alkylphenol isomere mixture

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-30 untill 2021-11-26 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Version: 2009

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Version: 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling method: 1.0, 10.6 and 51.5 mg/L at 0 and 72 hours and 16 mg/L at 0 and 24 hours, control at 72 hours
Sample storage conditions before analysis: The samples were analysed immediately after sampling.

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. 100.4 mg of the test item were added to a 1 litre portion of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 µm and an aseptic filter Sartobran sterile capsules (membrane material: cellulose acetate) with a pore size of 0.45 µm (pre-filter) + 0.2 µm (main filter).
The solution was dissolved and no particles were still visible (no Tyndall effect was observed). The pH was measured to be 7.8

To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 500 mL. 100 mL of the prepared solutions were taken per replicate and 0.772 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Maintenance
and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a particle counter, Z2, Beckmann Coulter.
Preparation of pre cultures : Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (annex 1).
Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mg/L CaCO3.

Test temperature:

During the test a temperature range of 21 - 24 °C with a maximum fluctuation of +/- 2 °C was maintained in the test vessels.

pH:

7.8 - 8.7

Nominal and measured concentrations:

1.0, 2.2, 4.8, 10.6, 23.4 and 51.5 mg/L (nominal) corresponding to 0.601, 7.125, 32.829 and 33.760 mg/L (measured initial concentrations)
The measured test item concentrations for the nominal concentrations 2.2, 4.8 and 23.4 mg/L were estimated from the analytical mean (64.1 %) determined with actual measurements of the nominal test item concentrations 1.0, 10.6 and 51.5 mg/L at test start.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type (delete if not applicable): closed: covered with cotton stoppers
- Material, size, headspace, fill volume: 100 mL test medium
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was
maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range
400 to 700 nm. Temperature was measured and recorded daily.
- Pre-cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Growth medium (OECD medium of OECD TG 201, annex 1) was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light: 8 h dark
- Light intensity: in the range 60 to 120 µE x m-2 x s-1 or an equivalent range of 4000 to 8000 lux,
was measured. The light intensity was checked before the start of the study
- Cell density measurements: Cell densities were measured in a particle counter (Z2, Beckmann
Coulter) by taking aliquots from each test flask, which were not replaced. Cell densities were
determined by means of a microscopic counting chamber by taking small aliquots from each
test flask, which were not replaced.
- Experimental design: 6 test concentrations plus 1 control, 3 replicates per concentration, 3
replicates per control. The initial cell density in the test cultures was approximately 5000 cells
per millilitre. Additionally the highest test concentration without algae was investigated.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

MAIN TEST
In the main test, the algae were exposed to the test item added to dilution water at a range of concentrations for a period of 72 hours. Defined concentrations of the test item led to a certain inhibition of algal yield and growth rate at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. The 72 hour EC 10 and EC 50 were calculated by probit analysis.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see details on results

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see details on results

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see details on results

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see details on results

Details on results:

NOEC/LOEC determinations for growth rate could not be performed due to mathematical reasons. In a regulatory context EC 10 values are generally regarded as surrogates for the NOEC. In the present case, 10 % effect on growth rate is effectuated by a concentration > 3.1 mg/L and < 7.1 mg/L and therefore NOEC and LOEC were read directly from these results (NOEC = 3.1 mg/L and LOEC = 7.1 mg/L).

Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU).

The results are expressed in terms of measured initial concentrations.
Measured test item exposure concentrations ranged from 59.5 % to 68.0% of their nominal target values at 0 hours and from 52.7 % to 61.6 % of their nominal target values at 72 hours.

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

Validity criteria fulfilled:

yes

Remarks:

-Factor of the biomass parameter: 178.0, thus >16 -Mean coefficient of variation (control from 0 to 72 h): 7.7%, thus <35% -Coefficient of variation of the mean specific growth rate (control from 0 to 72 h): 0.9%, thus <7%

Conclusions:

A study was performed to assess the toxicity of 'reaction products of m-cresol and propene, high boiling alkylphenol isomers' on the growth rate and the yield of the freshwater algal species Desmodesmus subspicatus. The following results (based on growth rate, index r) were obtained:

ErC 50 (0-72 h): 5.0
ErC 10 (0-72 h): 3.4
NOEC [r]: 3.1#
LOEC [r]: 7.1#

# NOEC/LOEC determinations for growth rate could not be performed due to mathematical reasons. In a regulatory context EC 10 values are generally regarded as surrogates for the NOEC. In the present case, 10 % effect on growth rate is effectuated by a concentration > 3.1 mg/L and < 7.1 mg/L and therefore NOEC and LOEC were read directly from these results.

Executive summary:

A study was performed to assess the adverse effects of Reaction products of m-cresol and propene, high boiling alkylphenol isomers on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 1.0, 3.2, 10, 32 and 100 mg/L of Reaction products of m-cresol and propene, high boiling alkylphenol isomers dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and a folded filter (pore size 7-12 µm) and an aseptic filter (pore size 0.45 µm
(pre-filter) + 0.2 µm (main filter).

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r) and yield (y), relative to control cultures grown under identical conditions. The following values were determined:

Results [mg/L]:

ErC 50 (0-72 h): 5.0

ErC 10 (0-72 h): 3.4

NOEC [r]: 3.1#

LOEC [r]: 7.1#

# NOEC/LOEC determinations for growth rate could not be performed due to mathematical reasons. In a regulatory context EC 10 values are generally regarded as surrogates for the NOEC. In the present case, 10 % effect on growth rate is effectuated by a concentration > 3.1 mg/L and < 7.1 mg/L and therefore NOEC and LOEC were read directly from these results.

The results are expressed in terms of measured initial concentrations.
Measured test item exposure concentrations ranged from 59.5 % to 68.0% of their nominal target values at 0 hours and from 52.7 % to 61.6 % of their nominal target values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of Reaction products of m-cresol and propene, high boiling alkylphenol isomers on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC)
No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 1.0, 3.2, 10, 32 and 100 mg/L of Reaction products of m-cresol and propene, high boiling alkylphenol isomers dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and a folded filter (pore size 7-12 µm) and an aseptic filter (pore size 0.45 µm
(pre-filter) + 0.2 µm (main filter).

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r) and yield (y), relative to control cultures grown under identical conditions. The following values were determined based on the mean measured start concentrations:

Results [mg/L]:

ErC 50 (0-72 h): 5.0

ErC 10 (0-72 h): 3.4

NOEC [r]: 3.1#

LOEC [r]: 7.1#

# NOEC/LOEC determinations for growth rate could not be performed due to mathematical reasons. In a regulatory context EC 10 values are generally regarded as surrogates for the NOEC. In the present case, 10 % effect on growth rate is effectuated by a concentration > 3.1 mg/L and < 7.1 mg/L and therefore NOEC and LOEC were read directly from these results.

The results are expressed in terms of measured initial concentrations.
Measured test item exposure concentrations ranged from 59.5 % to 68.0% of their nominal target values at 0 hours and from 52.7 % to 61.6 % of their nominal target values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

5 mg/L

EC10 or NOEC for freshwater algae:

3.4 mg/L