2-ethylhexyl 3,5,5-trimethylhexanoate

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:

23-07-2012-11-02-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Determination of algal biomass:
A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (excitation 440 nm, emission 680 nm) (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the single test concentration to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 235.12 mg of the test item in 2350 mL of test water in a glass stoppered 2 L Erlenmeyer flask (closed system).
This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 48 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
After the 48-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The negative pressure of the filtration unit was reduced as far as possible to avoid losses of volatile components of the test item during filtration. The filter was pre-conditioned with 200 mL of the dispersion. The undiluted filtrate containing the maximum concentration of dissolved test item was used as the test medium.
The test medium was prepared just before the start of the test (= start of exposure).
The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000 [OECD].

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 23 - 22 °C (start - end following 72 hours).

pH:

The pH was 7.9 - 8.3 in the control and 7.8 - 8.3 in the undiluted fitrate.

Dissolved oxygen:

not reported

Salinity:

Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:
Composition of dilution water (AAP Medium):
Ingredients Concentration
Macro-nutrients NaHCO3 250.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace elements H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L

The test was performed in a closed system, because the test item was determined to be volatile (see 3.5). To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water. Furthermore, the NaHCO3 concentration was increased in order to provide an additional carbon source. The modified medium was prepared according to the test guidelines and according to ISO Guidance Document (ISO (1999))[ISO]. The pH was 7.5.

Nominal and measured concentrations:

In the range finding test, the analytically determined concentrations of test item in the test media (undiluted filtrate = 100 mg/L loading rate) was found to be below the Limit of Quantification of 10 ug/L. Therefore, during the test period of 72 hours in the main test no analytical investigations were possible and not required.
The biological results were related to the loading rate of test item concentrations of the test.

Details on test conditions:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 - 23 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7700 Lux (range: 6980 to 8240 Lux, measured at nine places in the experimental area). The light intensity was within a ±15%-deviation from the average light intensity as recommended by the guideline.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test item had no toxic effect on the growth of the algae (average growth rate and yield of Pseudokirchneriella subcapitata ) during the test period of 72 hours up to its solubility limit in the test water under the present test conditions.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the undiluted filtrate and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.93 mg/L (Harlan Study Number D64298), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71- 1.7 mg/L).

Reported statistics and error estimates:

The average growth rate and yield at the test concentration were compared to the control values by Student t-test [Sachs, 1984].

In the control, the biomass increased by a factor of 237 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 10%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.7%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. The pH raised in the control from 7.9 at test start to 8.3 at test end fulfilling the requirement of the OECD guideline, the pH of the control medium should not increase by more than 1.5 units during the test.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the undiluted filtrate and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Validity criteria fulfilled:

yes

Conclusions:

NOEC (72h, algae): >= 100 mg/L (loading rate)

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 48 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion was used as the test medium. This corresponds to a theoretical concentration of 100 mg/L. Additionally, a control was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

No analytical work was performed due to the specific properties of the test item (low solubility in water).

In the study, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to its solubility limit in the test water under the present test conditions and thus EC50 and LOEC were >100 mg/L and the NOEC >= 100 mg/L (both for growth rate and yield).

Description of key information

In conclusion, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to its solubility limit in the test water under the present test conditions. 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonicated for 15 minutes and continuously stirred at room temperature in the dark over 48 hours, followed by filtration of the dispersion. The undiluted filtrate was used as the test medium, corresponding to a theoretical concentration of 100 mg/L. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

No analytical work was performed due to the specific properties of the test item (low solubility in water).

In the study, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to its solubility limit in the test water under the present test conditions and thus EC50 and LOEC were >100 mg/L and the NOEC >= 100 mg/L (both for growth rate and yield).