear

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:

From Feb. 2022 to Jul. 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

test was conducted utilizing modified exposure media, closed test system, and reduced inoculation density. Test with reference substance under same conditions indicates that these deviations from test guidelines do not affect the results.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: 99.842 %
- Water solubility: 0.7 mg/L ± 0.06 mg/L

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
exposure start (0h): uninoculated, replicate 7; end of exposure (72 h) combined inoculated replicates of each test group and replicate 0 uninoculated from each test group
- Sampling method: For each concentration, two retained samples were be taken at each time point.
- Sample storage conditions before analysis: freezer (at approx.-20 °C)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: according to OECD 23 (poorly water soluble)
- Loading rate: 0 (control), 1, 3.2, 10, 32, and 100 mg/L
- Controls: analytical monitoring technical not feasible
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no, clear appearance and negative Tyndall effect

Test organisms (species):

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

Details on test organisms:

-Test species and strain: Raphidocelis subcapitata KORSHIKOV(SAG 61.81)
- Age (on study day 0): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Supplier: Collection of algal cultures in University of Göttingen/Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23°C

pH:

7.5-10.1

Nominal and measured concentrations:

Nominal: 1, 3.2, 10, 32, 100 and control; Measured: analytically concentration verification is technical not feasible therefore nominal concentrations were used according to the guidance OECD 23

Details on test conditions:

TEST SYSTEM
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps, 100 ml fill volume
- Test volume: 300 mL
- Initial cells density: 0.3 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of other replicates: 1 per test group for concentration control analysis; 1 per test group uninoculated for fluorescence background determination and test substance stability

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes (2.8 to 7.0 by 1M HCl)
- Photoperiod: permanent illumination
- Light intensity and quality: 5378 - 5661 lux (mean: 5489 lux, coefficient of varation: 2.71%) at a wavelength of 400 - 700 nm
- Stirring rate: Continuous (approx. 130 rpm)
- Control of conditions: pH (0 and 72 h); Fluorescence (0, 24, 48 and 72 h), light (0 and 72 h)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorometer, correlation between fluorescence and cell density was determined by direct microscopic count (two counts in a Neubauer haemocytometer. These data were used to derive a linear correlation between fluorescence and cell density.
- Other: Algal morphology (microscopically)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: <= 3.2
- Justification for using less concentrations than requested by guideline:
- Range finding study; yes according to GLP but without status
- Results used to determine the conditions for the definitive study: E(fluorescence)C50 >100 mg/L; ErC50 >100 mg/L

CULTURING APPARATUS
-Details on culturing apparatus used: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorphenol

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material loading rate

Basis for effect:

growth rate

Details on results:

OBSERVATIONS
- Algae morphology: no remarkable observations
- Dissolution behavior: clear test solution, green coloration caused by algae after 48 h

Results with reference substance (positive control):

- Results with reference substance valid? yes (ErC50 = 0.9 – 4.4 mg/L after 72 hours)
- EC50: 2.13 mg/L
- Other: conducted under same modified conditions as the study

Reported statistics and error estimates:

TOXRAT Professional 2.10 was used for statistical evaluation. The data are illustrated using plots of percent inhibition (response) versus concentration. ECx values and confidence limits were calculated by probit analysis (Finney, 1971). The LOEC was determined by comparing the means of the calculated yield or growth rate of the various concentration levels with the control using Dunnett’s multiple t-test (onesided). The NOEC was the next tested concentration below the LOEC

Table 1: Effect concentrations in mg/ L based on loading rate obtained after 72 h

Algal Yield [mg/L]		Algal Growth Rate [mg/L]
EyL10 confidence limits 95%:	>100 –	ErL10 confidence limits 95%:	>100 –
EyL20 confidence limits 95%:	>100 –	ErL20 confidence limits 95%:	>100 –
EyL50 confidence limits 95%:	>100 –	ErL50 confidence limits 95%:	>100 –
NOEyL	≥100	NOErL	≥100
LOEyL	>100	LOErL	>100

Validity criteria fulfilled:

yes

Conclusions:

No inhibition of algal growth was observed up the highest tested concentration.

Executive summary:

An experimental study according to OECD guideline 201 and GLP regulations was conducted to assess the toxicity of the test item to aquatic algae. The test design was slightly modified due to poor water solubility of the test item (UVCB) according to the OECD guidance 23. The study was performed in a closed system and the inoculum density was reduced. An additional reference toxicant test was conducted with the same method modifications described in this study (modified exposure media, closed test system, and reduced inoculation density) to determine any effect on algal response to toxic stress. The ErC50 (72 h) of the control substance 3,5 Dichlorphenol was 3.05 mg/L. These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study.

Cultures of Raphidocelis subcapitata were exposed to the test item at loading rates of 0 (control), 1, 3.2, 10 32 and 100 mg/L under static conditions for 72 h in a closed system. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. After 72 h of exposure no growth inhibition was determined up to the highest tested concentration. Therefore, the effect values were >100 mg/L.

The concentration of test substance in test media could not be verified analytically, therefore the effect concentrations are based on the loading rate used to prepare the test substance WAF (Water accommodated fraction) solutions. According to guidance in OECD 23, for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or in the case of water accommodated fractions on the loading rate.

Description of key information

There is a high probability that the test item is not acutely harmful to aquatic algae (EL50 (72 h) > 100 mg/L.

Key value for chemical safety assessment

Additional information

An experimental study according to OECD guideline 201 and GLP regulations with Raphidocelis subcapitata is available to assess the toxicity of the test item to aquatic algae. Since the test item is a complex mixture (UVCB) with poor water solubility

 the test design was slightly modified according to the OECD guidance 23. The study was performed in a closed system and the inoculum density was reduced.

The functionality and validity of the test system was confirmed by an additional test with a reference substance conducted with the same method (modified exposure media, closed test system, and reduced inoculation density). The ErC50 (72 h) of the control substance 3,5 Dichlorphenol was 3.05 mg/L. These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study.

Cultures of Raphidocelis subcapitata were exposed to the test item at loading rates of 0 (control), 1, 3.2, 10 32 and 100 mg/L under static conditions for 72 h in a closed system. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. After 72 h of exposure no growth inhibition was determined up to the highest tested concentration. Therefore, the effect values were >100 mg/L.

The concentration of test substance in test media could not be verified analytically, therefore the effect concentrations are based on the loading rate used to prepare the test substance WAF (Water accommodated fraction) solutions. According to guidance in OECD 23, for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or in the case of water accommodated fractions on the loading rate.

In conclusion no adverse effect on algae growth or morphology was observed up to the highest tested concentration which indicates that the test item is not acutely harmful to aquatic algae with a high probability.